Confirmation of presumptive Legionella colonies on culture media according to ISO 11731:2017: Principles, problems and practice

Aims: The ISO 11731 norm, published in 2017, describes a method to identify and enumerate Legionella based exclusively on the confirmation of presumptive colonies by subculturing them on BCYE and BCYE-cys agar (BCYE agar without L-cysteine). Methods and results: Despite this recommendation, our laboratory has kept confirming all presumptive Legionella colonies by combining the subculture method with the latex agglutination and polymerase chain reaction (PCR) assays. Here, we show that the ISO 11731:2017 method adequately performs in our laboratory according ISO 13843:2017. We compared the performance of the ISO method in detecting Legionella in typical and atypical colonies (n = 7156) from health care facilities (HCFs) water samples to that of our combined protocol and we found a 2.1% false positive rate (FPR), underscoring the importance of combining agglutination test and PCR with subculture to achieve optimal confirmation. Lastly, we estimated the water system disinfection cost for HCFs (n = 7), that due to false positive results, would displayed Legionella values exceeding the threshold of risk acceptance established by the Italian guidelines. Conclusions: Overall, this large-scale study indicates that the ISO 11731:2017 confirmation method is error-prone, leading to significant FPRs and higher costs for HCFs due to remedial actions on their water systems.