In Myanmar, a patient infected with P. knowlesi was first detected in Northern Myanmar . This patient was a camp worker. In 2008, investigation of 146 blood samples collected in southern Myanmar near Yunnan province of China revealed that four samples were positive for P. knowlesi and that 28 samples showed co-infection of P. knowlesi with P. falciparum, P. vivax, or both . In Thailand, the first case of P. knowlesi infection was reported in 2004, of a patient who lived in Bangkok, Thailand and had recently visited the southern province of Prachuap Khiri Khan near the Myammar border . In addition, 10 and 23 more P. knowlesi infections have been identified in 2006-2007 and 2008-2009, respectively in Tak, Chantaburi, Prachuap Khiri Khan, Yala and Narathiwat provinces [10, 11]. Interestingly, all of these provinces are located near the Myanmar border (Tak and Prachuap Khiri Khan), Cambodia border (Chantaburi), and Malaysia border (Yala and Narathiwat). In this study, two cases of P. knowlesi infection detected at the Vector Borne Disease Control Center in Ranong province, which is close to the Myanmar border are reported. One patient was a Thai who worked in a rubber plantation in Koh Song, in the Kawthoung District of Myanmar and the other was Myanmese who worked as a fisherman in the same area. Koh Song is close to Ranong province (Figure 1), therefore, transmission of this malaria parasite species near this Thai border is suspected. Recently, there has been a report that a French tourist was infected with P. knowlesi at Koh Payam (Figure 1) near Ranong province . Wild macaques presence in the forest fringe near this tourist's bungalow were expected to be reservoirs of P. knowlesi. It is possible that human carriers of P. knowlesi exist in this and surrounding areas and monkey-to-human transmission of P. knowlesi may be taking place. Most primats observed in Thailand are long-tailed macaques (Macaca fascicularis) . Increasing in natural forest invasion disturbs habitats of these macaques and has caused them to migrate to forest fringes, temples or parks in urban areas near human settlements. In addition, some macaques are considered as pets to humans . Malaria among long-tailed macaques in southern Thailand was evaluated in Ranong and Prachuap Khiri Khan provinces by amplification and sequencing of the SSU rRNA and the mitochondria cytochrome b genes. Non-human primate malaria (P. inui and P. coatneyi) and Hepatocystis species were detected only in wild macaques in Ranong mangrove forests where anopheline mosquitoes were abundant . However, P. knowlesi was not detected in these macaques. Nevertheless, transmission of P. knowlesi from monkey to human cannot be ruled out.
It is considered important to correctly identify the malaria species and clarify the mode of transmission for establishing appropriate preventive measures. In Thailand, P. falciparum and P. vivax are most frequently detected in patients . In this study, malaria parasites in blood samples from a Thai and a Myanmese patient were probably in the late erythrocyte stage, so they were originally identified as P. vivax instead of P. malariae by microscopy. Thus, health care workers in this area who are responsible for identification of malaria species should be trained appropriately so that malaria species including P. knowlesi can be correctly identified.
The nucleotide sequences of the csp gene of P. knowlesi obtained from the two patients were identical. Comparison of these two P. knowlesi csp gene sequences and the other Plasmodium csp gene sequences deposited in GenBank confirmed that the P. knowlesi sequences obtained from the Thai and Myanmese patients were closely related to P. knowlesi derived from both monkeys and humans (Figure 3). Blood from these patients was collected in the same month (June 2008) and it is possible that they may share the same origin. More in-depth epidemiological studies, including entomological investigations should be undertaken around Ranong and surrounding areas to determine the incidence of P. knowlesi and to understand the mode of transmission of this malaria parasite in the Thai-Myanmar border areas.