Final Report Summary - VIROBATHE (Methods for the concentration and detection of adenoviruses and noroviruses in European bathing waters with reference to the revision of the bathing water ...)
The ultimate aim of the VIROBATHE project was to provide a reliable method for the analysis of the European Union's recreational waters in order to detect noroviruses and adenoviruses. Therefore, the objectives of the project were to:
- compare methods for norovirus and adenovirus detection in recreational waters;
- derive a combined concentration and detection technique to provide a reproducible system of testing recreational waters for the target viruses;
- provide evidence-based support for norovirus and adenovirus testing of environmental samples in respect of their role as the appropriate viral indicator of faecal pollution;
- prepare the technology for new Member States as part of the development of their environmental and social programmes;
- share technology between laboratories to achieve wider competence in the virological analysis of environmental materials.
The project was performed in two phases. Phase 1 was the comparative evaluation of detection and concentration techniques to derive a method suitable for routine environmental monitoring and Phase 2 was the surveillance of recreational water sites using the combined method.
Phase 1 focused on the development of a combined concentration-detection procedure for the two target viruses specified by the Commission (adenoviruses and noroviruses). The Commission specified that a rapid test was developed and this implied the use of the polymerase chain reaction (PCR) technique, which detects the specific genetic material of the target viruses. A test was also developed which would detect if any adenoviruses found were infectious. Quantitative detection by quantitative real-time (RT-PCR) was also developed for adenoviruses and RT-PCR for noroviruses was also included. Concerning the concentration techniques, three methods were evaluated:
- concentration by adsorption of virus to negatively charged membranes and elution with skimmed milk solution;
- concentration by adsorption to negatively charged membranes and elution with beef extract solution;
- concentration by adsorption to glass wool and elution with beef extract solution.
From the comparison of the concentration methods it was apparent that there were few differences between the methods tested when evaluated for recovery of virus, whether detected by infectivity assay or molecular means. However, on the basis of statistical analysis of the data for fresh water matrices a concentration process of adsorption by membrane filtration followed by elution with skimmed milk solution gave the best recovery. For salt water there was even less difference between the methods tested but that adsorption by membrane filtration followed by elution with beef extract solution was marginally better.
The first activity of phase 2 was to train all scientists and technical staff in the combined concentration/detection procedure. This was accomplished in a four-day training workshop. The surveillance phase was done over five months and included the bathing season of 2006. Recreational waters were chosen to reflect current recreational activities, including boating, swimming, surfing and potential pollution. Samples were taken and target viruses were widely present. Some of the samples contained infectious adenoviruses as detected by the integrated cell culture (ICC) PCR method. Furthermore, adenoviruses were found to be statistically associated with human faecal pollution in fresh water. Moreover, analysis by category and parametric statistics showed that there were significant associations between adenovirus occurrence and FIO levels in fresh waters and that it may be possible to link the virus occurrence to enterococci levels and thus to a parameter recognised as a measure of health risk. Sample numbers were too low to be certain of such associations for marine waters.
Further results of the project were the following:
- noroviruses were found to be not a practical target for surveillance as they were not sufficiently abundant nor was the RT-PCR robust;
- further work was needed to optimise a method for marine waters;
- the RT-PCR for noroviruses GII worked well, however a means of monitoring process and assay efficiency should be investigated;
- further work on nucleic acid extraction was needed to optimise recovery and further work was needed to reduce inhibition of the PCR by components, especially in fresh water;
- human adenoviruses were common, the serotypes of virus varied, but adenovirus 41 was common, and norovirus GGII was detected more often than GGI.
The evidence from VIROBATHE provided a springboard for further work to link virus occurrence with health effects of recreational water use, either through correlation with enterococci or by epidemiological studies based on projects such as EPIBATHE. VIROBATHE showed that the idea of using viruses as an indicator of pollution and as a valid parameter in a Bathing Water Directive moved from the 'conceptual' at the beginning of the project, where the methods were shown to work in the laboratory, to the 'feasible' stage, where structured field studies furnished evidence that viruses can be detected in surveillance programmes.
