Malaria seminar

SEMINAR ON MALARIA

SPEAKERS:• KOUSIK KARMAKAR• ARCHISMAN CHATTERJEE• SUDAKSHINA DAS• BISWADEEP DAS• DEBASRITA BHATTACHARJEE• DEBJYOTI GHOSH

Prepared & Designated By:- SAYAN BANERJEEfrom the 2nd year(5th sem) students of MLDMC&H

from the 2nd year(5th sem) students of MLDMC&H

MALARIA−SAYAN

BANERJEE


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INTRODUCTION (incl.

NOMENCLATURE &

HISTORY)

LIFE CYCLE

OF CAUSING PARASITE

MORPHOLOGY OF

THE PARASITE

CLINICAL FEATURES(incl. SPECIAL

FEATURES & COMPLICATI

ONS)

LAB DIAGNOSI

S

TREATMENT &

PREVENTION

RECENT ADVANCE

SBIBLIOGRAP

HY

SCHEME OF DISCUSSION


INTRODUCTION TO

MALARIA

-Kousik Karmakar


About MALARIA• 40% of the world’s population lives in endemic areas.• 3-500 million clinical cases per year.• 1.5-2.7 million deaths (90% Africa).• Increasing problem (re-emerging disease)→

resurgence in some areas . drug resistance ( mortality).

• causative agent = Plasmodium species → protozoan parasite . member of Apicomplexa . 4 species infecting humans .

• transmitted by female anopheles mosquitoes .

• P. falciparum - malignant tertian malaria• P. vivax - benign tertian malaria• P. malariae - quartan malaria• P. ovale - ovale malaria• P. knowlesi - simian malaria


TAXONOMICAL POSITION OF Plasmodium

Kingdom: ChromalveolataSuperphylum: Alveolata

Phylum: ApicomplexaClass: Aconoidasida

Order: HaemosporidaFamily: Plasmodiidae

Genus: PlasmodiumSpecies: vivax, falciparum, malariae, ovale, knowlesi


NOMENCLATUR

E• Name is derived from Italian ‘Mal’= bad; ‘aria’ = air

• Thus the meaning is bad air of ‘Malaria’


HISTORY

Malaria – Early History

The symptoms of malaria were described in ancient Chinese medical writings. In 2700 BC, several characteristic symptoms of what would

later be named malaria were described in the Nei Ching.


Hippocrates and Malaria

Hippocrates, a physician born in ancient Greece, today regarded as the "Father of Medicine", was the first to describe the manifestations of the

disease, and relate them to the time of year and to where the patients lived.



History – Events on Malaria• 1880 − Charles Louis Alphonse Laveran discovered malarial parasite in wet mount.• 1883 − Methylene blue stain – Marchafava.• 1885 − Golgi described the blood stage(erythrocytic schizogony) of malarial

parasite– Golgi cycle.• 1898 – Amigo & Grassi described the life cycle. • 1891− Polychrome stain- Romanowsky.• 1898−Roland Ross - Life cycle of parasite transmission, wins Nobel Prize in

1902(SSKM HOSPITAL , KOLKATA).• 1948 − Site of Exoerythrocytic development in Liver by Shortt and Garnham.


Major Developments in 20th Century

• 1955 - WHO starts world wide malaria eradication programme using DDT

• 1970 – Mosquitos develop resistance to DDT Programme fails• 1976 – Trager and Jensen in vitro cultivation of parasite


Pioneered the Events on Malaria

Laveran Ronald Ross

Charles Louis Alphonse Laveran

Charles Louis Alphonse Laveran, a French army surgeon stationed in Constantine, Algeria, was the first to notice parasites in the

blood of a patient suffering from malaria . This occurred on the 6th of November 1880. For his discovery, Laveran was awarded the

Nobel Prize in 1907.



Nature of parasite as Drawn by Laveran

Ronald Ross

In August 20th, 1897, Ronald Ross, a British officer in the Indian Medical Service, was the first to demonstrate that malaria

parasites could be transmitted from infected patients to mosquitoes For his discovery, Ross was awarded the Nobel Prize in 1902.


