Management of Legionella in Water Systems (2020) / Chapter Skim
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3 Quantification of Legionnaires' Disease and Legionella
3 Quantification of Legionnaires' Disease and Legionella
Pages 89-164
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From page 89... ...
framework can be designed using an estimate of the concentration of Legionella pneumophila (the pathogen most likely to cause disease) associated with a particular source (e.g., showerhead, hot tub, cooling tower)
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From page 90... ...
Most hospitals do not routinely culture sputum for Legionella, although some academic health centers routinely culture bronchoscopy specimens in patients with pneumonia of unknown etiology. Culture methods are critically important to epidemiologic investigations because molecular analysis can link clinical isolates to environmental samples to document the source of the exposure.
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pneumophila serogroup 6) using validated reagents on specimens collected three to six weeks apart.
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These are the National Notifiable Disease Surveillance System (NNDSS) and the Supplemental Legionnaires' Disease Surveillance System (SLDSS)
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The SLDSS includes potential environmental exposures, such as whether a case is travel- associated or whether an individual had exposure to hot tubs, respiratory therapy equipment, or a healthcare or senior-living facility. However, these data are often incomplete and not timely, and they frequently do not identify the potential environmental source of exposure.
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For decades, legionellosis programs both in states and at the CDC have been given low priority compared to other preventable infectious diseases, including communicable respiratory conditions. Furthermore, because the programs were initially focused on outbreak detection and control, the CDC and other public health agencies did not build expertise and capacity in fields that are needed to understand legionellosis prevention and control (e.g., building water systems, environmental engineering, and industrial hygiene)
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The waterborne disease outbreak reporting system is important because outbreaks are most likely to be investigated for environmental sources. A limitation of the NORS program for legionellosis is that the database (and hence the categories of setting, water types, and water exposure)
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The patient interview collects information on the patient's home, work, and other addresses, presenting symptoms, and health history, along with information on known water exposures, travel, and healthcare visits during the ten days before onset of symptoms (the typi cal disease incubation period)
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These data indicate that any rigorous effort to better understand the sources of exposure that cause individual legionellosis cases will require well-funded, coordinated studies involving medical centers, laboratories, and health departments in areas with capacity for the consistent collection and cultivation of both clinical and environmental Legionella cultures for a substantial proportion of cases. This is resource intensive because most sporadic cases involve multiple possible environmental sources of Legionella exposure, and environmental isolates that do not match clinical isolates may still require on-going public health follow-up when they indicate possible disease risk from a potential environmental source.
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From page 98... ...
It is likely that trends in treatment of outpatients with Legionnaires' disease and Pontiac fever follow trends similar to the hospitalization data. Reported rates of legionellosis are lower in some areas of the United States (e.g., the West)
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QUANTIFICATION OF LEGIONNAIRES' DISEASE AND LEGIONELLA 99 A B C FIGURE 3-2 Rates of reported legionellosis cases by state for (A)
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Cases not recognized as part of an outbreak are considered sporadic. In the United States, waterborne disease outbreaks in the NORS system are subdivided into whether the outbreak source was identified as potable water, recreational water (treated or untreated)
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. Data from the VHA between 2014 and 2016 show that the rate of Legionnaires' disease significantly increased among veterans receiving VHA healthcare services but with no exposure to a VHA healthcare facility during the disease incubation period (from 0.9 to 1.47/100,000 enrollees)
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in which a total of 28 environmental sources were identified. Potable water from single family homes, large building water systems, and car travel appeared to contribute to a substantial proportion of the sporadic Legionnaires' disease cases.
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From page 103... ...
are examined by water type, i.e., whether the outbreak is associated with drinking water, treated or untreated recreational water, or another water system. During 2013 to 2014, 19 states reported 42 outbreaks associated with drinking water; Legionella was implicated in 57 percent of the outbreaks (see Figure 3-4)
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. Outbreaks have also been attributed to wastewater treatment plants (Kusnetsov, 2010; Loenenbach et al., 2018)
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may increase risk for contamination of building water systems and other water devices or equipment. Contaminated environmental sources, from dental hygiene equipment to street cleaning machines, continue to be newly identified (Ricci et al., 2012; Schönning et al., 2017; Valero et al., 2017)
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The purpose of the investigation was to describe patient demographic characteristics and comor bidities, identify environmental exposures, and implement control measures. Reporting and Case Follow-up Physicians and clinical laboratories are required to report positive Legionella test results to DOHMH.
