Detection and Characterization of Hepatitis E Virus Genotype 3 in Wastewater and Urban Surface Waters in Germany.

In highly populated areas, environmental surveillance of wastewater and surface waters is a key factor to control the circulation of viruses and risks for public health. Hepatitis E virus (HEV) genotype 3 is considered as an emerging pathogen in industrialized countries. Therefore, this study was carried out to determine the prevalence of HEV in environmental waters in urban and suburban regions in Germany. HEV was monitored in water samples using quantitative RT-PCR (RT-qPCR) and nested RT-PCR without or with virus concentration via polyethylene glycol precipitation or ultracentrifugation. By RT-qPCR, 84-100% of influent samples of wastewater treatment plants were positive for HEV RNA. Genotypes HEV-3c and 3f were identified in wastewater, with HEV-3c being the most prevalent genotype. These data correlate with subtypes identified earlier in patients from the same area. Comparison of wastewater influent and effluent samples revealed a reduction of HEV RNA of about 1 log10 during passage through wastewater treatment plants. In addition, combined sewer overflows (CSOs) after heavy rainfalls were shown to release HEV RNA into surface waters. About 75% of urban river samples taken during these CSO events were positive for HEV RNA by RT-qPCR. In contrast, under normal weather conditions, only around 30% of river samples and 15% of samples from a bathing water located at an urban river were positive for HEV. Median concentrations of HEV RNA of all tested samples at this bathing water were below the limit of detection.

Development and testing of a fractionated filtration for sampling of microplastics in water.

A harmonization of sampling, sample preparation and detection is pivotal in order to obtain comparable data on microplastics (MP) in the environment. This paper develops and proposes a suitable sampling concept for waterbodies that considers different plastic specific properties and influencing factors in the environment. Both artificial water including defined MP fractions and the discharge of a wastewater treatment plant were used to verify the derived sampling procedure, sample preparation and the subsequent analysis of MP using thermal extraction-desorption gas chromatography - mass spectrometry (TED-GC-MS). A major finding of this paper is that an application of various particle size classes greatly improves the practical handling of the sampling equipment. Size classes also enable the TED-GC-MS to provide any data on the MP size distribution, a substantial sampling property affecting both the necessary sampling volume and the optimal sampling depth. In the artificial water with defined MP fractions, the recovery rates ranged from 80 to 110%, depending on the different MP types and MP size classes. In the treated wastewater, we found both polyethylene and polystyrene in different size classes and quantities.

Comparison of Microbial Removal Between Slow Dead-End Versus Tangential Sand Filtration.

Both river bank filtration and dead-end sand filtration are becoming increasingly applied in rural areas to improve the quality of fecally contaminated water. To evaluate the capacity of both treatments to remove E. coli, fecal streptococci, and somatic and K13-phages, this study investigates their concentrations in diluted wastewater after short-distance tangential sand filtration and dead-end sand filtration. Bacteria were almost undetectable in both systems after 60 cm depth, and at a pore-water velocity of 1 m/d. Both phages underwent removal of 2.5 logs by tangential filtration, whereas dead-end filtration removed 5.1 logs and 3.9 logs of K13-phages and somatic phages, respectively. After discounting removal by the schmutzdecke, observed only in the dead-end filtration, both systems removed phages similarly. It is concluded that short-distance river bank filtration, alone, does not meet WHO requirements for drinking water. However, the concomitant reduction of suspended solids renders the filtered water amenable to further treatment steps.

A new approach to testing the efficacy of drinking water disinfectants.

New disinfection procedures are being developed and proposed for use in drinking-water production. Authorising their use requires an effective test strategy that can simulate conditions in practice. For this purpose, we developed a test rig working in a flow-through mode similar to the disinfection procedures in waterworks, but under tightly defined conditions, including very short contact times. To quantify the influence of DOC, temperature and pH on the efficacy of two standard disinfectants, chlorine and chlorine dioxide, simulated use tests were systematically performed. This test rig enabled quantitative comparison of the reduction of four test organisms, two viruses and two bacteria, in response to disinfection. Chlorine was substantially more effective against Enterococcus faecium than chlorine dioxide whereas the latter was more effective against the bacteriophage MS2, especially at pH values of >7.5 at which chlorine efficacies already decline. Contrary to expectation, bacteria were not generally reduced more quickly than viruses. Overall, the results confirm a high efficacy of chlorine and chlorine dioxide, validating them as standard disinfectants for assessing the efficacy of new disinfectants. Furthermore, these data demonstrate that the test rig is an appropriate tool for testing new disinfectants as well as disinfection procedures.

[Environmental health relevance of airborne microorganisms in ambient and indoor air]. / Umweltmedizinische Relevanz von luftgetragenen Mikroorganismen im Außen- und Innenbereich.

