Genetic differentiation among Aedes aegypti populations from different eco-geographical zones of India.

The present study explicitly evaluated the genetic structure of Aedes aegypti Linn, the vector of dengue, chikungunya, and Zika viruses, across different geo-climatic zones of India and also elucidated the impact of ecological and topographic factors. After data quality checks and removal of samples with excess null alleles, the final analysis was performed on 589 individual samples using 10 microsatellite markers. Overall findings of this study suggested that, Ae. aegypti populations are highly diverse with moderate genetic differentiation between them. Around half of the populations (13 out of 22) formed two genetic clusters roughly associated with geographical regions. The remaining nine populations shared genetic ancestries with either one or both of the clusters. A significant relationship between genetic and geographic distance was observed, indicating isolation by distance. However, spatial autocorrelation analysis predicted the signs of long-distance admixture. Post-hoc environmental association analysis showed that 52.7% of genetic variations were explained by a combination of climatic and topographic factors, with latitude and temperature being the best predictors. This study indicated that though overall genetic differentiation among Ae. aegypti populations across India is moderate (Fst = 0.099), the differences between the populations are developing due to the factors associated with geographic locations. This study improves the understanding of the Ae. aegypti population structure in India that may assist in predicting mosquito movements across the geo-climatic zones, enabling effective control strategies and assessing the risk of disease transmission.

Vivax malaria: a possible stumbling block for malaria elimination in India.

Plasmodium vivax is geographically the most widely dispersed human malaria parasite species. It has shown resilience and a great deal of adaptability. Genomic studies suggest that P. vivax originated from Asia or Africa and moved to the rest of the world. Although P. vivax is evolutionarily an older species than Plasmodium falciparum, its biology, transmission, pathology, and control still require better elucidation. P. vivax poses problems for malaria elimination because of the ability of a single primary infection to produce multiple relapses over months and years. P. vivax malaria elimination program needs early diagnosis, and prompt and complete radical treatment, which is challenging, to simultaneously exterminate the circulating parasites and dormant hypnozoites lodged in the hepatocytes of the host liver. As prompt surveillance and effective treatments are rolled out, preventing primaquine toxicity in the patients having glucose-6-phosphate dehydrogenase (G6PD) deficiency should be a priority for the vivax elimination program. This review sheds light on the burden of P. vivax, changing epidemiological patterns, the hurdles in elimination efforts, and the essential tools needed not just in India but globally. These tools encompass innovative treatments for eliminating dormant parasites, coping with evolving drug resistance, and the development of potential vaccines against the parasite.

Characterization of midgut microbiome of Anopheles stephensi Liston.

BACKGROUND & OBJECTIVES: Anopheles stephensi is an important vector of malaria in South East Asia. The abundance and diversity of gut microbiota in the disease vectors affect their development, digestion, metabolism and immunity. The immatures of An. stephensi engulf microbes from their aquatic environment. The present study investigates midgut microbiota of wild and laboratory populations and compares it with their habitat bacterial diversity to study transstadial transmissibility. METHODS: The gut microbes from immatures, adults and water samples were cultured at ambient conditions on different media. The colony and biochemical characteristics, and 16S rRNA gene sequencing of gut microbes were studied. RESULTS: Altogether, 298 bacterial isolates were characterized as 21 genera belonging to four major Phyla viz., Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. In the field population-1, Proteobacteria and Firmicutes accounted for 49% and Actinobacteria constituted 51% of the bacterial isolates. In field population-2, Bacteroidetes and Firmicutes accounted for 99% of the isolates. In the laboratory populations, Firmicutes constituted 77%, while Proteobacteria 23% of the isolates. Additionally, 9 genera occurred in the breeding habitats, 13 in the larval midgut, 6 in pupal midgut, 9 in male midgut and 10 in the female midgut. INTERPRETATION & CONCLUSION: This is a unique study on diversity of microbiota of An. stephensi from breeding water, developmental stages and adults. Different culture media used enhanced the isolation of diverse bacteria. The presence of Micrococcus and Leucobacter in different life stages indicates their adaptation in An. stephensi as symbionts which need further evaluation for their role in paratransgenesis.

Clinical and epidemiological characterization of severe Plasmodium vivax malaria in Gujarat, India.

