PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies.

Study of Shellfish Growing Area During Normal Harvesting Periods and Following Wastewater Overflows in an Urban Estuary With Complex Hydrography.

Viral testing combined with hydrographic studies is considered standard good practice in determining microbiological impacts on shellfish growing areas following wastewater overflows. In this study, norovirus genogroup I and II, indicators of viral contamination (F-RNA bacteriophage genogroup II (F-RNA GII), crAssphage, pepper mild mottle virus) and Escherichia coli were monitored during periods of normal harvesting and following overflows in two commercial shellfish growing areas in Otago Harbour (Aotearoa New Zealand). Dye tracing, drogue tracking and analysis of particle tracking modelling were also undertaken to assess the dispersion, dilution and time of travel of wastewater discharged from a pump station discharge that impacts the growing areas. Norovirus was not detected in any of the 218 shellfish samples tested. PMMoV and crAssphage were more prevalent than F-RNA GII as determined by RT-qPCR. The dye study indicated long residence time of the waters (≥5 days) in the embayment impacted by the discharge. No relationships were found between the concentrations of viral indicators or E. coli and wastewater dilution, distance between the discharge and the growing areas or time since the last overflow. For the three spills studied (≤327 m3), there was little evidence of microbiological impact on the growing areas. This was likely associated with a deep shipping channel that enhances water flushing in the harbour and reduces contaminant transport to the growing areas. We recommend flexibility in the approach for closure/reopening growing areas impacted by spills, particularly for small duration/volume spills and when norovirus is not present in the community.

Persistence of Infectious Human Norovirus in Estuarine Water.

Norovirus is the predominant cause of viral acute gastroenteritis globally. While person-to-person is the most reported transmission route, norovirus is also associated with waterborne and foodborne illness, including from the consumption of contaminated bivalve molluscan shellfish. The main cause of shellfish contamination is via the bioaccumulation of norovirus from growing waters impacted by human wastewater. However, data on the persistence of infectious norovirus in the environment are limited due to a lack of a human norovirus culture method in the past. In this study, we applied the recently established method of norovirus replication in human intestinal enteroids to determine the persistence of norovirus in artificial estuarine water at 25 ppt for up to 21 days at 4 °C and 16 °C in the dark. Infectious norovirus was detected for up to 21 days. The relative infectivity declined from 100 to 3% at day 21, with decay rate constants of 0.07 day-1 at 4 °C and 0.17 day-1 at 16 °C. There was no decrease in norovirus titres as measured by reverse transcription-droplet digital PCR (RT-ddPCR), confirming the lack of the relationship between norovirus infectivity and direct detection by PCR. The results confirm that norovirus can remain infectious for at least 3 weeks in an estuarine water environment, presenting associated health risks.

Canadian perspectives in multiple myeloma on the use of steroids in clinical practice based on patient and healthcare provider interviews.

Corticosteroid (steroid) medications are associated with challenging adverse effects that can negatively impact patient quality of life. However, owing to a long legacy of effective use in treatment protocols, they remain a cornerstone of multiple myeloma (MM) care. We conducted a roundtable with Canadian healthcare providers (HCPs) with diverse healthcare backgrounds and involvement in MM care as well as with patients with MM. Our goal was to develop clear guidance for steroid management aimed at improving patient quality of life, taking into account patient perspective and experiences with managing the disease. Our recommendations, which are based on the insights acquired from this discussion, can be categorized to the following areas: steroid prescribing, dosing, and modifications; managing adverse effects; and patient-HCP communication. These recommendations can be used by the entire multi-disciplinary hematology team to improve patient quality of life while being treated with steroid medication for multiple myeloma.

A Framework for Public Health Authorities to Evaluate Health Determinants for Wastewater-Based Epidemiology.

