Pathogenic Enteric Viruses Research Articles

Protocol for detection of pathogenic enteric RNA viruses by regular monitoring of environmental samples from wastewater treatment plants using droplet digital PCR

BackgroundThe present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses, explicitly focusing on norovirus genogroup Ⅱ (GⅡ), astrovirus, rotavirus, Aichi virus, sapovirus, hepatitis A and E viruses in wastewater treatment plants through droplet digital PCR (ddPCR). Enteric viruses are of significant public health concern, as they are the leading cause of diseases like gastroenteritis. Regular monitoring of environmental samples, particularly from wastewater treatment plants, is crucial for early detection and control of these viruses. This research aims to improve the understanding of the prevalence and dynamics of enteric viruses in urban India and will serve as a model for similar studies in other regions. Our protocol objective is to establish a novel ddPCR-based methodology for the detection and molecular characterization of enteric viruses present in wastewater samples sourced from Bhopal, India. Our assay is capable of accurately quantifying virus concentrations without standard curves, minimizing extensive optimization, and enhancing sensitivity and precision, especially for low-abundance targets. MethodsThe study involves fortnightly collecting and analyzing samples from nine wastewater treatment plants over two years, ensuring comprehensive coverage and consistent data. Our study innovatively applies ddPCR to simultaneously detect and quantify enteric viruses in wastewater, a more advanced technique. Additionally, we will employ next-generation sequencing for detailed viral genome identification in samples tested positive for pathogenic viruses. ConclusionThis study will aid in understanding these viruses’ genetic diversity and mutation rates, which is crucial for developing tailored intervention strategies. The findings will be instrumental in shaping public health responses and improving epidemiological surveillance, especially in localities heaving sewage networks.

Urban wastewater-based epidemiology for multi-viral pathogen surveillance in the Valencian region, Spain

Wastewater-based epidemiology (WBE) has lately arised as a promising tool for monitoring and tracking viral pathogens in communities. In this study, we analysed WBE's role as a multi-pathogen surveillance strategy to detect the presence of several viral illness causative agents. Thus, an epidemiological study was conducted from October 2021 to February 2023 to estimate the weekly levels of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Respiratory Syncytial virus (RSV), and Influenza A virus (IAV) in influent wastewater samples (n = 69). In parallel, a one-year study (October 2021 to October 2022) was performed to assess the presence of pathogenic human enteric viruses. Besides, monitoring of proposed viral fecal contamination indicators crAssphage and Pepper mild mottle virus (PMMoV) was also assessed, along with plaque counting of somatic coliphages. Genetic material of rotavirus (RV), human astrovirus (HAStV), and norovirus genogroup I (GI) and GII was found in almost all samples, while hepatitis A and E viruses (HAV and HEV) only tested positive in 3.77 % and 22.64 % of the samples, respectively. No seasonal patterns were overall found for enteric viruses, although RVs had a peak prevalence in the winter months. All samples tested positive for SARS-CoV-2 RNA, with a mean concentration of 5.43 log genome copies per liter (log GC/L). The tracking of the circulating SARS-CoV-2 variants of concern (VOCs) was performed by both duplex RT-qPCR and next generation sequencing (NGS). Both techniques reliably showed how the dominant VOC transitioned from Delta to Omicron during two weeks in Spain in December 2021. RSV and IAV viruses peaked in winter months with mean concentrations 6.40 and 4.10 log GC/L, respectively. Moreover, the three selected respiratory viruses strongly correlated with reported clinical data when normalised by wastewater physico-chemical parameters and presented weaker correlations when normalising sewage concentration levels with crAssphage or somatic coliphages titers. Finally, predictive models were generated for each respiratory virus, confirming high reliability on WBE data as an early-warning system and communities illness monitoring system. Overall, this study presents WBE as an optimal tool for multi-pathogen tracking reflecting viral circulation and diseases trends within a selected area, its value as a multi-pathogen early-warning tool stands out due to its public health interest.

