Antigenic repertoire of Plasmodium vivax transmission-blocking vaccine candidates from the Indian subcontinent

<p>Abstract</p> <p>Background</p> <p>Genetic polymorphism is an inevitable component of a multistage infectious organism, such as the malaria parasite. By means of genetic polymorphism, parasite opts particular polymorph and reveals survival advantage. <it>Pvs25 </it>and <it>pvs28 </it>are sexual stage antigen genes, expressed at the ookinete stage inside the mosquito gut, and considered as potential transmission-blocking vaccine candidates. This study presents sequence variations in two important transmission blocking antigen genes <it>pvs25 and pvs28 </it>in the field isolates of <it>P. vivax </it>from the Indian subcontinent.</p> <p>Methods</p> <p>One hundred microscopically diagnosed <it>P. vivax </it>isolates were collected from five geographical regions of India. <it>Pvs25 and pvs28 </it>genes were PCR amplified and sequenced to assess sequence variation among field isolates.</p> <p>Results</p> <p>A total of 26 amino acid substitutions were observed in Pvs25 (10) and Pvs28 (16) among field isolates of <it>P. vivax</it>. Tandem repeat polymorphism observed in <it>pvs28 </it>shows 3-6 tandem repeats in the field isolates. Seven and eight novel amino acid substitutions were observed in Pvs25 and Pvs28, respectively in Indian isolates. Comparison of amino acid substitutions suggests that majority of substitutions observed in global isolates were also present in Indian subcontinent. A single haplotype was observed to be major haplotype among isolates of Delhi, Nadiad, Chennai and Panna except in isolates of Kamrup. Further, population comparison analyses suggest that <it>P. vivax </it>isolates inhabiting in north-eastern region (Kamrup) were distantly related with the isolates from remaining parts of the country. Majority of the amino acid substitutions observed in Indian isolates were more identical to the substitutions reported from isolates of Thailand and Bangladesh.</p> <p>Conclusion</p> <p>Study uncovered many new amino acid substitutions as well as a predominance of single haplotype in Indian subcontinent except in north-eastern region of the country. The amino acid substitutions data generated in this study from different geographical regions of the Indian subcontinent could be helpful in designing a more effective anti-malarial transmission-blocking vaccine.</p

Molecular epidemiology of Plasmodium vivax anti-folate resistance in India

<p>Abstract</p> <p>Background</p> <p>Sulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in <it>dihydrofolate reductase </it>(<it>dhfr</it>) and <it>dihydropteorate synthatase </it>(<it>dhps</it>) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in <it>dhfr/dhps </it>genes among Indian sub-continent.</p> <p>Methods</p> <p>Microscopically diagnosed one hundred <it>Plasmodium vivax </it>field isolates were collected from five widely separated geographical regions of India. <it>Dhfr </it>and <it>dhps </it>genes were PCR amplified and sequenced. Previously published mutations data were collected and analyzed using Chi square test to identify geographical cluster of mutant/wild type genotypes.</p> <p>Results</p> <p>Sequence analysis revealed single (S58R), double (S58R/S117N) and quadruple (F57L/S58R/T61M/S117T/) point mutations at <it>dhfr </it>and single (A383G) to double (A383G/A553G) mutations at <it>dhps </it>in <it>P. vivax </it>field isolates. In addition, three new mutations were also observed at <it>dhfr</it>. Both, <it>dhfr </it>and <it>dhps </it>genes revealed tandem repeat variations in field isolates. <it>Dhps </it>revealed very low mutation frequency (14.0%) compared to <it>dhfr </it>(50.70%). Comparative analysis revealed a progressive increase in frequency of quadruple mutant <it>dhfr </it>genotype (p < 0.001) within five years in north-eastern state (Kamrup, Assam). Frequency of <it>dhfr </it>genotypes revealed three distinct geographical clusters of wild (northern India), double mutant (southern India), and quadruple mutant (north-eastern and island regions of India) on the Indian sub-continent.</p> <p>Conclusion</p> <p>Study suggests that SP may be susceptible to <it>P. vivax </it>in India, except Andaman and north-eastern state. The distinction of geographical regions with sensitive and resistant parasite phenotypes would be highly useful for designing and administering national anti-malarial drug policy.</p

Allelic dimorphism of Plasmodium vivax gam-1 in the Indian subcontinent

BACKGROUND: Genetic 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. METHODS: A 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. RESULTS: Both 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. CONCLUSION: Study shows limited diversity of Pvgam-1 marker in Indian isolates with well representation of both Belem and Chesson type alleles

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

Background: Multi-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. Methods: 18S 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. Results: 18S 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. Conclusions: The 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

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

<p>Abstract</p> <p>Background</p> <p>Multi-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 <it>Plasmodium vivax </it>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 <it>P. vivax </it>by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers.</p> <p>Methods</p> <p><it>18S SSU rRNA S-type </it>gene was amplified from 420 <it>Plasmodium vivax </it>field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of <it>P. vivax </it>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.</p> <p>Results</p> <p><it>18S SSU rRNA S-type </it>gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of <it>Plasmodium vivax </it>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 <it>P. vivax </it>genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, <it>AE </it>= 14.6 ± 2.0) and heterozygosity (<it>He </it>= 0.697-0.924, <it>AE </it>= 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.</p> <p>Conclusions</p> <p>The 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.</p

Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India

<p>Abstract</p> <p>Background</p> <p>Molecular techniques have facilitated the studies on genetic diversity of <it>Plasmodium </it>species particularly from field isolates collected directly from patients. The <it>msp-1 </it>and <it>msp-2 </it>are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the <it>msp-1 </it>gene and the central repetitive domain (block3) of the <it>msp-2 </it>gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among <it>Plasmodium falciparum </it>population of India, this report evaluates the extent of genetic diversity in the field isolates of <it>P. falciparum </it>in eastern and north-eastern regions of India.</p> <p>Methods</p> <p>A study was designed to assess the diversity of <it>msp-1 </it>and <it>msp-2 </it>among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa.</p> <p>Results</p> <p><it>P. falciparum </it>isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of <it>msp-1 </it>and <it>msp-2</it>. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the <it>P. falciparum </it>populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of <it>msp-1 </it>was observed to be monomorphic with an allele size of 150/160 bp. The observed 80–90% sequence identity of Indian isolates with data of other regions suggests that Indian <it>P. falciparum </it>population is a mixture of different strains.</p> <p>Conclusion</p> <p>The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of <it>msp-1 </it>(block 2) and <it>msp-2 </it>(central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.</p

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

Background: Multi-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. \ud \ud Methods: 18S 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. \ud \ud Results: 18S 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. \ud \ud Conclusions: The 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