Mycobacterium tuberculosis (M. tb) serves as the main pathogen responsible for Tuberculosis (TB). It predominantly targets the lungs and leads to a persistent infectious disease. The spread of drug-resistant tuberculosis...Mycobacterium tuberculosis (M. tb) serves as the main pathogen responsible for Tuberculosis (TB). It predominantly targets the lungs and leads to a persistent infectious disease. The spread of drug-resistant tuberculosis and the exacerbation of economic burdens due to co-infections with Human Immunodeficiency Virus (HIV)/M. tb pose significant challenges in prevention and treatment. The BCG vaccine is currently the only approved (TB) vaccine, but its protective effect is limited for adults. In this research, we engineered the fusion protein gene RPC4, incorporating four crucial antigens from M. tb. The study revealed that the IFN-γ levels in the peripheral blood of infected patients significantly surpassed those in healthy individuals. To assess the immune response of RPC4 as a BCG-enhanced vaccine following initial immunity, researchers administered it alongside the novel adjuvant DIMQ to immunize mice. Experiments revealed that the BCG + RPC4/DIMQ vaccine induces a substantial immunogenic response in the mice.
Previously, a slot blot or an indirect enzyme-linked immunosorbent assay (ELISA) using a synthetic or purified MTP antigen, conceptually demonstrated IgG antibody induction in pulmonary TB patients, albeit with small sam...Previously, a slot blot or an indirect enzyme-linked immunosorbent assay (ELISA) using a synthetic or purified MTP antigen, conceptually demonstrated IgG antibody induction in pulmonary TB patients, albeit with small sample sizes and differing sensitivity. Therefore, we evaluated an IgG MTP ELISA in larger populations from The Gambia (n = 549), Uganda (n = 161), and South Africa (n = 193), comprising human immunodeficiency virus (HIV) positive and negative, with microbiologically confirmed active TB. The association between the IgG level and demographic characteristics was determined by multivariate logistic regression. The sensitivity (44.8-61.2 %) and specificity (33.4-78.5 %) varied in the three cohorts. Anti-MTP antibody titres differed between the TB positive and negative groups within the South African and The Gambian cohorts (p < 0.001), but not in Uganda (p = 0.35). Antibodies were detected in HIV positive and negative patients and were reduced at 6-month follow-up after treatment (p > 0.067). The study verified previous findings that anti-MTP antibodies, and therefore MTP antigen, are produced during active TB. However, the accuracy of the MTP-IgG ELISA was low, and is therefore not suitable as a target product profile in the high burden TB areas investigated. Further studies are needed to clarify the variable reactivities in different geographical areas.
BACKGROUND: We assessed the impact of centrifugation on recovery of Mycobacterium tuberculosis (M.tb). METHODS: We used 0.5 McFarland from the 2 weeks M. tb, H37Rv culture and homogenized sputum for our experiments. Samp...BACKGROUND: We assessed the impact of centrifugation on recovery of Mycobacterium tuberculosis (M.tb). METHODS: We used 0.5 McFarland from the 2 weeks M. tb, H37Rv culture and homogenized sputum for our experiments. Samples were decontaminated by 2 % NaOH for 20 min and with PBS for controls. Decontaminated aliquots were centrifuged at 2000×g, 3000×g and 6000×g for 40 min and inoculated on MGIT and LJ media. MGITs were incubated into the BACTEC MGIT 960 Systems following BD manuals and data analyzed on GraphPad Software. RESULTS: The positivity (days) for M. tb, H37Rv in MGIT and LJ decreased from 20.4 to 17.7 and from 47.6 to 26.6 at 2000×g and 6000×g, respectively; P > 0.05. For controls, MGIT and LJ positivity (days) decreased from 19 to 10 and from 39.2 to 11.2 at 2000×g and 6000×g, respectively; P > 0.05. MGIT positivity was 6(60 %) at 2000×g and 8(80 %) at 6000×g, corresponding to mean (±SD) of 13.7 ± 6.7 and 9.06 ± 4.6 days, respectively for sputum. LJ positivity was 1(10 %) at 2000×g and 7(70 %) at 6000×g. MGIT contamination for controls (sputum) was over 50 % and 80 % for LJ. CONCLUSION: Higher centrifugation speed improves yield and sensitivity of TB culture.
