plasmodium ii mbbs dr ekta chourasia microbiology

PLASMODIUMPLASMODIUM

II MBBSII MBBS

Dr Ekta ChourasiaDr Ekta ChourasiaMicrobiology Microbiology

26/04/0726/04/07 Dr Ekta Chourasia, MicrobiologyDr Ekta Chourasia, Microbiology

TaxonomyTaxonomy

Phylum ApicomplexaPhylum Apicomplexa

Subphylum SporozoaSubphylum Sporozoa

Genus PlasmodiumGenus Plasmodium

Disease MalariaDisease Malaria

Geographical Tropical & Geographical Tropical &

distribution distribution

Subtropical countriesSubtropical countries


Genus PlasmodiumGenus Plasmodium

Consists of 4 species:Consists of 4 species:1.1. P. vivaxP. vivax

2.2. P. falciparumP. falciparum

3.3. P. malariaeP. malariae

4.4. P. ovaleP. ovale


Landmarks in the evolution of MalariaLandmarks in the evolution of Malaria

1880 – 1880 – LaveranLaveran identified the identified the malarial parasitemalarial parasite in in an unstained smearan unstained smear

1885 – 1885 – GolgiGolgi described the described the blood stage blood stage (erythrocytic schizogony) of malarial(erythrocytic schizogony) of malarial parasite – Golgi cycleparasite – Golgi cycle

1898 – 1898 – Amigo & GrassiAmigo & Grassi described the described the life cyclelife cycle1891 – 1891 – RomanowskyRomanowsky introduced the introduced the staining staining

methodmethod1897 – 1897 – Ronald RossRonald Ross while in Calcutta, India, while in Calcutta, India,

demonstrated Anopheles sp. of mosquitoes asdemonstrated Anopheles sp. of mosquitoes as vectors of malaria. vectors of malaria. Got Got Nobel prizeNobel prize for his work in 1902 for his work in 1902


Transmission & Life CycleTransmission & Life Cycle

Definitive host Female Anopheles mosquitoDefinitive host Female Anopheles mosquito

Intermediate host ManIntermediate host Man

Infective form SporozoitesInfective form Sporozoites

Portal of entry SkinPortal of entry Skin

Mode of transmission Bite of an infected mosquitoMode of transmission Bite of an infected mosquito

Site of localization First in liver cells & then in Site of localization First in liver cells & then in

RBCs RBCs


Phases of Development in ManPhases of Development in Man

2 phases of development2 phases of development

1.1. Inside the liver (tissue phase)Inside the liver (tissue phase) Pre- erythrocytic schizogonyPre- erythrocytic schizogony – no clinical – no clinical

symptoms, no pathological damagesymptoms, no pathological damage Exo- erythrocytic schizogonyExo- erythrocytic schizogony – cause of relapse – cause of relapse

2.2. Inside the RBCs (erythrocytic phase)Inside the RBCs (erythrocytic phase) Erythrocytic schizogonyErythrocytic schizogony – cause of malarial – cause of malarial

paroxsymsparoxsyms Gametogony Gametogony – infects mosquito– infects mosquito


Morphological forms seen in HumansMorphological forms seen in Humans

In liver:In liver:1.1. SporozoitesSporozoites2.2. Pre erythrocytic schizontsPre erythrocytic schizonts

3.3. Merozoites – infect RBCsMerozoites – infect RBCs

In RBCs :In RBCs :1.1. Trophozoites – ring formTrophozoites – ring form2.2. Schizonts Schizonts 3.3. Merozoites – released by the rupture of schizonts Merozoites – released by the rupture of schizonts

– infect other RBCs– infect other RBCs4.4. Gametocytes – micro and macro gametocytesGametocytes – micro and macro gametocytes


Morphological forms seen in MosquitoMorphological forms seen in Mosquito

Further differentiation & development of Further differentiation & development of gametocytes take place in mosquitogametocytes take place in mosquito

1.1. Macro gametes (female gametes)Macro gametes (female gametes) – each macro – each macro gametocyte develops in to one macro gamete in gametocyte develops in to one macro gamete in the mid gut of mosquitothe mid gut of mosquito

2.2. Micro gametes (male gametes)Micro gametes (male gametes) – one micro – one micro gametocyte produces 6 to 8 micro gametes by gametocyte produces 6 to 8 micro gametes by exflagellation.exflagellation.

3.3. ZygoteZygote – – OokineteOokinete – – OocystOocyst – rupture – release of – rupture – release of SporozoitesSporozoites – predilection to salivary glands. – predilection to salivary glands.


