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CDE Elite Legionella Testing Laboratory


 

Legionella Testing Laboratory at EMSL

What is Legionella?

The Legionella organism is a gram negative, rod shaped bacterium that can cause pneumonia (Legionnaires' Disease) or a flu like illness (Pontiac fever). It was first identified and recognized as causing disease during the outbreak that occurred in conjunction with the American Legion Convention in Philadelphia in 1976. There are over 40 species ofLegionella and 18 of those can cause disease. It is estimated that the species Legionella pneumophila causes most of the infections. 

Legionella is a fastidious organism, meaning it has specific growth requirements that need to be met in order for it to survive and grow. Some of these growth requirements are:

  1. Temperature above 68° F
  2. Iron
  3. L Cysteine
  4. Biofilm (particularly protozoans)

Unlike some other bacteria, it can survive at lower dissolved oxygen levels and is somewhat resistant to chlorine disinfection. Particular copper and silver ion concentrations can kill the bacterium. Certain plastics and organics can provide nutrients for growth. These attributes make our modern day plumbing systems a good habitat for the organism. 

 

Legionella Overview

Legionnaires' disease (the most severe form of Legionellosis) is a form of pneumonia that is caused by inhalation or aspiration of bacteria that belong to the family Legionellaceae. This family includes 48 species and 70 serogroups. Legionellosis is most commonly linked to exposure to Legionella pneumophila, however, other species (i.e., L. micdadei, L. anisa ) can cause the disease as well. The name Legionella pneumophila was coined after the original outbreak of the disease at the American Legion convention in Philadelphia in 1976.L. pneumophila has many sub-groups called serotypes. L. pneumophila serotype 1 and 6 are commonly associated with Legionnaires disease.

ECOLOGY
Legionella are commonly found in aquatic environments and some species have been found in soil. The organisms are found in a wide range of environmental conditions and are relatively resistant to low pH, dissolved oxygen levels, and routine chlorination techniques for drinking water. Temperatures above 104° F promote rapid multiplication of the organism. The organisms are consistently found in the biofilm that forms in aquatic environments, cooling towers and potable water systems.

EPIDEMIOLOGY
The risk factors for Legionellosis in decreasing order of importance are heavy smoking, chronic lung disease, the elderly, and the immunosuppressed. A recent study has documented 76 cases of pediatric Legionellosis in children under 1 year of age or children with underlying medical conditions such as malignancy or immunosuppression. Legionellosisis not contagious; there is no evidence that the disease can be transmitted form person to person. Exposure must be thru inhalation or aspiration of contaminated, aerosolized water. Once a person has Legionnaires disease, getting it a second time is extremely rare.

MONITORING GUIDELINES
The US Centers for Disease Control (CDC) recommends routine monitoring for Legionella in all bone marrow and organ transplant hospitals nationwide. Routine monitoring in healthcare facilities is recommended or required in several states such as NY, TX, MD, Los Angeles County and Allegheny County PA. Canada has guidelines for monitoring healthcare facilities. The American Society of Heating, Refrigeration and Air Conditioning Engineers (ASHRAE) recommends routine monitoring of building water supply systems.

SAMPLING and ANALYSIS
Take a 1000ml sample for potable water and a 250 ml sample for non-potable water. (Be sure to use sterile bottles with a chlorine neutralizing agent.) It is also recommended to take sterile swab samples of biofilm in areas where it is present. Sample should be shipped overnight to the lab on freezer packs. Culturable analysis either by the US Center for Disease Control or the International Standard Organization is the "gold standard" and requires 10-14 days. Testing by Polymerase Chain Reaction (PCR) takes 2-3 days and is very useful for providing fast, presumptive results to reduce liability during an outbreak. Isolating Legionella from environmental samples is difficult. Make sure you use only an experienced lab with qualified analysts and a sound quality control program. 

