Biomédica
ISSN 0120-4157
Revista del Instituto Nacional de Salud
Volumen 28, Suplemento No. 1, Agosto, 2008
Bogotá, D.C., Colombia, S.A.
Proceedings of the
X International Congress on
Paracoccidioidomycosis
A Centennial Celebration
August 7-10, 2008
Intercontinental Hotel
Medellín, Colombia
Cover figures
Scanning electron microscope image of a conidium liberated from Paracoccidioides brasiliensis
mycelium. Magnification: approximately 12.000 X. Unpublished photography by WA Samsonoff,
MR Edwards, ME Salazar, LE Cano, A. Restrepo. New York State Department of Health,
Wadsworth Center for Laboratories and Research, Albany, NY, USA. and Corporación para
Investigaciones Biológicas (CIB), Medellin, Colombia.
Composite figure obtained by periodic observations of a microculture inoculated with P. brasilienisis
conidia. When freshly prepared, conidial structures - still attached to the parent mycelium - can be
seen in the lower portion of the illustration. After 72 hours of incubation at 36° C, multiple budding
yeast cells can be observed in the upper portion of the figure. Magnification: 1.000 X. Illustration
prepared by Martha Urán and Diana Molina, Corporación para Investigaciones Biológicas (CIB),
Medellin, Colombia.
Biomédica Instituto Nacional de Salud
Volumen 28, Suplemento No. 1, Bogotá, D.C., Colombia - Agosto, 2008
COMITÉ EDITORIAL
RUBÉN SANTIAGO NICHOLLS
Instituto Nacional de Salud
Bogotá, D.C., Colombia
EDITORES ASOCIADOS
LUIS ALBERTO GÓMEZ
Instituto Nacional de Salud
Bogotá, D.C., Colombia
CARLOS ARTURO HERNÁNDEZ
Bogotá, D.C., Colombia
ÁNGELA RESTREPO
Corporación para Investigaciones Biológicas
Medellín, Colombia
MARÍA CRISTINA FERRO
Investigadora Emérita
Instituto Nacional de Salud
Bogotá, D.C., Colombia
MIGUEL A. GUZMÁN
Bogotá, D.C., Colombia
RICARDO SÁNCHEZ
Universidad Nacional de Colombia
Bogotá, D.C., Colombia
LEONARD MUNSTERMANN
Yale University School of Medicine
New Haven, CN, Estados Unidos
OMAR SEGURA
Instituto Nacional de Salud
Bogotá, D.C., Colombia
SECRETARIA DEL COMITÉ LINDA GRACE MOLANO
COMITÉ CIENTÍFICO
ARNOLDO BARBOSA RAMÍREZ
Hospital Clínic, Universidad de
Barcelona, Barcelona, España
Centro de Investigação Em Saude
Da Manhiça, Manhiça, Mozambique
ANTONIO BERMÚDEZ
Instituto Nacional de Salud
Bogotá, D.C., Colombia
JORGE H. BOTERO
Universidad de Antioquia
Medellín, Colombia
VÍCTOR CÁRDENAS
University of Texas
El Paso, TX, Estados Unidos
ALBERTO CONCHA-EASTMAN
Organización Panamericana
de la Salud
Washington, D.C., Estados Unidos
ZOILO CUÉLLAR
Academia Nacional de Medicina
Bogotá, D.C., Colombia
LUIS GABRIEL CUERVO
Organización Panamericana de la
Salud
Washington, D.C., Estados Unidos
PATRICIA DEL PORTILLO
Corpogén
Bogotá, D.C., Colombia
ANDRÉS DE FRANCISCO
Foro Global para la Investigación en
Salud
Ginebra, Suiza
ENRIQUE GONZÁLEZ
University of Texas Health Science
Center at San Antonio
San Antonio, TX, Estados Unidos
JOHN MARIO GONZÁLEZ
Universidad de los Andes
Bogotá, D.C., Colombia
FERNANDO DE LA HOZ
Universidad Nacional de Colombia
Bogotá, D.C., Colombia
FELIPE GUHL
Universidad de los Andes
Bogotá, D.C., Colombia
JOSÉ LUIS DI FABIO
Organización Panamericana de la
Salud
Washington, D.C., Estados Unidos
ANTONIO IGLESIAS
Universidad Nacional de Colombia
Bogotá, D.C., Colombia
JORGE HERNANDO DONADO
Universidad Pontificia Bolivariana
Medellín, Colombia
JOSÉ FIGUEROA
World Health Organization
Ginebra, Suiza
JORGE JARA
BID/Secretaría de Salud
Tegucigalpa, Honduras
ERNESTO JARAMILLO
Organización Mundial de la Salud
Ginebra, Suiza
LUIS FERNANDO GARCÍA
Universidad de Antioquia
Medelín, Colombia
MARCELO LABRUNA
Universidade de São Paulo
São Paulo, Brasil
ALBERTO GÓMEZ
Pontificia Universidad Javeriana
Bogotá, D.C., Colombia
JAIRO LIZARAZO
Hospital Universitario Erasmo Meoz
Cúcuta, Colombia
JUAN GUILLERMO MCEWEN
Corporación para Investigaciones
Biológicas
Medellín, Colombia
VÍCTOR E. REYES
University of Texas Medical Branch
Galveston, TX, Estados Unidos
NANCY GORE SARAVIA
CIDEIM
Cali, Colombia
ROBERTO MENDOZA
The Hospital for Sick Children
Toronto, Ontario, Canada
GERZAÍN RODRÍGUEZ
Investigador Emérito
Instituto Nacional de Salud
Universidad de la Sabana
Bogotá, D.C., Colombia
ROBERT TESH
University of Texas
Galveston, TX, Estados Unidos
ALVARO MONCAYO
Universidad de los Andes
Bogotá, D.C., Colombia
GUSTAVO ROMÁN
University of Texas
Houston, TX, Estados Unidos
RICARDO NEGRONI
Hospital de Infecciosas
Francisco Javier Muñiz
Buenos Aires, Argentina
PEDRO ROMERO
Ludwig Institute for Cancer Research, Lausanne branch
Lausana, Suiza
MARÍA TERESA OCHOA
University of California Los Ángeles
Los Ángeles, CA, Estados Unidos
JUAN P. OLANO
University of Texas Medical Branch
Galveston, TX, Estados Unidos
BLANCA RESTREPO
University of Texas
Brownsville, TX, Estados Unidos
ORLANDO TORRES-FERNÁNDEZ
Instituto Nacional de Salud
Bogotá, D.C., Colombia
BRUNO TRAVI
University of Texas
San Antonio, TX, Estados Unidos
GUSTAVO VALBUENA
University of Texas
Galveston, TX, Estados Unidos
ÁLVARO RUIZ
Pontificia Universidad Javeriana
Bogotá, D.C., Colombia
JUAN MIGUEL VILLALOBOS
Universidade Federal de Rondônia
Porto Velho, Brasil
GIOCONDA SAN BLAS
Instituto Venezolano de
Investigaciones Científicas
Caracas, Venezuela
JOHN WALKER
Cideim
Cali, Colombia
ÁLVARO SANABRIA
Universidad de la Sabana
Fundación Abood Shaio
Bogotá, D.C., Colombia
MOISÉS WASSERMAN
Investigador Emérito
Instituto Nacional de Salud
Universidad Nacional de Colombia
Bogotá, D.C., Colombia
® Instituto Nacional de Salud
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INSTITUTO NACIONAL DE SALUD
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Proceedings of the
X International Congress on
Paracoccidioidomycosis
A Centennial Celebration
August 7-10, 2008
Intercontinental Hotel
Medellín, Colombia
CORPORACIÓN PARA
INVESTIGACIONES
BIOLÓGICAS
La Ciencia al Servicio de la Vida
Carrera 72A No 78B - 141 / Tel: (4) 441 08 55 / Fax: (4) 441 55 14
http://www. cib.org.co / e-mail: cib@cib.org.co / A.A 7378 / Medellín-Colombia
Committees
Organizing Committee
Juan Guillermo McEwen O. President
Luz Elena Cano R. General Secretary
Angela Restrepo M. General Coordinator
Diego Miguel Sierra B. 1st Treasurer
María del Pilar Jiménez A. 2nd Treasurer
Juliana Correa T. Secretary
Scientific Committees
International
Ricardo Negroni - Argentina
Vera L. Calich - Brazil
Eva Burger - Brazil
Marcelo Franco - Brazil
Rosana Puccia - Brazil
Gioconda San-Blas - Venezuela
Beatriz L. Gómez - United States
Agostinho de Almeida - Portugal
Colombia
Ana María García C.
Angel A. González M.
Angela M. Tobón O.
Angela Restrepo M
Beatriz H. Aristizabal B.
Catalina de Bedout G.
Juan G. McEwen O.
Luz Elena Cano R
Martha E. Urán J.
Myrta Arango A.
Editorial Commitee
Myrta Arango A.
Catalina de Bedout G.
Juan Eugenio Ochoa M.
Martha E. Urán J.
Financial Commitee
Lavive Rebaje de Álvarez
Diego Miguel Sierra B.
María del Pilar Jiménez A.
Gisela María Acevedo T.
Marcela Gallego B.
Logistic Agency
Entorno Comunicaciones:
María Eugenia Puerta V.
Olga Lucía Jaramillo E.
Graphic Design and Cover
Juan Eugenio Ochoa M.
Nelcy López M.
Diana Molina M.
Web page
Martha E. Urán J.
www.pcm2008.org.co
Powered by Interconsulting S.A.
www.interconsulting.com.co
Invited Speakers
Argentina
• Ricardo Negroni. Unidad de Micología, Hospital de Infecciosas Francisco Javier Muñiz, Buenos
Aires, Argentina.
Brazil
• André Amaral. Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidade
de Brasilia, Brasilia, DF, Brazil.
• Angela M.V.C. Soãres. Departamento de Microbiologia e Imunologia, Instituto de Biociências,
Universidade Estatal de São Paulo, Botucatú, SP, Brazil.
• Bodo Wanke. Laboratorio/Serviço de Micologia Médica, Centro de Pesquisa Hospital Evandro
Chagas (IPEC), Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
• Carla Pagliari. Departamento de Patologia, Facultade de Medicina, Universidade de São Paulo,
São Paulo, SP, Brazil.
• Carlos Taborda. Departamento de Microbiologia, Instituto de Ciências Biomédicas II (ICB-II),
Universidade de São Paulo (USP), São Paulo, SP, Brazil.
• Celia M. Soãres. Laboratorio de Biologia Molecular, Instituto de Ciências Biologicas, Universidade
Federal de Goiãnia, Goiãs, Brazil.
• Darcy Roberto Andrade Lima. Professor do Instituto de Neurologia da Universidade Federal do
Rio de Janeiro, Brazil e Director Associado de Pesquisas, ICS, Vanderbilt University, TN, USA.
• Eduardo Bagagli. Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatú, SP, Brazil.
• Eva Burger. Departamento da Imunologia, Instituto de Ciências Biomédicas (ICB), Universidade
de São Paulo (USP), São Paulo, SP, Brazil.
• Flavio Queiroz-Telles. Departamento de Saúde Comunitária, Universidade Federal do Paraná,
Curitiba, PR, Brazil.
• Gil Benard. Laboratorio de Alergia Clinica e Experimental e Imunologia. Departamento de Dermatologia, Faculdade de Medicina da Universidade de São Paulo (FMUSP), SP, Brazil.
9
• Henrique L. Lenzi. Departamento de Patologia, Fundação Oswaldo Cruz, FioCruz, Rio de Janeiro,
RJ, Brazil.
• Ildinete Silva Pereira. Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasilia, DF, Brazil.
• João Santana Silva. Departamento de Bioquimica e da Imunologia, Escola de Medicina, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
• José Daniel Lopes. Disciplina de Imunologia, Departamento de Microbiologia, Imunologia e
Parasitologia, Centro de Microscopia electrônica, Universidade Federal de São Paulo (UNIFESP),
São Paulo, SP, Brazil.
• Larissa Fernandes. Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidade de Brasilia, Brasilia, DF, Brazil.
• Ligia B. Vizeu. Departamento de Geografia, Faculdade de Filosofia, Letras e Ciências Humanas.
Universidade de São Paulo, São Paulo, SP, Brazil.
• María Adelaide Millington. GT de Pneumonias e Micoses Sistemicas, Ministerio de Saúde,
Brasilia, DF, Brazil.
• Maria Aparecida Shikanai-Yasuda. Laboratorio de Imunologia, Departamento de Doênças Infecciosas e Parasitarias, Faculdade de Medicina, Universidade de São Paulo (FMUSP), São Paulo,
SP, Brazil.
• Maria Heloisa Blotta. Departamento de Patologia Clínica, Faculdade de Ciências Médicas,
Universidade Estatal de Campinas (UNICAMP), Campinas, SP, Brazil.
• Maria José Mendes-Giannini. Departamento de Análises Clínicas, Faculdade de Ciências
Farmacêuticas, UNESP, Araraquara, SP Brazil.
• Maria Sueli Felipe. Departamento de Biologia Celular, Instituto de Ciências Biologicas, Universidade de Brasilia, Brasilia, DF, Brazil.
• Rinaldo Põncio-Mendes. Departamento de Medicina, Faculdade de Medicina, Universidade
Estatal de São Paulo, Botucatú, SP, Brazil.
• Roberto Martinez. Departamento de Molestias Infecciosas da Faculdade de Medicina de Ribeirão
Preto, Universidade de São Paulo. Ribeirão Preto, SP, Brazil.
• Rosana Puccia. Departamento de Microbiologia, Imunologia e Parasitologia, Divisão de Biologia
Celular, Universidade Federal do São Paulo, São Paulo, SP, Brazil.
• Rosely Zancopé Oliveira. Fundação Oswaldo Cruz, Instituto de Pesquisa Clínica Evandro Chagas.
Rio de Janeiro, RJ, Brazil.
• Vera L. Calich. Departamento da Imunologia, Instituto de Ciências Biomédicas (ICB), Universidade
de São Paulo (USP), SP, Brazil.
10
Central America
• Eduardo Arathoon. Director Médico Clínica Familiar “Luis Angel García”, Asociación de Salud
Integral. Hospital General San Juan de Dios, Guatemala City, Guatemala.
• Marion C. de Martin. Departamento de Microbiología, Sección de Micología, Universidad de
Panamá, Panamá, Republic of Panamá.
Colombia
• Ana María García. Corporación para Investigaciones Biológicas (CIB) and Universidad Pontificia
Bolivariana (UPB), Medellín, Colombia.
• Angel González M. Corporación para Investigaciones Biológicas (CIB), and Escuela de Microbiología,
Universidad de Antioquia (U de A), Medellín, Colombia.
• Angela M. Tobón. Corporación para Investigaciones Biológicas (CIB) and Hospital La María,
Medellín, Colombia.
• Angela Restrepo M. Corporación para Investigaciones Biológicas (CIB), Medelllín, Colombia.
• Manuel Elkin Patarroyo. Instituto de Inmunología, Bogotá, Colombia.
• Luz E. Cano. Corporación para Investigaciones Biológicas (CIB) and Escuela de Microbiología,
Universidad de Antioquia (UdeA), Medellín, Colombia.
Europe
• Agostinho João Almeida. Life and Health Sciences Research Institute (ICVS) School of Health
Sciences, University of Minho, Campus de Gualtar, Braga, Portugal.
• Christopher Kibbler. Department of Medical Microbiology, Royal Free Hospital, London, United
Kingdom.
• Neil AR Gow. School of Medical Sciences, Institute of Medical Sciences. University of Aberdeen,
Aberdeen, UK.
11
United States of America
• Beatriz L. Gómez. Mycotic Diseases Branch, Centers for Disease Control and Prevention (CDC),
Atlanta, GA, USA.
• Daniel Matute. Department of Ecology and Evolution, University of Chicago, Chicago, IL, USA.
• Garry T. Cole. Department of Biology, University of Texas at San Antonio, San Antonio, TX,
USA.
• John E. Bennett. Clinical Mycology Section, Laboratory of Clinical Investigation, National Institute
of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
• John R. Graybill. Department of Medicine, University of Texas Health Science Center at San
Antonio, San Antonio, TX, USA.
• John W. Taylor. Department of Plant and Microbial Biology, University of California, Berkeley, CA,
USA.
• Joshua Fierer. Research Service, VA San Diego Healthcare, San Diego, CA. Department of Medicine, Division of Infectious Diseases School of Medicine, University of California San Diego, USA.
• Karl V. Clemons. Division of Infectious Diseases, Santa Clara Valley Medical Center, California
Institute for Medical Research, and Division of Infectious Diseases and Geographic Medicine,
Department of Medicine Stanford University, San Jose, CA, USA.
• Mary Brandt. Mycology Disease Branch, Centers for Disease Control and Prevention (CDC),
Atlanta, GA, USA.
• Mia Champion. The Broad Institute of Massachusetts, Institute of Technology, Cambridge, MA,
USA.
• Steven K. Ault. Communicable Diseases Unit Area of Health Surveillance and Disease Management, Pan American Health Organization, Regional Office of the World Health Organization,
Washington DC, USA.
• Tom Chiller. Epidemiology Unit, Mycotic Disease Branch, National Center for zoonotic, vector borne
and enteric diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA.
• William B. Goldman. Department of Molecular Microbiology, Washington University, St. Louis,
MO, USA.
Venezuela
• Gioconda San-Blas. Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela.
• Gustavo Niño. Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones
Científicas (IVIC), Caracas, Venezuela.
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Chairpersons
Thursday, August 07
Symposium 1.
P. brasiliensis genomics and bioinformatics
Chairs: Celia M. Soãres and Daniel Matute
Symposium 2.
Understanding the immune responses to dimorphic fungi in experimental
animal hosts
Chairs: John R. Graybill and Luz Elena Cano
Friday, August 08
Symposium 3.
Virulence factors and host-parasite interactions
Chairs: Gioconda San-Blas and Angel González
Symposium 4.
Immune responses in paracoccidioidomycosis: Cellular and molecular
mechanisms
Chairs: Maria José Mendes-Giannini and María del Pilar Jiménez
Symposium 5.
Granuloma and fibrosis: Tissue markers of paracoccidioidomycosis
Chairs: Marcello Fabiano Franco and María Mercedes Patiño
Symposium 6.
Fungal cellular biology aspects
Chairs: Joshua Fierer and Agostinho J. Almeida.
Saturday, August 09
Symposium 7.
The endemic mycoses under consideration by public health agencies
Chairs: Andrea Vicari and Gilberto Álvarez
Symposium 8.
Clinical and epidemiological considerations on the endemic mycoses
Chairs: Tom Chiller and Ángela Restrepo
Symposium 9.
New approaches to the clinical and laboratory diagnosis of systemic
mycoses
Chairs: Maria José Mendes-Giannini and Elizabeth Castañeda
Symposium 10.
Advances on antifungal antibiotics
Chairs: Ricardo Negroni and Carlos Andrés Agudelo
Sunday, August 10
Symposium 11.
Agriculture and paracoccidioidomycosis
Chairs: María Adelaide Millington and Donald L. Greer
Symposium 12.
Evolutive biology of P. brasiliensis
Chairs: John W. Taylor and Gustavo Niño
13
Scholarship Winners
Traveling Award contributed by Drs. Karl V. and Lynda Clemons to one of the ten best abstracts
to be presented in the Congress by a Latin American graduate student
• Bonfim, C.V.
Laboratório de Imunologia Molecular e Celular. Departamento de Patologia
Clínica, Faculdade de Ciências Médicas -UNICAMP- Campinas, SP, Brazil
e-mail: camilinha@gmail.com
TLR-2, TLR-4 and DECTIN-1 expression in human macrophages and neutrophils stimulated by
Paracoccidioides brasiliensis.
Scholarships auspiced by the
X International Congress on Paracoccidioidomycosis
• Ferreira, M.C.
Department of Clinical Pathology
Faculty of Medical Science – UNICAMP – Campinas, SP, Brazil
e-mail: mcf@fcm.unicamp.br
Increased T cell regulatory activity in patients with paracoccidioidomycosis
• Estefanía, M.N. Martins.
Laboratório de Radiobiologia, CDTN / CNEN, Departamento de Bioquímica e Imunologia, UFMG,
Belo Horizonte, MG, Brazil
e-mail: estefaniabio@yahoo.com.br
Evaluation of the protection induced by the immunization with radioattenuated yeast cells of Paracoccidioides brasiliensis in animal model
• Borges, C.L.
Instituto de Ciências Biológicas,
UFG, Goiânia, Brazil
e-mail: clayton@icb.ufg.br
Formamidase of Paracoccidioides brasiliensis: cytolocalization, purification and analysis of the
native protein.
• Felonato, M.
Depto. de Imunologia, Instituto de Ciências Biomédicas,
Universidade de São Paulo, SP, Brazil.
e-mail: felonato@gmail.com
CD28 costimulatory molecule deficiency results in more severe Paracoccidioides brasiliensis infection but protects mice from life-threatening immunopathology.
15
• Bernardino, S.
Instituto de Ciências Biomédicas
Universidade de São Paulo, São Paulo, Brazil
e-mail: sibern@icb.usp.br
Nitric oxide but not TREG cells plays a major immunoregulatory role in a pulmonary model of fungal
infection
• Costa, T.A.
Instituto de Ciências Biomédicas
Universidade de São Paulo, Brazil.
e-mail: tacosta@usp.br
IL-10 deficiency determines a better fungicidal ability associated with overproduction of IFN-gama
and nitric oxide by Paracoccidioides brasiliensis infected macrophages
• Silva, J.F.
Faculdade de Ciências Farmacêuticas de Araraquara, UNESP, SP, Brazil
e-mail: silvajf@fcfar.unesp.
Gp43 isoforms of Paracoccidioides brasiliensis as ligands of laminin, fibronectin and collagen type I.
• Simoneide, S.S.
Instituto de Ciências Biológicas
Universidade de Brasília, Brasília, DF, Brazil
e-mail: simoneide.silva@gmail.com
Comparative analysis of gene expression during macrophages infection by pathogenic fungi (Paracoccidioides brasiliensis and Histoplasma capsulatum).
• Theodoro, R. C.
Depto. de Microbiologia e Imunologia
IBB- Unesp, Botucatu, SP, Brazil
e-mail: raquel@ibb.unesp.br
Phylogenetic analysis of PRP8 intein and mycological features in Paracoccidioides brasiliensis
species complex.
16
Editorial
In 1971 the Pan American Health Organization (PAHO) convened the First Pan American Symposium on Paracoccidioidomycosis in Medellin, Colombia. It was in this city and country that
many concepts -now classic- were revived and where many important advances in the area
were made. In 2008, 37 years later, we meet again in the same city and country to work on the
same subject, this time provided with new knowledge and renewed hopes for progress.
In 1908 Adolfo Lutz, a physician trained in Switzerland and who held his medical practice
in São Paulo, Brazil, described for the first time a peculiar disorder afflicting two patients and
that resembled a fungal disorder, coccidioidomycosis, described sixteen years earlier (1892)
by Posadas in Argentina. Lutz examined tissue sections from Posadas’ original case and noticed that the endogenous sporulation, characteristic of Coccidioides immitis, was absent from
his own patients concluding that the Argentinian and the Brazilian mycoses were two different
entities. Additionally, Lutz isolated the etiologic agent in its mycelial form and without naming it
wrote in his report that “… the tissue forms are different from the ones observed in cultures…”.
Thus, Lutz’ keen mind allowed him to discover a new disease, a new agent and its dimorphic
properties, all in a matter of a few months. From the very beginning, Lutz had set an example
for all the researchers who were to follow, including most of us.
One hundred years have elapsed by since Lutz’ discovery, and this century has provided
those of us, interested in paracoccidioidomycosis and Paracoccidioides brasiliensis, a wealth
of findings but most importantly, a multitude of doors open to future research, and a richness of
questions still unsolved. After all, it is the question that matters.
Nowadays the fungus has become an important research tool due to its versatility and endurance and, why not, its capricious moods. Yet patients continue to experiment the “slings and
arrows” inflicted by the fungus and suffer its ill effects when their vital organs are attacked.
Judging by the abstracts that will be presented during this X International Congress on Paracoccidioidomycosis, a great deal of progress has been accomplished in all fields of knowledge
on this and related mycoses. It appears that several of the previously existing “icebergs” are
now conquerable with thorough research. It remains to be determined whether or not such fresh
knowledge will solve some of the most significant problems still standing. Be as it may, we will
advance further only if we join efforts in searching common goals.
17
Along these lines of thought and accepting that, presently, scientific progress can be achieved only
through cooperative efforts and merging of the individual expertise, the X International Congress on
Paracoccidioidomycosis represents the forum where such progress can be achieved. We invite you
to profit from this opportunity in order to establish cooperative studies with your colleagues and find
common grounds for exploration of those significant points that mean so much to patients, to science
and to human kind’s progress.
All the members of the Corporación para Investigaciones Biológicas (CIB) heartily welcome
you to our city, to our country. We feel honored with your presence and trust that the program
we have prepared for you will prove satisfactory so that when you leave you may take with you
kind remembrances and new knowledge.
Ángela Restrepo M, Ph.D.
MedellÍn, Colombia, August 7, 2008.
18
Scientific Program
Wednesday, August 06
15:00-18:00
Arrival and Registrations
Thursday, August 07
7:00-7:45
Registrations
Displaying Posters
7:00-7:30
7:30-7:45
Note: Posters can be visited all throughout the congress; presenters will make themselves available for discussion from 7:00 to 8:00 and from 18:00 to 19:00 hours Friday
08 to Saturday 09 August.
Welcome greetings: Luz E. Cano.
General Secretary of the Congress. Corporación para Investigaciones Biológicas (CIB) y
Escuela de Microbiología, Universidad de Antioquia (UdeA), Medellín, Colombia.
Symposium 1. P. brasiliensis genomics and bioinformatics
Chairs: Celia M. Soãres and Daniel Matute
7:45-8:05
Comparative analysis of Paracoccidioides brasiliensis and other dimorphic fungi:
Mia Champion.
The Broad Institute of Massachusetts, Institute of Technology, Cambridge, MAS, USA.
8:05-8:25
Comparative genomics in Coccidioides species:
John W. Taylor.
Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA.
8:25-8:45
Post-genomic contribution to the knowledge on the host-pathogen interaction:
Maria Sueli Felipe. Departamento de Biologia Celular, Instituto de Ciências Biologicas,
Universidade de Brasilia, Brasilia, DF, Brazil.
8:45-9:05
Genes involved in morphogenesis and cell wall synthesis and regulation in P. brasiliensis:
Gustavo Niño.
Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones
Científicas (IVIC), Caracas, Venezuela.
9:05-9:25
Questions
9:25-9:45
Coffee break
Symposium 2. Understanding the immune responses to dimorphic fungi
in experimental animal hosts
Chairs: John R. Graybill and Luz Elena Cano
9:45-10:05
Experimental animal models and their contribution to the study of fungal pathogenesis:
Karl V. Clemons.
Division of Infectious Diseases, Santa Clara Valley Medical Center, California Institute
for Medical Research, and Division of Infectious Diseases and Geographic Medicine,
Department of Medicine Stanford University, San Jose, CA, USA.
19
10:05-10:25
Toll like receptors and the adaptor molecule MYD88 play an important role in pulmonary
paracoccidioidomycosis: Vera L. Calich.
Departamento da Imunologia, Instituto de Ciências Biomédicas (ICB), Universidade de
São Paulo (USP), SP, Brazil.
10:25-10:45
In vivo protection against P. brasiliensis infection with monoclonal antibodies:
Carlos Taborda.
Departamento de Microbiologia, Instituto de Ciências Biomédicas II (ICB-II), Universidade
de São Paulo (USP), São Paulo, SP, Brazil.
10:45-11:05
P10 immunization using Salmonella enterica’s flagellin as adjuvant:
Carlos Taborda and Luis R. Travassos.
Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de Ciências Biomédicas II (ICB-II), Universidade de São Paulo (USP), São Paulo, SP, Brazil.
11:05-11:25
Pattern recognition receptors in the host immune response to Coccidioides spp.:
Joshua Fierer.
Research Service, VA San Diego Healthcare, San Diego, CA. Department of Medicine, Division of Infectious Diseases School of Medicine, University of California San Diego, USA.
11:25-11:45
A strategy for the generation of a recombinant vaccine against coccidioidomycosis:
Garry T. Cole.
Department of Biology, University of Texas at San Antonio San Antonio, TX, USA.
11:45-12:00
Questions
12:00-17:00
18:00-19:00
Silleteros’ parade
Registrations
Opening Ceremony, “A Centennial Celebration”
Opening lecture: Henrique L. Lenzi. Departamento de Patologia, Fundação Oswaldo
Cruz, FioCruz, Rio de Janeiro, RJ, Brazil.
19:00-21:00
Official greetings: Juan Guillermo McEwen. President of the Congress. Corporación
para Investigaciones Biológicas (CIB) and Facultad de Medicina, Universidad de Antioquia Medellín, Colombia;
Diego Miguel Sierra B. Director General Corporación para Investigaciones Biológicas
(CIB), Medellín, Colombia.
Birthday Celebration (Cocktail)
Friday, August 08
Posters
7:00-8:00
Wake-up call: Tropical posters, do not miss this opportunity to enjoy fresh fruits while
studying the posters. A good mix.
Note: Posters can be visited all throughout the Congress; presenters will make themselves available for discussion from 7:00 to 8:00 and from 18:00 to 19:00 hours Friday
08 to Saturday 09 August.
20
Symposium 3. Virulence factors and host-parasite interactions
Chairs: Gioconda San-Blas and Angel González
8:00-8:20
Histoplasma virulence mechanisms with parallels in paracoccidioidomycosis:
William B. Goldman. Department of Molecular Microbiology, Washington University,
St. Louis, MO, USA.
8:20-8:40
Integrated genomics and proteomics strategies bring new insight into P. brasiliensis
response upon host interaction: Celia M. Soãres.
Laboratorio de Biologia Molecular, Instituto de Ciências Biologicas, Universidade Federal
de Goiãnia, Goiãs, Brazil.
8:40-9:00
Adhesion and extracellular matrix proteins: Maria José Mendes-Giannini.
Departamento de Análises Clinicas, Faculdade de Ciências Farmacêuticas, UNESP,
Araraquara, SP, Brazil.
9:00-9:20
Virulence insights from the P. brasiliensis transcriptome: Ildinete Silva Pereira.
Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de
Brasília, Brasilia, DF, Brazil.
9:20-9:40
Role of melanin in certain dimorphic fungi: Beatriz L. Gómez.
Mycotic Diseases Branch, Centers for Disease Control and Prevention (CDC), Atlanta,
GA, USA.
9:40-10:00
Questions
10:00-10:20
Coffee break
Symposium 4. Immune responses in paracoccidioidomycosis:
Cellular and molecular mechanisms
Chairs: Maria José Mendes-Giannini and María del Pilar Jiménez
10:20-10:40
Microbicidal mechanisms exerted by macrophages against P. brasiliensis conidia:
Angel González M.
Corporación para Investigaciones Biológicas (CIB), y Escuela de Microbiología, Universidad de Antioquia (U de A), Medellín, Colombia.
10:40-11:00
Human cells and molecules involved in the microbicidal mechanisms against P.brasiliensis:
Angela M.V.C. Soãres.
Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade
Estatal de São Paulo, Botucatú, SP, Brazil.
11:00-11:20
The role of CD8+ cells in human paracoccidioidomycosis:
Maria Heloisa Blotta
Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estatal
de Campinas (UNICAMP), Campinas, SP, Brazil.
11:20-11:40
Antigen-specific immunosuppression in paracoccidioidomycosis: adverse effect or desired
outcome?: Gil Benard.
Laboratorio de Alergia Clinica e Experimental e Imunologia. Departamento de Dermatologia, Faculdade de Medicina da Universidade de São Paulo (FMUSP), SP, Brazil.
21
11:40-12:00
Several reasons for the immunosuppression observed in paracoccidioidomycosis:
João Santana Silva.
Departamento de Bioquimica e da Imunologia, Escola de Medicina, Universidade de São
Paulo, Ribeirão Preto, SP, Brazil.
12:00-12:20
Questions
Lunch with professors. Historical accounts: Coccidioidomycosis
Coordinator: María del Pilar Jiménez A.
Coccidioidomycosis Discovery in Argentina: Ricardo Negroni.
Unidad de Micología, Hospital de Infecciosas Francisco Javier Muñiz, Buenos Aires,
Argentina.
12:20-13:20
A history of research on coccidioidomycosis in the U.S.: Garry T. Cole.
Department of Biology, University of Texas at San Antonio, San Antonio, TX, USA.
Coccidioidomycosis: Recent records in Latin America: Bodo Wanke.
Laboratorio/Serviço de Micologia Médica, Centro de Pesquisa Hospital Evandro Chagas
(IPEC), Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
13:20-14:00
Break
Symposium 5. Granuloma and fibrosis:
Tissue markers of paracoccidioidomycosis
Chairs: Marcello Fabiano Franco and María Mercedes Patiño
14:00-14:20
B-1 cells modulate the kinetics of granuloma formation induced by P. brasiliensis:
José Daniel Lopes. Disciplina de Imunologia, Departamento de Microbiologia, Imunologia
e Parasitologia, Centro de Microscopia electrônica, Universidade Federal de São Paulo
(UNIFESP), São Paulo, SP, Brazil.
14:20-14:40
Extracellular matrix modulation in paracoccidioidal granuloma: Eva Burger.
Departamento da Imunologia, Instituto de Ciências Biomédicas (ICB), Universidade de
São Paulo (USP), São Paulo, SP, Brazil.
14:40-15:00
Pulmonary fibrosis in an experimental model of paracoccidioidomycosis: Modulation
through therapeutic intervention: Luz E. Cano.
Corporación para Investigaciones Biológicas (CIB) y Escuela de Microbiología, Universidad de Antioquia (UdeA), Medellín, Colombia.
15:00-15:20
Immune reactive cells in skin lesions of patients with paracoccidioidomycosis:
Carla Pagliari. Departamento de Patologia, Facultade de Medicina, Universidade de
São Paulo, São Paulo, SP, Brazil.
15:20-15:40
Questions
15:40-16:00
Coffee break
Symposium 6. Fungal cellular biology aspects
Chairs: Joshua Fierer and Agostinho J. Almeida.
16:00-16:20
22
Immune recognition of the Candida cell wall: The taste of fungus:Neil AR Gow.
School of Medical Sciences, Institute of Medical Sciences. University of Aberdeen,
Aberdeen, UK.
16:20-16:40
Cell signaling in morphogenesis and virulence of P. brasiliensis: Larissa Fernandes.
Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidade de
Brasilia, Brasilia, DF, Brazil.
16:40-17:00
Paracoccidioides brasiliensis conidia to yeast transition analysis of certain genes involved
in the process: Ana María García.
Corporación para Investigaciones Biológicas (CIB) and Universidad Pontificia Bolivariana,
Medellín, Colombia.
17:00-17:20
CDC42p controls yeast-cell shape and virulence in P. brasiliensis:
Agostinho João Almeida.
Life and Health Sciences Research Institute (ICVS) School of Health Sciences, University
of Minho, Campus de Gualtar, Braga, Portugal.
17:20-17:40
Differential analysis of extracellular vesicles and of possible cell wall-associated proteins
in isolates of P. brasiliensis: Rosana Puccia.
Departamento de Microbiologia, Imunologia e Parasitologia, Divisão de Biologia Celular,
Universidade Federal do São Paulo, São Paulo, SP, Brazil.
17:40-18:00
Questions
18:00-19:00
Posters
Saturday, August 09
7:00-8:00
Registration for the new attendants to the Clinical Symposium
Posters
7:00-8:00
Wake-up call: Tropical posters, do not miss this opportunity to enjoy fresh fruits while
studying the posters. A good mix.
Note: Posters can be visited all throughout the Congress; presenters will make themselves available for discussion from 7:00 to 8:00 and from 18:00 to 19:00 hours Friday
08 to Saturday 09 August.
Symposium 7. The endemic mycoses under consideration by public health
agencies
Chairs: Andrea Vicari and Gilberto Álvarez
8:00-8:20
Paracoccidioidomycosis in the panorama of the neglected tropical diseases:
Steven K. Ault. Communicable Diseases Unit Area of Health Surveillance and Disease
Management, Pan American Health Organization, Regional Office of the World Health
Organization, Washington DC, USA.
8:20-8:40
The evolving public health importance of mycotic diseases: Tom Chiller. Epidemiology
Unit Mycotic Disease Branch, National Center for zoonotic, vector borne and enteric
diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA.
8:40-9:00
Epidemiology issues in invasive fungal infections: A United States prospective:
Mary Brandt. Mycology Disease Branch, Centers for Disease Control and Prevention
(CDC), Atlanta, GA, USA.
23
9:00-9:20
Paracoccidioidomycosis in Brazil – surveillance and control program:
María Adelaide Millington.
GT de Pneumonias e Micoses Sistemicas, Ministerio de Saúde, Brasilia, DF, Brazil.
9:20-9:40
Questions
9:40-10:20
Coffee break
Symposium 8. Clinical and epidemiological considerations on the endemic
mycoses
Chairs: Tom Chiller and Ángela Restrepo
10:20-10:40
The impact of histoplasmosis in HIV-infected patients:
John R. Graybill. Department of Medicine, University of Texas Health Science Center
at San Antonio, San Antonio, TX, USA and
Eduardo Arathoon. Clinica Familiar “Luis Angel García”, Asociación de Salud Integral.
Hospital General San Juan de Dios, Guatemala City, Guatemala.
10:40-11:00
The coexistence of HIV infection and paracoccidioidomycosis: Roberto Martinez.
Departamento de Molestias Infecciosas da Faculdade de Medicina de Ribeirão Preto
Universidade de São Paulo. Ribeirão Preto,SP, Brazil.
11:00-11:20
Treatment compliance in paracoccidioidomycosis: Rinaldo Põncio-Mendes.
Departamento de Medicina, Faculdade de Medicina, Universidade Estatal de São Paulo,
Botucatú, SP, Brazil.
11:20-11:40
Guidelines for the treatment of paracoccidioidomycosis: Flavio Queiroz-Telles.
Departamento de Saúde Comunitária, Universidade Federal do Paraná, Curitiba, PR,
Brazil.
11:40-12:00
Questions
Lunch with professors. Historical accounts: Histoplasmosis
Coordinator: Beatriz L. Gómez G.
12:00-13:00
13:00-14:00
Histoplasmosis: Discovery in Panama: Marion C. de Martin.
Departamento de Microbiología, Sección de Micología, Universidad de Panamá, Panamá,
Republic of Panamá.
Histoplasmosis in the United States: John R. Graybill.
Department of Medicine, University of Texas Health Science Center at San Antonio, San
Antonio, TX, USA.
Break
Symposium 9. New approaches to the clinical and laboratory diagnosis
of systemic mycoses
Chairs: Zoilo Pires de Camargo and Elizabeth Castañeda
14:00-14:20
24
The value and the promise of molecular biological tools in the diagnosis of aspergillosis
and candidiasis: Beatriz L. Gómez.
Mycotic Diseases Branch, Centers for Disease Control and Prevention (CDC), Atlanta,
GA, USA.
14:20-14:40
Molecular diagnostic approaches in paracoccidioidomycosis and histoplasmosis:
Rosely Zancopé Oliveira. Fundação Oswaldo Cruz, Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brazil.
14:40-15:00
Histopathology of paracoccidioidomycosis in the postgenomic era – reflections and perspectives: Henrique L. Lenzi. Departamento de Patologia, Fundação Oswaldo Cruz,
FioCruz, Rio de Janeiro, RJ, Brazil.
15:00-15:20
Clinico-immunological and immunopathological correlations in different clinical forms of
the paracoccidioidomycosis:
Maria Aparecida Shikanai-Yasuda. Laboratorio de Imunologia, Departmento de Doênças
Infecciosas e Parasitarias, Faculdade de Medicina, Universidade de São Paulo (FMUSP),
São Paulo, SP, Brazil.
15:20-15:40
Clinical diagnosis in paracoccidioidomycosis: Its defferentiation from similar entities:
Angela M. Tobón. Corporación para Investigaciones Biológicas (CIB) y Hospital La
María, Medellín, Colombia.
15:40-16:00
Questions
16:00-16:20
Coffee break
Symposium 10. Advances on antifungal antibiotics
Chairs: Ricardo Negroni and Carlos Andrés Agudelo
16:20-16:40
Coccidioidomycosis as seen in a nonendemic area: John E. Bennett.
Head, Clinical Mycology Section, Laboratory of Clinical Investigation, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
16:40-17:00
New and experimental antifungal drugs: Gioconda San-Blas.
Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones
Científicas (IVIC), Caracas, Venezuela.
17:00-17:20
Nanobiotechnology: Advances in antifungal therapy. André Amaral.
Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidade de
Brasilia, Brasilia, DF, Brazil.
17:20-17:40
The epidemiology of Candida and Aspergillus infections: Clinical and molecular aspects:
Christopher Kibbler.
Department of Medical Microbiology, Royal Free Hospital, London, United Kingdom.
17:40-18:00
Questions
18:00-19:00
Important notice: Posters should be removed from the exhibit hall after discussion.
Sunday, August 10
Symposium 11. Agriculture and paracoccidioidomycosis
Chairs: María Adelaide Millington and Donald L. Greer
7:30-8:00
P. brasiliensis growth and infective propagule production in clayey and sandy soil:
Eduardo Bagagli. Departamento de Microbiologia e Imunologia, Instituto de Biociências,
Universidade Estadual Paulista, Botucatú, SP, Brazil.
25
8:00-8:20
On the relationship between land use and paracoccidioidomycosis: Coffee, sugar cane
and other cultures: Ligia V. Barrozo.
Departamento de Geografia , Faculdade de Filosofia, Letras e Ciências Humanas. Universidade de São Paulo, São Paulo, SP, Brazil.
Influence of alternating coffee and sugar cane agriculture in the incidence of paracoc-
8:20-8:40
cidioidomycosis in Brazil: Flavio Queiroz-Telles. Departamento de Saúde Comunitária,
Universidade Federal do Paraná, Curitiba, PR, Brazil.
Chemical components derived from coffee and their impact on health:
8:40-9:00
Manuel Elkin Patarroyo.
Instituto de Inmunología, Bogotá, Colombia.
Impact of regular coffee consumption on alcohol intake and depressive feelings among
9:00-9:20
students: Darcy Roberto Andrade Lima.
Professor do Instituto de Neurologia da Universidade Federal do Rio de Janeiro, Brazil
e Director Associado de Pesquisas, ICS, Vanderbilt University, TN, USA.
9:20-9:50
Questions
9:50-10:10
Coffee break
Symposium 12. Evolutive biology of P. brasiliensis
Chairs: John W. Taylor and Gustavo Niño
10:10-10:30
What can we know about speciation in P. brasiliensis?: Daniel Matute.
Department of Ecology and Evolution, University of Chicago, Chicago, IL, USA.
Is P. brasiliensis an unique species? Maria Sueli Felipe.
10:30-10:50
Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidade de
Brasilia, Brasilia, DF, Brazil.
Climate variability and acute/subacute paracoccidioidomycosis in a hyperendemic area
10:50-11:10
in Brazil: Ligia V. Barrozo.
Departamento de Geografia, Faculdade de Filosofia, Letras e Ciências Humanas, Universidade de São Paulo, São Paulo, SP, Brazil.
Overview of the biogeography of P. brasiliensis: Eduardo Bagagli.
11:10-11:30
Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade
Estadual Paulista, Botucatú, SP, Brazil.
11:30-11:40
Questions
Farewell lunch
Poster awards and Adjourn: Ángela Restrepo M. Corporación para Investigaciones
Biológicas (CIB), Medelllín, Colombia.
11:40-14:30
Concert
Lunch
26
Symposia Index
Page
Symposium 1. P. brasiliensis genomics and bioinformatics
43
Comparative analysis of Paracoccidioides brasiliensis and other dimorphic fungi:
Mia Champion.
43
Comparative genomics in Coccidioides species:
John W. Taylor.
44
Post-genomic contribution to the knowledge on the host-pathogen interaction:
Maria Sueli Felipe.
45
Genes involved in morphogenesis and cell wall synthesis and regulation in P. brasiliensis:
Gustavo Niño.
47
Symposium 2. Understanding the immune responses to
dimorphic fungi in experimental animal hosts
49
Experimental animal models and their contribution to the study of fungal pathogenesis:
Karl V. Clemons.
50
Toll like receptors and the adaptor molecule MYD88 play an important role in pulmonary
paracoccidioidomycosis: Vera L. Calich.
51
In vivo protection against P. brasiliensis infection with monoclonal antibodies:
Carlos Taborda.
52
P10 immunization using Salmonella enterica’s flagellin as adjuvant:
Carlos Taborda and Luis R. Travassos.
53
Pattern recognition receptors in the host immune response to Coccidioides spp.:
Joshua Fierer.
55
A strategy for the generation of a recombinant vaccine against coccidioidomycosis:
Garry T. Cole.
57
Symposium 3. Virulence factors and host-parasite
interactions
59
Histoplasma virulence mechanisms with parallels in paracoccidioidomycosis:
William B. Goldman.
59
Integrated genomics and proteomics strategies bring new insight into P. brasiliensis
response upon host interaction: Celia M. Soãres.
60
Adhesion and extracellular matrix proteins:
Maria José Mendes-Giannini.
61
Virulence insights from the P. brasiliensis transcriptome:
Ildinete Silva Pereira.
62
Role of melanin in certain dimorphic fungi:
Beatriz L. Gómez.
27
65
Cellular and molecular mechanisms
67
Microbicidal mechanisms exerted by macrophages against P. brasiliensis conidia:
Angel González M.
68
Human cells and molecules involved in the microbicidal mechanisms against P.brasiliensis:
Angela M.V.C. Soãres.
68
The role of CD8+ cells in human paracoccidioidomycosis:
Maria Heloisa Blotta
70
Antigen-specific immunosuppression in paracoccidioidomycosis: Adverse effect or desired
outcome?: Gil Benard.
71
Several reasons for the immunosuppression observed in paracoccidioidomycosis:
João Santana Silva.
73
Lunch with professors
Historical Accounts: Coccidioidomycosis
73
Coccidioidomycosis Discovery in Argentina:
Ricardo Negroni.
74
A history of research on coccidioidomycosis in the U.S.:
Garry T. Cole.
74
Coccidioidomycosis: Recent records in Latin America:
Bodo Wanke.
77
28
Symposium 4. Immune responses in paracoccidioidomycosis:
Symposium 5. Granuloma and fibrosis:
Tissue markers of paracoccidioidomycosis
79
B-1 cells modulate the kinetics of granuloma formation induced by P. brasiliensis:
José Daniel Lopes.
80
Extracellular matrix modulation in paracoccidioidal granuloma:
Eva Burger.
82
Pulmonary fibrosis in an experimental model of paracoccidioidomycosis: Modulation
through therapeutic intervention: Luz E. Cano.
84
Immune reactive cells in skin lesions of patients with paracoccidioidomycosis:
Carla Pagliari.
85
Symposium 6. Fungal cellular biology aspects
87
Immune recognition of the Candida cell wall: The taste of fungus:
Neil AR Gow.
87
Cell signaling in morphogenesis and virulence of P. brasiliensis:
Larissa Fernandes.
89
Paracoccidioides brasiliensis conidia to yeast transition analysis of certain genes involved
in the process: Ana María García.
91
CDC42p controls yeast-cell shape and virulence in P. brasiliensis:
Agostinho João Almeida.
92
Differential analysis of extracellular vesicles and of possible cell wall-associated proteins
in isolates of P. brasiliensis: Rosana Puccia.
93
Symposium 7. The endemic mycoses under consideration
by Public Health Agencies
95
Paracoccidioidomycosis in the panorama of the neglected tropical diseases:
Steven K. Ault.
95
The evolving public health importance of mycotic diseases:
Tom Chiller.
95
Epidemiology issues in invasive fungal infections: A United States prospective.
Mary Brandt.
96
Paracoccidioidomycosis in Brazil – surveillance and control program:
María Adelaide Millington.
99
Symposium 8. Clinical and epidemiological considerations
on the endemic mycoses
101
The impact of histoplasmosis in HIV-infected patients:
John R. Graybill and Eduardo Arathoon.
102
The coexistence of HIV infection and paracoccidioidomycosis:
Roberto Martinez.
103
Treatment compliance in paracoccidioidomycosis:
Rinaldo Põncio-Mendes.
105
Guidelines for the treatment of paracoccidioidomycosis:
Flavio Queiroz-Telles.
107
Lunch with professors
Historical accounts: Histoplasmosis
107
Histoplasmosis: Discovery in Panama: Marion C. de Martin.
109
Histoplasmosis in the United States:
John R. Graybill.
111
Symposium 9. New approaches to the clinical and laboratory
diagnosis of systemic mycoses
113
The value and the promise of molecular biological tools in the diagnosis of aspergillosis
and candidiasis: Beatriz L. Gómez.
114
Molecular diagnostic approaches in paracoccidioidomycosis and histoplasmosis:
Rosely Zancopé Oliveira.
114
Histopathology of paracoccidioidomycosis in the postgenomic era - reflections and perspectives: Henrique L. Lenzi.
29
30
116
Clinico-immunological and immunopathological correlations in different clinical forms of
the paracoccidioidomycosis: Maria Aparecida Shikanai-Yasuda.
117
Clinical diagnosis in paracoccidioidomycosis: Its defferentiation from similar entities:
Angela M. Tobón.
119
Symposium 10. Advances on antifungal antibiotics
121
Coccidioidomycosis as seen in a nonendemic area:
John E. Bennett.
121
New and experimental antifungal drugs:
Gioconda San-Blas.
123
Nanobiotechnology: Advances in antifungal therapy.
André Amaral.
124
The epidemiology of Candida and Aspergillus infections: Clinical and molecular aspects:
Christopher Kibbler.
125
Symposium 11. Agriculture and paracoccidioidomycosis
127
P. brasiliensis growth and infective propagule production in clayey and sandy soil:
Eduardo Bagagli.
127
On the relationship between land use and paracoccidioidomycosis: Coffee, sugar cane
and other cultures: Ligia V. Barrozo.
129
Influence of alternating coffee and sugar cane agriculture in the incidence of Paracoccidioidomycosis in Brazil: Flavio Queiroz-Telles.
130
Chemical components derived from coffee and their impact on health:
Manuel Elkin Patarroyo.
132
Impact of regular coffee consumption on alcohol intake and depressive feelings among
students: Darcy Roberto Andrade Lima.
133
Symposium 12. Evolutive biology of P. brasiliensis
135
What can we know about speciation in P. brasiliensis?: Daniel Matute.
136
Is P. brasiliensis an unique species? Maria Sueli Felipe.
137
Climate variability and acute/subacute paracoccidioidomycosis in a hyperendemic area
in Brazil: Ligia V. Barrozo.
138
Overview of the biogeography of P. brasiliensis: Eduardo Bagagli.
Poster Index
1 Clinical Aspects / Case Reports
1-01
p 141
1-02
p 141
Clinical epidemiology of paracoccidioidomycosis patients in the region of Botucatu
(São Paulo State, Brazil).
Lopes DF; Carvalho LR ; Mendes RP.
Paracoccidioidomycosis in patients with and without HIV infection. Comparative
study
Paniago AM; Oliveira Al; Pegorare Al; Aguiar ESA; Aguiar JIA; M.r.; Chang RVC; MottaCastro ARC; Wanke B.
1-03
Paracoccidioidomycosis in Mexico
López-Martínez R; Velasco-Castrejón O; Bonifaz A; Cazarín-Barrientos; Saul A; Arenas
R; Padilla-DesgarennesMC.
1-04
Paracoccidioidomycosis in Bolivia
Vargas F.
1-05
Epidemiological and clinical features of patients with Paracoccidioides brasiliensis
attending at a University Hospital from Huila - Colombia, Between 1999-2007
Molano VM; Gualtero S; Duran LF; Gómez CA; Diaz G.
1-06
Paracoccidioidomycosis: Frequency, distribution, and hospitalization flows due to
the endemic in Brazil (1998-2007).
Coutinho ZF; Travassos CMR; Wanke B; Oliveira EXG; Valle ACF; Coimbra CEA.
p 142
p 142
p 143
p 143
1-07
p 144
1-08
p 144
1-09
p 145
1-10
p 145
1-11
p 146
1-12
p 146
Epidemiological and clinical profile of 137 patients with paracoccidioidomycosis in
Uberaba, Minas Gerais, Brazil.
Neves FF; Gerolin GP; Tavares MG; Castro-Silva MH; Lopes GP; Michelin MA; Silva A;
Vergara MI.
Paracoccidioidomycosis associated to HIV/AIDS infection in Uberaba, Minas Gerais,
Brazil.
Gerolin GP; Tavares MG; Castro-silva MH; Lopes GP; Michelin MA; Silva-Vergara MI;
Neves FF.
Acute severe paracoccidioidomycosis in an urban-living boy with a single exposure
episode: Evidence for a long subclinical evolution before full-blown disease
Benard G, Barata LCB.
First description of a fatal adult respiratory distress syndrome in a severe acute
paracoccidioidomycosis patient.
Ravanini JN; Silva LFF; Goular S; Benard G.
Sub-acute paracoccidioidomycosis in a 49 year-old woman.
Myiamoto D, Kono ASG; Benard G; Yoshida M; Giarolla I; Shikanai-Yasuda MA.
Paracoccidioidomycosis presenting as evanescent pleural effusion associated to
focal consolidation: An uncommon presentation.
Costa AN; Junior SAD; Takagaki TY; Kairalla RA; Carvalho CRR.
31
Nested-PCR, immunoblotting and double immunodiffusion in meningoencephalitis
1-13
p 147
by P. brasiliensis without radiological alterations of the central nervous system
Almeida RAMB; Marques SA; Mendes RP; Moris DV; Bosco SMG; Macoris SAG; Bagagli
E; Vicentini-Moreira AP; Kohara VS; Cavalcante RS, Chaves CR; Calvi ESA.
1-14
p 147
1-15
p 148
1-16
p 148
1-17
p 149
Cutaneous sarcoidic lesions and moriform stomatitis lesions in a young patient:
Two sides but not the same coin?
Fairbanks N; Kono A; Yoshida M; Valente NYS; Shikanai-Yasuda MA; Benard G.
Adrenal insufficiency by paracoccidioidomycosis (PCM) - series of cases
Bredt CSO; Bredt Jr GI; Duarte PAD; Pedott F; Pereira T; De CamposMyiamoto L; Ribeiro
AA; Soliva E Jr; Volpolini LC.
Adrenal insuficiency associated to paracoccidioidomycosis: Report of three autopsy
cases.
Mantilla JC; Serrano A.
Scintigraphic evaluation of enteric protein loss in patients with active
paracoccidioidomycosis.
Griva BI; Mendes RP.
1-18
Biliary obstruction due to acute paracoccidioidomycosis: Analisys of two cases
1-19
Serious hepatic damage in paracoccidioidomycosis and its treatment
p 149
p 150
1-20
p 150
1-21
p 151
1-22
p 151
Kono ASG; Yoshida M; Giarolla I; Jukemura I; Benard G; Shikanai-Yasuda MA.
Lazo R; Barrezueta J; Lazo J.
Acute respiratory failure in AIDS patients: Role of open lung biopsy to the diagnosis
of paracoccidioidomycosis (PCM)
Bredt CSO; Bredt GI Jr; Campos M; Pavan D; Duarte PAD; Pedott F; Pereira T; De Campos
L; Moraes DM; Richetti MA.
Paracoccidioidomycosis in a renal transplant recipient: Case report and review of
the literature
Adriana SG; Galvão MM; Yoshida M; Giarolla I; França FOS; Benard G; Lanhez LE; Shikanai-Yasuda MA.
Pericardic paracoccidioidomycosis (PCM) associated with pulmonary neoplasia
- Case Report
Bredt CSO; Bredt GI Jr; Duarte PAD; Luiz AA; Pedott F; Pereira T; De Campos L Myiamoto;
Ribeiro AA; Soliva E Jr; Volpolini LC.
1-23
Disseminated Herpes simplex infection in a patient with acute/subacute paracoccidioidomycosis
Souza BS; Duarte LX; Moraes MPT; Coelho KYR; Griva BI; Mendes RP.
1-24
Lung paracoccidioidomycosis: Analysis of clinical, tomographic and functional
impairment after adequate treatment
Costa AN; Fernandes CJCS; Suesada M; Salge JM; Kairalla R; Carvalho CRR.
p 152
p 152
2 Diagnosis
2-01
p 155
32
Diagnosis of paracoccidioidomycosis in patients attended In routine services of a
university hospital
Moreto TC; Marques MEA; Oliveira MISC; Moris DV; Carvalho LR; Mendes RP.
2-02
p 155
2-03
p 156
Serological diagnosis of paracoccidioidomycosis: Evaluation of negative serum
samples on immunodiffusion test from patients with confirmed disease
Moreto TC; Vicentini Moreira AP; Passos AN; Kohara VS; Carvalho LR, Mendes RP.
Combined use of Paracoccidioides brasiliensis recombinant 27 and 40-kilodalton
antigens in an enzyme-linked immunosorbent assay for immunodiagnosis of paracoccidioidomycosis
Fernandes VC; Coitinho JB; Fonseca JH; Veloso JMR; Araújo SA; Pedroso EP; Goes AM.
2-04
Laboratory measurements for therapeutic monitoring of paracoccidioidomycosis
patients with different clinical forms
Cavalcante R; Moris DV; Carvalho LR; Mendes RP.
2-05
Espirometric evaluation in patients with paracoccidioidomycosis (PCM)
Bredt CSO; Bredt GI Jr; Duarte PAD; Pedott F; Pereira T; De Campos L; Suzin F.
p 156
p 157
3 Epidemiology / Ecology
3-01
Determination of Paracoccidioides brasiliensis reservarea mapping the migratory
story of 128 patients
Moreto TC; Barrozo LV; Bueno RA; Moris DV; Mendes RP.
3-02
Space-time cluster of acute/subacute paracoccidioidomycosis bearing relationship
with the 1982-83 El Niño episode in a hyperendemic area in Brazil
Barrozo LV, Mendes RP; Marques SA; Benard G; Silva MES; Bagagli E.
3-03
Spatial distribution of chronic paracoccidioidomycosis in a hyperendemic area in
Brazil
Barrozo LV, Gonzalez CR; Santana MS; Mendes RP; Marques SA; Bagagli E.
3-04
Liquid urease test in human and animal isolates of Paracoccidioides brasiliensis
Macoris SAG, Bosco SMG; Theodoro RC; Richini-Pereira V, Bagagli E.
p 161
p 161
p 162
p 162
4 Genomic / Proteomic / Cellular Biology / Molecular Biology
4-01
p 165
Toward a molecular genetic system for the pathogenic fungus Paracoccidioides
brasiliensis
Almeida AJ; Carmona JA; Cunha C; Carvalho A; Rappleye CA; Goldman WE; Hooykaas
PJ; Leão C; Ludovico P; Rodrigues F.
4-02
RNA interference: A potential tool to study gene function in Paracoccidioides brasiliensis - strategies to construct silencing cassettes
Fernandes L; Paes HC; Teixeira MM; Rodrigues ACJ; Viana DF; Torres FA; Felipe MSS.
4-03
Development of vector-based RNA interference (RNAi) for ade2 silencing on the
pathogenic fungus Paracoccidioides brasiliensis
Polez VPP; Fernandes L; Torres FAG; Felipe MSS.
4-04
Protocol for establishment of Paracoccidioides brasiliensis RNA recovery from human pneumocytes cell culture
Oliveira A; Silva SS; Souza CR; Felipe MS.
4-05
Evidence for positive selection in putative virulence factors within the Paracoccidioides brasiliensis species complex
Matute DR; Quesada-Ocampo LM; Rauscher JT; McEwen JG.
p 165
p 166
p 166
p 167
33
4-06
Phylogenetic analysis of PRP8 intein and mycological features in Paracoccidioides
brasiliensis species complex
Theodoro RC, Bagagli E; Trinca LA.
4-07
A high degree of polymorphism and recombination of the GP43 gene among phylogenetic species of Paracoccidioides genus
Teixeira MM; Felipe MSS.
4-08
Virtual screening of Paracoccidioides brasiliensis genome – a new drug design
approach
Abadio AKR, Carvalho MJA, Martins NF, Felipe MSS.
p 167
p 168
p 168
4-09
p 169
4-10
p 169
4-11
p 170
Two subunits of β-1,3-glucan synthase complex: Gene expression (RHO1 and FKS1)
and functionality (RHO1) in Paracoccidioides brasiliensis
Sorais F; Niño-Vega G; San-Blas G.
β-1,3-glucan synthase: Recombinant protein, cytolocalization, activity and studies
under stress condition
Tomazett PK; Félix CR; Barbosa MS; Santana JM; Faria FP; Báo SN; Soares CMA;
Pereira M.
4-12
The α-amylase (AMY1) gene in Paracoccidioides brasiliensis, identification and
semiquantitative expression
Camacho E; Niño-vega G; San-blas G.
4-13
Mating type genes MAT1-1 and MAT1-2 and sexual reproduction in Paracoccidioides
brasiliensis
Torres I; García AM; Mcewen JG; Restrepo A; Arango M.
4-14
Molecular evidences of sexual reproduction in P. brasiliensis
Teixeira MM; Paes HC; Fernandes L; Silva SS; Felipe MSS.
4-15
α-1,3-glucanase is important in the remodelling of Paracoccidioides brasiliensis
cell wall
Villalobos H; Niño-vega G; San-blas G.
4-16
Characterization of Paracoccidioides brasiliensis Chs3 by overexpression in Saccharomyces cerevisiae
Barreto L; Sorais F; Niño-Vega G; San-blas G.
4-17
Autophagy in the human pathogenic fungus Paracoccidioides brasiliensis during
dimorphism
Pedroso SM; Campos CIB; Nóbrega FG, Morais FV.
4-18
Niche specific regulation of Paracoccidioides brasiliensis genes in the infectious
process
Bailão AM; Borges CI; Chagas RF; Salem-Izaac SM; Pereira M; Soares CMA.
4-19
Differential gene expression in virulent and avirulent isolates of Paracoccidioides
brasiliensis
Arruda C; Alves BCA, Calich VIG; Moreira-Filh CA
p 170
p 171
p 171
p 172
p 172
p 173
p 173
p 174
34
Formamidase of Paracoccidioides brasiliensis: Cytolocalization, purification and
analysis of the native protein
Borges CI; Barbosa MS; Parente JA; Feitosa LS; Santana JM; Báo SN; Sousa MV; Richart
CAO; Soares CMA.
4-20
Gp43 isoforms of Paracoccidioides brasiliensis as ligands of laminin, fibronectin
and collagen type I
Silva JF; Benard G; Mendes-Giannini MJS.
4-21
Transcription regulation of the PbGP43 gene with nitrogen in the human pathogen
Paracoccidioides brasiliensis
Rocha AA, Puccia R.
4-22
Identification of Paracoccidioides brasiliensis adhesins through representational
difference analysis
Nogueira SV; Bailão AM; Borges CI; Pereira M; Mendes-Giannini MJ; Winters M; Soares CMA.
4-23
Adhesion profiles and extracellular matrix ligands of Paracoccidioides brasiliensis
isolates obtained from armadillos (Dasypus novemcinctus)
Silva RP; Bagagli E; Mendes-Giannini MJS.
p 174
p 175
p 175
p 176
4-24
p 176
Use of in vivo-induced antigen technology in the identification of Paracoccidioides
brasiliensis proteins potentially expressed during infection
Rezende TCV; Dantas SFIM; Bailão AM; Castro NS; Tomazett MV; Deepe GS Jr.; Pereira
M; Soares CMA.
4-25
Changes in the transcription levels of Paracoccidioides brasiliensis CHS5 and CHS4
genes, mycelial phase, respond to external osmolarity
Niño-vega G; Sorais F; San-blas G.
4-26
A Paracoccidioides brasiliensis aconitase is induced by ethanol and acetate and
repressed by glucose as carbon sources
Brito WA, Castro NS; Pereira M; Soares CMA.
4-27
Comparative analysis of gene expression during macrophages infection by pathogenic fungi (Paracoccidioides brasiliensis and Histoplasma capsulatum)
Silva SS; Felipe MSS.
4-28
A serine protease S08 of Paracoccidioides brasiliensis is induced during infection
of machophages and nitrogen starvation
Parente JA; Bailão AM; Philippsen HK; Santana JM; Pereira M; Soares CMA.
4-29
The high affinity copper transporter of Paracoccidioides brasiliensis is up-regulated
during macrophage infection
Santos RS; Bailão AM; Borges CI; Salem-Izacc SM; Dantas SFIM; Deepe GS Jr.; Soares CMA.
4-30
Alternative oxidase is transcriptionally stimulated at 36°C and is required for mycelium to yeast transition and viability of Paracoccidioides brasiliensis
Junqueira NM; Souza GND; Alves HY; Santos MP; Di Benedette JPT; Campos CBL.
4-31
Transcriptional profile of Paracoccidioides brasiliens is induced by oenothein B,
and antifungal agent from Brazilian savannah plant Eugenia uniflora
Zambuzzi PF; Rezende RV; Borges CI; Ferri PH; Santos SC; Soares CMA; Pereira M.
p 177
p 177
p 178
p 178
p 179
p 179
p 180
4-32
p 180
4-33
p 181
Transcriptional profile of murine macrophages infected with pathogenic fungus
Histoplasma capsulatum
Silva SS; Passos-Silva DG; Teixeira SMR; Junta C; Medeiros AI; Silva CI; Faccioli FH;
Passos GAS; Felipe MSS.
Analysis and identification of three P. brasiliensis genes possibly implicated in
virulence
Hernandez O; Garcia AM; Restrepo A; McEwen JG.
35
4-34
p 181
Internalization of magnetic nanoparticles by Paracoccidioides brasiliensis yeast
cells
Amaral AC; Braun S; Nunes ES; Bocca AI; Lima ECD; Báo SN; Azevedo RN; De Morais
PC; Felipe MSS.
4-35
Molecular typing and evolution of the Paracoccidioides genus
Teixeira MM; Paes HC; Fernandes L; San-blas G; Felipe MSS.
4-36
Adhesion and expression of ligands of extracellular matrix (ECM) by different Paracoccidioides brasiliensis isolates
Silva JF, Benard G, Mendes-Giannini MJS.
4-37
The heath shock factor of Paracoccidioides brasiliensis and its complementation
of Saccharomyces cerevisiae
Paes HC, Matos LF, Teixeira MM, Torres FAG,Felipe MSS.
p 182
p 182
p 183
5 Immunology: Patients / Experimental Animals
5-01
p 187
5-02
Paracoccidioidomycosis patients dendritic cells: Effect of Paracoccidioides brasiliensis antigens on surface molecules expression
Sato PK; Oshiro TM; Diogo CI; Passos EC; Sadahiro A; Shikanai-Yasuda MA.
5-03
Altered expression of STATs 1,3,4,5 and their modulation by cytokines in mononuclear cells from paracoccidioidomycosis patients
Romano CC; Mendes-Giannini MJS; Duarte AJS; Benard G.
5-04
Increased T cell regulatory activity in patients with paracoccidioidomycosis
Ferreira MC; Blotta MHSI; Mamoni RI.
5-05
Polymorphisms analysis of CTLA-4 gene in paracoccidioidomycosis patients
Lozano VF; Paes HC; Teixeira MM; Lins TCI; Vieira RG; Blotta MHSI; Goes AM; Pereira
RW; Bocca AI; Felipe MSS.
5-06
Lymphoproliferation, levels of antibodies and cytokines to AB2-β-anti-idiotipic antibodies in patients with paracoccidioidomycosis (PCM)
Furuchó CR; Oliveira CCU; Bertossi DRT; Fonseca CA; Shikanai-Yasuda MA.
5-07
Paracoccidioidomycosis: Study of cytotoxic immune response in cutaneous and
mucosal lesions
C. Pagliari; N.v. Pereira; M. I. S. Duarte; And M.n. Sotto.
p 187
p 188
p 188
p 189
p 189
p 190
5-08
p 190
5-09
p 191
36
Phenotypic and functional characterization of human dendritic cells induced by low
and high-virulence strains of Paracoccidioides brasiliensis
Fornazim MC; Mamoni RI; Spago MC; Oliveira RTD; Gabetta CS; Nowill AE; Blotta
MHSI.
Pulmonary abnormalities induced by Paracoccidioides brasiliensis in a mouse
model: Application of high resolution computed tomography to evaluation and follow-up of lesions
Lopera DE; Naranjo TW; Duque DJ; Diaz-Granados LR; Jiménez RA; Restrepo A, Zuluaga
AF; Cano LE; Patiño J; Hidalgo JM.
Alterations in pulmonary mechanisms during experimental infection by Paracoccidioides brasiliensis
Fracon JF, Bocca AL; Siqueira IM; Zin WA; Faffe DS; Jerônimo MS; Soares MAS; Russo
RC; Figueiredo F.
5-10
TLR-2, TLR-4 and Dectin-1 expression in human macrophages and neutrophils
stimulated by Paracoccidioides brasiliensis
Bonfim CV; Mamoni RI; Blotta MHSI.
5-11
Absence of TLR-2 results in less severe paracoccidioidomycosis but increased
inflammatory response caused by PMN and TH17 cells
Loures FV; Calich VIG.
5-12
MYD88 is a dispensable factor in resistance to Paracoccidioides brasiliensis in a
murine model of blood-borne disseminated infection
González A; Yañez A; Gozalbo D; Gil ML.
5-13
The influence of β-2 integrin in the fungal burden in Paracoccidioides brasiliensis
infected mice
Nunes J; Catão E; Fraga CIF; Amaral A; Figueiredo F; Felipe MSS; Faccioli LH; Bocca AL.
p 191
p 192
p 192
p 193
5-14
p 193
5-15
p 194
5-16
p 194
5-17
p 195
Surface antigens increase susceptibility to Paracoccidioides brasiliensis infection
via interleukin-4 production
Oliveira LL; Cavassani KA; Rocha FA; Vancim JO; Moreira AP; Campanelli AP; Milanezi
CM; Martinez R; Rossi M; Oliveira EB; Silva JS.
FBP plays a role in the imunosuppression on the course of Paracoccidioides brasiliensis infection
Mariano VS; Oliveira LI; Coltri KC; Vendruscolo PE; Ferraz DB; Roque-barreira MC;
Panunto-Castelo A.
Paracoccidioides brasiliensis inhibits human neutrophils apoptosis
Acorci MJ, Dias-Melicio LA; Golim MA; Bordon-Graciani AP, Peraçoli MTS; Soares
AMVC.
Prostaglandin inhibits Paracoccidioides brasiliensis killing by human monocytes:
Reversal by activation with IFN-γ, TNF-α and GM-CSF due to increased H2O2 production
Bordon-Graciani AP; Dias-Melicio LA; Acorci MJ; Peraçoli MTS; Soares AMVC.
5-18
Production of leukotriene B4 by different strains of Paracoccidioides brasiliensis
Dias-Melicio LA, Biondo GA; Bordon-Graciani AP; MJ Acorci; Peraçoli MTS, Soares
AMVC.
5-19
Comparative analysis of the infection evolution between isolates Pb 18 and Pb 01
of the Paracoccidioides brasiliensis
Fraga CIF; Siqueira IM; Ribeiro AM; Nunes J; Figueiredo F; Felipe MSS; Bocca AL.
p 195
p 196
5-20
p 196
Paracoccidioides brasiliensis infection determines dendritic cells to differentiate to
the plasmocytoid subpopulation which induces a more severe pulmonary infection
when transfered to resistant mice
Pina A; Calich VIG.
5-21
IL-10 deficiency determines a better fungicidal ability associated with overproduction
of IFN-γ and nitric oxide by Paracoccidioides brasiliensis infected macrophages
Costa TA; Calich VIG.
5-22
CD28 costimulatory molecule deficiency results in more severe Paracoccidioides
brasiliensis infection but protects mice from life-threatening immunopathology
Felonato M; Calich VIG.
p 197
p 197
37
5-23
Nitric oxide but not TREG cells plays a major immunoregulatory role in a pulmonary
model of fungal infection
Bernardino S; Calich VIG.
5-24
Role of CCR4 in the experimental infection by Paracoccidioides brasiliensis: Migration control of CD4+CD25+ T cells to lesion site
Rocha FA; Oliveira LI; Massafera-Tristão FS; Milanezi CM; Silva JS.
5-25
Granulomatous inflammation and fibrosis in mice with chronic pulmonary paracoccidioidomycosis: Immune-related associations
Naranjo TW, Lopera DE; Duque JJ; Diaz-Granados LR; Restrepo A; Cano LE.
5-26
Alterations of granuloma architecture, cellularity and severity of the early infection
in murine paracoccidioidomycosis by IFN-γ, celecoxib or lumiracoxib treatment
Molina RFS; Nishikaku AS; Albe BP; Burger E.
p 198
p 198
p 199
p 199
5-27
p 200
Therapeutic activity of a DNA vaccine using the gene Hsp65 from Mycobacterium
leprae for experimental paracoccidioidomycosis
Ribeiro AM, Souza ACO; Amaral AC; Fraga CIF; Nunes J; Salles BC; Coelho-Castelo
AAM; Figueiredo F; Silva CI; Felipe MSS; Bocca AI.
5-28
Evaluation of the protection induced by the immunization with radioattenuated yeast
cells of Paracoccidioides brasiliensis in animal model
Martins EMN, Bernardo S; Reis AMG; Antero SRA.
5-29
Modulation of experimental paracoccidioidomycosis by monoclonal antibodies
against the major diagnostic antigen, Gp43
Pinto FA; Puccia R; Da Silva CJ; Muñoz JE; Travassos LR; Taborda CP.
5-30
The influence of Fonsecaea pedrosoi cell wall in peritoneal macrophage activation
Nóbrega YKM; Lozano VF; Figueiredo F; Bocca AL.
p 200
p 201
p 201
6 Treatment
6-01
p 205
6-02
Posaconazole for the treatment of paracoccidioidomycosis: First clinical results
Tobón AM; Agudelo CA; Restrepo A.
6-03
Variable effect of caspofungin on the mycelial and yeastlike phases of several strains
of Paracoccidioides brasiliensis
Rodríguez-Brito S; Niño-vega G; San-Blas G.
6-04
Assessment of the efficacy of voriconazole, comparativily to other antifungals, in
paracoccidioidomycosis experimental of the rats
Granzoto DS; Vitali LH; Martinez R.
p 205
p 206
p 206
6-05
p 207
38
Timing of itraconazole therapy onset and its impact on the progression of fibrosis
induced by Paracoccidioides brasiliensis conidia: Results in an experimental model
of paracoccidioidomycosis
Lopera DE; Naranjo TW; Duque DJ; Diaz-Granados LR; Jiménez RA; Restrepo A, Zuluaga
AF; Cano LE.
