Plasmodium-Specific Molecular Assays Produce Uninterpretable Results and Non-Plasmodium spp. Sequences in Field-Collected Anopheles Vectors

The Malaria Research and Reference Reagent Resource-recommended PLF/UNR/VIR polymerase chain reaction (PCR) was used to detect Plasmodium vivax in Anopheles spp. mosquitoes collected in South Korea. Samples that were amplified were sequenced and compared with known Plasmodium spp. by using the PlasmoDB.org Basic Local Alignment Search Tool/n and the National Center for Biotechnology Information Basic Local Alignment Search Tool/n tools. Results show that the primers PLF/UNR/VIR used in this PCR can produce uninterpretable results and non-specific sequences in field-collected mosquitoes. Three additional PCRs (PLU/VIV, specific for 18S small subunit ribosomal DNA; Pvr47, specific for a nuclear repeat; and GDCW/PLAS, specific for the mitochondrial marker, cytB) were then used to find a more accurate and interpretable assay. Samples that were amplified were again sequenced. The PLU/VIV and Pvr47 assays showed cross-reactivity with non-Plasmodium spp. and an arthropod fungus (Zoophthora lanceolata). The GDCW/PLAS assay amplified only Plasmodium spp. but also amplified the non-human specific parasite P. berghei from an Anopheles belenrae mosquito. Detection of P. berghei in South Korea is a new finding.