Domestic hot-water boilers harbour active thermophilic bacterial communities distinctly different from those in the cold-water supply.

Running cold and hot water in buildings is a widely established commodity. However, interests regarding hygiene and microbiological aspects had so far been focussed on cold water. Little attention has been given to the microbiology of domestic hot-water installations (DHWIs), except for aspects of pathogenic Legionella. World-wide, regulations consider hot (or warm) water as 'heated drinking water' that must comply (cold) drinking water (DW) standards. However, the few reports that exist indicate presence and growth of microbial flora in DHWIs, even when supplied with water with disinfectant residual. Using flow cytometric (FCM) total cell counting (TCC), FCM-fingerprinting, and 16S rRNA-gene-based metagenomic analysis, the characteristics and composition of bacterial communities in cold drinking water (DW) and hot water from associated boilers (operating at 50 - 60 °C) was studied in 14 selected inhouse DW installations located in Switzerland and Austria. A sampling strategy was applied that ensured access to the bulk water phase of both, supplied cold DW and produced hot boiler water. Generally, 1.3- to 8-fold enhanced TCCs were recorded in hot water compared to those in the supplied cold DW. FCM-fingerprints of cold and corresponding hot water from individual buildings indicated different composition of cold- and hot-water microbial floras. Also, hot waters from each of the boilers sampled had its own individual FCM-fingerprint. 16S rRNA-gene-based metagenomic analysis confirmed the marked differences in composition of microbiomes. E.g., in three neighbouring houses supplied from the same public network pipe each hot-water boiler contained its own thermophilic bacterial flora. Generally, bacterial diversity in cold DW was broad, that in hot water was restricted, with mostly thermophilic strains from the families Hydrogenophilaceae, Nitrosomonadaceae and Thermaceae dominating. Batch growth assays, consisting of cold DW heated up to 50 - 60 °C and inoculated with hot water, resulted in immediate cell growth with doubling times between 5 and 10 h. When cold DW was used as an inoculum no significant growth was observed. Even boilers supplied with UVC-treated cold DW contained an actively growing microbial flora, suggesting such hot-water systems as autonomously operating, thermophilic bioreactors. The generation of assimilable organic carbon from dissolved organic carbon due to heating appears to be the driver for growth of thermophilic microbial communities. Our report suggests that a man-made microbial ecosystem, very close to us all and of potential hygienic importance, may have been overlooked so far. Despite consumers having been exposed to microbial hot-water flora for a long time, with no major pathogens so far been associated specifically with hot-water usage (except for Legionella), the role of harmless thermophiles and their interaction with potential human pathogens able to grow at elevated temperatures in DHWIs remains to be investigated.

Co-incidence of BA.1 and BA.2 at the start of Singapore's Omicron wave revealed by Community and University Campus wastewater surveillance.

Wastewater surveillance (WWS) has been globally recognised to be a useful tool in quantifying SARS-CoV-2 RNA at the community and residential levels without biases associated with case-reporting. The emergence of variants of concern (VOCs) have given rise to an unprecedented number of infections even though populations are increasingly vaccinated. This is because VOCs have been reported to possess higher transmissibility and can evade host immune responses. The B.1.1.529 lineage (Omicron) has severely disrupted global plans to return to normalcy. In this study, we developed an allele-specific (AS) RT-qPCR assay which simultaneously targets the stretch of deletions and mutations in the spike protein from position 24-27 for quantitative detection of Omicron BA.2. Together with previous assays that detect mutations associated with Omicron BA.1 (deletion at position 69 and 70) and all Omicron (mutation at position 493 and 498), we report the validation and time series of these assays from September 2021 to May 2022 using influent samples from two wastewater treatment plants and across four University campus sites in Singapore. Viral RNA concentrations at the treatment plants corroborate with locally reported clinical cases, AS RT-qPCR assays revealed co-incidence of Omicron BA.1 and BA.2 on 12 January 2022, almost two months after initial BA.1 detection in South Africa and Botswana. BA.2 became the dominant variant by the end of January 2022 and completely displaced BA.1 by mid-March 2022. University campus sites were similarly positive for BA.1 and/or BA.2 in the same week as first detection at the treatment plants, where BA.2 became rapidly established as the dominant lineage within three weeks. These results corroborate clinical incidence of the Omicron lineages in Singapore and indicate minimal silent circulation prior to January 2022. The subsequent simultaneous spread of both variant lineages followed strategic relaxation of safe management measures upon meeting nationwide vaccination goals.

