Annalaura Carducci

Surveillance of adenoviruses and noroviruses in European recreational waters

Abstract Exposure to human pathogenic viruses in recreational waters has been shown to cause disease outbreaks. In the context of Article 14 of the revised European Bathing Waters Directive 2006/7/EC (rBWD, CEU, 2006) a Europe-wide surveillance study was carried out to determine the frequency of occurrence of two human enteric viruses in recreational waters. Adenoviruses were selected based on their near-universal shedding and environmental survival, and noroviruses (NoV) selected as being the most prevalent gastroenteritis agent worldwide. Concentration of marine and freshwater samples was done by adsorption/elution followed by molecular detection by (RT)-PCR. Out of 1410 samples, 553 (39.2%) were positive for one or more of the target viruses. Adenoviruses, detected in 36.4% of samples, were more prevalent than noroviruses (9.4%), with 3.5% GI and 6.2% GII, some samples being positive for both GI and GII. Of 513 human adenovirus-positive samples, 63 (12.3%) were also norovirus-positive, whereas 69 (7.7%) norovirus-positive samples were adenovirus-negative. More freshwater samples than marine water samples were virus-positive. Out of a small selection of samples tested for adenovirus infectivity, approximately one-quarter were positive. Sixty percent of 132 nested-PCR adenovirus-positive samples analysed by quantitative PCR gave a mean value of over 3000 genome copies per L of water. The simultaneous detection of infectious adenovirus and of adenovirus and NoV by (RT)PCR suggests that the presence of infectious viruses in recreational waters may constitute a public health risk upon exposure. These studies support the case for considering adenoviruses as an indicator of bathing water quality.

Virus hazards from food, water and other contaminated environments

Abstract Numerous viruses of human or animal origin can spread in the environment and infect people via water and food, mostly through ingestion and occasionally through skin contact. These viruses are released into the environment by various routes including water run‐offs and aerosols. Furthermore, zoonotic viruses may infect humans exposed to contaminated surface waters. Foodstuffs of animal origin can be contaminated, and their consumption may cause human infection if the viruses are not inactivated during food processing. Molecular epidemiology and surveillance of environmental samples are necessary to elucidate the public health hazards associated with exposure to environmental viruses. Whereas monitoring of viral nucleic acids by PCR methods is relatively straightforward and well documented, detection of infectious virus particles is technically more demanding and not always possible (e.g. human norovirus or hepatitis E virus). The human pathogenic viruses that are most relevant in this context are nonenveloped and belong to the families of the C aliciviridae, A denoviridae, H epeviridae, P icornaviridae and R eoviridae. Sampling methods and strategies, first‐choice detection methods and evaluation criteria are reviewed.

Assessing airborne biological hazard from urban wastewater treatment

The production of microbial aerosols by urban sewage treatment plants may have wide hygienic implications which call for careful evaluation: exposure to such aerosols may in fact represent a health hazard for plant workers and nearby residents alike. This paper describes the results of a study analysing the degree of microbial contamination at diAerent levels of treatment at several plants in the City of Leghorn (Livorno, Italy). Monthly aerosol samples were collected with an agar impact sampler from January to November 1996, from diAerent sites at an activated sludge plant, an anaerobic sludge plant and a wastewater washing station. The total bacterial and coliform counts were determined, and pathogenic enteric bacteria and viruses were determined. These same parameters were also measured in wastewater and sludge samples obtained at the same sites. The results revealed that high-grade airborne contamination existed at several of the studied sites. In particular, pathogenic enteric bacteria (Salmonella enteritidis and S. boydii) were isolated in 2% of the samples (540 l per sample), reovirus in 46% and enterovirus in 9% (1800 litres per sample in indoor environment and 3000 l outdoor), always in association with the former virus. The biological parameters measured had no evident correlation with meteorological factors such as temperature, relative humidity or wind characteristics. Viral contamination proved to be quite wide-spread and detectable even in the presence of low levels of bacterial contamination. Although virological analysis have been only qualitative, and the diAerent volumes examined for viruses and bacteria cannot allow us to appraise with accuracy the association between these two parameters, the viral presence along with low bacteria contamination suggests more dedicated studies to address with greater accuracy the quantitative aspects of this association. However the monitoring performed allowed for a determination of the areas of greatest potential risk for plant workers, and the preventive measures most suitable to guaranteeing their safety. # 2000 Elsevier Science Ltd. All rights reserved

