Molecular Identification and Characterization of Simian Malaria Parasites Isolated from Human Blood Samples from Malaysia

Plasmodium knowlesi, a parasite normally seen in macaques, is the main cause of malaria in Malaysia where human cases of a different simian malaria parasite, P. cynomolgi, has been described as well. Zoonotic malaria is a threat especially to people who live in the forest or forest fringes, and people who venture into the forest for work or leisure purposes. Staff at Universiti Malaya (UM) analysed 645 human blood samples collected from indigenous communities around Malaysia for malaria parasites. Following DNA extraction, PCR assays and sequencing of the nuclear small subunit (SSU) rRNA genes, they found both human and simian malaria parasites, including P. knowlesi, P. cynomolgi, P. inui and P. coatneyi in 102 of these samples. Since P. inui and P. coatneyi haven’t been previously described in natural infections of humans, it was necessary to confirm these results by nested PCR assays and by sequencing another gene at an independent laboratory. This study aims to identify simian malaria parasites from these human blood samples by nested PCR assays and by sequencing the cytochrome c oxidase subunit 1 (COX1) genes of Plasmodium. DNA was re-extracted at UM from 15 randomly selected human blood samples with simian malaria infections other than P. knowlesi from the 102 samples mentioned above. These were sent blind, together with five malaria-negative samples, to Universiti Malaysia Sarawak (UNIMAS) for further analysis. These simian malaria parasites were isolated from human indigenous communities from five states (Perak, Kelantan, Negeri Sembilan, Melaka, and Sarawak) in Malaysia. All the 20 gDNA samples sent to UNIMAS were screened and tested negative for the presence of macaque DNA upon receipt and also after concluding the sequencing of COX1 genes, ruling out contamination with macaque blood prior to or during succeeding experiments. Nested PCR assays with genus-specific PCR primers followed by species-specific primers were iii iv undertaken targeting the Plasmodium SSU rRNA gene. A single infection of P. inui was identified in one sample while double infections of P. inui and P. cynomolgi were found in nine out of the 20 other samples. Novel PCR primers for Plasmodium COX1 genes were designed and PCR assay parameters were optimised. Partial COX1 genes of the Plasmodium-positive samples were amplified using a single step or a hemi-nested PCR assay, then cloned and sequenced. The total of 41 partial Plasmodium COX1 sequences generated were subjected to phylogenetic analyses with 40 referral sequences, using both Neighbour-Joining and Bayesian Maximum Clade Credibility methods. The identity of the Plasmodium species in each sample was inferred from the phylogenetic analyses, with both of the phylogenetic trees generated having similar topology. Nine samples were identified as being infected with P. cynomolgi and one with P. inui-like parasites, one with a double infection of P. inui-like and P. simiovale, and one with a triple infection with P. cynomolgi, P. simiovale and P. inui-like. For two out of the 20 samples, the species of Plasmodium could not be inferred following phylogenetic analysis. Differences noted when PCR assay and sequencing results for both laboratories were compared may be due to the usage of different or separate DNA extractions from each human blood sample. Other reasons for the differences in results could be because species-specific primers were used when sequencing the SSU rRNA genes at UM while universal primers for Plasmodium spp. were utilized when sequencing the COX1 genes at UNIMAS. This study is the first description of naturally acquired human infections of P. inui-like and P. simiovale. It indicates that human infections with zoonotic malaria parasites are widely distributed in Malaysia and their prevalence is probably underestimated