Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

BackgroundThe analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal FindingsWe have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/SignificanceThe performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.背景 针对克氏锥虫（Trypanosoma cruzi）定量的高灵敏度、精准且标准化的实时荧光定量PCR方法的分析验证，对于为近期感染诊断以及治疗效果监测提供可靠的实验室工具至关重要。 方法与主要结果 我们基于TaqMan技术建立并验证了多重实时荧光定量PCR（qPCR）检测方法，旨在通过单管反应同时定量克氏锥虫卫星DNA与内参扩增对照（internal amplification control, IAC）。内参扩增对照可排除因样本处理过程中存在抑制物质或DNA丢失所导致的PCR假阴性结果。以加标克氏锥虫CL-Brener毒株的未煮沸胍盐EDTA抗凝全血作为起始样本时，该检测方法的检出限（limit of detection, LOD）为0.70个寄生虫当量/mL，定量限（limit of quantification, LOQ）为1.53个寄生虫当量/mL。本方法针对不同临床分期与流行病学场景的恰加斯病（Chagas disease）患者血液样本展开了评估：1. 16名来自口腔传播暴发疫情的委内瑞拉患者；2. 63名罹患慢性恰加斯病的玻利维亚患者；3. 34名慢性恰加斯病阿根廷患者；4. 27名血清学阳性母亲所生新生儿；5. 1名接受感染供体获取的尸体肾移植后发生感染的血清学阴性受体。 结论与意义 本检测方法的各项性能参数支持其应用于血清学方法无法提供有效信息的克氏锥虫感染早期评估场景，例如经口腔污染途径的近期感染、先天性传播或接受血清学阳性供体器官移植后的感染情况，同时也可用于对接受病因治疗的恰加斯病患者进行治疗监测。