Assessing viral freshwater hazard using a toxicokinetic model and Dreissena polymorpha

The detection all pathogenic enteric viruses in water is expensive, time-consuming, and limited by numerous technical difficulties. Consequently, using reliable indicators such as F-specific RNA phages (FRNAPH) can be well adapted to assess the risk of viral contamination of fecal origin in surface waters. However, the variability of results inherent to the water matrix makes it difficult to use them routinely and to interpret viral risk. Spatial and temporal variability of surface waters can lead to underestimate this risk, in particular in the case of low loading. The use of bivalve mollusks as accumulating systems appears as a promising alternative, as recently highlighted with the freshwater mussel Dreissena polymorpha, but its capacity to accumulate and depurate FRNAPH needs to be better understood and described. The purpose of this study is to characterise the kinetics of accumulation and elimination of infectious FRNAPH by D. polymorpha in laboratory conditions, formalised by a toxico-kinetic (TK) mechanistic model. Accumulation and depuration experiments were performed at a laboratory scale to determine the relationship between the concentration of infectious FRNAPH in water and the concentration accumulated by D. polymorpha. The mussels accumulated infectious FRNAPH (3–5.4 × 104 PFU/g) in a fast and concentration-dependent way in only 48 h, as already recently demonstrated. The second exposure demonstrated that the kinetics of infectious FRNAPH depuration by D. polymorpha was independent to the exposure dose, with a T90 (time required to depurate 90 % of the accumulated concentration) of approximately 6 days. These results highlight the capacities of D. polymorpha to detect and reflect the viral pollution in an integrative way and over time, which is not possible with point water sampling. Different TK models were fitted based on the concentrations measured in the digestive tissues (DT) of D. polymorpha. The model has been developed to formalise the kinetics of phage accumulation in mussels tissues through the simultaneous estimation of accumulation and depuration rates. This model showed that accumulation depended on the exposure concentration, while depuration did not. Standardized D. polymorpha could be easily transplanted to the environment to predict viral concentrations using the TK model defined in the present study to predict the level of contamination of bodies of water on the basis of the level of phages accumulated by the organisms. It will be also provide a better understanding of the dynamics of the virus in continental waters at different time and spatial scales, and thereby contribute to the protection of freshwater resources.

A new understanding of somatic coliphages belonging to the Microviridae family in urban wastewater

Somatic coliphages (SC) and F-specific RNA coliphages (FRNAPH) have been included in regulations or guidelines by several developed countries as a way of monitoring water safety and the microbiological quality of shellfish harvesting waters. SC are highly diverse in their morphology, size and genome. The Microviridae family contains three genera of phages (Alphatrevirus, Gequatrovirus, and Sinsheimervirus), all having a capsid of similar morphology (icosahedral) and size (25–30 nm in diameter) to that of common pathogenic enteric viruses. Three PCR assays specific for each genus of Microviridae were designed to study these phages in raw and treated wastewater (WW) in order to gain knowledge about the diversity and prevalence of Microviridae among SC, as well as their inactivation and removal during WW treatments. Among the four wastewater treatment plants (WWTPs) monitored here, two WWTPs applied disinfection by UV light as tertiary treatment. First, we noticed that Microviridae represented 10 to 30 % of infectious SC in both raw and treated WW. Microviridae appeared to behave in the same way as all SC during these WW treatments. As expected, the highest inactivation, at least 4 log10, was achieved for infectious Microviridae and SC in both WWTPs using UV disinfection. PCR assays showed that the highest removal of Microviridae reached about 4 log10, but the phage removal can vary greatly between WWTPs using similar treatments. This work forms the basis for a broader evaluation of Microviridae as a viral indicator of water treatment efficiency and WW reuse.

An extended colloid filtration theory for modeling Escherichia coli transport in 3-D fracture networks

Microbial transport in fractured carbonate rock using enhanced solutions is a significant and neglected research topic in the literature. We propose an extended colloid filtration theory (CFT) combined with a particle-tracking following streamlines (PTFS) model for the rapid prediction of breakthrough curves (BTCs) and plumes of pathogens in three-dimensional (3-D) discrete fracture networks (DFNs). We adapted CFT in porous media to pathogen transport in fractures containing Terra Rossa (soil) deposits. As an example of the model capability, a simulation was used to predict the 3-D motion field and Escherichia coli count in groundwater originating from the Forcatella managed aquifer recharge (MAR) Facility (Brindisi, Italy) using a DFN composed of 3,900 fractures. In arid regions, MAR facilities are significant for sustaining basic human needs, such as freshwater supply for drinking and crop production. The Markov chain Monte Carlo (MCMC) technique was applied to E. coli counts in the collected water samples to increase data representativeness. The pathogen transport coefficients were further supported by batch filtration tests carried out in the CNR/IRSA Laboratory (Bari, Italy). The mean E. coli attachment rate coefficient of 0.15 × 10−8 m2 d–1 (sticking efficiency = 1.1 × 10−8 m) resulted in a 2.1 log10 removal in 600 m of reclaimed water filtration. The simulation output visualized the E. coli 3-D pathways in groundwater and the positions of contaminated groundwater spring outflows on Forcatella Beach. The simulation results agreed with the mean MCMC output of E. coli concentrations in bathing water under unperturbed geochemical and environmental flow and transport conditions. However, results indicate that concentrations of pathogenic strains, parasites, and enteric viruses may enter the marine environment of MAR sites during flood periods.

