Legionella

Legionella are environmental-source bacterial pathogens found throughout natural and human-made aquatic environments, including potable and non-potable building water systems. They are found most frequently in domestic hot water and cooling towers. More than seventy different species of Legionella have been identified and named, many with numerous serotypes.

Legionnaires disease

Legionellosis is a broad term for illness caused by Legionella bacteria. Legionnaires’ disease (LD) is a severe form of pneumonia, which usually requires hospitalization. Approximately 9% of all reported LD cases are fatal. Healthcare-associated LD cases have a mortality rate of about 25%. LD results from Legionella infection of human immune cells in the deep, alveolar regions of the lungs. In most cases, infection is the result of inhalation or aspiration of very small, often invisible water droplets that are carrying Legionella. Sources of Legionella-contaminated aerosols most often associated with LD include domestic hot water systems in hotels, hospitals and long-term care facilities. Other sources of exposure include ice machines, cooling towers, ornamental fountains, spas, hot tubs and recreational whirlpool spas. LD is not transmitted person-to-person.

Most Legionella species are believed to have the potential to cause Legionnaires’ disease (LD). Most reported cases of LD in the northern hemisphere are caused by the species L. pneumophila which has at least 15 serogroups. Of all the serogroups of L. pneumophila, serogroup 1 is responsible for most reported cases. Other, non-pneumophila Legionella species have been definitively linked to cases of LD including L. micdadei, L. bozemani and L. longbeachae. In the southern hemisphere, L. longbeachae is the species responsible for most LD.

Legionella Testing

The infectious dose necessary for Legionella to cause LD is unknown: there is no known “safe” density of Legionella in water; there also is is no known “dangerous” level. Legionella test results represent the number of Legionella in a sample at the time the sample was collected, including any increases or decreases due to sample handling, preparation, and transport. Results of Legionella testing have not been correlated with risk and are not predictive of LD.

In general, in the absence of suspected disease, water should not be tested for Legionella without a plan. Routine testing for Legionella is not a substitute for water management.

Three important uses of Legionella screening and testing include:

Evaluation - Results of Legionella screening and tests on water samples collected from throughout a building water system are used to establish a baseline understanding of the microbial condition of the system and inform the development of the water management plan.

Validation - Legionella testing is an important tool for confirmation (validation) that a water management program is effectively controlling Legionella growth, consistent with the Standard of Care established by ASHRAE Standard 188, Legionellosis: Risk Management for Building Water systems.

Investigation - Testing water samples for Legionella is an essential tool in Legionella case/outbreak investigations.

Negative Screening by qPCR

In an emerging best practice, negative screening of samples by quantitative polymerase chain reaction (qPCR) with follow-on culture testing of qPCR-positive samples is used, because samples with qPCR-negative test results are extremely unlikely to yield positive results with culture testing – that is, they have very high negative predictive value (NPV). Because qPCR results typically are ready within 24 hours, valuable information can be available long before culture results.

Responding to Legionella test results

In the absence of disease, Legionella test results should not automatically trigger remedial or other actions except as may be required by regulations. If results from routine validation testing do not meet the Legionella control objectives of the water management plan, the following steps should be followed:

Review the sample collection, handling, and testing procedures to confirm that the results are not due to error.
Confirm that building water system equipment is in good working order and functioning as intended.
Review records to confirm that the water management program was implemented as designed (verification).
Review assumptions about operating conditions, such as the physical and chemical characteristics of the water supplied to the building.
Re-evaluate fundamental aspects of the water management plan that could affect results of Legionella
After careful review, re-evaluation, and possible adjustment of the water management plan, consider whether remedial treatment is needed.

Water Management Plans

The US Centers for Disease Control and Prevention (CDC) has determined that 9 in 10 outbreaks of LD were caused by problems preventable with more effective management of building water systems.

First published in 2015, ANSI/ASHRAE Standard 188, Legionellosis: Risk Management for Building Water Systems, (ASHRAE 188) established the Standard of Care for management of potable and non-potable water systems in buildings.

ASHRAE 188 methodology has seven key elements:

Water Management Team - Establish a multi-disciplinary team with the skills, authority and responsibility to develop and implement the water management plan.
Describe the Water System - Develop a description of the building water system, including schematics, with sufficient detail to enable a step-by-step analysis of the system for conditions that support microbial growth.
Analysis of the Building Water System - Conduct a systematic, step-by-step analysis of the building water system for conditions that could support microbial growth.
Control Measures - Based on the Analysis of the Building Water System, determine the type, location and parameters (control limits) of control measures necessary to manage conditions that, in the absence of control, could be growth supportive.
Monitoring & Corrective Actions - Establish procedures to monitor control measures and, if required, to restore control measures to within the specified control limits.
Confirmation (verification and validation) - Establish procedures to confirm that the water management plan is being implemented as designed (verification) and that, when implemented as designed, the water management plan is effective in achieving its objectives (validation).
DOCUMENTATION - Establish procedures to document and communicate all activities pertaining to implementation and confirmation of the water management plan.

