Imwong Mallika, Hanchana Sarun, Malleret Benoit, Rénia Laurent, Day Nicholas P J, Dondorp Arjen, Nosten Francois, Snounou Georges, White Nicholas J
Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
J Clin Microbiol. 2014 Sep;52(9):3303-9. doi: 10.1128/JCM.01057-14. Epub 2014 Jul 2.
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.
“低传播”地区疟疾的流行病学情况一直被低估。分子检测方法显示疟疾的流行率高于传统显微镜检查或快速诊断检测，但这些方法通常检测的是手指刺血获得的毛细血管血样（约5微升），因此无法检测到每毫升低于200个的寄生虫密度。使用这些方法会低估真正的寄生虫携带率。为了描述低传播环境下疟疾的流行病学特征并制定消除策略，需要更灵敏的定量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18S RNA的高灵敏度“大容量”定量PCR（qPCR）方法适用于≥250微升的血样，并进行了高通量规模化处理。通过评估分析灵敏度和特异性、诊断灵敏度和特异性、效率、精密度、分析和诊断准确性、检测限、假阳性的根本原因分析以及稳健性对这些方法进行了验证。基于疟原虫属物种18S RNA的大容量qPCR方法的PCR效率高达90%至105%。大量血液中的寄生虫DNA浓度给出了一致的分析检测限（LOD）为每毫升22个寄生虫（95%可信区间，21.79至74.9），这比传统显微镜检查灵敏约2500倍，比目前用于滤纸血斑的PCR方法灵敏50倍。诊断特异性为99.75%。使用自动化程序，每周能够处理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/毫升）的非常灵敏且特异的高通量大容量qPCR方法。
