Entero- and parechovirus distributions in surface water and probabilities of exposure to these viruses during water recreation.

Numerous studies have reported quantitative data on viruses in surface waters generated using different methodologies. In the current study, the impact of the use of either cell culture-based or molecular-based methods in quantitative microbial risk assessment was assessed. Previously and newly generated data on the presence of infectious human enteroviruses (HEV) and enterovirus and parechovirus RNA were used to estimate distributions of virus concentrations in surface waters. Because techniques for the detection of infectious human parechoviruses (HPeV) in surface waters were not available, a 'Parallelogram Approach' was used to estimate their concentrations based on the ratio infectious HEV/HEV RNA. The obtained virus concentrations were then used to estimate the probability of exposure for children during recreation in such virus contaminated surface waters. Human enterovirus cell culture/PCR ratios ranged from 2.3 × 10(-3) to 0.28. This broad range of ratios indicates that care should be taken in assuming a fixed ratio for assessing the risk with PCR based virus concentrations. The probabilities of exposure to both enteroviruses and parechoviruses were calculated, using our Parallelogram Approach for the calculation of infectious parechoviruses. For both viruses it was observed that the detection method significantly influenced the probability of exposure. Based on the calculated culture data, PCR data, and the ingestion volume, it was estimated that the mean probabilities of exposure, of recreating children, to surface water containing viruses were 0.087 (infectious enteroviruses), 0.71 (enterovirus particles), 0.28 (parechovirus particles) and 0.025 (calculated infectious parechoviruses) per recreation event. The mean probabilities of exposure of children recreating in surface water from which drinking water is produced to infectious enteroviruses were estimated for nine locations and varied between 1.5 × 10(-4) - 0.09 per recreation event. In this study, the use of the rotavirus dose response relationship as a surrogate was avoided. Instead, the probabilities of exposure were derived as a function of the distributions of the calculated doses. Our 'Parallelogram Approach' was used to estimate the unavailable infectious parechovirus concentrations using Monte Carlo simulations, and the exposure assessment carried out showed that virus concentrations present in surface waters could pose a health risk for children and other vulnerable populations.

Viable Legionella pneumophila bacteria in natural soil and rainwater puddles.

AIMS: For the majority of sporadic Legionnaires' disease cases the source of infection remains unknown. Infection may possible result from exposure to Legionella bacteria in sources that are not yet considered in outbreak investigations. Therefore, potential sources of pathogenic Legionella bacteria--natural soil and rainwater puddles on roads--were studied in 2012. METHODS AND RESULTS: Legionella bacteria were detected in 30% (6/20) of soils and 3·9% (3/77) of rainwater puddles by amoebal coculture. Legionella pneumophila was isolated from two out of six Legionella positive soil samples and two out of three Legionella positive rainwater samples. Several other species were found including the pathogenic Leg. gormanii and Leg. longbeachae. Sequence types (ST) could be assigned to two Leg. pneumophila strains isolated from soil, ST710 and ST477, and one strain isolated from rainwater, ST1064. These sequence types were previously associated with Legionnaires' disease patients. CONCLUSIONS: Rainwater and soil may be alternative sources for Legionella. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of clinically relevant strains indicates that rainwater and soil are potential sources of Legionella bacteria and future research should assess the public health implication of the presence of Leg. pneumophila in rainwater puddles and natural soil.

Environmental surveillance of human parechoviruses in sewage in The Netherlands.

The circulation of human parechoviruses (HPeVs) in the population was studied by environmental surveillance comprising of molecular analyses of sewage samples (n = 89) that were collected from 15 different locations in The Netherlands. Samples were taken from sewage originating from schools (n = 9) or from parts of municipalities (n = 6) during the Dutch school year 2010-2011. At 13/15 locations HPeV1, HPeV3, or HPeV6 RNA was detected at least once; however, sequence diversity did not reflect associations in time or place. A higher percentage of positives was observed in the samples originating from the municipalities. It was demonstrated that HPeV circulated in the studied population to a higher extent than would be expected from the current knowledge on infections predominating in young children.

Reduction of bacteriophage MS2 by filtration and irradiation determined by culture and quantitative real-time RT-PCR.

