How can the complex epidemiology of malaria in India impact its elimination?

Malaria is a human health hazard in the tropical and subtropical zones of the globe and is poised to be eliminated by the year 2030. Despite a decrease in incidence in the past two decades, many endemic countries, including India, report cases regularly. The epidemiology of malaria in India is unique owing to several features of the Plasmodium parasites, Anopheles vectors, ecoepidemiological situations conducive to disease transmission, and susceptible humans living in rural and forested areas. Limitations in public health reach, and poor health-seeking behaviour of vulnerable populations living in hard-to-reach areas, add to the problem. We bring all of these factors together in a comprehensive framework and opine that, in spite of complexities, targeted elimination of malaria in India is achievable with planned programmatic approaches.

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.

Does malaria epidemiology project Cameroon as 'Africa in miniature'?

Cameroon, a west-central African country with a ~ 20 million population, is commonly regarded as 'Africa in miniature' due to the extensive biological and cultural diversities of whole Africa being present in a single-country setting. This country is inhabited by ancestral human lineages in unique eco-climatic conditions and diverse topography. Over 90 percent Cameroonians are at risk of malaria infection, and ~ 41 percent have at least one episode of malaria each year. Historically, the rate of malaria infection in Cameroon has fluctuated over the years; the number of cases was about 2 million in 2010 and 2011. The Cameroonian malaria control programme faces an uphill task due to high prevalence of multidrug-resistant parasites and insecticide-resistant malaria vectors. Above all, continued human migration from the rural to urban areas as well as population exchange with adjoining countries, high rate of ecological instabilities caused by deforestation, poor housing, lack of proper sanitation and drainage system might have resulted in the recent increase in incidences of malaria and other vector-borne diseases in Cameroon. The available data on eco-environmental variability and intricate malaria epidemiology in Cameroon reflect the situation in the whole of Africa, and warrant the need for in-depth study by using modern surveillance tools for meaningful basic understanding of the malaria triangle (host-parasite-vector-environment).

Genetics of chloroquine-resistant malaria: a haplotypic view

The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.

Genetics of chloroquine-resistant malaria: a haplotypic view.

The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.

Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum.

Mutations in the Pfcrt gene that change the resulting amino acids and form different haplotypes are common and correlate with the prevalence of chloroquine resistant (CQR) field isolates of the malaria parasite, Plasmodium falciparum. This correlation provides opportunities to infer the global evolutionary history of CQ resistance by analysing CQR Pfcrt haplotype data. We collated data on the Pfcrt haplotypes from different global studies and performed evolutionary genetic analysis to present comprehensive and comparative information on the global distribution of five major CQR-Pfcrt haplotypes and evolutionary inter-relationships among 38 different countries. Using the haplotype diversity data, inter-continental genetic differentiation was also ascertained.

Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum

Mutations in the Pfcrt gene that change the resulting amino acids and form different haplotypes are common and correlate with the prevalence of chloroquine resistant (CQR) field isolates of the malaria parasite, Plasmodium falciparum. This correlation provides opportunities to infer the global evolutionary history of CQ resistance by analysing CQR Pfcrt haplotype data. We collated data on the Pfcrt haplotypes from different global studies and performed evolutionary genetic analysis to present comprehensive and comparative information on the global distribution of five major CQR-Pfcrt haplotypes and evolutionary inter-relationships among 38 different countries. Using the haplotype diversity data, inter-continental genetic differentiation was also ascertained.

Comparative evolutionary analyses of beta globin gene in eutherian, dinosaurian and neopterygii taxa.

BACKGROUND & OBJECTIVES: Comparative genomics and evolutionary analyses of conserved genes have enabled us to understand the complexity of genomes of closely related species. For example: ß-globin gene present in human hemoglobin is one such gene that has experienced many genetic changes in many related taxa and produced more than 600 variants. One of the variant, HBS causes sickle-cell anemia in humans but offers protection against severe malaria due to Plasmodium falciparum. In the present study, we characterized and performed evolutionary comparative analyses of the ?-globin gene in different related and unrelated taxa to have a comprehensive view of its evolution. METHODS: DNA and protein sequences of ß-globin gene were downloaded from NCBI and characterized in detail in nine eutherian (Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Bos taurus, Canis familiaris, Equus caballus, Oryctolagus cuniculus), a dinosaurian (Gallus gallus) and a neopterygii (Danio rerio) taxa. Three more eutherian (Papio anubis, Ovis aries and Sus scrofa) taxa were included for an analysis at the protein level but not included at the gene level owing to lack of genomic information. Computational and phylogenetic analyses were performed using evolutionary comparative approach. RESULTS: Results of comparative and phylogenetic analyses revealed less conservation of genetic architecture of ß-globin compared to its protein architecture in all eutherian taxa. Both dinosaurian and neopterygii taxa served as outgroups and varied at gene and protein levels. INTERPRETATION & CONCLUSION: Most remarkably, all primates from eutherian taxa including P. anubis showed only nine codon position differences and an absolute similarity between H. sapiens and P. troglodytes. Absolute conservation of coding region in Equus caballus (horse) was observed. The results were discussed with an inference on the role of evolutionary forces in maintaining such close similarities and variations across closely related taxa. Further, the need to utilize more comparative approaches in understanding the disease causing genes' evolution in closely related taxa is hoped for.

Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India.

Inferring the origin and dispersal of the chloroquine-resistant (CQR) malaria parasite, Plasmodium falciparum, is of academic and public health importance. The Pfcrt gene of P. falciparum is widely known as the CQR gene and two major haplotypes of this gene (CVIET and SVMNT) occur widely across CQR-endemic regions of the globe. In India, studies to date of the Pfcrt gene have indicated the widespread prevalence of the SVMNT haplotype (prevalent in the South America and Papua New Guinea), whereas the CVIET haplotype, primarily found in southeast Asia, was not detected at a high frequency in India. This distribution pattern of the two most common CQR-Pfcrt haplotypes in India is quite surprising. Thus, in order to understand probable evolutionary and migration patterns of the CQR-Pfcrt haplotypes into India, we generated new sequence data of exon 2 of the Pfcrt gene and collected published information on the CQR-Pfcrt haplotype data from India, Papua New Guinea, southeast Asia and South America, and performed several population and evolutionary genetic analyses. Among several interesting findings, statistically significant longitudinal clines for the CVIET and SVMNT haplotypes (in opposite directions) in India, and the clustering of India and Papua New Guinea under the SVMNT-specific clade in the phylogenetic tree, are the two most remarkable aspects of the data. It also appears that both the SVMNT and CVIET haplotypes in India have migrated from southeast Asia. In particular, whereas the Indian CVIET haplotype has a southeast Asian origin, the SVMNT haplotype, prevalent in India, seems to have originated in Papua New Guinea and entered India through southeast Asia.

Malaria parasite genome scan: insights into antimalarial resistance.

Genome scan and genotype-phenotype association study offer excellent opportunities to unearth drug/vaccine targets in human pathogens including malaria parasites. A recently conducted such study in worldwide isolates in the most devastating malaria parasite Plasmodium falciparum has reported important genomic information on genetic basis of antimalarial resistance. Several unknown genes were also found to be under strong influence of natural selection. The findings provide important insights into the malaria parasite genome evolution in general and to use this information to develop more focused malaria control strategies, in particular.

Evolutionary insights into duffy gene in mammalian taxa with comparative genetic analysis.

BACKGROUND & OBJECTIVES: Evolutionary analyses of genes conserved across taxa are keys to understand the complexity of gene and genome variation. Since malaria is a highly infectious human disease and its susceptibility in human is genetically controlled, characterization and evolutionary analyses of such genes are of prime importance to understand genetic mechanisms of disease susceptibility. In the present study we have characterized and performed comparative genomic analyses of the human Duffy gene responsible for malaria pathogenesis in nine different mammalian taxa. METHODS: DNA sequences of human duffy gene were downloaded from public domain and have been characterized in detail and compared with eight other different mammalian taxa (Pan troglodytes, Macaca mulatta, Pongo pygmaeus, Rattus norvegicus, Mus musculus, Monodelphis domestica, Bos taurus and Canis familiaris). Comparative and evolutionary analyses were performed using statistical software and tools. RESULTS: We observed that the genetic architecture of this gene was entirely different across all the nine taxa and a close similarity between Homo sapiens and Pan troglodytes (chimpanzee) was evident for several aspects of this gene. Comparisons on several aspects, such as ratio of coding and non-coding regions, total gene length number and size of introns and difference of number of nucleotides in human and chimpanzees have revealed interesting features. Phylogenetic inferences based on the Duffy gene among nine different taxa were found to be different than other genes previously studied. INTERPRETATION & CONCLUSION: Most remarkably, human and chimpanzee were only 0.75% different in this gene. The results were discussed on the similarities between human and chimpanzee and gain of introns in human-chimpanzee clade with an inference on the role of evolutionary forces (mainly natural selection) in maintaining such variations across closely-related mammalian taxa.

Why is it important to study malaria epidemiology in India?

