Phylogeographic Evidence for 2 Genetically Distinct Zoonotic Plasmodium knowlesi Parasites, Malaysia

The number of malaria cases in Malaysia steadily decreased from a peak of 59,208 in 1995 to 3,850 confirmed cases in 2013; of these, 80% were reported in the 2 states of Malaysian Borneo and the remainder in 6 of the 11 states of Peninsular Malaysia (Figure 1) (1). In Malaysia, the simian malarial parasite species Plasmodium knowlesi is now the dominant species infecting humans and is >2 times more prevalent than P. falciparum or P. vivax. Humans were found to be susceptible to P. knowlesi when this species was experimentally transmitted to man in 1932, the year in which it was first described (2,3). In 1965, the first confirmed case of a naturally acquired infection in humans was recorded (4). The next naturally acquired confirmed cases were reported in 2004, when a stable focus of P. knowlesi was discovered in Sarawak, 1 of 2 states that make up Malaysian Borneo (5). Thereafter, transmission of P. knowlesi to humans occurred in the second state, Sabah (6,7), and in neighboring countries (8,9). The natural hosts of P. knowlesi are principally the long-tailed (Macaca fascicularis) and pig-tailed (M. nemestrina) macaques (10), 2 species that are widely distributed in the Southeast Asia countries in which cases of P. knowlesi have been recorded. To date, human-to-human transmission has not been observed. Infections in humans can cause severe disease that can be fatal (9,11), underscoring the public health concern raised by this zoonotic simian parasite. The 2 states of Malaysian Borneo appear to be the epicenter of zoonotic P. knowlesi infections: 1,391 cases in Malaysian Borneo and 423 cases in Peninsular Malaysia were recorded in 2012. A total of 1,407 PCR-confirmed cases were reported during 2004–2013 in Malaysia, which contrasts with the low number of cases (n = 136) reported from neighboring countries (9): Cambodia (n = 1), China (n = 36), Indonesia (n = 1), Myanmar (n = 14), the Philippines (n = 5), Singapore (n = 2), Thailand (n = 36), and Vietnam (n = 32). The reasons for this uneven distribution remain unclear. Geographic variation in mosquito species and human social factors could be an explanation; it is also possible that the parasite populations circulating on the island of Borneo are distinct from those found in continental Malaysia. The P. knowlesi strains that had been studied in earlier years displayed distinct biologic characteristics, which in some cases led malariologists to propose distinct P. knowlesi subspecies (12). Such differences could indicate that local ecologic factors are influential and that each P. knowlesi subspecies became a zoonosis independently in each geographic area. To explore whether the P. knowlesi populations in Malaysia differed and independently became zoonoses, we focused on 2 genes that have been extensively used for phylogenetic studies (13–16): 1 nuclear, encoding the type A small subunit ribosomal 18S RNA (PkA-type 18S rRNA), and 1 mitochondrial, encoding the cytochrome oxidase subunit I protein (PkCOX1). Using samples collected from humans and macaques in both regions of the country, we generated the relevant sequences, compared them to those published previously (17), and conducted phylogenetic and population genetic analyses.