Other modes of transmissionOther modes of transmissionSporozoite- induced- malariaSporozoite- induced- malaria : injection of an : injection of an emulsion of salivary glands of mosquito containing emulsion of salivary glands of mosquito containing sporozoitessporozoites

Trophozoite- induced- malariaTrophozoite- induced- malaria : injection of blood : injection of blood from a malarial patient containing the asexual from a malarial patient containing the asexual forms of erythrocytic schizogony e.gforms of erythrocytic schizogony e.g..

1.1. Transfusion malariaTransfusion malaria – when persons with latent – when persons with latent infection are used as donorsinfection are used as donors

2.2. Congenital malariaCongenital malaria – transmission through some – transmission through some placental defects (a healthy placenta acts as a placental defects (a healthy placenta acts as a physiological barrier)physiological barrier)

3.3. Drug addictsDrug addicts – by using same syringe – by using same syringe


Incubation periodIncubation period

P. vivax P. vivax

P. ovale 10 to 14 daysP. ovale 10 to 14 days

P. falciparum P. falciparum

P. malariae 18 days to 6 P. malariae 18 days to 6

weeksweeks


Pathogenicity Pathogenicity Infection causes intermittent fever – MalariaInfection causes intermittent fever – Malaria

Each of the 4 species causes a characteristic Each of the 4 species causes a characteristic fever:fever:

P. vivaxP. vivax Benign Benign tertiantertian/ vivax malaria/ vivax malaria

P. falciparumP. falciparum Malignant Malignant tertiantertian/ falciparum/ falciparum

malaria, black water fevermalaria, black water fever

P. malariaeP. malariae QuartanQuartan malaria malaria

P. ovaleP. ovale TertianTertian/ Ovale malaria / Ovale malaria


Clinical FeaturesClinical FeaturesSeries of febrile paroxysmsSeries of febrile paroxysms – fever is caused – fever is caused by the release of merozoites & toxins from by the release of merozoites & toxins from ruptured erythrocytic schizont which in turn ruptured erythrocytic schizont which in turn causes the release of cytokines.causes the release of cytokines.

Quartan malaria – every 72 hrsQuartan malaria – every 72 hrs Tertian malaria - every 48 hrs Tertian malaria - every 48 hrs

* each paroxysm has 3 stages - * each paroxysm has 3 stages - cold stagecold stage (rigors), (rigors), hot stagehot stage (high temp., body & joint (high temp., body & joint pains, vomiting & diarrhoea) and pains, vomiting & diarrhoea) and perspirationperspiration stage stage (fall in temp.) (fall in temp.)


Clinical FeaturesClinical Features

AnaemiaAnaemia – due to breakdown of RBCs, – due to breakdown of RBCs, particularly occurs in falciparum malariaparticularly occurs in falciparum malaria

Splenomegaly Splenomegaly – all forms – all forms


Falciparum MalariaFalciparum MalariaMost widespreadMost widespread

Accounts for 80% of malaria cases Accounts for 80% of malaria cases worldwideworldwide

Most pathogenic of human malaria Most pathogenic of human malaria speciesspecies

Untreated infections - severe disease & Untreated infections - severe disease & even death, particularly in young children, even death, particularly in young children, pregnant woman & non immune adults.pregnant woman & non immune adults.


Falciparum malariaFalciparum malariaSevere falciparum malaria is Severe falciparum malaria is associated withassociated with

1.1. Pernicious malaria /cerebral malariaPernicious malaria /cerebral malaria

2.2. Blackwater feverBlackwater fever

3.3. AnaemiaAnaemia

4.4. HypoglycaemiaHypoglycaemia

5.5. Hypotension Hypotension

6.6. Complications in pregnancyComplications in pregnancy


Pernicious MalariaPernicious Malaria

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

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

Occurs as a result of capillary blockage.Occurs as a result of capillary blockage.


Black Water FeverBlack Water Fever

Occurs in Occurs in previously infectedpreviously infected subjects subjects

Can also occur in Can also occur in non immune adultsnon immune adults with with severe falciparum malaria, and also as a severe falciparum malaria, and also as a complication of quinine therapycomplication of quinine therapy..

A rare but acute condition characterised by A rare but acute condition characterised by sudden & sudden & massive hemolysismassive hemolysis of parasitised & of parasitised & non parasitised RBCs followed by fever and non parasitised RBCs followed by fever and haemoglobinuriahaemoglobinuria..

