1. Molecular diagnosis of respiratory viruses and its impact on clinical management Prof G Kudesia Sheffield Teaching Hospitals NHS Trust

2. Cell Culture  Widely used  Result in 7-14 days or longer

3. Adenovirus CPE in RMK

4. Un-infected RMK

5. Live cells required Cytopathic effect needs to be confirmed by specific tests. Technical expertise Time delay Catch ‘all’

6. Antigen detection by Immunofluorescence  Rapid  Relatively insensitive  Not suitable for all speciemn types  Subjective

7. Serology Technically demanding Insensitive Acute and convalescent serum sample

8. Respiratory Viruses: Diagnosis Pre 1990’s. Advantages Disadvantage s Tissue Culture  “Open” Technique  Sensitive  Further characterisation  Evidence of active infection  Not applicable to all  Time consuming  viable virus only  Contamination/to xins Serology  Detects current and past infection (immunity)  Important for fastidious viruses  Prolonged testing time Antigen detection  Rapid  Detects non-viable virus  Can test large numbers of samples  Not applicable to all  interference

9. Polymerase Chain Reaction (PCR)-Xeroxing DNA!  Kary Mullis  Won the NobelPrize in 1993 for describing the methodology in 1985 to replicate DNA in a test tube.

11. PCR

12. Impact of PCR testing on respiratory virus investigations

13. Comparison of cell culture with PCR for Influenza A and B and RSV-200 specimen tested winter 2006/07-Sheffield VirusRSV Flu A Rhino HMPV Total PCRPositive382932 11 110 Cell Culture Positive12(32%)17(59%) 1 (3%) 0 30 (27%) (27%) Total382932 11 110

14. Respiratory PCR from Children –winter 07/08(haematology/oncology)

15. Advantages of PCR over traditional methods-R Gunson, Glasgow Positive Flu A RhinoRSVTotal Culture162220 DIF14n/a620 Total Isolation 202830 PCR41141570 Sensitivity (Iso Vs PCR) 49%14%53%43%

16. Improved detection rate

17. Improvements in TRT

18. Clinical impact  Influenza  Treatment  Prophylaxis  Outbreak Management  Control of infection  Immunocompromised  Treatment  Control of infection

19. Treatment/prophylaxis for influenza-start within 48 hours  Oseltamivir Treat- 75 mg twice a day x 5 daysTreat- 75 mg twice a day x 5 days Prophylaxis- 75mg once a day x10 daysProphylaxis- 75mg once a day x10 days  Speed for laboratory confirmation of essence  PCR testing was invaluable in the late influenza B activity this winter- both for outbreak and individual patient management

20. Flu outbreak- SVC west Scotland Hospital X Patient A – flu positive Index case Doctor A – flu positive Doctor B – flu positive Patient B – flu positive Patient C – flu positive Nurse – flu positive Occurred out with flu season Flu virus sequenced Phylogenetic tree created

21. Shown to be H3 Wisconsin Sequence flu strain originated from Patient A – the index case

22. Flu outbreak Hospital Y Patient D – flu positive Doctor C – flu positive Patient E – flu positive Flu virus sequenced Phylogenetic tree created Was there a connection with hospital X?

23. No connection between Hospital X and Y Tree may have looked like this Hospital Y Hospital X BUT THERE WAS A CONNECTION

24. Showed both flu outbreaks were connected All were H3 Wisconsin What was the connection?

25. Hospital Y Patient D – flu positive Doctor C – flu positive Patient E – flu positive Patient C from hospital X was transferred to hospital Y Hospital X Patient A – flu positive Index case Doctor B – flu positive Patient B – flu positive Patient C – flu positive Nurse – flu positive Doctor A – flu positive

26. Molecular epidemiology for outbreak sequencing  Implications Shows connections between patients/staffShows connections between patients/staff Raises infection control issuesRaises infection control issues  Patient transferred while ill  Why were staff infected Re-evaluate hospital proceduresRe-evaluate hospital procedures  E.g. masks, gowns, gloves, hand washing

27. A case of Respiratory infection in BMT-Sheffield  37 year old male post BMT  Presented with GVHD in December 07  Third week of march 08- respiratory symptoms- ? Infection, ? Respiratory GVHD  Respiratory and PCP PCRs- HMPV PCR positive 25/3, 7/4  Not treated initially but subsequently treated with I/V and nebulised Ribavirin due to deterioration in respiratory symptoms.  Died 14/4

28. Post-mortem histology of lung  Sections from both the lungs show fibrin and macrophages in the alveolar spaces  along with focal squamous metaplasia. There are scattered large bizzare cells  with basophilic inclusions in the cytoplasm. The features are those of an  organizing pneumonia with virocytopathic effect suggesting of viral aetiology.

