Ana Maria de Roda Husman

Fate and transport of surface water pathogens in watersheds

Pathogens present in animal fecal deposits excreted to land undergo a poorly defined process of dispersion, transport or attenuation, and inactivation. The transport of pathogens overland in surface runoff is clearly responsible for event-related increases in the concentrations of in-stream waterborne pathogens in many watersheds. However, there are significant knowledge gaps concerning the precise mechanisms of pathogen transport. This article reviews the fate and transport of pathogens in watersheds supplying drinking water, from their deposition in feces and septic seepages on land to their dispersion in major tributaries. Pathogens considered representative of those associated with waterborne disease included enteric viruses derived from human fecal contamination, bacterial pathogens represented by Escherichia coli O157:H7, and the protozoan pathogens Cryptosporidium and Giardia. References to suitable model and index organisms for these pathogens are described. The key processes determining the fate and transport of pathogens within watersheds are discussed in the context of changing agricultural practices, climate, and scale factors. A generic conceptual model for watershed processes is described in light of the knowledge gaps identified from this review. Future areas for fundamental research were identified and included: (1) inactivation kinetics of pathogens in soil and fecal matrices; (2) characterization of the particle sizes with which pathogens are transported; (3) characterization of pathogen properties and watershed-specific features that affect terrestrial transport and attenuation; and (4) the inactivation and sedimentation of pathogens during their initial introduction to the aquatic environment. Such information is critical to advance the assessment of pathogen total maximum daily loads (TMDL), determining management priorities and appropriate control points, as well as integrating pathogens within the broader watershed hydrologic models.

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.

Sources of Hepatitis E Virus Genotype 3 in the Netherlands

Four subtypes have been detected, and pathogenicity, zoonotic potential, or stability may vary between subtypes.

Non-travel related Hepatitis E virus genotype 3 infections in the Netherlands; A case series 2004 – 2006

BackgroundHuman hepatitis E virus (HEV) infections are considered an emerging disease in industrialized countries. In the Netherlands, Hepatitis E virus (HEV) infections have been associated with travel to high-endemic countries. Non-travel related HEV of genotype 3 has been diagnosed occasionally since 2000. A high homology of HEV from humans and pigs suggests zoonotic transmission but direct molecular and epidemiological links have yet to be established. We conducted a descriptive case series to generate hypotheses about possible risk factors for non-travel related HEV infections and to map the genetic diversity of HEV.MethodsA case was defined as a person with HEV infection laboratory confirmed (positive HEV RT-PCR and/or HEV IgM) after 1 January 2004, without travel to a high-endemic country three months prior to onset of illness. For virus identification 148 bp of ORF2 was sequenced and compared with HEV from humans and pigs. We interviewed cases face to face using a structured questionnaire and collected information on clinical and medical history, food preferences, animal and water contact.ResultsWe interviewed 19 cases; 17 were male, median age 50 years (25–84 y), 12 lived in the North-East of the Netherlands and 11 had preexisting disease. Most common symptoms were dark urine (n = 16) and icterus (n = 15). Sixteen ate pork ≥ once/week and six owned dogs. Two cases had received blood transfusions in the incubation period. Seventeen cases were viremic (genotype 3 HEV), two had identical HEV sequences but no identified relation. For one case, HEV with identical sequence was identified from serum and surface water nearby his home.ConclusionThe results show that the modes of transmission of genotype-3 HEV infections in the Netherlands remains to be resolved and that host susceptibility may play an important role in development of disease.

Calicivirus Inactivation by Nonionizing (253.7-Nanometer-Wavelength [UV]) and Ionizing (Gamma) Radiation

ABSTRACT Noroviruses (previously Norwalk-like viruses) are the most common viral agents associated with food- and waterborne outbreaks of gastroenteritis. In the absence of culture methods for noroviruses, animal caliciviruses were used as model viruses to study inactivation by nonionizing (253.7-nm-wavelength [UV]) and ionizing (gamma) radiation. Here, we studied the respiratory feline calicivirus (FeCV) and the presumed enteric canine calicivirus (CaCV) and compared them with the well-studied bacteriophage MS2. When UV irradiation was used, a 3-log10 reduction was observed at a fluence of 120 J/m2 in the FeCV suspension and at a fluence of 200 J/m2 for CaCV; for the more resistant phage MS2 there was a 3-log10 reduction at a fluence of 650 J/m2. Few or no differences were observed between levels of UV inactivation in high- and low-protein-content virus stocks. In contrast, ionizing radiation could readily inactivate MS2 in water, and there was a 3-log10 reduction at a dose of 100 Gy, although this did not occur when the phage was diluted in high-protein-content stocks of CaCV or FeCV. The low-protein-content stocks showed 3-log10 reductions at a dose of 500 Gy for FeCV and at a dose of 300 for CaCV. The inactivation rates for both caliciviruses with ionizing and nonionizing radiation were comparable but different from the inactivation rates for MS2. Although most FeCV and CaCV characteristics, such as overall particle and genome size and structure, are similar, the capsid sequences differ significantly, making it difficult to predict human norovirus inactivation. Adequate management of UV and gamma radiation processes for virus inactivation should limit public health risks.

