One day WorkshopYUMS

|Tissue and Blood Protozoan diseases

Malaria & Leishmaniasis

Mohammad Amin Ghatee

Ph.D in medical Parasitology

MALARIA THE KILLER DISEASEMALARIA THE KILLER DISEASE

•

•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.

• 500 million people suffer from malaria

•Over one million of people die each year.

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.

HOSTSHOSTS

DEFINITIVE HOST:DEFINITIVE HOST: Anopheline female Mosquito (sexual Anopheline female Mosquito (sexual reproduction)reproduction)

INTERMEDIATE HOST:INTERMEDIATE HOST: Humans (asexual and sexual phases) 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 phasestages 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 A mature ookineteookinete. A number of organelles are shown. . A number of organelles are shown. The nucleus can be seen at the lower end of the organism. The nucleus can be seen at the lower end of the organism. There are abundant ribosomes in the area above the There are abundant ribosomes in the area above the nucleus and endoplasmic reditulum can also be seen. The nucleus and endoplasmic reditulum can also be seen. The zygote is surrounded by a three-layered pellicle. The apical zygote is surrounded by a three-layered pellicle. The apical complex at the upper end includes numerous rhoptries and complex at the upper end includes numerous rhoptries and micronemes (dark spots). micronemes (dark spots). Image from Sinden RE. 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 OOCYSTTHE OOCYST

SEM which shows two oocysts on the outer wall of SEM which shows two oocysts on the outer wall of the midgut of a mosquito. These contain the midgut of a mosquito. These contain developing developing P. gallinaciumP. gallinacium sporozoites. 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 tertiana: 48h between fevers (P. vivax and ovale)

• Malaria quartana: 72h between fevers (P. malariae)

• Malaria tropica: irregular high fever (P. falciparum)

Malaria Paroxysm

Disease Severity Pv Po Pm Pf Paroxysm Severity

moderate to severe

mild mild to

moderate severe

Average (per mm3)

20,000 9,000 6,000 50,000-500,000

Maximum (per mm3)

50,000 30,000 20,000 2,500,000

Anemia ++ + ++ ++++ Duration

Disease Infection

3-8 w 5-8 y*

2-3 w 12-20 m*

3-24 w >20 y

2-3 w 6-17 m

Complications renal cerebral**

*true relapses ( recrudescence) due to dormant hypnozoite stage in liver **plus many other organs

Malaria the disease

Irritability, loss of reflexes, neurological symptoms similar to menigitis, coma20% fatality

Progressive severe dropof hematocrit, poor oxygenSupply for organs andtissues

Dwindling urine, high ureaLevel in serum

Cerebral malaria Severe anemia Renal failure

3 Severe manifestations

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 PossiblePathophysiology

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

• Black water fever• Bone marrow suppression

Spleen

Other severe complications

• Pulmonary oedema

• Renal insufficiency (nephrotic syndrome)– P. malariae

• Haemolysis

• Thrombocytopaenia, DIC

• Superinfections (secondry infection)– Septicaemia

• 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.

Immunity and Resistance in malaria

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

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

Malaria Diagnosis

1.Touch 3 drops of blood to a clean slide.

2.Spread the drops to make a 1 cm circle.

3.Touch a fresh drop of blood to the edge of another slide.

6.Wait for both to dry before fixing and staining.

5.Pull the drop of blood across the first slide in one motion.

4.Carry the drop of blood to the first slide and hold at 45degree angle.

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

TrophozoiteSchizont

Gametocytes

Plasmodium falciparum

Rings: double chromatin dots, multiple infections in same red cell

Gametocytes: mature (M)andimmature (I) forms

Trophozoites: compact

Schizonts: 16-24 merozoites(rarely seen in peripheral blood)

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

Plasmodium vivax

Trophozoites: ameboid; deforms the erythrocyte

Gametocytes: round-oval Schizonts: 12-24 merozoites

Rings

Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots))

Plasmodium ovaleInfected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)

“malariae - like parasite in vivax - like erythrocyte”

Rings

Trophozoites: compact

Schizonts: 6-12 merozoites;(usually 8) dark pigment

Gametocytes: round-oval

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

Plasmodium malariae

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

• 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

Selected Anti-Malarials

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

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)

Plasmodium falciparum & Plasmodium malariae infections

Vaccine

• Radiated sporozoite

• Sporozoite recombinant proteins

• Spf66: synthethic polypeptides of merozoites

• RTS,S: recombinant HBV surface protein and parasite proteins