Comparison of the Legiolert™/Quanti-Tray® MPN test for the enumeration of Legionella pneumophila from potable water samples with the German regulatory requirements methods ISO 11731-2 and ISO 11731.

Due to the promising results of a previous study of the performance of the novel MPN method (Legiolert™/Quanti-Tray®) compared to ISO 11731-2, this study was performed to compare Legiolert for Legionella pneumophila with the German regulatory requirements methods ISO 11731-2 (100 ml membrane filtration) and ISO 11731 (1 ml direct plating) for the enumeration of L. pneumophila and Legionella spp. from potable water. Data from a multi-laboratory study according to ISO 17994 showed that Legiolert yielded on average higher counts of L. pneumophila than the ISO 11731-2 method, but the comparison with ISO 11731 was inconclusive due to the number of samples needing to be tested. Likewise, comparisons of the MPN method for 100 ml to the highest result of either ISO 11731 or ISO 11731-2 according to Federal Environmental Agency recommendation (2012) yielded no conclusive difference, regardless of whether non-pneumophila species of Legionella were included in the evaluation. The MPN method has a high specificity for L. pneumophila of 97.9% which compares favourably to the specificity of 95.3% quoted for ISO 11731. The new method represents a significant improvement in the enumeration of L. pneumophila from drinking water and related samples.

Distribution of Escherichia coli, coliphages and enteric viruses in water, epilithic biofilms and sediments of an urban river in Germany.

Fecal contamination of surface water is commonly evaluated by quantification of bacterial or viral indicators such as Escherichia coli and coliphages, or by direct testing for pathogens such as enteric viruses. Retention of fecally derived organisms in biofilms and sediments is less frequently considered. In this study, we assessed the distribution of E. coli, somatic coliphages, and enteric viruses including human adenovirus (HAdV), enterovirus (EV), norovirus genogroup GII (NoV GII) and group A rotavirus (RoV) in an urban river environment in Germany. 24 samples each of water, epilithic biofilms and sediments were examined. E. coli and somatic coliphages were prevalent not only in the flowing water, but also in epilithic biofilms and sediments, where they were accumulated compared to the overlying water. During enhanced rainfall, E. coli and coliphage concentrations increased by approximately 2.5 and 1 log unit, respectively, in the flowing water, whereas concentrations did not change significantly in epilithic biofilms and sediments. The occurrence of human enteric viruses detected by qPCR was higher in water than in biofilms and sediments. 87.5% of all water samples were positive for HAdV. Enteric viruses found less frequently were EV, RoV and NoV GII in 20.8%, 16.7% and 8.3% of the water samples, respectively. In epilithic biofilms and sediments, HAdV was found in 54.2% and 50.0% of the samples, respectively, and EV was found in 4.2% of both biofilm and sediment samples. RoV and NoV GII were not detected in any of the biofilms and sediments. Overall, the prevalence of enteric viruses was in the order of HAdV > EV > RoV ≥ NoV GII. In conclusion, epilithic biofilms and sediments can be reservoirs for fecal indicators and enteric viruses and thus should be taken into consideration when assessing microbial pollution of surface water environments.

From Lab to Lake - Evaluation of Current Molecular Methods for the Detection of Infectious Enteric Viruses in Complex Water Matrices in an Urban Area.

Quantitative PCR methods are commonly used to monitor enteric viruses in the aquatic environment because of their high sensitivity, short reaction times and relatively low operational cost. However, conclusions for public health drawn from results of such molecular techniques are limited due to their inability to determine viral infectivity. Ethidium monoazide (EMA) and propidium monoazide (PMA) are capable to penetrate the damaged or compromised capsid of the inactivated viruses and bind to the viral nucleic acids. We assessed whether dye treatment is a suitable approach to improve the ability of qPCR to distinguish between infectious and non-infectious human adenovirus, enterovirus and rotavirus A in surface water of an urban river and sewage before and after UV disinfection. Like the gold standard of cell culture assays, pretreatment EMA-/PMA-qPCR succeeded in removing false positive results which would lead to an overestimation of the viral load if only qPCR of the environmental samples was considered. A dye pretreatment could therefore provide a rapid and relatively inexpensive tool to improve the efficacy of molecular quantification methods in regards to viral infectivity.

