Norovirus Update. David Lees Cefas, Weymouth

Transcription

1 Norovirus Update David Lees Cefas, Weymouth European Union Reference Laboratory for Monitoring Bacteriological and Viral Contamination of Bivalve Molluscs

2 What influences virus risk? Circulation of virus in population NoV Season Virus strain Community and institutional outbreaks HAV Sporadic (high risk) Virus loading to sewerage system Effluent (post- UV disinfection) Storm tank Influent (screened) Activated sludge (post-clarifiers) Activated sludge (settlement tank) Sewage treatment (STW) Efficiency of physical removal Disinfection Reliability (maintenance) Location of discharge Sewer bypasses (CSO) Frequency/duration Volume Location Virus load discharged to marine environment Illness in consumer Contamination levels in final products (FBO monitoring) Virus viability Cooking Immune status Amount consumed Epidemiological investigation (meal setting) Depuration effectiveness Water temperature Virus loading Tank design Biology? Contamination levels in harvested products (FBO monitoring) Contamination of shellfish Proximity of discharge to shellfish Sewage dilution/dispersion Water temperature Sunlight Meteorological factors Environmental reservoirs Shellfish species

3 What influences virus risk? Circulation of virus in population NoV Season Virus strain Community and institutional outbreaks HAV Sporadic (high risk) Virus loading to sewerage system Risk factors study (Defra) Effluent (post- UV disinfection) Storm tank Influent (screened) Activated sludge (post-clarifiers) Activated sludge (settlement tank) Virus load discharged to marine environment Sewage treatment (STW) Efficiency of physical removal Disinfection Reliability (maintenance) Location of discharge Sewer bypasses (CSO) Frequency/duration Volume Location Illness in consumer Contamination levels in final products (FBO monitoring) Virus viability Cooking Immune status Amount consumed Epidemiological investigation (meal setting) Depuration effectiveness Water temperature Virus loading Tank design Biology? Contamination levels in harvested products (FBO monitoring) Contamination of shellfish Proximity of discharge to shellfish Sewage dilution/dispersion Water temperature Sunlight Meteorological factors Environmental reservoirs Shellfish species

4 The knowledge base Desk study of risk factors for NoV in shellfish water catchments in England and Wales (Carlos Campos: Defra funded) Levels of NoV and E. coli in oysters from 31 sampling points Results Conclusions Future work Catchment resident population Catchment population density Catchment urbanised area Water temperature Fluvial distance from sampling point to discharge Tidal range Mean high water springs Linear correlation (Pearson s r) analyses Linear regression Akaike Information Criterion Rainfall (day of sampling/cumulative 7 days prior to sampling) River flows (day of sampling/cumulative 7 days prior to sampling) Base flow index Number of discharges (continuous/intermittent) to shellfish water Number of discharges (continuous/intermittent) in the catchment Size (volume) of continuous discharges Number of trade discharges in the catchment Frequency/duration of sewage spills

5 Geometric mean norovirus (GI+GII) (copies/g) Geometric mean norovirus (GI+GII) (copies/g) Resident human population in the catchment Dry weather flow (litres/second) Total NoV levels in shellfish generally increase with human population in the catchment and volume of sewage discharged into shellfish waters

6 Environmental risk factors E. coli Rainfall Norovirus (GI+GII) River flows Water temperature Correlation coefficient: (-1.0) (-0.8) (-0.8) (-0.6) (-0.6) (-0.4) (-0.4) (-0.2) (-0.2) (0.0) (0.0) (0.2) (0.2) (0.4) (0.4) (0.6) (0.6) (0.8) (0.8) (1.0) Human population Population density Size of discharge Tidal range Distance from discharge Catchment area Number of continuous discharges

7 The knowledge base Aims Conclusions Correlation between number of sewage spills (CSO discharges) and NoV levels in oysters Future work Geometric mean norovirus (GI+GII)(copies/g) Regression 95% CI S R-Sq 65.3% R-Sq(adj) 61.0% Report has received stakeholder comment Now with Defra for peer review prior publication Average number of sewage spills 60