29. Human Metapneumovirus  Discovered in 2000.  Paramyxoviridae  Negative sense, Single stranded RNA  Two genotypes A and B

30. Clinical Problems  Upper respiratory infection  Lower respiratory infection  Non-specific symptoms  Fatalities reported in BMT patients

31. Objective  To determine the incidence  1 st September 2005 to 31 May 2006

32. Methods  Data collection-retrospectively  Descriptive methods

33. Results Specimen type NO of specimens NPA205 BAL73 ETS22 Others48 Total348

34. The incidence of Respiratory Pathogens Total Aden13 MPV11 FluA11 FluB17 Para8 RSV25 Meas1 Total 86 (25%)

35. ageward Cond ition Clinic al SpecOutcome 1<1PICUBronNPA 2<1PICUBronNPA 32PICUBronNPA 43M3ALLURTINPA 55M3ALLNasalNPA 65M3OncoCoryzaNPADischarged 710M3ALLNPADischarged 838E2HIVDyspNPA 939E2AtypiNPADischarged 1054P3BMTCoryzaNPA 1168ITUNPA

36. Finding  HMPV -4 th commonest respiratory pathogen  Affected all age groups  Detected in patients with both upper and lower respiratory tract infections  Some patients discharged before results were available  Further studies for clinical significance

37. New viruses- human Bocavirus (HBoV)  Identified in 2005  DNA virus belonging to family Parvoviridae  Found in respiratory secretions from children with and with out respiratory symptoms  Exact role in respiratory infections to be still worked out

38.  How feasible is it to introduce PCR in routine diagnosis

39. Multiplex Real Time PCR Multiplex 1 Influenza A Influenza B Influenza C MatrixNSMatrix Multiplex 2 HMPV A HMPV B Parainfluenza 1 FusionFusionHN Multiplex 3 Parainfluenza 2 Parainfluenza 3 Parainfluenza 4 HNHNFusion Multiplex 4 HuCoV 229E HuCoVOC43 HuCoV NL63 NucleocapsidNucleocapsid 1a gene Multiplex 5 RSV A RSV B RhinoNPNP5-UTR

40. Small numbers of tests/More pathogens detected YearFormat N o of tests Tests performed (cumulative) 2000-01 Gel based nested 4 influenza A, B; RSV; adenovirus; picornavirus 2001-02 2002-03 influenza A, B; RSV; adenovirus; picornavirus; 2003-04 Real time PCR 5 influenza A, B; RSV; adenovirus; Rhinovirus; PF1, 2, 3 2004-055 influenza A, B; RSV A + B; adenovirus; rhinovirus; PF1, 2, 3; coronavirus NL63, 229e, OC43; HuMPV 2005-066 influenza A, B, C; RSV A + B; adenovirus; rhinovirus; PF1, 2, 3, 4; coronavirus NL63, 229e, OC43; HuMPV A + B 2007-085 influenza A, B, C; RSV A + B; adenovirus; rhinovirus; PF1, 2, 3, 4; coronavirus NL63, 229e, OC43; HuMPV A + B; M pneumoniae

41. But……………..: –Post amplification processing –Contamination –Prolonged testing time –Non-automated –Expensive to implement/expertise needed –Qualitative (difficult to quantify) Advantages of PCR The utilisation of PCR conferred many advantages: –Highly sensitive/specific Applicable to RNA or DNA virusesApplicable to RNA or DNA viruses Rapid (turn around time of 24-48 hours)Rapid (turn around time of 24-48 hours) Can detect multiple virusesCan detect multiple viruses Products can be sequenced for epidemiological/resistance study.Products can be sequenced for epidemiological/resistance study. Improved patient management and disease surveillanceImproved patient management and disease surveillance Submitted by R Gunson June 2003 Submitted by R Gunson June 2003

42. – Closed system No post-amplification processing No post-amplification processing Rapid Rapid Reduced contamination Reduced contamination Automation/high throughput/Cost effective Automation/high throughput/Cost effective Real time PCR: Unlike conventional PCR: –Amplicon is visualised as the amplification progresses. – Exponential rather than endpoint analysis – More tests /less reagents/standardised cycling conditions – Increased sensitivity/specificity Disadvantages of real time PCR: –Risk of false negative reactions (due to miss-matches). –Number of amplicons detected is limited by the number of fluorophores. –Expensive to implement Submitted by R Gunson June 2003 Submitted by R Gunson June 2003

43. Examples of the benefits of real time PCR assays in viral respiratory infection  Gueudin et al: Developed a real time PCR to detect, subgroup, and quantitate RSV A and BDeveloped a real time PCR to detect, subgroup, and quantitate RSV A and B  RSV A and B to be responsible to differing disease severities  Found higher viral loads in more severe infections  Elden et al: Developed a real time PCR for simultaneous detection of influenza A and B.Developed a real time PCR for simultaneous detection of influenza A and B.  Rapid diagnosis allowed timely therapeutic and infection control intervention  Quantitation could be used to examine the effects of antiviral therapy  Mackay et al: Developed a sensitive real time PCR for Human metapneumovirusDeveloped a sensitive real time PCR for Human metapneumovirus  Most sensitive assay currently available  Puhakka et al: Examined the effect of zanamivir on the viral load of influenzaExamined the effect of zanamivir on the viral load of influenza  Viral loads were reduced significantly Submitted by R Gunson June 2003 Submitted by R Gunson June 2003

44. Summary  PCR for respiratory viruses are sensitive and specific  Positivity rate of 50% or greater  Cell culture sensitivity 30-50% compared to PCR (for viruses that can be cultured)  Detection rate of PCR improved further as many viruses not culturable.

45. Summary- continued  Several viruses can be tested for at the same time by multiplex PCR  In-house PCR cost effective compared to cell culture  PCR effective epidemiological tool in investigation of outbreaks  Rapid and sensitive assay aids in clinical management of respiratory infections.