Contextualizing Wastewater-Based surveillance in the COVID-19 vaccination era.

SARS-CoV-2 wastewater-based surveillance (WBS) offers a tool for cost-effective oversight of a population's infections. In the past two years, WBS has proven to be crucial for managing the pandemic across different geographical regions. However, the changing context of the pandemic due to high levels of COVID-19 vaccination warrants a closer examination of its implication towards SARS-CoV-2 WBS. Two main questions were raised: 1) Does vaccination cause shedding of viral signatures without infection? 2) Does vaccination affect the relationship between wastewater and clinical data? To answer, we review historical reports of shedding from viral vaccines in use prior to the COVID-19 pandemic including for polio, rotavirus, influenza and measles infection and provide a perspective on the implications of different COVID-19 vaccination strategies with regard to the potential shedding of viral signatures into the sewershed. Additionally, we reviewed studies that looked into the relationship between wastewater and clinical data and how vaccination campaigns could have affected the relationship. Finally, analyzing wastewater and clinical data from the Netherlands, we observed changes in the relationship concomitant with increasing vaccination coverage and switches in dominant variants of concern. First, that no vaccine-derived shedding is expected from the current commercial pipeline of COVID-19 vaccines that may confound interpretation of WBS data. Secondly, that breakthrough infections from vaccinated individuals contribute significantly to wastewater signals and must be interpreted in light of the changing dynamics of shedding from new variants of concern.

The one health perspective to improve environmental surveillance of zoonotic viruses: lessons from COVID-19 and outlook beyond.

The human population has doubled in the last 50 years from about 3.7 billion to approximately 7.8 billion. With this rapid expansion, more people live in close contact with wildlife, livestock, and pets, which in turn creates increasing opportunities for zoonotic diseases to pass between animals and people. At present an estimated 75% of all emerging virus-associated infectious diseases possess a zoonotic origin, and outbreaks of Zika, Ebola and COVID-19 in the past decade showed their huge disruptive potential on the global economy. Here, we describe how One Health inspired environmental surveillance campaigns have emerged as the preferred tools to monitor human-adjacent environments for known and yet to be discovered infectious diseases, and how they can complement classical clinical diagnostics. We highlight the importance of environmental factors concerning interactions between animals, pathogens and/or humans that drive the emergence of zoonoses, and the methodologies currently proposed to monitor them-the surveillance of wastewater, for example, was identified as one of the main tools to assess the spread of SARS-CoV-2 by public health professionals and policy makers during the COVID-19 pandemic. One-Health driven approaches that facilitate surveillance, thus harbour the potential of preparing humanity for future pandemics caused by aetiological agents with environmental reservoirs. Via the example of COVID-19 and other viral diseases, we propose that wastewater surveillance is a useful complement to clinical diagnosis as it is centralized, robust, cost-effective, and relatively easy to implement.

Monitoring human arboviral diseases through wastewater surveillance: Challenges, progress and future opportunities.

