Invasive non-tuberculous mycobacteria (NTM) infections are emerging worldwide in patients undergoing open-chest cardiac bypass surgery exposed to contaminated heater-cooler units (HCUs). Although this outbreak has been investigated by culturing bacteria isolated from HCU aerosol and water samples, these conventional methods have low-analytic sensitivity, high rates of sample contamination and long turnaround time (TAT).To develop a simple and effective method to detect NTM in HCUs by real-time PCR, with a short laboratory TAT and reliable culture results.A total of 281 water samples collected from various HCUs at 7 Italian hospitals were simultaneously screened for NTM by a propidium monoazide (PMA)-PCR assay and by conventional culture testing. The results were analyzed with culture testing being the reference method.i) The agreement between culture testing and PMA-PCR was 85.0% with a Ct cut-off value of < 38 vs.80.0% with a Ct of < 43, with a moderate Cohen's kappa coefficient; ii) the Ct cut-off value of < 42 was deemed more suitable for predicting positive specimens; iii) given the low concentration of target DNA in water samples, the minimum volume to be tested was 1 lt.We highly recommend the use of PMA-PCR for fast detection of NTM from environmental samples in order to ascertain whether HCUs may represent a potential source of human exposure to NTM. This reliable and simple method reduces laboratory TAT compared to conventional methods (1-2 days vs. 8 weeks, respectively), thereby improving control strategies and effective management of HCUs.