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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.

The isolation of non-tuberculous mycobacteria (NTM) from cultures is particularly laborious due to the potential overgrowth of coexisting non-acid fast bacilli. To reduce the overgrowth of these non-mycobacterial organisms, a decontamination step with NaOH or cetylpyridinium chloride is highly recommended before plating the samples on the culture medium. However, due to their toxicity, decontamination solutions tend to decrease NTM recovery from clinical and environmental samples. Here, we tested an alternative method for NTM recovery based on the use of NTM Elite agar, a selective medium that does not require a decontamination step. Using NTM Elite agar, we were able to detect non-tuberculous mycobacteria in 27.7% (30/108) of water samples analyzed. The average time to NTM detection was 18 days, but some strains required longer to grow, perhaps due to the stressful environmental conditions (periodical disinfection of devices). NTM Elite agar's effectiveness in inhibiting background flora was proven by the isolation of NTM from samples with and without background flora, showing no statistically significant differences in detection rates for different total viable counts of background flora (p = 0.4989). In conclusion, our findings indicate that effective NTM recovery from HCU- and ECMO-derived water samples can be achieved via filtration and direct culture of the filters on NTM Elite agar. This simple procedure can speed up laboratory work and provide an improved method, successfully resulting in low contamination and high detection rate, in addition to being less time-consuming. Its sensitivity and lack of a decontamination step make this protocol particularly useful for monitoring the effectiveness of device disinfection in hospital settings, even in the presence of low NTM loads. Reading timeframes should probably be extended to 7 weeks (i.e., well beyond the standard 4 weeks advised by the manufacturer), in order to isolate even the slow-growing mycobacteria. However, an extended incubation period is not necessary for exclusion of M. chimaera contamination of the devices, as M. chimaera isolation times do not generally exceed 3 weeks.

The methods employed to detect non-tuberculous mycobacteria on environmental samples are essentially those classically used in clinical microbiology, which envisage a decontamination step to reduce the overgrowth of non-mycobacterial organisms before plating them on the culture medium. The aim of this study was to propose alternative culture techniques to improve non-tuberculous mycobacteria detection in environmental samples. We used artificially contaminated samples to compare the membrane filter washing procedure against direct plating of membrane filters on culture media in relation to M.chimaera and M.chelonae recovery efficiency. Moreover, we compared the efficacy of NTM Elite agar in inhibiting the growth of aquatic bacteria with that of cetylpyridinium chloride and N-acetyl-L-cysteine sodium hydroxide decontamination treatments. The washing procedure yielded a low release of both mycobacterium strains (6.6% for Mycobacterium chimaera and 7.5% for Mycobacterium chelonae) from the membrane filters; on the contrary, direct plating of membrane filters led to a 100% cell recovery. Water sample pretreatment with N-acetyl-L-cysteine sodium hydroxide (1%), despite achieving complete suppression of non-acid fast bacilli, caused a reduction in mycobacteria growth. Decontamination with cetylpyridinium chloride (0.005%) was found to be ineffective against Methylobacterium spp. and Burkholderia multivorans. NTM Elite agar was ineffective against B. multivorans, but it inhibited the growth of all other aquatic bacteria. Our results indicate that NTM Elite agar provides a valid alternative method of recovering non-tuberculous mycobacteria from environmental samples. It does not involve a decontamination step and provides greater recovery efficiency by skipping the washing step and directly plating the filters on the media.

We evaluated the diagnostic performances of 2 media (BCYE, MWY) on 951 Legionella-positive hospital water samples. MWY allowed detecting Legionella in 89.2% of samples, but in 10.8% (103/951) Legionella was found on BCYE plates only. In samples where Legionella was isolated with other microorganisms (663/951), MWY was essential to detect the majority of positive samples (349/663, 52.6%), as fewer plates resulted unreadable; however, in those containing Legionella only, a higher frequency of positive samples was recorded with BCYE (94.8%, 273/288) compared to MWY (85.1%, 245/288). Considering the 484 concordant positive samples, overall Legionella counts were significantly higher on BCYE (P = 0.0029), with 47% of samples showing higher counts on BCYE compared to MWY plates. Furthermore, discordant samples (positive on only one medium) showed different relative proportions between Legionella pneumophila and non-pneumophila, the latter being found more frequently on BCYE only (P = 0.0296).Our findings confirm the appropriateness of the ISO 11731:2017 update.

