1. MERS (Corona-EMC) JUNE 2013 CountryReports (deaths) France2 (1) Italy3 (0) Jordan2 (2) Katar2 (0) Saudi Arabia39 (24) Tunesia2 (0) Great Britain3 (2) United Arabian Emirates1 (1) total54 (30)

2. 19.06.2013 | Rolf Kaiser, Ortwin Adams New Technology Challenges for Old Diseases

3. 19.06.2013| Rolf Kaiser, Ortwin Adams Viral respiratory Diseases Rhinovirus Coronavirus Parainfluenzavirus Respiratory Syncytial Virus (RSV) Human Metapneumovirus Adenovirus Bocavirus Influenzavirus A and B

4. 19.06.2013| Rolf Kaiser, Ortwin Adams

5. Influenzavirus A und B (5-15%) Rhinovirus (30-50%) Coronavirus (10-25%) Parainfluenzaviruses (5%) RSV (5%) h. Metapneumovirus ( 10%) Adenovirus (5-10%) Bocavirus (0-5%) Respiratory Viruses Common cold(?) “Erkältung” Flu “Grippe”

6. 1rst step of evolution: Before Swine-Flu: No diagnostics / (rapid) antigen tests (bedside tests) only Flu 2nd step of evolution After Swine-Flu: More diagnostics / Molecular based assays (PCR) Flu 3rd step of evolution Multiplex Molecular based assays (PCR) Flu and RSV and …and …

7. 19.06.2013| Rolf Kaiser, Ortwin Adams Influenza A, B Parainfluenza 1,2,3 RSV A, B; Adenovirus Human Metapneumovirus A,B Coronavirus 229E,OC43, NL63 Rhinovirus, Enterovirus Human Bocavirus M. pneumonia., C.pneumonia., B. pertussis. In house multiplex PCR (TaqMan /LightCycler) O. Adams

8. 19.06.2013| Rolf Kaiser, Ortwin Adams I. Multiplex Tagged Target-Specific PCR xMAP™ Read PE B B II. Universal Array Sorting and Detection xTAG TM RVP FAST 3.5 h (including extraction), 1 h hands on xTAG RVP FAST – streamlined workflow xTAG RVP FAST now combines several steps necessary with xTAG RVP to 2 key steps Speeds up time to result – 3.5h Reduce hands on time - <1h Reduce sample transfer steps to 1

9. 19.06.2013| Rolf Kaiser, Ortwin Adams Now 18 viruses/subtypes in RVP FAST versus 20 in RVP: Changes: Removal of influenza H5 (avian influenza) Removal of SARS (not required) RSV A & B not discriminated (not clinically important to discriminate) Addition of Human Bocavirus (the last target needed to bring coverage of xTAG RVP to 98% circulating respiratory viruses) xTAG RVP FAST – viruses/subtypes detected xTAG RVPxTAG RVP FAST Influenza A Influenza A H1 Influenza A H3 Influenza A H5 Influenza B RSV ARSV RSV B Coronavirus 229E Coronavirus OC43 Coronavirus NL63 Coronavirus HKU1 Coronavirus SARS Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Parainfluenza 4 Human Metapneumovirus Entero-Rhinovirus Adenovirus Human Bocavirus

10. 19.06.2013| Rolf Kaiser, Ortwin Adams Detail sample Positive call

11. 19.06.2013| Rolf Kaiser, Ortwin Adams

12. 19.06.2013| Rolf Kaiser, Ortwin Adams

13. 19.06.2013| Rolf Kaiser, Ortwin Adams #Patienten IDErgebnis in-house PCRErgebnis RVP FastBemerkung 110301714ØEntero-/Rhinovirus- 210301715ØØ- 310298863BocavirusEntero-/Rhino und Bocavirus- 410291005ØØ- 510304447AdenoØno eluate, only rest from tube (<1µl) 610300410ØØ- 710301705ØØ- 810291002ØØ- 910312536ØØ- 1010310926RSV and EnterovirusØWrong storage 1110312927BocavirusHMPV und Bocavirus- 1210312929Bocavirus - 13Run controle--- 14Negativ-controle--- 15Beads (controle)---

14. The Respiratory Virus Network: an initiative to collect and provide data on respiratory virus diseases via internet Kaiser R. 1 and Adams O. 2 1 Universität Köln, Institut für Virologie, Köln 2 Universität Düsseldorf, Institut für Virologie, Düsseldorf

15. RespVir-Network: Characteristics established by members of the clinical virology section of the Gesellschaft für Virology (GfV) collect data on the detection rates of respiratory viruses on different sites in germany, austria, and switzerland. meanwhile more than 250,000 tests of 27,000 patients are collected. main focus: frequency of respiratory viruses in hospitalized patients ("inpatients") supplement the existing surveillance systems which usually focus on community aquired respiratory infections ("outpatients") → Is it clear what is “going around” already? To what extent?

