Allelic dimorphism of Plasmodium vivax gam-1 in the Indian subcontinent
© Prajapati et al; licensee BioMed Central Ltd. 2006
Received: 14 July 2006
Accepted: 24 October 2006
Published: 24 October 2006
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.
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.
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.
Study shows limited diversity of Pvgam-1 marker in Indian isolates with well representation of both Belem and Chesson type alleles.
Plasmodium vivax is the most widespread human malaria parasite and is responsible for 70–80 million cases annually . In India, it is the most prevalent of human malaria parasites, contributing 50–55% cases of malaria each year. P. vivax infections are rarely fatal but impact heavily on personal health placing a considerable economic burden upon individuals and communities .
Malaria eradication programmes are facing a formidable challenge due to the spread of drug resistance and the complex population genetic structure of malaria parasites. Genetic polymorphism is an inevitable component of a complex organism, especially in multistage infectious organisms such as malaria parasites. Understanding the genetic population structure of the parasites may provide valuable information for effective malaria control strategies. There have been few studies on genetic polymorphism in P. vivax, first because more work has been done on the more virulent human malaria parasite, Plasmodium falciparum, and secondly because of the non-availability of continuous in-vitro cultures for P. vivax. Recently, the development of molecular tools, such as PCR, has made analysis of field samples possible and easier, thus strengthening research on P. vivax. The availability of the P. vivax genome sequence in the public domain has further facilitated this work.
P. vivax sub-populations have been reported between temperate zone type (Belem strain) and tropical zone type (Chesson strain) on the basis of different markers such as 18 S SSU rRNA , relapse pattern , and Pvcsp gene . Markers used for exploring population genetic structure of P. vivax are limited, namely the Pvmsp-1 , Pvmsp-3α , Pvcsp , Pvdbp , Pvama1 , Pvgam-1  and microsatellite markers . Pvgam-1 is expressed during the gametocyte stage and an antibody raised against the corresponding antigen has been reported to completely block the development of parasites in the mosquito midgut and is reported as potential transmission blocking vaccine candidate . Polymorphism in the C-terminal part of the gene was reported which is due to deletion of 33, 57 & 84 bp guanine-rich motif and is not covered by epitopic region of the PvGAM-1 antigen . However, the validation of Pvgam-1 as a genetic marker has been limited so far [13, 14]. This paper reports the prevalence of Chesson and Belem type alleles in field isolates from India, on the basis of PCR genotyping of Pvgam-1 in different epidemiological regions.
Materials and methods
Blood samples were collected during spot surveys or from patients attending a clinic for malaria diagnosis at headquarters in Delhi or at field units, between 2000 and 2004. Microscopically diagnosed P. vivax-positive blood was spotted on autoclaved Whatman filter paper (3 mm) strips and dried blood spots were stored at 4°C. Blood smears were stained with JSB stain  and examined at a 1,000 × magnification. The study protocol was approved by the ethics committee of the National Institute of Malaria Research, and all blood spots were collected with the consent of the patients.
Genomic DNA extraction and PCR amplification
Genomic DNA was extracted from three punches of Whatman filter paper with blood spots using QIAamp mini DNA kit (Cat. No.51306, Qiagen, Germany). DNA was eluted in 100 μl sterile triple distilled water. Genomic DNA was analysed using species-specific primers of Tirasophon  to confirm the P. vivax positivity in the samples. Only isolates having confirmed P. vivax infection by PCR were included in the study. All amplification reactions were carried out in a final volume of 20 μl, which included 1 μl template genomic DNA in primary PCR and 0.5 μl of primary product as template in nested PCR. PCR primers and the protocols used for amplification of Pvgam-1 were those of Snewin et al .
Sequencing of PCR products
PCR products were purified from agarose gel by using gel extraction kit (QIAquick, Cat. No. 28706). Sequencing was done by cycle sequencing method using Big Dye terminator method with ready to use kit (3.1 version, Perkin Elmer Corp) and run on the ABI Prism 310 automated DNA sequencer. Each sample was sequenced by forward and reverse primers to confirm deletion/point mutations in the study fragment of Pvgam-1. Sequences were analysed using DNASTAR (Lasergene, USA) software. All DNA sequences were aligned using ClustalW method.
