Two-year evaluation of Legionella in an aging residential building: Assessment of multiple potable water remediation approaches.

Legionella is an opportunistic waterborne pathogen that is difficult to eradicate in colonized drinking water pipes. Legionella control is further challenged by aging water infrastructure and lack of evidence-based guidance for building treatment. This study assessed multiple premise water remediation approaches designed to reduce Legionella pneumophila within a residential building located in an aging, urban drinking water system over a two-year period. Samples (n = 745) were collected from hot and cold-water lines and quantified via most probable number culture. Building-level treatment approaches included three single heat shocks, three single chemical shocks, and continuous low-level chemical disinfection in the potable water system. The building was highly colonized with L. pneumophila with 71 % L. pneumophila positivity. Single heat shocks had a statistically significant L. pneumophila reduction one day post treatment but no significant L. pneumophila reduction at one week, two weeks, and four weeks post treatment. The first two chemical shocks resulted in statistically significant L. pneumophila reduction at two days and four weeks post treatment, but there was a significant L. pneumophila increase at four weeks following the third chemical shock. Continuous low-level chemical disinfection resulted in statistically significant L. pneumophila reduction at ten weeks post treatment implementation. This demonstrates that in a building highly colonized with L. pneumophila, sustained remediation is best achieved using continuous low-level chemical treatment.

Applications of Quantitative Microbial Risk Assessment to Respiratory Pathogens and Implications for Uptake in Policy: A State-of-the-Science Review.

BACKGROUND: Respiratory tract infections are major contributors to the global disease burden. Quantitative microbial risk assessment (QMRA) holds potential as a rapidly deployable framework to understand respiratory pathogen transmission and inform policy on infection control. OBJECTIVES: The goal of this paper was to evaluate, motivate, and inform further development of the use of QMRA as a rapid tool to understand the transmission of respiratory pathogens and improve the evidence base for infection control policies. METHODS: We conducted a literature review to identify peer-reviewed studies of complete QMRA frameworks on aerosol inhalation or contact transmission of respiratory pathogens. From each of the identified studies, we extracted and summarized information on the applied exposure model approaches, dose-response models, and parameter values, including risk characterization. Finally, we reviewed linkages between model outcomes and policy. RESULTS: We identified 93 studies conducted in 16 different countries with complete QMRA frameworks for diverse respiratory pathogens, including SARS-CoV-2, Legionella spp., Staphylococcus aureus, influenza, and Bacillus anthracis. Six distinct exposure models were identified across diverse and complex transmission pathways. In 57 studies, exposure model frameworks were informed by their ability to model the efficacy of potential interventions. Among interventions, masking, ventilation, social distancing, and other environmental source controls were commonly assessed. Pathogen concentration, aerosol concentration, and partitioning coefficient were influential exposure parameters as identified by sensitivity analysis. Most (84%, n=78) studies presented policy-relevant content including a) determining disease burden to call for policy intervention, b) determining risk-based threshold values for regulations, c) informing intervention and control strategies, and d) making recommendations and suggestions for QMRA application in policy. CONCLUSIONS: We identified needs to further the development of QMRA frameworks for respiratory pathogens that prioritize appropriate aerosol exposure modeling approaches, consider trade-offs between model validity and complexity, and incorporate research that strengthens confidence in QMRA results. https://doi.org/10.1289/EHP12695.

Quantitative Microbial Risk Assessment Framework Incorporating Water Ages with Legionella pneumophila Growth Rates.

Water age in drinking water systems is often used as a proxy for water quality but is rarely used as a direct input in assessing microbial risk. This study directly linked water ages in a premise plumbing system to concentrations of Legionella pneumophila via a growth model. In turn, the L. pneumophila concentrations were used for a quantitative microbial risk assessment to calculate the associated probabilities of infection (Pinf) and clinically severe illness (Pcsi) due to showering. Risk reductions achieved by purging devices, which reduce water age, were also quantified. The median annual Pinf exceeded the commonly used 1 in 10,000 (10-4) risk benchmark in all scenarios, but the median annual Pcsi was always 1-3 orders of magnitude below 10-4. The median annual Pcsi was lower in homes with two occupants (4.7 × 10-7) than with one occupant (7.5 × 10-7) due to more frequent use of water fixtures, which reduced water ages. The median annual Pcsi for homes with one occupant was reduced by 39-43% with scheduled purging 1-2 times per day. Smart purging devices, which purge only after a certain period of nonuse, maintained these lower annual Pcsi values while reducing additional water consumption by 45-62%.