Nobel Prizes in Malaria

The discovery of this parasite in mosquitoes earned the British scientist Ronald Ross the Nobel Prize in Physiology or Medicine in 1902. In 1907, Alphonse Laveran received the Nobel prize for his

findings that the parasite was present in human blood.



Chloroquine (Resochin) (1934, 1946)

• Chloroquine was discovered by a German, Hans Andersag, in 1934 at Bayer I.G. Farbenindustrie A.G. laboratories in Eberfeld, Germany. He named his compound resochin. Through a series of lapses and confusion brought about during the war, chloroquine was finally recognized and established as an effective and safe antimalarial in 1946 by British and U.S.


LIFE CYCLE OF

Plasmodium spp.

-Archisman Chatterjee


Life Cycle• sporozoites injected during mosquito feeding• invade liver cells• exoerythrocytic schizogony (merozoites)• merozoites invade RBCs• repeated erythrocytic schizogony cycles• gametocytes infective for mosquito• fusion of gametes in gut• sporogony on gut wall in hemocoel• sporozoites invade salivary glands


Life Cycle(contd…)


Invasive Stages

Merozoite• erythrocytesSporozoite• salivary glands• hepatocytesOokinete• epithelium


Anopheles(female)

Transmission• sporozoites injected with saliva • enter circulation• trapped by liver (receptor-ligand)


Exoerythrocytic Schizogony

• hepatocyte invasion• asexual replication• 6-15 days• 1000-10,000 merozoites• no overt pathology


Hyponozoite Forms• some EE forms exhibit delayed replication (ie, dormant)• merozoites produced months after initial infection• only P. vivax and P. ovale

relapse = hypnozoite

recrudescence = subpatentt


Erythrocytic Stage• intracellular parasite undergoes

trophic phase• young trophozoite called ‘ring form’• ingests host hemoglobin

cytostome food vacuole hemozoin (malarial pigment)


Erythrocytic Schizogony• nuclear division = begin schizont stage• 6-40 nuclei• budding merozoites = segmenter• erythrocyte rupture releases merozoites• blood stage results in disease symptoms


Gametocytogenesis• alternative to asexual replication• induction factors not known

drug treatment ↑ #'s(increases) immune response ↑ #'s(increases)

• ring gametocyte Pf : ~10 days others: ~same as schizogony

• sexual dimorphism microgametocytes macrogametocytes

• no pathology• infective stage for mosquito


Gametogenesis• occurs in mosquito gut• ‘exflagellation’ most obvious

3X nuclear replication 8 microgametes formed

• exposure to air induces temperature (2-3oC) pH (8-8.3) result of pCO2

• gametoctye activating factor in mosquito xanthurenic acid


Sporogony• occurs in mosquito (9-21 d)• fusion of micro- and macrogametes • zygote ookinete (~24 hr)• ookinete transverses gut epithelium ('trans-invasion')


Sporogony(contd…)•ookinete oocyst

between epithelium and basal lamina•asexual replication sporozoites• sporozoites released


Sporogony(contd…)• sporozoites migrate

through hemocoel• sporozoites 'invade'

salivary glands


A brief recapitulation


TUTORIAL VIDEO


MORPHOLOG

Y -Sudakshina

Das


P. falciparumStages found in blood Appearance of Erythrocyte

(RBC)Appearance of Parasite

Ring normal; multiple infection of RBC more common than in other species

delicate cytoplasm; 1-2 small chromatin dots; occasional appliqué (accollé) forms

Trophozoite normal; rarely, Maurer’s clefts (under certain staining conditions)

seldom seen in peripheral blood; compact cytoplasm; dark pigment

Schizont normal; rarely, Maurer’s clefts (under certain staining conditions)

seldom seen in peripheral blood; mature = 8-24 small merozoites; dark pigment, clumped in one mass

Gametocyte distorted by parasite crescent or sausage shape; chromatin in a single mass (macrogametocyte) or diffuse (microgametocyte); dark pigment mass


