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 to 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), which due to false positive results, would display 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.

Antibody responses to BNT162b2 SARS-CoV-2 mRNA vaccine among healthcare workers and residents of long-term care facilities: A cohort study in Northern Italy.

Background and Aims: Long-term care facilities (LTCFs) have been severely impacted by COVID-19, with a disproportionate amount of SARS-CoV-2 infections and related deaths occurring among residents. Methods: This study is part of an ongoing multicenter, prospective cohort study conducted among healthcare workers (HCWs) and residents of 13 LTCFs in Northern Italy designed to evaluate SARS-CoV-2 specific immunoglobulin class G (IgG) titers before and following vaccination with Pfizer/BNT162b2 SARS-CoV-2 mRNA vaccine (two doses of vaccine, 21 days apart). Serum samples were obtained from participants (t0) before vaccination, and (t1) 2 weeks after and analyzed to determine anti-S1 IgG antibodies. Results: Five hundred and thirty-four participants were enrolled (404 subjects participated in both blood draws). Seropositivity was 50.19% at t0 and 99% at t1, with a significant difference in IgG titers. A higher proportion of residents were seropositive at t0 compared with HCWs, with significantly higher IgG titers among residents at both t0 and t1. Pre-existing immunity also had a significant effect on postvaccination IgG titers. However, a significant difference in titers at t1 between HCWs and residents considering only participants seropositive at t0 was found, with higher median titers among previously seropositive residents. Conclusion: Findings of this study provide scientific evidence endorsing the policy of universal vaccination in LTCFs.

Serological Responses up to 9 Months following COVID-19 mRNA Vaccination in Residents and Health-Care Workers of Long-Term Care Facilities: A Multicenter Prospective Cohort Study in Northern Italy.

Long-term care facilities (LTCFs) were severely affected by COVID-19, in particular in Northern Italy. We aimed to assess antibody responses among residents and healthcare workers (HCWs) of 13 LTCFs through serum samples collected at three time points: prior to, two weeks, and 9 months after receiving Pfizer/BNT162b2 SARS-CoV-2 mRNA vaccine (respectively t0, t1, and t2). IgG antibodies targeted towards the S1 domain of the spike protein were measured, and results were expressed in binding antibody units (BAU/mL). Friedman's average rank test was performed to compare antibody titres between the three time points. Two logistic regression models were built to identify independent predictors of (1) developing and (2) maintaining a significant antibody response to vaccination, using a previously identified threshold. In total, 534 subjects were enrolled (371 HCWs and 163 residents). The antibody titres at t1 were the highest; at t2, the IgG titres significantly decreased, remaining however 10 times higher compared to titres at t0. Previous infection was the only significant predictor of developing and maintaining a response over threshold in both models. Results of this study provided further insights on the humoral response elicited by vaccination, and on host factors determining variations in its magnitude and kinetics.

A New Culture Method for the Detection of Non-Tuberculous Mycobacteria in Water Samples from Heater-Cooler Units and Extracorporeal Membrane Oxygenation Machines.

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.

Seroprevalence of infection-induced SARS-CoV-2 antibodies among health care users of Northern Italy: results from two serosurveys (October-November 2019 and September-October 2021).

OBJECTIVES: The objective was to estimate the seroprevalence of SARS-CoV-2 in autumn 2019 (before case zero was identified in Italy) and 2021 among residual sera samples from health care users in the Piedmont region of northwestern Italy. METHODS: Two serosurveys were conducted. Using a semiquantitative method, samples were tested for the presence of immunoglobulin G (IgG) antibodies against the S1 domain of the spike protein. Samples with positive test results from the 2019 survey were independently retested using a multiplex panel to detect IgG antibodies against the receptor binding domain, S1 and S2 domains, and nucleocapsid. Samples with positive test results from the 2021 survey underwent repeat testing with enzyme-linked immunosorbent assay to detect anti-nucleocapsid IgG antibodies. Prevalence rates according to gender and age groups, together with their respective 95% confidence intervals (CIs), were calculated. RESULTS: Overall, the proportion of samples with positive test results was 2/353 in 2019 and 22/363 in 2021, with an estimated seroprevalence of 0.27% (95% CI 0-1.86) and 6.21% (95% CI 3.9-9.31) in 2019 and 2021 respectively. CONCLUSION: Results of this study support the hypothesis that the virus was circulating in Italy as early as autumn 2019. The role of these early cases in broader transmission dynamics remains to be determined.

A cross-sectional study of SARS-CoV-2 seropositivity among healthcare workers and residents of long-term facilities in Italy, January 2021.

