Hema Joshi

The complexities of malaria disease manifestations with a focus on asymptomatic malaria

Malaria is a serious parasitic disease in the developing world, causing high morbidity and mortality. The pathogenesis of malaria is complex, and the clinical presentation of disease ranges from severe and complicated, to mild and uncomplicated, to asymptomatic malaria. Despite a wealth of studies on the clinical severity of disease, asymptomatic malaria infections are still poorly understood. Asymptomatic malaria remains a challenge for malaria control programs as it significantly influences transmission dynamics. A thorough understanding of the interaction between hosts and parasites in the development of different clinical outcomes is required. In this review, the problems and obstacles to the study and control of asymptomatic malaria are discussed. The human and parasite factors associated with differential clinical outcomes are described and the management and treatment strategies for the control of the disease are outlined. Further, the crucial gaps in the knowledge of asymptomatic malaria that should be the focus of future research towards development of more effective malaria control strategies are highlighted.

World Antimalarial Resistance Network (WARN) III: Molecular markers for drug resistant malaria

Molecular markers for drug resistant malaria represent public health tools of great but mostly unrealized potential value. A key reason for the failure of molecular resistance markers to live up to their potential is that data on the their prevalence is scattered in disparate databases with no linkage to the clinical, in vitro and pharmacokinetic data that are needed to relate the genetic data to relevant phenotypes. The ongoing replacement of older monotherapies for malaria by new, more effective combination therapies presents an opportunity to create an open access database that brings together standardized data on molecular markers of drug resistant malaria from around the world. This paper presents a rationale for creating a global database of molecular markers for drug resistant malaria and for linking it to similar databases containing results from clinical trials of drug efficacy, in vitro studies of drug susceptibility, and pharmacokinetic studies of antimalarial drugs, in a World Antimalarial Resistance Network (WARN). This database will be a global resource, guiding the selection of first line drugs for treating uncomplicated malaria, for preventing malaria in travelers and for intermittent preventive treatment of malaria in pregnant women, infants and other vulnerable groups. Perhaps most important, a global database for molecular markers of drug resistant malaria will accelerate the identification and validation of markers for resistance to artemisinin-based combination therapies and, thereby, potentially prolong the useful therapeutic lives of these important new drugs.

Polymorphisms of TNF-enhancer and gene for FcγRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population

BackgroundSusceptibility/resistance to Plasmodium falciparum malaria has been correlated with polymorphisms in more than 30 human genes with most association analyses having been carried out on patients from Africa and south-east Asia. The aim of this study was to examine the possible contribution of genetic variants in the TNF and FCGR2A genes in determining severity/resistance to P. falciparum malaria in Indian subjects.MethodsAllelic frequency distribution in populations across India was first determined by typing genetic variants of the TNF enhancer and the FCGR2A G/A SNP in 1871 individuals from 55 populations. Genotyping was carried out by DNA sequencing, single base extension (SNaPshot), and DNA mass array (Sequenom). Plasma TNF was determined by ELISA. Comparison of datasets was carried out by Kruskal-Wallis and Mann-Whitney tests. Haplotypes and LD plots were generated by PHASE and Haploview, respectively. Odds ratio (OR) for risk assessment was calculated using EpiInfo™ version 3.4.ResultsA novel single nucleotide polymorphism (SNP) at position -76 was identified in the TNF enhancer along with other reported variants. Five TNF enhancer SNPs and the FCGR2A R131H (G/A) SNP were analyzed for association with severity of P. falciparum malaria in a malaria-endemic and a non-endemic region of India in a case-control study with ethnically-matched controls enrolled from both regions. TNF -1031C and -863A alleles as well as homozygotes for the TNF enhancer haplotype CACGG (-1031T>C, -863C>A, -857C>T, -308G>A, -238G>A) correlated with enhanced plasma TNF levels in both patients and controls. Significantly higher TNF levels were observed in patients with severe malaria. Minor alleles of -1031 and -863 SNPs were associated with increased susceptibility to severe malaria. The high-affinity IgG2 binding FcγRIIa AA (131H) genotype was significantly associated with protection from disease manifestation, with stronger association observed in the malaria non-endemic region. These results represent the first genetic analysis of the two immune regulatory molecules in the context of P. falciparum severity/resistance in the Indian population.ConclusionAssociation of specific TNF and FCGR2A SNPs with cytokine levels and disease severity/resistance was indicated in patients from areas with differential disease endemicity. The data emphasizes the need for addressing the contribution of human genetic factors in malaria in the context of disease epidemiology and population genetic substructure within India.

