Michael Berg, Ph.D. Legionella and Legionellosis: New Regulations and Detection Methods Participants will be in listen only mode. 9 a.m. (PST) Download the PDF: http:// www.emlab.com/m/media/legionella-webinar.pdf Presented by:
Michael Berg, Ph.D.
Legionella and Legionellosis: New Regulations and
Detection MethodsParticipants will be in listen only mode.
9 a.m. (PST)
Download the PDF:http:// www.emlab.com/m/media/legionella-webinar.pdf
Presented by:
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Continuing Education Units (CEUs)
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Click on the survey link in the �Thank you� email. (sent 1 hour after this webinar)• Complete survey within 24 hours.
• You will receive an email in 2-3 weeks when your certificate is ready.
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Outline
• Overview on Legionella and Legionellosis
• New Guidelines and Regulations
• Detection Systems– Culture test (ISO11731 & CDC)
– IDEXX Legiolert
– qPCR testing
• Sampling
Image from wikipedia: http://de.wikipedia.org/w/index.php?title=Datei:LegionellaPneumophila.jpg&filetimestamp=20060417163014
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Basic Biology
• Gram negative rod-shaped bacterium
• At least 61 species and 70 serogroups have been described
• Widely distributed natural inhabitant of water
• Survives and multiplies as intracellular parasite (e.g. in Acanthamoeba)
Acanthamoeba; Soucre: https://en.wikipedia.org/wiki/Acanthamoeba
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Legionella Bacteria
Photo Credit: https://unsplash.com/
Common in Aquatic Systems
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Temperature Requirements
Temperature Growth or Survival
35 - 46ºC 95 - 115ºF Optimum temp. for growth
< 20ºC < 68ºF Predominantly dormant but viable
> 50ºC > 122ºF 90% kill in 2 hours
> 60ºC > 140ºF 90% kill in 2 min
>70ºC > 158ºF 100% rapid kill
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Basic Pathology
Legionella is the causative agent of Legionellosis Legionellosis takes two distinct forms…1. Pontiac fever: respiratory illness without pneumonia, symptoms resemble acute influenza.
2. Legionnaires’ disease: symptoms include fever, chills, cough, muscle aches, headache, tiredness, loss of appetite, loss of coordination (ataxia), and occasionally diarrhea and vomiting.
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Disease Causing Legionella Species
L. anisa L. erythra L. longbeachae L. pneumophila
L. birminghamensis L. feeleii L. lytica L. sainthelensi
L. bozemanii L. gormanii L. maceachernii L. steeleiL. cardiaca L. hackeliae L. micdadei L. tusconensisL. cincinnatiensis L. jordanis L. nagasakiensis L. wadsworthiiL. clemensonensis L. lansingensis L. oakridgensis L. dumoffiiL. londiniensis L. parisiensis
Legionella species that have been associated with infections and disease (source: ISO11731:2017)
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Legionella Species Isolated from Patients
Legionella species isolated from consecutive patients with community-acquired pneumonia (84.2% of the L. pneumophila were SG1). Data source: Yu et al., JID 2002;186
Legionellaspecies
% isolated Cases
L. pneumophila 91.5% 465
L. longbeachae 3.9% 20
L. bozmanii 2.4% 12
L. micdadei 0.6% 3
L. dumoffii 0.6% 3
L. feeleii 0.4% 2
L. wadsworthii 0.2% 1
L. anisa 0.2% 1
Unknown 0.2% 1
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Guidelines and Regulations
• CDC Review and Toolkit– “Vital Signs” review – June 2016– Toolkit for Water Management Programs (June 2016 updated in
June 2017)
• ASHRAE– ANSI/ASHRAE Standard 188-2015 (Legionellosis: Risk
Management for Building Water Systems), updated 2018– ASHRAE Guideline 12-2000R (Draft for public review posted
July 2017
• European Guidelines (June 2017)– European Technical Guidelines for the Prevention, Control and
Investigation of Infections caused by Legionella species
• New York Regulations (2016)
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Legionella – CDC Review (June 2016)
Source: https://www.cdc.gov/legionella/surv-reporting.html
Reported cases of legionellosis, US, 2000-2016
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Legionella – CDC review
CDC reports:
ü 4x increase of legionellosisbetween 2000 and 2014
ü 10% fatality rateü 9 in 10 cases were caused
by problems preventable with more effective water management
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Legionella – CDC review
Water management program
1) Establish management team
2) Describe building water system
3) Identify areas where Legionellacould grow and spread
4) Decide where control measures are applies and how to monitor them
5) Establish ways to intervene when control limits are not met
6) Make sure the program is effective
7) Document and communicate all activities
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ANSI/ASHRAE Standard 188
Purpose… to establish minimum Legionellosis risk management
requirements for building water systems.
