Moreira Otacilio C, Fernandes Alice Gomes, Gomes Natalia Lins da Silva, Dos Santos Carolina Messias, Jacomasso Thiago, Costa Alexandre Dias Tavares, Nascimento Lucas de O Rossetti, Hasslocher-Moreno Alejandro Marcel, do Brasil Pedro Emmanuel Alvarenga Americano, Morello Luis Gustavo, Marchini Fabricio Klerynton, Krieger Marco Aurelio, Britto Constança
Real Time PCR Platform RPT09A, Laboratory of Molecular Virology and Parasitology, Oswaldo Cruz Institute/Fiocruz, Rio de Janeiro 21040-900, Brazil.
Laboratory of Molecular Biology and Endemic Diseases, Oswaldo Cruz Institute/Fiocruz, Rio de Janeiro 21040-900, Brazil.
Life (Basel). 2023 May 24;13(6):1236. doi: 10.3390/life13061236.
In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 10 to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected belonging to all six discrete typing units (DTUs-TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials.
在缺乏用于控制恰加斯病治愈的经过验证的生物标志物的情况下，基于聚合酶链反应（PCR）的诊断正被用作早期指示治疗失败的主要工具。然而，由于它被认为是一种重复性复杂的技术，主要是因为难以建立准确的对照来保证反应质量，恰加斯病诊断中PCR的使用仅限于专业中心。为了推广恰加斯病的分子诊断及其应用，近年来市场上推出了基于定量PCR（qPCR）的新型诊断试剂盒。在此，我们展示了NAT恰加斯试剂盒（恰加斯病核酸检测试剂盒）用于检测和定量疑似恰加斯病感染患者血样中的锥虫的验证结果。该试剂盒由针对锥虫卫星核DNA的TaqMan双重反应和一个外源性内部扩增对照组成，报告范围为10至0.5个寄生虫当量/毫升，检测限（LOD）为0.16个寄生虫当量/毫升血液。此外，NAT恰加斯试剂盒检测到了属于所有六个离散型别单元（DTUs - TcI至TcVI）的锥虫，与使用商业试剂进行的内部实时PCR类似，后者已被选为恰加斯病qPCR验证国际共识中性能最佳的检测方法。在此展示的临床验证中，与内部实时PCR共识检测方法相比，该试剂盒显示出100%的灵敏度和100%的特异性。因此，完全按照国际药品生产质量管理规范（GMP）标准在巴西生产的NAT恰加斯试剂盒，似乎是一种极佳的选择，能够在公共和私人诊断中心实现恰加斯病的分子诊断，并有助于监测参与临床试验的接受病原学治疗的患者。
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