Legionella is one of the most important opportunistic pathogens affecting human health today. It is widespread in the environment, a serious threat to public health and, over time, can have a negative impact on the economy.
This bacterium, which can cause a life-threatening form of pneumonia if left unchecked, first achieved worldwide notoriety in the 1970s when legionnaires’ disease broke out among members of the American Legion during a Philadelphia convention.
It can potentially grow and spread in man-made water systems, such as cooling towers, hotel and healthcare water networks, as well as spas, showers, hot tubs, and jacuzzis, in addition to being present in natural bodies of water like rivers, lakes, and streams.
It readily proliferates in stagnant water systems, often integrating with biofilms, in temperatures between 20 and 50°C. In some cases, wastewater treatment plants have been viewed as high-risk in causing legionnaires’ disease, but such testing is rarely undertaken, despite non-potable water systems being considered as widely neglected in this regard.
Some experts speculate that rising global temperatures due to climate change may have something to do with Legionella’s ability to thrive, often indicated by increased cases being detected in summer months.
Legionella comprises over 58 different species, with Legionella pneumophila being the species most frequently linked to outbreaks. Scientists continue to keep an eye on the health risks posed by this bacterium, especially in the light of lessons from the Covid-19 pandemic. In fact, for almost 40 years now, Legionella has dominated the testing at Latis Scientific, a SUEZ company, accounting for 50% of all samples taken.
Unfortunately, the incidence of legionellosis is still hugely underreported, with only about 10% of actual cases being reported globally. According to a study by Barskey et al. (2002), there is significant evidence to back up the claim that true legionellosis cases are rising worldwide.
Underreporting of cases is also attributed to a number of factors, including a lack of testing due to inadequate laboratory equipment in lower-income countries, early treatment of pneumonia infections, and failure to take cultures or taking them after antibiotic administration.
A stealthy, disruptive microbe
When people inhale small droplets (aerosols) containing the bacterium, and these get into their lungs, they could potentially develop infection, experiencing a variety of symptoms including cough, shortness of breath, fever, and headaches, among others. Severe cases may result in respiratory failure, kidney failure, and/or septic shock.
Inhaling Legionella, however, does not automatically result to infection and disease. It is in anthropogenic water systems (e.g. large stagnant tanks of water with lots of iron, which boosts Legionella growth) that lead to the proliferation of the bacterium to high concentrations capable of causing disease. People who are immunocompromised may be more susceptible to infection from lower concentrations compared to the general population.
A serious Legionella outbreak not only endangers people’s lives, but could also result in the loss of revenue or employment as authorities may be forced to close a facility due to an outbreak.
This is one of the reasons laws are strictly enforced in many countries that want to stop the disease’s spread. The levels of risk assessment criteria, implementation, enforcement, and sanctions, however, vary widely.
Staying cool and compliant
In the United Kingdom, the Health and Safety Executive’s (HSE) “L8 Approved Code of Practice” mandates Legionella testing and requires quarterly full risk assessments for hot and cold-water systems in businesses and healthcare facilities.
These guidelines suggest that hot water storage cylinders must store water at temperatures of 60°C or above, while hot water distribution should be at least 50°C. Additionally, cold water should be stored and distributed below 20°C. Remedial action is only necessary when Legionella levels exceed 100 colony forming units (cfu) per litre.
Meanwhile, European Union member states adhere to guidelines outlined by the European Commission, such as those set forth in the European Drinking Water Directive. In many cases, disinfection is only required when Legionella levels exceed 10,000 cfu/l. Immediate remedial action to decrease bacterial concentration is only required when levels between 1,000 and 10,000 cfu/l affect more than 20% of samples. However, this only applies for L. pneumophila and not Legionella species (L. spp.) as a whole.
In France, regulations specify two levels for L. pneumophila in healthcare settings. Medium-risk patients, such as the elderly and/or smokers, are recommended to have levels below 1,000 cfu/l, while severely immunocompromised individuals should have levels below 250 cfu/l for both Legionella spp. and L. pneumophila.
