Molecular Techniques for
the Identification of
Pathogenic Fungi
Wieland Meyer PhD
Molecular Mycology Laboratory
Westmead Hospital/The University of Sydney
WM, Westmead Hospital, Sydney, Australia
Issues in Mycology
 Significant
increase opportunistic fungal infections due to
the increase in immunocompromised hosts
 Cosmopolitan
environmental fungi have emerged as
causes of potentially life-threatening diseases
 Many
emerging pathogenic fungi are inherently resistant
to antifungal drugs
 A number
of pathogenic fungi are non-viable in tissue
samples
 Traditional identification techniques lack sensitivity &
specificity, are slow, labor-intensive and require skilled
personnel
 Clinical
and economic consequences
WM, Westmead Hospital, Sydney, Australia
Conventional Fungal
Identification Techniques
Identification Method
Concerns
Culture ID
- only in 26% positive if < 1cfu/ml blood
Serological Tests
- limited value (e.g. Iatron Crypto Kit)
- suboptimal sensitivity & specificity
Germ-tube Formation
- useful screening test only for C. albicans
(does not differentiate C. dubliniensis)
Carbohydrate Assimilation
- limited species in the databases
(e.g. Vitek & API strips)
- can misidentify certain pathogenic species
Morphological Characters
- subjective measure,
- high degree of skills required
WM, Westmead Hospital, Sydney, Australia
Fungal Identification is Currently
Based on the Analysis of:
Morphological Characters
 Culture and Microscopy
Physiological/Biochemical Characters
 e.g. Vitek and API
 These techniques are often time-consuming,
labour intensive and difficult to interpret.
WM, Westmead Hospital, Sydney, Australia
Results Obtained with VitekYBC and API
Total number of isolates tested
# of isolates included in the database
# of isolates correctly identified
# of isolates incorrectly identified
WM, Westmead Hospital, Sydney, Australia
Vitek-YBC
API ID32C
81
80
55
48 (87.3%)
7 (12.7%)
69
50 (76.8%)
16 (23.2%)
Culture-ID
 Blood
cultures positive in only 20-58% of Invasive
Candidaisis  Only in 26% positive if < 1cfu/ml blood,
and 10% of Aspergillosis
Serological Tests e.g.:
Iatron
Cryptococcus Kit
 95% accurate with Cryptococcus neoformans
Serotype specific antisera, problems AD strains
Iatron
Candida Check Kit
 95% accurate with Candida specific antisera
In general suboptimal sensitivity and specificity
WM, Westmead Hospital, Sydney, Australia
Ideal ID/Diagnostic Test
- Sensitive and specific
- High positive predictive value
- High negative predictive value
- Useful for monitoring
- Simple, rapid and inexpensive
WM, Westmead Hospital, Sydney, Australia
Why ?
Confirmation of medical diagnosis, choice of therapy,
follow-up and prevention are critical to a successful
infectious disease management.
- Facilitate earlier diagnosis
- Initiating earlier intervention with aggressive
antifungal treatment to improve patient outcome
- Reduce empiric use of antifungal agents
How ?
- Detection of fungal genomic sequences
WM, Westmead Hospital, Sydney, Australia
Why Molecular Methods?
Phenotypic Characters are unstable and
can change with environmental changes
 Identification methods based on
Genotypic Characteristics would be
advantageous and potentially more
accurate, reproducible, simple and rapid
WM, Westmead Hospital, Sydney, Australia
Proposed Applications of DNA Protocols
Organism detection in blood
Organism detection in body fluids
Organism detection in tissue
Molecular ID
Identification of the fungal agent
Quantification of the fungal load
Monitoring of antifungal treatment
WM, Westmead Hospital, Sydney, Australia
DNA Technology
 DNA
probes
– Southern Hybridization
– In-situ Hybridization
– Microarray
– Macroarray
– Reversed line blot
 Karyotyping
WM, Westmead Hospital, Sydney, Australia
PCR
primers
– SSCP
– Genotyping
– Panfungal PCR
– Multiplex PCR
– Nested PCR
– Real Time PCR
– Sequencing
– PCR Fingerprinting
– AFLP
– PCR-RFLP
Samples used: Blood:
Should be collected in tubes containing EDTA (1mg/ml)
!! To not use heparin as anticoagulant !!
Biopsy Tissue:
!! Fresh tissue is always better !!
Paraffin embedded tissue is not always amenable to PCR amplification
because of DNA modification due to cross-linking induced by the
fixative or fixation time!
In addition time-dependent physical degradation of DNA in paraffinembedded tissue limits the length of the DNA fragment amplified.
WM, Westmead Hospital, Sydney, Australia
Targets for PCR, PCR-RFLP,
Sequencing& In-situ Hybridization:
Universal fungal primers for:
- Multi-copy Genes

 rDNA gene cluster 18S, ITS1/2, 5.8S, 28S, 5S, IGS
- Single-copy Genes
 Actin, Alkaline Protease (ALP), Chitin Synthase,GP43, Lanosterol -  demethylase (LIA1), URA5, Secreted Aspartic Protease (SAP), Beta glucan
synthetase (FKS), Histone, etc.

