LABORATORY FOR VIROLOGY

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1 LABORATORY FOR VIROLOGY CENTRE FOR COMMUNICABLE DISEASES NATIONAL INSTITUTE OF PUBLIC HEALTH OF THE REPUBLIC OF SLOVENIA Bohoričeva 15, 1000 Ljubljana, Slovenia NATAŠA BERGINC Community Network of Reference Laboratories for Human Influenza - CNRL European Influenza Surveillance Scheme -EISS WHO Global Influenza Surveillance Network System - WHO GISN WHO National Influenza Centre - WHO NIC ACTIVITIES - INFLUENZA Virological surveillance of seasonal Influenza: season (national Influenza surveillance established in 1999) interseason (from 2006 on) Laboratory preparedness for pandemic Influenza: from 2006 SAMPLES sample sources: -network of 45 primary care physicians and -Department for Infectious Diseases, Clinical Centre, Ljubljana thelaboratory annualy provides: - swabs, transport medium, instructions for swabbing - prepaid envelopes for shipping of samples, insturctions for shipping of samples - questionnaire (patient personal data, clinical symptoms, data about vaccination and uptake of antiviral drugs) sample types: nose and throath swabs from patients with clinical symptomps of influenza like illness 1

2 GRIPA NAVODILO ZA ODVZEM IN POŠILJANJE BRISOV Transportna gojiščavplastičnih epruvetkah hranimo do uporabe v hladilniku pri + 4ºC. Bris žrela in nosu odvzamemo najkasneje v štirih dneh po pojavu bolezenskih znakov. ŽRELO: S sterilno lopatico potisnemo jezik navzdol, da so tonzile (ali lože) in žrelo dobro vidni. S sterilnim suhim brisom močno pobrišemo zadnjo steno žrela, tonzile ali tonzilarne lože. Bris vrtimo in rahlo pritiskamo. Pazimo, da se ne dotaknemo sluznice lic ali jezika. NOS: Sterilni suhi bris (izjemoma pomočen v transportno gojišče) vstavimo približno 2cm globoko v nosnico (pri majhnih otrocih 1 cm) in močno pobrišemo. Pazimo, da se ne dotaknemo kože in začetnega dela nosnice. Bris nato potopimo v gojišče v plastično epruvetko, s škarjami odrežemo plastično palčko in zapremo epruvetko. Na epruvetko napišemo ime in priimek bolnika ter označimo ali gre za bris nosa ali za bris žrela (npr. BN oz. BŽ). Izpolnimo anketni/spremni list. instructionsfor swabbingand shipping V kuverto z naslovom laboratorija za viruse damo anketni/spremni list in obe epruvetki z brisi ter pošljemo po pošti. Do pošiljanja shranimo epruvetke z brisi ali kar cel paketek v hladilniku pri + 4ºC. Če je le mogoče, materiala ne pošiljamo v petek, da se ne bi čez vikend zadržal na pošiti. Za vse dodatne informacije nas lahko pokličete na telefonsko številko: Hvala za sodelovanje! instructions for swabbing and shipping LABORATORY DIAGNOSTICS I Screening of samples for presence of respiratory viruses MOLECULAR DIAGNOSTIC METHODS: RNA extraction from swabs One-step multiplex RT-PCR (conventional): set of pair of primers for amplification of target sequences of INFLUENZA A, INFLUENZA B, RSV, ADENOVIRUSES, ENTEROVIRUSES (Reference: Gröndahl et al, 1999) Detection of RT-PCR products with agarose gel electrophoresis questionnaire Confirmation of positives with microwell hybridisation analysis (PCR ELISA) 2

