The “Malaria Evolution in South Asia” (MESA) program project is an International Center of Excellence for Malaria Research (ICEMR) sponsored by the US National Institutes of Health. This US–India collaborative program will study the origin of genetic diversity of malaria parasites and their selection on the Indian subcontinent. This knowledge should contribute to a better understanding of unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and Plasmodium vivax infections. In this first of two reviews, we highlight malaria prevalence in India. In particular, we draw attention to variations in distribution of different human-parasites and different vectors, variation in drug resistance traits, and multiple forms of clinical presentations. Uneven malaria severity in India is often attributed to large discrepancies in health care accessibility as well as human migrations within the country and across neighboring borders. Poor access to health care goes hand in hand with poor reporting from some of the same areas, combining to possibly distort disease prevalence and death from malaria in some parts of India. Corrections are underway in the form of increased resources for disease control, greater engagement of village-level health workers for early diagnosis and treatment, and possibly new public–private partnerships activities accompanying traditional national malaria control programs in the most severely affected areas. A second accompanying review raises the possibility that, beyond uneven health care, evolutionary pressures may alter malaria parasites in ways that contribute to severe disease in India, particularly in the NE corridor of India bordering Myanmar Narayanasamy et al., 2012.

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North-east India, being a corridor to South-east Asia, is believed to play an important role in transmitting drug resistant Plasmodium falciparum malaria to India and South Asia. North-east India was the first place in India to record the emergence of drug resistance to chloroquine as well as sulphadoxine/pyrimethamine. Presently chloroquine resistance is widespread all over the North-east India and resistance to other anti-malarials is increasing. In this study both in vivo therapeutic efficacy and molecular assays were used to screen the spectrum of drug resistance to chloroquine and sulphadoxine/pyrimethamine in the circulating P. falciparum strains. A total of 220 P. falciparum positives subjects were enrolled in the study for therapeutic assessment of chloroquine and sulphadoxine/pyrimethamine and assessment of point mutations conferring resistances to these drugs were carried out by genotyping the isolates following standard methods. Overall clinical failures in sulphadoxine/pyrimethamine and chloroquine were found 12.6 and 69.5% respectively, while overall treatment failures recorded were 13.7 and 81.5% in the two arms. Nearly all (99.0%) the isolates had mutant pfcrt genotype (76T), while 68% had mutant pfmdr-1 genotype (86Y). Mutation in dhps 437 codon was the most prevalent one while dhfr codon 108 showed 100% mutation. A total of 23 unique haplotypes at the dhps locus and 7 at dhfr locus were found while dhps-dhfr combined loci revealed 49 unique haplotypes. Prevalence of double, triple and quadruple mutations were common while 1 haplotype was found with all five mutated codons (F/AGEGS/T) at dhps locus. Detection of quadruple mutants (51I/59R/108N/164L) in the present study, earlier recorded from Car Nicobar Island, India only, indicates the presence of high levels of resistance to sulphadoxine/pyrimethamine in north-east India. Associations between resistant haplotypes and the clinical outcomes and emerging resistance in sulphadoxine/pyrimethamine in relation to the efficacy of the currently used artemisinin combination therapy are discussed.

Understanding the epidemiological features and metrics of malaria in endemic populations is a key component to monitoring and quantifying the impact of current and past control efforts to inform future ones. The International Centers of Excellence for Malaria Research (ICEMR) has the opportunity to evaluate the impact of malaria control interventions across endemic regions that differ in the dominant Plasmodium species, mosquito vector species, resistance to antimalarial drugs and human genetic variants thought to confer protection from infection and clinical manifestations of plasmodia infection. ICEMR programs are conducting field studies at multiple sites with the aim of generating standardized surveillance data to improve the understanding of malaria transmission and to monitor and evaluate the impact of interventions to inform malaria control and elimination programs. In addition, these epidemiological studies provide a vast source of biological samples linked to clinical and environmental “meta-data” to support translational studies of interactions between the parasite, human host, and mosquito vector. Importantly, epidemiological studies at the ICEMR field sites are integrated with entomological studies, including the measurement of the entomological inoculation rate, human biting index, and insecticide resistance, as well as studies of parasite genetic diversity and antimalarial drug resistance.

The genetic diversity in Plasmodium falciparum antigens is a major hurdle in developing an effective malaria vaccine. Protective efficacy of the vaccine is dependent on the polymorphic alleles of the vaccine candidate antigens. Therefore, we investigated the genetic diversity of the potential vaccine candidate antigens i.e. msp-1, msp-2, glurp, csp and pfs25 from field isolates of P.falciparum and determined the natural immune response against the synthetic peptide of these antigens. Genotyping was performed using Sanger method and size of alleles, multiplicity of infection, heterogeneity and recombination rate were analyzed. Asexual stage antigens were highly polymorphic with 55 and 50 unique alleles in msp-1 and msp-2 genes, respectively. The MOI for msp-1 and msp-2 were 1.67 and 1.28 respectively. A total 59 genotype was found in glurp gene with 8 types of amino acid repeats in the conserved part of RII repeat region. The number of NANP repeats from 40 to 44 was found among 55% samples in csp gene while pfs25 was found almost conserved with only two amino acid substitution site. The level of genetic diversity in the present study population was very similar to that from Asian countries. A higher IgG response was found in the B-cell epitopes of msp-1 and csp antigens and higher level of antibodies against csp B-cell epitope and glurp antigen were recorded with increasing age groups. Significantly, higher positive responses were observed in the csp antigen among the samples with ≥42 NANP repeats. The present finding showed extensive diversity in the asexual stage antigens.

