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

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 and 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 studies, respectively. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasional non-detectable results. This 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.


Introduction
For quantification purposes, standard curves were plotted with 1/10 serial dilutions of total 150 DNA obtained from a GEB seronegative sample spiked with 10 5 par. eq./mL LL014-1-R1 Cl1 Samples that yielded positive results by SL-IR-II PCR but were non-detectable by 24Sα-167 rDNA PCR were reported as belonging to the TcII/V/VI group. Those samples that amplified 168 the 140 bp of 24Sα-rDNA fragment but had non-detectable results for A10 PCR were 169 reported as belonging to TcII/VI group. Those samples amplifying both bands of 125 and 140 from Cochabamba, Tarija, and Aiquile cohorts at baseline, and between baseline and follow-  In this trial, qPCR was firstly assayed in duplicate from each S1 and S2 DNA extract. When 194 both replicates gave non-detectable qPCR results from one of these DNA extracts, a third qPCR replicate was analyzed from the corresponding sample. When the third replicate was 196 included, qPCR positivity increased from 54.8 to 60.5% (for S1) and from 53.6 to 59.2% (for 197 S2) in samples collected from the Cochabamba cohort, and from 59.5 to 63.4% (S1) and from 198 55.3 to 60.7% (S2) in those collected from the Tarija cohort (Table 1,  In the DNDi-CH-E1224-001 trial, the comparison of qPCR positivity obtained after testing 202 individual S1 or S2 samples did not give significant differences ( When S1 and S2 gave non-detectable qPCR results, a third sample, named S3 was taken 208 seven days later. The analysis of PCR positivity obtained using three serial samples 209 (S1+S2+S3) compared to that obtained from individual samples demonstrated higher 210 sensitivity for both Cochabamba (60.5 vs 76.2%, p< 0.001) and Tarija cohorts (63.4 vs 211 77.8%, p< 0.001). Finally, qPCR positivity obtained after testing S1+S2 versus that obtained 212 after testing S1+S2+S3 increased by 6.5% (N= 19/294)  On the other hand, no statistical difference was observed in qPCR positivity by testing 216 individual S1, S2, or S3 samples in the MSF-DNDi PCR sampling optimization study (Table   217 1, p> 0.05). Computing the cumulative qPCR positivity obtained for S1+S2 (85.1%) in 218 comparison to the positivity obtained for S1 (10 mL of blood, 73.5%, p< 0.01) or S2 alone 219 (5 mL of blood, 76.9%, p< 0.05) increased sensitivity. This was also true of the cumulative qPCR positivity obtained for S1+S2+S3 (90.2%) compared to that obtained for the individual 221 samples (S1, p< 0.001; S2, p< 0.001; and S3, 72.7%, p< 0.001). Comparison of the 222 cumulative qPCR positivity obtained from S1+S2+S3 with respect to S1+S2 showed an 223 increase of 5.1% ( Because both studies used the same qPCR method performed in the same laboratory, a 230 hypothesis for this geographical variability in qPCR positivity could be related to diversity of 231 parasitic strains or parasitic loads in the populations studied, and/or to a higher endemicity 232 and exposure to the vector in Aiquile, and therefore a potential risk of reinfection. In order to 233 investigate this, the distribution of T. cruzi DTUs was investigated by genotyping 180 qPCR 234 positive samples from these localities with Ct values below 33.   Table 3 shows the cumulative qPCR findings obtained from all three serial blood samples 255 during screening and monitoring of all treatment branches in both clinical trials.

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In the MSF-DNDi PCR sampling optimization study, the strategy involving serial sampling 315 analysis allowed an increase in detection of treatment failure of up to 32.1% (S1+S2+S3) at 316 the end of follow-up in comparison to that detected from individual samples (S1, 9.5%, p< cumulative treatment failure between S1 and S2 (p< 0.05), whereas no differences were found 319 between S3 and S1 or S2 (Fig 3F, p> 0.05). There was an increase of 7.3% in cumulative 320 treatment failure detected after testing S1+S2+S3 versus that detected after testing S1+S2 321 (24.8%) (Fig 3F, p> 0.05).

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Analysis of cumulative treatment failure tested at EOF in E1224 treatment arms did not show 324 differences between E1224 LD and SD, and between them and the placebo arm (Fig. 3, p>   325 0.05). In contrast, the E1224 HD arm showed a lower qPCR positivity than LD (p< 0.01), SD 326 (p< 0.05) and placebo (p< 0.05). In addition, the E1224 BZN arm showed lower qPCR 327 positivity than all other arms (p< 0.001).

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No statistically significant differences were observed between both BZN treated cohorts  Impact of serial sampling strategies on qPCR sensitivity. 334 In recent years, several clinical trials to evaluate anti-parasitic treatments for CD were carried 335 out using different sampling strategies and PCR protocols, and variable rates of PCR 336 positivity were obtained [11,12,20].

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The present analyses shows that qPCR sensitivity was significantly improved at baseline in 339 the DNDi-CH-E1224-001 trial when two blood samples were collected and each DNA extract 340 was analyzed in duplicate by qPCR. The addition of the third blood sample and third qPCR 341 replicate in the subset of patients who had non-detectable PCR results in S1 and S2, gave a 342 small, but non-statistically significant improvement in positivity. The limited data available thus far is insufficient to determine the clinical relevance of this small increase in sensitivity 344 in the evaluation of treatment response. In fact, the samples with only one out of three PCR 345 positive results were non-quantifiable. As treatment was expected to reduce further the 346 parasite burden in those patients with non-quantifiable baseline qPCR results, reducing the 347 chance of detecting treatment failure, three blood samples and qPCR triplicates were tested 348 during post-treatment follow-up.

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In the MSF-DNDi PCR sampling optimization study, the use of 5 mL of blood, instead of 10 351 ml as starting sample for qPCR analysis, as well as the collection of a third blood sample 352 seven days after the first two samples instead of a few minutes later, did not modify the 353 overall clinical sensitivity [15,16].

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In conclusion, these findings support the use of a lower volume of blood, collected during one 356 visit, for qPCR testing purposes.  Median parasitic loads were higher in Aiquile than in Cochabamba or Tarija, although the 367 differences did not reach statistical significance (Table 1 and Fig 1). This could be due to the rural nature of the Aiquile area compared to the cities of Cochabamba and Tarija. In a recent 369 study of pregnant women from Bolivia, it was observed that the differences in seroprevalence 370 for T. cruzi infection were above all related to the area in which the patients lived most of 371 their lives. Hyper-endemic hotspots were observed where prevalence surpassed 60% and one 372 of the affected areas was the municipality of Aiquile, with 66% seroprevalence [21]. In areas 373 where vector infestation was higher, the seroprevalence of CD was also higher [21]. The qPCR based study of the DNDi-CH-E1224-001 clinical trial demonstrated that BZN was 387 a better parasiticidal drug than E1224 in monotherapy, and that in turn, E1224 HD had higher 388 efficacy than the other E1224 regimens (Fig 2). Treatment with BZN gave a better 389 parasitological response in the urban cohorts of the DNDi-CH-E1224-001 trial than in the 390 rural patients from the MSF-DNDi PCR sampling study, although no significant differences    Positives 44 (100%) 3 (6.8%) 0 (0.0%) 2 (4.7%) 2 (4.5%) Quantifiables 11 (25.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%)