1st Area Medical Laboratory Participation in the FDA Specificity Evaluation of the JBAIDS Influenza A/H5 Detection System SSG Edward Loudenclos IV NCOIC, Bio/Endemic Disease Section

AGENDA Evaluation Abstract Background: A/H5 influenza Background: the JBAIDS analyzer H5 Project Overview The 1 st AML? Sample Processing and Testing THE EXPERIENCE! Results and Summary Acknowledgements and References

Evaluation Abstract The possibility of an emerging A/H5 pandemic is cause for great concern. Historically, influenza pandemics have killed countless millions. Fast and early detection impacts treatment and quarantine, and is CRITICAL!

Real Time PCR is recognized as the standard testing method for A/H5 by the CDC. The JBAIDS is a deployable Real Time PCR analyzer operated by DoD Personnel. Idaho Technology Inc. re-optimized the CDC A/H5 Flu Panel for use on the JBAIDS analyzer.

BACKGROUND: A/H5 Influenza

A/H5 Influenza (Avian Flu) Primarily infects birds, but can infect humans. Infected birds have been reported in at least 40 different countries. Human cases reported in at least 15 different countries. Mortality rate is approximately 61%! Death toll world-wide would exceed 1918 pandemic.

H5 Maps

Primary mode of transmission bird to human, but possibility of mutation exists for human to human transmission. Risk factors that promote possible pandemic: – Poor Infrastructure – Economics – Lack of surveillance – Lack of public awareness and education

Rapid detection of humans infected with H5 provides the following benefits: – Patient isolation, prevents possibility of additional exposures. – Early treatment. – Viral Characterization (did it mutate?!) What PCR assays are available now for detecting H5?

CDC Human Influenza Virus Real-time RT-PCR Detection and Characterization panel (rRT-PCR Flu Panel). – Detects several different flu types, including A/H5 – Sample extraction and purification can be done using different kits (i.e. Qiagen) – Manual or Automated extraction. ABI 7500 FAST

ABI 7500 FAST Real Time PCR using CDC Flu Panel Pros CDC Validated FDA Cleared Reliable Results Sensitivity Cons Size Not mobile Consumables (temperature issues) Operator Training How do we address these problems?

BACKGROUND: The JBAIDS Analyzer

JBAIDS Joint Biological Agent Identification and Diagnostic Systems DoD accepted Platform for both identification and diagnostic confirmation of biological agents and pathogens of concern. Can operate in fixed medical facilities, or deployed mobile units. Developed by Idaho Technology, Inc.

JBAIDS: Specifications Approximately 36 lbs packed. 110/220v power switching capability. Rugged, carbon fiber case. 1-meter drop test. – Meets MILSTD 810F requirements Easy user-level maintenance, replacement parts issued with instrument. 32-sample capacity Includes “Tough-book” laptop and software. Real-time PCR for both DNA and RNA. Cycle times vary (40 minutes for DNA, 70 minutes for RNA due to RT step)

JBAIDS: Extraction ITI produces Extraction kits for use with the JBAIDS analyzer: – SWIPE: Nasal Swabs (DNA), Surface Swabs (DNA and RNA), live culture (DNA and RNA), Powder (DNA and RNA) Pus swabs, lymph node aspirates. – VIBE: Whole Blood (RNA), Nasal Swabs (RNA), Sputum (DNA and RNA). – SCOOP: Stool and Soil samples. – QFLOW DNA: Whole Blood(DNA), Air in PBC (DNA), Water, Milk, Salad, Ground Beef, Tuna Salad, Gastric Washings. – QFLOW RNA: Air in PBS(RNA). – PLATINUM PATH: All Sample Types, can extract DNA and RNA simultaneously (Uses Mag-Bead Technology).

JBAIDS: Reagents Vaccinia (Orthopox) Yersina pestis (Plague) Bacillus anthracis (Anthrax) Francisella tularensis Brucella melitensis Rickettsia prowazekii Burkholderia spp. Variola (Smallpox) Coxiella burnetii Ebola Marburg Eastern, Western, and Venezuelan equine encephalitis FDA Cleared Reagents: – Anthrax IVD – Plague IVD – Tularemia IVD Kits are freeze-dried, lyophilized reagents. Can be stored at Room Temp.