Detection of coliphages and enteroviruses in sewage and aerosol from an activated sludge wastewater treatment plant.

Coliphages and enteroviruses were monitored over 12 months in sewage and air adjacent to an activated sludge plant. Both showed temporal variation but the mean count of phages in enterovirus‐positive samples was not significantly different from that in enterovirus‐negative samples. Hence coliphages are not necessarily a good indicator of enteroviruses in sewage and aerosols.

Enteric virus detection in Adriatic seawater by cell culture, polymerase chain reaction and polyacrylamide gel electrophoresis

Abstract Forty samples of sea and estuary water were collected from a 40 km strip along the Adriatic coast of Italy between June 1994 and September 1995. Each sample consisted of 10 l of water. Routine bacteriological analyses were carried out and viral particles concentrated on cross-flow membranes; the concentrated water samples, equally divided into two parts, were used to infect both BGM and Hep-2 cells. Lysates from all cell cultures were further tested for the presence of enteroviruses by reverse-transcribed polymerase chain reaction (RT-PCR) and reoviruses by polyacrylamide gel electrophoresis (PAGE). The results showed widespread viral contamination of the waters tested, particularly in late summer. Under our experimental conditions, BGM cells were more efficient than Hep-2 in recovering viruses. In fact, enteroviruses were detected in up to 33% and reoviruses in 80% of BGM infected with seawater, compared to 8% and 53%, respectively, for the Hep-2 cells. In estuarine samples, enteroviruses were detected in 30% and reovirus in 54% of BGM, compared to 23% and 30% of Hep-2. Twenty nine out of 40 samples showed the presence of infectious particles on the basis of the CPE appearance; after identification of the isolated viruses, only 13 turned out to be specifically contaminated by enteroviruses. Of the latter, five were below the bacteriological standards set by the Italian legislation in line with the EEC Directive 76/160 IEEC for bathing waters.

Environmental survey to assess viral contamination of air and surfaces in hospital settings

Summary The presence of pathogenic viruses in healthcare settings represents a serious risk for both staff and patients. Direct viral detection in the environment poses significant technical problems and the indirect indicators currently in use suffer from serious limitations. The aim of this study was to monitor surfaces and air in hospital settings to reveal the presence of hepatitis C virus, human adenovirus, norovirus, human rotavirus and torque teno virus by nucleic acid assays, in parallel with measurements of total bacterial count and haemoglobin presence. In total, 114 surface and 62 air samples were collected. Bacterial contamination was very low (<1cfu/cm2) on surfaces, whereas the ‘medium’ detected value in air was 282cfu/m3. Overall, 19 (16.7%) surface samples tested positive for viral nucleic acids: one for norovirus, one for human adenovirus and 17 (14.9%) for torque teno virus (TTV). Only this latter virus was directly detected in 10 air samples (16.1%). Haemoglobin was found on two surfaces. No relationship was found between viral, biochemical or bacterial indicators. The data obtained confirm the difficulty of assessing viral contamination using bacterial indicators. The frequent detection of TTV suggests its possible use as an indicator for general viral contamination of the environment.

