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Pandemic H1N1 (2009) laboratory response. Singapore. 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
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Pandemic H1N1 (2009) laboratory response Singapore
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
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
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
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
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
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
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
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
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!
Surveillance testing • Before pandemic: weekly data, 20-50 samples/week • Pandemic • *daily* monitoring • Achieve statistical confidence to detect 1% change = 160 samples/ day
Surveillance testing • Based on ILI symptoms • Sentinel sites • Polyclinics (government clinics) • Private GPs • Emergency departments
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
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Daily proportion (%) of influenza A/H1N1/2009 among all samples based on influenza surveillance of polyclinics, GP clinics, and hospitals
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.
Continued importance of H3N2 • In May 2009, we were in the middle of an H3N2 epidemic • Vaccine failures in longstay residents caused influenza clusters
Emergence of new H3N2 in 2009 associated with vaccine failure
Oseltamivir-resistant H1N1 (2009) with H275Y • View 3D structure with a lot of features
Mutations in seasonal H3N2 isolates Blue : residues within 3A of antibody in complex structure 2VITCyan: residues within 3A of antibody in complex structure 1KENGreen: residues within 3A of antibody in complex structure 1EO8Yellow residues: mutations in your patient/sample