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The New York State Department of Health Wadsworth Center and the NYC Public Health Laboratory tested cooling tower water samples for the presence of Legionella using PCR and culture methods. Use of PCR allowed for the rapid screening of samples to prioritize culture and cooling tower remediation.
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pneumophila serogroup 1. This estimate is conservative; other have found that the UAT only detects of 80 percent of L
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pneumophila serogroup 1 disease, which was not captured in the EPIC study.6 This consideration increased the rate to 6.17/100,000. The Committee then assumed that 10 percent of all legionellosis cases are healthcare-associated (see previous sections of this chapter)
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For example, monitoring of cooling towers or hospitals, in the absence of cases of disease, has largely focused on whether or not to implement water treatment. Presence/absence approaches, where positive results initiate action, have frequently been used rather than quantitative measures.
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Outbreak Investigation • PCR/PCR -- Rapidly identify suspect sites q Suspect sources? Cooling As soon as possible when an Water Culture needed for for further testing towers, hot and cold taps, outbreak is suspected comparison to patient • Culture -- Confirm viable Legionella showerheads, hot tubs, Numbers would be isolates • erogroup, sequence typing, whole-genome S decorative fountains, etc.
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From page 112... ...
While culture methods have remained the gold standard, they may need to be adapted or supplemented with other methods to assist in developing risk estimates and informing outbreak investigations. Depending on the application, it is likely that combinations of methods will be used in the future.
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TABLE 3-2 Comparison of Methods for Environmental Legionella Monitoring Discerns Form of Potential for Potential for Serogroups/ Bacteria Method Quantification Isolation Level of Use1 Sequence Types? Measured Pros Cons Culture Methods ISO Yes Yes Routine Yes Culturable Standardized Time to results; may underestimate VBNC, Historical data other serogroups and species, risks CDC Yes Yes Routine Yes Culturable Standardized Time to results; may underestimate VBNC, Historical data other serogroups and species, risks AHPA Yes Yes Routine Yes Culturable Standardized Time to results; may underestimate VBNC, Historical data other serogroups and species, risks Molecular Methods2 PCR No No Research, used No Inactivated, Can support sequencing Need to process gels with cultivation VBNC Culturable qPCR Yes No Research, potential No Inactivated Rapid results Measures inactivated cells; less historical for diagnostics and VBNC Greater sensitivity and specificity use surveillance Culturable ddPCR Yes No Research, potential No Inactivated Rapid results Measures inactivated cells; few studies for diagnostics and VBNC Greater sensitivity and specificity using and comparing the method surveillance culturable Emerging Methods Next No No Research No Inactivated Provides info on how bacteria relate Takes special expertise, instrumentation Generation VBNC to microbial community More cost and time to obtain results Sequencing Culturable Amoeba No Yes Research Yes Culturable Improves isolation of difficult-to- Adds at least 3 days to cultivation Co-culture culture strains Liquid culture- Yes Yes Research, potential No Culturable Simple set-up, may be specific to Lp 7 days for results based MPN for routine use More difficult to confirm EMA-PCR Yes No Research No Viable Can be used with molecular tools Not proven to work with disinfection PMA-PCR Yes No Research No Viable Can be used with molecular tools Not proven to work with disinfection Flow Yes Yes Research, potential Yes Inactivated Simple set-up, specific to Lp Early commercial release, limited Cytometry for routine use VBNC serogroups based on antibodies validation, higher detection limit Culturable 1Categories include Routine, Research, Potential for Routine, or Potential for Diagnostics and Surveillance; 2Molecular tools require special instruments, training, and expertise; AHPA: American Public Health Association; ddPCR: digital droplet PCR; EMA: ethidium monoazide; MPN: most-probably-number; PMA: propodium monoazide; VBNC: 113 Viable-but-Non-Culturable.
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with 491 potable water samples and 846 non-potable water samples. A third study (Rech et al., 2018)
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Another drawback of this MPN method is that colonies are not readily available for molecular discrimination assays. As new methods develop, there is a need for greater systematic study and reporting of information, including a full description of the types of samples compared, characterization of the genera and species eliciting false positives, and genetic characterization of the Legionella spp.
