Sukla Biswas

Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project

We have investigated the genetic diversity of the gene encoding the apical membrane antigen-1 (AMA-1) in natural populations of Plasmodium falciparum from western Kenya and compared it with parasite populations from other geographic regions. A total of 28 complete sequences from Kenya, Thailand, India, and Venezuela field isolates were obtained. The genetic polymorphism is not evenly distributed across the gene, which is in agreement with the pattern reported in earlier studies. The alleles from Kenya exhibit 20 and 30% more polymorphism than that found in Southeast Asia and Venezuelan alleles, respectively. Based on the gene genealogies derived from sequencing data, no evidence for allele families was found. We have found evidence supporting limited gene flow between the parasite populations, specifically, between the Southeast Asian and Venezuelan isolates; however, no alleles could be linked to a specific geographic region. This study reveals that positive natural selection is an important factor in the maintenance of genetic diversity for AMA-1. We did not find conclusive evidence indicating intragenic recombination is important in the generation of the AMA-1 allelic diversity. The study provides information on the genetic diversity of the AMA-1 gene that would be useful in vaccine development and testing, as well as in assessing factors that are involved in the generation and maintenance of the genetic diversity in P. falciparum.

A study of genetic diversity in the gene encoding the circumsporozoite protein (CSP) of Plasmodium falciparum from different transmission areas—XVI. Asembo Bay Cohort Project

We have investigated the genetic diversity of the gene encoding the CS protein. A total of 75 complete and 96 partial sequences are studied. We find high levels of genetic polymorphisms as evidenced by 50 and 24 alleles at the Th2R and Th3R epitopes, respectively. Overall, we find that African isolates are more polymorphic as compared with parasites from other geographic regions. We conclude that the uneven geographic polymorphism may have an adverse impact on the effectiveness of vaccines based on this antigen alone. We find extensive polymorphism in the repeat allotypes, or RATs. In order to explore how the protein structure may impose restrictions in the number of repeats, we have simulated the stability of the structure of the tandem repeat region. Our analysis suggests that the protein structure may play an important role in the observed polymorphism in the number of CS repeats in Plasmodium falciparum. We explored the linkage and recombination events among the polymorphic sites. We found that putative recombination events overlap with linked sites. We discuss how this pattern is explained by the action of positive natural selection, where the recombination events detected are convergent mutations. We conclude that it is inappropriate to use linkage-recombination patterns on genes under positive selection for assessing the structure of parasite populations.

Prevalence of point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum isolates from India and Thailand: a molecular epidemiologic study.

Pyrimethamine‐sulfadoxine (PS) is used as a second‐line treatment for P. falciparum malaria patients who fail to respond to chloroquine. Resistance to these drugs has been shown to encode with point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes. Our aim was to assess the comparative rate of point mutation occurring in DHFR and DHPS genes among P. falciparum isolates from India and Thailand where the use of PS is at a different rate. We used the mutation‐specific polymerase chain reaction (PCR) technique and mutation‐specific restriction digestion to determine the prevalence of DHFR and DHPS gene mutations at codons 16, 51, 59, 108, 164 and at 436, 437, 581 and 613, respectively. In the 89 clinical isolates from India, in the case of the DHFR gene, we found 71 of S108N, 10 of N51I, 28 of C59R and four of I164L types. Among the 50 isolates from Thailand the rate of point mutations in the DHFR gene was higher at four codon positions. We found 47 of S108N, 18 of N51I, 23 of C59R and 12 of I164L types. None of the isolates from either country possessed the paired mutations S108T and A16V. Mutations of the DHPS gene were less frequent among the Indian isolates: 4.5% showed DHPS gene mutation, two of S436F, A437G, A613T and two of S436F, A613T; whereas 66% (33/50) of the Thai isolates had mutated at codons 436, 437, 581 and 613 which include 13 of S436F, 15 of A437G, 19 of A581G and 25 of A613S/T, ranging from single to quadruple mutant types. Among the Indian isolates, DHFR point mutations were very frequent and 85/89 had a wild type DHPS genetic profile. The pattern of mutations in the samples from Thailand was different, as most were associated with point mutations in DHFR and DHPS genes.

