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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
from the 2nd year(5th sem) students of MLDMC&H
READ THINK UNDERSTAND REMEMBERVARIFY THAT
YOU REMEMBER COMPLETELY
NOW CLICK TO MOVE AHEAD TO NEXT STEP
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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
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INTRODUCTION TO
MALARIA
-Kousik Karmakar
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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
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TAXONOMICAL POSITION OF Plasmodium
Kingdom: ChromalveolataSuperphylum: Alveolata
Phylum: ApicomplexaClass: Aconoidasida
Order: HaemosporidaFamily: Plasmodiidae
Genus: PlasmodiumSpecies: vivax, falciparum, malariae, ovale, knowlesi
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NOMENCLATUR
E• Name is derived from Italian ‘Mal’= bad; ‘aria’ = air
• Thus the meaning is bad air of ‘Malaria’
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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.
from the 2nd year(5th sem) students of MLDMC&H
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.
from the 2nd year(5th sem) students of MLDMC&H
from the 2nd year(5th sem) students of MLDMC&H
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.
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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
from the 2nd year(5th sem) students of MLDMC&H
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.
from the 2nd year(5th sem) students of MLDMC&H
from the 2nd year(5th sem) students of MLDMC&H
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.
from the 2nd year(5th sem) students of MLDMC&H
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.
from the 2nd year(5th sem) students of MLDMC&H
from the 2nd year(5th sem) students of MLDMC&H
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.
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LIFE CYCLE OF
Plasmodium spp.
-Archisman Chatterjee
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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
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Life Cycle(contd…)
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Life Cycle(contd…)
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Life Cycle(contd…)
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Invasive Stages
Merozoite• erythrocytesSporozoite• salivary glands• hepatocytesOokinete• epithelium
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Anopheles(female)
Transmission• sporozoites injected with saliva • enter circulation• trapped by liver (receptor-ligand)
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Exoerythrocytic Schizogony
• hepatocyte invasion• asexual replication• 6-15 days• 1000-10,000 merozoites• no overt pathology
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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
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Erythrocytic Stage• intracellular parasite undergoes
trophic phase• young trophozoite called ‘ring form’• ingests host hemoglobin
cytostome food vacuole hemozoin (malarial pigment)
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Erythrocytic Schizogony• nuclear division = begin schizont stage• 6-40 nuclei• budding merozoites = segmenter• erythrocyte rupture releases merozoites• blood stage results in disease symptoms
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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
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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
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Sporogony• occurs in mosquito (9-21 d)• fusion of micro- and macrogametes • zygote ookinete (~24 hr)• ookinete transverses gut epithelium ('trans-invasion')
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Sporogony(contd…)•ookinete oocyst
between epithelium and basal lamina•asexual replication sporozoites• sporozoites released
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Sporogony(contd…)• sporozoites migrate
through hemocoel• sporozoites 'invade'
salivary glands
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A brief recapitulation
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TUTORIAL VIDEO
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MORPHOLOG
Y -Sudakshina
Das
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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
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from the 2nd year(5th sem) students of MLDMC&H
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
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from the 2nd year(5th sem) students of MLDMC&H
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
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from the 2nd year(5th sem) students of MLDMC&H
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
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from the 2nd year(5th sem) students of MLDMC&H
False-colored electron micrograph of a Plasmodium sp. sporozoite
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CLINICAL FEATURES
-Biswadeep Das
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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.
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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.
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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).
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• 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
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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 .
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from the 2nd year(5th sem) students of MLDMC&H
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 )
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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.
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Major Manifestations of Malaria
Cerebral malaria
Anemia
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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.
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• 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
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LABORATORY
DIAGNOSIS
−Debasrita Bhattacharje
e
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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
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• 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
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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
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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)
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+ 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 →
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P. vivax
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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)
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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
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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
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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
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P. falciparum
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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)
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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
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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
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P. ovale
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Fig.1: Normal red cellFigs.2-5: Young trophozoites (Rings)Figs.6-15: TrophozoitesFigs.16-23: SchizontsFig.24: Macrogametocytes (female)Fig.25: Microgametocyte (male)
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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
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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
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P. malariae
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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)
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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.
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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
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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
J) Gametocyte in thin blood smear
J) Gametocyte in thin blood smear
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• 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
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Principle of QBC System
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Trophozoites of P. falciparum stained with AO in the QBC UV fluorescence method
Trophozoites of P. falciparum stained with BCP in the fluorescence method
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• 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
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• 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
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• 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
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• 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
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Parts of RDT strip Steps in RDT
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PRINCIPLE INVOLVED
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Plastic cassette format of RDT
Results
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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
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• 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
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from the 2nd year(5th sem) students of MLDMC&H
TREATMENT
&
PROPHYLAXI
S−Debjyoti
Ghosh
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Classification according to the stages of the life cycle affected
Chemical classification
Anti-malarial drugs
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Sites of drug action in MALARIA
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TREATMENT OF CLINICAL CASES
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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) .
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Bed-netMosquito repellant
cream
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• 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
from the 2nd year(5th sem) students of MLDMC&H
• 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…)
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Overview of P. knowlesi
RECENT ADVANCES may include the discovery of Plasmodium knowlesi
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• 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
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• 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
from the 2nd year(5th sem) students of MLDMC&H
from the 2nd year(5th sem) students of MLDMC&H
ANYQUESTIONS???