Applying QMRA and DALY to assess health risks from river bathing.

To estimate the health impact of bathing in urban river waters a two-step risk assessment was conducted using the example of the Ruhr River in North-Rhine Westphalia (Germany). The risk of acquiring gastrointestinal illness (GI) due to bathing in the Ruhr River was the focus of this analysis. Referring to the WHO guidelines for safe recreational water environments, risk was defined as the probability of occurrence x severity of harm. Thus, the probability of acquiring GI by bathing in the Ruhr River has been calculated by means of the quantitative microbial risk assessment (QMRA) method. Additionally to this, harm was operationalized by using the DALY metric, quantifying the impact of disability for public health. The calculation of the DALYs based on the QMRA results, disease and lethality data of the population, duration of diseases, disability weights and a demographic profile of a regionally determined potential bathing population. DALYs were calculated for norovirus gastroenteritis, rotavirus gastroenteritis, cryptosporidiosis and giardiasis. The calculated DALYs were set into relation to other risks of daily life. Furthermore the effect of age weighting and time discounting for this site-specific population was considered. The viral load caused the main part of the environmental burden of disease by bathing in the river. The calculated DALYs are significantly lower than DALYs for all cause GI in Germany, which reach 1.19 DALY/1000, or DALYs accepted for an official EG designated bathing water (2.579 DALYs/1000 persons) but on a comparable level with the DALY for drowning (0.26 DALY/1000 Persons). The DALY concept provides a complementary tool to the QMRA for evaluating and comparing health risks arising from a specific environment for a specific population and behaviour and for comparing with other health risks of daily life.

Reducing pathogens in combined sewer overflows using ozonation or UV irradiation.

Fecal contamination of water resources is a major public health concern in densely populated areas since these water bodies are used for drinking water production or recreational purposes. A main source of this contamination originates from combined sewer overflows (CSOs) in regions with combined sewer systems. Thus, the treatment of CSO discharges is urgent. In this study, we explored whether ozonation or UV irradiation can efficiently reduce pathogenic bacteria, viruses, and protozoan parasites in CSOs. Experiments were carried out in parallel settings at the outflow of a stormwater settling tank in the Ruhr area, Germany. The results showed that both techniques reduce most hygienically relevant bacteria, parasites and viruses. Under the conditions tested, ozonation yielded lower outflow values for the majority of the tested parameters.

Use of ethidium monoazide and propidium monoazide to determine viral infectivity upon inactivation by heat, UV- exposure and chlorine.

Despite the great sensitivity of PCR in monitoring enteric viruses in an aquatic environment, PCR detects viral nucleic acids of both infectious and noninfectious viruses, limiting the conclusions regarding significance for public health. Ethidium monoazide (EMA) and propidium monoazide (PMA) are closely related membrane impermeant dyes that selectively penetrate cells with compromised membranes. Inside the cells, the dye can intercalate into nucleic acids and inhibit PCR amplification. To assess whether EMA and PMA pretreatment is a suitable approach to inhibit DNA amplification from noninfectious viruses upon heat treatment, UV exposure or chlorine treatment, viruses were measured by qPCR, EMA-qPCR, PMA-qPCR and cell culture titration. EMA/PMA-qPCR of UV- and heat-treated viruses did not correlate with the results of the cell culture assay. However, the data from EMA/PMA-qPCR of chlorine-inactivated viruses was consistent with the cell culture infectivity assay. Therefore, a dye treatment approach could be a rapid and inexpensive tool to screen the efficacy of chlorine disinfection, but it is not able to distinguish between infectious and noninfectious viruses inactivated via heat treatment or UV irradiation. Indeed, different viruses may have different trends and mechanisms of inactivation; thus, the assay must be evaluated for each virus separately.

