Ndung'u Loise, Langat Benard, Magiri Esther, Ng'ang'a Joseph, Irungu Beatrice, Nzila Alexis, Kiboi Daniel
PAUSTI, Jomo Kenyatta University of Agriculture and Technology, Nairobi, 00200, Kenya.
KEMRI- Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute (KEMRI), Nairobi, 00200, Kenya.
Wellcome Open Res. 2017 Jun 20;2:44. doi: 10.12688/wellcomeopenres.11768.2. eCollection 2017.
The human malaria parasite has evolved complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite ANKA as a surrogate of to investigate the mechanisms of amodiaquine resistance. : We used serial technique to select amodiaquine resistance by submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Submission of ANKA to amodiaquine pressure yielded resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED ) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80ºC for one month, the resistant parasite remained stable with an ED of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of revealed His95Pro mutation. No variation was identified in or nucleotide sequences. Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters; , and and Ca /H antiporter. Selection of amodiaquine resistance yielded stable "multidrug-resistant'' parasites and thus may be used to study common resistance mechanisms associated with other antimalarial drugs. Genome wide studies may elucidate other functionally important genes controlling AQ resistance in .
人类疟原虫已经进化出复杂的耐药机制以应对所有现有的抗疟药物。迄今为止，阿莫地喹-青蒿琥酯联合用药是治疗非复杂性疟疾的首选药物之一。在这种联合用药中，作用时间短的青蒿琥酯与作用时间长的阿莫地喹搭配使用，而阿莫地喹的耐药性可能迅速出现，尤其是在高传播地区。在此，我们使用啮齿动物疟原虫ANKA作为人类疟原虫的替代物来研究阿莫地喹耐药机制。我们采用连续传代技术，通过让疟原虫持续接触阿莫地喹来筛选对其的耐药性。然后我们采用4天抑制试验来监测耐药性的出现并确定交叉耐药谱。最后，我们通过PCR扩增、测序以及对靶向基因的mRNA转录本进行相对定量分析，对耐药疟原虫进行基因分型。将ANKA疟原虫置于阿莫地喹压力下，在三十六代内产生了耐药疟原虫。敏感株和耐药株降低90%虫血症（ED90）的有效剂量分别为4.29mg/kg和19.13mg/kg。在-80ºC冷冻一个月后，耐药疟原虫保持稳定，ED90为18.22mg/kg。对阿莫地喹耐药的疟原虫对氯喹（6倍）、蒿甲醚（10倍）、伯氨喹（5倍）、哌喹（2倍）和卤泛群（3倍）也耐药。对……的序列分析显示His95Pro突变。在……或……核苷酸序列中未发现变异。阿莫地喹耐药还伴随着关键转运蛋白、……、……和Ca²⁺/H⁺反向转运蛋白的高mRNA转录本。筛选出对阿莫地喹耐药的疟原虫产生了稳定的“多药耐药”疟原虫，因此可用于研究与其他抗疟药物相关的共同耐药机制。全基因组研究可能会阐明其他控制……中阿莫地喹耐药性的功能重要基因。 （注：原文中部分“……”处信息缺失，无法准确完整翻译）
