Surendra Kumar Prajapati

Plasmodium vivax in India

Four Plasmodium species cause malaria in humans: Plasmodium vivax is the most widespread and results in pronounced morbidity. India (population >1 billion) is a major contributor to the burden of vivax malaria. With a resurgence in interest concerning the neglected burden of vivax malaria and the completion of the P. vivax genome, it is timely to review what is known concerning P. vivax in India. The P. vivax population is highly diverse in terms of relapse patterns, drug response and clinical profiles, and highly genetically variable according to studies of antigen genes, isoenzyme markers and microsatellites. The unique epidemiology of malaria in India, where P. vivax predominates over Plasmodium falciparum, renders this location ideal for studying the dynamics of co-infection.

Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India.

BackgroundMolecular techniques have facilitated the studies on genetic diversity of Plasmodium species particularly from field isolates collected directly from patients. The msp-1 and msp-2 are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the msp-1 gene and the central repetitive domain (block3) of the msp-2 gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among Plasmodium falciparum population of India, this report evaluates the extent of genetic diversity in the field isolates of P. falciparum in eastern and north-eastern regions of India.MethodsA study was designed to assess the diversity of msp-1 and msp-2 among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa.ResultsP. falciparum isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of msp-1 and msp-2. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the P. falciparum populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of msp-1 was observed to be monomorphic with an allele size of 150/160 bp. The observed 80–90% sequence identity of Indian isolates with data of other regions suggests that Indian P. falciparum population is a mixture of different strains.ConclusionThe present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of msp-1 (block 2) and msp-2 (central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.

Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase

BackgroundIn order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions.MethodsThe P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network.ResultsSynonymous and non-synonymous single nucleotide polymorphisms (SNPs) within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel). SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N) dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T) and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T) mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu.ConclusionIt was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts sharply with the selective sweep of rare antifolate resistant alleles observed in the P. falciparum populations in Asia and Africa. The finding of multiple origins of resistance-conferring mutations has important implications for drug policy.

Therapeutic responses of Plasmodium vivax and P. falciparum to chloroquine, in an area of western India where P. vivax predominates.

Abstract In 2003–2005, following an increase in the local incidence of human malaria, the therapeutic efficacy of chloroquine (CQ) in the treatment of Plasmodium vivax and P. falciparum malaria was evaluated in the Anand district of Gujarat state, in western India. After oral administration of CQ, clinical and parasitological responses were measured over a follow-up period of 28 days, following the standard protocol of the World Health Organization. Most of the recurrent infections were checked, by genotyping, to see whether they were the result of treatment failure or re-infection during the follow-up. At the primary health centre (PHC) in Deva, all 57 P. vivax cases included in the study responded to CQ within 3 days. At the Pansora PHC, however, only 59 [90.8%, with a 95% confidence interval (CI) of 83.7%–97.8%] of the 65 P. vivax cases appeared to respond completely, recurrent infections being observed in the other six cases (9.2%; CI=2.2%–16.3%). Of the four recurrent infections checked by genotyping, however, only two appeared to be the result of true treatment failure. Twenty-seven (81.8%; CI=67.2%–94.4%) of the 33 P. falciparum cases who were enrolled in the study, all from Pansora PHC also showed apparent treatment failure, with one early failure, 17 late clinical failures and nine late parasitological failures. All 23 P. falciparum cases that showed apparent treatment failure and were investigated by genotyping appeared to be true cases of failure, none showing any evidence of re-infection during follow-up. The mean parasite-clearance times for those infected with P. falciparum, both those considered CQ-sensitive and the treatment failures, exceeded 2 days. These results indicate the presence of CQ-resistant P. vivax and P. falciparum in Anand district. The high frequency of CQ failure against P. falciparum observed in this study led to a change in the drug policy at the Pansora PHC, with artemisinin-based combination therapy now being used for the first-line treatment of P. falciparum malaria. Chloroquine remains the recommended first-line treatment for P. vivax infections in the area but the treatment failure seen in at least two P. vivax cases indicates a need for further monitoring of the therapeutic efficacy of CQ against such infections, in central Gujarat and elsewhere.

