Centre for Medical Parasitology, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.
Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
Malar J. 2019 Jul 26;18(1):252. doi: 10.1186/s12936-019-2894-8.
Large-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging.
Between 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13.
The study found a high frequency of the pfmdr1 codon 86N at 88-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation.
This study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.
大规模监测抗疟药物耐药性的分子标志物是一种有吸引力的耐药监测方法，可以补充在后勤方面具有挑战性的治疗效果研究。
在 2014 年至 2017 年间，本研究从几内亚比绍比绍的两个卫生中心采集了用于常规临床护理的疟疾快速诊断检测 (RDT)。为了获得流行病学见解，RDT 与年龄和性别患者数据一起收集。从两个地点中的一个（n=2184）采集了一部分阳性 RDT 进行疟原虫 DNA 含量检测。那些通过 PCR 检测为疟原虫 DNA 阳性的人（n=1390）用于文库制备、定制设计的双重索引和针对 PfCRT、Pfmdr1、PfDHFR、PfDHPS 和 Pfk13 的下一代 Miseq 靶向测序。
该研究发现 pfmdr1 密码子 86N 的频率很高，为 88-97%，CVMNK 单倍型的 pfcrt 野生型显著减少，pfdhfr/pfdhps 四重突变体的水平升高，2014 年至 2017 年间从 33%到 51%不等。未发现表明对青蒿素耐药的多态性。人口统计学数据表明，很大一部分年轻人（66%，四分位间距 11-28 岁）患有疟原虫感染。虽然总共在单个 Illumina Miseq 流动池中成功分析了 5532 个基因片段，但文库制备的 PCR 阳性率因不同的扩增子而有很大差异，从 13%到 87%不等。此外，在进行文库制备之前，对样本进行疟原虫 DNA 含量的预筛选是必要的。
本研究证明了使用剩余的临床材料（使用过的 RDT）进行大规模分子监测的概念，同时还包含了在进行此类监测时方法和分析方面的固有复杂性。在提取 DNA 之前 RDT 的储存以及感染的寄生虫血症等因素可能会影响是否可以成功分析寄生虫 DNA，并且被认为是数据产量不理想的部分原因。然而，鉴于在基础设施差、经济困难、缺乏受过教育的工作人员甚至政治动荡加剧仍然是进行临床研究的主要障碍的情况下，对抗疟药物耐药性进行分子监测是必要的，因此，尽管输出不理想，从使用过的 RDT 中获取必要的数据仍然是一种可行、负担得起且合理的方法。