Antimalarial drug resistance is a major global challenge in malaria control and elimination. Mutations in six different genes of Plasmodium falciparum (crt, mdr1, dhfr, dhps, ATPase6 and K-13 propeller) that confer resistance to chloroquine, sulphadoxine-pyrimethamine and artemisinin-based combination therapy were analyzed in samples from Chhattisgarh. Seventy-eight percent of the samples were found to have a pfcrt mutation (53% double, 24% triple and 1% single mutant), and 59% of pfmdr1 genes were found to have an N86Y mutation. Double mutations were recorded in pfdhfr gene among 76% of the samples while only 6% of the samples harbored mutant genotypes in pfdhps. No mutation was found in the K-13 propeller gene, while only one sample showed a mutant genotype for the PfATPase6 gene. The Tajima test confirmed that there is no role of evolutionary natural selection in drug resistance, and gene pairwise linkage of disequilibrium showed significant intragenic association. The high level of pfcrt mutations suggests that parasite resistance to chloroquine is almost at a fixed level, whereas resistance to SP is evolving in the population and parasites remain sensitive to artemisinin derivatives. These findings provide potential information and understanding of the evolution and spread of different drug resistance alleles in Chhattisgarh.

In north-eastern India, Anopheles minimus, An. dirus and An. fluviatilis are considered the three major vectors of the parasites causing human malaria. The role in transmission of the other Anopheles species present in this region is not, however, very clear. To examine the vectorial role of the more common anopheline mosquitoes, the heads and thoraces of 4126 female Anopheles belonging to 16 species (collected using miniature light traps set in human dwellings in a foothill village in the Jorhat district of Assam state) were tested, in ELISA, for the circumsporozoite proteins (CSP) of Plasmodium falciparum or the VK-210 and VK-247 polymorphs of P. vivax. Sixty-five pools of head-thorax homogenates, representing 10 different species of Anopheles, were found reactive, giving an overall minimum prevalence of infection (MPI) of 1.58%, with a 95% confidence interval (CI) of 1.21%-2.0%. Among the CSP-reactive pools of mosquitoes, 31% were positive only for P. falciparum, 45% only for P. vivax VK 247, 6% only for P. vivax VK 210, and 18% for both P. falciparum and P. vivax VK 247. The results indicate that not only the proven vector, An. minimus s.l. (MPI = 0.71%), but also several species of Anopheles previously considered unimportant in the epidemiology of malaria, especially An. aconitus (MPI = 3.95%), An. annularis (MPI = 5.8%), the An. hyrcanus group (MPI = 0.48%), An. kochi (MPI = 1.28%), the An. philippinensis-nivipes complex (MPI = 0.94%), and An. vagus (MPI = 3.87%), are important vectors in the foothills of Assam.

To estimate the vectorial capacity of Anopheles dirus, the main vector of forest malaria in the northeastern region of India, in order to gain an understanding of entomological factors related to malaria transmission in forest-fringe areas of Assam, India, an isolated village in the tropical rain forest-fringed area in the district of Dibrugarh, Assam, under the influence of An. dirus alone was studied. Data on various entomological variables required for computation of the vectorial capacity were generated in each month from June 1999 to May 2000 in the field using standard techniques. Malaria prevalence was also studied during the same period in the study village and correlated with the estimated vectorial capacity of An. dirus. Vectorial capacity of An. dirus was highest, 0.779 for Plasmodium vivax (Pv) and 0.649 for Plasmodium falciparum (Pf), during the hot-monsoon season (June-September) and decreased to 0.08 (Pv) and 0.07(Pf) in the temperate postmonsoon season (October-November) before attaining zero values in the cool-dry season (December-February). With increasing temperature in the temperate premonsoon season (March-May), vectorial capacity recorded was 0.119 and 0.82 for Pv and Pf, respectively. Significant positive correlation was seen between the estimated vectorial capacity of An. dirus and the number of new Pf (r = 0.86, p < 0.001) and Pv (r = 0.69, p < 0.02) cases in the study village in different months. Thus, this study highlights the pattern of malaria transmission by An. dirus in a forest-fringe area of Assam that begins in March, peaks in July/August, subsides by November, and remains interrupted between December and February. Measures for controlling malaria in forest-fringe areas should be scheduled accordingly.