Surveillance of Artemisinin Resistance in Plasmodium falciparum in India Using the kelch13 Molecular Marker

ABSTRACT Malaria treatment in Southeast Asia is threatened with the emergence of artemisinin-resistant Plasmodium falciparum. Genome association studies have strongly linked a locus on P. falciparum chromosome 13 to artemisinin resistance, and recently, mutations in the kelch13 propeller region (Pfk-13) were strongly linked to resistance. To date, this information has not been shown in Indian samples. Pfk-13 mutations were assessed in samples from efficacy studies of artemisinin combination treatments in India. Samples were PCR amplified and sequenced from codon 427 to 727. Out of 384 samples, nonsynonymous mutations in the propeller region were found in four patients from the northeastern states, but their presence did not correlate with ACT treatment failures. This is the first report of Pfk-13 point mutations from India. Further phenotyping and genotyping studies are required to assess the status of artemisinin resistance in this region.

Molecular epidemiology of Plasmodium vivax anti-folate resistance in India

BackgroundSulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in dihydrofolate reductase (dhfr) and dihydropteorate synthatase (dhps) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in dhfr/dhps genes among Indian sub-continent.MethodsMicroscopically diagnosed one hundred Plasmodium vivax field isolates were collected from five widely separated geographical regions of India. Dhfr and dhps genes were PCR amplified and sequenced. Previously published mutations data were collected and analyzed using Chi square test to identify geographical cluster of mutant/wild type genotypes.ResultsSequence analysis revealed single (S58R), double (S58R/S117N) and quadruple (F57L/S58R/T61M/S117T/) point mutations at dhfr and single (A383G) to double (A383G/A553G) mutations at dhps in P. vivax field isolates. In addition, three new mutations were also observed at dhfr. Both, dhfr and dhps genes revealed tandem repeat variations in field isolates. Dhps revealed very low mutation frequency (14.0%) compared to dhfr (50.70%). Comparative analysis revealed a progressive increase in frequency of quadruple mutant dhfr genotype (p < 0.001) within five years in north-eastern state (Kamrup, Assam). Frequency of dhfr genotypes revealed three distinct geographical clusters of wild (northern India), double mutant (southern India), and quadruple mutant (north-eastern and island regions of India) on the Indian sub-continent.ConclusionStudy suggests that SP may be susceptible to P. vivax in India, except Andaman and north-eastern state. The distinction of geographical regions with sensitive and resistant parasite phenotypes would be highly useful for designing and administering national anti-malarial drug policy.

Allelic dimorphism of Plasmodium vivax gam-1 in the Indian subcontinent

BackgroundGenetic polymorphism is an inevitable component of a complex organism especially in multistage infectious organisms such as malaria parasites. Understanding the population genetic structure of the parasites would provide valuable information for effective malaria control strategies. Recently, the development of molecular tools like PCR has made analysis of field samples possible and easier and research on Plasmodium vivax has also been strengthened. Not many reports are available on the genetic polymorphism of P. vivax from the Indian sub-continent. This study evaluates the extent of diversity in field isolates of India with respect to Pvgam-1.MethodsA study was designed to assess the diversity of Pvgam-1 among field isolates from India, using a nested PCR assay. Field isolates were collected from different regions of the country and the observed variability was confirmed by sequencing data.ResultsBoth Belem and Chesson type alleles were present either exclusively or in mixed form among isolates of all 10 study sites. The Belem type allele was predominant, occurring in 67% of isolates. The proportion of isolates showing the mixed form (both Belem and Chesson type alleles occurring together in the same isolate) was about 13 overall (up to 38.5% in some isolates). Sequencing of the PCR-amplified Belem and Chesson type alleles confirmed the PCR results. Among the 10 study sequences, 11 polymorphic sites and four singleton variations were observed. All the nucleotide substitutions were non-synonymous.ConclusionStudy shows limited diversity of Pvgam-1 marker in Indian isolates with well representation of both Belem and Chesson type alleles.

