Malaria

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MALARIA THE KILLER DISEASE
In Africa, malaria kills one
child in 20 before the age of
five (STI
• Every year, more than 500 million people become
severely ill with malaria.
• More than 1 million people die each year. The
majority of victims are children and pregnant women.
WHO says malaria kills 3,000 children under five
every day (one African child every 30 seconds)
• Sub-Saharan Africa bears 90 per cent of the burden.
History of malaria
• The periodic fever episodes
characteristic for malaria have been
described by physician early on in
human history
• People recognized a connection
between malaria and swamps
• It was thought that swamps exude a
miasma or poison which causes the
disease
Giovanni Maria Lancisi (1654-1720).
He first described a characteristic black pigmentation of the brain and
spleen in the victims of malaria. Lancisi linked malaria with poisonous
vapours of swamps or stagnant water on the ground. In 1717, in
his monograph titled Noxious Emanations of Swamps and Their
Cure, he speculates that malaria was due to minute "bugs" or
"worms" which entered the blood and revived the old idea that
mosquitoes might play a role.
History of malaria
Dr. Alphonse Laveran, a military
doctor in France’s Service de
Santé des Armées (Health
Service of the Armed Forces).
• Based on epidemiological
considerations Alphonse
Laveran concluded that
“Swamp fevers are due to a
germ”
• In 1880 he discovered
parasite life cycle stage
(gamonts and gametes) in the
blood of patients with fever
which were absent in samples
from healthy individuals
• After the development of
methylene blue (1899) it was
possible to identify all the
malaria species.
History of malaria
• Camillo Golgi, an Italian
neurophysiologist, established that
there were at least two forms of the
disease, one with tertian
periodicity (fever every other day)
and one with quartan periodicity
(fever every third day).
• He prepared high quality
micrographs and described the
asexual replication of the parasite
within the red blood cell
• He observed that fever coincided
with the rupture and release of
merozoites into the blood stream.
• He was awarded a Nobel Prize in
Medicine for his discoveries in
neurophysiology in 1906.
History of malaria
Ronald Ross was the first
to demonstrate that a
mosquito could transmit a
(bird) malaria parasite.
• Patrick Manson (discovered
mosquito transmission of
Wucheria) proposed same
transmission pathway for
malaria.
• With Manson’s
encouragement and advise,
Ross finds that the mosquito
can get the parasites from
human when feeding.
• He works with bird malaria in
Calcutta and discovers how
the parasite is passed from
mosquito to the birds (saliva).
• He received the Nobel Prize
for his work
History of malaria
• Battista Grassi
demonstrates that
mosquitoes are also
involved in the
transmission of human
malaria
• He identifies the major
vectors and life cycles for
different Plasmodium
species in Italy
• Enemies with Ross, he
was ignored by the
Nobel committee.
Nobel Prizes for Malaria Related Research
• Ronald Ross, 1902: For his work on malaria, he shows how the parasite enters
the organism. He laid the foundation for successful research on this disease and
methods of combating it. Ronald Ross demonstrated the oocyst of malarial parasite
in the gut wall of a mosquito on August 20, 1897 in Secunderabad, India.
• Alphonse Laveran, 1907: "In recognition of his work on the role played by
Ronald Ross (1857-1932)
protozoa in causing diseases". Laveran was the first to notice parasites in the blood
of a patient suffering from malaria on November 6, 1880 at Constantine, Algeria.
• Julius Wagner-Jauregg, 1927: "For his discovery of the therapeutic value of
malaria inoculation in the treatment of dementia paralytica". Wagner-Jauregg
developed methods for treating general paresis (advanced stage of neurosyphilis) by Laveran (1845-1922)
inducing fever through deliberate infection of patients with malaria parasites. This
method was used in the 1920s and 1930s. In the 1940s, the advent of penicillin and
more modern methods of treatment made such "malaria therapy" obsolete.
• Paul Hermann Müller, 1948: "For his discovery of the high efficiency of DDT as a
contact poison against several arthropods”
Jauregg (1857-1949)
• Camilo Golgi, 1906: Golgi shared the Nobel Prize with Santiago Ramon Cajal for
their studies on the structure of the nervous system. Golgi made significant
contributions to malaria research as well
Muller(1899-1965)
Malaria is caused by plasmodium parasites
The parasite
spends part of
its life cycle
inside the red
blood cells
Humans act as
intermediate hosts
where sexual and
asexual forms of the
parasite are found
The parasite is transmitted by the bite of
the female anopheline mosquito which
acts as the definitive host
Plasmodium species
(Malarial Parasites)
• Plasmodium falciparum: malignant tertian malaria. Tropics.