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Finally, the Legionella is isolated on BCYE agar. Amoebae co-culture methods have not been standardized and have primarily been used in the research arena and in reference laboratories in Europe for water and clinical samples.
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The use of L pneumophila serogroup 1-specific primers is relatively new, but appears to be gaining momentum since it was first introduced (Mérault et al., 2011)
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For L pneumophila serogroup 1, the agreement was poor for drinking water (10 percent, 5 percent, and 0 percent positive by qPCR, culture, or both, respectively)
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. In general, these studies showed that when dis infected water samples were exposed to PMA or EMA, the gene copy numbers of Legionella calculated were between the number of Legionella colony forming units obtained by culture and the number of gene copies obtained with qPCR without PMA or EMA exposure.
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pneumophila in drinking water samples taken from public sport facilities was analyzed by RT-qPCR, cultivation, and qPCR. For 86 percent of the samples, the results with RT-qPCR and cultivation were consistent.
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as well as estimate their relative abundance. In one laboratory study of domestic hot water, qPCR and amplicon-sequencing-based methods estimated Legionella spp.
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First, the water system to be sampled must be identified, such as cooling towers, residences, public buildings such as hotels, resorts, hospitals, drinking water, and wastewater. In particular, points thought to be most vulnerable to Legionella growth and where potential for human exposure is high should be prioritized.
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For large buildings, the building manager will need to identify the potential locations where Legio nella may be present and propagate, based on the number of potable water systems and the number of distribution components. Examples are as follows: • Potable sources: Some building configurations have multiple water mains.
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pneumophila serogroup 1. The detection limit should be carefully documented, addressing both the volume collected and concentrated.
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2. Cooling towers do not continuously circulate water even when they are in operation, which provides conditions ideal for Legionella growth.
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concentration data on cooling towers, residences, hotels and resorts, recreational venues, hospitals, cruise ships, and drinking water and wastewater treatment plants. The data were generated using either culture methods that quantify colony forming units and include cells that grow and produce colonies on solid agar, or qPCR for which the data are referred to as gene copies and that include live, VBNC-like, and dead cells with intact DNA.
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L pneumophila serogroup 6 was isolated from both the patient and his home potable water system as confirmed by pulsedfield gel electrophoresis (a method used to fingerprint DNA from bacteria)
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. Levels were found at 105 GC/L for Legionella spp.
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Low concentrations of L pneumophila serogroup 1 were found, between 40 and 620 GC/L, in around 50 percent of the positive samples; yet on occasion, a high level was found up to 105 GC/L, creating an average of 1.97 × 103 GC/L with a median of 62 GC/L.
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pneumophila serogroup 7 5 200 3,350 L pneumophila serogroup 8 1 50 L
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pneumophila serogroup 1. Table 3-4 shows the concentration data collected TABLE 3-4 Attack Rates, Case Numbers, and Legionella Concentrations Measured in Recreational Waters During Selected Outbreaks Venue Attack Rate (%)
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Hospitals There is great concern about Legionella infections in hospitals because of their susceptible populations. As mentioned in Box 3-6, in many large hospitals Legionella monitoring has been undertaken to confirm that water treatment is suppressing bacterial growth in the premise plumbing.
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pneumophila serogroups 2-14, and L anisa, respectively.
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pneumophila serogroup 1 ranging from 6.3 percent to 71.4 percent positive in one of the wings of the hospital. The premise plumbing was treated with 60°C water for ten minutes, yet positive samples were still detected (5/89, 5.6 percent)
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pneumophila serogroup 1 concurrent with other serogroups, respectively. In another similar study, 12 cruise ships were found to be negative for Legionella, while 28 ferries were sampled and found to be positive 81 percent of the time (Mouchtouri and Rudge, 2015)
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The source for the outbreak was a cooling tower that r eceived river water into which a biological wastewater treatment plant discharged (Maisa et al., 2015)
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Since 1954, the municipal water source was Flint River water treated at the Flint Water plant. In 1967, the city began purchasing water from Detroit Water and Sewerage Department (DWSD)
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Indeed, disease cases stemming from one hospital in Flint decreased dramatically after a biocide system was installed in the hospital. Nonetheless, while the Flint outbreak is an example of the failure of an important barrier -- treatment of the building water system -- it is also unique in highlighting the role of drinking water utilities in creating conditions con ducive to Legionella proliferation in premise plumbing.