Airborne microorganisms occur ubiquitously in the ambient air. Besides allergic and irritative-toxic effects, they can cause infections after inhalation. Occupational studies have shown that an increased incidence of respiratory diseases is found in adequately exposed workers. In addition to respiratory diseases, severe systemic infections can also occur in particular cases, such as in the case of a hantavirus infection that is recognized as an occupational disease. In studies from environmental medicine, respiratory diseases have also been observed in residents living in the vicinity of livestock facilities and evaporative cooling towers. In the latter case, an infection risk may be caused by inhalation of legionella-contaminated aerosol from the exhaust air of such systems.Currently, there are no health-related exposure limits for airborne microorganisms released from such facilities. Environmental risk assessment can be carried out on the basis of the guideline VDI 4250 part 1, which relies on an excess of natural background concentration by facility-specific emissions. For the approval practice, the LAI-Leitfaden Bioaerosole is a uniform, standardized method for the determination and assessment of bioaerosol exposure.In indoor spaces, only a few mold types, such as Aspergillus fumigatus are able to trigger infections by local or systemic infection of the human organism. In particular, persons with an immune deficiency or allergies must be informed about the risks of mold exposure in indoor air. In general, mold growth in indoor spaces is a hygienic problem and must not be accepted as a matter of principle.

Virus removal vs. subsurface water velocity during slow sand filtration.

In an attempt to obtain a conservative estimate of virus removal during slow sand and river bank filtration, a somatic phage was isolated with slow decay and poor adsorption to coarse sand. We continuously fed a phage suspension to a 7-m infiltration path and measured the phage removal. In a second set of experiments, we fed the phage suspension to 1-m long columns run at different pore water velocities. Using the data obtained, a mathematical model was constructed describing removal vs. pore water velocity (PWV), assuming different statistical distributions of the adsorption coefficient λ. The bimodal distribution best fit the results for PWVs higher than 1 m/d. It predicted a removal of approximately 4 log10 after 50 days infiltration at 1 m/d. At PWVs below 1 m/d the model underestimated removal. Sand-bound phages dissociated slowly into the liquid phase, with a detachment constant kdet of 2.6 × 10⁻5. This low kdet suggests that river bank filtration plants should be intermittently operated when viral overload is suspected, e.g. during flooding events or at high water-marks in rivers, in order for viruses to become soil-associated during the periods of standstill. Resuming filtration will allow only a very slow virus release from the soil.

Removal of Surrogate Bacteriophages and Enteric Viruses from Seeded Environmental Waters Using a Semi-technical Ultrafiltration Unit.

Experiments to determine the removal of viruses in different types of water (surface water from two reservoirs for drinking water treatment, treated groundwater and groundwater contaminated with either 5 or 30 % of wastewater) by ultrafiltration were performed with a semi-technical ultrafiltration unit. Concentrations of human adenoviruses (HAdVs), murine norovirus (MNV), and the bacteriophages MS2, ΦX174 and PRD1 were measured in the feed water and the filtrate, and log removal values were calculated. Bacteria added to the feed water were not detected in the filtrates. In contrast, in most cases viruses and bacteriophages were still present in the filtrates: log removal values were in the range of 1.4-6.3 depending on virus sizes and water qualities. Best removals were observed with bacteriophage PRD1 and HAdVs, followed by MNV and phages MS2 and ΦX174. Virus size, however, was not the only criterion for efficient removal. In diluted wastewater as compared to drinking water and uncontaminated environmental waters, virus removal was clearly higher for all viruses, most likely due to higher membrane fouling. For quality assessment purposes of membrane filtration efficiencies with regard to the elimination of human viruses the small bacteriophages MS2 and ΦX174 should be used as conservative viral indicators.

Attenuation and colloidal mobilization of bacteriophages in natural sediments under anoxic as compared to oxic conditions.

Redox conditions are known to affect the fate of viruses in porous media. Several studies report the relevance of colloid-facilitated virus transport in the subsurface, but detailed studies on the effect of anoxic conditions on virus retention in natural sediments are still missing. Therefore, we investigated the fate of viruses in natural flood plain sediments with different sesquioxide contents under anoxic conditions by considering sorption to the solid phase, sorption to mobilized colloids, and inactivation in the aqueous phase. Batch experiments were conducted under oxic and anoxic conditions at pH values between 5.1 and 7.6, using bacteriophages MS2 and PhiX174 as model viruses. In addition to free and colloid-associated bacteriophages, dissolved and colloidal concentrations of Fe, Al and organic C as well as dissolved Ca were determined. Results showed that regardless of redox conditions, bacteriophages did not adsorb to mobilized colloids, even under favourable charge conditions. Under anoxic conditions, attenuation of bacteriophages was dominated by sorption over inactivation, with MS2 showing a higher degree of sorption than PhiX174. Inactivation in water was low under anoxic conditions for both bacteriophages with about one log10 decrease in concentration during 16 h. Increased Fe/Al concentrations and a low organic carbon content of the sediment led to enhanced bacteriophage removal under anoxic conditions. However, even in the presence of sufficient Fe/A-(hydr)oxides on the solid phase, bacteriophage sorption was low. We presume that organic matter may limit the potential retention of sesquioxides in anoxic sediments and should thus be considered for the risk assessment of virus breakthrough in the subsurface.