Toward better monitoring of human noroviruses and F-specific RNA bacteriophages in aquatic environments using bivalve mollusks and passive samplers: A case study.

Monitoring pathogenic enteric viruses in continental and marine water bodies is essential to control the viral contamination of human populations. Human Noroviruses (NoV) are the main enteric viruses present in surface waters and foodstuff. In a context of global change, it is currently a challenge to improve the management of viral pollutions in aquatic environments and thereby limit the contamination of vulnerable water bodies or foodstuffs. The aim of this study is to evaluate the potential of specific accumulation systems for improving the detection of NoV in water bodies, compared to direct water analyses. Passive samplers (Zetapor filters) and three species of bivalve molluscan shellfish (BMS) (Dreissena polymorpha, Mytilus edulis and Crassostreas gigas) were used as accumulation systems to determine their performance in monitoring continental and marine waters for viruses. F-specific RNA bacteriophages (FRNAPH) were also analyzed since they are described as indicators of NoV hazard in many studies. During a one-year study in a specific area frequently affected by fecal pollution, twelve campaigns of exposure of passive samplers and BMS in continental and coastal waters were conducted. Using suitable methods, NoV (genome) and FRNAPH (infectious and genome) were detected in these accumulation systems and in water at the same time points to determine the frequency of detection but also to gain a better understanding of viral pollution in this area. The reliability of FRNAPH as a NoV indicator was also investigated. Our results clearly showed that BMS were significantly better than passive samplers and direct water analyses for monitoring NoV and FRNAPH contamination in water bodies. A dilution of viral pollution between the continental and the coastal area was observed and can be explained by the distance from the source of the pollution. Viral pollution is clearly greater during the winter period, and stakeholders should take this into consideration in their attempts to limit the contamination of food and water. A significant correlation was once again shown between NoV and FRNAPH genomes in BMS, confirming the reliability of FRNAPH as a NoV indicator. Moreover, a strong correlation was observed between NoV genomes and infectious FRNAPH, suggesting recent viral pollution since infectious particles had not been inactivated at sufficient levels in the environment. More generally, this study shows the value of using BMS as an active method for improving knowledge on the behavior of viral contamination in water bodies, the ranking of the contamination sources, and the vulnerability of downstream water bodies.

Community intervention trial for estimating risk of acute gastrointestinal illness from groundwater-supplied non-disinfected drinking water.

By community intervention in 14 non-disinfecting municipal water systems, we quantified sporadic acute gastrointestinal illness (AGI) attributable to groundwater. Ultraviolet (UV) disinfection was installed on all supply wells of intervention communities. In control communities, residents continued to drink non-disinfected groundwater. Intervention and control communities switched treatments by moving UV disinfection units at the study midpoint (crossover design). Study participants (n = 1,659) completed weekly health diaries during four 12-week surveillance periods. Water supply wells were analyzed monthly for enteric pathogenic viruses. Using the crossover design, groundwater-borne AGI was not observed. However, virus types and quantity in supply wells changed through the study, suggesting that exposure was not constant. Alternatively, we compared AGI incidence between intervention and control communities within the same surveillance period. During Period 1, norovirus contaminated wells and AGI attributable risk from well water was 19% (95% CI, -4%, 36%) for children <5 years and 15% (95% CI, -9%, 33%) for adults. During Period 3, echovirus 11 contaminated wells and UV disinfection slightly reduced AGI in adults. Estimates of AGI attributable risks from drinking non-disinfected groundwater were highly variable, but appeared greatest during times when supply wells were contaminated with specific AGI-etiologic viruses.

