Molecular diagnosis of respiratory viruses and its impact on clinical management

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.

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

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

15. Advantages of PCR over traditional methods-R Gunson, Glasgow

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

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

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

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

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

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

26. Molecular epidemiology for outbreak sequencing • Implications • Shows connections between patients/staff • Raises infection control issues • Patient transferred while ill • Why were staff infected • Re-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 • 1st September 2005 to 31 May 2006

32. Methods • Data collection-retrospectively • Descriptive methods

33. Results

34. The incidence of Respiratory Pathogens

36. Finding • HMPV -4th 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

40. Small numbers of tests/More pathogens detected

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

42. Submitted by R Gunson June 2003 Real time PCR: Unlike conventional PCR: • Amplicon is visualised as the amplification progresses. • Closed system • No post-amplification processing • Rapid • Reduced contamination • Automation/high throughput/Cost effective • Exponential rather than endpoint analysis • Increased sensitivity/specificity • More tests /less reagents/standardised cycling conditions 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

43. Submitted by R Gunson June 2003 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 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. • 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 metapneumovirus • Most sensitive assay currently available • Puhakka et al: • Examined the effect of zanamivir on the viral load of influenza • Viral loads were reduced significantly

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.