Tibet, Nyingchi Plasmodium Parasite Molecular Biology Identified

Object: To identify the Plasmodium.spp of the blood samples on filter paper isolated from Linzhi Prefecture of Tibet, China, and calculate the residents infected rate (RIR), making a comprehensive analysis on these malaria-endemic areas. To detect the polymorphism of Plasmodium vivax merozoite surface protein-1 (PvMSP-1) and the Plasmodium vivax circumsporzoite protein (PvCSP), for developing scientific malaria control strategies and measures.Methods: (1) Blood samples on filter paper were collected in Beibeng Town, Linzhi Prefecture in 2006. Chelex-100 boiled method was used to extract their DNAs. Then multiple-PCR was used to amplify DNAs. All the positive samples were re-validated by nested-PCR. (2) The RIR was calculated based on the PCR results. (3) The DNAs were extracted from the blood samples of the vivax malaria patients in Linzhi and Huaiyuan (Anhui Province) in 2007, using the Chelex-100 boiling method. (4) Nested-PCR was done to detect the PvCSP and PvMSP-1. All the amplified DNA products were sequenced. (5) The sequences of PvCSP and PvMSP-1 in Linzhi were compared to GenBank by BLASTN and BLASTX. Then N-J trees were built for all the sequenced genomic fragments and the sequences retrieved from GenBank. Finally, Fisher's exact tests were done between the PvMSP-1 genotype frequency in Linzhi and other 3 areas in China.Results: (1) Three hundred and eighty-eight samples were collected in Beibeng Town, Linzhi Prefecture. Among them, 3 P.v positive samples were identified, and the RIR is 0.77%. (2) Thirty-five blood samples were collected from the patients in Linzhi Prefecture and another one in Anhui. (3)The genomic DNA sequences of PvMSP-1 and PvCSP were amplified from the 39 P.v samples. The DNA sequences of PvMSP-1 can be clustered to four groups: A (Sal-1 group), B, C, and D (Belem group). While the amino acid sequences can be clustered to three groups: the Sal-1 group, the IIPR group, and the Belem group. The constituent ratios of genotypes in Linzhi Prefecture are different from that in Yunnan, Hainan and Anhui provinces (p-value=0.0163, 0.1752, P=0.0037 respectively). The PvCSP sequences in Linzhi Prefecture can be clustered as a single group and are similar to VK210 strains. Conclusion: (1) All the Plasmodium.spp positive samples in Linzhi Prefecture were Plasmodium vivax. No Plasmodium falciparum was identified in the samples. The results suggest that Plasmodium vivax is the major cause for the malaria epidemiology in Linzhi Prefecture of Tibet, China. A long-term observation is needed to determine whether there existed any case of Plasmodium falciparum. (2) This is the first systematic analysis on the polymorphism of PvCSP and PvMSP-1 in Linzhi. The sub-types of the P.v strains were classified on molecular level. It provides a base-line data for the molecular epidemiological study in Linzhi Prefecture.