Amphotericin B-PLGA-DMSA nanoparticle: A new alternative to treat paracoccidioidomycosis
Amaral AC; Bocca AI; Ribeiro AM; Nunes J; Falcomer CI; Peixoto DIG; Simioni AR; Pinto FI;
Lacava ZGM; Azevedo RB; Titze-De-Almeida R; Tedesco AC; De Morais PC; Felipe MSS.
6-06
p 207
6-07
p 208
6-08
p 208
6-09
p 209
6-10
p 209
6-11
p 210
Fungicidal activity of amphotericin B-magnetic fluid conjugate in vitro against Paracoccidioides brasiliensis
Medeiros PB; Amaral AC, Saldanha CA; Bocca AI; Guilherme RB; Silva JR; Nunes ES;
Lima ECD; Azevedo RB; De Morais PC; Felipe MSS.
PLGA-P10 conjugate improved 20-times the immunological protection of this peptide
against Paracoccidioides brasiliensis
Amaral AC, Marques AF; Muñoz JE; Bocca AI; Simioni AR; Titze-De-Almeida R; Tedesco
AC; De Morais PC; Taborda CP; Travassos LR; Felipe MSS.
Biological activity of the endophytic fungi UFMGCB551 against Paracoccidioides
brasiliensis
Campos FF; Rosa LH; Johann S; Cota BB; Rosa CA; Sá NP, Cisalpino PS; Zani CI.
Antagonistic effect of farnesol, a Candida albicans quorum sensing molecule, on
Paracoccidioides brasiliensis growth and morphogenesis
Derengowski LS; Braz SV; De-Souza-Silva C; Mello-De-Sousa TM; Báo SN; Kyaw CM,
Silva-Pereira I..
Killing of Paracoccidioides brasiliensis yeast cells by magnetic hyperthermia
Amaral AC; Braun S; Nunes ES; Bocca AI; Lima ECD; Azevedo RB; De Morais PC; Felipe
MSS.
Antifungal activity of plants extracts used in Brazilian traditional medicine against
Paracoccidioides brasiliensis
Johann S; Watanabe GA; Cota BB; Siqueira EP; Zani CI; Pizzolatti MG; Cisalpino PS;
Resende MA.
6-12
Treatment of murine paracoccidioidomycosis (PCM) with trimethoprim-sulfamethoxazole combination (TMP-SMX)
Moura F; Lima CRG; Moreto TC; Carvalho LR; Bueno RA; Moris DV; Mendes RP.
6-13
Therapeutic DNA vaccine in BALB/c and B10A mice against experimental paracoccidioidomycosis
Gomes-Rittner GM; Muñoz JE; Taborda CP; Travassos LR.
6-14
Adrenal function in paracoccidioidomycosis patients treated with azolic derivatives:
Results after prolonged post-therapy follow up
Tobón AM; Restrepo CA; Villa CA; Agudelo CA; Quiceno W; Restrepo A..
6-15
The association tuberculosis-paracoccidioidomycosis: Impact on the outcome of
theraphy
Tobón AM; Agudelo CA; Tabares AM; Restrepo A.
p 210
p 211
p 211
p 212
7 Other mycoses
7-01
Susceptibility to fluconazole and voriconazole of Candida species isolated from intensive care units patients in several hospitals in Medellín, Colombia (2001 - 2007)
Zuluaga A; De Bedout C; Agudelo CA; Hurtado H; Arango M; Restrepo A; González A.
7-02
Therapeutic study of gomesin in mice infected with Candida albicans
Rossi DCP; Muñoz JE; Taborda CP ; Daffre S.
p 215
p 215
39
7-03
Study of genotypic and phenotypic characteristics among Cryptococcus gattii serotype B - VGII molecular type clinical isolates from Cúcuta, Colombia and isolates
of the outbreak in Vancouver, Canada. G.Torres; and P. Escandon.
7-04
Microecology of Blastomyces dermatitidis: The ammonia hypothesis
DJ Baumgardner
p 216
p 216
7-05
p 217
7-06
An analysis of histoplasmosis in an endemic, resource poor area with a high HIV
prevalence rate — Guatemala, 2007
Miramontes R; Samayoa B; Herrera A; Scheel C; Gómez BI; Arathoon E; Chiller T.
7-07
Detection of DNA in peripheral blood from a patient with the ocular histoplasmosis
syndrome: A case report
Hernández JM; Muñoz C; Hernández D; Montoya C; González A.
p 217
p 218
40
Evaluation of an antigen-capture ELISA to detect Histoplasma capsulatum antigenuria
in immunocompromised patients
Scheel CM; Samayoa B; Benjamin L; Riley P; Lindsley M; Herrera A; Raxcacoj G; Lima
S; Miramontes R; Chiller TM; Brandt M; Arathoon E; Gómez BL.
Symposium
1
P. brasiliensis:
Genomics and Bioinformatics
Comparative analysis of Paracoccidioides brasiliensis
and other dimorphic fungi
Mia Champion
Broad Institute of MIT 2 Harvard
e-mail: champion@broad.mit.edu
Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis (PCM), one of the most
important human systemic mycosis in Latin America. In collaboration with the Dimorphic Fungal
Genomes Consortium, we have sequenced three strains of Paracoccidioides brasiliensis that are
representatives of distinct clades in the species tree: Pb01, Pb03, and Pb18. The genome assemblies
of these strains contain 33Mb, 29Mb, and 30Mb respectively, and Pb03 contains 9,063 predicted
genes. The Pb18 genome assembly has been anchored to an optical map consisting of 5 linkage
groups, providing a chromosomal context for this assembly. Analysis of syntenic regions as well as
regions of unique genomic content between the Paracoccidiodes strains, including the abundance
and distribution of repeat content, will be presented. Comparison to other dimorphic fungi as well
as more distant fungal genomes will enable description of gene families specific to dimorphic fungi.
These analyses will provide novel insights into the diversity between three Paracoccidioides strains
and contribute to our broader understanding of the larger group of dimorphic fungal pathogens.
Comparative genomics of Coccidioides species
Thomas J. Sharpton1, Jason Stajich1, Garry T. Cole2, and John W. Taylor1
1 University of California, Plant and Microbial Biology, Berkeley, CA 94720-3102 -USA
2 University of Texas, Biology, San Antonio, TX 78249 - USA
e-mail: jtaylor@berkeley.edu
Coccidioides species are ascomycetous fungi found in hot, dry areas of the New World. They associate with endemic small mammals and have been the focus of much research because, like
Paracoccidioides brasiliensis, they can cause life-threatening disease in otherwise healthy humans.
We are comparing genomes of Coccidioides species and their relatives to focus the tools of evolutionary biology on the problem of fungal adaptation to an animal host. Variation in gene sequences
and microsatellite repeats has been used to recognize two species of Coccidioides, C. immitis and
C. posadasii, and at least five populations therein. Through the efforts of the Coccidioides community, the Broad Institute and TIGR, genomes have been released for each Coccidioides species
and their close relative, Uncinocarpus reesii. This collection of genomes provides a rich resource
for comparative genomics, conditional upon the quality of annotation. With annotated genomes for
Coccidioides and Uncinocarpus, we have searched for genes specific to Coccidioides or the outgroup,
Uncinocarpus, for genes showing unusual rates of evolution, and for genes showing the effects of
strong selection. Recent effort by the Broad institute has produced sequence for 13 more Coccidioides genomes, making it possible to challenge hypotheses about selection based on comparison
of pair-wise genome comparison through the use of population based methods for detecting selection. We will discuss how all of the evolutionary results relate to adaptation and how these broader
hypotheses may, in turn, be falsified.
43
Post-genomic contribution to the knowledge on the
host-pathogen interaction
Maria Sueli Felipe, Aldo Henrique Tavares and Simoneide Souza Silva
Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil.
e-mail : msueli@unb.br or msueliunb@gmail.com
The genome constitutes the informational core of all biological processes and the study of living organisms depends heavily on our ability to access its contents. For many decades, the experimental
approach consisted of isolating single genes or regulatory elements and characterizing each one at
a time by means of loss-of- function and/or gain-of function experiments; or by identifying effectors
(proteins, cofactors or metabolites) and studying their roles and interactions. In keeping with the
informational flow in the cell, genome has been closely followed by transcriptome and proteome.
The study of pathogens and their interaction with hosts are of special interest. Computer-aided data
mining has enabled unambiguous identification of open reading frames, and transcriptional profiling
has yielded relevant information concerning differential gene expression. Microarray technology, in
conjunction with statistical and experimental validation, has been used to identify patterns of gene
expression—selected according to previous genome or transcriptome information—when the microbe
is confronted with conditions of interest, such as ex vivo interaction with specific cells or exposure
to therapeutic agents, signaling molecules or stressors. The fungus P. brasiliensis is one organism
that has benefited from these approaches. The yeast form of P.brasiliensis acts as a facultative
intracellular pathogen being able to survive and replicate within the phagosome of nonactivated
murine and human macrophages. This ability has been proposed to be crucial to the development
of disease. In this talk, I will outline the current post-genomic contribution to the knowledge on the
host-pathogen interaction, with focus on differential expression analysis of P. brasiliensis-macrophage cells. Recently, by using cDNA microarray technology we evaluated the early transcriptional
response of this fungus to the environment of peritoneal murine macrophages in order to shed light
on the mechanisms used by P. brasiliensis to survive within phagocytic cells. Of the 1152 genes
analyzed, we identified 152 genes that were differentially transcribed. Overall, our data provided
advances on the comprehension of the plasticity of response and indicate that P. brasiliensis is able
to undergo fast and dramatic expression profile microenvironment changes, as will be discussed
below. In order to better understand the complex interaction between host cells and P. brasiliensis
we have analyzed also by cDNA microarray the transcriptional profile of 624 previously selected murine immuno-regulatory genes following fungal infection of macrophages for 6, 24 and 48 hours. Our
microarray data have identified many genes that are modulated in particular those related to inflammatory mechanisms, membrane proteins, transcription regulation and apoptosis. Inflammation is a
powerful protective mechanism coordinated and controlled by cytokines and chemokines. Increased
chemokine gene expression has been detected by microarray analysis in macrophages infected with,
Candida albicans, Aspergillus fumigatus and Mycobacterium tuberculosis. Accordingly, our results
show that genes encoding the chemokines Ccl21, Ccl22 and Cxcl1, all of which are associated with
pro-inflammatory response, were significantly increased in peritoneal macrophages in response to
P. brasiliensis-Pb01, at the early stage of infection. Macrophage genes encoding membrane proteins
involved in adhesion and phagocytosis were also induced by P. brasiliensis. In this context, we have
observed significant induction of Cd14 and Clec1b. This differential expression pattern is probably
modulated by the pathogen and its ultimate result is an improved effectiveness of invasion and/or
macrophage activation. In addition, the gene for the C-type lectin-like receptor 1b (Clec1b), which is
also membrane-related, was induced at all times of macrophage infection with P. brasiliensis, which
44
has been confirmed by real-time RT-PCR. Matrix metalloproteases (Mmps) are known to be involved
in tissue remodelling, cleavage of cell-surface receptors and chemokine activation. Considering their
important role in collagen and cellular matrix remodelling, it has been suggested that induction of these
metalloproteases might be an important mechanism to facilitate fungal invasion. The up-regulation of
Mmp17 and Adam8 was observed in macrophages infected by P. brasiliensis and probably increase
the effectiveness of host cell parasitism. These data fit with the above described induction of adhesion and phagocytosis-related genes, since P. brasiliensis is a facultative parasite that can survive
and replicate in non-activated macrophages. Increased phagocytosis (Cd14 and Clec1b) and the
stimulation of matrix remodeling (Mmp17 and Adam8) are expected to contribute to a more efficient
invasion by the fungus of cell and tissue alike. Apoptosis plays a significant role in the regulation of
pathogenesis. In our experimental approach, several genes have been found to be responsive and
up-regulated by P. brasiliensis, including caspases 2, 8 and 3; this phenomenon has been correlated with a phenotypic marker of infection resistance, probably as an efficient manner to eliminate
the fungus without tissue damage. Recently, our group used cDNA microarray to evaluate the early
transcriptional response of P. brasiliensis to the inner environment of peritoneal murine macrophages
at six hours of infection. Thus, considering the analyses of differential gene expression for host and
pathogen, at six hours, we propose a model for the P. brasiliensis-macrophage interaction that discusses the events at molecular level during early infection. In response to the harsh macrophage
microenvironment P. brasiliensis up-regulates genes (sod3 and hsp60) related to the detoxification
of oxidative radicals and amino acid biosynthesis (metG). In addition, genes encoding five enzymes
of the glycolytic pathway have been found to be down-regulated, suggesting a glucose-poor environment. Macrophages at the same time point up-regulate genes related to inflammation (chemokines
and cytokines-Ccl21, Cxcl1 and Ccl22) and phagocytosis-related (Clec1b) genes, probably as an
effort to counteract infection by the fungus. In summary, our results demonstrate that P. brasiliensis
is a potent inducer of molecules involved in the initial host response to this organism. The kinetic
approach used in microarray study has elucidated a coordinate and temporal basis of host defense
molecules elicited against P. brasiliensis infections. These expression data described should provide
a foundation for further studying the pathogenesis of this infectious agent and a better knowledge
on the host-pathogen interaction.
Financial support – CNPq, FAP-DF, FUB.
Genes involved in morphogenesis and cell wall synthesis
and regulation in P. brasiliensis
Gustavo Niño-Vega
Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela
e-mail: gnino@ivic.ve
Currently, our group is working on the analysis of genes involved either in the synthesis (α-1,3-glucan synthase, chitin synthases), hydrolysis (α-1,3-glucanase) or regulation (Rho genes) of cell wall
α-1,3-glucan and β-1,3-glucan in Paracoccidioides brasiliensis, and also on genes participating in
morphological events by other mechanisms (septins, actin).
The α-glucan synthase gene of P. brasiliensis (PbrAGS) has been cloned in our laboratory (GenBank accession number AY780573). Analysis of its sequence suggests a resemblance to previously
reported fungal AGS (Proc Natl Acad Sci USA 95: 9161–9166, 1998; Fungal Genet Biol 42: 165-177,
2005) As with other α-glucan synthases, three distinct domains can be identified in the PbrAgs1 se-
45
quence. The first domain (aa 1-1073) –located extracellularly, according to computer models- shows
significant sequence similarities to α-amylases belonging to family 13 of the glycosyl hydrolases
(Biochem J 280: 309–316, 1991) and is denoted as an amylase homologous domain (AHD). In other
fungi, it has been suggested that this domain is involved in remodelling newly formed α-glucan or
crosslinking to the existing cell wall (Proc Natl Acad Sci USA 95: 9161–9166, 1998). The second
domain (aa1097–1990) is flanked by two hydrophobic sequences (aa1074–1096 and aa1991–2008).
It is predicted to be intracellular and contains a glycogen or starch synthases homologous domain
(G/SSHD). Therefore, it is likely that it encodes the glucan synthase domain of PbrAgs1. The third
domain (aa1097–2431) is predicted to span the membrane 8 times. This multi-pass transmembrane
region (MTM) is thought to be involved in the transport of the α-glucan chain across the plasma
membrane (Proc Natl Acad Sci USA 95: 9161–9166, 1998). In accordance with the exclusive presence of α-1,3-glucan in the yeast-like phase of the fungus, northern analyses show that the PbrAGS1
gene is only expressed in the yeast-like phase of P. brasiliensis .
Recently, Marion et al. (Mol Microbiol 62: 970-983, 2006) reported that an α-(1,4)-amylase is
essential for α-(1,3)-glucan production and virulence in Histoplasma capsulatum. Interruption of
the AMY1 gene responsible for the α-(1,4)-amylase by insertional mutagenesis, resulted in loss of
α-(1,3)-glucan and loss of virulence (Mol Microbiol 62: 970-983, 2006), a phenotype similar to disruption of domain 1 of the H. capsulatum AGS1 gene. These results have prompted us to search
for a similar gene in P. brasiliensis.
Also, a P. brasiliensis α-1,3-glucanase-like gene sequence has recently been cloned in our laboratory. It has high identity to fungal α-1,3-glucanases (also called mutanases due to their hydrolyzing
activity towards “mutan”, the so called α-1,3-glucan from Aspergillus nidulans and other fungi. Preliminary analysis of the gene sequence and comparison with other fungal α-1,3-glucanases suggest
that we have so far cloned and sequenced about two thirds of the gene.
So far, information on genes necessary for the establishment and morphology of the pathogenic
phase in P. brasiliensis is not certain, although valuable and increasing information on genes preferentially expressed on this phase is been given by the groups working on the construction of expressed
sequence tags (ESTs) libraries on this fungus (Yeast 20:263-271, 2003; Eukariotic Cell 2:34-48,
2003; Mol. Gen. Genomics 271:667-677, 2005; J. Biol. Chem. 280:24706-24714, 2005). Here we
describe the cloning and complete sequence of CHS5, a gene encoding a class V chitin synthase,
from the pathogenic fungus P. brasiliensis, and its arrangement within the genome in a head-to-head
configuration with CHS4, both genes sharing a common 5’UTR. Also, changes in transcript levels
for both genes, following alteration of external osmolarity, suggest a common regulatory mechanism
of transcription. The exploration by northern analises of other genes related to synthesis of cell wall
components (CHS3 and FKS1) reveals higher expression in the yeast phase of the fungus: contrary
to other CHS genes, CHS3 is only expressed in the pathogenic yeast phase and at the end of the
mycelium-to-yeast transition in P. brasiliensis, while we found, to our surprise, that FKS1 (the only
β-1,3-glucan synthase gene so far reported for P. brasiliensis (Yeast 16:451-462, 2000) has a also
higher expression in the yeast phase, which, made us wonder if β-1,3-glucan, even when represents
a small percentage of the yeast cell wall components, would be essential for its maintenance.
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Symposium
2
Understanding the immune Responses
against dimorphic Fungi in experimental
animal Hosts
Experimental animal models and their contribution to the study
of fungal pathogenesis
Karl V. Clemons, Ph.D.
California Institute for Medical Research, San Jose, CA; Division of Infectious Diseases,
Santa Clara Valley Medical Center, San Jose, CA; and
Department of Medicine, Division of Infectious Diseases and Geographic Medicine,
Stanford University, Stanford, CA USA.
e-mail: clemons@cimr.org
What makes a fungus cause disease in humans, and how that disease progresses are questions
that have been under study for decades. Although in vitro studies allow us to examine specific
questions and fungus host interactions, they cannot replicate the conditions found in vivo. Thus,
animal models, as surrogates of human disease, have been used historically in studies of fungal
pathogenesis. These investigations encompass virulence, as well as host response to obtain the
overall picture of the evolution of a fungal disease. Critical to the study of pathogenesis are the
choice of animal, route of infection, parameters for evaluation and how closely a model mimics that
disease in humans. Although numerous laboratory animal species have been used, mice are the
most commonly used species currently, and have numerous advantages with respect to affordability,
defined genetics, immunological reagents, and availability of gene-knockouts, deficient in a single
cytokine or gene-product. Our ability to modulate host response, severity of infection and genetically
manipulate the host or the infecting organism allow for the study of specific questions. However,
no single model, or even strain of animal, will be useful to answer all questions. For example, our
laboratory has demonstrated that mucosal candidiasis in SCID mice mimics mucosal infection in
humans with AIDS. The mucosal surfaces of the gastrointestinal tract are heavily colonized, but
disseminated disease does not occur. However, mucosal candidiasis established in normal mice
pretreated with 5-fluorouracil results in dissemination, which is similar to that seen clinically in patients receiving cancer chemotherapy. Furthermore, in studies on the potential of Saccharomyces
cerevisiae to be a pathogen, we found that virulence potential changed depending on the strain of
mouse used in the model. These data raised the possibility of different strategies of virulence might
exist among various strains of the same species of fungus. Pathogenesis is greatly influenced by
the host response. The use of gene-knockout mice has aided our study of the importance of various
host proteins such as collectins and cytokines. For example, we examined the role played by IL-10
in systemic aspergillosis, where we found IL-10 deficient mice more resistant to infection, but could
not demonstrate a role for IL-10 when mice were treated with anti-IL-10 receptor. In other studies,
we have shown how gender influences the outcome of the establishment of disease by demonstrating the estradiol in vivo blocks the initial transition of the conidia of Paracoccidioides brasiliensis
into the tissue yeast-form, and that the infecting inoculum is rapidly cleared from the pulmonary tissues of female BALB/c mice, whereas the organism multiplies and disease progresses in the male
counterparts. Animal models also allow us to examine host events associated with fungal infection.
Meningitis in humans due to Coccidioides is a severe and lethal disease, where patients often have
stroke events and associated vasculitis. We developed a rabbit model of coccidioidal meningitis
that also results in the development of vasculitis of the large arterial vessels of the CNS. Thus far,
we have found associations of increased MMP-9, IL-6, interferon-γ, and iNOS in vasculitic vessel
tissue, all of which may exacerbate the inflammatory process of vasculitis. Overall, animal models
of fungal infection are valuable tools in addressing questions concerning the infectious process and
contribute to our deeper understanding of occurrence, evolution and how they might be controlled
or better, prevented and eliminated.
49
Toll like receptors and the adaptor molecule MYD88 play an
important role in pulmonary paracoccidioidomycosis
Vera Lucia G. Calich and Flávio V. Loures
Departamento de Imunologia, Instituto de Ciências Biomédicas,
Universidade de São Paulo, Brazil, CP 66.208
e-mail: vlcalich@icb.usp.br
Recognition of invading microorganisms by the innate immune system is a first step to control or
eliminate an infectious process from the host. The initial interaction between immune cells and
microorganisms is mediated by several types of receptors which recognize molecular patterns of
pathogens and are collectively called pathogen recognition receptors (PRR). The Toll-like Receptors
(TLRs) constitute a molecular family that recognizes a wide range of microbes and their products
known as pathogen-associated molecular patterns. TLRS are expressed in diverse innate immune
cells such PMN, macrophages, dendritic cells and lymphocytes. Their activation triggers a signaling
cascade that results in an inflammatory response through production of proinflammatory cytokines
and up-regulation of costimulatory molecules expression leading to initiation of antigenic-specific
adaptative immune response. As described for other microorganisms, TLRs were shown to be
involved in host defense against different fungal pathogens. The contribution of individual TLRs to
the immune response against pathogenic fungi depends on several factors such as the route of infection, the fungal morphotype or fungal species. Alveolar macrophages are the first host cells that
interact with Paraccocidioides brasiliensis, however, the initial fungal-host cells interaction is poorly
known. Mannose and complement receptors appear to play a role in innate immunity to P.brasiliensis
infection, but the role of TLR2, TLR4 and MyD88 in paracoccidoidomycosis was never investigated.
Recent findings from our laboratory demonstrated that, compared with the normal strain (C3H/
HePas), macrophages from TLR4-deficient mice (C3H/HeJ) had a lower phagocytic ability which
appears to influence the decreased number of viable P. brasiliensis yeasts recovered after 72h cocultivation. Deficient macrophages secrete lower levels of nitric oxide (NO), IL-12, and MCP-1 but
produced equivalent amounts of TNF-α. In contrast, IL-10 was synthesized in higher amounts by
TLR4-deficient macrophages. Consistent with in vitro results, 96h after in vivo pulmonary infection
TLR4-deficient mice presented decreased fungal loads in the lungs associated with lower levels of
NO and pro-inflammatory cytokines (IL-12, and GM-CSF). Similar results were observed at week
11 after infection of TLR4-mutant mice: decreased CFU counts associated with low IL-12 levels but
high IFN-γ secretion. Paralleling its mild infection, the deficient strain secreted low levels of IgG1,
IgG2b and IgM P.brasiliensis specific isotypes. Cytospin preparations of lung infiltrating leukocytes
at week 2 of infection showed an increased number of PMN neutrophils associated with decreased
numbers of lymphocytes and monocytes. Although differences in CFU counts and synthesis of some
inflammatory mediators occur in TLR4-deficient mice, such differences were not sufficient to alter
their mean survival times.
We have also comparatively analyzed P.brasiliensis infection of TLR2-normal (WT) and TLR2-KO
mice in a C57BL/6 background. In vitro infection of KO macrophages resulted in lower phagocytic
indexes and decreased recovery of viable yeasts after 72 h co-cultivation. Macrophage supernatants
presented low levels of NO and MCP-1. Compared with WT mice, 48h and 2 weeks after infection KO
mice presented diminished pulmonary fungal loads and NO, and these findings were associated with
some differences in the levels of pro- and anti-inflammatory cytokines. Lung infiltrating leukocytes
at weeks 2 and 4 of infection showed a decreased proportion of macrophages but an increased
number of PMNs and lymphocytes. Interestingly, TLR2 KO mice secreted higher levels of IL-6,
TGF-β, IL-23 and IL-17 indicating an enhanced Th17 immunity. In addition, a decreased number of
50
CD4+CD25+FoxP3+ T cells were detected in TLR2 KO mice. At week 11 of infection TLR2-deficient
and normal mice presented similar humoral immunity but the former strain showed increased fungal
burden in the lungs. Despite this difference, equivalent mortality rates were showed by both mouse
strains.
When MyD88 KO macrophages were in vitro infected with P. brasiliensis yeasts for 72h, increased
number of viable fungi was recovered in comparison with normal cells. This diminished fungicidal
ability paralleled a decreased synthesis of NO and IL-12. The early in vivo infection reproduced the
in vitro findings: higher fungal loads were found in MyD88 KO mice associated with lower levels of
pulmonary NO and IL-12. This appears to indicate that absence of MyD88 molecule causes profound
effects in cell activation resulting in more severe infection. Mortality studies confirmed the higher
susceptibility of MyD88 KO mice to P. brasiliensis infection, since their mean survival time is significantly lower than that of WT mice.
As a whole, TLR deficiency caused less severe infections associated with altered secretion of NO,
cytokines and chemokines, resulting in altered cellular influx to the site of infection. In both PRR-deficient strains, the lung inflammatory infiltrate was composed of a diminished number of macrophages
associated with an increased presence of PMNs. Possibly the phagocytic and killing abilities of these
cells would contribute to the decreased fungal inoculum at the site of infection. Furthermore, the low
level of MCP-1 was parallel to the decreased number of lung infiltrating monocytes. An interesting
observation was the enhanced Th17 immunity associated with severe inflammatory lesions and
decreased number of regulatory T cells (CD4+CD25+ FoxP3+) in the lungs of TLR2 KO mice.
Mortality studies showed that TLR deficiency was not able to change the late course of infection since no differences were observed between TLR KO and TLR-normal mice. Compensatory
mechanisms appear to abolish the immunological differences caused by PRR-deficiencies. The
same was not true for MyD88 deficiency which causes higher mortality of infected mice. The more
severe infections of MyD88-deficient macrophages and mice are probably due to the intact fungal
recognition mediated by the expression of normal PRR but impaired ability of cell activation resulting
in diminished fungicidal ability.
In summary, our studies suggest that MyD88 deficiency is more important than TLR2 or TLR4
deficiency and P. brasiliensis yeasts appear to use TLRs as a virulence mechanism facilitating
the access of fungal cells into murine macrophages. Despite their TLR-mediated activation, macrophages are not able to control fungal growth, and the final balance between fungal growth and
activation of the immune system appears to control disease outcome. Furthermore, our studies have
also suggested that, besides TLR, other PRR play a role in the host immune response against by
P.brasiliensis infection.
Financial support provided by FAPESP
In vivo protection against P. brasiliensis infection
with monoclonal antibodies
Carlos P. Taborda1, Rosana Puccia2 and Luis R. Travassos2
1
Institute of Biomedical Sciences, Departament of Microbiology,
University of São Paulo, São Paulo, Brazil.
2
Departament of Microbiology, Immunology and Parasitology,
Federal University of São Paulo, São Paulo, Brazil
e-mail: taborda@usp.br
The treatment of paracoccidiodomycosis patients with antifungal drugs is the accepted therapy,
even if after prolonged periods of drug administration there is no assurance of complete cure and
with relapses being frequent events. The protective function of antibodies against fungal infections is
51
controversial. Recently, several studies have established that some antibodies are protective against
fungi as exemplified by two monoclonal antibodies that have entered clinical trials for cryptococcosis
and candidiasis treatment. Protective role for antibodies against Paracoccidioides brasiliensis is still
doubtful. In the present work the effects of monoclonal antibodies against the major diagnostic antigen (gp43) have been analyzed in vitro and in vivo. Passive administration of some mAbs before
and after intratracheal or intravenous infections resulted in reduction of fungal burden and decreased
pulmonary inflammation. Protection mediated by mAb 3E, the most efficient mAb in the reduction
of fungal burden, was associated with increased phagocytosis of yeast cells by macrophages. The
ingestion of opsonized yeast cells led to increased NO production by macrophages and enhanced
levels of IFN-γ in the lung of infected mice. The reactivity of mAb 3E against a panel of gp43 derived
peptides suggested that the sequence NHVRIPIGWAV contains its binding epitope. The protection
of paracoccidiodomycosis has always been attributed to a robust cellular immune response whereas
antibodies have been associated to bad prognosis. Here, we show that specific antibodies to a major
antigen of P. brasiliensis can be protective against a challenge of virulent fungi. A better understanding
of the parameters that influence antibody-mediated immunity will increase our knowledge of vaccine
efficacy and host susceptibility to infection.
P10 immunization using Salmonella enterica’s
flagellin as adjuvant
Luis R. Travassos1, Luis C.S. Ferreira2, Catarina J.M. Braga2 and Carlos P.Taborda2
Departament of Microbiology, Immunology and Parasitology,
Federal University of São Paulo, São Paulo, Brazil.
2
Institute of Biomedical Sciences, Departament of Microbiology,
University of São Paulo, São Paulo, Brazil
e-mail: taborda@usp.br
1
The major diagnostic antigen of P. brasiliensis (gp43) was cloned and sequenced. A peptide
sequence, known as P10 contained a T-cell epitope able to induce an IFN-γ-mediated Th-1
response. Association of P10 immunization with chemotherapy was studied in Balb/c mice intratracheally infected with P. brasiliensis with very encouraging results. Early studies on the gp43
and P10 always used complete Freund’s adjuvant (CFA) which is not acceptable for human use.
Alternative ways of delivering P10 and using other adjuvants were investigated. In the present
work, we studied the adjuvant properties of flagellin FliCd from Salmonella enterica Müenchen.
P10 was genetically fusioned in the central region of flagellin FliCd or just mixed together with
free flagellin and nasally administered in Balb/c mice. Our results showed that administration
of P10 mixed with flagellin FliCd promoted a better protection than the p10-fusioned flagellin or
the peptide alone in CFA. The analysis of colony-forming units indicated a reduction of fungal
burden in the lung with significant production of IL-12 and IFN-γ in lung homogenates. These
results may have an impact in P10 vaccination, with the perspective of using an intranasal vaccine of high immunological potency.
52
Pattern recognition receptors in the host immune
response to Coccidioides spp.
Maria del Pilar Jiménez-A.1,2,3, Suganya Viriyakosol2, Lory Walls2, Sandip K. Datta4,
Theo Kirkland 4,5, Sigrid E.M. Heinsbroek6, Gordon Brown7 and Joshua Fierer 2,4.
1 Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia.
2 Research Service, VA San Diego Healthcare, San Diego, CA.
3 Programa de Doctorado en Ciencias Médicas,
Universidad Pontificia Bolivariana (UPB)-CIB-CES. Medellín, Colombia.
4 Department of Medicine, Division of Infectious Diseases School of Medicine,
University of California San Diego, USA.
5 Department of Pathology, UC San Diego School of Medicine, San Diego, CA, USA.
6 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
7 Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, South Africa.
e-mail: pjimenez@cib.org.co
Coccidioidomycosis is a systemic mycosis endemic in the Sonoran deserts in the new world. The etiological agents are Coccidioides immitis and Coccidioides posadasii (Coccidioides spp). Coccidioides
spp., are dimorphic fungi that grow as a mold in the desert soil. Infections are acquired by inhaling
spores (arthroconidia) that then germinate in the host and transform into spherules, the pathognomonic
tissue form of the fungus. The vast majority of infections are either asymptomatic or self-limited, which
implies that innate immunity is quite effective under most circumstances. However, when exposure
is massive, a very high percentage of the resulting infections are symptomatic. African-Americans
and Filipinos are at least 10 times more likely to develop disseminated Coccidioidomycosis than
are Caucasians, even if the primary pneumonia is self-limited. This suggests that there is a genetic
basis for the extra-pulmonary dissemination of this infection in humans. There have been a number
of studies of acquired immunity to Coccidioides, both in humans and in experimental animals. It is T
cell dependent and the patients that recover spontaneously develop a Th1 type immune response.
Particularly, in the animal models have been established that there are different susceptibility patterns
in congenic strains of mice. C57BL/6J and BALB/c mice are the most susceptible strains and there is
a correlation between susceptibility and the production of Th2 cytokines. DBA/2N mice are the most
resistant and there is a correlation between resistance and the production of Th1 cytokines.
In contrast to what is known about acquired immunity, very little is known about the innate immune response to this fungus. Pattern Recognition Receptors (PRRs) play an important role in the
orchestration of the innate immune response by recognizing microbial molecules that are conserved
accross broad taxa and that are denominated Pathogen Associated Molecular Patterns (PAMPs).
Several cell wall components of Coccidioides spp. might act as PAMPs by receptors such as Dectin1, Toll like receptors (TLRs), or the Mannose Receptor (MR).
Dectin-1 is the β1-3 glucan receptor that is expressed on macrophages and dendritic cells. β-glucan accounts for a large percentage of the weight of a Coccidioides spherule. The mannose receptor (MR) is a C-type ligand expressed on antigen presenting cells that is involved in endocytosis
of mannosylated proteins and participates in the homeostasis of glycoproteins. Coccidioides spp.
contain significant amounts of mannose and 3-O-methylmannose in its cell wall. TLRs are a family of
53
conserved non-phagocytic receptors that mediate cellular responses to PAMPs and other ligands. In
the case of immune response to fungi, the contribution of individual TLRs might vary depending
on fungal species and fungal morphotypes.
Few studies have been done to determine how Coccidioides spp interact with the innate immune
system. It has been reported that spherules activate T cells to secrete pro-inflammatory cytokines
and chemokines such as TNFα and MIP-2, and that this response is both Dectin-1 and TLR2
dependant. One study done with human adherent peripheral blood mononuclear cells (PBMC)
shown that the MR mediates the cellular immune response to TK27, a glycosylated antigen. It is
known that mannose is the principal monosaccharide of T27K.
The goal of this work was to study the role of several PRRs: Dectin-1, MR and TLRs in the innate immune response to Coccidioides spp., both in vivo and in vitro experimental models.
We used formalin-killed spherules and 1,3-β-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and
resistant (DBA/2) mice. DBA/2 macrophages produced more TNF-α and IL-6 than macrophages
from C57BL/6 mice, and the amount of TNF-α made was dependent on both TLR2 and Dectin1. mDCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2
mDCs. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice
expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most
of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKSs
than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34
C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms,
respectively. Also, we found that resistant (DBA/2) mice had higher levels of Dectin-1 mRNA and
more Dectin-1+ cells in their infected lungs than did susceptible (B6 and BALB/c) mice. These
results suggest that Dectin-1 plays an important role in innate immunity to coccidioidomycosis
and that different isoforms and differential expression of this lectin contribute to susceptibility of
mice to coccidioidomycosis. As Dectin-1 is expressed on both macrophages and DC it affects the
cytokine responses to spherules.
We infected MR KO and did quantitative mycology on the lungs and spleens. We found that
MR KO mice infected either i.p. or intra-nasally, in both cases we recovered significantly higher
numbers of C.immitis than in control mice. These results suggest that MR is a pattern recognition
receptor that plays an important role in resistance to C.immitis.
We infected MyD88 -/-, TLR2-/-, TLR4-/-, TLR9 -/- and IL-1R-/- mice with C. immitis. We found
that MyD88 deficient mice on a C57BL/6 background were more susceptible to C. immitis. However, TLR4, TLR9, and TLR2 deficient mice were not more susceptible than C57BL/6 mice to
intra-nasal (i.n.) infection with C. immitis as measured by numbers of CFU cultures from lungs and
spleens 14 days after i.n. infection. IL-1R-/- mice were more susceptible to dissemination but not
pulmonary infection, and they were not as susceptible as MyD88 -/- mice. Thus, IL-1R played a
significant role in resistance to infection, but does not completely account for the MyD88 result.
We found that both TLR2 and Dectin-1 were necessary for TNF-α secretion from macrophages
stimulated by β-glucan. The apparent resistance of TLR2 deficient mice may be explained by a
defect in the Dectin-1 response to coccidioidomycosis in B6 mice.
All these results suggest that an effective host response to Coccidioides spp requires a collaborative recognition of distinct Coccidioides spp cell wall components by different innate immune receptors. Genetic polymorphisms in one or more components may explain the increased
susceptibility of some individuals to this infection.
54
A strategy for the generation of a recombinant vaccine against
coccidioidomycosis
Garry T. Cole
Department of Biology and South Texas Center for Emerging Infectious Diseases.
University of Texas at San Antonio, San Antonio, Texas 78249 U.S.A.
Coccidioides is an airborne fungal pathogen that can cause mild to severe respiratory disease (coccidioidomycosis; San Joaquin Valley fever) in immunocompetent individuals. The fungus inhabits
desert soil in the Southwestern U.S. between West Texas and Southern California, as well as certain
arid regions of Mexico, Central and South America. About 8 million people reside in the endemic
regions of United States. Although rarely a life-threatening disease, Coccidioides causes significant
morbidity in more than 40% of infected individuals, and is responsible for high health care costs related
to long term treatment. Although about half of the Coccidioides-infected individuals experience only
mild discomfort and do not seek medical intervention, a large body of clinical evidence suggests that
reactivation of the respiratory disease may occur months to years after the original insult. Recovery
from symptomatic infection typically confers lifelong immunity to reinfection, suggesting that vaccination against coccidioidomycosis is feasible. We have shown that disease-susceptible BALB/c mice
can be fully protected against coccidioidal infection by vaccination with a genetically engineered,
avirulent strain of the pathogen. A detergent-extracted protein fraction of the parasitic cell wall of
this mutant strain protects mice against pulmonary coccidioidomycosis. We have applied a novel
strategy to develop an epitope-driven vaccine based upon a subset of cell wall-derived antigens of the
live vaccine strain of Coccidioides that interface with the host immune system and elicit a protective
response against coccidioidal infection. We have identified candidate T-cell-reactive proteins in this
subset of antigens that drive a robust immune response against pulmonary challenge of transgenic
mice which express human MHC class II molecules. These protein vaccine candidates were selected
on the basis that they contain promiscuous epitopes with high affinity for human MHC II complexes,
stimulate an in vitro proliferative response of peripheral blood mononuclear cells isolated from skin
test-positive patients, and provide protection (survival and reduction of fungal burden) to mice infected
intranasally with a lethal inoculum of Coccidioides spores. We argue that this epitope-driven vaccine
can simultaneously direct the immune response to multiple dominant and subdominant epitopes and,
thereby, induce robust and durable protection against coccidioidomycosis.
55
Symposium
3
Virulence Factors and
host-parasite Interactions
57
Histoplasma virulence mechanisms with parallels
in paracoccidioidomycosis
William Goldman
University of North Carolina at Chapel Hill, NC, USA.
e-mail: goldman@med.unc.edu
Histoplasma capsulatum is one of the best-studied dimorphic fungal pathogens, and most biological
work has focused on specific characteristics that enable the yeast form to be a successful intracellular
parasite of macrophages. New molecular genetic tools have allowed us to evaluate the expression
and prove the importance of two yeast phase-specific factors: CBP, a secreted protein that is essential for virulence, and α-(1,3)-glucan, a virulence-associated cell wall polysaccharide. However,
the mechanisms that explain their role in fungal pathogenicity have remained a complete mystery
until recently.
We have now used NMR to solve the structure of CBP, revealing that this protein is a proteaseresistant homodimer and a member of the saposin family of lipid- and membrane-binding proteins.
It is likely that CBP is involved in lipid binding, lipid metabolism, and/or membrane remodeling in
the phagolysosomal compartment in which Histoplasma resides. We have taken two approaches
to study α-(1,3)-glucan: the first is a forward genetics strategy, using Agrobacterium-mediated insertional mutagenesis, to identify genes implicated in the regulation, synthesis, and processing of
α-(1,3)-glucan. The second approach uses reverse genetics, combining fungal gene disruption with
mammalian RNA-interference, to study the genes involved in production of and response to α-(1,3)glucan. This work has revealed that α-(1,3)-glucan on the surface of Histoplasma yeasts masks
recognition of the underlying β-glucan by dectin-1, a macrophage pattern-recognition receptor that
is critical in the innate immune response to fungi.
Integrated genomics and proteomics strategies bring new insight into
Paracoccidioides brasiliensis response upon host interaction
Celia M.A. Soares
Laboratório de Biologia Molecular, ICB,
Universidade Federal de Goiás, Goiânia, Goiás, Brasil.
e-mail: celia@icb.ufg.br
Paracoccidioides brasiliensis is a major fungal pathogen of humans. The fungus can thrive in a range
of niches within its human host. These niches can vary in many aspects, such as availability of nutrients and ambient pH. Also the fungus may have the ability of modifying the ambient by its metabolic
activity, implying that patterns of fungal gene expression could vary temporal and spatially in host
niches. Genome transcriptional expression profiles and proteomics of microbes during infection
provides information about the host microenvironment to which the microbe is exposed, as well as
the gene expression changes that enable the microorganism to adapt to its host niche. The aim of
the current study was the identification of genes and proteins expressed during invasion of organs
such as liver, spleen as well as in routes of fungal dissemination. Also the global influence of iron
and copper deprivation and extracellular matrix proteins (ECM) in fungal mRNAs and protein levels
was investigated. We observed that depending on the site of infection, P. brasiliensis adapts to the
environment. P. brasiliensis can modify its global transcriptional profile in a temporal way, as well as
59
can adopt a metabolic style on dependence of the host niche. For instance, the fungus can modify
its global transcriptional profile within 10 min of exposure to human blood and suffer a new global
adaptation after 60 min of incubation. Genes expressed in response to liver include those related
to the glycolytic style, to the cell-wall remodeling and to lipid synthesis. Interestingly upon exposure
to human plasma the fungus shifts to a starvation mode. Other experimental conditions that mimic
characteristics of certain host environments, such as iron and copper starvation, as well as fungal
adhesion to ECM components induce a global modification in the transcriptional and proteomic profiles. By analyzing and comparing transcriptional and proteomics patterns we identified genes and
metabolic routes that could be relevant to the fungal survival in the host milieu. Those genes include,
for example, those coding for proteins of iron and copper transport systems, those coding for predictable novel adhesins such as enolase, as well as those coding for enzymes of specific metabolic
routes over expressed in host niches. The methodological approaches also led to the detection of
specific pathways related to P. brasiliensis virulence.
Financial support: CNPq, FINEP.
Adhesion and extracellular matrix proteins
Maria José Mendes-Giannini
Departamento de Análises Clinicas, Faculdade de Ciências Farmacêuticas,
UNESP, Araraquara, São Paulo, Brazil.
e-mail: giannini@fcfar.unesp.br
Paracoccidioides brasiliensis possesses multiples factors that can cause damage to the host and
that can contribute to its virulence phenotype. With the objective of understanding the mechanisms
that regulate the interaction of this fungus with host cells, our study emphasis the role of molecules
that can represent new microbial targets to the infection control. For this reason, interaction between
P. brasiliensis and epithelial cells were evaluated, with an emphasis on the adherence, induction
of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The
success of colonization is a complex event, generally involving a ligand coded by the pathogen (adhesins) and a cell receptor. An important aspect in the interaction between P.brasiliensis and your
host is the ability to adhere to matrix extracelular components (ECM). This fungal employs a series of
strategies to colonize and to disseminate, including ligands. The understanding and identification of
these molecules it is very important in the discovery of efficient treatments for the systemic mycoses.
Differential adhesion to the epithelial cells and extracellular matrix (ECM) were verified depending
on the strain and the fungal growth conditions. In this study, proteomic approaches will allow the
characterization of adhesins under different circumstances. An increase of adhesin expression was
evident from freshly fungal isolated from human or animal passage as well as armadillos and that
possessed a higher adhesion capacity. This occurrence could be associated with host adaptation
and related to the virulence of P. brasiliensis, showing the potential of this fungal in the host-parasite
interaction.
FAPESP, PADC-FCF and CNPq
60
Virulence insights from the P. brasiliensis transcriptome
Ildinete Silva-Pereira
Departamento de Biologia Celular, Instituto de Ciências Biológicas,
Universidade de Brasília, Brasília/DF - Brazil
e-mail: ildinetesp@gmail.com or xocolau@unb.br
The ecology of Paracoccidioides brasiliensis, the etiologic agent of Paracoccidioidomycosis, is poorly
understood. However, host infection is probably acquired by inhalation of airborne propagules derived
from the mycelial saprophytic form of this fungus. Once in the lungs, this microorganism undergoes
a dimorphic transition, converting to the yeast form, an essential step for the establishment of the
pulmonary infection, which alternatively can be eradicated, contained in a granuloma, or disseminate
throughout the body. These different outcomes of the fungus-host interaction will mainly depend on
the host immunological response and the fungal virulence.
The major host defense mechanism against P. brasiliensis infection is the cell-mediated immune
response, characterized by the production of TNF-α, IL-12 and IFN-γ that activate macrophages.
However, in the absence of such cytokines, or in susceptible hosts, this fungus is able to survive
and replicate within the phagolysosome of nonactivated murine and human macrophages. Thus, the
facultative intracellular life style of P. brasiliensis must be compatible with the inhospitable microenvironment imposed by phagocytic cells. Only recently, we have started to better understand the
factors required for intracellular persistence of this fungus.
In the last decade, genomic approaches have proved to be a landmark in the characterization of
fungal virulence factors, becoming a starting point for the knowledge of its molecular pathogenesis.
A number of potential virulence factors and events are considered important for invasive fungi, including dimorphism, growth at elevated temperatures, adherence to host cells, cell wall components
and enzymes production.
Genomic and transcriptome sequencing efforts, coupled to sophisticated molecular biological tools,
have been used to find direct evidence of whether a given factor is required for fungal virulence. In
order to consider a gene as a virulence determinant, the infection caused by the null mutant must
be attenuated, when compared to those caused by the wild-type and reconstituted strains. This approach, which is based on the “Molecular Koch’s postulates”, was originally described for bacteria.
In P. brasiliensis, the lack of efficient gene-disruption and transformation systems preclude the study
of putative virulence genes based on these postulates.
In this sense, we performed in the P. brasiliensis transcriptome a search for orthologs of virulence
genes found in other pathogenic fungi. BLAST comparative analyses were done among PbAESTs
(P. brasiliensis Assembled Expressed Sequence Tag) and the sequences deposited in GenBank.
As a result, the putative virulence PbAESTs were grouped into five classes, cell wall-, metabolism-,
and detoxification-related, secreted factors, and other determinants.
Besides the previously described α- and β-glucan synthases, orthologs to chitin synthase and
mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. Among
the metabolism-related putative virulence PbAESTs, we have identified orthologs of the glyoxylate
cycle enzymes. This metabolic pathway allows the utilization of C2 compounds as carbon sources
to gluconeogenesis, and is involved in the virulence of bacteria and fungi. Further studies, employing
semiquantitative RT-PCR analyses, revealed that these genes were upregulated when P. brasiliensis was recovered from murine macrophages, without any further in vitro growth. The induction of
this cycle, in response to macrophages microenvironment, suggests that P. brasiliensis uses the
glyoxylate cycle as an important adaptive metabolic pathway. In addition, the levels of glyoxylate
cycle transcripts were also increased in response to the cultivation of P. brasiliensis in a medium
with acetate instead of glucose as the carbon source.
61
Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. With respect to the enzymes involved in the intracellular survival of P. brasiliensis,
orthologs to superoxide dismutase, thiol peroxidase, and an alternative oxidase, were also found.
Recently, by using cDNA microarray technology, we evaluated the early transcriptional response
of this fungus to the environment of peritoneal murine macrophages, in order to shed light on the
mechanisms used by P. brasiliensis to survive within phagocytic cells. The most significantly induced
gene, sod3, encodes for a putative Cu,Zn SOD, which is an enzyme involved in the elimination of
superoxide anions. In this context, the upregulation of sod3 expression in internalized P. brasiliensis,
as well as after in vitro menadione treatment, provide evidences that sod3 may be needed for the
elimination of exogenously and endogenously generated superoxides. Overall, these data reveal a
transcriptional plasticity of P. brasiliensis in response to the harsh environment of macrophages, which
may contribute to the adaptation and consequent survival of this pathogen within these cells.
Collectively, our results suggest that P. brasiliensis possesses a vast arsenal of putative virulence
genes allowing its survival in the different host environments.
Another important issue related to virulent fungi is the increased incidence of systemic mycoses
in healthy and in immunocompromised individuals. Since the treatment of those infections is still
hampered by the high costs, drug side effects and the development of resistant fungal strains, the
discovery of new treatment approaches is of prime relevance.
Recent data of the literature reveal that farnesol, one of the quorum sensing molecules of Candida
albicans, acts as an antagonistic molecule inhibiting growth and virulence of bacteria and fungi. In
this sense, our group has performed experiments assessing the effects of farnesol on P. brasiliensis
growth and morphogenesis. This isoprenoid, also present in many essential oils, was able to inhibit P.
brasiliensis growth and, when employing concentrations that do not compromise cell growth, it also
affected the P. brasiliensis dimorphic transition. Despite of the remaining of intact cell wall, and the
unaffected cell permeability, P. brasiliensis cells treated with farnesol exhibited a fully cytoplasmic degeneration, as reveled by electron microscopy. No synergistic effect between farnesol and fluconazole
was observed. Although our data clearly demonstrated the in vitro antimicrobial activity of farnesol
against P. brasiliensis, additional studies involving animal models of experimental paracoccidioidomycosis need to be performed to assess its potential use as an adjuvant therapeutic agent.
Role of melanin in certain dimorphic fungi
Beatriz L. Gómez1*, Marta E. Uran2, R. Morris-Jones3, Luz E. Cano2, Angela
Restrepo2, Arturo Casadevall4, Frank Odds5, Christopher Kibbler1,
and Joshua D. Nosanchuk4
University College London,2Corporación para Investigaciones Biológicas, Medellín, Colombia,
King’s College London, 4Albert Einstein College of Medicine, NY, 5University of Aberdeen, UK.
*Current address: Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta. GA USA,
e-mail: bgomez@cdc.gov
1
3
Melanin pigments are enigmatic compounds produced by organisms in all biological kingdoms,
including a wide variety of pathogenic fungi, bacteria, and helminths. Melanin synthesis has been
associated with virulence for a variety of pathogenic microbes and, in the last 10 years, increasing attention has been directed to the study of melanization in human pathogenic fungi. For some
microbes melanin appears to contribute to virulence by reducing their susceptibility to host defense
mechanisms and by altering host immune responses. Microbial melanization can interfere with the
activity of antimicrobial drugs in vitro, and may potentially result in clinical resistance, particularly to
62
certain antifungal drugs. The effect of melanin on microbial susceptibility to drugs and host defenses
may be an important consideration in the selection and development of antimicrobial therapy. Work
conducted by different groups has characterized melanogenesis in the important dimorphic fungal
pathogens Paracoccidioides brasiliensis, Histoplasma capsulatum, Sporothrix schenckii, Blastomyces
dermatitidis, Coccidioides posadassi and more recently in Candida albicans. These findings and
their possible clinical impact will be discussed and reviewed in this presentation with special focus
on recent developments in P. brasiliensis, the etiological agent of paracoccidioidomycosis (PCM).
Both conidia and yeast cells of this thermally dimorphic fungal pathogen produce melanin or melanin-like compounds in vitro and in vivo. Our results demonstrate that P. brasiliensis synthesizes and
polymerizes melanin during infection and generates antibodies, principally IgG but not IgM, against
conidia and yeast melanins, as detected in sera and BALs such as reported previously for other fungi.
The mouse anti-P.brasiliensis melanin MAbs obtained by our group and the significance of antibody
response in vivo will be useful in the study of P. brasiliensis melanization, particularly in regards
to passive immunization for prolonged survival of infected mice and/or modulation of the immune
response against this fungal infection. Another important and recent contribution (da Silva MB et al,
2006) indicates that P. brasiliensis production of melanin may contribute to virulence by reducing the
uptake of yeast cells by peritoneal and alveolar macrophages. This would enhance the resistance
of fungal cells to attack by the immune effector system. Melanized cells were observed to be less
susceptible to potent antifungal drugs, in particular amphotericin B, ketoconazole, fluconazole, itraconazole and sulfamethoxazole. We are currently investigating potential role(s) of melanogenesis
in the pathogenesis of P. brasiliensis infection.
63
Symposium
4
Immune Responses in
paracoccidioidomycosis:
cellular and molecular Mechanisms
Microbicidal mechanisms exerted by macrophages against
Paracoccidioides brasiliensis conidia
A. Gonzalez1,2, Erika Caro1, Marta E. Uran1, Damaris Lopera1 and Luz Elena Cano1,2
1
Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB);
2
Microbiology School, Universidad de Antioquia, Medellín, Colombia.
e-mail: agonzalezm@cib.org.co
It is known that macrophages play an essential role in the control of Paracoccidioides brasiliensis
infection. In this sense, our laboratory has demonstrated that these phagocytic cells once activated
with interferon-gamma (IFN-γ) or tumor necrosis factor alpha (TNF-α), exhibit either a fungicidal or
fungistatic activity against P. brasiliensis conidia mediated by a nitric oxide (NO) dependent or independent system, respectively. In addition, we also showed that the NO-mediated fungicidal mechanism
exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of iron
interaction.
Recently, we have demonstrated that the microbicidal activity exerted by IFN-γ activated murine
peritoneal macrophages is mediated by a combination of both nitrogen and oxygen reactive species.
Thus, IFN-γ-activated macrophages inhibited the conidia-to-yeast transition process, as well as their
viability. When co-cultures of macrophages and P. brasiliensis conidia were treated with superoxide
dismutase (SOD) and 5’-aminosalicilic acid (ASA) (O2- inhibitors), as well as with aminoguanidine (AG)
(NO inhibitor), the conidia-to-yeast transition process was reverted indicating that both O2- and NO
were involved in the fungistatic activity. Nonetheless, the fungicidal effect was observed only when
SOD and AG were added simultaneously to the co-cultures. Therefore, when P. brasiliensis conidia
were exposed to 3-morpholinosydnonimine (SIN-1), a peroxynitrite (ONOO-) donor, a fungicidal/fungistatic activity against the fungus was observed; in addition, immunohistochemical studies showed
that nitrotyrosine (an ONOO- nitration marker) was detected only in infected macrophages.
The effect of the antimicrobial peptide, lysozyme, against P. brasiliensis was also investigated.
When murine alveolar macrophages of the MH-S cell line were activated with TNF-α the transition process was inhibited and the macrophages exerted an important fungicidal effect against P.
brasiliensis conidia. However, when a selective inhibitor of lysozyme was added, these microbicidal
effects were reverted. Therefore, when P. brasiliensis propagules were exposed directly to different
concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of this enzyme
facilitated the conidia to yeast transition process while inflammatory concentrations impaired such
a process. Interestingly, when yeast cells were exposed to lysozyme, irrespective of concentration,
the multiple-budding ability was strongly impaired. Furthermore, ultra-structural changes such as
subcellular degradation, lipid vacuoles fusion, and damage to lamellar structures with interruption of
the fibrilar layer, were observed in lysozyme-exposed conidia.
In vivo preliminary studies have shown that in mice infected with P. brasiliensis conidia and for
the first 4 days post-infection, bronchoalveolar lavage fluids showed an increase in TNF-α production and/or in its mRNA levels, as observed in mouse lungs; simultaneously, lysozyme expression in
phagocytic cells was augmented with a parallel decreasing of the fungal loads. Contrariwise, during
these first days post-infection the inducible nitric oxide synthase mRNA levels and NO production
became detected only at their basal levels.
All these findings indicate that the various microbicidal mechanisms exerted by activated macrophages are operating in an integrated manner to attain control of P. brasiliensis infection especially during the initial process, and that these mechanisms are depending on the specific cytokine
involved.
Financial support: Colciencias, Banco de la República, U. de Antioquia.
67
Human cells and molecules involved in the microbicidal
mechanisms against Paracoccidioides brasiliensis
Angela M.V.C. Soares
Department of Microbiology and Immunology, Bioscience Institute
São Paulo State University- Unesp- Botucatu, São Paulo- Brazil
e-mail: acsoares@ibb.unesp.br
In the last years our laboratory has studied the modulatory and effector molecules involved in P.
brasiliensis killing by human phagocytic cells. First, experiments were carried out with the objective
to study the effect of the cytokine IFN-γ on activation process of monocytes for high (Pb18) and low
(Pb265) virulent P. brasiliensis killing. Nonactivated cells displayed a very low fungicidal activity
against both strains of the fungus. This activity was increased after priming cells with the cytokine.
However, killing against low virulent strain was significantly higher than that detected with low virulent. These results were explained by higher capacity of this strain to induce TNF-α production by
human monocytes. Further studies were carried out with the objective to study the modulatory effect
of prostaglandins upon fungicidal activity of monocytes against the two strains. It was concluded
that prostaglandins release by human monocytes after Pb18 and Pb265 challenge, inhibit TNF-α
production by human cells. To compensate this inhibitory mechanism the cells must be activated
with cytokines such as IFN-γ. However this compensatory effect was more efficient to Pb265, since it
induces higher TNF-α production when compared to Pb18. More recently we showed that activated
monocytes kill P. brasiliensis by a mechanism dependent on H2O2 release. Then, we asked if the
inhibitory effect of prostaglandins on human P. brasiliensis killing could involve H2O2 inhibition. For
this, human monocytes were no activated or activated with IFN-γ, TNF-α or GM-CSF in the presence
or absence of indomethacin, challenged with Pb18 or Pb265 and evaluated for fungicidal activity and
H2O2, TNF-α, IL-6 and IL-10 release. We concluded that TNF-α inhibition by prostaglandins, lead
human monocytes to release lower levels of H2O2. However, this effect was reverted by cytokines
activation, with monocytes activated with TNF-α or GM-CSF and challenged with Pb265 showing
higher results when compared to the other treatments. In view of these results, we asked if NO,
another metabolite involved in P. brasiliensis killing, could be modulated by prostaglandins. Two
approaches for NO detection were used: the quantification of this metabolite in culture supernatants
and evaluation of inducible nitric oxide synthase (iNOS) mRNA expression by real-time RT-PCR.
Differently of the assays with H2O2, a clear association between prostaglandins and NO inhibition
was not detected, and other assays have been made to better characterize this effect.
The role of CD8+ cells in human paracoccidioidomycosis
Ronei Luciano Mamoni, Lanny C. Burlandy-Soares, Maria Heloísa S.L. Blotta
Departamento de Patologia Clínica – FCM – UNICAMP, Campinas, SP, Brazil
e-mail: heblotta@fcm.unicamp.b
Cellular immune response mediated by Ag-specific IFN-γ secreting CD4+ T cells has long been established as an essential component of protective immune response against P. brasiliensis. However,
there is an increasing evidence of the participation of CD8+Tcells in both the murine and human P.
brasiliensis infection. A protective role for CD8+ T cells was suggested in experimental PCM since its
68
depletion induces a more severe and/or disseminated disease in both, resistant and susceptible mice.
Moreover, an elevated number of CD8+ T cells is detected in the lungs of patients with pulmonary
PCM, in addition to high concentration of MIP-1α in BAL, a chemokine known to selectively attract
this cell subset. Ag specific CD8+ T cells can act as cytokine-secreting cells producing IFN-γ and
TNF-α, which stimulate macrophage fungicidal activity. Moreover CD8+cells are able to kill infected
cells through the release of cytolitic molecules such as perforin, granzymes and granulysin.
In this study we analysed cytotoxic granules expression as well as fungicidal and cytotoxic activity
of CD8+ cells from patients with PCM, infected individuals living in the endemic area, but without any
clinical manifestation of the disease (PI), as well as controls (C). The ex-vivo evaluation of intracellular granules expression by flow cytometry showed that PCM patients presented a lower frequency
of CD8+ cells expressing granzyme A, B and perforin compared to PI individuals. Granulysin mRNA
expression was elevated in PBMC from controls and PI individuals and decreased in PCM patients,
mainly in the ones with severe forms of the disease. In accordance, sera from PCM patients showed
reduced levels granulysin concentration as compare to PI and controls individuals, which reverse
after antifungal treatment.
To determine the fungicidal activity, CD8+ and CD56+ cells were cultured with P. brasiliensis yeast
cells (high virulent, Pb18 and low virulent, Pb265). CD56+ and CD8+cells from PI individuals were
able to inhibit the growth of P. brasiliensis, against Pb18 and Pb265, and in higher degree than PCM
patients. In the control group only CD56+ cells were able to inhibit the growth of both strains of P.
brasiliensis. In general Pb265 was more susceptible to lysis than Pb18.
It has been demonstrated that lymphocyte-mediated antifungal activity was critically dependent
on IL-15, a cytokine with growth factor activity for T cells and NK cells. IL-15 is a potent chemoattractant and controls both proliferation and survival of naive and memory-phenotype CD8+ T cells. To
determine whether IL-15 could enhance the fungicidal activity, purified CD8+and CD56+ cells isolated
from patients with PCM, PI and control individuals were stimulated or not with IL-15 in the presence
of yeast cells. Cells from PI and patients with PCM displayed an increased fungicidal activity against
Pb265 after IL-15 treatment. In controls IL-15 addition resulted in increased fungicidal activity only
in CD56+ cells against both, Pb265 and Pb18. When the CD8+ and CD56+cells were pretreated
with SrCl2 to induce degranulation and deplete their granules, the ability to kill P. brasiliensis (Pb18)
was abrogated. However, pretreatment with concanamycin and EGTA, which inhibit the perforin
cytotoxicity pathway, had only a slight effect over the ability to kill Pb18. These data indicates that
the fungicidal activity against P. brasiliensis is mainly mediated by granules, positively induced by
IL-15 and more elevated in PI individuals than in patients with PCM. .
CD8+ and CD56 cells from PI individuals were able to kill target cells (CD14+) infected with Pb265
in a dose dependent manner and more intensively than cells from PCM patients. The cytotoxic activity was specific since cells from controls did not display considerable activity even in high numbers.
Only CD8+ cells showed a dose-dependent capacity to kill Pb18-infected cells while CD56+ cells
were able to kill target cells infected with Pb265, but not with Pb18. In general the percentage of
lysis was slightly higher in relation to Pb265 compared to Pb18 yeast cells and CD8+ cells exhibited
a greater cytotoxic potential than CD56+ cells. Similar to fungicidal activity the cytotoxic response
against infected cells by CD8+ and CD56+ cells was positively influenced by IL-15 and in a dosedependent manner. Cells from PI individuals presented a higher response as compared to PCM
patients. Moreover, the response of both CD8+ and CD56+ cells to Pb265 was stronger than to
Pb18. In relation to the control group IL-15 had a modest effect on CD56+ cells. When CD8+ and
CD56+ from PI individuals were pretreated with SrCl, CMA or EGTA their ability to lysis target cells
was significantly reduced, showing the dependency of granules and perforin.
CD8+ and CD4+ cells from PI individuals were able to specifically proliferate in response to P.
brasiliensis antigen or yeast cells (Pb18 and Pb265). Interestingly when CD8+ cells were cultivated in
the presence of CD4+ cells an increased proliferative response of CD8+ cells was detected, showing
69
a role for CD4+ cells in the growth of CD8+cells. The addition of IL-15 into the cultures stimulated
with P. brasiliensis yeast cells replaced the effect of CD4+ cells on CD8+ proliferation. Additionally,
treatment of CD8+CD4+ co-cultures with anti-IL-15 partially inhibited the positive effect of CD4+ cells
on CD8+ proliferative response. These data indicate that CD4 growth effect on CD8+ T cells might
be mediated, at least in part, by IL-15.
In summary we found that CD8+ cells from patients with PCM express lower levels of cytotoxic
molecules (granzyme A, B, perforin and granulysin) and diminished fungicidal and cytotoxic activity
against P. brasiliensis yeast cells compared to PI individuals. In general low virulent Pb265 was
more susceptible to lysis than high virulent Pb18 yeast cells. These factors, in association to other
mechanisms that compromise cellular immunity, may account for a deficient cytotoxic activity, and,
therefore reduced protective response against the fungus.
Antigen-specific immunosuppression in
paracoccidioidomycosis: Adverse effect or desired outcome?
Camila R. Cacere 1, A.J.S. Duarte 1, Maria José S. Mendes-Giannini 2, Gil Benard1,3
Laboratory of Dermatology and Immunodeficiencies,
Medical School of the University of São Paulo.
2
Laboratory of Clinical Mycology, Pharmaceutical Sciences Faculty, São Paulo State University.
3
Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases,
Hospital das Clínicas da Faculdade de Medicina da USP (HC FMUSP), Brazil,
e-mail: mahong@usp.br
1
The T-cell hypoproliferative reactivity observed in the immune response to P. brasiliensis antigens of
patients with active paracoccidioidomycosis probably contributes to the failure of the host in controlling
the infection, leading to a disseminated disease. It is, however, largely reversible with treatment in most
patients. The mechanisms leading to this hyporresponsiveness are not well known. We have previously
demonstrated that patients’ mononuclear cells in presence of gp43 exhibit enhanced apoptotic levels.
I an attempt to explain such findings, we hypothethized that these cells were inadequately activated
due to altered costimulatory molecules expression, such as CD80, CD86, CD28, CD152, ICOS e
PD-1. Expression of these molecules were evaluated on T-cells and monocytes of the peripheral
blood of patients with active, disseminated PCM (n = 71), and healthy individuals with a past history
of treated and cured PCM (n = 24). These cells were cultured in presence of a Candida albicans
metabolic antigen (CMA), gp43, or kept without exogenous stimuli for 4 days. Our results show that
the expression of CD28 was comparable between patients an controls’ cells, and that CD152, PD-1
e ICOS, all of which known to deliver negative costimulatory signaling and to arrest cell cycle entry,
were overexpressed in patients’ T-cells. In parallel, we performed additional experiments where the
respective costimulatory signalings were blocked by addition of blocking antibodies specific to each of
these molecules. Whatever the blocking antibody used, there was no reversal of the hypoproliferative
state of patients’ T-cells. However, while the expression of the CD80 and CD86 molecules on monocytes was similar between controls and patients, their expression on T-cells was significantly higher
in patients. Adding the respective blocking antibodies at day zero of the culture, we could observe
that both the gp43 and the CMA responses were inhibited, but differentially according to the antibody
employed. In both patients and controls the blocking CD86 signaling decreased the response to gp43
and CMA of patients and controls, while blocking of CD80 signaling decreased only the response to
gp43, and only in the control group. These data suggest that different antigens may have different
costimulatory requirements for antigen presentation. Addition of the antibodies at day 4 of culture
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did not restore the lymphoproliferative response or modified the response of the controls. Our results
suggest that the hypothesis, raised from other models of prolonged foreign antigen exposure, that
repetitive and persistent in vivo exposure to fungal antigens, which also occurs in patients with PCM,
lead the T-cells to a adaptive tolerant state, which is hardly reverted in vitro. We then investigated
the fate of such putatively tolerized patients’ cells, by analyzing the role that apoptosis may have
in this tolerant state. We observed that expression of the anti-apoptotic molecule Bcl-2 was lower
in patients’ cells, even when the cells were in vitro reestimulated with CMA and gp43, suggesting
that the cells are more susceptible to undergo apoptosis. When then analyzed the expression of the
active form of the caspases 8 and 9 molecules. We first analyzed their expression on cells kept in
cultures for 4 days with or without stimuli. Unexpectedly, we observed that controls’ cells, and not
patients’ cells, exhibited higher levels of expression of both molecules. To explore further these data,
we tested the hypothesis that the patients’ cells were already undergoing apoptosis at an earlier
than 4 days stage. Caspases expression were therefore analyzed ex vivo. In fact, we observed that
TCD3+ cells exhibited markedly enhanced caspase 8 and expression as compared to controls’ cells.
These findings may help to explain why we failed to redress the proliferative responses to gp43 in
the experiments where blocking antibodies were added: these cells would be committed to apoptotic
death, thus refractory to in vitro manipulations, as described for adaptively tolerant T-cells.
Several reasons for the immunosuppression
observed in paracoccidioidomycosis
Ana Paula Moreira1, Karen A. Cavassani1, Ana P. Campanelli1; Fabrine S. Massafera
Tristão1, F.A. Rocha1, L.L. Oliveira1, L. Panagio1, Roberto Martinez1 and Joao S. Silva1
Medical School of Ribeirão Preto; Ribeirão Preto, Brazil
e-mail: jsdsilva@fmrp.usp.br
The long-term persistence of pathogens in the host is a hallmark of certain infectious diseases, including the Paracoccidioides brasiliensis that causes the paracoccidioidomycosis (PCM). Although
several reasons could be responsible for the persistence of this fungus in the host, one is the profound
immunosuppression that occurs in the infected patients. Evaluating the mechanisms that lead to the
remarkable T cell unresponsiveness to antigens in paracoccidioidomycosis we described that they
are not associated with imbalanced cytokine production or with defective CD28 or Fas expression.
However, only T lymphocytes from patients expressed higher levels of CTLA-4 and were Annexin
V+ and FasL+. As the addition of anti-FasL to cultures of peripheral blood mononuclear cells (PBMC)
resulted in decreased levels of apoptosis, we concluded that an activation-induced cell death, triggered through the Fas-FasL pathway, could be responsible for this modulation. Moreover, the blockage of CTLA-4 and FasL resulted in increased production of interferon-gamma, and the concomitant
inhibition of FasL and CTLA-4, but not of transforming growth factor-beta, resulted in significant T
cell proliferation in response to phytohemagglutinin. Together, we concluded that apoptosis mediated by Fas-FasL and engagement of CTLA-4 are involved in modulation of the immune response
in patients infected with P. brasiliensis. However, as the inhibition of CTLA-4 and blockage of Fas
and FasL interaction only partially reversed the T cell response, it revealed that others mechanisms
certainly were involved in this phenomenon. Therefore, as natural regulatory T CD4+CD25+ (Treg)
cells are involved in the control of immune responses in the infections with several pathogens, the
role of these cells in the control of lesions of patients with PCM was investigated. We found that the
71
leukocyte subsets found in the lesions were similar in patients and controls, except for CD11c+CD1a+
cells. However, a higher frequency of CD4+CD25+ T cells expressing CTLA-4, glucorticoid-inducible
TNFR, membrane-bound TGF-β and Foxp3 were observed in PBMC of patients. In accordance, these
cells exhibited stronger suppressive activity when compared with those from controls (94.0 vs 67.5%
of inhibition of allogeneic T cell proliferation). It was observed a higher frequency of Treg in PBMC
of patients and chemokines receptors (CCR4 and CCR5) accumulate in the P. brasiliensis-induced
lesions. Indeed, the secreted CCL17 and CCL22, both associated with the migration of Treg cells to
peripheral tissues, were also detected in the biopsies. Because Tregs are in the lesions of patients
with PCM, the migration of Treg cells is dependent on the axis chemokine-chemokine receptors,
and CCR5 ligands are produced in P. brasiliensis-induced lesions, we investigated the role of CCR5
in the control of the infection. The results showed that CCR5-/- mice are more efficient in controlling
fungal growth and dissemination and exhibited smaller granulomas than wild-type (WT) mice. In the
absence of CCR5, the percentage of Treg expressing Foxp3, glucocorticoid-induced TNFR (GITR),
CD103, CD45low, and CTLA-4 in the granulomas was significantly decreased. Interestingly, P. brasiliensis infection resulted in an absence of T cell proliferation in response to Con A in WT but not
CCR5-/- mice that was abrogated by anti-CTLA-4 mAb and anti-GITR mAb. Moreover, the adoptive
transfer of CD4+CD25+ but not CD4+CD25- T cells from infected WT to infected CCR5-/- mice resulted
in a significant increase in fungal load. Altogether, these data provide the first evidence that Treg
cells play a role in controlling local and systemic immune response in patients with a fungal-induced
granulomatous disease advancing our understanding about the immune regulation in human chronic
diseases. Also, CCR5 is a key receptor for the migration of Treg cells to the site of P. brasiliensis
infection in mice, leading to down-modulation of effector immune response and the long-term presence of the fungus in the granulomas.
72
Historical accounts
Coccidioidomycosis
Coccidioidomycosis: Discovery in Argentina
Ricardo Negroni
Mycology Unit. Hospital de Infecciosas Francisco Javier Muñiz
e-mail: ricnegroni@hotmail.com
The history of coccidioidomycosis in Argentina began in 1892, when Dr. Bengolea asked a medical
student, Alejandro Posadas, to perform a histopathologic study of a skin biopsy taken from a verrucous lesion of patient with tentative diagnosis of fungoid mycosis. Alejandro Posadas was working
under the direction of Professor Roberto Wernicke, who was to head of histopathology laboratory of
“Hospital de Clínicas”. The patient was Domingo Ezcurra, a 36-year-old, cavalry soldier, who was
born in Argentina. Ezcurra presented a chronic disseminated form of coccidioidomycosis, with a very
show progression until his death in 1898.
Posadas and Wernicke found that the biopsy presented a chronic inflammatory response which
was identical to the granulomatous reaction observed in tuberculosis, but they visualized a lot of cystic- like parasitic forms very similar, but not equivalent to those usually found in the protozoan of the
Coccidia gender. They also described the existence of two types of spherules, those of proliferative
progression containing protospors inside the spherule and those of cystic appearance with mature
endospors, and they related these two types of parasitic forms to the host’s defensive capacity.
They were able to reproduce this infection in several animal species, using monkeys, rats, mice,
rabbits, cats, dogs and guinea pigs. Monkeys were the most susceptible hosts. After subcutaneous
inoculation of maceration of human biopsy, they developed an acute disease, with severe pulmonary lesions which presented a fatal outcome in three weeks. The microscopic examination of the
lesions exhibited great amount of proliferative spherules in histopathology. In other animal species
the evolution of the experimental infection was chronic, cutaneous and lymph nodes involvement
was often observed at histopathology. They considered that the lung was the portal of entry of coccidioidomycosis and they tried to establish cultures in several natural culture media, but these were
systematically discarded as contaminations due to the growth of mould. Perhaps the only mistake
made in this outstanding research.
73
A history of research on coccidioidomycosis in the U.S.
