The use of capture-recapture methods to provide better estimates of the burden of norovirus outbreaks from seafood in England, 2004-2011.

Norovirus (NoV) is the greatest cause of infectious intestinal disease in the UK. The burden associated with foodborne outbreaks is underestimated in part because data are dispersed across different organisations. Each looks at outbreaks through a different lens. To estimate the burden of NoV from seafood including shellfish we used a capture-recapture technique using datasets from three different organisations currently involved in collecting information on outbreaks. The number of outbreaks of NoV related to seafood including shellfish in England was estimated for the period of 2004-2011. The combined estimates were more than three times as high (N = 360 using Chao's sample coverage approach) as the individual count from organisation three (N = 115), which captured more outbreaks than the other two organisations. The estimates were calculated for both independence and dependence between the datasets. There was evidence of under-reporting of NoV outbreaks and inconsistency of reporting between organisations, which means that, currently, more than one data source needs to be used to estimate as accurately as possible the total number of NoV outbreaks and associated cases. Furthermore, either the integration of reporting mechanisms or simplifying the process of reporting outbreaks to organisations is essential for understanding and, hence, controlling disease burden.

Evaluation of the protection against norovirus afforded by E. coli monitoring of shellfish production areas under EU regulations.

EC Regulation 854/2004 requires the classification of bivalve mollusc harvesting areas according to the faecal pollution status of sites. It has been reported that determination of Escherichia coli in bivalve shellfish is a poor predictor of norovirus (NoV) contamination in individual samples. We explore the correlation of shellfish E. coli data with norovirus presence using data from studies across 88 UK sites (1,184 paired samples). We investigate whether current E. coli legislative standards could be refined to reduce NoV infection risk. A significant relationship between E. coli and NoV was found in the winter months (October to February) using data from sites with at least 10 data pairs (51 sites). We found that the ratio of arithmetic means (log10 E. coli to log10 NoV) at these sites ranged from 0.6 to 1.4. The lower ratios (towards 0.6) might typically indicate situations where the contribution from UV disinfected sewage discharges was more significant. Conversely, higher ratios (towards 1.4) might indicate a prevalence of animal sources of pollution; however, this relationship did not always hold true and so further work is required to fully elucidate the factors of relevance. Reducing the current class B maximum (allowed in 10% of samples) from 46,000 E. coli per 100 g (corresponding NoV value of 75750 ± 103) to 18,000 E. coli per 100 g (corresponding NoV value of 29365 ± 69) reduces maximum levels of NoV by a factor of 2.6 to 1; reducing the upper class B limit to 100% compliance with 4,600 E. coli per 100 g (corresponding NoV value of 7403 ± 39) reduces maximum levels of NoV by a factor of 10.2 to 1. We found using the UK filtered winter dataset that a maximum of 200 NoV corresponded to a maximum of 128 ± 7 E. coli per 100 g. A maximum of 1,000 NoV corresponded to a maximum of 631 ± 14 E. coli per 100 g.

The development of LENTICULES™ as reference materials for noroviruses.

AIMS: To investigate the potential for LENTICULES™ to act as reference materials (RMs) for noroviruses (NoV) [genogroups I (GI) and II (GII)] by determining their homogeneity and stability characteristics. METHODS AND RESULTS: NoV used in this study originated from human faecal material, screened for the absence of other faecally transmitted pathogens. The norovirus strains present in the faecal material were characterized by sequencing, and samples containing GI and GII strains representative of genotypes commonly circulating in the community were selected. RMs were produced utilizing modified lenticulating technology. A batch comprising 500 LENTICULES™ containing both norovirus genogroups was produced according to ISO Guide 34. The batch was tested and quantified using an ISO 17025 accredited quantitative real-time RT-PCR assay. Sufficient homogeneity was established using procedures described by Fearn and Thompson (2010), while stability at less than -15°C and ambient temperature (17-22°C) was assessed over 52 weeks and 7 days, respectively. CONCLUSIONS: Lenticulation was shown to be an effective means of preservation of detectable NoV. LENTICULES™ were sufficiently homogeneous and stable throughout medium-term frozen and short-term storage at room temperature to serve as RMs. Virus LENTICULES™ have the advantages of being easy to manipulate, provide assigned values and do not require the manipulation of high titre clinical material. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that norovirus LENTICULES™ can be used as stable RMs for quantitative real-time RT-PCR assays. They can be utilized as in-run positive extraction controls and potentially for method calibration and to enable more easy comparison of data generated by the variety of differing norovirus determination methods that have emerged in recent years. LENTICULES™ have the potential to provide essential elements of laboratory quality assurance systems for laboratories implementing these new methods for virus testing in foodstuffs and for those running routine analyses.

