WHY DO WE DO RESEARCH? MONITORING FOR LEGIONELLA IN BUILDING WATER SYSTEMS: 7/20/2015. Learning Objectives. Insights From New Research

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1 MONITORING FOR LEGIONELLA IN BUILDING WATER SYSTEMS: Insights From New Research Janet E. Stout, PhD Director, Special Pathogens Laboratory Research Associate Professor University of Pittsburgh Learning Objectives WHY DO WE DO RESEARCH? To Find Answers to Practical Problems Understand: The effect of sample collection methods and transport (hold time) Laboratory testing for Legionella: it s not always apples to apples when comparing laboratories The role of Legionella environmental monitoring in hospital/building risk assessments 1

2 Legionellosis in the U.S. Legionellosis cases have increased substantially over 200% in last 10 years THE CHALLENGE OF LEGIONNAIRES DISEASE IS PREVENTION Outbreaks continue to occur (hospital water systems, cooling towers, fountains) What is the most Important source of exposure for Legionella? Myth: Legionella Is Everywhere Potable water especially in hospitals (and other buildings) with complex hot water systems, is the most important source of Legionella transmission. Published studies find 20 70% of buildings colonized with Legionella Legionella is not ubiquitous (everywhere)! 2

3 Objectives Understand the effects of sample collection and transport (hold time) on results of Legionella testing. Laboratory Results Depend on Sample Collection & Processing The Problem A lack of consensus regarding different approaches to sample collection and processing for Legionella testing has created confusion. QUESTION Should You Flush the Outlet Before Sample Collection? 3

4 QUESTION How Much Water Should Be Collected (What Volume)? Study Design We evaluated the effects of two sample collection variables on the recovery of Legionella from a water sample Flushing the outlet for 2 minutes prior to sample collection (CDC Method) Volume of sample collected and filter concentrated (100mL, 200mL and 1L) Study Design Hot water was collected from 16 sinks weekly for 15 weeks in two buildings. Building 1 had high average colony forming units per milliliter (CFU/ml) (>100) Building 2 had lower average CFU/ml (<50). 4

5 No significant difference between 100 ml or 200 ml no flush samples and 1000 ml flush samples in percentage of positive Legionella culture results after direct plating or filter concentration Conclusions: Flush/No Flush? No Flush Collect immediately after opening faucet or shower valve Flushing reduces recovery Conclusions: Sample Volume Large volume (1000 ml) collection and processing of water samples for Legionella culture does not significantly improve recovery of Legionella; it will significantly increase sample collection and processing time as well as shipping costs. Collection and processing of smaller volume samples for Legionella environmental surveillance can be done without significant loss in sensitivity. 5

6 Meaning for You Collection of 1L samples is not necessary to asses water system colonization There is reduction in recovery with filtration and the greater the volume the greater the loss Flushing an outlet prior to sample collection will result in reduced recovery Biofilm loosely adherence bacteria are flushed away QUESTION Does the time it takes to ship samples to the lab (hold time) significantly change results (cause false results)? Study Design Total of 2544 samples analyzed Split samples: tested half immediately and half after 24hr Immediate testing of replicate samples = testing variation 24hr hold time = effect of hold time Flanders, W., et al Water Research (2014) doi: /j.watres Water Research vol 62;

7 Study Results Difference in replicates between T=0 and processed next day was only 0.12 log and the change in CFU only 7.5 CFU/mL Flanders, W., et al Water Research (2014) doi: /j.watres Conclusion No significant increase or decrease in Legionella viability when cultured within the time recommended from collection to processing, i.e. 48 hr of collection. SPL Research on Sample Collection & Transport Testing for Legionella in Healthcare Facilities: Evaluation of the Reproducibility of Legionella Test Results and the Impact of Time on Viability and Variability Sue M. Mietzner MS, Andrea J. Schaeffer, Mohamed H. Yassin MD, Marilyn M. Wagener MS, Janet E. Stout Ph.D. APIC 40th Annual Conference. Ft Lauderdale, FL June 8-10,

8 Study Questions Will results of Legionella culture be consistent from samples collected from the same locations over time? Will the time between sample collection and processing significantly affect the result? Study Design Legionella culture performed on 155 water samples. Hot water samples (500 ml) were collected from 12 sites in a hospital building 2 times a week for 6.5 weeks. Samples were processed on site, immediately after collection (T=0), T=1h, T=24h and T=48h. Table. Mean recovery of L. pneumophila from 12 sink faucets sampled at 0, 1, 24 and 48 hours. Time (hr) Recovery of L.pneumophila Mean log cfu/ml Mean difference p-value NA NA < a b <0.05 c a T0 vs T1; b T0 vs T24; c T0 vs T48 Mean Log CFU/ml T=0 hr T=1 hr T=24 hr T=48 hr Mean Log CFU/mL Over 48 hrs per Site Site Number 8

9 Conclusion Recovery of Legionella from outlets was consistent both in log CFU/mL and proportion of sites positive. No significant increase or decrease in Legionella viability when cultured within the time recommended from collection to processing, i.e. 48hr of collection. Reduction in viability over time between sample collection and culture processing was not greater than the day-to-day variation, even at 48hr after collection. Meaning for You You can have confidence that: Sampling the water system of a building can give you a meaningful snapshot of the colonization status. Variability in recovery of Legionella between sample collection and transport to the lab is insignificant and will not give false results. Laboratory Testing: Apples To Apples or Apples To Oranges? Detection Methods Culture isolation Direct visualization Molecular methods 9

