High-throughput molecular diagnosis of circumsporozoite variants VK210 and VK247 detects complex Plasmodium vivax infections in malaria endemic populations in Papua New Guinea.

Malaria is endemic in lowland and coastal regions of Papua New Guinea (PNG), and is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Infection by P. vivax is attributed to distinct strains, VK210 and VK247, which differ in the sequence of the circumsporozoite protein (pvcsp). Here, based upon sequence polymorphisms in pvcsp, we developed a post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) to distinguish these P. vivax strains. This diagnostic assay was designed to detect the presence of both VK210 and VK247 P. vivax strains simultaneously in a high-throughput 96-well format. Using this assay, we analyzed human blood samples from the Wosera (n=703) and Mugil (n=986) regions to evaluate the prevalence of these P. vivax strains. VK210 and VK247 strains were found in both study sites. In the Wosera, single infections with VK210 strain were observed to be most common (41.7%), followed by mixed-strain (36.8%) and VK247 single-strain infections (21.5%). Similarly, in Mugil, VK210 single-strain infections were most common (51.6%), followed by mixed-strain (34.4%) and VK247 single-strain infections (14%). These results suggest that the distribution of P. vivax infections was similar between the two study sites. Interestingly, we observed a non-random distribution of these two P. vivax strains, as mixed-strain infections were significantly more prevalent than expected in both study sites (Wosera and Mugil χ(2)p-value<0.001). Additionally, DNA sequence analysis of a subset of P. vivax infections showed that no individual pvcsp alleles were shared between the two study sites. Overall, our results illustrate that PNG malaria-endemic regions harbor a complex mixture of P. vivax strains, and emphasize the importance of malaria control strategies that would be effective against a highly diverse parasite population.