P. vivaxStages found in blood Appearance of Erythrocyte (RBC) Appearance of Parasite

Ring normal to 1-1/4 X, round; occasionally fine Schüffner’s dots; multiple infection of RBC not uncommon

large cytoplasm with occasional pseudopods; large chromatin dot

Trophozoite enlarged 1-1/2–2 X; may be distorted; fine Schüffner’s dots large amoeboid cytoplasm; large chromatin; fine, yellowish-brown pigment

Schizont enlarged 1-1/2–2 X; may be distorted; fine Schüffner’s dots Large , may almost fill RBC; mature = 12-24 merozoites ; yellowish-brown, coalesced pigment

Gametocyte enlarged 1-1/2–2 X; may be distorted; fine Schüffner’s dots round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or diffuse (microgametocyte); scattered brown pigment


P. ovaleStages found in blood Appearance of Erythrocyte

(RBC)Appearance of Parasite

Ring normal to 1-1/4 X, round to oval; occasionally Schüffner’s dots; occasionally fimbriated; multiple infection of RBC not uncommon

sturdy cytoplasm; large chromatin

Trophozoite normal to 1-1/4 X; round to oval; some fimbriated; Schüffner’s dots

compact with large chromatin; dark-brown pigment

Schizont normal to 1-1/4 X; round to oval; some fimbriated; Schüffner’s dots

mature = 6-14 merozoites with large nuclei, clustered around mass of dark-brown pigment

Gametocyte normal to 1-1/4 X; round to oval; some fimbriated; Schüffner’s dots

round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or more diffuse (microgametocyte); scattered brown pigment


P. malariaeStages found in blood Appearance of Erythrocyte (RBC) Appearance of Parasite

Ring normal to 3/4 X sturdy cytoplasm; large chromatin

Trophozoite normal to 3/4 X; rarely, Ziemann’s stippling (under certain staining conditions)

compact cytoplasm; large chromatin;occasional band forms; coarse, dark-brown

pigment

Schizont normal to 3/4 X; rarely, Ziemann’s stippling (under certain staining conditions)

mature = 6-12 merozoites with largenuclei, clustered around mass of coarse,dark-brown pigment; occasional rosettes

Gametocyte normal to 3/4 X; rarely, Ziemann’s stippling (under certain staining conditions)

round to oval; compact; may almost fillRBC; chromatin compact, eccentric(macrogametocyte) or more diffuse

(microgametocyte); scattered brown pigment


False-colored electron micrograph of a Plasmodium sp. sporozoite


CLINICAL FEATURES

-Biswadeep Das


Malaria

Symptoms

• Symptoms of malaria may include fever,

chills, vomiting, diarrhoea, cough, stomach,

pain and muscular aches and weakness.

• If infected with the malaria parasite,

Plasmodium results in the most severe form

of malaria and if left untreated, it can cause

serious illnesses like seizures, mental

confusion, kidney failure, coma and death.


How Malaria present Clinically?(Clinical Malaria)Stage 1

Chills for 15 mins to 1 hour. Caused due to rupture from the host red cells escape into Blood. Preset with nausea, vomiting, headache.

Stage 2 Fever may reach upto 400c may last for several hours starts invading newer red cells.

Stage 3 patient starts sweating, concludes the episode.

Cycles are frequently Asynchronous. Paroxysms occur every 48 – 72 hours. In P. malariae pyrexia may lost for 8 hours or more and temperature may exceed 410c.


Special Clinical Features• characterized by acute febrile attacks (malaria paroxysms)

periodic episodes of fever alternating with symptom-free periods.• manifestations and severity depend on species and host status

immunity, general health, nutritional state, genetics.• recrudescences and relapses can occur over months or years.• can develop severe complications (especially P. falciparum).


• paroxysms associated with synchrony of merozoite release .

• between paroxysms temperature is normal and patient feels well .

• P. falciparum may not exhibit classic paroxysms (continuous fever).

Malaria Paroxysm

tertian malariaquartan malaria


gametocytes

erythrocytic schizogony• 48 hr in Pf, Pv, Po• 72 hr in Pm

Recurrences in Malaria May result from –

reinfection or due to certain events related to the parasite’s life cycle.