The next stage would be to move from 'feasible' to 'operational', where quantitative determinations of target viruses (probably adenoviruses) would be reconciled with levels of known faecal indicators (probably enterococci) so that meaningful discussions on the formulation of a viral parameter based on sound scientific data could be held.
- compare methods for norovirus and adenovirus detection in recreational waters;
- derive a combined concentration and detection technique to provide a reproducible system of testing recreational waters for the target viruses;
- provide evidence-based support for norovirus and adenovirus testing of environmental samples in respect of their role as the appropriate viral indicator of faecal pollution;
- prepare the technology for new Member States as part of the development of their environmental and social programmes;
- share technology between laboratories to achieve wider competence in the virological analysis of environmental materials.
The project was performed in two phases. Phase 1 was the comparative evaluation of detection and concentration techniques to derive a method suitable for routine environmental monitoring and Phase 2 was the surveillance of recreational water sites using the combined method.
Phase 1 focused on the development of a combined concentration-detection procedure for the two target viruses specified by the Commission (adenoviruses and noroviruses). The Commission specified that a rapid test was developed and this implied the use of the polymerase chain reaction (PCR) technique, which detects the specific genetic material of the target viruses. A test was also developed which would detect if any adenoviruses found were infectious. Quantitative detection by quantitative real-time (RT-PCR) was also developed for adenoviruses and RT-PCR for noroviruses was also included. Concerning the concentration techniques, three methods were evaluated:
- concentration by adsorption of virus to negatively charged membranes and elution with skimmed milk solution;
- concentration by adsorption to negatively charged membranes and elution with beef extract solution;
- concentration by adsorption to glass wool and elution with beef extract solution.
From the comparison of the concentration methods it was apparent that there were few differences between the methods tested when evaluated for recovery of virus, whether detected by infectivity assay or molecular means. However, on the basis of statistical analysis of the data for fresh water matrices a concentration process of adsorption by membrane filtration followed by elution with skimmed milk solution gave the best recovery. For salt water there was even less difference between the methods tested but that adsorption by membrane filtration followed by elution with beef extract solution was marginally better.
The first activity of phase 2 was to train all scientists and technical staff in the combined concentration/detection procedure. This was accomplished in a four-day training workshop. The surveillance phase was done over five months and included the bathing season of 2006. Recreational waters were chosen to reflect current recreational activities, including boating, swimming, surfing and potential pollution. Samples were taken and target viruses were widely present. Some of the samples contained infectious adenoviruses as detected by the integrated cell culture (ICC) PCR method. Furthermore, adenoviruses were found to be statistically associated with human faecal pollution in fresh water. Moreover, analysis by category and parametric statistics showed that there were significant associations between adenovirus occurrence and FIO levels in fresh waters and that it may be possible to link the virus occurrence to enterococci levels and thus to a parameter recognised as a measure of health risk. Sample numbers were too low to be certain of such associations for marine waters.
Further results of the project were the following:
- noroviruses were found to be not a practical target for surveillance as they were not sufficiently abundant nor was the RT-PCR robust;
- further work was needed to optimise a method for marine waters;
- the RT-PCR for noroviruses GII worked well, however a means of monitoring process and assay efficiency should be investigated;
- further work on nucleic acid extraction was needed to optimise recovery and further work was needed to reduce inhibition of the PCR by components, especially in fresh water;
- human adenoviruses were common, the serotypes of virus varied, but adenovirus 41 was common, and norovirus GGII was detected more often than GGI.
The evidence from VIROBATHE provided a springboard for further work to link virus occurrence with health effects of recreational water use, either through correlation with enterococci or by epidemiological studies based on projects such as EPIBATHE. VIROBATHE showed that the idea of using viruses as an indicator of pollution and as a valid parameter in a Bathing Water Directive moved from the 'conceptual' at the beginning of the project, where the methods were shown to work in the laboratory, to the 'feasible' stage, where structured field studies furnished evidence that viruses can be detected in surveillance programmes.
The next stage would be to move from 'feasible' to 'operational', where quantitative determinations of target viruses (probably adenoviruses) would be reconciled with levels of known faecal indicators (probably enterococci) so that meaningful discussions on the formulation of a viral parameter based on sound scientific data could be held.
