Legionella Testing Methods: Quantitative PCR (qPCR) Versus Culture

How to test for Legionella? That’s a common question and one that is best answered by a truly independent Legionella company like Legionella Control Systems.

A traditional culture (spread plate) test has historically been the gold standard for Legionella testing. However, Legionella quantitative Polymerase Chain Reaction (quantitative PCR, Legionella qPCR) has grown rapidly as a supplemental or replacement test for the Legionella culture test.

The Legionella culture and qPCR tests vary in their technical approach, detection levels, and interpretation, so this provides an overview and closer examination to help with the understanding and relevant use of these Legionella tests.

Overview of Legionella Testing: Culture versus qPCR

• Traditionally, monitoring of Legionella populations in water samples relied on colony growth on selective media. This is known as the Legionella culture test.
• Quantitative polymerase chain reaction (Legionella qPCR) is a DNA-based method that allows the creation of many copies of a small section of DNA or RNA. Legionella qPCR was developed to aid in the rapid detection of Legionella DNA in water samples in response to the slowness of the traditional culture method. The results of a Legionella culture test can take 7-14 days, causing delays in response times to outbreaks of Legionnaires’ disease.
• Legionella testing can be part of the identification of potential risks, step 4 in a water management program.
• Exact values of qPCR that predict the safety of water have not been published. However, it has been suggested that when a PCR analysis shows a low number of genomic units (GU) of DNA detected (100-2000 GU/ml), it is likely that the Legionella in the samples are dead. When the GU numbers are higher, such as 15,000 or more, the likelihood of positive cultures increases dramatically.

Characteristics of Legionella Culture Testing Versus qPCR Legionella Testing

The CDC (Centers for Disease Control) notes that the Legionella test method may vary by the type of water system and the reason for testing. The CDC describes the following characteristics of the Legionella culture versus Legionella quantitative PCR test methods:

Culture for Legionella Testing

• Detects viable bacteria
• Detects all Legionella species
• Results typically reported in colony forming units (CFU) per volume with a limit of detection ~10 CFU/mL
• Yields isolate for additional characterization
• Results typically reported in 7–14 days
• Is subject to skill, experience, and procedural rigor of the laboratory
• May be preferred for evaluating growth trends

PCR (qPCR) for Legionella Testing

• Detects Legionella-specific DNA or RNA
• May not differentiate between live and dead bacteria
• Results typically reported in genomic units (GU) which is not directly equivalent to CFU
• Results are typically reported in 2–48 hours
• Is useful for negative screening
• May be preferred for evaluating whether remediation was successful

Studies Evaluating Legionella Culture Testing Versus qPCR Legionella Testing

A study by M.J. Donohue entitled “Quantification of Legionella pneumophila by qPCR and culture in tap water with different concentrations of residual disinfectants and heterotrophic bacteria” highlighted two key aspects of culture versus qPCR:

• Legionella culture testing underestimates L. pneumophila detection frequency and concentration
• Legionella quantitative PCR testing is more sensitive than culture for L. pneumophila detection

More specifically, the study noted the limitations of each Legionella testing approach:

• The Legionella culture method may not detect all live L. pneumophila microorganisms, including those that are viable but nonculturable cells (VBNC) or amoeba encysted. The Legionella culture method often underestimates the levels of Legionella bacteria (L. pneumophila) in a water sample.
• The Legionella qPCR method can detect both alive and damaged/dead cells by detecting decaying DNA. As a result, the method can overestimate the Legionella bacteria (L. pneumophila) concentrations. The qPCR test does not differentiate between live and dead Legionella bacteria.

A study by Collins et al entitled “Evaluation of Legionella real-time PCR against traditional culture for routine and public health testing of water samples” found that the high NPV of qPCR supports its use to rapidly screen out negative samples without culture. As a result, qPCR is a valuable tool for routine and public health testing. (NPV, also called negative predictive value, is the likelihood that an individual with a negative test result is truly unaffected and/or does not have the particular gene mutation in question.)

“Real-time PCR to supplement gold-standard culture-based detection of Legionella in environmental samples,” a study by Collins et al noted that while the Legionella culture testing method remains the standard for the enumeration of environmental Legionella, it has several drawbacks including long incubation and poor sensitivity, causing delays in response times to outbreaks of Legionnaires’ disease. It concluded that qPCR can be used as a supplementary technique to screen out negative samples and allow more rapid indication of positive samples, that is, in a timely manner not possible with culture.

Which is Best? Legionella Culture Testing Versus qPCR Legionella Testing

As the above information suggests, both the Legionella culture and qPCR tests are useful in consideration of their limitations and advantages. Which is appropriate for your Legionella testing? Contact an independent Legionella testing expert at Legionella Control Systems. Our team will help you evaluate your situation, facility, Legionella water quality monitoring, and timing needs, and recommend the best Legionella testing option that works for your budget.