A seafood risk tool for assessing and mitigating chemical and pathogen hazards in the aquaculture supply chain.

Intricate links between aquatic animals and their environment expose them to chemical and pathogenic hazards, which can disrupt seafood supply. Here we outline a risk schema for assessing potential impacts of chemical and microbial hazards on discrete subsectors of aquaculture-and control measures that may protect supply. As national governments develop strategies to achieve volumetric expansion in seafood production from aquaculture to meet increasing demand, we propose an urgent need for simultaneous focus on controlling those hazards that limit its production, harvesting, processing, trade and safe consumption. Policies aligning national and international water quality control measures for minimizing interaction with, and impact of, hazards on seafood supply will be critical as consumers increasingly rely on the aquaculture sector to supply safe, nutritious and healthy diets.

Human norovirus RNA persists in seawater under simulated winter conditions but does not bioaccumulate efficiently in Pacific Oysters (Crassostrea gigas).

Norovirus (NoV) is the principal agent of bivalve molluscan shellfish-associated gastroenteric illness worldwide. Currently, noncultivable human NoVs can be detected in bivalve molluscan shellfish by using molecular methods such as real-time reverse transcription PCR assays (qRT-PCR). In addition to infectious viruses, this methodology may also detect noninfectious NoV, including fragments of the NoV genome. This study addresses, in part, the implications of qRT-PCR results for the detection of NoV in shellfish in the absence of an infectivity assay. To evaluate environmental persistence, the stability of a short fragment of the NoV genome, spanning the qRT-PCR target in the open reading frame 1/2 junction, was assessed in seawater under artificial environmental conditions simulating winter in the United Kingdom (1 mW/cm² UV irradiation, 8°C) during a 4-week period. Detectable RNA levels decreased exponentially (T90 of approximately 141 h); however, sequences were still detectable for up to 2 weeks. The ability of Pacific oysters (Crassostrea gigas) to bioaccumulate NoV particles (from human feces) and RNA fragments was also compared using qRT-PCR. Oysters exposed to NoV particles subsequently were positive for NoV by qRT-PCR at levels several orders of magnitude in excess of the theoretical limit of detection, whereas oysters exposed to similar quantities of NoV RNA were either negative or positive at significantly lower levels. Therefore, although noninfectious fragments of NoV RNA may persist in the environment under winter conditions, this type of material will not be efficiently bioaccumulated by Pacific oysters and should not significantly contribute to positive qRT-PCR results.

Rapid identification and differentiation of agricultural faecal contamination sources using multiplex PCR.

AIMS: To develop a quick, easy-to-use, robust and sensitive multiplex PCR assay to detect common sources of agricultural faecal contamination using a combination of bacterial and eukaryote-specific PCR targets. METHOD AND RESULTS: A novel multiplex PCR method was developed that utilizes primers specific for a conserved region of the eukaryote cytochrome-B gene as well as a universal 16S rRNA and the E. coli-specific uidA gene. This multiplex PCR assay was capable of identifying faecal amendments from pig, sheep, cow and goat sources in 24/30 (80%) of amended water samples. CONCLUSIONS: The method was capable of accurately identifying common agricultural sources. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure described here is simple, rapid (<5 h) and can be used as a first step in microbial source tracking studies, particularly where agricultural faecal contamination is suspected.

Validation of EN ISO method 15216 - Part 1 - Quantification of hepatitis A virus and norovirus in food matrices.

Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness, with common vehicles including bivalve molluscan shellfish, soft fruit and various vegetables. Outbreaks of viral illness due to contamination of the surfaces of foods, or food preparation surfaces by for example infected food handlers are also common. Virus analysis of food matrices can contribute towards risk management for these hazards and a two-part technical specification for determination of Hepatitis A virus and norovirus in food matrices (ISO/TS 15216:2013) was published jointly by the European Committee for Standardisation and the International Organization for Standardization in 2013. As part of the European Mandate No. M381 to validate 15 standards in the field of food microbiology, an international validation study involving 18 laboratories from 11 countries in Europe was conducted between 2012 and 2014. This study aimed to generate method characteristics including limit of detection, limit of quantification, repeatability and reproducibility for ISO 15216 - Part 1, the method for quantification, in seven food matrices. The organization and results of this study, including observations that led to improvements in the standard method are presented here. After its conclusion, the method characteristics generated were added to the revised international standard, ISO 15216-1:2017, published in March 2017.

Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100 copies/g norovirus and > 10 pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.

A One-Year Survey of Norovirus in UK Oysters Collected at the Point of Sale.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a food safety risk and a potential contributor to the overall burden of gastroenteritis in the community. The United Kingdom (UK) has comprehensive national baseline data on the prevalence, levels, and seasonality of norovirus in oysters in production areas resulting from a previous two-year study (2009-2011). However, previously, data on final product as sold to the consumer have been lacking. As part of a wider project to establish the overall burden of foodborne norovirus in the UK, this study aimed to address this data gap. A one-year survey of oysters collected from the point-of-sale to the consumer was carried out from March 2015 to March 2016. A total of 630 samples, originating in five different European Union Member States, were collected from 21 regions across the UK using a randomised sampling plan, and tested for norovirus using a method compliant with ISO 15216-1, in addition to Escherichia coli as the statutory indicator of hygiene status. As in the previous production area study, norovirus RNA was detected in a high proportion of samples (68.7%), with a strong winter seasonality noted. Some statistically significant differences in prevalences and levels in oysters from different countries were noted, with samples originating in the Netherlands showing lower prevalences and levels than those from either the UK or Ireland. Overall, levels detected in positive samples were considerably lower than seen previously. Investigation of potential contributing factors to this pattern of results was carried out. Application of normalisation factors to the data from the two studies based on both the numbers of norovirus illness reports received by national surveillance systems, and the national average environmental temperatures during the two study periods resulted in a much closer agreement between the two data sets, with the notably different numbers of illness reports making the major contribution to the differences observed in norovirus levels in oysters. The large majority of samples (76.5%) contained no detectable E. coli; however, in a small number of samples (2.4%) levels above the statutory end product standard (230 MPN/100 g) were detected. This study both revealed the high prevalence of norovirus RNA in oysters directly available to the UK consumer, despite the high level of compliance with the existing E. coli-based health standards, while also highlighting the difficulty in comparing the results of surveys carried out in different time periods, due to variability in risk factors.

A computer-aided instructional module for radiological physics.

A computer program and instructional module have been written to assist the physicist in radiation therapy physics instruction. The program, written in Pascal, presents the student with a screen of instructional material, followed by a multiple choice question pertaining to that material. A correct response allows the student to proceed to the next screen. If the answer is incorrect, another screen is presented which explains to the student why the answer is incorrect. Then the original screen and question are repeated. The program is general and can be used with any instructional module. It also is transportable, with a minimum of machine-dependent instructions.

Ultrastability and enhanced stiffness of approximately 6 nm TiO2 nanoanatase and eventual pressure-induced disorder on the nanometer scale.

At 300 K ultrafine approximately 6 nm grain-size nanoanatase retains its structural integrity up to 18 GPa. There is progressive pressure-induced structural disorder to a highly disordered state at P>18 GPa. Signatures of short-range order persist to well beyond 18 GPa in both the synchrotron-x-ray diffraction and Raman data. Molecular dynamics simulations suggest disorder initiated in the surface shell of a nanograin with crystallinity being retained in the core. A bulk modulus B0=237+/-3 GPa for the nanoanatase (approximately 30% higher than the bulk value) is derived from the P-V data, concordant with the MD calculations.