Genus- or species-specific primers
 18S, ITS1/2, 28S rDNA, Mitochondrial DNA, Histone
e.g. Candida, Cryptococcus, Aspergillus
WM, Westmead Hospital, Sydney, Australia
Tandem Repeat of the Ribosomal Gene Cluster
WM, Westmead Hospital, Sydney, Australia
Vilgalis Lab, Duke University, NC, USA
Species-Specific Amplification
1 = Cryptococcus neoformans
2 = Cryptococcus albidus
3 = Candida albicans
WM, Westmead Hospital, Sydney, Australia
Mitchell et al. JCM 1994 32(1) 253-255
PCR Amplification of Specific Genes




Clinical specimen
Direct PCR of a single colony
form a primary isolation plate,
tissue sample or clean culture
Agarose gel electrophoresis
ID via comparison with the
data base
 Species Level
ITS1, 5.8S, ITS2 region
WM, Westmead Hospital, Sydney, Australia
Nicolas Latouche
Commercial Fungal Molecular Identification Kits
Fungal ID Kits for:
- Blastomyces dermatitidis
- Coccidioides immitis
- Histoplasma capsulatum
- Single-stranded DNA probe targeted to the ribosomal RNA
- Selection reagent differentiates between non-hybridized and hybridized probe
- Labelled DNA:RNA hybrids measured in a GEN-PROBE luminometer
WM, Westmead Hospital, Sydney, Australia
Protocol of the
Kits
WM, Westmead Hospital, Sydney, Australia
Product of amplification using Histone loci
• Four members of histone gene family: H2A, H2B, H3, and H4
• The genes are organized into two pairs of genes separated by divergent
promoter regions
• Histone genes are very highly conserved between species
Unique species-specific sequence
Highly conserved coding region
Highly conserved coding region
Histone H3/H4 gene pair is arranged differently between yeast and humans
H3
H4
yeast
Human
H4
H3
Primers designed to amplify yeast H3-H4 region should not amplify human sequences
WM, Westmead Hospital, Sydney, Australia
MicroBioGen
Nested PCR Histone locus
K. marxianus ID
•use SYBR green to detect PCR product
Z. rouxii ID
•use species specific primers to do
nested amplification
S. cerevisiae ID
•use degenerate consensus primers to
amplify unknown
S. cerevisiae cells
WM, Westmead Hospital, Sydney, Australia
MicroBioGen
PCR-RFLP
(Restriction Fragment Length Polymorphism Analysis)






Clinical specimen
Direct PCR of a single colony
form a primary isolation plate,
tissue sample, or clean culture
Agarose gel electrophoresis
Digestion with restriction
enzymes
Agarose gel electrophoresis
ID via comparison with the data
base
 Species Level
WM, Westmead Hospital, Sydney, Australia
ITS1, 5.8S, ITS2 region
Nicolas Latouche
RFLP MAPS
Candida albicans/dubliniensis
Atlas of Clinical Fungi de Hoog
et al 2000
RFLP maps available from:
 “Atlas
of Clinical Fungi” 2nd Edition 2000
by: GS de Hoog, J Guarro, J Gené & MJ Figueras
Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
 BioloMICS
at: www.cbs.knaw.nl
by: V. Robert
WM, Westmead Hospital, Sydney, Australia
Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands and BioAware, Belgium
Real Time PCR
 ABI

7700 System (TaqMan)
Reporter Dye and Quencher Probe Detection
 Quantitative DNA and Species Detection
 Roche

LightCycler
SYBR Green Detection
 Quantitative DNA Detection
Diagnostics
e.g. Candida sp. detection Guiver et al. J. Clin. Pathol. 2001 54:362-366
e.g. Pneumocystis carinii detection Kaiser et al. (2001) J. Microbiol. Meth. 45:113-118

Hybridization Probes (Donor Fluor and Acceptor Fluor) Detection
 Quantitative DNA and Species Detection
e.g. C. albicans and A. fumigatus detection Loeffler et al. (2000) JCM 38:586-590
WM, Westmead Hospital, Sydney, Australia
 All
necessary reagents for amplification and detection of
Candida albicans
 Targets the ITS region
 Wide range of biological specimens including swabs,
sputum, urine as well as blood cultures and isolated
colonies
 Detect Candida albicans in less than 3 hours using the
PCR workflow system
 Runs under a common thermal profile with other
Microbiology specific kits eg. Enterococcus,
Pseudomonas and Staphylococcus kits
WM, Westmead Hospital, Sydney, Australia
Diagnostics
LightCycler Candida Kit MGRADE
Multilocus approaches: PCR-Fingerprinting
RAPD (Random Amplified Polymorphic DNA)
AFLP (Amplified Fragment Length Polymorphism)