The mounting evidence supporting the capacity of Plasmodium vivax to cause severe disease has prompted the need for a better characterization of the resulting clinical complications. India is making progress with reducing malaria, but epidemics of severe vivax malaria in Gujarat, one of the main contributors to the vivax malaria burden in the country, have been reported recently and may be the result of a decrease in transmission and immune development. Over a period of one year, we enrolled severe malaria patients admitted at the Civil Hospital in Ahmedabad, the largest city in Gujarat, to investigate the morbidity of severe vivax malaria compared to severe falciparum malaria. Patients were submitted to standard thorough clinical and laboratory investigations and only PCR-confirmed infections were selected for the present study. Severevivax malaria (30 patients) was more frequent than severe falciparum malaria (8 patients) in our setting, and it predominantly affected adults (median age 32 years, interquartile range 22.5 years). This suggests a potential age shift in anti-malarial immunity, likely to result from the recent decrease in transmission across India. The clinical presentation of severe vivax patients was in line with previous reports, with jaundice as the most common complication. Our findings further support the need for epidemiological studies combining clinical characterization of severe vivax malaria and serological evaluation of exposure markers to monitor the impact of elimination programmes.

A Proteogenomic Analysis of Haptoglobin in Malaria.

SCOPE: Haptoglobin (Hp), an acute phase inflammatory protein is associated with malaria pathogenesis in several proteomics and genomics studies. The Hp gene has two co-dominant alleles: Hp1 and Hp2 that produce three genotypes: Hp1/Hp1, Hp1/Hp2 and Hp2/Hp2. EXPERIMENTAL DESIGN: In this study, validation of the proteomics data with Multiple Reaction Monitoring Mass Spectroscopy (MRM-MS) is performed and the association of the Hp gene variants with severe, non-severe malaria and community (healthy) controls using genotyping PCRs and DNA sequencing is analysed. RESULTS: Highly significant values of Hp is observed in the MRM assay that show a correlation with severity of malaria and is clearly distinguished from another febrile disease, dengue. Moreover, the Hp2/Hp2 genotype is seen in high percentages in non-severe malaria patients (74%) and community controls (72%) whereas patients diagnosed with severe malaria show only (31%) of this genotype. Sequencing of the Hp promoter region reveals three SNPs along with 10 unique haplotypes, out of which five are associated with non-severe and three with severe malaria populations (χ2  = 130; df = 18; p < 0.0001). CONCLUSION AND CLINICAL RELEVANCE: This proteo-genomic study focuses on the correlation of the Hp protein and gene with malaria, thus highlighting the pivotal role of this acute phase immune gene in malaria pathogenesis.

Mitochondrial population genomic analyses reveal population structure and demography of Indian Plasmodium falciparum.

Inference on the genetic diversity of Plasmodium falciparum populations could help in better management of malaria. A very recent study with mitochondrial (mt) genomes in global P. falciparum had revealed interesting evolutionary genetic patterns of Indian isolates in comparison to global ones. However, no population genetic study using the whole mt genome sequences of P. falciparum isolates collected in the entire distribution range in India has yet been performed. We herewith have analyzed 85 whole mt genomes (48 already published and 37 entirely new) sampled from eight differentially endemic Indian locations to estimate genetic diversity and infer population structure and historical demography of Indian P. falciparum. We found 19 novel Indian-specific Single Nucleotide Polymorphisms (SNPs) and 22 novel haplotypes segregating in Indian P. falciparum. Accordingly, high haplotype and nucleotide diversities were detected in Indian P. falciparum in comparison to many other global isolates. Indian P. falciparum populations were found to be moderately sub-structured with four different genetic clusters. Interestingly, group of local populations aggregate to form each cluster; while samples from Jharkhand and Odisha formed a single cluster, P. falciparum isolates from Asom formed an independent one. Similarly, Surat, Bilaspur and Betul formed a single cluster and Goa and Mangalore formed another. Interestingly, P. falciparum isolates from the two later populations were significantly genetically differentiated from isolates collected in other six Indian locations. Signature of historical population expansion was evident in five population samples, and the onset of expansion event was found to be very similar to African P. falciparum. In agreement with the previous finding, the estimated Time to Most Recent Common Ancestor (TMRCA) and the effective population size were high in Indian P. falciparum. All these genetic features of Indian P. falciparum with high mt genome diversity are somehow similar to Africa, but quite different from other Asian population samples.

Mitochondrial genome sequence diversity of Indian Plasmodium falciparum isolates.

We have analysed the whole mitochondrial (mt) genome sequences (each ~6 kilo nucleotide base pairs in length) of four field isolates of the malaria parasite Plasmodium falciparum collected from different locations in India. Comparative genomic analyses of mt genome sequences revealed three novel India-specific single nucleotide polymorphisms. In general, high mt genome diversity was found in Indian P. falciparum, at a level comparable to African isolates. A population phylogenetic tree placed the presently sequenced Indian P. falciparum with the global isolates, while a previously sequenced Indian isolate was an outlier. Although this preliminary study is limited to a few numbers of isolates, the data have provided fundamental evidence of the mt genome diversity and evolutionary relationships of Indian P. falciparum with that of global isolates.

