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. 2017 Nov 27;12(11):e0188550.
doi: 10.1371/journal.pone.0188550. eCollection 2017.

First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease

Juan C Ramírez  1 , Rudy Parrado  2 , Elena Sulleiro  3 , Anabelle de la Barra  2 , Marcelo Rodríguez  4 , Sandro Villarroel  2 , Lucía Irazu  4 , Cristina Alonso-Vega  5 , Fabiana Alves  5 , María A Curto  1 , Lineth García  2 , Lourdes Ortiz  6 , Faustino Torrico  7 , Joaquim Gascón  8 , Laurence Flevaud  9 , Israel Molina  3 , Isabela Ribeiro  5 , Alejandro G Schijman  1
Affiliations

First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease

Juan C Ramírez et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Comparison of intra- and inter-laboratory SatDNA qPCR results for proficiency testing panels analysis based on T. cruzi stocks.
A: K98 (TcIa); B: Sylvio X10 Cl1 (TcId); C: LL014-1-R1 Cl1 (TcV); D: CL-Brener (TcVI); Ct: Cycle threshold; SatDNA qPCR: Satellite DNA Real-Time PCR; par. eq./mL: parasite equivalents in 1 mL of blood.
Fig 2
Fig 2. Comparison of intra- and inter-laboratory SatDNA qPCR results for proficiency testing panels analysis based on number of panel.
A: 1 par. eq./mL; B: 10 par. eq./mL; C: 100 par. eq./mL; Ct: Cycle threshold; SatDNA qPCR: Satellite DNA Real-Time PCR; par. eq./mL: parasite equivalents in 1 mL of blood.
Fig 3
Fig 3. Comparison of quantifiable SatDNA qPCR results for retesting samples analysis.
Parasitic loads obtained by LabB and Core Lab (A) and Bland-Altman bias plot as a measure of the degree of agreement between the results of both laboratories (B). par. eq./mL: parasite equivalents in 1 mL of blood; SatDNA qPCR: Satellite DNA Real-Time PCR; SD: standard deviation.
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