16. Linz RespVir-Network: Sites agreed to input data into the database: germany: 21 sites austria: 4 sites switzerland: 1 site further activites and aims: sequencing and genotyping e.g. adenovirus – Hannover influenzavirus – Cologne RSV – Würzburg qualitiy controls e.g. interlaboratory tests weak points: different techniques different spectrums of detected viruses

17. RespVir-Network –data from Feb 2011 to Feb 2013 20112012 20112012

20. RespVir-network: example of a monthly report (january 2013)

21. RespVir-network: RSV-incidence feb 2011-feb 2012 february 2011 marchapril may june augustseptember november december february 2012

22. RespVir-network: RSV-incidence feb 2012-jan 2013 february 2012 marchapril may june augustseptember november december january 2013

23. RespVir-network: RSV-incidence since Dec 2009 RSV saison 2010-2011: week 44 – week 17 RSV saison 2011-2012: week 50 – week 20 RSV saison 2012-2013: week 44 – week 21

24. Ref: Buda et al: AGI-RKI Influenza Wochenbericht 8.KW 2013 (Feb. 2013) AGI: focus on outpatients RespVir-network: focus on (pediatric) inpatients comparable data Arbeitsgemeinschaft Influenza (AGI): INF/RSV

25. RespVir-network: influenza A and B activity since july 2012

26. Arbeitsgemeinschaft Influenza (AGI): Praxisindex AGI: focus on outpatients RespVir-network: focus on inpatients Ref: http://influenza.rki.de

27. www.rki.de

28. RespVir-network: rhinovirus incidence since Dec 2009 Rhinoviruses: weakest activity in july and august

29. Why viral Diagnostics? 4. January, Friday, 22.30 Girl 8 Months old (normal development, no risk factors) feaver, cough, reduced general condition (AZ) reduced food intake Diagnosis: acute interstial Pneumonia

30. Which ward? Which room? Hospitalization Cohorts?? (RSV, Influenza, usw) 4 hospitalizations within the past 4h due to respiratory infections Why viral Diagnostics?

31. Bronchitis: 25 11 4 2 3 Bronchiolitis: 23 Pneumonia: 24 2 6 2 0 RSV Boca Rhino Entero hMPV Correlation of virus detection and clinical symptoms (Dat from Unikinderklinik Düsseldorf) Why viral Diagnostics?

32. 8 months female (normal development, no risk factors) feaver, cough, reduced general condition (AZ) reduced food intake Diagnosis : Pneumonia Hospitalization Diagnosed virus:Rhinoviren Why viral Diagnostics?

33. No Antibiotics !!! Specific antiviral therapy for Influenza (Tamiflu, Relenza) Shorter time of hospitalization Relevant for DRG-coding The reimbursement for RSV positive cases is higher Symptomatic Therapy Cohorts Consequnces of virus diagnostics: Why viral Diagnostics?

34. Next steps: Access and contribution to the network-database to the Netherlands (H.Niesters, Groningen) open access to the database (online statistics, incidence reports etc.) including relevant bacterial pathogens (M. pneumoniae, B. pertussis, C. pneumoniae) english and other languages (NL) version of database network for viral and bacterial gastroenteritis

35. Detection of respiratory pathogens by conventional and molecular diagnostics Virus isolation in cell culture ("gold standard"??) Virus antigen detection (IFT, EIA) Real-time-PCR (monoplex) Real-time-PCR (duplex/triplex) Real-time-PCR (multiplex) Amplification with detection on Luminex: ResPlex II Panel v2.0 (Qiagen) MultiCode-PLx (EraGen Biosciences) RVP fast (Luminex)

36. Detection of respiratory pathogens by conventional and molecular diagnostics Virus isolation in cell culture ("gold standard"??) Virus antigen detection (IFT, EIA) Real-time-PCR (monoplex) Real-time-PCR (duplex/triplex) Real-time-PCR (multiplex) Amplification with detection on Luminex: ResPlex II Panel v2.0 (Qiagen) MultiCode-PLx (EraGen Biosciences) RVP fast (Luminex)

37. Conclusions Molecular diagnostic tools are now available to assess better the epidemiology of respiratory viruses This year Influenza and RSV were the drivers of epidemia Tamiflu/Relenza are specific drugs for Flu; Synagis for RSV Flu and RSV diagnostics have therapeutic consequenses The RespVir Network is an instrument to get an online-insight into the epidemiology of respiratory viruses (mainly hospitalized patients). The RespiVir Network induced a standardization of methods and spectrum of investigated viruses

