One day Workshop
YUMS
|Tissue and Blood Protozoan diseases
Malaria & Leishmaniasis
Mohammad Amin Ghatee
Ph.D in medical Parasitology
MALARIA THE KILLER DISEASE
• 500 million people
suffer from malaria
•Over one million of
people die each year.
•
•The majority of victims are children and
pregnant women. (one African child every
30 seconds)
• Sub-Saharan Africa bears 90 per cent of
the burden.
Plasmodium species which
infect humans
Plasmodium falciparum (tertian)
Plasmodium vivax (tertian)
Plasmodium malariae (quartian)
Plasmodium ovale (tertian)
Geographical distribution and incidence of malaria
Figure 23.10
Plasmodium species
• Plasmodium falciparum: Tropics. Accounts for 50% of all
malaria cases. Most pathogenic.
• Plasmodium vivax: Tropics, subtropics, and some temperate
regions.. 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: Tropics. About 7% of all malaria cases.
• Plasmodium ovale: West Africa. Rare.
HOSTS
DEFINITIVE HOST: Anopheline female Mosquito (sexual
reproduction)
INTERMEDIATE HOST: Humans (asexual and sexual phases)
Malaria Life Cycle
Types of Infections
• Recrudescence
– exacerbation of persistent undetectable parasitemia, due
to survival of erythrocytic forms, no exo-erythrocytic
cycle (P.f., P.m.)
– Blood origin
– Can be occurred along the life time
• Relapse
– reactivation of hypnozoites forms of parasite in liver,
separate from previous infection with same species
(P.v. and P.o.)
– Tissue (liver) origin
– Up to 3-5 years after primary infection
Erythrocytic phase
stages of parasite in RBC
•
•
•
•
Young trophozoites or ring form
Tropohozoite
forms merozoites
Schizogeny to forms merozoites releasing
merozoites into blood stream.
• Merozoites invade other RBCs and schizongeny is
repeated
• Parasite density increases until host’s immune
response slows it down
• Merozoites may develop into gametocytes
(gametogony), the sexual forms of the parasite
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
Development in the mosquito:
the ookinete
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
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)
Schizogenic periodicity and fever
patterns
• Schizogenic periodicity is length of asexual
erythrocytic phase
– 48 hours in P.f., P.v., and P.o. (tertian)
– 72 hours in P.m. (quartian)
• Initially may not see characteristic fever
pattern if schizogony not synchronous.
• With synchrony, periods of fever or febrile
paroxsyms assume a more definite 3
(tertian)- or 4 (quartian)- day pattern.
Clinical presentation
• Early symptoms
–
–
–
–
–
–
–
–
–
Headache
Malaise
Fatigue
Nausea
Muscular pains
Slight diarrhea
Slight fever
Photophobia
Anorexia
• Could mistake for influenza or gastrointestinal
infection
Paroxysm of malaria
Malaria Paroxysm
• Malaria tertiana: 48h
between fevers (P. vivax
and ovale)
• Malaria quartana: 72h
between fevers (P.
malariae)
• Malaria tropica: irregular
high fever (P. falciparum)
Disease Severity
Pv
Po
Pm
Pf
moderate
Paroxysm
mild to
mild
severe
Severity
moderate
to severe
Average
50,00020,000
9,000
6,000
500,000
(per mm3)
Maximum
50,000
30,000
20,000
2,500,000
(per mm3)
Anemia
++
+
++
++++
Duration
Disease
3-8 w
2-3 w
3-24 w
2-3 w
Infection
5-8 y*
12-20 m*
>20 y
6-17 m
Complications
renal
cerebral**
*true relapses ( recrudescence) due to dormant hypnozoite
stage in liver **plus many other organs
Malaria the disease
3 Severe manifestations
Cerebral malaria
Irritability, loss of reflexes,
neurological symptoms
similar to menigitis, coma
20% fatality
Severe anemia
Progressive severe drop
of hematocrit, poor oxygen
Supply for organs and
tissues
Renal failure
Dwindling urine, high urea
Level in serum
Knobs and cytoadherence
• Cytoadhrence and rosetting
correlates with the presence of
“knobs” (left column) on the
surface of the infected RBC
• The right column shows a RBC
infected with a knob-less strain
which does not cause cerebral
malaria
• Knobs are made up of parasite
derived proteins
knobs
knob-less
Cerebral Malaria Possible
Pathophysiology
cytoadherence

cerebral ischemia

hypoxia,
metabolic effects,
cytokines (eg, TNF-)