The connection between genetic mutations linked to delamanid resistance and phenotypic resistance remains unclear. We assessed the phenotypic effects of delamanid-resistant mutations in the Mycobacterium tuberculosis H37...The connection between genetic mutations linked to delamanid resistance and phenotypic resistance remains unclear. We assessed the phenotypic effects of delamanid-resistant mutations in the Mycobacterium tuberculosis H37Rv strain through gene disruption using homologous recombination and complementation tests. Delamanid resistance was assessed by determining the minimum inhibitory concentration (MIC) via the 7H9 microdilution method. Sanger sequencing identified mutations, and conservation of the mutated residues was predicted through multiple sequence alignments of orthologs. A total of 116 isolates with MIC ≥0.025 μg/mL were analyzed, among which mutations were identified in the ddn and fbiA genes. Isogenic strains were generated based on these mutations. The ddn or fbiA isogenic strains with Ala77Val, Gly81Ser, Asn25fs, and Leu104Phe in fbiA had MICs ≥0.8 μg/mL, indicating resistance. In contrast, the ddn isogenic strain with Pro12Ala had an MIC of 0.012 μg/mL, showing susceptibility, while Gly96Asp in fbiA had an MIC of 0.1 μg/mL, indicating resistance. All mutations, except for Pro12Ala, were conserved in the protein sequences of both FbiA and Ddn and their mycobacterial orthologs. The characterization of these mutations provides insights into the mechanisms of delamanid resistance, which may inform the development of optimized treatment strategies.
The aim of our paper is to demonstrate a case (KD429) with tuberculous meningitis (TBM) from the 2nd-3rd‒century‒CE Carpathian Basin. The skeleton of KD429 was subject to a detailed macromorphological evaluation, focusin...The aim of our paper is to demonstrate a case (KD429) with tuberculous meningitis (TBM) from the 2nd-3rd‒century‒CE Carpathian Basin. The skeleton of KD429 was subject to a detailed macromorphological evaluation, focusing on the detection of pathological lesions likely related to tuberculosis (TB). It was the presence of endocranial alterations, especially the TB-specific granular impressions, based on which the diagnosis of TBM was established in KD429. Besides KD429, only eight cases with TB have been published from the Sarmatian-period (1st-5th centuries CE) Carpathian Basin. Reports of archaeological cases with TB, like KD429, can provide invaluable information about the spatio-temporal distribution of the disease in the past. Nonetheless, to get a more accurate picture about the burden that TB may have put on the Sarmatians, the systematic macromorphological (re-)evaluation of their osteoarchaeological series would be advantageous. Interestingly, the skeleton of KD429 was unearthed from not a grave-pit but a storage pit from the archaeological site of Kiskundorozsma-Daruhalom-dűlő II (Hungary). At the current state of research, the motive behind the exclusion of KD429 from the "normal" burial custom cannot be determined; therefore, it remains an open question whether their disease (TBM) played a role in it or not.
Inhaled therapy of tuberculosis (TB) by a Dry Powder Inhalation (DPI) comprising mycobacteriophage D29 and TM4 was non-inferior to oral anti-tuberculosis therapy (ATT) with isoniazid and rifampicin in a mouse model of in...Inhaled therapy of tuberculosis (TB) by a Dry Powder Inhalation (DPI) comprising mycobacteriophage D29 and TM4 was non-inferior to oral anti-tuberculosis therapy (ATT) with isoniazid and rifampicin in a mouse model of infection with Mycobacterium tuberculosis (Mtb). No pharmaceutical phage product of mycobacteriophages is approved for large-scale production. We scaled up preparation and downstream processing of phages, developed DPI formulations, and established methods for determining identity, purity, assay, stability, and critical quality attributes (CQA). We carried out cell-based assays of intracellular bactericidal activity and pharmacokinetics and comparative efficacy in Mtb-infected mice. Daily doses of the DPI containing ∼10 Plaque Forming Units/dose DPI reduced Mtb colony forming units (CFU) in the lungs from 6.4 ± 0.3-log to 4.8 ± 0.7-log in four weeks, while oral human equivalent doses (HED) of isoniazid and rifampicin reduced CFU to 3.8 ± 0.8-log. Combining inhaled phages with oral drugs sterilized the lungs of one of four mice and reduced group mean CFU to 2.3-log. Inhalations significantly upregulated tumor necrosis factor (TNF) in lung tissue to ∼1500 pg/ml of homogenate, improved organ morphology, and reduced histopathology. The HD DPI may be a useful adjunct to oral drugs. Dose-finding animal efficacy studies are required before assessing preclinical safety.
