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
Paroxysm moderate mild to
mild severe
Severity to severe moderate
Average 50,000-
20,000 9,000 6,000
(per mm3) 500,000
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 Severe anemia Renal failure

Irritability, loss of reflexes, Progressive severe drop Dwindling urine, high urea
neurological symptoms of hematocrit, poor oxygen Level in serum
similar to menigitis, coma Supply for organs and
20% fatality tissues
 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)

• Erythrophagocytosis in
spleen
• Immune mediated
response
Spleen
• 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 Carry the drop of blood
blood to a clean to the first slide and hold
slide. at 45 degree angle.

2. 5.
Spread the drops to Pull the drop of blood
make a 1 cm circle. across the first slide in
one motion.

3. 6.
Touch a fresh drop Wait for both to dry
of blood to the edge before fixing and
of another slide. 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 • THIN 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
– 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

Trophozoite
Schizont

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 Trophozoites: ameboid; deforms the erythrocyte

Schizonts: 12-24 merozoites 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) Gametocytes: round-oval

dark pigment
 Plasmodium malariae
Infected erythrocytes: size normal to decreased (3/4X), Zeimanns dots

Trophozoite: Trophozoite: Schizont: Gametocyte:

compact typical 6-12 merozoites round; coarse,
band form (usually 8); dark pigment
coarse, dark pigment
; (“rosettes”)
 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