8 What influences virus risk? Circulation of virus in population NoV Season Virus strain Community and institutional outbreaks HAV Sporadic (high risk) Virus loading to sewerage system Storm tank Influent (screened) Virus behaviour field studies (Defra, FSA) Effluent (post- UV disinfection) Activated sludge (post-clarifiers) Activated sludge (settlement tank) Virus load discharged to marine environment Sewage treatment (STW) Efficiency of physical removal Disinfection Reliability (maintenance) Location of discharge Sewer bypasses (CSO) Frequency/duration Volume Location Illness in consumer Contamination levels in final products (FBO monitoring) Virus viability Cooking Immune status Amount consumed Epidemiological investigation (meal setting) Depuration effectiveness Water temperature Virus loading Tank design Biology? Contamination levels in harvested products (FBO monitoring) Contamination of shellfish Proximity of discharge to shellfish Sewage dilution/dispersion Water temperature Sunlight Meteorological factors Environmental reservoirs Shellfish species

9 FSA Project FS246004/Defra Project WT1529 Fate and behavior of norovirus in shellfish waters Carlos Campos, Cefas Field study investigation of sewage inputs and their impacts 1. Coastal embayment (deep, open coastal) receiving low freshwater inputs and impacted by single source of contamination 2. Estuary receiving substantial freshwater inputs and impacted by several sources of contamination (stormwater, secondarytreated and UV disinfected effluent) Agencies funding the work: Cefas also collaborating with: and members of the industry and local authorities.

10 Characterisation of norovirus in sewage and treated effluents Types of sewage treatments investigated: Storm overflows, influent screened, primary settlement tank (PST), humus tanks, sand filtration, biological aerated filters, UV disinfection. Preliminary results: Extreme variability in NoV concentrations in sewage influents. The highest levels were detected in storm overflows (not significantly different from crude influent). Significant differences between primary and secondary-treated effluents. Of all of the above, activated sludge is the most effective in removing NoV.

11 Optimised activated sludge In this plant, we found 2.8log 10 removal for NoV GI and 3log 10 removal for GII. The process is a two-stage nitrification/de-nitrification process with internal circulation.

12 Background The knowledge base Aims Methods Conclusions Future work Apportionment of NoV loads from intermittent vs continuous discharge (study site 2) 1.E E+13 1.E+13 1.E+12 1.E E+10 1.E+10 1.E E E+08 1.E+08 E. coli (CFU/day) (settled storm) Norovirus (GI+GII) (copies/day) (settled storm) E. coli (CFU/100ml) (UV disinfected) Norovirus (GI+GII) (copies/g) (UV disinfected) Storm tank discharges represented the majority (>98% )of the NoV load discharged to the catchment during the study period (study site 2)

13 Field studies: research approach Placement of shellfish cages in network of sampling stations at different locations downstream from the sources of pollution (covering maximum distance of 13km). Sampling campaigns targeting dry- and wet-weather. Norovirus (genogroups I and II) and E. coli quantified in mussels, Pacific oysters and native oysters. Shellfish monitoring combined with hydrographic studies (drogue tracking and dye tracing) to establish connectivity in the shellfish waters. Other parameters considered in the programme: STW: sewage flows, sewage retention times, UV applied dose. Environment: water temperature, salinity, rainfall, CSO spills, river flows. Sewage sampled from different stages of treatment process Bags with 11kg native oysters deployed at 6 stations in the estuary Samples tested for E. coli and norovirus (GI and GII)

14 Concentrations of norovirus and E. coli as a function of distance from sewage outfalls (study site 2) E. coli (MPN/100g) E. coli LoD Fluvial distance from sewage outfall (km) Regression 95% CI R-Sq 30.2% R-Sq(adj) 27.9% Norovirus Norovirus (GI+GII) (copies/g) Fluvial distance from sewage outfall (km) LoD Regression 95% CI R-Sq 37.4% R-Sq(adj) 35.4%

