Background: A global outbreak of invasive Mycobacterium chimaera infections has occurred since 2015 in patients undergoing open chest cardiac surgery. Currently, 15 cases has been reported in Italy. The Ministry of Health issued in 2019, the document contain the recommendations for the prevention of M.chimaera infection, including clinical surveillance and microbiological monitoring of HCUs. In Decree 3822/2019, the Tuscany Region issued recommendations for microbiological surveillance by applying both the cultural and biomolecular methods in order to detect early contamination by M. chimaera Aim: verify the use of a PMA-Real-Time PCR technique, to assess the efficacy of the disinfection procedure applied to the HCUs and to evaluate the presence of viable but non-culturable state of M.chimaera after water treatment. Methods: In two Tuscan cardiac surgery centers, water samples collected from HCUs were analyzed according to the regional protocol. All samples were treated by BLU-V Viability PMA kit (Qiagen Germantown, MD, USA), according to manufacture instruction. The extraction and Real-Time was performed using QIAamp DNA Mini Kit a QuantiNova PCR kit (Qiagen), respectively, to amplify a sequence of the ITS1 region. Results: From November 2018 to July 2019, 24 HCUs were monthly sampled (n.11 3T and n.3 1T LivaNova, n.1 TCM-Sarns Terumo and n.9 HCU40 Maquet) for a total of 82 samples. M.chimaera was detected in 12/82 (15%) (strain CP015272.1). From 2020-2022, with the replacement of all HCUs Livanova with HCUs Maquet, 100% (102/102) of the samples resulted negative by culture method, but 20.5% (21/102) of which were positive by PMA-Real Time PCR. Conclusions: The presence of VBNC state in disinfected HCUs Results in an overestimation of the disinfection efficacy and the resuscitation/regrowth of the VBNC in the HCU water system could further increase the infection risks in patients undergoing open chest cardiac surgery.