coma

death
Severe anaemia - pathogenesis
• Erythrocyte destruction
during schizogony (destruction
of both parasitized and
nonparasitized erythrocytes)
Spleen
• Erythrophagocytosis in
spleen
• Immune mediated
response
• Black water fever
• Bone marrow suppression
Other severe complications
• Pulmonary oedema
• Renal insufficiency (nephrotic syndrome)
– P. malariae
• Haemolysis
• Thrombocytopaenia, DIC
• Superinfections (secondry infection)
– Septicaemia
Immunity and Resistance in malaria
• Protective immunity to malaria is primarily
a premunition.
• In highly endemic areas, infants are
protected by maternal antibodies, and young
children are at greatest risk after weaning.
Genetically resistance factors
• Sickle cell anemia, favism, and thalassemia
can cause resistance to infection by P. f
-Duffy blood groups and P. v
Modes of transmission
• 1. Natural or biological transmission
• 2. Accidental transmission:
blood transfusion
sharing of needles by IV drug users
3. Maternal transmission
Malaria Diagnosis
Clinical Diagnosis:
• Symptoms: fever, chills,
headache, malaise, etc.
• History of being in
endemic area
• Splenomegaly and anemia
as disease progresses
Malaria Diagnosis
Laboratory diagnosis:
• Microscopic demonstration of parasite in
blood smear (distinguish species)
• thick film: more sensitive
• thin film: species identification easier
• Fluorescent microscopy
• antigen detection ‘dipstick’
• Serology
• Polymerase Chain Reaction
1.
4.
Touch 3 drops of
blood to a clean
slide.
Carry the drop of blood
to the first slide and hold
at 45 degree angle.
2.
5.
Spread the drops to
make a 1 cm circle.
Pull the drop of blood
across the first slide in
one motion.
3.
Touch a fresh drop
of blood to the edge
of another slide.
6.
Wait for both to dry
before fixing and
staining.
Malaria Blood Smear
• Remains the gold standard for diagnosis
• Giemsa stain
• distinguishes between species and life cycle stages
• parasitemia is quantifiable
• Requirements: equipment, training, reagents,
supervision
• Simple, inexpensive yet labor-intensive
• Accuracy depends on laboratorian skill
Interpreting Thick and Thin Films
• THICK FILM
–
–
–
–
–
–
–
–
lysed RBCs
larger volume
0.25 μl blood/100 fields
blood elements more
concentrated
good screening test
positive or negative
parasite density
more difficult to diagnose
species
• THIN FILM
–
–
–
–
fixed RBCs, single layer
smaller volume
0.005 μl blood/100 fields
good species
differentiation
– requires more time to read
– low density infections can
be missed
Malaria Parasite Erythrocytic Stages
Ring form
Schizont
Trophozoite
Gametocytes
Plasmodium falciparum
Infected erythrocytes: normal size, maurers cleft, discrimination of P. falciparum from
other species is a important, because P. falciparum in blood of non-immune case is a
medical emergency.
M
I
Gametocytes: mature (M)and
immature (I) forms
Rings: double chromatin dots, multiple
infections in same red cell
Schizonts: 16-24 merozoites
(rarely seen in peripheral blood)
Trophozoites: compact
Plasmodium vivax
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots))
Rings
Schizonts: 12-24 merozoites
Trophozoites: ameboid; deforms the erythrocyte
Gametocytes: round-oval
Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)
“malariae - like parasite in vivax - like erythrocyte”
Trophozoites: compact
Rings
Schizonts: 6-12 merozoites;(usually 8)
dark pigment
Gametocytes: round-oval
Plasmodium malariae
Infected erythrocytes: size normal to decreased (3/4X), Zeimanns dots
Trophozoite:
compact
Trophozoite:
typical
band form
Schizont:
6-12 merozoites
(usually 8);
coarse, dark pigment
; (“rosettes”)
Gametocyte:
round; coarse,
dark pigment
Malaria Serology – antibody detection
Antibodies to asexual parasites appear some
days after invasion of RBCs and may
persist for months
• Positive test indicates past infection
• Not useful for treatment decisions
– Investigating congenital malaria, esp. if
mom’s smear is negative
Malaria Antigen Detection
• Immunologic assays to detect specific antigens
• Commercial kits now available as
immunochromatographic rapid diagnostic tests
(RDTs), used with blood
• P. falciparum histidine-rich protein 2 (PfHRP-2)
• parasite LDH (pLDH)
• Monoclonal and polyclonal antibodies used in
antigen (Ag) capture test
• Species- and pan-specific Ab
• Cross reactivity with rheumatoid factor
reportedly corrected
Detection of Plasmodium antigens
Polymerase Chain Reaction (PCR))
• Molecular technique to identify parasite
genetic material
• Uses whole blood collected in
anticoagulated tube (200 µl) or directly
onto filter paper (5 µl)
• Definitive species-specific diagnosis now
possible
Treatment
•
•
•
•
Chloroquine
Primaquine
Quinine
Artemisinins
Selected Anti-Malarials
• Blood schizonticides:
1-Rapid acting:
Chloroquine, Amodiaquine, Quinine, Quinidine, Mefloquine,
Atovaquone, Artemisinin
2- Slow acting: Antifolates(Fansidar, proguanil) , Clindamycin,
Tetracyclines, Proguanil
Tissue schizonticides: Primaquine , Proguanil
Gametocidal: Primaquine
Anti – relapsing: Primaquine
Chinchona the source of quinine
• Peruvian Indians appear to have
been the first to know about the
medicinal effects of quinine.
• They chewed Chinchona bark
while working in the mines as
forced laborers for the Spanish
Malaria Drug Therapy in Iran
Plasmodium falciparum & Plasmodium
malariae infections
First day: Chloroquine 600 mg (4 tab), 6 h later: 300 mg
Second day: Chloroquine 300 mg
Third day: Chloroquine 300 mg & Primaquine 45 mg (3 tab)
Vaccine
• Radiated sporozoite
• Sporozoite recombinant proteins
• Spf66: synthethic polypeptides of
merozoites
• RTS,S: recombinant HBV surface protein
and parasite proteins