Development of a Luminex assay for the simultaneous detection of human enteric viruses in sewage and river water.

Real time PCR (qPCR) is increasingly being used for viral detection in aquatic environments because it enables high specificity and sensitivity of detection. However, the limited number of fluorescent reporter dyes restricts its multiplex application. In this study, a multiplex Luminex assay was established for the simultaneous detection of human adenovirus (HAdV), human polyomavirus (HPyV), enterovirus (EV), rotavirus (RoV), norovirus GI (NoVGI) and norovirus GII (NoVGII). Different river water and wastewater samples were tested for the viruses using both qPCR and the multiplex Luminex xMAP assay. HAdV and HPyV were the most abundant in all environmental samples. HAdV was detected in all river water and wastewater samples, and HPyV was detected in 79% of river water and 95.8% of wastewater samples. The multiplex xMAP assay revealed high specificity and no cross-reactivity. Using the multiplex Luminex assay, the viral detection rates in river water samples were lower than the rates obtained by qPCR for all viruses. Conversely, in wastewater samples, the viral detection rates were the same for both methods. In addition, the analytical sensitivity of the monoplex Luminex assay was comparable to or lower than qPCR. Results suggest that the multiplex Luminex assay could be a reliable method for the simultaneous detection of viral pathogens in wastewater.

Methods to detect infectious human enteric viruses in environmental water samples.

Currently, a wide range of analytical methods is available for virus detection in environmental water samples. Molecular methods such as polymerase chain reaction (PCR) and quantitative real time PCR (qPCR) have the highest sensitivity and specificity to investigate virus contamination in water, so they are the most commonly used in environmental virology. Despite great sensitivity of PCR, the main limitation is the lack of the correlation between the detected viral genome and viral infectivity, which limits conclusions regarding the significance for public health. To provide information about the infectivity of the detected viruses, cultivation on animal cell culture is the gold standard. However, cell culture infectivity assays are laborious, time consuming and costly. Also, not all viruses are able to produce cytopathic effect and viruses such as human noroviruses have no available cell line for propagation. In this brief review, we present a summary and critical evaluation of different approaches that have been recently proposed to overcome limitations of the traditional cell culture assay and PCR assay such as integrated cell culture-PCR, detection of genome integrity, detection of capsid integrity, and measurement of oxidative damages on viral capsid protein. Techniques for rapid detection of infectious viruses such as fluorescence microscopy and automated flow cytometry have also been suggested to assess virus infectivity in water samples.

Evaluation of pepper mild mottle virus, human picobirnavirus and Torque teno virus as indicators of fecal contamination in river water.

A reliable indicator is needed to predict and reduce the risk of infection associated with fecal contamination of surface water. Since Pepper mild mottle virus (PMMoV), human picobirnaviruses (hPBV) and Torque teno virus (TTV) have been detected at substantial levels in human feces, we explored whether detection of nucleic acids of these viruses is a suitable indicator of fecal contamination in river water. From September 2008 to December 2009, water samples (n = 111) were collected from the Ruhr and Rhine rivers and from the influents and effluents of a wastewater plant (n = 12). Quantitative real time (RT-) PCR was used to determine the abundance of PMMoV, hPBV, and TTV in comparison to human adenoviruses (HAdV) and human polyomaviruses (HPyV) that are frequently detected in surface water and were previously proposed as indicators. While PMMoV was detected in all river water samples, the other viruses were detected less frequently. The concentration of the studied viruses in positive river water ranged from 5 × 10(1) to 1.07 × 10(6) genome equivalents per liter (gen.equ./l). All wastewater samples were positive for PMMoV, HAdV and HPyV, while TTV and hPBV were detected in 6/12 and 3/12 of samples, respectively. To determine if PMMoV is specific to human-derived fecal waste, fecal samples from human (n = 20) and animal (n = 53) were also tested. In contrast to the ubiquity of PMMoV in human feces (19/20) the virus was only detected at low concentration in a minority of the animal fecal samples tested (7/15 from chicken, 1/10 from Geese and 1/6 from cows). Therefore, in this setting TTV and hPBV do not seem to be suitable indicators of fecal contamination in water. Whereas, the high excretion level and dissemination of PMMoV in human sewage and river water suggest that PMMoV could be a promising indicator of fecal pollution in surface water.