Clinical specimen
Pure culture
DNA extraction
PCR amplification with Mini- or Microsatellite specific primers
Agarose gel electrophoresis
ID via comparison with the data bank

Species and Strain Level





Homology
Primer
M13
(GACA)4
Intra-species
75-95%
74-94%
Inter-species
5-25%
6-26%
(Data obtained from 80 Candida species and 150 strains)
Primer: M13
WM, Westmead Hospital, Sydney, Australia
Meyer et al. (1997) Electrophoresis, 18: 1548-1559
PCR Fingerprinting Database Development

DNA extraction, PCR fingerprinting and gel running conditions
standardized

Reference profiles
- 70 anamorph-teleomorph pairs
- type cultures and clinical strains (>300 individual strains)

Pattern analysis via GelcomparII and

Integrated database/web access via
Collaboration is welcome:
WM, Westmead Hospital, Sydney, Australia
w.meyer@usyd.edu.au
Heide-Marie Daniel/Krystyna Marszewska/Vincent Robert
Fungal ID via Sequencing






Clinical specimen
Direct PCR of a single colony from a primary isolation plate,
tissue sample or clean culture
DNA extraction
PCR amplification
Sequencing
ID via comparison with the EMBL or GenBank data bases
 Species and Strain Level
WM, Westmead Hospital, Sydney, Australia
rDNA gene cluster
18S rDNA or SSU
1800 bp
SR1R
5.8S rDNA
159 bp
SR6R/IT S1
V1/2
V 3/4
V5
ITS3 LR1/ITS4
D1
V7 V8 V9
5.8S/ITS2
IGS
28S rDNA or LSU
3396 bp
LROR
D2
D3
D 4/5
D 6/7a/7b
D8
D 9/10
D 11/12
LR16
ITS 1 ITS 2
361 bp 231 bp
LR12
IGS
Heide-Marie Daniel/Wieland Meyer
ITS1 Sequence
variability
ITS2
ITS1 Discriminatory
power
ITS2
LSU
5.8S
5.8S
LSU
SSU
SSU
WM, Westmead Hospital, Sydney, Australia
Sequence Data Bases
EMBL
at: www.ebi.ac.uk