Garry T. Cole
Department of Biology and South Texas Center for Emerging Infectious Diseases,
University of Texas at San Antonio, San Antonio, Texas 78249, U.S.A.
The history of coccidioidomycosis begins south of the U.S. border. In January 1891, a 32-year-old
soldier was brought from Northern Argentina to the University Hospital in Buenos Aires with a lesion
on his right cheek. The condition was believed to be an unusual case of mycosis fungoides because
of a coccidia-like parasite associated with the apparent malignant neoplasm. In 1893, a similar
case was observed in the San Joaquin Valley of Southern California which involved an immigrant
Portuguese laborer from the Azores. The causative agent in both cases was Coccidioides, originally
thought to be a protozoan, but correctly identified as a fungus by Ophuls and Moffitt in 1900 at Stanford Medical School. Fewer than 500 cases of coccidioidomycosis were reported by the California
Department of Health up to 1935 and “Valley fever” was considered a rare infection. This concept
changed dramatically after a series of epidemiologic studies conducted in the San Joaquin Valley of
Southern California between 1937 and 1950 led by Gifford, Dickson and Smith. The earlier impression
that coccidioidomycosis was a rare and often fatal disease, shifted as a result of these studies to a
new perception that this mycosis in endemic regions of the U.S. is common and may present as a
benign infection or a disseminated, potentially fatal disease. The stage was set to attract researchers
interested in the mycology, ecology, immunology, diagnosis and treatment of coccidioidomycosis.
Some of the major pioneering investigators and their contributions to our current understanding of
the nature of this disease and the biology of its causative agent will be reviewed.
Coccidioidomycosis: Recent records in Latin America
Bodo Wanke1, Márcia S. Lazéra1, A. Deus Filho2, Luciana Trilles1, Maria A. Salmito2,
Liline M.s. Martins2, Regina C.l. Macêdo1, Claudia C.f.bezerra1, And Kelsen D. Eulálio, 2
1Evandro Chagas Clinical Research Institute, Fiocruz,
Rio de Janeiro, Brazil. 2Federal University Of Piauí, Brazil.
e-mail: bodo.wanke@ipec.fiocruz.br
Coccidioidomycosis and paracoccidioidomycosis are systemic mycoses that occur endemically
only in the Western Hemisphere. While paracoccidioidomycosis is confined to Latina America,
coccidioidomycosis exhibits a broader distribution, reaching its most important endemic area in the
southwestern United States (US), where it presents an estimated annual incidence of 150,000 new
infections, nearly 60,000 primary acute and 750 disseminated cases.
Since coccidioidomycosis is not a reportable disease, its true incidence is unknown. Consequently,
statistics on its prevalence and incidence are either fragmentary or simply not available. However,
some recent data have been reported in Latin America.
Mexico has the largest and most important endemic region following the US, reflecting an estimated
annual incidence of 1,500 new cases of primary and 15 cases of disseminated coccidioidomycosis.
In comparison, the estimated incidence in the US is 40 times fold higher than in Mexico. Moreover,
similarly to the US, an incresed incidence is expeted in some Mexican regions, mainly those situated
next to the US border.
Although clinical cases and epidemiological surveys have demonstrated endemic areas in Central
America (Guatemala, Honduras, Nicaragua and El Salvador) and South America (Colombia, Paraguay
and Bolivia), no recent records have been published.
74
In South America, from Venezuela to Argentina, coccidioidomycosis is endemic in several discontinuous semi-desertic regions, but in all these areas the prevalence and incidence rates are quite lower
than in North America. Venezuela appears to have the highest prevalence and incidence of the mycosis
in South America. Unfortunately, the only recent information point to an annual incidence ranging
from 0 to 13 (mean 4.3) cases/year from 1984 to 2004 (San Blas 2007, personal communication).
In argentina, where the first case was reported in 1892, coccidioidomycosis is endemic in large
semidesertic regions extending from Jujuy in the North to Neuquén in the South. Despite many studies on coccidioidomycosis along the last 110 years, there are no precise data on its true incidence.
However, a recent multicenter serodiagnostic study points to possibly at least 10 new cases/year.
The most recent endemic region of coccidioidomycosis was discovered in the NE semi-arid region of Brazil. Since 1978 more than 100 human cases were recorded in the states of Piauí, Ceará,
Maranhão, and Bahia. An attempt to estimate the incidence in the state of Piauí during a 9 years
period (1999-2007) diagnosed 81 new cases, out of them nearly 90% had the primary acute and
10% had disseminated forms of coccidioidomycosis. Although the annual distribution is not uniform,
an approximate annual incidence of 9 cases/year can be estimated only for Piauí State. However,
adding other states where the mycosis has been demonstrated and the remaining semi-arid region
of northeastern Brazil, the incidence should be estimated as twice higher. The infection was also
demonstrated in dogs and armadillos (Dasypus novemcinctus), and Coccidioides posadasii was
isolated from the soil of four armadillo burrows. The excavated sites caused outbreaks of acute
pulmonary coccidioidomycosis in armadillo hunters. Thus, so far the most important risk activity for
acquiring coccidioidomycosis was armadillo hunting with digging them out of their burrows, in nearly
90% of the patients.
Looking to the estimated incidence in North America (US and Mexico) and comparing their rates
with that estimated outside, as in Venezuela, Argentina and Brazil, primary and disseminated coccidioidomycoses are less important in the latter countries. In comparison, Mexico presents an estimated
incidence 30 times fold higher than the three above mentioned South American countries together.
It shoud be considered that the above estimated incidence rates are based on imprecise and even
not comparable data, frequently obtained by different and not standardized methods, pointing to the
necessity of a Pan American surveillance programme on coccidioidomycosis.
The same importance should be given to soil studies, attempting the demonstration of contaminated foci in wild animal burrows and ancient Indian burial sites, in order to determine the major
environmental risk factors. To illustrate this, in Brazil curiously it was found that areas endemic for
paracoccidioidomycosis (humid climate) are next to areas endemic for coccidioidomycosis (arid
climate) and, additionally, for both mycoses the nine-banded armadillo Dasypus novemcinctus has
been demonstrated as, so far, the most important non-human host.
Key words: coccidioidomycosis, epidemiology, incidence, prevalence, ecology.
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Symposium
5
Granuloma and Fibrosis:
Tissue Markers of
paracoccidioidomycosis
B-1 cells modulate the kinetics of granuloma formation
induced by P. brasiliensis
José Daniel Lopes, Ana Flavia Popi and Mario Mariano.
Department of Microbiology, Immunology and Parasitology,
Universidade Federal de São Paulo, São Paulo, Brasil.
The concept, origin, fate and biological significance of granulomatous inflammation are still controversial. Kindler and others objectively defined the typical granulomatous lesion induced by Mycobacterium
tuberculosis as an isolated newly formed organ originating through specific assembly of differentiated macrophages named epithelioid cells. The multicellularity, cell pleomorphism, participation of
the immune system modulating the lesion and the complexity of factors involved in the mediation of
granuloma formation limit the comprehensive interpretation of the process. Strong evidence indicates
that most macrophages in granulomas derive from blood monocytes. It is unclear, however, to what
extent proliferation, migration and activation of other cell types influence the onset of lesions. The
characteristic of Paracoccidioidomycosis (PCM), a disease produced by P. brasiliensis, is the typical
granulomatous formation in immunologically non-compromised patients. However, the nature and
function of cells which participate in their formation are as yet poorly understood.
Results from our and other laboratories have implicated B-1 cells in the pathogeny of inflammation,
tumor growth and immune regulation. B-1 cells are a unique type of hematopoietic cells, present in
the peritoneal and pleural cavities of mice, that can be identified by CD11b+IgMhiIgDlow phenotype
and can be further subdivided by differential expression of cell-surface antigen CD5 into B-1a (CD5+)
and B-1b cells (CD5–). These cells migrate to distant sites of inflammation, produce large amounts of
autoreactive IgM and secrete large amounts of IL-10. After migration, B-1 cells home at distant sites
of inflammation and become macrophage-like cells. However, the influence that these cells might
have on the kinetics and fate of the inflammatory process is not known.
Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate
the possible influence B-1 cells could have on macrophage activities in vitro. Results showed that
peritoneal macrophages from Xid mice, a strain deprived of B-1 cells, have higher phagocytic indexes
for zymosan particles when compared with macrophages from wild-type mice. Experiments using
co-cultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1
cells down-regulate macrophage activities. As B-1 cells are one of the main sources of interleukin
(IL)-10, we demonstrated that adherent peritoneal cells from Xid mice produce significantly lesser
amounts of this cytokine when compared with cells from wild-type mice. When B-1 cells from IL10 knock-out mice and macrophages from wild-type mice were co-cultured in transwell plates, the
phagocytic index of macrophages was not altered, demonstrating that B-1 cells influence the effector
functions of macrophages in vitro via IL-10 secretion.
Gp43, the main antigen secreted by P. brasiliensis, the causative agent of PCM, is a high mannose
glycoprotein. The role played by gp43 in the pathogenesis of the disease is not completely known.
We have described the influence of this molecule on the interaction between peritoneal murine
macrophages and Pb. Phagocytosis of Pb, live or heat-killed, by adherent peritoneal cells from both,
B10.A (susceptible) and A/Sn (resistant) mice, was evaluated. Addition of different concentrations of
gp43 to the culture medium inhibited, in a dose-dependent pattern, phagocytosis of live or heat-killed
Pb by peritoneal macrophages from both B10.A and A/Sn mice. Gp43 also inhibits phagocytosis of
zymosan particles but did not interfere with the uptake of opsonized sheep red blood cells. It was
also shown that both gp43 and heat-killed Pb have an inhibitory effect on the release of NO by zymosan stimulated macrophages. Finally, it was demonstrated that gp43 inhibits the fungicidal ability
of macrophages from both lineages. Preliminary results showed that peptides derived from gp43,
79
chosen for being exposed at the molecule surface, were equally able to produce these effects, as
well as to increase H2O2 expression by bone-marrow derived macrophages. Based on these data,
it was suggested that gp43 can be considered one of the evasion mechanisms for the installation
of primary infection in susceptible hosts. We developed a granuloma model in vitro using beads to
evaluate the role of isolated mouse peritoneal macrophages and B-1 cells. We also investigated
granuloma formation in the presence of gp43 which is secreted exocellularly.
To determine whether B-1 cells, macrophages, or both, participate in granuloma formation, peritoneal cells from Xid mice, were used. Granuloma-like structures were not formed with Xid peritoneal
cells or with cells from wild type mice that had their peritoneal and pleural cavities irradiated before
the cultures were established. Granulomas were observed either when total adherent peritoneal cells
or when isolated B-1 cells were added to macrophage cultures. The data strongly suggest that an
interaction of B-1 cells and macrophages plays an important role in granuloma-like formation in this
experimental model and that the presence of gp43 strongly stimulates this response.
Protective immunity in PCM is mainly mediated by cellular immunity nevertheless, the role of B
cells in the process, in particular B-1 cells, is poorly investigated. The participation of B-1 cells in resistance or susceptibility of BALB/c and BALB/Xid mice to P. brasiliensis (Pb) pulmonary infection was
investigated. BALB/Xid, which lacks B-1 cells, exhibited higher resistance to infection when compared
with BALB/c mice. However, adoptive transfer of B-1 cells to BALB/Xid mice drastically increased the
susceptibility of these animals to Pb infection. The fungal burden in BALB/c and B-1-reconstituted
BALB/Xid was significantly higher as compared to BALB/Xid strain. Compact, well-organized granulomas were observed in the lungs of BALB/Xid mice, whereas large lesions with necrotic centers with
a plethora of fungi developed in BALB/c mice. It was also shown that B-1 cells impair phagocytosis
of Pb by macrophages in vitro via secretion of IL-10, which was increased upon stimulation with a Pb
antigen, gp43. Finally, in vivo blockade of IL-10 led to a better control of infection by the susceptible
B10.A mice. These findings demonstrate that B-1 cells play a role in the mechanisms of resistance/
susceptibly to Pb infection in murine models, most likely via production of IL-10.
Extracellular matrix modulation in paracoccidioidal granuloma
Eva Burger1, and Angela Satie Nishikaku1
1 Intituto do Ciencias Biomedicas, USP, Sâo Paulo - Brasil.
e-mail: evaburger@usp.br
In the isogenic murine model of paracoccidioidomycosis, resistance is associated with sustained immune response, in direct correlation with cure, whereas susceptibility is parallel to depressed immunity,
leading to a progressive disease and, ultimately, death. Among other differences that characterize
these profiles, the lesions present in the omentum show remarkable differences between the two
strains: progressive increase of the areas in the lesions where Paracoccidioides brasiliensis (Pb)
yeast cells can be identified in the susceptible mouse strain (B10.A) in contrast to the initial increase
but further stabilization of lesional areas containing fungi in the resistant mouse strain (A/J). The
architecture of the lesions of susceptible and resistant mice shows, respectively, loose and compact
granulomas with different cell populations, cytokines and estracellular matrix (ECM) components.
Animals of either strain inoculated with a low virulence Pb isolate originated residual granulomas
after some period of infection.
The components of the ECM participate in the formation and organization of the granulomas and
also of the mechanisms of tissue response developed in the attempt to contain infectious agents
such as Pb. The balance between synthesis and degradation of ECM components in the granulomas
can be influenced by diverse cytokines and enzymes that can be produced in the process of tissue
destruction and repair (necrosis and fibrosis) occurring during the infection. The disbalance of these
80
mechanisms can lead to permissiveness for the dissemination of Pb while its efficient modulation
can contain the fungus and contribute for the restoration of the homeostasis of the organism.
The fundamental role of gamma-interferon (IFN-γ) in anti-fungal protective immunity as well as its in
situ expression and antifibrotic effects in fungal granulomas have been reported. Osteopontin (OPN)
is a glycoprotein of great importance in cell migration and activation, and also in the tissue remodeling observed in granulomatous lesions. Lately OPN has been considered as an important cytokine
for the development of cellular immunity and induction of granulomatous responses. This chronic
tissue inflammatory process can be associated to a balance between synthesis and degradation of
the ECM components, This phenomenon involves the participation of proteolytic enzymes, such as
matrix metalloproteinases (MMPs), particularly MMP-9 and MMP-2, and also products secreted by
the fungus, that altogether can regulate the processes of tissue destruction and repair, as well as
the different cellular events occurring during the granulomatous infection with Pb.
In the present work, we studied by immunohistochemistry the expression of IFN-γ, OPN and
MMP-9 in the granulomatous response developed in susceptible or resistant mice at 15 and at 120
days after infection (dai) by the ip route with Pb and compared with the pattern of lesions produced.
Gelatinolytic activity of MMP-2 and MMP-9 was evaluated by zymography and nitric oxide (NO) and
OPN levels were studied, respectively, by Griess Reaction and ELISA.
IFN-γ (+) lymphocytes were detected at the periphery of granulomas in both mouse strains and
quantitative analysis showed a significant increase in such cells in compact granulomas of A/J mice
at 120 dai compared to 15 dai with the highly virulent Pb isolate. The number of positive cells in
compact granulomas of A/J mice at 120 dai was significantly higher than that observed in loose,
multifocal granulomas of B10.A at the same time.
We found marked OPN expression in macrophage-like and multinucleated giant cells in the center of the lesions and also in a lesser degree in the extracellular matrix. At 15 dai with the virulent
fungal isolate, OPN-positive cells were more numerous and intensely immunostained in the loose
granulomas of B10.A than in the compact granulomas of A/J; at this time high fungal load and low
NO levels were observed in B10.A mice. At 120 dai, increased OPN expression was detected in
compact granulomas of resistant mice; at this time point these animals showed higher NO and lower
fungal load than susceptible mice. Residual lesions associated to low OPN expression, high NO and
control of fungal dissemination were observed in both mouse strains at 120 days infection with the
slightly virulent isolate.
At 15 dai, all infected mice with the highly virulent Pb isolate showed disorganized granulomas
and particularly three gelatinase bands with molecular weights of 106.2 kDa, 95.2 kDa and 64.2 kDa,
probably corresponding respectively to pro-MMP-9 and to the active forms of MMP-9 and MMP-2.
The pro-MMP-9 bands were more intense when compared to active-MMP in B10.A, whereas the
same intensity of the two forms was detected in A/J. These bands were not observed in non-infected
control mice. At 120 dai, B10.A showed loose lesions with many viable fungi and had a similar pattern of MMP-9 and MMP-2 activity as detected at 15th day, whereas A/J developed compact lesions
enclosing fungi with altered morphology and also produced MMP-9 and MMP-2 bands similar to
those detected at 15th day, with more pro-MMP-9 activity and less MMP-2 activity. In the infection
with the slightly virulent Pb, MMP-9 and MMP-2 activity was observed in both mouse strains at 15
dai and no gelatinase bands were observed at 120 dai. Addition of metalloproteinase inhibitors,
EDTA and 1.10-phenanthroline completely abolished gelatinase activities, confirming the presence
of MMPs. Expression of MMP-9 was observed in mononuclear cells in MGCs and also in fungal cells
in omentum of infected mouse of both strains.
The present work suggests that OPN may be associated with more severity in an early phase of
Pb infection and with a degree of control of progressive infection and that OPN may exert anti-inflammatory effects by inhibiting the expression of NO These data strongly suggest that OPN is involved
in granuloma formation, contributing to its development and organization pattern, depending on
the resistance pattern of the mouse strains and also on the virulence of the P. brasiliensis isolates.
Also, this study demonstrates for the first time the presence of MMPs in paracoccidioidomycosis,
suggesting their influence in the organization pattern of granulomatous lesions and in the fungal
dissemination.
81
Pulmonary fibrosis in an experimental model of paracoccidioidomycosis:
Modulation through therapeutic intervention
Damaris E. Lopera1, Tonny W. Naranjo1, Andres F. Zuluaga2, Jose M. Hidalgo3, Jairo
Patiño3, Roberto Jiménez1,4, Lucia R. Díaz-Granados5, John J. Duque6,
Angela Restrepo1 and Luz Elena Cano1,7
1 Grupo Micología Médica y Experimental,
Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia.
2 Grupo GRIPE, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia.
3 Departamento de Radiología, Hospital Universitario San Vicente de Paúl, Medellín-Colombia.
4 University of Nebraska, Lincoln, Nebraska, USA.
5 Facultad de Medicina, Universidad Pontificia Bolivariana, UPB, Medellín, Colombia.
6 Departamento de Patología, Facultad de Medicina,
Universidad de Antioquia, Medellín, Colombia.
7 Grupo de Microbiología Molecular, Escuela de Microbiología,
Universidad de Antioquia, Medellín, Colombia.
e-mail: lcano@cib.org.co
Background: Fibrosis, a condition resulting from progressive chronic diseases, does not have an effective therapy and, consequently, it gradually diminishes the functionality of the affected organ sometimes
reaching an irreversible stage. Patients with paracoccidioidomycosis (PCM) often develop pulmonary
fibrosis and exhibit important respiratory limitations, even if an adequate course of therapy with Itraconazole (ITZ) is given; the latter effectively controls the active stages of this mycosis but does not hinder
lung fibrosis. A previous retrospective clinical study evaluated chest radiographs from 47 ITZ-treated
patients with prolonged post-therapy follow-ups and reported that, at diagnosis, infiltrative lesions were
observed in 93.6% of patients and fibrosis in 31.8%. Later on, at the end of the observation period, none
of these patients had cleared; on the contrary, fibrosis had developed de novo in 11 patients (25%), so
that by the end of the follow-up period this residual process was seen in 53.2% of patients. Due to this
high frequency of fibrosis in patients with PCM, it appears desirable to count with new therapies capable
of avoiding and/or controling fibrosis.
Aims: Based on these clinical findings, we carried out a study in an experimental model of PCM in
order to evaluate by radiologic (tomography), immunologic (cytokines dosing) and histologic analyses
the effects of two combination therapies (ITZ+pentoxifylline or ITZ+prednisolone) on the progression of
the mycosis and on the development of pulmonary fibrosis.
Material and methods: Male BALB/c mice (n=10 animals/group/evaluation period) were intranasally
(i.n.) infected with 4x106 Paracoccidioides brasiliensis (Pb) conidia and distributed in seven experimental
groups: (i) non-treated, (ii, iii, iv) treated for 8-weeks with ITZ alone or in combination with Prednisolone
(PD) or Pentoxifylline (PTX), initiating the treatment before fibrosis consolidation (4th week). Groups
(v,vi,vii) were treated in the same manner as the last three groups but beginning treatment when fibrosis
was already present (8th week of infection). Negative controls were inoculated i.n. with PBS. The follow-up
began before inoculation and continued monthly until the 16th week. At each time period, n=10 animals/
group were subjected to conventional high resolution computed tomography (HRCT), a non-invasive
tool for the in vivo monitoring the course of the lung’s parenchymatous lesions, and then sacrificed with
half of them (n=5) being used for immunological assays and the other half for histologic studies of their
pulmonary tissues. We determined by ELISA the levels of TNF-α, TGF-γ, IL-13, IL-1β, IFN-γ and of PGE2
in lung homogenates supernatants. The histopathologic changes occurring in the pulmonary tissue were
evaluated by two independent pathologists who determined the lung area presenting granulomatous,
inflammatory response (H&E) and registered the appearance (thin or thick) of collagen and reticulin fibers
scoring their relative increase in relation to controls as 0=no changes, 1=minor, 2=moderate or 3=major
changes. Fibrosis was defined as the observation of thick collagen fibers around inflammatory lesions
in the presence of reticulin fibers whereas incipient fibrosis was taken as presence of only thin fibers.
Two-way ANOVA was used to determine differences among groups.
Results: Initially, we standardized and adapted the conventional HRCT technique using two groups
82
of mice (PBS-untreated as negative controls and Pb-infected untreated as positive controls). For this
purpose we used a multi-slice CT-scanner Lightspeed (General Electric, EU of 16 channels) for human
patients, to characterize and monitor the course of the lung’s parenchymatous lesions, pulmonary density
(pδ, expressed as Hounsfield units, HU) and volume alterations. We observed that the major changes
occurred in the upper lung fields where the Pb-infection induced peri-bronchial and parenchymatous
consolidations that increased significantly (p≤0.001) the pδ in those zones (-263.2±29.6, -191.2±25.1,
-269.5±43.5 and -356.5±33.6 HU for 4, 8, 12 and 16 weeks post-infection, respectively) with respect
to pδ of negative controls that remained constant around -432.7±24.2HU. Additionally, multiple peripheral nodules were noticed in 60% of the mice at the end of the follow-up. In sharp contrast, the groups
treated with ITZ alone or in combination with PD or PTX normalized the pδ with values similar those of
the negative controls.
Histopathologic analyses showed that Pb-infection induced a significant (p≤0.001) increase of the
peri-bronchial granulomatous inflammatory response that involved 10%-35% of the lung’s area, with
values of 10±7, 22±15, 20±11 and 15±10% at 4, 8, 12 and 16 weeks, respectively. In contrast, after one
month post therapy, groups (ii) (iii) and (iv) showed a significant decrease (p≤0.01) of the areas exhibiting inflammatory response (9.3±3%, 4±1.5% and 1.8±0.6% for ITZ, ITZ+PD and ITZ+PTX, respectively
at 8th week) and also at the end of the follow-up (3.5±1, 2.5±1.2 and 1.3±0.6%). When therapy had been
started 8-weeks post-challenge, ITZ alone also reduced significantly (p≤0.05) the inflammatory area after
four-week of treatment (11±3% Vs 20±11); however, such a reduction was more significant (p≤0.001)
with both combination therapies (6.7±3%).
Eighty percent of the Pb-infected mice showed incipient pulmonary fibrosis after 4 weeks post-infection; later on (8–12 weeks), this process progressed to well-established fibrosis in 60% of the mice and
reached 100% at 16-weeks. In group (ii) at 4-weeks post-treatment, a significant reduction (p≤0.05) in
the score assigned to thin reticulin and collagen fibers deposition was observed along with a score=0
for thick fibers (p≤ 0,05). Consequently, at 16-weeks, 80% of these mice had incipient fibrosis, none
(0%) had well-established sequelae while 20% had no sings of fibrosis. Notably, ITZ+PD and ITZ+PTX
when initiated at the 4th week, were more efficient in diminishing fibrosis than ITZ alone; at the end of the
follow-up period these therapies avoided either form of fibrosis in 60% and 80% of mice, respectively,
in contrast with 20% when ITZ was given alone. The remainder mice presented incipient fibrosis. Group
(v), presented a significant reduction (p≤0,05) in the score assigned to thin reticulin and collagen fibers
deposition. This effect was observed after 2 months, whereas combination treatment in groups (vi) and
(vii) induced the same effect from the first month of treatment. Additionally, ITZ-therapy started at 8
weeks, was unable to completely prevent the progress to severe fibrosis and at the end of the followup, 40% had incipient fibrosis, 20% had severe fibrosis and 40% of the mice had no apparent fibrosis.
Instead, combination treatments, prevented the appearance of severe fibrosis in groups (vi) and (vii). The
immunological assays showed that Pb-infection induced in the lungs a significantly (p=0.01) increase
in pro-inflammatory cytokines (TNF-α, IL-1β) and in PGE2, although each molecule presented different
kinetics. TNF-α levels were higher during all observation periods (p=0.001), IL-1β levels increased until
the 8-week and then decreased to reach normal levels while and PGE2 increased only at 8th-week postinfection. Pro-fibrotic cytokines (IL-13, TGF-β also increased but only at 8-weeks post infection. IFN-γ
levels did not experience significant changes during the studied periods. All therapies normalized levels
of all cytokines studied and, also of PGE2, except for TGF-β. This molecule, in contrast, increased significantly (p=0.05), especially in groups that received combination treatment.
Conclusions: These preliminary results suggest that in Pb-infected mice: 1) the progression to severe
pulmonary fibrosis can be avoided by early ITZ treatment; 2) ITZ+PD and ITZ+PTX started after 4 weeks
of infection, avoided either form of fibrosis in 60% and 80% of mice, respectively and in contrast with
20% when ITZ was given alone, and 3) Even when the combination therapies were started during the
chronic periods of infection (8 weeks), they also avoided the development of severe pulmonary fibrosis.
These experimental results open an important and interesting possibility for avoiding severe fibrosis in
the human patients by the early administration of the combination therapies proposed.
Financial support: Colciencias (Project No. 2213-04-16439), Corporación para Investigaciones Biológicas (CIB), University of Antioquia, Radiology Department of Hospital Universitario San Vicente de Paúl.
83
Immune reactive cells in skin lesions of patients
with paracoccidioidomycosis
Carla Pagliari1,2, Naiura V. Pereira1,2, Maria I.S. Duarte1 and Mirian N. Sotto2.
1 Departamento de Patologia e 2 Departamento de Dermatologia,
Faculdade de Medicina da USP, São Paulo - Brasil.
e-mail: cpagliari@usp.br
The study of cutaneous lesions in PCM is highly important considering the frequent involvement of
skin in the disease. The skin is considered an immune organ because several of its components
play a role in immune response, such as dendritic cells, keratinocytes, macrophages, mast cells and
endothelial cells. We studied two populations of dendritic cells in PCM skin lesions. The first one
was the Langerhans cell (disclosed by anti-CD1a antibody) which is known as antigen presenting
cell in many cutaneous diseases. The PCM skin lesions were classified, according to characteristics
of granulomatous response, as G1 (well-organized granuloma), G2 (poorly-organized granuloma)
and G3 (both kind of granuloma in the same lesion). The Langerhans cells were distributed evenly
throughout the epidermis of all lesions, but displayed an evident decrease in size and number in G1
and G2. In G3 this cell population was similar to normal skin control group. These data led to the
supposition that Langerhans cells could be deactivated by products of P. brasiliensis as an escape
mechanism, or the cells could suffer phenotypic alterations and not express CD1a antigen in G1 and
G2 lesions. The other group of dendritic cells studied was Factor XIIIa+ dermal dendrocytes (FXIIIa
DD). These cells were hypertrophic, abundant and distributed around the granuloma in all three
groups. G3 presented the higher number of these cells. Through a double immunostaining technique
we detected the P. brasiliensis in the cytoplasm of such cells. This result indicates that FXIIIa DD play
a role as phagocytic cells in PCM. FXIIIa DD due to their constitutive immunophenotype could act as
antigen presenting cells in PCM. For a better understanding of the difference in cutaneous granuloma
formation, we studied some cytokines related to Th1 and Th2 pattern. G1 was characterized by the
presence of a high number of IFN-gamma expressing cells. G2 lesions exhibited a high number of
cells expressing IL5 and IL10. G3 presented an equal number of both kinds of cytokines. TNF-alpha
expressing cells were detected in all groups, and in higher number in G3. Some cells expressing
this cytokine were dendritic and observed around the granuloma. This finding suggests that they
could be FXIIIa DD taking part in the granuloma formation. By double immunostaining technique we
demonstrated that mast cells, present in PCM lesions, express IL10. This finding suggests that mast
cells could play a role as IL10 producing cells in cutaneous lesions of PCM.
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Symposium
6
Fungal cellular biology Aspects
Immune recognition of the Candida cell wall: The taste of a fungus
Neil A.R.Gow
School of Medical Sciences, University of Aberdeen,
Institute of Medical Sciences, Aberdeen AB25 2ZD, UK
Candida albicans is the most common agent of life-threatening human disease due to a fungus. The
outer layer of the cell wall of C. albicans is heavily enriched in glycosylated proteins that is the immediate point of contact and interaction with the human host. The inner cell wall layer contains the
two structural polysaccharides, chitin and β-1,3 glucan to which the mannoproteins are attached. We
have constructed a series of mutant strains with alterations in C. albicans O- and N-linked mannans
and used these to explore the role of the glycans on fungal pathogenesis. In a collaborative project
along with M. Netea and B-J Kullberg in Nijmegen we then used a combination of defined mutants
of the pathogen surface and in pathogen pattern recognition receptors along with receptor-blocking
agents to explore how C. albicans is recognised by the innate immune system. Cytokine production
by human mononuclear cells or murine macrophages was markedly reduced when stimulated by
C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated
by mannose receptor protein binding to N-linked mannosyl residues, and Toll-like receptor 4 binding
to O -linked mannosyl residues. Residual cytokine production was mediated by recognition of β-1,3
glucan by the dectin-1/TLR2 receptor complex. In conclusion, recognition of C. albicans by monocytes/macrophages is mediated by three recognition systems each of which senses a specific layer
of the C. albicans cell wall. Recent advances will also be described on how Candida is recognised
by DC cells and macrophages and how differences between fungal species are detected by cells
of the innate immune system. The integrity of the cell wall and the action of cooperative binding
to multiple PAMP molecules are also important in determining the nature of the innate recognition
mechanism. In addition new evidence will also be shown that chitin in the cell wall plays a role in
innate immune interactions.
Cell signaling in morphogenesis and virulence of P. brasiliensis
Larissa Fernandes
Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil.
e-mail: larissaf@unb.br
Signalling pathways are commonly used by an extensive array of biological molecules to regulate
various cell processes including growth, differentiation, and proliferation. Signalling systems in fungi
also mediate cell polarity, mating, and pheromone control, and hence play a role in determining
cell morphogenesis. Pathogenic organisms sense and respond to the harsh conditions imposed by
the host, and do so by activating the conserved components of signalling pathways that culminate
in the expression of genes responsible for virulence and differentiation of the pathogen in order to
survive and establish disease. Even though many virulence factors have been reported in several
fungi, all signalling events and cascade components that elicit the activation their virulence factors
remain unknown.
Transcriptome analysis and reverse annotation revealed several components of signalling pathways in P. brasiliensis known to regulate cell events, such as morphogenesis and virulence. Pathways identified are i) mitogen-activated protein kinases (MAP Kinases) that control cell integrity, cell
87
wall construction, mating and osmo-regulation; ii) the cAMP/PKA system, which regulates fungal
morphogenesis and virulence; iii) the Ras protein, a small GTPase involved on the cross-talking of
cascades; iv) calcium-calmodulin-calcineurin, which controls cell survival under oxidative stress, high
temperature, and membrane/cell wall perturbation; and v) TOR – the target of rapamycin pathway,
controlling cell growth and proliferation. These components were found to be similar to those of other
pathogenic fungi such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans
(Felipe et al., 2005 and Fernandes et al., 2005).
Post-transcriptome studies using array technology revealed the transcriptional modulation of many
components of signalling cascades of P. brasiliensis. Among 2,583 genes modulated during the differentiation from mycelium to yeast, Nunes et al. (2005) identified PKA, G protein α and β-subunits,
calmodulin and calcineurin as positively regulated. In addition, Bastos et al. (2007), on evaluating
early morphogenesis of P. brasiliensis transition, detected a variety of signalling components that are
modulated, such as the histidine kinase (drk1), a conserved dimorphism related kinase that is very
well known in other dimorphic fungi (Nemecek et al., 2006); a MAPKinase; the β-subunit calcineurin
and others. Considered as a whole, such studies reveal the ability of P. brasiliensis to modulate
signalling pathways to sense the environment and induce morphological changes accordingly.
Experimental studies on components of signalling cascades are still scant on P. brasiliensis
literature. The evidence that cAMP, an intermediate molecule of the PKA system, has a role in the
dimorphic transition that is closely related to virulence was first reported in 1985 by Paris and Duran,
who observed that exogenous cAMP inhibits rather than induces the process of filamentation in this
pathogen. Recently, Chen et al. (2007) cloned and analysed PKA/cAMP signalling components in
the course of the morphological switch from mycelium to yeast. Furthermore, Carvalho et al. (2003)
characterized calmodulin, which regulates the protein phosphatase calcineurin. Inhibitors of the
calmodulin pathway impaired the transition from mycelium to yeast cells in P. brasiliensis. In order
to understand the main role of Ras-GTPases in this pathogen, Fernandes et al. (2008) identified
and characterized two ras genes: ras1 and ras2. As these genes are involved in cell morphogenesis
and differentiation, response to heat shock and nutrient deprivation, we decided to evaluate the
transcriptional response of ras1 and ras2 during the temperature-dependent dimorphic switch from
mycelium to yeast, during heat shock at 42 ºC and during in vivo macrophage infection. Both genes
were down-regulated inside macrophages upon infection and only ras1 expression decreased at
42 ºC. In contrast, ras genes did not show any transcriptional variation during the differentiation.
The fact that Ras proteins are attached to the membrane via farnesylation prompted the use of a
farnesyltransferase inhibitor to investigate the importance of that process to vegetative growth and
dimorphic transition. Farnesylation blockage interfered with vegetative growth of yeast cells, stimulating germinative tube production even at 37 ºC. During yeast to mycelium transition the inhibitor
increased filamentation in a dose-dependent manner, indicating that impaired farnesylation favours
the mycelium form of P. brasiliensis. These data strongly suggest that ras genes are closely involved
in the morphogenesis of P. brasiliensis.
The data from in silico analyses and these first experimental studies on signalling components of
P. brasiliensis indicate that this fungal pathogen has co-opted conserved pathways to regulate the
appearance of morphotypes required for its virulence, as have the other pathogenic microorganisms. Despite the scarce information about the mechanisms and regulatory connections that control
signalling cascades, those studies promoted an improvement of our understanding of the plasticity
of P. brasiliensis in response to its microenvironment. Certainly, that information will serve to clarify
how this fungus orchestrates signalling networks to promote morphogenesis, pathogenicity and the
onset of disease. Besides, understanding signalling events should enable the development of new
drugs and therapies against this pathogen.
88
Paracoccidioides brasiliensis conidia to yeast transition:
Analysis of certain genes involved in the process
Ana M. García1, 2, Orville Hernandez1,3 , Angela Restrepo M4, Juan G. McEwen 1,5
1 Unidad de Biología Celular e Inmunogenética,
Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia.
2 Escuela de Ciencias de la Salud, Universidad Pontificia Bolivariana (UPB), Medellín, Colombia.
3 Departamento de Biología, Universidad de Antioquia. Medellín, Colombia.
4 Unidad de Micología Médica y Experimental,
Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia.
5 Facultad de Medicina, Universidad de Antioquia Medellín, Colombia
e-mail: agarcía@cib.org.co
Background: Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM),
is a thermodimorphic fungus. The mycelial form produces asexual conidia capable of undergoing
thermal transition to either the yeast or the mycelia forms. Dimorphism constitutes not only P. brasiliensis main physiological characteristic but also represents a crucial feature when considering fungal
adaptation to its main host, man, since the infective propagules (conidia or mycelia fragments) must
convert into yeast cells in order to survive and proliferate in tissues for such host.
The ability to differentiate morphologically from mould to yeast, or from yeast to mould is one feature
that links many fungal pathogens. In P. brasiliensis diverse virulence factors have been described
with thermal dimorphism being identified as one of the most important features. At present, this
morphological differentiation has commanded attention for its putative impact on the pathogenesis
of invasive fungal infections. Thus, transition appears to be a clue step in the pathogenesis of the
disease and, consequently, constitutes a suitable target for hindering the infectious process.
Until recently, little was known about the molecular machinery involved in P. brasiliensis morphogenesis and on the genes specifically involved in fungal transition. In 2005, however, Nunes et al.
used broad range approaches to study the M-Y transition process by microarray technology and
Bastos et al.(2007) constructed a M-Y transition process EST library while Garcia et al (submitted)
worked in the C-Y transition process. The later study is the first attempt to correlate P. brasiliensis
C-Y morphological changes with concomitant gene expression modifications. These studies have
pinpointed genes involved in diverse mechanisms probably essential to this adaptation process.
Garcia et al work (submitted) detected certain genes of interest associated to various cellular
pathways known to be relevant to transition, such as the following:
• Cholestenol delta isomerase (also known as emopamil binding protein) a gene involved in the
ergosterol pathway and essential for plasma membrane sterol building during growth and transition,
as well as representative gene in this process.
• Flavoprotein ubiquinone oxidoreductase (ETF-QO) a protein belonging to the electron-transfer
system that plays an important role as an essential component in fatty acid metabolism. Apparently,
it is an exclusive protein not detected in other libraries but in the fungus Yeast and C-Y transition
process.
• Putative response regulator receiver SKN7p a protein belonging to the transmembrane signal-transduction mechanisms most commonly seen in bacteria and fungi, in the “two-component
system”. This protein is associated with oxidative stress tolerance and is a transcriptional factor for
89
heat shock proteins, processes of huge importance during transition especially when occurring in
the host environment.
• Guanine nucleotide binding proteins, important members of the signal-transduction machinery.
Alpha and beta subunits have been described before in the Yeast and Mycelia libraries and their
importance in thermal transition has been described, but the Gamma subunit is being described for
the first time in the C-Y library.
Protein tyrosine phosphatase-Pyp, a phosphatase that regulates numerous biological processess by
hydrolyzing the protein-associated phosphate monoesters (dephosphorylation), which participate
in regulation of cell cycle, growth and proliferation, as well as in cytoskeletal integrity. This gene may
be involved in regulation of the cellular and cell remodeling cycles induced by the stress response
during the the thermal change to which conidia are submitted during their transition to yeast cells.
Recently, the complete genome of three P. brasiliensis isolates has been published in the web and
the annotation of one of them, strain pb03, reveled at least the 90% of all expressed proteins.
Aims: Analyze some genes involved in the P. brasiliensis conidia to yeast (C-Y) transition process, based on the knowledge revealed by publication of the fungus’ whole genome, by The Broad
Institute.
Methods: We used as probes partial sequences of certain genes integrated to the C-Y EST library
in a search conducted with the aim of finding complete gene sequences in the Pb03 annotated genome (including introns and exons, as well as expected mRNA products). These sequences were
searched for and found in the data base. PCR primers were designed to amplify in strain ATCC
60855 (the same used for construction of the C-Y EST library), the longest fragment possible and
then comparing its sequences with those previously described in the Broad Institute data base, to
confirm the existence of each gene.
Results: Search for the complete sequences in P. brasiliensis strain Pb03 annotated genome
resulted in the finding of ORFs (Open Reading Frames) for most of them; complete sequences with
introns and exons were also found. Similarity values were high, between 90 to 100%, with an E value
equal to 0.0 for all of them, so that the probability of at random results was zero, confirming the
existence of these genes in P. brasiliensis. PCR amplification and sequencing of the following genes
was possible:a Cholestenol delta isomerase, a Flavoprotein ubiquinone oxidoreductase (ETF-QO),
a putative response regulator receiver SKN7p (annotated as transcription factor prr1), a Guanine
nucleotide binding protein gamma 13 (annotated as a predicted protein), a putative protein tyrosine
phosphatase (Pyp1). among others, These findings confirm the existence of these genes in P.
brasiliensis strain 60855. Clustal W showed the highest similarity between the sequences of these
genes in strain 60855, as well as in strains with known genomes (Pb01, Pb18 and Pb03).
Conclusions: The genes coming from the specific C-Y transition library and their presence in the
genome as confirmed in the Pb03 complete genomic sequence and also, in strain ATCC 60855 PCR
amplification, confirm the special connotation of our previous work. As stated, a description of some P.
brasiliensis genes obtained from conidia, the infectious propagules causing paracoccidioidomycosis,
were shown to correspond to functions related to their transitional process.
At the moment and with encouraging results, qPCR is being standardized to estimate the relative expression of some of these genes during the C-Y process with emphasis in the differences in
their expression when compared to the Mycelia and Yeast forms; these studies may reveal possible
candidate genes useful for genetic manipulation of P. brasiliensis. For instance, generation of stable
mutant knockouts in transition-related genes, plus modulation of their expression levels using RNA
interference (RNAi), could be fundamental tools in the understanding of their role in the dimorphic
process itself and/or their relevance as virulence factors.
90
CDC42p controls yeast-cell shape and virulence
in Paracoccidioides brasiliensis
Agostinho J. Almeida1, Celia Cunha1†, Belem Sampaio-Marques1†, Jenny A.
Carmona1, Agostinho Carvalho1, Iran Malavazi2, H.Y. Steensma3,
D.I. Johnson4, E. Logarinho1, Cecilia Leão1, Gustavo H. Goldman2,
A.G. Castro1, Paula Ludovico1, Fernando Rodrigues1
1. Life and Health Sciences Research Institute (ICVS),
School of Health Sciences, University of Minho, Braga, Portugal.
2. Faculdade de Ciências Farmacêuticas de Ribeirão Preto,
Universidade de São Paulo, Brazil.
3. Institute of Biology, Leiden University, Leiden, The Netherlands.
4. Department of Microbiology and Molecular Genetics,
University of Vermont, Burlington, Vermont, USA.
† These authors contributed equally for this work.
e-mail: ajalmeida@ecsaude.uminho.pt
Previous work in our laboratory The multiple budding nature of Paracoccidioides brasiliensis
yeast cells has been suggested to follow alternative control mechanisms during cell growth.
As the Rho-like GTPase Cdc42p is a pivotal molecule to establish and maintain polarized cell
growth, we evaluated the role of this protein in the polymorphic morphology and the virulence
of the yeast-form of this pathogenic fungus.
The isolated PbCDC42 gene functionally complements ∆cdc42 S. cerevisiae and triggers a
loss of spatiotemporal control of cell division in a temperature-dependent manner.
By using antisense technology we knocked-down PbCDC42 in P. brasiliensis yeast cells
showing that, despite it does not eliminate the multiple budding phenotype, it promotes a
more organized and controlled cell growth by decreasing cell size and the typical polymorphism of wild-type cells.
Moreover, an 88% decrease in the expression levels of PbCDC42 significantly increases
phagocytosis and abrogates virulence of this dimorphic pathogenic fungus in a mice
model.
To the best of our knowledge, we provide for the first time genetic evidences that establish the
central role of polymorphic morphologies in the pathogenesis of P. brasiliensis, thus opening
new therapeutic targets for the treatment of paracoccidioidomycosis.
Acknowledgments:
Almeida, A.J., Sampaio-Marques, B. and Carvalho, A. were financially supported by a fellowship (SFRH/BPD/33035/2006, SFRH/BI/15406/2005, and SFRH/BD/11837/2003).
The work was mostly supported by a grant from FCT, Portugal (POCTI/ESP/45327/2002)
and partially by FAPESP, Brazil.
91
Differential analysis of extracellular vesicles and of
possible cell wall-associated proteins in isolates
of Paracoccidioides brasiliensis
Rosana Puccia
1
Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, São Paulo, Brasil
e-mail: rpuccia@unifesp.br
Evidences accumulate on the existence of a vesicular transport mechanism of molecules from the
intra to the extracellular milieu in fungi and parasites. These vesicles are apparently similar to exosomes, which are derived from the exocytic fusion of multivesicular endosomes with the cell surface
in mammalians. Here we describe the isolation of exosome-like vesicular structures from supernatant
fluids of Paracoccidioides brasiliensis yeast cells. Pb339, Pb18 and Pb3 were cultivated in defined
medium enriched or not with fetal calf serum and membranous vesicular structures were isolated
following ultracentrifugation (100,000g). We are showing some microscopic, antigenic and enzymatic
features of these fractions. We are also presenting preliminary data on the identification of differentially expressed cell wall-associated proteins when comparing Pb18 and Pb3 isolates, yeast phase,
cultivated in F12/glucose medium supplemented or not with fetal bovine serum (FBS). This work
counts on groups of collaborators from UNIFESP, USP, UFRJ, in Brazil, and from the University of
Texas in El Paso, USA.
Financial support: FAPESP, CNPq, and NIH (grant # 5G12RR008124).
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Symposium
7
The endemic Mycoses
under consideration
by public health Agencies
The evolving public health importance of mycotic diseases
Tom Chiller
National Center for Zoonotic, Vectorborne and Enteric Diseases
Centers for Disease Control and Prevention, Atlanta, Georgia
e-mail: tnc3@cdc.gov
The emergence of new fungal pathogens and the resurgence of mycotic diseases that had previously been uncommon is a serious and growing public health problem. Defining public health’s role
in mycology is a unique challenge.
Fungal diseases are generally not notifiable to public health; therefore very few hard data are
available on incidence and prevalence. Data that exist are fragmentary and there are serious deficiencies in surveillance systems and our ability to detect infections. Given these challenges, it is difficult
to understand the true burden of disease which leads to a lack of awareness by the general public.
There many other competing public health priorities to consider and in order to get the resources
we need to address fungal diseases; we need to initiate global efforts to estimate the burden of these
diseases. We must start by improving surveillance of these infections and then work to develop cost
effective intervention strategies.
Epidemiologic issues in invasive fungal infections:
A United States perspective
Mary E. Brandt
Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta GA USA
e-mail: mbb4@cdc.gov
Fungal infections are among the most difficult to diagnose, identify, and manage in the immunocompromised patient population. Candida is the fourth leading cause of nosocomial bloodstream infection
in the United States, with an estimated national cost of $200-800 million US dollars per year. Although
its incidence is decreasing in the United States and Europe, Candida albicans remains the leading
cause of candidemia in the United States. Non-albicans species, particularly C. glabrata, are seen
increasingly more frequently in some patient populations. The proportions of these species vary in
different geographic regions of the United States. In general, Candida albicans, tropicalis, and parapsilosis remain susceptible to most azole antifungal agents. Candida glabrata remains a species of
concern due to its ability to acquire azole resistance quickly, with about 10% of incident C. glabrata
isolates resistant to fluconazole. Candida krusei remains a species of low prevalence and inherent
resistance to azole antifungals. Invasive pulmonary aspergillosis (IPA) remains a disease impacting
transplant patients, occurring in approximately 6-11% of allogeneic stem cell transplants, 6-13% of
lung transplants, and 1-6% of heart and liver transplants in the US. The TransNet consortium (Transplant Associated Infection Surveillance Network) is a group of 25 transplant institutions in the US
formed to conduct surveillance for invasive fungal infections in transplant patients, to determine risk
factors for these infections, and to assess the impact of prevention programs in these patients. The
incidence and timing of IPA after transplant has been studied in this network. The incidence of IPA
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after stem cell transplant (HCT) is 1.7% for all types of HCTs. The timing of IPA varies as well, with
about half of cases occurring after 90 days after transplant. Aspergillus fumigatus (43%) remains the
major agent of IPA followed by Aspergillus flavus and niger (8% each). Aspergillus terreus (5%) is a
species of concern due to amphotericin B resistance. The 90-day mortality of IPA in HCT patients is
50%. The incidence of IPA in solid organ transplant patients is 0.7% by 12 months post-transplant.
Some studies have reported an increase in the incidence of zygomycotic infections after transplant.
The TransNet network is studying this question. It is not clear whether this phenomenon is due to
increased survival after transplantation, or due to an increase in prophylaxis with azole drugs, which
have limited activity against zygomycetes.
Paracoccidioidomycosis in Brazil:
Surveillance and control Program
Maria Adelaide Millington
Head of the Systemic Mycoses Program
Secretariat of Health Surveillance, Ministry of Health, Brasília,Brasil.
e-mail:adelaide.millington@saude.gov.br
The Secretariat of Health Surveillance, Ministry of Health from Brazil is structuring (initiating) the
Program for Surveillance and Control of Systemic Mycoses to determine the magnitude of these
infections in the country.
The Program mainly aims to estimate the prevalence and geographic distribution of Paracoccidioidomycosis (PCM), Histoplasmosis, Coccidioidomycosis and Cryptococcosis. Also other objectives
are: to estimate the mortality rates of each of these diseases, to make early diagnosis and timely
and properly handle (management) of the cases, to detect and control outbreaks, through adoption
of prevention and control measures, to support and promote the scientific-technical development in
the area, providing for the network of health services new diagnostic tools, treatment and control of
these diseases.
The Program will initiate the efforts with Paracoccidioidomycosis (PCM) because it is an indigenous
mycoses, endemic in Brazil and represents an important Public Health problem by its high potential
disability and premature deaths it causes, if not appropriately diagnosed and treated. Studying the period 1980-1995, an author showed an annual mortality average rate of 1487 deaths/106 of inhabitants,
and shows that the PCM is the eighth cause of chronic disease mortality, greater than leishmaniasis
mortality and the highest rate among the systemic mycoses. Most cases have been reported in the
south, southeast and center-west of Brazil where It is endemic among the populations of rural area.
PCM has been reported in areas of recent colonization most subject to deforestation, as in parts of
the Amazon Region, where can be considered an emerging systemic mycosis.
PCM is caused by the dimorphic fungus Paracoccidioides brasiliensis, that appears to be predominantly in the chronic form in adults and in the acute or subacute forms when it affects children,
adolescents and young adults. The PCM is acquired through inhalation of fungal particles dispersed
in the air, from sites with contaminated soil.
In Brazil, the systemic fungal infections are not reportable diseases, so they are not object of
routine epidemiological surveillance and only in the states of Paraná, Rondônia and more recently
Mato Grosso do Sul, the PCM is a reportable disease. In these states the health care is organized,
with reference services well defined, and it is subsidizing the Program in other Brazilian states.
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Several difficulties have been identified in the care process, ranging from the clinical and laboratory
diagnosis to thel availability of specific medication, which often makes the adherence to treatment
hard to do .
Then, we don’t know the real magnitude of PCM, where are the endemic areas, the prevalence
rates. The data of incidence and morbidity available today are based on limited reports of clinical
cases and epidemiological investigations.
Since 2005, the Ministry of Health, through the Department of Health Surveillance has invested in
designing and implementing the Program for Surveillance and Control of PCM, developed in partnership with experts from Teaching and Research Institutions in health.
Among the strategic measures for the structuring the Program, these are highlighted:
• Development of a surveillance system to know the epidemiological situation, development of
case reporting, research, control measures.
• Organization of the healthcare network, with basic care units and reference hospitals and standardization of clinical management and provision of medications.
• Organization of the Public health laboratories, with adequate infrastructure and equipment,
development of technical standards for collecting, packaging and transportation of samples, standardization of diagnostic methods and acquisition of laboratory supplies;
• Training of human resources - physicians, radiologists, pathologists and laboratory technicians
The surveillance system will be implemented gradually, in eight (8) of the 27 states, which presents
high prevalence rates of PCM, and have some organization to attend these patients.
The activities already taken were the training of technicians of the Public Health Laboratories in
mycological and serological diagnosis, standardization of treatment, and the acquisition and provision
of drugs - Itraconazole, Sulfamethoxazole + Trimethoprim for injection, conventional and liposomal
Amphotericin B. It is worth to emphasize that all the standardization were based on Brazilian Consensus on Paracoccidioidomycosis prepared by the Brazilian Society of Infectious Diseases, Brazilian
Society of Tropical Medicine and São Paulo Infectious Diseases Society.
The main activities planned for 2008 are highlighted below:
• Establishment of a technical advisory group for the various systemic mycoses.
• Preparation and publishing of clinical and epidemiological protocols for the deployment of surveillance in the states of Paraná, São Paulo, Rio de Janeiro, Minas Gerais, Mato Grosso do Sul, Mato
Grosso, Goiás and Rondônia.
• Standardization of epidemiological research and information system
• Definition of reference health services at the various states
• Training of human resources (doctors and technicians surveillance and Lab.)
• Drafting Technical Laboratory Manual for diagnosis of Systemic Mycoses
• Distribution of the Antigen of P.brasiliensis to standardize diagnosis
• Making available Technical Advisors to the states involved
• Facilitating technical cooperation with other countries for improving diagnoses
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Symposium
8
Clinical and epidemiological
Considerations on the
endemic Mycoses
The impact of histoplasmosis in HIV-infected patients
John R Graybill1, Eduardo Arathoon2, Blanca Samayoa2.
1. University of Texas Health Science Center, San Antonio, TX USA,
2. Clinica Familiar Luis Angel Garcia, Guatemala City
e-mail: jrgraybill@aol.com
Histoplasma, Coccidioides, and Paracoccidioides are genera with pathogenic species sharing many
features, including infection by inhalation, dimorphic morphology, a protective response dependent
on T lymphocytes, generation of antibodies which are useful for diagnosis but not protective, and
the propensity to disseminate in situations where T cell mediated immunity is depressed such as
in HIV infected patients. However, of these Histoplasma capsulatum has the greatest impact and
Paraccidioides brasiliensis the least impact in HIV infected patients. Reasons are complex, unclear,
and may relate at least partly to the likelihood of exposure to environmental organisms, and to the
immune status of the host.
Most patients with histoplasmosis acquire the infection late in the course of HIV disease, when
immune responses are severely depressed. The illness may take the form of progressive primary
disease with widespread dissemination following on the heels of primary infection. However, subjects
infected years before are also at risk for H capsulatum to escape immune confinement as their HIV
progresses. In the early years of the HIV pandemic, dramatic increases of histoplasmosis occurred in
the endemic regions of the United States. However, cities like San Antonio, Texas, formerly thought
on the periphery or west of the endemic region, and bothered by very few cases of primary disease,
experienced dramatic increases in histoplasmosis. San Antonio (and other cities) is now thought well
within an enlarged Histoplasma endemic zone in the USA. In the 1980’s and early 1990’s, hospitals
such as ours increased from scattered cases to as many as 20-25 cases per year. Histoplasmosis
often appeared late in a difficult course of poorly effective HIV treatment. However, with the advent
of HAART in the United States, our case rate decreased by more than half, to 5-7 cases per year.
Further, most patients with histoplasmosis now present at their initial evaluation, and are then found
to be HIV positive. Diagnosis is often made by bone marrow aspirate, sometimes also positive blood
or sputum smears, and confirmed by positive urine and/or blood antigen measurements before
cultures turned positive. At present, effective treatment consists of induction with amphotericin B
(commonly liposomal amphotericin B) for treatment of the most seriously ill patients, followed by a
switch to itraconazole. In the less seriously ill, itraconazole might be started initially. Fluconazole has
been effective in some patients, but the emergence of resistance has made it a second line agent.
Perhaps not surprisingly, MIC values, which increased in patients failing fluconazole, also rose to
voriconazole. There have been some patients successfully treated with voriconazole, but this may
not be wise. Posaconazole, much more like itraconazole, appears effective in vitro (including the
voriconazole resistant isolates) and clinically in a limited salvage experience. Histoplasmosis is now
a pathogen of intermediate importance in AIDS patients in the USA.
This situation in the United States is not mirrored in the third world, where access to HAART is
limited, where it is not possible to perform rapid diagnosis using an antigen test, and where tuberculosis accompany histoplasmosis in >5% of cases, and where the histoplasmosis remains common.
Diagnosis of cryptococcosis is straightforward, whereas diagnosis of histoplasmosis must rely on
clinical suspicion confirmed by cultures (which even with the lysis technique which require more than
a week, during which the patient may succumb) or identification by histopathology. Optimal culture
methods are not available in many settings, and histopathology and fungal stains may not be used
aggressively. Small countries such as Guatemala (population 13 million) have about 30 cases of
histoplasmosis diagnosed per year, versus 45 with cryptococosis and 70 with tuberculosis in one
101
clinic with a total patient population of 700-800. There few experts in HIV associated diseases, and
many patients likely undiagnosed. Late presentation and delay of diagnosis may account for mortality
over 30%. Latin American constitutes far the greatest reservoir for histoplasmosis, but regions such
as South Africa also have marked increases of patients infected with H capsulatum, which remains
a major pathogen.
What can be done to ameliorate the problems of histoplasmosis in Latin America? First, more rapid
diagnosis of HIV infection and initiation of HAART will prevent the deterioration of immunity which
facilitates histoplasmosis. Second, Histoplasma antigen detection must be readily available in central
laboratories of countries in endemic regions. A test is under development at the CDC in Atlanta, Georgia. Aggressive biopsies and cytochemistry are also important. Third, every patient with suspected
tuberculosis or histoplasmosis should be investigated for both infections. The use of rifampicin in
treatment of concurrent tuberculosis might necessitate extended treatment with amphotericin B in
patients with dual infection. Fourth, in these usually very ill patients, treatment should be initiated
rapidly, sometimes empirically while diagnostics are in process. A short course of amphotericin B
should be followed by change to a triazole as soon as the patient is stable. Finally, as the patient
improves with reduction of viral load and rise of CD4, sudden worsening may be related to the immune response syndrome rather than progressive disease this happens with both tuberculosis and
histoplasmosis. Therefore not rush to change anti-infectives for documented disease until you know
what is transpiring.
The coexistence of HIV infection and paracoccidioidomycosis
Roberto Martinez
Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo-Brazil
e-mail: rmartine@fmrp.usp.br
The first cases of paracoccidioidomycosis associated with acquired immunodeficiency syndrome were
reported in 1989. Since then, approximately 150 cases of the co-infection Paracoccidioides brasiliensis
– HIV were related. The co-infected patients lived mainly in the Southeast and in the Center-West
regions of Brazil, but a few reports described cases in other Latin America countries.
A large number of patients acquired both infections in the metropolitan area of Ribeirão Preto, São
Paulo state, Brazil, where paracoccidioidomycosis is hiperendemic. The 73 patients diagnosed from
1989 to 2007 with P. brasiliensis – HIV co-infection, compared with the number of cases of AIDS in
the same period, resulted in a prevalence estimated at 1.7%. Most co-infection patients had a number
of blood T CD4+ lymphocytes lower than 50/µL, and 20/25 (80%) had more than 30,000 copies/mL of
the HIV1 RNA in the plasma. Only 59% of the 27 patients who were aware of being infected by HIV
were taking antiretroviral drugs, and 41% were in prophylaxis with trimethoprim-sulfamethoxazole
against Pneumocystis jiroveci.
The P. brasiliensis – HIV co-infected patients were compared with 1000 cases of endemic paracoccidioidomycosis with respect to epidemiological and clinical data. Mean age was lower in the
co-infected patients (33,4 x 41,9 years old), but a nonsignificant difference was observed in relation
to gender. Among HIV-infected patients 27% had some type of occupation in the rural area versus
53% of the endemic disease cases.
HIV co-infected patients had more disseminated and rapidly progressing lesions of P. brasiliensis
than the individuals HIV non-infected. They had more fever, skin lesions, lymphadenopathy, and
splenomegaly. Pulmonary involvement were very common in both cohorts. Lesions of the mucosa
of the mouth, nose or larynx were more frequent in patients with endemic paracoccidioidomycosis.
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The conjunct data from 66 cases previously reported of paracoccidioidomycosis associated with
HIV/AIDS showed a great frequency of lesions suggestive of fungal hematogenic dissemination, in
particular to lymphnodes (52%), spleen (18%), and skin (41%); pulmonary involvement was observed
in 55% of the cases.
The diagnosis of the fungal infection in HIV-infected patients was confirmed by histopathological
examination (94%) and by isolating P. brasiliensis (42%), particularly from a skin lesion biopsy. The
sera of the co-infected patients had lower reactwity in a test for detection of anti – P. brasiliensis
antibodies. The initial treatment of paracoccidioidomycosis – HIV/AIDS patients consisted in general
of HAART and amphotericin B, trimethoprim-sulfamethoxazole or fluconazole. In comparison to
endemic disease no difference in response to treatment was observed but late relapses were more
common in the HIV-infected patients. In some reports the lethality attributed to paracoccidioidomycosis is near to 30%.
Paracoccidioidomycosis should be classified as opportunistic disease in HIV/AIDS patients because of its clinical and epidemiological characteristics, that are different from clinical forms of the
endemic disease.
Treatment compliance in paracoccidioidomycosis
Rinaldo Poncio Mendes1, Daniela V. Moris1, L.C. Carvalho2, E.F. Oliveira3,
and Ricardo Cavalcante1.
Tropical Diseases Area, Botucatu Medical School,
2
Biostatistic Department, Biosciences Institute,
3
Information Technology Service, Botucatu University Hospital
São Paulo State University (UNESP)
1
Introduction. Paracoccidioidomycosis (PCM), a widespread systemic mycosis in Latin America, requires long treatment in its most frequent clinical forms. Its treatment aims clinical cure, regression
of specific antibodies serum levels to normal and recovery of the specific cell mediated immune
response, to reduce long-term sequelae and relapses. Behavioral changes and long-term treatment
compliance are crucial to reach these goals.
Compliance, the act of taking medications as prescribed, is a highly complex clinical behavior
and remains problematic for evaluation. Medication nonadherence can take many different forms,
leading to therapeutic failure.
Methods. Patients with confirmed PCM by the identification of typical Paracoccidioides brasiliensis yeast forms and/or specific serum antibodies by double agar gel immunodiffusion test, with both
acute/subacute or chronic form, assisted at the Tropical Area Service – Botucatu Medical School
– UNESP, were eligible for the study. Overall, 97.4% of the 77 eligible subjects who were contacted
agreed to participate and answered a specific questionnaire.
The following compliance indices were evaluated: 1) Global attendance compliance (GAC) – percentage of presence in the appointments in a period; 2) Individual clinic attendance compliance
(ICAC) – percentage of appointments of each patient in a specific period; only patients with at least
10 attendances in the Paracoccidioidomycosis Outpatient Service were evaluated; 3) Antifungal
compliance (AFC) – percentage of evaluations with appropriate sulfonamide serum levels (≥70µg/
mL in the initial treatment and ≥50µg/mL in the consolidation therapy); only patients receiving cotrimoxazole with at least 10 sulfonamide measurements during the follow-up period were included; 4)
Serological curve (SC) – evaluation of specific antibodies serum levels by agar gel immunodiffusion
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test during the follow-up period; only patients with at least 10 measurements were included; 5) Oneweek self-report compliance (OWSRC) – percentage of drug intake in the last week of treatment; all
interviewed patients receiving antifungal drugs were included; 6) Last appointment self-report compliance (LASRC) – percentage of drug intake in the period between the last two evaluations. Indices
80% or higher were considered patient compliance. ICAC, AFC, OWSRC, and LASRC analyses
were carried out according to this cutoff. SC was evaluated as appropriate when descending, or
inappropriate when stable or ascending (dichotomous parameter). Compliance was analysed as to
clinical form, education level, family help, and alcohol intake. Adherence measures were evaluated
alone and in combination.
Unpaired t test and analysis of variance/Tukey test for continuous variables, x2 and Fisher’s exact
test for proportions, and Pearson’s test for correlations were used, with level of significance set at
p<0.05.
Results. Global attendance compliance, evaluated in 3,195 scheduled appointments at the Paracoccidioidomycosis Outpatient Service for 65 months, from January 2003 through May 2008, was
88.2% with no variation as to year distribution (85.5% to 89.7%, p>0.05).
Individual clinic attendance compliance, studied in 132 patients with at least 10 routine appointments, showed a 90.3% average and revealed that 85.6% came to at least 80.0% of these clinical
evaluations. ICAC presented no variation as to clinical presentation: 90.8%±11.9% in the chronic
and 88.2%±9.9% in the acute/subacute form (p>0.05).
Antifungal compliance, evaluated in 95 patients treated with trimethoprim-sulfamethoxazole
combination, was 42.1%. A positive correlation was observed between antifungal compliance and
individual clinic attendance compliance (r=0.27; p=0.037).
Serological curve, studied in 96 patients, showed descending curves in 90.9% of them and stable/
ascending in 9.1%. A direct correlation between antifungal compliance and serological curve, i.e.,
higher frequency of adequate sulfonamide serum levels in patients decreasing the antibody serum
levels, was observed in this cohort (x2=4.6; p=0.032). Average of AFC was higher (p=0.016) in patients
with descending serological curves (66.6±29.6) than those with ascending ones (42.6±35.2).
One-week self-report compliance (28 patients) did not correlate with antifungal compliance (p=0.12).
Last appointment self-report compliance did not correlate with antifungal compliance (23 patients;
r=0.10; p>0.05) but revealed a linear correlation with individual clinic attendance compliance (30
patients; r=0.48; p=0.02).
Alcohol intake did not interfere with one-week self-report compliance (28 patients; p>0.05) or with
serological curve (70 patients; p>0.05). However, individual clinic attendance compliance was mildly
higher in patients who denied alcohol intake (75 patients; 97.0% versus 93.3%; p=0.04). Education
level was in general low, but individual clinic attendance compliance was lower in patients who had
never attended school (p=0.02). Family help did not play a role in individual clinic attendance compliance and serological curve (p>0.05).
Compliance was evaluated in 96 patients combining individual clinic attendance compliance,
antifungal compliance and serological curve indices. Three indices were appropriate in 34 (35.4%)
patients, two in 46 (47.9%), only one in 14 (14.6%), and none in 2 (2.1%). Thus, 80 of 96 patients
(83.3%) revealed high compliance.
The main causes of failure in antifungal intake were blood tests (24.3% of the cases), forgetfulness
(16.2%), adverse effects to medication (16.2%), lack of medication (16.2%), and alcohol intake to
avoid an alcohol-antifungal compound mixture (13.5%).
Discussion. Noncompliance to treatment is a main obstacle to cure and may result in disease
progression and resistant microorganisms. Despite the importance of compliance in clinical care
and research, rigorous study of measurement techniques is uncommon. Evaluation of compliance
to long-term treatment is complex and does not have a gold standard parameter. Patient self-report
has been used extensively to assess medication-taking, but it tends to overestimate compliance.
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Although drug serum levels are an objective measure of drug intake, they provide only a snapshot
of behavior and can be affected by factors other than adherence.
Indices obtained by using a questionnaire and laboratory charts, with drug measure (sulfonamide
serum level) and antifungal effect (serological curve) were compared. Our study revealed correlation
between individual clinic attendance compliance and serological curve. However, descending serological curves were observed in patients with lower sulfonamide serum levels. These findings could
be explained by intake failure in the appointment day, when fasting patients are submitted to blood
tests. Thus, patients are told to take only the medication before having their blood draw. It can also be
hypothesized that adequate sulfonamide serum level was overestimated. Alcohol intake, family help
and education level played a mild role in treatment compliance. The high values of combined compliance indices provided a more accurate evaluation, and demonstrated high quality of medical care.
Guidelines for the treatment of paracoccidioidomycosis
Flavio Queiroz-Telles
Serviço de Infectologia, Hospital de Clinicas, Universidade Federao do Paraná
e-mail: queiroz.telles@uol.com.br
Evidence-based guidelines for the management of patients with Paracoccidioidomycosis (PCM)
were prepared by an Expert Panel of the Brazilian Societies of Infectology and Tropical Diseases.
The guideline was published two years from now (Ver. Soc. Bras. Med. Trop. 39(3):297-310, 2006)
aiming to help the clinicians to manage most of the clinical forms of the disease. PCM is considered
to be the most important systemic endemic mycosis affecting South America. The prevalence of this
infection varies among regions of endemicity, and it is estimated that the incidence of PCM ranges
from 1 to 3 cases per 100,000 inhabitants in regions where the disease is endemic. However, because the reporting of PCM is not compulsory, it is difficult to determine accurately the number of
people affected by the disease. Without treatment, the natural evolution of the disease is typically
death. In patients with immunosuppression, such as AIDS, the infection can progress to full-blown
disseminated disease.
P. brasiliensis differs from other pathogenic fungi, because it is a very sensitive organism when
exposed to antifungal drugs; even sulfonamides can inhibit its growth. A large therapeutic armamentarium is available for patients with PCM.
Several classes of antimycotic drugs have been used in the treatment of this systemic mycosis,
including sulfonamides (sulfadiazine, sulfadoxine, sulfamethoxypyridazine, cotrimazine, and cotrimoxazole), Amphotericin B, azoles (ketoconazole, itraconazole, fluconazole and voriconazole), and
terbinafine. The cure rates achieved with these various drugs have ranged between 69% and 100%.
To date, no antifungal resistance has been demonstrated in PCM.
Itraconazole is considered the standard treatment for mild and moderate clinical forms of PCM.
This recommendation is based on the results from a non comparative study of 47 patients, most of
who had multifocal disease and received itraconazole for a mean duration of 6 months. A scoring
system indicated a favorable response in 43 patients (89%). In another trial, 90 patients with proved
PCM were treated with cotrimoxazole or itraconazole and evaluated for clinical and microbiological;
response, safety and tolerability. The final analysis showed that itraconazole is more effective and better tolerated than cotrimoxazole (trimethoprim-sulfamethoxazole) in the treatment of mild to moderated
PCM. In addition, the mean duration of treatment with itraconazole is significantly shorter (7 months
versus 24 months, p<0.0001). The rate of recurrence of patients treated with itraconazole may vary
from 0% to 15% after a median time of 12 months, depending on co-morbidities, such as alcoholism
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and AIDS, as well as on the itraconazole dose (100 mg/day vs. 200 mg/day). Conventional or lipid
formulations of amphotericin B are indicated to treat severe disseminated cases in patients who are
intolerant to other agents or have refractory infections. Besides its toxicity, the rate of relapse with
amphotericin B is generally higher than with itraconazole (20%–30% of cases).
After clinical and microbiological diagnosis, all patients must be evaluated for clinical classification (acute/sub acute, chronic or residual form) and graded to severity (mild, moderate and severe
clinical form). Itraconazole is used at the daily dose of 200 mg during 6 to 12 months, according to
the clinical, mycological, serological and radiological response. The second therapeutic option is
cotrimoxazole (160 to 240 mg of thrimetoprim combined with 800 to 1,200 mg of sulphametoxazole
BID) from 12 to 24 months. Young children can be treated with cotrimoxazole oral solution (08 to 10
mg per kg of thrimetoprim combined with 40 to 50 mg of sulphametoxazole BID). For patients with
neurologic involvement, cotrimoxazole or fluconazole can be used although the former compound
was used in most the reported cases.
Among the new triazoles, posaconazole and isavuconazole were not investigated in human PCM
to date. But voriconazole a second generation triazole was compared to itraconazole in terms of
efficacy and safety in a controlled randomized clinical trial. Fifty tree patients with proved mild to
moderate forms of PCM were included. The response rate among treatment-evaluable patients was
100% for both group of patients and no relapses were observed after 8 weeks of follow-up. Both
drugs were well tolerated but adverse events were slighter more frequent in the voriconazole arm.
Because voriconazole is a fluconazole molecular derivative, it presents excellent CNS penetration
and may be an adequate antifungal drug for neuroPCM.
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Historical accounts
Histoplasmosis
Histoplasmosis: Discovery in Panama
Marion Clarke Martin
Microbiology Department, Faculty of Medicine,
University of Panama--Panama
e-mail: marionma38@yahoo.com
The discovery of histoplasmosis in Panama is closely linked to the history of the Republic of
Panama, since the first case of this disease, worldwide, was diagnosed by Samuel T. Darling in
1905 in a Martinique man employed in the construction of the Panama Canal. Panama became
an independent republic after separation from Colombia in 1903, and in 1904, a second attempt
at building the Canal was made by the United States of America, after the French failed at this
endeavor in the nineteenth century. Since the discovery of this disease on the Isthmus of Panama
a little over a hundred years ago, cases spanning the complete spectrum of histoplasmosis -from
the asymptomatic infection through a mild flu-like illness to full-blown, disseminated disease as
described in the first diagnosed case -have been documented in the population of all ages, from
infants to senior citizens, and even in a dog (1945).
During the eight months following the first case, Darling diagnosed two additional cases. Twenty
years later (1926), the fourth case worldwide was identified in Minnesota, USA, and in 1934
Robert De Monbreun isolated the etiologic agent, the dimorphic fungus Histoplasma capsulatum.
Christie and Peterson demonstrated for the first time in 1945 that skin sensitivity to histoplasmin
was common in Tennessee in persons with lung calcifications and negative tuberculin reactions;
and by the following year, 1946, it was evident that histoplasmosis was most likely a very common,
usually asymptomatic infection in endemic areas, and not a rare, deadly illness.
In light of the foregoing, investigators in Panama began work in this field, and Mastellari, a specialist in chest diseases, demonstrated (1948) histoplasmin sensitivity prevalence in 48% of children between 5 and 14 years of age. Various studies published between 1950 and 1964 have
established the prevalence of histoplasmin sensitivity at 50 – 85% of the Panamanian population;
and later work done by us has generally confirmed these data. However, in the 80s we noticed
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that the prevalence of histoplasmin sensitivity appeared to be dropping, and documented that it
was in the range of 11 to 18% of populations of children and young adults, respectively. None
of these subjects had a diagnosis of the illness histoplasmosis; their skin reactivity was just a
fortuitous finding.
Having stated that the infection was largely asymptomatic among Panamanians, it is appropriate
to point out that an active search for acute, primary cases of the disease was carried out between
1954 and 1957 at Gorgas Military Hospital in the former Panama Canal Zone, and based on clinical history, careful physical examination, histoplasmin test and chest x-ray a series of 45 cases
was identified. This work confirmed what we now take for granted -clinical findings and x-rays
are not specific for histoplasmosis, since these signs are also found in tuberculosis, influenza,
pneumonia due to Mycoplasma or viruses, and other respiratory infections.
In addition to the three original cases of disseminated disease diagnosed by Darling in three
West Indian immigrants contracted to work on the Canal enterprise, this form of the illness has
also been found in Panamanian mestizos, native Indians and whites, usually the very young and
the aged; and AIDS patients, in whom it is considered to be the reactivation of a dormant infection. Unusual manifestations of histoplasmosis in patients in Panama include perianal abscess
in a 40-year-old man, generalized lymphadenopathy, broncholithiasis, bronchial histoplasmosis,
histoplasmomas, chronic cavitary pulmonary histoplasmosis, laryngeal histoplasmosis.