Molecular analysis of canine circovirus in dogs from animal shelters in Belém, Pará, northern Brazil: first detection at the amazon region.

The canine circovirus (CanCV) is a single-stranded DNA virus that has become an important emerging virus associated with gastroenteritis in dogs worldwide. In the present study, the CanCV was detected by PCR in 15% (22/147) of dogs from animal shelters in Belém, between 2019 and 2020. We observed an association between the CanCV infection and the presence of diarrhea in animals younger than one year of age (p > 0.01). The Brazilian strains were grouped in Chinese genotypes, with 99.54 to 100% nucleotilde homology. The GMRF Bayesian Skyride used the molecular clock model, which was the best suited technique to plot the dataset. The most recent common ancestor (TMRCA) was estimated in 2017, with the evolution rate of 1.6 x 10-3 s/s/y. The viral family diversity was also investigated, with emphasis on the families of the enteric pathogenic viruses Parvoviridae, Picornaviridae and Astroviridae, which were detected in the CanCV positive pooled samples. This study highlights the importance of the CanCV as an emergent virus that causes diarrhea in Brazilian dogs. The results found herein contribute to the understanding of the role of CanCV in enteric diseases and in the evolutionary molecular characterization of the circulating genotypes. Furthermore, we increased the understanding of the fecal virome in dogs with diarrhea, providing data for the monitoring and prevention viral gastroenteric diseases in domestic animals.

Identification of enteroviruses along Lake Victoria shoreline - a potential indicator of sewage pollution.

E n t e r i c v i r u s e s a r e m a i n l y t r a n s m i t t e d b y t h e f a e c a l - o r a l r o u t e a n d h a v e b e e n l i n k e d t o s e v e r a l d i s e a s e s i n c l u d i n g g a s t r o e n t e r i t i s a n d r e s p i r a t o r y i n f e c t i o n s . T h e i r p r e s e n c e i n s u r f a c e w a t e r s h a s b e e n exacerbated by p o l l u t i o n f r o m a v a r i e t y o f p o i n t s o u r c e s s u c h a s s e w a g e d i s c h a r g e . W e s t u d i e d t h e occurrence o f e n t e r o v i r u s e s i n w a t e r s a m p l e s f r o m L a k e V i c t o r i a i n K e n y a t o i n v e s t i g a t e i f t h e r e w a s a l i n k b e t w e e n s e w a g e p o l l u t i o n a n d d e t e c t i o n o f e n t e r o v i r u s e s ( E V s ) t o b u i l d a b a s e l i n e f o r a n enteric viruses monitoring platform for this region. We analysed 216 samples collected over 6 months from six different locations along the Homa Bay Pier. The six sampling locations comprised of three sites (P3, P5, P6) located <500 m from a local sewage treatment plant and pit latrines while three other sites (P1, P2, P4) were located at approximately 0.5 to 3 Km. EVs were concentrated using glass wool adsorption elution protocol and identified using the nested reverse transcription-polymerase chain reaction. The odds ratio was performed to determine whether the location of the sources of sewage pollution near the lake was associated with the EVs contamination. Five out of 108 (5 %) samples collected from the sites (P3, P5 and P6 were EV positive, while 2 % (2/108) of samples from P1, P2 and P4 were EV positive. The presence of the EVs was associated with the distance from the possible sources of faecal contamination (odds ratio 20.28 and 4.86, confidence interval 2.42, and 0.95) for pit latrines and the sewage treatment plant respectively. The result from this study indicates that sewage discharge at the shoreline of Lake Victoria may have been the source of EVs contamination. Data from this study could significantly contribute to informing risk management on sewage pollution in Lake Victoria and it is important to continue monitoring this lake for potentially pathogenic enteric viruses.