Estimating the burden of illness caused by domestic waterborne Legionnaires' disease in Canada: 2015-2019.

Legionellosis is a disease caused by the bacterium Legionella that most commonly presents as Legionnaires' disease (LD), a severe form of pneumonia. From 2015 to 2019, an average of 438 LD cases per year were reported in Canada. However, it is believed that the actual number of cases is much higher, since LD may be underdiagnosed and underreported. The purpose of this study was to develop an estimate of the true incidence of illnesses, hospitalizations, and deaths associated with LD in Canada. Values were derived using a stochastic model, based on Canadian surveillance data from 2015 to 2019, which were scaled up to account for underdiagnosis and underreporting. Overall, there were an estimated 1,113 (90% CrI: 737-1,730) illnesses, 1,008 (90% CrI: 271-2,244) hospitalizations, and 34 (90% CrI: 4-86) deaths due to domestically acquired waterborne LD annually in Canada from 2015 to 2019. It was further estimated that only 36% of illnesses and 39% of hospitalizations and deaths were captured in surveillance, and that 22% of illnesses were caused by Legionella serogroups and species other than Legionella pneumophila serogroup 1 (non-Lp1). This study highlights the true burden and areas for improvement in Canada's surveillance and detection of LD.

Pilot-scale assessment reveals effects of anode type and orthophosphate in governing antimicrobial capacity of copper for Legionella pneumophila control.

Copper (Cu) is sometimes applied as an antimicrobial for controlling Legionella in hot water plumbing systems, but its efficacy is inconsistent. Here we examined the effects of Cu (0 - 2 mg/L), orthophosphate corrosion inhibitor (0 or 3 mg/L as phosphate), and water heater anodes (aluminum, magnesium, and powered anodes) on both bulk water and biofilm-associated L. pneumophila in pilot-scale water heater systems. Soluble, but not total, Cu was a good predictor of antimicrobial capacity of Cu. Even after months of exposure to very high Cu levels (>1.2 mg/L) and low pH (<7), which increases solubility and enhances bioavailability of Cu, culturable L. pneumophila was only reduced by ∼1-log. Cu antimicrobial capacity was shown to be limited by various factors, including binding of Cu ions by aluminum hydroxide precipitates released from corrosion of aluminum anodes, higher pH due to magnesium anode corrosion, and high Cu tolerance of the outbreak-associated L. pneumophila strain that was inoculated into the systems. L. pneumophila numbers were also higher in several instances when Cu was dosed together with orthophosphate (e.g., with an Al anode), revealing at least one scenario where high levels of total Cu appeared to stimulate Legionella. The controlled, pilot-scale nature of this study provides new understanding of the limitations of Cu as an antimicrobial in real-world plumbing systems.

Epidemiology and direct health care costs of hospitalised legionellosis in New Zealand, 2000-2020.

BACKGROUND: Legionellosis is a collective term used for disease caused by Legionella species which result in community and hospital acquired pneumonia worldwide. The aim of this analysis was to describe the epidemiology of legionellosis hospitalisations in Aotearoa New Zealand (NZ) over a 21-year period and quantify the health care costs. METHOD: This study combined national legionellosis notification and hospital discharge data that were linked via the National Health Index (NHI) to provide a more complete dataset of hospitalised cases. The direct cost of hospital care was estimated by multiplying the diagnosis-related group cost-weight by the national price and inflating to 2020/2021 values. RESULTS: There were 1479 records matched across notifications and discharge databases, including 990 with principal and 489 with additional diagnosis of legionellosis. Incidence rose to an average of 143 cases per annum for 2016-2020, a rate of 3·2/100,000. The median LOS was 6 days (IQR 4-13·5) with direct costs of $2·1 million per annum over that period. Rates were highest in those aged 65 years and above, male, and of European/Other ethnicity. Hospitalisations showed a peak in spring and summer. CONCLUSION: The rate of hospitalised legionellosis in New Zealand rose from 2000 to 2015, largely reflecting improved diagnosis. This preventable disease results in substantial health care costs. Greater efforts are needed to identify and control sources of exposure. Surveillance could be improved by routine integration of notification and hospital discharge data.