Mitochondrial genome sequence diversity of Indian Plasmodium falciparum isolates

We have analysed the whole mitochondrial (mt) genome sequences (each ~6 kilo nucleotide base pairs in length) of four field isolates of the malaria parasite Plasmodium falciparum collected from different locations in India. Comparative genomic analyses of mt genome sequences revealed three novel India-specific single nucleotide polymorphisms. In general, high mt genome diversity was found in Indian P. falciparum, at a level comparable to African isolates. A population phylogenetic tree placed the presently sequenced Indian P. falciparum with the global isolates, while a previously sequenced Indian isolate was an outlier. Although this preliminary study is limited to a few numbers of isolates, the data have provided fundamental evidence of the mt genome diversity and evolutionary relationships of Indian P. falciparum with that of global isolates.

New insights into the evolutionary history of Plasmodium falciparum from mitochondrial genome sequence analyses of Indian isolates.

Estimating genetic diversity and inferring the evolutionary history of Plasmodium falciparum could be helpful in understanding origin and spread of virulent and drug-resistant forms of the malaria pathogen and therefore contribute to malaria control programme. Genetic diversity of the whole mitochondrial (mt) genome of P. falciparum sampled across the major distribution ranges had been reported, but no Indian P. falciparum isolate had been analysed so far, even though India is highly endemic to P. falciparum malaria. We have sequenced the whole mt genome of 44 Indian field isolates and utilized published data set of 96 genome sequences to present global genetic diversity and to revisit the evolutionary history of P. falciparum. Indian P. falciparum presents high genetic diversity with several characteristics of ancestral populations and shares many of the genetic features with African and to some extent Papua New Guinean (PNG) isolates. Similar to African isolates, Indian P. falciparum populations have maintained high effective population size and undergone rapid expansion in the past with oldest time to the most recent common ancestor (TMRCA). Interestingly, one of the four single nucleotide polymorphisms (SNPs) that differentiates P. falciparum from P. falciparum-like isolates (infecting non-human primates in Africa) was found to be segregating in five Indian P. falciparum isolates. This SNP was in tight linkage with other two novel SNPs that were found exclusively in these five Indian isolates. The results on the mt genome sequence analyses of Indian isolates on the whole add to the current understanding on the evolutionary history of P. falciparum.

Whole mitochondrial genome sequence of an Indian Plasmodium falciparum field isolate.

Mitochondrial genome sequence of malaria parasites has served as a potential marker for inferring evolutionary history of the Plasmodium genus. In Plasmodium falciparum, the mitochondrial genome sequences from around the globe have provided important evolutionary understanding, but no Indian sequence has yet been utilized. We have sequenced the whole mitochondrial genome of a single P. falciparum field isolate from India using novel primers and compared with the 3D7 reference sequence and 1 previously reported Indian sequence. While the 2 Indian sequences were highly divergent from each other, the presently sequenced isolate was highly similar to the reference 3D7 strain.

Comparative genomic analysis of simple sequence repeats in three Plasmodium species.

Simple sequence repeats (SSRs) are known to be responsible for genetic complexities and play major roles in gene and genome evolution. To this respect, malaria parasites are known to have rapidly evolving and complex genomes with complicated and differential pathogenic behaviors. Hence, by studying the whole genome comparative SSRs patterns, one can understand genomic complexities and differential evolutionary patterns of these species. We herein utilized the whole genome sequence information of three Plasmodium species, Plasmodium falciparum, Plasmodium vivax, and Plasmodium knowlesi, to comparatively analyze genome-wide distribution of SSRs. The study revealed that despite having the smallest genome size, P. falciparum bears the highest SSR content among the three Plasmodium species. Furthermore, distribution patterns of different SSRs types (e.g., mono, di, tri, tetra, penta, and hexa) in term of relative abundance and relative density provide evidences for greater accumulation of di-repeats and marked decrease of mono-repeats in P. falciparum in comparison to other two species. Overall, the types and distribution of SSRs in P. falciparum genome was found to be different than that of P. vivax and P. knowlesi. The latter two species have quite similar SSR organizations in many aspects of the data. The results were discussed in terms of comparative SSR patterns among the three Plasmodium species, uniqueness of P. falciparum in SSR organization and general pattern of evolution of SSRs in Plasmodium.