Transport and removal of viruses in saturated sand columns under oxic and anoxic conditions--Potential implications for groundwater protection.

To protect groundwater as a drinking water resource from microbiological contamination, protection zones are installed. While travelling through these zones, concentrations of potential pathogens should decline to levels that pose no risks to human health. Removal of viruses during subsurface passage is influenced by physicochemical conditions, such as oxygen concentration, which also affects virus survival. The aim of our study was to evaluate the effect of redox conditions on the removal of viruses during sand filtration. Experiments in glass columns filled with medium-grained sand were conducted to investigate virus removal in the presence and absence of dissolved oxygen. Bacteriophages MS2 and PhiX174, as surrogates for human enteric viruses were spiked in pulsed or in continuous mode and pumped through the columns at a filter velocity of about 1m/d. Virus breakthrough curves were analyzed by calculating total viral elimination and fitted using one-dimensional transport models (CXTFIT and HYDRUS-1D). While short-term experiments with pulsed virus application showed only small differences with regard to virus removal under oxic and anoxic conditions, a long-term experiment with continuous dosing revealed a clearly lower elimination of viruses under anoxic conditions. These findings suggest that less inactivation and less adsorption of viruses in anoxic environments affect their removal. Therefore, in risk assessment studies aimed to secure drinking water resources from viral contamination and optimization of protection zones, the oxic and anoxic conditions in the subsurface should also be considered.

Monitoring of adenovirus serotypes in environmental samples by combined PCR and melting point analyses.

BACKGROUND: Human adenoviruses are promising candidates for addressing health risks associated with enteric viruses in environmental waters. Relatively harmless but common, these DNA viruses persist within the population and are generally considered extremely stable, remaining infectious in water for long periods of time. Group-specific or single species detection of human adenoviruses in environmental samples is usually based on polymerase chain reaction assays. Simultaneous identification of specific species or serotypes needs additional processing. Here we present a simple molecular approach for the monitoring of serotypic diversity in the human adenovirus populations in contaminated water sites. METHODS: Diversity patterns of human adenoviruses in environmental samples, collected in an outdoor artificial stream and pond simulation system, were analyzed using a closed tube polymerase chain reaction method with subsequent melting point analysis. RESULTS: Human adenovirus serotype 41 was the most prominent adenovirus serotype detected in environmental water samples, but melting point analyses indicated the presence of additional adenovirus serotypes. CONCLUSIONS: Based on investigations with spiked and environmental samples, a combination of qPCR and melting point analysis was shown to identify adenovirus serotypes in sewage contaminated water.

Overview of the study design, participation and field work of the German Environmental Survey on Children 2003-2006 (GerES IV).

The German Federal Environment Agency carried out its fourth German Environmental Survey (GerES IV), which is the first survey on children only and the environment-related module of the German Health Interview and Examination Survey for Children and Adolescents (German acronym: KiGGS), conducted by the Robert Koch Institute (RKI). The German Environmental Surveys are nationwide population studies conducted to determine the exposure to environmental pollutants, to explore exposure pathways and to identify sub-groups with higher exposure. GerES IV was conducted on randomly selected 1790 children aged 3-14 years from the cross-sectional sample of KiGGS. The participants of GerES IV lived in 150 sampling locations all over Germany. Field work was carried out from May 2003 to May 2006. The response rate in GerES IV was 77.3%. Due to the fact that participation in GerES IV was limited to children that had previously participated in the KiGGS study, the total response rate in GerES IV resulted in 52.6%. Response rates did neither differ significantly between West and East Germany, nor between different community sizes, age groups and gender. The basic study programme included blood samples, morning urine, tap water and house dust as well as comprehensive questionnaire-based interviews. In addition, subgroups were studied with regard to "noise, hearing capacity and stress hormones", "chemical contamination of indoor air" and "biogenic indoor contamination". A key element of the field work in GerES IV was a home visit to carry out interviews, conduct measurements and collect samples. An exception was blood sampling which was carried out within KiGGS. The quality of field work, data collection, evaluation, and chemical, biological and physical analyses was successfully evaluated by internal and external quality assurance. This comprehensive overview aims at giving other research groups the opportunity to compare different study designs or to adapt their own design to get comparable results.

