Giraldo L E, Acosta M C, Labrada L A, Praba A, Montenegro-James S, Saravia N G, Krogstad D J
Corporación Centro Internacional de Entrenamiento e Investigaciones Médicas, Cali, Colombia.
Am J Trop Med Hyg. 1998 Jul;59(1):124-8. doi: 10.4269/ajtmh.1998.59.124.
Several point mutations in the dihydrofolate reductase (DHFR) gene of Plasmodium falciparum have been correlated with in vitro anti-folate drug resistance of laboratory and field isolates. Furthermore, two different point mutations that generate amino acid substitutions at the same position of the enzyme have been observed in all the isolates studied to date. These point mutations change a serine (Ser-108) in the wild type to an asparagine (Asn-108 mutation) or to a threonine (Thr-108 mutation). Using the polymerase chain reaction (PCR), it is possible to identify isolates that present these mutations. We used a mutation-specific PCR to screen 71 samples from several geographic locations of Colombia for the Asn-108 mutation (pyrimethamine resistance). In this initial screening 53 of 71 yielded amplification product with the DHFR mutation-specific primers. We further analyzed the 18 samples that did not amplify using a mutation-specific nested PCR. Of those 18 samples, seven amplified with primers specific for the Thr-108 mutation (proguanil resistance), one with the wild type (Ser-108), and 10 did not amplify. Of these 10 samples, three were identified as P. falciparum using a species-specific diagnostic nested PCR base on sequences from the small ribosomal RNA subunit gene. Overall, 51.6% of the samples amplified for the Asn-108 mutation, 10.9% for the Thr-108 mutation, 35.9% with the wild type specific primer, and 4.8% did not amplify with any of the DHFR primers. We observed variability in the frequency of the mutation between the different geographic location. The frequency of the Asn-108 and Thr-108 mutations in the state of Narifio was 25% each, while in Valle del Cauca the frequencies were 59% and 11%, respectively. These results contrast with observations in Brazil in which the Asn-108 mutation was found in 90% of the blood samples screened.
恶性疟原虫二氢叶酸还原酶（DHFR）基因中的几个点突变已与实验室和现场分离株的体外抗叶酸药物耐药性相关。此外，在迄今为止研究的所有分离株中都观察到了两个不同的点突变，它们在酶的同一位置产生氨基酸替代。这些点突变将野生型中的丝氨酸（Ser-108）变为天冬酰胺（Asn-108突变）或苏氨酸（Thr-108突变）。使用聚合酶链反应（PCR），可以鉴定出存在这些突变的分离株。我们使用突变特异性PCR从哥伦比亚几个地理位置的71个样本中筛选Asn-108突变（乙胺嘧啶耐药性）。在这次初步筛选中，71个样本中有53个用DHFR突变特异性引物产生了扩增产物。我们进一步使用突变特异性巢式PCR分析了18个未扩增的样本。在这18个样本中，7个用Thr-108突变特异性引物扩增（氯胍耐药性），1个用野生型（Ser-108）扩增，10个未扩增。在这10个样本中，3个使用基于小核糖体RNA亚基基因序列的物种特异性诊断巢式PCR鉴定为恶性疟原虫。总体而言，51.6%的样本扩增出Asn-108突变，10.9%扩增出Thr-108突变，35.9%用野生型特异性引物扩增，4.8%用任何DHFR引物都未扩增。我们观察到不同地理位置之间突变频率存在差异。纳里尼奥州Asn-108和Thr-108突变的频率均为25%，而在考卡山谷，频率分别为59%和11%。这些结果与在巴西的观察结果形成对比，在巴西，90%的筛查血样中发现了Asn-108突变。
