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Unlike culture analysis where inter and intra-laboratory variability is high, PCR results are reproducible, accurate, precise, and very sensitive. The detection limit is theoretically a single DNA fragment. PCR measures the DNA associated with both viable and non-viable Legionella. (The culture method only measures viable bacteria which will grow on the selective media.)
The primary disadvantage of PCR is the potential for sample matrix effects. The presence of common divalent cations in the sample such as calcium, magnesium, or silver, and the divalent form of copper will cause false negative results unless the samples are processed properly. This requires that the lab have a strict Quality Assurance program that includes positive, negative, and sample matrix controls.
Another disadvantage of PCR is that it is a species specific test. While most PCR labs can identify L. pneumophila, there may be other species colonizing your water system or causing the disease that you would like identified.
Non-microbiologists often confuse the terms genus, species, serotype and strain. These are independent terms for the identification of organisms and each is used to reach a successively more specific level of identification. (i.e., Legionella pneumophila, serotype 1, Philadelphia 1 is the identification of the bacteria that caused the 1976 outbreak in Philadelphia.)
The culture method provides quantification and identification of Legionella species and serotypes. Currently, the limited number of commercial labs using PCR will only identify to species level. Species and serotype identification is insufficient for determining the actual source of the contamination during an outbreak. During an epidemiological investigation, it is necessary to employ strain identification to determine if the bacteria in the clinical samples match the bacteria found in the environmental samples.
Currently in the US, Pulsed Field Gel Electrophoresis (PFGE) is most commonly used to identify strains within L. pneumophila serogroup 1. However, a newer molecular technique, Sequence Based Typing, is used by CDC and the European Working Group for Legionella Infections (EWGLI) for subtyping L. pneumophila serogroup 1. EWGLI has proposed the use of SBT as the standard method for strain identification for travel related outbreaks in the European Union.
Intent of the Risk Assessment
The intent of your Legionella risk assessment will determine the type of data you need. Proactive monitoring is conducted to determine the effectiveness of an existing maintenance program in the absence of suspected cases of legionellosis. With this type of monitoring a qualitative, present/absent result or a quantitative result of Legionella spp. is sufficient. Species and serotype identification is optional. Strain identification or subtyping is not needed.
Reactive monitoring is conducted when a suspected or confirmed case of legionellosis occurs. In this situation, species and serotype quantification and identification is necessary. If the case or outbreak was diagnosed as L. pneumophila serotype 1, strain identification will useful to link clinical isolates to the environmental samples to identify the source.
Whether your risk assessment is proactive or reactive, the results should indicate non-detectable amounts of the bacteria. This is the OSHA recommended performance goal.
The actual concentration provides useful information concerning the degree of contamination. However it should be understood that the concentrations are relative and are not an absolute number. Bacterial populations are in always in flux; bacterial cells are multiplying, dying, or dormant. Since bacteria multiply logarithmically, an order of magnitude difference (10x) in the results is significant.
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A difference of a few CFUs or a low single digit multiplication of results is not significant. The goal is to demonstrate a history of non-detectable results over time.
To recap, the analytical method used determines the type and accuracy of the results. While using BCYE agars to isolate Legionella is the recognized “gold standard” worldwide, there are still some labs using other methods. Also, the reagents and methods used for Legionella identification are not standardized. This makes comparing lab results very difficult. Be sure to identify the isolation method the lab is using as well as the identification methods used. This will ensure you obtain the information you need.
About the Author:
Ms. Miskowski has 30 years experience in the areas of Microbiology, Laboratory Management, and Industrial Hygiene with a focus on aerobiology and exposure to pathogens. She is Business Development Manager with EMSL Analytical, Inc., Westmont, NJ. She may be reached at 800-220-3675 x1218 or dmiskowski@emsl.com references>>