Scope… applies to human-occupied commercial, institutional,
multiunit residential, and industrial buildings - excluding
single-family homes
Intended use… building owners and managers as well as individuals
involved in design, installation, commissioning etc. of
centralized building water systems and components
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ANSI/ASHRAE 188 – Relevance
Applicable to buildings with the following features:• multiple housing units with one or more centralized potable water heater
systems, • more than 10 stories high • healthcare facilities where patient stays exceed 24 hours, • buildings containing one or more areas for the purpose of housing or
treating occupants receiving treatment for burns, chemotheraphy for cancer, or solid organ transplantation etc.
• buildings containing one or more areas for the purpose of housing or treating occupants that are immunocompromised, at-risk, are taking drugs that weaken the immune system, have renal disease, have diabetes or have chronic lung disease etc.
• buildings identified by the owner or designee as being for the purpose of housing occupants over the age of 65 years.
…when any one of the risk factors applies
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ANSI/ASHRAE 188 – Compliance
Are building owners required to comply with ASHRAE 188?
• ASHRAE Standard 188 is a set of standards, not legislation!
• It provides a robust and effective Legionella risk management system.
• It provides protection against allegations of wrongdoing or negligence, should a Legionella outbreak occur.
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ASHRAE 188 – Water Management Program
Elements of a water management program (Fig.1: ANSI/ASHRAE 188)
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ASHRAE 188 – Water Management Program (cont’d)
Program Team – Identify persons responsible for program development and implementation
Health CareHospitals etc.
Facility DirectorAdministrator
Health & SafetyInfection control
Environm. ServicesMedical DirectorChief Engineer
InstitutionalHotels, Casinos etc.
Facility DirectorMaintenance
HousekeepingHealth & Safety
IndustrialPharma, Food
etc.
Plant ManagerMaintenanceEngineering
Health&Safety
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ASHRAE 188 – Water Management Program (cont’d)
• Flow diagram• Analyze: determine
critical control points
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Water System (Description)
https://www.cdc.gov/legionella/downloads/toolkit.pdf
(Example only)
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ASHRAE 188 – Water Management Program (cont’d)
Access Point /Process Unit
Action Item Control Limit Method Frequency
Cooling tower (CCP1)
Legionella <100 cfu/ml Culture test Quarterly
HPC <10,000 cfu/ml Culture test Weekly
Disinfection See microbial action limits
Ozone treatment
As needed (see microbial thresholds)
Off-line cleaning Visual appearance
Visual inspection
Annually (late fall)
On-line cleaning Visual appearance
Visual inspection
Annually (early spring)
…
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Control Measures
• Legionella control measures– Temperature control– Supplemental disinfection/treatment– Flushing– Recirculation– Filtration– Cleaning and maintenance
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ASHRAE Guideline 12-2000R
Proposed Revision of Guideline 12-2000, Managing the Risk of Legionellosis Associated with Building Water Systems (2nd public review Feb 2018)
Purpose: …to provide information and guidance for control of
legionellosis associated with building water systems.
Scope: …applies to human-occupied commercial, institutional,
multiunit residential, and industrial buildings - excluding single-
family homes. It is also intended for use in the implementation of ANSI/ASHRAE Standard 188.
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ASHRAE Guideline 12-2000R (cont’d)
Guideline 12-2000R provides details on water systems management including
• Potable water systems• Ornamental water features• Heated while pools spas/hot tubs• Cooling towers and evaporative condensors• Misters, humidifiers, air washers, direct evaporative air coolers• Indirect evaporative air coolers• Air handling equipment• Other building water systems
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ASHRAE Guideline 12-2000R (cont’d)
Annex list
• Annex A: Bibliography
• Annex B: Guidance on US regulations on drinking water treatment and on chemicals used for potable and non-potable water treatment
• Annex C: Testing for Legionella
– C1: Sample selection, collection and transport (general recommendation: 250 ml collection for routine management, 1 liter for investigative sampling)
– C2: Laboratories should be accredited (e.g. meet ISO 17025:2015)
– C3: Test methods (culture, PCR, other)
– C5: Responses to Legionella test results
• Annex D: Guidance on PPE for use when there is risk of exposure to contaminated aerosols
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European Guidelines
https://ecdc.europa.eu/sites/portal/files/documents/Legionella%20GuidelinesFinal%20updated%20for%20ECDC%20corrections.pdf
Part I: Procedures for the risk assessment, environmental investigation and the control and prevention of Legionella in water systems.