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

BackgroundMulti-drug resistance and severe/complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers.Methods18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages.Results18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 ± 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 ± 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster.ConclusionsThe global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin.

Antigenic repertoire of Plasmodium vivax transmission-blocking vaccine candidates from the Indian subcontinent

BackgroundGenetic polymorphism is an inevitable component of a multistage infectious organism, such as the malaria parasite. By means of genetic polymorphism, parasite opts particular polymorph and reveals survival advantage. Pvs25 and pvs28 are sexual stage antigen genes, expressed at the ookinete stage inside the mosquito gut, and considered as potential transmission-blocking vaccine candidates. This study presents sequence variations in two important transmission blocking antigen genes pvs25 and pvs28 in the field isolates of P. vivax from the Indian subcontinent.MethodsOne hundred microscopically diagnosed P. vivax isolates were collected from five geographical regions of India. Pvs25 and pvs28 genes were PCR amplified and sequenced to assess sequence variation among field isolates.ResultsA total of 26 amino acid substitutions were observed in Pvs25 (10) and Pvs28 (16) among field isolates of P. vivax. Tandem repeat polymorphism observed in pvs28 shows 3-6 tandem repeats in the field isolates. Seven and eight novel amino acid substitutions were observed in Pvs25 and Pvs28, respectively in Indian isolates. Comparison of amino acid substitutions suggests that majority of substitutions observed in global isolates were also present in Indian subcontinent. A single haplotype was observed to be major haplotype among isolates of Delhi, Nadiad, Chennai and Panna except in isolates of Kamrup. Further, population comparison analyses suggest that P. vivax isolates inhabiting in north-eastern region (Kamrup) were distantly related with the isolates from remaining parts of the country. Majority of the amino acid substitutions observed in Indian isolates were more identical to the substitutions reported from isolates of Thailand and Bangladesh.ConclusionStudy uncovered many new amino acid substitutions as well as a predominance of single haplotype in Indian subcontinent except in north-eastern region of the country. The amino acid substitutions data generated in this study from different geographical regions of the Indian subcontinent could be helpful in designing a more effective anti-malarial transmission-blocking vaccine.

Insights into the invasion biology of Plasmodium vivax.

