2.6. External Quality assurance of qPCR performance

An external quality assurance of qPCR performance was built-up at the Research & Development Department of INP to evaluate the methodology implemented. Three proficiency testing panels made of seronegative human blood samples spiked with 1·5 (P102 sample), 15 (P103 sample) and 150 (P104 sample) parasite equivalents/mL of T. cruzi (a Tc V strain isolated from a cCD case [4]) and a negative blood control without parasites (P101 sample) were evaluated in three laboratories using different thermocyclers: ABI 7500 (Applied Biosystem, California, USA), Light Cycler 96 (Roche Diagnostic Rotkreuz, Switzerland), CFX96 Touch Real Time PCR Detection System (Bio Rad laboratories, California, USA). Each operator ran the assays from each panel in duplicates and in two consecutive days. The Cts were registered and qualitative results were expressed as non-detectable or positive. The reports were sent to the reference laboratory for statistical analysis. Intra- and inter-laboratory qPCR results from the proficiency testing panels were analyzed by SPSS Statistics software. The Cohen kappa coefficient was used to analyze the closeness of the agreement and the differences between qualitative qPCR results obtained from the samples tested at the three participating laboratories. The Analyse-It software for Microsoft Excel 5.30.2 Build 7069-17990 was used for this analysis. The performances of each participating Laboratory were compared in terms of the Z score, in accordance with ISO 13528:2015(E) [11]. The Z-score was negative or positive when data points were below or above the mean, respectively.