Viral Removal by Wastewater Treatment: Monitoring of Indicators and Pathogens

The discharge of treated civil wastewater into natural waters or their reuse in industry and agriculture involves virological risks for the exposed population. Although European and Italian regulations do not require routine viral analysis of treated wastewater, a better understanding of viral contamination and resistance to treatments is needed to assess and control such risks. To this end, a wastewater treatment plant was monitored by analysing the sewage at the plant entry and exit points in order to quantify the initial presence and eventual reduction of adenovirus, Torque Teno virus, Hepatitis A virus, rotavirus, enterovirus, norovirus genogroups I and II, somatic coliphages, Escherichia coli and enterococci. The results reveal that treated water may still contain infectious human viruses and thereby represent a potential health hazard. No significant correlations were found between bacterial indicators and the viruses considered, confirming their inadequacy for virological risk assessment, while the best indicators for virus inactivation in recycled waters seem to be adenovirus, followed by somatic coliphages.

A new RT-PCR method for the identification of reoviruses in seawater samples

The frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. In order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (RT-PCR) was set up to detect reoviruses. Two primers were engineered to amplify a 538 base pair fragment of the Sigma 2 gene. Reovirus strains obtained from ATCC (Jones, Lang, Dearing, Abney, NC-TEV, SV59 and SV12) were used as references. Twenty-four samples of 101 were collected from two beaches of the Adriatic sea and 12 from the neighbourhood of Fano Harbour Channel. The presence of environmental reoviruses was tested on both concentrated seawater samples and lysates of BGM cells infected with the concentrated seawater samples. The new method was used in parallel with the detection of a 3:3:4 electrophoretic pattern of reovirus RNA in polyacrylamide gel electrophoresis (PAGE). Enterovirus and bacteria were also screened in compliance with EEC directives. No enteroviruses were isolated, and it was not attributable to reovirus interference. All the reovirus found by PAGE (8/72) were confirmed by RT-PCR, while several genomes (14/72) were detected only by RT-PCR. Presumptive methods of virus identification, that is CPE on BGM cells and haemagglutination test, were not able to detect them. The specificity of RT-PCR products was checked by direct nucleotide sequence analyses of the amplicons. The phylogenetic analyses showed heterogeneous taxa including human and animal reoviruses, with strong evidence that they were spreading consistently from the Harbour-Channel. This novel approach for reovirus detection will be very useful as a trace route of faecal pollution; more importantly, it could be very useful in contributing to the creation of a databank of circulating enteric viruses.

Issues Concerning Survival of Viruses on Surfaces

Viruses are the causative agents of an estimated 60% of human infections worldwide. The most common viral illnesses are produced by enteric and respiratory viruses. Transmission of these viruses from an infected person or animal to a new host can occur via several routes. Existing studies strongly suggest that contaminated fomites or surfaces play an important role in the spreading of viral diseases. The potential of viral spreading via contaminated surfaces depends particularly on the ability of the virus to maintain infectivity whilst it is in the environment. This is affected by a combination of biological, physical and chemical factors. This review summarises current knowledge about the influence of environmental factors on the survival and spread of viruses via contaminated surfaces.

Viral contamination of aerosol and surfaces through toilet use in health care and other settings.

Background The airborne spreading of enteric viruses can occur through the aerosol and droplets produced by toilet flushing. These can contaminate the surrounding environment, but few data exist to estimate the risk of exposure and infection. For this reason environmental monitoring of air and selected surfaces was carried out in 2 toilets of an office building and in 3 toilets of a hospital before and after cleaning operations. Methods To reveal the presence of norovirus, enterovirus, rhinovirus, human rotavirus, and Torque teno virus and to quantify human adenovirus and bacteria counts, molecular and cultural methods were used. Results On the whole, viruses were detected on 78% of surfaces and in 81% of aerosol. Among the researched viruses, only human adenovirus and Torque teno virus were found in both surface and air samples. In several cases the same adenovirus strain was concurrently found in all matrices. Bacterial counts were unrelated to viral presence and cleaning did not seem to substantially reduce contamination. Conclusions The data collected in our study confirm that toilets are an important source of viral contamination, mainly in health care settings, where disinfection can have a crucial role in preventing virus spread.