Sample Collection and Shipping Procedures

  1. Personal safety and precautions should be observed during sampling. Avoid breathing aerosols that may be contaminated with Legionella bacteria. Avoid generating aerosols or water mists during sampling of the water system. If you have been fit tested, wear a respirator equipped with a HEPA cartridge, goggles, and nitrile gloves.
  2. Prepare or obtain sterile, screw-capped plastic bottles for sampling. Sodium thiosulfate is routinely added to the bottle as a preservative and halogen (chlorine)-neutralizing agent. A 1 liter water sample is recommended for potable water sampling and 250 ml for non-potable water sampling.
  3. For drinking or potable water, such as water fountains, faucets, and shower heads, collect two samples if possible. Collect the "pre-flush or first draw" sample by draining the first 1000 ml of water from the faucets or flush drains into a bottle. Allow the water to run for approximately 60 seconds and collect the second draw of 1000 ml of water.Leave a one-inch air space on top of the water sample.
  4. When sampling faucet aerators and showerheads, swabs of faucet aerators and inside the faucet and shower heads should be taken. When sampling cooling towers, whirlpool spas or fountains, look for areas of biofilm and take a swab sample of the biofilm. We will provide sterile swabs for this purpose.
  5. For non-drinking or non-potable samples from such sources as cooling towers, chillers, , surface water in reservoirs, sprinklers, etc., collect 250 ml water from the bottom or side of the vessel or reservoir. If taking a cooling tower sample, also consider taking a sample in the pack column. Leave a one-inch space on top of the sample. Record any biocide used in water treatment when collecting non-drinking water. If the water treatment includes the use of chlorine, be sure you use bottles with the sodium thiosulfate preservative. If sampling whirlpool spas, consider taking a swab sample of any biofilm as well as a sample of the sand filter.
  6. Label sample number on the bottle and record on the sample data sheet. Use a distinctive number for each sample. Complete all sample information on a sample data sheet for your own record. Send a copy with the samples to the laboratory.  You can download a copy of the Chain of Custody form this website.
  7. Tightly cap the bottles. Make sure that water does not leak during shipping and transporting. Taping of bottle around the cap and neck with electric vinyl tape is recommended. Place taped bottles in a clean plastic bag.

Place the samples in insulated boxes or on freezer packs to protect specimens from extreme temperature fluctuations in the summer months. NEVER USE ICE OR DRY ICE.Stuff the box with foam chips to cushion, and seal the box securely for shipping. Send samples by overnight express carrier. Schedule sampling between Monday and Friday so that samples can be delivered to the laboratory no later than Saturday. Take holidays into consideration.

Legionella Test Codes

CDC Culturable Methods

M210 Level 1 – Presumptive Legionella Detection – Presumptive enumeration or presence/absence

M211 Level 2– Identification, Enumeration, and individual serotyping of L. pneumophila, Serogroups 1-14

M212 Level 3 – Identification and Enumeration of L. pneumophila plus L. anisa, L. bozmanii, L. dumoffii L. gormanii, L. jordanis, L. longbeachae, L. micdadei, L. macheachernii, L. sainthelensis.  Individual serotyping included.

M213 Level 4 – CDC Heat Enrichment Plus Level 3 Identification for Samples Suspected of Containing High Levels of Protozoans

M 214 Pure Culture Preparation and Storage

M 215 Pure Culture Preparation and Shipping

Polymerase Chain Reaction (PCR) Legionella Panels

M163 – Broad Screen Legionella Panel
Presence/absence of 50 species of Legionella including L. pneumophila.  Species are combined and include:  L. pneumophila : 16S rRNA gene and mip gene, 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 

Polymerase Chain Reaction (PCR) Legionella Panels
The test codes below include M212 Level 3 culture, if positive*

M162 Presence/absence of L. pneumophila, L. micdadei, L. maceachernii, L. sainthelensis/cincinnatiensis.

M103 Presence/absence of L. pneumophila

M102 Presence/absence of L. micdadei

M104 Presence/absence of L. sainthelensis/cincinnatiensis

M101Presence/absence of L. maceachernii

Legionella pneumophila serotype 1 Strain Identification

M029 DNA Sequence Based Typing  

Remediating Legionella

Remediating Legionella from potable and non-potable water is no easy matter and may require several attempts before the treatment is successful for achieving non-detectable results.  Unfortunately due to the nature of these systems and the bacteria, there are very few permanent solutions. The only permanent solutions for preventing the bacteria from amplifying in a potable water require the installation of expensive and maintenance intensive equipment for delivery of chlorine, chlorine dioxide or copper silver ion treatment.

The proper design, maintenance, and temperature of a potable water system is the first defense for preventing the amplification of Legionella. Maintaining hot water above 135 degrees Fahrenheit and cold water less than 68 degrees Fahrenheit and eliminating dead legs or low flow areas goes a long way for prevention.  However this is not always feasible. There are several procedures that can be taken for emergency remediation, or routine treatment of a potable water system.  However, it needs to be understood that these are temporary solutions since the bacteria will rebound within a few weeks.  These cleaning protocols are listed below:

  1. Heat treatment of hot water tanks and the complete water system includes raising the temperature of the system to 157 degrees Fahrenheit. This temperature needs to be maintained in the tank for 3 hours. The hot water needs to be drawn through all the outlets at 157 degrees Fahrenheit starting with the outlets closest to the hot water tank.. Sequentially work away from the hot water tank drawing water at 157 degrees Fahrenheit to all the outlets at a trickle flow rate for 3 hours. After this is completed, flush the hot water from the system and return it to normal operating conditions. If the capacity of the system is too low for heat treatment, then chlorination should be used.
  2. Chlorination is accomplished by draining the hot water tank and manually cleaning it of debris. Remove all deposits by scraping followed by wet vacuuming of the tank. Fill the tank with clean water and add chlorine until 50 ppm of free chlorine is obtained through all the outlets including deadlegs and low use points and risers. Soak at this level of free chlorine for 16 hours. Test to ensure that a minimum of 30 ppm free chlorine is obtained at all outlet points for the 16 hours. After this has been accomplished, flush the chlorinated water from the system and test to ensure that less than 2 ppm chlorine remains in the system.

