Technical Notes

Comparison of Legionella testing methods

The gold standard for detection of Legionella is the lab culture method. The culture method is still used by most laboratories in the world. It requires selective media and prolonged incubation periods. A concentration stage is followed by decontamination with heat and acid, which may involve loss of the target bacteria. The incubation stage itself takes 10-14 days to provide results and does not identify the virulent phase of the bacterium when the bacterium is viable but non-culturable.

There are also other rapid tests on the market, most of which are based on DNA detection, and go under the title PCR (Polymerase Chain Reaction) tests. PCR tests are not on-site. They require a sample to be sent to a specialist laboratory where sophisticated equipment and highly trained staff can implement the analysis, and interpret and report the results. Although testing is rapid the time from sample collection to receiving the results is typically greater than 24 hours, because of transport of water samples to the lab.

See the Comparison table comparing the advantages and disadvantages of the most important analysis methods existing on the market.

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Is speed the most important feature of a Legionella test?

The diagnostics industry has for some time now been moving away from the laboratory and culture testing towards on-site, rapid testing. The advantages in testing on-site and in real time are clear to everyone and include: better and more frequent monitoring of cases, immediate knowledge supporting fast action, user involvement and responsibility and more efficient use of expert time. These all support more focused, flexible and individually tailored treatments. In the environmental sector they promote better public health outcomes.

For reasons best known to the UK HSE this is not yet the case in the UK Legionella sector. It is still considered better to wait 10-14 days to know you have a serious Legionella infestation, or that your corrective action has worked, or even worse, not worked.

In the case of Legionella testing speed of the test has to include transport of the sample to a lab.

The Hydrosense Legionella test is the fastest test on the market – providing a result in 25minutes. However, its most important feature is that it provides a result on-site in real time. No need for a return visit from an expert technician to carry out treatment. Hydrosense smartphone app reader and connected data portal ensure that people can communicate, see results and manage them in real time.

Detecting live and dead Legionella cells

The Hydrosense test is very good at detecting Legionella antigen. This is its greatest utility. It is also highly specific for Legionella pneumpohila serogroup 1. (LpSG1) This means that if you get a positive result then you have a problem and should take action as this is the most virulent species of the Legionella genus. Further the test detects LpSG1 in its most virulent phase, when it is viable, dangerous, but not culturable. i.e. cannot be detected by culture methods.

Some people suggests that antigen tests detect both live and dead cells. However, the test detects antigen. Antigen by itself does not persist in a managed water system. Research shows that even at very low levels of chlorine Legionella antigen is no longer detectable within minutes, confirming that a positive result is an indicator of a serious problem.

Download further information at Persistence of Legionella antigen under biocides 140213 v4.

Validation of Hydrosense Legionella test

The Hydrosense innovative method has been recognised and independently validated by both commercial and research institutions around the world including China.

KWR, the Dutch Watercycle Research Institute, carried out research comparing three techniques for the analysis of Legionella contamination of water systems.

Development of a fast and reliable monitoring system enables prompt treatment of contaminated systems. Of the methods tested, q-PCR and culture testing both require access to a lab and as such, cannot be performed on site and incur a delay. The Hydrosense test, referred to as Fastpath in the report, can be used on site by untrained operators and gives a reliable result in 25 minutes.

Culture tests for Legionella are reliant on the bacterium being in the correct phase of its lifecycle: it is not always detectable by culture, and it is not detectable by culture when in its most virulent form. In addition, culture tests can take up to two weeks to return results.

The Hydrosense test utilises lateral flow technology, as commonly used in pregnancy tests, to detect the presence of Legionella pneumophila serogroup 1 antigen in a water sample. The test may be used directly on the water sample or combined with filtration to achieve higher levels of sensitivity. The test is intended for use in the field with minimal levels of operator training, and requiring no other specialist equipment.

The KWR report states:

“given the speed and simplicity of the test, [this method] would appear to be a useful addition to the set of instruments available to the process operator responsible for the daily management of cooling water systems. The method’s specificity makes it a useful addition to ATP measurements, colony number measurements or dip slides that are already used for monitoring water systems. The method’s speed and simplicity make it exceptionally interesting for monitoring in emergencies.”

Water Temperature control and sampling

In many parts of the world water temperature is used as the primary mechanism for control of Legionella. Water should be in the range <20degC (cold water) to control growth; or >55deg C (hot water) to kill the bug. When water is in the middle range 20-55degC i.e. when mixed for use, Legionella grows quickly and risks are higher. Consequently, cold water stores the bug and can contaminate a system at any time that control is relaxed or hot and cold water streams are mixed.

The 55degC water temperature is a control measure not a testing paradigm. It does not matter what temperature you sample at. The water sample temperature will drop significantly as the water is carried to the lab. If this is done quickly the bug load won’t vary much. If it takes more than a few hours, the warmer the sample the higher the bug load result. Which is one of the reasons on site testing gives a more accurate result. The actual bug load won’t vary much over the few minutes that it takes to carry out an on-site test.

It is actually quite difficult to run a water system at a constant high temperature. If you measured the temperature at near and far points from the heat source (boiler) you would see quite large variations in temperature. This is one of the reasons tests are required, because the whole system is not receiving the same treatment.

For good control the cold water system needs to be below 20degC. If this is not the case then the cold water system is the greater source of risk. Of course when the water is mixed the temperature is in the ideal growth range 20-55degC. Mixer taps are notorious sources of bugs. The biofilm builds up and the bug load with it. Flushing with hot water helps control this, but you need to test to check control is being maintained.