Two types of recurrences known in malaria: 1. Recrudescence –

seen in P. falciparum & P. malariae . due to persistence of blood infection (some erythrocytic forms evade host immunity) even after clinical illness has

subsided. The numbers may increase later, leading to reappearance of clinical symptoms . Occur mostly up to one year or two in P. falciparum but in P. malariae, it can occur even after decades .

2. Relapse − Occurs due to a special form of parasites – hypnozoites. Hypnozoites are the sporozoites that remain dormant after infecting liver . Activated from time to time to initiate pre erythrocytic schizogony - Exoerythrocytic schizogony .



Severe falciparum malaria(complications)

Falciparum Malaria→– Most widespread.– Accounts for 80% of malaria cases worldwide.– Most pathogenic of human malaria species.

Severe malaria→ Cerebral/Pernicious malaria −

Impaired consciousness/coma Repeated generalized convulsions

Renal failure (Serum Creatinine >3 mg/dl) − BLACK WATER FEVER Jaundice (Serum Bilirubin >3 mg/dl) Severe anaemia (Hb <5 g/dl) Pulmonary oedema/acute respiratory distress syndrome Hypoglycaemia (Plasma Glucose <40 mg/dl) Metabolic acidosis Circulatory collapse/shock (Systolic BP <80 mm Hg, <50 mmHg in

children) Abnormal bleeding and Disseminated intravascular coagulation(DIC) Haemoglobinuria Hyperpyrexia (Temperature >106˚F or >42˚C) Hyperparasitaemia (>5% parasitized RBCs )


Cerebral/Pernicious malaria

• Definition: refers to a series of phenomenon occurring during infection with P. falciparum which, if not effectively treated, threatens the life of the patient within 1 to 3 days.

• In children & non immune adults, can cause coma & death – Cerebral malaria.

• Occurs as a result of capillary blockage, which occurs following this chain of phenomenons– Infected RBCs have special tendancy to form

‘ROSETTES’ by adhering to the healthy RBCs by means of some KNOBS (CYTOADHERENCE)→ These rosettes will block the brain capillaries.

Anaemia • Can be severe & occur rapidly,

particularly in young children • Occurs due to →

– destruction of parasitised RBCs – phagocytosis & destruction in the spleen

– Decreased production of RBCs in the bone marrow.


Major Manifestations of Malaria

Cerebral malaria

Anemia


BLACK WATER FEVER

• Occurs in previously infected subjects • Can also occur in non immune adults with severe falciparum malaria,

and also as a complication of quinine therapy. • A rare but acute condition characterised by sudden & massive

hemolysis of parasitised & non parasitised RBCs followed by fever and haemoglobinuria.

• Often fatal due to renal failure.• Difficult to find the parasites in the blood following a hemolytic attack. • Urine appears dark red to brown black due to the presence of free Hb. • Clinical features – fever, rigor, aching pains in the loin, icterus, bilious

vomiting, circulatory collapse, haemoglobinuria & acute renal failure. • Treatment – Chloroquine, blood transfusion, peritoneal dialysis in ARF.


• CONGENITAL MALARIA:– It is very rare and occurs in < 5% of affected pregnancies. Placental barrier and maternal Ig G antibodies which cross the placenta

may protect the foetus to some extent .– However, it is much more common in non-immune population and the incidence goes up during epidemics of malaria. – Fetal plasma Quinine and Chloroquine levels are about one third of simultaneous maternal levels and this subtherapeutic drug level

does not cure the infection in the foetus . – All four species can cause congenital malaria, but it is proportionately more with P. malariae . – The new born child can manifest with fever, irritability, feeding problems, hepato-splenomegaly, anaemia, jaundice etc. – The diagnosis can be confirmed by a smear for M.P. from cord blood or heel prick, anytime within a week after birth (or even later if

post-partum, mosquito-borne infection is not likely). – Differential diagnoses include Rh. incompatibility, infections with C.M.V., Herpes, Rubella, Toxoplasmosis, and syphilis .

