In Vitro Sensitivity Of Plasmodium Vivax To Artesunate In China-myanmar Border

Objectives:To investigate artesunate susceptibility of the Plasmodium vivax isolates in China-Myanmar border and to analyze association between drug sensitivity and Pvcrt-o, Pvmdr1 gene polymorphismMethods:(1) P. vivax-infected blood samples which were confirmed by microscopy and PCR were collected and short term cultured in vitro. The sensitivity of P. vivax to artesunate was determined by calculating the IC50 results of each P. vivax isolates.(2)PCR was used to amplify specific fragments of Pvcrt-o, after sequencing, gene mutation sites were determined by comparing with referential strain of Pvcrt-o sequence(AF314649).(3) Investigate the relation of IC50 values of P. vivax isolates to artesunate between mutated and wild type of Pvcrt-o, Pvmdr1.Results :(1) The artesunate sensitivity(IC50) of 37 P. vivax samples in China-Myanmar border distributed range from 0.1196 to 11.0300 n M, and its 95%Confidential Interval(CI) was 2.172-4.271 n M. The geometric mean was 3.045 n M, the Median was 3.468 n M. The geometric mean results were higher than the literature reported in Indonesia, South Korea and central China.(2) After successful amplification and sequencing of Pvcrt-o gene from above 37 P. vivax samples. The sequencing results were compared to referential strain(AF314649). There are 5isolates were found carrying R317 W mutation(14.0%), three isolates were found carrying E299 K mutation(5.4%), one isolates was found carrying G298 E mutation( 2.7%), one isolates was found carrying S279 C mutation( 2.7%).(3) There was no significant difference of IC50 to artesunate between the Pvcrt-o gene W317 mutation isolates with R317 wild type isolates(P>0.05). There was also no significant difference of IC50 to artesunate between the Pvcrt-o gene K299 mutation isolates with E299 wild type isolates(P>0.05).(4) According to our previous research on Pvmdr1 gene of same samples used in this research, there were 22 Pvmdr1 gene sequencing results used for comparison. There was no significant difference of IC50 to artesunate between the Pvmdr1 gene F976 mutation isolates with Y976 wild type isolates(P>0.05). There was also no significant difference of IC50 to artesunate between the Pvmdr1 gene L1076 mutation isolates with F1076 wild type isolates(P>0.05). And there were no significant differences of IC50 were found between F976, L1076 double mutation and wild type P. vivax samples.Conclusions:(1) There were signs of decreasing susceptibility of P. vivax isolates to artesunate in China-Myanmar border area.(2) There were four mutations(R317W,E299 K, G298 E and S279C) in Pvcrt-o gene of P. vivax isolates found in China Myanmar border areas.(3) There were not relation found between decreasing susceptibility of P. vivax isolates to artesunate and the mutations in Pvcrt-o, Pvmdr1(R317W and E299 K in Pvcrt-o, F1076 L and Y976 F in Pvmdr1) in China-Myanmar border areas.