Can legionellosis be considered an occupational risk in the healthcare sector? A systematic review and meta-analysis.

OBJECTIVES: According to current knowledge about legionella transmission, healthcare workers (HCWs) are at an increased risk of exposure. The aim of this research was to systematically review the literature about HCWs' exposure to legionella and establish whether there is an occupational risk. STUDY DESIGN: This was a systematic review and meta-analysis. METHODS: PubMed, Scopus and Web of Science were searched to identify studies regarding the occupational risk of legionellosis for HCWs. Keywords used in the search were 'Legionella pneumophila', 'occupational medicine', 'occupational' and 'risk'. Selected studies were reviewed to assess the quality and meta-analysed. Finally, the nine epidemiological principles of Bradford-Hill criteria were used to assess whether legionellosis could be considered an occupational risk for HCWs. RESULTS: The search strategy retrieved 124 studies, and 10 studies were included in the present review. The overall study quality was low. The pooled odds ratio estimate was 2.45 (95% confidence interval: 1.52-3.96). The assessment using Bradford-Hill criteria showed that only two criteria (plausibility and coherence) were met, which is insufficient to establish an occupational risk. CONCLUSIONS: This systematic review suggests that there is a higher risk of legionella exposure for HCWs, but there is currently no clinical evidence. Further studies with appropriate study design are needed to determine whether legionella infection is an occupational risk for HCWs.

An application for relating Legionella shower water monitoring results to estimated health outcomes.

Exposure models are useful tools for relating environmental monitoring data to expected health outcomes. The objective of this study was to (1) compare two Legionella shower exposure models, and (2) develop a risk calculator tool for relating environmental monitoring data to estimated Legionella infection risks and Legionnaires' Disease (LD) illness risks. Legionella infection risks for a single shower event were compared using two shower Legionella exposure models. These models varied in their description of partitioning of Legionella in aerosols and aerosol deposition in the lung, where Model 1 had larger and fewer aerosol ranges than Model 2. Model 2 described conventional vs. water efficient showers separately, while Model 1 described exposure for an unspecified shower type (did not describe it as conventional or water efficient). A Monte Carlo approach was used to account for variability and uncertainty in these aerosolization and deposition parameters, Legionella concentrations, and the dose-response parameter. Methods for relating infection risks to illness risks accounting for demographic differences were used to inform the risk calculator web application ("app"). Model 2 consistently estimated higher infection risks than Model 1 for the same Legionella concentration in water and estimated deposited doses with less variability. For a 7.8-min shower with a Legionella concentration of 0.1 CFU/mL, the average infection risks estimated using Model 2 were 4.8 × 10-6 (SD=3.0 × 10-6) (conventional shower) and 2.3 × 10-6 (SD=1.7 × 10-6) (water efficient). Average infection risk estimated by Model 1 was 1.1 × 10-6 (SD=9.7 × 10-7). Model 2 was used for app development due to more conservative risk estimates and less variability in estimated dose. While multiple Legionella shower models are available for quantitative microbial risk assessments (QMRAs), they may yield notably different infection risks for the same environmental microbial concentration. Model comparisons will inform decisions regarding their integration with risk assessment tools. The development of risk calculator tools for relating environmental microbiology data to infection risks will increase the impact of exposure models for informing water treatment decisions and achieving risk targets.

Quantitative Microbial Risk Assessment Applied to Legionella Contamination on Long-Distance Public Transport.

The quantitative microbial risk assessment (QMRA) framework is used for assessing health risk coming from pathogens in the environment. In this paper, we used QMRA to evaluate the infection risk of L. pneumophila attributable to sink usage in a toilet cabin on Italian long-distance public transportation (LDT). LDT has water distribution systems with risk points for Legionella proliferation, as well as premise plumbing for drinking water, but they are not considered for risk assessment. Monitoring data revealed that approximately 55% of water samples (217/398) were positive for L. pneumophila, and the most frequently isolated was L. pneumophila sg1 (64%, 139/217); therefore, such data were fitted to the best probability distribution function to be used as a stochastic variable in the QMRA model. Then, a sink-specific aerosolization ratio was applied to calculate the inhaled dose, also considering inhalation rate and exposure time, which were used as stochastic parameters based on literature data. At L. pneumophila sg1 concentration ≤100 CFU/L, health risk was approximately 1 infection per 1 million exposures, with an increase of up to 5 infections per 10,000 exposures when the concentrations were ≥10,000 CFU/L. Our QMRA results showed a low Legionella infection risk from faucets on LDT; however, it deserves consideration since LDT can be used by people highly susceptible for the development of a severe form of the disease, owing to their immunological status or other predisposing factors. Further investigations could also evaluate Legionella-laden aerosols from toilet flushing.

