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 1st 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
• 600-950 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 1-2-3 VIBE Extraction Kit (on JBAIDS)
– IT 1-2-3 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
1st Area Medical Laboratory!
How the 1st 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, 1st 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 2nd or 3rd 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
• 1st 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, 2005. 353(13): p. 1374-85.
• WHO, Avian Influenza Update.
http://www.who.int/csr/disease/avian_influenza/en/
, 2008.
• CDC, Key Facts About Avian Influenza (Bird Flu)
and Avian Influenza A (H5N1) Virus.
http://www.cdc.gov/flu/avian/geninfo/facts.htm, 2007.
• For up-to-date information on either H5 and
H1 Influenza:
• World Health Organization
– http://www.who.int/en/
• Centers for Disease Control and Prevention
– http://www.cdc.gov/
QUESTIONS?