12 research outputs found
Usefulness of Serial Blood Sampling and PCR Replicates for Treatment Monitoring of Patients with Chronic Chagas Disease
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-CHE1224-001 (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
First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease
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
Genetic polymorphism of Trypanosoma cruzi bloodstream populations in adult chronic indeterminate Chagas disease patients from the E1224 clinical trial
Background: The role that the genetic diversity of natural Trypanosoma cruzi populations plays in response to trypanocidal treatment of chronic Chagas disease (CD) patients remains to be understood. We analysed the genetic polymorphisms of parasite bloodstream populations infecting chronic CD patients enrolled in the E1224 clinical trial. Methods: A total of 506 baseline and post-treatment follow-up samples from 188 patients were analysed. T. cruzi satellite DNA (satDNA) was amplified and sequenced using cruzi1/cruzi2 primers, and samples with TcI/ III, TcII, TcIV or hybrid satDNA sequences were identified. Minicircle signatures were obtained after kinetoplast DNA amplification using 121/122 primers and restriction enzyme digestion. Genetic distances between baseline and post-treatment minicircle signatures were estimated using the Jaccard coefficient. Results: At baseline, 74.3% TcII, 17.9% hybrid and 7.8% TcI/III satDNA sequences were found, whereas at the end of follow-up the distribution was 55.2% TcII, 35.2% hybrid and 9.5% TcI/III. The placebo arm was the treatment group with the highest variation of satDNA sequences between baseline and post-treatment follow-up. Genetic distances between baseline and post-treatment minicircle signatures were similar among all treatment arms. No association between minicircle signature variability and satDNA type distribution was found. Conclusions: Genetic variability of T. cruzi bloodstream populations during post-treatment follow-up did not differ from that observed during chronic infection in the absence of treatment, suggesting that there were no selection events of E1224-resistant parasite populations. This is the first report documenting the genetic polymorphism of natural T. cruzi populations in chronic patients in the context of clinical trials with trypanocidal drugs.Fil: Ramirez Gomez, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Acevedo, Gonzalo Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Torres, Carolina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones en Bacteriología y Virología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Parrado, Rudy. Universidad Mayor de San Simón; BoliviaFil: De La Barra, Anabelle. Universidad Mayor de San Simón; BoliviaFil: Villarroel, Sandro. Universidad Mayor de San Simón; BoliviaFil: García, Lineth. Universidad Mayor de San Simón; BoliviaFil: Gascon, Joaquim. Universidad de Barcelona; EspañaFil: Ortiz, Lourdes. Universidad Autónoma Juan Misael Saracho; BoliviaFil: Torrico, Faustino. Colectivo de Estudios Aplicados y Desarrollo Social; BoliviaFil: Ribeiro, Isabela. Drugs for Neglected Diseases Initiative; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin
First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease
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
First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease
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
Comparison of quantifiable SatDNA qPCR results for retesting samples analysis.
<p><b>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).</b> par. eq./mL: parasite equivalents in 1 mL of blood; SatDNA qPCR: Satellite DNA Real-Time PCR; SD: standard deviation.</p
Comparison of intra- and inter-laboratory SatDNA qPCR results for proficiency testing panels analysis based on number of panel.
<p>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.</p
Comparison of intra- and inter-laboratory SatDNA qPCR results for proficiency testing panels analysis based on <i>T</i>. <i>cruzi</i> stocks.
<p>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.</p
Accordance and concordance analysis of SatDNA qPCR qualitative results for proficiency testing panels.
<p>Accordance and concordance analysis of SatDNA qPCR qualitative results for proficiency testing panels.</p