In Panama, in the 50s and 60s there was a great deal of research relating to the Histoplasma
agent, which gave rise to several isolations of the fungus from soil in bat caves, from the air in
such caves, and from bat tissues. This work was largely carried out by the Middle America Research Unit (MARU) located in Ancon on the Canal Zone, with the cooperation of Panamanian
collaborators and scientists.
Histoplasmosis in Panamanians most often presents as a benign, flu-like illness, usually diagnosed
long after the initial infection during skin testing surveys with histoplasmin. Skin reactivity to this
antigen has diminished markedly (11 to 18%) from the values obtained in surveys conducted up
to the 1970s (in some communities as high as 100%), at least as regards the population residing
in the big cities, eg Panama and Colon; probably the prevalence of infection continues to be high
in rural areas, which have not experienced the changes found in the large cities. We postulate
that this dramatic reduction in the prevalence of histoplasmin sensitivity may be explained by
the following:
1. Bat-proof houses are now being built, so that no guano is available to favor the multiplication
of H. capsulatum in the environment.
2. Big-city dwellers have small plots of land, so that patios are reduced in size, therefore.
3. No chickens are raised, whose droppings facilitate the survival and reproduction of H. capsulatum , and
4. Few or no old trees can be found in which bats can sleep during the day and therefore contaminate the space with their guano.
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Histoplasmosis in the United States
John R. Graybill
University of Texas Health Science Center, San Antonio, TX USA
e-mail: jrgraybill@aol.com
Just like Coccidioides, Histoplasma was first appreciated in Latin America, and just like Coccidioides,
Histoplasma had an initial false identity as a protozoan. And just like Coccidioides, Histoplasma was
initially considered a very rare pathogen. And just like Coccidioides, initial Latin American discoveries
were followed by discovery in the USA. But not very rapidly…it took more than 30 yearsafter Darling
for a dedicated pathologist, WA DeMonbreun, to recover H capsulatum from blood and tissue, and
to culture the yeast phase using enriched medium. In the next 2 decades investigators at a single
university elucidated most of what we know about the epidemiology of histoplamosis and its clinical
manifestations. Primary disease and the sequellae of abnormal healing, acute, subacute, or chronic
dissemination, chronic pulmonary disease, and hypersensitivity reaction were all relatively rapidly
defined. By the 1960’s histoplasmosis was appreciated as a common infection and a well known
disease in states bordering the Mississippi River Valley. Major outbreaks in Indianapolis also contributed greatly to our understanding of primary infection and successful host response. In the 1980’s,
HIV/AIDS provided a number of hosts with very depressed immune defenses. AIDS patients, infected
with either fewer organisms or with less virulent organisms, and extending the appreciated endemic
range of histoplasmosis. With recent control of AIDS in the USA, the relative importance of this disease has decreased. However, histopasmosis remains prominent throughout Latin America, where
Histoplasma is still a major pathogen of patients with AIDS, and still carries a high mortality.
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Symposium
9
New Approaches to the
clinical and laboratory Diagnosis
of systemic Mycoses
The value and the promise of molecular biological tools
in the diagnosis of aspergillosis and candidiasis
Beatriz L. Gómez, Ph.D
Mycotic Diseases Branch,
Centers for Disease Control and Prevention,
Atlanta, GA USA
e-mail: bgomez@cdc.gov
Improvements in medical intervention have led to significant increases in cases of invasive fungal
infections (IFIs) in a growing immunocompromised population. Early diagnosis is still a challenge
for clinicians, with delays or missed diagnosis increasing mortality. Diagnosis of most IFIs is
difficult, particularly for invasive aspergillosis, with classical culture techniques providing poor
sensitivity. Radiological investigations are a useful adjunct but provide nonspecific and transient
results, and histopathology, despite providing definitive evidence of IFI, cannot always identify
the causative organism. Therefore, indirect diagnostic means have been developed.
Whereas detection of antigen has gained enough reliability to be included in consensus criteria
for IFI (European Organization for Research and Treatment of Cancer, Mycoses Study Group,
EORTC/MSG), PCR has not, despite more than 10 years of molecular-based diagnostics developments and publications.
The main reason is the absence of consensus technique(s) and of commercial kits. However,
this limitation has become less strict as a result of emerging real time PCR assays and the
strong efforts by different groups working together to standardize these methods. Additionally,
kits are starting to be commercially available. Well-conducted comparisons should facilitate
selection from the various available DNA extraction techniques, targets for amplification within
the fungal genes and primers, and from the clinical specimens relevant to the given infection.
For the more promising choices, sensitivity and specificity of the PCR test can be assessed in
well-designed prospective studies. Efforts are underway towards this important goal. It is also
important to regularly run quality controls to ensure reliability of the tests.
Real-time PCR is a quick method for quantifying how much target sequence was present at
the start of the reaction. Second and very important for the routine laboratory, real-time PCR
decreases enormously the risk of false positive results. Indeed, there is no post-amplification
handling, thus minimizing the risk of contamination from the environment. Another advantage
for routine laboratories is the fast turnaround time of less than 3 h. Additionally, fully automated
nucleic acid extraction techniques eliminate the need for vacuum pumps, centrifugation, or other
manual steps that may result in cross-contamination.
In this review the benefits and limitations of recently published assays are compared, and the
requirement for both international standards and a consensus protocol that is sensitive enough
for diagnosis of invasive fungal infections will be discussed.
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Molecular diagnostic approaches in
paracoccidioidomycosis and histoplasmosis
R.M. Zancopé-Oliveira1, C.M.A. Soares2, T. C.V. Rezende2, K.P. Castro2,, M.A. Almeida1, A.J.Guimarães1, H.L.M Guedes3 , C.V. Pizzini1.
1
Laboratório de Micologia-Setor de Imunodiagnóstico,
Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz.
Avenida Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brasil.
Tel. (55-21) 3865-9640 e-mail: rosely.zancope@ipec.fiocruz.br
2
Laboratório de Biologia Molecular, Universidade Federal de Goiás, Goiânia, Goiás, Brasil.
3
Laboratório de Bioquímica de Proteínas e Peptídeos, Instituto Oswaldo Cruz,
Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
Endemic mycoses can be challenging to diagnose, and accurate interpretation laboratory data is
important to ensure the most appropriate treatment for the patients. Although the definitive diagnosis
of paracoccidioidomycosis (PCM), and histoplasmosis (HP) the most frequent endemic mycoses in
the world, is represented by direct diagnosis performed by micro and/or macroscopic observation
of the fungi, the isolation of the etiologic agents is time-consuming and lacking in sensitivity. The
accepted limitations associated with classic techniques for the diagnosis of endemic mycoses have
lead to the emergence of many non-culture-based methodologies. Analysis of the humoral immune
response to fungal infections has played, and will continue to play, a key role in their diagnosis and
immune surveillance. Although rapid genome detection methodologies, such as PCR, are beginning to replace immune assays for the diagnosis of systemic mycoses, they are not suitable for all
applications, especially the surveillance of the immune status of human populations. Several immunological methods have proven useful for the endemic mycoses diagnosis; however, they are often
time consuming and many lack sensitivity and specificity, partially attributed to the use of unpurified
antigenic complexes fromeither whole fungal cells or their culture filtrates, which give cross reactivity.
Emphasis has shifted, however, to clinical immunoassays using highly purified and well-characterized
antigens including recombinant antigens. Here we shall review the limitations of current conventional
tools for measuring immune responses and outline principles for the design and production of novel
diagnostic reagents for Paracoccidiodomycosis and Histoplasmosis. Methods for the production of
diagnostic antigens by recombinant systems are described and their relative merits and disadvantages will be discussed.
Histopathology of paracoccidioidomycosis in the postgenomic era:
Reflections and perspectives
Henrique Leonel Lenzi
Laboratory of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
The histopathologic study of Paracoccididodes brasiliensis, considered now as a member of the
new family Ajellomycetaceae (Untereiner et al. Mycology 96:812-861, 2004) is presenting some
new challenges: 1) Different isolates have revealed a great degree of genetic variability in the
pathogenesis; 2) The existence of four different phylogenetic species recently demonstrated (S1,
PS2, PS3 and Pb01-like) (Matute et al. J Clin Microb 44: 2153-2157, 2006; Mol Biol Evol 23: 6573, 2006; Carrero et al. Fungal Genet Biol 45: 605-612, 2008); 3) Pb exhibits epigenomic changes
depending on the habitat (Silva et al. Microbes Inf 10: 12-20, 2007; Derengowski et al. Med Mycol
46: 125-134, 2008); 4) Limitations of the traditional static granuloma concept, not considered as a
dynamic lesion, that follows the properties and mechanisms of adaptative complex systems; 5) The
114
need to consider the P. brasiliensis-host interactions as determinant of a new system (cohabitome),
deeply dependent on the genetic characteristics of the fungi and of the hosts; 6) The importance
of analyzing the tissue in a three-dimensional perspective and not just in two-dimensional plan;
7) The inadequacy of the Euclidean geometry as form of measuring most of the complex biological phenomena like the tissue fibrosis; 8) The limitations of the reductionist view of the traditional
immunology, causing excessive “fragmentation” of the biological phenomena . Based on the above
considerations, the pathology of postgenomic era should apply multidisciplinary approaches, showing a profound integration with Cellular and Molecular Biology through the uses of High resolution
photonic microscopies (laser confocal and two or multiphotons microscopies), laser microdissection techniques and tissue microarrays. The high resolution microscopies allow to do virtual
tomographic sections of the lesions, providing a three-dimensional analysis of the cellular and
extracellular matrix components, obtaining adequate serial sections to further tissue modeling by
different technical procedures. These types of microscopies easily enable multichanel fluorescent
analysis with a very sophisticated system of spectra discrimination and can be applied also in
samples obtained by tissue microarray, defining histogenomic and histoproteomic characteristics,
contextualizing the genomic and proteomic in the cells and tissues (=morphological localizome).
Laser microdissections techniques (laser-manipulated microdissection. LMM) will allow verifying
the epigenomic expression of the P. brasiliensis according to the “metastatic” localization in different sites of the organism. In fact, LMM constitutes the best bridge between the Pathology and The
Molecular Biology and can be applied in histological specimens, including the fungi, alive cells (ex
vivo) and in culture cells, using the following technical preparations: fixed and/or frozen specimens,
alive or fixed cells, stained or non stained material. The specimens can be analyzed by bright-field
or multichanel fluorescence microscopies, including confocal and two photons microscopies. After
the laser capture, the DNA can be analyzed by PCR, genetic mutation, single nucleotide polymorphism, fingerprinting, loss of heterozygoty, fluorescent in situ hybridization, DNA microarrays. The
RNA can be detected by mRNA isolation, reverse transcriptase-PCR, expression analysis and
microarrays. The proteins can be distinguished by two-dimensional electrophoresis (2D-PAGE),
surface-enhanced desortion–ionization-time of flight (SELDI -TOF), matrix-assisted laser desortion
ionization-TOF (MALDI-TOF), IMMUNOBLOT, nano-scale liquid chromatography tandem mass
spectrometry (nLC/MS/MS) and protein microarrays. The tissue microarrays (TMA) offer the great
advantage of compare simultaneously, in the same assay, a large number of specimens from different places of the same individual or from several individuals (human or experimental animals).
Imaging MALDI mass spectrometry has been successfully used to obtain the distribution of proteins
in thin tissue slices (Stoeckli et al. Nat Med 7:493-496, 2001). This procedure can be expanded by
adding a third dimension allowing the 3-D mapping of proteins in specific regions of the interested
tissue by imaging serial sections. The 3-D distribution of targeted proteins can be an important tool
for the understanding of disease. The Pb-Host interactions can be now better studied with some
genetic sophisticated details in vivo using genetic engineering of laboratory animals, obtaining
transgenic, knock-out, knock-in, knock-down (iRNA) animals and animals tagged with fluorescent
protein gene (fluorescent animals).
It is important to stress out that most of these referred procedures were not yet applied to the
paracoccidiodomycosis histopathology due to a clear dissociation between the molecular and
the morphological studies (the “disease” of the specialization) and due to the very expensive
cost of these high-throughput modern technologies. This new approach will require a compulsory
cooperative work in national and international levels and the change of the way of thinking about
the host-parasite relationship, looking under the perspective of Systems Biology and Systems
Pathology, considering that the Pb-Host interaction configures the emergence of a new system,
the Pb-Host Cohabitome.
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Clinico-immunological and immunopathological correlations in
different clinical forms of the paracoccidioidomycosis
M. A.Shikanai-Yasuda 1,2, A. Sadahiro3 , Pagliari, C4., Duarte, M. I. S.4, M. Yoshida 2
1 Departamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da USP,
2 Ambulatório de Micoses Sistêmicas da Clínica de Moléstias Infecciosas e Parasitárias e Laboratório de
Imunologia (LIM 48) do Hospital das Clínicas da Faculdade de Medicina da USP;
Av. Enéias de Carvalho Aguiar, 500. Térreo, Sala 4 CEP 05403 000
3 Instituto de Ciências Biológicas, Universidade Federal da Amazônia;
4. Depto. Patologia da Faculdade de Medicina da USP
e-mail: dmip.shikanai@hcnet.usp.br, lim48imuno@yahoo.com.br
After inhalation of conidia and their phagocytosis by alveolar macrophages, inflammatory infiltrate with
neutrophils, followed by mononuclear infiltrate with macrophages and lymphocytes was observed in
experimental model. On dependence of cellular recruitment and organization of granulomas around
the yeast, fungus may disseminate through peribronchial lymphatics. In the majority of the cases this
fungus-host interaction is represented by an asymptomatic infection, as the expression of resistant
pole of the infection. Lymphocytes from infected individuals secret gamma interferon in the presence of P. brasiliensis antigens and positive late hipersensitivity skin test to paracoccidioidin and,
more specifically to gp43, is usually associated to the presence of mononuclear infiltrate to in the
infected tissues. Fungal dissemination through phagocytic mononuclear system, compromising the
lymphnodes, liver, spleen, bone marrow occurs in the acute phase of the disease, as the result of
imbalance of cytokine secretion, increased levels of IL10, IL4 and depressed levels of IFN gamma
produced by patients´ lymphocytes stimulated by fungal antigens concomitantly to high levels of
anti- P. brasiliensis antibodies. IgG4 and IgG2 were more commonly present in high or moderate
levels in some patients with acute and chronic form, respectively. Lower avidity of anti-P. brasiliensis
antibodies was associated to recidives of patients whereas higher avidity was associated with control
of the disease.
In the infected tissues, high levels of IL10, TGFβ and FAs L and CTLA-4 expression in T cells correlated with loose granulomas unable to avoid fungal dissemination. In chronic cases, reactivation
occurs after a long period of asymptomatic infection in different organs and tissues and was expressed
by unifocal or multifocal lesions in the lungs, mucosa, skin, lymph nodes, Central Nervous System,
adrenal, bones etc. Different levels of anti-P. brasiliensis antibody and different spectrum of cellular
immunity are correlated with extension of the disease; usually increased levels of IL10 and different
levels of IFN γ were produced by peripheral lymphomononuclear cells. In parallel, local immunity
depends on the organ and on the period of the disease and the duration of the treatment. Different
expression of cytokines, chemokines or cellular markers was observed in areas containing necrosis, granuloma or perivascular infiltrate. In human infected lymphnodes, fungi germination in small
granuloma and in the interior of giant Langhans cells, and CD20, CD4 > CD8 lymphocytes, IFγ and
small amount of IL4 and IL10 were observed in the granulomatous inflammatory infiltrate. In Central
Nervous System, great amount of TNFα is observed around necrotic area, CD34, CD68, NK, CD4
were observed in perivascular spaces and TGFβ is associated with fibrogenesis. Skin lesion of HIV
patient with paracoccidioidomycosis show intense germination of fungi, presence of macrophage,
NK,TNFα, expression of IL10, IL4 and IFγ, and CD8>CD4 associated to low level of peripheral blood
CD4/µl. In human lesions, regulatory T cells exhibit suppressor activity and produce IL1, TGF β. In the
tissues, TGF β may represent regeneration of large areas of necrosis. CCR5 chemokine receptors
were also expressed in the cells of the inflammatory infiltrate (Moreira et al, 2008).
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Lymphomononuclear cells of 29 patients with Paracoccidioidomycosis recognizes at least one of five
peptides selected with the TEPITOPE algorithm), including the peptide from gp43 at the sequence
position 180-194 (Iwai et al, 2007). The immunogenicity of forty one synthetic peptides of 43 kDa
glycoprotein was also tested in “in vitro” lymphoproliferation assay of mononuclear cells from 44
cured patients, and 19 controls. Ten GP43 peptides without the sequence position 180-194 were
recognized by 77.3% cured patients; differences among clinical forms were detected on lymphoproliferative response to P15, P16, P22, P28, P29 and P30; higher levels were more commonly seen
in acute cases (Sadahiro, 2007a). Interferonγ and TNFα were preferentially induced by G2 group
of peptides (P6 to P10) whereas IL10 was observed after G6 and G7 peptides stimulation (P26 to
P30 and P31 to P35, respectively. Regarding HLA frequency, DRB1*11 allele was associated with
unifocal form, localized disease, suggesting its possible influence in the outcome of the host-parasite
interaction in paracoccidioidomycosis (Sadadahiro et al, 2007b). The inclusion of new immunogenic
gp43 peptides may represent a strategy for new therapeutical approach associated with antifungal
drugs aiming to control severe disseminated forms of paracoccidioidomycosis.
Financial support FAPESP 02/06481-6
Clinical diagnosis in paracoccidioidomycosis:
Its differentiation from similar entities
Angela Tobón, MD.
Internal Medicine. Hospital La María and
Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia.
e-mail:atobon@cib.org.co
Paracoccidioidomycosis (PCM) is characterized by systemic involvement leading to development of
lesions in various organs and sites such as lungs, mucous membranes, skin, intestinal tract, adrenal
glands, central nervous system, lymph nodes, liver, spleen and larynx, among others. Such an broad
range of lesions demands from the clinician a high level of suspicion concerning this entity so that
appropriate differential diagnosis, supported by pertinent laboratory tests, may be established thus
allowing instauration of prompt specific therapy.
Pulmonary manifestations (cough, expectoration, hemoptysis, dyspnea) accompanied by constitutional symptoms are common to almost all infectious and non-infectious lung pathologies. In most
cases, these manifestations lead to consider tuberculosis in the first place, a disorder that can be
suspected by the presence of apical fibrocavitary lesions revealed by the X-rays and diagnosed by
simple acid-fast bacilli stains. In cases like these but when no acid-fast bacilli are found in respiratory
secretions, it would be advisable to consider PCM especially if chest X-rays reveal mixed infiltrates,
predominantly alveolar, located in the central and lower lung fields often respecting the apices. Due to
the high frequency of tobacco smoking among the PCM population, cytology of sputum or bronchoalveolar lavage fluids is recommended to discard lung carcinoma, especially of the bronchioalveolar
type, as its radiographic representation may resemble that of PCM.
Other mycoses such as histoplasmosis in its disseminated, chronic progressive form frequently
exhibiting nodular interstitial involvement and apical cavitations accompanied by skin and mucosae
lesions, represent a challange to the laboratory personnel who should keep up with the suspicion
while attempting to confirm it through properly chosen tests due to the fact that the clinical presentation of this disorder is much alike that of PCM.
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Mucosal and skin lesions induced by P. brasiliensis are recognized by their ulcerated and crusty appearence, as well as by their thick, well-defined borders often accompanied by important soft tissue
edema. Their localization in the lips, oral and nasal mucosae appear to be characteristic of PCM but
they are not patognomonic as long as other pathologies including histoplasmosis, carcinomatous
processess and the Kaposii syndrome may give rise to similar lesions. They may also resemble
leishmaniosis or sporothrichosis especially when lesions appear in the limbs’ surface.
One of the clinical problems of great importance due to its severe prognosis is the involvement of
the adrenal glands which is as frequent in PCM as in tuberculosis. CT studies evidence hypertrophy
with or without calcifications but these observations will not allow a differential diagnosis between
the two entities; biopsy and anatomopathologic studies, as well as microbiological and mycological
tests, are required to confirm the etiology. The observation of concurrent lung involvement and/or
muco-cutaneous lesions may avoid the need of the above diagnostic invasive procedure.
Involvement of the CNS by P brasiliensis, seldom recognized clinically but frequently observed at
autopsy, is another problems when the specific diagnosis is searched for. The single nodular cerebral
lesion, the multiple ring lesion revealed by the contrast medium, as well as the perilesional edema,
can be found in diseases as varied as toxoplasmosis, cryptococcosis, tuberculomas, lymphoma and
ischemic cerebral disease.
The temporal profile of the neurologic signs and symptoms, the patient’s immune status and the
concommitance of the general clinical manifestations contribute to orient the diagnosis but only the
study of the tissues obtained through stereotactic biopsy would allow the establishment of the proper
diagnosis. Presence of lung and/or mucocutaneous lesions and reactivity of the patient’s serum in
front of the fungal antigen, would also constitute important guides to define diagnosis and allow the
instauration of specific therapy.
The study ulcers in the larynx causing severe pain, as well as their presence in the intestinal lumen,
the latter accompanied by diarrhea and constitutional symptoms in young adults, may surprise the
physician by revealing the characeristic P. brasiliensis multiple budding cells. These abnormalities
should be taken into consideration when defining their cause.
Lastly, the presence of lymph node hypertrophy of the various chains, especially of the cervical ones,
accompanied or not by liver and spleen megalies, should lead to consider disseminated infectious
or neoplastic disorders presenting common general symptoms that do not allow specific diagnosis.
Marked hypertrophy of the cervical lymph nodes can be observed in tuberculosis, lymphoma and in
the juvenile, disseminated form of PCM. Only the judicious use of biopsy materials or of secretions
obtained from such lesions would allow to determine the correct diagnosis.
This review pretends to alert the clinician about the systemic nature characterizing PCM, an entity
that presents numerous and varied organic pathologies resembling many other entities that although
more frequent in their presentation may not have the reserved prognosis of PCM when its diagnosis
and treatment are not promptly undertaken, thus allowing for the development of sequelae, special
lung fibrous scarring.
FInancial support: Hospital La Maria, Corporación Investigaciones Biológicas (CIB).
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Symposium
10
Advances on antifungal Antibiotics
Coccidioidomycosis as seen in a nonendemic area
John Bennett,M.D.
Laboratory of Clinical Investigation,
National Institute of Allergy and Infectious Diseases,
Bethesda, Maryland USA.
The diagnosis of coccidioidomycosis in the USA is rarely considered outside of the southwestern endemic areas, leading to delays in diagnosis. Acute pneumonia in recent travelers is rarely considered.
More serious is the delay in the diagnosis of persistent lesions in the lung, bone and central nervous
system. Absence of a standard serologic diagnostic test has created a plethora of commercial tests
with uncertain and often confusing interpretation Cases referred to the National Institutes of Health
in Bethesda, Maryland show the variety of clinical manifestations of coccidioidomycosis seen outside
the endemic area, including acute pneumonia, chronic solitary cavities, chronic fibrocavitary disease
and disseminated infection in bone and central nervous system. Development of syrinx in the spinal
cord is a uncommon but dreaded complication of coccidioidal meningitis. Obstructive hydrocephalus
responds to CSF shunting but complicates administration of intrathecal amphotericin B. Voriconazole
was effective in one patient with meningitis but the unpredictable blood levels of this drug present a
challenge in seriously ill patients.
New and experimental antifungal drugs
G. San-Blas
Instituto Venezolano de Investigaciones Científicas,
Centro de Microbiología y Biología Celular,
Apartado 20632, Caracas 1020A, Venezuela.
e-mail: sanblasg@ivic.ve
Preferred antifungals for the treatment of paracoccidioidomycosis are sulfamethoxazol-trimethoprim,
itraconazole, amphotericin B. Treatment is lengthy, the drugs may have undesirable side effects,
and some are costly. Therefore, the search for more selective and efficient antifungals to treat this
and other mycoses continues.
1. Lipopeptide derivatives, new antifungals in clinical use
The fungal cell wall is a unique structure with no equivalent in mammalian cells. Because of that,
cell wall glucans and chitin should be specific targets for antifungal action, inasmuch as substances
blocking their syntheses may lead to fragile cell walls and ensuing cell death. One such specific group
of substances is the echinocandin class of lipopeptide antifungals [caspofungin acetate (Cancidas®,
Merck), anidulafungin (Eraxis®, Pfizer Inc.), micafungin (Mycamine®, Astellas Pharma US)]. They
target the synthesis of β-1,3-D-glucan synthase (GS), the activity of which is essential for the assembly of a functional cell wall in many fungi. The enzyme is a multisubunit complex, which includes
an integral membrane protein and a regulatory subunit, encoded by members of the FKS and RHO1
gene families, respectively. Echinocandins such as caspofungin have activity against important
fungal pathogens, including Candida and Aspergillus spp., a result that prompted FDA authorization
in 2001 for clinical use of caspofungin in the treatment of those candidiasis or aspergillosis cases,
refractory to treatment with polyenic or azolic antifungal drugs. However, values for Candida show
ranges of action depending on the species, e.g., Candida albicans (0.03->16 µg/ml), C. krusei (0.032 µg/ml), C. glabrata (0.03-2 µg/ml), C. tropicalis (0.06-1 µg/ml), or C. parapsilosis (0.5-16 µg/ml).
Limited in vitro studies indicate that echinocandins have high MICs for zygomycetes, Fusarium spp.,
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and Cryptococcus neoformans. MICs for caspofungin in the pathogenic yeastlike phases of several
dimorphic fungi, fluctuate according to species and strains, e.g., Blastomyces dermatitidis (0.5-8
µg/ml) or Histoplasma capsulatum (<0.125 µg/ml; 0.5-4 µg/ml).
We tested caspofungin in vitro against P. brasiliensis. In this fungal species, β-1,3-glucan constitutes 36% and 5% of the mycelial (M) and yeastlike (Y) walls, respectively. Instead, Y cells synthesize
α1,3-glucan as the main wall component (45%), a polysaccharide absent in the M cell wall. Therefore,
we hypothesized a minimal, if any, effect of caspofungin in the pathogenic Y phase. However, tests
on 5 P. brasiliensis isolates [IVIC Pb73 (ATCC 32071), IVIC Pb300 (soil), IVIC Pb377 (armadillo),
IVIC Pb381 and IVIC Pb444 (patients); caspofungin concentrations 0; 0.1; 0.5 and 1.0 µg/ml)] indicated that yeast growth was partially inhibited, albeit at different proportions, depending on the
isolate: Pb73 (65%) > Pb300 (35%) > Pb381 (27%) > Pb444 (21%) > Pb377 (17%) (values at 1.0 µg
caspofungin/ml). The mycelial phase, as expected, was highly susceptible to caspofungin, inhibition
fluctuating between 74% (Pb381) and 81% (Pb73). In no case, a 100% inhibition was achieved, even
at concentrations as high as 16 µM caspofungin, when 91% inhibition was the observed value for
strain Pb73, M phase. Scanning electron microscopic observations indicated structural modifications
in the cell walls of both phases as a consequence of caspofungin action. Such results may indicate
variability in P. brasiliensis cell wall composition, particularly with regards to the participation of β-1,3glucan and the related activity of its synthesizing enzyme (GS). Experiments to solve this biological
question are currently under way. But from a clinical point of view, caspofungin does not appear to
be a promising antifungal for the treatment of paracoccidioidomycosis.
2. Azasterols and sterol hydrazones, experimental drugs
The sterol biosynthetic pathway has been largely studied for the search of antifungals. Azoles and
allilamines act on differents steps of this pathway. However, they may interfere with similar steps in
the host. Hence, we have undertaken the search for drugs that may act on more specific steps in the
sterol pathway. While mammals synthesize C27 cholestane-based members of the steroid family,
pathogenic fungi, protozoa and plants require the presence of sterols (typically ergosterol and 24alkyl analogs) which act as irreplaceable essential growth factors for these organisms. The enzyme
responsible for the addition of these alkyl groups to carbon C-24 and for the regulation of carbon
flow in the sterol pathway is the ∆(24)-sterol methyl transferase (SMT). There is no such enzyme in
the pathway of cholesterol biosynthesis, and thus SMT inhibition should selectively block ergosterol
biosynthesis, affecting only fungal cells, and theoretically bypassing any undesirable blockage in the
synthesis of the host’s cholesterol. The crucial and specific role of SMT has stimulated interest on the
biorational design of SMT inhibitors for potential clinical or agrochemical use as antifungal agents.
SMT inhibitors such as azasterols and sterol hydrazones (AZA1-AZA3; H1-H3) have proven highly
effective as antiproliferative agents against protozoa and some fungi, among them, Paracoccidioides brasiliensis. In the presence of AZA-1, a dose-dependent inhibition of P. brasiliensis growth (Y
phase) was observed from 0.1 to 5 µM, to reach 100% growth arrest at the latter concentration and
above. AZA-2, instead, was only able to inhibit growth by 60% at the highest concentration used in
these experiments (10 µM). AZA-3 was the most powerful drug, as a concentration of 0.5 µM was
able to completely inhibit fungal growth. Lipid analyses indicated that in control cells, main sterols
were brassicasterol (69.1 %), ergosterol (26.8 %) and lanosterol (4.1 %). On exposure to AZA-1,
ergosta-5,7,24(28)-trien-3β-ol (17.1%) and lanosterol (11%) accumulated, while AZA-2 led to an
important accumulation of ergosta-5,7,22,24(28)-tetraen-3β−ol (50.5 %). With AZA-3, instead, an
important accumulation of lanosterol (34.5 %) was observed, as a result of SMT inhibition. Concurrent with the accumulation of these intermediates, final products of the sterol pathway (ergosterol
and brassicasterol) decreased substantially. Similarly, sterol hydrazones (H1, H2, and H3) generated a dose-dependent effect in fungal growth, which were active at nanomolar concentrations, also
related to SMT inhibition. These drugs were active in the following sequence: AZA3 > AZA1 > H3
≥ H1 >> H2 >> AZA2.
Acknowledgement: Merck, Sharp & Dohme (Caracas, Venezuela), for partial support in the caspofungin experiments.
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Nanobiotechnology: Advances in antifungal therapy
A.C. Amaral1, 2 and Felipe M.S.1
2
1
Instituto de Ciências Biológicas, Universidade de Brasília, UnB, Brasília, Brazil
Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, UCB, Brasília, Brazil
e-mail: amaralandre@yahoo.com.br
Nanotechnology presents enormous potential to prepare new antifungal drugs. This science works
at 10-9 part of a meter; in this scale it is possible to design a drug-delivery system to drive the drug to
the infection site or to maintain drug levels at a therapeutic concentration over long periods of time
(Nat. Rev. Cancer, 6:688-701). Although AMB is a potent drug to combat pathogens, its use is limited
by severe adverse side effects as fever, anorexia and nephrotoxicity. Lipid-based nanopreparations
containing AMB represent a promise to minimize the adverse side effects caused by this drug, and
three of these formulations are commercially available for clinical use: Abelcet®, Amphotec®, and
Ambisome®. These lipid preparations reduce the nephrotoxicity by its link to plasmatic proteins and
can be safely used in patients with various conditions predisposing to renal impairment (Int. J. Antimicrob. Agents, 27S:S12-S16, 2006). Even improving the therapeutic index of AMB, some drawbacks
still remain, as for example, all of these formulations lack the ability to release AMB at controlled
rates and, accordingly, it has to be daily administered. Polymeric controlled release systems offer
the flexibility in terms of architecture and drug loading allowing designing different combination to
conjugate drugs and targeting molecules to be released at controlled rates. Polymers used to prepare
these nanocarriers can be natural (e.g. chitosan and alginate) or synthetic (e.g. PLGA-poly(lactico-glicolic acid)) and they have been choose due to its biocompatibility and biodegradability. PLGA
is a co-polymer formed from lactic (PLA) and glycolic (PGA) acids with the ability to control drug
release due to its polymeric physicochemical properties (molecular weight, hydrophilicity) and the
ratio PLA:PGA (Indian. J. Exp. Biol. 38:746-52, 2000). PLGA has presented satisfactory results for
the controlled delivering of different classes of drugs, including for the fungistatic drug voriconazole.
It has also been shown to improve drug efficacy in the treatment of experimental candidiasis (Int. J.
Pharm. 352(1-2):29-35, 2008). In our laboratory we have design, prepared and tested in vivo some
strategies to develop new antifungal drugs to treat mycosis. Based on the principles of nanomedicine,
we have prepared and tested a sustained delivery system to D-AMB within PLGA polymeric blends
which reduced to one third the number of drug administration (patent filled INPI # 0700446-0, Antmicrob. Agents Chemother. paper submitted). This preparation (NanoAnf) was tested in the murine
model of chronic paracoccidioidomycosis (PCM), which involve mainly the lungs. Nanoparticles were
functionalized with dimercaptosuccinic acid (DMSA) that presents preferential lung tropism (J. Magn.
Magn. Mater. 293:277-282, 2005). The decrease in the number of administrations was possible as
these nanoparticles were able to carry the total D-AMB dose in a single injection. NanoAnf at 6mg/Kg
each 3 days was able to control the clinical signs of PCM and pulmonary infection, as observed by
the CFU decrease in lungs. Therapeutic effects were comparable to those obtained with D-AMB at
2mg/Kg administered once a day. Moreover, NanoAnf was able to prevent body weight loss observed
in the D-AMB-treated group during the first month. After two months, the animals presented a slight
decrease on body weight, suggesting a delay on the onset of the adverse side effects as observed in
other study with lipid nanoparticles containin D-AMB (Transpl. Infect. Dis. 1(4):273-83, 1999). Other
symptomes, such as piloerection and hypotrihosis observed during long time use of this drug were
prevented by our formulation. Yet, even exceed the initial AMB amount extrapolating tolerable limit,
NanoAnf have not caused renal and liver toxicities in the animals, probably because of the AMB
gradual release from the nanoparticles and by its site-targeting provided by DMSA in the formulation.
In addition, we have investigated others polymeric strategies against fungal infections. One consisted
in the combination of an immune protector peptide P10 entrapped within PLGA nanoparticles with
antifungal chemotherapy against PCM (paper in preparation). The combining therapy started with a
four weekly administration of 20µg of the P10 peptide in complete Freud adjuvant (CFA) or 1µg, 5µg,
10µg, 20µg, or 40µg of the P10 peptide within PLGA nanoparticles, and 15mg/kg sulfametoxazole
administrated daily for 30 days. The antifungal therapy efficacy, evaluated by the fungal burden tis-
123
sue assay, revelead that P10-PLGA conjugate were 20-times more efficient when compared with
administration of P10 in CFA during the first 30 days of treatment. PLGA nanoparticle containing 1µg
of P10 presented the same adjuvancity on therapy than 20µg of the peptide with CFA. It was marked
by an increase on IFN-γ level and by the presence of more compact granulomas in lungs. After 60
days from the beggining of treatment regimens, that is 30 days without sulfametoxazole therapy,
we observed that 10µg of P10 entrapped within PLGA nanoparticle, such as 20µg in CFA, was efficient to avoid the recidive of the disease. This approach proved to be an efficient aid as adjuvant
to sulfametoxazol, a drug responsible for several cases of fungal resistance. Besides improving the
sulfametoxazol efficacy, this strategy has been shown to be a safe alternative to use its chemotherapy.
Other experiments are in progress to evaluate the antifungal potential of conventional drugs in different polymeric nanoparticles constructions against vaginal candidiasis. Our findings indicate that
nanotechniques are powerful tools in the formulation of antifungal agents either by decreasing the
number of drug administration or by improving the potential of the antifungal agents. In conclusion,
nanobiotechnology has contributed to the advances in antifungal therapy. The hybrid conjugates that
combine synthetic polymer with the conventional drugs can help on fight against the fungal diseases by
shortening the find of new antifungal molecules. There is no doubt that these ever more sophisticated
nano drug delivery systems will provide the improved treatments to combat the infectious diseases,
assuring a safely and less painful therapy. Financial support: CNPq and FAP/DF.
The epidemiology of Candida and Aspergillus infections:
Clinical and molecular aspects
C.C.Kibbler
Centre of Medical Microbiology, University College London, UK
e-mail: c.kibbler@medsch.ucl.ac.uk
The understanding of the epidemiology of these important opportunistic invasive fungal infections
(IFIs) has increased considerably over the last decade. Until recently, most of the available data
were from small, often single centre studies, or largely confined to the US. However, increasing
numbers of studies are being conducted at a national level around the world and the largest study
of candidaemia carried out so far has recently been published under the auspices of the European
Confederation of Medical Mycology and took place in seven countries.
These studies are expanding our knowledge of risk factors, prognostic factors and mortality and
it is becoming possible to compare incidences across the world. Geographical differences in epidemiology may be explained by differences in patient populations, different interventions in underlying
disease and differences in measurement and case definitions. The latter has been addressed to a
considerable extent by the EORTC/MSG criteria for diagnosis of IFIs and these have recently been
revised and will be discussed in this presentation.
Molecular studies have helped uncover new species responsible for invasive disease and sequencing techniques used in unravelling mould taxonomy are now finding their way the routine laboratory,
where they may be used for identification. An international working party lead by the CDC is now in
the process of setting out guidelines for this approach.
Typing methodologies such as multi locus sequence typing are beginning to provide an understanding of the global distribution of Candida species, demonstrating geographical clades, providing
evidence of microevolution and allowing transmission routes to be more closely elucidated.
A number of different molecular typing methods have shown the wide biodiversity of Aspergillus
species, although some studies have managed to determine the source of invasive isolates from the
environment or water supplies.
Much of what we have learnt will be invaluable in deciding where to target resources, in conducting
clinical trials, formulating risk-reduction strategies and investigating outbreaks.
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Symposium
11
Agriculture and
Paracoccidioidomycosis
P. brasiliensis growth and infective propagula
production in clayey and sandy soil
E. Bagagli1, Terçarioli G.R.1, Barrozo L.V.2
1 Departamento de Microbiologia e Imunologia,
Instituto de Biociências, Unesp, Botucatu, SP, Brasil.
2 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil.
e-mail: bagagli@ibb.unesp.br
Although the existence of some controversies, including the hypothesis that P. brasiliensis occurs in
heterothermic animals from fresh water environments (Conti-Diaz, 2007), several pieces of evidence
point to the soil as its main saprobic habitat. In such environment, the fungus must develop and
produce the infective propagula, the arthrospores or simply conidia. The influence of soil texture and
chemical composition in fungus development has been little understood. For example, in Botucatu
PCM endemic area, while infected armadillos occur in places containing both sandy and clayey soil
type (Bagagli et al. 2003), the human infection seems to be more prevalent in regions where clayey
soil is more abundant (Simões et al, 2004).
We have then observed experimentally that the fungal growth was similar in clayey and sandy soil
textures, and that no growth occur when the soil composition contained high values of Exchangeable
Aluminum (H+Al) and, consequently, a low Bases Saturation value (V%). The fungal growth was
higher in soil saturated with water than in soil with moderate humidity, determined by its field capacity
(FC). There was no growth in low humidity soil, such as in the one that contains only half of its FC. It
was also observed that some isolates did not produce conidia while in others the conidia production
was relatively high, mainly when the fungus was grown on relatively poor substrates containing soil
extracts, and that this feature (conidia production) could be associated with the genetic group of the
isolates (abundant in S1 and rare in PS2 genetic group), according to Terçarioli et al, 2007.
In conclusion, the major incidence of paracoccidioidomycosis in clayey soil areas may be due to
the widespread use of this soil type in agricultural activity than sandy soil. High humidity is necessary for P. brasiliensis develop in soil. Some soil conditions, in particular those that contain elevated
Exchangeable Aluminum (H+Al) may inhibit or limit fungus growth. The intensity of conidia production
among the isolates in soil extract agar appears to be dependent of its genetic group.
Financial support: Fapesp (Proc 06/03597-4)
On the relationship between land use and paracoccidioidomycosis:
Coffee, sugar cane and other cultures
L.V. Barrozo1, Santana, M.S.1, Gonzalez, C.R.1, Mendes, R.P.2, Marques, S.A.2,
Benard, G.3, Bagagli, E.4
1 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil.
2 Faculdade de Medicina de Botucatu, UNESP, São Paulo – Brasil.
3 Faculdade de Medicina, USP, São Paulo – Brasil.
4 Instituto de Biociências, UNESP, São Paulo – Brasil.
e-mail: lija@usp.br
Introduction and Objectives: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the
thermal dimorphic fungus Paracoccidioides brasiliensis. It is currently accepted that the portal are
the lungs and the air-borne propagules of the fungus infects the respiratory tract of humans after
inhalation. It is recognized that the disease occurs more frequently in patients involved with soilrelated activities, mainly agricultural workers. Land use has been frequently associated with PCM
127
since some agricultural activities produce more aerosols, increasing the opportunity of human
infection. Nevertheless, to date, it is not clear if some specific cropland increases the risk of
acquiring PCM. Thus, the aim of this work is to compare the areas of the most important land
use types and PCM prevalence in a highly endemic area in Botucatu neighboring, São Paulo
State, Brazil.
Methods: The spatial pattern of the series of chronic cases was studied through the application of an inference test for the detection of high and low clusters. Chronic PCM data
corresponded to cases seen in the Infectious Diseases and Dermatology Sectors of the UHUNESP. Only the cases with residence in the study area (n = 280) were selected. A spatial
scan statistic was applied. Cases were assumed to be Poisson distributed, adjusted according to age and gender, with constant risk over space under the null hypothesis. The analysis
was set to include up to 50% of the total population at risk and to find clusters of excess and
low risks. Data of area for coffee, sugar cane, orange plantation, reforestation (Eucaliptus
and Pinus) and pasture (natural and cultivated) were acquired from the Instituto de Economia
Agrícola de São Paulo, from 1983 to 2006. Population data per municipality by year were
obtained from the SEADE Foundation. The percentages of each land use type in relation to
the area of the municipality were calculated. Exploratory analyses of the evolution of land
use types were made to observe each culture in the studied period. The mean areas of each
land use type for the high and low clusters were calculated and compared through t test and,
in the absence of homoskedasticity, through the non-parametric Welch’s test. Also bivariate
regression analyses were performed between prevalence and each percentage of land use
for the 44 municipalities.
Results: Three important spatial clusters were found. The first high cluster included the
following municipalities: Botucatu, São Manuel, Pratânia, Pardinho, Areiópolis, anhembi, Igaraçu do Tietê, Bofete and, Itatinga (P = 0.0001). This cluster presented a mean incidence of
2.9 annual cases/100,000 inhabitants. Cerqueira César comprised another high cluster (P =
0.0012), with 5.8 annual cases/100,000 inhabitants. The low cluster included: Espírito Santo
do Turvo, Paulistânia, Cabrália Paulista, Santa Cruz do Rio Pardo, Lucianópolis, Águas de
Santa Bárbara, Agudos, Óleo, Piratininga, Iaras and Bernardino de Campos (P = 0.0001), with
0.6 annual cases/100,000 inhabitants. The comparison of the percentage of areas for each
culture between the high and low clusters showed that sugar cane plantation and reforestation
are statistically higher (P<0.0001 and P<0.0001, respectively) in the high cluster while pasture
was higher in the low cluster (P<0.0001). Coffee and orange cultures did not present significant differences between the high and low risk areas (P = 0.89 and P = 0.85). Any bivariate
regression analysis revealed significant association with PCM prevalence (P > 0.05).
Discussion: Coffee, tobacco, sugar cane and cotton plantations, riparian forests and alteration of natural forest and other land use types (e.g., mining, wood harvesting) as well, have
been pointed out as associated with the distribution of PCM. In our analyses, land use has
been statistically different for sugar cane, reforestation and pasture between the two opposite
clusters of the disease. This result could suggest that those land use types are associated
with PCM. Regression analyses did not allow the establishment of any direct relationship,
although some land use types have been statistically associated in other geographic areas in
South America. This fact suggests the complexity of the issue and that the association is not
completely clear. PCM seems to be dependent on the degree of exposition to any soil-related
activity, the number of persons involved and the environmental conditions, which allows the
growth of the fungus. Studies addressing this issue should be developed in other endemic
areas.
Supported by FAPESP, PRP/IC-USP and CNPq.
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Influence of alternating coffee and sugar cane agriculture
in the incidence of paracoccidioidomycosis in Brazil
Flavio Queiroz-Telles
Hospital de Clinicas, Universidade Federal do Paraná,
Curitiba, Paraná, Brasil
Paracoccidioidomycosis (PCM) is a chronic or subacute, granulomatous systemic fungal infection
caused by the thermally dimorphic fungus Paracoccidioides brasiliensis. It occur in most of the Latin
America countries, with higher incidence in Brazil, Colombia and Venezuela, followed by Argentina,
Peru, Equator, Uruguay, and Paraguay. So far, no cases have been reported in Nicaragua, Chile,
and the Antilles. Sporadic cases in the United States, some European countries, and Japan have
occurred in individuals who came from endemic Latin American areas. PCM is observed as a natural
infection mostly in humans but sporadic infections have been reported in wild as in domestic animals.
There is uncertainty about the natural habitat of P. brasiliensis but is assumed that the fungus is a
soil saprobe. This evidence comes from sporadic fungal isolations from soil samples and armadillos
internal organs.
Several investigators have indicated that PCM is more prevalent among rural workers engaged in
intensive agriculture. The higher frequency of the disease in men can be attributed to their exposure
to the fungus’ habitat (soil) through agricultural work. The predominance of sugar cane, cotton, and
coffee plantations in the endemic regions favors contact between the rural worker and soil particles
and plants, in which the fungus is found. There are some data supporting that coffee and tobacco
plantations may constitute ecological variables to PCM infection. Records of Colombian PCM patients,
collected by Calle et al, suggest that beside geo-climatic factors, coffee and tobacco plantations may
be linked to PCM infection. In addition, P. brasiliensis has been isolated from the soil of coffee crops
in Venezuela and Brazil, but attempts to re isolate it from the same sources have failed. In the past,
the coffee monoculture usually occurred in several South America countries preceded by a progressive deforestation and this was associated with human PCM by several authors.
In Brazil, with time, soil depletion led to gradual substitution of coffee plantations for pastures, corn,
wheat, soybean, vegetable gardening, fruit growing and others. Historically, the sugar cane cultivation
in Brazil have been an important economic source but mainly associated to sugar production. This
scenario has changed during the last and actual decades due to the energetic requirements, mainly
because of petrol prices. Nowadays, the sugar cultivation area has significantly increased, especially
in some areas of the hinterland of the state of Sao Paulo, located in Brazil Southern Region. Sugar
cane plantation is usually related to wide pesticide aspersion and plant burning. The use of fire may
significantly elevate the soil temperature. In addition, agricultural insecticides, herbicides and fungicides are widely used in most crops, including sugar cane. The combination of these agricultural
practices in sugar cane cultivation may affect the saprobiotic life of several soil micro organisms,
including P. brasilieinsis.
In a recent article, Ono et al demonstrated that most of the agricultural pesticides may inhibit the
in vitro growth of P. brasiliensis, what may explain, at least in part, the difficulty in its isolation from
agricultural soil. Although new PCM cases are still reported in agricultural areas, data of contemporary
incidence and prevalence of this disease is lacking, since the reporting of PCM is not compulsory.
In the future, the epidemiological data related to new cases of PCM, especially from acute/subacute
forms will help to define if PCM is declining in areas where sugar cane cultivation predominates.
129
Analysing coffee’s chemical composition and
effects upon human health
Manuel Elkin Patarroyo
Coffee is one of the most widely know beverages, drunk by people all around the world and know for
acting as a stimulant of people’s psycogenic activity. Coffee is one of the most important agricultural
products for the social and economical growth of producing countries. Due to this psycogenic activity
and its economical, political and social relevance, coffee has been the subject of extensive analysis
(chemical and biological concerning its psycogenic activity) aimed at understanding the mechanisms
underlying such effect, its action or relation to the nervous system, as well as the positive or harmful
effects that its consumption might have on human health. It has become need thus, to gain a deeper
knowledge of its chemical components in order to understand its biological activity.
Coffee, same as any other biological product, has a huge biological and chemical complexity, with
more than 100 substances and chemical components being identified to date, some of which have
been clearly identified and their biological function has been defined.
When studying the chemical characteristics of human beings, several factors must be taken into
account, including their genetical origin, degree of biological development, environmental framework,
type of analysis being applied and such method’s ability to reach valid conclusions etc. Such same
restrictions applied to coffee.
Regarding coffee thereby, variables such as the variety’s degree of maturity (Robusta, Arábica,
etc.), the way they are prepared, analysis methods being employed and their intrinsic limitation, etc.;
may account for different between interpretations (another variable) and may even contract be contradictory in some occasions.
Soluble coffee contains a larger amount (expressed as a percentage of dried weight) of minerals
(2 to 3 times) and caffeine (4 or 5 times), compared to other substances susceptible to degradation
(processed products) where such components disappear or diminish drastically their concentration,
as occurs for instance with trigonelline, aliphatic lipids (10%) and oligosaccharides.
Such variation in composition is not only important in terms of the products’ physicochemical characteristics, but also concerning its organoleptic features such aroma, taste, stimulating activity, etc.
Differences between the Arábica and Robusta varieties, mainly regarding the of carbohydrates
content of green coffee beans (in relation to the variation in dried weight), and most of all in the content
of sucrose; regarding this later aspect, Arábica content of sucrose is found to be 2 or 3 times larger
than the one in Robusta, which has a key effect on taste related characteristics (2).
Roasted coffees have a higher level of lipid content (around 16% in Arábica and 11% in Robusta),
associated in two coffee specific diterpenes (cafestol and kahweol), which are the ones releasing
their most volatile products along the roasting process and whose level might be help determining the
proportion of Arábica and Robusta coffee chosen for preparing blended coffees, given that kahweol
is present in Robusta but not in Arábica coffee (4).
The protein content of both varieties is practically the same, being between 8.8% to 12.2% in green
coffee beans, but the content of free amino acids is highly scarce, ranging between 0.2% to 8%. By
the same token, around 20% to 40% is lost during the roasting process (depending on its intensity),
as result of protein’s denaturation. Some amino acids’ proportion might increase during roasting,
whilst some other amino acids, as arginine, cysteine, serine and threonine, might disappear as a
consequence of this process (5,6).
Caffeine is the major alkaloid substance contained in coffee. Its concentration (in milligrams/Kg/dried
weight) varies; being Arábica’s (9.000-14.000) practically half of Robusta’s concentration (15.00026.000). All other alkaloids in coffee, such as theobromine and theophylline, are found in much lesser
proportions.
Nitrogenous bases are divided in two large groups: those remaining stable after raosting as ammoniac, betaine and choline, and some instable, such as trigolline (present in around 0.6% to 1.2%
130
in Arábica and 0.9% Robusta varieties), which are decompose and turn into nicotinic acid or niacin.
The content of free acid is relevant for coffee’s organoleptic properties, mainly when it results in
the formation of acetic, citric or phosphoric acid. Phosphoric acid content (same as pyroglutamic acid
derived from glutamic acid) becomes considerably higher during the roasting process (9).
Quinic and vinidinic acids’ content, which varies between 0.3% and 0.5% in green beans and
increases during treatment, but from a biological point of view its byproducts are more important.
One of them is n-chlorogenic acid, whose proportion and byproduct depend on the bean’s degree of
roasting (10,11).
Coffee quality depends largely on the relative proportion of monochlorogenic and dichlorogenic
acid. To much dichlorogenic acid might result in a metallic and bitter taste associated with a coffee
that has been heated for to long. Such flavor results from an increase in quinic acid, lactone formation
and diminishes of pyridine concentration (11).
Regarding coffee’s mineral content, the one found in large proportion is potassium (around 80 milligrams in a cup of instant coffee). Although cupper concentration tends to be low, its content is much
more higher in Robusta coffee, which led to argue that such content might explain this variety’s higher
resistance to fungi due to copper’s fungicidal activity.
It can be say then:
• Coffee’s composition is very complex, including more than a thousand substances;
• Not all coffee components that have been identified possess physiological effects, being caffeine
the most widely known among them.
• In order to validate experimental results of studies done in animals, the following criteria must be
considered:
• Dose used
• Length of coffee’s administration;
• Metabolic characteristic of each animal species
• Caffeine is the most widely-known alkaloid amongst those known to produce significant physiological effects; others such as theophylline and theobromine cause also significant biological effects.
• Coffee’s metabolism is complex. Its physiological effects may in part be explained by three mechanisms:
• Antagonism of adenosine receptors;
• Phophorodiesterase inhibition; and
• Intracellular calcium mobilization
• The only neurological effects being clearly demonstrated so far are increased mental alertness
and delayed of onset of sleep. Coffee content produces vasoconstriction of brain vessels, which
is why it is present in several pharmaceutical products used for migraine treatment. Caffeine also
boosts the analgesic effect of some drugs,
• Coffee enlarges short-term memory, alertness and mental sharpness;
• Coffee regular consumption in moderate quantities does not affect normal cardiovascular functioing
or systolic and diastolic arterial pressure
• The gastric or intestinal intolerance regularly attributed to coffee is usually linked to some
people’ssensibility to coffee and has not been successfully reproduced in any experimental
study;
• Coffee exerts a cholecystokinetic action and increases pancreatic secretion;
• Coffee regular consumption does not cause respiratory apparatus associated diseases;
• Endocrine functions are not modified by consuming coffee;
• Consuming coffee does not have any important effect on muscle function;
• It has not been demonstrated that consuming coffee increases the risk of bone fractures;
• Coffee is a good source of potassium, magnesium and fluoride;
• Coffee consumption increases energy metabolism in the hours following its ingestion, but does not
modify the total energy expenditure;
• Coffee consumption has no harmful effects on reproduction or fertility;
• Coffee (in the regular amounts consumed by human) has no teratogenic effect; and
• In the habitual amounts consumed by humans, has no genotoxic, mutagenic or carcinogenic potential.
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Impact of regular coffee consumption on alcohol intake
and depressive feelings among students
Santos, Roseane Maria M1, Le, Thaovy2 and Lima, Darcy Roberto A3
Assistant Professor, Department of Pharmaceutical Sciences,
South University School of Pharmacy, Savannah, Georgia
2
Pharm. D Candidate, South University School of Pharmacy, Savannah, Georgia
3
Professor, Instituto de Neurologia, Universidade Federal do Rio de Janeiro, Brazil
e-mail: drlima@cafeesaude.com.br
1
Recent studies have associated regular coffee intake with beneficial effects in diseases such as adult
diabetes, Parkinson’s disease, cancer and cirrhosis as well as on suicidal tendencies. Results of our
recent pilot study have led us to hypothesize that compounds known to possess opioid antagonist
properties occur naturally in coffee and could be beneficial helpful in the control alcoholism.
Objective: Evaluate the relationship between the consumption of coffee and alcoholic beverages
with the development of signs and symptoms of apathy and depression within student population.
Method: A cross-sectional study utilizing a standardized questionnaire regarding coffee intake,
depressive feelings and alcohol consumption was completed anonymously. The population studied
was somewhat heterogeneous, composed of students from different parts of the country and backgrounds and of different ethnicity.
Results: The incidence of apathy and/or depressive feelings associated with regular alcohol intake relationship was found to range from 0 % to 9 % and 0 % to 31 % on a daily and weekly basis
respectively. Moderate daily coffee intake varied widely (from 10 to 64%) and was age-related. Our
data suggests a strong inverse association between regular to moderate coffee intake and depressive feelings and to alcohol intake compared with non-coffee drinkers.
Implications: Coffee and its health related effects do not seem to be exclusively dependent on
caffeine. Properly roasted coffee has antioxidant poliphenolic chlorogenic acids and lactones with
opioid antagonist activity. This might well explain the diverse and controversial data published so
far, either positive or negative, about coffee and health.
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Symposium
12
Evolutive Biology of P. brasiliensis
What can we know about speciation in P. brasiliensis?
Daniel R. Matute
Department of Ecology and Evolution, The University of Chicago
1101 E. 57 Street, Chicago, IL 60637
e-mail: dmatute@uchicago.edu
The definition of a species has been a major impediment to studies of speciation in organisms other
than animals. In some cases botanists and microbiologists have often expressed doubt that species
even exist, because of frequent reports of interspecific hybrids, phenotypic variation that does not
assort into discrete categories and because of the inherent philosophical difficulties to define species
in these clades. Nevertheless, like the formation of animal species, speciation in other organisms
including fungi is characterized by the evolution of barriers to genetic exchange between previously
interbreeding populations. Defining species boundaries (and taxonomy in general) in fungi has many
difficulties. The main issue is that morphological characters are still the most common way to assess
the existence of different species without taking into account that species may become genetically
isolated before the development of morphological autopomorphies. A novel approach that has been
used recently used is the use of phylogenetic methods to estimate the genetic diversity and determine
the existence of groups reproductively isolated by assessing the degree of gene flow between cryptic
groups. By this approach it has been estimated that the use of phenotype as the sole parameter to
define fungal species highly underestimates their number when compared to species recognized by
phylogenetics. Phylogenetic species recognition using concordance of multiple nuclear genes has
enjoyed widespread use with fungi earlier than other groups like plants and animals, mainly because
of their lack of phenotypic characters, making nucleic acid characters attractive. However, in animals,
the use of multiple nuclear markers is becoming increasingly popular. Dettman et al. (2003) (Dettman, Jacobson, and Taylor 2003) proposed a simple approach to identify independent evolutionary
lineages and phylogenetic species from multilocus genealogies. Species boundaries in Neurospora
sp. that were recognized by this approach have been shown to be in good agreement with those
identified by mating tests (Dettman et al. 2003).
P. brasiliensis has shown extensive genetic variability when analyzed by molecular tools such
as random amplified polymorphic DNA, restriction fragment length polymorphism (Soares et al.
1995; Nino-Vega et al. 2000), and electrophoretic karyotyping (Montoya et al. 1997). These results
have been the ground to study and characterize natural genetic variation in this fungus. Recently,
the existence of three genetically distinct evolutionary lineages in P. brasiliensis was demonstrated
through analysis of DNA sequence data for multiple genes (Matute et al. 2006). These groups are
currently designated S1 (species 1), PS2 (phylogenetic species 2), and PS3 (phylogenetic species
3). Additional support for these lineages comes from variation in virulence and expression levels of
antigenic proteins previously found between P. brasiliensis isolates which are now known to belong
to S1 and PS2 groups (Carvalho et al. 2005). Moreover, other isolate with a considerable genomic
divergence has also been detected by the use of the genealogical phylogenetic species recognition
(Carrero et al. 2008).
So far, all the studies on genetic diversity in P. brasiliensis have focused on the phylogenetic reconstruction of genealogies but certainly none of them has tried to use the biological species concept
that is considered the gold standard for speciation and diversity inventories. This issue is not restricted
to P. brasiliensis but to all those fungi that have not known sexual stage what makes difficult to apply
canonical approaches to the study of speciation. Yet, inability to demonstrate the sexual stage does
not mean that it does not exist. Many fungi, P. brasiliensis amongst them, have been concluded to
have a sexual stage by population genetic methods although we have no morphological evidence for
their sexual reproduction. In this talk I want to stress out the need for reconciling the biological species
concept and the phylogenetic concepts in fungi in order to understand speciation processes.
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Is Paracoccidioides brasiliensis an unique specie?
M.S. Felipe and Teixeira M.M.
Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil.
e-mail: msueli@unb.br or marcus.teixeira@gmail.com
Paracoccidioidomycosis is a systemic illness with an area of occurrence covering most of Latin
America, with highest prevalence in Brazil, Venezuela and Colombia, and that affects mainly
individuals living in the countryside. Studies on difference isolates of the causative agent, Paracoccidoides brasiliensis, have revealed a great degree of genetic variability in this pathogen. The
Pb01 isolate, in particular, differs from the three previously described phylogenetic species by the
genealogical concordance (S1, PS2 and PS3; Matute et al., 2006) and its taxonomic classification
in the genus Paracoccidioides remains undefined. In the present work, sixteen isolates genotypically similar to Pb01 have been identified by analysis of a molecular marker from the first intron
of the hsp70 gene; these isolates have been named “Pb01-like”.
Using the method of recognition of phylogenetic species by genealogic concordance (GCPSR),
this work aimed at looking into the existence of a systematically significant group of “Pb01-like”
isolates from a total of 88 samples. For phylogenetic analysis the methods of Maximum Parsimony
and Bayesian analysis were applied to thirteen (13) polymorphic regions, including individual and
concatenated loci. This enabled the grouping of the “Pb01-like” isolates in a distant phylogenetic
group distinct from S1, PS2 and PS3. A high number of fixed polymorphisms were identified
between the “Pb01-like” cluster and that composed of the other three species, which suggests a
blockage of all genetic flow between them.
The speciation event that defined the new phylogenetic group is sympatric relative to S1 and PS2.
The “Pb01-like” cluster and the group that includes S1, PS2 and PS3 are highly divergent and
have been genetically isolated for about 19 million years. Recombination analysis revealed that
recombination events occur independently inside the two groups, which suggests reproductive
isolation. Two isolates from the “Pb01-like” group, 769 and 133, have quite distinctive genotypes;
in the phylograms and in the recombination analysis these isolates share alleles with isolates from
the S1 and PS2 phylogenetic species respectively.
In keeping with GCPSR, the “Pb01-like” clade can be considered a new phylogenetic species
distinct from the other three, since the clade corresponding to “Pb01-like” isolates is strongly supported by all phylogenetic trees independently and concatenated generated from polymorphism
data of the thirteen loci, with values of posterior probability (1.0) and of bootstrap agreement
(100%) highly significant.
Exclusive characteristics were identified for the “Pb01-like” group, such as the morphology of yeast
cells and conidia (Bagagli et al., personal communication). In conjunction with molecular phylogeny
data, the formal description of a new species for the genus Paracoccidioides should be accompanied by a renaming of the group, in the light of the medical relevance of this pathogen in Latin
America. A new nomenclature would obviously ease information exchange among pathologists
and researchers. We suggest formally that the phylogenetic species encompassing the “Pb01-like”
clade should be named Paracoccidioides lutzii, as a tribute to medical mycologist Adolpho Lutz,
who first described human pathogen P. brasiliensis exactly one hundred years ago.
Financial support – CNPq, FAP-DF, FUB.
136
Climate variability and acute / subacute paracoccidioidomycosis
in a hyperendemic area in Brazil
L.V. Barrozo1, Mendes, R.P.2, Marques, S.A.2, Benard, G.3, Silva, M.E.S.1, Bagagli, E.4
1 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil.
2 Faculdade de Medicina de Botucatu, UNESP, São Paulo – Brasil.
3 Faculdade de Medicina, USP, São Paulo – Brasil.
4 Instituto de Biociências, UNESP, São Paulo – Brasil.
e-mail: lija@usp.br
Introduction and Objectives: Paracoccidioides brasiliensis has rarely been identified in the natural
environment, which makes difficult to characterize its ecological niche. The geographical distribution
of the disease is associated with specific environmental conditions such as mild temperatures, fertile
soils and high humidity. Many studies have focused on defining the niche of the fungus based on the
location of residence of the patient. However, since acute/subacute cases present shorter latency
periods, they may allow spatial and temporal analyses. To date, variations in incidence have been
observed in Colombia, among the Suruí Amerindian population in the Amazonian region of Brazil
and in Argentina after the built of a hydroelectric power. Besides land use changes, which causes
soil disturbance and the exposure of larger numbers of individuals, P. brasiliensis responds to the
moisture content and temperature; therefore, a relationship between climate conditions and PCM in
its acute/subacute form is suggested. To date, studies that characterize the space-time pattern of the
series of acute PCM and its relationship with climate variables are not yet available. The aim of this
work was to analyze the series of acute/subacute cases admitted to the University Hospital of the São
Paulo State University (UH-UNESP), located in a highly endemic area in Botucatu neighboring, São
Paulo State, Brazil, from 1966 to 1999 and the possible associations between the acute/subacute
PCM incidence and climate factors.
Methods: Acute/subacute PCM data corresponded to the cases seen in the Infectious Diseases
and Dermatology Sectors of the UH-UNESP. Stepwise regression of the 1966-1999 annual data was
used to model acute/subacute PCM incidence from lagged variables: antecedent precipitation, air
temperature, soil water storage, absolute and relative air humidity and, Southern Oscillation Index
(SOI). Previously, all data were linearly detrended.
Results: Exploratory bivariate analyses showed that incidence correlates positive and linearly with
air temperature (r = 0.45, P < 0.05) and with absolute air humidity (r = 0.50, P < 0.01) in the year
preceding diagnosis. Backward and forward stepwise analyses aided to select the most important
variables for explaining the acute/subacute PCM incidence. Multiple regression analyses resulted
in two models: the first explains 44% (P < 0.001) of the incidence in this period, taking into account
only the 1-year lagged absolute air humidity and 3-year lagged SOI and, the second additionally
includes the soil water storage of 3 years before incidence, explaining 49% (P < 0.0001) of the PCM
variance in the yearly series.
Discussion: The higher incidence could then be understood as resulting from positive absolute
air humidity of the year of exposure, and negative SOI and positive soil water storage of the 2 years
before exposure. Although it is recognized that PCM distribution is driven by environmental variables
and human activities, to date any data based attempt has been made to verify the links between
fluctuations in PCM incidence and climate. This first approach of explaining acute/subacute PCM
incidence based on climate variability suggests some relevant implications to the biology of P. brasiliensis. Greater absolute humidity is found to be significant in the year before increase in incidence,
or according to our assumptions, in the probable year of infection. Although laboratory studies have
not been carried out to observe the influence of humidity on conidial liberation of P. brasiliensis, a
parallelism can be made with the two other closest pathogenic fungi (e.g., Blastomyces dermatitidis,
Histoplasma capsulatum) due to some similarities among them. Higher soil water storage two years
before exposure may explain the already suggested relationship between P. brasiliensis and rainfall/
humidity favoring fungus growth in this period. How SOI may affect the growth of P. brasiliensis is a
137
matter of conjecture. As a global index, SO may be a proxy for others local environmental variables
not measured (e.g., dew, fog drip), which could also increase humidity in soil surface two years before
exposure. Teleconection patterns defined by ENSO events throughout the globe establish different
conditions of atmospheric circulation, influencing regional humidity convergence and divergence. By
its turn, it defines preferable regions for development of precipitation. This result suggests that there
is fungal growth after increase in precipitation in the longer term, as has been noted for Coccidioides
sp (Comrie 2005), and probably greater spore release with increase in absolute humidity in the short
term. We can hypothesize that an antecedent period of high moisture is necessary to the fungus to
establish itself in the soil. Human activity then dislodge the superficial portion of the soil, exposing
the filamentous, spore producing, form of the fungus. If the absolute humidity is high enough at that
moment, the spores are wet and then freed and aerolized.
Supported by FAPESP and CNPq.
Overview of the biogeography of P. brasiliensis
E. Bagagli, Teodoro R.C, and Gimenes Bosco S.M.
Departamento de Microbiologia e Imunologia,
Instituto de Biociências, Unesp, Botucatu, SP, Brasil.
e-mail: bagagli@ibb.unesp.br
The geographic distribution of P.brasiliensis is located mainly in South America, where the fungus has
evolved in the latest millions of years, as others Ajellomycetaceae family members. Although details
about the fungus ecology are not yet completely revealed, several pieces of evidence indicate that P.
brasiliensis presents two distinct ecological niches, one inside hosts as individual yeast cells, and one
in the saprobe environment as mycelia that produces the infective propagula. While P. brasiliensis
has been rarely isolated from saprobe sources, it has been frequently recovered from clinical samples
and from tissues of armadillos, a dwelling soil mammal. Human cases of Paracoccidioidomycosis still
represent the most important sources of information about the spatial distribution of the pathogen in
the endemic areas, and have been described in patients from distinct biomes, such as in rain- and
semideciduos forest, open fields, and savannas, in a wide range of biotic and abiotic factors.
In the latest years, it becomes clear that P. brasiliensis does not represent a unique biological entity, but a complex of different genetic groups that has diverged separately as cryptic species. Four
distinct genetic groups have already been detected (S1, PS2, PS3 and Pb01-like) and the divergence
time among the groups was estimated in 8 to 19 millions of years, according to Matute et al, 2007 and
Teixeira et al 2008, respectively. While S1, a paraphyletic group, appears to be the most ubiquous,
occurring in different regions and countries of Latin America, the remaining ones, apparently monophyletic groups, seem to be geographically more localized: PS2 has been detected sympatrically with
S1 in Southeast region of Brazil, PS3 as clonal in Colombia, and Pb01-like as a well diverged group in
Central-west region of Brazil. Some mycological features, such as the intensity of conidia production,
appear to be associated with the genetic group and, in this manner, contributing to the different infection
rates. For example, conidia production tend to be high in S1 and very low in PS2 group isolates, and
the infection incidence rates of S1 and PS2 genotypes in human and armadillos, in Botucatu endemic
area, is nearly 5:1, respectively.
Important points to be addressed: In which condition and circumstances sexual reproduction is occurring in S1, PS2 and Pb01-like groups? What kinds of biological barriers could be acting to avoid
interbreeding between S1 and PS2, the sympatric groups? Considering that each species has its own
ecological niche, what is the role of parasitism for the species divergence and also for defining the effective ecological niche? The evaluation of representative numbers of isolates from different regions of
Latin America, both by molecular and mycological approaches, must be carried out in order to provide
a more realistic picture of P. brasiliensis biogeography.
Financial support: Fapesp (Proc 06/03597-4)
138
Poster Session
1
Clinical Aspects / Case Reports
1-01
Clinical epidemiology of paracoccidioidomycosis patients in the region of Botucatu (São
Paulo state, Brazil)
D.F. Lopes1, L.R. Carvalho2, R.P. Mendes1.
Tropical Diseases Area, Botucatu Medical School; São Paulo State University – UNESP. 2Department of Biostatistics, Biosciences
Institute. São Paulo State University – UNESP. faccioli_lopes@yahoo.com.br
1
Introduction: This study was carried out in order to clearly differentiate acute/subacute-AF from chronic-CF
form and to evaluate aspects of medical attention to paracoccidioidomycosis patients.
Methods: Clinical records of 100 confirmed paracoccidioidomycosis patients with AF or CF attended at
the Tropical Diseases Area – Botucatu Medical School were reviewed. Information collected on standardized
questionnaire included age, sex, occupation, smoking history, clinical manifestations, previous treatment, and
hospitalizations. Chi-square and Fisher’s exact test were used in the statistical analysis; significance was set up
at p<0.05.
Results: This study showed higher prevalence of males (84% of the cases), rural workers (84%), and CF
(73%). Many (97.3%) CF patients were older than 29, while only 26% of AF belonged to this age-group. CF
was more prevalent among males (97.3 %) while AF showed no difference as to gender (48.2% males, 51.8%
females). Lungs, in CF and lymph nodes, in AF, were the most frequently involved organs. Co-morbidities were
rare, except for smoking (83%). Symptomatology over 4 months was observed in 69.8% of CF but in 48.2% of AF
[p=0.09]. The main complaints were fever and lymph node enlargement in AF; dyspnea, cough and expectoration
in CF. Generalized adenomegaly predominated in AF, however it was localized, mainly in the cephalic segment
and neck, in CF. Pulmonary semiological findings were usually mild, predominating the hyperinflation syndrome.
Splenomegaly was more frequent in AF (22.2%) than in CF (4.1%). Four patients were misdiagnosed as tuberculosis
and received antimycobacterial treatment, without improvement. Treatment of 30 patients was previously started
by other Services. Hospitalization (median length of 22 days, ranging from 2 to 109) was recommended for 75%
of the patients. Readmissions were frequent (39.6%), specially for complex laboratory tests. Median of treatment
duration was 4.3 years, and therapy discontinuation was observed in 19% of the patients, usually after marked
clinical improvement or clinical cure.
Comments: These data show the protean clinical presentations of paracoccidioidomycosis, the high length of
symptomatology in many AF cases, the difficulties to maintain patients’ compliance after clinical improvement, and
the needs of hospitalization for initial therapy and laboratory evaluation, leading to high financial and social costs.
1-02
Paracoccidioidomycosis in patients with and without HIV infection. Comparative study
A.M.M. Paniago1; A.L.Oliveira1; A. L. Pegorare1; E.S.A.Aguiar1; J.I.A. Aguiar1; M.R.Chang1; R.V. Cunha1; A.R.C MottaCastro1; B. Wanke2.
1 Universidade Federal de Mato Grosso do Sul, Campo Grande-MS –Brasil. 2 Instituto de Pesquisa Clínica Evandro Chagas-FIOCRUZ,
Rio de Janeiro – Brasil. e-mail: anapaniago@terra.com.br
Introduction and Objectives: The association of paracoccidioidomycosis (PCM) and HIV infection in Latin
America, although consistently reported in the endemic areas of this mycosis, is relatively low in contrast to the
higher incidence of other systemic endemic mycoses, e.g. histoplasmosis and coccidioidomycosis. Moreover, little
is known on the incidence and clinical manifestations of PCM among the HIV infected population. The objective
of this study is to compare the epidemiological and clinical characteristics of PCM in HIV seropositive and HIV
seronegative patients.
Methods: 329 patients with PCM attended during the period of 1992 to 2007 at the University Hospital of
the Federal University of Mato Grosso do Sul, in Campo Grande, Brazil, were evaluated. Clinical manifestations,
demographic data, co-morbidity and outcome were compared in both a) HIV seropositive group (n=14) and b) HIV
seronegative group (n=315).
Results: The prevalence of HIV infection among PCM patients was 4.3%. The HIV seropositive patients are
younger (mean age 36.1 years old versus 48 years old), showed more fever (71.4% versus 35.2%), lymph nodes
involvement (85.7% versus 41%), hepatomegaly (42.5% versus 5.1%) and lower levels of hemoglobin (mean of
10.5mg/dl versus 13.1mg/dl). In addition, the HIV seropositive group showed higher lethality rate (57.1% versus
3.5%) and more tuberculosis co-morbidity (42.9% versus 5.4%).
Conclusions: These results suggest that PCM, when associated with aids, behaves clinically as an
opportunistic disease.
Financial support: Programa Nacional de DST/Aids and UNESCO.
Presenting author’s: Anamaria Mello Miranda Paniago; anapaniago@terra.com.br, Rua das Paineiras, 68, Bairro
São Francisco, Campo Grande-MS, CEP: 79.010-070. Phones: 55 (67) 2109-8545; 55 (67) 9912-0164 (mobile)
141
1-03
Paracoccidioidomycosis in Mexico
R López-Martínez, 2O Velasco-Castrejón, 3A Bonifaz, 3J Cazarín-Barrientos, 3A Saul, 4R Arenas, 5MC Padilla
Desgarennes
1
1- Departamento de Microbiología y Parasitología. Facultad de Medicina, Universidad Nacional Autónoma de México. México D.F.