“a serious Legionella outbreak not only endangers people’s lives, but could also result in the loss of revenue or employment”
Although many of these regulations primarily target healthcare and other major industries, in Germany, enforcement also covers domestic water systems in residential homes. These protocols were carefully implemented as the world emerged from the Covid-19 pandemic.
During the UK’s Covid-19 lockdown, many buildings were temporarily closed, leading to a surge in Legionella testing upon reopening to comply with regulations. Today, routine testing is primarily driven by clients’ legal requirements in the regulated market.
US epidemiologist and legionnaires’ disease expert Dr. Janet E. Stout lamented the lack of risk assessment of legionnaires’ disease in hospitals and other buildings. “Testing for Legionella is the only direct way to know the risk, whether Legionella is present or not,” she was quoted as saying during a Pennsylvania State Senate hearing in 2021.
“…We shouldn’t limit testing to only L. pneumophila, especially in healthcare facilities, because outbreaks have been documented in those facilities,” she added.
Analytical methods on steroids
In terms of environmental quality monitoring worldwide, the prevalent analytical methods are primarily based on traditional culture following the ISO 11731:2017, considered as the gold standard.
Water samples from the suspected contamination source are cultured and observed in the lab for environmental quality monitoring, with results taking approximately 10-13 days to be finalised.
“testing for Legionella is the only direct way to know the risk, whether Legionella is present or not”
While culture-based methodologies offer numerous advantages, they also have some limitations, including the following:
- The slow growth rate of bacteria necessitates long incubation times;
- Such methods do not detect viable but non-culturable bacteria (VBNC), which may pose a risk to public health; and
- It can be challenging to isolate Legionella in samples containing high levels of other microorganisms.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) techniques have been identified as useful for detecting Legionella spp. and L. pneumophila in clinical and environmental samples, overcoming the limitations of culture-based methods. PCR offers high sensitivity, specificity, low detection limits, and rapidity, with quantitative PCR (qPCR) allowing for accurate quantification. By amplifying specific DNA regions, PCR detects and accurately confirms the presence or absence of the target microorganism in a sample, making it useful when Legionella is suspected but not detected by other methods. Compared to culture-based methods, PCR results can be obtained in just hours.
PCR testing is not typically considered the “gold standard” for Legionella testing, so clients usually require a compelling reason to use this method. For high-risk areas such as cruise ships, however, where there is a risk of Legionella infection in spas and hot tubs due to stagnant water tanks with iron, proactive PCR sampling can be useful.
Like culture, PCR has valuable technical advantages, but also has limitations. Two major limitations include the potential presence of PCR inhibitors, which can prevent the detection of Legionella in the sample, and the inability of PCR to differentiate between live and dead cells as DNA can persist for long periods after cell death.
From sample to solution
At Latis Scientific, the culture method accounts for over 99% of Legionella sample testing, with 15,000 to 20,000 samples processed monthly. Upon request, the lab can perform qPCR for faster results, but culture testing may still be required for regulatory compliance.
After a client requests testing, samples are transported to Latis Scientific within 24 hours and kept at 6°C to 20°C. The registration team assigns a sample number and tests on the Laboratory Information Management System (LIMS). Results are input, checked, and reported to the client.
Latis Scientific’s digitised logistics system, Samplefy, speeds up the process by allocating information before the lab receives the sample. In addition to their testing services, Latis Scientific offers expert consultancy drawing on 40 years of experience in managing and remediating risk from Legionella and other bacteria across various settings.
The scientific community pushes the boundaries of innovation to overcome the restrictions and challenges of PCR and culture-based methodologies.
Numerous alternative methods for Legionella testing are being proposed but validation of technical performance through specific protocols like those outlined in ISO 13843:2017 is necessary. Moreover, if these methods are intended for regulatory purposes, they must be accepted as an alternative to the ‘gold standard’ by the relevant regulatory body.
Breaking the mold with culture-based alternatives
Most Probable Number (MPN) is a culture-based alternative method that focuses on L. pneumophila and provides results in seven days instead of 10-13 days. It is user-friendly, with inexpensive and non-specialised equipment, and can be processed and interpreted by those with minimal technical experience, potentially eliminating the need to send samples to a laboratory.