• TRANSFUSION MALARIA(when persons with latent infection are used as donors) • MALARIAL NEPHROPATHY• TROPICAL SPLENOMEGALY

Special forms of malaria


LABORATORY

DIAGNOSIS

−Debasrita Bhattacharje

e


Diagnosis schemePeripheral blood smear(THICK & THIN smear), stained with Giemsa stain (or alternatively Leishman’s/Wright’s/Field’s stain)

Quantitative Buffy Coat(QBC) method- by Acridine Orange staining

Fluorochrome staining for rapid identification(<1 min.) in low parasitaemia

Immuno-fluorescence test(IFT)Indirect Haemagglutination(IHA) test

ELISAPCR

Malarial antigen test by immunochromatographic method-Rapid Detection Test(RDT)

Culture


• Firstly THIN & THICK smear is being prepared and after staining with Giemsa, Wright’s, Field’s, or, Leishman’s stain it is seen under light microscope.

• Thin films:– Dry– Fix– Stain– Used to determine the species entire thin film should be examined about 20-40 minutes for an experienced observer.

• Thick films:– Dry– Do not fix but dehaemoglobinate– Stain

Peripheral blood smear examination


Interpreting Thick and Thin Films

THICK FILM 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

– lysed RBCs– larger volume– 0.25 μL blood/100 fields– more difficult to diagnose species– good screening test– positive or negative– parasite density


Thick smears• The number of parasites/μl of blood is determined

by enumerating the number of parasites in relation to the standard number of WBCs/μl (8000).

Thin smears • The percent of infected RBCs is determined by

enumerating the number of infected RBCs in relation to the number of uninfected RBCs. A minimum of 500 RBCs total should be counted.

Estimating Parasite Density (PARASITAEMIA)


+ 1-10 parasites per 100 HPF

++ 11-100 parasites per 100 HPF

+++ 1-10 parasites per each HPF

++++ > 10 parasites per each HPF

Estimating Parasite Density (Alternate Method)

Count the number of asexual parasites per high-power field (HPF) on a thick blood film →


P. vivax


Fig.1: Normal red cellFigs.2-6: Young trophozoites (ring stage parasites)Figs.7-18: TrophozoitesFigs.19-27: SchizontsFigs.28 and 29: Macrogametocytes (female)Fig.30: Microgametocyte (male)


F) Mature trophozoites. Note the resemblance to

the band forms of P. malariae. The enlarged

size of the infected RBCs helps distinguish the two

species

B) Ameboid ring in an enlarged and distorted

infected RBC Schüffner’s dots are visible

A) Ring in a thick blood smear

The “halo” is suggestive of Schüffner’s dots

C) Ring forms in a thin blood smear

D) Trophozoite in thick blood smear

E) Large, ameboid trophozoites in thin blood smears

Note the presence of Schüffner's dots


J) Ookinetes in thin blood smears.

Ookinetes may form if blood is allowed to sit too long before processing

H) Gametocyte in thin blood smear

G) Gametocyte in a thick blood smear

H) Gametocyte in thin blood smear

H) A pair of gametocytes in a thin

blood smear

I) Ookinete in a thick smear


M) Mature schizont in thin blood smear

K) Schizont in a thick blood smear

L) Schizont in thick blood smear

L) Immature schizont in a thin

blood smear

M) Mature schizont in thin blood smear

K) Schizont in thick blood smear


P. falciparum


Fig.1: Normal red cellFigs.2-18: Trophozoites (among these,Figs.2-10 correspond to ring-stage trophozoites)Figs.19-26: Schizonts (Fig.26 is a ruptured schizont)Figs.27,28: Mature macrogametocytes (female)Figs.29,30: Mature microgametocytes (male)


Thin, delicate rings in a thin blood smear

Note the double chromatin dot in the infected RBC at top, and

the accolle form in the infected RBC at bottom

Images from a thick blood smear showing more rings

Note the classic “headphones” appearance

of many of the rings

Rings and some developing trophozoites seen in thin smears Note also the presence of