Accounts for 50% of all malaria cases. Most pathogenic.
• Plasmodium vivax: causes benign tertian malaria. Tropics,
subtropics, and some temperate regions. Mostly found in Asia. About 43%
of all malaria cases. Some Africans are refractory to infection because the
lack the red cell receptor that the parasite use to enter.
• Plasmodium malariae: quartan malaria. Tropics. About 7% of
malaria cases
• Plasmodium ovale: mild tertian malaria. West Africa,
occasionally East Africa. Rare. It was used to treat syphilis.
HOSTS
DEFINITIVE HOST:Sexual stages of the parasite. Anopheline
female Mosquito (sexual reproduction). 400 species and one-tenth
are potential malaria vectors
INTERMEDIATE HOST: Humans (asexual and sexual phases)
PARASITE FORMS
Merozoite: invades erythrocytes
Sporozoite invades
mosquito salivary glands and
liver cells
Ookinete invades mosquito
gut epithelial cells
Mosquito forms: gametocytes,
Oocyst, ookinete, sporozoite
Blood forms:
merozoites, rings,
trophozoite, schizonts
Malaria Life Cycle
Oocyst
Sporozoites
Mosquito Salivary
Gland
Zygote
After an
indeterminate
number of asexual
generations, some
merozoites become
gametocytes
Preerythrocytic
cycle
Gametocyte
s
IRC ruptures
Female anopheline
mosquito injects
sporozoites which
invade liver cells
In the liver parasite
changes into
trophozoite
releasing more
merozoites
Erythrocytic
cycle
Merozoite transform
into trophozoite, ring
stage, schizont.
Merozoites are
released into the
blood
Plasmodium life cycle in the mosquito
Gametocytes develop into gametes:
macrogametocyte to macrogamete.
Microgametocyte displays
exflagellation to form microgametes
10-12 days: sporozoites
enter salivary glands
9-11 days: sporozoites
exit oocyst in
hemolymph
25-50 hours: ookinete
transforms into oocyst
in midgut epithelium
15-60 min after blood
meal: male and female
gametes form zygote,
which develops into
ookinete
24-40 hours: ookinete
penetrates and crosses
blood meal sac
Sporozoite development takes 10-14 days depending on the plasmodium species.
Mosquito remains infective for life
Anopheles spp. live long enough to feed on human blood repeatedly
Infection appears to stimulate mosquito to feed more frequently
Exoerythrocytic Cycle
Plasmodium sporozoites (green)
are deposited under
the skin of the vertebrate host
through the bite of an infected
female Anopheles mosquito. After
injection into the skin, the
sporozoites move through the
dermis until they contact blood
vessels (red) and move into the
circulatory system, which allows
them to travel to the liver. A small
proportion of sporozoites can enter
the lymphatic system (yellow).
Nature Reviews Microbiology (2006): 4-849
The sporozoite
After traversing several host
cells the sporozoite settles in
one and develops and
replicates within a welldelimited parasitophorous
vacuole. Each invading
sporozoite develops and
multiplies inside a hepatocyte,
forming the schizont, which is
made up of thousands of
merozoites.
In and out. The sporozoite traverse the
cytosol of the host cell.
Nature Reviews Microbiology (2006): 4-849
Hypnozoites in P. vivax and P.ovale. Dormant forms that seem to
be responsible for relapse months or years later. Same section of
liver observed by immunofluorescence showing a fluorescent liver
schizont but also a smaller fluorescent hypnozoite
The sporozoite
• 10-15 m long
• 2 sporozoite surface proteins contain hepatocyte adhesive domains (CSP
circumsporozoite protein and TRAP thrombospondin related anonymous
protein)
• Both proteins bind to glycosoaminoglycans on the surface of hepatocytes and
this binding is important for entry
SEM of a sporozoite of Plasmodium cynomolgi. Reaction between the surface antigen (circumsporozoite protein (CSP) and
immune serum. The sporozoite would normally have a smooth surface, but the formation of antigen-antibody complexes has
created this roughened appearance. The first synthetic malaria vaccine was based upon the CSP repeat sequence in P.
Exoerythrocytic Cycle
• Schizonts take 5-7 days to develop
• formation of >10,000 merozoites
• upon rupture of hepatocyte, released
merozoites will invade rbc’s
The final step involves the release of
merozoites (green) into the bloodstream. The
signal(s) that trigger the release remain
unknown. Plasmodium merozoites are
released by the formation of merozoite-filled
vesicles (merosomes), which bud off from the
infected hepatocytes into the sinusoidal lumen.