Novel 2-phenylquinolin-4(1H)-one threaded 1,2,3- triazoles were designed, synthesized and evaluated for in vitro activity against Mycobacterium tuberculosis which could be putatively through inhibition of carbonic anhydr...Novel 2-phenylquinolin-4(1H)-one threaded 1,2,3- triazoles were designed, synthesized and evaluated for in vitro activity against Mycobacterium tuberculosis which could be putatively through inhibition of carbonic anhydrase β. Molecules were synthesized in simple Schottan Baumann reaction for amide synthesis. Purified compounds were screened for antitubercular and antibacterial activities. Among them, 1-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-phenylquinolin-4(1H)-one 9j with 2-methoxy at the ortho position of phenyl ring indicated significant antitubercular activity with MIC value of 6.25, 3.12 and 3.12 μg/ml antimicrobial activity against Mycobacterium tuberculosis, gram positive and gram negative strain. The molecular docking and dynamics studies demonstrated that the compound 9j occupied the Zn-binding site of the enzyme with docking energy of -6.2 kcal mol. In silico ADME studies indicated that the synthesized compounds have good drug likeliness. The findings explore and present a potential series of antimycobacterial agents in the hope of developing new and advanced therapeutics for tuberculosis.
Tuberculosis, a significant public health concern in Central African Republic lacks whole-genome-based identification and typing of the Mycobacterium tuberculosis complex strains circulating in populations in that countr...Tuberculosis, a significant public health concern in Central African Republic lacks whole-genome-based identification and typing of the Mycobacterium tuberculosis complex strains circulating in populations in that country. Here, we investigated 68 rifampin-resistant clinical isolates collected in 2024 from eight districts in Bangui and surrounding regions. The analysis revealed that all isolates were M. tuberculosis stricto sensu, distributed across nine lineages: L4.1.2.1 Haarlem (n = 20), L4.6 Euro-American (n = 17), L4.6.1.2 Uganda (n = 13), L4.6.2.2 Cameroon (n = 12), and L4.1.1.1 X-Type (n = 2), and single isolates in L4.1 (Euro-American), L4.6.1 (Uganda), L4.3.1 (LAM), and L3 (Delhi-CAS). The antibiotic resistance profile showed that 9/68 (13.2 %) of the M. tuberculosis isolates were susceptible, while 59/68 (86.7 %) exhibited at least one predicted antibiotic resistance. These data provide new insights into tuberculosis transmission in Central African Republic in contrast to reports from neighboring countries, including the absence of Mycobacterium bovis, hence zoonotic tuberculosis and other factors. This preliminary study limited to rifampin-resistant isolates, nevertheless paves the way for a genome-based survey of tuberculosis in Central African Republic which is essential for enhancing the management and control of the deadly tuberculosis that is a public health concern in the country.
Feizi S, Cooksley CM, Reyne N
… +12 more, Boog B, Finnie J, Shaghayegh G, Hon K, Ramezanpour M, Psaltis AJ, Wormald PJ, Cmielewski P, McCarron A, Donnelley M, Parsons D, Vreugde S
Animal models that can mimic progressive granulomatous pulmonary disease (PD) due to non-tuberculous mycobacteria (NTM) have not been established in rats to date. These models could assist with the study of the pathophys...Animal models that can mimic progressive granulomatous pulmonary disease (PD) due to non-tuberculous mycobacteria (NTM) have not been established in rats to date. These models could assist with the study of the pathophysiology of NTM-PD as well as the preclinical development of new therapies. In the present study, an immunocompetent rat model of progressive Mycobacterium abscessus (MABs)- PD was developed using MABs originating from a patient with cystic fibrosis. MABs was embedded in agarose beads and delivered intratracheally to the lungs of Sprague Dawley rats two times at a one-week time interval. The bacterial burden of lysed lungs, spleen and liver was assessed by calculating colony forming units (CFUs) on day 28. Lung CFUs indicated a ∼1.2-2 log total CFU increase compared to the initial total bacterial load instilled into the lungs. In all infected rats, multinodular granulomatous inflammatory lesions containing MABs were found in the lung. These findings support the establishment of an immunocompetent MABs PD rat model, characterised by an increase in mycobacterial burden over time and a chronic granulomatous inflammatory response to the MABs infection.
