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

恶性疟原虫氯喹抗性转运蛋白(PfCRT)第76位密码子Lys突变为Thr突变与CQ抗性有关，该基因可作为监测CQ抗性疟疾分布和频率的分子标记。在本研究中，利用实时荧光定量聚合酶链式反应技术，在低氯喹压力(80 Nm)下对耐药株(显示K76T的K1株)和敏感株(显示76K的FCR-3株)混合培养的抗性株进行了体内筛选。然后，我们利用来自菲律宾、泰国和越南南部的恶性疟原虫分离株，分析了pfcrt基因72-76密码子和pfcrt两侧的3个微卫星(MS)DNA标记的突变频率。来自泰国的39株分离株具有相同的K76T，但来自菲律宾的13株分离株中有6株表现为混合型。39株越南分离株中，27株为CQ敏感基因，编码CVMNK，另外10株为CQ抗性类型(CVIET、CVIDT、CVMDT)。对越南分离物的进一步分析表明，CQ敏感分离物的MS DNA标记具有较高的多态性，而CQ抗性分离物的MS DNA标记具有较低的多态性。其中一个CQ抗性分离物(CVIET)的MS DNA图谱与来自泰国的K1(CQ抗性株系；CVIET)不同。其他CQ抗性分离物类型(CVIDT、CVMDT)具有相同的MS DNA图谱，而这些图谱与CQ敏感分离物或K1不同。这些结果表明，越南CQ抗性分离物的起源可能不是由于从泰国引进的分离物。相反，推测CQ抗性突变可能发生在越南境内。

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.