High-throughput ultrasensitive molecular techniques for quantifying low-density malaria parasitemias.
High-throughput ultrasensitive molecular techniques for quantifying low-density malaria parasitemias.
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DOI：
10.1128/jcm.01057-14
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发表时间：
2014-09
影响因子：
9.4
通讯作者：
White NJ
中科院分区：
文献类型：
--
作者：
Imwong M;Hanchana S;Malleret B;Rénia L;Day NP;Dondorp A;Nosten F;Snounou G;White NJ
疟疾在“低传播”地区的流行情况被低估了。分子检测方法显示疟疾的流行率高于常规显微镜或快速诊断测试，但这些方法通常评估手指针刺毛细血管血液样本（105 μl），因此无法检测到小于200/ml的寄生虫密度。它们的使用低估了真正的寄生虫携带率。为了描述低传播环境中疟疾的流行病学特征并制定消除策略，需要更敏感的定量PCR（qPCR）来识别和量化低密度疟疾寄生虫血症。一种基于疟原虫属18 S RNA的高灵敏度“高容量”定量PCR（qPCR）方法适用于≥250 μl的血样体积，并按高通量进行缩放。通过评估分析灵敏度和特异性、诊断灵敏度和特异性、效率、精确度、分析和诊断准确性、检测限、假阳性的根本原因分析和稳健性来验证这些方法。基于疟原虫18 S RNA的高容量qPCR方法提供了90 - 105%的高PCR效率。大量血液中寄生虫DNA的浓度得到了一致的分析检测限（LOD）为22个寄生虫/ml（95% CI，21.79至74.9），这是约2,500倍的灵敏度比传统的显微镜和50倍的灵敏度比目前使用的PCR方法从滤纸血斑。诊断特异性为99.75%。使用自动化程序，每周可以处理700份血样。开发并验证了一种用于检测低密度寄生虫血症（>20个寄生虫/ml）的非常灵敏和特异的高通量高容量qPCR方法。
The epidemiology of malaria in “low-transmission” areas has been underestimated. Molecular detection methods have revealed higher prevalences of malaria than conventional microscopy or rapid diagnostic tests, but these typically evaluate finger-prick capillary blood samples (∼5 μl) and therefore cannot detect parasite densities of <200/ml. Their use underestimates true parasite carriage rates. To characterize the epidemiology of malaria in low-transmission settings and plan elimination strategies, more sensitive quantitative PCR (qPCR) is needed to identify and quantify low-density malaria parasitemias. A highly sensitive “high-volume” quantitative PCR (qPCR) method based on Plasmodium sp. 18S RNA was adapted for blood sample volumes of ≥250 μl and scaled for high throughput. The methods were validated by assessment of the analytical sensitivity and specificity, diagnostic sensitivity, and specificity, efficiency, precision, analytical and diagnostic accuracies, limit of detection, root cause analysis of false positives, and robustness. The high-volume qPCR method based on Plasmodium sp. 18S RNA gave high PCR efficiency of 90 to 105%. Concentrations of parasite DNA from large volumes of blood gave a consistent analytical detection limit (LOD) of 22 parasites/ml (95% CI, 21.79 to 74.9), which is some 2,500 times more sensitive than conventional microscopy and 50 times more sensitive than currently used PCR methods from filter paper blood spots. The diagnostic specificity was 99.75%. Using automated procedures it was possible to process 700 blood samples per week. A very sensitive and specific high-throughput high-volume qPCR method for the detection of low-density parasitemias (>20 parasites/ml) was developed and validated.