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1 EMSL Analytical, Inc.-Copyright 2011
NE Regional AIHA Conference
Legionella – Analytical Developments
Dec 5, 2014
Diane Miskowski
EMSL Analytical Inc. Cinnaminson, NJ
856-303-2528 [email protected]
Legionella Species That Cause Disease
• L. pneumophila (Lp) – causes about 80 to 90% of Legionnaires’ Disease in the US. 75% caused by Lp serotype 1 (Lp1).
• Under diagnosed. Urinary Antigen test –positive after day 5. Only looks for Lp1
• In Australia, 80% of LD cases are caused by L. longbeachae
• A hospital in NYC had an outbreak caused by L. micdadeii
• Another hospital in NYC had an outbreak caused by Lp serotype 6
• Hospital in NC had an outbreak due to L. feeleii
Legionella Species That Cause Disease
• L. pneumophila , L. anisa, L. oakridgensis, L. bozemanii, L. birminghamensis, L, brunensis, L. cherri, . L. cincinnatiensis, . L. dumoffii, L. erythra, L. fairfieldensis, L. feelei, L. gormanii, L. gratiana, L. hackeliae, L. israelensis, L. jamestownensis. L. jordanis, L. lansingensis, L. longbeachae, L. maceachernii, L. micdadei, L. moravica, L. parisiensis, L. quinlivanii, L. rubrilucens, L. santicrucis, L. sainthelensis, L. spiritensis, L. steigerwaltii, L. tucsonensis. L. wadsworthii, L. worsliensis
• Veterans Administration requires individual serotyping of Lp or complete species identification
Proper Analysis
• Culture is Gold Standard in US. CDC/ISO Method
- Culture methods-10 species and individual serotyping of
L. pneumophila 1-14
• Molecular Methods
- PCR Broad screen for 50 Legionella species combined, +/-, no individual identification/quantification
- DNA sequencing of isolates for strain ID compare clinical outbreak strains to env outbreak strains
Isolating Legionella from Environmental Samples
• Isolation
- Buffered Charcoal Yeast Extract Agar (BCYE)
- With and Without Antibiotics
• Presumptive Presence – plating suspected isolates on BCYE With and Without Iron and L-cysteine
• Confirmation/Identification- different results depending on type of kit purchased
- Direct Fluorescent Antibody Technique (DFA)
- Latex Agglutination
EMSL Analytical, Inc.-Copyright 2013
7 EMSL Analytical, Inc.-Copyright 2013
EMSL Analytical, Inc.-Copyright 2013
L. rubrilucens
L. bozmanii
Interpreting Lab Results
• Can’t draw conclusions based on one sample or one sample set
• Non -detectable samples
- Limited sample number or volume
– Testing unconcentrated samples
– Culturing wastewater or potting soil samples without heat pre-enrichment
– Failing to run positive and negative control samples on media used
– Collecting unrepresentative samples
– Improper collection or handling of samples
• Positive samples – Relatively common
– Not uncommon to find more than one type of Legionella
– No agreement on concentrations that are deemed hazardous (infectious dose is unknown; likely variable from person to person)
Field Test Kits
• Not CDC Elite Proficient. CDC will not accept into the Legionella proficiency program
• No validation of test method or procedure
• No validation of media used in the kits
• Is manufacturing of the media standardized?
• Reproduceability of the testing results not studied sufficiently
• Limit of Quantitation is not sensitive enough
How to Choose a Lab • CDC Elite Certified or UK HSE proficient.
• Goal is to use an independent, third party testing lab to ensure no conflict of interest
• Ask lab what method they use and what the limits of detection are –different methods may mean different results
• Beware of Labs that offer risk level in their reports
• Beware of consultants or water treatment providers doing their own Legionella testing.
• Beware of using field test kits for Heterotrophic Plate Count and Legionella
• Do not rely on HPC, pH, and/or free residual chlorine testing in lieu of Legionella testing
Thank You!
Questions?
Diane Miskowski
856-303-2528
or
www.legionellatesting.com
References
• Legionella Regulations from HSE, WHO, South Australia, New South Wales
• Legionnaires Disease, OSHA Technical Manual, USDOL-OSHA, Washington, DC
• Guidelines for the Prevention of Nosocomial Pneumonia, Morbidity and Mortality Weekly Reports, CDC, Atlanta, GA, January 3, 1997
• Hospital Associated Legionellosis, State of New York Department of Health, July, 2005
• Approaches to Prevention and Control of Legionella Infection, Allegheny County Health Department, Allegheny County, PA January, 1997
References
• Report of the Maryland Scientific Working Group to Study Legionella in Water Systems in Healthcare Institutions, MD Department of Health and Mental Hygiene, Baltimore, MD, June 14, 2000
• Procedures of the Recovery of Legionella from the Environment, US Centers for Disease Control, January, 2005
• Water Quality-Detection and Enumeration of Legionella, ISO 11731 1&2
• Legionella Human Health Criteria Document, USEPA, November, 1999
Break – 30 minutes