Comparative Study and Analytical Verification of PCR Methods for the Diagnosis of Congenital Chagas Disease.

Congenital infection is currently the first cause of new cases of Chagas disease in Argentina and nonendemic areas worldwide. Its diagnosis is of utmost importance to guarantee curative treatment. To improve such diagnosis, a transfer process of PCR tests to the national laboratory network has been initiated. We performed a comparative study of four PCR assays [two end-point PCR and two duplex real-time quantitative PCR (qPCR) procedures] to detect Trypanosoma cruzi DNA in blood samples. Because satellite DNA and kinetoplastid DNA qPCR methods showed the best performance and the use of two different molecular targets for confirmatory purposes has been recommended, these methods were selected to perform the transfer process and, in consequence, subjected to an analytical verification protocol based on international guidelines. The anticipated reportable range was verified between 0.25 and 10 parasite equivalents per milliliter of blood (par. eq./mL) for both qPCR methods, and the limit of detection was estimated to be 0.87 (95% CI, 0.62-1.24) and 0.43 (95% CI, 0.32-0.59) par. eq./mL for satellite DNA and kinetoplastid DNA qPCR methods, respectively. In addition, both qPCR methods showed trueness and verified precision in the highest and the lowest concentrations tested. This work provides critical knowledge of the technology transfer process planned to cover laboratories of the regional network with known installed facilities.