Molecular methods are increasingly applied for virus detection in environmental samples without rendering data on viral infectivity. Infectivity data are important for assessing public health risks from exposure to human pathogenic viruses in the environment. Here, treatment efficiencies of three (drinking) water treatment processes were estimated by quantification of the indicator virus bacteriophage MS2 with culture and real-time reverse transcription polymerase chain reaction (qRT-PCR). We studied the virus reduction by slow sand filtration at a pilot plant. No decay of MS2 RNA was observed, whereas infectious MS2 particles were inactivated at a rate of 0.1 day(-1). Removal of MS2 RNA and infectious MS2 particles was 1.2 and 1.6 log10-units, respectively. Virus reduction by UV and gamma irradiation was determined in laboratory-scale experiments. The reduction of MS2 RNA based on qRT-PCR data was negligible. Reduction of infectious MS2 particles was estimated at 3.0-3.6 log10-units (UV dose up to 400 or 800 J/m(2)) and 4.7-7 log10-units (gamma dose up to 200 Gray). As shown in this study, estimations of viral reduction, both inactivation and removal, obtained by molecular methods should be interpreted carefully when considering treatment options to provide virus-safe drinking water. Combining culture-based methods with molecular methods may provide supplementary information on mechanisms of virus reduction.

Isolation of Legionella pneumophila from pluvial floods by amoebal coculture.

Viable Legionella pneumophila bacteria were isolated by amoebal coculture from pluvial floods after intense rainfall and from water collected at sewage treatment plants. Several isolated L. pneumophila strains belonged to sequence types that have been previously identified in patients.

Feasibility of quantitative environmental surveillance in poliovirus eradication strategies.

The progress of the Global Polio Eradication Initiative is monitored by acute flaccid paralysis (AFP) surveillance supplemented with environmental surveillance in selected areas. To assess the sensitivity of environmental surveillance, stools from (re)vaccinated elderly persons with a low seroprevalence and from wastewater were concurrently collected and analyzed in the Netherlands over a prolonged period of time. A total number of 228 healthy individuals with different levels of immunity were challenged with monovalent oral polio vaccine serotype 1 or 3. Poliovirus concentrations were determined by the titration of fecal suspensions on poliovirus-sensitive L20B cells and of sewage concentrates by L20B monolayer plaque assay. Almost half of the individuals (45%) shed poliovirus on day 3 after challenge, which peaked (57%) on day 8 with an average poliovirus excretion of 1.3 × 10(5) TCID(50) per g of feces and gradually decreased to less than 5% on day 42. The virus concentrations in sewage peaked on days 6 to 8 at approximately 100 PFU per liter, remained high until day 14, and subsequently decreased to less than 10 PFU per liter on day 29. The estimated poliovirus concentration in sewage approximated the measured initial virus excretion in feces, within 1 log(10) variation, resulting in a sensitivity of detection of 100 infected but mostly asymptomatic individuals in tens of thousands of individuals. An additional second peak observed in sewage may indicate secondary transmission missed by enterovirus or AFP surveillance in patients. This enables the detection of circulating poliovirus by environmental surveillance, supporting its feasibility as an early warning system.

Presence of enteric viruses in source waters for drinking water production in The Netherlands.

The quality of drinking water in The Netherlands has to comply with the Dutch Drinking Water Directive: less than one infection in 10,000 persons per year may occur due to consumption of unboiled drinking water. Since virus concentrations in drinking waters may be below the detection limit but entail a public health risk, the infection risk from drinking water consumption requires the assessment of the virus concentrations in source waters and of the removal efficiency of treatment processes. In this study, samples of source waters were taken during 4 years of regular sampling (1999 to 2002), and enteroviruses, reoviruses, somatic phages, and F-specific phages were detected in 75% (range, 0.0033 to 5.2 PFU/liter), 83% (0.0030 to 5.9 PFU/liter), 100% (1.1 to 114,156 PFU/liter), and 97% (0.12 to 14,403 PFU/liter), respectively, of 75 tested source water samples originating from 10 locations for drinking water production. By endpoint dilution reverse transcription-PCR (RT-PCR), 45% of the tested source water samples were positive for norovirus RNA (0.22 to 177 PCR-detectable units [PDU]/liter), and 48% were positive for rotavirus RNA (0.65 to 2,249 PDU/liter). Multiple viruses were regularly detected in the source water samples. A significant correlation between the concentrations of the two phages and those of the enteroviruses could be demonstrated. The virus concentrations varied greatly between 10 tested locations, and a seasonal effect was observed. Peak concentrations of pathogenic viruses occur in source waters used for drinking water production. If seasonal and short-term fluctuations coincide with less efficient or failing treatment, an unacceptable public health risk from exposure to this drinking water may occur.

Seroprevalence and molecular detection of hepatitis E virus in wild boar and red deer in The Netherlands.