Legionella contamination of a cold-water supplying system in a German university hospital - assessment of the superheat and flush method for disinfection.

INTRODUCTION: In case of a contamination of water-supplying systems in hospitals with legionella, usually chemical disinfection measures are used for remediation. Unfortunately, it is reported, that these methods may not be sustainable, have an impact on water quality, and can even fail. As an alternative, the superheat and flush method does not need any special equipment, can be initiated in a short lead of time and does not affect the water quality. However, evidence on this disinfection measurement against legionella is lacking. We therefore investigated and report on the effectiveness and long-term results of the superheat and flush disinfection method. METHODS: During routine periodical examinations, a rising count of legionella was detected in the cold-water supplying system at a German university hospital. Adapted to an analysis of risks, effort and benefit, the superheat and flush procedure was applied twice within 6 months. RESULTS: While 33 out of 70 samples had a higher legionella count than the legal threshold of 100 CFU/100 mL (CFU - Colony Forming Units) before the first disinfection was carried out, this number could be reduced to 1 out of 202 samples after the first intervention. Additionally, in contrast to previously published studies, the effect was long-lasting, as no relevant limit exceedance occurred during the following observation period of more than two years. CONCLUSION: The superheat and flush disinfection can provide an economic and highly effective measure in case of legionella contamination and should be shortlisted for an eradication attempt of affected water-supplying systems in hospitals.

Assessment of hygienic conditions of recreational facility restrooms: an integrated approach.

INTRODUCTION: Microbiological quality of recreational environments included restrooms, is generally assessed by water and surface monitoring. In this study, an environmental monitoring, conducted in spring, of swimming pool restrooms of a recreation center located in the Marche region has been carried out. Seven water samples and seven surface swabs were collected. Moreover, six air samples have been included. The aim of this study was to evaluate if air microbiological monitoring, along with molecular detection in real-time PCR, could give additional useful information about the hygienic conditions of the facility. METHODS: Heterotrophic Plate Count (HPC) both at 22°C (psychrophilic) and 37°C (mesophilic) was determined by separate cultures in all samples. The presence of Legionella pneumophila and Pseudomonas aeruginosa was evaluated by both culture and real-time PCR. RESULTS: The analysis of shower water recorded a HPC load of mesophilic bacteria (37°C) more than 10-fold higher in men restroom, respect to women's one (> 100 vs < 10 CFU/ml), while in air samples was between < 100 and > 500. Concerning pathogen presence, both species Legionella pneumophila and Pseudomonas aeruginosa were detected only in men restroom, but in different sample types by using different methods (culture and real-time PCR). CONCLUSIONS: Air sampling may offer the advantage of giving more representative data about microbial presence in restrooms, including bacterial species transmitted through aerosol, like Legionella. Moreover, the concurrent use of molecular and microbiological detection in an integrated approach could offer the advantage of greater sensitivity.

Risk assessment and disease burden of legionella presence in cooling towers of Iran's central hospitals.

Regular monitoring and measurement of Legionella in tower water and preventive measures against contamination are particularly important in hospitals. This study aimed at risk assessment and disease burden because of legionella presence in cooling towers of Iran's central hospitals. Then its correlation with temperature, pH, turbidity, residual chlorine, and EC was investigated by the Pearson test. The health risk and burden of diseases caused by Legionella exposure were determined using QMRA and DALY models. Statistical analysis and modeling were performed in MATLAB2018. Of the total samples, 30-43% was infected with Legionella. The mean concentrations in hospital A and B were 5-102.5 ± 10 and 5-89.7 ± 0.7 CFU/L, respectively. Among environmental factors, turbidity and pH were the most effective factors in increasing and decreasing Legionella concentration, respectively. According to the QMRA model, the risks of Legionella infections and annual mortality in both hospitals were 0.2-0.3, 0-0.19, 2-2.9 × 10-5, and 0-0.7 × 10-5, respectively, which was higher than the acceptable risk range for Legionella (10-4-10-7). However, the trend of its change was negatively correlated with time (RB = - 0.77). According to the results, the concentration of Legionella and the exposure risk in both hospitals were higher than the permissible range, which is necessary to decrease to 0.1 current concentrations.