JBAIDS H5 Project Overview

Overview: Clinical Testing Locations 3 Tests Sites: Naval Health Research Center San Diego, CA Brooke Army Medical Center San Antonio, TX Tripler Army Medical Center Honolulu, HI

Overview: Sample Criteria Specimens Two sample types: – Throat Swabs – Nasopharyngeal Swabs 0.6mL-1.5mL sample volume Specimens previously tested using standard diagnostic methods De-identified (no Personally Identifiable Information)

Overview: Sample Flow Sick. Flu-like Symptoms Samples obtained Lab results negative for Flu Samples De-identified Sample aliquots for extraction

Overview: Extraction De-identified Specimens split 3 ways to be extracted using the following kits: – IT VIBE Extraction Kit (on JBAIDS) – IT Platinum Path Extraction Kit (on JBAIDS) – Qiagen QIAamp RNA mini Kit (on ABI 7500 FAST) Positive and Negative extraction controls are processed with every specimen batch. All extracted samples must be sent to analysis the day of extraction.

Overview: Results Specimens were tested against 3 targets: – 2 targets specific to A/H5 (Asian Lineage) – Control target Human RNase P gene (functioned as Extraction and Inhibition control) ALL results from the processed specimens must match regardless of extraction method and PCR analyzer used.

Here comes the 1 st Area Medical Laboratory!

How the 1 st AML got involved Tripler Army Medical Center. – Small number of people JBAIDS trained. – Staff had multiple duties. Clinical Mission > Research Mission. Time requirements for extraction and testing necessitated Full-time staffing. Oh, and…

LTC Hamilton at TAMC submitted request for assistance through Pat Craig at JPEO-CBMS. CBMS issued request to COL Freund, Commander, 1 st AML. Two Soldiers Selected: – SSG Loudenclos – SGT Harris TDY in HAWAII from 30 NOV 09 – 23 DEC 09

Sample Processing and Testing

Extraction

Reagent Prep

Analysis

Results

The Hawaiian Experience!

The Results!

Total number of samples evaluated: 986 – 518 Throat Swabs – 468 Nasopharyngeal Swabs JBAIDS Influenza A/H5 Detection Kit resulted with a Clinical Specificity of 99% and with a 95% confidence. (This met target goals!) All findings, results, and data were submitted to the FDA for review on: 29 JAN 2010

Future Goals The JBAIDS Program Office is currently working on taking the full Influenza test panel (A,B, seasonal A/H1, season A/H3, swine A and 2009 A/H1 assays) through 510(k) Anticipated FDA clearance is 2 nd or 3 rd Quarter 2011.

Summary A/H5 Avian Flu could pose a grave pandemic threat. Rapid diagnostic testing necessary to help prevent spread of infection. Real-time PCR is set as standard for testing. ITI’s H5 Flu kit can provide same level of sensitivity and confidence as currently accepted methods.

Acknowledgements Tripler Army Medical Center: – LTC Hamilton, CPT Barrera, Mr. and Miss. Eberly Idaho Technology, Inc: – Sean Phipps, Beth Lingenfelter, Sarah Jeffs JPEO-CBMS and JBAIDS Program Office: – Pat Craig, James Karaszkiewicz, Jennifer McLaughlin 1 st Area Medical Laboratory: – COL Freund, SGM Krueger, LTC Pell, CPT Pierson

References Beigel, J.H., et al., Avian influenza A (H5N1) infection in humans. N Engl J Med, (13): p WHO, Avian Influenza Update CDC, Key Facts About Avian Influenza (Bird Flu) and Avian Influenza A (H5N1) Virus. info/facts.htm, info/facts.htm

For up-to-date information on either H5 and H1 Influenza: World Health Organization – Centers for Disease Control and Prevention –

QUESTIONS?