| Summary: | This study was performed to comparably assess two commercial real-time PCR assays for the identification of <i>Trypanosoma cruzi</i> DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either <i>T. cruzi</i> or apathogenic <i>Trypanosoma rangeli</i> were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for <i>T. cruzi</i> and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both <i>T. cruzi</i> and <i>T. rangeli</i> without further discrimination. To discriminate between <i>T. cruzi</i>- and <i>T. rangeli</i>-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94) <i>T. cruzi</i>-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite <i>T. rangeli</i>. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with <i>T. rangeli</i> in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of <i>T. cruzi</i> was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of <i>T. cruzi</i> from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic <i>T. rangeli</i> according to the RealStar assay may be a disadvantage in areas of co-circulation with <i>T. cruzi</i>, while the test performance of the two compared assays will be quite similar in geographic settings where <i>T. rangeli</i> infections are unlikely.
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