The development of reference materials for norovirus and hepatitis A viruses

Transcription

1 The development of reference materials for norovirus and hepatitis A viruses The Future of Reference Materials- Science and Innovation JRC-IRMM, Geel, 24 th November 2010 Centre for Environment, Fisheries and Aquaculture Science Dr Rachel Rangdale European Union Reference Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs

2 The development of reference materials for norovirus and hepatitis A viruses Introduction to issues Bivalve shellfish Sewage contamination Viral pathogens Methods and proficiency testing for viruses Development of LENTICULES Use of LENTICULES in proficiency testing Matrix samples

3 Common cockle (Cerastoderma edule) Pacific oyster (Crassostrea gigas ) Bivalve Molluscan Shellfish Common mussel (Mytilus edulis) Manila clam (Tapes phillipinarum)

4 Bivalve shellfish growing areas

5 Coastal population Point source discharges Non-point source discharges

6 Shellfish hygiene controls (EU legislation) Control of sewage pollution (Shellfish Waters Directive 79/923/EEC) Classification and monitoring of harvesting areas (854/2004) Commercial processing (853/2004) (depuration, relaying, cooking) End-product standards (2073/2005)

7 Major pathogenic viruses associated with sewage pollution Norovirus Relatively mild gastroenteritis, often including nausea, diarrhoea, vomiting, fever and abdominal pain Incubation period of 1 to 4 days, duration of about 2 days generally followed by complete recovery The most common cause of infectious intestinal disease in outbreaks and in the community Hepatitis A Virus Extended incubation period of about 4 weeks (range 2 to 6) Serious debilitating disease with fever, headache, nausea, vomiting, diarrhoea, abdominal pain and jaundice Self-limiting and rarely causes death but patients may be incapacitated for several months Neither virus can be routinely cultured using conventional tissue culture techniques

8 Virus Morphology Bar is 50 nanometers Calicivirus Norovirus virus Astrovirus Bar is 100 nanometers Enterovirus (e.g. polio) and hepatitis A Rotavirus

9 Illness Associated with Bivalve Molluscan Shellfish (BMS) Numbers of recorded outbreaks associated with Aetiology of illness associated with BMS in England and Wales Unknown 48.6% BMS in England and Wales Salmonella DSP 2.9% 2.9% Astrovirus 5.7% NoV 40.0% Data compiled by HPA Communicable Disease Surveillance Centre

10 Importance of development of standard methods for detection in foodstuffs No standard method exists for the detection of norovirus and hepatitis A viruses in foodstuffs, including bivalve shellfish. The European Committee on Normalisation (CEN) are developing a standard method for use on all foodstuff (CEN TC275 WG 6 TAG4) Introduction of viral assessments foreshadowed in legislation (EC No. 882/2004, EC No. 854/2004, EC 2073/2005). EC No. 882/2004 requires quality framework (incl. accreditation etc) for Official Control laboratories. Virus reference materials are required to support both developing methodology and for future quality systems

11 CEN TC275 WG 6 TAG4 -Framework for method Horizontal method (all foodstuffs included) Viruses of primary focus: Norovirus Hepatitis A virus Matrices of primary focus: Hard surfaces Salad crops Soft fruits Bottled water Bivalve shellfish

12 Framework for horizontal method for detection of norovirus and hepatitis A virus in food by RT- PCR : update from 6 th CEN TAG4 meeting Sample extraction Nucleic acid extraction Amplification and confirmation Hard surfaces Swabbing 10cm 2 portions into 0.5 ml PBS Raw vegetables and soft fruit Bottled water Elute 25g sample in 40 ml glycine buffer ph 9.5, clarify (pectinase for soft fruit) 10,000g, PEG precipitate (10,000g), (chloroform:butanol step for soft fruit), resuspend pellet in water to 0.5ml Filter concentrate 1-5 L +ve nylon membrane, elution in glycine 1% BE buffer, adjust to ph8, centrifugal concentration, adjust to 0.5ml Volume 500µl Magnetic silica and GITC (modification of Boom et al., 1990) Volume 5µl probed based one step real time reverse transcription PCR (NoV GI, NoV GII, HAV) Result Interpretation and quantitation Bivalve shellfish 2.0g excised digestive gland equal volume of proteinase K (30 units/mg), incubate 37C and 65C, centrifuge 3000g, take 0.5ml Extraction control EMCV (Mengo virus) added at the first possible point according to the extraction Amplification inhibition control IC RNA (Nov GI, NoV GII, HAV) DNA quantitation curve (Nov GI, NoV GII, HAV) Acceptance criteria (following validation studies)

13 Laboratories undertaking virus (norovirus and/or hepatitis A) testing in BMS Implications of virus testing Closure of fisheries Enhanced purification Opening of fisheries Retail purchasing policies Restrictions on imports/exports Certification of safe products To support virus testing the EU-RL developed virus proficiency testing

