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.

Cost-Effectiveness of Targeted Prophylaxis among Allogenic Stem Cell Transplant Recipients.

Bloodstream infections (BSI) are life-threatening complications for onco-hematologic patients. Fluoroquinolones prophylaxis (FQP) was recommended for patients with neutropenia. Later, it was correlated with increased resistance rates among this population and its role became debated. While the role of FQ prophylaxis is still being studied, its cost-effectiveness is also unknown. The objective of this study was to evaluate the costs and effects associated with two alternative strategies (FQP vs. no prophylaxis) for patients with hematological malignancies undergoing allogenic stem cell transplant (HSCT). A decision-tree model was built integrating retrospectively collected data from a single transplant center, part of a tertiary teaching hospital in Northern Italy. Probabilities, costs and effects were considered in the assessment of the two alternative strategies. Probabilities of colonization, BSIs, extended-spectrum beta lactamase (ESBL) and Klebsiella pneumoniae carbapenemase (KPC) BSIs and mortality associated with infection, as well as median duration of length of stay (LOS) were calculated based on data collected between 2013 and 2021. The center applied the strategy of FQP between 2013 and 2016, and of no prophylaxis between 2016 and 2021. Data on 326 patients were collected during the considered time period. Overall, the rates of colonization, BSI, KPC/ESBL BSI, and mortality were 6.8% (95% confidence interval (CI) 2.7-13.5), 42% (9.9-81.4) and 20.72 (16.67-25.26), respectively. A mean bed-day cost of 132€ was estimated. Considering no prophylaxis vs. prophylaxis, the difference in costs ranged between additional 33.61 and 80.59€ per patient, whereas the difference in effects ranged between 0.11 and 0.03 life-years (LYs) lost (around 40 and 11 days). Given the small differences in terms of costs and effects between the two strategies, no prophylaxis seems an appropriate choice. Furthermore, this analysis did not consider the broader effect on hospital ecology of multiple doses of FQP, which could provide further support for the strategy of no prophylaxis. Our results suggest that the necessity for FQP in onco-hematologic setting should be determined based on local antibiotic resistance patterns.

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.

Impact of an empiric antimicrobial therapy manual on antimicrobial usage and multidrug resistant organism trends in a large Italian teaching hospital.

Aim: To evaluate the changes in antimicrobial consumption and multidrug-resistant microorganism trends after introducing an empiric antimicrobial therapy manual to support antimicrobial stewardship. Methods: A 4-year prospective interventional study assessed the effect of introducing an empiric antimicrobial therapy manual in medical and surgical wards during two periods: pre-intervention period (January 2015-May 2017) and post-intervention period (June 2017-December 2019). Outcomes included microorganism trends of bloodstream infections (BSI) for Klebsiella pneumoniae carbapenemase-producing bacteria (KPC), extended spectrum beta-lactamase ESBL-E. coli, meticillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. Also, Clostridioides difficile infection (CDI) episodes were included. Rates were normalised per 1000 patient-days (PD). Antimicrobial consumption was assessed as defined daily dose (DDD)/1000 PD in interrupted time series analysis. Results: In medical wards, we observed a significant decrease in the consumption of piperacillin-tazobactam and a decrease in the trends of tigecycline and vancomycin consumption. In surgical wards, there was a significant decrease in consumption of fluoroquinolones and piperacillin-tazobactam. This decrease was maintained in trend for all the antimicrobials but was significant for tigecycline only. In medical wards, there was a significant reduction of MRSA and C. albicans. In surgical wards, we observed a decrease in MRSA, ESBL-E. coli, C. albicans and CDI. KPC cases decreased by 22.5% in medical wards and 74.3% in surgical wards. Conclusion: The results suggest that a persuasive educational approach to antimicrobial stewardship, with the introduction of an empiric antimicrobial manual and continuous education, resulted in reductions in both antimicrobial use and healthcare-associated BSI caused by multidrug-resistant organisms. More studies with longer follow up are needed to investigate the effect of antimicrobial stewardship on clinical outcomes.

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.

Using Microbiological Sampling to Evaluate the Efficacy of Nasofibroscope Disinfection: The Tristel Trio Wipes System in Ear-Nose-Throat (ENT) Endoscopy.

