Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the...Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the disease is estimated to be 350～500 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. Since prevention of malaria epidemics hasn’t achieved tremendous success, emergence of primate malaria infection in human makes the situation even 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 belongs to primate malaria. P. knowlesi usually infects monkeys in nature and is shown to be infective 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 for atypical form of P. vivax 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 the rings 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-third 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 conclude 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℃ 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.falciparum, 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 patient was infected with P. knowlesi, not P. vivax. A pair of primer was designed according to the reported msp-1 partial sequence of P. knowlesi. The partial msp-1 encoding gene was amplified by PCR from the extracted DNA. The amplified fragments were digested by endonuclease, cloned into pET-32a vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the partial msp-1 gene was identical to the sequence of P. knowlesi, which was deposited in the GenBank previously. The recombinant plasmid pET-32a/msp-1 was transformed into E. coli BL21(DE3), and then the positive recombinant clone was expressed by induction with IPTG. The expressed E. coli BL21(DE3) was analyzed by SDS-PAGE gel electrophoresis. A 31 kDa fusion protein band could be discerned. E. coli BL21(DE3) containing recombinant msp-1 was disintegrated with supersonic, then centrifuged. SDS-PAGE gel electrophoresis showed that the msp-1 is expressed in E. coli as precipitation. The protein was purified by Ni-NTA affinity chromatography column. Western blot result showed that the purified protein could react with the blood serum from the patient infected with P.falciparum and its molecular weight accorded with the theoretical expectation. This is the first report of naturally human infection of P. knowlesi in China,and also established the basis for molecular diagnosis and differentiation of Plasmodium species. Further working idea for identifying the P. vivax multinucleatum, and the importance of human infection of simian malaria are discussed.展开更多
文摘Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the disease is estimated to be 350～500 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. Since prevention of malaria epidemics hasn’t achieved tremendous success, emergence of primate malaria infection in human makes the situation even 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 belongs to primate malaria. P. knowlesi usually infects monkeys in nature and is shown to be infective 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 for atypical form of P. vivax 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 the rings 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-third 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 conclude 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℃ 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.falciparum, 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 patient was infected with P. knowlesi, not P. vivax. A pair of primer was designed according to the reported msp-1 partial sequence of P. knowlesi. The partial msp-1 encoding gene was amplified by PCR from the extracted DNA. The amplified fragments were digested by endonuclease, cloned into pET-32a vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the partial msp-1 gene was identical to the sequence of P. knowlesi, which was deposited in the GenBank previously. The recombinant plasmid pET-32a/msp-1 was transformed into E. coli BL21(DE3), and then the positive recombinant clone was expressed by induction with IPTG. The expressed E. coli BL21(DE3) was analyzed by SDS-PAGE gel electrophoresis. A 31 kDa fusion protein band could be discerned. E. coli BL21(DE3) containing recombinant msp-1 was disintegrated with supersonic, then centrifuged. SDS-PAGE gel electrophoresis showed that the msp-1 is expressed in E. coli as precipitation. The protein was purified by Ni-NTA affinity chromatography column. Western blot result showed that the purified protein could react with the blood serum from the patient infected with P.falciparum and its molecular weight accorded with the theoretical expectation. This is the first report of naturally human infection of P. knowlesi in China,and also established the basis for molecular diagnosis and differentiation of Plasmodium species. Further working idea for identifying the P. vivax multinucleatum, and the importance of human infection of simian malaria are discussed.