BACKGROUND: Wastewater-based epidemiology (WBE) is rapidly developing as a powerful public health tool. It can provide information about a wide range of health determinants (HDs), including community exposure to environmental hazards, trends in consumption of licit and illicit substances, spread of infectious diseases, and general community health. As such, the list of possible candidate HDs for WBE is almost limitless. Consequently, a means to evaluate and prioritize suitable candidates for WBE is useful, particularly for public health authorities, who often face resource constraints. OBJECTIVES: We have developed a framework to assist public health authorities to decide what HDs may be appropriate for WBE and what biomarkers could be used. This commentary reflects the experience of the authors, who work at the interface of research and public health implementation. DISCUSSION: To be suitable for WBE, a candidate HD should address a public health or scientific issue that would benefit from better understanding at the population level. For HDs where information on individual exposures or stratification by population subgroups is required, WBE is less suitable. Where other methodologies are already used to monitor the candidate HD, consideration must be given to whether WBE could provide better or complementary information to the current approach. An essential requirement of WBE is a biomarker specific for the candidate HD. A biomarker in this context refers to any human-excreted chemical or biological that could act as an indicator of consumption or exposure to an environmental hazard or of the human health state. Suitable biomarkers should meet several criteria outlined in this commentary, which requires background knowledge for both the biomarker and the HD. An evaluation tree summarizing key considerations for public health authorities when assessing the suitability of candidate HDs for WBE and an example evaluation are presented. https://doi.org/10.1289/EHP11115.

Reductions of human enteric viruses in 10 commonly used activated carbon, polypropylene and polyester household drinking-water filters.

Drinking-water treatment in non-networked rural communities relies on the use of point-of-use (PoU) household filters. Source waters treated by PoU filters are often microbially contaminated, but information about human enteric virus reductions in these filters is limited. This study evaluated human rotavirus, adenovirus and norovirus reductions in 10 commonly used, new PoU carbon, polypropylene and polyester microfilters. The viruses were spiked into chlorine-free tap water (pH 8.0, ionic strength 1.22 mM), and 3 sequential challenge tests were conducted in each filter under a constant flow rate of 1 L/min. In most of the filters investigated, the norovirus and adenovirus reductions were similar (P > 0.49). Compared with the norovirus and adenovirus reductions, the rotavirus reductions were significantly lower in the carbon filters (P ≤ 0.009), which may relate to rotavirus's higher zeta potential and lower hydrophobicity. Virus reductions appeared to be dictated by the filter media type through electrostatic and hydrophobic interactions; the effects of filter media pore sizes on virus reductions via physical size-exclusion were very limited. The virus reductions in the carbon filters were significantly greater than those in the polypropylene and polyester filters (P ≤ 0.0001), and they did not differ significantly between the polypropylene and polyester filters (P > 0.24). None of the filters met the "protective" rotavirus reduction level (≥3 log10) required for household drinking-water treatment. Our study's findings highlight a critical need for additional water treatment when using PoU microfilters, for example, water boiling or ultraviolet radiation, or the use of effective surface-modified filter media to prevent drinking-waterborne infections from enteric viruses.

Sensitivity of wastewater-based epidemiology for detection of SARS-CoV-2 RNA in a low prevalence setting.

To assist public health responses to COVID-19, wastewater-based epidemiology (WBE) is being utilised internationally to monitor SARS-CoV-2 infections at the community level. However, questions remain regarding the sensitivity of WBE and its use in low prevalence settings. In this study, we estimated the total number of COVID-19 cases required for detection of SARS-CoV-2 RNA in wastewater. To do this, we leveraged a unique situation where, over a 4-month period, all symptomatic and asymptomatic cases, in a population of approximately 120,000, were precisely known and mainly located in a single managed isolation and quarantine facility (MIQF) building. From 9 July to 6 November 2020, 24-hr composite wastewater samples (n = 113) were collected daily from the sewer outside the MIQF, and from the municipal wastewater treatment plant (WWTP) located 5 km downstream. New daily COVID-19 cases at the MIQF ranged from 0 to 17, and for most of the study period there were no cases outside the MIQF identified. SARS-CoV-2 RNA was detected in 54.0% (61/113) at the WWTP, compared to 95.6% (108/113) at the MIQF. We used logistic regression to estimate the shedding of SARS-CoV-2 RNA into wastewater based on four infectious shedding models. With a total of 5 and 10 COVID-19 infectious cases per 100,000 population (0.005% and 0.01% prevalence) the predicated probability of SARS-CoV-2 RNA detection at the WWTP was estimated to be 28 and 41%, respectively. When a proportional shedding model was used, this increased to 58% and 87% for 5 and 10 cases, respectively. In other words, when 10 individuals were actively shedding SARS-CoV-2 RNA in a catchment of 100,000 individuals, there was a high likelihood of detecting viral RNA in wastewater. SARS-CoV-2 RNA detections at the WWTP were associated with increasing COVID-19 cases. Our results show that WBE provides a reliable and sensitive platform for detecting infections at the community scale, even when case prevalence is low, and can be of use as an early warning system for community outbreaks.

