Njokah Muturi J, Kang'ethe Joseph N, Kinyua Johnson, Kariuki Daniel, Kimani Francis T
Jomo Kenyatta University of Agriculture and Technology (JKUAT), P.O. Box 62000-00200, Nairobi, Kenya.
Kenya Medical Research Institute (KEMRI), Centre for Biotechnology Research and Development (CBRD), P.O. Box 54840-00200, Nairobi, Kenya.
Malar J. 2016 Jul 22;15(1):381. doi: 10.1186/s12936-016-1443-y.
Anti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance.
Here, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast.
Exposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene.
This data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro and that this phenotype is achieved by mutations in Pfmdr1, the genetic changes that are also underpinning lumefantrine resistance. This finding is of practical importance, because artemisinin drugs in Kenya are used in combination with lumefantrine for the treatment of malaria.
Artemisinin resistance phenotype as has been shown in this work, is a decrease in parasites susceptibility to artemisinin derivatives together with the parasite's ability to recover from drug-induced dormancy after exposure to drug dosage above the in vivo clinical concentrations. The study surmises that Pfmdr1 may play a role in the anti-malarial activity of artemisinin.
抗疟药物是疟疾预防和治疗的主要重点。以青蒿素为基础的联合疗法（ACT）是世界卫生组织推荐的在疟疾流行地区治疗恶性疟原虫疟疾的一线疗法。此外，在氯喹治疗失败的间日疟原虫疟疾治疗中，ACT的使用也越来越多。青蒿素耐药寄生虫的出现是全球疟疾控制项目面临的严重威胁。因此，治疗和干预策略的成功高度依赖于对耐药性遗传基础的理解。
在此，在体外诱导恶性疟原虫对青蒿素产生耐药性，使其体内水平高于临床相关浓度，并对分子单倍型进行研究。使用QIAamp mini DNA试剂盒提取基因组DNA。通过PCR扩增Pfk13、Pfcrt和Pfmdr1基因的DNA序列，并成功对扩增子进行测序。通过标准双向测序追踪单核苷酸多态性，并在Codon code Aligner Version 5.1和NCBI blast中对照野生型序列读取转录本。
将寄生虫株D6和W2暴露于青蒿素后，寄生虫对青蒿素（W2和D6）和卤泛群（仅D6）的敏感性降低。这些寄生虫对多种青蒿素的半数抑制浓度（IC50）升高，对青蒿素具有两倍以上的耐药性；然而，用标准方法获得的耐药指数明显低于从50μg/ml 48小时药物压力下恢复的寄生虫株预期值。寄生虫敏感性的变化与Pfmdr-1 85K突变有关，这是一种此前从未报道过的突变。保留了Pfcrt-CVMNK基因型（Pfcrt密码子72-76），值得注意的是，该研究在Pfk13基因的编码序列中未检测到任何据报道会降低恶性疟原虫体内敏感性的多态性。
这些数据表明，恶性疟原虫在体外有能力对青蒿素衍生物产生耐药性，并且这种表型是由Pfmdr1突变实现的，这些基因变化也是卤泛群耐药性的基础。这一发现具有实际重要性，因为肯尼亚的青蒿素药物与卤泛群联合用于治疗疟疾。
如本研究所示，青蒿素耐药表型是寄生虫对青蒿素衍生物的敏感性降低，以及寄生虫在暴露于高于体内临床浓度的药物剂量后从药物诱导的休眠中恢复的能力。该研究推测Pfmdr1可能在青蒿素的抗疟活性中起作用。
