Real Time PCR for the Evaluation of Treatment Response in Clinical Trials of Adult Chronic Chagas Disease: Usefulness of Serial Blood Sampling and qPCR Replicates.

A serial blood sampling procedure was developed 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: 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 three qPCR replicates were tested per sample, the first two samples 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 sampling strategy increased cumulative 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 regimes, respectively; and from 4.6 to 15.9% and 9.5 to 32.1% for the benznidazole (BZN) arm in the DNDi-CH-E1224-001 and MSF-DNDi PCR sampling optimization clinical studies, respectively. The monitoring of samples from patients treated with placebo in the DNDi-CH-E1224-001 E1224 trial revealed in a proportion of patients fluctuations in parasitic loads and occasional non-detectable results. This sampling strategy enhanced PCR sensitivity to detect treatment failure and has the potential for improving recruitment capacity in Chagas disease clinical trials.