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A Quarterly Legionnaires' disease incidence in Genesee County, MI, 2010–2016.
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The more limited set of studies for which concentrations were reported demonstrates that higher concentrations of Legionella are associated with higher disease risk. For example, the studies of Legionella outbreaks associated with cooling towers suggest that duration of the outbreak, but not the total number of cases, is related to Legionella concentrations BOX 3-10 Wastewater Treatment Plant Identified as a Source for Legionnaires' Disease A wastewater treatment plant in Norway was identified as a source for Legionnaires' disease, leading to 56 cases and ten deaths in 2005, and five cases and two deaths in 2008 (Borgen et al., 2008; Nygård et al., 2008)
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Their work reported Legionella concentrations ranging from 8.4 × 104 to 1.6 × 106 CFU/L with an average of 8.0 × 105 CFU/L. An outbreak associated with a wastewater treatment plant showed that Legionella concentrations from the aerators ranged from 2.0 × 106 to 2.2 × 109 CFU/L with an average of 1.1 × 109 CFU/L (Loenenbach et al., 2018)
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, recreational water outbreaks (Leoni et al., 2018) , wastewater treatment plant outbreaks (Loenenbach et al., 2018)
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pneumophila serogroup 1; the relative potency of
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(A discussion of these and putative viability assays is found earlier in this chapter.) Exposure estimates are necessary to produce good risk estimates, and the number of samples collected in a monitoring program and their detection limits should be sufficient to determine exceedance or compliance with an acceptable risk value.
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FIGURE 3-12-1 Estimation of mean number given fraction of positive samples for Poisson vs. nega tive binomial distributions.
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in Legionella spp. concentrations in flushed samples between sampling points within a single building and, for a given point, between hours in a day or between weeks.
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pneumophila; in this analysis, the monitoring results from several water reuse facilities were used (Johnson et al., 2018) in which Legionella spp.
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In developing U.S. drinking water regulations for virus and protozoa, EPA was informed by an annual risk level of 10−4 infections/year (Regli et al., 1991)
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i=1 For an exposure that is relatively continuous to a large population, an annual risk level may be an appropriate approach to control. This could be pertinent to exposures such as large industrial cooling towers.
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practice has been to use a 1/10,000 annual infection endpoint as a measure of acceptability in drinking water (Rose et al., 1991) , WHO has promoted use of a 10−6 DALY annual risk as an endpoint because of the increased severity of Legionnaires' disease.
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This includes (1) characteristics of the cooling tower, including aerosol generation rate and height, (2)
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The Committee estimates 52,000 to 70,000 cases of Legionnaires' disease in the United States each year. There are many sources of Legionella risk in engineered water systems, from cooling towers to premise plumbing to hot tubs.
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Although all cases will not receive thorough environmental investigations, at a minimum it should be discerned whether a case may be associated with a healthcare facility, accommodation site, hot tub or other well-recognized potential source, as well as some information about the building water system and any known deficiencies (e.g., water main breaks) during the incubation period.
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The available data suggest that cooling towers, hot tubs, showers, and wastewater treatment plants can be hot spots for growth of Legionella and exposures. This data set could be improved by adopting standardized molecular methods that allow for greater quantitation and more rapid results.
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2013. Clinical application of a multiplex real-time PCR assay for simultaneous detection of Legionella species, Legionella pneumophila, and Legionella pneumophila serogroup 1.
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and Legionella pneumophila at a biological treatment plant. Environmental Science and Technology 42:7360-7367.
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2014. Widespread molecular detection of Legionella pneumophila serogroup 1 in cold water taps across the United States.
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2008. M A PCR-based method for monitoring Legionella pneumophila in water samples detects viable but non ultivable c legionellae that can recover their cultivability.
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1983. Distribution and seasonality of Legionella pneumophila in cooling towers.
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2010. Two Legionnaires' disease cases associated with industrial wastewater treatment plants: a case report.
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2011. Specific real time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples.
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2016. Genomic resolution of outbreak-associated Legionella pneumophila serogroup 1 isolates from New York State.
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2018. Comparison of the Legiolert™/Quanti-Tray® MPN test for the enumeration of Legionella pneumophila from potable water samples with the German regulatory requirements methods ISO 11731–2 and ISO 11731.
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1988. Investigation of Legionella pneumophila in drinking water.
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