Imitation of ß-lactam binding enables broad-spectrum metallo-ß-lactamase inhibitors.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

Application of crAssphage, F-RNA phage and pepper mild mottle virus as indicators of human faecal and norovirus contamination in shellfish.

Shellfish growing waters contaminated with inadequately treated human wastewater is a major source of norovirus in shellfish and poses a significant human health risk to consumers. Microbial source tracking (MST) markers have been widely used to identify the source (s) of faecal contamination in water but data are limited on their use for shellfish safety. This study evaluated the source specificity, sensitivity, occurrence and concentration of three viral MST markers i.e. cross-assembly phage (crAssphage), F-specific RNA bacteriophage genogroup II (F-RNA phage GII) and pepper mild mottle virus (PMMoV) using animal faeces (n = 119; 16 animal groups), influent wastewater (n = 12), effluent wastewater (n = 16) and shellfish (n = 33). CrAssphage, F-RNA phage GII and PMMoV had source specific values of 0.97, 0.99 and 0.91, respectively. The sensitivity of MST markers was confirmed by their 100% detection frequency in influent wastewaters. The frequency of detection in effluent wastewater ranged from 81.3% (F-RNA phage GII) to 100% (PMMoV). Concentration of F-RNA phage GII was one log10 (influent wastewater) and 2-3 log10 (effluent wastewater) lower than crAssphage and PMMoV, respectively. Despite lower prevalence of F-RNA phage GII in oysters and mussels compared to crAssphage and PMMoV, concentrations of the three MST markers were similar in mussels. As an indicator of norovirus contamination in shellfish, crAssphage and PMMoV had greater predictive sensitivity (100%; [95% CI; 81.5%-100%)]) and F-RNA phage GII had greater predictive specificity (93.3%; [95% CI; 68.1%-99.8%]). In contrast, crAssphage and F-RNA phage GII have similar accuracy for predicting norovirus in shellfish, however, PMMoV significantly overestimated its presence. Therefore, a combination of crAssphage and F-RNA phage GII analysis of shellfish could provide a robust estimation of the presence of human faecal and norovirus contamination.

Global Trends in Norovirus Genotype Distribution among Children with Acute Gastroenteritis.

Noroviruses are a leading cause of acute gastroenteritis (AGE) among adults and children worldwide. NoroSurv is a global network for norovirus strain surveillance among children <5 years of age with AGE. Participants in 16 countries across 6 continents used standardized protocols for dual typing (genotype and polymerase type) and uploaded 1,325 dual-typed sequences to the NoroSurv web portal during 2016-2020. More than 50% of submitted sequences were GII.4 Sydney[P16] or GII.4 Sydney[P31] strains. Other common strains included GII.2[P16], GII.3[P12], GII.6[P7], and GI.3[P3] viruses. In total, 22 genotypes and 36 dual types, including GII.3 and GII.20 viruses with rarely reported polymerase types, were detected, reflecting high strain diversity. Surveillance data captured in NoroSurv enables the monitoring of trends in norovirus strains associated childhood AGE throughout the world on a near real-time basis.

A multi-platform metabolomics approach to identify possible biomarkers for human faecal contamination in Greenshell™ mussels (Perna canaliculus).

Bivalve molluscs have the potential to bioaccumulate microbial pathogens including noroviruses from aquatic environments and as such, there is a need for a rapid and cheap in-situ method for their detection. Here, we characterise the tissue-specific response of New Zealand Greenshell™ mussels (Perna canaliculus) to faecal contamination from two different sources (municipal sewage and human faeces). This is done with the view to identify potential biomarkers that could be further developed into low cost, rapid and sensitive in-situ biosensors for human faecal contamination detection of mussels in growing areas. Tissue-specific metabolic profiles from gills, haemolymph and digestive glands were analysed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Clear differentiation of metabolic profiles was observed among treatments in each tissue type. Overall, energy pathways such as glycolysis, citrate cycle and oxidative phosphorylation were downregulated across the three mussel tissues studied following simulated contamination events. Conversely, considerable sterol upregulation in the gills was observed after exposure to contamination. Additionally, free pools of nucleotide phosphates and the antioxidant glutathione declined considerably post-exposure to contamination in gills. These results provide important insights into the tissue-specific metabolic effects of human faecal contamination in mussels. This study demonstrates the utility of metabolomics as a tool for identifying potential biomarkers in mussels.

