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Background: Undiagnosed tuberculosis (TB) patients hospitalized because of comorbidities constitute a challenge to TB control in hospitals. We aimed to assess the impact of introducing highly sensitive fluorescent microscopy for examining sputum smear to replace conventional microscopy under a high TB risk setting. Methods: We measured the impact of switch to fluorescent microscopy on the smear detection rate of culture-confirmed pulmonary TB, timing of respiratory isolation, and total non-isolated infectious person-days in hospital at a high-caseload medical center (approximately 400 TB cases annually) in Taipei. Multivariable Cox regression was applied to adjust for effects of covariates. The effect attributable to the improved smear detection rate was determined using causal mediation analysis. Results: After switch to fluorescence microscopy, median non-isolated infectious duration decreased from 12.5 days to 3 days (P<0.001). Compared with conventional microscopy, fluorescence microscopy increased sputum smear detection rate by two-fold (for all patients: from 22.8% to 48.1%, P<0.001; for patients with cavitary lung lesion: from 43% to 82%, P = 0.029) and was associated with a 2-fold higher likelihood of prompt respiratory isolation (odds ratio mediated by the increase in sputum smear detection rate: 1.8, 95% CI 1.3–2.5). Total non-isolated infectious patient-days in hospital decreased by 69% (from 4,778 patient-days per year to 1,502 patient-days per year). Conclusions: In a high TB caseload setting, highly sensitive rapid diagnostic tools could substantially improve timing of respiratory isolation and reduce the risk of nosocomial TB transmission. [ABSTRACT FROM AUTHOR]