Nature Reviews Microbiology (2006): 4-849
Merozoites rapidly enter erythrocytes
At schizogony,
the parasite
undergoes
multiple rounds
of mitosis to
generate nuclei
that are
assembled into
the daughter
merozoites.
At 48 hours, the
infected
erythrocyte
ruptures to
release
merozoites.
Merozoites invade erythrocytes and for
this the apical organelles of the merozoite
(dense granules, rhoptries and
micronemes) must be oriented first.
Invasion results in the formation of an
intracellular 'ring'-stage that is surrounded
by a parasitophorous
vacuolar
membrane (PVM).
After approx. 24–30 hours the
trophozoite stage is reached.
Rings and trophozoites export
parasite proteins into the
cytoplasm and membrane of the
erythrocyte, and into structures
called the Maurer's clefts.
PfEMP1, P. falciparum
erythrocyte membrane protein 1
The merozoite
The merozoite is briefly present in the blood
between leaving one red blood cell and
entering the next one. Lemon shape, Aprox
1 by 1.6 m micron.
In this scanning electron micrograph, a merozoite
(the small sphere) is attached to an erythrocyte at
the start of invasion.(Image from Bannister, L.H. "Malaria",
Topics in Inernational Health,(1998) The WellcomeTrust, CABI
Publishing, CAB International)
Cowman and Crabb, (2006) Cell 124: 755
The merozoite
RBC cell surface
molecules act as
receptors for merozoite
ligands:
• P. vivax: Duffy antigen
binding protein binds to
the duffy antigen
• P. falciparum: EBA-175
binds to glycophorin A,
unknown ligands bind to
glycophorin B and
receptor “X”
This is TEM which shows the invasion of a red blood cell by a merozoite. The apical
complex (at the left side of the merozoite) is attached to the red cell. One of the rhoptries is
visible close to the apical complex. Observe dense granules (Image from Aikawa M. "Malaria", Topics
in Inernational Health,(1998) The WellcomeTrust, CABI Publishing, CAB International)
Plasmodium blood forms: the ring stage
• 1st 14-16 hours
spent as ring
stage, or young
trophozoite
• little to no Hb
degradation
• only form seen in
blood films of P.
falciparum
A ring stage of the cup-like
form showing the nucleus
(n), surrounded by
ribosomes and some
endoplasmic reticulum.
(A Brief Illustrated Guide to the Ultrastructure of Plasmodium falciparum Asexual Blood Stages. L. H. Bannister, J. M.
Hopkins, R. E. Fowler, S. Krishnac and G. H. Mitchell, Parasitology Today, 16 (10), pages: 427-433)
THE TROPHOZOITE
• It is the form found inside the erythrocyte after 10-18 hours post-infection
• It interact with the host cell in a very sophisticated way: 1) it takes up the
red blood cell hemoglobin and digests it inside a food vacuole. Many of the
drugs in use target this compartment; 2) it transports proteins from the
parasite to the surface of the erythrocyte for its own benefit.
The digestive vacuole of the
trophozoite
• site of Hb digestion
(50-100% digested in
< 10 hrs)
• several proteases
required
(plasmepsins and
cysteine proteases)
THE SCHIZONT
• Schizogony: form of asexual
reproduction in which multiple
mitoses take place, followed by
cytokinesis resulting in multiple
daughter cells
• multiple mitoses produce 20-24 nuclei
• once nuclei & organelles replicated
cytokinesis occurs
• rupture of RBC membrane releases
merozoites
TEM which shows a schizont of Plasmodium yoelii
(rodent malaria parasite). Merozoites surround a
residual body (the circular area to the right lower
center). Within the apical areas of the merozoites we
can see up to two densely-staining rhoptries with
finer micronemes. Note the irregular appearance of
the red blood cell (RBC)
(Image from Ellis DS, Malaria", Topics in Inernational Health,(1998)
The WellcomeTrust, CABI Publishing, CAB International)
Schizont from Plasmodium falciparum
Gamogony
27, 28: Mature macrogametocytes (female); 29, 30: Mature microgametocytes (male).
• Formation of gametocytes
• trigger for this is unknown, in in
vitro culture it is stress related.