Sediment re-suspension as a potential mechanism for viral and bacterial contaminants

Pathogenic enteric viruses and bacteria tend to occur in higher concentrations and survive longer in aquatic sediments than suspended in the water column. Re-suspension of these organisms can result in a significant degradation of overlying water quality. Additionally, the re-suspension of microbial pathogens in artificial irrigation canals could endanger the consumption of fresh and ready-to-eat produce. Irrigation water has been implicated in numerous fresh produce outbreaks over the last 30 years. This study aimed to quantify the proportions of bacterial and viral re-suspension from sediment in a recirculating flume with varying velocities. MS2 coliphage and Escherichia coli were found to re-suspend at rates that were not significantly different, despite organism size differences. However, E. coli re-suspension rates from sand and clay were significantly different. This suggests that likely sediment-associated particles were recovered with the organisms attached. Similar re-suspension rates are hypothesized to be due to the dynamics of sediment transport, rather than that of the organisms themselves. This study also indicated that the re-suspension of sediment at very low velocities (e.g., less than 10 cm/s), could impact the microbiological quality of the overlaying water. Results from this study conclude that sediment could be a viable mechanism for irrigation water contamination.

Structural Organizations of Qβ and MS2 Phages Affect Capsid Protein Modifications by Oxidants Hypochlorous Acid and Peroxynitrite.

Pathogenic enteric viruses and bacteriophages such as Qβ and MS2 are transmitted through the fecal-oral route. However, oxidants such as peroxynitrite (ONOOH) and hypochlorous acid (HClO) can prevent new infection by inactivating infectious viruses. Their virucidal effect is well recognized, and yet predicting the effects of oxidants on viruses is currently impossible because the detailed mechanisms of viral inactivation remain unclear. Our data show that ONOOH and HClO cross-linked the capsid proteins and RNA genomes of Qβ and MS2 phages. Consistently, the capsids appeared intact by transmission electron microscopy (TEM) even when 99% of the phages were inactivated by oxidation. Moreover, a precise molecular study of the capsid proteins shows that ONOOH and HClO preferentially targeted capsid protein regions containing the oxidant-sensitive amino acid C, Y, or W. Interestingly, the interaction of these amino acids was a crucial parameter defining whether they would be modified by the addition of O, Cl, or NO2 or whether it induced the loss of the protein region detected by mass spectrometry, together suggesting potential sites for cross-link formation. Together, these data show that HClO and ONOOH consistently target oxidant-sensitive amino acids regardless of the structural organization of Qβ and MS2, even though the phenotypes change as a function of the interaction with adjacent proteins/RNA. These data also indicate a potential novel mechanism of viral inactivation in which cross-linking may impair infectivity.

Co-contamination of food products from family farms in an environmental disaster area in Southeast Brazil with pathogenic bacteria and enteric viruses.

In the present study, we evaluated the degree of contamination of fresh vegetables, cheeses and jellies from disaster area in Brazil with bacteria and enteric viruses. Food samples (n = 350) were tested for Escherichia coli, Salmonella spp., Listeria monocytogenes, Staphylococcus spp., and enteric viruses (rotavirus A (RVA), human adenovirus (HAdV), hepatitis A virus (HAV), and human norovirus (HNoV). E. coli was present in 56% of the samples, Salmonella spp. was present in 14% of the samples, L. monocytogenes and Staphylococcus spp. (coagulase-positive) were present in 36% of the samples. The enteric viruses RVA and HAdV were detected in cheeses and vegetables.

Detection of potentially pathogenic enteric viruses in environmental samples from Kenya using the bag-mediated filtration system.

Enteric virus environmental surveillance via a highly sensitive method is critical, as many enteric viruses have low infectious doses and can persist in the environment for extended periods. This study determined the potential of the novel bag-mediated filtration system (BMFS) to recover human enteric viruses and pepper mild mottle virus (PMMoV) from wastewater and wastewater-impacted surface waters, examined PMMoV use as a fecal contamination indicator in Kenya, and identified potential BMFS process controls. From April 2015 to April 2016, BMFS samples were collected from seven sites in Kenya (n = 59). Enteroviruses and PMMoV were detected in 100% of samples, and human adenovirus, human astrovirus, hepatitis A virus, norovirus GI, norovirus GII, sapovirus, and human rotavirus were detected in the majority of samples. The consistent detection of enteroviruses and PMMoV suggests that these viruses could be used as indicators in similarly fecally contaminated sites and BMFS process controls. As contamination of surface water sources remains a global issue, enteric virus environmental surveillance is necessary. This study demonstrates an effective way to sample large volumes of wastewater and wastewater-impacted surface waters for the detection of multiple enteric viruses simultaneously.

Molecular detection of human adenovirus in urban wastewater in Egypt and among children suffering from acute gastroenteritis.

