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Imparting training is one of the mandatesof Malaria Research Centre. Expertiseavailable for providing training indifferent techniques is listed in Box 2. Toeducate school children about malariaand malaria parasites, organizing dem-onstrations and exhibitions has been oneof the regular activities of the ParasiteBank. Several scientists/research scholarswere given training for one week to fourmonths in different techniques (Details inAnnexure 2). Many who got trained atthe Parasite Bank have established facili-ties for in vitro culture in their respectiveinstitutes.

ActivitiesActivities

Foreign visitors being explained about theactivities of the parasite bank

WHO fellows from the Republic of Korea whoreceived training in the Parasite Bank

Human Resource DevelopmentHuman Resource Development

Training for regional malaria officers onmalaria microscopy

Dr. V.P. Kamboj, Director, Central DrugResearch Institute during his visit to theParasite Bank

Health education on malaria for schoolchildren

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ActivitiesActivities

Demonstration of in vitro drug sensitivity of malaria parasites

Box 2: Training Facilities Available

l Plasmodial species identification by microscopy, PCR & byimmunological techniques.

l Collection, cryopreservation, revival and transportation ofmalaria parasite isolates/strains.

l In vitro cultivation of erythrocytic stages of P. falciparum.

l Short-term cultivation of P. vivax and other species ofPlasmodium.

l In vitro cultivation of exo-erythrocytic stages of P. vivax.

l In vitro testing for sensitivity of P. falciparum isolates toantimalarials.

l In vitro and in vivo screening of medicinal plant extracts forantiplasmodial properties.

l Polyacrylamide gel electrophoresis for various enzyme markersof plasmodia species.

l Molecular characterization of plasmodia species/isolates usingPCR, RFLP and sequencing in relation to genetic polymorphismand drug resistance.

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Providing malaria parasites to thescientific community has been one of themajor activities of the Prasite Bank.

Following biological material is beingsupplied to various Institutes, Universitiesand other Research Organizations(Details in Annexure 3).

l Adapted and non adaptedcryopreserved parasites isolates

l Parasite isolates resistant/sensitiveto chloroquine

l Parasite isolates with rosettingproperties

l Parasite isolates with differentcytoadherence properties

l Isolates with different erythrocyteinvasion properties

l Sera/plasma from malaria positivepatients

l Different stages of parasites such asmerozoites, ring forms, gametocytesfrom culture

l Sporozoites harvested fromartificially fed mosquitoes

l Different species of avian, simianand rodent plasmodia

l Rodent plasmodia infected rats/mice

l Sera/plasma from respectivevertebrate hosts

ActivitiesActivities

Cryopreservation of malaria parasites inliquid nitrogen

Supply of Biological MaterialsSupply of Biological Materials

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Research outputsResearch outputs

The malaria parasite respsitory at the Centre is a rich national resource for humanand non-human plasmodial species and strains. Isolates preserved have been usedby researchers from MRC and from other Institutes. A brief summary of research car-ried out at MRC is given below and publications are listed in Annexure 4.

For the first time in India, pre-erythrocyticcycle of P. vivax was developed in hepatomacell line. Mosquitoes fed on P. vivax infectedblood through artificial membrane feedingapparatus were dissected for sporozoites ondays 8-10 after feeding . Sporozoites har-vested were inoculated to the hepatoma cellline and on day 7, fully grown schizontswere seen.

Research OutputsResearch Outputs

P. vivax mature pre-erythrocytic schizont inin vitro hepatoma cell line

P. vivax mature erythrocytic cycle schizont inin vitro culture

Pre-erythrocytic Cycle of Pre-erythrocytic Cycle of P. vivaxP. vivax

Erythrocytic Stages of Erythrocytic Stages of P. vivaxP. vivax

Procedure has been standardized for main-taining erythrocytic stages of P. vivax in invitro short term culture of 2-3 cycles (7-8days) (Usha Devi et al., 2000). This shortduration in vitro culture system can be usedfor testing sensitivity of field isolates to chlo-roquine and for screening plant extracts/newmolecules for antimalarial properties againstP. vivax parasites.

Screening of hepatoma cell line for in vitrocultivation

Characterization of malaria parasites by PCRtechnique

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With an objective to understand the ge-netic structure of the isolates being col-lected from different geographical areas/varied eco-epidemiological situations,parasite genomic variations were studiedby enzyme and DNA markers. Study re-vealed that Indian isolates of P.falciparum exhibit high level of genetic di-versity with respect to the markers stud-ied (Fig. 2).