Assessment of flow cytometry for microbial water quality monitoring in cooling tower water and oxidizing biocide treatment efficiency.

The control of Legionella proliferation in cooling tower water circuits requires regular monitoring of water contamination and effective disinfection procedures. In this study, flow cytometry was assessed to monitor water contamination and disinfection treatment efficiency on bacterial cells regarding nucleic acid injury (SYBR® Green II), cell integrity (SYBR® Green II and propidium iodide) and metabolism activity (ChemChrome V6). A total of 27 cooling tower water samples were analyzed in order to assess water contamination levels regarding viable populations: standard culture, ATP measurement and flow cytometry methods were compared. Flow cytometry and plate counts methods showed a significant correlation for changes in concentrations despite a 1 to 2-log difference regarding absolute quantification. Concerning intracellular activity, the use of two different flow cytometers (FACSCanto™ II and Accuri™ C6) showed no statistical difference while a difference was observed between flow cytometry and usual methods (culture and ATP measurement). The standard culture and flow cytometry methods were also compared for in vitro bacteria inactivation measurements in the presence of 3 different types of oxidizing biocides commonly used for cooling tower disinfection. Reductions observed ranged between 1 and 2 log depending on (1) the detection method, (2) the bacterial population origin and/or (3) the active biocide molecule used. In conclusion, flow cytometry represents an efficient, accurate and fast approach to monitor water contamination and biocide treatment efficiency in cooling towers.

Quantitative Microbial Risk Assessment and Opportunist Waterborne Infections⁻Are There Too Many Gaps to Fill?

Quantitative microbial risk assessment (QMRA) is a relatively new approach in identifying health risks associated with the ubiquitous presence of pathogens and opportunists in the human environment. The methodology builds on experimental and meta-analytical data to identify measurable factors that contribute to, and can quantify, the likely extent of disease given a particular exposure. Early modelling was particularly focused on food-borne disease, and subsequently water-borne disease, with the emphasis focused on ingestion and its role in enteric disease. More recently, there has been a focus on translating these principles to opportunist waterborne infections (OWI) with primary focus on Legionella spp. Whereas dose and susceptibility are well documented via the ingestion route of exposure there is considerably less certainty regarding both factors when understanding Legionella spp. and other OWI. Many OWI can arise through numerous routes of transmission with greatly differing disease presentations. Routes of Legionella spp. infection do not include ingestion, but rather aspiration and inhalation of contaminated water are the routes of exposure. The susceptible population for OWI is a vulnerable sub-set of the population unlike those associated with enteric disease pathogens. These variabilities in dose, exposure and susceptibility call in to question whether QMRA can be a useful tool in managing risks associated with OWI. Consideration of Legionella spp. as a well-documented subject of research calls into question whether QMRA of OWI is likely to be a useful tool in developing risk management strategies.

Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.

Viability qPCR, a new tool for Legionella risk management.

BACKGROUND: Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. METHODS: The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. RESULTS: In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. CONCLUSIONS: The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy.

Evaluation of timing of re-appearance of VBNC Legionella for risk assessment in hospital water distribution systems.

BACKGROUND: In this study we estimated the presence of Legionella species, viable but non-culturable (VBNC), in hospital water networks. We also evaluated the time and load of Legionella appearance in samples found negative using the standard culture method. METHODS: A total of 42 samples was obtained from the tap water of five hospital buildings. The samples were tested for Legionella by the standard culture method and were monitored for up to 12 months for the appearance of VBNC Legionella. RESULTS: All the 42 samples were negative at the time of collection. Seven of the 42 samples (17.0%) became positive for Legionella at different times of monitoring. The time to the appearance of VBNC Legionella was extremely variable, from 15 days to 9 months from sampling. The most frequent Legionella species observed were Legionella spp and L. anisa and only in one sample L. pneumophila srg.1. CONCLUSIONS: Our study confirms the presence of VBNC Legionella in samples resulting negative using the standard culture method and highlights the different time to its appearance that can occur several months after sampling. The results are important for risk assessment and risk management of engineered water systems.

Legionella risk assessment in cruise ships and ferries.

