Kachin Wa Plasmodium Falciparum Chloroquine Resistance In Five Different Anti-malarial Drugs In Vitro Assay

[Objective] To explore the application of pfcrt of plasmodium falciparum isolates from Kachin and Wa State, Myanmar.[Method]1. Amplify the specified gene fragment of MSP1,MSP2, and Glurp separately with nested PCR, then do genotyping research so as to select single-infected isolates.2. Amplify two fragments of pfcrt with nested PCR, do sequence determination and compare with that of3D7, and finally we find the mutation sites.[ResμLts]1. Amplification of MSP2of52isolates by doing nested PCR, then do gel electrophoresis with the obtained PCR amplification products, and finally we sift39single-infected isolates.2.Amplification of pfcrt of39single-infected isolates, we found mutation at site71,72,74,75,76and220, and the mutation rate was respectively23.1%,23.1%,92.3%,89.7%,100%,100%. The discovered mutant types includedV71V, C72S, M74I, N75E, K76T, A220S.[Conclusion]1. Genotype MSP1,MSP2,Glurp of the39single-infected isolates, of which13isolates from Kachin State and26isolates from Wa State.2. Of these39isolates we found mutations at site71,72,74,75,76and220. And the mutation rate of the genotype of isolates CVIETS from Kachin State are much higher than that of Wa State(P=0.003<0.05). [Objective] To do drug-resistance assay of the falciparum isolates obtained from China-Myanmar border areas-Wa State and Kechin State, Myanmar., we select five antimalarials:chloroquine, piperaquine, quinine, Artesunate and dihydroartemisinine to do this experiment;[Methods] CμLturing the sing-infected isolates, then after synchronizing the life cycle of P.falciparum we do drug-resistance assay with the five above-mentioned antimalarials and finally obtain the ICsovalues.[ResμLts] The IC50values of13isolates from Kachin State on the five above-mentioned antimalarials was respectively690.9±243.2nM for Chloroquine,65.71±23.8nM for piperaquine,1028±466.5nM for quinine,18.8±10.5nM for Artesunate and4.6±3.2nM for dihydroartemisinine; while the IC50values of26isolates from Wa State on the five antimalarials was respectively340.1±156.9nM for chloroquine,35.7±21.2nM for piperaquine,385.4±161.8nM for quinine,9.4±5.8nM for Artesunate and2.5±1.6nM for dihydroartemisinine; As a control group, we selected8international-standard isolates to do this drug-resistance assay, that is to obtain the IC50values of3D7, HB3,7G8, Dd2, D10, K1and D6on the five antimalarials which was respectively205.5±225.9nM for chloroquine,15.3±13.2nM for piperaquine,387±267.9nM for quinine,14±6.6for Artesunate and2.4±1.8nM for dihydroartemisinine.[Conclusion]1. Chloroquine:Of the39isolates there is only one was intermediate-sensitivity, and the other38isolates(IC50≥100nM) were seriously resistant to chloroquine. It is worth mentioning, the ICsovalues of isolates N11-1733and N11-966were both above1000nM which belong to the referential severe-resistance. There is positive correlation between the ex vivo resistance of chloroquine and the polymorphism of pfcrt (P<0.005)2.Piperaquine:There is only one of the39isolates, that is F07-12, remained sensitive to piperaquine (IC50<7.5nM),while the other38isolates are resistant to piperaquine(IC50>12.8nM), of which the isolate N11-966was seriously resistant to piperaquine(IC50>100nM).3.Quinine:17of39isolates has decreased sensitivity to quinine(IC50>500nM), of which isolate F07-12was severely resistant to quinine (IC50>2000nM). While22isolates were still sensitive to this antimalarial(IC50<500nM).4. Artesunate:On account that different referential control value may cause much effect on our experiment conclusion, we defined the threshold value of resistance using statistical method and finally obtained the control value29.6nM. Of these39isolates there were38isolates still sensitive(ICso<29.6nM) and one isolate had decreased its sensitivity(IC50>40nM).5. Dihydroartemisinine:For this antimalarial, we also used formula (Mean±2×SD) to define the critical value of resistance and obtained the control value7.7nM. There were37of the39isolates were still sensitive(ICso<7.7nM), and the other2had already decreased their sensitivity(IC50≥7.7nM).6. Cross-resistance:There existed cross-resistance between chloroquine and piperaquine, and it also occurred among the other three antimalrial drug-quinine, Artesunate and dihydroartemisinine.7. We acknowledged that isolates from Wa State and Kachin State were all resistant to CQ, PPQ and QN to some extent and the sensitivity of isolates from Wa State were more sensitive than that from Kachin State; While sensitivity to AS and DHA between the isolates from the two areas were different, that isolates from Wa State were more sensitive than Kachin State. [Objective] Compare the effect on the ICsovalue of isolates that previously treated with two different synchronization methods-4℃refrigeration and5%Sorbitolum.[Methods] With the statistical method Hotelling T2, we use the two above-mentioned synchronization methods to treat the previously-selected8falciparum isolates separately. After that, do drug-resistance assay with4antimalarials-chloroquine, piperaquine, dihydroartemisinine and quinine, and finally obtained the IC50value.[Results] The IC50values of8isolated treated with4℃refrigeration synchronization method were respectively270.8±189.7nM for chloroquine,22.2±12.2nM for piperaquine,1.5±0.9nM for dihydroartemisinine and398.4±162.8nM for quinine; While the IC50values of8isolated treated with5%Sorbitolum synchronization method were respectively261±178.7nM for chloroquine,25.4±15.8nM for piperaquine,1.4±1.0nM for dihydroartemisinine and380.7±162.6nM for quinine.[Conclusions] After did statistical test of Hotelling T2, we made the conclusion that there were no significant differences of the IC50values of8isolates which were separately treated with the two synchronization methods(P=0.894>0.05).