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 dmiskowski@emsl.com

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