15 Current work Field studies at site 1 (open coastal) Hydrographic dye tracing and drogue tracking studies at both sites to study correlation between sewage effluent dilution and NoV contamination Analysis of data to inform recommendations on: Possible approaches to exclusion zones around sewage outfalls Active management of harvesting operations around contamination events Sampling strategies for effective virus monitoring Optimisation of sewage treatment technologies Injection of Rhodamine dye in the STW discharge

16 What influences virus risk? Circulation of virus in population NoV Season Virus strain Community and institutional outbreaks HAV Sporadic (high risk) Virus loading to sewerage system Effluent (post- UV disinfection) Storm tank Influent (screened) Activated sludge (post-clarifiers) Activated sludge (settlement tank) Sewage treatment (STW) Efficiency of physical removal Disinfection Reliability (maintenance) Location of discharge Sewer bypasses (CSO) Frequency/duration Volume Location NoVAS UK retail study (FSA) Virus load discharged to marine environment Illness in consumer Contamination levels in final products (FBO monitoring) Virus viability Cooking Immune status Amount consumed Epidemiological investigation (meal setting) Depuration effectiveness Water temperature Virus loading Tank design Biology? Contamination levels in harvested products (FBO monitoring) Contamination of shellfish Proximity of discharge to shellfish Sewage dilution/dispersion Water temperature Sunlight Meteorological factors Environmental reservoirs Shellfish species

17 Assessing the contribution made by the food chain to the burden of UK-acquired norovirus infection Sarah J O Brien PARTNERS Centre for Environment, Fisheries and Aquaculture Science Food and Environment Research Agency Leatherhead Food Research Public Health England Stericycle Expert Solutions University of East Anglia University of Liverpool UK Food Standards Agency

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19 ORGANISATION OF WORK PROGRAMME Work Package 1 (SJO B) Systematic literature review Feasibility study for gathering new data Work Package 2 (AK) Developing and implementing capsid integrity assay Work Package 3 (DL/JK) Acquisition of new data on NoV contamination levels in oysters at retail Work Package 4 (NC/Md A) Acquisition of new data on NoV contamination levels in raspberries and lettuces at retail Work Package 5 (MI-G/DWGB) Prevalence of NoV in catering environments in outbreak and non-outbreak premises. Work Package 6 (PRH) Develop QMRA to develop an overall assessment of the contribution that NoV makes to food-related illness in the UK.

20 WP3 Retail level study of norovirus in oysters Survey plan based on market research by Stericyle Identified 376 outlets for oysters in 22 representative cities/regions Plan finalised following stakeholder consultation Random selection of outlets to be sampled by month 52 samples per month 12 month survey period (total 630 samples) All samples obtained from retail outlets (categories: fishmongers, supermarkets, restaurant, wholesale, internet sales) Sampling Stericyle undertake all sampling (pre-ordering often necessary) Stericyle communication with outlet supported by explanatory letter from FSA Cool chain transport to Cefas Blind analysis for E.coli and norovirus according to accredited ISO methods All product information (health mark etc) held by Stericyle Sampling started mid-march 2015 Reporting E.coli non-conformities immediately to FSA All sample results reported back to sample providers at end of study NoVAS final report 2017

21 What influences virus risk? Circulation of virus in population NoV Season Virus strain Community and institutional outbreaks HAV Sporadic (high risk) Virus loading to sewerage system EU harmonised baseline study Effluent (post- UV disinfection) Storm tank Influent (screened) Activated sludge (post-clarifiers) Activated sludge (settlement tank) Virus load discharged to marine environment Sewage treatment (STW) Efficiency of physical removal Disinfection Reliability (maintenance) Location of discharge Sewer bypasses (CSO) Frequency/duration Volume Location Illness in consumer Contamination levels in final products (FBO monitoring) Virus viability Cooking Immune status Amount consumed Epidemiological investigation (meal setting) Depuration effectiveness Water temperature Virus loading Tank design Biology? Contamination levels in harvested products (FBO monitoring) Contamination of shellfish Proximity of discharge to shellfish Sewage dilution/dispersion Water temperature Sunlight Meteorological factors Environmental reservoirs Shellfish species

