Background
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 [
1]. 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 [
2]. 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 [
6].
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 [
18]. 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 [
28]. The
pvmsp 1 gene has been used to determine whether an infection is a result of a new infection or a relapse [
11] 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 [
21]. 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 [
29]. 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 [
22], 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 [
23]. 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 [
19] and
pvcs genotyping [
18]. 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 [
28]. 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 [
11],
pvmsp 1 and
pvmsp 3-alpha display high levels of diversity [
24]. By contrast, relatively low genetic diversity of
pvmsp 1 has been detected in the re-emerging vivax malaria focus in Korea [
31]. 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 [
27]. 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 [
37]. 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.
Discussion
The results showed that P. vivax parasites from Kolkata demonstrated an extremely high prevalence of VK210 type in the pvcs gene (with only one VK247 type), high polymorphism in both merozoite surface proteins (pvmsp 1 and pvmsp 3 alpha), and low rates (10.6%) of multiple genotype infection.
The predominance in Kolkata isolates of VK210
pvcs gene type (99.3%) has not been seen elsewhere to the same extent. Recent studies in Thailand have found rates of 70.5% [
35], 78% [
24] and 90% [
28], though these results were in sharp contrast to those of earlier studies in which VK247 was found to be the predominant type; 83% in Thai and 90% in Papua New Guinea samples by PCR/Oligo Probe [
23]. Another study of Indian strains has shown the VK210 type to be predominant, but also demonstrated (using a different technique) significant numbers of VK247 type [
33]. This phenomenon may be attributed to selection by host immune pressure on a particular genotype, and/or the preferential production of sporozoites carrying a specific variant [
18], such as VK210 for
pvcs in a mosquito species. These differences may also be due to sampling biases or regional temporal fluctuations of individual genotypes frequencies. The frequency of absence of the pre-repeat insertion of VK210 in the
pvcs gene was 4.7% (7/150) and that of the post-repeat region of the gene 56% (84/150) in Kolkata. In the absence of a multicentre study involving various regions of India it is difficult to define the extent to which the observed difference was in any way related to the functional aspects of the Kolkata strain.
In this study, two major types of the
pvmsp 1 marker containing 35 alleles were found. A similar degree of diversity (36 allelic types) was also found in the study from Thailand using the same laboratory protocol [
28], and with a different protocol in Papua New Guinea and Indian strains [
16,
42]. For
pvmsp 3-alpha, the present study reveals 37 alleles with three size variants cut by two restriction enzymes. The frequencies of the three
pvmsp 3-alpha types were consistent with those found in Papua New Guinea and Thailand [
18,
19]. A recent study using a different protocol revealed 16 size and sequence polymorphic allele in Indian strains [
37]. This high degree of polymorphism in the merozoite surface protein (
pvmsp 1 and
pvmsp 3-alpha) is similar to that found in other studies from other regions.
All three markers show marked genetic diversity in Kolkata strains, with 11 alleles for
pvcs, 35 alleles for the second fragment of
pvmsp 1, and 37 alleles for
pvmsp 3-alpha. This compares with another study of Indian vivax isolates which revealed 2 sequence variants of
pvcs, 9 size variants in
pvgam 1, and 16 size variants with sequence polymorphism in
pvmsp 3-alpha gene [
37].
The frequency of multiple genotype infections of
P. vivax malaria has been estimated in many regions but it is difficult to compare these values due to differences in sampling and genotyping methods. The proportion of mixed gene infections estimated in Papua New Guinea, India, and Thailand ranges from 30% to 65% [
16,
20,
24,
25]. The present study with a relatively large sampling size, shows an overall 10.6% for multiple genotypes (1.3% for
pvcs, 0.7% for
pvmsp 1 and 8.6% for
pvmsp 3 alpha). This rate may reflect a limitation in the sensitivity of PCR for the detection of multiple genotype infections, despite the high degree of polymorphism seen.
According to the Calcutta School of Tropical Medicine Report 2005, the malaria clinic treats around 6,000 malaria positive cases annually, of which an average of 65% are caused by
P. vivax. The rate of mixed infection with both
P. vivax and
P. falciparum during the last few years has declined (1.0% in 1997, 0.7% in 1998 and 0.1% in 2001) [
6]. The samples from Kolkata were collected from an urban area where vivax malaria remains chloroquine-sensitive, but where recurrences are frequent. Only a few cases were contracted outside of central Kolkata. The high degree of polymorphism and low level of multiple genotype infection probably reflects the nature of this endemic setting. More study is needed to assess whether these discrepancies reflect true differences between disease populations, or are due to differences in sample sizes or the laboratory methodology [
20,
38].
Authors' contributions
JRK was involved in all stages of this study. SP and AN designed the study and were responsible for the day-to-day supervision of patient recruitment and clinical management. MI was responsible for the supervision of the molecular genetic study and NT for the laboratory work. AM and MA participated in the recruiting of patients. KC and APN participated in the coordination of laboratory work. NJW and NPD helped compose the manuscript and gave constructive advice. All authors read and approved this final manuscript.