Lars Jurzik

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.

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.

Methods to detect infectious human enteric viruses in environmental water samples

Abstract 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.

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.

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.

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.

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.

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.

Microbial and viral pathogens in freshwater: current research aspects studied in Germany

Continuous research in the field of water and health is important to ensure high standards of hygiene and to improve microbial risk assessment and water management. Various waterborne outbreaks induced by pathogenic bacteria, viruses and protozoa have been reported in recent decades. Pathogens derived from human and animal feces have been played frequently a key role in contaminations of freshwater. Reasons for the presence of pathogens in drinking water systems are insufficient wastewater treatment, contaminated raw water or damaged water supply systems. Numerous routes of transmission and the distribution of individual pathogens by raw water and drinking water constitute potential hazards to public health. Recently, different research activities have been initiated in Germany, which focus on the examination of pathogens in water to establish a modern microbial risk assessment according to the high-tech strategy employed by the federal government. This review article provides a compilation of the pathogens that are important in German water bodies (wastewater, surface water, groundwater, and drinking water) and includes coverage of German research on pathogens and technologies for reducing these pathogens in water, challenges to research, and recent developments in concentration and detection methods for pathogens. Finally, current knowledge gaps and ongoing research questions are highlighted.