Effect of disinfection on detection of indicator organisms with RT-PCR - Executive Summary - Use of disinfection methods can occasionally lead to differences between culture and RT-PCR results in the detection of indicator organisms

The recently approved RT-PCR method in certain situations detects faecal indicators that are not detected by the regular culture methods, particularly when disinfection steps are used in the treatment process. Measurements in practice and laboratory experiments demonstrate that this detection with RT-PCR of RNA of inactivated organisms can occur in cases where UV or low concentrations of Cl2 or ClO2 are used. It is not expected that detection of indicator organisms will also occur following the use of higher concentrations of Cl2, for instance in the case that pipes are flushed after emergencies, and certainly not in the case of longer contact times, as laid down in the Drinking Water Hygiene Code. RT-PCR can be effectively used in all situations to demonstrate the absence of indicator organisms.

Interest: insight into the occurrence of differences between culture and RT-PCR for correct assessment – As an indicator of the presence of a faecal
contamination in drinking water, E. coli and enterococci are detected by means of a culture. Drinking water utilities and laboratories have invested a great deal in the development, implementation and validation of alternative methods to determine the presence of faecal contaminants more quickly. The use of RT-PCR makes it possible to demonstrate the presence of RNA of E. coli and enterococci selectively and quickly: with RT-PCR the analysis result is available within 4-6 hours. This makes rapid reaction possible following emergencies, or in the case of contaminations after work activities on the pipe network, and thus limits the health hazards for the customer. After the validation of the RT-PCR method, the Human Environment and Transport Inspectorate (ILT) granted temporary approval for the use of RT-PCR as an alternative method for the detection of E. coli.

During the application of the RT-PCR method in practice at the Evides drinking water utility, E. coli and enterococci were detected in a portion of the samples, while the regular culture methods in these samples did not detect any indicator organisms. It is possible that RNA of E. coli and enterococci is still detected after the indicator organisms have already been inactivated by treatment disinfection steps – such as UV and chlorine dioxide. To enable a correct interpretation of the measurement results, research was conducted into the effects that disinfection steps (UV, ClO2and Cl2) have on the outcome of RT-PCR analyses.

Approach: measurements in practice and laboratory experiments – In order to understand the differences between culture and RT-PCR results:
– analyses were conducted on samples from the different treatment steps at Evides, in which UV and ClO2 are applied as disinfection steps;
– laboratory experiments were conducted on artificially contaminated water samples, which were subjected to disinfection steps under controlled conditions.

Report – This research is described in the report Effect van desinfectie op detectie van indicatororganismen met RT-PCR (BTO 2019.005).