Maurer’s clefts, which are often seen in older ring forms. Maurer’s clefts stain best with

an alkaline pH of 7.2—7.6

Gametocytes in a thick blood smear

Two gametocytes in a thin smear

Gametocytes in thin smear the one on the right is

undergoing exflagellation


Another schizont in a thin blood smear

Trophozoites in a thick blood smear

Mature, compact trophozoites in a thin

blood smear

Mature schizont in a thin blood smear

Ruptured schizont in a thin blood smear

Gametocytes in thin blood smears Note the presence of “Laveran’s bib” (black arrow),

which is not always visible


P. ovale


Fig.1: Normal red cellFigs.2-5: Young trophozoites (Rings)Figs.6-15: TrophozoitesFigs.16-23: SchizontsFig.24: Macrogametocytes (female)Fig.25: Microgametocyte (male)


Ring forms, developing and

compact trophozoites in a thin blood smear

Rings in fimbriated RBCs in thin blood

smears

Ring in a thick blood smear

Rings in thin blood smears

Note the multiply-infected RBCs

Trophozoite in a thick blood smear

Compact trophozoites in fimbriated RBCs in thin

blood smears Schüffner’s dots are

also visible


Schizonts in thin blood smears

Note the infected RBCs are oval

Gametocyte in a thin blood smear

The infected RBC shows some fimbriation

Gametocyte in a thick blood smear

Macrogametocytes in thin blood smears

Notice how they nearly fill the infected RBCs

Coarse pigment, a discrete red nucleus and Schüffner’s dots

can be seen

Microgametocyte in thin blood smear

Note the diffuse pigment

Schizonts in thick blood smears


P. malariae


Fig.1: Normal red cellFigs.2-5: Young trophozoites (rings)Figs.6-13: TrophozoitesFigs.14-22: SchizontsFig.23: Developing gametocyteFig.24: Macrogametocyte (female)Fig.25: Microgametocyte (male)


B) Ring in thin blood smears

A) Ring in a thick blood smear

C) Trophozoite in a thick blood smear

D) Band-form trophozoites in thin

blood smears.

B) Ring in thin blood smears

D) Band-form trophozoites in thin

blood smears.


F) “Basket-form” trophozoite in a thin smear

E) “Basket-form” trophozoite in a thick smear

G) Schizont in thick blood smears

Note the classic “rosette” appearance of the

merozoites

H) Schizonts in thin blood smears

H) Schizonts in thin blood smears

The schizont has the appearance of a rosette

pattern

F) “Basket-form” trophozoite in a thin smear


I) Gametocytes in thick blood smear

J) Gametocyte in a thin blood smear

J) Gametocyte in thin blood smear

I) Gametocytes in thick blood smear




• Useful for screening large numbers of samples .• Quick, saves time .• Requires centrifuge, special stains .• Malaria parasite floresce green yellow against dark red –black background .• 3 main disadvantages→

Species identification and quantification difficult . High cost of capillaries and equipment . Can’t store capillaries for later reference .

Quantitative Buffy Coat(QBC) method


Principle of QBC System


Trophozoites of P. falciparum stained with AO in the QBC UV fluorescence method

Trophozoites of P. falciparum stained with BCP in the fluorescence method


• Fluorescent Microscopy• Modification of light microscopy • Fluorescent dyes detect RNA and DNA that is contained in

parasites• Nucleic material not normally in mature RBCs• Kawamoto technique:

– Stain thin film with acridine orange (AO)– Requires special equipment – fluorescent microscope– Nuclei of malaria parasites floresce bright green and cytoplasm red.– Staining itself is cheap– Sensitivities around 90%

Fluorochrome staining for rapid identification(<1 min.) in low parasitaemia

fluorescent microscope

malaria parasites


• Indirect fluorescent antibody (IFA) test. The fluorescence indicates that the patient serum being tested contains antibodies that are reacting with the antigen preparation (here, Plasmodium falciparum parasites).

