Sensitivity Assessment of a Multiplex and Real-Time PCR Protocols for the Detection of Malaria in External Quality Control Samples in the Malaria Reference Center in Greece

Parasitologia Pub Date : 2024-05-17 DOI:10.3390/parasitologia4020013

N. Tegos, Christos Goumenopoulos, Anastasia Mpimpa, Vasilios Papavasilopoulos, S. Beleri, Eleni Patsoula

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Abstract

Background: Accurate malaria diagnosis constitutes a challenging task, necessitating the need for the implementation of targeted and effective diagnostic tools. The purpose of the current study was to evaluate the effectiveness of two different molecular methodologies in terms of sensitivity for the detection of External Quality Assessment (EQA) Plasmodium samples. Methods: A total of 104 lyophilized blood samples from 14 different UK-NEQAS (National External Quality Assessment Site) (2016–2021) and eight WHO-NEQAS distributions (2017–2020) were analyzed. An in-house multiplex PCR protocol, followed by single target real-time PCR protocols for all five Plasmodium species, was implemented. Results: The multiplex PCR had a success rate of 10/16 and 20/28 for P. vivax and P. falciparum species, respectively. On the other hand, the respective results for real-time PCR had a success rate of 13/16 (P. vivax), 28/28 (P. falciparum), 5/8 (P. malariae), 8/10 (P. ovale), and 10/14 (P. knowlesi). Plasmodium falciparum samples displayed the highest sensitivity of detection, 0.02 parasites/μL. Plasmodium vivax samples displayed a 0.1 parasites/μL cutoff value, greater than the respective value for whole blood samples, while P. ovale species displayed a respective cutoff value of 0.05 parasites/μL. Due to the limited number of tested samples, data obtained for P. malariae and P. knowlesi species samples were inconclusive. Conclusions: Real-time PCR comprises a credible molecular methodology in terms of sensitivity assessment and detection of low parasitemia levels of Plasmodium sp. in EQA lyophilized blood samples.

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希腊疟疾参考资料中心检测外部质量控制样本中疟疾的多重和实时 PCR 方案灵敏度评估

背景：准确诊断疟疾是一项具有挑战性的任务，因此需要采用有针对性的有效诊断工具。本研究旨在评估两种不同分子方法在检测外部质量评估（EQA）疟原虫样本灵敏度方面的有效性。研究方法分析了来自 14 个不同的英国-NEQAS（国家外部质量评估点）（2016-2021 年）和 8 个 WHO-NEQAS 分布点（2017-2020 年）的共 104 份冻干血样。实施了内部多重 PCR 方案，然后针对所有五个疟原虫物种实施了单目标实时 PCR 方案。结果显示对于间日疟原虫和恶性疟原虫，多重 PCR 的成功率分别为 10/16 和 20/28。另一方面，实时 PCR 的成功率分别为 13/16（间日疟原虫）、28/28（恶性疟原虫）、5/8（疟原虫）、8/10（卵形疟原虫）和 10/14（克雷西疟原虫）。恶性疟原虫样本的检测灵敏度最高，为 0.02 寄生虫/μL。间日疟原虫样本的临界值为 0.1 个寄生虫/μL，高于全血样本的临界值，而卵形疟原虫样本的临界值为 0.05 个寄生虫/μL。由于检测的样本数量有限，疟疾疟原虫和克雷西疟原虫样本的数据尚无定论。结论就灵敏度评估和检测 EQA 冻干血样中低寄生虫血症水平的疟原虫而言，实时 PCR 是一种可靠的分子方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Parasitologia

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