Application of dried blood spot sample pooling strategies for Plasmodium 18S rRNA biomarker testing to facilitate identification of infected persons in large-scale epidemiological studies.
Application of dried blood spot sample pooling strategies for Plasmodium 18S rRNA biomarker testing to facilitate identification of infected persons in large-scale epidemiological studies.
复制标题
应用干血斑样本合并策略进行疟原虫18S rRNA生物标志物检测，以便于在大规模流行病学研究中识别感染者。
DOI：
10.1186/s12936-021-03907-8
复制
发表时间：
2021-10-07
期刊：
影响因子：
3
通讯作者：
Murphy SC
中科院分区：
文献类型：
--
作者：
Chang M;Johnston S;Seilie AM;Hergott D;Murphy SC
疟原虫18 S rRNA是检测人体血液中疟原虫感染的敏感生物标志物。干血斑（DBS）是疟疾现场研究的一种实用样本类型，用于收集、储存和运输大量血液样本进行诊断检测。混合测试是降低试剂成本和劳动力的常见方法。本研究检查了DBS的疟原虫18 S rRNA生物标志物检测的性能，提高了合并样本的检测灵敏度，并创建了图形用户界面（GUI）程序以促进最佳合并。使用疟原虫18 S rRNA定量三重逆转录聚合酶链反应（qRT-PCR）测定和简化的双重测定，对来自临床标本、恶性疟原虫体外培养物和恶性疟原虫Armored RNA®的不同寄生虫密度的DBS样品进行了检测。评价了DBS样品精密度、线性、检测限（LoD）和不同储存温度下的稳定性。创建了新颖的GUI来模拟两阶段层次结构、方阵和三阶段层次结构合并策略，其中样本具有不同的阳性率和估计的测试计数。对来自居住在患有隐性感染的流行地区的78份DBS样本进行了池测试并进行去卷积以识别阳性病例。测定性能显示DBS在4 × 107至5 × 102个寄生虫/mL范围内呈线性，与液体血液样本具有强相关性（r2 > 0.96）。与液体血液样本相比，DBS rRNA拷贝/mL略有定量减少。DBS的分析灵敏度估计为5.3 log拷贝18 S rRNA/mL血液（28个估计寄生虫/mL）。当在环境温度下储存一个月时，适当保存的DBS表现出最小的18 S rRNA降解。省略人mRNA靶标的简化双链qRT-PCR试验显示出改善的分析灵敏度，1个寄生虫/mL血液，并针对合并进行了优化。最佳合并规模因患病率而异。两阶段分层汇总方案的DBS试点研究证实了先前通过测试单个DBS确定的结果。疟原虫18 S rRNA生物标志物测定可应用于现场研究中收集的DBS。已建立简化的疟原虫qRT-PCR检测试剂盒和GUI，为检测大量DBS样本提供有效方法。在线版本包含补充材料，可通过10.1186/s12936-021-03907-8获得。
Plasmodium 18S rRNA is a sensitive biomarker for detecting Plasmodium infection in human blood. Dried blood spots (DBS) are a practical sample type for malaria field studies to collect, store, and transport large quantities of blood samples for diagnostic testing. Pooled testing is a common way to reduce reagent costs and labour. This study examined performance of the Plasmodium 18S rRNA biomarker assay for DBS, improved assay sensitivity for pooled samples, and created graphical user interface (GUI) programmes for facilitating optimal pooling. DBS samples of varied parasite densities from clinical specimens, Plasmodium falciparum in vitro culture, and P. falciparum Armored RNA® were tested using the Plasmodium 18S rRNA quantitative triplex reverse transcription polymerase chain reaction (qRT-PCR) assay and a simplified duplex assay. DBS sample precision, linearity, limit of detection (LoD) and stability at varied storage temperatures were evaluated. Novel GUIs were created to model two-stage hierarchy, square matrix, and three-stage hierarchy pooling strategies with samples of varying positivity rates and estimated test counts. Seventy-eight DBS samples from persons residing in endemic regions with sub-patent infections were tested in pools and deconvoluted to identify positive cases. Assay performance showed linearity for DBS from 4 × 107 to 5 × 102 parasites/mL with strong correlation to liquid blood samples (r2 > 0.96). There was a minor quantitative reduction in DBS rRNA copies/mL compared to liquid blood samples. Analytical sensitivity for DBS was estimated 5.3 log copies 18S rRNA/mL blood (28 estimated parasites/mL). Properly preserved DBS demonstrated minimal degradation of 18S rRNA when stored at ambient temperatures for one month. A simplified duplex qRT-PCR assay omitting the human mRNA target showed improved analytical sensitivity, 1 parasite/mL blood, and was optimized for pooling. Optimal pooling sizes varied depending on prevalence. A pilot DBS study of the two-stage hierarchy pooling scheme corroborated results previously determined by testing individual DBS. The Plasmodium 18S rRNA biomarker assay can be applied to DBS collected in field studies. The simplified Plasmodium qRT-PCR assay and GUIs have been established to provide efficient means to test large quantities of DBS samples. The online version contains supplementary material available at 10.1186/s12936-021-03907-8.
登录
查看更多内容
影响因子：
9.4
作者：
Murphy, Sean C.;Daza, Glenda;Coombs, Robert
通讯作者：
Coombs, Robert
影响因子：
16.6
作者：
Slater, Hannah C.;Ross, Amanda;Okell, Lucy C.
通讯作者：
Okell, Lucy C.
影响因子：
3
作者：
Jiram, Adela Ida;Ooi, Choo Huck;Alias, Nor Wahida
通讯作者：
Alias, Nor Wahida
影响因子：
1.9
作者：
McMahan CS;Tebbs JM;Bilder CR
通讯作者：
Bilder CR
影响因子：
3
作者：
Das S;Peck RB;Barney R;Jang IK;Kahn M;Zhu M;Domingo GJ
通讯作者：
Domingo GJ