Budde K, Lange C, Reimann M
… +5 more, Zielinski N, Meiwes L, Köhler N, DZIF TB cohort study group, Carballo PS
Tuberculosis (Edinb)
· 2025 May · PMID 40048961
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The diagnosis of tuberculosis largely relies on the detection of Mycobacterium tuberculosis (M. tuberculosis) via pathogen-specific DNA or bacterial culture from sputum samples. As the only point-of-care test so far, uri...The diagnosis of tuberculosis largely relies on the detection of Mycobacterium tuberculosis (M. tuberculosis) via pathogen-specific DNA or bacterial culture from sputum samples. As the only point-of-care test so far, urinary lipoarabinomannan (LAM) has been endorsed by the World Health Organization for the diagnosis of tuberculosis in people living with HIV. In this study, the electrochemiluminescence LAM research assay (EclLAM) was used to measure LAM in the urine of HIV-sero-negative individuals with pulmonary tuberculosis and to monitor anti-tuberculosis treatment. Urine samples from 18 patients with microbiologically confirmed tuberculosis were analyzed before and after the initiation of anti-tuberculosis therapy and 17 healthy controls via the S4-20/A194-01 antibody pair. The assay identified 13/18 (72.2 %) patients with tuberculosis and was negative in 17/17 (100.0 %) controls (AUC 0.88). The results of the reactive urine LAM tests correlated with the detection of M. tuberculosis growth in culture (r = 0.94, p < 0.05). In conclusion, the LAM-specific antibody assay is promising to fulfill the WHO target product profile for the diagnosis of tuberculosis.
Mycobacterium abscessus (M. abscessus) is an emerging pathogenic mycobacterium that mainly causes pulmonary infections, especially in immunocompromised patients. This bacterium shows exhibits intrinsic resistance to many...Mycobacterium abscessus (M. abscessus) is an emerging pathogenic mycobacterium that mainly causes pulmonary infections, especially in immunocompromised patients. This bacterium shows exhibits intrinsic resistance to many anti-tuberculosis drugs, posing significant challenges for both patients and clinicians, thereby raising the need for innovative drug discovery. In this study, we selected phenylalanine-tRNA ligase beta unit (PheT) as a model target and used CRISPR interference to evaluate its essentiality as a therapeutic target against M. abscessus. The results show that genetically disruption of PheT leads to clear growth inhibitory phenotypes both in vitro and in vivo. Further transcriptome analysis revealed differential expression of host genes in response to PheT gene silencing, including genes involved in the cell cycle, apoptotic signaling, and inflammatory responses. Overall, PheT gene plays a crucial role in M. abscessus infection, and its silencing may represent a druggable therapeutic strategy for treating this infection.
Despite significant advances in research over the past century, Tuberculosis (TB) remains a formidable global health concern. TB granulomas are organized structures composed of immune cells, that serve as the body's prim...Despite significant advances in research over the past century, Tuberculosis (TB) remains a formidable global health concern. TB granulomas are organized structures composed of immune cells, that serve as the body's primary defense against the spread of Mycobacterium tuberculosis (Mtb). The immune landscape of TB granulomas involves a complex array of immune cells, including CD4 and CD8 T cells, B cells, NK cells, and others, which collectively influence the fate of the granuloma. B cells contribute to the formation of the granuloma's germinal center, while the functional state of T cells-particularly their ability to control infection-dictates whether the granuloma is controlling or proliferative. The intricate interplay between T cells and the dynamic microenvironment of the granuloma plays a pivotal role in determining the outcome of the infection. However, several aspects of the immunological basis of tuberculosis are still unknown. This review delves into the immunological landscape of TB granuloma, focusing on the dynamic cellular interplay within the granuloma and its profound influence on disease pathogenesis.
