An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples.
An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples.
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DOI：
10.1186/s12936-015-1038-z
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发表时间：
2015-12-23
期刊：
影响因子：
3
通讯作者：
Plowe CV
中科院分区：
文献类型：
--
作者：
Adams M;Joshi SN;Mbambo G;Mu AZ;Roemmich SM;Shrestha B;Strauss KA;Johnson NE;Oo KZ;Hlaing TM;Han ZY;Han KT;Thura S;Richards AK;Huang F;Nyunt MM;Plowe CV
需要高度敏感、可扩展的诊断方法来指导消除疟疾的干预措施。虽然传统的显微镜检查和快速诊断测试（RDTs）适用于诊断有症状的疟疾感染，但需要更敏感的测试来筛查低密度的无症状感染，这些感染是旨在消除人类疟疾感染的整个水库的干预措施的目标。应用逆转录聚合酶链反应（RT-PCR）技术，建立了恶性疟原虫和间日疟原虫18 S核糖体RNA基因和核糖体RNA的多重检测方法。用样品保存缓冲液储存14天的模拟现场样品用于评估分析灵敏度和特异性。此外，从缅甸东南部的1750个现场样品进行了测试RDT和超灵敏的RT-PCR。在模拟田间条件下测定检测限（LoD）。当0.3 mL血液样本在28 °C和80%湿度下储存14天时，恶性疟原虫的LoD小于16个寄生虫/mL，间日疟原虫的LoD小于19.7拷贝/μL（使用质粒替代物），比RDTs低约10，000倍。在1739份同时进行RT-PCR和RDT检测的样本中，仅2份RDT阳性，24份为恶性疟原虫阳性，108份为间日疟原虫阳性，127份为间日疟原虫和/或恶性疟原虫阳性。这种超灵敏的RT-PCR方法是一种稳健的、经过现场测试的筛选方法，比RDTs灵敏得多。进一步优化可能会产生一个真正可扩展的工具，适用于广泛监测低水平的无症状恶性疟原虫和间日疟原虫寄生虫病。本文的在线版本（doi：10.1186/s12936-015-1038-z）包含补充材料，可供授权用户使用。
Highly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans. A reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR. Limits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80 % humidity, the LoD was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR. This ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomatic P. falciparum and P. vivax parasitaemia. The online version of this article (doi:10.1186/s12936-015-1038-z) contains supplementary material, which is available to authorized users.