Attenuation and transport of human enteric viruses and bacteriophage MS2 in alluvial sand and gravel aquifer media-laboratory studies.

Potable groundwater contamination by human enteric viruses poses serious health risks. Our understanding of virus subsurface transport has largely depended on studying bacteriophages as surrogates. Few studies have compared the transport behaviour of enteric viruses, especially norovirus, with phage surrogates. We conducted laboratory column experiments to investigate norovirus and bacteriophage MS2 (MS2) filtration in alluvial sand, and rotavirus, adenovirus and MS2 filtration in alluvial gravel aquifer media in 2 mM NaCl (pH 6.6-6.9) with pore velocities of 4.6-5.4 m/day. The data were analysed using colloid filtration theory and HYDRUS-1D 2-site attachment-detachment modelling. Norovirus removal was somewhat lower than MS2 removal in alluvial sand. The removal of rotavirus and adenovirus was markedly greater than MS2 removal in alluvial gravel. These findings concurred with the log10 reduction values, mass recoveries, attachment efficiencies and irreversible deposition rate constants. The modelling results suggested that the MS2 detachment rates were in the same order of magnitude as norovirus, but they were 1 order of magnitude faster than those of rotavirus and adenovirus. The attachment of viruses and MS2 was largely reversible with faster detachment than attachment rates, favouring free virus transport. These findings highlight the risk associated with continual virus transport through subsurface media if viruses are not inactivated and remobilising previously attached viruses could trigger contamination events. Thus, virus attachment reversibility should be considered in virus transport predictions in subsurface media. Further research is needed to compare surrogates with enteric viruses, especially norovirus, regarding their transport behaviours under different experimental conditions.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.

Synthesis and Structure-Activity Relationships of N-(4-Benzamidino)-Oxazolidinones: Potent and Selective Inhibitors of Kallikrein-Related Peptidase 6.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) and 34 ((5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) have single-digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32, effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.

Current and Emerging Technologies for the Detection of Norovirus from Shellfish.

Reports of norovirus infections associated with the consumption of contaminated bivalve molluscan shellfish negatively impact both consumers and commercial shellfish operators. Current virus recovery and PCR detection methods can be expensive and time consuming. Due to the lack of rapid, user-friendly and onsite/infield methods, it has been difficult to establish an effective virus monitoring regime that is able to identify contamination points across the production line (i.e., farm-to-plate) to ensure shellfish quality. The focus of this review is to evaluate current norovirus detection methods and discuss emerging approaches. Recent advances in omics-based detection approaches have the potential to identify novel biomarkers that can be incorporated into rapid detection kits for onsite use. Furthermore, some omics techniques have the potential to simultaneously detect multiple enteric viruses that cause human disease. Other emerging technologies discussed include microfluidic, aptamer and biosensor-based detection methods developed to detect norovirus with high sensitivity from a simple matrix. Many of these approaches have the potential to be developed as user-friendly onsite detection kits with minimal costs. However, more collaborative efforts on research and development will be required to commercialize such products. Once developed, these emerging technologies could provide a way forward that minimizes public health risks associated with shellfish consumption.

Evaluation of pepper mild mottle virus as an indicator of human faecal pollution in shellfish and growing waters.

Bivalve molluscan shellfish grown in areas impacted by human faecal pollution are at risk of being contaminated with multiple enteric viruses. To minimise the public health risks associated with shellfish consumption, determining the presence of faecal contamination in shellfish and their growing waters is crucial. In this study, we evaluated the use of pepper mild mottle virus (PMMoV) as an indicator of human faecal contamination in oysters, mussels, cockles and shellfish growing waters in New Zealand. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR) the presence, and where applicable, the concentration of PMMoV was determined in faeces from 11 different animal species, influent (untreated) wastewater, shellfish and shellfish growing waters. Non-human faecal samples (from seagull, Canada goose, black swan and dog) were RT-qPCR positive for PMMoV. The faecal source specificity of PMMoV was 0.83 (maximum value of 1) when 'detected but not quantifiable' (DNQ) values were used. However, when 'lower limit of quantification' (LLOQ) values were used, the specificity increased to 0.92. The PMMoV concentration in influent wastewater (n = 10) ranged from 6.3 to 7.7 log10 genome copies (GC)/L with a mean (±standard deviation) of 7.1 ±â€¯0.5 log10 GC/L. The overall occurrence of PMMoV in shellfish and shellfish growing waters from four different areas was 46/51 (90%) and 29/52 (56%), respectively. Of the cockles collected from an area known to be impacted by effluent wastewater, 14/14 (100%) contained PMMoV concentrations above the LLOQ. In contrast, only 13/37 (35%) shellfish and 6/52 (11.5%) growing water samples collected from three areas with low anthropogenic impact contained PMMoV concentrations above the LLOQ. The high concentration of PMMoV in influent wastewater indicates that PMMoV may be a promising indicator of human faecal contamination. The presence of PMMoV in shellfish and growing waters with a low anthropogenic impact may be of avian origin, and this needs to be considered if using PMMoV for monitoring shellfish and shellfish growing water quality in New Zealand.