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.

Plasmodium vivax: enzyme polymorphism in isolates of Indian origin

185 isolates of Plasmodium vivax were collected from patients visiting the malaria clinic run by the National Malaria Eradication Programme, Delhi, India. Percoll gradient centrifugation was used to concentrate P. vivax parasites from 0.4 to 0.5 ml of blood collected by finger prick. The parasite concentrate from each isolate was electrophoretically analysed for lactate dehydrogenase (LDH), NADP-dependent glutamate dehydrogenase (GDH), glucose phosphate isomerase (GPI) and adenosine deaminase (ADA). Variations were observed in GPI, GDH and ADA systems. Four electrophoretic forms of GPI and 5 each of GDH and ADA were observed. Electrophoretic mobilities of the different isoenzymic forms in P. vivax were identical to those reported for P. falciparum, indicating that the 2 species cannot be differentiated on the basis of electrophoretic patterns of the 4 enzyme systems studied.

Low efficacy of chloroquine: Time to switchover to artemisinin-based combination therapy for falciparum malaria in India

Drug resistance in Plasmodium falciparum poses a major threat to malaria control globally; including India. Chloroquine is still the most widely used drug in the country because of its safety and cost effectiveness. Although chloroquine resistance was first reported in 1973 in North Eastern India, the extent of the problem was realized only after the more intensive 28-day drug efficacy studies were used to monitor drug resistance. In the present study, efficacy of chloroquine in treatment of uncomplicated falciparum malaria was investigated using standard World Health Organization (WHO) procedures in three distinct epidemiological settings. The prevalence of molecular markers of drug resistance, Pfcrt K76T, Pfmdr1 N86Y, was also studied. A total of 374 children and adults with uncomplicated P. falciparum malaria were enrolled at six sites in four states, treated with chloroquine and follow-up was done for 28 days. The cumulative incidence of success of chloroquine at Day 28 by the Kaplan Meier analysis in the state of Orissa (District Sundargarh, CHC Bisra and Kuarmunda) was 57 (95% CI 43-68) and 54 (95% CI 40-66); in the state of Jharkhand (District Ranchi, PHC Angara and District Simdega, PHC Jaldega) it was 72 (95% CI 59-81) and 65 (95% CI 50-76); in the state of Goa (District North-Goa, Panaji Town), it was 20 (95% CI 10-2) and in the state of Rajasthan (District Udaipur, PHC Rishabdev), it was 96 (95% CI 85-99). Treatment failure was related to Pfcrt mutations but not Pfmdr mutations. Early treatment failure was observed only in 15.8% out of total failures, probably due to the semi-immune nature of the population. This type of response may give false perception about efficacy of the failing drug to patients, clinicians and National Authorities. In a large country like India it is not feasible to conduct in vivo studies in all districts and lack of direct correlation between molecular markers, in vitro studies and treatment outcome makes it difficult to predict the areas requiring change of policy. In this scenario, it is a challenge for National Programmes to make evidence-based revisions in the drug policy. However, considering the global, especially Southeast Asian, scenario and interpretation of available in vivo data, trends of mutations, availability of effective drugs and support of international donors, India should consider changing the first line treatment, at least for all diagnosed P. falciparum cases.

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.

Genetic structure of Plasmodium vivax isolates in India

Variations in the allelic composition of glucose phosphate isomerase (GPI), NADP-dependent glutamate dehydrogenase (GDH) and adenosine deaminase (ADA) enzyme systems of Plasmodium vivax were observed in isolates of Indian origin in 1985-1993. No significant difference was observed in allelic frequencies in different years. The data indicated random distribution of GPI, GDH and ADA alleles among the isolates, suggesting that loci for these enzymes were not linked. A high proportion of the isolates comprised at least 2 genetically distinct clones, the mean number of clones per isolate being 1.4. There was no significant difference in the number of oocysts in Anopheles stephensi fed on uniclonal and multiclonal isolates. No difference was observed in the proportions of uniclonal and multiclonal isolates during low and high transmission periods.