14 Problems associated with production of whole animal proficiency testing samples Bioaccumulation of Crassostrea gigas Bioaccumulation kinetics- temperature/time dependent log PCR units Time (hours) Sample variability (non-homogeneity) differential uptake by shellfish 40 CT C GI determinant C GII

15 Problems associated with distribution of whole animal proficiency testing samples Transport challenges Require frozen or refrigerated transportation (highly perishable) Restricted number of couriers licensed to transport material Very expensive (Weymouth to Belgium circa 400 Euros) Most countries have complex and different regulations requiring permits to import complex, difficult, often result in samples being detained or destroyed at borders

16 LENTICULES as reference materials for IQC and EQA Established use in bacterial preservation. LENTICULES are small convex discs containing biologically active material within a water-soluble substance. Utilise the preservation properties of nonreducing disaccharides. Easy to reconstitute and are stable at a range of temperatures. Provide defined reproducibly countable bacterial CFU/disc Used in internal and external quality assurance in microbiology, including EU-RL/HPA PT scheme for shellfish

17 LENTICULES as virus carriers? Currently no intact virus reference materials for norovirus and hepatitis A available- stable at ambient temperature Early studies with cultivable viral surrogate FRNA bacteriophage showed promising results. HPA patented Newcastle s lenticulating fluid formula was adapted to prevent PCR inhibition. Developed a preservation formula containing: -cellulose derivative -non-reducing sugars -glucose -bovine serum albumin -phosphate buffered saline

18 Virus characterisation sequencing capsid and polymerase region NoV. Sample description Source Sequence type Norovirus genogroup I Faecal material GI.4 capsid type; 96.2% sequence homology to virus (AB022679) Norovirus genogroup II Hepatitis A Faecal material Laboratory reference strain GII.4 capsid type; 99.7% sequence homology to Isumi strain (AB295790) strain HM175/43c

19 Generation of virus starting materials and pathogen screening safety Norovirus GI and GII faecal samples with high genome copies/g (>10 9 ) selected- from patients presenting symptoms. Screened for: Sapovirus Rotavirus Astrovirus Enteric Adenovirus E. coli Verotoxin 1 and 2 Salmonella Giardia Cryptosporidia Listeria monocytogenes Shigella

20 Virus quantitation For quantification of non-cultivable viruses; dilution series of a plasmid carrying the target sequence added to each plate Concentration of sample RNA in detectable copies/ul calculated Figure converted into sample concentration in detectable genome copies per g/ml/cm 2 of starting material Plasmid control serves as a generalised positive control for PCR portion of the test

21 Quantitation- For quantification; dilution series of a plasmid carrying the target sequence added to each plate

22 Stability and self-life testing- norovirus GI and GII, hepatitis A- 6 months 1 week Log 10 genome copies per lenticule

23 Homogeneity testing- norovirus GI and GII, hepatitis A Example of replicate testing of multiple LENTICULES from the same batch under repeatability conditions

24 Use of virus LENTICULES in pilot proficiency testing Produce of varying virus combinations and concentrations Quality control testing target genome copy quantities and homogeneity- satisfies requirements of ISO TS (PT samples) Distribute to selected laboratories with ability to quantify target viruses Analyse returned dataset Repeatability (within lab differences) <0.5 log Reproducibility (between lab differences) almost 4 logs Problems identified with quantitation standard curves Variations in quantities of GI participating laboratories in multi-lab study

25 Pilot study using shellfish matrix samples To produce norovirus (and HAV) matrix based samples for use as reference materials. Aliquots of digestive glands from characterized outbreak related Crassostrea gigas (Pacific oysters), originated in Korea, shipped from New Zealand. Shipped to IRMM for freeze-drying. Returned to Cefas for processing using ISO accredited qrt-pcr assay.

26 Matrix samples- preliminary results Log 10 genome copies Both genogroups of norovirus detectable from freeze dried digestive glands re-suspended in PBS, subject to standardised qrt-pcr testing Pre and post freeze dried digestive gland samples showed less than 0.5 log 10 reduction in genome copies. GI NoV Log 10 genome copies GII NoV Between replicate homogeneity acceptable (<0.26 log 10 ). Pilot distribution planned for February Pre-treatment Post -treatment

27 Conclusions Need for reference materials for norovirus and hepatitis A to support developing methodology (and future food safety legislation?). LENTICULES offer a suitable, stable, homogeneous matrix for virus preservation. Useful for routine quality control. Overcome stability and transport problems associated with whole bivalve shellfish but do not contain shellfish matrix. Preliminary data using freeze dried digestive glands shows promise for future matrix based material.

28 Acknowledgements Dr Julie Russell, FEPTU, HPA, Colindale, UK. IRMM, Geel, Belgium. European Commission, DG SANCO, EU-RL bivalve molluscs. Dr James Lowther, Justin Avant and Louise Stockley - Cefas, Weymouth, UK.