3 LABORATORY DIAGNOSTICS II VIRUS ISOLATION LABORATORY DIAGNOSTICS III INFLUENZA TYPING AND SUBTYPING Samples positive for INFLUEZNA A or INFLUENZA B in RT-PCR inoculated on Madine-Darby Canine Kidney (MDCK) cell tissue culture Virus propagation and checking for CPE Conforamtion/identification of virus isolates: - immunofluorescent methods: DIF, IIF - (confirmation with RT-PCR not routinely) Samples positive for other viruses (RSV, adenoviruses, enteroviruses) are inoculated in corresponding cell tissue cultures for virus isolation; identification and typisation of virus isolates is performed consequently. TYPING/SUBTYPING OF VIRUS ISOLATES IIF: hemagglutinin typing of virus isolates with specific monoclonal antibodies annually provided by WHO (H1, H3) HEMAGGLUTINATION INHIBITION TEST (HAI) typing/subtyping of virus isolates with specific antisera annually provided by WHO (A/New Caledonia/20/99 (H1N1) etc.) (Reference and test reagents: WHOn Influenza reagent kit for identification of Influenza isolates) Confirmation of results in the WHO Reference Laboratory (Mill Hill, London, UK) MOLECULAR TYPING Reverse transcription of RNA into cdna with random hexamers HEMAGGLUTININ SUBTYPING: cdna amplified in a nested multiplex conventional PCR (subtyping for H1 and H3 performed routinely) Reference: Stockton et all, 1998 NEURAMINIDASE SUBTYPING: cdna amplified in a multiplex conventional PCR (subtyping for N1 and N2 performed routinelly) (Reference: Poddar, 2002) DATA FLOW I DATA FLOW II NATIONAL REPORTING INTERNATIONAL REPORTING PHYSICIANS LABORATORY RESULTS LABORATORY RESULTS DATABASE WEEKLY - authorities at NIPH - regional public health units - Department for Infectious Diseases, Clinical Centre, Ljubljana EISS - weekly NATIONAL INTERNATIONAL MONTHLY - Ministry of Health - Ministry of Agriculture, Forestry and Food - Veterinary Faculty - National Veterinary Institute WHO Flu Net - weekly 3

4 ORGANISATION IN THE LABORATORY I PREPAREDNESS OF ADEQUATELY TRAINED EXPERT ON DUTY ALSO IN OUT OF OFFICE HOURS: adeqately trained expert on duty attainable by mobile phone: 24 hours/day 7 days/week alternation according the schedule upon emergency call: - arrival in the laboratory within a limited period of time - receipt of sample: shipped to the laboratory by adecuately trained courier - performance of specific laboratory diagnostic procedures - interpretation of laboratory results - reporting of results to the physician and other authorities ORGANISATION IN THE LABORATORY II STOCK OF LABORATORY MATERIAL FOR SPECIFIC LABORATORY DIAGNOSTIC PROCEDURES Stock of specific reagents: - H5 and N1 corresponding pair of primers for PCR (WHO, EISS) Positive controls for H5 and N1 -WHO, EISS Stock of nonspecific reagents and laboratory durables - reagents for PCR mix preparation, plastics Avaliability of adeqate PCR protocols (WHO, EISS) ORGANISATION IN THE LABORATORY III SAFETY OF LABORATORY STAFF Stock of safety / protective equipment for laboratory staff Continuous exercise for work with potentially high infectious materials: - enable staff members to use protective equipment correctly - enable staff members to perform laboratory procedurs correctly Encouragement of vaccination for laboratory staff LABORATORY DIAGNOSTIC METHODS ONLY BSL 2!!! ONLY MOLECULAR DIAGNOSTIC METHODS: RNA extraction from swabs One-step conventional RT-PCR: set of primers for amplification of target sequences from Influenza A/H5N1: -primers: > pair of primers for amplification of target sequence for H5 (source: WHO) > pair of primers for amplification of target sequence for N1 (source: WHO) - method reference PCR protocol: Recomended laboratory tests to identify avian influenza A virus in specimens from humans, WHO Geneva, 2005 Detection of RT-PCR products with agarose gel electrophoresis Interpretation of results Report of results to the physician and other authorities 4

5 ALARM!!! upon receipt of sample from bird-flu suspected patient!!! upon Influenza A positive result with RT PCR for a sample that couldn t be prooved as H3 or H1!!! when influenza A virus isolate reacts weakly or not at all with any of the antisera provided by WHO or EISS in HAI!!! Laboratory algorithm I H5N1 suspected case RNA extraction PCR for H5 PCR for N1 NEG two different PCRs with primers that cover all Inluenza A subtypes are performed POS sample is instantly sent to the WHO Reference Laboratory POS PCRs for HA (H1,3,5,7,9) and NA (N1,N2) determination are preformed both PCRs NEG sample is most probably NEG for Influenza A WHO Laboratory algorithm II FUTURE PLANS InfA, but not H3 or H1 Antigenic suspicious strain POS PCRs for HA (H1,3,5,7,9) and NA (N1, N2) determination is performed Confirmation of Influenza A in sample or isolate with PCR with different pairs of general primers. All tests NEG sampleis most probably NEG for Influenza A WHO HIGH PRIORITY: real time PCR technology as diagnostic method more specific, more sensitive, faster > fast and reliable results to trigger planned response measurements OTHER: monitoring for antiviral drug resistance enlargement of storing facilities: -70 C WHO Checklist for Influenza Pandemic Preparedness Planing WHO Protocol for Rapid Conatinment of an Emerging Influenza Pandemic 5

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