一种新型基于现场的分子检测方法，用于检测已验证的青蒿素耐药性 K13 突变体。

A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants.

机构信息

Medical Diagnostic Discovery Department (MD3), bioMérieux, Grenoble and Marcy l'Etoile, France.

Malaria Molecular Epidemiology Unit, Institute Pasteur in Cambodia, Phnom Penh, Cambodia.

出版信息

Malar J. 2018 Apr 24;17(1):175. doi: 10.1186/s12936-018-2329-y.

DOI:10.1186/s12936-018-2329-y

PMID:29690890

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5916714/

Abstract

BACKGROUND

Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas.

METHODS

A semi-automated workflow based on the easyMAG platform and the Argene Solution (bioMérieux, Marcy l'Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing).

RESULTS

By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients.

CONCLUSION

The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America.

摘要

背景

鉴于青蒿素耐药性从大湄公河次区域传播的风险，应加快在撒哈拉以南非洲进行前瞻性监测。用于监测的分子生物学技术依赖于通过聚合酶链反应（PCR）和 Sanger 测序方法检测已验证的 k13 突变体，但该方法通常在疟疾流行地区不可用。

方法

本研究开发了一种基于 easyMAG 平台和 Argene Solution（生物梅里埃，马西勒托伊勒）的半自动工作流程，作为一种可在国家级操作的基于现场的监测工具，该流程分四个步骤进行。从干血斑（DBS）中检测五种最常见和已验证的 k13 突变体（Y493H、I543T、R539T、F446I 和 C580Y）的这种工具的临床和分析性能与金标准方法（PCR 和 Sanger 测序）进行了比较。

结果

通过使用 ARMS（扩增受阻突变系统）策略，在 3 个单独的实时 PCR 双管（IC 作为内部对照/I543T、C580Y/Y493H 和 F446I/R539T）中找到了最佳的多重检测选项，检测限范围从 50（C580Y）到 6.25 个寄生虫/µL（Y493H）。在现场条件下，使用从柬埔寨、缅甸和非洲（乍得）收集的 642 份来自有症状患者和无症状个体的临床 DBS（干血斑），生物梅里埃开发的 K13 bMx 原型检测的总体敏感性和特异性均≥90%。在来自有症状患者的样本中，所有 k13 突变体的 ROC 曲线下面积估计值均大于 0.90。

结论

该研究开发的 K13 即用型 bMx 原型检测方法被最终用户认为是一种易于使用的检测方法（比 K13 参考检测方法用时更短），且易于解释，因此被认为需要较少的预算规划和更少的后勤限制。其优异的性能使该原型检测方法成为一种可靠的筛查工具，可用于被认为存在已验证的 k13 突变体出现或传播风险的疟疾流行国家。还需要进行更多的多地点研究，以评估 K13 bMx 原型检测在不同流行病学背景（如非洲、印度或南美洲）中的性能。