México. 2.- Unidad de Medicina Experimental. Facultad de Medicina. Universidad Nacional Autónoma de México. México D.F. México..
3.- Servicio de Dermatología. Hospital General de México, S.S.a. México D. F. México. 4.- Servicio de Dermatología y Micología.
Hospital General “Manuel Gea González”, S.S.a. México DF. México. 5.- Laboratorio de Micología. Centro Dermatológico “Ladislao de
la Pascua”. S.S.a. México DF. México.
e-mail: rlm@servidor.unam.mx, rlmmex@hotmail.com
Paracoccidioidomycosis is an endemic infection in Latin America. In the south countries most cases is
observed in Brazil, Colombia and Venezuela, with fewer cases in Central America and Mexico. Spite of in
Mexico the paracoccidioidomycosis cases are sporadic, disease is important due to both its severity and
diagnosis difficulty. Here 109 cases reported in a 57 years period within five dermatological hospitals are
presented. The disease evolution time before clinical diagnosis was between one and nine years, but in
most cases was of two years.
The most affected age group was that from 41 to 50 years; cases in children of 11 years and in older
than 72 years were also observed. The man/woman rate was 20/1. By occupation, the distribution was
farmers 88.7 %, housewives 4.8 % and others activities 6.5 %. In the Veracruz state, situated in the Gulf
of Mexico, 63 cases were observed. The most northern case was originated from San Luis Potosí state
(Huasteca potosina) at 23° North latitude. The primary pulmonary form was followed by skin dissemination
in 36.6 % and mucosal dissemination in 37.6%. The treatment of first observed cases was based on SMX/
TMP and amphotericin B. In the last years itraconazole only or in combination with other antifungal drug
was used obtaining a faster disease resolution.
1-04
Paracoccidioidomycosis in Bolivia
J. Vargas F.
Unidad de Micología, CENETROP, Santa Cruz-Bolivia. e-mail: drjvargasf@hotmail.com
Paracoccidioidomycosis is the most important systemic fungal infection in Bolivia not merely because it
is the most common, but because of its high potential to cause disability of the severity of its clinical forms
and its severe sequelae.
History: There is no medical literature sufficient to determine from where the Paracoccidioidomycosis is
known in Bolivia. The works of Morales Villazon (1916) and Veintemillas (1935) confuse this mycosis with
leishmaniasis. The first cases published and confirmed by laboratory testing belong to Sangueza (1965),
Galindo Decker (1969) and Rios Dalenz (1979) The creation of CENETROP in 1974 and the start of the
activities of the Mycology Unit in 1979 are two important milestones in the history of this disease in Bolivia,
to become the national focal point of Paracoccidioidomycosis.
Geographical distribution and frequency: Based on the origin of cases, it is considered that tropical
regions and valleys are the endemic zones of Paracoccidioidomycosis, covering 75% of Bolivian territory.
There are diagnosed 30 to 40 cases per year.
Clinical forms: Chronic-Multifocal: 80% Chronic-Unifocal: 16% Acute (child-young): 4%
Site of infection (chronic multifocal form): Lungs: 80% Lymph nodes: 78% Pharingeal mucosa: 60%
Skin: 58% Adrenal glands: 3% other sites 3%
Therapeutic experience: In 1988, a work was published about 30 cases treated with ketoconazole with
93% of good results (6), in 1992 at the V International Meeting of Paracoccidioidomycosis in Buenos Aires
were presented the results of 40 cases treated with itraconazole, with an efficacity of 95% Actually, the
drug itraconazole is the treatment of choice. Clinical pictures with the results obtained are shown.
142
1-05
Epidemiological and clinical features of patients with paracoccidiodomycosis attending at
a University Hospital from Huila - Colombia, between 1999-2007
Molano VM1, Gualtero S2, Duran LF1, Gómez CA2, Diaz G.1
1. Departamento Medicina Interna, Hospital Universitario Hernando Moncaleano Perdomo, Neiva-Colombia. 2. Unidad de Infectologia, Hospital
Universitario Hernando Moncaleano Perdomo, Neiva-Colombia. e-mail: victormolano2007@gmail.com - sandra.gualtero@gmail.com
Introduction: Paracoccidioidomicosis (Suramerican Blastomicosis or Lutz Splendore de Almeida disease) is a
endemic mycosis with highly concern in latinamerica countries. In Huila-Colombia there are not studies that describe
its behavior.
Objetive: describe clinical and epidemiological features from patients with Paracoccidioidomicosis brasiliensis that
recieve medical care at Hospital Universitario Hernando Moncaleano Perdomo, between january 1999 to september
2007.
Materials and methods: retrospective study starting from medical records of patients with Paracoccidiodomicosis
brasiliensis histological confirmed. A personal phone call was used to establish survival. Reservarea area and endemic
area concepts was used according to Borelli´s definition. EPI INFO 3.4 programs was used for statistical analysis.
Results: Ten (10) patients was identified. All of them were man with histopathological confirmed
Paracoccidiodomicosis. Chronic presentation was observed in 9 patients and only one had the juvenil type. 6 had
between 30 and 60 years old. 7 were farmers dedicated to coffee cultivation. 8 patients were heavy smokers and 1
had HIV infection. 2 refered armadillo intake at the past. 3 had lymphoid nodes and mucous compromised. 8 had
lung, skin and central nervous systems concomitant involment and 1 had ileal compromise. Four received treatment
with anfotericine B followed by itraconazol, 3 with ketoconazol and 1 with fluconazol. Seven patients was alive at
september 2007, 2 was dead in one it could not settle down.
Neiva city and the municipalities of Gigante, Acevedo, Argentina, Garzon, Oporapa, Rivera and Yaguara were
identified like probable reservarea area. Neiva city (4 cases), Gigante (2 cases), Acevedo, Garzón, Paico y Rivera (
1 case each one) were considered endemic area.
Conclusions: the factors associated with the patients are similar to those described in the literature. Important
regions was identified likely Endemic and reservarea area. This would allow to carry out active epidemiological
surveillance.
1-06
Paracoccidioidomycosis: Frequency, distribution, and hospitalization flows due to the
endemia in Brazil (1998-2007)
Coutinho, Z.F.(1), Travassos,C.M.R.(2), Wanke,B.(3), Oliveira, E.X.G.(4), Valle, A.C.F. (3), Coimbra Junior, C.E.A. (1).
National School of Public Health/FIOCRUZ, Brazil. (2) Institute of Scientific and Technological Communication and Information in Health
/FIOCRUZ. (3) Evandro Chagas Clinical Research Institute /FIOCRUZ. (4) Brazilian Institute of Geography and Statistics – IBGE.e-mail:
ziadir@centroin.com.br (presenting Author)
(1)
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis,
an endemic exclusive to the Americas. In Brazil, the mean annual PCM mortality rate was 1.49/million inhabitants from
1980 to 1995 (COUTINHO, 2002). This study analyzes the frequency, distribution, and case flow between the municipality
of residence and that of hospitalization for PCM patients. Hospitalization data were obtained from the Health Ministry
through the Hospital Information System of the National Health System (SIH-SUS) for January 1998 to May 2007.
Hospitalizations were selected with a principal diagnosis of blastomycosis (B40-ICD10) or paracoccidioidomycosis (B41ICD10), with blastomycosis considered equivalent to PCM. The period totaled 7,017 hospitalizations for PCM, of which
48.98% were concentrated in 21 hospitals and 82% were male patients. Spatial distribution of hospitalizations showed
distinct patterns between the different States and regions of Brazil: (a) the Southeast had 57.90% of hospitalizations,
followed by the Central-West with 13.34%; (b) in Piauí, 22.36% of the 626 hospitalized cases were residents of that
State, while 49.52% were from Maranhão and 24.92% from Pará. The States of Piauí, Maranhão, Pará, and Tocantins
had 12.64% of all hospital admissions; (c) Rondônia (257 admissions) and Mato Grosso (612) had 12.38% of all
hospitalizations; (d) Among 88 hospitalizations of residents from the Federal District, 56.81% were admitted there and
25% in the State of Goiás; (e) Santa Catarina (209 admissions) had more hospitalizations than Rio Grande do Sul (93);
(f) Most States in the North and Northeast had small caseloads. PCM hospitalization flows display places with large
displacement by residents to obtain hospital care. The North of Brazil exported nearly 30% of its PCM patients to other
regions. The pattern of States cited in items (b) and (c) appears to correspond to expansion of the agricultural frontier in
Amazonia. The current analysis shows the inadequacy of public policy for PCM care, identifies the healthcare system’s
limitations for adequately treating patients, and reflects the trend in the ecodynamics of the PCM endemic in light of
economic, environmental, and migratory changes.
Key words: paracoccidioidomycosis, epidemiology, mycosis, health policy
143
1-07
Epidemiological and clinical profile of 137 pacients with paracoccidioidomycosis in Uberaba,
Minas Gerais, Brazil
Neves, FF; Gerolin, GP;Tavares, MG ; Castro-Silva, MH; Lopes, GP;Michelin, MA;SilvA; Vergara, ML
Doenças Infecciosas e Parasitárias, 2. Pneumologia, 3. Imunologia e 4. Radiologia, Universidade Federal do Triângulo Mineiro – Uberaba,
MG, Brasil. E-mail: ffneves@bol.com.br
Introduction : After hundred years of the first report, paracoccidioidomycosis is still one of the most prevalent
systemic mycosis in Latin America countries. The follow-up of these patients commonly is difficult and the relapses
number cases is frequently unknown. The aim of this study is to present preliminar information of a cases serie of
patients diagnosed between 1988-2007.
Methods: Medical records of patients with paracoccidioidomycosis diagnosis performed in the teaching
hospital were reviewed and after this, patients were contacted and invited to perform a new clinical and laboratorial
assessment.
Results: During 19 years, 137 patients with paracoccidioidomycosis were diagnosed. From these, 108
(78,8%) were male, median age of 39,2 years. Between them, 45 (35,6%) were farmers and the median time
of evolution of their symptoms was of 4 months. The main clinical features were related to respiratory, lymphatic
and mucocutaneous involvement in 79 (56,6%), 84 (61,3%) and 63 (46%) cases respectively. Clinical picture
according to the present classification was: acute/subacute 29 (21,2%) cases localized chronic form 35 (25,5%),
disseminated chronic form 72 (52,5%) and mixed two (1,4%) cases. Twenty (14,5%) patients presented coinfection
with HIV. Most of cases were diagnosed by cytological or histopathologic exam of secretions and/or fragment
tissues. Lung involvement by X-ray plain was evidenced in 76 (55,1%) cases. Amphotericin B and/or itraconazole
or Trimethoprim-Sulfamethoxazole were administered to these patients. Twenty five (18,1%) cases presented
clinical relapse. At present, 76 (55,4%) of 137, are alive and seven of them are receiving therapy and 29 (21,1%)
died. No definitive information about the other 32 was yet obtained.
Comments: The clinical and epidemiological features of 137 patients with paracoccidioidomycosis described,
in this study are similar to other reports from Brazil and the high rate of relapse observed among them can be
explained by the poor adderence registered during the follow-up. Otherwise, 25 (14,5%) patients with HIV/AIDS
association were diagnosed and most them presented a low CD4 count, supporting that immunosuppresion is
a important risk factor to develop this mycosis. Otherwise 50% of the deaths occured in HIV patients and no
definitive cause for the others was obtained at present functional and laboratory assessment of patients alive is
currently performed.
1-08
Paracoccidioidomycosis associated to HIV/AIDS infection in Uberaba, Minas Gerais,
Brazil
Gerolin, GP1; Tavares, MG2; Castro-Silva, MH3; Lopes, GP4;Michelin, MA5; Silva-Vergara, ML, Neves, FF.
1.Doenças Infecciosas e Parasitárias, 2. Pneumologia, 3. ImunologiaE 4. Radiologia Universidade Federal do Triângulo Mineiro – Uberaba,
MG, Brasil.
E-mail: ffneves@bol.com.br
Introduction: The coinfection HIV/Paracoccidioides brasiliensis was described for the first time in 1989 in
Brazil. After this, near of two hundred cases has been reported and most severe clinical forms in. The aim of
this study is to present the main epidemiological and clinical features of this cases has been already described
patients with both infections. Methods: Medical records of HIV/AIDS patients with Paracoccidioidomycosis
diagnosed at teaching hospital from 1988 to 2007 were reviewed. Results: Twenty (14,5%) patients among
137 with Paracoccidioidomycosis presented the HIV infection. From these, 16 (80%) were male, median age of
34,7 years. Illicit drugs use and multiple partners were reported by most of them. The median time of symptoms
until mycological diagnosis was 2,9 months. The main clinical features were related to respiratory, lymphatic and
mucocutaneous involvement, in 13 (65%), 13 (65%) and 5 (25%) cases respectively. Clinical form was characterized
according to the current classification as acute/subacute in nine (45%) cases, chronic localizated in five (25%),
chronic disseminated in four (20%) and mixed in two (10%) patients. Lung involvement was evidencied by X-ray
plain in 10 (50%) cases. CD4 count values varieted from 5 to 349 cells/mm with mediacy of 88 cells. Trimethoprimsulfamethoxazole and itraconazole were the drugs more frequently prescribed. At present six patients (30%) are
alive. Comments : Despite the epidemiological overlap between HIV/AIDS and Paracoccidioidomycosis patients,
no substancial increase of number cases of this mycosis were observed, although it’s opportunistic character in
AIDS patients is to be determined yet. However, severe disseminated acute/subacute clinical form or mixed of
features of the two classical clinical forms were frequently observed. The epidemiological and clinical profile of the
patients described is according to the other series already reported.
144
1-09
Acute severe paracoccidioidomycosis in an urban-living boy with a single exposure episode:
Evidence for a long subclinical evolution before full-blown disease
G. Benard1,2 & Barata. L. C. B.3
Laboratory of Dermatology and Immunodeficiencies, Medical School of the University of São Paulo; 2 Systemic Mycoses Outpatient Clinic,
Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP (HC FMUSP), Brazil 3Hospital e Maternidade
Santa Catarina, São Paulo, Brazil mahong@usp.br
1
We describe a 16 year-old boy born who always lived in downtown São Paulo and developed a severe acute
paracoccidioidmycosis. He had no history of exposure in the previous ~4 years except from having gone to a oneday “ecological” hiking (January 25, 2007) in a riverside at the Vale do Paraíba, a well-known reservarea described
previously by Gonçalves and Londero (1998). He walked trough remnants of rain forests and along the border
of plantations. A sorethroat led him to a medical visit on March 9, where a blood film revealed an eosinophilia
(20%, 2499 eosinophils/mm3) and chest X-rays showed a left peri-hilar image compatible with a lymph node
enlargement; pulmonary parenchyma was unnoticeable. Neither alteration was investigated. By June, he referred
development of discrete acneiform lesions on the face. These lesions progressed, and by mid September he
was then evaluated by a dermatologist who diagnosed acne. He was treated accordingly. At this time he was still
doing good and following his studies regularly. By December, skin lesions were more pronounced and spread to
the upper trunk; fever and cervical, axilar, and retroauricular adenopathies and hepatosplenomegaly were noted,
together with weight loss, weakness, persistent eosinophilia, discrete anemia and hypergammaglobulinemia. He
was investigated for several hypotheses other then paracoccidioidomycosis. This diagnosis finally came out on
January through a cutaneous lesion biopsy. He received itraconazole, 300 then 400 mg/day, but after 3 weeks his
general condition continued to deteriorate, the inflamed lymph nodes were even more enlarged, some fistulizated,
and the hemoglobin dropped to 8.5 g/dL. He was then hospitalized and put on lipossomal amphotericin. He was
discharged 15 days later, somewhat improved, and was punt on sulfadiazine (100 mg/kg/day). He continues to
improve gradually. His hemoglobin is now 13.5 g/dL and he is reassuming his normal activities.
This case allows to estimate precisely the incubation time (or subclinical evolution) before the full blown acute
form disease manifests: 5 months.
Other recent travels referred by the patient were to USA and to mountain counties within São Paulo state
(altitude >1500 m above see level), which do not correspond to reservareas in our state.
1-10
First description of a fatal adult respiratory distress syndrome in a severe acute
paracoccidioidmycosis patient
Ravanini. J.N.,1 Silva L.F.F. 1, Goulart S., 2 Benard G. 2,3
Department of Pathology, Medical School of the University of São Paulo, Brazil, 2Systemic Mycoses Outpatient Clinic, Division of
Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP (HC FMUSP), Brazil, 3Division of Clinical Dermatology,
Clinics Hospital, Medical School of the University of São Paulo, Brazil, mahong@usp.br
1
We describe a 22 years-old patient who was born and always lived in São Paulo downtown, with a 5-months
history of progressive weakness and malaise and generalized adenophaties. During this period he lost 7 kg and
also complained of an initially non-productive cough that lately produced some sputum. He denied any drug
abuse and tobacco smoking habit. He used to go on week ends to a county near São Paulo from where some
patients with acute PCM have been referred in the last years. On admission he was in a good general condition,
eupneic, presented an hepatosplenomegaly and a generalized adenopathy involving the cervical, inguinal, axilar,
and occipital chains. Laboratory data showed marked leukocytosis, eosinophilia, abnormal liver function tests. He
was HIV negative. A thoracic CT scan mediastinal adenomegalies e several lytic lesions on ribs. A biopsy of a
cervical lymph node done previously at an outside service was inconclusive, but P. brasiliensis yeast forms were
visualized in sputum. Specific serology tests were also positive. When receiving the first dose of amphotericin B
he developed respiratory insufficiency, tachycardia hypotension. He was moved to the ICU immediately, where
he received antibiotics to an eventual hospital-acquired infection. His respiratory insufficiency worsened, he
was intubated, put of assisted mechanical ventilation. Chest X-rays showed the typical pattern of ARDS. His
respiratory insufficiency was refractory to the ventilatory support and he died after 4 days at the ICU. Autopsy
showed disseminated paracoccidiodidomycosis and histopathological findings in the lungs compatible with the
ARDS. All cultures were negative for bacterial growth and the autopsy did not disclose any finding suggestive of
other bacterial or viral infection.
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Sub-acute paracoccidioidomycosis in a 49 years-old woman
D. Myiamoto1, Kono Adriana. S. G .1, Benard. G.1,2, Yoshida. M.1, Giarolla. I.1, Shikanai-Yasuda. M. A.1,3
Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP(HCFMUSP),
Brazil. 2Division of Clinical Dermatology, HCFMUSP, Brazil .3 Department of Infectious and ParasiticDiseases, FMUSP, Brazil
masyasuda@yahoo.com.br
1
A 49 year-old patient complained since 2004 of occasional upper abdominal pain irradiating to flanks. This
symptom became more intense by 2006, and was then accompanied by fever and abdominal distension. An
ultrasound imaging revealed peri-pancreatic and peri-aortic nodules, as well as gallbladder microcalculi. The
patient was submitted to videolaparoscopic cholecistectomy during a peri-gallbladder lymph node was biopsed.
This biopsy revealed paracoccidioidomycosis. She had a good recovery of the surgical procedure but received
no treatment for the mycosis at this time. In the following two months she lost 20 kg, and then was referred to our
service.
The patients was born in São Paulo, where she lived most time, except for a short period between 1998 and
2000, during which she went on the weekends to a rural area close to São Paulo for resting purposes. This area
may be considered a reservarea. She developed her menopause at the age of 42 (thus before developing the
symptoms), and she had been diagnosed as having hypothyroidism at 46, to which she has been adequately
treated since then with levotyroxin .
On admission she presented hepatosplenomegaly and cervical lymph node enlargement. Acute phase
reactants and liver function tests were also abnormal. A thoracic CT scan showed mediastinal and supraclavicular
lymph node enlargements, while an abdominal TC scan revealed generalized and remarkable lymph node
enlargement and hepatosplenomegaly. She was treated with trimethoprim-sulphamethoxazole 160/800 mg twice
a day with a good response and since then has been on regular follow-up at outpatient service.
Conclusion: This case describes a 49 year old women who developed a typical acute/subacute form of the
disease, soon after her menopause. Paracoccidoidomycosis is rare among women, with cases being described
either before puberty, when the clinical presentation is the acute form of the disease, or after menopause, when
the clinical presentation is the chronic form of the disease. From the epidemiological point of view, it is possible to
admit the she acquired the disease recently, when frequenting a reservarea, and in a period when she was already
presenting estrogen levels reduction.
1-12
Paracoccidioidomycosis presenting as evanescent pleural effusion associated to focal
consolidation: An uncommon presentation
Costa AN1; Junior SAD1; Takagaki TY1; Kairalla RA1; Carvalho CRR1.
1.Pulmonary Division – Hospital da Clinicas - University of São Paulo Medical School, SP, BRASIL. e-mail: nathan.andre@gmail.com
(presenting Author).
Men, 72 years old, born and living in São Paulo, ex-smoker (15 pack/years; 30 years ago), presented with left
thoracic discomfort while exercises that began 2 months before, without associated systemic symptoms. While
excluding cardiologic and coronary causes of the pain, a left inferior lobe infiltrate associated to a moderate pleural
effusion was found, and he was then sent to a pneumologist. The computed tomography of the chest showed
parenquimal consolidation associated to moderate pleural effusion, without lymphadenopathy. One week later,
when he came back for a thoracocentesis, there was no more pleural effusion, neither in the physical exam nor in
the chest ultrasound. A bronchoscopic lavage was then performed, which revealed the typical yeast form of the
Paracoccidioides brasiliensis. Although the serology was negative, he was treated with Itraconazol 200mg/day,
with total remission of the symptoms after 2 months of treatment. We performed a new computed tomography two
months after the beginning of the treatment that showed only mild residual reticular infiltrates, no pleural effusion
nor consolidations. Discussion: Pulmonary paracoccidioidomycosis is one of the most important fungal infections
in Brasil, being responsible for 200 deaths/year and concentrating 80% of the cases in the world. Primary infection
occurs in the infant and the chronic form affects adults in the third to the fifth decades of life. Radiologically, the
findings are typically bilateral and symmetrical, with interlobular septal thickening, nodules, peribronchovascular
interstitial thickening, centrilobular opacities, ground-glass opacities, cavitations, consolidations, traction
bronchiectasis and paracicatricial emphysema. In this case, de presentation was atypical because of the focal
air space consolidation, absence of lymphadenopathy and the evanescent pleural effusion, besides the negative
serology. The microbiological confirmation and resolution of the clinical and radiological findings after imidazolic
use support the diagnosis.
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Nested-PCR, immunoblotting and double immunodiffusion in meningoencephalitis by
P. brasiliensis without radiological alterations of the central nervous system
R.A.M.B. Almeida1, S.A. Marques1, R.P. Mendes1, D.V. Moris1, S.M.G. Bosco2, S.A.G. Macoris2, E. Bagagli2, A.P. Vicentini
Moreira3, V.S. Kohara3, R.S. Cavalcante1, C.R. Chaves1 e S.A. Calvi1.
1. Botucatu Medical School, UNESP, São Paulo State University, Brazil. 2. Botucatu Biosciences Institute, UNESP, São Paulo State
University, Brazil. 3. Adolfo Lutz Institute, São Paulo, Brazil. e-mail: mip.ricardo@gmail.com.
Background: Meningoencephalitis by P. brasiliensis (Pb) affects 10 to 27% of paracoccidioidomycosis (PCM)
patients and its diagnosis is generally made by serological, radiological as well as clinical evidence of PCM in different
sites. Aim: To report an unprecedented case of meningoencephalitis by Pb, without CNS radiological alterations,
diagnosed by Nested-PCR. Case report: A man, 51 years old, presented PCM lesions in the oral mucosa, confirmed
by biopsy, DID = 1/32, paracoccidioidin = 20x18 mm and oral candidiasis, without additional symptoms. After one
week of cotrimoxazole, he started presenting dizziness, epigastralgia, anorexia and adynamia, followed by fever and
behavioral alteration 14 days later. The patient used depot corticosteroids for 11 months, due to contact dermatitis.
He was feverish, with meningeal signs and Glasgow = 8. There was oral candidiasis, without active PCM lesions.
Cerebrospinal fluid (CSF) showed 13 cells, predominantly lymphomononuclear, hyperproteinorrhachia (123 mg%) and
hypoglycorrhachia (34 mg%). Cytology for fungi, bacteria, mycobacteria, viruses, protozoa and neoplasias showed
negative results and there was no fungal, bacterial and mycobacterial growth. EEG demonstrated diffuse slowness,
without herpetic encephalitis pattern, and cranium CT was normal. Aciclovir was initiated and cotrimoxazole was stopped
due to suspected medication-associated meningoencephalitis and hepatotoxicity. Two days later, the clinical signs and
symptoms completely resolved. Serology for Epstein-Barr, Chagas’ disease, HIV, hepatitis B/C, and syphilis, as well
as the tuberculin test, was negative; IgG antibodies to CMV were positive and brain MRI was normal. DID resulted in
½. Serological tests for toxoplasmosis indicated recent infection contracted, however, more than six months before.
Nested-PCR reaction was performed using primers derived from the rDNA region of Pb, being positive in CSF and
biopsy of the initial lesion, and DID and immunoblotting in CSF were negative. The levels of TNF-alpha, INF-gamma
and IL-10 in the serum and CSF were high (TH0). Conclusions: The clinical evolution and the cytokines profile may
suggest hypersensitivity to the antigens released after the initiation of cotrimoxazole, corroborated by the immediate
improvement after the medication suppression. Meningoencephalitis by Pb without CNS radiological images was not
identified in literature, which demonstrates the importance of using Nested-PCR in etiologic diagnosis.
1-14
Cutaneous sarcoidic lesions and moriform stomatitis lesions in a young patient: Two sides
but not the same coin
N. Fairbanks1, A. Kono1, M. Yoshida1, N.Y. S. Valente2, M. A. Shikanai-Yasuda1,3, G. Benard1, 2
Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP), Brazil,
Division of Clinical Dermatology, HCFMUSP, Brazil, 3Department of Infectious and Parasitic Diseases, FMUSP, Brazil mahong@usp.br
1
2
A 24-year-old man from Barueri, a town nearby São Paulo city, was admitted for investigation of a relapsing,
ulcerated oral lesions and development of disseminated cutaneous plaques. He referred to have played in remnants
of rain forests around his birthplace since childhood. Paracoccidioidomycosis has been diagnosed by identification of
Paracoccidioides brasiliensis on direct examination of an oral lesion in 2001 (when 17 y-old). Since then he has been
using short courses (~15 days) of sulfametoxazol-trimetoprim on his on, with partial improvement. In 2006 he developed
skin lesions that also partially subsided with sulfa treatment to reappear soon after. On the present admission there
were multiples, infiltrated, sarcoid-type, erythematous plaques lesions on face, neck, thorax, arms and buttocks and a
two typical mulberry-like lesion on the palate. The aspect of the skin lesions suggested tuberculoid leprosy in reaction.
Evaluated by a hansenologist, a partially (although inconclusive) altered pattern of skin sensitivity tests and suspected
thickening of ulnar nerves pointed to this diagnosis. However, ultrasound imaging showed sural nerves within the
normal range. Lymphadenopathy and hepatosplenomegaly were absent. CT scan revealed normal lungs. Biopsy of
a skin lesion revealed sarcoidic paracoccidioidomycosis: compact granulomas with rare yeast cells. Simultaneously,
biopsy of the palate lesion revealed a granulomatous reaction with numerous eosinophils and plasmocytes, epidermic
microabcesses and numerous yeast-like cells. The suspected leprosy coinfection was discarded. This case highlights
two main points: a) the occasional difficulty in classifying the clinical presentation either as the acute/subacute or
chronic form. This patient presented oral lesions suggestive of the chronic form, but not pulmonary involvement, and a
skin involvement more commonly seen in young patients with good general condition but no systemic involvement. He
not only did not present the typical features of the acute form, but also had a 7-y history of relapsing manifestations; b)
the compartmentalization of the immune response; at the same time the oral lesion depicted a less organized reaction
with abundant yeast and eosinophils, the cutaneous lesions were more organized and compact, sometimes sarcoidiclike, denoting two different outcomes of the host-parasite relationship in the same patient depending on the site of
involvement.
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Adrenal insufficiency by paracoccidioidomicosys (PCM) - series of cases
C.S.O. Bredt 1 ; G.L.Bredt Jr1; P.A.D.Duarte1; F. Pedott 2 ; T. Pereira2; L. De Campos2. Myiamoto2; A.A.Ribeiro2; E.Soliva Jr2; L.C.Volpolini2
1 Medicine Associated Professor. Universidade Estadual do Oeste do Paraná-Cascavel-PR-Brazil, 2 Medicine Students- Universidade
Estadual do Oeste do Paraná- Paraná -Cascavel - Brazil. email: csakuma21@yahoo.com.br
Background: In endemic areas, PCM is the most frequent etiology of Addison’s disease. Paracoccidioides brasiliensis,
the causative agent of PCM, exhibits high tropism will be the adrenal glands, which results in low hormone reserves and in
more severe cases, in symptoms of primary adrenal insufficiency. Adrenal insufficiency in symptomatic you marry is described
in 10-15%. Methods: report cases of three patients with adrenal insufficiency caused by PCM. Results: 1st case: a 75years-old-woman, with fever, malaise, anorexia 10-kg weight lost. She was presented with hypothension and dehydrated.
Clinical examination disclosed abdominal masses in bilateral renal topography. CT scan of abdome demonstrated solid
injuries in the adrenal glands. Biopsy disclosed abscess due PCM. She was submitted to a bilateral adrenalectomia.
The patient was placed on antifungal therapy with B anfothericin and glucocorticoid replacement and made an excellent
symptomatic recovery. 2 nd case: a 43-years-old male had a 2-years history of progressive darkening of the skin and a 20kg weight loss, in addition to lethargy, anorexia, nausea and hypothension. X-ray of thorax demosntrated hilar infiltrated
bilateral. Sorologia for PCM 1:8 (IDD) and plasma cortisol levels was determined by radioimmunoassay of baseline blood
samples 1,3 ug/dL. Initiate treatment with Itraconazol and prednisona with important improvement. 3 rd case: a 47-yerasold- woman, presented weakness, cutaneous darkening and 12-kg weigth lost in 1 year beyond complaint of intense avidez
for salt. She looked attendance with persistent and dispnéia cough. X-ray of thorax presented multiple bilateral areas and
the lung biopsy disclosed PCM. Plasma cortisol levels was 1,72 ug/dL and 89,2 ACTH of pg/mL. Initiate treatment with
itraconazol and prednisona with good evolution. In all the cases described above there were significant improvement of the
symptoms and the quality of life. Conclusions: The authors conclude that an early detection of hipoadrenalism in patients
living in the endemic areas is necessary and very important to minimize further adrenal damage and consequently permits
to shorter hormonal treatment in these patients.
1-16
Adrenal insuficiency associated to paracoccidioidomycosis: Report of three autopsy cases
Mantilla J.C.1, and Serrano A.2
1 Hospital Universitario de Santander, HUS, Bucaramanga – Colombia. 2Universidad Industrial de Santander, UIS, Bucaramanga –
Colombia. e-mail: eterna888@hotmail.com
Paracoccidioidomycosis or South American Blastomycosis is an endemic fungal infection prevalente in Latin
America, which is caused by a dimorphic fungus, Paracoccidioides brasiliensis. This systemic infection, primarily
involves lungs, from where it spreads via lymphatic or blood to other organs such as the skin, mucous membranes,
lymph nodes, central nervous system and adrenal glands, among others. Objective: Describe the main pathologic
findings in three older adults with chronic progressive disease, who died at Hospital Universitario de Santander (HUS),
and who were diagnosed with paracoccidioidomycosis boy medical-scientific autopsy, performed in the Department
of Pathology, Medical School of Universidad Industrial de Santander (UIS). Materials and methods: It is presented
the clinical-pathological report or three autopsies performed in the Department or Pathology or UIS, Bucaramanga,
Colombia, during the years 2005 and 2006. Results: case: The first of these relates to a patient of 52 years of age, male
gender, with clinical symptoms of a month of evolution characterized by progressive loss of weight, chills, diaphoresis,
appearance of vesicles in the oral cavity, accompanied by bleeding, odynophagia, cough with white expectoration and
pints of blood and progressive dysphagia, who died as a result of multiple organic failure. The second, is a patient of 53
years of age, male gender, with clinical symptoms of is months of evolution characterized by occurrence of mass in the
left eye of progressive growing, who died due to a complex hydroelectrolitic imbalance characterized by dehydration
unwieldy and sustained hyperkalemia. The third case is about a man of 59 years of age, male gender, consulting por
clinicals symptoms of three months of evolution represented by aletered state of consciousness with subsequent inability
to walk, loss of sphincters control and hydroelectrolitic imbalance with hiperkalemia and dehydration unwieldy, which
will eventually produce death. Findings of necropsy: The first case shows corpse of an elderly, male gender, with
cervical, thoracic, abdominal and groin lymphadenopathy and multiple white-grayish nodules of firm consistency and
appearance fibrotic in soft tissue of the thorax, pleura, lungs, pericardium, kidneys, adrenal glands and right testicle. The
microscopic study shows inflammatory lesion characterized by multiple granulomas with abundant multinucleated giant
cells type langhans, in which cytoplasm are rounded birrefringent structures with multiple budding, which correspond to
Paracoccidioides brasiliensis. The second patient was an elderly, male, with small curvature of the stomach and around
the pancreas, fibrous adhesions in left hemithorax and yellowish-white nodules in lungs and adrenal glands, which
sere observed increased of size, histopathological examination revealed chronic inflammatory process with countless
granulomas with multinucleated giant cells type Langhans and fungal structures of Paracoccidioides brasiliensis in
its cytoplasm. The third case refers to an elderly, male gender, with findings of cervical lymphadenopathy and whitegrayish nodules of firm consistency and various sizes in meninges, lungs, liver and adrenal glands, microscopic
examination revealed a chronic granulomatous inflammatory lesion widely distributed in those organs, with multiple
budding yeast of Paracoccidioides brasiliensis inside the giant cell type Langhans. Conclusion: Three autopsy cases
of patients who died at HUS as a result of widespread infection by Paracoccidioides brasiliensis are presented. The
deep hydroelectrolitic imbalance due to an adrenal insufficiency secondary to granulomatous diffuse infiltration was the
direct cause of death in these cases.
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1-17
Scintigraphic evaluation of enteric protein loss in patients with active
paracoccidioidomicosis
B.L. Griva1, R.P. Mendes2.
1 Nuclear Medicine Service, University Hospital; 2 Tropical Diseases Área. Botucatu Medical School – São Paulo State University.
e-mail: tietemendes@terra.com.br (presenting Author)
Introduction: The acute subacute form of paracoccidioidomycosis is characterized by intense involvement
of organs rich in the phagocytic mononuclear system, such as lymph nodes, spleen, liver and bone marrow.
Some of these patients present intense involvement of the abdominal lymphatic system, with protein loss. These
patients can also reveal a malabsorption syndrome, becoming severe their prognostic. Methods: Ten patients
with the acute/subacute paracoccidioidomycosis form, confirmed by the identification of typical Paracoccidioides
brasiliensis yeast forms, were included in this study. Technetium-99m labeled human serum albumin abdominal
scintigraphy was performed in all patients. The radiopharmaceutical was prepared according to the manufacturer’s
specifications and the dose administered was 555 MBq IV. A control quality imaging was done immediately after
the injection using a gamma-camera equipment. Static imaging at 30 min., 1 h, 2 h, 3 h and hourly during the
day, if necessary, was performed until the visualization of the presence of radioactivity in the abdominal region.
A last imaging was done after 24 h of injection. Two independent nuclear medicine physicians did the qualitative
imaging evaluation. The exam was considered positive of enteric protein loss when radioactivity was seen in any
abdominal imaging with subsequent migration at later images. Results: All the patients presented protein loss in
the 15 exams carried out near the diagnosis and during their follow up. Persistence of protein loss was observed
in three patients 3, 5 and 7 years after treatment. Comments: These data show the sensibility of this scintigraphic
evaluation and reveal a long period of protein loss in some cases, explaining the delay in their recovery.
1-18
Biliary ostruction due to acute paracoccidioidomycosis: Analisys of two cases
A. S. G. Kono1, M. Yoshida.1, I. Giarolla1, J. Jukemura2, G. Benard1,3, M. A. Shikanai-Yasuda.1,4
Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP (HC
FMUSP), Brazil. 2Division of Clinical Surgery, HCFMUSP, Brazil. 3Division of Clinical Dermatology, HC FMUSP. Brazil. 4 Department of
Infectious and ParasiticDiseases, FMUSP, Brazil.
e-mail: masyasuda@yahoo.com.br
1
Paracoccidoidomycosis has been classified in chronic or acute/sub-acute form. The acute/sub-acute form is
characterized by a rapid course and reticuloendothelial system involvement. In the absence of specific therapy,
mortality is high. We describe two cases of acute/sub-acute PCM with abdominal lymphadenopathy and biliary
obstruction.
Case 1: A 32 years-old male was hospitalized in 2002 due to fever, cough and weigh loss. He also complained
of abdominal pain. He performed an abdominal CT scan that revealed generalized adenomegaly and hepatomegaly
of right lobe. A lymph node biopsy revealed chronic granulomatous inflammation with fungal elements compatible
with P. brasiliensis. The immunodiffusion test was positive and counterimmunoelectrophoresis titer was 1:512. He
received sulfadiazine 6 g/day with improvement. However, he evolved with icterus and increased hepatic function
tests and a progressive hepatomegaly, despite the gradual fall in serology titers during the six years follow-up. A
magnetic resonance performed during this follow-up revealed important dilatation of intra and extra-hepatic biliary
tract and hepatomegaly. Nowadays he is on waiting list for surgical intervention.
Case 2: A 34 years-old male was hospitalized in 2006 complaining of fever, cervical adenomegaly, abdominal
pain and ictericus. He was submitted to a lymph node biopsy that revealed paracoccidioidomycosis. He performed
cervical, thoracic and abdominal CT scans that revealed a prominent and generalized adenomegaly. His serology
for P. brasiliensis was reagent (immunodiffusion) with a counterimmunoelectrophoresis titer of 1:128. He
received trimetropim-sulphametoxazole. Patient evolved with important jaundice and increased hepatic function
tests. Another abdominal CT scan revealed biliary compression and common bile duct dilatation. Trimetropimsulphametoxazole was replaced by amphotericin but the patient presented azotemia and no improvement of the
jaundice. The sulfa treatment was re-started and the patient underwent a surgical procedure to decompress the
biliary tract. He evolved with complete total recovery. CT scan performed 6 months later didn’t show adenomegaly
or visceromegaly, biliary dilatation. Serology titer fell to 1:8.
Conclusions/Discussion: Although clinical treatment is the rule in PCM, clinicians must be aware of the
possibility of compression of the biliary tract. In these situations the surgical procedure can be crucial for the
recovery of the patient.
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Serious hepatic damage in paracoccidioidomycosis and its treatment
R Lazo 1 – 2, J Barrezueta 1 – 2, J Lazo 1 – 3
1.Centro de Investigaciones de Enfermedades Parasitarias y por Hongos. CIDRALAS – Ecuador, 2. Cátedra de Micología, Escuela de
Medicina, Facultad de Ciencia Médicas, Universidad de Guayaquil. – Ecuador, 3. Disciplinas de Biología Celular / Curso Post Graduación
en Patología de la Universidad Federal do Triangulo Mineiro – Uberaba, MG – Brasil.
e-mail: rlazo@cidralas.med.ec
With previous experiences on Sistemic Mycosis and Amphotericine B, we use this drug on patients with severe
hepatic compromised, that is hepatotoxic and nephrotoxic being a contraindicated treatment. We considered that
is an opportunity to report the use of Amphoticin B on seriously ill patients with cirrhotic syndrome. We receive
the request for to examine a male patient with 26 year, tractor driver on sugar cane plantations. After 25 days
of treatment for pulmonary tuberculosis in Pulmonary Diseases Hospital presented small pustules on the back.
It was made the direct microscopy examination of material obtained from the lesion and sputum, finding multiple
gemation yeasts , which features corresponding to Paracoccidioides brasiliensis. The Inmunodifusion test was
positive to Paracoccidioidomycosis. It was recommended the suppression of Isoniazina, and it was not accepted.
Few days after it presented marked abdominal distention. The paracentesis did not improved the symptoms. After
that, he was derived to our service and completely evaluated. The results of hepatic biopsy reported cirrhosis and
yeast with multiple sporulation stained by Grocott method. The same was reported in the ganglionic biopsy. By this
way, it was confirmed the linfatic ganglionic visceral dissemination and seriously ill. We administered Amphotericin
B, follow this Sulfametoxasole and then Sulfamotoxipiridazine. The clinical respond to the therapeutic was quick.
At the First Symposium of Paracoccidioidomycosis, Medellín 1971, we commented the first findings and we
proposed to study and to determine the relationship with Paracoccidioidomycosis cirrhotic syndrome. At 1982, in
the book Paracoccidioidomicose (Gildo del Negro et al.) is quoted by a new propose for the clinical diagnosis of
Paracoccidioidomycosis. At 1994, in the book Paracoccidioidomycosis (Marcelo Franco et al.) is quoted the same
propose. At 2006, in Thesis for Public Health Master grade, Lazo R. F. indicated the degree of therapeutic respond
to Amphotericine B, after review of 48 patients with systemic mycosis.
1-20
Acute respiratory failure in AIDS patients: Role of open lung biopsy to the diagnosis of
paracoccidioidomycosis (PCM)
C.S.O. Bredt 1,3; G.L.Bredt Jr1; M. Campos1; D. Pavan1; P. A.D. Duarte
D.M.Moraes3; ; M.A.Richetti 3
1;
F. Pedott2; T. Pereira,2; L. de Campos2 ; ;
1Associated professor. Universidade Estadual do Oeste do Paraná, Cascavel-PR- Brazil. 2 Medicine Student’s Universidade Estadual do
Oeste do Paraná, Cascavel-PR- Brazil. 3. Hospital Acquired Infections Practitioners. 3 e-mail: csakuma21@yahoo.com.br
Background: PCM is a systemic mycosis from Latin America. Rural regions in Paraná State (in Southern
Brazil) are notoriously endemic to this entity (Brazilian Society of Infectious Diseases). Methods: Case reports and
literature review of 3 cases of patients with co-infection of AIDS and PCM with Acute Respiratory Failure (ARF) in a
Rural University Hospital diagnosed by open lung biopsy. Results: 1st case: Male, 40 y old, heterosexual, from rural
region. He was admitted with a history of consumptive syndrome, fever, cough and and dispnea. He had previous
diagnosis of AIDS since 06 years ago without specific treatment. Chest X-Ray showed bilateral micronodular
interstitial infiltrate; pancitopenia; hypoxemia in the arterial gasometry at room air, CD4=103 mm3 and viral load:
59.400 cop/ml. It was started empiric treatment for pneumocystosis and tuberculosis. Lung biopsy revealed P.
brasiliensis. PCM serology was 1:8 (IDD). Treatment was done with Amphotericin B. 2nd case: Male, 32 y old,
homosexual, admitted with a neurological disease with a diagnosis of neurocryptococosis, HIV antibodies were
positive. His chest X-ray had bilateral reticulous-nodular infiltrate. Lung biopsy showed association of pulmonary
cryptococosis and PCM. 3rd case: Female, 36 y, AIDS diagnosis since 05 years ago, without specific treatment.
She was admitted at Emergency Dept with ARF. Chest X-ray with bilateral interstitial-alveolar infiltrates. Lung
biopsy revealed PCM. The three cases showed distinct radiological patterns, and in despite of all they received
treatment with Amphotericin B (the 1st choice drug for severe cases), all of them evaluated to death. Conclusion:
In despite of the great benefit of HAART (high active antiretroviral therapy) significantly reducing the incidence of
opportunistic infection (OI) in AIDS, there are still a lot of inpatients with non adherent patients or new diagnosis
with OI. The authors concluded that lung biopsy is very important to confirm etiology of pulmonary infections in
AIDS patients, especially in areas well known to be endemic to PCM.
150
1-21
Paracoccidioidomycosis in a renal transplant recipient:
Case report and review of the literature
Adriana S. G. 1, M. M. Galvão2, M. Yoshida1, I. Giarolla.1, F. O. S, França1, G. Benard1,3, L. E. Ianhez2, M. A., ShikanaiYasuda.1,4
Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP(HCFMUSP),
Brazil. 2Division of Urology, HCFMUSP, Brazil. 3Division of Clinical Dermatology, HCFMUSP, Brazil. 4 Department of Infectious and
ParasiticDiseases, FMUSP, Brazil.
e-mail: masyasuda@yahoo.com.br
1
Background and aims: Paraccoccidioidomycosis (PCM) is the most prevalent endemic mycosis in Latin
America but the disease is rare in immunocompromised patients, especially in transplant recipients. So far, there
are only seven cases reported in literature; all of them in renal transplant recipients. The aim of this report was to
describe another case of PCM.
Case report: A 59 years old male patient who received a cadaveric renal transplantation 13 years before was
hospitalized due to a recent history of cough and pulmonary nodules. The initial hypotheses were lung cancer
or lymphoma. During his stay in hospital, he was submitted to a trans-bronchial biopsy that revealed a chronic
granulomatous inflammation with fungal elements compatible with P. brasiliensis. He was in use of prednisone 20
mg/day, cyclosporine 300 mg/day. He received trimethoprim-sulphamethoxazole 160/800 mg twice a day and was
discharged with resolution of the cough and afebrile. After 18 months of follow-up he remains asymptomatic and
with complete resolution of the pulmonary lesions.
Conclusions/Discussion: This is the eighth case of PCM in renal transplant recipient reported to date. The
use of primary prophylaxis for P. jirovecii with trimethoprim-sulphamethoxazole may explain the scarcity of cases
of PCM in immunocompromised patients such as organ transplant recipients and aids patients. However, clinicians
must be aware of this possibility when evaluating patients from endemic areas.
1-22
Pericardic paracoccidioidomycosis (PCM) associated with pulmonary neoplasia:
Case report
C.S.O. Bredt 1 ; G.L.Bredt Jr1; P.A.D.Duarte1; A.A.Luiz1; F. Pedott 2 ; T. Pereira2; L. De Campos2. Myiamoto2;A.A.Ribeiro2;
E.Soliva Jr2; L.C.Volpolini2
1 Medicine Associated Professor. Universidade Estadual do Oeste do Paraná-Cascavel-PR-Brazil. 2 Medicine Students- Universidade
Estadual do Oeste do Paraná- Paraná -Cascavel - Brazil.
e-mail: csakuma21@yahoo.com.br
Background: PCM is the most common systemic mycosis of the South America. The chronic form has
manifestations pulmonary and / or extra pulmonary , that can be varied and hamper or delay the diagnosis.
Case report: Male, 57 years old, resident of the rural area, longtime smoker, there are 30 days shows
progressive dyspnea to the efforts, chest pain, high intensity with radiation to the back and left shoulder and
worsening in dorsal position and deep inspiration, 4-kg weight lost in this period. The examination showed up
hypotensive, tachycardia, cardiac auscultation with sound rhythmic and muffled without further changes. The ECG
showed diffuse repolarization disorder. The echocardiogram showed paradoxical movement of interventricular
septum, impairment of systolic and diastolic myocardial functions and pericardial effusion causing major cardiac
tamponade. The patient was submitted to a pericardiocentesis with biopsy and drainage of 1800 ml. The study
showed a pathological process chronic granulomatous compatible with paracoccidioidomycosis. The patient
was treated with anfotericina B. Twenty days after, the patient evaluated to dyspnea again and pulmonary
auscultation were abolished in both lung bases and lymph nodes supraclavicular palpable. The patient was then
subjected to thoracentesis whose cytological analysis showed consistent with adenocarcinoma cells. The biopsy
of supraclavicular lymph node also showed adenocarcinoma little differentiated tubular suggesting lung origin.
Conclusion: It is trying to do with the possibility of atypical presentations of PCM, especially in endemic areas
such as the western region of the state of Paraná in Brazil. Early diagnosis and appropriate treatment has a major
impact on quality of life.
151
1-23
Disseminated Herpes simplex infection in a patient with acute/subacute paracoccidioidomycosis
B.S. Souza1, I.X. Duarte2, M.P.T. Moraes2, K.Y.R. Coelho2, B.L Griva3, R.P. Mendes1.
1Tropical Diseases Área; 2 Department of Pathology, 3 Nuclear Medicine Service – Botucatu Medical School – São Paulo State University.
e-mail: tietemendes@terra.com.br (presenting Author)
Introduction: Paracoccidioidic infection and paracoccidioidomycosis are highly prevalent in Botucatu Region
(São Paulo State, Brazil), where severe cases and rare coinfections, such as this study, have been observed.
Case report: A 21-year-old white housewife was hospitalized in October 2000, with a 2-month history of
increasingly epigastric pain, cervical and inguinal lymph node enlargement, a 7.0kg-weight loss and, in the last
7 days, fever of 38-39oC, jaundice, nausea and vomiting. Physical examination revealed bad general condition,
weight of 43.5 kg, blood pressure of 100/60 mmHg, pulse rate of 102/min, respiratory rate of 31/min, temperature
of 37.6oC, conjunctival jaundice, cervical adenomegaly of the tumoral type, and hepatosplenomegaly. Abdominal
computed tomography showed mild dilatation of the intrahepatic biliary tree and enlarged lymph nodes - hepatic
hilum, peripancreas and retroperitoneum. Product of fine needle aspiration of a superficial lymph node showed
typical yeast P. brasiliensis forms. As her condition improved with amphotericin B she was followed up as outpatient,
with frequent readmissions due to lack of compliance to antifungal treatment and for nutritional support. An intestinal
protein loss was confirmed in October 2003. At her last admission she presented vomiting, pain in the right
hypochondrium, bad general condition, weight of 36.6 kg, malnourishment, dehydration, blood pressure of 100/70
mmHg, pulse rate of 120/min, and respiratory rate of 28/min. Active paracoccidioidomycosis, intestinal occlusion
and pneumonia were diagnosed and treated. However, she died on March 24, 2006, despite the introduction
of intravenous cotrimoxazole, appropriate diet, antibiotics and general measures of support. Autopsy showed
active paracoccidioidomycosis in lungs, epiglottis, larynx, liver, spleen, bone marrow, peritoneum, lymph nodes
and intestinal mucous membrane; intestinal perforation and purulent peritonitis; laryngitis and intense necrotizing
bronchopneumonia by Herpes simplex, positive for peroxidase antiperoxidase stain. Discussion: This case shows
the intense immunosupression induced by the acute/subacute paracoccidioidomycosis with abdominal lymph
nodes and bowels involvement, associated with protein malabsorption syndrome. Immunosupression triggered
the development of severe herpetic lesions in lungs and epiglottis, since she was infected by Herpes simplex, as
most of the population.
1-24
Lung paracoccidioidomycosis: Analysis of clinical, tomographic and functional impairment
after adequate treatment
Costa AN1, Fernandes CJCS1, Suesada M1, Salge JM1, Kairalla R1 and Carvalho CRR1.
1.University of São Paulo School of Medicine. Pulmonology Service. Interstitial Lung Diseases Group. São Paulo – Brasil. e-mail: nathan.
andre@gmail.com (presenting Author)
Paracoccidioidomycosis is the most commonly found systemic mycotic disease in South America, in which
lungs are the main organs affected. Eighty percent of world cases are found in Brazil and are responsible for
over 200 deaths per year. Nevertheless the extension of disability due to treated lung paracoccidioidomycosis
is not known. We report a case series of tomographic, quality of life, functional and ergo-spirometric evaluation
of adequately treated patients with lung paracoccidioidomycosis. In our service we have diagnosed in the period
of 1999 to 2005 eleven cases of lung paracoccidioidomycosis. Eight patients have completed the treatment and
performed the proposed protocol (CT scan, spirometric tests, diffusion capacity, 6MWT, cardiopulmonary exercise
test and Saint-George Respiratory questionnaire). The patients mean age was 63 ± 9 years-old and the period
between the end of the treatment and the protocol was 4 ± 2,4 years. Seven patients were former smokers. The
spirometric values found were FVC 3,98 ± 0,75 L (105 ± 14 % of predicted); FEV1 2,67 ± 0,47 L (89,2 ± 11 % of
predicted); TLC 6,24 ± 1,03 L (101 ± 8 % of predicted), RV 2 ± 0,67 L (90 ± 26 % of predicted) and diffusion capacity
of 84 ± 22 % of predicted . The mean distance performed in 6MWT was 500 ± 63 mts. The mean peak VO2 found
in the cardiopulmonary exercise test was 24,8 ± 5,1 ml/kg (90 ± 3 % of predicted). The relative SGRQ was 8,5 ±
7,26. The CT scan findings were micronodules (4 patients, one with calcified nodules), air trapping (4 patients),
apical retraction and fibrosis (3 patients) ground glass infiltrates, calcified hilar and mediastinal lymphonodes,
paraseptal and centrolobolar emphysema (2 patients), bronchial thickening, septal and peribronchovascular
interstitium thickening (1 patient).Therefore in this case series adequately treated lung paracoccidioidomycosis,
despite the radiologic sequelae, did not cause late disability in patients. Only a minor obstructive pattern was
found in lung functional tests without influence in the 6MWT, cardiopulmonary exercise test and in the quality of
life evaluated by the SGRQ.
152
Poster Session
2
Diagnosis
2-01
Diagnosis of paracoccidioidomycosis in patients attended in routine services of a university
hospital
T.C. Moreto1, M.E.A. Marques2, M.L.S.C. Oliveira2, D.V. Moris1, L.R. Carvalho3, R.P. Mendes1.
1 Tropical Diseases Área, 2 Department of Pathology – Botucatu Medical School – São Paulo State University.3 Biostatistic Department
– Biosciences Institute – São Paulo State University. e-mail: tamoreto@yahoo.com.br (presenting Author)
Introduction: Identification of appropriate laboratory measurements for confirmation of a clinical impression
is important in a routine paracoccidioidomycosis patients’ medical care and constituted the objective of this study.
Methods: Clinical records and laboratory cards of 401 paracoccidioidomycosis patients with acute/subacute or
chronic form attended in the Tropical Diseases Area – Botucatu Medical School (São Paulo State, Brazil) during the
period 1974-2008 were reviewed. Their laboratory records from Department of Pathology and Tropical Diseases
Research Laboratory were also reviewed. Direct mycological examination, cell block preparation stained by GomoriGrocott, histopathological examination of tissues stained by hematoxylin-eosin and Gomori-Grocott, and specific
antibodies serum levels carried out by double agar gel immunodiffusion test using P. brasiliensis culture filtrate as
antigen were evaluated before treatment. McNemar, Tukey and chi-square tests were used in the statistical analysis;
significance was set up at p<0.05. Results: Males (88.0%) and chronic form (76.8%) predominated. Patients
distribution according to period of attendance presented no differences. Identification of typical P. brasiliensis yeast
forms in clinical specimens was observed in 86% of the patients [confirmed cases] while 14% of them showed
only a positive serological test [probable cases]. Direct mycological examination carried out in 51 different tissue
specimens showed 74.5% of sensitivity; 62.5% of sensitivity was observed in 112 sputum samples. Cell block
preparation carried out in 483 sputum samples showed 55.3% of sensitivity. Histopathological examination carried
out in 239 tissues from different organs revealed 96.7% of sensitivity. Serological tests carried out in 351 patients
and 200 healthy controls paired according to gender showed 90% of sensitivity, 100% of specificity, 100% of
positive predictive value, 85% of negative predictive value and 94% of accuracy. Comparisons 2x2 of laboratory
measurements carried out in the same patient showed that sensitivity decreases from histopathological examination
to serological tests to cell block preparation and direct mycological examination; the last two assays showed no
differences in sensitivity. Comments: This study revealed that P. brasiliensis can be identified in almost all the
cases, mainly by histopathological examination, and the value of serological tests in a routine Service, including
during long periods, with exchange of technicians, biologists and pathologists.
2-02
Serological diagnosis of paracoccidioidomycosis: Evaluation of negative serum samples
on immunodiffusion test from patients with confirmed disease
T.C. Moreto1, A.P. Vicentini Moreira2, A.N. Passos2, V.S. Kohara2, L.R. Carvalho3 and R.P.Mendes 1.
1 Tropical Diseases Area, Botucatu Medical School – São Paulo State University. 2 Adolpho Lutz Institute, São Paulo State Health
Secretariat. 3 Department of Biostatistics, Biosciences Institute – São Paulo State University.
e-mail: tamoreto@yahoo.com.br (presenting Author)
Serological tests for detection of specific serum antibodies have been used in the diagnosis of
paracoccidioidomycosis. Double agar gel immunodiffusion test (DID) is the most used assay due to its easy
execution, low cost, high specificity, and direct correlation with disease severity. However, about 10% of confirmed
paracoccidioidomycosis patients tested negative to DID test. The objective of this study was the evaluation of
these cases. Serum samples from 32 patients with confirmed paracoccidioidomycosis but negative to DID test
before treatment were evaluated. These assays were carried out at the Research Laboratory of Tropical Diseases
(RLTD) using a filtrate culture antigen prepared at the Laboratory of Clinical Mycology – Pharmaceutical Sciences
Institute – UNESP (DIDr). DID tests were also performed at the same laboratory using culture filtrate from strains
Pb-113 (DID1) and Pb-339 (DID2), prepared at Adolpho Lutz Institute (ALI)-São Paulo. As controls, positive sera
from other 32 confirmed patients, paired according to clinical form and age, were analysed. The negative sera on
DIDr test were also submitted to immunoblotting tests at ALI, using gp43 and gp70 kDa fractions. Results were
compared 2x2 using the McNemar’s test and significance was set up at p<0.05. Analysis of positive and negative
serum (64 samples) on DIDr test showed the following results: 1) sensitivity using three different antigens was the
same: DIDr=50.0%; DID1=50.0%; DID2=48.4% (p>0.05); 2) differences in sensitivity were not detected between
laboratories when Pb-113 (53.1% and 50.0%) and Pb-339 (48.4% and 50.0%) antigens were used in ALI and RLTD
(p>0.05); 3) immunoblotting sensitivity was higher than DIDr and DID1 tests, with gp43 or gp70 (respectively, 45.8%
versus 100%; 45.8% versus 96.6%; 47.5% versus 96.6%; 47.5% versus 96.6%; p<0.001);. Studies with positive
serum on DIDr showed that immunoblotting (84.4%) and DID2 (78.1%) reveals the same sensitivity when gp43 was
used (p>0.05), but a lower sensitivity (0.0% versus 78.1%) with gp70 (p=0.02). These findings suggest that our
results with DIDr test are limited by the sensitivity of the method itself and are not related to the antigen. In addition,
negative serum on DID tests should be evaluated by immunoblotting with gp43, due to its higher sensitivity.
155
2-03
Combined use of Paracoccidioides brasiliensis recombinant 27 and 40-kilodalton antigens in an
enzyme-linked immunosorbent assay for immunodiagnosis of paracoccidioidomycosis
Fernandes. V.C.1, Coitinho, J. B.1, Fonseca, J.H. 1, Veloso, J.M.R.2, Araújo, S.A.2, Pedroso, E.P.2, and Goes. A.M.1.
1 Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, MG - Brasil. 2 Faculdade de
Medicina, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG - Brasil. e-mail: cfernandes.viviane@gmail.com
Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it’s usually
diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a
variety of immunological methods, such as complement fixation and immunodifusion. Although these approaches
are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures
of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and
may yield good results. Accordingly, an indirect enzyme linked immunosorbent assay (ELISA) was design by
combination of two P. brasiliensis recombinant antigens, 27kDa and 40-kDa-molecular-mass for diagnosis and
follow-up PCM patients. The cDNA of 40-kDa protein was cloned in pET-21a vector and was expressed in a
prokaryotic system as a protein with 378 amino acids. The cDNA of pb27 was cloned in pET-DEST 42 vector and
was also expressed in a prokaryotic system. A total of 109 PCM sera, 62 sera from patients with other diseases
and 22 sera from healthy individuals were studied. Detection of anti-pb27 and anti-pb40 antibodies in sera of
patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 96% with a
specificity of 100% in relation to control non-infected human sera and 93,5% to sera from patients with diverse
infections. These results demonstrated an increase in sensitivity and specificity compared to results when the
antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer a lot off
advantages over the use of single antigens for diagnosis of PCM. Patients undergoing treatment for more than 1
year showed a reduced antibody response against pb27. These results suggest that the presence of anti-pb27
antibodies might be an indicator of active disease. Financial support: FAPEMIG and CNPq
2-04
Laboratory measurements for therapeutic monitoring of paracoccidioidomycosis patients
with different clinical forms
R. Cavalcante1, D.V. Moris1, L.R. Carvalho2, R.P. Mendes1.
1Tropical Diseases Area – Botucatu Medical School – São Paulo State University; 2Biostatistic Department – Biosciences Institute – São
Paulo State University. e-mail: mip.ricardo@gmail.com (presenting Author)
Introduction: Treatment control and length of paracoccidioidomycosis are an evolving field. As clinical cure is
early reached and latent fungal cells persist after successful therapy, other criteria of cure have to be evaluated.
Thus, the study of clinical and laboratory parameters in the follow up of paracoccidioidomycosis patients with different
clinical forms is our objective. Methods: Clinical records of 88 patients with confirmed paracoccidioidomycosis
attended in Tropical Diseases Area – Botucatu Medical School and their laboratory cards were reviewed.
Classification of clinical forms was performed according to Mendes (1994). Specific antibodies serum levels were
carried out by double agar gel immunodiffusion test using culture filtrate as antigen. Erythrocyte sedimentation rate
(ESR), gamma-globulin (GG), α1-acid glycoprotein (α1GP), C-reactive protein (CRP) and mucoprotein (MCP) were
evaluated according to its sensitivity and time, in months, for regression to normal values. Data were presented as
medians. Kruskal_Wallis, chi-square and McNemar tests were used in the statistical analysis; significance was set
up at p<0.05. Results: Thirty patients presented the acute /subacute form (G1), 33 the chronic moderate (G2) and
25 the chronic severe (G3). The youngest patients belonged to G1 (G1=24years old, G2=43, G3=45; G1<(G2=G3),
p<0.05). Time to reach clinical cure were not different: G1=9.5, G2=8.0, G3=8.0; p>0.05. Pre-treatment serology
showed higher levels in the acute/subacute form [G1=1/128, G2=1/16, G3=1/64; G2<(G1=G3); p<0.05]. Time for
serological regression to ½ and to positive in undiluted serum, and to reach negative values didn’t differ among
groups: G1=11, G2=22.5, G3=29, p>0.05; G1=18, G2=17, G3=47, p>0.05; G1=28, G2=32, G3=44, respectively;
p>0.05. Sensitivity of the other laboratory tests were: ESR=72.7%, MCP=62.7%, CRP=54.1%, α1GP=50.0%,
and GG=49.4%; ESR>MCP>CRP=GG=α1GP. ESR showed higher sensitivity [G1>(G2=G3); p<0.05] and higher
intensity (G1=54.5 mm/1st hour, G2=30, G3=25; p<0.05) in the acute/subacute form. Comments: Clinical cure and
ESR regression to normal values are reached early in the treatment. Negative serological tests are observed after
clinical cure and normal ESR. ESR should be the non specific measurement of choice in treatment monitoring.
Calculation of treatment length to patients with negative serological tests can be based on the clinical form and the
difference of time to reach negative serology and normal ESR.
156
2-05
Espirometric evaluation in patients with paracoccidioidomycosis (PCM)
C.S.O. Bredt 1; G.L.Bredt Jr1; P. A.D. Duarte 1; F. Pedott2; T. Pereira,2; L. de Campos2 ; F. Suzin 3
1Associated professor. Universidade Estadual do Oeste do Paraná, Cascavel-PR- Brazil. 2 Medicine Student’s Universidade Estadual do
Oeste do Paraná, Cascavel-PR- Brazil. 3. Fisioterapeuta. e-mail: csakuma21@yahoo.com.br
Background: PCM is an endemic disease that causes a mortality rate of 1, 45 cases per million inhabitants
Brazilians. The chronic form represents the vast majority of cases, reaching mostly adults, knowing that the lung
is one of the body most affected the identification of pulmonary sequel by spirometry, helps to characterize the
seriousness of the framework and guidance therapy.
Methods: 22 patients treated with itraconazole by recommendations of The Brazilian Consensus of
Paracoccidioidomycosis (edited by The Brazilian Infectious Diseases Society – 2006 ) were included in this study.
The patients were submitted to a spirometry evaluation and was utilized the portable equipment. The parameters
evaluated were forced vital capacity (FVC), forced expiratory volume in one minute (FEV1), and the relationship
FVC/FEV1. The data were interpreted in accordance with the recommendations of the Brazilian guidelines for
pulmonary function tests, 2000. The assessment was made after 15 minutes of the use of salbutamol 400 µg.
Results: 22 patients were evaluated, 17 (77.27%) were male. There was standard obstructive in 16 patients
(72.72%) and restrictive pattern in 2 (12.5%). Only 2 patients had no disorder. The answer occurred after the
use of bronchodilators was significant only in 6 patients (27.27%), and not significant in 16 patients (72.72%).
Conclusion: The study concluded that most patients have pulmonary sequel of PCM spirometry and that is very
important not only to quantify the damage but also to direct the doctors the best clinical management of these
patients. The bronchodilators may be useful to minimize the obstruction pulmonary and consequently improve the
quality of life of these patients but most patients do not beneficial with the single use of this medication and needs
an alternative or adjunctive therapy.
157
Poster Session
3
Epidemiology / Ecology
3-01
Determination of Paracoccidioides brasiliensis reservarea mapping the migratory story of
128 patients
T.C. Moreto1, Barrozo L.V.2, Bueno R.A.1, Moris D.V.1, Mendes R.P.1
Tropical Diseases Department, Botucatu Medical School – São Paulo State University, Botucatu, Brazil. 2 Department of Geography,
School of Philosophy, Literature and Human Sciences – University of São Paulo, São Paulo, Brazil. e-mail: tamoreto@yahoo.com.br
1
Introduction and Objectives: The long latency period observed between infection and clinical manifestations
of disease makes it difficult to determine the fungal reservarea, i.e., area where the patients were infected. The aim
of this paper was to study the migratory profile of 128 paracoccidioidomycosis (PCM) patients assisted at Botucatu
University Hospital, to determine the fungal reservarea.
Methods: Interviews were carried out with 128 patients about their place of birth, municipality of residence
at the disease onset, first, second and third municipalities in the rural area of the longest periods of residence
during their entire lives. Using geoprocessing techniques, several maps were made: place of birth, place of the
last residence, place of birth matching the last residence and, place of birth matching the longest and the last
municipality of residence.
Results: Out of 128 patients, 101 (78.9%) were born in 58 municipalities in São Paulo State. All 128 patients
lived in a municipality in this State at the time of disease onset, corresponding to 56 municipalities. Sixty-five
patients (50.8%) had the same place of birth and last residence, and 87 (68.0%) had spent more time at the same
place of their last residence. Finally, 62 (48.4%) patients were born, had the longest and the last residence in 31
municipalities in São Paulo State.
Discussion: The migratory profile of our patients suggested as reservarea a geographic arrangement
predominantly in the SW-NE direction, confirming previous studies based only on their municipality of residence
at the disease onset.
3-02
Space-time cluster of acute/subacute paracoccidioidomycosis bearing relationship with
the 1982-83 El Niño episode in a hyperendemic area in Brazil
L.V. Barrozo1, Mendes, R.P.2, Marques, S.A.2, Benard, G.3, Silva, M.E.S.1, Bagagli, E.4
1 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil. 2 Faculdade de Medicina de Botucatu, UNESP, São Paulo
- Brasil. 3 Faculdade de Medicina, USP, São Paulo – Brasil. 4 Instituto de Biociências, UNESP, São Paulo -Brasil. e-mail: lija@usp.br
Introduction and Objectives: The aim of this work was to analyze the space-time pattern of the series of
acute/subacute cases, in a highly endemic area in Botucatu neighboring, São Paulo State, Brazil, from 1966-99,
through the application of an inference test for the detection of clusters. The possible associations between the
cluster and climate factors were also investigated.
Methods: PCM data (n = 91) corresponded to cases seen in the Infectious Diseases and Dermatology Sectors
of the UH-UNESP. A spatial scan statistic was applied. Cases were assumed to be Poisson distributed, adjusted
for age and gender, with constant risk over space and time under the null hypothesis. The analysis was set to
include up to 50% of the total population at risk and up to three years of temporal window. Maps and exploratory
analyses were made between incidence and soil water storage.
Results: A significant excess risk was found during 1983-85 (P = 0.0077). There was a non-significant cluster in
1998. The distribution of the total precipitation along time shows an important peak above two standard deviations
in the years 1982/83, preceding the high-risk period. Maps depict the spatial pattern of the soil water storage from
1981-86 and were overlaid by cluster localization, suggesting association between excess soil water storage in
the preceding years and the cluster.
Discussion: Our study area is in the region of ENSO (El Niño-Southern Oscillation) influence and experiences
general increase in precipitation during El Niño years, especially during intense events. The space-time cluster
seems to be related to the most intense El Niño event occurred during the past 50 years, in 1982/83. The second
most intense in 1997/98, could be also related with the non-significant cluster of 1998. Our model for acute/
subacute PCM incidence (r2=0.49, P<0.0001) shows that peak in incidence in 1985 is successfully simulated and
is best fitted when the variable soil water storage is taken into account, supporting the connection with the local
response to the El Niño in 1982/83. We suggest an investigation of this association in other endemic areas of
ENSO influence. Supported by FAPESP and CNPq.
161
3-03
Spatial distribution of chronic paracoccidioidomycosis in a hyperendemic area in Brazil
L.V. Barrozo1, Gonzalez, C.R.1, Santana, M.S.1, Mendes, R.P.2, Marques, S.A.2, Bagagli, E.3
1 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil. 2 Faculdade de Medicina de Botucatu, UNESP, São Paulo
– Brasil. 3 Instituto de Biociências, UNESP, São Paulo – Brasil.
e-mail: lija@usp.br
Introduction and Objectives: The aim of this work is to analyze the spatial pattern of the series of chronic
cases admitted to the University Hospital of the São Paulo State University (UH-UNESP), in a highly endemic
area in Botucatu neighboring, São Paulo State, Brazil, from 1966 to 2006, through the application of an inference
test for the detection of clusters.
Methods: Human chronic PCM data corresponded to cases seen in the Infectious Diseases and Dermatology
Sectors of the UH-UNESP. Only the cases with residence in the study area (n = 382) were selected. A spatial
scan statistic was applied. Cases were assumed to be Poisson distributed, adjusted according to age and
gender, with constant risk over space under the null hypothesis. The analysis was set to include up to 50% of the
total population at risk and to find clusters of excess and low risks. Results were mapped using geoprocessing
techniques.
Results: A significant excess risk was found in the following municipalities: Botucatu, São Manuel, Pratania,
Pardinho, Areiópolis, Anhembi, Igaraçu do Tietê, Bofete and Itatinga (P = 0,0001). This cluster presented a mean
incidence of 2.7 annual cases/100,000 inhabitants. A secondary cluster of excess risk occurred in Cerqueira
César (P = 0,001), with mean incidence of 4.5 annual cases/100,000 inhabitants. Important low risk cluster was
found in Cabrália Paulista, Lucianópolis, Espírito Santo do Turvo, Piratininga, Agudos and Santa Cruz do Rio
Pardo (P = 0,0001) with mean incidence of 0.4 annual cases/100,000 inhabitants.
Discussion: This approach of looking chronic cases separately can be very interesting because acute/
subacute cases may be associated with temporal variability deriving from land use changes or climate anomalies,
which do not represent the mean conditions of the entire period. In Brazil, estimates based on both clinical forms
indicate an annual incidence rate of 1-3 per 100,000 inhabitants. Comparing the incidences for chronic cases
found here with that, Cerqueira César could be considered highly endemic while the low risk cluster is below the
annual incidence rate in Brazil. Results indicate that this study area shows two opposite situations, which allows
comparing their conditions. Supported by FAPESP, PRP/IC-USP and CNPq.
3-04
Liquid urease test in human and animal isolates of Paracoccidioides brasiliensis
S. A. G. Macoris1, Bosco, S. M. G. 2, Theodoro, R. C. 2, Richini-Pereira, V. 2 and Bagagli, E. 2
1 Instituto Adolfo Lutz, São Paulo - Brasil. 2 Depto. de Microbiologia e Imunologia, IBB- Unesp, Botucatu, São Paulo - Brasil.
e-mail: massis@ibb.unesp.br
P. brasiliensis is a thermo-dimorphic fungus and causes Paracoccidioidomycosys, the most prevalent
human systemic mycosis in South America. Some aspects of its natural and parasitic life cycle are still
unknown. One important aspect of this interaction with the human and animal hosts is the virulence, which
was evaluated by some experimental models. The metabolism of urea is associated with other fungal
pathogens, such as Cryptococcus neoformans and Coccidioides immitis, with degrees of infection, leading
to tissue damage or compromised functionality of the affected tissue.
To determine the role of urease activity of P. brasiliensis, if this enzymatic system may have some
importance for its pathogenicity, we have evaluated this activity in different isolates of P. brasiliensis
obtained from human and animals hosts, belonging to the S1, PS2, PS3 and Pb01-like, genetic groups. It
was employed the Cristenssen Urea Test and liquid urease test, described by Maslen, in order to evaluate
the urease activity of these isolates. It was observed that the liquid urease test showed to be better rather
than Cristenssen test concerning to time and confiability of the results to discriminate the organism that
hydrolyze the urea substrate. The liquid urease test was also able to discriminate some isolates which
presented different levels of urease production. The association with genetic groups, virulence and source
of the isolates are discussed.
162
Poster Session
4
Genomics,
Proteomics,
cellular Biology and
molecular Biology
4-01
Towards a molecular genetic system for the pathogenic fungus
Paracoccidioides brasiliensis
Almeida, A.J.1, Carmona, J.A.1, Cunha, C.1, Carvalho, A.1, Rappleye, C.A.2†, Goldman, W.E.2, Hooykaas, P.J.3, Leão, C.1,
Ludovico, P.1, Rodrigues, F.1
1. Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal. 2. Department
of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA. 3. Institute of Biology, Leiden University,
Clusius laboratorium, Leiden, The Netherlands.
e-mail (presenting author): ajalmeida@ecsaude.uminho.pt
We herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis,
specifically a more efficient transformation and a gene expression system. We evaluated several parameters
that influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions and
host cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensis
mixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78±9 transformants/cocultivation (5±1 transformants/106 target cells). P. brasiliensis GFP-expressing isolates were also constructed by
insertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopy
and flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differences
in fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random single
gene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use in
targeted mutagenesis and linking mutations to phenotypes.