• 9-10 day development for P.
falciparum
• 36 hours development for P.
vivax
• in P. falciparum have
characteristic banana shape
Development in the mosquito
•
•
•
•
Upon ingestion with a blood meal,
both the micro and
macrogametocyte rapidly mature
Macrogamete is released from
ruptured rbc
Microgametocyte rapidly
undergoes multiple nuclear
divisions to form 8 gametes
Exflagellation
THE OOKINETE
• Fertilization and fusion of nuclei (diploid zygote)
• Transforms to a motile ookinete which migrates to outer midgut wall and encysts
A mature ookinete. A number of organelles are shown.
The nucleus can be seen at the lower end of the
organism. There are abundant ribosomes in the area
above the nucleus and endoplasmic reditulum can also
be seen. The zygote is surrounded by a three-layered
pellicle. The apical complex at the upper end includes
numerous rhoptries and micronemes (dark spots).
Image from Sinden RE. "Malaria", Topics in Inernational
Health,(1998) The WellcomeTrust, CABI Publishing, CAB
International
EM of P. gallinaceum ookinetes. An ookinete (o)
penetrating the perithrophic membrane (PT) 34 h
after a blood meal.Inset a: the ookinete is
surrounded by fine granular material. Inset b: The
ookinete pellicle consists of the ookinete plasma
membrane (PM), two inner membranes (IM-1 and
IM-2) and a dark fuzzy layer (DL). (Toril et al, J.
Protozool. 39(4) (1992), pp.449)
Development in the mosquito
• Encysted ookinete
transforms into oocycst
• 10-14 days of development
• reductional nuclear
division, haploid again
• multiplication to form
1000’s of sporozoites
THE OOCYST
SEM which shows two oocysts on the outer wall of
the midgut of a mosquito. These contain
developing P. gallinacium sporozoites.
Image from Guggehheim R."Malaria", Topics in Inernational
Health,(1998) The WellcomeTrust, CABI Publishing, CAB International)
Anopheles gambiae, the deadliest malaria vector
(top), and blue-colored Plasmodium oocysts,
appearing from the mosquito’s gut. (MOSQUITO
ENGINEERING:Building a Disease-Fighting Mosquito. Martin
Enserink/Science 2000 290: 440-441. (in News Focus)
The sporozoite invades the
mosquito salivary glands
Sporozoites which are released from
the oocysts come in contact, and
invade only the salivary glands.
The mechanism of invasion has been
studied but still is not well
characterized which are receptors for
sporozoite invasion. The figure shows
sporozoites entering the salivary
glands and assembling in bundles in
the secretory cavity . A small number
of parasites enter the secretory duct.
BL: basal lamina; DW: duct wall; N: nucleus; n:
nucleolus; PM: plasma membrane; SC:
secretory cavity. Current opinion in microbiology,
2009, 12:394
SEM of sporozoites from an opened
oocyst which has developed on the
outer surface of a mosquito
Anopheles stephensi midgut. The
parasite is Plasmodium falciparum.
(Image from Guggenheim R. "Malaria",
Topics in International Health,(1998) The
WellcomeTrust, CABI Publishing)
Chemotherapy of Malaria
Spectrum of activity of antimalarial drugs
Chloroquine
Pyrimethamine
Sulfadoxine
Quinine
Tetracycline
Primaquine
Artemisinin
Sporozoite
Liver stage
(exoerythrocytic)
-
-+
+
-
RBC stages
RBC stages
Asexual
sexual
(trophozoites) (gametocytes)
+
+
-+
+
-+
+
+
+
-
Other drugs: Clindamycin, azythromycin, atovaquone, Fluoroquinolones, others,
combinations
DIAGNOSIS
Malaria should be considered a potential medical emergency
Delay in diagnosis and treatment is a leading cause of death
Clinical diagnosis: patient’s symptoms and physical examination.
The first symptoms (fever, chills, sweats, headaches, muscle pains, nausea
and vomiting) are not specific.
In severe malaria, clinical findings are more striking (confusion, coma,
neurologic focal signs, severe anemia, respiratory difficulties.
Microscopic diagnosis:
Blood smear stained with Giemsa
Antigen detection
Molecular diagnosis: PCR
Serology: detection of antibodies
against parasites
EPIDEMIOLOGY
Important factors:
Female Anopheles mosquito
Humans
Malaria parasites
In rare cases malaria parasites can be transmitted
through blood transfusion, organ transplantation or
shared needles.
Animal reservoirs have no important role
Climate can influence all three components of the life cycle:
Rainfall
Ambient temperature: transmission
is greater in warmer areas
Human behavior
EPIDEMIOLOGY
• Anopheles mosquitos: species of anopheles present in the
area influence the intensity of transmission.
• The females that prefer to feed on humans indoors will be
more effective vectors.
