从使用过的疟疾 RDT 中回收 DNA 以检测疟原虫物种并评估恶性疟原虫遗传多样性：在马达加斯加的一项初步研究。

DNA recovery from used malaria RDT to detect Plasmodium species and to assess Plasmodium falciparum genetic diversity: a pilot study in Madagascar.

机构信息

Mention Biochimie Fondamentale et Appliquée, Faculté des Sciences, Université d'Antananarivo, Antananarivo, Madagascar.

Programme National de Lutte contre le paludisme, Ministère de la Santé Publique, Antananarivo, Madagascar.

出版信息

Malar J. 2022 Jul 26;21(1):227. doi: 10.1186/s12936-022-04246-y.

DOI:10.1186/s12936-022-04246-y

PMID:35883089

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9327223/

Abstract

BACKGROUND

Rapid diagnostic tests (RDT) are widely used for malaria diagnosis in Madagascar, where Plasmodium falciparum is the predominant species. Molecular diagnosis is essential for malaria surveillance, but requires additional blood samples for DNA extraction. Used RDTs is an attractive alternative that can be used as a source of DNA. Plasmodium falciparum genetic diversity and multiplicity of infection, usually determined by the genotyping of polymorphic regions of merozoite surface proteins 1 and 2 genes (msp1, msp2), and the repeated region RII of the glutamate-rich protein gene (glurp) have been associated with malaria transmission levels and subsequently with the impact of the deployed control strategies. Thus, the study aims to use RDT as DNA source to detect Plasmodium species, to characterize Plasmodium falciparum genetic diversity and determine the multiplicity of infection.

METHODS

A pilot study was conducted in two sites with different epidemiological patterns: Ankazomborona (low transmission area) and Matanga (high transmission area). On May 2018, used RDT (SD BIOLINE Malaria Ag P.f/Pan, 05FK63) were collected as DNA source. Plasmodium DNA was extracted by simple elution with nuclease free water. Nested-PCR were performed to confirm Plasmodium species and to analyse P. falciparum msp1, msp2 and glurp genes polymorphisms.

RESULTS

Amongst the 170 obtained samples (N = 74 from Ankazomborona and N = 96 from Matanga), Plasmodium positivity rate was 23.5% (40/170) [95% CI 17.5-30.8%] by nested-PCR with 92.2% (37/40) positive to P. falciparum, 5% (2/40) to Plasmodium vivax and 2.5% (1/40) to P. falciparum/P. vivax mixed infection. Results showed high polymorphisms in P. falciparum msp1, msp2 and glurp genes. Multiple infection rate was 28.6% [95% CI 12.2-52.3%]. The mean of MOI was 1.79 ± 0.74.

CONCLUSION

This pilot study highlighted that malaria diagnosis and molecular analysis are possible by using used malaria RDT. A large-scale study needs to be conducted to assess more comprehensively malaria parasites transmission levels and provide new data for guiding the implementation of local strategies for malaria control and elimination. Trial registration Retrospectively registered.

摘要

背景

快速诊断检测（RDT）在马达加斯加被广泛用于疟疾诊断，在那里恶性疟原虫是主要物种。分子诊断对于疟疾监测至关重要，但需要额外的血液样本进行 DNA 提取。使用 RDT 是一种有吸引力的替代方法，可以用作 DNA 来源。恶性疟原虫的遗传多样性和感染多重性，通常通过对裂殖子表面蛋白 1 和 2 基因（msp1、msp2）的多态区和谷氨酸丰富蛋白基因（glurp）的重复区 RII 的基因分型来确定，与疟疾传播水平有关，进而与所部署的控制策略的影响有关。因此，本研究旨在使用 RDT 作为 DNA 来源来检测疟原虫种类，表征恶性疟原虫的遗传多样性并确定感染多重性。

方法

在两个具有不同流行病学模式的地点进行了一项试点研究：Ankazomborona（低传播区）和 Matanga（高传播区）。2018 年 5 月，收集了用过的 RDT（SD BIOLINE 疟疾 Ag P.f/Pan，05FK63）作为 DNA 来源。通过用无核酸酶水简单洗脱来提取疟原虫 DNA。进行巢式 PCR 以确认疟原虫种类，并分析恶性疟原虫 msp1、msp2 和 glurp 基因的多态性。

结果

在所获得的 170 个样本中（Ankazomborona 74 个，Matanga 96 个），巢式 PCR 的疟原虫阳性率为 23.5%（40/170）[95%CI 17.5-30.8%]，92.2%（37/40）对恶性疟原虫阳性，5%（2/40）对间日疟原虫阳性，2.5%（1/40）对恶性疟原虫/间日疟原虫混合感染阳性。结果表明恶性疟原虫 msp1、msp2 和 glurp 基因高度多态性。多重感染率为 28.6%[95%CI 12.2-52.3%]。平均 MOI 为 1.79±0.74。