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The recent resurgence of Plasmodium vivax malaria requires close epidemiological surveillance and monitoring of the circulating parasite populations. In this study, we developed a combination of polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) method to investigate the genetic diversity of the P. vivax merozoite surface protein 3beta (PvMSP3beta) gene among four Asian parasite populations representing both tropical and temperate strains with dramatic divergent relapse patterns (N = 143). Using P. vivax field isolates from symptomatic patients, we have validated the feasibility of this protocol in distinguishing parasite genotypes. We have shown that PCR alone could detect three major size polymorphisms of the PvMSP3beta gene, and restriction analysis detected a total of 12 alleles within these Asian samples. Samples from different geographical areas differed dramatically in their PvMSP3beta allele composition and frequency, indicating that complex, yet different parasite genotypes were circulating in different endemic areas. This protocol allowed easy detections of multiple infections, which reached 20.5% in the samples from Thailand. It is interesting to note that samples from one temperate site in China collected during a recent outbreak of the disease also showed a high level of genetic diversity with multiple infections accounting for 5.6% of the samples. When combined with the PvMSP3alpha locus, this method provides better capability in distinguishing P. vivax genotypes and detecting mixed genotype infections.

During blood feeding, arthropod vectors inject saliva into vertebrate hosts. The saliva is biochemically complex and pharmacologically active, and may play an important role in pathogen transmission. To examine whether mosquito saliva could elicit humoral immune response in humans under natural conditions, we have collected sera from malaria patients, healthy villagers, and people from a non-malarious region in Thailand. Here we have demonstrated that anti-Anopheles salivary protein antibodies occurred predominantly in patients with acute Plasmodium falciparum or P. vivax malaria, whereas people from a non-malarious area had no such antibodies. Besides, antibody levels against mosquito salivary proteins in malaria patients were highly variable, which may be related to the levels of mosquito exposure. Despite variability, patients' sera with high IgG titers consistently detected several proteins in Anopheles dirus salivary gland protein extracts. Immunohistochemical staining of Anopheles salivary glands with human sera showed that the salivary gland-specific IgGs reacted strongly with the median lobe. Comparison using Anopheles and Aedes salivary proteins suggests that the anti-salivary protein antibodies detected in malaria patients were Anopheles-specific, consistent with the major malaria vector status of An. dirus in this area.