Finally, Legionella grows in the biofilm and escapes into the main water system intermittently. If the temperature is above 55degC throughout the whole system you should have control because this will kill the bug. Note this in minimum temperature NOT average. Wherever the temperature dips below 55degC the bug is not controlled. The bug will be continually re-introduced from cold water and the mains supply. It will also hide in dead legs and little used facilities where the water temperature will not be maintained above 55degC simply because fresh hot water Is not regularly flushing through these sections of pipework.

The Hydrosense on-site Legionella test can be used at temperatures up to 45degC. When sampling hot water simply leave to stand for a few minutes. The bug load won’t increase significantly in the few minutes it takes to cool. If you are using filtration, which can be dome at temperatures up to 100degC, the water sample will have cooled sufficiently during the filtration process to allow you to test without any problems.

Comparative virulence of Legionella species

Legionella pneumophila is a parasite of eukaryotic cells. It has evolved to survive and replicate in a wide range of protozoan hosts and can also infect human alveolar macrophages as an opportunistic pathogen causing Legionnaires disease. Crucially for the infection process, L. pneumophila uses a specialized secretion system to translocate bacterial proteins called effectors into host cells. A recent study reported in Nature Genomics provides valuable insight into the mechanism by which Legionella species can manipulate eukaryotic cellular processes to traffic and replicate in host cells and has implication on the virulence mechanisms of different species. See the full report.

38 species of Legionella were studied using comparative genomic sequencing methods to explore the diversity of these effectors which give an indication of the virulence of each of the species. Of the 38 species studied, Legionella pneumophila was found to have the largest number of effectors and the largest number of species specific effectors, which is consistent with the status of Lp as the most virulent of the Legionella species.

Legionella pneumophila serogroup 1 is widely regarded as the most virulent of the Legionella species. Unfortunately, the study did not differentiate between the various serogroups of Legionella pneumophila so there was no insight given into the comparative virulence of the various serogroups. The possibility of this study being carried out is intriguing.

How frequently to test for Legionella?

There are 2 parts to the answer. Regulatory and risk based. Actual testing frequency will be a trade-off between risk perception and outlay, unless mandated by regulations.

Regulatory is quite simple. You need to test as often as regulations require. For example, in the UK cooling towers are required to culture test every 3 months. The legal context would be dictated by the national employment law which tends to require that a company protects its staff against known risks. Legionella regulations tend to extend this protection to the general public and it is often assumed as part of the responsibilities of a company which takes us on to risk based testing.

Risks are particular to the facility (building or enterprise) and vary by sector. Hotels and medical facilities have higher risks than office blocks, because they have a reputation to protect and/or a more susceptible clientele.

The actual physical risks from a given water system increase with complexity and age of the water system.  The bigger the water system the more likely it is to have dead legs – little used stretches of pipework – and temperature disparities. These increase the risk of Legionella growing. So the bigger and older a facility the more often testing would be recommended. Giving a general rule is quite hard and facility managers would make their decision based on their perception of the risks.

Once a system is out of conformance –  the temperature is not being kept in the control range of less than 20degC and higher than 50deg C – then Legionella will grow very quickly and the risks will increase exponentially. While quarterly or annual testing would be fine for a well-run facility, monthly testing might be better for older water systems. Time of year does have a bearing on Legionella growth to the effect that testing is less necessary in the winter months.

Once a system needs to be brought back into conformance more frequent testing is recommended. Daily followed by weekly testing should be conducted until control can be shown to be re-established. The Hydrosense test provides proof of re-establishment of control, which could be handy in a court case.

Quantification of Legionella bacteria cells using the Hydrosense test

Traditionally Legionella quantification is measured using cfu/liter – colony forming units per liter. This is measured by culturing a sample on specialist agar plates and counting how many colonies are formed –hence the term. This method does not measure Viable, But Non- Culturable cells (VBNC). In the case of Legionella this is a serious shortcoming as the VBNC phase of Legionella is the virulent disease causing phase of the bacteria, and it is not even included in the basic method. Further it is difficult to exclude other species of bacteria from growing on the agar plates, so false positives are an issue with culture testing.

The Hydrosense test measures an antigen response, it does not measure colony forming units. There should be a direct correlation between antigen quantity and number of cells – note the number of cells will be higher than colony forming units, because the VBNC cells are included. Our lab studies show a direct relationship i.e. higher cell quantities show a stronger antigen response as indicated by a stronger test line. More precisely the Hydrosense test is also measuring VBNC cells, which is an improvement on the lab test.

The App test reader provides a way of measuring the concentration of antigen and consequently indicating the quantity of bacteria. A negative Hydrosense test result indicates that the Legionella concentration is less than the limit of detection. A Hydrosense test line result of 1 (Hazard Index 1) is the weakest recordable result and indicates cell count equivalent to the Limit of Detection (LOD) of the test type used. That is a Hazard index 1 would be equivalent to 100 CFU/liter if the filtration step is used, or 100,000CFU/litre if filtration concentration step is not used. Higher hazard indices indicate higher quantities of Legionella cells. The correlation is designed to be very closely linear and holds up to an HI of 10. Which would indicate a concentration of 10 times the LOD. Results higher than this are returned as Hazard Index 10. The response tends to HI 10 and so all concentrations higher than 10 times the LOD will return an HI of 10.