Biomarker research characterising the effect of anti-tuberculosis (TB) chemotherapy on systemic body response is still limited. In this study, we aimed to investigate fluctuations in circulating cell-free mitochondrial DNA (ccf-mtDNA) and circulating cell-free nuclear DNA (ccf-nDNA) copy number (CN) in blood plasma of patients with drug-susceptible TB (DS-TB) and to decipher factors related to these fluctuations. The results showed considerable changes in ccf-mtDNA CN in plasma samples before drug intake and 2 and 6 h afterwards, with high inter patient variability at each time point. Multivariate linear regression revealed that the dynamics of ccf-mtDNA CN was influenced by patients' age, ethambutol pharmacokinetics, and body-mass index (BMI)
ethambutol exposure emerged as the most significant factor. Very low ccf-nDNA CN in all three time points with little variation was observed
none factors were strongly associated with ccf-nDNA. In conclusion, our study revealed the effect of anti-TB chemotherapy, age and BMI on acute changes in circulating ccf-mtDNA CN in blood plasma and highlighted the systemic, mitochondria-related effects of ethambutol in patients with TB. Further studies with larger cohorts are needed to understand the biological relevance of ccf-DNA in patients with TB and to validate its application in TB treatment monitoring.