Journal Article > CommentaryFull Text
PLoS Negl Trop Dis. 4 June 2015; Volume 9 (Issue 6); e0003697.; DOI:10.1371/journal.pntd.0003697
Porras AI, Yadon ZE, Altcheh J, Britto C, Chaves GC, et al.
PLoS Negl Trop Dis. 4 June 2015; Volume 9 (Issue 6); e0003697.; DOI:10.1371/journal.pntd.0003697
Journal Article > ResearchFull Text
Antimicrob Agents Chemother. 3 December 2018; Volume 63 (Issue 2); DOI:10.1128/AAC.01191-18
Parrado R, Ramirez JC, de la Barra A, Alonso-Vega C, Juiz N, et al.
Antimicrob Agents Chemother. 3 December 2018; Volume 63 (Issue 2); DOI:10.1128/AAC.01191-18
This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time PCR (qPCR) for baseline detection and quantification of parasitic loads and post-treatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely DNDi-CH-E1224-001 (NCT01489228) and MSF-DNDi PCR sampling optimization study (NCT01678599). Patients from Cochabamba (N= 294), Tarija (N= 257), and Aiquile (N= 220) were enrolled. Three serial blood samples were collected at each time-point and qPCR triplicates were tested per sample. The first two samples were collected during the same day and the third one seven days later.A patient was considered PCR positive if at least one qPCR replicate was detectable. Cumulative results of multiple samples and qPCR replicates enhanced the proportion of pre-treatment sample positivity from 54.8 to 76.2%, 59.5 to 77.8%, and 73.5 to 90.2% in Cochabamba, Tarija, and Aiquile cohorts, respectively. This strategy increased the detection of treatment failure from 72.9 to 91.7%, 77.8 to 88.9%, and 42.9 to 69.1% for E1224 low, short, and high dosage regimens, respectively; and from 4.6 to 15.9% and 9.5 to 32.1% for the benznidazole arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The addition of the third blood sample and third qPCR replicate in patients with non-detectable PCR results in the first two samples, gave a small, non-statistically significant improvement in qPCR positivity. No change in clinical sensitivity was seen with a blood volume increase from 5 to 10 ml. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasional non-detectable results. In conclusion, serial sampling strategy enhanced PCR sensitivity to detecting treatment failure during follow-up and has the potential for improving recruitment capacity in Chagas disease trials, which require an initial positive qPCR result for patient admission.
Journal Article > ResearchFull Text
PLOS One. 27 November 2017; Volume 12 (Issue 11); e0188550.; DOI:10.1371/journal.pone.0188550
Ramirez JC, Parrado R, Sulleiro E, de la Barra A, Rodriguez M, et al.
PLOS One. 27 November 2017; Volume 12 (Issue 11); e0188550.; DOI:10.1371/journal.pone.0188550
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.