Reteng Patrick, Vrisca Visia, Sukarno Inka, Djarkoni Ilham Habib, Kalangi Jane Angela, Jacobs George Eduardo, Runtuwene Lucky Ronald, Eshita Yuki, Maeda Ryuichiro, Suzuki Yutaka, Mongan Arthur Elia, Warouw Sarah Maria, Yamagishi Junya, Tuda Josef
Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
Department of Medical Genome Sciences, University of Tokyo, Kashiwa, Chiba, 277-8562, Japan.
BMC Res Notes. 2017 Apr 4;10(1):147. doi: 10.1186/s13104-017-2468-1.
Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat; however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia.
Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%).
Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.
疟疾仍然是对人类健康的主要威胁之一。有效的抗疟药物的开发降低了这种威胁；然而，作为疟疾病因的耐药恶性疟原虫的出现令人担忧。抗疟药物氯喹曾被有效使用，但耐药寄生虫已在全球传播。有趣的是，据报道在某些情况下停用该药物会导致易感寄生虫数量增加。我们研究了在印度尼西亚停用氯喹后，恶性疟原虫中与抗疟药物氯喹耐药相关的基因组多态性的流行情况。
2010年从印度尼西亚北苏拉威西省的95名疟疾患者中采集血样。提取寄生虫DNA，并通过聚合酶链反应 - 限制性片段长度多态性（PCR - RFLP）分析pfcrt和pfmdr1。同时，使用Illumina MiSeq对相同基因进行多重扩增子测序。在通过显微镜诊断为恶性疟原虫感染的59例病例中，PCR - RFLP分析明确鉴定出44例pfcrt中的76T基因型。测序分析验证了这44例病例中鉴定出的基因型，并表明周围基因组区域的单倍型仅为SVMNT。成功获得了51个样本的pfmdr1结果，两种方法获得的基因分型结果完全一致。在pfmdr1中，45例（88.2%）观察到86Y突变基因型。
我们的结果表明，即使在该地区停用氯喹6年后，突变基因型的流行率仍然占主导地位。耐药相关位点的多样化单倍型，可能涉及适应度代价、氯喹的未经授权使用和/或停药后的短时间，可能是观察到的高流行率持续存在的原因。
