Naturally Acquired Plasmodium Knowlesi Malaria In Human

Malaria remains the one of the most devasting infectious diseases in the world. The annual incidence of the disease is estimated to be 300-500 million, and it causes about 1-3 million deaths each year, most of them are children. Due to the emergence of the drug resistance of the parasite and the insecticide resistance of mosquitoes, it becomes more difficult to control malaria. Furthermore, Simian malaria parasites infecting human were reported many times, which might cause severe public health problem: For prevention of malaria epidemics hasn' t achieved tremendous success, emerging of primate malaria infection in human makes the situation more complicated.Recently when we examine retrospectively the smears of "P. vivax" collected from Yunnan Province (the patient had spent sometimes in China-Myanmar border, logging), we found the similarity in morphology between the smears of the Yunnan patient and the naturally acquired P. knowlesi infection of human reported in Thailand, which belong to primate malaria. P. knowlesi usually infect monkeys in nature and is shown to be infectious to human occasionally from the report of oversea. It hadn' t drawn much attention of scientists, because its clinic symptoms were neither specific nor severe. The morphology of P. knowlesi in human infection could not easily differentiate with other Plasmodium species, especially with "P. vivax multinucleatum Chiang" , and P.malariae.We observed all stages of the erythrocytic cycle of the parasite in Giemsa-stained blood films. Early trophozoites appeared as ring forms as early trophozoites of P. falciparum in multi-infection, except thering were a little larger. Frequently, more than one ring forms were noted in erythrocytes and double or tri-chromatin dots were also seen. Late trophozoites were usually amoeboid, as that of P. vivax. Sometimes, the cytoplasm was compact, which occupied no more than two-thirds of the erythrocytes. Some late trophozoites had a trend to form "band" that are typical for P. malariae. Mature schizonts had a central cluster of 8 to 16 merozoites and did not fill the whole erythrocyte. Late trophozoites and schizonts were densely pigmented with dark brown/black malaria pigment. Gametocytes filled most part of the erythrocytes and malaria pigment was scattered. The structure of gametocytes was compatible with that of P. vivax, but smaller.From the observation above, we can't make a conclusion of which species of Plasmodium the patient was infected with. A DNA-based diagnosis with the polymerase chain reaction (PCR) method targeting the small subunit ribosomal RNA (SSU rRNA) genes of two human malaria species and P. knowlesi was introduced.The blood films were scraped using a clean blade. All the films were incubated overnight at 37°C and extracted with pheonl-chlorolform and DNA was precipitated with isopropanol. Using primers targeting SSU rRNA of different Plasmodium species, the extacted DNA was amplified by PCR with different primes for P. fale ipar urn, P. vivax and P. knowlesi, respectively. The primer pair specific for P. knowlesi produced a single fragment of 150 bp. The amplified fragments were digested by endonuclease, cloned into T-vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the amplified fragment was identical to the sequence of P. knowlesi. This result confirmed that the pationt was infected with P. knowlesi, not P. vivax.A pair of primer was designed according to the reported msp-l partial sequence of P. knowlesi. The partial msp-l encoding gene was amplified...