As featured in waterline Winter 2018-19
Rapid Microbiology Industry Liaison Group
by Elise Maynard
The Rapid Microbiology Industry Liaison Group (RMILG) is a sub-committee of the Water Management Society (WMSoc) Technical Committee. This group was formed in order to answer questions that had been asked by WMSoc members about certain new technologies but also to assist organisations such as the United Kingdom Accreditation Service (UKAS) and the Health and Safety Executive (HSE) by providing evidence to assist with future technical guidance updates.
The independent group members represent HSE, Public Health England (PHE), WMSoc and the Legionella Control Association (LCA). Their brief was to consider the strengths and weaknesses of a variety of new methodologies and to review various data from both manufacturers and independent sources. It was important to increase the awareness of techniques via the provision of technical publications in “Waterline”. A series of well-attended conferences were also held in order to communicate ongoing findings and to gauge the gaps in knowledge which needed to be addressed.
The major outcome has been the creation of a series Factsheets to guide users, which are now freely available via the WMSoc website and can be found here:
The HSE plan to link to these from their website, in the same way they already do for the Dry/Wet Cooling System Guidance document).
These factsheets cover technologies such as Polymerase chain reaction (PCR), immunomagnetic separation (IMS), most probable number (MPN) and Matrix-Assisted Laser Desorption/Ionisation Time of Flight (MALDIToF). Many of these techniques have been used for clinical sample testing for a number of years and are now being adapted and validated for environmental samples.
Culture techniques – still the Gold Standard?
There are a number of pros and cons regarding culture methods and these are referred to in many of the guidance documents. ISO 11731:2017 “Water quality — Enumeration of Legionella” specifies culture methods for the isolation and estimation of Legionella in all kinds of water samples including potable, industrial, waste and natural waters. The method can also be used for biofilms, sediments, etc. The document, however, is clear that not all Legionella species (spp) are culturable and therefore the methods described do not recover all species of Legionella. Viable But Non-Culturable (VBNC) bacteria have very low metabolic activity but are still alive and are able to become culturable once resuscitated. This could be via amoeba from within biofilms in water, or if inhaled via droplets, they can infect the lungs, allowing Legionella to WMSoc Factsheets Rapid Microbiology Industry Liaison Group recover and cause serious illness (Legionellosis). The new ISO standard refers to a matrix of testing procedures for the testing laboratory to choose from and this could differ from laboratory to laboratory, which makes comparison even more difficult. The positive side of culture is that when the organism does grow, it means that it can be used to profile and check against other isolates, either environmental or clinical. Cultures can also be stored for future checks – perhaps in outbreak scenarios.
ISO 11731:2017 typically requires a concentration step prior to culture and there are various options, such as centrifugation or filtration. This requires 500 – 1000 mL (1 L) samples to obtain a good Limit of Detection (LOD) i.e. the lowest quantity of bacteria that can be distinguished from the absence of that bacteria (a blank value) with a stated confidence level (generally 99%) and generally the larger the volume tested, the more sensitive the test. Laboratories should provide the limit of detection on the test report.
Matrix Assisted Laser Desorption Ionisation Time of Flight (MALDI ToF) depends on growing a culture prior to identification but can significantly reduce the length of time that it requires to get a confirmation of a particular species. Legionella, for example, can be identified very shortly after colonies appear on the plate and this can reduce the confirmation time from 10 days to around 5 days in some cases. The technique is highly specific and is available in many laboratories for both clinical and environmental samples.
Immuno-Magnetic Separation (IMS) requires the sample to be prepared according to the concentration step from ISO 11731:2017. There is an environmental test kit available for the detection of Legionella spp. which attaches microscopic magnets to live bacteria in order to enable detection. The result can be crudely measured by a colour change, or more accurately with the use of a photometer. This method can be advantageous in field laboratories which do not have the facilities to handle the growth and disposal of hazardous micro-organisms.
One Most Probable Number (MPN) technique for the detection of Pseudomonas aeruginosa has been accepted by the UK Standing Committee of Analysts (SCA) for the analysis of drinking water and is specified as a recommended method in the committee’s publication, The Microbiology of Drinking Waters Part 8 – Methods for the isolation and enumeration of Aeromonas and Pseudomonas aeruginosa (MoDW). MPN techniques are widely used within the municipal water plants for the detection of E.coli in drinking water. MPN technology has recently been accepted as ISO 16266-2:2018 Water quality — Detection and enumeration of Pseudomonas aeruginosa — Part 2: Most probable number method. Advantages of this technique are that there are no concentration steps, it utilises a liquid growth medium so detects live cells and encourages some VBNC to grow. The method can be quantitative and results presented as CFU. There is very little equipment required so the tests can easily be field based and results can be obtained in approximately 24 hr. A similar technique is now available for the detection of Legionella pneumophila, with results within 7 days.
