| Objective:To investigate Chloroquine of seven kinds of antimalarials susceptibility of Plasmodium Vivax in China-Myanmar Border, and to analyze related gene polymorphism.Methods:1.Short term culturing the single species infected Plasmodium vivax isolates, which confirmed by microscopy and PCR, then using in vitro microtest technique to calculate the IC50 values for Chloroquine, Artesunate, Dihydroarte-misinine, Mefloquine, Quinine, Pyronaridine Phosphate, Piperaquine Phosphate drug sensitivity.2. Insect plant DNA extraction of Plasmodium vivax, application of polymerase chain reaction (PCR) amplification Pvmdrl, Pvcrt-o, Pvdhfr, Pvdhps four genes, respectively. After sequencing, sequence alignment, with standard strains of Salvador I get its genetic mutations. Real-Time PCR method for Pvmdr1 gene copy number increase, compared with Pvaldolese gene, get its copy number.3. Analyse the correlation between in vitro drug sensitivity and gene polymorp-hism, Analyse the correlation between Pvmdr1 copy number and drug resistane. Results:1.In vitro drug sensitivity results:58 isolates of Plasmodium vivax had been cultrue in vitro,26 isolates development to the schizont stage. The geometric mean IC50 of Chloroquine, Artesunate, Dihydroartemisinine, Mefloquine, Quinine, Pyronaridine Phosphate, Piperaquine Phosphate were 65.52、9.65、7.42、33.15、76.73、 34.13、41.57, respectively.95% CI of geo. Mean are 41.76～102.8、6.07～15.34、 4.68～11.8、23.46～46.85、47.63～123.6、19.66～59.28、28.53～60.59, respectively. Median were 81.37、9.78、7.52、35.88、45.85、28.51、60.90, respectively. There were three insensitive isolates of Chloroquine, the insensitive Chloroquine rate was 11.45%.2. The gene polymorphism result:2.1 Pvmdrl gene:the 166 DNA samples were used for amplification of Pvmdrl gene the full-length 4606bp fragment. In this study,there were nine mutation of Pvmdrl, S513R、T529、G698S、M908L、T958M、 K997R、F1076L、S1358、K1393N, the rate were 11.45%、89.16%、98.8%、100%、 100%、0.6%、76.51%、17.47%、19.28%, respectively. But there has not been found mutations in Y976F. L1022、F1233、E1396. Y976F and F1076L loci mutation associated with chloroquine sensitivity mutations were 0、76.51%, respectively. The number of wild plants both two mutations were 39 (23.49%).2.2 Pvcrt-o gene:the 166 DNA samples were used for amplification of Pvcrt-o gene 1702 bp fragment. All samples in this study all happened in P297 synonymous mutations, no other mutations found.2.3 Pvdhps gene:the 166 DNA samples were used for amplification of Pvdhps gene 767 bp fragment. In this study, there were four mutations of Pvdhps, S382A/C、 A383G、K512M and A553G, there were two common mutation of Pvdhps, A383G、 A553G, the rate were 64.45%、33.12%, respectively. The common haplotype were SGKAV (37.35%), SGKGV (23.49%), and wild-type SAKAV (29.52%). Four mutation AGMGV, double mutation SAMGV and single mutation SAKGV rate were 3.61%、0.60%、5.42%, respectively.2.4 Pvdhfr gene:the 166 DNA samples were used for amplification of Pvdhfr gene 632 bp fragment. In this study, there were four mutations of Pvdhfr, F57L/I、S58R, T61M and S117N/T, there were three common mutation of Pvdhfr, S58R、T61M、S117N/T, the rate were 50%、38.55%、36.75%, respectively. The common haplotype were wild-type IPFSTSI (50%), IPLRMTI (34.94%). Double mutation IPFRTTI, IPFRTNI and three mutation IPLRMSI,the rate were 1.81%、9.64%、3.61%, respectively.2.5 Pvmdrl copy number:The result for 26 isolates of Real-Time PCR was all single copy.3.In vitro and molecular determination results of corcorrelation test results:The result of IC50 values analysis with Pvmdrl gene polymorphism show that there were no correlation between chloroquine IC50, the number of sensitive and insensitive isolates and seven mutations(P>0.05), respectively.Conclusions:1. It was noticed that isolates from the China-Myanmar border had reduced susceptibility to CQ.2. No corcorrelation was found between chloroquine IC50 values and Pvmdrl mutations in China-Myanmar border; No corcorrelation was found between the number of chloroquine sensitive and insensitive and Pvmdrl mutations in China-Myanmar border; 3. Pvmdrl copy number did not increase.4. No mutation was found of Pvcrt-o gene except P297 synonymous mutations in China-Myanmar border.5. The haplotypes of Pvdhfr and Pvdhps in China-Myanmar border were obviously different with those in Central China. |
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