Fig. 3: Distribution of family groups of MSP-1& 2 of P. falciparum isolates of the parasitebank

Genetic PolymorphismGenetic Polymorphism

Fig. 2: Agarose gel electrophoretograms showing size polymorphism of MSP-1 & 2 in P. falciparumisolates

In general a high proportion of isolates,more than 60%, were found to havemore than one genetically distinct para-site type i.e., multiclonal. Analysis re-vealed that isolates belong to a singlerandom mating population of P.falciparum and alleles studied are un-linked. The study also supported thefeasibility of using the molecular markersfor differentiating recrudescence fromfresh infection in P. falciparum (Joshi etal 1996, 2003). The distribution of iso-lates with different families of MSP-1 andMSP-2 is shown in Fig. 3

Research outputsResearch outputs

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Research outputsResearch outputs

P. falciparum isolates available in theparasite bank were used for thestandarization of Pulsed field gel electro-phoresis procedure for the separation ofall the 14 chromosomes of P. falciparum.A 72 hours run on PFGE with 270 and999 sec pulse has shown separation ofthe chromosomal complement (Fig. 4 ).

between chloroquine resistance and al-lelic variation in Pfmdr-1 gene. Similarlyin studies carried out in collaborationwith scientists from AIIMS, New Delhi andIISc, Bangalore on Pfcrt gene, high fre-quencies of mutations among Indian iso-lates were observed but there was a poorcorrelation with in vitro tested chloroquineresistance and sensitivity (Vathsala et al.,2004, Vinayak et al., 2003). Frequency ofmutations in DHFR and DHPS genes wasalso relatively high in field isolates com-pared to the sensitivity levels in SP drug inin vitro assays.

Since 1992 a total of 257 P. falciparumsamples from different regions weretested for sensitivity to chloroquine and167 (64.98%) were found to be resistantto chloroquine. In vitro tested chloroquineresistant and sensitive P. falciparum iso-lates were used to correlate with nucle-otide changes at positions 754, 1049,3598, 3622 and 4234 in the codingregion of Pfmdr-1 gene (Bhattacharya andPillai, 1999) (Table 6). The nucleotidechanges in the said positions were foundto be ambiguous showing a strongassociation but incomplete correlation

Table 6: Analysis of Point Mutations Observed in Pfmdr Gene of P. falciparum Isolates

Fig. 4: Pulsed field gel eletrophore- togram of

P. falciparum

Chromosomal Complement Chromosomal Complement

Molecular Markers Related to Drug ResistanceMolecular Markers Related to Drug Resistance

Nucleotide CQ sensitive CQ resistant isolates positions isolates

(13) (5) (1) (3) (1) (1) (3) (1) (3) (2) (2) (2) (1) (2)

754 - + - + + + + + - + + + - +

1049 - - + - + + - + + + - + + +

3598 - - + - + + - + - + + + +

3622 - - + - + - + - - + + + + +

4234 - - - + - - - + + + + - + +

Figures in parentheses are number of isolates; with mutation (+) or absence (-).

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Generally, sialic acid residues of glycoph-orin A are used as invasion receptors byPlasmodium falciparum. And in vitro invasionstudies demonstrated that some cloned P.falciparum lines use alternate receptors in-dependent of sialic acid residues of glyco-phorin A. In studies with ICGEB, NewDelhi aimed to define invasion profile ofIndian field isolates of P. falciparum, iso-lates collected and cryopreserved in theBank from Orissa, Chattisgarh, UttarPradesh and Rajasthan were used. Inin vitro growth assays and erythrocyte in-vasion assays, 12 of the 15 isolates

tested used alternate invasion pathways(Okoyeh et al., 1999) indicating usingalternate invasion pathways may not beuncommon in nature.

In another assay anti-EBA-175 R.II (F2)antibody raised against EBA-175 wasused to test its ability to inhibit invasionof erythrocytes by parasites. It showedabout 80% inhibition compared tocontrols indicating that this antibody ishighly effective in blocking erythrocyteinvasion by the parasites (Pandey et al.,2002).

Research outputsResearch outputs

The adhesion of P.falciparum infectederythrocytes in brain capillaries is impli-cated in cerebral malaria.Cytoadherence enables P.falciparum toavoid passage through the spleen whereinfected erythrocytes are destroyed. Theendothelial receptors used by P.falciparumfor cytoadherence include CD36, ICAM-1, CD31, V-selectin, E-selectin and

chondro-itan sulfate - A (CSA). In collabo-rative studies with ICGEB, a few P.falciparum field isolates were screened fortheir cytoadherence properties in vitro.And some of these isolates were identi-fied having cytoadherence properties toICAM-1, a molecule that can cause cere-bral/ complicated malaria (Chatto-padhyay et al., 2004).

Screening antiplasmodial activity of medicinal plants

Erythrocyte Invasion Studies in Erythrocyte Invasion Studies in P. falciparumP. falciparum

CytoadherenceCytoadherence

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Fig. 5 a&b: In vivo antimalarial effect of some medicinal plantsscreened at the parasite bank

a)a)

b)b)

Research outputsResearch outputs

Screening of Medicinal Plant Extracts for Antiplasmodial ActivityScreening of Medicinal Plant Extracts for Antiplasmodial Activity

As a part of drug (antimalarial) develop-ment research, isolates from the Bankare regularly used for primary screeningof medicinal plants (extracts) collectedfrom rural and tribal areas. About 25

plants were already screened for theantiplasmodial activity either in vitro in P.falciparum culture or in vivo animal mod-els. A few of the plant extracts tested areshowing encouraging results (Fig. 5a&b )(Usha Devi et al., 2001).