Arboviral diseases are caused by a group of viruses spread by the bite of infected arthropods. Amongst these, dengue, Zika, west nile fever and yellow fever cause the greatest economic and social impact. Arboviral epidemics have increased in frequency, magnitude and geographical extent over the past decades and are expected to continue increasing with climate change and expanding urbanisation. Arboviral prevalence is largely underestimated, as most infections are asymptomatic, nevertheless existing surveillance systems are based on passive reporting of loosely defined clinical syndromes with infrequent laboratory confirmation. Wastewater-based surveillance (WBS), which has been demonstrated to be useful for monitoring diseases with significant asymptomatic populations including COVID19 and polio, could be a useful complement to arboviral surveillance. We review the current state of knowledge and identify key factors that affect the feasibility of monitoring arboviral diseases by WBS to include viral shedding loads by infected persons, the persistence of shed arboviruses and the efficiency of their recovery from sewage. We provide a simple model on the volume of wastewater that needs to be processed for detection of arboviruses, in face of lower arboviral shedding rates. In all, this review serves to reflect on the key challenges that need to be addressed and overcome for successful implementation of arboviral WBS.

Rapid displacement of SARS-CoV-2 variant Delta by Omicron revealed by allele-specific PCR in wastewater.

On November 26, 2021, the B.1.1.529 COVID-19 variant was classified as the Omicron variant of concern (VOC). Reports of higher transmissibility and potential immune evasion triggered flight bans and heightened health control measures across the world to stem its distribution. Wastewater-based surveillance has demonstrated to be a useful complement for clinical community-based tracking of SARS-CoV-2 variants. Using design principles of our previous assays that detect SARS-CoV-2 variants (Alpha and Delta), we developed an allele-specific RT-qPCR assay which simultaneously targets the stretch of mutations from Q493R to Q498R for quantitative detection of the Omicron variant in wastewater. We report their validation against 10-month longitudinal samples from the influent of a wastewater treatment plant in Italy. SARS-CoV-2 RNA concentrations and variant frequencies in wastewater determined using these variant assays agree with clinical cases, revealing rapid displacement of the Delta variant by the Omicron variant within three weeks. These variant trends, when mapped against vaccination rates, support clinical studies that found the rapid emergence of SARS-CoV-2 Omicron variant being associated with an infection advantage over Delta in vaccinated persons. These data reinforce the versatility, utility and accuracy of these open-sourced methods using allele-specific RT-qPCR for tracking the dynamics of variant displacement in communities through wastewater for informed public health responses.

Making waves: Wastewater surveillance of SARS-CoV-2 in an endemic future.

Wastewater-based surveillance (WBS) has been widely used as a public health tool to monitor the emergence and spread of SARS-CoV-2 infections in populations during the COVID-19 pandemic. Coincident with the global vaccination efforts, the world is also enduring new waves of SARS-CoV-2 variants. Reinfections and vaccine breakthroughs suggest an endemic future where SARS-CoV-2 continues to persist in the general population. In this treatise, we aim to explore the future roles of wastewater surveillance. Practically, WBS serves as a relatively affordable and non-invasive tool for mass surveillance of SARS-CoV-2 infection while minimizing privacy concerns, attributes that make it extremely suited for its long-term usage. In an endemic future, the utility of WBS will include 1) monitoring the trend of viral loads of targets in wastewater for quantitative estimate of changes in disease incidence; 2) sampling upstream for pinpointing infections in neighborhoods and at the building level; 3) integrating wastewater and clinical surveillance for cost-efficient population surveillance; and 4) genome sequencing wastewater samples to track circulating and emerging variants in the population. We further discuss the challenges and future developments of WBS to reduce inconsistencies in wastewater data worldwide, improve its epidemiological inference, and advance viral tracking and discovery as a preparation for the next viral pandemic.

Mega festivals like MahaKumbh, a largest mass congregation, facilitated the transmission of SARS-CoV-2 to humans and endangered animals via contaminated water.