Guidance from CDC and requirements (for healthcare facilities) of the US Centers for Medicare & Medicaid Services (CMS) and The Joint Commission (TJC) include the implementation water management plans consistent with ASHRAE 188. In addition, some authorities having jurisdiction (AHJ), such as New York City Department of Health and Mental Hygiene (NYCDOHMH) and New York State Department of Health (NYSDOH) have rules and regulations that require water management plans.

Legionella test methods OFFERED BY EUROFINS

Molecular Methods – qPCR

OVERVIEW

Molecular methods are procedures that detect the genetic material of organisms. Polymerase chain reaction (PCR) is a method used to make millions to billions of copies of a specific sequence of DNA from a sample that contains very small amounts of that DNA. PCR and tests based on PCR, such as qPCR, are collectively called “Molecular Methods”. Molecular tests may target:

a broad class of microorganisms, such as Legionella spp., all species
a subset of related microorganisms, such as pneumophila, all serogroups
a specific microorganism, such as pneumophila, serogroup 1

Molecular methods, such as qPCR, can be used to detect a much larger variety of pathogens than culture methods. Molecular tests may not be able to discriminate between live and dead microorganisms. However, molecular tests can detect viable but non-culturable (VBNC) microorganisms that cannot be detected by culture but can still cause disease. qPCR test results typically are available within 24 hours.

qPCR tests offered by Eurofins include:

BWS Legionella Triplex™ qPCR

The BWS Legionella Triplex qPCR assay is a Legionella DNA test that simultaneously performs three different tests on one sample to detect and differentiate:

Legionella (all species of Legionella)
pneumophila, all serogroups
pneumophila, serogroup 1

Results are reported in genomic units per milliliter (GU/L).

Bio-Rad iQ-Check® qPCR

The Bio-Rad iQ-Check qPCR assay is a Legionella DNA test that detects Legionella spp. (all species of Legionella) but does not differentiate sub-species or serogroups. Results are reported in genomic units per milliliter (GU/L).

Culture Methods

OVERVIEW

Culture is a broad term for procedures that involve growing bacteria on artificial media. Historically, culture methods have been the most frequently used test methods to determine whether Legionella bacteria are detectable in a sample. Culture testing detects only live organisms that can be grown on media. Traditional culture-based testing can detect all Legionella species and serogroups. Some stressors can cause Legionella to enter a state where it can still cause disease but cannot be detected by culture methods, a state called “viable but nonculturable” (VBNC).

Culture methods offered by Eurofins include:

CDC Method - Traditional Culture

The CDC Method, published by the US Centers for Disease Control and Prevention (CDC) in 2005, describes isolation and enumeration of Legionella bacteria by spread plating filter-concentrated water samples onto selective agar. After incubation for 7-10 days, a series of confirmation steps are used to identify Legionella bacteria. Further testing may be used to differentiate all Legionella species and serogroups. Results are reported as colony forming units per milliliter (CFU/mL).

ISO Method - Traditional Culture

The International Organization for Standardization (ISO) method describes isolation and enumeration of Legionella bacteria by spread plating filter-concentrated water samples onto selective agar. Prior to plating, a portion of the sample is heat- and/or acid-treated to reduce background bacteria; another portion of the sample is plated without pre-treatment. After incubation for 10 days, a series of confirmation steps are used to identify Legionella bacteria. Further testing may be used to differentiate all Legionella species and serogroups. Results are reported as colony forming units per milliliter (CFU/mL).

New York State

The New York State (NYS) regulation Protection Against Legionella specifies use of ISO11731 and requires that all Legionella culture analyses are by a laboratory approved by the New York State Environmental Laboratory Approval Program (ELAP) to perform such analyses. NYS regulations require detection of Legionella spp. (all species).

New York City

New York City (NYC) regulations Local Law 77 and Chapter 8 Legionella Compliance specify use of ISO11731 and require that all Legionella culture analyses are by a laboratory approved by the New York State Environmental Laboratory Approval Program (ELAP) to perform such analyses. NYC regulations require detection of Legionella spp. (all species).

IDEXX Legiolert^®- Non-Traditional Culture

The IDEXX Legiolert test is a non-traditional culture method that detects Legionella pneumophila in water samples. This test is based on a bacterial enzyme detection technology that signals the presence of Legionella pneumophila through utilization of a substrate present in the Legiolert reagent. Legionella pneumophila cells grow rapidly and reproduce using the rich supply of amino acids, vitamins, and other nutrients present in the Legiolert reagent. Actively growing strains of Legionella pneumophila use the added substrate to produce a brown color indicator. The Legiolert Test detects Legionella pneumophila at 1 organism in 100 mL within 7 days. Results are reported as most probable number per 100 milliliters (MPN/100 mL).