Polymorphism in the gene encoding the Pfs48/45 antigen of Plasmodium falciparum. XI. Asembo Bay Cohort Project

We have investigated the genetic diversity of the gene encoding the transmission-blocking vaccine antigen Pfs48/45 of Plasmodium falciparum parasites from western Kenya and compared it with parasite populations from Thailand, India, and Venezuela. We report 44 complete new sequences. Overall, the antigen is less polymorphic as compared with other pre-erythrocytic and blood stage antigens. Contrary to other P. falciparum antigens, the number of synonymous substitutions per synonymous site exceeds the number of non-synonymous substitutions per non-synonymous site. We have found that the Pfs48/45 gene of Kenyan parasites is more polymorphic than parasites from other geographic origins. Our analysis reveals that positive natural selection is involved in the maintenance of the observed polymorphism. No evidence of intragenic recombination was found. F(st) values reveal high levels of gene flow between India and Thailand, however, there are strong constraints in gene flow among Kenyan, Southeast Asian, and Venezuelan parasites. No alleles could be linked to a specific geographic region. The results of this study suggest that this gametocyte antigen, like other asexual blood stage antigens, is under selection pressure.

Complete nucleotide sequence of the 6 kb element and conserved cytochrome b gene sequences among Indian isolates of Plasmodium falciparum

The malaria parasite contains a nuclear genome with 14 chromosomes and two extrachromosomal DNA molecules of 6 kb and 35 kb in size. The smallest genome, known as the 6 kb element or mitochondrial DNA, has been sequenced from several Plasmodium falciparum isolates because this is a potential drug target. Here we describe the complete nucleotide sequence of this element from an Indian isolate of P. falciparum. It is 5967 bp in size and shows 99.6% homology with the 6 kb element of other isolates. The element contains three open reading frames for mitochondrial proteins-cytochrome oxidase subunit I (CoI), subunit III (CoIII) and cytochrome b (Cyb) which were found to be expressed during blood stages of the parasite. We have also sequenced the entire cyb gene from several Indian isolates of P. falciparum. The rate of mutation in this gene was very low since 12 of 14 isolates showed the identical sequence. Only one isolate showed a maximum change in five amino acids whereas the other isolate showed only one amino acid change. However, none of the Indian isolates showed any change in those amino acids of cyb which are associated with resistance to various drugs as these drugs are not yet commonly used in India.

Inter‐Test Comparison Between Filter Paper Absorbed Blood Eluate and Serum for Malaria Serology by Enzyme Immunoassay: An Operational Feasibility

Abstract Antimalarial IgG and IgM were detected by enzyme immunoassay in finger‐stick blood samples collected in capillary tubes and also spotted onto Whatman filter paper. Assay was performed in 92 blood samples obtained from 53 falciparum malaria patients, representing 23 fever cases (malaria negative) and 16 healthy individuals. A simple indirect ELISA was done using Plasmodium falciparum lysate and MSP119 peptide as antigens. Total IgG and IgM contents were also estimated in individual sera and filter paper eluate by single radial immunodiffusion (SRID). Assay results of both serum and filter paper eluates were compared. The sensitivity and specificity of the assays for IgG measurement were comparable between serum and filter paper eluates (P < 0.001), whereas, in case of IgM, detection level was poor in filter paper eluates as observed by ELISA and SRID. The filter paper eluates may serve the purpose of antigen‐specific IgG detection in seroepidemiological surveys.

Allelic variation in the cg2 gene does not correlate with chloroquine resistance among Indian Plasmodium falciparum isolates

The cg2 gene of Plasmodium falciparum has been proposed to be associated with chloroquine resistance. Here we describe PCR amplification and sequencing of all the four repeat regions (kappa (kappa), gamma (gamma), psi (psi) and omega (omega)) of this gene, from Indian isolates. There were variant forms for each of these repeat regions (two for kappa and gamma, and three for psi and omega) among the 123 Indian isolates of P. falciparum. Among these isolates certain forms of psi and omega repeats were uniquely present while some of the reported forms of the kappa and omega repeats were absent. The pattern of combination of all four repeat regions of cg2 gene (genotype) was analysed from 52 isolates. A total of 11 different genotypes were observed among these cases, of which 10 were unique to Indian isolates. Certain genotypes were more common than others. The nucleotide sequencing of all the four repeat regions revealed that Indian isolates have some unique repeating units within the gamma and omega domains. Altogether, the PCR and sequencing results showed that there was an unrelatedness between cg2 repeats and chloroquine resistance.