Often Often fatalfatal due to due to renal failurerenal failure


Black Water FeverBlack Water FeverDifficult to find the parasites Difficult to find the parasites

in the blood following a in the blood following a hemolytic attack.hemolytic attack.

Urine appears Urine appears dark red todark red to brown blackbrown black due to the due to the presence of free Hb.presence of free Hb.

Clinical featuresClinical features – fever, rigor, aching pains in – fever, rigor, aching pains in the loin, icterus, bilious vomiting, circulatory the loin, icterus, bilious vomiting, circulatory collapse, haemoglobinuria & acute renal failure.collapse, haemoglobinuria & acute renal failure.Treatment Treatment – Chloroquine, blood transfusion, – Chloroquine, blood transfusion, peritoneal dialysis in ARF.peritoneal dialysis in ARF.


AnaemiaAnaemiaCan be severe & occur rapidly, Can be severe & occur rapidly, particularly in young childrenparticularly in young children

Occurs due to Occurs due to destruction of parasitiseddestruction of parasitised RBCs RBCs – phagocytosis & destruction in the – phagocytosis & destruction in the spleenspleen

Decreased production of RBCs in the Decreased production of RBCs in the bone marrow.bone marrow.


Falciparum malaria in PregnancyFalciparum malaria in Pregnancy

Can result in:Can result in:

Severe anemia Severe anemia

Low birth weight babiesLow birth weight babies

Greatest risk in 1Greatest risk in 1stst pregnancy pregnancy


Malaria caused by P.vivax, Malaria caused by P.vivax, P.ovale & P.malariaeP.ovale & P.malariae

Rarely life threateningRarely life threatening

Relapses/ recurrences are a featureRelapses/ recurrences are a feature

Recurrences in MalariaRecurrences in MalariaMay result from – reinfection or May result from – reinfection or

- due to certain events related - due to certain events related

to the parasite’s life cycleto the parasite’s life cycle


Recurrence of MalariaRecurrence of MalariaTwo types of recurrences known in malaria:Two types of recurrences known in malaria:

1.1. RecrudescenceRecrudescence – – seen in seen in P. falciparum & P. malariaeP. falciparum & P. malariae due to due to persistence of blood infectionpersistence of blood infection (some (some

erythrocytic forms evade host immunity) even after erythrocytic forms evade host immunity) even after clinical illness has subsided.clinical illness has subsided.

The numbers may increase later, leading to The numbers may increase later, leading to reappearance of clinical symptomsreappearance of clinical symptoms

Occur mostly up to one year or so but in P. malariae, Occur mostly up to one year or so but in P. malariae, it can occur even after decadesit can occur even after decades


Recurrence of MalariaRecurrence of Malaria2.2. RelapseRelapse

Occurs due to a special form of parasites Occurs due to a special form of parasites – – hypnozoiteshypnozoites..

Hypnozoites are the sporozoites that Hypnozoites are the sporozoites that remain dormant after infecting liverremain dormant after infecting liver

Activated from time to time to initiate pre Activated from time to time to initiate pre erythrocytic schizogony - erythrocytic schizogony - Exoerythrocytic Exoerythrocytic schizogonyschizogony


Genetic factors protecting Genetic factors protecting against Malariaagainst Malaria

Sickle cell anaemiaSickle cell anaemia – sickle celled RBCs are – sickle celled RBCs are removed by the spleen before the development removed by the spleen before the development of schizontsof schizonts

Ovalocytosis –Ovalocytosis – RBCs are rigid and they resist RBCs are rigid and they resist parasitic invasionparasitic invasion

Duffy blood group negativeDuffy blood group negative individuals – duffy individuals – duffy blood group Ag is the receptor for the blood group Ag is the receptor for the attachment of merozoites of P.vivaxattachment of merozoites of P.vivax


Genetic factors protecting Genetic factors protecting against Malariaagainst Malaria

Newborn infantsNewborn infants – natural protection for – natural protection for 11stst few months of life due to high conc. of few months of life due to high conc. of HbF in their RBCs.HbF in their RBCs.

Beta thalassaemiaBeta thalassaemia – protects against – protects against severe falciparum infectionsevere falciparum infection


Laboratory Diagnosis Laboratory Diagnosis

Microscopy Microscopy – detecting & identifying malarial – detecting & identifying malarial parasites in peripheral blood films.parasites in peripheral blood films.