Our surrounding environment has been influenced by the COVID-19 pandemic situation. The second wave of COVID-19 in India has proven to be more devastating and aggressive than the first wave of the pandemic, which led to recognizing India as one of the world's topmost worst-hit nations considering >4000 fatalities reported in a single day in May 2021. Such "resurgence and acceleration" of COVID-19 transmission has been fuelled by the MahaKumbh festival and political mass gathering (elections rallies) events, where the COVID-19 protocols have been ignored by millions of pilgrims/followers. The present review discusses only the consequences of this year's MahaKumbh festivals, the largest religious mass gathering on earth, which was held during the COVID-19 pandemic in India, and its impact on both the spread of SARS-CoV-2 among participants and their families and its influence on the quality of the river Ganga. This article tries to give readers outside of India an overview of how much impact of any such single large gathering of any relgion in any part of the world can drive coronavirus infections and effectively commence the second/third wave outbreak with this case study. Furthermore, the religious large scale celebration are widely accepted through out the world that have played a significant role in the spread of the pandemic into remote villages and towns all over the subcontinent/world, thus affecting many areas with insufficient healthcare facilities that have been relatively spared. This review also highlights the potential risk of transmission from infected humans into the aquatic environment of the river Ganga. Besides the obvious relevance of SARS-CoV-2, a large variety of other water-related disease vectors (bacteria, viruses, and protozoa) stemming from visitors to the religious congregation were introduced into the upstream regions of the Ganga river. Their sheer number is assumed to have had a severe influence on its delicate ecosystem, including endangered mammals such as the river Dolphins. The detailed epidemiological and clinical study on transmission routes of SARS-CoV-2 is the need of the hour to understand the pathogenesis of RNA virus infection and prevent the massive spreading of such infectious respiratory diseases. An interdisciplinary approach, rooted in evidence-based efficient learning, contextual strategies, and a streamlined unified approach should be adopted to help in the development of a proactive prevention model during future MahaKumbh festival (and similar religious gatherings) instead of just "picking up the pieces" in a conventional post-event model.

Pathogen performance testing of a natural swimming pool using a cocktail of microbiological surrogates and QMRA-derived management goals.

In recent decades, natural swimming pools (NSPs) have gained popularity in Europe, especially in Germany and Austria. NSPs differ from swimming pools in that they utilize biological treatment processes based on wetland processes with no disinfection residual. However, data are missing on the specific log-reduction performance of NSPs to address enteric virus, bacteria, and parasitic protozoa removal considered necessary to meet the North American risk-based benchmark (<35 illnesses per 1,000 swimming events) set by the USEPA for voluntary swimming. In this study, we examined Canada's first NSP at Borden Park, Edmonton, Canada, to address the following three questions: (1) Given normal faecal shedding rates by bathers, what is the total log reduction (TLR) theoretically needed to meet the EPA benchmark? (2) what is the in-situ performance of the NSP based on spiking suitable microbial surrogates (MS2 coliphage, Enterococcus faecalis, and Saccharomyces cerevisiae [Baker's yeast])? and (3) how much time is required to reach acceptable bather risk levels under different representative volume-turnover rates? A reverse-quantitative microbial risk assessment (QMRA) revealed that of the four reference pathogens selected (Norovirus, Campylobacter, Cryptosporidium, and Giardia), only Norovirus was estimated to exceed the risk benchmark at the 50th, 75th, and 95th percentiles, while Campylobacter was the only other reference pathogen to exceed at the 95th percentile. Log-reduction values (LRVs) were similar to previous reports for bacterial indicators, and novel LRVs were estimated for the other two surrogates. A key finding was that more than 24 h treatment time would be necessary to provide acceptable bather protection following heavy bather use (378 bathers/day for main pool and 26 bathers/day for children's pool), due to the mixing dynamics of the treated water diluting out possible residual pool faecal contamination. The theoretical maximum number of people in the pool per day to be below USEPA's 35 gastro cases in 1,000 swimming events was 113, 47, and 8, at the 50th, 75th, and 95th percentiles. Further, the use of ultra-violet disinfection to the pool return flow had little effect on reducing the treatment time required.

Characteristics and Injury Patterns of Road Traffic Injuries in Urban and Rural Uganda-A Retrospective Medical Record Review Study in Two Hospitals.