Disinfection and sterilization are needed for guaranteeing that medical and surgical instruments do not spread contagious microorganisms to patients. The aim of this study was to evaluate the efficacy of a simple manual technique of high-level disinfection (HLD) of flexible fiberoptic nasofibroscopes (FFNs) with wipes impregnated with a chlorine dioxide solution (Tristel Trio Wipes System-TTW) against a conventional automated washer machine (Soluscope ENT, Cimrex 12-AW). FFNs used in 62 patients undergoing endoscopy at an ENT clinic were sampled according to an aseptic procedure. For each nasoendoscopy, microbiological samples were taken at two times: (1) after a patient's nasoendoscopy and (2) immediately after high-level disinfection. Ten microliters of each prepared sample were inoculated onto specific culture media for the detection of nasopharyngeal flora microorganisms. The microbiological results obtained from 62 post-disinfection samples revealed bacterial growth on two FFNs disinfected with AW, and five FFNs disinfected with TTW, but this difference is not statistically significant. None of the isolates were pathogenic bacteria. Our results are different than the results obtained by two previously published studies on the TTW system. In both studies, sampling was carried out by swabbing the tip and the handle surface of FFNs. This sampling method was the least effective method means of detecting bacteria on a surface. It can be concluded that the two disinfection systems allow providers to obtain a reduction of the saprophytic and pathogenic microbial load.

Colonization of Dental Unit Waterlines by Helicobacter pylori: Risk of Exposure in Dental Practices.

Dental unit waterlines (DUWLs) can be considered one of the possible routes of H. pylori transmission, although its presence in DUWLs has not yet been investigated thoroughly. The present study aimed to discover the prevalence of H. pylori and oral streptococci (S. oralis and S. mutans) in DUWLs to evaluate the risk of exposure to human pathogens in dental practices. We collected the output water from 60 dental chair units (DCUs) in 26 private dentistry settings in Turin, searching for H. pylori and oral streptococci (OS) DNA, with a polymerase chain reaction (PCR) technique. At the same time, dentists completed a questionnaire about their DCUs, their main activities, the presence of anti-retraction devices, their attitudes about disinfection, etc. No dental chair unit tested was contaminated with H. pylori or S. mutans; only one dental chair was contaminated with S. oralis (1.7%). Considering the results, we can state that: (i) the lack of H. pylori DNA in water samples analyzed, suggests that municipal water is presumably treated with a sufficient chlorine level to inactivate DNA over time; (ii) the aspiration of oral fluids is limited by anti-retraction valves fitted distally to hand pieces; (iii) propidium monoazide qPCR (PMA-qPCR) could be a good technique to investigate and monitor potential environmental sources of infections such as DUWLs.

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.

The role of chemical products at low doses in preventing the proliferation of bacteria in dental unit waterlines: the ICX® experience.

In this study we evaluated (1) the efficacy of a protocol that combines hydrogen peroxide (shock treatment) and ICX® tablets (continuous treatment) for the control of microbial contamination in dental unit water lines, and (2) the in vitro antimicrobial activity of ICX® tablets on collection and wild strains isolated from dental chair output waters. To assess the treatment effectiveness, the microbial load in the output water samples of three dental chairs were investigated: one control chair received only shock treatment. In vitro bactericidal activity was tested against Staphylococcus aureus and Pseudomonas aeruginosa. Data obtained from samples collected from chairs treated with ICX® and shock treatment and data from the control chair did not differ significantly on the basis of microbial load. In the in vitro study, the product was unable to kill Gram-negative bacteria. These results show that the continuous introduction of ICX® was not effective in maintaining low counts of the heterotrophic bacteria in the output water of dental devices, and shock treatment may be needed more frequently than monthly.

The "bundle" approach to reduce the surgical site infection rate.

RATIONALE, AIMS, AND OBJECTIVES: In Italy, since 2008, the surveillance of surgical site infections (SSIs) has been conducted following ECDC recommendations, according to the protocol of the National System of Surveillance of Surgical Site Infections. In 2009, in Piedmont region, where the study was conducted, it was introduced a survey of a "bundle" for every patient under SSIs surveillance. The bundle includes 5 items: infection risk index calculation, preoperative shower, trichotomy, antibiotic prophylaxis, and body temperature control. The aim of this study is the evaluation of the incidence rate of the SSIs in relation to the implementation of the bundle from January 1st to December 31st, 2012. METHOD: This study is an observational study (retrospective cohort). The regional surveillance system collected 3314 surgical operations during the year 2012 from 37 hospitals. The represented surgical categories were hip prosthetic surgery (HPRO: 1992 cases) and colon surgery (COLO: 1322 cases). The bundle was implemented in 1114 and 671 operations, respectively. Univariate and multivariate analysis were conducted stratifying the sample for hip surgery and colorectal surgery, with the purpose to identify an association between the implementation of the bundle and a decrease of the rate of SSIs. RESULTS: From the analysis, the bundle resulted as a protective factor for the infection risk in colon surgery (odds ratio [OR], 0.55; 95% confidence interval [CI], 0.38-0.78). The main risk factors were American Society of Anesthesiologists score ≥ 3 (OR, 1.57; 95% CI, 1.10-2.24) and contamination class ≥ 3 (OR, 2.02; 95% CI, 1.37-2.97). In the hip surgery, the application of the bundle was not statistically associated to a decrease of the risk of infection. CONCLUSION: The use of surgical bundle seems to reduce significantly the SSIs rate in the colon surgery.

