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Introduction: Oral and/or tongue swabs have demonstrated ability to detect Mycobacterium tuberculosis (Mtb) in adults with pulmonary tuberculosis (TB). Swabs provide useful alternative specimens for diagnosis of TB using molecular assays however, the diagnostic pickup by culture requires further improvement and development. Several studies identified the presence of differentially culturable tubercle bacilli (DCTB) populations in a variety of clinical specimens. These organisms do not grow in routine laboratory media and require growth factors in the form of culture filtrate (CF) from logarithmic phase cultures of Mtb H37Rv., Methods: Herein, we compared the diagnostic performance of sputum and tongue swabs using Mycobacterial Growth Indicator Tube (MGIT) assays, Auramine smear, GeneXpert and DCTB assays supplemented with or without CF., Results: From 89 eligible participants, 83 (93%), 66 (74%) and 79 (89%) were sputum positive by MGIT, smear and GeneXpert, respectively. The corresponding tongue swabs displayed a lower sensitivity with 39 (44%), 2 (2.0%) and 18 (20%) participants respectively for the same tests. We aimed to improve the diagnostic yield by utilizing DCTB assays. Sputum samples were associated with a higher positivity rate for CF-augmented DCTB at 82/89 (92%) relative to tongue swabs at 36/89 (40%). Similarly, sputum samples had a higher positivity rate for DCTB populations that were CF-independent at 64/89 (72%) relative to tongue swabs at 26/89 (29%). DCTB positivity increased significantly, relative to MGIT culture, for tongue swabs taken from HIV-positive participants. We next tested whether the use of an alternative smear stain, DMN-Trehalose, would improve diagnostic yield but noted no substantial increase., Discussion: Collectively, our data show that while tongue swabs yield lower bacterial numbers for diagnostic testing, the use of growth supplementation may improve detection of TB particularly in HIV-positive people but this requires further interrogation in larger studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ealand, Sewcharran, Peters, Gordhan, Kamariza, Bertozzi, Waja, Martinson and Kana.)

Introduction: Routine efficacy assessments of new tuberculosis (TB) treatments include quantitative solid culture or routine liquid culture, which likely miss quantification of drug tolerant bacteria. To improve these assessments, comparative analyses using additional measures such as quantification of differentially culturable tubercle bacteria (DCTB) are required. Essential for enabling this is a comparative measure of TB treatment responses using routine solid and liquid culture with liquid limiting dilutions (LLDs) that detect DCTB in sputum., Methods: We recruited treatment-naïve TB patients, with and without HIV-infection, and serially quantified their sputum for DCTB over the course of treatment., Results: Serial sputum sampling in 73 individuals during their first 14 days of treatment demonstrated that clearance of DCTB was slower compared to routine solid culture. Treatment response appeared to be characterized by four patterns: (1) Classic bi-phasic bacterial clearance; (2) early non-responders with slower clearance; (3) paradoxical worsening with an increase in bacterial count upon treatment initiation; and (4) non-responders with no change in bacterial load. During treatment, LLDs displayed greater bacterial yield when compared with quantitative solid culture. Upon treatment completion, 74% [46/62] of specimens displayed residual DCTB and within this group, two recurrences were diagnosed. Residual DCTB upon treatment completion was associated with a higher proportion of MGIT culture, GeneXpert, and smear positivity at two months post treatment. No recurrences occurred in the group without residual DCTB., Discussion: These data indicate that DCTB assays detect distinct subpopulations of organisms in sputum that are missed by routine solid and liquid culture, and offer important alternatives for efficacy assessments of new TB treatments. The residual DCTB observed upon treatment completion suggests that TB treatment does not always eliminate all bacterial populations, a finding that should be investigated in larger cohorts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Peters, McIvor, Papadopoulos, Masangana, Gordhan, Waja, Otwombe, Letutu, Kamariza, Sterling, Bertozzi, Martinson and Kana.)