Incidence of enteric viruses in sewage, the efficacy of wastewater treatment plants to remove these viruses, and health effects from their release into the surface water are very important environmental issues in the microbiology field. One of the most pathogenic enteric viruses is adenovirus which can cause a serious disease such as gastroenteritis with low grade fever and mild dehydration in humans. In this study we performed qualitative polymerase chain reaction (PCR) analysis of HAdV on 60 stool samples from children with acute gastroenteritis admitted to Abu-Rish hospital and 96 environmental samples (32 raw sewage, 32 treated sewage, 32 sewage sludge) collected from Zenin wastewater treatment plant (WWTP). HAdV were detected in 17 (28.3%) of stool, 27 (84.4%) of raw sewage, 16 (50%) of treated sewage and 25 (78%) of sludge samples. The viral concentrations were in the range of 2.02 × 106-7.23 × 106, 8.7 × 105-4.3 × 106, 1.22 × 104-3.7 × 106 and 1.48 × 106-1.77 × 107 GC/mL in stool, raw sewage, treated sewage, and sludge, respectively. HAdV was detected throughout the whole year of sample collection. Moreover, our results suggested that males were more susceptible to adenovirus infections than females. The results indicate that the high incidence of HAdV in the treated sewage may cause adverse health effects.

Vomiting symptom of acute gastroenteritis estimated from epidemiological data can help predict river contamination by human pathogenic enteric viruses

Contamination of fresh water bodies by human enteric viruses from wastewater discharge is a well-established phenomenon. Here we propose a model of viral contamination of rivers based on acute gastroenteritis epidemiology and assess how well it can simulate in situ experimental monitoring. Noroviruses, rotaviruses, enteroviruses, adenoviruses and hepatitis A viruses were quantified by molecular methods after water concentration. Water flows were obtained from the Hydro databank and wastewater treatment plant (WWTP) data. Acute gastroenteritis cases based on medical prescriptions were recorded by the French public health agency. We estimated the total number of daily viral acute gastroenteritis cases and modeled virus shedding and fate in WWTPs and rivers. Simulated virus concentrations were compared to the weighted sum of measured concentrations. Seasonal variations in viral acute gastroenteritis were predicted from vomiting occurrence. All viruses except hepatitis A virus were widely detected in wastewaters and river, in concentrations reaching 10+6 genome copies·L−1 for adenoviruses in the Artière River. We were able to predict virus load in raw wastewater and in the Artière River. Estimated weighting coefficients showed the high impact of noroviruses GII. This model can thus serve to compare water treatment, discharge and reuse scenarios.

Viral tools for detection of fecal contamination and microbial source tracking in wastewater from food industries and domestic sewage

Alternative indicators may be more suitable than thermotolerant coliform bacteria to assess enteric virus pollution in environmental waters and their removal from wastewaters. In this study, F-specific RNA bacteriophages (F-RNAPh) showed to be potential viral indicators of fecal contamination when they were quantified from domestic and food-industrial effluents containing human, chicken, swine or bovine wastes. In addition, they showed to be resistant to the primary and secondary treatments of the wastewater treatment plants. The viable F-RNAPh count showed correlation with viable thermotolerant coliforms but also with human polyomaviruses (HPyV) quantified by a new molecular method. In domestic effluents, F-RNAPh and HPyV indicators significantly correlated with a human viral pathogen, norovirus, while the bacterial indicator did not, being then better predictors of the behavior of enteric pathogenic viruses. In addition, we assessed human, bovine and fowl microbial source tracking markers, based on the molecular detections of human polyomavirus, bovine polyomavirus, and fowl adenovirus, respectively. The techniques implemented extend the range of viruses detected, since they target different viral types simultaneously. These markers could be applied when multiple source pollution is suspected, contributing to making decisions on public health interventions.

Quantification of human adenovirus and norovirus in river water in the north-east of France.

