Identification and molecular characterisation of simian malaria parasites in wild monkeys of Singapore

Abstract

Plasmodium knowlesi is a simian malaria parasite currently recognized as the fifth cause of human malaria. Singapore reported its first local human knowlesi infection in 2007 and epidemiological investigations revealed that long-tailed macaques were the reservoir host of this blood parasite. Apart from P. knowlesi, long-tailed macaques are also natural host to P. coatneyi, P. fieldi, P. cynomolgi and P. inui, of which the latter two were also found to be infectious to humans under laboratory conditions. As there was no previous study of simian malaria parasites in Singapore?s macaques, this study aims to determine their prevalence for the risk assessment of zoonotic transmission of simian malaria parasites to the general human population. Detection and accurate identification of simian malaria parasites through microscopy is typically challenged by low parasitemia, mixed species infection in the natural hosts and overlapping morphological characteristics among the different simian Plasmodium species. A sensitive Plasmodium parasite screening polymerase chain reaction (PCR) assay and a simian malaria species-specific nested PCR assay were thus developed. The PCR primers for Plasmodium parasites screening were designed against the conserved regions in the small subunit ribosomal RNA (SSU rRNA) genes. These primers were able to detect the four human and five simian Plasmodium species parasites, and could be used in both conventional and real-time PCR. The simian Plasmodium species-specific nested PCR assay, on the other hand, was developed using the Plasmodium circumsporozoite protein (csp) gene. Plasmodium screening on 65 peri-domestic and 92 wild macaques revealed that the former group was uninfected, while 71.7% of the sampled wild macaques were infected. Peri-domestic macaques were found in areas near human habitations while wild macaques were caught in military forest where access is restricted to the general public. All five simian Plasmodium species were detected, with P. knowlesi having the highest prevalence (68.2%), followed by P. cynomolgi (60.6%), P. fieldi (16.7%), P. coatneyi (3.0%) and P. inui (1.5%). Co-infection with multiple species of Plasmodium parasites was also observed; double infection was detected in 23 (34.8%) macaques while five (7.6%) were infected with three Plasmodium species. Phylogenetic analysis of the non-repeat region of the Plasmodium csp gene from 15 infected macaques revealed high genotypic diversity of the parasites, reflecting a high intensity of malaria transmission among the macaques in the forest. On the other hand, all four local knowlesi cases had single P. knowlesi genotype which was identical to the P. knowlesi isolates of some macaques, suggesting that macaques were the reservoir hosts of the knowlesi malaria. Identical Plasmodium csp sequences shared by macaques caught at different timepoint also illustrates an ongoing sylvatic transmission. Despite these findings, the risk of zoonotic transmission of simian malaria parasites to the general population is assessed to be low as malaria parasites were absent among peri-domestic macaques, and all human knowlesi cases reported in Singapore were thus far occupational or travel related. However, to enable continuous risk assessment and surveillance, more studies will be required to determine the identity and distribution of the mosquito vector/s and the spatial distribution of the wild macaques.