Within-day variation of cell-free mitochondrial and nuclear DNA in blood plasma of patients with tuberculosis: associations with drug exposure and patient-related factors

Circulating cell-free (ccf) mitochondrial DNA copy number (mtDNA CN) is a biomarker of cellular injury or cellular stress, and was associated with inflammation, the response of innate immunity, and mortality in individuals with severe infections. In addition, ccf nuclear DNA (nDNA) levels are related to inflammatory cytokines and tissue damage. In this study, blood plasma samples were collected from patients with drug-susceptible tuberculosis (TB) receiving standard four-medication regimen (isoniazid, rifampicin, ethambutol and pyrazinamide) at three time points: before medication intake (0 st), and 2- and 6-hours after medication administration. DNA extraction was performed and absolute CN (per microliter of plasma) of both mtDNA and nDNA were quantified using the QX200 ddPCR System (Bio-Rad) through separate amplifications targeting the MT-ND1 gene region for mtDNA and the B2m gene region for nDNA. The QuantaSoft software version 1.0.596 was utilized for the analysis of reaction data. Statistical analysis was conducted using GraphPad Prism 5.0 software and XLSTAT, with a significance level set at α = 0.05. Ccf-mtDNA CN levels significantly changed after medication administration (p = 0.03). The main factors determining this change were the exposure to ethambutol and isoniazid (p = 0.03 and p = 0.04, respectively). In contrast, ccf-nDNA CN remained stable after TB drug consumption. However, ccf-nDNA CN levels in blood samples collected before medication administration showed a significant association with drug-related liver injury (p < 0.0001), patient smoking status (p = 0.007), and, to a lesser extent, with patient age and isoniazid exposure (p < 0.05). In conclusion, this study demonstrates the potential of ccf-mtDNA and ccf-nDNA as biomarkers for drug exposure, drug-induced liver injury, and several TB patient-related factors. Additional studies in larger cohorts are necessary.