38. Institute of Virology, University of CologneInstitute of Virology, University of Cologne Jens Verheyen (Uniklinik Essen), Melanie Balduin, Dörte Hammerschmidt, Monika Timmen-Wego, Monika Junk, Rolf Kaiser, Herbert PfisterJens Verheyen (Uniklinik Essen), Melanie Balduin, Dörte Hammerschmidt, Monika Timmen-Wego, Monika Junk, Rolf Kaiser, Herbert Pfister Institute of Virology, University of Duesseldorf, Institute of Virology, University of Duesseldorf, Ortwin Adams, Linda Bonzel Ortwin Adams, Linda Bonzel Medeora, KölnMedeora, Köln Andrej Wöhrmann, Norbert SchmeisserAndrej Wöhrmann, Norbert Schmeisser Treffen der LaborleiterTreffen der Laborleiter Barbara Gärtner, Benedikt WeissbrichBarbara Gärtner, Benedikt Weissbrich W.Gross, Pediatrician, BonnW.Gross, Pediatrician, Bonn Luminex Edwin RooversLuminex Edwin Roovers ABBOTTABBOTT Jenny PahneJenny Pahne www.medeora.de

39. Thanks to: Ortwin Adams (Institute for Virology, Düsseldorf) all members of the "RespVir-Netzwerk" in Germany, Austria and Switzerland working group "clinical virology " of the GfV (B. Gärtner and B. Weissbrich) B. Reinard (Abbott GmbH) and E. Roovers (Luminex) G. Schmeisser (Medeora) T. Mertens (GfV) and P. Wutzler (DVV)

40. 19.06.2013| Rolf Kaiser, Ortwin Adams In-House-multiplex / FTD / xTAG-RVP-FAST test panel Sampletarget value (CT)Inhouse MultiFTDRVP FTD-1Ade (21)Ade FTD-2 Cor229E (26) Ade (35) Cor229E Ade Cor229E FTD-3 EV/Parecho (26) EV/Parecho Ade ? EV/Parecho Cor NL43 ? EV/Rhino FTD-4Inf-A (23)neg Inf-AHandling mistake FTD-5hMPV-A/B (31)hMPV-A hMPV-A/BhMPV FTD-6Rhino (26)Rhino EV/Rhino FTD-7 Para-2 (27,5) Rhino (31)Rhino Para-2 Rhino Para-2 EV/Rhino FTD-8Cor NL63 (20,5)neg Cor NL63 FTD-9Para-1 (34)neg Para-1 FTD-10Ade (22)Ade FTD-11Inf-A (22,7)neg Inf-AInf-A/H3 FTD-12Cor OC43 (21)Cor OC43

41. 19.06.2013| Rolf Kaiser, Ortwin Adams FTD-13RSV (22)RSV-ARSV FTD-14neg FTD-15Para-2 (26)negPara-2 FTD-16neg FTD-17RSV (24,5)RSV-BnegRSV FTD-18EV/Parecho (27)neg FTD-19Inf-B (25)Inf-B FTD-20neg FTD-21Para-3 (35)neg Para-3 FTD-22Rhino (Traces)Rhino EV/Rhino FTD-23 Cor NL63 (22) Ade (22,5)Ade Cor NL63 Ade FTD-24Para-4 (31)negPara-4 Panel part 2

42. 19.06.2013| Rolf Kaiser, Ortwin Adams Data from Cologne in-house vs RVP #extraction methodresultsin-house PCR resultsextraction method in-house PCR 1extraction with m2000sp RNA/DNA protocolnegative extraction with Siemens 2extraction with m2000sp RNA/DNA protocolnegative extraction with Siemens 3extraction with m2000sp RNA/DNA protocolEntero/Rhinovirus positiveRhinovirus positiveextraction with Siemens 4extraction with m2000sp RNA/DNA protocolnegative extraction with Siemens 5 extraction with Siemens * Entero/Rhinovirus positiveRhinovirus positiveextraction with Siemens 6 extraction with Siemens * negative extraction with Siemens 7 extraction with Siemens * Entero/Rhinovirus + Adenovirus (!!!) positiveRhinovirus positiveextraction with Siemens 8 extraction with Siemens * Flu A matrix positiveSwineflu positiveextraction with Siemens 9 extraction with Siemens * Flu A matrix positiveSwineflu positiveextraction with Siemens 10No extraction (plasmid control Coronavirus NL63 + OC43 + Entero/Rhionvirus + Metapneumovirus + Bocavirus positive plasmid control mixtureNo extraction (plasmid control 11 Lambda control * negative-- 12negative control (H 2 O)negative-- 13beadsnegative-- * no internal control added