Abstract
Real-Time PCR (qPCR) testing is recommended as both a diagnostic and outcome measurement of etiological treatment in clinical practice and clinical trials of Chagas disease (CD), but no external quality assurance (EQA) program provides performance assessment of the assays in use. We implemented an EQA system to evaluate the performance of molecular biology laboratories involved in qPCR based follow-up in clinical trials of CD. An EQA program was devised for three clinical trials of CD: the E1224 (NCT01489228), a pro-drug of ravuconazole; the Sampling Study (NCT01678599), that used benznidazole, both conducted in Bolivia; and the CHAGASAZOL (NCT01162967), that tested posaconazole, conducted in Spain. Four proficiency testing panels containing negative controls and seronegative blood samples spiked with 1, 10 and 100 parasite equivalents (par. eq.)/mL of four Trypanosoma cruzi stocks, were sent from the Core Lab in Argentina to the participating laboratories located in Bolivia and Spain. Panels were analyzed simultaneously, blinded to sample allocation, at 4-month intervals. In addition, 302 random blood samples from both trials carried out in Bolivia were sent to Core Lab for retesting analysis. The analysis of proficiency testing panels gave 100% of accordance (within laboratory agreement) and concordance (between laboratory agreement) for all T. cruzi stocks at 100 par. eq./mL; whereas their values ranged from 71 to 100% and from 62 to 100% at 1 and 10 par. eq./mL, respectively, depending on the T. cruzi stock. The results obtained after twelve months of preparation confirmed the stability of blood samples in guanidine-EDTA buffer. No significant differences were found between qPCR results from Bolivian laboratory and Core Lab for retested clinical samples. This EQA program for qPCR analysis of CD patient samples may significantly contribute to ensuring the quality of laboratory data generated in clinical trials and molecular diagnostics laboratories of CD.
Highlights
Chagas disease (CD), caused by the kinetoplastid flagellate Trypanosoma cruzi, has been considered to be “the most neglected of the neglected diseases” given the research and development gaps related to diagnosis and treatment [1]
This work aimed to evaluate the performance of Satellite DNA (SatDNA) Quantitative Real-Time PCR (qPCR) methods used as primary endpoints during three clinical trials of chronic CD patients conducted in Bolivia and Spain, through an external quality assurance (EQA) program specially devised for this purpose
This study included four panels of negative controls and samples spiked with four T. cruzi stocks belonging to three different Discrete Typing Units (DTUs) (TcI, TcV and TcVI) and two different TcI miniexon-based genotypes (TcIa and TcId), at three different concentrations
Summary
Chagas disease (CD), caused by the kinetoplastid flagellate Trypanosoma cruzi, has been considered to be “the most neglected of the neglected diseases” given the research and development gaps related to diagnosis and treatment [1]. The most widely applied qPCR standard operating procedure (SOP) for detection and quantification of T. cruzi DNA includes DNA extraction from 300 μL of guanidine-EDTAblood samples using glass-fiber commercial columns. This is followed by duplex qPCR using TaqMan probes targeted to T. cruzi Satellite DNA (SatDNA) and an internal amplification control (IAC) [6,7]. This method has been used to follow-up parasite response to treatment with different compounds and regimens, such as benznidazole [8] and E1224, a water-soluble ravuconazole pro-drug [9]
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