Suboptimal performance of rotavirus testing in a vaccinated community population should prompt laboratories to review their rotavirus testing algorithms in response to changes in disease prevalence.

Rotavirus vaccine has reduced disease prevalence in many countries. Consequently, we aimed to assess the reliability of a rotavirus immunoassay in the community population of Auckland and Northland, New Zealand. Between 22 October 2015 and 31 December 2016, 2873 fecal samples were tested by enzyme immunoassay (EIA, Rotascreen II, Microgen, UK) from 2748 patients (median age 8 years, range 0-101 years). Eighty-nine (3.1%) samples were reactive; 86 samples were tested by a second method. Rotavirus was confirmed in 49/86 (57%). Positive rotavirus EIAs were more likely to be confirmed in samples from cases ≥1 year of age (positive predictive value [PPV] 61%, 95% confidence interval [CI] 50-72%, P = 0.049) and in spring/summer (PPV 67%, 95% CI 55-78%, P = 0.003). Reactive rotavirus tests required confirmatory testing regardless of demographic, vaccine, or seasonal factors; a review of rotavirus testing algorithms may be necessary in other vaccinated community populations.

Recombinant GII.P16/GII.4 Sydney 2012 Was the Dominant Norovirus Identified in Australia and New Zealand in 2017.

For the past two decades, norovirus pandemic variants have emerged every 3⁻5 years, and dominate until they are replaced by alternate strains. However, this scenario changed in 2016 with the co-circulation of six prevalent viruses, three of which possessed the pandemic GII.4 Sydney 2012 capsid. An increased number of institutional gastroenteritis outbreaks were reported within the Oceania region in mid-2017. This study identified emerging noroviruses circulating in Australia and New Zealand in 2017 to assess the changing dynamics of the virus infection. RT-PCR-based methods, next generation sequencing, and phylogenetic analyses were used to genotype noroviruses from both clinical and wastewater samples. Antigenic changes were observed between the capsid of pandemic Sydney 2012 variant and the two new Sydney recombinant viruses. The combination of these antigenic changes and the acquisition of a new ORF1 through recombination could both facilitate their ongoing persistence in the population. Overall, an increased prevalence of GII.P16/GII.4 Sydney 2012 viruses was observed in 2017, replacing the GII.P16/GII.2 recombinant that dominated in the region at the end of 2016. This shift in strain dominance was also observed in wastewater samples, demonstrating the reliability of wastewater as a molecular surveillance tool.

Preliminary evaluation of BioFire FilmArray® Gastrointestinal Panel for the detection of noroviruses and other enteric viruses from wastewater and shellfish.

The BioFire FilmArray® Gastrointestinal Panel was evaluated for the rapid detection of adenovirus, astrovirus, norovirus, rotavirus and sapovirus from influent and effluent wastewater and shellfish. The multiplex BioFire FilmArray® Gastrointestinal Panel compared well to singleplex qPCR/RT-qPCR methods for the detection of adenovirus, astrovirus, rotavirus and sapovirus from influent and effluent wastewater samples. However, the BioFire FilmArray® Gastrointestinal Panel showed poor performance for the detection of norovirus, significantly underestimating its presence in wastewater and shellfish samples when compared with the singleplex norovirus GI and GII RT-qPCR assays. Therefore, improvement on detection efficiency for norovirus from environmental and food samples is necessary before using results from the FilmArray® Gastrointestinal Panel to assess associated public health risks.