Genetic diversity of Plasmodium vivax clinical isolates from southern Pakistan using pvcsp and pvmsp1 genetic markers

Plasmodium vivax malaria is an important public health problem causing an estimated 80–300 million clinical infections annually [1]. In Pakistan it is the common malarial species, contributing to 70% of the malaria burden [2]. Although highly prevalent, P. vivax has received scant scientific attention in Pakistan. Thus, paucity of baseline data exists on various aspects of P. vivax, such as population structure and drug resistance patterns.

Molecular epidemiological studies on genetic diversity of P. vivax have been based mainly on single copy polymorphic genes which code for parasite surface antigens such as gametocyte antigen-1 (pvgam-1), circumsporozoite protein (csp), merozoite surface protein-1 (msp-1) and merozoite surface protein 3 alpha (msp 3α) [3]. Briefly, CSP is a protein involved in binding of sporozoite to liver cells [3‐6]. It comprises of central domain of tandem repeated sequences flanked by two non-repeated conserved sequences [7‐10]. Two types of repeat elements, either VK210 or VK247 types are detected in clinical isolates of P. vivax and thus csp serves as a useful tool for genotyping [11, 12]. Similarly, MSP-1 protein is responsible for red cell invasion by the parasite [13]. Structurally, it is a mosaic organization of 13 regions of inter-allele conserved and extensively polymorphic variable blocks. Three main regions of sequence divergence, at variable blocks 2, 6–8 and 10 have been revealed through studies [14]. Size polymorphisms in the respective blocks can be detected through PCR, thus allowing detection of genetically distinct msp 1 populations within a region [15].

Molecular studies on circulating genotypes provide an insight into transmission intensity and parasite rate variation within a region. Usually, in areas of high malaria transmission, extensive genetic diversity is observed [16, 17]. However, no study as yet has been reported on genetic diversity and transmission intensity of P. vivax from southern Pakistan. This study aims to provide baseline epidemiological data on circulating pvcsp and pvmsp 1 allelic variants from three malaria-endemic areas of southern Pakistan, i e, Karachi, Baluchistan and Sindh.

This is the first study on genetic diversity of P. vivax clinical isolates from southern Pakistan and the results reveal that extensively diverse P. vivax populations are present in this region. By using pvcsp genetic marker, both VK210 and VK 247 types were found in parasite population from southern Pakistan with VK210 being the predominant type. However, the percent positivity of VK 247 type (14.5%) is close to the findings from Herat, Afghanistan (10.4%) [24] and south-eastern areas of Iran (17.5%) [25] but different from that reported from Northern Belt (FATA region) of Pakistan (2.7%) [26]. With respect to study areas, a similar pattern was observed with VK 247 type percent positivity being high in Baluchistan (39%) and Karachi (12.5%) but not in Sindh Province (4.8%). This sharp difference in the frequency of VK 247 type between southern and northern regions of Pakistan and between the respective study areas may be attributed to a number of factors such as co distribution of various vector species and subsequently increased susceptibility of these species to infection by VK 247 type, sampling biases, migration rates, host immune pressure and regional temporal fluctuations [25, 27]. It is possible that these factors, either alone or in conjunction with each other may be allowing gradual natural selection of VK 247 type, particularly in Baluchistan and Karachi.

A total of 16 allelic variants of pvcsp types were observed on the basis of size polymorphisms which is consistent with 15-21varients types observed in previous studies [5, 28]. However, other studies from Thailand and India reported ten and three variants respectively [15, 27]. Studies suggest that specific variant type infecting an individual may influence induced immune responses [29‐31]. Therefore, detection of diverse VK 210 and VK 247 types in this study may serve as a baseline for conducting further studies to understand the role of these variants in host response.

With pvmsp1, a total of 87 distinct genotypes could be distinguished through size polymorphisms in southern Pakistan (Table 2). With respect to F1, F2 and F3 fragments, 23, 41 and 23 allelic variants were detected in the three study areas respectively, indicating that extensive polymorphism has accumulated in msp-1 gene, particularly in F2 fragment. This result is inconsistent with that reported from Thailand and India [15, 27], in which the F2 fragment was found to be poorly polymorphic, with two allelic variants detected through size polymorphism and further distinction of F2 variants required RFLP analysis. The results of this investigation revealed that the F2 fragment in P. vivax isolates from Pakistan, is extensively polymorphic and can be genotyped easily using PCR only with no further allelic distinction through RFLP. However, further studies on F2 fragment from different areas of Pakistan and with larger sample size, is required to corroborate these results. Moreover, mixed genotype infections were observed frequently in all the three msp-1 fragments in Karachi, F2 and F3 fragments in Baluchistan and only in F3 fragment in Sindh (Table 2). The F3 mixed variants were observed predominantly in all three study areas indicating the predisposition of this block to mixed infections. Such high rate of mixed infections and prevalence of genetically diverse isolates signifies that pvmsp-1 gene is under selective pressure for its survival and transmission in Pakistan. This finding is important with respect to transmission intensity and surveillance of P. vivax in southern Pakistan.

The ability to detect genetically diverse variants in all the three fragments of pvmsp1 signifies that this marker can serve useful in identification of individual’s infections. In P. falciparum, recrudescence is identified when the genotype pattern for the two paired samples of a study participant is the same or any bands are shared between the two samples, while a new infection is indicated when the patterns are completely different [32]. Studies suggest that similar strategy can be used for identification of relapses/recrudescence in P. vivax. However, distinction between relapse and recrudescence is difficult due to the high variability in the genotypes of parasite during relapse/recurrence/new infections [19‐21]. In this study, three relapses and one recrudescence case has been identified on the basis of differences/similarities in the pvmsp-1 genetic marker. However, extensive polymorphism in P. vivax and small number of relapse samples collected during this study suggests interpretation of recurrent infection with caution.

Molecular characterization on population structure of P. vivax isolates from southern Pakistan has been explored for the first time in this study. The extensive polymorphism and diversity observed in pvcsp and pvmsp1 genetic markers is indicative of natural selective pressure on the parasite for its survival and transmission in the region. However, further studies from different areas of Pakistan are needed to determine the prevalent genotypes and the transmission intensity of P. vivax. Results from this study can serve as a baseline for conducting studies for effective malaria control.

Increased sequence variation is observed in P. vivax, signifying the high transmission rates and consequently the difficulties associated with malaria control in Pakistan.