Alex Eapen

Malaria in India: The Center for the Study of Complex Malaria in India

Malaria is a major public health problem in India and one which contributes significantly to the overall malaria burden in Southeast Asia. The National Vector Borne Disease Control Program of India reported ∼1.6 million cases and ∼1100 malaria deaths in 2009. Some experts argue that this is a serious underestimation and that the actual number of malaria cases per year is likely between 9 and 50 times greater, with an approximate 13-fold underestimation of malaria-related mortality. The difficulty in making these estimations is further exacerbated by (i) highly variable malaria eco-epidemiological profiles, (ii) the transmission and overlap of multiple Plasmodium species and Anopheles vectors, (iii) increasing antimalarial drug resistance and insecticide resistance, and (iv) the impact of climate change on each of these variables. Simply stated, the burden of malaria in India is complex. Here we describe plans for a Center for the Study of Complex Malaria in India (CSCMi), one of ten International Centers of Excellence in Malaria Research (ICEMRs) located in malarious regions of the world recently funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The CSCMi is a close partnership between Indian and United States scientists, and aims to address major gaps in our understanding of the complexity of malaria in India, including changing patterns of epidemiology, vector biology and control, drug resistance, and parasite genomics. We hope that such a multidisciplinary approach that integrates clinical and field studies with laboratory, molecular, and genomic methods will provide a powerful combination for malaria control and prevention in India.

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

GIS based malaria information management system for urban malaria scheme in India

A GIS based information management system has been developed to help Urban Malaria Control in India. The basic objective is to develop a model to assist planning and implementation of a suitable control measure. The system can help in: (i) identifying high receptive areas in time and space domain; (ii) identifying risk factors for high receptivity; (iii) monitoring and evaluating control measures. To demonstrate this system, information on 33 parameters and malaria cases has been attached to a digitised map of Dindigul, an urban town in Tamil Nadu. Functionalities of the system and its utility are described in this paper. A GIS based information management system ensures that if a localised spurt of the disease occurs, it can be associated rapidly with a likely cause, a specific vector, and a probable human source, so that appropriate preventive action can be taken to arrest any rising trend.

Characterizing microclimate in urban malaria transmission settings: a case study from Chennai, India

BackgroundEnvironmental temperature is an important driver of malaria transmission dynamics. Both the parasite and vector are sensitive to mean ambient temperatures and daily temperature variation. To understand transmission ecology, therefore, it is important to determine the range of microclimatic temperatures experienced by malaria vectors in the field.MethodsA pilot study was conducted in the Indian city of Chennai to determine the temperature variation in urban microclimates and characterize the thermal ecology of the local transmission setting. Temperatures were measured in a range of probable indoor and outdoor resting habitats of Anopheles stephensi in two urban slum malaria sites. Mean temperatures and daily temperature fluctuations in local transmission sites were compared with standard temperature measures from the local weather station. The biological implications of the different temperatures were explored using temperature-dependent parasite development models to provide estimates of the extrinsic incubation period (EIP) of Plasmodium vivax and Plasmodium falciparum.ResultsMean daily temperatures within the urban transmission sites were generally warmer than those recorded at the local weather station. The main reason was that night-time temperatures were higher (and hence diurnal temperature ranges smaller) in the urban settings. Mean temperatures and temperature variation also differed between specific resting sites within the transmission environments. Most differences were of the order of 1-3°C but were sufficient to lead to important variation in predicted EIPs and hence, variation in estimates of transmission intensity.ConclusionsStandard estimates of environmental temperature derived from local weather stations do not necessarily provide realistic measures of temperatures within actual transmission environments. Even the small differences in mean temperatures or diurnal temperature ranges reported in this study can lead to large variations in key mosquito and/or parasite life history traits that determine transmission intensity. Greater effort should be directed at quantifying adult mosquito resting behaviour and determining the temperatures actually experienced by mosquitoes and parasites in local transmission environments. In the absence of such highly resolved data, the approach used in the current study provides a framework for improved thermal characterization of transmission settings.

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.

Chloroquine efficacy studies confirm drug susceptibility of Plasmodium vivax in Chennai, India.

BackgroundAssessing the Plasmodium vivax burden in India is complicated by the potential threat of an emerging chloroquine (CQ) resistant parasite population from neighbouring countries in Southeast Asia. Chennai, the capital of Tamil Nadu and an urban setting for P. vivax in southern India, was selected as a sentinel site for investigating CQ efficacy and sensitivity in vivax malaria.MethodsCQ efficacy was evaluated with a 28-day in vivo therapeutic study, while CQ sensitivity was measured with an in vitro drug susceptibility assay. In both studies, isolates also underwent molecular genotyping to investigate correlations between parasite diversity and drug susceptibility to CQ. Molecular genotyping included sequencing a 604 base pair (bp) fragment of the P. vivax multidrug resistant gene-1 (Pvmdr1) for single nucleotide polymorphisms (SNPs) and also the amplification of eight microsatellite (MS) loci located across the genome on eight different chromosomes.ResultsIn the 28-day in vivo study (N=125), all subjects were aparasitaemic by Day 14. Passive case surveillance continuing beyond Day 28 in 22 subjects exposed 17 recurrent infections, which ranged from 44 to 148 days post-enrollment. Pvmdr1 sequencing of these recurrent infections revealed that 93.3% had identical mutant haplotypes (958M/Y976/1076L) to their baseline Day 0 infection. MS genotyping further revealed that nine infection pairs were related with ≥75% haplotype similarity (same allele at six or more loci). To test the impact of this mutation on CQ efficacy, an in vitro drug assay (N=68) was performed. No correlation between IC50 values and the percentage of ring-stage parasites prior to culture was observed (rsadj: -0.00063, p = 0.3307) and the distribution of alleles among the Pvmdr1 SNPs and MS haplotypes showed no significant associations with IC50 values.ConclusionsPlasmodium vivax was found to be susceptible to CQ drug treatment in both the in vivo therapeutic drug study and the in vitro drug assay. Though the mutant 1076L of Pvmdr1 was found in a majority of isolates tested, this single mutation did not associate with CQ resistance. MS haplotypes revealed strong heterogeneity in this population, indicating a low probability of reinfection with highly related haplotypes.

Ovicidal activity of Ageratum houstonianum Mill. (Asteraceae) leaf extracts against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae)

Objective To determine the repellent activity of hexane, ethyl acetate and methanol crude extracts of Ageratum houstonianum(A. houstonianum) leaves against adult Anopheles stephensi (An. stephensi), Aedes aegypti (Ae. aegypti) and Culex quinquefasciatus (Cx. quinquefasciatus) mosquitoes.

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.

Characterizing Antibody Responses to Plasmodium vivax and Plasmodium falciparum Antigens in India Using Genome-Scale Protein Microarrays.

Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world.

In vitro antioxidant activity of Ageratum houstonianum Mill. (Asteraceae)

Abstract Objective To determine the antioxidant property of Ageratum houstonianum leaves. Method The present study was conducted in three different solvent extracts of leaves of Ageratum houstonianum Mill. (Asteraceae) to evaluate the antioxidant properties such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals which were carried out at various concentrations under in vitro condition. Results It was found that ethyl acetate extract could scavenge both the oxidants at 500μg/mL with high percentage inhibition (88.26 ±0.35) of DPPH, and in the case of hydroxyl radicals the maximum percentage inhibition was 75.81 ±0.39, which were found to be greater in ethyl acetate extract than in positive controls such as Butylated hydroxytoluene (BHT) and ascorbic acid. The next higher inhibitory extract was found to be methanol. Conclusion This shows that the plant Ageratum houstonianum may be a potent source of natural antioxidant.