Distribution of Pvgam-1 alleles in Indian populations
Number of samples tested
Observed Allelic Composition of the isolates
Belem (550 bp)
Chesson (520 bp)
Belem + Chesson
Gautam Budh Nagar
Our results are different to earlier observations made among Korean and Thai isolates [13, 14]. Korean isolates had shown only Belem type allele while among Thai isolates Chesson type was the predominant allele (92%). Further, Imwong et al  observed PCR associated artifacts thus suggested Pvgam-1 not to be a dependable marker. In the present study, PCR associated artifacts were not observed in any of the isolates. Pvgam-1 is highly GC rich and GC rich regions are reported to be highly prone to illegitimate recombinations thus generating PCR artifacts .
Studies on genetic structure of P. vivax is limited mainly because of non-availability of suitable polymorphic markers. Snewin et al , added gam-1 to the list of available P. vivax markers namely csp, msp-1, msp-3α, dbp, ama-1 by describing a polymorphic region in the gam-1 gene (encoding a P. vivax transmission blocking candidate antigen) and reported four variants based on nucleotide sequence deletions among 10 Sri Lankan isolates. However, later monomorphic nature of gam-1 was observed among Korean isolates . On the other hand, among Thai isolates, both Belem and Chesson type alleles were observed with predominance (92%) of Chesson type allele (520 bp) .
Our observations of presence of both Belem and Chesson strains of P. vivax among Indian field isolates has been similar to observations of another study reporting presence of the both the strains temperate (Belem) and tropical (Chesson) in Indian population based on the relapsing pattern of vivax infection among the patient . Though the present study could not correlate molecular markers with clinical findings, but predominance of both Belem and Chesson type allele was observed in all the study sites and in isolates collected during spot surveys from malaria endemic as well as during outbreaks.
The polymorphic region of Pvgam-1 analysed in this study is known not to be covering the epitopic region responsible for transmission-blocking activity . However, the highly polymorphic nature of the C-terminal region of the fragment in the Belem type allele (11 sites out of 15 showing polymorphism) led to speculation that this region may be under host immune pressure and may also be involved in immunogenicity. In Chesson type alleles, no variation was observed in this region and the deletion of 33 bp repeats may be disrupting the peptide sequence required for antigenicity. This is supported by the conserved nature of this region in all four Chesson type allele (NM1, 62, 6210, 9086) sequenced in this study. Further functional studies are needed to prove whether or not this variable region is involved in immunogenicity.
The highly polymorphic nature of Indian P. vivax field isolates had previously been recognized by the relapse pattern , drug susceptibility  and enzymes polymorphism [19, 20]. Based on epidemiological features, Adak et al  suggested the existence of polymorphic P. vivax population in Delhi region of India and populations were characterized by three types of incubation periods following primary attack of malaria. Short term relapsing strains (Chesson) showed susceptibility to Primaquine and Bulaquine (anti-relapse drugs) while long term relapsing strains (temperate or St. Elizabeth) were not susceptible to these drugs .
Studies using biochemical and molecular markers have shown polymorphic forms of Glucose phosphate isomerase (6 alleles), glutamate dehydrogenase (7 alleles) and adenosine deaminase enzymes (5 alleles)  as well as size & sequence variations in msp-3α, csp, gam-1 . A recent study using csp, msp-1 and msp-3α in Kolkatta, eastern region, have also reported highly polymorphic nature of Indian P. vivax isolates .
The present study is first to reveal dimorphic nature of gam-1 in the field isolates studied from different parts of India. Pvgam-1 marker having two alleles with good frequency distribution among the isolates increases the probability of identifying mixed infections. Studies related to single nucleotide polymorphism, house keeping genes, microsatellite markers as well as drug resistance associated mutations, identification of single clone infection is a prerequisite. Thus, present study suggested Pvgam-1 marker could be a useful marker for the identification of multiple infections of different genotypes in addition to Pvmsp-1, Pvcsp &Pvmsp-3α. Advantage of Pvgam-1 marker is that its analysis is performed by means of simple PCR assays and does not require sequencing, hybridization or RFLP analysis as do other markers genes such as csp or msp-3α locus.
Data obtained in this study shows limited diversity of Pvgam-1 marker in Indian isolates with well representation of both Belem and Chesson type alleles.
The authors are thankful to the Indian Council of Medical Research (ICMR) for providing the facilities as well as partially funding the project under the genomics scheme. The authors would also like to thank the patients for their co-operation during the study. Thanks are due to staff members of National Institute of Malaria Research for their support and help in the field as well as laboratory work.
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