Comparative Assessment of Two Commercial Real-Time PCR Assays for the Diagnosis of Trypanosoma cruzi DNA in Serum

Abstract

This study was performed to comparably assess two commercial real-time PCR assays for the identification of Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma rangeli 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 T. cruzi 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 T. cruzi and T. rangeli without further discrimination. To discriminate between T. cruzi- and T. rangeli-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) T. cruzi-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite T. rangeli. 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 T. rangeli 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 T. cruzi was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic T. rangeli according to the RealStar assay may be a disadvantage in areas of co-circulation with T. cruzi, while the test performance of the two compared assays will be quite similar in geographic settings where T. rangeli infections are unlikely.

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“…This was associated with a very low detection threshold of only 1.5 target DNA copies per µL nucleic acid eluate, as assessed with a dilution series using DNA of the T. cruzi Tulahuen (DTU TcII) strain, as reported elsewhere [55]. Even more, NDO-PCR allowed for superior delineation of T. cruzi from non-pathogenic, but phylogenetically closely related, T. rangeli compared to competing in-house and commercial real-time PCR protocols in sequencing-controlled assessments [55,56]. Hybridization probe (as used for the kit commercialized by TibMolBiol)…”

“…In particular, the oligonucleotides used for the patent-protected NDO-PCR assay targeting kinetoplast minicircle DNA (GenBank accession number U07846.1) are provided in Table 1. Superior diagnostic accuracy as compared to other published or commercialized real-time PCR approaches was shown for the application with both hybridization probe variants [55,56], which are indicated in Table 1. In those evaluation assessments [55,56], excellent diagnostic accuracy values comprising sensitivity ranging between 92.3% and 97.9% as well as specificity ranging between 99.3% and 100% for the detection of T. cruzi DNA in human blood could be confirmed.…”

“…Superior diagnostic accuracy as compared to other published or commercialized real-time PCR approaches was shown for the application with both hybridization probe variants [55,56], which are indicated in Table 1. In those evaluation assessments [55,56], excellent diagnostic accuracy values comprising sensitivity ranging between 92.3% and 97.9% as well as specificity ranging between 99.3% and 100% for the detection of T. cruzi DNA in human blood could be confirmed. This was associated with a very low detection threshold of only 1.5 target DNA copies per µL nucleic acid eluate, as assessed with a dilution series using DNA of the T. cruzi Tulahuen (DTU TcII) strain, as reported elsewhere [55].…”

“…The NDO-PCR was performed in 2018 as an in-house assay, as described previously [55]. In 2020, the kit of the assay was professionally produced by TibMolBiol, Berlin, Germany (T. cruzi Light Mix, Ref 53-0755-96, Phocid Herpes Virus (PhHV) Extraction control, reference 66-0901-96, Lyophilized 1-step RT-PCR polymerase mix, Cat.-No 90-9999-96) and used with the nucleic acid eluates as reported [56]. Real-time PCR was run on a Rotor-Gene Q cycler (Qiagen, Hilden, Germany) with reaction conditions exactly as detailed in the manual of the T. cruzi Light Mix assay, Ref 53-0755-96 (TibMolBiol).…”

“…All samples were assessed in duplicate. Further details on the PCR approach are provided in detail in the evaluation studies [54][55][56] and summarized below. In particular, the oligonucleotides used for the patent-protected NDO-PCR assay targeting kinetoplast minicircle DNA (GenBank accession number U07846.1) are provided in Table 1.…”