Introduction. The increasing development of marine traffic has led to a rise in the incidence of legionellosis among travellers. It occurs in similar environments, especially closed and crowded, and aboard ships Legionella survives and multiplies easily in water pipes, spreading into the environment through air conditioning systems and water distribution points. Although in recent years in the construction of cruise ships preventive measures aimed at curbing the proliferation of Legionella (design, materials, focus on the operation and maintenance of the water system), have been taken account, little or no attention has been paid to small ships which, in many cases, are old and not well maintained. Objective. The aim of the study was to evaluate the frequency and severity of Legionella contamination in ferries and cruise ships in order to adopt more specific control measures. Materials and method. A prevalence study was carried out on 10 ferries and 6 cruise ships docking or in transit across the port of Messina (Sicily, Italy). Water and air samples collected from many critical points were tested for qualitative and quantitative identification of Legionella. Results and conclusions. Legionella pneumophila sg 1 was isolated from the samples of shower and tap water in 7 (70%) of the 10 ferries examined, and in 3 (33%) of the 6 cruise ships examined, and L. pneumophila sg 2-14 in 8 (80%) and 1 (16.7%) of these ships, respectively. No Legionella contamination was found in whirlpool baths, air and ice samples. In conclusion, the data obtained confirm higher levels of Legionella contamination in local ferries and cruise ships, underlining the need to adopt corrective actions more specific for these smaller vessels.

On-filter direct amplification of Legionella pneumophila for rapid assessment of its abundance and viability.

Guidelines and regulations to control Legionella pneumophila in cooling water systems of large buildings are evolving due to the increasing number of outbreaks. Rapid, on-site, simple, and sensitive quantification methods that are also able to assess viability may be extremely useful in monitoring and control. Culture-based methods for measuring L. pneumophila may take 4-10 days and qPCR-based methods are also slow, requiring at least a day from sample to result, albeit mainly due to the need for sample transport to a centralized laboratory. This study reports a rapid isothermal amplification method for L. pneumophila concentration and detection with live/dead differentiation under field conditions. Using an on-filter direct amplification (i.e., amplification of cells without DNA extraction and purification) approach with propidium monoazide (PMA), and a real time isothermal amplification platform (Gene-Z), L. pneumophila could be detected in 1-2 h at ∼1 cfu/100 ml of tap water. Signature sequences from 16S rRNA and cadA genes were used as genetic markers for L. pneumophila and loop-mediated isothermal amplification (LAMP) primers were designed using Primer Explorer V4. Result were also compared with direct amplification of cells spiked into distilled, tap, and cooling water samples as well as extracted DNA by qPCR. This method may be useful to managers of cooling water systems in large buildings for rapid detection of L. pneumophila. The overall approach of on-site sample concentration, on-filter amplification, and live/dead differentiation may be extended to other organisms where analytical sensitivity and speed are equally important.

Seasonal Assessment of Opportunistic Premise Plumbing Pathogens in Roof-Harvested Rainwater Tanks.

A seasonal study on the occurrence of six opportunistic premise plumbing pathogens (OPPPs) in 24 roof-harvested rainwater (RHRW) tanks repeatedly sampled over six monthly sampling events (n = 144) from August 2015 to March 2016 was conducted using quantitative qPCR. Fecal indicator bacteria (FIB) Escherichia coli (E. coli) and Enterococcus spp. were enumerated using culture-based methods. All tank water samples over the six events were positive for at least one OPPP (Legionella spp., Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Pseudmonas aeruginosa, or Acanthamoeba spp.) during the entire course of the study. FIB were positively but weakly correlated with P. aeruginosa (E. coli vs P. aeruginosa τ = 0.090, p = 0.027; Enterococcus spp. vs P. aeruginosa τ = 0.126, p = 0.002), but not the other OPPPs. FIBs were more prevalent during the wet season than the dry season, and L. pneumophila was only observed during the wet season. However, concentrations of Legionella spp., M. intracellulare, Acanthamoeba spp., and M. avium peaked during the dry season. Correlations were assessed between FIB and OPPPs with meteorological variables, and it was determined that P. aeruginosa was the only OPPP positively associated with an increased antecedent dry period, suggesting stagnation time may play a role for the occurrence of this OPPP in tank water. Infection risks may exceed commonly cited benchmarks for uses reported in the rainwater usage survey such as pool top-up, and warrant further exploration through quantitative microbial risk assessment (QMRA).