Cluster of Cases of Acute Hepatitis Associated with Hepatitis E Virus Infection Acquired in The Netherlands

Increasing evidence suggests that hepatitis E virus (HEV) infection may occur in developed countries and that swine may act as a reservoir. We report a cluster of 2 confirmed cases and 1 presumptive case of hepatitis associated with HEV. The typed strain from 1 case was related to HEV strains found in North America and Europe, and it was also related to a cluster of swine HEV strains found in The Netherlands. Our findings indicate that locally acquired HEV infections in industrialized countries may be overlooked. Routine testing for HEV infection in patients with acute hepatitis in The Netherlands should be considered before a diagnosis of autoimmune hepatitis is reached and steroid therapy is initiated.

The course of hepatitis E virus infection in pigs after contact-infection and intravenous inoculation.

BackgroundWorldwide, hepatitis E virus (HEV) genotype 3 is observed in pigs and transmission to humans is implied. To be able to estimate public health risks from e.g. contact with pigs or consumption of pork products, the transmission routes and dynamics of infection should be identified. Hence, the course of HEV-infection in naturally infected pigs should be studied.ResultsTo resemble natural transmission, 24 HEV-susceptible pigs were infected either by one-to-one exposure to intravenously inoculated pigs (C1-pigs; n = 10), by one-to-one exposure to contact-infected pigs (C2-pigs: n = 7; C3-pigs: n = 5) or due to an unknown non-intravenous infection route (one C2-pig and one C3-pig). The course of HEV-infection for contact-infected pigs was characterized by: faecal HEV RNA excretion that started at day 7 (95% confidence interval: 5–10) postexposure and lasted 23 (19–28) days; viremia that started after 13 (8–17) days of faecal HEV RNA excretion and lasted 11 (8–13) days; antibody development that was detected after 13 (10–16) days of faecal HEV RNA excretion. The time until onset of faecal HEV RNA excretion and onset of viremia was significantly shorter for iv-pigs compared to contact-infected pigs, whereas the duration of faecal HEV RNA excretion was significantly longer. At 28 days postinfection HEV RNA was detected less frequently in organs of contact-infected pigs compared to iv-pigs. For contact-infected pigs, HEV RNA was detected in 20 of 39 muscle samples that were proxies for pork at retail and in 4 of 7 urine samples.ConclusionThe course of infection differed between infection routes, suggesting that contact-infection could be a better model for natural transmission than iv inoculation. Urine and meat were identified as possible HEV-sources for pig-to-pig and pig-to-human HEV transmission.

Exposure assessment for swimmers in bathing waters and swimming pools.

Bathing water compliant with bathing water legislation may nevertheless contain pathogens to such a level that they pose unacceptable health risks for swimmers. Quantitative Microbiological Risk Assessment (QMRA) can provide a proper basis for protective measures, but the required data on swimmer exposure are currently limited or lacking. The objective of this study was to collect exposure data for swimmers in fresh water, seawater and swimming pools, i.e. volume of water swallowed and frequency and duration of swimming events. The study related to swimming in 2007, but since the summer of 2007 was wet and this might have biased the results regarding surface water exposure, the study was repeated relating to swimming in 2009, which had a dry and sunny summer. Exposure data were collected through questionnaires administered to approximately 19 000 persons representing the general Dutch population. Questionnaires were completed by 8000 adults of whom 1924 additionally answered the questions for their eldest child (< 15 years). The collected data did not differ significantly between 2007 and 2009. The frequency of swimming and the duration of swimming were different for men, women and children and between water types. Differences between men and women were small, but children behaved differently: they swam more often, stayed in the water longer, submerged their heads more often and swallowed more water. Swimming pools were visited most frequently (on average 13-24 times/year) with longest duration of swimming (on average 67-81 min). On average, fresh and seawater sites were visited 6-8 times/year and visits lasted 41-79 min. Dependent on the water type, men swallowed on average 27-34 ml per swimming event, women 18-23 ml, and children 31-51 ml. Data on exposure of swimmers to recreational waters could be obtained by using a questionnaire approach in combination with a test to measure mouthfuls of water for transformation of categorical data to numerical data of swallowed volumes of water. Previous assumptions on swimmer exposure were replaced with estimates of exposure parameters, thus reducing uncertainty in assessing the risk of infection with waterborne pathogens and enabling identification of risk groups. QMRA for Cryptosporidium and Giardia was demonstrated based on data from previous studies on the occurrence of these pathogens in recreational lakes and a swimming pool.

Isolation and detection of enterovirus RNA from large-volume water samples by using the NucliSens miniMAG system and real-time nucleic acid sequence-based amplification.

ABSTRACT Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.