Chemical and microbiological parameters as possible indicators for human enteric viruses in surface water.

There are still conflicting results on the suitability of chemical and microbiological parameters as indicators for the viral contamination of surface waters. In this study, conducted over 20 months, the abundance of human adenovirus, human polyomavirus, enterovirus, group A rotavirus and norovirus was determined in Ruhr and Rhine rivers, Germany. Additionally, prevalence of different possible indicators such as somatic coliphages, E. coli, intestinal enterococci, and total coliforms was also considered. Moreover, the chemical parameter TCPP (tris-(2-chloro-, 1-methyl-ethyl)-phosphate), characterized by environmental stability and human origin, was included. Furthermore, chemical parameters (fluoride, chloride, nitrate, nitrite, bromide, phosphate, and sulfate) which may influence the stability and subsequently the detection rates of viruses in aquatic environment were measured. Quantitative Real-Time (RT-)PCR and double agar layer test were used for the quantification of human enteric viruses and somatic coliphages, respectively. The analyses for E. coli, total coliforms, and intestinal enterococci were done with respect to the standard reference method. The chemical parameters were measured by liquid chromatography of ions and by gas chromatography-flame photometer detector (GC-FPD), respectively. We demonstrated that human adenovirus had the highest detection rate (96.3%), followed by somatic coliphages (73.5%), human polyomavirus (68.6%), and rotavirus (63.5%). However, norovirus GII and enterovirus were found in only 25.7 and 17.8%, respectively. The concentration of the viral genome ranged between 16 and 1.1 xs 10(6) gen. equ./l (genome equivalents/l) whereas the concentrations for TCPP ranged between 0.01 and 0.9 microg/l. The results of the Pearson correlation showed no association between TCPP and any other microbiological parameter. None of the other tested chemical parameters correlated negatively, and therefore they do not influence the stability of enteric viruses. We conclude that neither TCPP nor any other chemical or microbiological parameter can be used as a reliable indicator for the presence of enteric viruses in river water.

Role of HSP-90 for increased nNOS-mediated vasodilation in mesenteric arteries in portal hypertension.

AIM: To explore the role of heat shock protein-90 (HSP-90) for nitrergic vasorelaxation in the splanchnic circulation in rats with and without portal hypertension. METHODS: Neuronal nitric oxide synthase (nNOS) and HSP-90 were analyzed by immunofluorescence, western blotting and co-immunoprecipitation in the mesenteric vasculature and isolated nerves of portal-vein-ligated (PVL) rats and sham operated rats. In vitro perfused de-endothelialized mesenteric arterial vasculature was preconstricted with norepinephrine (EC(80)) and tested for nNOS-mediated vasorelaxation by periarterial nerve stimulation (PNS, 2-12 Hz, 45V) before and after incubation with geldanamycin (specific inhibitor of HSP-90 signalling, 3 microg/mL) or L-NAME (non-specific NOS-blocker, 10(-4) mol/L). RESULTS: nNOS and HSP-90 expression was significantly increased in mesenteric nerves from PVL as compared to sham rats. Moreover, nNOS and HSP-90 were visualized in mesenteric nerves by immunofluorescence and immunoprecipitation of nNOS co-immunoprecipitated HSP-90 in sham and PVL rats. PNS induced a frequency-dependent vasorelaxation which was more pronounced in PVL as compared to sham rats. L-NAME and geldanamycin markedly reduced nNOS-mediated vasorelaxation abrogating differences between the study groups. The effect of L-NAME and geldanamycin on nNOS-mediated vasorelaxation was significantly greater in PVL than in sham animals. However, no difference in magnitude of effect between L-NAME and geldanamycin was noted. CONCLUSION: HSP-90 acts as a signalling mediator of nNOS-dependent nerve mediated vascular responses in mesenteric arteries, and the increased nitrergic vasorelaxation observed in portal hypertension is mediated largely by HSP-90.

