DCLS Influenza Laboratory Testing

Denise M. Toney, Ph.D.Commonwealth of Virginia

Division of Consolidated Laboratory Services

H & M Panel – May 2009

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

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

Laboratory Tests for Seasonal and Novel Influenza Viruses

• 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

Influenza RNA

DCLS Influenza Surge Plan

Surveillance Testing Surge Testing

Real time RT-PCR,

DFA, culture

Real-time RT-PCR

only

Flu A/B/H1/H3

(H5 - with VDH consult)

Flu A/H5

(or current novel subtype)

Manual specimen prep Robotic specimen prep

One workshift/day 2 to 3 workshifts/day

1-20 specimens/day Up to 800 specimens/day

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

Novel Influenza Strain DetectionLaboratory Testing Timeline

2004 2005 2006 2007 2008 2009

CDC distributed rtRT-PCR assay for detection

and characterization of influenza through

APHL

CDC updated protocols to reduce variability and

to initiate efforts at obtaining FDA approval. Assay released through

MTA to state labs

CDC MTA assay received FDA approval to be used for diagnosis of human

Influenza infectionsIVD-rtRT-PCR Flu PanelA/H1, A/H3, A/H5 and B

CDC obtained EUA and

releases NEW swine flu assay

to states; Lab verification

required

MTA assay DOES NOT detect the influenza A/H1 (swine-like) viral subtype

DCLS validated the CDC rtRT-PCR

assay and implements for

seasonal surveillance

purposes

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

Interpretation of Swine Flu ResultsSeasonal

Flu A

Swine

Flu A

Swine

H1

Seasonal

H1

Seasonal

H3

RP DCLS Reportable Results

+ + + NT NT + Swine-like Influenza A/H1 detected

+ - - NT NT + Seasonal influenza A detected

Subtype not determined

Swine-like influenza A/H1 not detected

+ - - + - + Seasonal influenza A/H1 detected

Swine-like influenza A/H1 not detected

+ - - - + + Seasonal influenza A/H3 detected

Swine-like influenza A/H1 not detected

2 of 3 Positive NT NT + Equivocal for Swine-like Influenza A/H1

- - - NT NT + Seasonal and Swine-like Influenza A not detected

- - - NT NT - Inconclusive due to Specimen Quality

Note: Influenza B testing NOT PERFORMED

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?

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

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

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

Contact Information:Denise M. Toney, Ph.D.

600 North 5th StreetRichmond, VA 23219

Phone: 804-648-4480 ext 282Blackberry: 804-641-6960