Acknowledgments: Almeida, A.J. and Carvalho, A. and the majority of the work were financially supported by
a fellowship (SFRH/BD/8655/2002 and SFRH/BD/11837/2003) and a grant (POCTI/ESP/45327/2002) from FCT,
Portugal.
4-02
RNA interference: A potential tool to study gene function in Paracoccidioides brasiliensis
- strategies to construct silencing cassettes
L. Fernandes1, Paes H.C. 1, Teixeira M.M. 1, Rodrigues A.C.J. 1, Viana D.F. 1, Torres F.A1 and Felipe M.S.S. 1
Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília - UnB, Brasília, DF, Brazil.
email: larissaf@unb.br
1
Genetic manipulation is an important tool in general biology, evolution and the study of pathogenicity, as
well as to the identification of new therapeutic targets in fungal pathogens. Transformation systems have been
developed to a variety of fungi, including Paracoccidioides brasiliensis. Due to its high number of nuclei per cell,
slow growth and low efficient transformation ratios, classic experiments of gene knock-out seem unfeasible in this
fungus. The RNA interference mechanism appears a good strategy to circumvent these limitations. Through an in
silico search on P. brasiliensis genome database (Broad Institute) we identified all components of RNAi machinery
common to other fungi: Dicers (dcl1 and dcl2), RISC components ago1/qde2, ago2/sms2, the RNA-dependent
RNA polymerase, RdRP (qde1) and the DNA helicase (qde3). Sequence alignments revealed high degree of
conservation, indicating that the RNAi phenomenon should be present in this pathogen. Based on previous usage
of RNAi to study gene function in fungi, we decided to explore the construction of cassettes to silence gene
expression in P. brasiliensis. We cloned all cassettes on the pBluescript KS® between the cpc-1 promoter from
Neurospora crassa and the trpC terminator from Aspergillus nidulans, both of which have already been tested for
P. brasiliensis. Furthermore, the cassettes were amplified using specific primers, digested with BclII and cloned
into the pAD1625 vector, which is suited to Agrobacterium-mediated transformation and contains the dominant
selectable marker hph (Hygromycin B resistance). Goldoni (2005) demonstrated that the presence of an intron on
the loop increases the silencing efficiency in N. crassa by favouring hairpin formation in vivo after intron splicing.
We developed three different strategies to construct the silencing cassettes: (1) one contains a loop of 100 bp and
an intron of 163-bp; (2) another harbours just the loop and the last (3), just the intron. Another important feature of
these cassettes is that they constitute a chimerical vector, since they contain our gene of interest and also inverted
repeated fragments of ura3 gene to use 5- fluororotic acid counter-selection to select for recombinants. The use of
RNAi in P. brasiliensis will certainly contribute to a better understanding of this pathogen.
Financial support: CNPq.
165
4-03
Development of vector-based RNA interference (RNAI) for ade2 silencing on the pathogenic
fungus Paracoccidioides brasiliensis
Polez, V.P.P.1; Fernandes, L. 2; Torres, F.A.G 2 & Felipe, M.S.S. 2
Embrapa Recursos Genéticos e Biotecnologia, Brasília, Brazil. 2Instituto de Ciências Biológicas, Universidade de Brasília, UnB, Brasília,
DF, Brazil. e-mail: larissaf@unb.br
1
RNA interference (RNAi) has revealed a powerful tool for transcriptional gene silencing mediated by
sequence specific mRNA depletion. The multinucleated and multi-budded nature of the pathogenic fungus
Paracoccidioides brasiliensis have been pointed out as the reasons for the limited application of the gene
knock out. To implement the potential technique RNAi, we construct a vector carrying the inverted repeated
sequences of ade2 to silence this gene in P. brasiliensis, since the loss of function of ade2 gene, which
codifies a phosphoribosylamino-imidazole-carboxylase, promotes a red pigmentation of the colonies on limited
adenine medium, easily detected for visual screening. The silencing cassette for ade2 was amplified from the
pCR79 – originally used in Histoplasma capsulatum. It presents the CBP1 (calcium binding protein) promoter
from H. capsulatum and the ade2 inverted repeated from Aspergillus oryzae. The ade2 silencing cassette
was cloned into the pAD1625, a vector designed for Agrobacterium mediated transformation system, which
contains the Hygromicin B resistance marker, and was called pAD1627. The pAD1627 vector was transformed
into the EHA105 and LBA4404 Agrobacterium strains by electroporation. Further, those Agrobacterium
strains containing the silencing cassettes were used to genetic transform P. brasiliensis (Pb113) yeast cells.
On the BHI rich medium, we identified Hygromycin B resistant colonies which were also confirmed by PCR
amplification of the hph gene. The red pigmentation phenotype characteristic of the ade2 loss of function is
being investigated by replica-plating the Hygromycin B resistant colonies on DYP, which is a limited adenine
medium. The appearance of red colonies certainly will validate the RNA interference phenomenon in the multibudding yeast P. brasiliensis as a potential tool to the studies of gene function in this pathogen. Financial
support: CNPq, MCT, FUB.
4-04
Protocol for establishment of Paracoccidioides brasiliensis RNA recovery from human
pneumocytes cell culture
A. Oliveira1, Silva, S.S.1, Souza C.R.1 and Felipe M.S.1
1
Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazile-mail: HJaol@gmail.com (presenting Author).
Paracoccidioides brasiliensis is the etiological agent of the most common systemic mycosis in Latin America,
paracoccidioidomycosis. The yeast form of P. brasiliensis acts as a facultative intracellular pathogen, being
able to survive and replicate within macrophages and epithelial cells, such as pneumocytes, as a probable
mechanism to evade the immune system. Recently, our group used cDNA microarray technology to find out the
early transcriptional response of P. brasiliensis to the inner environment of peritoneal murine macrophages. This
approach revealed that in response to the harsh macrophage microenvironment P. brasiliensis up-regulates genes
related to the detoxification of oxidative radicals and amino acid biosynthesis. Also, genes encoding enzymes of the
glycolytic pathway was down-regulated, suggesting the ability of the fungus to survive in the hostile macrophage
microenvironment. To investigate the pathogen response to the non phagocytic cell we will analyze the interaction
between the fungus and A549 epithelial cells during infection by cDNA microarrays. In this context, the objective of
this study was to establish a protocol for recovery the P. brasiliensis RNA from human pneumocytes. P. brasiliensis
Pb01 (ATCC-MYA- 826) was co-cultivated with A549 cells in RPMI1640 medium during 6, 12, 24, 48, 72 and 96h
to determine the time in which more fungi could be recovered. Followed each time, the extracellular fungi were
removed by exhausting washing with PBS prewarmed at 37 oC. The percentage of fungi cells adhered and/or
internalized by pnemocytes was determined by CFU counting in BHI supplemented. The 48, 72 and 96h were
the most efficient times to recovery fungal cells. The A549 cells were lysed with a guanidine thiocyanate-based
solution and intact fungi were harvested by centrifugation to extract the fungal total RNA by the TRIzol® assay. We
have thus succeeded in recovering RNA from the fungus during pneumocyte infection, thus setting up the protocol
for future experiments.
Financial support: FAP-DF/CNPq and MCT/CNPq.
166
4-05
Evidence for positive selection in putative virulence factors within the Paracoccidioides
brasilensis species complex
Daniel R. Matute1, Lina M. Quesada-Ocampo2, Jason T. Rauscher3 and Juan G. McEwen4
Department of Ecology and Evolution, University of Chicago, 1101 East 57th Street, Chicago, Illinois 60637 2 Michigan State University.
Department of Plant Pathology 3 University of Puerto Rico, Rio Piedras. Department of Biology 4 Corporacion para Investigaciones
Biologicas (CIB), and University of Antioquia, Medellin, Ciolombia
1
Paracoccidioides brasilensis, a dimorphic fungus, is the causative agent of paracoccidiodomycosis, one of the
most importnat systemic mycosis in Latin America. Recently, the existence of three different genetically isolated
groups in P. Brasilensis was demonstrated, thus enabling comparative studies of molecular evolution among P.
Brasilensis lineages. Thirty two gene sequences coding for putative virulence factors were analyzed to determine
wheter or not they were under positive selection. Our maximum likelihood-based approach yielded evidence for
selection in 12 genes all involved in different cellular processes. This proportion is significantly higher than the
number of housekeeping genes under positive selection. An in-depth analysis of four of these genes showed
them to be either antigenic or involved in pathogenesis. In this paper, we present evidence inidicating that several
replacement mutations in the gp43 gene are under positive balancing selection. The other three genes (fks, cdc42
and p27) exhibit very little variation among the P. Brasilensis lineages and appear to be under positive directional
selection. Our results are consistent with the more general observations indicating that selective constrains are
variable across the genome, and that even in the genes under positive selection, only a few sites are altered.
We present our results within an evolutionary framework that may be applicable to studies on adaptation and
pathogenesis in P. Brasilensis and other pathogenic fungi.
4-06
Phylogenetic analysis of PRP8 intein and mycological features in Paracoccidioides
brasiliensis species complex
R. C. Theodoro1, Bagagli E.1 and Trinca L. A.2
1 Depto. de Microbiologia e Imunologia, IBB- Unesp, Botucatu, São Paulo - Brasil. 2 Depto. de Bioestatística, IBB-Unesp, Botucatu, São
Paulo - Brasil. e-mail: raquel@ibb.unesp.br
A recent species status investigation in Paracoccidioides brasiliensis described three cryptic species (S1,
PS2 and PS3). This study aimed to evaluate the potential of the PRP8 Intein, a parasitic genetic element, as
well as some mycological characteristics for the recognition of these species in P. brasiliensis. The PRP8 intein
sequence from a total of 22 P. brasiliensis isolates, 20 from the three genetic groups and 2 unidentified isolates,
were determined for phylogenetic analysis by Maximum-Parsimony, Maximum Likelihood and Bayesian Analysis.
Additionally, mycological analysis, such as conidia production (in Potato Dextrose Agar and Soil Extract Agar
media), mycelia-yeast transition and yeast morphometry were performed. All the isolates presented a full-length
intein, although the Endonuclease domain seems to be inactive due to substitutions in the second essential
aspartic acid residue. The phylogenetic analysis clearly separated the isolates from the three species and revealed
a significant difference between the isolate Pb01 and the remaining ones. The Pb01 isolate does not belong to any
of the three genetic groups, suggesting the occurrence of a new species. While most of the isolates from S1 group
produced high number of conidia (5-50/field), the isolates from PS2 and PS3 groups did not produce conidia.
Pb01 isolate produced few conidia per field (0-5), which were peculiar due to their longer shape when compared
to S1 group. Concerning the yeast morphology, the isolate Pb01 presented a large number of giant cells (>30µm
of diameter) and some isolates from PS2 group presented pseudo-hyphae at 36°C. The M-Y transition experiment
suggested that some elongated yeast isolates from PS2 take more time, in days, to start the transition. In conclusion
this study presented a reliable molecular marker for species recognition, including the new species Pb01-like and
pointed some potential morphological markers for species differentiation. If the groups are genetically separated,
they are supposed to accumulate some differences, reflecting in the occupation of new ecological niches or in
different strategies for survival in saprobic or host environment. Thus, the monitoring of the genotypes that are
causing PCM disease is extremely important for the comprehension of the different clinical aspects and treatment
responses.
Financial support: Fapesp (Grant Numbers: 06/03597-4 and 07/01306-5)
167
4-07
A high degree of polymorphism and recombination of the gp43 gene among phylogenetic
species of Paracoccidioides genus
Teixeira, M. M.1, and Felipe, M.S.S.1
1 Instituto de Biologia, UnB, Brasília - Brasil.
e-mail: marcus.teixeira@gmail.com
Background and aims: Paracoccidioidomycosis (PCM) diagnosis is mainly performed by using serological test
against the immunodominant antigen GP43, a molecule that is secreted under both in vitro and in vivo conditions.
Many studies demonstrate that the gp43 gene is highly variable among isolates and the genealogy of this locus
represents the real evolutionary scenario of the genus Paracoccidioides. Our aim is to investigate the polymorphism
and the possibility of recombination of this gene among the phylogenetic species S1, PS2, PS3 and the cryptic
isolated group “Pb01-like”. Methods: We amplified and sequenced fragments of gp43 gene (promoter+exon1
and exon2) from 17 isolates (“Pb01-like”) and compared to the 65 sequences previously deposited for the three
phylogenetic species of P. brasiliensis (S1, PS2 and PS3). The sequences were aligned using the ClustalW
algorithm and phylogenetic analyses were performed by parsimony and Bayesian methods for this locus. Also, we
have analysed and compared the deduced entire protein sequences of GP43 from isolates Pb18, Pb3, Pb eslava
and Pb01, each one representing the four phylogenetic groups. Recombination analysis was performed using the
split decomposition method that allowed the identification of recombination nets. Results: The DNA sequences
obtained from the four phylogenetic groups showed a high degree of polymorphism in the gp43 gene (π=0.03978).
In a total of 880 nucleotides analyzed 159 were polymorphic (18%). Phylogenetic analyses showed that genetically
isolated groups could be recognized by gp43 genealogy, suggesting that this molecular marker could be useful to
track phylogenetic species in the Paracoccidioides genus. The amino acid sequences of GP43 from isolates of the
four phylogenetic groups showed exclusive amino acid substitutions and indicates high genetic variability between
Pb01-like group and the other three phylogenetic species (S1, PS2 and PS3). Recombination analysis in the exon
2 of this gene revealed that recombination events occurs inter-species (“Pb01-like”- S1 - PS2), which is showed
by the nets connecting these phylogenetic species. Conclusions: Here we concluded that the gp43 gene may
have been a recombination hotspot during speciation in Paracoccidioides. This gene could be a potential marker
to distinguish between phylogenetic species in this genus. Financial support: CNPq, FAP-DF, FUB.
4-08
Virtual screening of Paracoccidioides brasiliensis genome – a new drug design approach
A.K.R. Abadio1, Carvalho M.J.A.1, Martins N.F. 2, and Felipe M.S.S.1
1 Instituto de Ciências Biológicas, Universidade de Brasília, Brasília – Brasil.
2 Embrapa Recursos Genéticos e Biotecnologia, Brasília – Brasil.
e-mail: anakarina.abadio@gmail.com
Introduction and aims: Paracoccidioides brasiliensis is the dimorphic fungus responsible for
paracoccidioidomycosis (PCM), one of the most important systemic mycosis in Latin America. Among the areas of
PCM incidence there are Brazil (80 % of the cases), Venezuela, Colombia and endemic regions that extend from
Mexico to Argentina. The mycosis current treatment with conventional drugs normally causes serious side effects,
such as nephrotoxicity. We focused in protein sequences of Aspergillus fumigatus (54 genes) and Candida
albicans (26 genes), previously described as essential genes, representing broad biological functions such as
cellular metabolism, cell wall organization and biogenesis, ergosterol biosynthesis, ribosome biogenesis, protein
modification. The objective of this work was to identify possible essential genes of P. brasiliensis and also in other
human pathogenic fungi as C. albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis
and A. fumigatus that are also absent in the human genome, by comparative genomic analysis.
Methods: These protein sequences were analysed through multiple sequence alignments against several
databases using a comparative approach. Next, a conservative domain screening was made by multiple alignments
using Clustal and the structural domains were identified using InterProtScan. Furthermore, phylogenetic studies
were performed.
Results and conclusions: The virtual screening allowed the selection of 8 candidate genes such as: NOB1
that encode an essential protein for processing the 20S pre-rRNA to the mature 18S rRNA; NOC3 that encode
a nuclear complex-associated protein required for ribosome maturation and transport. This approach strongly
suggests the potential use of those candidate genes to new antifungal drug development.
168
4-09
Formamidase of Paracoccidioides brasiliensis: Cytolocalization, purification and analysis
of the native protein
C.L Borges1, Barbosa, M.S..1, Parente, J.A.1, Feitosa, L.S.5, Santana, J.M.3, Báo, S.N4., Sousa, M.V. 4, Richart, C.A.O.4,
Soares, C.M.A. 1.
Instituto de Ciências Biológicas, UFG, Goiânia - Brasil. 2Instituto de Ciências Biológicas, UnB, Brasília – Brasil. 3Faculdade de Medicina,
UnB, Brasília – Brasil. 4Instituto de Biologia, UnB, Brasília – Brasil. 5Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP,
São Paulo, Brasil. e-mail: clayton@icb.ufg.br
1
Paracoccidioides brasiliensis is a thermally dimorphic fungus causing paracoccidioidomycosis, an endemic
disease widespread in Latin America. The dimorphic transition from the mycelial (22 ºC) to the yeast (37 ºC)
form is induced by a shift from the environmental temperature to that of the mammalian host. The formamidase
gene of P. brasiliensis was described as highly expressed in mycelia of P. brasiliensis, isolate Pb01. Formamide
aminohydrolase (formamidase, EC 3.5.1.49) catalyzes the highly specific hydrolysis of formamide to produce
ammonia and formate. In a previous work we identified the formamidase of P. brasiliensis, which reacts with
antibodies present in sera of P. brasiliensis infected patients. In this work we continue the characterization of
this highly expressed protein of P. brasiliensis. In this vein, the recombinant formamidase was used to produce
polyclonal antibody in mice, which showed high specificity in Western blot assays with total protein extract of P.
brasiliensis yeast cells. We sought to determine the cellular localization of the native protein in fungal yeast cells by
confocal and transmission electron microscopy. The P. brasiliensis formamidase was found in cytoplasm and cell
wall. Due to descriptions of the protein in other systems as a tetrameric molecule we also performed purification
of the native protein in two steps of column chromatography, DEAE sepharose and phenyl sepharose columns.
Fractions with formamidase activity were selected and analysed by PAGE, which revealed a protein with molecular
mass of 200 kDa. The purified protein was submitted to trypic digestion and confirmed by MALDI-TOF mass
spectrometry as a formamidase. Additionally, SDS-PAGE assay with boiled purified protein revealed a protein
with molecular mass of 50 kDa. Those results suggest that P. brasiliensis formamidase is a tetrameric protein.
The function of P. brasiliensis formamidase remains unclear. In order to elucidate the role of this molecule, the
cDNA encoding formamidase was used to screen a library constructed with P. brasiliensis yeast cDNAs using a
Saccharomyces cerevisiae two hybrid system. Proteins related with protein folding, processing and destination
were found, which can be related with the cell wall localization of formamidase of P. brasiliensis. A model for
formamidase interactions is provided. Supported by: FINEP/CNPq.
4-10
Two subunits of β1,3-glucan synthase complex: Gene expression (RHO1 and FKS1) and
functionality (RHO1) in Paracoccidioides brasiliensis
F. Sorais, Niño-Vega, G., and San-Blas, G.
Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A, Venezuela.
e-mail: fsorais@ivic.ve
A multigene family of six known RHO members (RHO1-RHO5, and CDC42) has been identified in yeasts.
Several functions have been ascribed to these GTPases, among which regulation of cell wall glucan synthesis,
organization of the actin cytoskeleton, regulation of secretion and control over polarity are highly relevant. Rho1 is
required for β-1,3-glucan synthase (Fks1) activity in yeast and Candida albicans. In Paracoccidioides brasiliensis,
little is known about this synthase complex, apart from the sequencing of its FKS1 gene (Pereira et al., 2000).
Therefore, we searched the gene coding for the second subunit of this enzymic complex, RHO1, and compare the
expression of both genes. Also, functionality of the P. brasiliensis RHO1 gene was demonstrated by complementing
a temperature-sensitive Saccharomyces cerevisiae rho1 null mutant with an expression vector containing the P.
brasiliensis RHO1 gene. P. brasiliensis RHO1 is 950 bp long, harbours four introns, and codes for a 191 amino
acid-long protein (accession number: AY392528). Gene expression comparison of both putative subunits of the β1,3-glucan synthase complex in P. brasiliensis, Fks1 and Rho1, were assessed by northern blot at intervals during
the mycelial (M) to yeast (Y) transition and in the extreme (M and Y) stages. Opposite patterns of expression
were seen in both genes, a result that may be linked to the fact that Rho1 plays roles in several events within the
cells, not only in controlling Fks1. To test whether P. brasiliensis RHO1 could complement a S. cerevisiae rho1
mutation, a temperature-sensitive mutant rho1-104 (S. cerevisiae strain HNY21) was transformed with the plasmid
construct pYES2-RHO1. As negative control, the mutant strain was transformed with pYES2. P. brasiliensis RHO1
successfully restored growth of S. cerevisiae rho1 mutant under restrictive temperature conditions.
Whether P. brasiliensis Rho1p regulates β-1,3-glucan biosynthesis, or any other function related to fungal
Rho1p orthologs, remains for further studies.
169
4-11
β1,3-glucan synthase: Recombinant protein, cytolocalization, activity and studies under
stress condition
P. K. Tomazett1, Félix, C. R2., Barbosa, M. S1., Santana, J. M3., Faria, F. P1., Báo, S. N4., Soares, C. M. A1., Pereira, M1.
1 Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia-GO-Brazil. 2 Laboratório
de Enzimologia, Instituto de Ciências Biológicas, Universidade de Brasília, Goiânia-GO-Brazil. 3 Faculdade de Medicina, Universidade de
Brasília, Goiânia-GO-Brazil. 4 Laboratório de Microscopia Eletrônica, Instituto de Ciências Biológicas, Universidade de Brasília, GoiâniaGO-Brazil. e-mail: patriciakt@hotmail.com
The cell wall of fungi is an essential and antigenic structure. The β-1,3-glucan polymer possibly gives
shape to the cell and its synthesis, which occurs at the plasma membrane, may be the result of the activity
of β-1,3-glucan synthase. In P. brasiliensis just one gene homologue of β-1,3-glucan synthase (PbFKS1) has
been characterized. Here, the catalytic subunit of glucan synthase (PbFKS1c) was used to obtain a recombinant
protein in Escherichia coli. The pGEX-4T-3 vector was used to produce the recombinant protein in fusion with
Glutathione S-transferase (GST) in its active form. The enzymatic assay of the recombinant and native glucan
synthase was performed using a radioisotopic method where the substrate UDP-glucose is radioactively labeled.
The purified recombinant PbFKS1c was used to generate specific rabbit polyclonal serum. This antibody was used
to perform an immunocitolocalization of glucan synthase in P. brasiliensis yeast cells. Glucan synthase activity is
currently assayed by the radioisotopic method. However, this method is expensive and requires special handling
and disposal methods for radioactive wastes. Based in a method described to soybean seeds galactinol synthase
we have adapted a new colorimetric method to determine the glucan synthase activity. The colorimetric method
has been standardized with the native glucan synthase. The reaction time, protein concentration and substrate
concentration have already been determined. The colorimetric assay is still being tested to the recombinant protein
PbFKS1c. The growth of P. brasiliensis was evaluated under stress conditions to determine the concentration in
which the fungus is sensible to the stressors agents calcofluor white, congo red, SDS, NaCl, KCl and sorbitol,
including cell wall damage and osmotic stress. By using the concentration in which the fungus is visually sensible
to those agents, the transcript level glucan synthase was evaluated by real time RT-PCR, and the enzymatic
activity was performed by using colorimetric method.
4-12
The α-amylase (amy1) gene in Paracoccidioides brasiliensis, identification and
semiquantitative expression
E. Camacho, Niño-Vega, G., and San-Blas, G.
Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,
Venezuela. e-mail: ecamacho@ivic.ve
In Paracoccidioides brasiliensis, an α-1,3-glucan is present as the outer layer of the cell wall in the yeastlike
phase, a polysacharide that is absent in the mycelial phase. α1,3-Glucan has been proposed as a virulence factor
not only in P. brasiliensis but also in Blastomyces dermatitidis and Histoplasma capsulatum. An α1,4-amylase is
essential for the synthesis of cell wall α1,3-glucan and expression of the virulence in H. capsulatum. With the
aim of exploring the possibility of similar functions in P. brasiliensis, we identified, isolated and characterized its
α-amylase (amy) gene.
P. brasiliensis IVIC Pb73 (ATCC 32071) was grown in PYG medium, at 23ºC (mycelial phase, M) or 37ºC
(yeastlike phase, Y). Genomic DNA was extracted from freeze-dried M cultures. Total RNA was extracted with
TRIzol from frozen Y cells. To amplify the gene, degenerate and gene-specific oligonucleotides were sequentially
used, the latter by means of a RACE reaction. Semiquantitative RT-PCR was applied for gene expression, using 3
µg total RNA as template, in 10-30 cycles, to determine the exponential phase in the expression of P. brasiliensis
amy1 and 18S genes.
A gene with high identity with H. capsulatum α-1,4-amylase, expressed preferentially in the pathogenic Y
phase, was identified. In silico amino acid analysis of the deduced protein led to the identification of all four
conserved regions of the αamylase family, the critical moieties for biological activity and amino acids associated
with the specificity to glucosidic α-1,4-linkages. Currently, the participation of such gene in the remodeling of the
cell wall architecture in P. brasiliensis, pathogenic Y phase is under study.
Acknowledgements: To FONACIT (Caracas, Venezuela) and ICGEB (Trieste, Italy) for partial financial support.
170
4-13
Mating type genes MAT1-1 and MAT1-2 and sexual reproduction in Paracoccidioides brasiliensis
Torres I1,5, García AM1,4, McEwen JG1,2,3, Gonzalez A4, Restrepo A1, Arango M1,2.
1. Unidad de Biología Celular e Inmunogenética-Corporación para Investigaciones Biológicas-CIB. Medellín, Colombia. 2. Departamento de
Microbiología y Parasitología-Universidad de Antioquia. Medellín, Colombia. 3. Departamento de Fisiología y Bioquímica -Universidad de Antioquia.
Medellín, Colombia. 4. Universidad Pontificia Bolivariana-Facultad de Medicina. Medellín, Colombia. 5. Estudiante de doctorado en Biología-Facultad
de Ciencias Exactas y Naturales- Universidad de Antioquia. Medellín, Colombia. 4. Escuela de Microbiología U de A. e-mail: isaurap10@gmail.com,
myrthaarango@yahoo.com.
Introduction: Paracoccidioides brasiliensis (Pb) taxonomic classification has not yet been defined.
Nevertheless, some morphological, molecular and phylogenetic studies have placed the fungus as a member of
the phylum Ascomycota even if its sexual cycle and mating type system remain unknown. The aim of this work was
to demonstrate the presence of both MAT genes in Pb and perform some crosses between potentially compatible
partners by plating in different culture media. Methods: 76 Pb isolates from a variety of sources (geographic,
clinical, environmental and phylogenetics species) were analyzed in an attempt to find mating type genes.
Specific primers were designed from Pb ESTs libraries and the MAT genes sequences derived from Histoplasma
capsulatum. Samples corresponding to the above isolates were assessed by PCR by means of these specific
primers and the mating genotype was assigned according to the amplicons’ size thus obtained. Some amplicons
were sequenced and analyzed by means of the BLAST and Expasy tools. Some isolates were selected according
to certain characteristics such as mating type, phylogenetic species, country of origin and were then crossed on
synthetic media, Results: The PCR assays were successful as the presence of both mating type genes was
made evident in all Pb isolates tested with an amplicon of 400pb corresponding to the MAT1-1 gen (heterothallic)
and fragments of 300pb and/or 1000pb corresponding to the MAT1-2 gen (heterothallic). Interestingly, there was
a group that showed both the MAT1-1 and the MAT1-2 amplicons (“homothallic”). Homology analysis from Pb
sequences performed with BLAST tools, showed a high grade of identity and importat e-values when compared
with MAT genes from others Ascomycetes. However, the same analysis in “homothallic” isolates showed that one of
these genes was a truncated copy, not being therefore true homothallism. Protein analysis (Expasy) revealed that
Pb MAT1-1 and MAT1-2 had the characteristic alpha and HMG boxes, respectively. When the crosses performed
on synthetic media were analyzed some structures resembling fruiting bodies were observed, but their content did
not correspond to the expected reproductive structures; there were neither asci nor ascospores. Conclusions:
This study allowed to develop a PCR assay that used specific primers and permited identification of MAT genes in
Pb isolates. In the future, this finding may allow to improve the taxonomic classification of this fungus The presence
of both genes suggest that sexual reproduction may occur in Pb and that, at the moment, additional crosses with
different partners, with emphasis in crossing the phylogenetic species, are under way, and may point towards a
more precise definition of the concept of biological species in the Pb population.
4-14
Molecular evidences of sexual reproduction in P. brasiliensis
Teixeira, M. M., Paes, H. C, Fernandes, L., Silva, S. S. and Felipe, M.S.S.
Instituto de Biologia, UnB, Brasilia - Brasil.e-mail: marcus.teixeira@gmail.com
Background and aims: The thermodimorphic fungus P. brasiliensis is the aetiological agent of
Paracoccidioidomycosis (PCM). The teleomorph form of this fungus has never been observed either in vitro or in
vivo. Recently, through phylogenetic analysis by concordance and non-discordance methods and by recombination
analysis, it was suggested that P. brasiliensis has a sexual stage in its life cycle (Matute et al., 2006). Here we
show that this pathogen has sex-suggestive features and contains homologues to all genes that comprise the
mating machinery in other fungal species. Methods: Based on the transcriptome of the isolate Pb01 and the
structural genome of the isolate Pb3 we designed two pairs of primers to amplify the transcriptional factors α-box
and HMG that correlate to both idiomorphs (MAT1.1 and MAT 1.2 respectively). We amplified the cognate regions
from 29 isolates that represent the three phylogenetic species of P. brasiliensis (S1, PS2 and PS3) and isolates
from “Pb01-like”. We also performed in silico analysis of the structural genome of Pb3 to search for potential
genes involved in mating as described in Histoplasma capsulatum, Aspergillus fumigatus and Saccharomyces
cerevisiae. We performed a direct blastX of these genes against the structural genome of P. brasiliensis isolate
Pb3. Results: For the 29 isolates we were able to amplify three populations: i) 15 isolates that present only the
α-box domain (MAT1.1); ii) 8 isolates that present only the HMG (MAT1.2); and iii) 6 isolates that present both
the α-box and HMG (MAT1.1 and MAT1.2). The in silico analysis showed that P. brasiliensis isolate Pb3 contains
the core genes involved in mating with high similarity to H. capsulatum, such as pheromone-processing enzymes
(ste14, ste24, ste6, ste13, ram1, ram2, kex1 and kex2), components of a pheromone-response pathway (GBA1,
ste4, ste18, ste20, ste11, ste7, fus3) and pheromone receptors (ste2 and ste3). Conclusion: These results
suggest that P. brasiliensis may undergo mating and then meiosis during its life cycle. This work opens new
avenues of investigation into the possibility of in vivo or in vitro mating in this pathogen.
171
4-15
α 1,3-glucanase is important in the remodelling of Paracoccidioides brasiliensis cell wall
H. Villalobos, Niño-Vega, G., and San-Blas, G.
Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,
Venezuela. e-mail: hvillalo@ivic.ve
Although not a frequent component of the fungal cell wall, α-1,3-glucan is present in Schizosaccharomyces
pombe, some Aspergilli and the yeastlike forms (not the mycelial ones) of Histoplasma capsulatum, Blastomyces
dermatitidis, and Paracoccidioides brasiliensis, among a few other species, where it plays decisive roles in
dimorphism and virulence. To degrade this polysaccharide and allow separation of the daughter cells, α-1,3glucanases are required. A strict control on such activity is important in order to prevent any possibility of autolysis.
Therefore, the study of this enzyme, its encoding gene and regulation are important for the understanding of
morphogenetic processes in these fungi.
This work aims to decode the full sequence of the agn1 gene in P. brasiliensis, verify the presence of introns,
analyze the deduced amino acid sequence, and quantify its expression by RT-PCR. In view of the observed boost
in cell wall α1,3-glucan when P. brasiliensis is grown in medium supplemented with horse serum (HS), agn1
expression is also measured in genomic material extracted under such experimental conditions.
The agn1 5’ region was obtained by means of oligonucleotides designed on a partial sequence from a HindIII
library (SMART™ RACE cDNA Amplification Kit). New oligonucleotides designed on agn1 end regions allowed the
amplification of the genomic and codifying sequences, and detection of introns.
The genomic sequence is 1494 bp long, interrupted by two introns, for an ORF 1370 bp long. Its expression is
currently under study. The deduced protein sequence is composed of 456 amino acids, with high similarity to other
fungal glucanases. The presence of a signal peptide and putative sequences for post-translational modifications
are detected in the deduced protein.
Acknowledgements: To FONACIT (Caracas, Venezuela) and ICGEB (Trieste, Italy) for partial financial
support.
4-16
Characterization of Paracoccidioides brasiliensis Chs3 by overexpression in Saccharomyces
cerevisiae
L. Barreto, Sorais, F., Niño-Vega, G., and San-Blas, G.
Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,
Venezuela. e-mail: lbarreto@ivic.ve
Cytoplasmic membrane-associated chitin synthases are responsible for the biosynthesis of cell wall chitin in
fungi. Six chitin synthases (Chs1-Chs6) have been detected in Paracoccidioides brasiliensis. Northern analyses
show that, contrary to other CHS genes, CHS3 is only expressed in the pathogenic yeast phase and at the
end of the mycelium-to-yeast transition in P. brasiliensis, suggesting a possible involvement in morphology and
virulence. Heterologous expression of the CHS3 cDNA under the regulation of the inducible GAL1 promoter in
Saccharomyces cerevisiae has been successfully performed, to be followed by determination of Chs3 enzymatic
activity.
The CHS3 gene (accession number: AF107623) contains a 3817 bp-long open reading frame, interrupted by
two introns (79 bp and 86 bp), and encodes for a protein 1220 amino acid-long, highly homologous to Class IV
chitin synthases. The first cDNA strand was synthesized by RT-PCR, using gene-specific primers designed on the
basis of the genomic sequence; it included an engineered NotI restriction site around the start codon and a second
engineered XbaI restriction site around the stop codon.
The CHS3 cDNA was cloned into NotI / XbaI sites of the yeast expression vector pYES2, and the plasmid
construct was used to transform a S. cerevisiae chs3 null mutant (ATCC 4003160), using the EasyCompTM
Transformation Kit (Invitrogen).
Transformants were grown in galactose-raffinose medium, where expression of the cloned cDNA was induced
under the GAL1 promoter. As controls, yeast cells transformed with the empty vector were used. Determination of
chitin synthase activity, according to Orlean (1987), is currently under progress in our laboratory.
172
4-17
Autophagy in the human pathogenic fungus Paracoccidioides brasiliensis during
dimorphism
Sarah M. Pedroso, Cláudia L. B. Campos, Francisco G. Nóbrega, Flavia V. Morais.
Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, Brazil.
e-mail: cbcampos@univap.br
The degradation of macromolecules and organelles in eukaryotic cells occurs by a highly conserved
process, named macro-autophagy (autophagy), through the action of the lysosomal/vacuole compartments.
This process is crucial for survival of several microorganisms, mainly in stress conditions, being involved in
many biological events such as development, differentiation and adaptation.
The human pathogenic fungus Paracoccidioides brasiliensis is the etiological agent of a deep mycosis,
paracoccidioidomycosis, which has to undergo mycelium to yeast dimorphism inside the host prior to the
establishment of the disease. There are many genes involved in autophagy in fungi, as previously reported
in Aspergillus species and Saccharomyces cerevisiae. In Paracoccidioides brasiliensis, some of them were
identified in studies of gene expression during mycelium to yeast transition, suggesting that autophagy may be
functional and may contribute to the differentiation of the fungus. In this work, we investigated the involvement
of autophagy in dimorphism of Paracoccidioides brasiliensis induced by temperature rise from 25oC to 36oC.
We show that autophagy is mostly absent in mycelia.
However, the amount of autophagic vacuoles, visualized by mono-dansylcadaverine labeling, progressively
increases during mycelium to yeast transition, but is low by the end of this process. Both in mycelia and
yeast cells at stationary phase, the level of autophagic vacuoles are much higher than in the actively growing
fungi, which suggest a role for autophagy during nutrient starvation. We also investigated the effect of two
autophagy inhibitors, N-ethylmaleimide (NEM) and 3-methyladenine (3-MA), on mycelium to yeast dimorphism.
NEM potently induced death during dimorphism, while 3-MA reduced autophagy and inhibited P. brasiliensis
dimorphism. Our results suggest a potential role for autophagy during P. brasiliensis dimorphism and nutrient
starvation. Key words: Paracoccidioides brasiliensis, autophagy, dimorphism.
4-18
Niche specific regulation of Paracoccidioides brasiliensis genes in the infectious
process
A.M. Bailão, Borges C.L., Chagas R.F., Salem-Izaac S.M., Pereira M. and Soares C.M.A.
Laboratório de Biologia Molecular, UFG, Goiania - Brasil.
e-mail: alexandre.bailao@gmail.com
The fungal pathogen Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis
(PCM). The disease may present a large spectrum of clinical manifestations with the involvement of different
organs and tissues. We have been studying genes that could be relevant to the fungal parasitic style and to the
infectious process. Our hypothesis is that during the parasitic phase it would occur genes whose expression
will perform a control role in the well-succeeded parasitic life style. Additionally, the fungus invades different
niches in the host, a factor that may induce expression of different set of genes.
Our results, obtained by transcriptome analyses and by confirmation experiments strongly suggest that
P. brasiliensis presents a metabolic program in which: (i) the nitrogen metabolism, the glicolytic pathway and
the lipid synthesis predominate during liver infection; (ii) a change to a lypolitic style and increased nitrogen
metabolism , as well as, cell wall remodeling are predominant during fungal dissemination through the
bloodstream; (iii) the transport of copper and iron are exacerbated during fungal infection in liver and during
dissemination through bloodstream. Due to the relevance to the fungal pathogenesis of understanding the
gene expression in host niches, and also considering the potential of this knowledge for improving the disease
therapy, studies had been conducted adopting genomic and proteomic strategies in order to extend the initial
studies and to evaluate the fungal global adaptation to host different micro environments. A model for fungal
adaptation to the host niches is presented.
Supported by: CNPq e FINEP
173
4-19
Differential gene expression in virulent and avirulent isolates of Paracoccidioides
brasiliensis
C. Arruda1, Beatriz C. A. Alves1,2, Vera L. G. Calich1 and C. A. Moreira-Filho1, 2
Department of Immunology, Biomedical Sciences Institute, University of São Paulo, São Paulo, SP, Brazil; 2 Albert Einstein Research and
Education Institute, Hospital Israelita Albert Einstein, São Paulo, SP, Brazil.
e-mail: celinaarruda@ol.com.br (presenting Author)
1
P. brasiliensis is a dimorphic fungus which causes paracoccidioidomycosis (PMC), a systemic granulomatous
disease. Different clinical manifestations are due to host related factors or to intrinsic characteristics of the fungus,
like virulence. Some virulence factors especially that involved in dimorphism have been proposed. This work
was focused on the identification of differentially expressed sequences between yeast forms of virulent and less
virulent Paracoccidioides brasiliensis (Pb) isolates by means of mRNA Differential Display.
A genetic murine model of paracoccidioidomycosis (PCM) characterized B10.A and A/J mice as susceptible
and resistant strains to Paracoccidioides brasiliensis (Pb) infection, respectively. The disease developed by these
mice strains mimics the severe and benign forms of human PCM. In the present work, two isolates of P. brasiliensis
Pb 18 (Pb 18V and Pb 18AV) were used to infect intratracheally (i.t.) B10, susceptible mice. The inoculation of
Pb 18V, a highly pathogenic sample, promotes a severe and disseminated infection in susceptible mice. On
the other hand, Pb18AV, obtained after several years of in vitro subculture of Pb 18, has low pathogenicity and
causes a restrained pulmonary infection in the same mice. Total RNA were obtained from lungs at weeks 4 and
8 post-infection and from in vitro cultures. The cDNA were synthesized using oligo-deoxythymidine-anchored
primers, and amplified by PCR with oligo dT plus random primers. The products were submitted to polyacrylamide
gel electrophoresis analysis. One band of approximately 250 bp expressed only by Pb 18V inoculated mice was
obtained with the random primer OPA 05. This fragment was cloned and sequenced. Its nucleotide sequence
showed no similarity with any fungus sequence described so far. RT-PCR experiments are being conducted in
order to quantify this differential gene expression.
This work was supported by research grants from FAPESP and CNPq.
4-20
Gp43 isoforms of Paracoccidioides brasiliensis as ligands of laminin, fibronectin and
collagen type I
Silva, J.F1 ; Benard, G2.; Mendes-Giannini, M.J.S1.
Faculdade de Ciências Farmacêuticas de Araraquara – UNESP – Brasil and
e-mail: silvajf@fcfar.unesp.
1
2
Faculdade de Medicina de São Paulo – USP – Brasil.
The capacity of the Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis (PCM), to
provoke human illness, and to cause mycosis with great variety of clinical manifestations, from localized forms
until spread disease which may cause death, depends probably on factors such as: fungal virulence, its ability in
interacting with the host superficial structures, invading them and on the host immunological response. Studies
of the correlation among some of the components (gp43) and their role in the pathogenicity are subjects of great
importance in paracoccidiodomycosis. It has also been shown that different fungal strains produce gp43 with at
least four isoform profiles with pIs ranging from 5.8 to 8.5. The 43 kDa glycoprotein, ligand of laminin, and the
peptide 1 (NLGRDAKRHL), corresponding to residues 76-85, competed with gp 43 and significantly inhibited
the adhesion of P. brasiliensis to the Vero cells by ~ 60 %. Aiming to understand the mechanisms that regulate
the interaction of this fungus with host epithelial cells, this study investigated the protein expression profile of
four isolates of P. brasiliensis and the capability of these proteins, in special gp43 isoforms, binding to different
compounds of extracellular matrix proteins (ECM). Depending on the isolate, proteins were expressed differentially.
The gp43 isoforms of pIs 6.0 and 6.3 (presented in Pb339 and Pb265 isolates) and pI 5.7 (presented only in Pb339
isolate) binding to laminin, while the gp43 isoforms of pI 6.7, 6.6 and 5.5 (presented only Pb339 isolate) binding to
fibronectin and collagen type I, confirming previous data and increasing our knowledge about the different isoforms
of the gp43 and their behaviour as adhesin and as ligand to ECM compounds. Then, the several gp43 isoforms
can binding to different ECM compounds, depending on the isolate and maybe this fact is very important from
better understanding of their role in the pathogenicity of P. brasiliensis.
Suppoted by: FAPESP; PADC-FCF; CNPq and CAPES
174
4-21
Transcription regulation of the PbGP43 gene with nitrogen in the human pathogen
Paracoccidioides brasiliensis
A. A. Rocha, and Puccia R.
Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, São Paulo, Brasil. e-mail: rpuccia@unifesp.br
The present work shows the involvement of NIT2-like binding motifs in transcription modulation of the
PbGP43 gene, which encodes an important antigen from the human pathogen Paracoccidioides brasiliensis.
This investigation has been motivated by the finding of a high number of NIT2-like motifs within the first 2,047
nucleotides of the PbGP43 5’ intergenic region from Pb339 isolate, recently cloned and sequenced by our group.
This fragment contains 23 NIT2-like sites, which form 4 putative clusters, two of them identical. Our analysis was
based on electrophoretic mobility shift assay (EMSA) with selected probes and real time reverse transcription
(RT)-PCR of PbGP43 from P. brasiliensis cultures exposed to, or depleted of, ammonium sulfate and glutamine.
The results suggested that at least some NIT2-like motifs are functional and that PbGP43 is modulated by nitrogen
primary sources. We have mapped 4 oligonucleotides containing TATC motifs that formed DNA-protein complexes
possibly involving a NIT2-like factor. This suggestion is based on the following observations: i) they formed more
intense EMSA bands with extracts from ammonium sulfate-depleted cells; ii) a point mutation in the TATC core
(to GATC) decreased shifted band intensity; iii) all the complexes migrated similarly. Both ammonium sulfate and
glutamine provoked rapid decrease in PbGP43 mRNA accumulation, but this effect could be reversed upon salt
depletion. This kind of modulation was observed not only in Pb339, but also in Pb3 and Pb18. The oligonucleotides
that prime amplification of a 2,047-bp PbGP43 intergenic fragment using Pb339 DNA elongated a fragment of
similar size with template from Pb18 and six other isolates, while for Pb2, Pb3, Pb4 and Pb5 the amplicon was
~1,500 bp and for Pb9 and Pb17 it was ~3,000 bp. The Pb18 genome sequence of this amplicon is 98% identical
to that of Pb339. In Pb3, the number of NIT2-like motifs and clusters is lower. This is the first report about PbGP43
transcription modulation with primary nitrogen sources. That will help understand the antigen biological function
and its modulation during infection.
Financial support: Fapesp and CNPq
4-22
Identification of Paracoccidioides brasiliensis adhesins through representational difference
analysis
S.V. Nogueira1; Bailão, A.M. 1; Borges; C.L.1; Pereira, M. 1; Mendes-Giannini, M.J. 2; Winters, M. 3;Soares, C.M.A.
1
1 Laboratório de Biologia Molecular,UFG,Goiás-Brasil. 2 Faculdade de Ciências Farmacêuticas, UNESP-Brazil. 3 Division of Infectious
Diseases, UC, College of Medicine, Ohio - USA. e-mail: alexandre.bailao@gmail.com
Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), the most prevalent
systemic mycosis in Latin America. As a dimorphic pathogenic fungus it grows as saprobic mycelium, resulting
in the formation of propagules, which initiates the infection in humans when inhaled into the respiratory tract.
Subsequently, in the lung, the mycelia propagules develop into yeast cells. Adhesion of microorganisms to host
cells and tissues represents a critical step in the process of infection. The propagules that lodge in the alveoli
adhere and invade the alveolar cells and/or the basal lamina. Alveolar basal lamina is composed of a specialized
extracellular matrix (ECM), in which laminin, types IV and V collagen, entactin, and fibronectin can be found. In
normal tissues, most ECMs are covered by epithelial or endothelial cells and hence are not available for binding.
However, any type of trauma that damages host tissues may expose the ECM and enable microbial colonization
and infection. The aims of the present study are to identify and select genes induced in P. brasiliensis adhered
to collagen, to promote heterologous expression of recombinant proteins and to characterize the predictable
adhesins. Representational difference analysis (RDA) was applied to two cDNA populations of P. brasiliensis: from
control yeast cells and from yeast cells in vitro adhered to collagen type I. Our analysis identified 47 transcripts
differentially expressed. Among them we found enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11), an
enzyme that catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP) in the
catabolic direction in the second half of the glycolytic pathway. Despite the absence of a signal sequence and
typical motifs required for membrane anchoring, the attachment of enolase to cell surfaces of some microorganisms
is well established. The cDNA was cloned into the pGEX-4T-3 vector and expressed in Escherichia coli pLYS cells
as a glutathione S-transferase fusion protein. The purified recombinant enolase was used to generate specific
mouse polyclonal and both were used in affinity ligand and binding assays to characterize enolase as an adhesin.
Also, biochemical characterization of the P. brasiliensis enolase was performed.
Supported by: Capes, CNPq, FINEP
175
4-23
Adhesion profiles and extracellular matrix ligands of Paracoccidioides brasiliensis isolates
obtained from armadillos (Dasypus novemcinctus)
Silva, R.P. 1, Bagagli, E. 2; Mendes-Giannini,M.J.S. 1
Faculdade de Ciências Farmacêuticas de Araraquara-UNESP-Brasil and 2Instituto de Biociências de Botucatu-UNESP-Brasil.
e-mail: rbtps@hotmail.com
1
Paracoccidioidomycosis (PCM) is human systemic mycoses caused by the thermodimorphic fungus P.
brasiliensis, with a wide distribution in Latin America, mainly in Brazil. The clinical manifestations include
cutaneous and systemic forms, and can attack various tissues, especially the lung. Recently, P. brasiliensis
strains with typical morphology have been isolated from Dasypus novemcinctus, confirming as the primary
natural reservoir of this fungus. Its geographic distribution is similar to that of human PCM. The isolates from
armadillos were virulent in the animal model, with dissemination to many organs. The isolates were classified
into virulence categories according to number of cfu per gram of tissue. The ability of the pathogen to interact
with the host superficial structures is essential to its virulence. P. brasiliensis expresses proteins that interact
in various ways with the extracellular environment and generally involves ligands produced by the pathogen
and is capable to adhere to extracellular matrix proteins (ECM). This approach has not been studied with the
armadillo’s isolates. Then, we studied the capacity of different P. brasiliensis armadillos isolates to adhere
to pulmonary epithelial cells (A549), as well as the protein profile of these isolates and ECM ligands. Our
data confirmed previous studies that more virulent isolates have greater adhesion capacity. The comparative
analysis of the “cell-free” isolates extracts showed that T10B1 (more virulent) presented remarkable difference.
The isolates also showed distinct patterns for the ECM ligands. T10B1 presented an elevated number of ligands
to fibronectin, I and IV collagen and similar to laminin. Therefore, one of the armadillo isolate has additional
ligands to three ECM proteins. This pattern may be related to microniche of the fungus in the host as well as,
the difference that may occur in the first contact with the human tissues.
FAPESP, PADC-FCF and CNPq
4-24
Use of in vivo-induced antigen technology in the identification of Paracoccidioides
brasiliensis proteins potentially expressed during infection
T.C.V. Rezende1, Dantas. S.F.I.M.1, Bailão. A.M.1, Castro. N.S.1, Tomazett. M.V.1, Deepe Jr. G.S.2, Pereira. M. 1 and
Soares. C.M.A.1*.
1 Laboratório de Biologia Molecular, UFG, Goiânia - Brazil. 2 Division of Infectious Diseases, UC, College of Medicine, Ohio - USA.
e-mail: celia@icb.ufg.br
In vivo induced antigen technology (IVIAT) is a technique that has been used in organisms for identification
of immunogenic proteins that may play a role in virulence or pathogenesis. The objective of this work was to
screen immunogenic proteins of Paracoccidioides brasiliensis potentially expressed during human infection.
Sera were collected from patients with paracoccidioidomycosis. Equal volumes of sera were pooled and
reacted with whole yeast cells and yeast cell lysates of the fungal isolate Pb01. We constructed a cDNA
expression library with RNAs of P. brasiliensis yeast cells recovered from livers of infected mice. Sera
adsorbed with P. brasiliensis yeast cells were used to screen the library. We identified 35 clones which
cDNAs encoded predicted proteins related to cell metabolism, transport, energy, transcription, protein fate,
signal transduction, biogenesis of cellular components. Of all positive clones identified by IVIAT we selected
two encoding the sera reactive aromatic - L- amino acid decarboxylase (DDC) and lumazine synthase (LS)
of P. brasiliensis for further studies. We characterized the complete cDNA of Pbddc and Pbls that were
overexpressed in an Escherichia coli host to produce high leves of recombinant fusion protein with GST. The
recombinant proteins DDC and LS were recognized by sera of patients with confirmed paracoccidioiomycosis
and not by sera of healthy individuals. Real time RT-PCR was used to analyze the expresion of the Pbddc
and Pbls in the two forms of P. brasiliensis and in yeast cells infecting macrophages. The accumulation of
both transcripts was higher in the yeast form and in yeast cells infecting murine macrophages. The results
suggest that PbDDC and PbLS can be considered immunogenic proteins up-regulated during the infective
process.
176
4-25
Changes in the transcription levels of Paracoccidioides brasiliensis CHS5 and CHS4 genes,
mycelial phase, respond to external osmolarity
G. Niño-Vega, Sorais, F., and San-Blas, G.
Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,
Venezuela. e-mail: fsorais@ivic.ve
Six chitin synthase (CHS) genes have been identified in the genome of Paracoccidioides brasiliensis.
From the deduced amino acid sequences of their encoded products, they belong in six out of seven (I-VII)
proposed Chs classes: PbrCHS1 (I); PbrCHS2 (II); PbrCHS3 (IV); PbrCHS4 (VII); PbrCHS5 (V) and PbrCHS6
(VI). Herein we report the arrangement of PbrCHS5 in a head-to-head configuration with PbrCHS4, both
genes sharing a common 5’UTR. Also, changes in transcription levels for both genes, following alteration of
external osmolarity, are studied.
The 5’UTR sequence comprised 3.8 kb between their respective translational start codons. In silico
analysis of the shared promoter region also yielded sequences that matched three potential sites for Rlm1p.
This fact made us wonder whether the transcription of PbrCHS4 and PbrCHS5 might be stimulated by hypoosmotic stress, as reported for their respective orthologous A. nidulans genes, csmB and csmA. To evaluate
changes in transcription levels in response to alterations of the external osmolarity, the fungus was grown
in its mycelial form in 0.3 M KCl-supplemented YPD medium. Lower transcription levels were found for both
PbrCHS4 and PbrCHS5 when the fungus was grown under hyper-osmotic conditions. Although a common
regulatory control for both genes, mediated by a Rlm1p-like transcription factor acting in a shared 5’UTR,
might be at work during osmotic stress in P. brasiliensis, the chromosomal head-to-head arrangement of
both genes does not necessarily imply a similar response to every external challenge. In fact, while both
have high expression in the mycelial form of the fungus, PbrCHS5 but not PbrCHS4 was expressed in the
yeast form, as previously reported (Niño-Vega et al., 2004).
Research into gene regulation of P. brasiliensis could help us to understand the relationship among
genes involved in cell wall synthesis, and their role in cell wall maintenance.
4-26
A Paracoccidioides brasiliensis aconitase is induced by ethanol and acetate and repressed
by glucose as carbon sources
W.A. Brito.1,2,3, Castro. N.S.3, Pereira. M.3 and Soares. C.M.A.3
1 Curso de Farmácia, UnUCET, UEG, Anápolis – GO – Brasil. 2 Curso de Farmácia, UniEVANGÉLICA, Anápolis – GO – Brasil. 3
Laboratório de Biologia Molecular, ICBII, UFG, Goiânia – GO – Brasil. e-mail: wesleyfarmacia@uol.com.br
Paracoccidioides brasiliensis is a thermal-dimorphic fungus, the causative agent of Paracoccidioidomycosis
(PCM), an important endemic mycosis in Latin America. In this work a protein species preferentially expressed
in yeast cells with a molecular mass of 82kDa and isoeletric point (pI) of 6.1 was isolated from the proteome
of P. brasiliensis and characterized as an aconitase (E.C. 4.2.1.3). Aconitase is an enzyme that catalyzes the
isomerization of citrate to isocitrate in both the tricarboxylic acid (TCA) cycle and the glyoxalate cycle (GC).
We report the cloning and characterization of the cDNA encoding the aconitase of P. brasiliensis (PbACO1).
The cDNA showed a 2400 bp open reading frame (ORF) and encoded a predicted protein with 779 amino
acids. The expression analysis of the PbACO1 gene was performed through quantitative real time RT-PCR
and results demonstrated an increasing expression during differentiation from mycelium to yeast cells. Real
time RT-PCR assays showed that PbACO1 transcript is over expressed when the fungus grows on media
with acetate and ethanol as carbon sources and is repressed by glucose. Those results point that PbACO1
may be required when glucose is unavailable and other precursors of acetyl-CoA such as ethanol or acetate
are available. These adaptations to nutrient-limited growth may involve a differential compartmentalization of
some enzymes. Since mitochondrial, peroxisomal and cytosolic aconitases has been described in fungi, we
obtained a polyclonal antibody against the purified recombinant PbACO1 in order to analyze the subcellular
localization of the molecule in P. brasiliensis yeast cells. The protein localization in yeast cells compartments
is under progress.
Supported by FINEP, CNPq.
177
4-27
Comparative analysis of gene expression during macrophages infection by pathogenic
fungi (Paracoccidioides brasiliensis and Histoplasma capsulatum)
Simoneide, S.S.1 and Felipe M.S.S.1
1
Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil. e-mail: simoneide.silva@gmail.com (presenting Author)
Paracoccidioides brasiliensis and Histoplasma capsulatum are the human pathogenic thermo-dimorphic
fungi that cause the paracoccidioidomycosis and histoplasmosis, respectively. The establishment of these
mycoses occurs by propagules inhalation in where they will be in contact with the lung epithelial cells and
the fungi are phagocytosed by resident macrophages. Once inside the phagocytic cells, the pathogens
may be eliminated or create a favorable microenvironment in where they could multiplicate and establish
the disease. In the present work we investigated by microarray technology and compared the kinetic
profile of ex vivo peritoneal macrophage infected with P. brasiliensis and H. capsulatum. Total RNA from
infected and non-infected macrophages by these fungi at different times were extracted with the TRIzol®
reagent (Invitrogen, USA) and hybridized onto arrays glass. After normalization, the Significance Analysis
of Microarrays software (SAM - http://www.stat.standford.edu) was used to assess the significant variations
in gene expression between infected and non-infected macrophages. Significantly modulated genes were
identified by passing both a statistical tests q value <5% and FDR <5%. The two pathogens present a
different kinetic infection profile, since H. capsulatum and P. brasiliensis is internalized by macrophages in 2
and 6 hours, respectively. The transcriptional comparative analysis between both fungi showed modulated
macrophages genes involved with inflammatory process, membrane proteins related, transcriptional
regulation, signal transduction and apoptosis. The results observed in this work indicate that the pathogens
were able to induce a similar pattern of gene modulation categories in macrophages, such as the induction
of genes that codify to chemokines related to the cell migration (monocytes and neutrophils), facilitating the
invasion and maintenance the fungi inside the non-activated phagocyte. By the other hand the macrophages
induced genes related with fungicide activity such as Txk, responsible for a positive regulation of IFN-� gene,
probably in an attempt to eliminate these pathogens. In summary, our results demonstrate that P. brasiliensis
and H. capsulatum are potent inducer of molecules involved in the initial host response to this organism.
These expression data obtained by microarray experiments should provide a foundation for further studying
the paracoccidioidomicosis and histoplasmosis and especially for a better knowledge on the host-pathogen
interaction.
Financial support: FAP-DF/CNPq and MCT/CNPq.
4-28
A serine protease S08 of Paracoccidioides brasiliensis is induced during infection of
machophages and nitrogen starvation
J.A. Parente1, Bailão A.M. 1, Philippsen H.K. 1, Santana, J.M.2, Pereira M. 1, and Soares C.M.A. 1
1 Instituto de Ciências Biológicas, UFG, Goiânia – Brasil. 2Faculdade de Medicina, UnB, Brasília – Brasil. e-mail: juparente@gmail.com
Paracoccidioides brasiliensis is a thermodimorphic fungus and the etiologic agent of paracoccidioidomycosis
(PCM), a human systemic disease prevalent in Latin America. Proteases are an important class of proteins
with many functions in cells, such as nitrogen acquisition and facilitation of fungal invasion in host tissues.
A cDNA encoding for a subtilase serine protease S08 (Pbsp) was induced during infection of P. brasiliensis
yeast cells in mice and during incubation of yeast cells in human blood and plasma. The deduced amino acid
sequence (PbSP) was obtained and characterized. The Pbsp transcript level was analyzed by Real Time
RT-PCR. Pbsp transcripts were found to be more expressed in yeast cells when compared to mycelium.
The Pbsp was also induced in yeast cells during macrophages infection. Azocasein assays were performed
and revealed higher levels of protease activity after the incubation of yeast cells in medium depleted of
nitrogen sources, as well as in culture supernatants. The protein level was evaluated by Western blot assays
using the polyclonal antibody produced to the recombinant PbSP, confirming that PbSP is induced during
nitrogen starvation. The results suggest the importance of serine protease in the adaptative mechanisms of
P. brasiliensis to the host during the infectious process and in the strategies used by the fungus for nitrogen
acquisition.
Financial Support: Finep, CNPq and CAPES.
178
4-29
The high affinity copper transporter of Paracoccidioides brasiliensis is up-regulated during
macrophage infection
R.S. Santos1, Bailão. A.M.1, Borges. C.L.1, Salem-Izacc. S.M. 1, Dantas. S.F.I.M.1, Deepe Jr. G.S.2 and Soares. C.M.A.1*.
1- Laboratory of Molecular Biology, Department of Biochemistry and Molecular Biology, Universidade Federal de Goiás, Goiânia - Brazil.
2 – Division of Infectious Diseases, College of Medicine, University of Cincinnati, Ohio - USA. e-mail: celia@icb.ufg.br
Paracoccidioidomycosis (PCM) is a granulomatous mycosis caused by the dimorphic fungus Paracoccidioides
brasiliensis. The fungus occurs as mycelium at 26ºC and as yeast at 36ºC. The fungus invades the human body
through inhalation of propagules, which can establish infection once they undergo phase transition to the yeast
cells in the pulmonary alveolar epithelium. The success of host tissues colonization is intimately associated to
pathogen capacity on uptake essential nutrients from the host milieu. Copper is one of those nutrients and the
ability in acquiring this metal is considered a virulence factor. Copper is an important cofactor for a wide range
of enzymes involved in many biological processes such as respiration, cell growth, iron uptake and oxidative
stress. Transcriptional studies have shown that ctr3 (high affinity copper transporter) of P. brasiliensis is induced
in yeast cells recovered from liver of infected mice. In the present study we isolated and characterized the cDNA
and gene sequences coding to CTR3 of P. brasiliensis. The cDNA showed a 582 bp open reading frame (ORF)
encoding a predicted protein with 193 amino acids, predicted molecular mass of 21.5 kDa and of pI 8.6. The
genomic sequence presented four exons interrupted by three introns. The transcriptional behavior of the ctr3 gene
was analyzed during exposure of P. brasiliensis yeast cells to conditions of depletion of iron, copper and zinc by
semi-quantitative RT-PCR. It was shown a significant increase in the transcription level of the ctr3 gene in the
combined absence of the three metals, in absence of copper as well as in absence of iron. Real time RT-PCR was
used to analyze the expression of ctr3 in the two fungal morphological forms of P. brasiliensis and in yeast cells
infecting macrophages. The ctr3 expression was upregulated in the yeast phase and in yeast cells residing into
macrophage phagosomes. Taken all together those data suggest the importance of ctr3 and of the copper/iron
uptake system during the infective process. In order to better understand the biological function of this molecule,
proteic interactions of CTR3 are under progress using a Saccharomyces cerevisiae two hybrid system.
Financial support: MCT/CNPq, CAPES, FINEP and FUNAPE-UFG.
4-30
Alternative oxidase is transcriptionally stimulated at 36°C and is required for mycelium to
yeast transition and viability of Paracoccidioides brasiliensis
Natália M. Junqueira, Guilherme N. D. Souza, Hilsamara Y. Alves, Mariana P. Santos, João Paulo T. Di Benedette,
Claudia B. L. Campos
IP&D, Universidade do Vale do Paraiba, São José dos Campos, SP, Brazil. e-mail: cbcampos@univap.br
Alternative oxidase (AOX) is an enzyme present in the mitochondrial respiratory chain of plants, fungi, many
types of yeasts and some protistis, but absent in animal cells. Likewise cytochrome oxidase, AOX reduces the
molecular oxygen to H2O at the final step of the respiration process. AOx activation has been associated with
a fine tuning of ATP production and control of cellular redox balance under environmental stress conditions.
Recently, AOX has also been associated with virulence in C. neoformans. In this work, we studied the role of AOX
in temperature-induced mycelium to yeast (M-Y) transition of P. brasiliensis using biochemical, molecular and
cellular biology approaches. The AOX inhibitor salicyl hydroxamic acid (SHAM) at 0.5 mM reduced proliferation
and viability of P. brasiliensis during M-Y transition without effecting dimorphism. At 2 mM, SHAM impaired
dimorphism and killed the fungus. In yeast cells, SHAM also delayed proliferation, but fungi remained viable. In
oxygen consumption experiments performed with antimycin A, a respiratory complex III inhibitor, the total capacity
of the AOX pathway was slightly increased in mycelia at 36°C compared to 25°C. At 36°C, the rate of oxygen
consumption and generation of H2O2 by mitochondria was increased in mycelia, but respiration was coupled,
which suggest that AOX might be controling energy production while prevents excessive ROS generation.
Quantitative real time PCR showed that AOX transcripts highly increased 15 min after temperature shift to 36°C.
The profile of AOX expression during M-Y transition followed a bimodal curve, increasing early after temperature
shift, decreasing to the basal level at 24 hours and rising again from 48 hours until day 7. In yeast cells, AOX
transcripts were about 30x the basal mycelial level. Our data show that alternative oxidase is biochemically and
transcriptionally stimulated in P. brasiliensis in response to temperature shift to 36°C, contribute to survival during
M-Y transition and to proliferation of yeast cells. We propose that AOX is required to orchestrate metabolic demand
while avoid oxidative stress. Considering that AOX is absent in mammals, this enzyme might be a promizing target
for therapeutical proposals.
This work was supported by Fapesp. Key words: Paracoccidioides brasiliensis, alternative oxidase,
mitochondria, dimorphism.
179
4-31
Transcriptional profile of Paracoccidioides brasiliensis induced by oenothein B, an
antifungal agent from Brazilian Savannah plant Eugenia uniflora
P. F. Zambuzzi1, Rezende, R. V.1, Borges, C. L.1,Ferri, P. H.2, Santos, S. C.2, Soares, C. M. A.1 and Pereira, M1
1 Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiania-GO-Brazil. 2 Laboratório de
Bioatividade Molecular, Instituto de Química, Universidade Federal de Goiás, Goiania-GO-Brazil. e-mail: mani@icb.ufg.br
Paracoccidioides brasiliensis is a soil-borne fungus and the causative agent of paracoccidioidomycosis, a
systemic mycosis that cause chronic and progressive infection. The long-time treatment, the high toxicity of the
drugs, and the appearance of resistant or multi-resistant strains have imposed the need for a permanent search
and development of new therapeutic approaches. A large variety of compounds extracted from plants has been
used to the discovery of new antimicrobial agents. The active compound oenothein B, purified from leaves of
Eugenia uniflora, a Brazilian Savannah plant, has been evaluated on the growth, viability and gene expression
of P. brasiliensis. That compound interferes with yeast cell morphology and inhibits glucana synthase transcripts
accumulate; suggesting that oenothein B can be a good candidate to antifungal agent. Aiming elucidate the
mechanism of action of oenothein B on P. brasiliensis, was realized Representational Difference Analysis (RDA).
P. brasiliensis yeast cells were grown on MMcM (Mc Veigh & Morton) medium in the presence (tester) and in the
absence (driver) of oenothein B for 90 min, at 37oC. After extraction of total RNA, the cDNA was obtained and
used to RDA experiments according with modified protocol previously described by Pastorian et al. (2000). RDA
experiment originated 280 ESTs that were successfully sequenced and originated 28 contig and 24 singlets. Using
the BLATX program, was possible classified the ESTs in agreement with the functions. The analyses indicated the
presence of transcripts with functions related with cell wall and membrane, elongation and transcription factors and
hypothetic proteins. The elucidation of the action mechanism of oenothein B will facilitate the design and synthesis
of related compounds with enhanced pharmacological profiles.
4-32
Transcriptional profile of murine macrophages infected with pathogenic fungus Histoplasma
capsulatum
Simoneide.S.S1, Passos-Silva. D. G.2., Teixeira. S.M.R2, Junta, C.3,4, Medeiros. A. I.6, Silva. C. L.5, Faccioli. L. H.6, Passos.
G.A.S.3,4, Felipe. M.S.S.1
Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil. 2 Departamento de Bioquímica e Imunologia, UFMG,
Belo Horizonte, MG, Brazil. 3 Departamento de Genética, USP, Ribeirão Preto, SP, Brazil. 4 Faculdade de Odontologia, USP, Ribeirão
Preto, SP, Brazil. 5 Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto 14040-900, SP, Brazil. 6 Faculdade de Ciências
Farmacêuticas, USP, Ribeirão Preto, SP, Brazile-mail: simoneide.silva@gmail.com
1
Histoplasma capsulatum is the thermo-dimorphic fungus that causes histoplasmosis. The yeast form of P.
brasiliensis acts as a facultative intracellular pathogen, being able to survive and replicate within macrophages as
a probable mechanism to evade the immune system. In the present work the kinetic profile of murine macrophages
infected with H. capsulatum was investigated. A clinical isolate of the fungus was used for macrophage infection.
Total RNA from infected and non-infected macrophages at 6, 24 and 48 hours was extracted with the TRIzol®
reagent (Invitrogen, USA) and hybridized against glass arrays. After normalization, the Significance Analysis of
Microarrays software (SAM- http://www-stat.standord.edu/) was used to assess the significant variations in gene
expression between experimental and control conditions. Genes were considered to be modulated if they crossed
two statistical thresholds (q value <5%; FDR <5%). A total of 4,500 murine macrophage genes were analyzed.
We identified 320 differentially transcribed genes, which were grouped into 12 functional categories. An increase
was especially evident in the transcription of a number of pro-inflammatory molecules (Ccl2, Ccl3, Il15, Ccl17 and
Ccr7) and membrane proteins (Col27a1, Itga5, Tmem115 and Tmem48), all of which are involved in adhesion and
phagocytosis. We observed also the repression at 24 hours of genes whose products may cause apoptotic events.
For the first time, a comprehensive gene expression tool is used for the expression analysis of macrophage genes
when these cells interact with P. brasiliensis. The results showed in this work suggest that the pathogens have
induced in macrophages a dynamic changes in gene expression that in the early times (two and 24 hours) can
create favorable conditions for fungal persistence. However, at 48 hours macrophages induced genes related
to fungicidal activity, probably in an attempt to eliminate the fungus. Finally, the identification of genes that are
differentially expressed by macrophages upon exposure to H. capsulatum, as described here, should result in a
better understanding of this systemic mycosis.
Financial support: FAP-DF/CNPq and MCT/CNPq.
180
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Analysis and identification of three P. brasiliensis genes possibly implicated in virulence
O. Hernandez1, AM Garcia1 A Restrepo1 and JG McEwen1,2.
1 Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia. 2 Facultad de Medicina Universidad de Antioquia, Medellín
– Colômbia. e-mail: orvillehr@hotmail.com
In general, fungal dimorphism is considered an essential trait for successful adaptation of pathogenic fungi
to the human host, and this ability that has been studied among different dimorphic fungi and characterized
by several physiological and structural modifications. P. brasiliensis conidia are consider to be the infectious
particles which once inhaled into the lungs begin their morphological transition to the yeast phase. This process
has been previously studied, both morphologically and at the transcriptome level either during batch culture
growth or in the infection process itself. Some of the major alterations described in the transcriptome profile
during the morphological switch include differential expression of genes involved in the rearrangement of cell wall
composition, stress response and diverse metabolic pathways. Nonetheless, conidia-to-yeast transition remains
greatly uncharacterized and, by this reason, it is important to analyze the relevance of specific proteins during such
process. Recently, the complete genome and the corresponding annotation of P. brasiliensis strain Pb 03 has
been published by the Broad Institute with 90% of all possibly proteins expressed by the fungus being incorporated
into this genome. The aims of this work was to analyze some important gene sequences considered as virulence
factors in other fungi, such as: Glucan synthase, involved in the generation of beta-glucan, important in the cell
wall -and an alternative oxidase, involved in the electron transport chain. The latter provides an alternative route
for electrons passing through the electron transport chain to reduce oxygen and glyceraldehide 3 phosphate
deshidrogenase (GAPDH), catalyzes the sixth step of glycolysis and serves thus to break down glucose for energy
and carbon molecules. Based in the Pb 03 genome “in silico” analyses were made using the partial sequences
of the genes mentioned above; additionally, PCR primers were designed to amplify these genes in strain ATCC
60855, and the PCR products were sequenced and compared with the database published by the Broad Institute
in order to confirm their presence. Results: All the sequences corresponding to virulent genes searched for were
found with similar levels above 90% and e-value of 0.0 for all of them. The probability of these results being at
random is near zero, confirming the existence of these genes in P brasiliensis. ClustalW analysis showed high
similarity between the sequence of these genes in 60855 and Pb03 strains. These results made of these genes
suitable targets for knockout–knockdown studies aimed at confirming their role in pathogenicity.
4-34
Internalization of magnetic nanoparticles by Paracoccidioides brasiliensis yeast cells
A.C. Amaral1, 3, Braun S.1, Nunes E.S.4, Bocca A.L.1, Lima E.C.D.4, Báo S.N.1, Azevedo R.B.1, de Morais P.C.2 and Felipe M.S.S.1
Instituto de Ciências Biológicas e 2Física, Universidade de Brasília, UnB, Brasília, Brazil. 3Ciências Genômicas e Biotecnologia,
Universidade Católica de Brasília, UCB, Brasília, Brazil. 4Instituto de Química, Universidade Federal de Goiás, UFG, Goiânia, Brazil.
e-mail: amaralandre@yahoo.com.br
1
Introduction and aim: Functionalized magnetic nanoparticles can be used for several applications in biology,
such as cell labeling, isolation and purification of specific molecules or cells from a culture (J. Biosci. Bioeng.,
100(1):1-11, 2005). When properly functionalized these nanoparticles, with average size between 3 and 20nm can
easily penetrate the cells. Once inside the cell, this magnetic nanoparticle can separate or drive the cell using an
external magnetic field. Functionalization is possible by linear linker molecules with reactive organic groups at both
ends. One group is attached to the nanoparticle surface and the other is used to link biocompatible molecules,
antibodies, fluorophores, etc (J. Nanob., 2:3, 2004). Here we report the internalization of magnetic nanoparticles
by P. brasiliensis Pb18 yeast cells. Methods: To investigate the internalization of nanoparticles, the yeast cells
(3 × 104 cells/mL) were incubated during 1, 4 and 8 hours in YPD medium containing maghemite nanoparticles
functionalized with dimercaptosuccinic acid (1014 particles/mL), followed by routine techniques for transmission
electron microscopy (TEM). To evaluate the toxicity, after the exposure times to magnetic particles 3 × 102 cells were
plated in BHI supplemented with 4% horse serum, 5% P. brasiliensis 192 culture filtrate and 40mg/L Gentamicin.
The plates were incubated at 36 ºC and CFU was counted at 21st day post-plating to asses the cell viability. Results
and discussion: The TEM has revelead the uptake of magnetic nanoparticles by the P. brasiliensis Pb18 after
4 and 8 hours of incubation. The majority of nanoparticles were found between the cell wall and the plasmatic
membrane, and a very small number were observed within the cytoplasm. These magnetic nanoparticle uptakes
did not interfere with the cell viability, once no differences on CFU between the treated groups and controls were
noted. These findings could be useful, for example, to accompaning the establishment of the paracoccidioidomycosis
in animal model. Since the magnetic nanoparticles have the capability to be used as contrast agents in magnetic
resonance imaging (J. Biosci. Bioeng., 100(1):1-11, 2005), animals can be infected with P. brasiliensis containing the
nanoparticles to investigate the kinectic of the fungal infection. Financial support: CNPq.