In the ongoing Second Decade of Action for Road Safety, road traffic crashes pose a considerable threat especially in low-income countries. Uganda shows a vast burden of non-fatal injuries and resides at the top range of countries with the highest death rates due to unsafe roads. However, little is known about the differences in road traffic associated injuries between urban and rural areas and potential influence factors. Here, we used a cross-sectional study conducted by a retrospective medical record review from trauma cases admitted in 2016 to hospitals in rural and urban areas in Uganda. Injury severity scores were calculated and descriptive analysis was carried out while multivariate logistic regression was applied to assess significant covariates. According to the 1683 medical records reviewed, the mean age of trauma patients in the dataset under investigation was 30.8 years with 74% male. The trauma in-hospital mortality was 4% while prevalence of traumatic injuries is 56.4%. Motorcycle users (49.6%) and pedestrians (33.7%) were identified as the most vulnerable groups in both urban and rural setting while mild injuries of extremities (61.6%) and the head/neck-region (42.0%) were registered most. The frequency of road traffic injuries was homogenous in the urban and rural hospitals investigated in this study; interventions should therefore be intensified ubiquitously. The identification of significant differences in road traffic crash and injury characteristics provides the opportunity for specific programmes to decrease the socio-economic and health burden of unsafe roads. In addition to law enforcement and introduction of a Systems Thinking approach to road safety including infrastructural and educational concepts, the strengthening of trauma care and health resources is recommended.

Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications - A systematic review.

Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-one recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009-2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 µM; incubation on ice or at room temperature (20 - 25 °C) for 5-120 min; and dye activation using LED or high light (500-800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever.

Global Water, Sanitation and Hygiene Research Priorities and Learning Challenges under Sustainable Development Goal 6.

MOTIVATION: Sanitation and Water for All (SWA) is a global partnership addressing challenges to universal water, sanitation, and hygiene (WaSH) access. Shortly following adoption of the United Nations' Sustainable Development Goals, the Research and Learning (R&L) constituency of SWA undertook a systematic study to determine global research priorities and learning needs. PURPOSE: We aimed to identify priority topics where improved knowledge would aid achievement of Goal 6, by developing a global WaSH research agenda, and to describe evidence-use challenges among WaSH professionals. APPROACH AND METHODS: We delivered a tailored, semi-structured electronic questionnaire to representatives from countries, R&L institutions, and other SWA partners (external support agencies, civil society, and private sector). The survey gathered views from 76 respondents working in an estimated 36 countries across all world regions. Data were analyzed quantitatively and qualitatively to identify patterns and themes. FINDINGS: Most responses indicated lowered confidence on at least one Goal 6 target area, especially managing untreated wastewater and faecal sludge. Both brief and lengthy information formats were valued. WaSH information was perceived as conflicting or unreliable among non-R&L constituencies, suggesting differences in perceptions and information-seeking approaches. While the R&L constituency appeared saturated with learning and training opportunities, others perceived barriers to participating (e.g. not receiving notice or invitation). Research and other WaSH activities were frequently constrained by upward accountability to funders, while stakeholders were inconsistently included in research processes. POLICY IMPLICATIONS: This study offers insight into perceived research and decision challenges related to Goal 6 targets. It develops a unified research agenda focused on high priority topics, and recommends renewed attention to evidence synthesis, learning and implementation support, research engagement, and multisectoral coordination.

Evaluating Microbial and Chemical Hazards in Commercial Struvite Recovered from Wastewater.