Triacylglycerol (TAG) is the major storage lipid of mycobacteria. Mycobacterium tuberculosis (Mtb) genome encodes 15 triacylglycerol synthases (Tgs), which are speculated to differ in substrate preference, suggesting spe...Triacylglycerol (TAG) is the major storage lipid of mycobacteria. Mycobacterium tuberculosis (Mtb) genome encodes 15 triacylglycerol synthases (Tgs), which are speculated to differ in substrate preference, suggesting specific physiological roles. In this study, we investigated the role of a Tgs, Rv3371, in the context of infection. Rv3371 knock-out (KO) Mtb was attenuated in mice, with corresponding poor fitness inside macrophages. The KO was more sensitive to free long-chain fatty acids, but was more tolerant to oxidative and nitrosative stresses. Enzyme kinetics of Rv3371 showed its preference for propionyl-CoA. Excess propionate in growth medium retarded the growth of the KO more significantly than the wild type and complemented mutant. This suggests an additional role of Rv3371 in reducing toxic levels of propionate in Mtb by synthesising propionyl TAG. Moreover, chemical inhibition of methylcitrate cycle caused a decrease in methyl-branched lipids in the KO. Overall, the results suggest a role of Rv3371 in Mtb survival in the host through its roles beyond TAG storage.
Most of Mycobacterium tuberculosis(Mtb) infection result in the formation of granulomas, which are often rich in immune cells, with subsequent clinical symptoms. However, the role of the immune system in the formation of...Most of Mycobacterium tuberculosis(Mtb) infection result in the formation of granulomas, which are often rich in immune cells, with subsequent clinical symptoms. However, the role of the immune system in the formation of tuberculosis granuloma structures has not been fully revealed. Here we first analyzed single-cell transcriptome and microenvironment spatial characteristics to reveal the contribution of immune cells to granuloma expansion with validation by immunofluorescence. We then integrated published peripheral blood transcriptome data for Mtb-infected patients and healthy controls. Immune cell profiles were deconvoluted and results were validated on a local cohort using flow cytometry. At the same time, an in-depth evaluation of the changes in the population and function of multiple peripheral blood immune cells during tuberculosis infection were conducted to define correlation with granuloma area. Finally, we screened 6 cytokines (IL6, IL8, IL10, IFNγ, TNFα, TGFβ) through machine learning bioinformatics and analyzed their correlation with the size of tuberculosis granuloma. Based on these findings, we confirmed that the dynamic variation in proportion of immune cells in peripheral blood and the levels of cytokine profiles are closely related to the occurrence and development of tuberculosis granuloma. This study provides a theoretical basis for the molecular mechanism of tuberculosis granuloma.
Mycobacterium tuberculosis (Mtb) is a crucial and destructive intracellular pathogen responsible for causing tuberculosis (TB), a disease of substantial morbidity and mortality. Mtb capsular polysaccharides can misdirect...Mycobacterium tuberculosis (Mtb) is a crucial and destructive intracellular pathogen responsible for causing tuberculosis (TB), a disease of substantial morbidity and mortality. Mtb capsular polysaccharides can misdirect the host's immune response pathways, resulting in additional challenges in TB treatment. These capsule polysaccharides are biosynthesized by a series of stealth proteins including CpsY. Our prior investigations elucidated the structural and functional information of the central domain (aa 201-520) of CpsY within Mtb. However, within the host milieu, it is the full-length iteration of CpsY, rather than its truncated form CpsY, that assumes pivotal roles in immune evasion. Consequently, investigating the functional mechanism of full-length CpsY is extremely important. Here, we found that the indispensable role of four conserved regions (CR1-CR4) in governing the phosphotransferase activity of full-length CpsY. Notably, the deletion of S2 (ΔS2) dramatically increased the activity compared to the wild-type (WT) full-length CpsY, thereby revealing S2 in the regulatory dynamics governing the inactivation and activation of full-length CpsY. The gene cpsY helps Mtb to survive in macrophages. Our findings were useful for the development of vaccines and immunotherapies targeting Mtb.
