Legionella analysis

Real Time PCR detection of Legionella

Introduction

BioVisible has developed and used an advanced DNA-testkit for the detection of Legionella bacteria for the past 8 years. The QuickScan (qPCR) supplements and improves the shortcomings of the current NEN6265 plate count method for the detection of Legionella bacteria with regards to speed and reliability. The NEN6265 is the accredited method for legionella detection. The Dutch Ministry of VROM (public affairs) granted permission to use the QuickScan for the release of contaminated sites. The Legionella fast service is a combination of the rapid QuickScan (results in 1 day) and the NEN6265 (results in 12 days).
This service incorporates the speed and reliability of the QuickScan and the accredited NE6265 method. This document is meant to provide background information regarding the Legionella fast service as it is performed by BioVisible. It is important that the customer can choose between the different techniques and is informed about interpretation of the results and the positive benefits of our fast service.

NEN 6265

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Legionella pneumophila colony

The current method for the detection of Legionella bacteria, the NEN6265, uses the ability of Legionella bacteria to grow on specific media. Colonies appear after approximately 7 days of growth. The method processes 0,5 litres of water resulting in a lower limit of detection of 50 Colony Forming Units (CFU)/litre. Growing Legionella bacteria on plates has several disadvantages;

  • Culture methods assume that the organisms will grow and produce classical characteristics within a specified period.

  • Environmental parameters for growth of Legionella in water pipes are far from ideal. A survival strategy of Legionella is switching of energy consuming processes. Recovery of growth of the Legionella bacteria on the NEN6265 plates is often a problem. Organisms that are not culturable under the specific growth conditions imposed in the plates remain undetected. It has been reported in several scientific publications that the majority of Legionella species are not able to grow on the prescribed NEN6265 media. Loss of culturability may not guarantee loss of pathogenicity. If pathogenicity persists, pathogens in the dormant 'viable but non culturable (VBNC)' state may, in fact, pose a severe threat.

  • Amoebas are often present in the water system. Legionella propagates in amoeba and a single amoeba can contain up-to 300 Legionella bacteria. This can case a severe under estimation of the incidence of Legionella in the water system.

  • Visual inspections of NEN6265 plates after 7 days (first screening) is a human process and therefore probe to differences in interpretation and human error. Also, overgrowth of the plates by other organisms (bacteria and or moulds) can severely obstruct or completely prohibit correct analysis of the plates.

  • 50 CFU/litre is the lower limit of detection of the NEN6265 method. Statistical errors and dilution deficiencies can influence this lower limit of detection.

Suffice it to say that the NEN6265 method harbours several important shortcomings. Clearly this is illustrated by the enormous deviation in results from (internationally) performed proficiency testing schemes in which different laboratories perform Legionella analysis on identical samples. Values, in identical samples, range from less than 50 CFU/litre to 100,000 CFU/litre.

QuickScan

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Real time PCR plot different concentrations Legionella.

This real time PCR method is not dependent on the physiological state of the organism. After isolation of the bacteria from the water sample the DNA (genetic material of the bacteria) is extracted. This DNA is subsequently amplified using the Polymerase Chain reaction (PCR). PCR enables a logarithmic increase of the target Legionella DNA. If the reaction is started with the DNA of 1 Legionella bacterium within 20 min more than 1 million copies of this target DNA are produced. The amplification process is monitored (in real time) by the addition of a Legionella specific fluorescent probe. If the number of Legionella bacteria in the sample was high a quick increase of the fluorescence is observed.

The Legionella QuickScan is validated with optimally growing Legionella bacteria. The validation consists of the quantification of the number of Legionella bacteria in a water sample using the NEN6265 (expressed in CFU/litre) compared to the quantification of the number of Legionella bacteria, in the same sample, using the QuickScan (expressed in Legionella DNA Units, LDU). Under laboratory conditions the number of CFU/litre is identical to the number of LDU/litre. The QuickScan has several important advantages compared to the NEN6265 method:

  • Results are available within 3-4 hours instead of 12-14 days. The QuickScan is reliable, accurate and not subject to interpretation.

  • The test is more sensitive 10 LDU/litre.

  • The results are not dependent on the physiological state of the Legionella bacteria. Also bacteria, which are present inside amoebas, are correctly identified and enumerated.

  • Less organic waste is produced therefore the test is less damaging to the environment.

  • The QuickScan can be used on heavily contaminated water, which can not be processed using the NEN6265.

  • Large scale high through-put analysis is possible


Practice

In practice it is possible that in water samples the NEN6265 does not detect the presence of Legionella bacteria but the QuickScan is clearly positive. This can be caused by a number of reasons;
* The Legionella are not able to grow on the provided NEN6265 media. * The bacteria are in the VNBC state (viable but not culturable). * Amoebas are present containing large numbers of Legionella bacteria, which are not detected using the NEN6265.

BioVisible has performed a large number (>2000) of analysis on samples from water supplies. The NEN6265 was compared with the Legionella QuickScan. In all cases that the NEN6265 was positive the QuickScan was also positive. The QuickScan detects the presence of viable and not viable Legionella.
Live/Dead
A common question is if the QuickScan can discriminate between live and dead Legionella. With the QuickScan only the live non-compromised bacteria are detected. When a bacterium dies the cell wall will be degraded and the DNA is liberated from the organism. Once the DNA is liberated from the organism the QuickScan test can not detect this DNA anymore. Release of the DNA from dead bacteria is dependent on environmental conditions; in general it is assumed that it takes a few days. A positive result from the QuickScan therefore indicates that live Legionella bacteria were present in the water supply in the past few days; the conditions were favorable for Legionella growth. The water system should be cleaned. Experiments have clearly shown that after treatment of the water supply with chlorine or heat treatment no legionella could be detected with the QuickScan.

Interpretation of the Legionella QuickScan test results

From the exhaustive validation BioVisible has done comparing the NEN6265 with the QuickScan it was evident that in all cases the Legionella QuickScan detects equal or higher amounts of Legionella bacteria in comparison with the NEN6265 plate method (LDU vs. CFU). The QuickScan reports the presence of the Legionella bacteria in the correct amounts.
The table below provides the interpretation of the QuickScan results:

table

Green = Suspicious Both methods did not detected Legionella or had detected a lower amount of Legionella bacteria. There is a good reason to assume that the water supply was clear at time of sampling. (Therefore there is no guarantee of remaining absence of Legionella bacteria.)

Yellow = Suspicious Both methods showed a light increment in the amount of Legionella bacteria. The amount of Legionella bacteria in the water supply circumstance is a risk for much greater infection. It’s necessary to flush the water supply.

Red = Contaminated Alarming: Legionella bacteria are clearly present. Cleaning the water supply is obligatory. Direct and frequent measurements of the water supply give information about the results of the cleaning procedure. By using the QuickScan as a monitoring tool it is easy to determine the exact location and the amount of bacterial infection. The QuickScan makes it possible to see the effects of cleaning within 4 hours. Dutch ministry of VROM (Ministry for Housing, Regional Development and the Environment) has accepted this method to release installations after they were cleared.



The Legionella Fast Service is only available for the Netherlands.

Contact

For questions, please contact Mr. J. de Vogel: j.vogel@biovisible.com