Surveillance of adenoviruses and noroviruses in European recreational waters.

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.

German Environmental Survey for Children (GerES IV)--first results.

German Environmental Surveys (GerESs) are large scale population studies which have been carried out on adults in 1985/86, 1990/92 and 1998 and on children aged 6-14 years in 1990/92. GerES IV is the first survey focussing exclusively on children [Becker, K., Schulz, C., Babisch, W., Dürkop, J., Roskamp, E., Seiwert, M., Szewzyk, R., Ullrich, D., Seifert, B., 2005. German Environmental Survey for Children (GerES IV) 2003-2006. Pullut. Atmos. 188, 475-479]. GerES IV included a representative sample of 1790 children aged 3-14 of the participants of the National Health Interview and Examination Survey on Children and Adolescents. The primary goal of GerES IV is not only to analyse and document the extent, distribution and determinants of German children's exposure to environmental pollutants but to discover links between environmental exposure and health. Results will help develop preventive measures and advance further research. They might provide the basis for environmental and public health policy decisions. Precondition to achieve this task is a description of the data on exposures and the data on health outcomes. This work is currently performed at the Federal Environment Agency. First results show a remarkable decrease of the blood lead level of German children aged 6-14 years from 32.3 microg/l in 1990/92 (GerES II) to 16.3 microg/l in GerES IV which is the lowest mean lead concentration determined in German studies so far. None of the children had a value exceeding 100 microg/l. In GerES IV, the following health-related issues will be primarily examined: the relationship between sensitisation against mould spores and the occurrence of mould in households, irritation of eyes and respiratory system caused by formaldehyde, other aldehydes, or total volatile organic compounds (TVOC); the impact of non-occupational noise on hearing loss, stress and sleep disturbances, and the connection between contact allergies, nickel and scents. 9.5% of the children showed a sensitisation to at least one of the moulds examined (Penicillium (notatum) chrysogenum, Aspergillus versicolor, Wallemia sebi, Eurotium spp., Alternaria alternata). The most frequent sensitisation was against Penicillium chrysogenum. GerES IV might broaden the knowledge in terms of environmental causes of health outcomes. Children of smoking mothers showed higher mean cotinine concentrations than children living with a smoking father, regardless whether they smoked daily or occasionally. Results from the GerES IV pilot study showed a relation between environmental tobacco smoke (ETS) exposure and an increased susceptibility to infections and inflammations of the middle ear.

A randomized controlled trial assessing infectious disease risks from bathing in fresh recreational waters in relation to the concentration of Escherichia coli, intestinal enterococci, Clostridium perfringens, and somatic coliphages.

We performed epidemiologic studies at public freshwater bathing sites in Germany to provide a better scientific basis for the definition of recreational water quality standards. A total of 2,196 participants were recruited from the local population and randomized into bathers and nonbathers. Bathers were exposed for 10 min and had to immerse their head at least three times. Water samples for microbiological analysis were collected at 20-min intervals. Unbiased concentration-response effects with no-observed-adverse-effect levels (NOAELs) were demonstrated for three different definitions of gastroenteritis and four fecal indicator organisms. Relative risks for bathing in waters with levels above NOAELs compared with nonbathing ranged from 1.8 (95% CI, 1.2-2.6) to 4.6 (95% CI, 2.1-10.1), depending on the definition of gastroenteritis. The effect of swallowing water provided additional evidence for true dose-response relationships. Based on the NOAELs, the following guide values for water quality are suggested: 100 Escherichia coli, 25 intestinal enterococci, 10 somatic coliphages, or 10 Clostridium perfringens per 100 mL. Recreational water quality standards are intended to protect the health of those consumers who are not already immune or resistant to pathogens that may be associated with indicator organisms. In contrast to current World Health Organization recommendations, we concluded that standards should be based on rates of compliance with NOAELs rather than on attributable risks determined above NOAELs, because these risks depend mainly on the unpredictable susceptibility of the cohorts. Although in theory there is no threshold in real concentration-response relationships, we demonstrated that a NOAEL approach would be a more robust and practical solution to the complex problem of setting standards.

[Strategies and targets for establishing a multicenter trial: "Identification of indoor relevant moulds"]. / Strategien und Ziele der Etablierung eines Ringversuchs. "Differenzierung von innenraumrelevanten Schimmelpilzen".

Quality control is universally recognised as fundamentally important in ensuring that diagnostic laboratories are performing appropriate tests at acceptable levels of competence. Therefore, the working group "Quality assurance--Fungi in Indoor Environments" which was initiated by the Public Health Service Baden-Württemberg (LGA BW) started with the external quality assurance in the diagnosis of indoor fungi in autumn 2001. Up to now we carried out the third mailing based on pure cultures. The results are reported and will be discussed. Exchanging experience on the international level is intended.