Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays

Abstract

BackgroundPolymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process.Methodology/Principal findingsWe assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately.Conclusions/SignificanceThis is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

Plasma-Derived Extracellular Vesicles as Potential Biomarkers in Heart Transplant Patient with Chronic Chagas Disease

“…After transplantation and in the context of immunosuppression therapy ( Table 1 ), quantitative PCR (qPCR) was performed weekly to detect T. cruzi in the blood ( Tc -qPCR) ( 7 ). First benznidazole treatment was started when several consecutive and positive Tc -qPCRs confirmed Chagas disease reactivation.…”

ARCHITECT Chagas® as a single test candidate for Chagas disease diagnosis: evaluation of two algorithms implemented in a non-endemic setting

“…A sample was considered valid when the internal control of amplification was amplified and positive when the Cycle threshold (Ct) of the target was 40. For details of both the DNA extraction and amplification protocols, see Abras et al [19]. S1).…”

Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation

“…Several studies using different in-house PCR methods depicted better performance than the current diagnostic cCD algorithm [ 36 – 39 ]. However, evaluation of commercial methods suitable for clinical practice are urgently needed and there are only a few reports on the use of nucleic acid amplification kits [ 9 , 15 , 40 , 41 ]. Our data show the diagnostic potential of the Loopamp Trypanosoma cruzi prototype kit for early detection of congenital transmission.…”