Malaria remains one of the most important communicable diseases in the world. Despite enormous control efforts over many decades malaria is still a significant health problem. It is estimated that around 300–500 million cases occur each year with one to three million deaths. The problem is compounded by multiple drug resistance in Plasmodium falciparum and chloroquine resistance in Plasmodium vivax . The global burden of malaria due to P. vivax is 70–80 million cases annually. Vivax malaria is usually a non-lethal infection but its prolonged and recurrent infection can have major deleterious effects on personal well-being, growth and on the economic performance at the individual, family, community and national levels . The recent emergence of chloroquine-resistant strains is of great concern [3–5].
P. vivax causes about 60–65% of all malaria infections in India [6, 7, 42]. The frequency of relapse after a standard course of chloroquine and primaquine treatment is 23–40% depending on the duration of follow-up in India [7, 36]. P. vivax and P. falciparum are prevalent in all age groups but their prevalence is highly seasonal and differs between the species; longitudinal studies in India show a winter peak for P. falciparum and a summer peak for P. vivax [7, 8]. Chloroquine appears to remain an effective drug in the treatment of P. vivax malaria in Kolkata .
The majority of studies on the genetic structure of Plasmodium have focused on P. falciparum, using polymorphic markers such as the merozoite surface protein-1(msp -1), -2 (msp -2), glutamate-rich protein (glurp) [9, 14]. A similar approach has been adopted for P. vivax but it has been less well-studied at the molecular level than P. falciparum . Three polymorphic P. vivax genes have been widely used for molecular epidemiological studies. The pvcs gene has a central repeat domain that varies in sequence and number of repeat units [10, 41]. Two major types, VK 210 and VK247, have a worldwide distribution and four subtypes from VK210 and two subtypes from VK 247 can be differentiated by restricted enzyme digestion to show polymorphisms in both the pre- and post- repeat region . The pvmsp 1 gene has been used to determine whether an infection is a result of a new infection or a relapse  and used to genotype isolates of different strains from different geographical regions [12, 13, 15–17]. The polymorphic pvmsp 3-alpha gene was also studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis [19, 20]. The pvmsp 3-alpha gene encodes a merozoite surface protein with an alanine-rich central domain that is predicted to form a coiled-coil tertiary structure . There is added interest in the pvmsp3 antigen family as immunogens and vaccine candidates. Use of the pvmsp 3-alpha gene as genetic marker has been recently validated [19, 20, 24, 30, 39, 40]. Other genetic markers used for P. vivax are the apical membrane antigen 1 (Ama-1), gametocyte antigen 1 (gam 1) and msp 3-beta. The PvAma-1 is a protein essential for erythrocyte invasion, which shows limited sequence polymorphism [26, 34]. The potential of pvgam 1 as a molecular marker for genotyping is compromised by artifacts associated with amplification of this region . The pvmsp 3-beta gene is a member of a family of related merozoite surface proteins containing a central alanine-rich central region with significant genetic diversity [22, 30]. Despite this high level of sequence diversity certain physical properties of the encoded protein are maintained, particularly the ability to form coiled-coil tertiary structure , which may limit genetic studies.
Studies using single or combined pvcs, pvmsp 1 and pvmsp 3-alpha genotyping have assessed the genetic diversity of P. vivax isolates from various regions. A study on pvcs identified that the VK247 genotype was widely distributed and was the predominant form in Thai and Papua New Guinea isolates but its prevalence was much lower in Mexico . Another study, by contrast, revealed that the VK210 type in dimorphic pvcs gene was found in the majority of the parasites in Thai strains [24, 10]. A recent single gene study of pvmsp 3-alpha revealed that it was highly polymorphic, and that three major types of the pvmsp 3-alpha locus could be distinguished [32, 39]. Moreover earlier studies revealed a high prevalence of multiple genotype infection as determined by pvmsp 3-alpha  and pvcs genotyping . Recently a combined pvcs and pvmsp 1 study showed a lower rate of multiple genotype infections than an earlier study and high polymorphism in Thai strains . In hyperendemic areas, intragenic recombination and high genetic diversity have been reported in pvmsp 1 and pvmsp 3-alpha [16, 20, 39]. Even in hypoendemic areas, such as Thailand and Brazil , pvmsp 1 and pvmsp 3-alpha display high levels of diversity . By contrast, relatively low genetic diversity of pvmsp 1 has been detected in the re-emerging vivax malaria focus in Korea . Little is known about the genetic diversity among parasite populations in India, where most vivax malaria in the world occurs. Earlier studies carried out using isoenzyme typing was consistent with the random mating nature of vivax malaria isolates in India . Recent studies on the polymorphism of pvcs, pvgam 1 and pvmsp 3 alpha in Indian isolates have revealed two types of pvcs and nine size variations of pvgam 1 and high polymorphism in the pvmsp 3 alpha gene . Therefore, the highly polymorphic, single-copy, unlinked genes, pvcs, pvmsp 1 and pvmsp 3-alpha were selected for this study of genetic diversity of P. vivax in Kolkata.