Detection and quantification of human bocavirus in river water.

Human bocavirus (HBoV) was recently discovered in children with respiratory-tract infection and has been detected frequently in faecal specimens from children with gastroenteritis. The present study addresses for the first time, to our knowledge, the prevalence of HBoV in river water. By using a newly developed real-time PCR targeting a conserved region of the NP1 gene of HBoV, virus levels in water samples were determined. Moreover, partial sequence analysis of the NP1 gene of HBoV and comparative phylogenetic analysis were performed. HBoV was detected in 40.8 % of collected water samples. The virus level ranged between 3x10(1) and 2x10(3) genome equivalents l(-1). Therefore, the present study suggests that river water could play a role in the spread of HBoV. However, further work should be done to determine the actual risk of infection via surface water.

Detection of human viruses in rivers of a densly-populated area in Germany using a virus adsorption elution method optimized for PCR analyses.

Transmission of viruses via surface water is a major public health concern. To determine the viral concentration in rivers of a densely-populated area in Germany, the virus adsorption elution (VIRADEL) method was optimized for downstream PCR applications. Using a high-salt alkaline phosphate buffer for elution, the median recovery efficiency from spiked 1l water samples ranged from 21.3% to 100% for JC polyomavirus, human adenovirus type 5, Echovirus 11, and norovirus genogroup I. Analyses of 41 water samples collected during the winter 2007/08 from the rivers Ruhr and Rhine yielded detection rates 97.5% for adenoviruses and human polyomavirus (JC, BK), and 90% for group A rotaviruses. Noroviruses genogroup II were detected in 31.7% of the samples and only one sample was positive for enteroviruses. Virus concentrations ranged from 9.4 to 2.3x10(4) gen.equ./l. However, the genome equivalents/liter determined for the RNA viruses and their detection frequency are only lower limits, since the concentration procedure leads to carry-over of inhibitors of the reverse transcription step. Sequence analyses of the PCR products revealed that the adenovirus and rotavirus PCRs used could cross-react with animal viruses from the respective virus families. These results suggest that detection of human polyomavirus genomes is the most sensitive and specific marker for contamination of surface water with viruses from human sewage. Although we could routinely detect nucleic acids of viral pathogens in river water by the PCR-optimized VIRADEL method, threshold levels of viral nucleic acids above which there is a risk of infection with viruses derived from human remain to be determined.

Colonization of patients and contamination of the patients' environment by MRSA under conditions of single-room isolation.

Meticillin-resistant Staphylococcus aureus (MRSA) are endemic in hospitals worldwide and present a major concern in hospital hygiene. The aim of the present study was to investigate the relationship between patients' MRSA colonization of the body and the frequency of environmental contamination. Twenty-five MRSA-positive hospitalized surgical patients and their environment in isolation rooms were screened on four occasions over a 14-day period. Out of 1099 samples from patients, 330 (30.0%) were MRSA-positive. The median number of MRSA-positive body sites per screening decreased significantly from the 1st (3, range 1-9) to the 14th (2, range 0-9, p=0.011) day of isolation. Contamination was found in 45% of the 100 environmental sampling dates and MRSA was detected in a low proportion of the 1000 environmental surface samples: 105/1000 (10.5%). The number of positive results for each sampling date decreased from the 1st (median 1, range 0-8) to the 14th (median 0, range 0-3, p=0.21) day of isolation. The results show a very strong correlation between the number of MRSA-positive body sites of individual patients and the MRSA contamination of the patient's hospital room (r=0.700, p<0.001). Pulsed-field gel electrophoresis (PFGE) analysis demonstrated a 98% agreement between patient and environmental samples. MRSA colonization of the groin area correlates most strongly with colonization of the body and environment. Seventy-five of 240 (31%) samples taken in rooms of patients with colonization of the groin were MRSA-positive, whereas only 27 of 760 (3.6%) samples taken in rooms of patients without colonization of the groin produced positive results (odds ratio 12.3; 95% confidence interval, 7.7-20). It is concluded that MRSA patients without colonization of the groin have a relatively low risk of environmental spread of MRSA and thus a reduced risk of transmission.