Malaria is a major vector-borne disease in India. Based on vast geographic areas with associated topographic and climatic diversity, the variable malaria epidemiology in India is associated with high parasite genetic diversity and rapidly evolving drug resistance, differential distribution of vector species and emerging insecticide resistance and underlying human genetic diversity and past evolutionary histories. Further, changing climatic patterns have possibly changed malaria epidemiology to a great extent. The outcome of these changes is an increased incidence of Plasmodium falciparum over the P. vivax malaria in recent years. Accordingly, the drug and insecticide application policy in India has changed too. The above facts and associated rapid shifting trend of malaria epidemiology makes India a hot-spot for malaria research.

Characterization & evolutionary analysis of human CD36 gene.

BACKGROUND & OBJECTIVE: Understanding evolutionary genetic details of immune system genes responsible for infectious diseases is of prime importance concerning disease pathogenecity. Considering malaria as a devastating disease in the world including India, detail evolutionary understanding on human immune system gene is essential. The primary aim of this study was to initiate work on one such gene, the human CD36 gene responsible in malaria pathogenesis. METHODS: DNA sequences of the human CD36 gene was retrieved from public domain and fine-scale details were characterized. Both comparative and evolutionary analyses were performed with sequences from six other taxa (5 mammalian one avian) where CD36 homologs are present. Different statistical analyses were also performed. RESULTS: Differential distribution in number and length of exons and introns was detected in CD36 gene across seven taxa. The CpG islands were also found to be distributed unevenly across the gene and taxa. Neighbour-joining tree was constructed and it was observed that the chimpanzee and human are diverged at the CD36 gene relatively recently. The chicken, Gallus gallus was found to be diverged from rest of the taxa significantly. Also copy number variation was observed across different taxa. INTERPRETATION & CONCLUSION: Comparative genomic study of a human immune system gene CD36 show relationships among different taxa at the evolutionary level. The information can be of help to study genetic diversity in malaria endemic zones and to correlate it with malaria pathogenecity.

Genetic characterization and evolutionary inference of TNF-α through computational analysis

TNF-α is an important human cytokine that imparts dualism in malaria pathogenicity. At high dosages, TNF-α is believed to provoke pathogenicity in cerebral malaria; while at lower dosages TNF-α is protective against severe human malaria. In order to understand the human TNF-α gene and to ascertain evolutionary aspects of its dualistic nature for malaria pathogenicity, we characterized this gene in detail in six different mammalian taxa. The avian taxon, Gallus gallus was included in our study, as TNF-α is not present in birds; therefore, a tandemly placed duplicate of TNF-α (LT-α or TNF-β) was included. A comparative study was made of nucleotide length variations, intron and exon sizes and number variations, differential compositions of coding to non-coding bases, etc., to look for similarities/dissimilarities in the TNF-α gene across all seven taxa. A phylogenetic analysis revealed the pattern found in other genes, as humans, chimpanzees and rhesus monkeys were placed in a single clade, and rats and mice in another; the chicken was in a clearly separate branch. We further focused on these three taxa and aligned the amino acid sequences; there were small differences between humans and chimpanzees; both were more different from the rhesus monkey. Further, comparison of coding and non-coding nucleotide length variations and coding to non-coding nucleotide ratio between TNF-α and TNF-β among these three mammalian taxa provided a first-hand indication of the role of the TNF-α gene, but not of TNF-β in the dualistic nature of TNF-α in malaria pathogenicity.

Genetic characterization and evolutionary inference of TNF-alpha through computational analysis.

TNF-alpha is an important human cytokine that imparts dualism in malaria pathogenicity. At high dosages, TNF-alpha is believed to provoke pathogenicity in cerebral malaria; while at lower dosages TNF-alpha is protective against severe human malaria. In order to understand the human TNF-alpha gene and to ascertain evolutionary aspects of its dualistic nature for malaria pathogenicity, we characterized this gene in detail in six different mammalian taxa. The avian taxon, Gallus gallus was included in our study, as TNF-alpha is not present in birds; therefore, a tandemly placed duplicate of TNF-alpha (LT-alpha or TNF-beta) was included. A comparative study was made of nucleotide length variations, intron and exon sizes and number variations, differential compositions of coding to non-coding bases, etc., to look for similarities/dissimilarities in the TNF-alpha gene across all seven taxa. A phylogenetic analysis revealed the pattern found in other genes, as humans, chimpanzees and rhesus monkeys were placed in a single clade, and rats and mice in another; the chicken was in a clearly separate branch. We further focused on these three taxa and aligned the amino acid sequences; there were small differences between humans and chimpanzees; both were more different from the rhesus monkey. Further, comparison of coding and non-coding nucleotide length variations and coding to non-coding nucleotide ratio between TNF-alpha and TNF-beta among these three mammalian taxa provided a first-hand indication of the role of the TNF-alpha gene, but not of TNF-beta in the dualistic nature of TNF-alpha in malaria pathogenicity.