Real time Polymerase Chain Reaction (rtPCR), also known as quantitative (q)PCR, is capable of detecting a wide range of organisms and in water microbiology it tends to be used for detection of L. pneumophila serogroup 1 & Legionella spp. Different manufacturers have different options and test kits available. PCR requires a concentration and an extraction step to access the cellular DNA i.e. the genetic “fingerprint”. The method depends on creating multiple replicates of the DNA until sufficient is available to detect and should be conducted according to ISO 12869:2012. PCR has had mixed reviews in the past due to the fact that the early tests tended to pick up both live and dead cells, but this is not necessarily the case now.
HSE advice is published on the HSE website www.hse.gov.uk/legionnaires/faqs.htm which is based on a trial performed in conjunction with PHE. HSG 274 and HSG 282 recommend routine Legionella sampling is undertaken as part of the checks on the effectiveness of control regimes in cooling towers, evaporative condensers and spa pools, as well as in some hot and cold water systems and other risk systems. Although they currently note that analytical culture by UKAS-accredited laboratories remains the gold standard method for the detection of Legionella in environmental samples, they do recognise that Legionella culture methods do have certain acknowledged disadvantages including:
- Long incubation period
- Poor reproducibility
- Poor sensitivity
- Inability to detect VBNC
- Inhibition due to other competing organisms
There are currently three key areas where HSE recognises the benefits and one key limitation of the use of the qPCR assay as an alternative to the traditional Legionella culture-based methods:
Rapid detection of Legionella bacteria – the high negative predictive value (NPV) of qPCR means that it is suitable for use as a negative screening tool to rapidly rule out potential sources, for example, in an outbreak situation, and to support public health investigators in prioritising resources.
Indication of the effectiveness of cleaning and disinfection – the high NPV of qPCR, means that negative qPCR results may be a useful indicator for the restarting of system implicated in the source of an outbreak following remedial actions, such as cleaning and disinfection.
Complementary tool for the rapid routine monitoring of Legionella trends at dutyholder sites – it is important that data from such tests can be properly interpreted, to enable informed decisions on the effectiveness of control measures and these should be made by a competent person. It is important to note that the units of measurement for qPCR (genomic units/L) are not directly comparable with the action and alert levels for culture (CFU) published in HSG 274.
HSG 274 part 2 advises that Legionella testing should be performed in UKAS accredited labs with current ISO Legionella standard methods within their scope of accreditation. In addition, the labs should take part in appropriate proficiency testing schemes (accredited to ISO/ IEC 17043:2010). This standard defines the competence of providers and the development and operation of proficiency testing schemes. HSG 274 part 2 does, however, recognise that alternative quantitative testing methods may be used as long as they have been validated using ISO 17994:2014 and meet the required sensitivity & specificity.
- Sensitivity – also called the true positive rate or probability of detection) measures proportion of positives that are correctly identified as such.
- Specificity – (also called the true negative rate) measures proportion of negatives that are correctly identified as such.
ISO 17994:2014 specifies an evaluation procedure for comparing two methods with established performance characteristics according to ISO/TR 13843 and intended for the quantification of the same target group or species of microorganisms.
HTM 04-01 Guidance HTM 04-01 (Safe water in healthcare premises) part B notes that all microbiological measurements should be:
- By approved methods and/or
- Performed by UKAS accredited labs for the method being used
Why and Where to Test?
It is important to have a good sampling plan in place, based on risk. This could be due to the requirements of the Water Safety Management Plan, compliance, trend analysis and /or routine monitoring. Outbreaks will need an escalation plan and culture should be part of this as isolates may be required. Where to test may depend on compliance requirements, for example, access to some industrial sites is highly controlled and testing is easier performed in-house. Equipment outlay may also be another factor. Sampling is a critical factor and personnel undertaking this task should be appropriated trained and competency checked.
Appropriate personnel training and records of such activities will be required if the data is to be used for accurate monitoring and also for compliance. Knowledge of sampling standards such as ISO 5667 and ISO 19458 are advantageous for the creation of sampling plans. BS 6068, BS 7592 and BS 8554 will assist in ensuring that accurate samples are taken. Aseptic technique will also be required for both sampling and testing, especially within healthcare premises.
Test standards include ISO 17025, ISO 11731, ISO 16266-2, ISO 17994 and ISO 17043 and a working knowledge of which ones apply will be required. Positive and negative controls should be used whether testing in a laboratory or in a field site, as well as keeping good records such as operator details, timings, product batch numbers, use by dates and manufacturer quality control data.
There are a variety of training methods available from both the manufacturers and from independent training bodies, with associated accreditation or CPD. WMSoc has created a specific course (W038) based on ISO 16266-2:2018 Water quality — Detection and enumeration of Pseudomonas aeruginosa — Part 2: Most probable number method, which also covers the various sampling standards: ISO 5667, ISO 19458, BS 6068, BS 7592 and BS 8554 and which is City and Guilds accredited.
Next steps for the RMILG are to recruit new group members, a number of which have already been identified and to create a comparative matrix between the technologies that have already been appraised. Factsheets for new technologies will also be created as manufactures engage and provide the relevant information. The group constantly liaise and inform the regulators and it is anticipated that the information contained in the factsheets will be used to inform future policy and guidance. Waterline and the Waterline website will be used to communicate any progress to our members.