Usefulness of Serial Blood Sampling and PCR Replicates for Treatment Monitoring of Patients with Chronic Chagas Disease

Abstract

This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time quantitative PCR (qPCR) for baseline detection and quantification of parasitic loads and posttreatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely, DNDi-CH-E1224-001 (ClinicalTrials.gov registration no. NCT01489228) and the 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 for each sample. The first two samples were collected during the same day and the third one 7 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 pretreatment 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 nondetectable 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 occasionally nondetectable results. In conclusion, a 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.

Keywords: Chagas disease; PCR; Trypanosoma cruzi; benznidazole; clinical trials; ravuconazole; treatment monitoring.

Copyright © 2019 Parrado et al.

Figures

FIG 1

Study diagram and schedule of qPCR assessments.

FIG 2

Distribution of parasitic loads in peripheral blood samples of pretreated chronic Chagas disease patients from DNDi-CH-E1224-001 and MSF-DNDi PCR sampling optimization clinical studies. CBBA, Cochabamba.

FIG 3

Distribution of parasitic loads during baseline and follow-up of the different groups of treatment of DNDi-CH-E1224-001 and MSF-DNDi PCR sampling optimization clinical studies. (A) E1224, placebo arm; (B) E1224, low-dose arm; (C) E1224, short-dose arm; (D) E1224, high-dose arm; (E) benznidazole arm from the DNDi-CH-E1224-001 trial; (F) benznidazole arm from the MSF-DNDi PCR Sampling Optimization Study. BL, baseline; 2 M, 4 M, 6 M, and 12 M, 2, 4, 6, and 12 months from the beginning of the study.

FIG 4

Cumulative therapeutic failure during the follow-up of the different treatment groups of DNDi-CH-E1224-001 and MSF-DNDi PCR sampling optimization clinical studies. (A) E1224, placebo arm; (B) E1224, low-dose arm; (C) E1224, short-dose arm; (D) E1224, high-dose arm; (E) benznidazole arm from the DNDi-CH-E1224-001 trial; (F) benznidazole arm from the MSF-DNDi PCR Sampling Optimization Study. S1 to S3, samples 1 to 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.