Part II: Methods for the investigation and control of an outbreak of Legionnaires’ in a hotel, other accommodation sites and other public buildings.
Part III: Technical guidelines for the control and prevention of Legionella in water systems.
Part IV: Treatment methods for different water systems.
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NY Regulations (Cooling Towers) – Subpart 4-1
Scope: All owners of cooling towers shall comply…
Registration: Registration online;
…the maintenance program and plan shall include the
following elements:
(1) a schedule for routine bacteriological culture sampling and
analysis to assess microbiological activity at intervals not to
exceed 30 days while the cooling tower is in use, …
(2) a schedule for routine Legionella culture sampling and analysis
within 14 days of seasonal start-up and, thereafter, at intervals
not to exceed 90 days…
Notification: …the owner of a cooling tower shall notify the local
health department within 24 hours of receipt of a Legionella culture
sample result that exceeds 1,000 colony forming units per milliliter
(CFU/mL).
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Result Interpretation – Cooling Towers
• Appendix 4-A (NY Regulations) – Interpretation of Legionella Culture Results from Cooling Towers
Results (cfu per ml) Approach
<20 (or no detection) Maintain treatment program and Legionella monitoring
≥20 Review treatment program
<100 (but >20)
<1000 (but >100)
Institute immediate online disinfectionRetest water in 3 -7 daysContinue retest and treatments until 2 consecutive acceptable tests are obtainedFurther investigate treatment program in addition
≥1000 Review treatment programInstitute immediate online decontaminationContinue retest and treatments until 2 consecutive acceptable tests are obtained
https://regs.health.ny.gov/volume-title-10/1339572150/appendix-4-interpretation-legionella-culture-results-cooling-towers
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NYC Regulations (Cooling Towers)
Requirement New York CityLocal Law 77
(2015 )
New York CityChapter 8 Rules
New York State
Test each cooling tower every 90 days.
XDOHMH specifiestesting “everythree months.”
XTest must be forLegionella; notifyDOHMH within 24hours if high levels found (those greaterthan or equal to 1,000 CFU/mL).
XLegionella testing must have been done by 9/16/15 unless done 30days prior.Otherwise, HPC (heterotrophic platecount) dip slide orculture test allowed.
Perform cleaning at least two times peryear.
X
Perform microbial monitoring.
X (weekly) X
Examples only – more details at: https://www1.nyc.gov/assets/doh/downloads/pdf/cd/cooling-tower-FAQs.pdf
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NY Regulations (Health Care Facilities) – Subpart 4-2
Scope: All general hospitals and residential health care facilities as
defined in Article 28 of the Public Health Law (collectively, “covered
facilities”) shall comply…
Sampling and Management Plan: …all covered facilities shall
adopt and implement a Legionella culture sampling and management
plan for their potable water systems.
Plan must include
(1) Legionella culture sampling sites
(2) provisions requiring Legionella culture sampling and analysis at
intervals not to exceed 90 days for the first year following adoption
of the sampling and management plan. Thereafter, the plan shall
include provisions for annual Legionella culture sampling and
analysis
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Result Interpretation - Health Care Facilities
• Appendix 4-B (NY Regulations) – Interpretation of Routine Legionella Culture Results from Covered Facilities
% positive Legionella test sites Approach< 30% Maintain environmental assessment
and Legionella monitoring in accordance with the sampling and management plan.