• Not practical for routine diagnosis of acute malaria because:– Delayed development of antibody– persistence of antibodies– Serology does not detect current infection but rather

measures past experience• Valuable epidemiologic tool• Useful for

– Identifying infective donor in transfusion-transmitted malaria

– Investigating congenital malaria, esp. if mom’s smear is negative

– Retrospective confirmation of empirically-treated non-immunes

Immuno-fluorescence test(IFT) ELISA


• Malaria Serology .• Antibody detection .• Immunologic assays to

detect host response . • 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 .• Valuable epidemiologic tool• Useful for →

– Identifying infective donor in transfusion-transmitted malaria .

– Investigating congenital malaria, esp. if mom’s smear is negative .

– Diagnosing, or ruling out, tropical splenomegaly syndrome .

– Retrospective confirmation of empirically-treated non-immunes .

Indirect Haemagglutination(IHA)

test


• Card/cassette/dipstick

• HRP2• HRP2 & aldolase• pLDH Pf & pan • pLDH Pf & Pv• HRP2, pLDH pan• HRP2, pLDH pan & pLDH Pv• Aldolase

– A: HRP-2 (histidine-rich protein 2) (ICT) – B: pLDH (parasite lactate dehydrogenase)(Flow)– C: HRP-2 (histidine-rich protein 2) (PATH)

"COMBO" tests

Malarial antigen test by immunochromatographic method-Rapid

Detection Test(RDT)

Amrad ICT Pf immunochromatographic

test device for detection of P. falciparum HRP-2

OptiMAL immunochromatographic

test device for the detection of pLDH


Parts of RDT strip Steps in RDT


PRINCIPLE INVOLVED


Plastic cassette format of RDT

Results


Comparison between Microscopy & RDTs Disadvantages

• The use of the RDT does not eliminate the need for malaria microscopy– Cannot detect mixed infections– may not be able to detect infections

with lower parasitaemia– Cannot detect P. ovale and P. malariae– microscopy is needed to quantify

parasitaemia


• Molecular technique to identify parasite genetic material

• Uses whole blood collected in anticoagulated tube or directly onto filter paper

• Threshold of detection 5 parasites/µl • Definitive species-specific diagnosis now

possible• Can identify mutations – try to correlate to

drug resistance • Parasitaemia not quantifiable• May have use in epidemiologic studies• Requires specialized equipment, reagents, and

training

Lane S: Molecular base pair standard (50-bp ladder). Black arrows : size of

standard bands. Lane 1: P. vivax (size: 120 bp).

Lane 2: P. malariae (size: 144 bp). Lane 3: P. falciparum (size: 205 bp).

Lane 4: P. ovale (size: 800 bp).

PCR

• use of a candlejar• RPMI 1640 -which turned out

to be the best of the commercial media

Magnetic collection of P. falciparum infected blood

Culture



TREATMENT

&

PROPHYLAXI

S−Debjyoti

Ghosh


Classification according to the stages of the life cycle affected

Chemical classification

Anti-malarial drugs


Sites of drug action in MALARIA


TREATMENT OF CLINICAL CASES


PROPHYLAXIS• PERSONAL:

– Bed-net– Mosquito repellant cream etc.

• CHEMOPROPHYLAXIS: – for travellers to endemic zone from the non-endemic zone, it is required.– Chloroquine 300 mg (base) or 5 mg/kg weekly. ln travelers, start one week

before with a loading dose of 10 mg/kg and continue till one month after return from endemic area. The last dose should be 25mg/kg over 3 days along with primaquine 15 mg/day for 14 days.

• VACCINES: RTS, S/A SO2 vaccine- the most promising.• CONTROL:

– Early diagnosis & treatment .– Vector control– National programme- NVBDCP(National Vector Borne Disease Control

Programme) .


Bed-netMosquito repellant

cream


• Drug disco very efforts directed towards the liver and transmission stages are in their infancy but are receiving increasing attention as targeting these stages could be instrumental in eradicating malaria.

• The Central Drug Research Institute, Lucknow, India, is investigating the trioxane CDRI-97/78 in Phase-I studies. The key step in the construction of the trioxane core is an ene reaction between an allylic alcohol and singlet oxygen, to give peroxide.

