Legionella Under Control: From Culture to qPCR

Legionella detection improvements have remained a key focus, alongside the need to optimize the management of water resources. In response to these challenges, on July 2, 2024, Spain published Royal Decree 614/2024, which amends Royal Decree 487/2022, the Spanish regulation establishing health requirements for the prevention and control of legionellosis.

Why was a new Royal Decree necessary?

Legionella surveillance continues to be a challenge and an important public health issue at European level, due to the continued increase in reported cases. According to the latest annual report published by the ECDC, based on 2024 data, there was an increase of 3.4 cases per 100,000 inhabitants compared with the previous year, as well as 32 outbreaks in 8 European countries. France, Germany, Italy and Spain accounted for 71% of all reported cases.

The new Spanish Royal Decree provides further detail on certain definitions that did not previously cover specific situations or scenarios, such as the responsibilities of the person or entity responsible for an installation. It also specifies requirements concerning sampling procedures, terminal points, and technical requirements for certain priority and susceptible installations.

These amendments affect both the Legionella Prevention and Control Plan (PPCL) and the Legionella Health Plan (PSL), two elements of Spain's national framework for Legionella prevention and control. As a result, these plans will need to be reviewed and updated accordingly.

Has anything changed regarding microbiological control?

The analysis of Legionella using the culture method in accordance with ISO 11731 has not been modified by the Spanish regulatory update. However, some aspects of the analytical process have changed, including sample collection.

Under the previous Spanish regulation, sampling could only be carried out under the responsibility of, or by, the laboratory responsible for performing the analysis. The updated legislation establishes that this activity must be carried out by entities accredited according to ISO 17025, with a deadline of January 1, 2030 to obtain the required accreditation.

Legionella analysis using culture media

Detection is performed according to ISO 11731, an international standard for the detection and enumeration of Legionella. Depending on the nature of the sample, certain steps may be required before isolation:

• Concentration (filtration or centrifugation)
• Dilution
• Pre-treatment (thermal or acid treatment)
• Direct plating

Regarding filtration, the ISO standard recommends using a PES membrane for elution or an MCE membrane when the membrane is placed directly onto the agar plate.

Several culture media can be used, with BCYE agar and GVPC agar being the main ones.

Although culture remains the primary analytical method under the Spanish framework, the procedure is lengthy and complex. In addition, its sensitivity is lower for species other than L. pneumophila, the main species responsible for human infections.

To address these limitations, particularly in urgent situations, molecular techniques can provide valuable complementary information. The updated Spanish regulation references ISO 16140-2 for the validation of alternative methods, as real-time quantitative PCR (qPCR) has become increasingly established as a rapid and reliable tool for the detection and quantification of Legionella in water samples.

A complementary approach: qPCR and culture

Rather than viewing qPCR and culture as independent techniques, it currently makes sense to consider them as complementary tools.

qPCR enables the rapid screening of a large number of samples and provides information within a few hours about the presence and concentration of Legionella genetic material. This can be particularly valuable in situations where rapid decisions are required, such as when an outbreak is suspected or during an epidemiological investigation.

However, qPCR detects DNA and, therefore, a positive result alone cannot determine whether the detected bacteria are viable. In this context, culture remains essential in certain situations, particularly when it is necessary to confirm the presence of viable microorganisms, recover isolates, and subsequently identify and characterize them microbiologically.

For this reason, in the event of an outbreak, a particularly useful strategy may be to use qPCR as an initial screening tool, while reserving culture for the confirmation and characterization of positive results or for samples that, due to their characteristics, require a more comprehensive microbiological investigation.

This rapid approach makes it possible to obtain information within a few hours, enabling much faster decision-making. It also offers:

• High sensitivity and specificity
• Detection and quantification of Legionella, even in complex samples
• Greater speed and simplicity

At Condalab, our Condagene^® range includes detection kits that allow you to detect and quantify:

• Legionella spp.
• Legionella pneumophila
• Legionella spp. & Legionella pneumophila

In addition to our detection kits, we offer extraction kits specifically designed for water samples, as well as the equipment required to perform qPCR in your laboratory.

What does the future hold for qPCR in Legionella detection?

Molecular techniques are expected to play an increasingly important role in Legionella surveillance and control. Their speed, ability to process large numbers of samples, and capacity to provide quantitative results make them highly valuable tools, particularly in situations that may pose a public health risk.

In this context, ISO 12869, which establishes requirements for the detection and quantification of Legionella spp. and L. pneumophila using PCR-based methods in water samples, provides an international methodological framework that contributes to the standardization and application of these techniques.

Nevertheless, it is unlikely that qPCR will completely replace culture. The ability of culture to demonstrate the presence of viable bacteria and, above all, to obtain isolates that can subsequently be characterized remains essential in certain situations.

The most likely scenario, therefore, is greater integration of both methodologies, with qPCR being used more routinely as a screening and surveillance tool, while culture is used when it is necessary to confirm microorganism viability or carry out more in-depth microbiological and epidemiological characterization.