To date, sources of hepatitis E virus (HEV) in the Netherlands, including swine and wild boar, have been identified, but no direct attribution to Dutch hepatitis E cases have been demonstrated. Other animal sources may exist. To identify these species, HEV RNA detection by RT-PCR is required, but complicated. A preselection based on serology may be useful. Therefore, wildlife species were studied by serology and molecular methods. Using a species-independent double-antigen sandwich ELISA, HEV-specific antibodies were detected in sera from 12% of 1029 wild boar (Sus scrofa scrofa), in 5% of 38 red deer (Cervus elaphus) and in none of 8 studied roe deer (Capreolus capreolus). Differences in background signals were observed between species and accounted for by fitting finite mixture distributions. HEV RNA was detected in 8% of 106 wild boars, in 15% of 39 red deer and in none of 8 roe deer. In conclusion, HEV was shown to be present in European red deer for the first time. This preselection based on species-independent serological assays may be beneficial to identify new potential animal reservoirs of HEV. The consumption of Dutch undercooked wild boar and red deer meat may lead to human exposure to HEV.

Confirmation of the presence of Trichobilharzia by examination of water samples and snails following reports of cases of cercarial dermatitis.

In case of cercarial dermatitis after exposure to fresh water lakes, managers responsible for bathing water quality wish to confirm the presence of Trichobilharzia, which substantiates taking measures to protect bathers from further exposure. A novel approach, including concentration of suspected water samples by filtration and PCR detection of the parasite, is proposed. This approach has been applied to bathing sites with a history of cercarial dermatitis, sampled from 2005 to 2008. Examination of snails, the standard procedure for confirmation of the parasite's presence, and analysis of water samples, appear to be complementary procedures that enhance the chance of parasite detection in implicated bathing water. Water analysis is particularly valuable when snails cannot be found; it confirmed the presence of Trichobilharzia on 25% of sampling days with reported skin conditions and no snails found. PCR of the ToSau3A repeat directly confirmed the parasite in the water. The application of the combination of analysis of water samples and examination of snails is suggested when cases of (presumptive) cercarial dermatitis are reported or when lakes with a history of cercarial dermatitis are inspected prior to the bathing season, in order to guide interventions to prevent (further) cases of swimmers' itch.

Detection of infectious rotavirus in naturally contaminated source waters for drinking water production.

AIMS: To assess public health risks of rotavirus via drinking water consumption, a cell culture-PCR assay was developed and optimized for the detection of infectious environmental rotavirus strains in naturally contaminated source waters for drinking water production. METHODS AND RESULTS: Infectious rotavirus concentrations were estimated by an optimized cell culture-PCR assay as most probable numbers by using the presence or absence of replicated virus in different sample volumes. Infectious rotavirus was detected in 11 of 12 source water samples in concentrations varying from 0.19 (0.01-0.87) to 8.3 (1.8-34.0) infectious PCR detectable units per litre (IPDU/l), which was not significantly different from the concentrations of infectious enterovirus in these samples. CONCLUSIONS: In 55% of the samples, rotavirus genomes were 1000 to 10 000 times (3 log(10)-4 log(10)) more abundantly present than infectious rotavirus particles, whereas in the remaining 45% of the samples, rotavirus genomes were less than 1000 times (<3 log(10)) more abundantly present. SIGNIFICANCE AND IMPACT OF THE STUDY: The broad variation observed in the ratios of rotavirus RNA and infectious particles demonstrates the importance of detecting infectious viruses instead of viral RNA for the purposes involving estimations of public health risks.

Long-term inactivation study of three enteroviruses in artificial surface and groundwaters, using PCR and cell culture.

Since the transmission of pathogenic viruses via water is indistinguishable from the transmission via other routes and since the levels in drinking water, although significant for health, may be too low for detection, quantitative viral risk assessment is a useful tool for assessing disease risk due to consumption of drinking water. Quantitative viral risk assessment requires information concerning the ability of viruses detected in drinking water to infect their host. To obtain insight into the infectivity of viruses in relation to the presence of virus genomes, inactivation of three different enteroviruses in artificial ground and surface waters under different controlled pH, temperature, and salt conditions was studied by using both PCR and cell culture over time. In salt-peptone medium, the estimated ratio of RNA genomes to infectious poliovirus 1 in freshly prepared suspensions was about 10(0). At 4 degrees C this ratio was 10(3) after 600 days, and at 22 degrees C it was 10(4) after 200 days. For poliovirus 1 and 2 the RNA/infectious virus ratio was higher in artificial groundwater than in artificial surface water, but this was not the case for coxsackievirus B4. When molecular detection is used for virus enumeration, it is important that the fraction of infectious virus (based on all virus genomes detected) decays with time, especially at temperatures near 22 degrees C.

Cercarial dermatitis in the Netherlands caused by Trichobilharzia spp.