Background: This study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEa +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEa +AB medium with that of the highly selective MWY medium.

Results: Legionella spp. was detected in 48.2 and 47.1% of the samples by BCYEa +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%.

Conclusions: According to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp. count.

According to recent investigations, the risk of M. chimaera contamination of heater-cooler units (HCUs) has reached global proportions. Our aim was to field evaluate a protocol for early detection of M. chimaera contamination. We assessed the presence of viable M. chimaera in 395 water samples obtained from 48 devices (HCUs and extracorporeal membrane oxygenation) by Real Time PCR. Thirty devices were NTM positive, of which 14 were contaminated with M. chimaera. The most frequently contaminated devices were the Stockert 3T. Noteworthy, Stockert 3T devices were positive for M. chimaera. In conclusion, this study introduces novel PMA-PCR designed to specifically detect M. chimaera in HCUs and ECMO devices; this method can replace the culture method for continuous microbiological surveillance. The timely detection of M. chimaera contamination can then be used to improve effective management of the devices.

The quality control of culture media used for Legionella spp. isolation and enumeration is paramount to achieve a satisfactory degree of comparability among water testing results from different laboratories. Here, we report on a comparative assessment of the sensitivity and selectivity of MWY and BCYEa media supplied by two different manufacturers (i.e., Xebios Diagnostics GmbH and Oxoid Ltd) for the detection of Legionella spp. from environmental water samples. Even though our analysis showed an excellent agreement between the recovery rates of the four media tested (90.5%), the quantitative recovery of Legionella spp. colonies using Xebios media was significantly greater than that achieved by Oxoid media (P = 0.0054). Furthermore, the sensitivity of detection was significantly higher when samples were plated on MWY Xebios agar (P = 0.0442), while the selectivity of MWY appeared to be the same regardless of the manufacturer. Furthermore, MWYXebios agar favored the growth of much larger colonies compared to those observed on MWYOxoid agar. Finally, MWYXebios medium enhanced the recovery of non-pneumophila Legionella species. Collectively, our findings demonstrate that quality control is crucial to ensure high selectivity and sensitivity of the culture media used for the detection and enumeration of Legionella spp. from environmental water resources. As water remediation measures strictly depend on Legionella spp. recovery, culture protocol standardization, as well as quality control of the culture media, is essential to achieve intra- and interlaboratory reproducibility and accuracy.

Background: 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.

Aim: To develop a simple and effective method to detect NTM in HCUs by real-time polymerase chain reaction (PCR), with a short laboratory turnaround time and reliable culture results.

Methods: A total of 281 water samples collected from various HCUs at seven Italian hospitals were simultaneously screened for NTM by a propidium monoazide (PMA)-PCR assay and by conventional culture testing. The results were analysed with culture testing as the reference method.

Findings: (i) The agreement between culture testing and PMA-PCR was 85.0% with a cycle threshold (CT) cut-off value of <38 vs 80.0% with a CT of <43, with a moderate Cohen's ?-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 L.

Conclusion: The use of PMA-PCR for fast detection of NTM from environmental samples is highly recommended in order to ascertain whether HCUs may represent a potential source of human exposure to NTM. This reliable and simple method reduces laboratory turnaround time compared to conventional methods (one to two days vs eight weeks, respectively), thereby improving control strategies and effective management of HCUs.

Legionella spp. are ubiquitous in aquatic habitats and water distribution systems, including dental unit waterlines (DUWLs). The aim of the present study was to determine the prevalence of Legionella in DUWLs and tap water samples using PMA-qPCR and standard culture methods. The total viable counts (TVCs) of aerobic heterotrophic bacteria in the samples were also determined. Legionella spp. were detected and quantified using the modified ISO 11731 culture method. Extracted genomic DNA was analysed using the iQ-Check Quanti Legionella spp. kit, and the TVCs were determined according to the ISO protocol 6222. Legionella spp. were detected in 100% of the samples using the PMA-qPCR method, whereas these bacteria were detected in only 7% of the samples using the culture method. The number of colony forming units (CFUs) of the TVCs in the DUWL and tap water samples differed, with the bacterial load being significantly lower in the tap water samples (p-value = 0). The counts obtained were within the Italian standard range established for potable water in only 5% of the DUWL water samples and in 77% of the tap water samples. Our results show that the level of Legionella spp. contamination determined using the culture method does not reflect the true scale of the problem, and consequently we recommend testing for the presence of aerobic heterotrophic bacteria based on the assumption that Legionella spp. are components of biofilms.

Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified.Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 105 cells ml-1. A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R2 = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (?log10 = 3.74 ± 0.39 log10 GU ml-1). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 105 cells ml-1 were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal.When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

Quantitative polymerase chain reaction (qPCR) offers rapid, sensitive, and specific detection of Legionella in environmental water samples. In this study, qPCR and qPCR combined with propidium monoazide (PMA-qPCR) were both applied to hot-water system samples and compared to traditional culture techniques. In addition, we evaluated the ability of PMA-qPCR to monitor the efficacy of different disinfection strategies. Comparison between the quantification obtained by culture and by qPCR or PMA-qPCR on environmental water samples confirms that the concentration of Legionella estimated by GU/L is generally higher than that estimated in CFU/L. Our results on 57 hot-water-system samples collected from 3 different sites show that: i) qPCR results were on average 178-fold higher than the culture results (? log10=2.25), ii) PMA-qPCR results were on average 27-fold higher than the culture results (? log10=1.43), iii) propidium monoazide-induced signal reduction in qPCR were nearly 10-fold (? log10=0.95), and that iv) different degrees of correlations between the 3 methods might be explained by different matrix properties, but also by different disinfection methods affecting cultivability of Legionella. In our study, we calculated the logarithmic differences between the results obtained by PMA-qPCR and those obtained by culture, and we suggested an algorithm for the interpretation of PMA-qPCR results for the routine monitoring of healthcare water systems using a commercial qPCR system (iQ-check real-time PCR kit; Bio-Rad, Marnes-la-Coquette, France).

Evaluation of the potential risk associated with Legionella has traditionally been determined from culture-based methods. Quantitative polymerase chain reaction (qPCR) is an alternative tool that offers rapid, sensitive and specific detection of Legionella in environmental water samples. In this study we compare the results obtained by conventional qPCR (iQ-Check™ Quanti Legionella spp.; Bio-Rad) and by culture method on artificial samples prepared in Page's saline by addiction of Legionella pneumophila serogroup 1 (ATCC 33152) and we analyse the selective quantification of viable Legionella cells by the qPCR-PMA method. The amount of Legionella DNA (GU) determined by qPCR was 28-fold higher than the load detected by culture (CFU). Applying the qPCR combined with PMA treatment we obtained a reduction of 98.5% of the qPCR signal from dead cells. We observed a dissimilarity in the ability of PMA to suppress the PCR signal in samples with different amounts of bacteria: the effective elimination of detection signals by PMA depended on the concentration of GU and increasing amounts of cells resulted in higher values of reduction. Using the results from this study we created an algorithm to facilitate the interpretation of viable cell level estimation with qPCR-PMA.

The study aim was to determine retrospectively whether the parallel use of 2 media [buffered charcoal yeast extract (BCYE) and medium of Wadowsky and Yee (MWY)] to isolate Legionella spp. from water samples taken from hospital water supply systems increased the sensitivity of the culture method as compared with methods/protocols in which only seeding on a selective medium is used. We analyzed the results obtained from 931 positive water samples. In 484 of the 931 positive water samples, Legionella spp. was isolated in the presence of other microorganisms; in 83% (400/484), we used MWY to count suspected colonies, which gave a lower number of unreadable plates. In the 447 samples containing only Legionella spp., the highest frequency of positive samples (93%, 418/447) was obtained with BCYE, whereas seeding on MWY yielded 78% (348/447) (P < 0.001). Evaluation of the influence of the media on the Legionella spp. counts obtained by the 2 media showed that BCYE agar produced significantly higher counts than MWY (P < 0.001). The major conclusions that may be drawn from our data are as follows: 1) BCYE gives a high recovery rate of positive samples (93%) and a much greater yield of Legionella spp. than MWY; 2) BCYE was necessary for the detection of non-L. pneumophila spp. which grew poorly on selective media; 3) selective media [MWY or GVPC (glycine, vancomycin, polymyxin B, and cycloheximide)] were necessary for the recovery of Legionella spp. when the non-selective medium (BCYE) was difficult to interpret because of contaminating background flora. The use of different media is recommended for routine water tests in hospitals.