In vitro and in vivo antibacterial activities of GV129606, a new broad-spectrum trinem.

GV129606 is a new parenteral trinem antibiotic belonging to the beta-lactam class. It combines broad-spectrum activity (against gram-negative and -positive bacteria, aerobes and anaerobes), with high potency and resistance to beta-lactamases. Comparative in vitro and in vivo antibacterial activities were determined for GV129606 against more than 400 recent clinical isolates (aerobes, including beta-lactamase producers, and anaerobes), using representative antibacterial agents (meropenem, piperacillin, ceftazidime, cefpirome, ciprofloxacin, and gentamicin for aerobes and metronidazole, cefoxitin, piperacillin, and clindamycin for anaerobes). Against methicillin-susceptible staphylococci and streptococci, GV129606 and meropenem were the most active of the drugs tested. GV129606 showed an MIC for 90% of strains tested (MIC90) ranging from < or =0.015 to 0.06 microg/ml against methicillin-susceptible staphylococci and Streptococcus sanguis, Streptococcus pyogenes, and Streptococcus agalactiae. Against penicillin-susceptible and -resistant Streptococcus pneumoniae isolates, GV129606, meropenem, and cefpirome showed MIC90s of < or =0.015 and 1 microg/ml, respectively. Meropenem was the most active compound against members of the family Enterobacteriaceae with MIC90s of < or =0.5 microg/ml. Against these species, GV129606 possessed activity superior to those of piperacillin, ceftazidime, cefpirome, and gentamicin, with MIC90s of < or =8 microg/ml, but its activity was two- to sixfold less than that of ciprofloxacin (with the exception of Proteus rettgeri and Providencia stuartii). Haemophilus spp., Moraxella catarrhalis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa were also included in the spectrum of GV129606. GV129606 showed good antianaerobe activity, similar to metronidazole. It was stable against all clinically relevant beta-lactamases (similar to meropenem). The in vitro activity was confirmed in vivo against septicemia infections induced in mice by selected gram-positive and -negative bacteria with 50% effective doses (ED50s) of < or =0.05 and < or =0.5 mg/kg of body weight/dose, respectively. GV129606 was as effective as meropenem against septicemia in mice caused by ceftazidime-resistant Pseudomonas aeruginosa, exhibiting an ED50 of 0.33 mg/kg/dose.

In vivo pharmacodynamics of HMR 3270, a glucan synthase inhibitor, in a murine candidiasis model.

In vivo pharmacokinetic/pharmacodynamic characterization for numerous antibacterial compounds has had a significant impact upon optimal dosing regimen design and the development of in vivo susceptibility breakpoints. More recently, similar characterization has been undertaken for antifungal drug classes. Very little is known of these pharmacodynamic relationships for the new echinocandin class of compounds. We utilized a neutropenic murine model of disseminated candidiasis to describe the time course antifungal activity of HMR 3270, a new glucan synthase inhibitor. Single-dose in vivo time kill studies with four 16-fold escalating doses demonstrated concentration-dependent killing when drug levels in serum were more than four times the MIC. Postantifungal effects were dose dependent, ranging from 8 to 80 h duration. Multiple dosing regimen studies utilized six total doses, four dosing intervals, and a treatment duration of 6 days. Shortening the dosing interval from every 144 h (q144h) to q36h resulted in a fourfold rise in the dose necessary to achieve a net fungistatic effect. The peak/MIC ratio most strongly correlated with treatment outcomes (peak/MIC ratio, R(2) = 98%; ratio of the area under the concentration-time curve from 0 to 24 h to the MIC, R(2) = 79%, percentage of time above the MIC, R(2) = 61%). Studies were also conducted with five additional Candida albicans isolates to determine if a similar peak/MIC ratio was associated with efficacy. In vivo concentration-dependent killing was similarly observed in studies with each of the additional isolates. The peak/MIC ratio necessary to produce efficacy was relatively similar among the strains studied (P = 0.42). The peak/MIC ratio (mean +/- standard deviation) necessary to achieve a fungistatic effect was 3.72 +/- 1.84, and the ratio necessary to achieve maximal killing was near 10.