Long-term care facilities (LTCFs) are high-risk settings for SARS-CoV-2 infection. This study aimed to describe SARS-CoV-2 seropositivity among residents of LTCFs and health-care workers (HCWs). Subjects were recruited in January 2021 among unvaccinated HCWs of LTCFs and hospitals and residents of LTCFs in Northern Italy. Information concerning previous SARS-CoV-2 infections and a sample of peripheral blood were collected. Anti-S SARS-CoV-2 IgG antibodies were measured using the EUROIMMUN Anti-SARS-CoV-2 QuantiVac ELISA kit (EUROIMMUN Medizinische Labordiagnostika AG). For subjects with previous COVID-19 infection, gender, age, type of subject (HCW or resident), and time between last positive swab and blood draw were considered as possible determinants of two outcomes: the probability to obtain a positive serological result and antibody titer. Six hundred and fifty-eight subjects were enrolled. 56.1% of all subjects and 65% of residents presented positive results (overall median antibody titer: 31.0 RU/ml). Multivariable models identified a statistically significant 4% decrease in the estimated antibody level for each 30-day increase from the last positive swab. HCWs were associated with significant odds for seroreversion over time (OR: 0.926 for every 30 days, 95% CI: 0.860-0.998), contrary to residents (OR: 1.059, 95% CI: 0.919-1.22). Age and gender were not factors predicting seropositivity over time. Residents could have a higher probability of maintaining a seropositive status over time compared to HCWs.

Chemical susceptibility testing of non-tuberculous mycobacterium strains and other aquatic bacteria: Results of a study for the development of a more sensitive and simple method for the detection of NTM in environmental samples.

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.

The use of BCYE medium for the detection of Legionella in environmental water samples: an appropriate update to ISO 11731:2017 standard?

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.

Comparison of BCYEα+AB agar and MWY agar for detection and enumeration of Legionella spp. in hospital water samples.

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

Real-time PCR, the best approaches for rapid testing for Mycobacterium chimaera detection in heater cooler units and extracorporeal membrane oxygenation.

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.

Persistence of Legionella in Routinely Disinfected Heater-Cooler Units and Heater Units assessed by Propidium Monoazide qPCR.

BACKGROUND: Evidence to date indicates that heater-cooler units (HCUs) and heater units (HUs) can generate potentially infectious aerosols containing a range of opportunistic pathogens such as Mycobacterium chimaera, other non-tuberculous mycobacterial (NTM) species, Pseudomonas aeruginosa and Legionella spp. Our purpose was to determine the extent of Legionella contamination and total viable count (TVC) in HCUs and HUs and to analyze the relationship by water system design of devices of two different brands (LivaNova vs. Maquet). METHODS: Legionella spp. were detected and quantified by our optimized PMA-qPCR protocol; TVCs were assessed according to ISO protocol 6222. Analyses were performed in the first sampling round and after six months of surveillance. RESULTS: Overall, Legionella spp. was detected in 65.7% of devices. In the second sampling round, Legionella positivity rates were significantly lower in water samples from the Maquet devices compared to the LivaNova ones (27.3% vs. 61.5%). LivaNova HCUs also yielded more Legionella, and aquatic bacteria counts than Maquet in both first and second-round samples. CONCLUSIONS: We recommend that all surgical patients and staff exposed to aerosols from thermoregulatory devices should be followed up for Legionella infection and that microbiological surveillance on such devices should be conducted regularly as precautionary principle.

Sensitivity and Selectivity of Two Commercially Available Media for Legionella spp. Recovery from Environmental Water Samples.

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 BCYEα 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.

Colonization by Pseudomonas aeruginosa of dental unit waterlines and its relationship with other bacteria: suggestions for microbiological monitoring.

Pseudomonas aeruginosa is an environmental bacterium, ubiquitous in aquatic habitats and water distribution systems, including dental unit waterlines (DUWLs). We investigated the prevalence of P. aeruginosa in DUWLs from private dental settings. We also analyzed the relationship between P. aeruginosa contamination and the presence of Legionella spp. and total viable count (TVC) in order to suggest a simple and inexpensive protocol to test the quality of water from DUWLs. We detected and quantified P. aeruginosa both by culture and by a PMA (propidium monoazide)-qPCR method. Overall, we detected P. aeruginosa in 17 samples using the PMA-qPCR and in 11 samples using the culture. All culture-positive samples were positive with the PMA-qPCR too, with an agreement between the two methods of 93% and a Cohen's kappa coefficient of κ = 0.747 (good concordance). Comparing results with results of our previous study, we noted that (a) P. aeruginosa was isolated only from DUWLs with high TVC and (b) five out of six Legionella-positive samples were negative for Pseudomonas spp. Our final suggestion is that the cleanliness of DUWLs should be assessed by TVC because it is a good indicator of the presence of pathogens such as Legionella spp. and P. aeruginosa.