Dataset Information

Performance Evaluation of a Commercial Real-Time PCR Assay and of an In-House Real-Time PCR for Trypanosoma cruzi DNA Detection in a Tropical Medicine Reference Center, Northern Italy.

ABSTRACT: Chagas disease, a neglected protozoal disease endemic in Latin America, is also currently considered an emerging threat in nonendemic areas because of population movements. The detection of Trypanosoma cruzi DNA is increasingly being considered as important evidence to support Chagas disease diagnoses. However, further performance evaluation of molecular assays is useful for a standardization of strategy considering the whole process in routine diagnosis, especially for the different settings such as endemic and nonendemic countries. Seventy-five samples were collected from subjects screened for Chagas disease in Italy. The DNA was isolated from blood using automated extraction. We evaluated the performance of the commercial RealCycler® CHAG kit (pmPCR) based on satellite DNA (SatDNA) and of an in-house real-time PCR (ihPCR) targeting Sat and kinetoplast (k) DNAs, using the concordance of two serology assays as a reference standard. The sensitivity of kDNA and SatDNA tests by ihPCR and SatDNA by pmPCR were 14.29% (95% confidence interval (CI) 6.38 to 26.22), 7.14% (95% CI 1.98 to 17.29), and 7.14% (95% CI 1.98 to 17.29), respectively. Specificity was 100% for all PCR assays and targets. Overall, our results suggest that the preferred approach for clinical laboratories is to combine the kDNA and SatDNA as targets in order to minimize false-negative results increasing sensitivity.

SUBMITTER: Longoni SS

PROVIDER: S-EPMC7692395 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Performance Evaluation of a Commercial Real-Time PCR Assay and of an In-House Real-Time PCR for <i>Trypanosoma cruzi</i> DNA Detection in a Tropical Medicine Reference Center, Northern Italy.

Longoni Silvia Stefania SS Pomari Elena E Antonelli Alberto A Formenti Fabio F Silva Ronaldo R Tais Stefano S Scarso Salvatore S Rossolini Gian Maria GM Angheben Andrea A Perandin Francesca F

Microorganisms 20201030 11

Chagas disease, a neglected protozoal disease endemic in Latin America, is also currently considered an emerging threat in nonendemic areas because of population movements. The detection of <i>Trypanosoma cruzi</i> DNA is increasingly being considered as important evidence to support Chagas disease diagnoses. However, further performance evaluation of molecular assays is useful for a standardization of strategy considering the whole process in routine diagnosis, especially for the different sett ...[more]

PMID: 33143253

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Project description: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.

Project description:BackgroundChagas disease is a neglected illness, with limited treatments, caused by the parasite Trypanosoma cruzi. Two drugs are prescribed to treat the disease, nifurtimox and benznidazole, which have been previously reported to have limited efficacy and the appearance of resistance by T. cruzi. Acquisition of drug-resistant phenotypes is a complex physiological process based on single or multiple changes of the genes involved, probably in its mechanisms of action.ResultsThe differential genes expression of a sensitive Trypanosoma cruzi strain and its induced in vitro benznidazole-resistant phenotypes was studied. The stepwise increasing concentration of BZ in the parental strain generated five different resistant populations assessed by the IC(50) ranging from 10.49 to 93.7 μM. The resistant populations maintained their phenotype when the BZ was depleted from the culture for many passages. Additionally, the benznidazole-resistant phenotypes presented a cross-resistance to nifurtimox but not to G418 sulfate. On the other hand, four of the five phenotypes resistant to different concentrations of drugs had different expression levels for the 12 genes evaluated by real-time PCR. However, in the most resistant phenotype (TcR5x), the levels of mRNA from these 12 genes and seven more were similar to the parental strain but not for NTR and OYE genes, which were down-regulated and over-expressed, respectively. The number of copies for these two genes was evaluated for the parental strain and the TcR5x phenotype, revealing that the NTR gene had lost a copy in this last phenotype. No changes were found in the enzyme activity of CPR and SOD in the most resistant population. Finally, there was no variability of genetic profiles among all the parasite populations evaluated by performing low-stringency single-specific primer PCR (LSSP-PCR) and random amplified polymorphic DNA RAPD techniques, indicating that no clonal selection or drastic genetic changes had occurred for the exposure to BZ.ConclusionHere, we propose NTR as the major marker of the appearance of resistance to BZ.

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Dataset Information

Performance Evaluation of a Commercial Real-Time PCR Assay and of an In-House Real-Time PCR for Trypanosoma cruzi DNA Detection in a Tropical Medicine Reference Center, Northern Italy.

Publications

Performance Evaluation of a Commercial Real-Time PCR Assay and of an In-House Real-Time PCR for <i>Trypanosoma cruzi</i> DNA Detection in a Tropical Medicine Reference Center, Northern Italy.

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