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Molecular typing and evolution of the Paracoccidioides genus
Teixeira, M. M.1, Paes, H. C. 1, Fernandes L. 1, San-Blas, G. 2 and Felipe, M.S.S.1
1 Instituto de Biologia, UnB, Brasilia - Brasil. 2 Instituto Venezoelano de Investigaciones Cientificas, IVIC – Venezuela.
e-mail: marcus.teixeira@gmail.com
Background and aims: Paracoccidioidomycosis is a systemic illness with an area of occurrence covering
most of Latin America, affecting mainly young male individuals living in the countryside. Studies on different
isolates of the causative agent, Paracoccidoides brasiliensis, have revealed a great degree of genetic variability
in this pathogen. The Pb01 isolate, in particular, differs from the three phylogenetic species (S1, PS2 and PS3)
previously described by the genealogical concordance method (GCPSR). This work aimed at looking into the
existence of a systematically significant group of “Pb01-like” isolates through GCPSR. Methods: 88 isolates were
analyzed by phylogenetic methods of Maximum Parsimony and Bayesian analysis in thirteen (13) polymorphic
regions, including individual and concatenated loci. The polymorphisms were evaluated and the divergence time
of speciation was measured between the phylogenetic groups. We also assessed the possibility of recombination
both within each group and between them by split decomposition analysis. Results: Phylogenetic data enabled
the grouping of “Pb01-like” isolates in a clade distinct from S1, PS2 and PS3. In keeping with GCPSR, the “Pb01like” clade can be considered a new phylogenetic species, since the clade corresponding to “Pb01-like” isolates
is strongly supported by all phylogenetic trees – separately and in a concatenated analysis – generated from
polymorphism data of the thirteen loci, with highly significant values of posterior probability (1.0) and bootstrap
agreement (100%). A high number of fixed polymorphisms were identified between the “Pb01-like” cluster
and that composed of the other three species, which suggests a blockage of genetic flow between them. The
speciation event that defined the new phylogenetic group is sympatric relative to S1 and PS2. The “Pb01-like”
cluster and the group that includes S1, PS2 and PS3 have been genetically isolated for about 19 million years.
Recombination analysis revealed that recombination events occur independently inside the two groups, which
suggests reproductive isolation. Conclusion: Added to the morphological exclusive data for this genetic isolated
group (Bagagli, E., personal communication) we suggest that the phylogenetic species encompassing the “Pb01like” clade should be named Paracoccidioides lutzii, as a tribute to medical mycologist Adolpho Lutz, who first
described human pathogen P. brasiliensis one hundred years ago. Financial support: CNPq, FAP-DF, FUB.
4-36
Adhesion and expression of ligands of extracellular matrix (ECM) by different
Paracoccidioides brasiliensis isolates
J.F Silva1, G. Benard2, M.J.S. Mendes-Giannini1.
Faculdade de Ciências Farmacêuticas de Araraquara – UNESP – Brasil and 2Faculdade de Medicina de São Paulo – USP – Brasil.
e-mail: silvajf@fcfar.unesp.
1
Paracoccidioides brasiliensis is a dimorphic fungus and the causative agent of paracoccidioidomycosis, a mycosis
usually characterized by multiple organ involvement and a chronic evolution. P. brasiliensis virulence can be attenuated
or lost after subsequent cycles of subculturing for prolonged periods and reestablished after passage in animals or in
epithelial cells cultures. The success of colonization is a complex event, generally involving a ligand encoded by the
pathogen (adhesins) and a cell receptor. An important aspect in the interaction between P.brasiliensis and its host is
the ability to adhere to matrix extracelular components (ECM). The understanding of this process and identification
of these molecules may eventually provide new targets for more efficient treatment strategies. The aim of this study
was to investigate the protein expression profile of P. brasiliensis isolates during infection of pulmonary epithelial cells
(A549), and more specifically, the pattern of proteins expressed by four different isolates (Pb01, Pb113, Pb339 and
Pb265) with ability to ligate to ECM components, before and after passage in cell cultures. All P. brasiliensis isolates
presented different capacity to adhere to epithelial cells, but specially before cell cultures passage. The profiles of four
P. brasiliensis “cell-free” extracts from repeatedly subcultured strains and from reisolated strains were analyzed by twodimensional electrophoresis. Differentially expressed proteins were identified. Furthermore, a large series of ligands
to ECM components was observed: 62 ligands to collagen type I, 23 to fibronectin, and 18 to laminin, of a total of 420
proteins. The isolates Pb01 and Pb339 presented fibronectin ligands only after reisolation from epithelial cells, apparently
showing that these ligands may have a role in pathogenicity. The same occurred with Pb01 in relation to collagen.
Pb113, even after reisolation, did not present laminin ligands, whereas Pb265 did not present fibronectin ligands. Thus,
this study suggested that different patterns of the ECM ligands can occur depending on the isolate and that multiple
proteins may function as adhesins, depending on the challenged posed by the microenvironment. Expressive number of
proteins, with characteristics of ECM ligands, was observed mainly after passage in epithelial cultures. These differential
profile ligands could be associated with the ability of this fungus to express different proteins in fungal-host interaction.
Suppoted by: FAPESP; PADC-FCF; CNPq and CAPES
182
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The heath shock factor of Paracoccidioides brasiliensis and its complementation of
Saccharomyces cerevisiae
Paes HC, Matos LF, Teixeira MM, Torres FAG,Felipe MSS.
Laboratorio de Biologia Molecular, Instituto de Biologia Celular, Universidade de Brasilia, Distrito Federal, Brasil.
e-mail: sorumbatico@gmail.com (presenting author)
The heath shock factor, a ubiquitous transcription factor of eukaryotes, is responsible for the regulation of
several stress response genes in baker´s yeast and metazoans, but has not been thoroughly studied in filamentous
or dimorphic fungi so far. It might be of importance in the control of morphogenetic programmes in the latter
group, in view of its evolutionary specialization to respond to changes in temperature. Therefore, we set out
to characterize its homologue in P. brasiliensis, and have identified, cloned and sequenced it. The hsf gene is
present in a sole copy in this fungus, and the corresponding sequence is the largest heat shock factor found so far.
The ORF codes for protein 840 residues long, wich contains easily identifiable DNA binding and oligomerization
domains. However, is presents other regions that are conserved among other Eurotiomycetidae as Coccidioides,
Histoplasma and aspergilli, but that bear no resemblence to gous expression of hsf in S. cerevisiae was abble to
rescue the loss of the native gene, wich would normally be lethal to the yeast. The recombinant strain presented a
growth defect that we have beeb unable to account so far. The functional evidence of trans – complementation has
prompted us to device gel shift experiments to further assess the behavior of this protein, and these are currently in
progress. We intend to examine the binding of this new hsf to canonical and non-canonical heat shock elements
(HSEs) from promoters of several genes of P. brasiliensis and to the classic HSE described in S. cerevisiae. It may
be that genes other than heat shock proteins respond to this factor in P. brasiliensis , including differential genes
for dimorphic transition and determinants of virulence – a possibility we also envision to investigate.
183
Poster Session
5
Immunology:
Patients / Experimental Animals
5-01
Phenotypic and functional characterization of human dendritic cells induced by low and
high-virulence strains of Paracoccidioides brasiliensis
2Fornazim MC, 1Mamoni RL, 2Spago MC, 1Oliveira RTD, 2Gabetta CS, 2Nowill AE, 1Blotta MHSL.
1Laboratório de Imunologia Celular e Molecular - Departamento de Patologia Clínica – FCM - UNICAMP e 2Laboratório de Imunologia
Celular - CIPOI –FCM - UNICAMP Campinas – São Paulo – Brasil.
e-mail: fornazim@unicamp.br
Paracoccidioidomycosis (PCM) is the most important deep mycosis in Latin America. Dendritic cells (DCs)
play an essential role in the initiation and modulation of the immune response, since cells they are the only
professional antigen-presenting cells able to stimulate naive T lymphocytes. To determine whether DCs become
phenotypically and functionally competent in the presence of P. brasiliensis, peripheral blood monocytes obtained
from healthy individuals were differentiated in the presence of GM-CSF and IL-4 for 7 days and pulsed in vitro
with yeast cells (Pb18, high virulence and Pb265, low virulence) for 24 hours. After that, DCs were stained
with monoclonal antibodies against surface antigens and analyzed by flow cytometry. Cell cultures (DCs and T
lymphocytes) supernatants were stored at -20°C for cytokines determination (TNF-a, IL-6, IL-12p40, IL-10 e IFNg) by ELISA. DCs pulsed with the both strains of the fungus present morphology and phenotype characteristic of
mature cells with increased expression of CD83, CD80, CD86 and CCR7 and decreased phagocytic capacity. The
expression of CD83 and the production of inflammatory cytokines such as TNF-a, IL-6 e IL-12p40 were higher
in DCs pulsed with low virulence yeast cells (Pb265) compared with Pb18. Moreover, DCs pulsed with Pb265
were also able to induce higher levels of T lymphocytes proliferation and IFN-g production than those generated
in the presence of Pb18. Altogether the results indicate that DCs generated in the presence of the fungus exhibit
phenotype of mature APC (expression of CD83, CD80, CD86 and CCR7), are able to produce pro-inflammatory
cytokines as well as to stimulate adaptive immune response inducing the
CD4+ T lymphocyte proliferation and IFN-g production. Furthermore, the higher response obtained with the
strain of lower virulence demonstrates the ability of DCs to sense small changes in pathogens and to appropriately
modulate the adaptive immune response.
5-02
Paracoccidioidomycosis patients dendritic cells: Effect of Paracoccidioides brasiliensis
antigens on surface molecules expression
P. K. Sato1, Oshiro. T.M.2, Diogo. C.L.2, Passos. E.C.3, Sadahiro. A.4, Shikanai-Yasuda. M.A.2,5
1 Pós-graduação do Depto de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da USP(FMUSP), São Paulo, Brasil.
2 Laboratório de Investigação Médica em Imunologia (LIM-48) do Hospital das Clínicas da FMUSP, São Paulo, Brasil. 3 Curso de
Aprimoramento Fundap do Depto Moléstias Infecciosas e Parasitárias da FMUSP, São Paulo, Brasil. 4 Instituto de Ciências Biológicas
- Universidade Federal do Amazonas, Manaus, Brasil 5 Depto de Moléstias Infecciosas e Parasitárias da FMUSP, São Paulo, Brasil.
e-mail: lim48imuno@yahoo.com.br
Introduction: Paracoccidoidomycosis (PCM) is an endemic infectious disease caused by the pathogenic
and dimorphic fungus Paracoccidioides brasiliensis (P. brasiliensis). Previous studies have shown that Th1
immune response confers protection in PCM whereas Th2 is associated with susceptibility. Recently, as antigen
presenting cells, dendritic cells (DCs) able to activate Th1 response were described in resistant mice infected with
P. brasiliensis. However, their role in human cellular response for this disease is not clear.
Objective: To investigate the effect of P. brasiliensis 43KDa-glicoprotein (gp43) and cell-free antigen (CFA)
on DCs’ surface molecules expression.
Materials and Methods: Monocyte-derived DCs were generated from peripheral blood of cured PCM patients
(positive paracoccidioidin skin test and serum specific antibody titles measured by CIE≤1: 4). Then they were
pulsed with CFA or gp43 and/or activated with inflammatory recombinant cytokine TNF-α, after differentiation
(CD11c+, CD1a+, CD14-). DCs’ surface molecules expression was verified by flow cytometry.
Results: Both Gp43 and CFA stimuli induced DCs maturation resulting on high expression of CD86, HLA-DR
and DC-Sign molecules. Gp43 stimulus is associated with higher percentage of positive cells for these markers
when compared with TNF-α activation or CFA + TNF-α activation.
Conclusions: DCs were more efficiently activated by GP43 than CFA. CFA components may be common to
other fungi resulting in non-specific and weaker maturation of DCs.
Financial support: Fapesp 04/14955-3 e FFM.
187
5-03
Altered expression of stats 1,3,4,5 and their modulation by cytokines in mononuclear cells
from paracoccidioidomycosis patients
1,2
Romano C.C.; 2Mendes-Giannini, M.J.S., 1Duarte, A.J.S. 1Benard. G.
Universidade Estadual de Santa Cruz- UESC, Ilhéus, Bahia, Brasil. 2Laboratório de Investigação Médica 56- FMUSP, São Paulo, Brasil.
3
UNESP, Araraquara, São Paulo, Brasil. e-mail: ccromano@uesc.br (presenting Author)
1
Introduction/Objective: We have previously shown a specific imbalance in the IL-12/INFγ/IL-10 regulation
in PCM patients. In this regard, it has recently been shown that cytokines modulate the expression and activity of
the STATs family of proteins. In this study we analysed the expression of inactive and active STATs 1, 3, 4 and 5
proteins by intracellular cytometry.
Methods/Results: The mononuclear cells of cured controls and patients with PCM was gotten through gradient
of Ficoll and the cells cultured for 72h with medium plus ionomycin and PMA (basal) or stimulated with gp43. We
observe higher levels of gp43 STAT 1 (inactive and active ) in the cured controls when compared with the patients
cells (25±3,7 controls vs 13,8±3,9 patients)( active: 28.4 ± 2.8 controls vs 8.8 ± 1.3 patients).Similarly, the gp43
STAT 4 expression was higher in the cured control in relation to patients for the two isoforms (26,6±3,2 vs 19,8±4,6
for inactive and 33,4 ±4,5 vs 10,5±3,3 for active). However, the active STAT 3 expression was significantly bigger
in patients’ group when compared with controls (7,0±2,2 vs 37,3±8,5 for basal; and 8,8±2,2 vs 35,6±8,7 for gp43).
We also observed bigger expression of inactive STAT5 in the patients cells, when compared with control (8,7±3,2
vs 24.1±4,5, for basal; 10.9±1,8 vs 22,5±4.6 for gp43). Upon gp43 stimulus, only controls’ cells were able to
activate this factor of transcription (17,2± 4,4 STAT 5 active for basal; 28,5±7,5 STAT 5 active for gp43). In vitro
neutralization of endogenous IL-10 resulted in increase of gp43- active STAT 1 in patients to levels displayed by
the cured subjects and IL-12 addition restored STAT 4 expression in PCM patients to levels expressed by the
cured subjects for the two isoforms.
Conclusion: These data suggest that the PCM infection can inhibit the activation of transcription factors
as STAT1 and 4, which is important for IFN-g and IL-12 production. In parallel, the activation of STAT3 and
constitutive expression of STAT5 in cells of patients related, respectively, with the control of lymphocytes activation
and protection against apoptosis.
5-04
Increased T cell regulatory activity in patients with paracoccidioidomycosis
M.C. Ferreira, M.H.S.L Blotta, R.L. Mamoni.
Department of Clinical Pathology - Faculty of Medical Science – UNICAMP – Campinas / SP – Brazil.
e-mail: mcf@fcm.unicamp.br (presenting Author)
Introduction and Aims: It has been shown that Paracoccidiodomycosis (PCM) patients present a suppressed
cellular immune response characterized by negative DHT to P. brasiliensis antigens, elevated apoptosis of
activated lymphocytes, high expression of CTLA-4, and production of IL-10 and TGF-β. These data point toward
the involvement of regulatory T cells (Tregs), which have emerged as a crucial T cell population regulating the
immunological response in several infectious and non-infectious diseases. The aim of this study was to verify
whether Tregs are involved in the immunosuppression observed in PCM patients, through the analysis of the
number and the phenotype, as well as the functional activity of Treg cells (CD4+CD25+ T cells) in peripheral blood
of patients before and after antifungical treatment.
Methods and Results: Ex vivo expression of mRNA for FoxP3 in PBMC evaluated by qRT-PCR was higher in
PCM patients with active disease than in the healthy controls. The number and phenotype of Treg cells (CD4+CD25+
T cells) were determined by flow cytometry. In accordance to mRNA analysis, the results showed that before the
treatment, PCM patients present higher number of Treg cells (CD4+CD25+) than patients after treatment or healthy
controls. Furthermore, we detected an increased expression of CTLA-4, LAP-1 and GITR on TCD4+CD25+ of
patients with active disease in relation to healthy individuals. In order to compare the functional activity of Tregs,
cells obtained from patients (with active or treated disease) and healthy controls, we analyzed the suppressive
effect of these cells in cultures of PBMC stimulated with the mitogen ConA. The results demonstrate that the Tregs
from patients with active disease exhibited stronger regulatory activity than cells from control or treated patients in
a dose dependent manner.
Conclusions: The elevated expression of Foxp3 mRNA and increased number of Treg cells in peripheral
blood of patients with active disease, which presented higher expression of regulatory markers and suppressive
activity in vitro, suggest an elevated regulatory activity during the disease and indicate the potential role of Tregs
in the immunosuppression observed in PCM. Supported by FAPESP and CNPq
188
5-05
Polymorphisms analysis of CTLA-4 gene in paracoccidioidomycosis patients
V. F. Lozano¹, Paes H.C.², Teixeira M.M.¹, Lins T.C.L.³, Vieira R.G.³, Blotta M.H.S.L.4, Goes A.M.5, Pereira R.W.3, Bocca
A.L.¹,2, Felipe M.S.S1,2.
¹ Faculdade de Medicina, UNB, Brasilia – Brasil. ²Instituto de Biologia, UNB, Brasília – Brasil. 3Pós-graduação em Ciências Genômicas e
Biotecnologia, UCB, Brasília – Brasil. 4 Faculdade de Ciências Médicas, UNICAMP, Campinas – Brasil. 5Instituto de Ciências Biológicas,
UFMG, Belo Horizonte - Brasil.
e-mail: vivianefurlan74@yahoo.com.br (presenting Author)
The CTLA-4 protein is mainly expressed in activated T cells and plays an essential role in the immune response
through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated to
several autoimmune pathologies and, more recently, to neoplastic and infectious illnesses. Paracoccidioidomycosis
(PCM) is a systemic mycosis caused by the dimorphic pathogen Paracoccidoides brasiliensis. Its symptoms are
associated to various factors, including altered secretion of cytokines (higher IL-10 and lower IFN-γ production),
hypergammaglobulinaemia and suppression of cellular immunity. This low responsiveness is also attributed to a
higher expression of CTLA-4 in the cells of patients relative to control individuals. This work aimed at studying
possible associations of PCM and two single nucleotide polymorphisms (SNPs) of CTLA-4, -318C/T in the promoter
and +49A/G in exon 1. To this end, 74 PCM patients and 76 controls from different regions of Brazil had their
allelic and genotypic frequencies determined. The comparison of genotypic and allelic frequencies had not shown
significant differences between patient and control groups that could link either CTLA-4 SNP to PCM. The analysis
of results concerning haplotypic frequencies revealed a strong linkage disequilibrium (D’=1) between SNPs -318
and +49 for both groups. Nevertheless, the analysis did not reveal significant differences of haplotypic frequencies
between groups, which reinforce the absence of correlation between those SNPs and PCM. Furthermore, the
genetic structure of the patient and control groups was also evaluated in order to eliminate the bias of ancestry.
There was a predominance of European over Amerindian and African ancestries in both groups. From the results
it was possible to determine that the population used in this study was genetically homogeneous, and that the
absence of an association between polymorphisms of CTLA-4 and PCM cannot be attributed to ancestral bias.
This work shows that there is no association of SNPs -318 and +49 of CTLA-4 and resistance and/or susceptibility
to paracoccidioidomycosis.
Financial Suport: CNPq
5-06
Lymphoproliferation, levels of antibodies and cytokines to ab2-β anti-idiotipic antibodies
in patients with paracoccidioidomycosis (PCM)
C.R. Furuchó1, Oliveira1 C.C.U., Bertossi1, D.R.T., Fonseca1, C.A., Shikanai-Yasuda, M.A1,2.
1 Laboratório de Investigação Médica em Imunologia (LIM-48), Hospital das Clínicas da Faculdade Medicina da USP(FMUSP), São Paulo,
Brasil. 2 Departamento Moléstias Infecciosas e Parasitárias, FMUSP, São Paulo, Brasil.
e-mail: lim48imuno@yahoo.com.br
The development of specific, sensitive, and easily accessible methods for monitoring of antibody levels and
analysis of cellular immune response represent important strategy for diagnosis and host-parasite studies in
paracoccidioidomycosis. The performance of an anti-idiotipic 7.B12 (Ab2-β) antibody was compared with gp43
in lymphoproliferative and serological tests in patients with PCM. Methods: 51 patients with PCM, 36 previously
treated/cured (CT) patients and 14 with active disease (AD). The criteria for inclusion were: positive mycological
and histopathological tests and/or high levels of serum antibodies (CIE>1/32) to P. brasiliensis and negative CIE
for 25 healthy individuals (CO). Ab2-β (1 and 4µg/ml) and gp43 (1µg/ml) were employed in lymphoproliferative
assay, PHA (5µg/ml) was used as mitogen. IgG, IgG1, IgG2, IgG3, IgG4 ELISA anti- gp43 (5µg/ml) and anti-Ab2-β
(15µg/ml) were analysed. Results: Ab2-β induced lymphoproliferation in patients with PCM. The specificity was
better using 1µg/ml of Ab2-β, however, the sensitivity was higher using 4µg/ml. Analyze of lymphoproliferation
using gp43 showed that CT and CO groups are different. There was no cross reactivity in CO group using Ab2-β
(1 and 4 µg/ml) and gp43; there was no difference between levels of IL-10 secreted by cells from CT, AD or CO
groups. Levels of IFN-γ from CT and AD groups were related to the presence of lymphoproliferation and low levels
of IL-10; specificity for gp43 or Ab2-β was higher than 95%. Higher Ig G and IgG2 anti-gp43 and IgG anti-Ab2-β
were registered in CT and AD than in the CO group; IgG4 levels of anti-gp43 were significantly higher in the CT
than CO group, and the highest levels was observed in the AD group. Conclusions: The Ab2-β antibody showed
a good specificity but lower sensitivity than gp43 in the analysis of lymphoproliferation and humoral immune
response. Financial support: CNPq 472809/04
189
5-07
Paracoccidioidomycosis: Study of citotoxic immune response in cutaneous and mucosal lesions
C. Pagliari1,2, N. V. Pereira1,2 , M. I. S. Duarte1 and M.N. Sotto2.
Laboratório da Disciplina de Patologia de Moléstias Transmissíveis 1 Departamento de Patologia e 2 Departamento de Dermatologia. Faculdade de
Medicina da USP, São Paulo – Brasil. e-mail: cpagliari@usp.br
Some studies have related the role of CD8+T cells and Natural-killer (NK) cells in immune response in PCM,
both capable to produce immune mediators. Experimentally, NK cells are associated to a depressed immune
response. Studies focusing CD8+T cells in PCM suggested their role in the control of fungi due to perforin and
granzyme production. Our objective was to contribute to the study of in situ PCM immune response addressing the
participation of NK cells, CD8+T cells and granzyme B expression. Sixty biopsies of PCM skin and mucosa were
classified according to tissue reaction: presence of compact granulomas (G1), poorly-organized granulomas (G2)
and both kinds of granuloma in the same lesion (G3). CD8+T cells, NK cells and granzyme B were demonstrated
by immunohistochemistry. The cells were quantified and the results compared by Mann-Whitney test. There was
a statistically significant increase in the number of CD8+T cells over NK cells in cutaneous G1 and G2 lesions.
There was no statistical difference regarding such cells in G3 lesions, although CD8+ T cells were abundant in
such lesions. In mucosal lesions, CD8+T cells were increased in number over NK cells in all groups (p<0.0001).
Cells expressing granzyme B in skin lesions were increased in G2 and G3 when compared to G1 (p=0.0003 and
0.0068, respectively). The number of granzyme expressing cells did not differ statistically in mucosal lesions in the
three kinds of tissue reaction, although it was higher in G2 and G3. Our results suggest the participation of CD8+
T cells and NK cells in PCM cutaneous and mucosal lesions. The predominance of CD8+ T cells over NK cells may
represent a more effective in situ immune response against the fungi. The high number of granzyme B expressing
cells in G2 and G3 in situ reaction corroborates this possibility.
5-08
Pulmonary abnormalities induced by Paracoccidioides brasiliensis in a mouse model:
Application of high resolution computed tomography to evaluation and follow-up of lesions
DE. Lopera1, TW. Naranjo1, JJ. Duque2, Diaz-Granados LR 3, RA. Jiménez4, A. Restrepo1, AF. Zuluaga 5, LE. Cano
1
JM.Hidalgo7
Grupo de Micología Médica y Experimental, Corporación para Investigaciones Biológicas, CIB.
, J. Patiño
1,6
7,
and
Departamento de
Patología, Facultad de Medicina, Universidad de Antioquia (UdeA). 3 Facultad de Medicina ,Universidad Pontificia Bolivariana. 4 University of
Nebraska, Lincoln,, Nebraska, USA. 5 Grupo GRIPE, Facultad de Medicina, UdeA. 6 Grupo de Microbiología Molecular, Escuela de Microbiología,
UdeA. 7Facultad de Medicina, UdeA. 8Departamento de Radiología, Hospital Universitario San Vicente de Paúl, Medellín – Colombia. e-mail:
dlopera@cib.org.co
2
Background and aim: Chest High-Resolution Computed Tomography (cHRCT) is a non-invasive tool for
assessing changes in lung structures. It permits to rapidly acquire serial images of whole lung, qualitative and
quantitative analyses, 3D-reconstructions, and others. We determined here the value of this technique in the
follow-up of the course of Paracoccidioidomycosis (PCM) in an experimental model that reproduces a chronic
granulomatous disease of pulmonary origin and attempted to characterize lung parenchymal alterations and assess
changes occurring in tissue density. Materials and methods: Two groups of male BALB/c mice were intranasally
inoculated with (i) PBS (control group, n=25) or (ii) 4x106 Paracoccidioides brasiliensis (Pb) conidia (n=25). Animals
were scanned by cHRCT (n=5), sacrificed at 0, 4, 8, 12 or 16-weeks after inoculation, and their lungs removed
and analyzed by histopathology. Whole-lung evaluation was performed to determine parenchymal alterations;
then, slides from the upper, central and lower regions (slides 5, 12 and 18 below the apex) were used to quantify
lung density in Hounsfield Units (HU), followed by 3-D reconstructions to determine the air volume (Advantage
Workstation 4.3, USA). Results: At 4-weeks, 100% of Pb-infected mice showed peri-bronchial consolidations in
the left apical zone associated with significant increase in upper lung density as compared with controls (-286±44
versus -426±12,6 HU, p<0.05). At weeks 8 and 12, bilateral consolidation had progressed to involve the upper
and middle regions with a statistically significant increase in density (-185±64 versus -443.8±12 HU, p<0.001). In
the damaged lobes, lack of air volume was corroborated by failure to perform 3-D reconstruction of the affected
areas. At 16-weeks, the number of mice presenting central consolidations tended to decrease (20%), as well as
the density in their pulmonary lesions (-355±46 versus -441±11, p>0.05). However, multiple peripheral nodules
were noted in 60% of the mice at this end-point. Granulomatous reaction was observed in all animals from weeks
4 to 16. Conclusions: cHRCT is a useful, precise and non-invasive technique for the follow-up and quantification
of P. brasiliensis-induced damage in an animal model. The radiological findings in this study provide further insight
into the pathogenesis of experimental PCM. The early left apical infiltrates became bilateral and symmetrical, but
later on tended to slight down-ware while multiple peripheral nodules became more prominent. Furthermore, the
marked increase in density of the apical lesions impeded aeration of that zone. The cHRCT images have revealed,
for the first time, the early distribution and dissemination trends in experimental PCM.
Financial support: Colciencias (Proyecto 2213-04-16439), HUSVP, UdeA, CIB.
190
5-09
Alterations in pulmonary mechanics during experimental infection by Paracoccidioides
brasiliensis
J.F. Fracon 1, Bocca A.L.2, Siqueira I. M.2Zin W.A.3, Faffe D.S.3, Jerônimo M.S. 2, Soares M.A.S.4, Russo R.C.5, Figueiredo F.1
1- University Católica of Brasília – UCB - Brazil; 2 –University of Brasília – UnB - Brazil, 3- Federal University of Rio de Janeiro – UFRJ
- Brazil; 4- Sabin Laboratory – DF - Brazil; 5-Federal University of Minas Gerais – UFMG - Brazil.
Paracoccidioidomycosis, a systemic mycoses restricted to Latin America, is caused by dimorphic fungus
Paracoccidioides brasiliensis. When the patients are treated with specific therapy there are improvements but
lesions usually remain as sequelae. The remission is often accompanied by significant pulmonary fibrosis. In
the present work, mice were infected intravenously with variable yeast cells of P. brasiliensis and were analyzed
the morphology and pulmonary mechanics in the course of the experimental infection caused by the fungus,
at different days pos infection. The ventilated, static (Est), dynamic (Edyn), elastances, their differences (ℵE),
resistive (ℵP1), viscoelastic/inhomogeneous (ℵP2) pressures and the total variation of pressures (ℵPtot) of the
lungs, were obtained by end-inflation occlusion method. Arterial blood samples were collected for arterial blood
gas analysis of the parameters: hidrogenionic potential (pH), partial pressures of oxygen (PaO2), carbon dioxide
(PaCO2), oxygen saturation (SaO2), base excess (BE) and bicarbonate (HCO3). After the collecting data the
animals were sacrificed and the lungs were removed and were analyzed for morphology and for the concentration
of chemokines. Histopathology revealed progressive inflammatory response, leading to granuloma formation, with
increase in the fungus counts and formation of fibrosis, gradual to the development of infection, compromising
the pulmonary tissue. The dosage of chemokines detected the presence of: MIP-1 α, RANTES, MIG and KC,
demonstrating significant increase directly proportional to the time of infection. The presence of mist acidosis
followed by metabolic acidosis, indicating alterations more pronounced in the acute phase, was observed in the
arterial blood. In the evaluation of pulmonary mechanics showed increases in static and dynamic elastances
and increases in viscoelastic/inhomogeneous pressure and in the total variation of pressures of the lungs. This
results indicate that the lung injury is progressive, with the establishment of tissue fibrosis, probably determining a
restrictive functional pattern as a result of lower distensibility of lung tissue.
Financial Suport: CNPq.
5-10
TLR-2, TLR-4 & DECTIN-1 expression in human macrophages and neutrophils stimulated
by Paracoccidioides brasiliensis
Bonfim, CV; Mamoni, RL; Blotta, MHSL.
Laboratório de Imunologia Molecular e Celular. Departamento de Patologia Clínica – Faculdade de Ciências Médicas – UNICAMP
e-mail: camilinha@gmail.com
The pattern of the immune responses to P. brasiliensis determines the disease progression and clinical
outcome. Innate immune response is mediated by phagocytic cells, such as macrophage and neutrophils, which
ingest and kill invading pathogens and then trigger the adaptive immune system through the secretion of cytokines
and chemokines. C-type like lectin receptors (CLR) and Toll-like receptors (TLRs) are the two main PRRs involved
in fungus recognition. Therefore, the purpose of the present study was to evaluate the expression of TLR-2, TLR4 and dectin-1 (CLR) in macrophage and neutrophils from healthy individuals by flow cytometry analysis, after
stimulation with Pb18 (high virulence) and Pb265 (low virulence) yeasts of P. brasiliensis. As positive controls
we used specific ligands to TLR-4 (LPS), TLR-2 and dectin-1 (zymosan). Our results demonstrated a decreased
TLR-2 expression on macrophages and neutrophils as soon as 30 minutes after yeast cells stimulation. This
decrease was similar to the one caused by zymosan stimulation. A marked decrease of TLR4 expression was
detected only after 60 minutes of stimulation and in this case, it was possible to verify that Pb265 was able to
induce a higher decrease of TLR-4 expression than Pb18. The low-virulence isolate (Pb265) also provided a
higher decrease on dectin-1 (a β-glucan receptor) expression in both cells, but mainly in macrophages, showing an
elevated potential to activate the host’s defense response. The highest decrease of the dectin-1 expression was
observed on macrophages after 30 minutes of stimulation with yeast cells. Altogether, our data suggest that the
decrease of TLR-2, TLR-4 and dectin-1 on phagocytes surface can be related to the recognition and internalization
of the fungus, resulting in activation of the immune response.
Supported by FAPESP and CNPq
191
5-11
Absence of TLR2 results in less severe paracoccidioidomycosis but increased inflammatory
response caused by PMN & TH17 cells
Loures, FV & Calich, VLG
Instituto de Ciências Biomédicas, USP, São Paulo, Brasil.
Email: loures@icb.usp.br
Macrophages are the first host cells that interact with Paraccocidioides brasiliensis. Toll-like receptors (TLRs)
present in macrophages recognize molecular patterns of microorganisms and influence innate and adaptative
immunity. However, the role of TLR2 in paracoccidoidomycosis was never investigated. The aim of our work was
to characterize the involvement of TLR2 in murine paracoccidioidomycosis using TLR2-/- and C57BL/6 control
(WT) mice. In vitro cultivated macrophages, primed or not by IFN-γ, were challenged with yeasts and fungicidal
activity, cytokines and nitric oxide (NO) production assessed 48h later.
Mice were in vivo infected by the intratracheal route with 1x106 yeasts. After 48h, 2 and 11 weeks, the mice
were sacrificed and severity of infection was evaluated by CFU counts, presence of NO and cytokines in lung
homogenates besides flow cytometric analysis of lung infiltrating leucocytes. We verified that macrophages from
TLR2-/- mice secreted lower levels of NO, MCP-1 and IL-10 and have a decreased ability to interact with fungus
than those from WT mice resulting in decreased number of viable yeasts recovered 48h postinfection. In vivo,
diminished fungal burdens and NO levels were detected in the lungs of toll-deficient mice at all postinfection
periods assayed. Augmented levels of IL-17 were detected in the lungs of TLR2-/- mice by 48h postinfection.
At week 2, higher amounts of IL-17 and IL-23 associated with decreased levels of MCP-1 were found. At
week 11, increased IL-23 was associated with decreased concentrations of MCP-1, IL-10 and IL-12. At both
postinfection periods, increased numbers of polymorphonuclear (PMN) cells were observed in the lungs of TLR2−/−.
Furthermore, by week 2 decreased number of macrophages associated with augmented numbers of lymphocytes
were recovered from TLR2-/- mice relative to WT mice.
Lungs of TLR2-/- mice presented decreased number of activated CD4+ and CD8+ T cells at weeks 2 and
11 although lower numbers of regulatory CD4+CD25+FoxP3+ T cells were detected in this strain by week 11.
Equivalent patterns of mortality and lung lesions were observed in both mouse strains. Conclusion: absence of
TLR2 results in less severe infection but increased inflammatory response and lung pathology caused by PMN
and TH17 cells.
5-12
MYD88 is a dispensable factor in resistance to Paracoccidioides brasiliensis in a murine
model of blood-borne disseminated infection
Angel González1, 2, Alberto Yañez3, Daniel Gozalbo3 and Maria Luisa Gil3.
Microbiology School, Universidad de Antioquia, Medellín-Colombia. 2Medical and Experimental Mycology Group, Corporación para
Investigaciones Biológicas (CIB), Medellín-Colombia. 3Departamento de Microbiología y Ecología, Universitat de Valencia, València,
Spain. e-mail: agonzalezm@cib.org.co
1
We aimed at determining the role of MyD88, the universal toll-like receptor (TLR) adaptor protein, in murine
defenses against Paracoccidioides brasiliensis yeast infection.
Wild-type (WT) and MyD88-deficient mice infected i.v. with P. brasiliensis yeast cells had an equivalent fungal
burden as measured by colony forming units, as well as similar levels of pro-inflammatory cytokines (IL-1β, IL-6,
IL-12p70, TNF-α and MIP-2), Th1 (IFN-γ) and of Th2 (IL-4) in tissue homogenized supernatants. In both types of
mice the in vitro production of TNF-α, IFN-γ and IL-12p70, by antigen-stimulated splenocytes from mice previously
infected with the fungus were also similar.
Production of Th1 cytokines correlated with a similar frequency of IFN-γ producing CD4+ T cells. Recruitment
of neutrophils to the peritoneal cavity of i.p. infected mice was not affected in TLR2, TLR4 and WT mice, but
significantly decreased in MyD88-deficient mice. Additionally, in response to P. brasiliensis yeasts the in vitro
production of TNF-α by peritoneal macrophages from MyD88-, TLR2- and TLR4-deficient mice, was undiminished,
as compared with TNF-α production by macrophages from WT mice, even in the presence of laminarin.
Taken together, these data suggest that recognition of P. brasiliensis yeast occurs independently of the adaptor
molecule MyD88, as well as of other molecules such as TLR2, TLR4 and dectin-1.
192
5-13
The influence of ß2 integrin in the fungal burden in Paracoccidioides brasiliensis infected mice
J. Nunes2, Catão. E.1, Fraga C. L. F. 1Amaral.A1, Figueiredo. F.2, Felipe M. S.S.1 Faccioli. L. H. 3, Bocca A. L. 1,2
Instituto de Ciências Biológicas - Dept. de Biologia Celular, Brasília, Brazil, 2Área de Patologia - Faculdade de Medicina, Brasília, Brazil,
Dept. Análises Clínicas, Toxicológicas e Bromatológicas - Faculdade de Ciências Farmacêuticas de Ribeirão Preto, São Paulo, Brazil.
e-mail: albocca@unb.br
1
3
Paracoccidioides brasiliensis is a facultative intracellular fungus that causes aracoccidioidomycosis (PCM),
a chronic and granulomatous disease. As in other systemic mycoses, the host’s principal defense mechanism
against PCM is the cellular immunity mediated mainly by IFN-gamma activation of macrophages. Macrophages
appear to play a fundamental role in this infection acting as the first line of defense for organism. The first interaction
between macrophages-fungi is very important in this mycosis progression and some molecules are involved in
this interaction, like ß2 integrin. To investigate the importance of ß2 integrin to progression of the disease we
used gene knockout mice (KO) of ß2 integrins and C57Bl/6 (WT) both intravenous infected with the virulent P.
brasiliensis strain (Pb18). We analyzed the points 15, 30, 45, 60 days after infection. After 15 days of infection, in
ß2KO mice, no fungal cells were detected in the liver of mice, althougth we observed more fungus in the lung of KO
mice than in the control mice. After 30 and 60 days of infection, The KO mice there were less fungal burden when
compared with the control (WT mice). The spleen cell proliferation stimulated by concanavalin-A is increased in all
points of analyzes when compared with the WT. We observed also that macrophages infected with P. brasiliensis
in vitro had decreased in internalized yeast after 24h of co-culture. Recently was demonstrated that P. brasiliensis
regulates up-genes related to inflammation and phagocytosis, and this genes can be involved in fungi survive in
macrophages. Our data suggest that ß2 integrins that are probably involved with the fungi internalization contribute
with the survival of the fungi inside macrophages and this phagocytosis serve as a protected environment for the
P. brasiliensis. Financial Suport: CNPq.
5-14
Surface antigens increase susceptibility to Paracoccidioides brasiliensis infection via
interleukin-4 production
Oliveira L.L.1, Cavassani K.A.1, Rocha F.A.1; Vancim J.O.1; Moreira A.P.1; Campanelli A.P.1,4; Milanezi C.M.1, Martinez R.2;
Rossi M3, Oliveira E.B.1; and Silva J.S.1
1 Department of Biochemistry and Immunology, 2 Internal Medicine and 3 Department of Pathology, School of Medicine of Ribeirão PretoUSP, Ribeirão Preto, SP; 4 Department of Biological Sciences, Bauru Dental School, University of São Paulo, Bauru, SP; Brazil.
e-mail: licursi@usp.br
Introduction and Objectives: The paracoccidioidomycosis (PCM) is characterized by a chronic inflammatory
granulomatous reaction. The Th1/Th2 paradigm of acquired immunity to fungi is essential for a better understanding
of the immunoregulation which occurs during fungal infections. Classical studies on the immune responses
developed by patients with polar forms of paracoccidioidomycosis (PCM) have demonstrated a Th1-biased
immune response in the asymptomatic and mild forms of PCM, whereas a Th2 pattern has been associated with
the severe disease. In this work, we evaluated the ability to induce specific suppression in a murine model by the
inoculation of surface/secreted P. brasiliensis antigens (sPbAg). These results can provide new information that
may be important in the understanding of immunoregulatory perturbations observed in paracoccidioidomycosis.
Methods and Results: C57BL/6 and IL-4KO mice were immunized three times by subcutaneous route (three
weeks apart) with sPbAg. Yeast cells (Pb18) were shook vigorously (in PBS) for 30s in vortex and the supernatant
(sPbAg) was used in the assays. Control mice were injected with PBS. Two weeks after the last boost, the animals
were challenged (iv) with viable yeast forms of P. brasiliensis. The results showed the inoculation of sPbAg prior
to infection resulted in a severe granulomatous lesions and fungal dissemination to the all organs analyzed.
Clearly, increased fungal growth after sPbAg inoculation was dependent of IL-4, since high level of this cytokine
was found in the homogenates of lungs, spleen and liver of mice injected with sPbAg, but not in the control group.
Moreover, the injection of sPbAg did not affect the course of infection in mice deficient of IL-4, suggesting that the
exacerbation of PCM after inoculation of sPbAg is mediated by IL-4. Conclusion: Based on these data, our results
demonstrated that sPbAg is able to increased secretion of IL-4 and this fact impaired Th1 responses associated
with the cure of PCM. So is suggested that sPbAg play important role in the modulation mechanisms for the
installation of primary infection in hosts. Financial support: CNPq, FAPESP.
193
5-15
FBP plays a role in the imunosuppression on the course of Paracoccidioides brasiliensis
infection
Mariano, V.S.1, Oliveira, L.L.1; Coltri, K.C.1; Vendruscolo, P.E.1, Ferraz, D.B.1, Roque-Barreira, M. C.1 and PanuntoCastelo, A.2
1 Faculty of Medicine of Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil. 2 School of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP,
Brazil.
e-mail: vaniasmariano@usp.br
Paracoccidioidomycosis (PCM) is a chronic, granulomatous and progressive disease, which is associated
with various degrees of suppressed cell-mediated immunity. In the present study we described the isolation in
the NaCl 1M-eluted fraction (FBP) fraction upon affinity chromatography on immobilized fetuin of the soluble
antigen fraction from P. brasiliensis. Although these fetuin-binding proteins were divalent cation-dependent,
they did not present either enzymatic or lectin activity.
Electrophoresis analysis of FBP revealed a major protein of 60kDa and four minor proteins of 50, 35, 24 e
14kDa. In a blotting assay, we showed that 60kDa protein was bound by pulmonary extracellular matrix from
mouse, suggesting that FBP can play important role in the paracoccidioidomycosis (PCM) and, consequently,
be a therapy target. When mice were treated 21 days postinfection with FBP or saline emulsified in complete
Freund adjuvant (CFA), a strong adjuvant to protective Th1 immune response against PCM, we observed that
CFA-treated mice were protected, whereas FBP+CFA-treated mice reversed the protection.
Although lungs from FBP+CFA-treated mice presented organized granulomas with few compromised
tissue, there was a large number of viable fungi inside the granuloma. Consequently, the CFU number in the
organs of these mice was higher than that from CFA-treated mice. In addition, lung homogenates from FBP
mice had high levels of TGF-β, a known immunosuppressor cytokine. In vitro analysis, FBP had a proliferative
effect under B cells and induced IL-10 production.
On the basis of these results, we hypothesized that FBP suppressed the protector immune response
triggered by CFA, leading to persistent viable fungi infection inside the granulomas. Moreover, high levels
of TGF-β were observed in the pulmonary epithelium from FBP+CFA-treated mice. Altogether, these results
indicate that FBP plays an important role in the imunosuppression produced on course of P. brasiliensis
infection, and open perspectives of intervention in this suppressive process, leading to a beneficial effect on
the infection severity. Support: CAPES, CNPq, FAPESP.
5-16
Paracoccidioides brasiliensis inhibits human neutrophils apoptosis
M.J. Acorci1, Dias-Melicio L.A.1, Golim M.A2, Bordon-Graciani A.P.1, Peraçoli M.T.S.1, and Soares A.M.V.C.1
Department of Microbiology and Immunology, Biosciences Institute, São Paulo State University, Botucatu, São Paulo, Brazil.
Blood Center, School of Medicine, São Paulo State University, Botucatu, São Paulo, Brazil.
e-mail: ladiasmelicio@ibb.unesp.br
1
2
Botucatu
Paracoccidiodomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents
a wide spectrum of clinical manifestations. Because of the great number of neutrophils (PMNs) found in the
P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development
of the infection. This fungus is found intracellularly in PMNs and monocytes/macrophages, suggesting that it
is capable of evading damage and surviving inside these cells.
Thus, the goal of this study was evaluate if P. brasiliensis induce PMNs apoptosis suppression, and
if this process was related to IL-8 production, a chemokine involved in apoptosis delay. PMNs apoptosis
and intracellular levels of IL-8 were analyzed by flow cytometry, and culture supernatants IL-8 levels were
evaluated by ELISA. We showed that Paracoccidioides brasiliensis inhibited human neutrophils apoptosis.
We also demonstrated that PMNs challenged with the fungus produced high levels of IL-8.
These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to
escape killing, P. brasiliensis can extend the life span of PMNs by inducing autocrine IL-8 production, probably
making these cells suitable for survival and multiplication
194
5-17
Prostaglandin inhibits Paracoccidioides brasiliensis killing by human monocytes: Reversal
by activation with IFN-γ, TNF-α & GM-CSF due to increased H2O2 production
A.P. Bordon-Graciani, Dias-Melicio L.A., Acorci M.J., Peraçoli M.T.S., and Soares A.M.V.C.
Department of Microbiology and Immunology, Biosciences Institute, São Paulo State University, Botucatu, São Paulo, Brazil.
e-mail: ladiasmelicio@ibb.unesp.br
Paracoccidioides brasiliensis (Pb), the etiological agent of paracoccidioidomycosis, is a dimorphic fungus
that survives within nonactivated human monocytes/macrophages. Previous studies have demonstrated that
the lack of fungicidal activity by nonactivated human monocytes is associated to fungus capacity of inducing
prostaglandins release, since a significative fungicidal activity was detected after monocytes treatment with
indomethacin (INDO), a cyclooxigenase inhibitor. Thus the purpose of this work was to assess if the inhibitory
effect of prostaglandins could be reversed by cells activation with IFN-γ, TNF-α and GM-CSF.
Moreover, we asked if this process could be associated with alterations on the levels of killing effector
molecules such as NO and H2O2 and/or cytokines such as TNF-α, IL-6 and IL-10. Peripheral blood monocytes
obtained from 18 healthy donors were treated only with INDO or activated with IFN-γ, TNF-α or GM-CSF in
presence or absence of INDO for 18h, and further challenged with high (Pb18) or low (Pb265) virulent strain of
Pb for 4h. Then, cultures were evaluated for fungicidal activity and for H2O2 and NO release, and expression of
inducible nitric oxide synthase (iNOS) mRNA by real-time RT-PCR. The concentrations of TNF-α, IL-6 and IL-10
on supernatants of cocultures were evaluated by ELISA.
As expected, nonactivated cells lacked fungicidal activity against both strains. Killing activity against two
strains was significantly increased after cells incubation with INDO and/or cytokines. However, Pb265 killing
was always higher than that detected for Pb18. A clear association between killing and increased H2O2 and TNFα levels was observed. The iNOS mRNA expression showed interesting results, indicating a modulatory effect
of prostaglandin upon this expression via TNF-α production, what could indicate the role of NO in this process.
Nevertheless, both strains were capable to inhibit the NO production.
Taken together, these results strongly suggest that human monocytes challenged with Pb release
prostaglandins that inhibit the capacity of these cells to produce adequate levels of H2O2 and TNF-α. This effect
may be compensated by cells incubation with cytokines that activates them to release higher levels of these
mediator with consequent Pb killing.
5-18
Production of leukotriene B4 By different strains of Paracoccidioides brasiliensis
L.A. Dias-Melicio, Biondo G.A., Bordon-Graciani A.P., Acorci M.J., Peraçoli M.T.S., and Soares, A.M.V.C.
Department of Microbiology and Immunology, Biosciences Institute, São Paulo State University, Botucatu, São Paulo, Brazil.
e-mail: ladiasmelicio@ibb.unesp.br
Leukotrienes (LT) are eicosanoids derived from arachidonic acid (AA) metabolism and produced via
lipoxygenase (LOX) followed by conversion into leukotriene B4 (LTB4). Host cells are one source of eicosanoids
during fungal infection; however, another potential source of eicosanoids is the fungal pathogen itself.
Independently of the source, the leukotrienes production is critical for the fungal survival and/or growth and for
the modulation of host immune response.
Thus, the purpose of this study was to investigate if different strains of P. brasiliensis (Pb18, Pb265, Bt79,
Bt192) have the capacity to produce LTB4 in vitro, and if this production is related to the fungal survival and/or
growth. Additionally, we evaluated if these strains could utilize exogenous source of AA to this production. The
four strains were cultured during 4 and 8 hours, and after the LTB4 levels were measured in supernatants culture
by ELISA.
The results demonstrated that all strains produced high levels of LTB4 at 4 hours of culture, however, after
8 hours of culture, almost all strains showed significant decrease in LTB4 levels. The addition of AA in the
fungal cultures significantly increased the LTB4 production in all cultures. The treatment of fungal cultures with
MK 886, an inhibitor of leukotrienes synthesis, significantly inhibited the synthesis of this mediator, as well as
decreased the fungal viability. Then, our results show that P. brasiliensis produce LTB4 utilizing endogenous and
exogenous sources of AA and this production is involved in the fungus survival.
195
5-19
Comparative analysis of the infection evolution between isolates Pb 18 and Pb 01 of the
Paracoccidioides brasiliensis
C. L. F. Fraga1, Siqueira I. M.2, Ribeiro, A.M. 2, Nunes J.1, Figueiredo F.1,3, Felipe M. S.S.1,2, Bocca A.L.1,2
Pós-graduação em Patologia Molecular, Faculdade de Medicina, UNB, Brasilia – Brasil. 2Departamento de Biologia Celular, Instituto de
Biologia, UNB, Brasília – Brasil. 3Área de Medicina, Universidade Católica de Brasilia, Brasilia – Brasil. e-mail: albocca@unb.br
1
There is a remarkable genetic diversity between different isolates of Paracoccidioides brasiliensis. After the
finding of genetic variability within the species, it was concluded that the Pb 01 isolate occupied a special position
in the group, differing from the Pb 18 isolate. However, all the knowledge about the immune response during the
development of the Paracoccidioidomycoses in experimental models is based on protocols that use either the
virulent strain Pb 18 or the non-virulent strain Pb 265, obtained from the state of São Paulo, Brazil. Little is known
about the Pb01 strain, specially regarding its virulence and the induced immune response in experimental animal
models. The aim of this study was to verify the immune response in mice infected with Pb 01 compered to the Pb18
strain which is known the pathogenicity in vivo. For that purpose, mice C57Bl/6 was infected with 1x107 cells/ml e.v.
and the evolution of the disease was analyzed, by histopathological studies, counting colony-forming units (CFUs)
as well as by spleen cells proliferation on days 15th, 30th, 45th, 60th, 75th and 100th post-infection. It was observed
that Pb18 and Pb01 strain have different pathogenicity in the Paracoccidioidomycosis infection. Compared with
Pb01 strain, Pb18 caused more inflammatory tissue reaction such as a decrease lymphoproliferative activity. The
lung from mice infected with Pb01 revealed a lower progressive inflammatory response, leading to few granuloma
formations after 45 days of infection and a significant reduction in pulmonary fungal burden when compared with
mice infected with Pb18. In additional, the analysis of the immune response is in progress, measuring the levels of
IL2, IL4, IL10, IL12, IFNγ, TNFα, IgGs and NO, in the supernatants of cell cultures.
Financial Support: CNPq.
5-20
Paracoccidioides brasiliensis infection determines dendritic cells to differentiate to the
plasmocytoid subpopulation which induces a more severe pulmonary infection when
transferred to resistant mice
A. Pina, and Calich, V.L.G.
Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil
e-mail: adripina@usp.br
B10.A and A/J mice were previously characterizes as susceptible and resistant strains to pulmonary
Paracoccidioides brasiliensis (Pb) infection. Resident alveolar macrophages (AM) and dendritic cells (DC), the
first cells that interact with Pb in lungs play a fundamental role in the immunity that further develops. Differently
from expected, resident AM from susceptible mice present an increased ability to kill fungal cells and to secrete
pro-inflammatory cytokines. Thus, the aim of this work was to analyze the role of another antigen presenting cell,
the dendritic cell, in the genetic resistance to Pb.
Bone-marrow derived DC from B10.A and A/J mice were in vitro challenged with Pb yeasts (1:25 fungus:DC
ratio) or soluble Pb antigen (100 µg/ml) and cytokines and oxide nitric (NO) were determined 48h later in culture
supernatants. Costimulatory molecules were analyzed by flow cytometry and some DC preparations were used
to study their stimulatory activity using homologous and heterologous naïve lymphocytes previously stained with
CSFE. Pb-activated DC of B10.A mice produced higher levels of IL-12, TNF-α and NO when compared with DC of
A/J mice. Viable yeast cells were more efficient activators than the soluble Pb antigen. DC from A/J mice presented
augmented co-expression of CD11c and B220 whereas Pb yeasts were able to induce high expression of CD11c+
CD11b+ in B10.A DCs. This indicates that A/J cells preferentially maturate to a plasmocytoid subpopulation but
the myeloid subset was observed with B10.A cells. Differently from B10.A cells, A/J DCs induced IL-2 secretion
and a high lymphoproliferation of homologous lymphocytes. Previous treatment of resistant but not susceptible
mice with A/J DCs led to a more severe infection as determined by organ fungal loads, secretion of cytokines
and activation of regulatory T cells. B10.A DCs, however, did not substantially alter the pattern of infection and
adaptative immunity of both mouse strains. Thus, differently from expected, DCs from resistant mice are able to
transfer a pattern of immunity which results in more severe infection.
FAPESP
196
5-21
IL-10 deficiency determines a better fungicidal ability associated with overproduction of
IFN-γ and nitric oxide by Paracoccidioides brasiliensis infected macrophages
Costa, T.A.1 and Calich, V.L.G.1
1 Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil. e-mail: tacosta@usp.br
In systemic mycosis, the interaction between tissue macrophages and fungal cells play a fundamental role
in the protective mechanisms of innate immunity and influences adaptative immunity that further develops.
These phagocytes exert their function by ingesting and inactivating invading organisms, producing cytokines and
interacting with other cells of the adaptive immune system. Several clinical observations suggest that there is an
inverse relationship between interferon (IFN)-γ and IL-10 production in patients with fungal infections. Studies in
a pulmonary model of paracoccidioidomycosis demonstrated that the resistance in mice is linked to a preferential
secretion of IFN-γ whereas susceptibility is associated with very low levels of this cytokine. Both susceptible and
resistant mice produced IL-10, a macrophage deactivating cytokine, throughout the course of the disease. To
better understand the role of IL-10 in murine PCM, macrophages from IL-10-deficient mice (IL-10 KO) and their
normal (WT) C57BL/6 counterparts were comparatively studied after P.brasiliensis infection. IFN-γ (10,000 pg/
mL) primed and unprimed peritoneal macrophages (1x106 cells/well) of both mouse strains were co-cultivated in
vitro with 4x103/mL P.brasiliensis yeasts (1:50 fungus : macrophage ratio). 48h after infection fungicidal activity
was assessed by CFU counts, and supernatants used to measure the levels of nitric oxide (Griess reagent) and
cytokines (ELISA). It was verified that macrophages from IL-10 KO mice produced higher levels of NO than those
from WT (WT, 1.35±9.35 µM; IL-10 KO 60.31±1.47 µM, p<0.05). Compared with IL-10-deficient macrophages,
higher number of viable yeast cells was recovered from normal macrophages after 72h co-cultures. Thus,
4.82±0.07 log10 viable yeasts were recovered from normal macrophages whereas 4.10±0.07 log10 were obtained
from IL-10-deficient cells (p<0,05). Very interestingly, the increased fungicidal ability of IL-10 KO macrophages
could be correlated with their increased ability to secrete IFN-γ (KO 1407.02±126,27; WT <3,2). In addition, the
macrophage chemotactic protein (MCP)-1 was also produced in higher levels by IL-10 deficient macrophages (KO
3083.08±86.57; WT 1637.34±137.48). Our findings suggest that endogenous secretion of high levels of IFN-γ
determines the better fungicidal ability of IL-10 KO macrophages and demonstrate the deleterious role that this
cytokine can play in the resistance mechanisms to P.brasiliensis infection.
5-22
CD28 costimulatory molecule deficiency results in more severe Paracoccidioides brasiliensis
infection but protects mice from life-threatening immunopathology
M. Felonato and Calich, V.L.G.
Depto. de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brasil. e-mail: felonato@gmail.com
CD28 is a costimulatory molecule of T cells that interacts with B7-1/B7-2 antigens expressed by antigen
presenting cells. As immunoprotection in PCM is mainly mediated by T cell immunity, we investigated the impact
of CD28 deficiency in the severity of pulmonary infection of mice. The effect of CD28 deficiency was also studied in
the immunoprotection induced by a previous subcutaneous (s.c.) infection. Therefore, CD28KO and WT C57BL/6
mice were i.t. infected with 1x106 yeast cells and sacrificed 72 hours, 2 and 10 weeks post-infection. Fungal loads
were analyzed by CFU counts and cytokines and antibodies by ELISA. By week 2, compared with WT counterparts,
KO male mice presented higher pulmonary fungal burdens accompanied by decreased IgM, and low hepatic IL-2,
and GM-CSF. IL-10 and GM-CSF were present in elevated amounts in the lung of CD28-deficient mice. At week
10, the increased fungal burden of CD28KO was associated with decreased antibody production (IgM, IgG1,
IgG2a, IgG2b) and diminished secretion of pulmonary IL-12 and hepatic IL-12, IFN-γ, IL-10 and GM-CSF. IL-5 was
the only cytokine present in elevated levels in the lungs of CD28KO mice. Both, CD28 KO and WT mice developed
equivalent immunoprotection when pre-immunized by the s.c. route. Immunoprotection, in addition, was shown to
be antibody-independent since the phenomenon was seen in KO mice despite their impaired ability of antibody
production. Interestingly, mortality studies revealed that exacerbated fungal burdens were not deleterious to KO
mice since this mouse strain presented higher survival times when compared with WT mice. Again, CD28 KO
survivors showed diminished ability of antibody secretion. In conclusion, our work showed for the first time that
the CD28 molecule exerts an important regulatory role in PCM immunity and its deficiency leads to a more severe
infection associated with decreased production of antibodies and cytokines probably due to the deficient T cell
activation. Moreover, our studies demonstrated that high fungal burdens in the absence of effector adaptative
immunity are not deleterious to P.brasiliensis infected hosts, clearly indicating the dual role played by the immune
response, which can protect but can also induce life-threatening pathology. Supported by FAPESP
197
5-23
Nitric oxide but not treg cells plays a major immunoregulatory role in a pulmonary model
of fungal infection
S. Bernardino and Calich V.L.G.
Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil
e-mail: sibern@icb.usp.br
Paracoccidioidomycosis (PCM), caused by the inhalation of Paracoccidioides brasiliensis spores, is a major
pulmonary fungal disease in South America. The protection in PCM is mainly mediated by cellular immunity.
Inducible nitric oxide synthase (iNOS) is a nitric oxide (NO) generating enzyme which plays an important role in
clearance of microbial infections, but its overproduction can lead to immunessupression of cellular immunity. We
investigated the role of NO in the acute and chronic phases of the disease using C57BL/6 iNOS KO and their WT
counterparts infected by the intratracheal route with 1 million yeasts. Compared with WT, at week 2 post infection
iNOS KO mice presented decreased fungal loads and high levels of TNF-alpha in the lungs. These findings
were associated with increased number of infiltrating pulmonary CD4+CD69+, CD4+CD25+, CD8+CD69+ T cells and
activated alveolar macrophages expressing CD40+, CD80+, CD86+ , Iak and CD11 molecules. In contrast, at week
10 post-infection KO mice presented higher fungal burdens in the lungs which, however, were still associated with
elevated number of CD4+CD69+ and CD4+CD25+ T cells, macrophages expressing CD80+ and CD40+ molecules
but an equivalent number of regulatory T cells TCD4+CD25+FoxP3+. In agreement, iNOS KO mice developed well
organized pulmonary granulomas which control the fungal dissemination and resulted in similar mortality rates
for both mouse strains. Early in vivo TNF-alpha depletion was able to abrogate the CFU differences between KO
and WT mice observed at week 2 of infection. Furthermore, this treatment resulted in increased and precocious
mortality of KO, but not WT mice. In conclusion, despite its importance in fungal clearance, NO exerts an important
suppressive effect in cellular immunity and TNF-alpha production which plays a fundamental role in the organization
of lesions, control of yeasts dissemination and host survival.
Supported by FAPESP
Key words: Paracoccidioidomycosis, nitric oxide, TNF-alpha, T cells, granuloma
5-24
Role of CCR4 in the experimental infection by Paracoccidioides brasiliensis: Migration
control of CD4+CD25+ T cells to lesion site
Rocha F.A. 1, Oliveira L.L.1, Massafera Tristão FS1; Milanezi C.M.1 and Silva J.S.1
1 Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto-USP, Ribeirão Preto, SP; Brazil
e-mail: nandaffer@usp.br
Introduction and Objectives: Paracoccidioidomycosis (PCM) is a chronic and granulomatous disease
caused by the inhalation of conidia forms of Paracoccidioides brasiliensis. The infection results in the formation of
granulomas containing viable yeast cells that are the fungal sources for disease reactivation. Because CD4+CD25+
regulatory T cells (Tregs) are in the lesions of patients with paracoccidioidomycosis, the migration of Treg cells
is dependent on the axis chemokine-chemokine receptors, and CCR4 ligands are produced in P. brasiliensisinduced lesions, we aimed to evaluate the role of this receptor during the P. brasiliensis infection in CCR4-/- and
C57BL/6 wild-type mice. Methods: Mice were infected with 1x106 yeast cells of a P. brasiliensis strain (Pb18)
and analyzed weekly until 8 weeks. The lung, liver and spleen from mice were used for histopathology and flow
cytometry analyses, and for quantification of viable fungal. We observed an increase in fungal burdens in the lungs
of CCR4-/- mice in the early infection. The cell surface expression of CD3, CD4, CD8, CD19, NK, Gr1, CD103,
CTLA-4 (CD152), CD11c, CD11b and GITR, and intracellular Foxp3 were assessed by flow cytometry, for best
chraracterization of leukocytes isolated from the lungs. The histopathological section of organs were stained with
H&E and analysed for Morphology and morphometry of granulomas. Conclusion: Probably, the aspects of the
maintenance of chronicity during the infection with P. brasiliensis can be influenced by CD4+CD25+ T cells expressing
CCR4. The potent suppressor activity of Tregs that migrated to the granulomas, in response to chemokine ligands
produced in the lesions, became this chemokine receptor an important target in immunotherapeutic strategies to
control PCM.
Financial support: FAPESP, CAPES, CNPQ, FAEPA
198
5-25
Granulomatous imflammation and fibrosis in mice with chronic pulmonary Paracoccidioidomycosis:
Immune-related associations
Naranjo TW1, Lopera DE1, Duque JJ2, Diaz-Granados LR3, Restrepo A1 and Cano LE1,4
1 Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia. 2 Departamento de Patología, Facultad
de Medicina, Universidad de Antioquia. 3 Facultad de Medicina, Universidad Pontificia Bolivariana, Medellín-Colombia. 4 Molecular Microbiology Group, Escuela
de Microbiología, Universidad de Antioquia, Medellín – Colombia. e-mail: tnaranjo@cib.org.co
Background: In our experimental model of pulmonary paracoccidioidomycosis (PCM), infection is induced by
the intranasal (i.n.) inoculation of Paracoccidioides brasiliensis conidia. This model has the advantage of reproducing
several aspects of the human disease allowing to study pulmonary tissue response at different post-infection periods
taking into consideration aspects as diverse as cytokine production, inflammatory response, fibrosis development,
treatments evaluation and others. Aim: To determine if during the chronic stages of experimental pulmonary PCM an
immune-related association could be established between the granulomatous inflammation and fibrosis Materials
and methods: BALB/c male mice, 7-8 weeks old, were inoculated i.n. with 4x106 P.brasiliensis viable conidia, control
animals received PBS. Ten mice were sacrificed at different times, 2h, 4, 8, 12 and 16 weeks post-inoculation. The
lungs of 5 mice/period were embedded in paraffin, sectioned and stained with H&E, Masson’s trichromic and reticulin
for histologic analysis; two independent pathologists evaluated granulomatous inflammation and fibrosis. Fibrosis
was defined as the observation of thick collagen fibers around inflammatory lesions in the presence of reticulin fibers.
The lungs of the remaining 5 mice/period were homogenized in 2 ml of proteases’ inhibitors cocktail and resultant
supernatants were frozen at -70oC for cytokine (TNF-α, IFN-γ, IL-13, IL-1β, TGF-β determination and PGE2 assays
by commercial ELISAs. Results: Sequential histopathologic analyses showed that P.brasiliensis infection induced
a significant (p≤0.001) increase of the peribronchial granulomatous inflammation with involvement of 10 to 35%
of the lung’s area, with values of 10±7, 22±15, 20±11 and 15±10% at 4, 8, 12, and 16 weeks, respectively. Proinflammatory cytokine levels, (TNF-α, IL-1β and PGE2 were increased in same periods where high inflammatory
responses were observed. In addition, pro-fibrotic cytokines (IL-13, TGF-β) showed a significant increase during the
periods immediately before establishment of fibrosis (8 weeks post-infection); this fibrous process progressed until the
16 weeks post-infection. IFN-γ levels did not show significant increase during the studied periods. Conclusion: This
study reveals association, albeit not direct, causality between pulmonary histopathologic evidence (granulomatous
inflammatory response, fibrosis) and cytokine behavior. Financial support: CIB, UdeA, Colciencias (Project
No.2213-04-16439).
5-26
Alterations of granuloma architecture, cellularity and severity of the early infection in murine
paracoccidioidomycosis by IFN-γ, celecoxib or lumiracoxib treatment
R.F.S. Molina, Nishikaku. A.S, Albe. B.P, Burger. E.
Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo – Brasil. e-mail: raphaelmolina@usp.br
In the present study we sought to interfere in the fibrotic process, which worsens the quality of life in patients with
paracoccidioidomycosis (PCM). We infected intraperitoneally B10.A female mice, susceptible to Paracoccidioides
brasiliensis (Pb), with the highly virulent Pb18 isolate. We treated these animals with IFN-γ (with antifibrotic and
fungicidal activity) or the anti-inflammatories drug Lumiracoxib or Celecoxib (which affect neutrophil migration
and possibly inhibit components of the extracellular matrix - ECM). Control groups received no drug treatment.
After 15 days of inoculation, we collected the spleen, liver, lungs and omentum. We observed that the omentum
of mice treated with either anti-inflammatory drug had more than twice the size and number of lesions compared
with the same organ of only infected animals or those treated with IFN-γ. Through analysis of histological slides
stained with HE, Giemsa, Grocott and Picrosirius we observed, in omentum of the non-treated infected mice, few,
loose granulomas with thin collagen fibers. These lesions had Pb with typical morphology confined in granulomas,
extensive neutrophil infiltration, numerous giant cells and few eosinophils. Mice treated with IFN-γ showed few,
compact granulomas, with large quantity of thin and coarse collagen fibers circumscribing few Pbs with morphology
ranging from typical to atypical, and major presence of neutrophils, giant cells and eosinophils. Mice treated with
either anti-inflammatory drug showed a large number of loose granulomas, with reduced numbers of thin collagen
fibers. In this group there was a high number of fungi with typical morphology disseminated throughout the tissue,
reduction of neutrophils and eosinophils, but presence of many giant cells. We recovered more viable Pb from the
omentum of animals treated with either anti-inflammatory drug than from the other groups, and the other hand,
mice treated with IFN-γ yielded the fewest number of Pb. Our results suggest that in susceptible animals to PCM,
at this period of infection, the treatment with IFN-γ restrained the infection through segregation within compact
granulomas by ECM fibers, causing a decrease of fungal load, and the treatment with anti-inflammatory drugs did
not contain the infection but, instead, facilitated fungal dissemination in the entire tissue examined.
Financial Support: FAPESP – 06/60091-6 and 07/56745-3; CNPq – 307492/2006-0.
199
5-27
Therapeutic activity of a DNA vaccine using the gene hsp65 from Mycobacterium leprae
for experimental paracoccidioidomycosis
A. M. Ribeiro1, Souza A. C. O.2, Amaral A.C.5, Fraga C. L. F. 1, Nunes J.1, Salles B. C.2, Coelho-Castelo A. A. M.3,
Figueiredo F.4, Silva C. L.3, Felipe M. S. S.2, Bocca, A. L.1
Área de Patología, Faculdade de Medicina, UNB, Brasilia – Brasil. 2Departamento de Biologia Celular, Instituto de Biologia, UNB, Brasília
– Brasil. 3 Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, USP, São Paulo – Brasil. 4Área de
Medicina, Universidade Católica de Brasilia, Brasilia – Brasil. 5Ciências Genômicas e Biotecnología, Universidade Católica de Brasilia,
Brasilia – Brasil.
e-mail: ribeiroalice@yahoo.com.br
1
Heat shock proteins are recognized as important molecules in the modulation of the immune system and
are highly conserved among different organisms. The DNA-hsp65 vaccine from Mycobacterium leprae has been
shown to have prophylactic and immunotherapeutic effects against various diseases, for instance, tuberculosis,
arthritis and leishmaniasis. In this work, we evaluated the effectiveness and immunomodulatory potential of the
DNA-hsp65 treatment in model BALB/c mice infected with Paracoccidioides brasiliensis, the etiological agent of
Paracoccidioidomycosis, the most important endemic mycosis in Latin America.
The DNA-hsp65 vaccine conferred protection against this pathogen for therapeutic assays, as indicated by:
1) a significant reduction in pulmonary fungal burden; 2) a decrease in pulmonary damage and in the presence
of collagen in granulomas (revealed by histological analyses of the pulmonary tissue); 3) the reestablishment of
spleen cells proliferation; increase levels of IL-12, IFN-γ, TNF-α and IgG2a and unaltered levels of IL-4, IL-10
and IgG1 compared with the control mice. Together, these findings indicate that, in mice, the treatment with the
DNA-hsp65 vaccine protect mice against paracoccidioidomycosis. Previously we demonstrated that DNA-hsp65
immunization protects mice against paracoccidioidomycosis. Our results open new perspectives on prevention
and treatment of other systemic mycoses.
Financial Suport: CNPq.
5-28
Evaluation of the protection induced by the immunization with radioattenuated yeast cells
of Paracoccidioides brasiliensis in animal model
Estefânia M.N. Martins1,2, Bernardo S. Reis2, Alfredo M. Goes2 and Antero S. R. Andrade1
Laboratório de Radiobiologia, CDTN / CNEN, Belo Horizonte – Brasil. 2 Departamento de Bioquímica e Imunologia, UFMG, Belo Horizonte
– Brasil.
e-mail: estefaniabio@yahoo.com.br (presenting Author)
1
Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin
America. Currently there is no effective vaccine. In our laboratory yeast cells of P. brasiliensis were attenuated by
gamma irradiation, which lose the reproductive ability, while retaining the morphology, the synthesis and secretion
of proteins, the oxidative metabolism and the expression of the antigens present in the native yeast. The aim of
present work was evaluate the protection elicited by the immunization of BALB/c mice with radioattenuated yeast
cells of P. brasiliensis.
BALB/c mice were divided in two groups that were immunized one or two times, respectively. For each group
the mice were divided in three sub-groups that were challenged 30, 45 and 60 day after immunization. Recovery of
CFUs, histopathological analysis and cytokines determination (IFN - γ, TNF - α, IL - 10 and IL – 5) were performed
one and three months post challenge. The sera were collected weekly to evaluate the IgG antibody titers and the
IgG1 and IgG2a pattern in the course of infection. To evaluate the type of elicited immune response the cytokines
were determined by real time PCR. The mice immunized once presented a significant reduction in the CFUs
recovery when examined 30 days after the challenge.
However, 90 days post challenge the number of CFUs recovered from all organs increased. The use of two
immunizations increased the protector effect and a remarkable protection was verified 90 days post challenge,
showing that a long lasting protection able to eliminate the fungi cells of the tissues was elicited. At the same time
the levels of IgG2a and IFN - γ, were high while a very low production of IL-10 and IL-5 was verified, suggesting
that a Th1 pattern was dominant. The current study showed the efficacy of radioattenuated yeast cells of P.
brasiliensis for induction of protection in experimental PCM.
200
5-29
Modulation of experimental paracoccidioidomycosis by monoclonal antibodies against the
major diagnostic antigen, GP43
F. A. Pinto1, R. Puccia,2 C. J. da Silva1, J. E. Muñoz1, L. R. Travassos2, C. P. Taborda1
Instituto de Ciências Biomédicas, Departamento de Microbiologia, Universidade de São Paulo, São Paulo, Brasil. 2Departamento de
Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brasil. e-mail: fel-ipe@hotmail.com
1
Background: Antifungal chemotherapy is required for paracoccidioidomycosis (PCM) treatment, though
even after prolonged administration there is no assurance of complete destruction of the fungus. The period of
treatment depends on the drug used and the severity of disease. The role of antibody-mediated immunity in
host resistance to P. brasiliensis is less recognized. In several other systems, however, there is considerable
evidence that administration of monoclonal antibodies (mAbs) can modify the course of disease in mice infected
with fungi such as Cryptococcus neoformans, Candida albicans, Histoplasma capsulatum, Pneumocystis spp,
Fonsecaea pedrosoi and Aspergillus spp. In the present study a panel of monoclonal antibodies against gp43 was
utilized for evaluating the effect of passive immunization in mice intravenously infected with a virulent strain of P.
brasiliensis.
Methods: BALB/c mice were immunized with 1mg of the monoclonal antibody (mAb) 3E, 32H or an irrelevant
one by intraperitoneal route, 24 h before the intravenous infection with 3x105 yeast cells of P. brasiliensis (Pb18).
A maintenance dose was given every week for a month. A week after the last immunization, mice were sacrificed
and lung, spleen and liver were removed for measuring the fungal burden, cytokine levels (IL12, IL4, IL10 and
IFN-γ) and histology.
Results: Animals immunized with mAb 3E showed significant reduction of colony forming units (CFU) in lung
and spleen compared to the groups that received mAb 32H or irrelevant mAb. Histological analysis confirmed the
results obtained in the CFU assay although a significant difference in the cytokine levels between the groups was
not observed.