Human adenoviruses (HAdVs) are a major cause of infection and have been proposed as viral indicators of water quality. Human noroviruses (NoV) are the main cause of viral acute gastroenteritis. Quantitative data on the environmental prevalence of both viruses are needed. The genomes of HAdVs enteric adenovirus type 41 (HAdV41) and noroviruses of genogroups I and II (NoV GGI and GGII) were quantified over a 6-month period in a river located in north-eastern France. The samples were collected downstream from the discharge of a wastewater treatment plant. The viruses were concentrated using a glass wool method and the viral genomes were quantified using digital droplet PCR (ddPCR). All river water samples (15/15) were positive for the genomes of HAdVs, HAdV41, NoV GGI and NoV GGII. Concentrations of HAdVs, HAdV41 and NoV GII genomes were similar and HAdV41 represented ~ 80% of HAdVs. Infectious HAdVs were quantified in these samples using an integrated cell culture-quantitative PCR method (ICC-qPCR); they were detected in 93% (14/15) and quantified in 53% (8/15) of the samples. Thus, infectious HAdVs represented 0.3 to 12.2% of total HAdV particles detected by ddPCR. Infectious HAdV41 particles were found in 73% (11/15) of the samples. This common presence of pathogenic enteric viruses underlines the impact of wastewater discharge on quality of surface waters and may constitute a threat for human health. The relative abundance of genome of HAdV41 underlines the need for studies focusing on the specific detection of its infectious forms along water cycle.

Evaluation of the activity of disinfectants against coliform bacteria group strains isolated from a sewage treatment plant (ETE-Ipanema)

Municipal wastewater usually presents pathogenic enteric bacteria, viruses and intestinal parasites. Primary, secondary and tertiary wastewater treatment eliminates 90-99.9% of these microorganisms and if a more efficient reduction of microorganisms is needed, disinfection with chemical compounds must be done. The extraordinary genetic variability of bacteria gives them adaptation ability against theses chemical agents, which indiscriminate use brings about serious health problems. In this study, chemical agents commonly used in disinfection procedures like chlorhexidine gluconate, sodium hypochlorite and quaternary ammonium compounds were investigated for their activity against bacteria from the coliform group, isolated from a sewage treatment plant, using the macrodilution method. It was observed that the addition of organic matter interferes in sensitivity levels and also that the lower the concentration and the exposure time, the higher the resistance. Sodium hypochlorite was the most effective against the coliforms, once inhibited their growth in every exposure time and at every disinfectant concentration used . Quaternary ammonium compounds and chlorhexidine gluconate were also effective, although a higher percentage of resistance was observed for both.

Relationship between Enteric Pathogen and Acute Gastroenteritis Disease Severity: A Prospective Cohort Study

Background: Acute gastroenteritis is one of the top reasons children are brought for emergency department care. Little is known about the association between specific pathogens and disease severity in children with vomiting/diarrhea in the outpatient setting. Recent advances in diagnostic approaches enabling the simultaneous detection of common pathogenic enteric bacteria, viruses, and parasites have become readily available. Clinicians can request testing be performed to rapidly and comprehensively identify etiologic pathogens. While such knowledge can be used to optimize therapy, it also has the potential to predict disease severity. Such knowledge can aid clinical …

Persistent Norovirus Contamination of Groundwater Supplies in Two Waterborne Outbreaks.

Microbiological contamination of groundwater supplies causes waterborne outbreaks worldwide. In this study, two waterborne outbreaks related to microbiological contamination of groundwater supplies are described. Analyses of pathogenic human enteric viruses (noroviruses and adenoviruses), fecal bacteria (Campylobacter spp. and Salmonella spp.), and indicator microbes (E. coli, coliform bacteria, intestinal enterococci, Clostridium perfringens, heterotrophic plate count, somatic and F-specific coliphages) were conducted in order to reveal the cause of the outbreaks and to examine the effectiveness of the implemented management measures. Moreover, the long-term persistence of noro- and adenovirus genomes was investigated. Noroviruses were detected in water samples from both outbreaks after the intrusion of wastewater into the drinking water sources. In the outbreak I, the removal efficiency of norovirus genome (3.0 log10 removal) in the sand filter of onsite wastewater treatment system (OWTS) and during the transport through the soil into the groundwater well was lower than the removal efficiencies of E. coli, coliform bacteria, intestinal enterococci, and spores of C. perfringens (6.2, 6.0,>5.9, and>4.8 log10 removals, respectively). In the outbreak II, cleaning of massively contaminated groundwater well and drinking water distribution network proved challenging, and noro- and adenovirus genomes were detected up to 3months (108days). The long-term persistence study showed that noro- and adenovirus genomes can remain detectable in the contaminated water samples up to 1277 and 1343days, respectively. This study highlights the transport and survival properties of enteric viruses in the environment explaining their potency to cause waterborne outbreaks.