Enhanced Y1-receptor-mediated vasoconstrictive action of neuropeptide Y (NPY) in superior mesenteric arteries in portal hypertension.

BACKGROUND/AIMS: Vascular hyporeactivity to catecholamines contributes to arterial vasodilation and hemodynamic dysregulation in portal hypertension. Neuropeptide Y (NPY) is a sympathetic neurotransmitter facilitating adrenergic vasoconstriction via Y1-receptors on the vascular smooth muscle. Therefore, we investigated its role for vascular reactivity in the superior mesenteric artery (SMA) of portal vein ligated (PVL) and sham operated rats. METHODS: In vitro perfused SMA vascular beds of rats were tested for the cumulative dose-response to NPY dependent on the presence and level of alpha1-adrenergic vascular tone (methoxamine MT: 0.3-10 microM). Moreover, the effect of NPY (50 nM) on vascular responsiveness to alpha1-adrenergic stimulation (MT: 0.3-300 microM) was evaluated. Y1-receptor function was tested by Y1-selective inhibition using BIBP-3226 (1 microM). RESULTS: NPY dose-dependently and endothelium-independently enhanced MT-pre-constriction in SMA. This potentiation was increasingly effective with increasing adrenergic pre-stimulation and being more pronounced in PVL rats as compared to sham rats at high MT concentrations. NPY enhanced vascular contractility only in PVL rats correcting the adrenergic vascular hyporeactivity. Y1-receptor inhibition completely abolished NPY-evoked vasoconstrictive effects. CONCLUSIONS: NPY endothelium-independently potentiates adrenergic vasoconstriction via Y1-receptors being more pronounced in portal hypertension improving mesenteric vascular contractility and thereby correcting the splanchnic vascular hyporeactivity. This makes NPY a superior vasoconstrictor counterbalancing arterial vasodilation in portal hypertension.

Up-regulation of nNOS and associated increase in nitrergic vasodilation in superior mesenteric arteries in pre-hepatic portal hypertension.

BACKGROUND/AIMS: Splanchnic arterial vasodilation in portal hypertension has been attributed largely to vascular NO overproduction. Three NO-synthase (NOS) isoforms have been identified of which e(ndothelial)-NOS has been found up-regulated and i(nducible)-NOS not expressed in the splanchnic circulation in portal hypertension. So far, n(euronal)-NOS has not been investigated and hence, the current study evaluates nNOS-expression and nNOS-mediated vasorelaxation in a model of portal vein-ligated rats (PVL). METHODS: Mesenteric vasculature of PVL and sham rats was evaluated for nNOS-protein (immunohistochemically and Western blotting). In vitro perfused de-endothelialized mesenteric arterial vasculature was pre-constricted with norepinephrine (EC(80)) and tested for nNOS-mediated vasorelaxation by periarterial nerve stimulation (PNS, 2-12 Hz, 45V) before and after incubation with the NOS-inhibitor L-NAME (10(-4)M). RESULTS: nNOS was localized to the adventitia of the mesenteric arterial tree showing more intense staining and increased protein expression in PVL as compared to sham rats. PNS induced a frequency-dependent vasorelaxation, which was more pronounced in PVL rats. L-NAME abolished this difference in nerval-mediated vasorelaxation, the effect being significantly greater in PVL than in sham animals. CONCLUSIONS: Perivascular nNOS-protein expression is enhanced in mesenteric arteries in portal hypertension mediating an increased nerval NO-mediated vasorelaxation. This nNOS-derived NO overproduction may play an important role in the pathogenesis of arterial vasodilation in portal hypertension.