Amoeba co-culture, which utilises Acanthamoeba spp., increases culturability of bacteria (i.e. this may give positives where bacteria living inside other organisms fail to grow with standard culture), but may only provide qualitative results.
Unlocking legionella via molecular-based technologies
Digital Droplet PCR (ddPCR) is one of the easiest ways to run rapid and accurate quantification of genomic targets with minimal technical expertise and fewer reagents. It enables absolute target DNA quantification and is more costly than standard thermocyclers used in qPCR.
Lateral flow testing is designed for detecting Legionella from environmental samples, including other L. pneumophila serogroups and other Legionella spp. While rapid and user-friendly, its detection limits have been considered too high to detect low concentrations. Recent studies have improved its performance by combining it with additional sample concentration steps.
Fluorescence in situ hybridisation (FISH) is another molecular method based on the use of fluorescent genomic probes that bind to specific genomic regions to detect any species of Legionella or just L. pneumophila.
New kids on the ‘emerging technologies’ block
Flow cytometry (FC), combined with immunomagnetic separation, can be useful in environmental testing due to its increased recovery compared to standard culture. Although FC allows for real-time detection and quantification of bacteria, it is typically used to detect all viable bacteria, not just Legionella, though Legionella-specific systems are available in the market.
Next-generation sequencing (NGS) is a promising molecular method used to determine bacterial diversity and abundance, including Legionella, and trace the source of infections. However, data interpretation requires a considerable learning curve.
Loop-mediated isothermal amplification (LAMP) is a low-cost alternative to PCR that can be performed at a constant temperature without the need for expensive equipment. However, it shares some limitations with other PCR methods.
Microcolony enumeration (MICA) is a culture-based method that relies on detecting microscopic colonies before they are visible, offering potential advantages in reducing errors, resources, and costs while improving efficiency. However, it suffers from the same limitations as culture-based methods, including low recovery rates and the inability to detect non-culturable cells that can still cause disease.
Viability PCR uses chemicals that bind with the DNA of dead cells and free DNA, preventing the PCR reaction. This method detects dead cells without potential health risks, but may still quantify bacteria killed with methods that leave the cell intact, such as UV treatment.
The methods that Latis Scientific routinely offers are typically driven by regulations and guidelines, including culture and qPCR, which are accredited under ISO 17025:2017 to ensure quality and technical competence. It also has an experienced and knowledgeable innovation team that develops, tests, and validates new Legionella methodologies such as MALDI-TOF, amoeba co-culture, and flow cytometry.
The ideal Legionella test should have good technical specifications, be affordable, fast, easy to perform with minimal expertise, and easy to interpret. The scientific community aims to develop new technologies that move testing into the field, reducing transport and waiting times. In the long term, sensor technologies that provide real-time information via an integrated digital system would be beneficial. Analytical services are undergoing a metamorphosis, evolving from a descriptive approach to a more impactful and predictive one. The digital transformation plays a pivotal role in this process. Integrating new and disruptive analytical systems with digital tools such as artificial intelligence can significantly reduce health risks from pathogens like Legionella and prevent outbreaks. This transformative approach has the power to revolutionise public health and pave the way for a safer future.
Barskey, A.E.; Derado, G.; Edens, C.; ‘Rising Incidence of Legionnaires’ Disease and Associated Epidemiologic Patterns, United States, 1992-2018’. Emerging Infectious Diseases. 2022;28(3), pp. 527-38.
For over 160 years, SUEZ has been delivering essential services that protect and improve people’s quality of life by providing water and waste services with innovative solutions. With 40,000 employees in 40 countries, SUEZ helps its customers create value over the entire lifecycle of their assets and services, and drive ecological transition.
Latis Scientific, a part of the SUEZ Group, is a top provider of laboratory services for various industries, including water treatment and water hygiene consultants, healthcare providers, medical device manufacturers, local authorities, and industrial water consultants. With a modern laboratory in London, expert staff, and a refrigerated logistics network, Latis Scientific specializes in high-level technical expertise and consultancy while maintaining the quality of the science performed.
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