≥ 30% Immediately institute short term control measuresRe-sample no sooner than 7 days and no later than 4 weeksImplement long term control measures to ensure ≤30% positive sitesIf ≥30% positive sites, repeat short term control measures
https://regs.health.ny.gov/volume-title-10/11428922/appendix-4-b-interpretation-routine-legionella-culture-results-covered
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OSHA Guidelines
https://www.osha.gov/dts/osta/otm/legionnaires/sampling.html#Protocol
General information - sampling protocol - recommended action
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Action Cooling Tower Domestic Water Humidifier
1 100 CFU/ml 10 CFU/ml 1 CFU/ml
2 1,000 CFU/ml 100 CFU/ml 10 CFU/ml
• OSHA technical manual…– Action 1: Prompt cleaning and/or treatment of the
system.
– Action 2: Immediate cleaning and take prompt steps to prevent employee exposure.
Suggested Action Limits – OSHA
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Action Limits – Europe
European Working Group on Legionella Infections (EWGLI)
Legionella bacteria CFU per liter
Action required
Hot and cold water systemsMore than 1000
up to 10,000• At 10 – 20% positive samples, re-sample, review control
measures, conduct risk assessment.• If most samples are positive, in addition consider disinfection.
>10,000 • Identify remedial actions, immediate review control measures, conduct risk assessment, identify remedial actions.
Cooling towers
More than 1000 less than 10,000
• Review program operation. Review of control measures and risk assessment should be carried out to identify any remedial actions.
>10,000 • Conduct risk assessment and implement corrective action.
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CMS memorandum (cont’d)
Expectations for Healthcare Facilities and SurveyorsCMS expects Medicare certified healthcare facilities to have water management policies and procedures to reduce the risk of growth and spread of Legionella and other opportunistic pathogens in building water systems. An industry standard1 calling for the development andimplementation of water management programs in large or complex building water systems to reduce the risk of legionellosis was published in 2015 by American Society of Heating, Refrigerating, and Air Conditioning Engineers (ASHRAE). In 2016, the CDC and its partnersdeveloped a toolkit to facilitate implementation of this ASHRAE Standard(https://www.cdc.gov/legionella/maintenance/wmp-toolkit.html). Environmental, clinical, and epidemiologic considerations for healthcare facilities are described in this toolkit.
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Test Methods
• Culture testing– Traditional spread plate (ISO11731, CDC)– IDEXX Legiolert
• PCR– ISO12869– BioRad iQ-Check
• Immuno-magnetic Method (IMM)
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Culture (Traditional Spread Plate)
Ø Concentration of (clean) water samples
Ø Treatment to reduce background (heat, acid)
Ø Plating on selective media
Ø Isolate and confirm colonies
Ø Isolate can be stored
Ø Detects all viable colonies of species that grow on Legionella media
Ø “Gold Standard” – only method recognized for NY regs (ISO11731:2017); application of thresholds are based on enumeration by culture
Summary
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IDEXX Legiolert
Ø Culture test
Ø Uses Quanti-Tray system (MPN)
Ø Easy set up
Ø Results in 7 days
Ø Detects Legionella pneumophila
Ø Correlates well with traditional culture
https://www.idexx.com/water/legiolert-info.html
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IDEXX Legiolert vs. Traditional Culture Test
* EMLabP&K procedure following ISO11731
Legiolert Traditional Culture*Turn-around-time 7 - 8 days 10 – 14 days (presumptive
reporting at 7 daysSpecificity Detects Legionella pneumophila
onlyDetects and differentiatesmost Legionella species
Sensitivity Potable water: 0.1 per mlNon-potable water: 10 per ml
Potable water: 0.1 per mlNon-potable water: 4 per ml
Accreditations and Certifications
AIHA, CDC ELITE AIHA, CDC ELITE, NY ELAP
Isolate Recovery Indirect (sub-culture) Direct
Industry Acceptance Working toward alternative “Gold Standard”
Current “Gold Standard”
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PCR Testing – ISO12869 continued
• qPCR detection and quantification
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Legionella PCR
• Extract DNA from concentrated water sample• Add chemicals and run qPCR• Extraction + qPCR = 4 hours
• Quick turn-around-time• Inhibitors can be an issue
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BioRad iQ-Check
ü Kit provides validated standards
ü Streamlined and easy to use
ü Validated against ISO12869
ü Provides a solid solution for quick
TAT Legionella testing
ü Problems: free DNA and non-
viable cells (enumeration does
not always correlate well with
culture)
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Validation
Published validation…
