Merozoite surface protein-3 alpha as a genetic marker for epidemiologic studies in Plasmodium vivax: A cautionary note

Background: Plasmodium vivax is the most widespread of the human malaria parasites in terms of geography, and is thought to present unique challenges to local efforts aimed at control and elimination. Parasite molecular markers can provide much needed data on P. vivax populations, but few such markers have been critically evaluated. One marker that has seen extensive use is the gene encoding merozoite surface protein 3-alpha (MSP-3α), a blood-stage antigen known to be highly variable among P. vivax isolates. Here, a sample of complete msp-3α gene sequences is analysed in order to assess its utility as a molecular marker for epidemiologic investigations. Methods. Amplification, cloning and sequencing of additional P. vivax isolates from different geographic locations, including a set of Venezuelan field isolates (n = 10), yielded a sample of 48 complete msp-3α coding sequences. Characterization of standard population genetic measures of diversity, phylogenetic analysis, and tests for recombination were performed. This allowed comparisons to patterns inferred from the in silico simulation of a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) protocol used widely. Results: The larger sample of MSP-3α diversity revealed incongruence between the observed levels of nucleotide polymorphism, which were high in all populations, and the pattern of PCR-RFLP haplotype diversity. Indeed, PCR-RFLP haplotypes were not informative of a population's genetic diversity and identical haplotypes could be produced from analogous bands in the commonly used protocol. Evidence of frequent and variable insertion-deletion mutations and recurrent recombination between MSP-3α haplotypes complicated the inference of genetic diversity patterns and reduced the phylogenetic signal. Conclusions: The genetic diversity of P. vivax msp-3α involves intragenic recombination events. Whereas the high genetic diversity of msp-3α makes it a promising marker for some epidemiological applications, the ability of msp-3α PCR-RFLP analysis to accurately track parasites is limited. Local studies of the circulating alleles are needed before implementing PCR-RFLP approaches. Furthermore, evidence from the global sample analysed here suggests such msp-3α PCR-RFLP methods are not suitable for broad geographic studies or tracking parasite populations for an extended period of time.