    According to the International Plumbing Code, this chlorination needs to be conducted for all new systems. Chlorination needs to conducted following major alterations to an existing system, if a system tests positive for Legionella, or after major upsets, water main breaks or breakdowns in a municipal   treatment system, or after a municipal water main system has been flushed.

Several studies have indicated that routine testing of a potable water system will identify a potential risk.  These studies suggest that it is not the concentration of the Legionella that is the best determination of risk, but rather the overall percentage of positive outlets.  Therefore, the goal is to establish a history of non-detectable results over time.

Remediating cooling towers is successful as long as the treatment is conducted in conjunction with an ongoing maintenance program of the tower.  Currently, the cleaning protocols for cooling towers can be found in the American Society of Refrigeration, Air Conditioning and Heating Engineers (ASHRAE), the Wisconsin Emergency Protocol (later withdrawn by Wisconsin but the revised version can still be found in OSHA LegionellaTechnical Document), and UK Health and Safety Executive Directive for Water Treatment HS (G) 70.  These protocols emphasize the need for routine maintenance, inspection, manual cleaning of system components and water treatment by professionals. Minimizing microbial growth, scale, corrosion, and sediment accumulation in the cooling tower components is critical for preventing the amplification of Legionella.

One approach for cleaning is a modification of the UK Water Treatment Method HS (G) 70 and includes the following:

  1. Chlorinate to 5-10 ppm for 5 hours with biodispersant; test for chlorine residual every 30 minutes
  2. Completely drain the system
  3. Manually clean the sump, tower pack, distribution system and drift eliminators to remove all deposits. Multi-celled systems can be cleaned sequentially.
  4. Refill the system.
  5. Chlorinate to 5-10 ppm for 5 hours with biodispersant; test for chlorine residual every 30 minutes
  6. Completely drain the system; refill
  7. Re-sample for Legionella after 2 weeks.

For systems having existing online chlorination, this first response is used to reduce (not eliminate) a positive Legionella result:

  1. Maintain 5-10 ppm chlorine for 24-48 hours using a biodispersant
  2. Re-sample for Legionella after the chlorine level drops below 0.5 ppm
  3. Re-sample again after 2 weeks

In both scenarios it is important to review your treatment program. There is a reason you got a positive Legionella result in the first place.

Chlorination is the most effective method for emergency cleaning but excessive chlorination will reduce the life of the system components.  Deposits and biofilm reduce the efficacy of chlorination.  Routine cleaning and chlorination will reduce the presence ofLegionella but it is not permanent solution. The organisms will re-grow in a few weeks.

Unless you change your cooling tower maintenance and treatment program, Legionella will re-appear. An overall maintenance program should include routine shut down of the system to manually clean and flush the system components. Treatment should include the use of corrosion inhibitors, biodispersants (chemicals used to breakdown biofilm) and oxidizing and non-oxidizing biocides. Non-oxidizing biocide treatment will penetrate the biofilm accumulation on the tower components.  Since Legionella multiply in the protozoans within the biofilm, controlling the biofilm is critical to controlling Legionella.  The non-oxidizing biocides need to be rotated frequently to eliminate the development of resistant bacteria.

Improperly maintained hot tubs and whirlpool spas are increasing being associated with legionellosis.  The Association of Pool and Spa Professionals has comprehensive guidelines for maintenance that are available for purchase (www.apsp.org). Considerations for the proper maintenance of these features include the following: Single-use systems should be completely drained between use and stored dry. Non single-use systems should be treated daily and cleaned weekly.  These should be cleaned, treated and stored dry at the end of the season.  Water treatment and filtration is essential whether these systems use potable or salt water.  Heavy bather load will increase the need for cleaning, treatment, and filtration.  Other organisms that can cause disease in these systems include Mycobacterium aviumPseudomonas aeruginosaStaphylococcus aureusNaegleria fowleri and organisms that are associated with fecal contamination caused by children in diapers.

In summary, all the remediation protocols mentioned here are for a short term, immediate response; they should not be considered as permanent solutions.  If you return to operating your potable and non-potable water systems as before, the problem will return.  Continuous, proper, routine maintenance and treatment is the only way to prevent the amplification of Legionella.


Accredited and CDC Elite Certified Legionella Testing Labs at EMSL 

Cinnaminson NJ
New York, NY
Houston, TX
San Leandro, CA
Indianapolis, IN
Mississauga, Ontario 


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