Plasmodium vivax is the most widely distributed human malaria parasite outside sub Sahara regions of Africa causing huge morbidity and occasionally being severe and fatal (Kochar et al., 2005; Tjitra et al., 2008). Invasion of host erythrocytes is essential for development of disease and the process varies greatly among different malaria parasites. Merozoites of P. vivax and P. berghei (a rodent malaria parasite) primarily invade reticulocytes (Kitchen, 1938; Cromer et al., 2006) whereas P. falciparum invades both reticulocytes and mature erythrocytes (Pasvol et al., 1980; Mitchell et al., 1986). Erythrocyte invasion by malaria parasites is a complex and multi-step process involving interaction between parasite ligands and host cell receptors. The molecules involved in host-parasite interactions of P. falciparum are well characterized and engage multiple invasion pathways (Hadley et al., 1987; Cowman and Crabb, 2006). Conversely, host-parasite interactions of P. vivax are poorly understood. Erythrocyte invasion by P. vivax is mediated by a single receptor expressed on the surface of erythrocytes and reticulocytes called Duffy receptor, or Duffy antigen receptor for chemokines (DARC) (Horuk et al., 1993). During the invasion process, Duffy receptor is recognized and bound by Duffy binding protein (DBP) of P. vivax. DBP is a 140 kD protein located within micronemes of merozoites (Fang et al., 1991; Adams et al., 1992) and it belongs to the family of Duffy binding like erythrocyte binding proteins (DBL-EBP). DBP is comprised of five regions based on conserved cysteine residues, and region II confers adherence to Duffy receptor on the erythrocyte surface. The absence of Duffy receptor (Duffy negative trait) confers resistance to P. vivax infection (Miller et al., 1976). The Duffy negative trait is conferred by a point mutation in the promoter region of the Duffy receptor gene, which abolishes erythroid expression of this receptor (Tournamille et al., 1995). Fixation of the Duffy negativity trait, and the absence of P. vivax infection in human populations of the African continent, supports the hypothesis suggesting that P. vivax cannot infect Duffy negative individuals (Miller et al., 1976). In addition to DBP, other P. vivax proteins involved in recognition and binding of reticulocytes have been identified, however, their respective receptors on reticulocytes are not defined. These parasite proteins include reticulocyte-binding proteins (RBP) and merozoite surface protein-1 (MSP-1) (Galinski et al., 1992; Cantor et al., 2001; Rodriguez et al., 2002). The specificity in binding of reticulocytes is conferred by the RBP family. Members of the RBP family are found in malaria parasites of humans, simians, and rodents (Galinski et al., 1992; Keen et al., 1994; Rayner et al., 2000, 2004). Major functions of RBPs can be observed during the initial process of erythrocyte selection/recognition and invasion (Galinski et al., 1992). Two members (RBP-1 and RBP-2) of the RBP family have been characterized so far from P. vivax and are promising vaccine candidates (Galinski and Barnwell, 1996). However, genome sequences of several malaria parasites of human, primate, and rodent have revealed many putative RBP family-like genes. Comparative genetic analysis of members of the RBP family suggests that the family is highly evolved and conserved across malarial parasites where each parasite species encodes a range of 4–15 members (Aurrecoechea et al., 2009). P. vivax encodes 11 members of the RBP gene family that are believed to provide recognition and specificity in the reticulocyte binding process. Characterization of DNA sequence polymorphisms of a few members of the RBP gene family from worldwide field isolates (Rayner et al., 2005; Kosaisavee et al., 2012; Prajapati et al., 2012) reveals a level of genetic polymorphisms commonly observed in promising vaccine candidate genes of malarial parasites. This suggests that members of RBP are recognized by the host immune system. Interestingly, several recent reports show infection of P. vivax in Duffy-negative individuals in African (Ryan et al., 2006; Mendes et al., 2011; Wurtz et al., 2011) and American continents (Cavasini et al., 2007a,b; Carvalho et al., 2012). These reports are highly intriguing considering the presumed dependence of P. vivax on Duffy receptor for invasion. Thus, Duffy receptor may not be the only gateway for P. vivax and alternative invasion mechanisms may exist (Mons, 1990). It is important to understand how, and under what circumstances, P. vivax infects to Duffy negative individuals. P. vivax is the only human malaria parasite that invades reticulocytes. Each of the 11 proteins encoded by the RBP gene family has a cognate receptor on the surface of reticulocytes that is essential for P. vivax invasion. Therefore, understanding the invasion biology of P. vivax requires functional characterization of RBPs and their receptor on the reticulocyte surface. Unlike P. falciparum, the study of P. vivax is associated with technical and experimental difficulties that have impeded progress in understanding aspects of parasite genetics, such as population genetics. A major technical problem with P. vivax is the lack of an in vitro culture system. Other experimental constraints include the need of ample reticulocytes for long-term culture, a poor understanding of the biology of reticulocytes and their receptors, and a remarkably low parasitemia in P. vivax-infected individuals. Consequently, functional studies aiming to uncover various aspects of P. vivax biology such as invasion, reproduction, virulence, and development are poorly understood. Despite these limitations, extensive efforts aimed at developing a continuous culture system for P. vivax have resulted in establishment of short-term in vitro invasion and maturation assays (Chotivanich et al., 2001; Udomsangpetch et al., 2007; Russell et al., 2008), in addition to a methods for cryopreservation of both P. vivax and cord blood reticulocytes (Borlon et al., 2012). These advances provide important tools for unraveling the unique biology of P. vivax invasion, such as identifying receptors for RBPs on reticulocytes using pull-down assays. This kind of study would not only uncover molecular mechanism underlying invasion biology but also open new avenues to look for alternative routes for P. vivax invasion. Considering P. vivax can infect Duffy negative individuals, functional characterization of RBPs and their receptors on reticulocytes will provide new insights into the biology of P. vivax invasion. Given that P. vivax cannot invade mature erythrocytes, understanding the role of RBPs in invasion process is critical for developing a new generation of treatment therapies.