1. Pandemic H1N1 (2009) laboratory response Singapore

2. Containment phase –All patients with travel history to affected countries go to one hospital (TTSH) –Later more hospitals opened up –Severe symptoms or high-risk groups: admit to hospital –Others wait at Emergency Department for laboratory test results: 6-8 hours

3. Transition and mitigation phase –Test only patients in high-risk groups, or those sick enough to be admitted to hospital –Influenza-like illness (ILI): stay home, MC x 7 days –ILI in vulnerable groups: empiric treatment with antiviral

4. Laboratory testing methods PCR for influenza A –H1N1 (2009) + H3 + H1 (seasonal) –US CDC protocol (modified for primers, machines, reagents) –Self-designed real-time and end-point PCR –Roche kit Virus isolation (MDCK) for positive PCR cases

5. Quality assurance of H1N1 PCR Test with virus samples –Seasonal and H1N1(2009) –Other respiratory viruses –Samples known to be positive for seasonal flu Control strain from Melbourne WHO CC External quality assurance (9 labs) –Known positive samples / RNA sent to participating labs First cases – M gene sequencing New testing labs – first 20 cases require re- testing by NPHL

6. Sample collection Nasopharyngeal swabs –Preferred flocked swabs in universal transport medium (UTM) Alternatives –Combined throat + nose swab –Throat swab only Diagnostic testing: 2 swabs taken within 10 minutes –Concordance almost 100%, so reduced to one swab

7. Testing laboratories NPHL (National Public Health Laboratory) –Obtain test protocol –Recommend and distribute primers/ probes –Obtain control strains; make RNA –Perform sequencing and virus isolation –Re-test problem cases 6 hospital labs with PCR capability –TTSH, SGH, KKH, NUH, AH, CGH

8. Increasing lab capacity Bought more –Robotic extractors (Qiagen, EasyMag, Qiacube, liquid handler) –Thermocyclers Hired temporary and permanent staff Use other labs: National Environment Agency, Defence Science Organization Improve workflow

9. Increasing lab capacity Working hours –Extended 8 am to 12 midnight + weekends –One hospital team: 24 hour service Before pandemic –<50 influenza PCR samples/ day At the peak –>1 000 samples/day

10. Testing objectives Diagnostic testing: 6-8 h turnaround time Test before discharge Surveillance testing Coverage for major international meetings Coverage for mass sports events –Asian Youth Games + other non-recommended situations!

11. Surveillance testing Before pandemic: weekly data, 20-50 samples/week Pandemic –*daily* monitoring –Achieve statistical confidence to detect 1% change = 160 samples/ day

12. Surveillance testing Based on ILI symptoms Sentinel sites –Polyclinics (government clinics) –Private GPs –Emergency departments

13. Surveillance testing Support decision-making Examples –0 to 1% : ? Signal of community spread – transit into mitigation * –>15% : justifies empiric use of antivirals in susceptible groups –Future: return to 10-15% - tailing of pandemic? Monitor other parameters as well e.g. total ARI (acute respiratory illness) attendance *Estimated 20 000 ARIs/ day, est. 4 000 ILIs/ day, therefore 1% = 40 cases 5% = 200 cases; x6 for private GPs

14. Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

16. Daily proportion (%) of influenza A/H1N1/2009 among all samples based on influenza surveillance of polyclinics, GP clinics, and hospitals

17. Note: N refers to daily no. tested.

18. Daily proportion (%) of influenza A/H1N1/2009 among flu A positive samples based on influenza surveillance of polyclinics, GP clinics, and hospitals Note: n refers to daily no. tested positive for Flu A.

19. Combined Daily Influenza Surveillance and Flu A Typing Results Note: N refers to daily no. of samples tested and n refers to daily no. tested positive for Flu A.

20. Daily Influenza Surveillance and Flu A Typing Results Note: N refers to daily no. of samples tested and n refers to daily no. tested positive for Flu A.

21. Daily Influenza Surveillance and Flu A Typing Results Note: N refers to daily no. of samples tested and n refers to daily no. tested positive for Flu A.

22. Continued importance of H3N2 In May 2009, we were in the middle of an H3N2 epidemic Vaccine failures in longstay residents caused influenza clusters

23. Position7886160174205276277 Vaccine strain and 2008 isolates ELNKNIR 2009 isolatesKIKN/RKMQ % (Jan to Feb)(n=9)22Kindergarten sample only 22562200 %(Mar to May)(n=16)69 81753164 2008 Isolates000R was seen in Nov and Dec 000 Emergence of new H3N2 in 2009 associated with vaccine failure

24. Input your sequence (a.a or n.a) Free web tool from Bioinformatics Institute, A*Star, Singapore http://flumap.inmegen.gob.mx

25. Automatic select reference sequence Generate report of mutations vs reference sequences

26. View 3D structure with a lot of features Oseltamivir-resistant H1N1 (2009) with H275Y

27. Mutations in seasonal H3N2 isolates Blue : residues within 3A of antibody in complex structure 2VIT Cyan: residues within 3A of antibody in complex structure 1KEN Green: residues within 3A of antibody in complex structure 1EO8 Yellow residues: mutations in your patient/sample