Concentrating parasitesConcentrating parasites in venous blood by in venous blood by centrifugation when they can not be found in centrifugation when they can not be found in blood filmsblood films

Using a Using a rapidrapid malaria Ag or enzyme malaria Ag or enzyme detection detection testtest

Other testsOther tests – Hb, PCV, Blood glucose, total – Hb, PCV, Blood glucose, total WBC & platelet count.WBC & platelet count.


Examination of Blood filmExamination of Blood filmCollection of bloodCollection of blood

- best prepared directly from capillary blood- best prepared directly from capillary blood

- in EDTA bulb (used within 30 mins)- in EDTA bulb (used within 30 mins)

Time of collectionTime of collection

- as soon as possible if malaria is suspected- as soon as possible if malaria is suspected

- before administering antimalarials- before administering antimalarials

- during pyrexial phase- during pyrexial phase


Types of Blood filmTypes of Blood film

Two types:Two types:

1.1. Thick filmsThick films : :

- 30 to 40 times - 30 to 40 times more sensitivemore sensitive than thin films than thin films

- more suitable for - more suitable for detection of malarialdetection of malarial parasiteparasite when they are few in numberwhen they are few in number

- blood is not fixed, RBCs are lysed during - blood is not fixed, RBCs are lysed during staining (only parasitic forms will be seen)staining (only parasitic forms will be seen)


Types of Blood filmTypes of Blood film2. Thin films :2. Thin films : - to - to confirm the Plasmodium speciesconfirm the Plasmodium species - assists in the identification of - assists in the identification of mixed mixed

infectionsinfections - blood is fixed, parasites are seen within - blood is fixed, parasites are seen within

the RBCs the RBCs - also helps in assessing the - also helps in assessing the response toresponse to

treatmenttreatment especially in areas where especially in areas where drug drug resistance is suspected (by counting resistance is suspected (by counting the the number of parasitised RBCs before & number of parasitised RBCs before &

after the treatment)after the treatment)


Making of Thin & Thick filmsMaking of Thin & Thick films


Fixation & StainingFixation & Staining

FixationFixation – thin films are fixed with absolute – thin films are fixed with absolute alcohol for 1 to 2 mins. alcohol for 1 to 2 mins.

Staining Staining – films are stained with – films are stained with Romanowsky stain: giemsa, Romanowsky stain: giemsa,

field’s, wright’s field’s, wright’s

Giemsa Giemsa – 10% solution for 10 mins– 10% solution for 10 mins


Reporting of Blood filmReporting of Blood filmLook for the Look for the different morphologicaldifferent morphological formsforms of parasite in blood smear: of parasite in blood smear:

1.1. Trophozoites / ring formsTrophozoites / ring forms2.2. Schizont Schizont 3.3. Gametocytes Gametocytes

Identify speciesIdentify species – differences in the – differences in the characteristics of morphological forms in characteristics of morphological forms in different speciesdifferent species


Trophozoites / Ring formsTrophozoites / Ring formsCharacter Character P. vivaxP. vivax P. falciparumP. falciparum

Size Size 2.52.5µµ (1/3 (1/3rdrd of RBC) of RBC) 1.25 to 1.5 1.25 to 1.5 µµ

Cytoplasm Cytoplasm Thick opposite to Thick opposite to nucleusnucleus

Uniform thicknessUniform thickness

NucleusNucleus One/ ringOne/ ring Can have >1Can have >1

Number of ringsNumber of rings One ring/ RBCOne ring/ RBC >1/ RBC>1/ RBC

Location in Location in RBCsRBCs

Always inside Always inside RBCsRBCs

Inside as well as on the Inside as well as on the surface (accole’ forms)surface (accole’ forms)

Type of RBC Type of RBC infectedinfected

Preferentially Preferentially young RBCs & young RBCs & reticulocytesreticulocytes

All typesAll types


Thin Blood Film Thick Blood Film

Ring Forms / Trophozoites


SchizontSchizontCharacterCharacter P. vivaxP. vivax P. falciparum P. falciparum

Size of RBCSize of RBC Increases to Increases to twice its sizetwice its size

Does not Does not changechange

No of No of merozoitesmerozoites

1616 8 to 328 to 32

Arrangement Arrangement of merozoitesof merozoites

Symmetric in Symmetric in form of rosetteform of rosette

Asymmetrical Asymmetrical

Presence in Presence in peripheral peripheral bloodblood

Present Present Absent Absent


P. vivax P.falciparum


Gametocytes (male & female) Gametocytes (male & female)

Character Character P. vivaxP. vivax P. falciparumP. falciparum

Shape – MaleShape – Male

Female Female

SphericalSpherical

SphericalSpherical

CresenticCresentic

Sausage shapedSausage shaped

Nucleus- MNucleus- M

FF

Central, diffuseCentral, diffuse

Peripheral,smallPeripheral,small

Central, diffuseCentral, diffuse

Central,compactCentral,compact

Infected RBCInfected RBC Enlarged Enlarged Deformed, with Deformed, with its membrane its membrane stretched.stretched.