22 EU Harmonised Baseline Survey Recommendation by EFSA, supported by EU CAs and producers Informed by questionnaire among Member State CAs Target oysters Norovirus and hepatitis A virus analysis using ISO method (other viruses at MS discretion) Include E.coli analysis Production areas and end-products but not imports Official sampling (at current RMPs, and in establishments) Sampling design to be established (at least 12 months duration) Collect supporting environmental information Control of quality of analysis (EURL and NRLs) EFSA working group lead/coordinate (with MS CAs, EURL, NRLs) EU co-funding EFSA will report Objectives (draft) Establish levels of NoV (and occurrence of HAV) in classified areas, and end-products, representative of production in each MS to understand impact of possible legislative limits

23 What influences virus risk? Circulation of virus in population NoV Season Virus strain Community and institutional outbreaks HAV Sporadic (high risk) Virus loading to sewerage system Effluent (post- UV disinfection) Storm tank Influent (screened) Activated sludge (post-clarifiers) Activated sludge (settlement tank) Sewage treatment (STW) Efficiency of physical removal Disinfection Reliability (maintenance) Location of discharge Sewer bypasses (CSO) Frequency/duration Volume Location Virus load discharged to marine environment Illness in consumer Contamination levels in final products (FBO monitoring) Virus viability Cooking Immune status Amount consumed Epidemiological investigation (meal setting) Depuration effectiveness Water temperature Virus loading Tank design Biology? Contamination levels in harvested products (FBO monitoring) NoVAS study (FSA) Viruses in marine environment (NERC) Contamination of shellfish Proximity of discharge to shellfish Sewage dilution/dispersion Water temperature Sunlight Meteorological factors Environmental reservoirs Shellfish species

24 Virus viability ISO methods (PCR) target virus RNA - this does not establish viability Norovirus cannot be cultured (although has been recent report) STW disinfection, exposure to sunlight, other environmental pressures, may inactivate virus (hypothesis!) Accepted need for progress on methods for confirming virus viability Approaches Culture Virus capsid integrity Virus genome integrity Surrogates (eg bacteriophage, murine norovirus)

25 Capsid Integrity Assay: Angus Knight

26 Capsid Integrity Assay Laboratory 1 Laboratory 2 Laboratory 3 Cq GI-3 GI-6 GI-7 GII-1 GII-4 GII-5 * Figure 3. ΔCq values obtained from the 0.1% stool samples using the capsid integrity assay. ΔCq values were measured by subtracting the Cq obtained following treatment at 80⁰C for 2 min. and RNase I digestion from that obtained from unheated (4⁰C) controls. * denotes a ΔCq > 8.

27 Capsid Integrity Assay Standardised method among partner laboratories Reproducible results among 3 labs on clinical samples Results sometimes difficult to interpret (ribonucleic acid complexes can confound results) Does not detect inactivation due to UV (eg in STW disinfection) Application to oyster matrix not straightforward Work ongoing by Leatherhead Food Research NoVAS samples being archived (frozen) for separate study

28 New approaches for the quantitative detection of human pathogenic viruses within the freshwater-marine continuum NERC (FSA) Environment and Human Health Programme Bangor University lead Professor Davey Jones Partners: Uni Cambridge, Uni Liverpool, Cranfield Uni, CEH, Cefas Start date May 2015, 3 years Development of robust technologies for human viruses that can be used for risk management (shellfish and bathing waters) WPs on: Quantitation in different environmental matrices, integrated sampling, viability, deep sequencing, field validation, modelling and prediction, technical guidance for risk management

29 WP5: Establishing viral infectivity from environmental samples Uni Cambridge (Goodfellow), Cranfield Uni (Nocker), Cefas (Lees) Genome integrity Viral genome capture (using VPg or biotinylated oligos) (Cambridge) MiniION nanopore sequencing technology (Cefas) Capsid integrity Affinity capture (using pig mucin) (Cambridge) Propidium monoazide exclusion (Cranfield and Cefas) Virus culture (Cambridge) Surrogates Murine norovirus (Cambridge) FRNA Bacteriophage (Cefas)

30 Questions?

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