A genomic epidemiological research on a drug resistant malaria spreading in Asian countries.

恶性疟原虫氯喹耐药转运蛋白（PfCRT）第76位密码子由Lys突变为Thr与氯喹耐药相关，基因型可作为监测氯喹耐药疟疾分布和频率的分子标记。本研究从耐药菌株的混合培养中筛选耐药菌株，（K1，其显示K76 T）和敏感菌株使用实时PCR分析，在体内很好地观察到在低氯喹压力（<80 nM）下的FCR-3（显示76 K）。我们使用来自菲律宾，泰国，越南南部。39株泰国分离株K76 T基因型完全相同，13株菲律宾分离株中有6株为混合型。39株越南分离株中，27株为CQ敏感基因型（编码CVMNK），10株为CQ耐药基因型（编码CVIDT、CVET、CVMDT）。对越南分离物的MS DNA分析表明，敏感菌株的MS DNA多态性较高，而抗性菌株的MS DNA多态性较低。其中一个CQ抗性分离株（CViet）与泰国的K1（CQ抗性株; CViet）具有不同的MS DNA图谱。其他CQ抗性菌株类型（CVIDT、CVMDT）的MS DNA带型相同，且与CQ敏感菌株和K1菌株的MS DNA带型不同。这些结果表明，越南的CQ抗性菌株的起源可能不是由于从泰国引进的菌株。相反，这表明CQ耐药突变可能发生在越南。

A mutation from Lys to Thr in the codon 76 of Plasmodium falciparum chloroquine resistant transporter protein (PfCRT) is associated with CQ resistance, and the genotype is used as a molecular marker to monitor the distribution and frequency of the CQ resistant malaria. In the present study, selection of the resistant strain in mixed cultures of a drug resistant strain (K1 which shows K76T) and a susceptible strain (FCR-3 which shows 76K) under a low chloroquine pressure (<80nM) was nicely observed in vivo using real-time PCR analysis.Then, we analyzed the frequency of the mutation in codon 72-76 of the pfcrt gene and 3 microsatellite (MS) DNA markers flanking the pfcrt, using P. falciparum isolates from the Philippines, Thailand, and southern part of Vietnam. Thirty-nine isolates from Thailand showed identical K76T but 6 out of 13 isolates from the Philippines showed mixed type. Twenty-seven of 39 isolates from Vietnam showed CQ susceptible genotype coding CVMNK, while other 10 did CQ resistance type (CVIET, CVIDT, CVMDT). The other 2 isolates were proved to be mixed with CQ susceptible/resistant.In the further analysis of the Vietnam isolates, the MS DNA markers of the CQ susceptible isolates were revealed to be highly polymorphic, while those of CQ resistant isolates were less polymorphic. One of the CQ resistant isolates (CVIET) had a different MS DNA pattern from K1 (CQ resistant strain; CVIET) from Thailand. The other CQ resistant isolate types (CVIDT, CVMDT) shared the same MS DNA patterns and these patterns were different from those of the CQ susceptible isolates nor K1. These results showed that the origin of the CQ resistant isolates in Vietnam might not be due to an introduction of the isolates from Thailand. Instead, it is suggested that the CQ resistant mutations possibly occurred within Vietnam.