Department of Biochemistry & Biotechnology, University of Gujrat, Gujrat, Pakistan.
Department of Medical Laboratory Technology, University of Haripur, Haripur, KPK, Pakistan.
Malar J. 2018 Dec 17;17(1):471. doi: 10.1186/s12936-018-2620-y.
In Pakistan, artesunate (AS) in combination with sulfadoxine-pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan.
A total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing.
The prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R + 108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R + 108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance.
The absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS + SP being effective in Pakistan.
在巴基斯坦，青蒿琥酯（AS）联合磺胺多辛-乙胺嘧啶（SP）是治疗无并发症恶性疟原虫疟疾的推荐疗法。监测抗疟药物耐药性的分子标志物对于早期发现和遏制寄生虫对治疗的耐药性至关重要。目前，尚无关于来自巴基斯坦的恶性疟原虫分离株的青蒿素耐药性（K13 突变）的分子标志物的数据。在这项研究中，估计了巴基斯坦不同地区与 SP 和青蒿素耐药性相关的突变的流行率。
从 2016 年 1 月至 2017 年 2 月，从巴基斯坦的 16 个不同地点共采集了 845 份经显微镜或快速诊断检测呈疟原虫阳性的血液样本。其中 300 份样本经 PCR 检测呈恶性疟原虫阳性。通过焦磷酸测序鉴定恶性疟原虫二氢叶酸还原酶（pfdhfr）和二氢蝶酸合成酶（pfdhps）基因的多态性，通过 Sanger 测序鉴定 Pfk13 基因的螺旋桨结构域中的多态性。
PfDHFR 108N 和 59R 突变的流行率分别为 100%和 98.8%，而 PfDHFR 50R 和 51I 突变的流行率分别为 8.6%。PfDHFR 位置 164 未观察到突变。在 PfDHPS 中，436、437 和 613 位突变的流行率分别为 9.9%、45.2%和 0.4%。PfDHPS 位置 540 和 581 未发现突变。双 PfDHFR 突变体（59R+108N）的流行率范围为 93.8%至 100%，而在巴基斯坦不同地区，同时携带 PfDHFR 59R+108N 突变和 PfDHPS 437G 突变的寄生虫流行率范围为 9.5%至 83.3%。PfK13 螺旋桨结构域观察到 9 个非同义突变和 4 个同义突变，均与验证有助于青蒿素耐药性的突变无关。
缺乏高度耐药的 PfDHFR/PfDHPS 五重突变体寄生虫以及缺乏与青蒿素耐药性相关的 PfK13 突变与 AS+SP 在巴基斯坦有效是一致的。