Outbreaks of cercarial dermatitis which occurred in recreational lakes in the Netherlands were studied and a method for direct rapid detection of the parasite Trichobilharzia in water samples was developed. A standardized questionnaire with questions on health complaints and exposure was distributed to individuals who developed symptoms of cercarial dermatitis after visiting fresh water lakes. Snails from the suspected lakes were examined for the presence of Trichobilharzia by microscopy and PCR. Water samples were concentrated by filtration and examined by PCR. Water quality was tested according to European Bathing Water Directive 76/160/EEG. Trichobilharzia was detected in snails and water samples from lakes which met European bathing water standards. Despite a response of 25.5%, epidemiological data suggested that longer and more frequent exposure to the water resulted in increased reporting of symptoms of cercarial dermatitis and confirmed the importance of exposure as a risk factor. A novel method for direct detection of Trichobilharzia, which includes concentration of water samples by filtration and detection of the parasite by PCR proved to be a valuable and simple tool for confirmation of presumptive outbreaks, particularly when snails could not be found in the suspected water and public health protecting measures were necessary.

Mixed plaques: statistical evidence how plaque assays may underestimate virus concentrations.

Even at very low concentrations human pathogenic viruses may result in infection and possibly subsequent disease. Ideally, viruses are quantified by use of cell culture assays to determine their infectivity. Plaque assays are common tools for enumeration of viruses in inocula and this process is straightforward when a plaque results from the offspring of a single infectious virus particle. In the course of a study on the usefulness of sewage monitoring for surveillance of polio-virus transmission, sewage samples containing a mixture of two live polio vaccine strains (type 1 and type 3) were analyzed. The total poliovirus concentration in plaque forming units (pfu) was estimated by means of a monolayer plaque assay on L20B cells. Subsequent typing of virus directly by neutralisation of virus from excised plaques revealed the occurrence of plaques containing both type 1 and type 3 virus. This means that there must be plaques that originate from more than one initial infectious virus particle. As a consequence, the estimated virus concentration is incorrect. We present statistical methods that utilize these mixed plaque counts to estimate the concentrations of either virus type in our sewage samples. We can also calculate a correction factor for the error in virus concentration, which would result from equating a pfu to a single infectious particle. Since many quantitative methods in microbiology are based on colony counts, we conclude that such counts should be interpreted with caution, especially when data are used in quantitative microbial risk assessment to estimate the public health impact.

Presence of noroviruses and other enteric viruses in sewage and surface waters in The Netherlands.

Since virus concentrations in drinking waters are generally below the detection limit, the infectious risk from drinking water consumption requires assessment from the virus concentrations in source waters and removal efficiency of treatment processes. In this study, we estimated from reverse transcription-PCR on 10-fold serially diluted RNA that noroviruses, the most prevalent waterborne gastroenteritis agents, were present at 4 (0.2 to 38) to 4,900 (303 to 4.6 x 10(4)) PCR-detectable units (PDU) per liter of river water (ranges are given in parentheses). These virus concentrations are still high compared with 896 to 7,499 PDU/liter of treated sewage and 5,111 to 850,000 PDU/liter in raw sewage. Sequencing analyses designated human norovirus GGII.4 Lordsdale as the most prevalent strain in the sampling period 1998 to 1999 in both sewage and surface waters. Other GGII strains were also very abundant, indicating that the majority of the virus contamination was derived from urban sewage, although very divergent strains and one animal strain were also detected in the surface and sewage waters. Rotaviruses were also detected in two large rivers (the Maas and the Waal) at 57 to 5,386 PDU/liter. The high virus concentrations determined by PCR may in part be explained by the detection of virus RNA instead of infectious particles. Indeed, reoviruses and enteroviruses that can be cultured were present at much lower levels, of 0.3 to 1 and 2 to 10 PFU/liter, respectively. Assuming 1% of the noroviruses and rotaviruses to be infectious, a much higher disease burden than for other viruses can be expected, not only because of the higher levels but also because of these viruses' higher infectivity and attack rates.

Molecular detection of Norwalk-like caliciviruses in sewage.

In this study, Norwalk-like virus (NLV) RNA was detected by reverse transcriptase PCR (RT-PCR) in sewage water concentrates. Sequence analysis of the RT-PCR products revealed identical sequences in stools of patients and related sewage samples. In 6 of 11 outbreak-unrelated follow-up samples, multiple NLV genotypes were present. Levels as high as 10(7) RNA-containing particles per liter were found. These data show that high loads of NLVs may be present in sewage and warrant further studies addressing the efficacy of NLV removal by sewage water treatment processes.