Conclusion: The anti-gp43 mAb 3E but not mAb32H or the irrelevant mAb significantly reduced the fungal
burden in BALB/c mice intravenuouly infected with P. brasiliensis, The present work also raises the possibility of
a potential therapeutic use of mAb3E.
Supported by Fapesp and CNPq.
5-30
The influence of Fonsecaea pedrosoi cell wall in peritoneal macrophage activation
Y.K.M. Nóbrega1, Lozano V.F.1, Figueiredo. F.3, Bocca A. L. 1,2
1
Área de Patologia - Faculdade de Medicina, Brasília, Brazil, 2Instituto de Ciências Biológicas - Dept. de Biologia Celular, Brasília, Brazil,
3
University Católica of Brasília – UCB - Brazil.
e-mail: albocca@unb.br
Chromabastomycosis is a chronic supporative granulomatous mycosis that exists all around the world. The
Fonsecaea pedrosoi is the most frequent etiologic agent in the worls. The infection begins with the trauma and the
implantation of conidia or hyphae fragments in the subcutaneous tissue producing the initial lesion. Inside the host,
a fungus structure differentiates into sclerotic forms allowing the establishment of the disease.
Considering the importance of an efficient immune cellular response, through the interaction between the
cells of immunologic system with the components of the fungus wall, the objective of the work was to analyze
the influence of the fractions from the F. pedrosoi cellular wall in the activation of murine peritoneal phagocytes.
Our results demonstrated that after 4 hours after the inoculation of the solutions the migration was constituted
predominantly by neutrophils and after 72 hours predominantly by macrophages.
The F2 fraction and the inactivate fungus stimulates predominantly B lymphocytes migration. The F1 and F2
fraction induced the high production of IL-12 and IL-10 in macrophages respectively. The macrophages stimulated
with F2 fraction showed a decreased phagocytosis index and an increased production in IL-10 production. Our
results suggest that, in chromoblastomycosis, the cell wall components presents in F1 or F2 fraction contribute to
induce a Th1 response or Th2 response.
Financial Suport: CNPq.
201
Poster Session
6
Treatment
6-01
Timing of itraconazole therapy onset and its impact on the progression of fibrosis
induced by Paracoccidioides brasiliensis conidia: Results in an experimental model of
paracoccidioidomycosis
DE. Lopera1, TW. Naranjo1, JJ. Duque2 , Diaz-Granados LR 3, RA. Jiménez 4, A. Restrepo1, AF. Zuluaga 5 and LE. Cano 1,6,
Grupo de Micología Médica y Experimental, Corporación para Investigaciones Biológicas, CIB. 2 Departamento de Patología, Facultad de
Medicina, Universidad de Antioquia, UdeA. 3 Facultad de Medicina ,Universidad Pontificia Bolivariana. 4 University of Nebraska, Nebraska
– USA. 5 Grupo GRIPE, Facultad de Medicina, UdeA. 6Grupo de Microbiología Molecular, Escuela de Microbiología, UdeA, Medellín –
Colombia. e-mail: dlopera@cib.org.co
1
Background and aim: The active stages of Paracoccidioidomycosis (PCM) are usually controlled by Itraconazole
(ITZ) therapy; however, patients often (~30-50%) develop pulmonary fibrosis and exhibit important functional respiratory
limitations. Our aim was to determine if in an experimental model of PCM, fibrous sequelae could be avoided or modified
depending on the time of initiation of ITZ therapy. Materials and methods: Male BALB/c mice (n=50) were infected
intranasally with 4x106 Paracoccidioides brasiliensis (Pb) conidia and distributed in three experimental groups: (i) nontreated mice (control), (ii) orally treated with ITZ (1mg/day by 8-weeks) starting at 4-weeks of infection (before fibrosis
consolidation) or (iii) orally treated with ITZ starting at 8-weeks of infection (when fibrosis is well-established). Sacrifice was
every four weeks from 0 to 16 and lungs were processed histologically using Masson’s trichrome and reticulin stainings.
Then, two pathologists registered the appearance (thin or thick) of collagen and reticulin fibers and scored their relative
increase respect to control as 0=no changes, 1=minor, 2=moderate or 3=major. Fibrosis was defined as the observation of
thick collagen fibers around inflammatory lesions in the presence of reticulin fibers whereas incipient fibrosis was taken as
presence of only thin fibers. Results: At 4-weeks post-infection, 80% of the Pb infected mice showed incipient pulmonary
fibrosis that, later on, (8 and 12-weeks) progressed to well-established fibrous sequelae in 60% of the animals, attaining
100% of them by 16-weeks. Group (ii) showed an early and significant reduction (p≤0,05) in the score assigned to thin
reticulin and collagen fibers deposition along with a score=0 to thick fibers (p≤ 0,05); consequently, at 16-weeks, 80% had
incipient fibrosis and none (0%) had well-established sequelae. In Group (iii) but only at the end of treatment (16-weeks),
a significant reduction in the score of thin fibers was observed, although formation of thick fibers was not completely
prevented. In fact, in sharp contrast with Group (ii), 40% of the mice had incipient fibrosis and 20% showed severe sequela.
Conclusions: The progression of pulmonary fibrosis in Pb-infected mice can be controled by an early ITZ treatment.
These experimental results open the possibility of avoiding fibrosis in the human patients by early therapy.
Financial support: Colciencias (Proyecto N° 2213-04-16439), UdeA, CIB.
6-02
Posaconazole for the treatment of paracoccidioidomycosis: First clinical results
Tobón AM1,2, Agudelo CA1,3,4, Restrepo A1
1 Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia. 2 Hospital La María, Medellín – Colombia 3 Universidad Pontificia
Bolivariana, Medellín – Colombia. 4 Hospital Pablo Tobón Uribe, Medellín - Colombia. e-mail: atobon@cib.org.co
The introduction of azolic derivatives in the treatment of paracoccidioidomycosis (PCM) drastically reduced the duration
of therapy, as well as the relapse rate. Posaconazole is a new antifungal azolic, liposoluble with a wide antifungal spectrum,
making an interesting option for PCM treatment. The results of posaconazol therapy in 4 adult PCM patients are presented.
Three of these patients had the chronic multifocal and one the subacute form. All patients were male, smokers, and had
a mean age of 52 years (range 24-73), One had sequelae of lung (fibrothorax, cor pulmonale) and kidney tuberculosis,
as well as adrenal insufficiency. In these chronic patients duration of their clinical course was 8.3.months; constitutional
and respiratory symptoms predominated accompanied by oral mucosae ulcerations. The X-rays revealed mixed bilateral
infiltrates and in one patient, a cavity in the upper field. As for the patient with the subacute form, an important gastrointestinal component was recorded with diarrhea of two months duration, involvement of the colon, lymph nodes, liver
and spleen. In the chronic patients diagnosis was accomplished by direct examination and isolation of Paracoccidioides
brasiliensis from clinical samples (sputum, oral exudates); these patients had reactive serological tests. In the subacute
form patient, diagnosis was made by lymph node and colon biopsies; his serologic tests were non-reactive. In these
four patients, the standard treatment with ketoconazole or itraconazole had failed, in one because concomitant anti-TB
therapy, another patient due to worsening of adrenal insufficiency and the last patient due to gynecomastia with clinical and
serologic failure. The juvenile case had failed to both amphotericin and itraconazol therapies. Once the failure was verified,
posaconazole at a 400 mg BID was instaurated. One of the patients with chronic disease died a month after therapy
initiation secondary to lung sequelae and adrenal insufficiency due to previous PCM and TB. Other two chronic patients
showed rapid clinical improvement and significant decreases in serologic titres after starting posaconazole. The infiltrates in
chest X ray´s dissapeared, leaving mild fibrotic sequelae. Both patients were followed up along three years, without relapse
of disease. The patient with juvenile form improved of his symptoms in the first month after start the posaconazole, followed
by disappearance of adenopathies; he remained without symptoms until end of therapy at six month. The colonoscopy
done at fifth month of therapy showed absence of any commitment. This report shows that posaconazole is a therapeutic
alternative for patients with PCM and failure of first treatment.
205
6-03
Variable effect of caspofungin on the mycelial and yeastlike phases of several strains of
Paracoccidioides brasiliensis
S. Rodríguez-Brito, Niño-Vega, G., and San-Blas. G.
Instituto Venezolano de Investigaciones Científicas (IVIC), Laboratorio de Micología, Apartado 21827, Caracas 1020A, Venezuela.
e-mail: srodrigu@ivic.ve
Caspofungin inhibits the activity of β-1,3-D-glucan synthase (GS), enzyme responsible for the synthesis
of β-1,3-glucan in the fungal cell wall. This polysaccharide is an essential component of this outer fungal cell
structure. In Paracoccidioides brasiliensis, β-1,3-glucan constitutes 36% and 5% of the mycelial and yeastlike
walls, respectively. Instead, the yeastlike cells synthesize α-1,3-glucan as the main wall component (45%), a
polysaccharide absent in the mycelial cell wall.
To study the effect of caspofungin on both morphological phases of P. brasiliensis, isolates IVIC Pb73 (ATCC
32071), IVIC Pb300 (soil), IVIC Pb377 (armadillo), IVIC Pb381 and IVIC Pb444 (patients), were grown for 4 days in
RPMI 1640 (GIBCO) medium, in the presence of caspofungin (0; 0.1; 0.5 and 1.0 µg/ml). Yeast cells were incubated
at 37ºC; cell density was followed by turbidimetry in Klett units, every 24 hours. Mycelia grew at 23ºC; growth was
measured daily by dry weight. At 1 µg/ml, caspofungin inhibited yeast growth in different proportions depending
on the isolate: Pb73 (65%) > Pb381 (52%) > Pb300 (35%) > Pb377 (22%) > Pb444 (21%) Considering the low
amount of β-1,3-glucan in the yeast cell wall of P. brasiliensis, the performance of caspofungin against the yeast
phase of P. brasiliensis was surprising. The mycelial phase, as expected, was highly susceptible to caspofungin,
inhibition fluctuating between 74% (Pb381) and 81% (Pb73). Scanning electron microscopic observations indicated
structural modifications in the cell walls of both phases as a consequence of caspofungin action.
We are currently working on cell wall composition analysis for both phases of the isolates under study, in
order to determine whether differences in P. brasiliensis cell wall composition, particularly with regards to the
participation of β-1,3-glucan and the related activity of its synthesizing enzyme (GS), could somehow explain the
observed isolate–dependent inhibition by caspofungin.
Acknowledgement: Merck, Sharp & Dohme (Caracas, Venezuela), for partial support.
6-04
Assessment of the efficacy of voriconazole, comparativily to other antifungals, in
paracoccidioidomycosis experimental of the rats
D. S. Granzoto 1, Vitali, L. H.1; Martinez, R.1
1 Departament of Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo-USP, Ribeirão Preto, Brazil.
emal: danielagranzoto@yahoo.com.br
In this study it was evaluated the effectiveness of voriconazole in comparison to ketoconazole, fluconazole,
itraconazole and sulfamethoxazole-trimethoprim in the experimental infection of female Wistar rats by
Paracoccidioides brasiliensis. The parameters used to quantify the response to the drugs were counts of colony
forming units (CFU) of Paracoccidioides brasiliensis in the lung and spleen and animal survival. The antifungal
treatment was started seven days after infection. The medications were administered by gavage for 6 to 15 days,
according to the experiment, the following daily doses in mg/kg weight: voriconazole-5, 7, 10 and 20; ketoconazole10, 12 and 15; fluconazole-6; itraconazole-4; sulfamethoxazole-trimethoprim-100, 120 and 150(sulfamethoxazole).
In evaluation of the effectiveness of the drug made by counting the CFU, the animals treated with voriconazole7mg/kg/dia showed a significant reduction of infection splenic compared to the control group (averages of 36,9x10³
and 68,8x10³/body). The daily dose of 10mg there was decrease of fungal infection in the lung (averages 20,1
x106and 96,5 x106). With a daily dose of 10mg reduced the fungal infection in the lung (averages 20,1 x106
and 96,5 x106) and in the spleen (averages of 9,1 x10³ and 108,9 x10³) compared to untreated rats. However,
itraconazole and fluconazole showed greater efficacy in reducing the fungal load in the lung and spleen than
voriconazole. In two studies, voriconazole (10mg/kg body weight/day) prolonged discreetly the survival of animals
treated with the drug. In relation to the control group, the period of mortality of 50% of the animals treated with
6 doses of voriconazole, fluconazole, itraconazole and sulfamethoxazole-trimethoprim was respectively, 16, 22,
22, 39, 28 and 26 days. With 12 doses, the values corresponding to 50% of deaths were, respectively, 16, 17,
14, 36, 25 and 32 days after inoculation. Voriconazole showed a dose-dependent effect, because there was a
less effective not significant in the dose recommended for clinical use (5 and 7mg/kg/day) in relation to the higher
dosages (10 and 20mg/kg/day). The treatment with voriconazole provided a reduction of the load fungal tissue
and increased survival of rats, and may represent a new therapeutic option in human paracoccidioidomycosis.
Keywords: Paracoccidioidomycosis, voriconazole, azoles, experimental infection.
206
6-05
Amphotericin B-PLGA-DMSA nanoparticle: A new alternative to treat Paracoccidioidomycosis
A.C. Amaral1, 3, Bocca A.L.1, Ribeiro A.M.1, Nunes J.1, Falcomer C.L.1, Peixoto D.L.G.1, Simioni A.R.4, Pinto F.L.4, Lacava
Z.G.M.1, Azevedo R.B.1, Titze-de-Almeida R.1, Tedesco A.C.4, de Morais P.C.2 and Felipe M.S.1
Instituto de Ciências Biológicas e 2Física, Universidade de Brasília, UnB, Brasília, Brazil. 3Ciências Genômicas e Biotecnologia,
Universidade Católica de Brasília, UCB, Brasília, Brazil. 4Instituto de Química, Universidade de São Paulo, USP, Ribeirão Preto, Brazil.
e-mail: amaralandre@yahoo.com.br
1
Introduction and aim: Biodegradable polymers used to prepare controlled release systems are attractive
by the possibility to control the release of a drug slow and gradual through pre-designing its properties, such as
degradation kinetics and incorporation of target-specific molecules. Polymers used in these nanocarriers can
be natural (e.g. chitosan and alginate) or synthetic (e.g. PLGA-poly(lacti-co-glicolic acid)) and they have been
choosing mainly because of its biocompatibility and biodegradability. During in vivo applications biocompatible
polymers are broken down into molecules that can take part in normal metabolic pathways to be removed from
the body (Trends in Biotech. 24(1):39-47, 2006; J. Interventi. Cardiol. 19(6):500-506, 2006). Here we describe
the antifungal efficacy of a newly developed formulation of desoxycholate amphotericin B (D-AMB) encapsulated
within PLGA-DMSA nanoparticles (NanoAnf) in the murine model of paracoccidioidomycosis (patent filled INPI #
0700446-0, Antmicrob. Agents Chemother., submitted). Methods: Balb/C mice infected by the intravenously route
with the virulent strain Pb18 of P. brasiliensis received NanoAnf (6mg/Kg of encapsulated D-AMB, ip, interval of
72hs, which is equivalent to 2.7mg/Kg of pure amphotericin B) or D-AMB (Fungizon® 2mg/kg, ip, interval of 24hs,
which is equivalent to 0.9mg/Kg of pure amphotericin B). The treatments started 30 days after the fungal infection
and run for 30 and 60 days. Results and discussion: NanoAnf treated animals presented a marked antifungal
efficacy noted by the decrease on the lung fungal burden, followed by absence of renal and hepatic abnormalities,
as well as genotoxic and cytotoxic effects. Also, the animals received this new preparation, presented better
parameters of healthy status, like absence of piloerection, hypotrichosis, and reduced the loss of body weight
observed in D-AMB-treated animals (12.4%) after 30 daily doses. Moreover, this preparation allows a sustained,
gradual drug release and lung tropism, due to the presence of DMSA, and a favorable 72h extended dosing interval.
In conclusion, our results indicate the novel D-AMB-coated PLGA-DMSA, NanoAnf, is a promising preparation
useful to treat paracoccidioidomycosis such as other fungal infections. Financial support: CNPq.
6-06
Fungicidal activity of amphotericin B-magnetic fluid conjugate in vitro against
Paracoccidioides brasiliensis
Medeiros P.B.1, Amaral A.C.1, 3, Saldanha C.A.1, Bocca A.L.1, Guilherme R.B.4, Silva J.R.4, Nunes E.S.4, Lima E.C.D.4,
Azevedo R.B.1, de Morais P.C.2 and Felipe M.S.1
Instituto de Ciências Biológicas e 2Física, Universidade de Brasília, UnB, Brasília, Brazil. 3Ciências Genômicas e Biotecnologia,
Universidade Católica de Brasília, UCB, Brasília, Brazil. 4Instituto de Química, Universidade Federal de Goiás, UFG, Goiânia, Brazil.
e-mail: amaralandre@yahoo.com.br
1
Introduction and aim: Functionalized magnetic nanoparticles can be used for several applications in biology,
mainly because its ability to be driven in body by an external magnetic field (J. Biosci. Bioeng., 100(1):1-11, 2005).
Properly functionalized these nanoparticles may aggregate biocompatible molecules and drugs to be delivery
direct to the site of infection and, once there, release the drug. Polymeric linear molecules, with reactive organic
groups at both ends, are used to functionalize nanoparticles. One group is attached to the nanoparticle surface
and the other may be used to link polymers creating a shell in which the drug is added. After conjugated with this
preparation, the drug must be efficiently released or be able to interact with the pathogen (J. Nanob., 2:3, 2004).
Here we report the in vitro tests using magnetic nanoparticles conjugate with amphotericin B (AMB) against P.
brasiliensis Pb01 and Pb18 yeast cells. Methods: The Minimum Inhibitory Concentration (MIC) were determined
by incubation of the yeast cells in RPMI1640 medium containing AMB conjugated with magnetic fluid in block
polymer (because of patent filled required, the type of polymers used are not detailed). After three days of exposure
times to magnetic particles, 3 × 102 cells were plated in BHI supplemented with 4% horse serum, 5% P. brasiliensis
192 culture filtrate and 40mg/L Gentamicin. The plates were incubated at 36 ºC and CFU was counted at 21st day
post-plating to asses the cell viability. Results and discussion: AMB conjugate with magnetic nanoparticles in
block polymer was able to interact with the fungal cells. Also, the AMB did not loss its fungicidal efficacy once
the MIC observed for both Pb01 and Pb18 was similar to that determined for the conventional D-AMB (0,5µg/ml).
We can envision that AMB preparation tested here presents a promising potential to be used in the treatment of
paracoccidioidomycosis. Although AMB is successful used to treat PCM, its use is limited because of its toxicity.
So, once conjugated with magnetic nanoparticle it could be delivered direct to the site of infection diminishing their
severe adverse effects by reduction on drug amount. Financial support: CNPq.
207
6-07
PLGA-P10 conjugate improved 20-times the immunological protection of this peptide against
Paracoccidioides brasiliensis
A.C. Amaral1, 3, Marques A.F.4, Muñoz J.E.4, Bocca A.L.1, Simioni A.R.6, Titze-de-Almeida R.1, Tedesco A.C.6, de Morais
P.C.2, Taborda C.P.4, Travassos L.R.5 and Felipe M.S.S.1
Instituto de Ciências Biológicas e 2Física, Universidade de Brasília, UnB, Brasília, Brazil. 3Ciências Genômicas e Biotecnologia,
Universidade Católica de Brasília, UCB, Brasília, Brazil. 4Departamento de Microbiologia, USP, São Paulo, Brazil. 5Departamento de
Microbiologia, Imunologia e Parasitologia, Unifesp, São Paulo, Brazil. 6Instituto de Química, Universidade de São Paulo, USP, Ribeirão
Preto, Brazil. e-mail: amaralandre@yahoo.com.br
1
Introduction and aim: Because of the dramatic increase in the incidence of systemic mycosis, extensive research
has been focused to design efficient adjuvant systems to develop vaccines to protect for fungal diseases (Nat Rev
Microbiol, 5:13-28, 2007). Peptide antigens are especially promissory to trigger an effective immune-protective
response against these infections. The P10 peptide, identified in the glycoprotein Gp43, the major antigen secreted by
Paracoccidioides brasiliensis, elicits an immunological protection against this fungus (Infect Immun, 66(2):786-793,
1998). Encapsulation of peptides within polymeric systems, such as nano and microparticles, are suitable to provide
its release at controlled rate under polymer degradation for a prolonged time. Polymers used in these nanocarriers
can be natural (e.g. chitosan and alginate) or synthetic (e.g. PLGA-poly(lacti-co-glicolic acid)) and they have been
choosing mainly because of its biocompatibility and biodegradability (Trends in Biotech, 24(1):39-47, 2006). Here
we investigate the adjuvant effects of the peptide P10 encapsulated within PLGA-DMSA polymeric blends combined
with sulfametoxazole to treat the paracoccidioidomycosis murine model. Methods: Balb/C mice were infected with
the virulent strain Pb18 of P. brasiliensis. After 30 days from fungal infection, the animals received 4 weekly doses of
the peptide P10 (20µg) with complete Freud adjuvant (CFA) or within PLGA-DMSA blend containing 1, 5, 10, 20, or
40µg of P10, followed by daily injections of 15mg/kg sulfametoxazole during 30 days. The animals were sacrificed on
days 30 and 60 from the beginning of treatments. Results and discussion: The results of fungal burden recovery
have shown that 1µg of P10 entrapped within PLGA were more efficient than 20µg of P10 with CFA as adjuvant
for sulfametoxazole during 30 days of treatment. Also, in these two groups, the histological analysis revealed the
formation of compact granulomas, probably as a consequence of the elevated levels of interferon-γ detected in these
groups when compared with the others. In conclusion, the peptide P10 conjugated with PLGA-DMSA improved its
immunological protection 20 times and proved to be an efficient aid to the sulfametoxazol chemotherapy to treat
paracoccidioidomycosis in murine model. Financial support: CNPq.
6-08
Biological activity of the endophytic fungi UFMGCB 551 against Paracoccidioides
brasiliensis
F.F. Campos1,2, L.H. Rosa3, S. Johann2, B.B. Cota2, C.A. Rosa1, N.P. Sá1, P.S. Cisalpino1, C.L. Zani2.
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil;
Laboratório de Química de Produtos Naturais, Centro de Pesquisas René Rachou, Fiocruz, Belo Horizonte, Brazil; 3Laboratório de
Microbiologia, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, MG, Brazil.
e-mail: fcampos@cpqrr.fiocruz.br
1
2
Endemic mycoses caused by dimorphic fungi Paracoccidioides brasiliensis remain a major public health
problem in several countries in Latin America, especially in Brazil, Venezuela, Colombia and Argentina. Some
compounds currently used for the control of P. brasiliensis infections are toxic and not efficacious, and infection
relapses may occur. The aim of the present study was to evaluate the effects of endophytic fungus UFMGCB 551 on
three different clinical isolates of P. brasiliensis (Pb18, Pb01, Pb608). Thus, the Minimal Inhibitory Concentrations
(MIC) of the crude extract, fractions, and two isolated compounds, were evaluated. The isolate UFMGCB 551 was
grown in potato dextrose agar (PDA) and inoculated at the center of 150 Petri dishes containing malt extract agar
(MEA). After incubating the plates for 14 days at 28 ± 2 ºC, their contents were pooled and extracted with ethyl
acetate to yield 1.6 g of crude extract. This extract was able to inhibit all three P. brasiliensis strains when tested
at 6.3 µg mL-1. One gram of this extract was subjected to high-speed co-current chromatography (HSCoCC) to
afford 134 fractions of 15 mL, which were pooled into 25 groups after their analysis by thin layer chromathograpy
(TLC). Group 18 completely inhibited fungal growth at 0.9 µg mL-1. This group was then fractionated by reversedphase HPLC to yield two pure compounds that inhibited all P. brasiliensis at concentration below 0.9 µg mL-1.
Thus, the bioassay-guided fractionation of the title fungus yielded compounds with strong inhibitory effect against
P. brasiliensis, for which new drugs are much needed. This work corroborates the potential of endophytic fungi as
source of new leads for drug development.
208
6-09
Antagonistic effect of farnesol, a Candida albicans quorum sensing molecule, on
Paracoccidioides brasiliensis growth and morphogenesis
L.S. Derengowski1, S.V. Braz2, C. De-Souza-Silva1, T.M. Mello-de-Sousa1, S.N. Báo2, C.M. Kyaw3 & I. Silva-Pereira1.
Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília/DF – Brazil. Laboratórios de 1Biologia
Molecular, 2Microscopia Eletrônica and 3Microbiologia. e-mail: lorena.bio@gmail.com
Farnesol, a quorum sensing molecule of Candida albicans, is a sesquiterpene produced by many other
organisms, and also found in several essential oils, possibly protecting plants from parasitic induced damages.
Recently, this sesquiterpene alcohol has been demonstrated to inhibit the growth of some microorganisms, such
as the human pathogens Staphylococcus aureus and Streptococcus mutans, and the plant pathogenic fungus
Fusarium graminearum, signaling its potential use as an antimicrobial agent. Farnesol also enhance microbial
susceptibility to antibiotics, indicating a putative application as an adjuvant therapeutic agent. This sesquiterpenoid
prevents C. albicans transition from yeast to mycelium, and compromises its biofilm formation. A recent study
showed that farnesol is employed by C. albicans in order to reduce competition with other microbes, since this
compound mediated apoptosis in the filamentous fungus Aspergillus nidulans and inhibited biofilm formation in
other Candida species.
The potential use of farnesol as an antimicrobial agent was investigated, and here, we report its activity
against the human pathogen Paracoccidioides brasiliensis. This isoprenoid was able to inhibit P. brasiliensis
growth and, when employing concentrations that not compromise cell growth, it also affected the P. brasiliensis
dimorphic transition. Electron microscopy shows that P. brasiliensis cells treated with farnesol exhibited a fully
cytoplasm degeneration. However, the cell wall remained intact, and the cell permeability was not affected.
However, no synergistic effect between farnesol and fluconazole was observed. Our results clearly showed
the in vitro antimicrobial activity of farnesol against P. brasiliensis. However, additional studies involving animal
models of experimental paracoccidioidomycosis need to be performed to assess their potential use as an adjuvant
chemotherapeutic agent.
6-10
Killing of Paracoccidioides brasiliensis yeast cells by magnetic hyperthermia
A.C. Amaral1, 3, Braun S1., Nunes E.S.4, Bocca A.L.1, Lima E.C.D.4, Azevedo R.B.1, de Morais P.C.2 and Felipe M.S.S.1
Instituto de Ciências Biológicas e 2Física, Universidade de Brasília, UnB, Brasília, Brazil. 3Ciências Genômicas e Biotecnologia,
Universidade Católica de Brasília, UCB, Brasília, Brazil. 4Instituto de Química, Universidade Federal de Goiás, UFG, Goiânia, Brazil. email: amaralandre@yahoo.com.br
1
Introduction and aim: Magnetic fluids are colloidal systems in which the magnetic nanoparticles, usually
magnetite or maghemite, are dispersed in a physiologic solution (J Mater Res, 13(10:)2975-2981, 1998). The
average size of the magnetic nanoparticle in a magnetic fluid varies between 3 and 20nm; and at this dimension
(10-9 part of the meter), matters show special characteristics, such as the ability to be directed to a body-site
specific by an external magnetic field or respond to a time-varying magnetic field, which result in the nanoparticles’
agitation, generating an increase on temperature up to 45-47 ºC. This is the principle of a technique called
magnetic hyperthermia, used to treat some kinds of tumors (J. Phys. D. Appl. Phys, 36:R167-181, 2003). In this
study we have subjected the P. brasiliensis Pb18 to the magnetic hyperthermia. Methods: The yeast cells (3 × 104
cells/mL) were incubated during 3 and 10 minutes under a time-variable magnetic field in YPD medium containing
maghemite nanoparticles functionalized with dimercaptosuccinic acid (1014 particles/mL). After the exposure to
the magnetic field the cells (3 × 102) were plated in BHI supplemented with 4% horse serum, 5% P. brasiliensis
192 culture filtrate and 40mg/L Gentamicin. The plates were incubated at 36 ºC and the Colony Forming Units
(CFU) was counted at 21st day post-plating to asses the cell viability. Results and discussion: Subjecting P.
brasiliensis yeast cells, co-cultured with magnetic fluid, to an alternating magnetic field killed the fungus. Cells
cultivated in YPD subjected to a magnetic field or incubated with magnetic fluid without magnetic field had not
the cell viability affected. These findings could be useful to evaluate the use of magnetic hyperthermia to treat
paracoccidioidomycosis. Since the magnetic nanoparticles are sensitive to the action of an external magnetic
field, they could be driven to the site of fungal infection and, once there, apply the alternate magnetic field. As
noted for cancer treatment, the magnetic hyperthermia technique is used against tumor cells, which are sensible to
increases on temperature without or minor commitment of the health cells (J. Phys. D. Appl. Phys, 36:R167-181,
2003). Financial support: CNPq.
209
6-11
Antifungal activity of plants extracts used in brazilian traditional medicine against
Paracoccidioides brasiliensis
S. Johann1, G.A. Watanabe1, B.B. Cota3, E.P. Siqueira3, C.L. Zani3; M.G. Pizzolatti2, P.S. Cisalpino1, M.A. Resende1,*
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; 2
Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil; 3 Laboratório de Química dos Produtos
Naturais, Centro de Pesquisas René Rachou, FioCruz, Belo Horizonte, Brazil. +
Corresponding author: sjohann@cpqrr.fiocruz.br
1
The antifungal activity of extracts of plants employed in Brazilian traditional medicine was assayed
against Paracoccidioides brasiliensis and their citotoxicity on murine macrophages was determined. The
extracts of ten plants were assessed for activity against three P. brasiliensis clinical isolates. Susceptibility
was determined by the broth microdilution method. The hexanic fractions of Piper regnellii and Baccharis
dracunculifolia have shown the lower inhibitory concentrations (MIC) (7.8-30 µg ml-1 and 7.8 µg ml-1,
respectively).
Additionally, neither exhibited cytotoxicity on murine macrophages. According to Gas chromatographymass spectrometry (GC-MS) analysis, the major components of the hexanic fraction of B. dracunculifolia were
1H-cycloprop[e]azulen-7-ol (17.57%), cyclododecanol (11.77%) and α-farnesene (8.15%). The antifungal
activity of the hexanic fractions of P. regnellii and B. dracunculifolia against this pathogenic fungus, without
evidences of cytotoxicity, supports further studies in search of new chemical structures with activity against
P. brasiliensis.
6-12
Treatment of murine paracoccidioidomycosis (PCM) with trimethoprim-sulfamethoxazole
combination (TMP-SMX)
F. Moura1, C.R.G. Lima1, T.C. Moreto1, L.R. Carvalho2, R.A. Bueno1, D.V. Moris1, R.P. Mendes1.
1 Tropical Diseases Área, Botucatu Medical School. 2 Department of Biostatistics, Biosciences Institute. São Paulo State University UNESP. e-mail: tietemendes@terra.com.br (presenting Author)
Introduction: Trimethoprim-sulfamethoxazole combination (TMP-SMX) has been widely used in the
treatment of paracoccidioidomycosis patients. However, only two experimental studies were carried out to
verify its efficacy and its capacity in eradicating the infection. The objective of this study was the evaluation
of the efficacy of different TMP-SMX regimens in the treatment of murine paracoccidioidomycosis.
Methods: Different TMP-SMX regimens, 200 mg SMX/kg once a day by gavage, were used to treat 145
male BALB/c mice infected with P. brasiliensis strain 18, comprising 6 groups: G1 – healthy controls; G2
– Infected controls; G3 – early introduction and long course treatment (140 days); G4 – early introduction
and short course (56 days); G5 – late introduction (at 28th day post-infection) and long course (112 days);
G6 – SMX serum levels evaluation. Treatment efficacy was evaluated by the number of colony-forming units
(CFU) from lung and spleen fragments at 2nd, 4th, 8th, 12th, 16th and 20th weeks post-infection; endpoint was a
decreased CFU. Kruskal-Wallis, Mann-Whitney, χ2, Fisher’s exact and Wilcoxon test made up the statistical
analysis, with level of significance set at p < 0.05.
Results: The cumulative mortalities of the mice revealed the following percentages of survival:
G1=100.0%, G2=78.9%, G3=89.3%, G4=71.4%, and G5=88.2%. Survival was not different in G2 and G4
(p>0.05), or G3 and G5 (p>0.05); however, survival rate was higher in G3+G5 than G2+G4 (p<0.01). Colonyforming units assays also showed that long course regimens (G3 and G5) were able to significantly decrease
the fungal load in the spleen and lungs, while the short course therapy (G4) was not (p<0.05). TMP-SMX
was more efficacious in the spleen than in the lungs (p<0.05). After administration by gavage SMX peaks at
6 hours (97.2 µg/mL), but its serum level decreases quickly, reaching 25.1 µg/mL at 12 hours. No survival
mice in any regimen were free of infection. Conclusions: Long courses of TMP-SMX are efficacious in the
treatment of murine paracoccidioidomycosis, which proved to be a good model for therapeutic evaluation.
Decrease of the fungal load was higher in the spleen than in the lungs. TMP-SMX should be administrated
every 12 hours.
210
6-13
Therapeutic DNA vaccine in BALB/c and B10A mice against experimental
paracoccidioidomycosis
G. M. Gomes Rittner1, J. E. Muñoz1, C. P. Taborda1 and Travassos L. R.2
1 Departamento de Microbiologia, Instituto de Ciências Biomedicas,USP, São Paulo, Brasil. 2 Departamento de Microbiologia, Imunologia
e Parasitologia, UNIFESP, São Paulo, Brasil. e-mail: travassos@unifesp.br
Background: Paracoccidiodomycosis (PCM) is a systemic granulomatous disease caused by the thermo-dimorphic
fungus Paracoccidioides brasiliensis. It is widespread in Latin America and found mainly in Brazil, Colômbia, Argentina
and Venezuela. Immunotherapy has been proposed as an adjuvant to PCM chemotherapy and to control relapses. A
DNA vaccine is a promising approach to Ag-specific immunotherapy and was shown to be protective in mice, encoding
the major antigen gp43 (Pinto et al., 2000). Plasmid construction is easily prepared, stable, and can be repeatedly
administered. Peptide 10 contains the T-cell epitope of gp43 and is protective against experimental infection in mice.
The aim of this work was to analyze a DNA-based vaccine encoding P10 or IL-12 in mice intratracheally infected with
P. brasiliensis. Methods: BALB/c and B10A mice were immunized with 100 µg of pCDNA3 encoding P10, IL-12 or
a mixture of the two plasmids. Animals were infected intratracheally with 3x105 yeast cells of virulent P. brasiliensis
Pb18. After 30 days, they were immunized again, once a week for 4 weeks. Colony forming units (CFU) and cytokine
production were measured in lungs after 1 week of treatment. Control groups of infected mice were immunized with
pCDNA3 without insert. Results: Significant reduction of CFU in the lungs of mice immunized with plasmid encoding
P10, IL-12 or plasmid mixtures encoding P10/IL-12 were observed. The cytokines in lungs showed enhanced levels of
IFN-β and IL-12. Conclusions: (I) DNA-based vaccine encoding P10 and/or IL-12 was very effective in reducing the
fungal load; (II) therapy using a mixture of plasmids encoding P10 and IL-12 was the most effective formulation; (III)
CFU reduction was associated with enhanced levels of IFN-β and IL-12, and reduced IL-4; (IV) DNA-P10 and DNA-IL12 mixed therapeutic treatment induced a Th-1 immune response in BALB/c and B10A mice that was protective against
intratracheal challenge with virulent P. brasiliensis; (V) B10A mice treatment was more effective than that with BALB/c
mice ending up with scarcely no lung CFU. Supported by Fapesp and CNPq.
6-14
Adrenal function in paracoccidioidomycosis patients treated with azolic derivatives: Results
after prolonged post-therapy follow-up
Tobón AM1,2, Restrepo CA3, Villa CA3, Agudelo CA1,3,4, Quiceno W4, Restrepo A1
Corporación para Investigaciones Biológicas (CIB), 2Hospital La María, 3Universidad Pontificia Bolivariana, 4Hospital Pablo Tobón Uribe,
Medellín, Colombia. e-mail: atobon@cib.org.co
1
Adrenal insufficiency is a rather frequent sequela of paracoccidioidomycosis (PCM). The presence of
Paracoccidioidomicosis brasiliensis in adrenal glands tissues has been previously reported in patients with this mycosis
even after their completion of specific therapy. Additionally, some of these patients exhibit reactive serologic tests and
show high antibody titers that persist after adequate treatment indicating continuity of an antigenic stimulus probably
linked to the presence of the fungus in these glands. This work attempted to study the situation of these glands after
prolonged post-therapy evaluation and tried to establish if a connection could exist between high anti-P. brasiliensis
antibody titers and the diagnosis of adrenal insufficiency in these patients who had been followed years after termination
of their antifungal treatment. In this study we included 28 PCM patients who had been previously treated for this disorder;
they were all males with a medium age of 55.3 years (SD+12.1) and were called for consultation after a mean of 13.32
(SD+9.22) years after being diagnosed. From these patients, 17 (60.7%) had the chronic multifocal form of the disease,
6 (21.4%) the subacute form, and 5 (17.8%) the chronic unifocal form. Eighteen (64.3%) patients had been treated with
itraconazole, 4 (14.3%) with ketoconazole, and 6 (21.4%) with other antifungals alone or in combination. The media of
basal cortisol was 13.45 (SD+4.81) µg/dl (N >5 µg/dl), after ACTH stimulation cortisol amounted to 34.36 (DS+7.41)
µg/dl (N >18 µg/dl) with an increase of 20.47 (DS+6.35) µg/dl (N >16.5 µg/dl) after ACTH stimulus. In 2 (7.1%) patients
adrenal insufficiency was demonstrate while in 8 (28.6%) the response to ACTH was below normal. P. brasiliensis was
seen in a biopsy of adrenal glands from a patient with insufficiency. At diagnosis, most patients (71.4%) had shown
high antibodies titers (≥1:32) which in 39.3% persisted up to the end of treatment and in 10.7% after the prolonged
post-therapy follow up. However, a statistically significant association between persistently high antibody titers at the
follow-up observation and low cortisol values post ACTH challenge was not demonstrated indicating that the fungal
antigenic stimulus, as shown by the presence of specific antibodies, appeared not to originate in the adrenals implying
no relation between adrenal malfunction and antibody production. The proportion of adrenal insufficiency (7.1%) found
after such an extended period of therapy cessation, as well as the subnormal response to ACTH stimulation in 28.6%
of our cases, confirm that adrenal damage is an important marker of paracoccidioidomycosis. It would be advisable to
check on this abnormality as soon as diagnosis is established so as to take the proper measures on time, thus avoiding
further damage to vital organs such as the adrenal glands.
211
6-15
The association tuberculosis - paracoccidioidomycosis: Impact on the outcome of
theraphy
Tobón AM1,2, Agudelo CA1,3,4, Tabares AM1, Restrepo A1.
Corporación para Investigaciones Biológicas, 2Hospital La María, 3Universidad Pontificia Bolivariana, 4Hospital Pablo Tobón Uribe,
Medellín, Colombia.
e-mail: carlosagudelo@yahoo.com
1
Tuberculosis (TB) and paracoccidioidomycosis (PCM) are both chronic diseases with overlaping clinical
presentations and which, additionally, may coexist in 10-13 % of the PCM cases. In patients with both diseases
treatment difficulties have been reported due to the induction of the P450 enzymatic system with increasing azolic
clearance. This work evaluates the results of antifungal therapy in patients with the TB - PCM coinfection and
compares the findings with those in patients with PCM alone.
The clinical records from 7 patients with dual infections who received simultaneously antifungal and anti-TB
medications were analyzed. Demographic data, serologic titers, and response to therapy based in a point system
evaluation, were analyzed. Their responses to therapy were compared with a weighted control group, formed by
PCM patients whose clinical form and antifungal therapy were alike.
The coinfected group consisted of 6 men and 1 woman, median age of 49.3 (SD+15.9) years with symptoms
having a duration of 232.1 (SD+184.3) days. All patients in the control group were men, with a median age of 42,0
(SD+14.9) years and with symptoms duration of 167.9 (SD+114.8) days. At diagnosis, the point system evaluation
score was 21.1 (SD+6.5) in the group with TB and 14.6 (SD+6.2) in control group. The mean treatment duration
was 8,7 (SD+3.6) months and the medium itraconazole dosage was 228.6 (SD+125.6) mg/day in the group with
TB and in the respectively of control group these figures were 7.3 (SD+2.2) months and 185.7 (SD+37.8) mg/day
of itraconazole. None of these features showed statistically significant differences between the two groups. The
response to therapy evaluated by point system and by serologic titers showed no statistically significant differences
between the TB coinfected and the PCM alone groups.
These findings indicate that antiTB medication does not affect the response to antifungal therapy in patients
with PCM coinfected with tuberculosis. However, the small number of cases studied precludes more precise
determinations.
212
Poster Session
7
Other Mycoses
7-01
Susceptibility to fluconazole and voriconazole of Candida species isolated from intensive
care units patients in several hospitals in Medellín, Colombia (2001 - 2007)
A. Zuluaga1, C. Bedout1, CA. Agudelo1,2,3, H. Hurtado1, M. Arango1,4, A. Restrepo1 and A. González. 1,5.
1 Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia. 2. Universidad Pontificia Bolivariana, Medellín. 3. Hospital Pablo
Tobón Uribe, Medellín. 4 Facultad de Medicina. Universidad de Antioquia. 5. Escuela de Microbiología. Universidad de Antioquia.
e-mail: azuluaga@cib.org.co
Disseminated candidiasis, an opportunist mycosis caused by different Candida species, has become an important
health problem that posses a threat for the lives of many patients, especially if they are immunosuppressed or
hospitalized in Intensive Care Units (ICUs). From 2001 to 2007, a transversal cohort study was done aiming
at determining the frequency and susceptibility to fluconazole and voriconazole of Candida species isolates sent
to the CIB for susceptibility studies; they had been obtained from different ICUs patient. Based on the protocols
recommended by the CLSI from the United States, document M44P, discs impregnated with these antifungal drugs
were used in the agar diffusion technique. A bivaried statistical analysis was done to determine the association
between the isolated Candida species and their resistance to each antimycotic. From 337 isolated analyzed, 196
corresponded to blood cultures, 111 to peritoneal fluids, 10 to deep organs biopsies, 9 to pleural fluids, 9 to closed
abscesses, and 2 to cerebrospinal fluids. These isolates were speciated as follows: 147 (43,6%) C. albicans, 79
(23,4%) C. tropicalis, 32 (9,5%) C. glabrata, 47 (13,9%) C. parapsilosis, 11 (3,3%) C. krusei and 12 (3,6%) C.
guilliermondii. The remaining isolates (2,7%) were distributed in 5 different species (C. famata, C. lusitaniae, C.
lipolytica, C. pelliculosa y Candida ssp). As it concerns fluconazole, 78,3% of all the above isolates were susceptible,
11,9% susceptible dose dependent (SDD) and 9,8% resistant. Regarding voriconazole, we observed that 94,1% of
the isolates were susceptible, 2,4% SDD and 3,6% resistant. The range of susceptibility of the various species to
fluconazole, was as follows: C. albicans 95,2%, 86% C. tropicalis, 65,6% C. glabrata and 59,6% C. parapsilosis. As it
concerns voriconazole, susceptibility was shown for 100% of C. albicans isolates, 96,2% of C. tropicalis, 93,6% of C.
parapsilosis and 84,4% of C. glabrata. The bivarieted analysis showed that the following isolates were significantly
associated with susceptibility to fluconazole, thus, C. albicans (OR: 10,6, IC 95% 4,7 – 24,06, p < 0.01) and C.
tropicalis (OR: 1,95, IC 95% 0,97 – 3,93, p 0,05), with SDD C. parapsilosis (OR: 3,2, IC 95% 1,50 – 6,88, p < 0,01)
and C. guilliermondii (OR: 1,01, IC 95% 1,15 – 13,99, p 0.04) and C. guilliermondii (OR: 11,03, IC 95% 3,33 – 36,6,
p < 0,01) and C. krusei (p < 0,01) with resistance. As for voriconazole, the isolations tested were significantly
associated with susceptibility, in the cases of C. albicans (p < 0,01), with SDD C. krusei (OR: 29,3, IC 95% 6,45
– 132,5, p < 0,01) and C. glabrata (OR: 7,88, IC 95% 2,34 – 26,52, p < 0,01) with resistance. These data indicate that
C. albicans is the species most frequently (43.6%) recovered from the different clinical samples; however, altogether,
the group formed by other species different to C. albicans also represents an important proportion (56,4%) of the
isolates recovered from the different clinical samples. In addition, these results reveal a remarkable change in the
frequency of non-albicans species, as well as the presence of new susceptibility patterns that demands the precise
identification of the causative organism. There is need to determine the susceptibility pattern of these emerging
isolates in order to take the correct therapeutical approach.
7-02
Therapeutic study of gomesin in mice infected with Candida albicans
D. C.P. Rossi1, J. E. Muñoz2, C. P. Taborda2 and Daffre S.1
1 Departamento de Parasitologia; 2 Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São
Paulo – Brasil. e-mail: rossi.d@usp.br
Background: Gomesin is a cationic antimicrobial peptide produced by the hemocytes of the spider Acanthoscurria
gomesiana. It has a molecular mass of 2270.4 Da, 18 amino acids and four cysteine residues forming two disulfide
bridges. Gomesin strongly affects bacterial growth and the development of filamentous fungi and yeast. It also
effective against Leishmania amazonensis (Silva Jr et al. JBC 2000, 275: 33464) and different life stages of the
Plasmodium spp. (Moreira et al., Exp Parasitol 2007, 116: 346). In addition, Gomesin causes death of Cryptococcus
neoformans through membrane permeabilization. In association with Fluconazole, it kills fungi and enhances the
activity of brain phagocytes (Barbosa et al., FEMS 2007 Microbiol Lett 274: 279). In the present study we evaluated
the efficacy of Gomesin on systemic candidiasis model. Methods: We used an experimental model of Balb/c mice
infected intravenously with 3 x 105 yeast cells of Candida albicans with high virulence (strain 78). Treatments with
Gomesin, Fluconazole, Anfotericin B and Gomesin in association with Fluconazole consisted of three doses (5mg/Kg)
at 1, 3 and 6 days post infection. At the seventh day post infection, the kidneys, spleen and liver were removed and
the CFU were counted. Results: Gomesin has a dose-dependent effect and the treatment significantly decreased the
number of CFU compared with control. Conclusions: Treatment with Gomesin is efficient in the systemic candidiasis
experimental model. This result suggests that Gomesin might be used as an alternative to the usual treatment,
especially when treating resistant pathogens.
215
7-03
Study of genotypic and phenotypic characteristics among C. gattii serotype B - VGII
molecular type clinical isolates from Cúcuta, Colombia and isolates of the outbreak in
Vancouver, Canada
G.Torres, and P. Escandon.
Grupo de Microbiología, Instituto Nacional de salud, INS, Bogotá D.C - Colombia. Tel. 2207700 ext. 445-446.
e-mail: getorresr@unal.edu.co
Introduction: Cryptococcus gattii serotype B - VGII pattern emerged as a primary pathogen in Vancouver,
Canada, causing a major epidemic of cryptococcosis in 2000 that affected immunocompetent humans
and animals. In Cúcuta, Colombia isolates of C. gattii of the same serotype and molecular type have been
reported.
Aim: Compare serotype B isolates from Cúcuta and isolates from the outbreak of Vancouver, with the
purpose of identifying similarities and differences in the virulence profile between the two groups of isolates.
Methods: 20 isolates molecular type VGII – mating type α, from the outbreak of Vancouver and 11 Colombian
isolates of C. gattii serotype B were studied, determining the molecular type by gene URA5 restriction. Factors
associated with virulence evaluated were: mating type, characteristics of the colonies, phenotypic switching, cell
and capsular diameter, enzyme activity (phenoloxidase and phospholipase) and growing at 37 °C.
Results: 6 / 11 Colombian isolates were molecular type VGII. These isolates were mating type a, and have
mucoid colonies; 5 showed phenotypic switching. 19/20 isolates from Vancouver presented smooth colonies
and phenotypic switching. The cell and capsular diameter was higher in Colombian isolates (P = 0.02). No
difference was observed in enzyme activity between the two groups of isolates (P = 0.083). The 26 VGII isolates
grew at 37 ° C.
Conclusion: The presence of C. gattii VGII molecular type in Colombia and the similarities observed in
some of the factors associated with virulence between the two groups of isolates, shows the need to do a
surveillance of the cases associated with this molecular type.
7-04
Microecology of Blastomyces dermatitidis: The ammonia hypothesis
D. J. Baumgardner
Center for Urban Population Health and Aurora University of Wisconsin Medical Group, Milwaukee, Wisconsin – USA.
e-mail: dennis.baumgardner@fammed.wisc.edu
Background: The precise microecology of Blastomyces dermatitidis is unknown. The fungus has been
associated with nitrogenous waste products and rapidly changing environmental conditions of water tension,
temperature, pH and potential chemical inhibitors. Ammonia accumulates in certain microenvironments, is toxic
to most fungi, yet may not be identified in processed soil samples.
Methods: Ammonia tolerance of B. dermatitidis was investigated by growth of two Wisconsin strains
(ATCC MYA-2585/6), a clinical and an environmental isolate, respectively, on phosphate and HEPES buffered
agar media supplemented with mineral salts, low (1 g/l) and high (20 g/l) dextrose and increasing amounts of
ammonium sulfate, at pH 7, in gas-impermeable bags at 20o C. Growth of soil fungi from 200 aqueous slurries
of fresh and frozen soil samples from the northern USA and Canada was tested on similar media.
Results: Moderate mold growth and sporulation of both strains of B. dermatitidis was observed at calculated
ammonia concentrations of 375-563 mmol/l when plates were inoculated with either mold or yeast forms. Fungal
growth was inhibited in virtually all soil samples at these ammonia levels when low dextrose concentrations were
utilized.
Conclusion: The ability of B. dermatitidis to survive and grow in organic carbon-poor, high ammonia
microenvironments may be important to the competitive success of this fungus. Such microenvironments
may include animal droppings or guano on sand soils, animal burrow latrine chambers and runoff from
ammonia fertilizers with nitrification inhibitors. This may have implications for other dimorphic fungi such as
Paracoccidioides brasiliensis.
Financial Support: St. Luke’s Foundation Donation from Mr. and Mrs. Charles Goldsworthy, Eagle River, WI.
216
7-05
Evaluation of an antigen-capture ELISA to detect Histoplasma capsulatum antigenuria in
immunocompromised patients
C.M. Scheel1, B. Samayoa2 L. Benjamin1, P. Riley1, M. Lindsley 1, A. Herrera 2, G. Raxcacoj2, S. Lima2 , R. Miramontes1,
T.M. Chiller1, M. Brandt1, E. Arathoon2, B.L.Gómez1
Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA. 2Clinica Familiar Luis Angel Garcia (CFLAG), Hospital
General San Juan de Dios, Guatemala City, Guatemala. e-mail: BGomez@cdc.gov
1
Background: Histoplasma capsulatum infection causes significant morbidity and mortality in HIV-infected
individuals, particularly those in developing countries without access to sophisticated diagnostics or highly-active
antiretroviral therapy. Furthermore, symptoms of histoplasmosis are non-specific, yet most deaths occur in the
first 2 weeks after diagnosis. Current diagnostic methods can be complex, expensive and slow. A simple, rapid
method to detect H. capsulatum infection would dramatically decrease time to diagnosis and treatment and reduce
morbidity and mortality. We evaluated an antigen-capture ELISA to detect antigen in urine of HIV patients with
histoplasmosis in Guatemala.
Methods: Urine samples were collected prior to treatment in HIV patients with culture-confirmed histoplasmosis
(n = 48) or non-histoplasmosis fungal and non-fungal diseases (n = 113). Urine from healthy controls (n = 83)
were used to define specificity. An antigen-capture ELISA was developed which utilizes polyclonal rabbit anti-H.
capsulatum antibody as both capture and detection reagent. A standard curve was included in each assay plate
to insure inter-assay reproducibility. Urine specimens were run twice on separate days and repeated a third time
if the coefficient of variance (CV) was greater than 20%.
Results: The H. capsulatum antigen-capture ELISA demonstrates a sensitivity of 81% for confirmed
histoplasmosis and a specificity of 96% (all other disease controls, 95.0%; healthy controls, 97%) when tested
against baseline urine specimens in these patient cohorts. Thirteen of the patients with follow-up urine specimens
showed decreased antigenuria during the course of antifungal chemotherapy.
Conclusions: In this analysis, the antigen ELISA assay shows high sensitivity and specificity as a simple rapid
diagnostic test for histoplasmosis in HIV-infected individuals. This assay has the potential to be easily adapted
to laboratories across the world. A simple test using urine would allow for the rapid diagnosis and initiation of
treatment. Longitudinal analysis of H. capsulatum antigenuria in serial specimens during therapeutic intervention
may prove useful for monitoring patient recovery in a clinical setting.
7-06
An analysis of histoplasmosis in an endemic, resource poor area with a high HIV prevalence
rate - Guatemala, 2007
R. Miramontes1, B. Samayoa2, A. Herrera2, C. Scheel1, B. L. Gómez1, E. Arathoon2 T. Chiller1.
Mycotic Diseases Branch, Centers for Disease Control and Prevention; Clinica Familiar Luis Angel Garcia (CFLAG), 2Hospital General
San Juan de Dios, Guatemala City, Guatemala. e-mail: TChiller@cdc.gov
1
Background: Disseminated histoplasmosis is a serious opportunistic infection in AIDS, often representing
the first manifestation of the syndrome in endemic regions. Central America is a known endemic area for
histoplasmosis; however little is known about the true prevalence in persons with AIDS. In Guatemala, barriers to
patient care such as lack of medication and extended travel to health facilities are common and rapid diagnostics
are not available.
Methods: A prospective cohort study of hospital patients was conducted from February 2005 - December
2007 in large public hospital in Guatemala City. Study criteria required that a patient be HIV-infected and
have three out of five of the following: Fever, pancytopenia, weight loss, radiological evidence consistent with
histoplasmosis, or skin/mucosal lesions suspicious for histoplasmosis. A histoplasmosis case was defined as
a positive Histoplasmosis capsulatum culture from a clinical specimen, or positive tissue sample suggestive of
histoplasmosis.
Results: Of the 279 patients that met surveillance criteria, 217 (78%) were enrolled in the study. A total of
63 (29%) of 217 patients met the case definition for histoplasmosis. There were 29 (46%) deaths among the 63
case patients. The median time to death was 17 days. A total of 11 (17%) case patients were co-infected with
tuberculosis and 4 of those were among reported deaths. The median CD4 cell count among case patients on
date of diagnoses was 25 (1-193).
Conclusion: The incidence of histoplasmosis in this cohort of patients with HIV in Guatemala is high.
Mortality occurred rapidly after admission and in more than a third of the patients. Patient care is complicated in
this setting by the lack of availability of a rapid diagnostic test. Rapid diagnosis is critical in this population. This
study highlights the importance of histoplasmosis as an opportunistic infection in persons with AIDS in Guatemala.
More work is needed to better define the burden of this disease in patients with HIV and its incidence among those
patients co-infected with TB in order to provide guidance for better diagnosis and more rapid treatment.
217
7-07
Detection of DNA in peripheral blood from a patient with the ocular histoplasmosis
syndrome: A case report
J. Hernández1, 3, C. Muñoz1, 2, D. Hernández, 3, 4,5 C. Montoya, 5 Á. González1,2
Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB), 2Microbiology School, Universidad de
Antioquia2 Medicine School Universidad Pontificia Bolivariana3, Hospital Pablo Tobón Uribe4, and Clínica Oftalmológica San Diego5,
Medellín Colombia. e-mail: jhernandez@cib.org.co
1
The ocular histoplasmosis syndrome (OHS) is a significant cause of vision loss in young and middle-aged
adults. Despite the fact that the direct role of the fungus Histoplasma capsulatum var capsulatum in this condition
has not been clearly demonstrated, some epidemiological studies appear to implicate the fungus in OHS. We
report here a case of OHS in an immunocompetent individual in whom a nested PCR in peripheral blood for H.
capsulatum turned out positive. A 37 years- old man indicated that previous to consultation he has had fever,
malaise, headache and anterior cervical lymphadenopaty for one week after which he noticed gradual improvement
of his symptoms but started to experience a sudden loss of his right eye visual acuity. The fluorescent angiography
and an optical coherent tomography informed the presence of a type II choroidal neo-vascular membrane in
the right eye. On the basis of these results, the ophthalmologist advanced a presumptive diagnosis of OHS.
Conventional laboratory tests such as complement fixation and immunodiffusion tests for antibodies were negative
at the beginning of infection which became positive after 8 weeks with 1:8 titters - A peripheral blood sample was
tested in a nested PCR for H. capsulatum var capsulatum using a set of primers known to amplify a DNA sequence
coding for the specific 100-kDa protein of this fungus (Hc100-PCR). The results were compared with a positive
control sample of H. capsulatum DNA and with a negative peripheral blood sample from a healthy individual.
Additionally, the PCR products were sequenced to confirm the amplified product obtaining an identity of over 97%.
The patient received intravitreal bevacizumab injection and Itraconazol therapy (200 mg 3 times daily for 3 days
and then 200 mg once or twice daily for 1 year). Median vision improves from 20/100 to 20/40 after 4-8 weeks of
treatment. In summary, we present a case of OHS with unusual manifestations. The molecular test used provided
evidence linking the ocular lesions with an earlier infection by H. capsulatum. In addition, the Hc100-nested PCR
represents an important and novel test for the diagnosis of histoplasmosis in human samples.
218
Author Index
A
Abadio AKR. 168
Acorci MJ. 194, 195
Adriana SG. 151
Agudelo CA. 205, 212, 215
Aguiar ESA. 141
Aguiar JIA. 141
Albe BP. 199
Almeida AJ. 91, 165
Almeida MA. 114
Almeida RAMB. 147
Alves BCA. 174
Alves HY. 123, 179, 181, 193, 200, 207, 208, 209
Andrade SR. 200
Arango M. 171, 215
Arathoon E. 101, 217
Araújo SA. 156
Arenas R. 142
Arruda C. 174
Azevedo RB. 181, 207, 209
B
Bagagli E. 127, 137, 138, 147, 161, 162, 167, 176
Bailão AM. 173, 175, 176, 178, 179
Báo SN . 169, 170, 181, 209
Barata LCB. 145
Barbosa MS. 169, 170
Barreto L. 172
Barrezueta J. 127, 137, 150, 161, 162
Baumgardner DJ. 216
Bedout C. 215
Benard G. 70, 127, 137, 145, 146, 147, 149, 151, 161,
174, 182, 188
Benjamin L. 217
Bennett J. 121
Bernardino S. 198
Bertossi DRT. 189
Bezerra CCF. 74
Biondo GA. 195
Blotta MHSL. 68, 187, 188, 189, 191
Bocca AL. 181, 189, 191, 193, 196, 200, 201, 207,
298, 209
Bonfim CV. 191
Bonifaz A. 142
Bordon-Graciani AP. 194, 195
Borges CL. 169, 173, 175, 179, 180
Bosco SMG. 147, 162
Braga CJM. 52
Brandt ME. 95, 217
Braun S. 181, 209
Braz SV. 209
Bredt Jr GL. 148, 150, 151, 157
Bredt CSO. 148, 150, 151, 157
Brito WA. 177
Brown G. 53
Bueno RA. 161, 210
Burger E. 80, 199
Burlandy-Soares LC. 68
C
Cacere CR. 70
Calich VGL. 50, 174, 192, 196, 197, 198
Calvi SA. 147
Camacho E. 170
Campanelli AP. 71, 193
Campos CLB. 173, 179
Campos FF. 208
Campos M. 150
Cano LE. 62, 67, 82, 190, 199, 205
Carmona JA. 91, 165
Caro E. 67
Carvalho A. 91, 165
Carvalho CRR. 146, 152
Carvalho LC. 103
Carvalho LR. 141, 155, 156, 210
Carvalho MJA. 168
Casadevall A. 62
Castro AG. 91
Castro KP. 114
Castro NS. 176, 177
Castro-Silva MH. 144
219
Catão E. 193
Cavalcante RS. 103, 147, 156
Cavassani KA. 71, 193
Cazarín-Barrientos J. 142
Chagas RF. 173
Champion M. 43
Chang MR. 141
Chaves CCR. 147
Chiller T. 95, 217
Cisalpino PS. 208, 210
Clemons KV. 49
Coelho KYR. 152
Coelho-Castelo AAM. 200
Coimbra Jr CEA. 143
Coitinho JB. 156
Cole GT. 43, 55, 74
Coltri KC. 194
Costa AN. 146, 152
Costa TA. 197
Cota BB. 208, 210
Coutinho ZF. 143
Cunha C. 91, 165
Cunha RV.141
D
Da Silva CJ. 201
Daffre S. 215
Dantas SFIM. 176, 179
Datta SK. 53
De Campos L. 148, 150, 151, 157
De Morais PC. 181, 207, 208, 209
De Souza-Silva C. 209
Deepe Jr GS. 176, 179
Derengowski LS. 209
Di Benedette JPT. 179
Dias-Melicio LA. 194, 195
Diaz G. 143
Diaz-Granados LR. 82, 190, 199, 205
Diogo CL. 187
Duarte AJS. 70, 188
Duarte IX. 152
Duarte MIS. 84, 116, 190
Duarte PAD. 148, 150, 151, 157
Duque JJ. 82, 190, 199, 205
Duran LF. 143
E
Escandon P. 216
Eulálio KD. 74
220
F
Faccioli LH. 180, 193
Faffe DS. 191
Fairbanks N. 147
Falcomer CL. 207
Faria FP. 170
Feitosa LS. 169
Felipe MS. 44, 123, 136, 165, 166, 168, 171,178,180,
181, 182, 183, 189, 193, 196, 200, 207, 208, 209
Félix CR. 170
Felonato M. 197
Fernandes CJCS. 152
Fernandes L. 87, 165, 166, 171
Fernandes VG. 156
Ferraz DB. 194
Ferreira LCS. 52
Ferreria MC. 188
Ferri PH. 180
Fierer J. 53
Figueiredo F. 191, 193, 196, 200, 201
Filho AD. 74
Fonseca CA. 189
Fonseca JH. 156
Fornazim MC. 187
Fracon JF. 191
Fraga CLF. 193, 196, 200
Franca FOS. 151
Furuchó CR. 189
G
Gabetta CS. 187
Galvãon MM. 151
García AM. 89, 171, 181
Gerolin GP. 144
Giarolla I. 146, 149, 151
Gil ML. 192
Gimenes-Bosco SM. 138
Goes AM. 156, 189, 200
Goldman GH. 91
Goldman WE. 59, 165
Golim MA. 194
Gomes Rittner GM. 211
Gómez BL. 62, 113, 217
Gómez CA. 143
González A. 67, 192, 215, 218
Gonzalez CR. 127, 162
Goulart S. 145
Gow NAR. 87
Gozalbo D. 192
Granzoto DS. 206
Graybill JR. 101, 109
Griva BL. 149, 152
Gualtero S. 143
Guedes HLM. 114
Guilherme RB. 207
Guimarães AJ. 114
H
Heinsbroek SEM. 53
Hernández D. 218
Hernández JM. 218
Hernández O. 89, 181
Herrera A. 217
Hidalgo JM. 82, 190
Hooyakaas PJ. 165
Hurtado H. 215
I, J
Ianhez LE. 151
Jerônimo MS. 191
Jiménez M del P. 53
Jiménez RA. 82, 190, 205
Johann S. 208, 210
Johnson DI. 91
Jukemura J. 149
Junior SAD. 146
Junqueira NM. 179
Junta C. 180
K
Kairalla RA. 146, 152
Kibbler CC. 62, 124
Kirkland T. 53
Kohara VS. 147, 155
Kono ASG. 146, 147, 149
Kyaw CM. 209
L
Lacava ZGM. 207
Lazèra MS. 74
Lazo J. 150
Lazo R. 150
Le T. 132
Leão C. 91, 165
Lenzi HL. 114
Lima CRG. 210
Lima DRA. 132
Lima ECD. 181, 207, 209
Lima S. 217
Lindsley M. 217
Lins TCL. 189
Logarinho E. 91
Lopera DE. 67, 82, 190, 199, 205
Lopes DF. 141
Lopes GP. 144
Lopes JD. 79
López-Martínez R. 142
Loures FV. 50, 192
Lozano VF. 189, 201
Ludovico P. 91, 165
Luiz AA. 151
M
Macêdo RCI. 74
Macoris SAG. 147, 162
Madeiros PB. 207
Malavazi I. 91
Mamoni RL. 68, 187, 188, 191
Mantilla JC. 148
Mariano M. 79
Mariano VS. 194
Marques AF. 208
Marques MEA. 155
Marques SA. 127, 137, 147, 161, 162
Martin MC. 107
Martinez R. 71, 102, 193, 206
Martins EMN. 200
Martins LMS. 74
Martins NF. 168
Massafera-Tristão FS. 198
Matos LF. 183
Matute DR. 135, 167
McEwen JG. 89, 167, 171, 181
Medeiros AI. 180
Mello de Souza TM. 209
Mendes RP. 103, 127, 137, 141, 147, 149, 152, 155,
156, 161, 162, 210
Mendes-Giannin MJS. 60, 70, 174, 175, 176, 182,
188
Michelin MA. 144
Milanezi CM. 193, 198
Millington MA. 96
Miramontes R. 217
221
Molano VM. 143
Molina RFS. 199
Montoya C. 218
Moraes DM. 150
Moraes MPT. 152
Morais FV. 173
Moreira AP. 71, 193
Moreira-Filho CA. 174
Moreto TC. 155, 161, 210
Moris DV. 103, 147, 155, 156, 161, 210
Morris-Jones R. 62
Motta-Castro ARC. 141
Moura F. 210
Muñoz C. 218
Muñoz JE. 201, 208, 211, 215
Myiamoto D. 146, 148, 151
N
Naranjo TW. 82, 190, 199, 205
Negroni R. 73
Neves FF. 144
Niño-Vega G. 45, 169, 170, 172, 177, 206
Nishikaku AS. 80, 199
Nóbrega FG. 173
Nogueira SV. 175
Nosanchuk J. 62
Nowill AE. 187
Nunes ES. 181, 207, 209
Nunes J. 193, 196, 200, 207
O
Odds F. 62
OIiveira EXG. 143
Oliveira AL. 141
Oliveira CCU. 189
Oliveira EB. 193
Oliveira EF. 103
Oliveira LL. 71, 193, 194, 198
Oliveira MLSC. 155
Oliveira RTD. 187
Oliveria A. 166
Oshiro TM. 187
P
Padilla-Desgarennes MC. 142
Paes HC. 165, 171, 182, 183, 189
Pagliari C. 84, 116, 190
Panagio L. 71
Paniago AMM. 141
222
Panunto-Castelo A. 194
Parente JA. 169, 178
Passos AN. 155
Passos EC. 187
Passos GAS. 180
Passos-Silva DG. 180
Patarrollo ME. 130
Patiño J. 82, 190
Pavan D. 150
Pedott F. 148, 150, 151, 157
Pedroso EP. 156
Pedroso SM. 173
Pegorare AL. 141
Peixoto DLG. 207
Peraçoli MTS. 194, 195
Pereira M. 173, 175, 176, 177, 178, 180
Pereira NV. 84, 190
Pereira RW. 189
Pereira T. 148, 150, 151, 157
Pereira M. 170
Philippsen HK. 178
Pina A. 196
Pinto FA. 201
Pinto FL. 207
Pizzini CV. 114
Pizzolatti MG. 210
Polez VPP. 166
Popi AF. 79
Puccia R. 51, 92, 175, 201
Q
Queiroz-Tellez F. 105, 129
Quesada-Ocampo LM. 167
Quiceno W. 211
R
Rappleye CA. 165
Rauscher JT. 167
Ravanini JN. 145
Raxcacoj G. 217
Reis BS. 200
Resende MA. 210
Restrepo A. 62, 82, 89, 171, 181, 190, 199, 205, 211,
212, 215
Restrepo CA. 211
Rezende RV. 180
Rezende TCV. 114
Rezende TCV. 176
Ribeiro AA. 148, 151
Ribeiro AM. 196, 200, 207
Richart CAO. 169
Richetti MA. 150
Richini-Pereira V. 162
Riley P. 217
Rocha AA. 175
Rocha FA. 71, 193, 198
Rodrigues ACJ. 165
Rodrigues F. 91, 165
Rodrígues-Brito S. 206
Romano CC. 188
Roque-Barreira MC. 194
Rosa CA. 208
Rosa LH. 208
Rossi DCP. 215
Rossi M. 193
Russo RC. 191
S
Sá NP. 208
Sadahiro A. 116, 187
Saldanha CA. 207
Salem-Izaac SM. 173, 179
Salge JM. 152
Salles BC. 200
Salmito MA. 74
Samayoa B. 101, 217
Sampaio-Marques B. 91
San-Blas G. 121, 169, 170, 172, 177, 182, 206
Santana JM. 169, 170, 178
Santana MS. 127, 162
Santos MP. 179
Santos RMM. 132
Santos RS. 179
Santos SC. 180
Sato PK. 187
Saul A. 142
Scheel CM. 217
Serrano A. 148
Sharpton TJ. 43
Shikanai-Yasuda MA. 116, 146, 147, 149, 151, 187,
189
Silva CL. 180, 200
Silva JF. 174, 182
Silva JR. 207
Silva JS. 71, 193, 198
Silva LFF. 145
Silva MES. 137, 161
Silva RP. 176
Silva SS. 44, 166, 171
Silva-Pereira I. 61, 209
Silva-Vergara ML. 144
Simioni AR. 207, 208
Simoneide SS.178, 180
Siqueira EP. 210
Siqueira IM. 191, 196
Soares AMVC. 68, 194, 195
Soares CMA. 59, 114, 169, 170, 173, 175, 176, 177,
178, 179, 180
Soares MAS. 191
Soliva Jr E. 148, 151
Sorais F. 169, 172, 177
Sotto MN. 84, 190
Sousa MV. 169
Souza ACO. 200
Souza BS. 152
Souza CR. 166
Souza GND. 179
Spago MC. 187
Stajich J. 43
Steensma HY. 91
Suesada M. 152
Suzin F. 157
T
Tabares AM. 212
Taborda CP. 51, 52, 201, 208, 211, 215
Nóbrega YKM. 201
Takagaki TY. 146
Tavares AH. 44
Tavares MG. 144
Taylor JW. 43
Tedesco AC. 207, 208
Teixeira MM.136, 165, 168, 171, 182, 183, 189
Teixeira SMR. 180
Terçarioli GR. 127
Theodoro RC. 138, 162, 167
Titze de Almeida R. 207, 208
Tobón AM. 117, 205, 211, 212
Tomazett MV. 176
Tomazett PK. 170
Torres FA. 165
Torres FAG. 166, 183
Torres G. 216
Torres I. 171
Travassos CMR. 143
Travassos LR. 51, 52, 201, 208, 211
Trilles L . 74
223
W
Trinca LA. 167
Tristão FSM. 71
U
Urán ME. 62
Urán ME. 67
Walls L. 53
Wanke B. 74, 141, 143
Watanabe GA. 210
Winters M. 175
V
Valente NYS. 147
Valle ACF. 143
Valpolini LC. 148, 151
Vancim JO. 193
Vargas J. 142
Velasco Castrejón O. 142
Veloso JMR. 156
Vendruscolo PE. 194
Viana DF. 165
Vicentini-Moreira AP. 147, 155
Vieira RG. 189
Villa CA. 211
Villalobos H. 172
Viriyakosol S. 53
Vitali LH. 206
224
Y
Yañez A. 192
Yoshida M. 116, 146, 147, 149, 151
Z
Zambuzzi PF. 180
Zancopé-Oliveira RM. 114
Zani CL . 208, 210
Zin WA. 191
Zuluaga A. 215
Zuluaga AF. 82, 190, 205
Acknowledgment and Gratitude
To the following Institutions, Organizations and Enterprises that gave their support to the
X International Congress on Paracoccidioidomycosis:
Academic and Governmental Sponsors
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Academia Colombiana de Ciencias Exactas, Físicas y Naturales
Alcaldía de Medellín – Secretaría de Salud Municipal
American Society for Microbiology (ASM)
Asociación Colombiana de Dermatología y Cirugía Dermatológica (ASOCOLDERMA)
Asociación Colombiana de Infectología, Capítulo Antioquia
Asociación Colombiana de Infectología, Capítulo Central
Asociación Colombiana de Parasitología y Medicina Tropical
Asociación Colombiana de Pneumología y Cirugía de Tórax, Capítulo Antioquia
Banco de la República - Fundación para la Promoción de la Investigación y la Tecnología
Biomédica, Revista del Instituto Nacional de Salud
Centers for Disease Control and Prevention, Department of Healt and Human Services (CDC)
Cooperación Técnica, Embajada de Brasil, Bogotá, D.C. – Colombia
Cooperativa Médica de Antioquia - COMEDAL
Empresas Públicas de Medellín – EPM
Federación Nacional de Cafeteros de Colombia
Gobernación de Antioquia – Dirección Seccional de Salud de Antioquia
Instituto Colombiano de Crédito y Estudios Técnicos en el Exterior – ICETEX
Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnología “Francisco José de Caldas”
COLCIENCIAS
Instituto Nacional de Salud - INS
International Society for Human and Animal Mycology (ISHAM)
Medical Mycological Society of the Americas (MMSA)
Metrosalud
Metro de Medellín
Organización Panamericana de la Salud, Colombia
Pan American Health Organization/ World Health Organization ( PAHO/WHO)
Servicio Nacional de Aprendizaje - SENA
Springer Verlag, Mycopathologia
The Academy of Science for the Developing World (TWAS)
Universidad de Antioquia, Facultad de Medicina y Escuela de Microbiología
Universidad Pontificia Bolivariana, Facultad de Medicina
225
Personal Contributors
Drs. Karl V. and Lynda Clemons
Drs. Steve and Maria Croker
Dr. Juan Esteban Gutierrez C.
Mrs. Catalina de Bedout G.
Drs. Moisés and Maria Helena Robledo
Members of the Medical & Experimental Mycology and Molecular Biology Units,
Corporación para Investigaciones Biológicas (CIB)
Commercial Sponsors
AM Limitada
ARC Análisis
Avianca
Bioinstrumental
Filtración y Análisis
Fundación Suramericana
Interconsulting S.A.
Janssen-Cilag
Merck Sharp & Dohme
Metroquirúrgicos
Pfizer S.A., Inc
Schering-Plough S.A.
226
Notes