Controlled struvite (NH4MgPO4·6H2O) precipitation has become a well-known process for nutrient recovery from wastewater treatment systems to alleviate the pressures of diminishing, finite rock phosphate reservoirs. Nonetheless, coprecipitation of potential microbial and chemical hazards is poorly understood. On the other hand, antimicrobial resistance (AMR) is a major global public health concern and wastewater is thought to disseminate resistance genes within bacteria. Fecal indicator bacteria (FIB) are typically used as measures of treatment quality, and with multiresistant E. coli and Enterococcus spp. rising in concern, the quantification of FIB can be used as a preliminary method to assess the risk of AMR. Focusing on struvite produced from full-scale operations, culture and qPCR methods were utilized to identify FIB, antibiotic resistance genes, and human enteric viruses in the final product. Detection of these hazards occurred in both wet and dry struvite samples indicating that there is a potential risk that needs further consideration. Chemical and biological analyses support the idea that the presence of other wastewater components can impact struvite formation through ion and microbial interference. While heavy metal concentrations met current fertilizer standards, the presence of K, Na, Ca, and Fe ions can impact struvite purity yet provide benefit for agricultural uses. Additionally, the quantified hazards detected varied among struvite samples produced from different methods and sources, thus indicating that production methods could be a large factor in the risk associated with wastewater-recovered struvite. In all, coprecipitation of metals, fecal indicator bacteria, antimicrobial resistance genes, and human enteric viruses with struvite was shown to be likely, and future engineered wastewater systems producing struvite may require additional step(s) to manage these newly identified public health risks.

A bioassay-based protocol for chemical neutralization of human faecal wastes treated by physico-chemical disinfection processes: A case study on benzalkonium chloride.

In situ physico-chemical disinfection of high risk faecal waste is both effective and widely used as a sanitation management strategy for infection prevention and control. Systematic tests where the performance of alternative physico-chemical disinfection methods is systematically compared and optimized must be based on reliable protocols. These protocol are currently not adequately addressing the neutralization related issues: the neutralization of the tested disinfectant after specified conditions of concentration and contact time (CT) is necessary to prevent continued disinfection after the intended contact time; moreover such neutralization is often necessary in practice and on a large scale to prevent adverse health and ecological impacts from remaining disinfectant after the target CT is achieved. Few studies adequately assess the extent of neutralization of the chemical disinfectant and are intended to optimize on-site disinfection practices for waste matrices posing high microbial risks. Hence, there is a need for effective and reproducible neutralization protocols in chemical disinfection trials and practice. Furthermore, for most of chemical disinfectants used in healthcare settings there is no practical methodology to reliably and conveniently measure the residual disinfectant concentration after its neutralization and also determine the optimum concentration of the neutralizer. Because some neutralizing compounds can themselves be toxic to the test microorganisms, it is necessary to optimize neutralization procedures in disinfection experiments for the development of infection control practices using accepted positive control microbes. In the presented work, a stepwise bioassay-based protocol using representative faecal indicator microbes is described for optimizing chemical disinfection and subsequent disinfectant neutralization of any infectious faecal waste matrix. The example described is for the quaternary ammonium compound benzalkonium chloride and its recommended chemical neutralizer in a high strength human faecal waste matrix.

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.

Relative Abundance of Human Bocaviruses in Urban Sewage in Greater Cairo, Egypt.

Human bocavirus (HBoV) is predominantly found in the respiratory tract infections and in the stool of patients with gastroenteritis symptoms. However, data on the prevalence of HBoV genotypes in environmental samples are limited. Here we addressed the prevalence of HBoV in sewage collected from three different wastewater treatment plants in Egypt. HBoV-1, HBoV-2, and HBoV-3 were detected, whereas none of the samples were positive for HBoV-4. The median concentration of HBoV in influent samples was 8.5 × 103 GC/l for HBoV-1, 3.0 × 104 GC/l for HBoV-2, and 2.5 × 104 GC/l for HBoV-3. The concentration was reduced but not completely removed in the effluent samples. The median concentration in the outlet samples was 2.9 × 103 GC/l for HBoV-1, 4.1 × 103 GC/l for HBoV-2, and 2.1 × 103 GC/l for HBoV-3. Moreover, no seasonality pattern of HBoVs was found. The high incidence of HBoV in sewage samples provided an evidence of its circulation in the local population. Although the role of HBoV in respiratory or gastro-intestinal infections still remains to be fully elucidated, the risk of infection via contaminated water should be taken into consideration.

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.