• Drugs that can reduce the formation of gametocyte s (gametocytogenesis), or can kill them (gametocytocides),are highly desirable but have been underexplored because of the lack of quantitative high throughput assays. These transmission blocking drugs could target endpoints such as:

A. The effective and complete killing of mature gametocytes once they are formed in the human host.B. The inhibition of the onward development of gametocytes into ookinetes and ultimately into sporozoites in the

mosquito. This assumes that enough drug from the blood sample reaches the gut of the mosquito.

RECENT ADVANCES IN DRUG DISCOVERY OF MALARIA

−Debjyoti Ghosh


• The pipeline for the blood stage is arguably the best in history, but still needs to be expanded. The last few years have seen an explosion of potent new chemotypes, and the new challenge is to assess the potential of these chemotypes. Ideally, the new drug should:

i. address drug-resistance issues,ii. have a rapid onset of action,iii. be safe, especially in children and pregnant women, andiv. cure malaria in a single dose.

The challenge is to find a drug that addresses all of these features. It is our hope that with the rich variety of new chemical entities, such a drug will be discovered. Nevertheless, drug discovery efforts should continue, as the artemisinins set a high standard of efficacy and safety.

• Drugs that target the liver and transmission stages have the potential to be transformation al, but research efforts have been hampered by the absence of high-throughput screens. New imaging techniques are beginning to solve this problem and open up novel avenues, with an innovative clinical compound having liver stage activity. The field of transmission-blocking agents is in its infancy, but may be the most transformative of all in achieving the ultimate goal of eradicating malaria.

RECENT ADVANCES IN DRUG DISCOVERY OF MALARIA(contd…)


Overview of P. knowlesi

RECENT ADVANCES may include the discovery of Plasmodium knowlesi


• Plasmodium knowlesi is a primate malaria parasite commonly found in Southeast Asia.

• It causes malaria in long-tailed macaques (Macaca fascicularis) ; but it may also infect humans, either naturally or artificially.

• Plasmodium knowlesi parasite replicates and completes its blood stage cycle in 24-hour cycles resulting in fairly high loads of parasite densities in a very short period of time. This makes it a potentially very severe disease if it remains untreated .

• Life cycle: Human stages: sporozoite →schizonts→ merozoite →

trophozoites→ schizont → merozoite; these stages of Plasmodium knowlesi are microscopically indistinguishable from Plasmodium malariae and the early trophozoites are identical to those of Plasmodium falciparum .

Mosquito stages: gametocyte → (microgamete or macrogamete) → zygote → ookinete → oocyst → sporozoites.

• This parasite is mostly found in South East Asian countries particularly in Borneo, Cambodia,Malaysia, Myanmar, Philippines, Singapore, Thailand and neighboring countries .

• Anopheles latens is attracted to both macaques and humans and has been shown to be the main vector transmitting P. knowlesi to humans.

• Two possible modes of transmission to humans have been proposed: either from an infected monkey to a human or from an infected human to another human.

• Symptoms: headache ,fever with chills & rigor, cold sweats, cough, vomiting, nausea and diarrhea.

• At least two subspecies of P. knowlesi known – P. knowlesi edesoni and P. knowlesi .

Macaca fascicularis


• Essentials of Medical Pharmacology BY K.D.Tripathi(7/E)• Lippincott Illustrated Reviews, Pharmacology(6/E)• Guidelines for Diagnosis and Treatment of Malaria in India 2014 BY

NATIONAL INSTITUTE OF MALARIA RESEARCH, NEW DELHI & NATIONAL VECTOR BORNE DISEASE CONTROL PROGRAMME, DELHI

• MALRIA, PLASMODIUM, P. knowlesi-WIKIPEDIA• MALARIA-CDC Health topics• YOUTUBE-MALARIA• GOOGLE IMAGES- PLASMODIUM LIFE CYCLE• BMCL Digest-Recent advances in malaria drug discovery BY Marco A.

Biamonte, Jutta Wanner, Karine G. Le Roch

BIBLIOGRAPHY


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Malaria seminar

Health & Medicine

Transcript of Malaria seminar