• Insecticide resistance is an important factor
• Biological characteristics influence a person’s malaria risk
• Two genetic factors are epidemiologically important: 1)
persons who have the sickle cell trait heterozygotes for the
abnormal hemoglobin S are resistant; 2) Persons who are
negative for the Duffy blood group have rbc resistant to
infection by P. vivax.
• Pregnancy: women who have developed protective
immunity against P. falciparum tend to lose this protection if
pregnant
• Behavioral factors:
• housing, use of bed nets, financial situation, standing
water, agricultural work, domestic animals, war migrations
MALARIA and PREGNANCY
• In high transmission areas, women have gained a level of immunity
to malaria that wanes during pregnancy. In these cases, malaria is
more likely to result in severe maternal anemia and delivery of lowweight infants
• In low transmission areas, women have developed no immunity to
malaria. In these cases malaria infections is more likely to result in
severe malaria disease, anemia, premature delivery and fetal
loss.
• As many as 200,000 fetal and infant
deaths and 2,500 maternal deaths
yearly attributed to increased malaria
susceptibility during pregnancy.
• Parasitized red cells adhere and
accumulate within the placenta.
Pregnant woman having a blood smear taken at
an antenatal clinic at the Maela Camp in Thailand
near the Myanmar border. Pregnant women are at
increased risk of malaria. Image contributed by the
Shoklo Malaria Research Unit, Mae Sot, Thailand.
Each year, approximately 50 million women
living in malaria-endemic countries
throughout the world become pregnant, of
whom over half live in tropical areas of Africa
with intense transmission of Plasmodium
falciparum.
MALARIA AN HIV
Worldwide Distribution of Malaria
Malaria and HIV overlap in Sub
Saharan Africa, Southeast Asia
and South America.
In 2003 HIV/AIDS caused the
deaths of approx. 2.9 M people of
whom 2.4 M lived in Africa
Worldwide Distribution of HIV, End of 2004
Malaria and HIV interaction studies
Summary of current status
• HIV-associated immunosuppression
contributes to more and worse malaria
and it’s consequences in adults,
pregnant women, and children.
• Malaria contributes to stimulus of HIV
replication and possibly(?) to its
consequences: disease progression,
transmission in adults, and mother to
child transmission.
• Co-infection with Malaria and HIV in
pregnant women contributes to anemia,
low birth weight, and their risk for poor
infant survival.
• Malarial anemia in children too frequently
requires blood transfusion and may still
lead to HIV
transmission
A clinician at the HIV clinic of the
Provincial General Hospital in Kisumu,
Kenya, examines a child hospitalized with
fever possibly caused by malaria.
(Courtesy Kenya Ministry of Health.)
MALARIA CONTROL
• Diagnosis and treatment
• Prevention of infection through vector control (use
of insecticide-treated mosquito nets (shown to reduce
all-cause child mortality by 20%-25%)
• Prevention of disease by administration of
antimalarial drugs to particularly vulnerable population
groups such as pregnant women
Ali Khamis Abbas, on behalf of the Zanzibar
Malaria Control Program, presents a longlasting insecticide-treated net to Consolata
John, Chairperson of the Zanzibar
Association of People with HIV/ AIDS Living
Positively. (Courtesy Patrick Kachur)
The nets from all houses in a Tanzanian village are given
their annual retreatment free of charge by a nurse from the
health centre. Photograph taken by T.J. Wilkes.
Malaria control
Mosquito Control and ACT (Artemisinin-based combined therapies) are both likely contributors to the
Reduction of Malaria in KwaZulu–Natal
ROLL BACK MALARIA
To provide a coordinated international approach to fighting malaria the
Roll Back Malaria (RBM) Global Partnership was launched in 1998 by the
World Health Organization, UNICEF, UNDP and the World Bank. RBM’s
goal is to halve the burden of malaria by 2010.
PRESIDENTIAL MALARIA INITIATIVE
• The President’s Malaria Initiative (PMI) represents an historic fiveyear expansion of U.S. Government (USG) resources to fight malaria in
the region most affected by the disease.
• The President committed an additional $1.2 billion in malaria funding to
this Initiative with the goal of reducing malaria-related deaths by 50
percent in 15 focus countries.
• This will be achieved by expanding coverage of highly effective malaria
prevention and treatment measures to 85 percent of the most vulnerable
populations – children under 5 years of age and pregnant women.
• This package of high-impact interventions includes insecticide-treated
mosquito nets (ITNs), indoor residual spraying (IRS) with
insecticides, intermittent preventive treatment for pregnant women
(IPTp), and artemisinin-based combination therapy (ACT).
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