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 in the US, it is very useful for quickly determining the presence or absence of Legionella in a sample.  Since same day 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 all serotypes of these species. 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. As a result of a new test procedure it is now possible to identify L. pneumophila serotype 1 using PCR.

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  is the identification of the genus (Legionella), species (pneumophila), serotype (1) and strain (Philadelphia) 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.  As of this writing, EMSL is the only commercial lab that has a validated PCR method for determining Legionella spp., Legionella pneumophila, and Legionella pneumophila serotype 1 (Lp1) in the same test. Species and serotype identification is not sufficient 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 and other species.  However, newer molecular techniques such as Sequence Based Typing (SBT) which looks at seven specific genes within Lp1, as well as Whole Genome Sequencing (WGS) which can discriminate between the roughly 3.4 million genes in the bacterium are used by CDC, some state health departments, and the European Working  Group for Legionella Infections (EWGLI) for subtyping L. pneumophila serogroup 1 and source identification.  The use of PFGE and SBT does not have enough discriminatory power to identify strains of Lp1 or to identify the genetic mutations in Lp1 or other species and serotypes (LaPierre, 2017). Therefore only WGS can be used for source tracking. As of this writing EMSL is the only commercial testing lab that can provide the raw data of WGS to be used for source tracking.

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 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 a confirmed case of Legionnaires’ disease occurs. In this situation, individual species and serotype quantification is necessary.  For an epidemiological investigation of an outbreak, only whole genome sequencing has the discriminatory power to determine if the molecular fingerprints of the environmental and clinical samples are related.

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

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 between sampling periods 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 from two different labs very difficult. Be sure to identify the isolation method the lab is using as well as the identification methods used. This will ensure you are drawing correct conclusions when comparing results and will also ensure you obtain the level of detailed information you need.