Delivery of written and verbal information on healthcare-associated infections to patients: opinions and attitudes of a sample of healthcare workers.

BACKGROUND: Patients education is considered a valuable mean to prevent and control healthcare-associated infections (HAIs). This cross-sectional study aims to assess declared practices of healthcare workers (HCWs) regarding the delivery of information about HAIs to patients. METHODS: A 14-item multiple-choice questionnaire was designed to assess the attitudes and declared practices of HCWs (physicians, nurses and nursing assistants). Between October 2012 and October 2013, we surveyed a sample of HCWs from 4 acute hospitals in Piedmont (North-western Italy). Written information was available at three hospitals (A, B and C) and verbal information at the last one (hospital D). RESULTS: We surveyed 288 HCWs (79 physicians, 124 nurses and 85 healthcare assistants). At hospital A, B and C, 128 (71.6%) HCWs declared that written information was usually delivered to any patient and 145 (66.5%) that nurses usually delivered it. Only 42 (26.3%) of them - 97.6% nurses -declared that they usually delivered written information to patients. Among all surveyed HCWs, 210 (72.9%) declared that patients also receive verbal information on HAI - mainly by nurses (70.8%) and physicians (50%) - but only 88 (29,2%) - 23.8% physician and 48.8% nurses - declared that they usually informed patients. Finally, 83 (27.7%) HCWs believed that they should decide whether or not to deliver information to patient case by case. CONCLUSIONS: A formal policy requiring to deliver written information is most likely not enough to induce HCWs to better inform patients about HAIs. Health Trusts might introduce more target actions to reinforce HCWs' practices, such as training and internal auditing.

Efficacy of a Low Dose of Hydrogen Peroxide (Peroxy Ag⁺) for Continuous Treatment of Dental Unit Water Lines: Challenge Test with Legionella pneumophila Serogroup 1 in a Simulated Dental Unit Waterline.

This study was designed to examine the in vitro bactericidal activity of hydrogen peroxide against Legionella. We tested hydrogen peroxide (Peroxy Ag⁺) at 600 ppm to evaluate Legionella survival in a simulated dental treatment water system equipped with Water Hygienization Equipment (W.H.E.) device that was artificially contaminated. When Legionella pneumophila serogroup (sg) 1 was exposed to Peroxy Ag⁺ for 60 min we obtained a two decimal log reduction. High antimicrobial efficacy was obtained with extended periods of exposure: four decimal log reduction at 75 min and five decimal log reduction at 15 h of exposure. Involving a simulation device (Peroxy Ag⁺ is flushed into the simulation dental unit waterlines (DUWL)) we obtained an average reduction of 85% of Legionella load. The product is effective in reducing the number of Legionella cells after 75 min of contact time (99.997%) in the simulator device under test conditions. The Peroxy Ag⁺ treatment is safe for continuous use in the dental water supply system (i.e., it is safe for patient contact), so it could be used as a preventive option, and it may be useful in long-term treatments, alone or coupled with a daily or periodic shock treatment.

Cultural and Molecular Evidence of Legionella spp. Colonization in Dental Unit Waterlines: Which Is the Best Method for Risk Assessment?

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.

Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

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 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 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 (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) 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).

Legionella in water samples: how can you interpret the results obtained by quantitative PCR?

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.

Overestimation of the Legionella spp. load in environmental samples by quantitative real-time PCR: pretreatment with propidium monoazide as a tool for the assessment of an association between Legionella concentration and sanitary risk.

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 (Δ log10=2.25), ii) PMA-qPCR results were on average 27-fold higher than the culture results (Δ log10=1.43), iii) propidium monoazide-induced signal reduction in qPCR were nearly 10-fold (Δ 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).