10 Culture Method Why Is Culture the Gold Standard? Industry standard/best practice Proficiency Programs Standards based ISO (1&2) ASTM D 5952 International HSE L8 ACP 2013 Laboratory-based and validated culture method is the industry standard Culture is more reliable than rapid tests DFA ICT Immunochromatographic Test PCR Dip slide Example: ICT Not Sensitive: Same Water Sample Different Results Methods for Legionella Testing & Identification: Did You Know That Labs Differ in Their Methods? 10

11 Culture Method Media for Isolation of Legionella spp. from Environmental Samples Processing Methods Pretreatment (heat and/or acid) Filtration (yes/no or how much) Culture media (types and how many) Identification (ID) Methods Only presumptive agglutination test? Definitive ID with DFA or sequencing for unusual species? DGVP Dyes 10 ug/ml Glycine 3.0 mg/ml Vancomycin 1.0 ug/ml Polymyxin B 50 U/ml CCVC Colistin 16 mg/l Cephalothin 4 mg/l Vancomycin 0.5mg/L Cycloheximide 80mg/L Pretreatment to Inhibit Competing Bacteria Acid buffer is a HCl-KCl solution at a ph of M hydrochloric acid plus 0.2 M potassium chloride, adjusted to ph 2.2 Heat treatment 50 o C 30 min Legionella Colony Morphology Colonies of Legionella pneumophila have a typical ground-glass, opalescent morphology 11

12 Molecular Methods: U.S. Centers for Disease Prevention (CDC) Molecular Methods For Legionella Testing: Are We There Yet? CDC does not currently recommend the routine use of genetic probes or PCR for detection of Legionella. Use of PCR for the identification of Legionella spp. is not recommended until more data regarding sensitivity and specificity of procedures are available Molecular Assay False-Positivity Organisms exhibiting cross-reaction are commonly found in water and may interact with previously designed PCR primers for Legionella detection The Microbiome The Future of Monitoring? 12

13 SPL Publication Available Online Is the Sky Falling if I Find Some Legionella? Interpreting Results: What Is an Acceptable Amount of a Contaminant? IS ZERO NECESSARY? 13

14 Establish Targets Action Levels Minimum Levels Goals Legionella Zero Is the Goal, But Not Necessary to Avoid Outbreaks Not All Legionella Are Dangerous Legionella Species Are Not Equal When it Comes to Causing Disease 58 named species to date Only half implicated in human disease Some species very common in the environment, but rarely cause illness, and almost exclusively in the immunocompromised 14

15 Legionella Species That Fluoresce Under UV Light Some fluoresce red and some fluoresce bluewhite Blue-white species L. anisa L. dumoffii L. gormanii L. bozemanii Legionella rubrilucens Keeping Risk in Perspective Legionella pneumophila, serogroup 1 is the primary disease-causing species Non-pneumophila species cause illness on rare occasions Keeping Risk in Perspective Conducting a system wide disinfection when only non-pneumophila species (such as L. anisa) are isolated would be a time consuming and costly proposition with very little evidence to support such an action Legionella Species Table 15

16 WHY TEST? Role of Testing in Legionella Risk Management Environmental Monitoring for Legionella: Key to Prevention Journal Am Water Works Assoc 2014; 106(10): Establish Baseline Legionella Distal Site Positivity And Demonstrate Disinfection Efficacy Essential Part of a Proactive Approach to Reducing Legionnaires Disease 120% 100% Monochloramine Start Date Distal Site Positvity 80% 60% 40% Distal Site Positivity 20% 0% Am. J. Infection Control 2005; 33(6): Date 16

17 Our Study Results - 48 Hospitals Proportion of hospitals performing Legionella environmental surveillance = 65% Environmental monitoring identified hospitals at risk for Legionnaires disease Proportion that started water treatment (disinfection) = 44% ASHRAE Proposes a Standard to Prevent Legionnaires Disease U.S. STANDARD TO CONTROL & PREVENT LEGIONNAIRES DISEASE American Society of Heating, Refrigerating and Air-conditioning Engineers 17

18 SPC 188: The Long and Winding Road Committee was authorized as a standard committee in June 2005, formally renamed SPC188 Jan Five Public Reviews of 188P The final document includes changes in response to comments on the 5 public review drafts over 5 years What Is ASHRAE Standard 188? Approved June 26, 2015 The purpose of this standard is to establish minimum Legionellosis risk management requirements for building water systems 18

19 Compliance The building shall be surveyed to determine whether it has one or more of the listed water systems and/or the factors described that relate to risk for Legionellosis. Water Systems Covered by 188 Potable and non-potable water systems, in the building or on the site Includes building water distribution systems (including centralized potable water heater systems) Cooling towers, evaporative condensers Whirlpools or spas ornamental fountains, misters, atomizers, air washes, humidifiers or other non-potable water systems or devices that release water aerosols in the building or on the site Legionella Testing in ASHRAE Standard 188 Copyright 2015 ASHRAE. Reprinted by permission 19

20 WMP Team Determines Approach to Testing The Program Team shall determine: Whether testing for Legionella shall be performed and if so how test results will be used to validate the Program. WMP Team Determines Approach to Testing The Program Team shall determine the testing approach including: Sampling frequency Number of samples Locations and sampling methods How Can You Use Legionella Testing in Water Safety Plans? Establish A Baseline Define Realistic Expectations Testing should be appropriate for the system being evaluated If treatment programs change (verify efficacy) or are implemented (pre & post test) Dependent on cooling tower operation (quarterly?) Building water systems (Hospital?) 20

21 Mark Twain It Ain t What You Don t Know That Gets You Into Trouble, It s What You Know For Sure That Just Ain t So. Mark Twain Mark Twain What You Know The effects of sample collection and transport (hold time) Laboratory testing for Legionella: Best practices/methods The role of Legionella environmental monitoring in hospital/building risk assessments THANK YOU Dr. Janet E. Stout Director, Microbiologist info@specialpathogenslab.com 21