Limit of detection dataset for PCR assays.

BackgroundZoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence.Methodology/Principal findingsAn established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with reverse transcription (RT). Assay specificities were assessed using clinical malaria samples and malaria-negative controls.The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi) and 1000-fold (P. vivax and P. cynomolgi).The Kamau et al. Plasmodium genus RT-qPCR assay was highly sensitive for P. knowlesi detection with a median LOD of ≤0.0002 parasites/μL compared to 0.002 parasites/μL for P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were enhanced for the Imwong et al. 18S rRNA (0.0007 parasites/μL) and Divis et al. real-time 18S rRNA (0.0002 parasites/μL) assays, but not for the Lubis et al. hemi-nested SICAvar (1.1 parasites/μL) and Lee et al. nested 18S rRNA (11 parasites/μL). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were moderately improved at 0.02 and 0.002 parasites/μL, respectively (1000-fold change). For DBS P. knowlesi samples the use of RT also markedly improved the Plasmodium genus qPCR LOD from 19.89 to 0.08 parasites/μL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi.Conclusions/SignificanceOur findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.

背景：在东南亚流行地区，均有诺氏疟原虫（Plasmodium knowlesi, P. knowlesi）与食蟹猴疟原虫（Plasmodium cynomolgi, P. cynomolgi）引发的有症状及无症状人兽共患感染病例报道。多数感染患者呈现低寄生虫血症，且常规显微镜检测阈值以下的感染占比尚不明确。优化检测限（limit of detection, LOD）的分子监测工具，可为人兽共患疟疾流行率的精准估算提供支撑。
方法/主要结果：本研究针对靶向18S核糖体RNA（18S rRNA）基因的已建立超敏感疟原虫属定量聚合酶链式反应（quantitative-PCR, qPCR）检测方法，分别采用经DNA/RNA Shield保存的总核酸分离物与存档干血斑（dried blood spots, DBS），针对诺氏疟原虫、食蟹猴疟原虫及间日疟原虫（Plasmodium vivax, P. vivax）开展有无逆转录（reverse transcription, RT）条件下的检测限评估。同时针对筛选得到的诺氏疟原虫特异性检测方法，以及参考用间日疟原虫、食蟹猴疟原虫特异性检测方法，通过逆转录步骤完成检测限测定。采用临床疟疾样本与疟疾阴性对照样本评估检测方法的特异性。
逆转录步骤可使疟原虫属的检测灵敏度提升最高达10000倍、诺氏疟原虫达2759倍、间日疟原虫与食蟹猴疟原虫达1000倍。
Kamau等建立的疟原虫属逆转录定量PCR（RT-qPCR）检测方法对诺氏疟原虫的检测灵敏度极高，其中位检测限≤0.0002个寄生虫/微升，而食蟹猴疟原虫与间日疟原虫的中位检测限均为0.002个寄生虫/微升。针对诺氏疟原虫的特异性PCR检测方法中，经逆转录步骤后，Imwong等建立的18S rRNA检测方法（0.0007个寄生虫/微升）与Divis等建立的实时18S rRNA检测方法（0.0002个寄生虫/微升）的检测限均得到优化，但Lubis等建立的半巢式SICAvar检测方法（1.1个寄生虫/微升）及Lee等建立的巢式18S rRNA检测方法（11个寄生虫/微升）未出现检测限提升。经逆转录步骤后，间日疟原虫与食蟹猴疟原虫特异性检测方法的检测限分别适度优化至0.02与0.002个寄生虫/微升（变化幅度达1000倍）。针对干血斑保存的诺氏疟原虫样本，逆转录步骤同样可使疟原虫属qPCR的检测限从19.89个寄生虫/微升显著提升至0.08个寄生虫/微升（变化幅度达249倍）；但存档时间超过6年的干血斑样本未检测到检测限提升。在190例临床感染样本与48例健康对照样本中，疟原虫属检测方法与诺氏疟原虫特异性检测方法对疟原虫及诺氏疟原虫的检测特异性均为100%。参考用间日疟原虫特异性引物已被证实可与食蟹猴疟原虫发生已知的交叉反应。
结论与意义：本研究结果表明，采用结合逆转录步骤的18S rRNA疟原虫属qPCR检测方法及种特异性巢式PCR方案，可实现人兽共患及人类疟原虫感染的高灵敏度监测。