GenBank
at: www.psc.edu/general/software/packages/genbank/genbank.html

BioloMICS
at: www.cbs.knaw.nl

International European rRNA database (Candida)
at: www.rrna.uia.ac.be.Isu

Sequence variation used for fungal ID



Sequence Analysis:
Intra-species variation:
Inter-species variation:
LSU rDNA
0 - 0.5%
0 - 17.5%
PLB1
0 - 0.8%
0.8 - 16%
URA5
0 - 0.3%
0.8 - 15%
(Data obtained from sequences of 82 Candida species)
WM, Westmead Hospital, Sydney, Australia
Heide-Marie Daniel/Nicolas Latouche/Stuart Jackson
Candida species
Not all
fungal species
are
sequenced!!!
e.g. GenBank 1.10.2002
WM, Westmead Hospital, Sydney, Australia
Candid a castelii
Candid a catenulata
Candid a ciferri
Candid a famata var.famata
Candida famata var. flareri
Candid a (Torulopsis ) glabrata
Candid a guilliermondii
Candid a humicola
Candid a inconspicua
Candid a intemedia
Candid a kefyr
Candid a krusei
Candid a lambica
Candid a lipolytica
Candid a lusitaniae
Candid a nitrativorans
Candid a norvegenisis
Candid a norvegica
Candid a paraps ilosis
Candid a (Torulopsis ) pintolopesii
Candid a pseudo tropicalis
Candid a pulcherrima
Candid a rugosa (var rugosa )
Candid a saitoana
Candid a sake
Candid a sphaerica
Candid a utilis
Candid a valida
Candid a viswanathii
Candid a zeylanoides
Candid a lusitaniae
18S
Y
Y
Y
Y
Y
ITS1
ITS2
28S
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
®
MicroSeq Workflow
or
Sample from colony or pure culture
PrepMan Ultra DNA isolation
< 30 minutes
MicroSeq PCR module (1 reaction)
< 2 hours
MicroSeq Cycle sequencing module (2 reactions)
< 2 hours
MicroSeq analysis software and rDNA database
< 30 minutes
Final identification report
WM, Westmead Hospital, Sydney, Australia
The Future
Extraction
DNA Extraction Robot
WM, Westmead Hospital, Sydney, Australia
Detection
Real Time PCR
Identification
Sequencing
DNA Array Technology
 Small
glass or silicon matrices on which potentially
thousands of short oligonucleotides can be immobilized
 e.g. - species-specific DNA probes
- antifungal resistance genes
- virulence genes
C. g.
C. p.
C. l.
C. k.
C. n.
C. li.
C. m.
C. t.
C. u.
Macroarray
C. glabrata
C. parapsilopsis
C. lipolytica
C. krusei
C. norvegensis
C. lusitaniae
C. multigemmis
C. tropicalis
C. utilis
WM, Westmead Hospital, Sydney, Australia
Microarray
PCR Methods for Fungal ID
Method
Culture
Requirements
Pattern
Stability
PCR-Amplification
of Specific Genes
single colony
tissue sample
very good
PCR-RFLP
single colony
Real Time PCR
Identification
Level
Turnaround
Time
very good
species
1 days
good
very good
very good
species
2 days
good
single colony
very good
very good
species
1-2 hours
good
PCR-Fingerprinting pure culture
(RAPD/AFLP)
very good
very good
(RAPD: good-poor)
species/strain
2 days
good
best
best
species/strain/
single mutation
2 days
expensive
Sequencing
single colony/
tissue sample
Reference Labs only!
WM, Westmead Hospital, Sydney, Australia
Reproducibility
Cost
Efficiency
Average Medical Mycology Lab
PCR Assay Sensitivity
 Multi-copy
e.g.
Genes:
18S rRNA
ITS
 Single-copy
e.g.
1-5 CFU
10 CFU/ml blood
Genes:
Actin
1.4- lanosterol demethylase
Heat-shock protein 90
WM, Westmead Hospital, Sydney, Australia
1-10 CFU
10-100 CFU/ml blood
10-100 CFU
Role of Molecular Diagnosis for Candidiasis
- Blood / Serum
+ve PCR
good
Consecutive +ve PCR
very good
- ve PCR
cannot justify ceasing therapy
WM, Westmead Hospital, Sydney, Australia
Precautions for clinical PCR

-
-
Standard approaches for the three major phases
of clinical PCR:
Sample preparation
Target and probe selection
PCR and post-PCR analysis
Positive and negative controls
 PCR contamination!!!
WM, Westmead Hospital, Sydney, Australia
Pitfalls of DNA based Identification

PCR reaction; contamination risk

e.g. Qiagen DNA extraction columns contaminated
with fungal DNA

Detection of false positives or false negatives

Disease causing agent or colonization
WM, Westmead Hospital, Sydney, Australia
Sources of Contamination
Pre-PCR Contamination
No routine sample collection methods have been established for PCRdiagnosis
Contaminating DNA can originate from:
Any persons skin, hair, door handles, surfaces in the laboratory
Clinical equipment (maybe sterile but not DNA free)
Reagents
Use only PCR grade!!
Taq polymerase (can contain procaryotic or eucaryotic DNA)
PCR components (e.g: gelatine, BSA)
PCR products from previous PCR reactions (e.g. if diagnostic PCR is
repeatedly performed)
WM, Westmead Hospital, Sydney, Australia
Floor Plan for a Clinical PCR Laboratory
WM, Westmead Hospital, Sydney, Australia
In summary, consistently reliable, universally applicable and
standardized methods for fungal ID are still to be established.
Many in-house DNA based fungal ID techniques exist:
 Problem all of them lack standardization
There is a lack of commercial interest to develop DNA based fungal ID systems,
because of:
- the limited market
- limited antifungal spectrum available
- high development costs
Commercially available DNA based ID kits exist only for:
- Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis
(AccuProbe Kits, Gen-Probe, USA)
- Candida
albicans (Roche)
- Universal Fungal ID via Sequencing MicroSeq (Applied Biosystems)
WM, Westmead Hospital, Sydney, Australia
Acknowledgements
Molecular Mycology Laboratory
Microbiogen/Macquarie University
Krystyna Marszewska
Dr. Phillip Bell
Mathew Huynh
Sarah Kidd
CBS, The Netherlands
Dr. Nicolas G. Latouche
Dr. Vincent Robert
Dr. Heide-Marie Daniel
David Yarrow
Dr. Catriona L. Halliday
WM, Westmead Hospital, Sydney, Australia
Molecular Mycology Reference Laboratory
Samples Should be Directed to:
Westmead Hospital
ICPMR
Darcy Road
Westmead, NSW 2145

Contact Persons:
Marked: Molecular Mycology Laboratory
Dr. Wieland Meyer
Ph.:
61-2-98456895
Fax:
61-2-98915317
E-mail: w.meyer@usyd.edu.au
More Info soon at: www.usyd.edu.au/~cidm
WM, Westmead Hospital, Sydney, Australia