P. falciparumP.vivax


Counting the % age of Counting the % age of parasitised RBCsparasitised RBCs

On thin blood filmsOn thin blood filmsWhen falciparum malaria parasitemia is highWhen falciparum malaria parasitemia is highMethod of counting:Method of counting:

1.1. Select an area where no of RBCs is roughly 250.Select an area where no of RBCs is roughly 250.2.2. Count the no of parasitised RBCs in 4 such fields i.e. Count the no of parasitised RBCs in 4 such fields i.e.

approximately 1000 RBCs.approximately 1000 RBCs.3.3. Divide by 10 to obtain the percentage.Divide by 10 to obtain the percentage.

*WHO – if it is >5%, then the parasitemia is *WHO – if it is >5%, then the parasitemia is heavy & prognosis is poor.heavy & prognosis is poor.


Buffy Coat preparationBuffy Coat preparation

To concentrate malarial parasiteTo concentrate malarial parasite

Centrifuge EDTA anticoagulated venous Centrifuge EDTA anticoagulated venous blood in a thin bore capillary tubeblood in a thin bore capillary tube

Buffy coat layer is formed between the Buffy coat layer is formed between the RBCs & the plasma.RBCs & the plasma.

Break the tube & transfer buffy coat & Break the tube & transfer buffy coat & RBCs to a slide - make a thin smear – air RBCs to a slide - make a thin smear – air dry – fix with ethanol – stain with Giemsa.dry – fix with ethanol – stain with Giemsa.


Quantitative Buffy CoatQuantitative Buffy Coat

Capillary tube is coated with an Capillary tube is coated with an anticoagulant & Acridine orange anticoagulant & Acridine orange fluorescent dyefluorescent dye

After centrifugation, the tube can be used After centrifugation, the tube can be used for two purpose:for two purpose:

1.1. Complete blood countComplete blood count

2.2. Identification of malarial parasite using a Identification of malarial parasite using a fluorescence microscope.fluorescence microscope.


Quantitative Buffy Coat


Rapid Diagnostic testsRapid Diagnostic tests

Developed to Developed to diagnose falciparum malariadiagnose falciparum malaria rapidly & without a microscope.rapidly & without a microscope.

Can also detect Can also detect vivax malariavivax malaria

Three tests are available commercially Three tests are available commercially

Detects either Detects either HRP2 Ag (Histidine rich protein)HRP2 Ag (Histidine rich protein) or or specific pLDH (parasite lactate dehydrogenase)specific pLDH (parasite lactate dehydrogenase)

Both HRP2 & pLDH are produced by the Both HRP2 & pLDH are produced by the parasites during their growth & differentiation in parasites during their growth & differentiation in RBCs.RBCs.


Rapid Diagnostic testsRapid Diagnostic tests

HRP2 testsHRP2 tests detection of P.falciparumdetection of P.falciparumTwo types of test – ParaSight FTwo types of test – ParaSight F

- ICT Malaria Pf- ICT Malaria Pf

pLDH test e.g. OptiMAL testpLDH test e.g. OptiMAL testDetection of P.falciparum & P.vivaxDetection of P.falciparum & P.vivaxProduced by all human malarial parasitesProduced by all human malarial parasitesDifferentiation of species is based on antigenic Differentiation of species is based on antigenic differences between pLDH isoforms. differences between pLDH isoforms.


Optimal test

ParaSightF test

ICT Malaria Pf / Pv


Stage specificity of antimalarial drugsStage specificity of antimalarial drugs

Stage of malarial Stage of malarial parasiteparasite

Antimalarial drugAntimalarial drug

Sporozoite Sporozoite Proguanil, PyrimethamineProguanil, Pyrimethamine

Hypnozoite Hypnozoite Primaquine Primaquine

Pre- erythrocytic Pre- erythrocytic schizontschizont

Proguanil, PyrimethamineProguanil, Pyrimethamine

Blood schizontBlood schizont Chloroquine, Quinine, Chloroquine, Quinine, ArtemisininArtemisinin

Gametocyte Gametocyte Primaquine Primaquine

plasmodium ii mbbs dr ekta chourasia microbiology

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