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Analytical Methods-Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a genetic test which looks for the deoxyribonucleic acid (DNA) that is specific for Legionella. While PCR is not considered the gold standard for Legionella analysis, it may be very useful for quickly determining the presence or absence of Legionella in a sample. Since same day qualitative results can be obtained, the quick turnaround time can be useful for confirming the presence of Legionella during an outbreak when time is critical.

 

 

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.

 

Other disadvantages of PCR are it cannot identify individually the 50 species known to cause disease and it cannot identify serotypes. While most PCR labs can identify L. pneumophila, there may be other species or serotypes 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.

 

In the US, Pulsed Field Gel Electrophoresis (PFGE) was the most commonly used method to identify strains within L. pneumophila serogroup 1. However, a newer molecular technique, Sequence Based Typing (SBT), is used by CDC and the European Working Group for Legionella Infections (EWGLI) for subtyping L. pneumophila serogroup 1.

 

Intent of Risk Assessment Monitoring

The intent of your Legionella risk assessment will determine the type of data you need. Proactive monitoring is conducted to determine baseline conditions and concentrations of Legionella or to validate 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 Legionnaires’ disease occurs. In this situation, individual species and serotype quantification is necessary. For an epidemiological investigation of an outbreak, whole genome DNA sequencing or SBT can determine if the genetic fingerprints of the environmental and clinical samples are related.

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Whether your risk assessment is proactive or reactive, the results should indicate non-detectable (ND) or negative 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. 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.

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