DCLS Influenza Laboratory Testing

1. DCLS Influenza Laboratory Testing Denise M. Toney, Ph.D. Commonwealth of Virginia Division of Consolidated Laboratory Services H & M Panel – May 2009

2. Division of ConsolidatedLaboratory Services (DCLS)Current Role in Influenza Testing • Virginia’s state laboratory serving a large number of local, state, and federal agencies • Tests human specimens for influenza virus year-round according to VDH guidelines: • Seasonal influenza surveillance (Sentinel physicians, OCME) • Investigation of respiratory illness outbreaks • Surveillance for novel influenza virus in humans • If case meets VDH and CDC clinical and epidemiologic criteria for possible novel influenza infection, including avian influenza • CDC and LRN protocols validated

3. DCLS - Public Health MissionPandemic Influenza Testing • NOT Diagnostic testing!!! • Determine when and where influenza viruses are circulating • Determine how much influenza activity is occurring • Identify types and strains of circulating influenza viruses in addition to the pandemic strain • Monitor for mutations in the influenza virus strains • Detect unusual events: • Infection by unusual viruses • Unusual syndromes caused by influenza viruses • Unusual large/severe outbreaks of influenza • Increased mortality

4. Laboratory Tests for Seasonal and Novel Influenza Viruses Influenza • Virus Isolation • Technically difficult; TAT issues • Good sensitivity and specificity • Typically not performed for avian or novel influenza (requires a BSL-3 laboratory with enhancements) • Immunofluorescence (DFA) • Rapid; limited specificity and sensitivity • PCR Techniques • Rapid and specific • Sensitivity depends on the test used, the influenza strain, and the type of specimen tested • Rapid antigen detection • not performed at DCLS Influenza RNA

5. DCLS Influenza Surge Plan

6. Existing DCLS Capabilities • Infrastructure available from ongoing preparedness efforts and planning • Multiple Biosafety level 3 (BSL3) suites • Dedicated molecular suite • Multiple real time PCR instruments and platforms • Laboratory Response Network (LRN) certified and CDC IVD qualified • Highly experienced molecular and virology staff • Statewide courier • 24 hours a day, 7 days a week emergency response capability • Dedicated collection kit preparation department • All staff classified as essential personnel

7. Novel Influenza Strain DetectionLaboratory Testing Timeline DCLS validated the CDC rtRT-PCR assay and implements for seasonal surveillance purposes CDC updated protocols to reduce variability and to initiate efforts at obtaining FDA approval. Assay released through MTA to state labs CDC obtained EUA and releases NEW swine flu assay to states; Lab verification required 2004 2005 2006 2007 2008 2009 CDC distributed rtRT-PCR assay for detection and characterization of influenza through APHL CDC MTA assay received FDA approval to be used for diagnosis of human Influenza infections IVD-rtRT-PCR Flu Panel A/H1, A/H3, A/H5 and B MTA assay DOES NOT detect the influenza A/H1 (swine-like) viral subtype

8. EUA CDC Swine Flu AssayDCLS Testing Algorithm Before assay release: • Specimens tested using culture, DFA and rtRT-PCR for influenza A/H1, A/H3 and B • Specimens positive for influenza A but negative by rtRT-PCR for seasonal H1 and H3 reported as probably swine-flu and forwarded to CDC for confirmation • Seasonal isolates and negatives were reported as usual After assay release: • Approved specimens tested using the 4 target EUA Swine flu rt-RT-PCR assay; viral culture performed • 4 Targets: Seasonal Flu-A, Swine Flu-A, Swine H1 and RP (control) • Seasonal Flu A (+) specimens reflex tested for seasonal subtypes • Influenza B testing not performed

9. Interpretation of Swine Flu Results Note: Influenza B testing NOT PERFORMED

10. Assay Implementation Issues • Required VDH screening of specimens prior to DCLS testing • SWF numbers  • Slight delay in obtaining testing reagents and supplies from CDC • Stock Pan-flu supplies beneficial • Swine-flu assay required “verification” using 5 positive specimens prior to receiving approval to report confirmatory results • No interpretation for results provided • EUA method – very restrictive as authorized • Not validated for DCLS preferred robotics • Seasonal surveillance role unclear • Flu Bs • Continue culture?

11. Current DCLS Pandemic Testing Capacity • Well…..it depends ???? • Capacity: ~800 PCR tests/day in a “perfect scenario” assuming limited targets, unlimited reagents, full staffing, no reporting deadlines • Capacity is dictated by critical testing reagents and supplies • Changing technologies, Limited shelf life, Restocking of expired reagents is expensive and cost ongoing • Specimen collection kits for rapid deployment • Implemented a LIMS to streamline result reporting and data tracking – ongoing • Cross-trained personnel in BSL-3 and PCR methods – ongoing

12. Challenges “thankfully” NOT encountered THIS TIME • Limitations or absence of lab reagents, supplies, PPE, etc • Worker absenteeism (ie. personal illness, family issues, quarantine) • Disruptions of medical and community services • Need to suspend other critical lab services • Exceeding bio-safety level 3 containment • High demand for diagnostic testing for the “worried well” • Successful HD screening of suspect cases (SWF) to limit testing and conserve critical reagents and supplies

13. DCLS - Public Health MissionPandemic Influenza Testing • NOT Diagnostic testing!!! • Determine when and where influenza viruses are circulating • Determine how much influenza activity is occurring • Identify types and strains of circulating influenza viruses in addition to the pandemic strain • Monitor for mutations in the influenza virus strains • Detect unusual events: • Infection by unusual viruses • Unusual syndromes caused by influenza viruses • Unusual large/severe outbreaks of influenza • Increased mortality

14. Contact Information: Denise M. Toney, Ph.D. 600 North 5th Street Richmond, VA 23219 Phone: 804-648-4480 ext 282 Blackberry: 804-641-6960