Development of a PCR Assay to Detect Low Level Trypanosoma cruzi in Blood Specimens Collected with PAXgene Blood DNA Tubes for Clinical Trials Treating Chagas Disease

Abstract

Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi’s discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease.

Targeting Multicopy Prophage Genes for the Increased Detection of Borrelia burgdorferi Sensu Lato (s.l.), the Causative Agents of Lyme Disease, in Blood

“…efficiency from both serial dilution experiments confirmed that our standard curve assay was robust and repeatable (Bustin et al, 2009). In this study, we defined the analytical limit of detection (LoD) as the lowest concentration where at least 95% of the technical replicates were positive in the Ter-qPCR (Wei et al, 2016). The LoD was calculated from serial dilutions of Terplasmids.…”

“…In this study, we defined the analytical limit of detection (LoD) as the lowest concentration where at least 95% of the technical replicates were positive in the Ter-qPCR ( Wei et al, 2016 ). The LoD was calculated from serial dilutions of Ter-plasmids.…”

“…25 showed copy numbers of 10, 2.8, and 0, respectively ( Supplementary Table). This qPCR variability is due to the stochastic effect of having a low number of PCR templates, and has been observed in PCR detection of low level bacteria in blood before (Wei et al, 2016). Markedly differences were observed in the mean copy number of early LD (2.4), and late LD (6.9), as well as healthy volunteers (0.8), which suggests a potential positive correlation between the severity of LD and the copy numbers, i.e., higher copy numbers in late LD patients, in contrast to lower copy numbers in early LD patients and healthy volunteers.…”

A retrospective study on the influence of siblings’ relatedness in Bolivian patients with chronic Chagas disease

“…Furthermore, some acquired conditions are known to modify the results of PCR assays in patients with Chagas disease, such as co-infection with helminths, infection with human immunodeficiency virus, immunosuppression and pregnancy [14–17]. In the last 15 years a strong effort has been made in validation and standardization of these techniques, reaching a sensitivity of 60–90% for the best performing methods, with a specificity of 100% [18, 19]. In our study, pre-treatment PCR was performed in 73% of our patients.…”

Chagas Disease Treatment Efficacy Biomarkers: Myths and Realities

“…False negatives occur due to fluctuations in parasitemia, the isolation of parasite in tissue or organs, and the intrinsic limit of detection of PCR and qPCR techniques [58]. Other distinct factors may contribute to the overall performance of PCR assays: the size of the serum sample for parasite DNA extraction, the sample collection tubes, the different PCR assay conditions, and the algorithm used to classify results can affect the evaluation of the samples, as was demonstrated by Wei et al [59] for the STOP CHAGAS clinical trial that evaluated posaconazole for the treatment of CD [60]. Nevertheless, PCR is a promising tool that can be easily performed in clinical settings and used for clinical trials; thus, the investment in improving PCR methodologies is worthwhile.…”