尼泊尔两个地区间日疟原虫和恶性疟原虫抗疟药物耐药性的分子标志物流行情况。
Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal.
机构信息
Centre for Medical Parasitology, Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
出版信息
Malar J. 2011 Apr 1;10:75. doi: 10.1186/1475-2875-10-75.
BACKGROUND
Sulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been used in treatment of falciparum and vivax malaria in Nepal. Recently, resistance to both drugs have necessitated a change towards artemisinin combination therapy (ACT) against Plasmodium falciparum in highly endemic areas. However, SP is still used against P. falciparum infections in low endemic areas while CQ is used in suspected cases in areas with lack of diagnostic facilities. This study examines the prevalence of molecular markers of CQ and SP resistance in P. falciparum and Plasmodium vivax to determine if high levels of in vivo resistance are reflected at molecular level as well.
METHODS
Finger prick blood samples (n=189) were collected from malaria positive patients from two high endemic districts and analysed for single nucleotide polymorphisms (SNPs) in the resistance related genes of P. falciparum and P. vivax for CQ (Pfcrt, Pfmdr1, Pvmdr1) and SP (Pfdhfr, Pfdhps, Pvdhfr), using various PCR-based methods.
RESULTS AND DISCUSSION
Positive P. vivax and P. falciparum infections were identified by PCR in 92 and 41 samples respectively. However, some of these were negative in subsequent PCRs. Based on a few P. falciparum samples, the molecular level of CQ resistance in P. falciparum was high since nearly all parasites had the Pfcrt mutant haplotypes CVIET (55%) or SVMNT (42%), though frequency of the Pfmdr1 wild type haplotype was relatively low (35%). Molecular level of SP resistance in P. falciparum was found to be high. The most prevalent Pfdhfr haplotype was double mutant CNRNI (91%), while frequency of Pfdhps double mutant SGEAA and AGEAA were 38% and 33% respectively. Combined, the frequency of quadruple mutations (CNRNI-SGEAA/AGEAA) was 63%. Based on P. vivax samples, low CQ and SP resistance were most likely due to low prevalence of Pvmdr1 Y976F mutation (5%) and absence of triple/quadruple mutations in Pvdhfr.
CONCLUSIONS
Based on the limited number of samples, prevalence of CQ and SP resistance at molecular levels in the population in the study area were determined as high in P. falciparum and low in P. vivax. Therefore, CQ could still be used in the treatment of P. vivax infections, but this remains to be tested in vivo while the change to ACT for P. falciparum seems justified.
背景
在尼泊尔，磺胺多辛-乙胺嘧啶（SP）和氯喹（CQ）一直用于治疗恶性疟原虫和间日疟原虫。最近，这两种药物的耐药性使得在高度流行地区必须改用青蒿素为基础的联合治疗（ACT）来治疗恶性疟原虫。然而，SP 仍用于低流行地区的恶性疟原虫感染，而 CQ 则用于缺乏诊断设施的地区的疑似病例。本研究检测了恶性疟原虫和间日疟原虫中 CQ 和 SP 耐药的分子标志物的流行情况，以确定体内耐药水平是否也反映在分子水平上。
方法
从两个高度流行地区的疟疾病例中采集指尖血样（n=189），并采用各种基于 PCR 的方法分析恶性疟原虫和间日疟原虫中与耐药相关的基因中的单核苷酸多态性（SNP），用于 CQ（Pfcrt、Pfmdr1、Pvmdr1）和 SP（Pfdhfr、Pfdhps、Pvdhfr）。
结果与讨论
通过 PCR 在 92 个样本中鉴定出阳性间日疟原虫和 41 个样本中的阳性恶性疟原虫。然而，随后的 PCR 中有些样本呈阴性。根据少数恶性疟原虫样本，恶性疟原虫中 CQ 耐药的分子水平很高，因为几乎所有寄生虫都具有 Pfcrt 突变单倍型 CVIET（55%）或 SVMNT（42%），尽管 Pfmdr1 野生型单倍型的频率相对较低（35%）。发现恶性疟原虫中 SP 耐药的分子水平很高。最常见的 Pfdhfr 单倍型是双突变 CNRNI（91%），而 Pfdhps 双突变 SGEAA 和 AGEAA 的频率分别为 38%和 33%。联合起来，四突变（CNRNI-SGEAA/AGEAA）的频率为 63%。根据间日疟原虫样本，CQ 和 SP 耐药率低可能是由于 Pvmdr1 Y976F 突变（5%）的流行率低以及 Pvdhfr 中不存在三/四突变。
结论
基于有限的样本数量，研究地区人群中恶性疟原虫和间日疟原虫中 CQ 和 SP 耐药的分子水平被确定为恶性疟原虫高，间日疟原虫低。因此，CQ 仍可用于治疗间日疟原虫感染，但这仍需在体内进行测试，而改用 ACT 治疗恶性疟原虫似乎是合理的。
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