Leprosy diagnosis is difficult to perform due to variable sensitivity and/or specificity of the tests. In addition, the collection of the blood samples requires laboratorial structure and trained professionals. In the pr...Leprosy diagnosis is difficult to perform due to variable sensitivity and/or specificity of the tests. In addition, the collection of the blood samples requires laboratorial structure and trained professionals. In the present study, the diagnostic efficacy of M1 chimeric protein, which was recently showed to be antigenic for leprosy using a serum-based ELISA, was evaluated against patient urine. Paired serum and urine samples were collected from patients with paucibacillary (PB) and multibacillary (MB) leprosy, tegumentary and visceral leishmaniasis, tuberculosis, Chagas disease, malaria, and HIV-infected subjects. Samples from healthy individuals and household contacts were also used. The protein and peptides used to compose it were used as antigens, and results showed that the four peptides presented good sensitivity and specificity to detect MB leprosy, while M1 protein showed sensitivity and specificity of 98.5 % and 100 %, respectively, to detect both PB and MB leprosy, when an urine-based ELISA was performed. Positive (PPV) and negative (NPV) predictive values were 100 % and 98.3 %, respectively. In a serum-based ELISA, sensitivity and specificity were 96.9 % and 100 %, respectively, with PPV and NPV of 100 % and 96.5 %, respectively. In conclusion, preliminary data suggest that M1 protein could be considered for diagnosis of leprosy by using patient urine.
In recent years, a rise in non-tuberculosis mycobacteria pulmonary disease (NTM-PD) has been reported in several countries. However, data for high-burden tuberculosis settings, including South Africa, is currently limite...In recent years, a rise in non-tuberculosis mycobacteria pulmonary disease (NTM-PD) has been reported in several countries. However, data for high-burden tuberculosis settings, including South Africa, is currently limited. In this study, we conducted a retrospective analysis of routine diagnostic data obtained from one diagnostic laboratory in South Africa between 2015 and 2019. During this period, samples from 275 individuals with suspected mycobacterial infection were tested using the GenoType Mycobacterium CM (Common mycobacteria) or AS (Additional species) line probe assay (LPA) (Brucker-Hain Life science, Nehren, Germany), yielding an NTM-positive result for 163 of these individuals. Interestingly, the positivity rate in respiratory samples declined from 93 % in 2015 to 79 % in 2019. Just over half of the positive samples were of respiratory origin, and the most common species identified in respiratory samples was Mycobacterium intracellulare/Mycobacteium avium complex (28.9 %), followed by M. avium (17.4 %). Where the mycobacterial species was not identified by the LPA, a higher proportion of the subsequent cultures were negative, suggestive of colonisation rather than infection. More than half of patients with a positive NTM-LPA were HIV positive (55.9 %), and this association declined slightly during the study period (62.5 %-50 %).
Khayumbi J, Sasser LE, McLaughlin TA
… +8 more, Ongalo J, Tonui J, Ouma SG, Campbell A, Odhiambo FH, Gandhi NR, Kiprotich C, Day CL
Tuberculosis (Edinb)
· 2025 Mar · PMID 39864237
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Infection with HIV is associated with dysregulated CD4 T cell responses to Mycobacterium tuberculosis (Mtb) and increased risk of developing tuberculosis. Mtb-specific CD4 T cells in people with HIV have diminished Th1 c...Infection with HIV is associated with dysregulated CD4 T cell responses to Mycobacterium tuberculosis (Mtb) and increased risk of developing tuberculosis. Mtb-specific CD4 T cells in people with HIV have diminished Th1 cytokine production capacity, thus we utilized a flow cytometry-based assay to measure CD40L expression by Mtb-specific CD4 T cells in a cytokine-independent manner. We evaluated the frequency and phenotype of Mtb-specific CD4 responses in Kenyan adults with latent Mtb infection and found that the majority of Mtb-specific CD4 T cells expressed CD40L in the absence of IFN-γ, regardless of HIV infection status. Expression of HLA-DR was increased on Mtb-specific CD4 T cells in people with HIV, compared to people without HIV. These data suggest expression of HLA-DR by Mtb-specific CD4 T cells may represent an early biomarker of increased mycobacterial antigen stimulation in people with HIV prior to the development of symptomatic tuberculosis disease.
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 D...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.