provides technical information on • Detection and quantification limits
• Efficiency and robustness
• Selectivity (inclusivity and exclusivity)
provides defensibility for legal cases
https://nf-validation.afnor.org/en/water-analysis/legionella-spp-legionella-pneumophila-pcr-all-water/
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PCR – Result Interpretation
Culture pos. Culture neg.PCR pos. 73 (31%) 148 (64%)PCR neg. 0 (0%) 11 (5%)
Cooling towers (L. spp test)
Culture pos. Culture neg.PCR pos. 278 (55%) 217 (43%)
PCR neg. 3 (1%) 7 (1%)
Hot/cold Water (L. spp test)
…very good predictor for negative samples
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PCR – Result Interpretation
L. spp L. pnCooling tower 2.03 0.71
Hot/Cold water 1.05 0.62
…PCR results (GU) are typically higher than culture (CFU)
Log difference between qPCR and culture test
Potential causes• Viable but non-culturable cells (NVBC)• Non-viable cells• High background of competing bacteria in culture• DNA duplication before cell division• …
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PCR Applications
qPCR Traditional Culture*Turn-around-time Same day 10 – 14 days (presumptive
reporting at 7 days
Specificity L. pneumophila, L. species Detects and differentiates most Legionella species
Sensitivity 80 GU per liter Potable water: 0.1 per mlNon-potable water: 4 per ml
Isolate Recovery No Yes
Industry Acceptance Accepted as alternative standard in Europe
Current “Gold Standard”, reference for threshold
* EMLabP&K procedure following ISO11731
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PCR Applications (cont’d)
Case Investigations
Cases Source Lpn +Culture/PCR
Days to resultsCulture/PCR
1 Birthing pool 0/4 10/14 Undet. 0/0 10/26 Cooling tower(s) 1/62 10/26 Undet. 0/0 N/A3 HCWS 2/2 4/11 Undet. 0/0 10/11 Spa pool 1/1 4/11 Undet. 0/0 10/28 Undet. 0/2 10/13 Undet. 0/0 10/2
Collins et al. (2017). Journal of Applied Microbiology 122, 1692—1703
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PCR Applications (cont’d)
Time critical testing for risk managementDrawback: • no conversion factor from qPCR to culture• Detection of dead cells by qPCR
Free-DNA Removal Solution (FDRS)
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Sampling for Legionella
Sample types
• Water samples• Swab samples• Air samples (not common)
Sampling instructions:www.osha.gov/dts/osta/otm/legionnaires/sampling.html
https://www.cdc.gov/legionella/downloads/cdc-sampling-procedure.pdf
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Sampling for Legionella (cont’d)
Respiratory Protection
https://www.cdc.gov/legionella/downloads/cdc-sampling-procedure.pdf
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Sampling – Water Samples
• 250 ml or 1 L polypropylene bottles
• Collect 250 ml - 1 L water
• Add sodium thiosulfate
• Warm water systems
• Collect pre- and post-flush sample
• Cold water systems may also contain Legionella bacteria
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CDC Sampling Instructions
https://www.cdc.gov/legionella/health-depts/environmental-inv-resources.html
Keep in mind: Focus of CDC procedure and sampling is for case investigations!
Considerations for case investigations and water management: • Sample volume and sample number • Test sensitivity• Species and serotype identifications• Isolate storage• Test method
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Sampling – Surface Swabs
• Swabs can be used to collect surface samples• Especially useful for investigating biofilms
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Sampling – Air Samples
• Air sampling is typically not recommended.
• Impaction samplers often collect fast growing fungi and bacteria which overwhelm slow growing Legionella.
• Concentration of Legionella in water is generally higher than in air and easier to detect.
Source: CDC 2005: Procedures for the Recoveryof Legionella from the Environment.
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Shipping Samples
• Overnight
• Temperature stable coolers
• Ship on ice or cold packs (ISO recommendation 2 - 8ºC)
• Check for local regulations
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Summary
• Legionella infections are an increasing concern in the US
• New guidelines and regulations have been put in place since 2015 to address water risk management
• New methods are introduced to provide additional tools for the detection of Legionella bacteria in the environment
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Continuing Education Units (CEUs)
To receive a certificate of attendance, you must complete the survey after the webinar:
• Click on the survey link in the �Thank you� email (sent 1 hour after this webinar).
• Complete survey within 24 hours.
• You will receive an email in 2-3 weeks when your certificate is ready.
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Thank you for joining us!
Questions About Legionella:[email protected]
Other Questions:[email protected]