Clinical Evaluation of the JBAIDS Influenza A & B - BioFire Defense
1
Presented at the Society of Armed Forces Medical Laboratory Scientists, April 2012 CONTACT INFORMATION Lou Banks Idaho Technology, Inc. [email protected] See all of ITI’s scientific posters by scanning the QR code to access the Scientific Poster Page Kevin Bourzac 1 , Sean Phipps 1 , Cynthia Phillips 1 , Andrew Hemmert 1 , Cynthia Andjelic 1 , Brook Danboise 2 , Dennis Faixs 3 , Jason Lane 4 , Vidhya Prakash 5 , Jennifer Russell 6 , Patricia Beverly 7 , Jennifer C. Dabisch 8 , Gary Uzzell 1 , and Beth Lingenfelter 1 1 Idaho Technology, Inc., Salt Lake City, UT, 2 Womack Army Medical Center, Fayetteville, NC, 3 Naval Health Research Center, San Diego, CA, 4 David Grant Medical Center, Farfield, CA, 5 Wright-Patterson Air Force Base, Dayton, OH, 6 Brooke Army Medical Center, San Antonio, TX, 7 United States Army Medical Materiel Development Activity, Fort Detrick, MD, 8 Joint Project Management Office for Chemical, Biological Medical Systems - Biosurveillance, Frederick, MD Clinical Evaluation of the JBAIDS Influenza A & B Detection Kit and JBAIDS Influenza A Subtyping Kit DISCLAIMER The views, opinions, and findings contained in this report are those of the author(s) and should not be construed as an official Department of Defense position, policy, or decision, unless so designated by other official documentation. INTRODUCTION The Joint Biological Agent Identification and Diagnostic System (JBAIDS), a real-time PCR- based system, is the United States Department of Defense’s Program of Record for diagnostic testing of infectious diseases of operational concern. The JBAIDS Influenza A & B Detection Kit is designed to detect influenza viruses A and B, and the JBAIDS Influenza A Subtyping Kit is designed to differentiate influenza A hemagglutinin subtypes seasonal influenza A H1, seasonal influenza A H3, and 2009 influenza A H1N1 when testing nasopharyngeal swabs (NPS) or nasopharyngeal washes (NPW). Here, we present selected analytical and clinical studies that were performed in support of 510(k) pre-market applications for JBAIDS Influenza A & B Detection kits and the Influenza A Subtyping kit to the US FDA. Two nucleic acid purification kits were also evaluated, both of which use magnetic bead-based technology. The IT 1-2-3™ Platinum Path™ Sample Purification kit, manufactured by Idaho Technology, Inc, is a manual extraction method and the Roche MagNA Pure Compact Nucleic Acid Isolation kit I is an automated system. PERFORMANCE TESTING FOOTNOTES This work was funded by the Armed Forces Health Surveillance Center (AFHSC) as the Requesting Agency under the Memorandum of Agreement (MOA) between AFHSC and the Chemical Biological Medical Systems Joint Project Management Office (CBMS JPMO). Limit of Detection The LoD for each target influenza assay was determined using both NPS and NPW samples spiked with quantified live virus. The LoD was the lowest concentration for which greater than 95% of samples yielded positive results for both sample matrices and both nucleic acid purification kits. Kit Assay(s) Influenza Type Strain LoD (EID 50 /mL) Influenza A & B Detection Kit Flu A Influenza A H1N1 A/New Caledonia/20/1999 50 Influenza A H3N2 A/New York/55/2004 5 Influenza A H1N1 2009 A/New York/18/2009 50 Flu B Influenza B B/Ohio/1/2005 5 Influenza B B/Florida/7/2004 10 Influenza A Subtyping Kit Flu A H1 Influenza A H1N1 A/New Caledonia/20/1999 50 a A/Hawaii/15/2001 5,000 Flu A H3 Influenza A H3N2 A/New York/55/2004 5 A/Wisconsin/67/2005 10 Flu A H1 2009/ Flu A Sw Influenza A H1N1 2009 A/New York/18/2009 1,500 A/California/7/2009 5,000 a The aliquot of A/New Caledonia/20/1999 tested contains 17-45 times more PCR target copies per EID 50 than the aliquot of A/Hawaii/15/2001 tested. Assay Design The JBAIDS Influenza assays are based on influenza assays from the ‘CDC Human Influenza Virus Real-time RT-PCR Detection and Characterization Panel (CDC rRT-PCR Flu Panel)’ and the ‘CDC Influenza 2009 A(H1N1)pdm RT-PCR Panel’. The JBAIDS assays were re-optimized to work with Idaho Technology’s proprietary freeze-dried PCR reagent formulation. Testing demonstrated that the assays detected a wide range of influenza strains, as shown in the tables below. JBAIDS Influenza A & B Detection Kit Assay Name Organisms Identified Flu A 28/28 Influenza A strains Flu B 11/11 Influenza B strains JBAIDS Influenza A Subtyping Kit Assay Name Organisms Identified Flu A H1 9/10 a Influenza A H1 strains Flu A H3 9/12 b Influenza A H3 strains Flu A H1 2009 Detected 13/13 pandemic influenza A H1 2009 strains Flu A SW a The influenzaA/1/ Denver/1/57 strain was not detected at a final concentration of 5,000 TCID 50 /mL. b FluAH3 assay, the following strains were not detected at the following concentration: A/ Aichi/2/68 at 114,000 TCID 50 /mL, A/Hong Kong/8/68 at 137,000 TCID 50 /mL, and A/MRC-2 recomb at 7,350 TCID 50 /mL. Exclusivity testing demonstrated that the assays were specific for influenza viruses and did not detect 36 non-influenza Viruses and Organisms (e.g. Coronaviruses, Rhinovirus, Parainfluenza viruses, Haemophilus influenzae, Mycoplasma pneumoniae, etc.). Additionally, with the following exceptions, we found that subtyping assays were not cross-reactive with incorrect subtypes. Influenza A H1N1 strains (A/Maryland/12/1991, A/Iowa/1/2006, and A/swine/ Wisconsin/125/1997) were detected by the Flu A Sw assay. These results may be explained because the first two of these strains were isolated from humans but have an origin of swine lineage and the third was isolated from swine. The Influenza A H3N2 virus strain A/SW/IA/1/99 (swine origin) was detected by both the Flu A H3 and Flu A Sw assays. Detection of swine Influenza A H3 viruses by the Flu A H3 assay may be attributed to the similarity between the hemagglutinin sequences for swine and human isolates. Reproducibility A multicenter reproducibility study was performed to determine the overall system reproducibility. Multiple sample panels were prepared by spiking NPS and NPW samples with representative influenza viruses at three designated concentrations (~3×LoD, ~1×LoD, ~1/20×LoD). Simulated NPS and NPW samples (HeLa cells in VTM) were used for influenza A H3N2 and 2009 influenza a H1N1 spiking. At each testing site, each panel was tested twice daily for five days. On each testing day, two users at each site purified and tested one aliquot of each sample, nine total samples per operator per day. One operator used the Platinum Path purification kit and the other used the Roche MagNA Pure purification kit prior to JBAIDS influenza testing. The Flu SC was also tested with each sample. Each concentration level was tested a total of 90 times. The JBAIDS Influenza A & B Detection and Influenza A Subtyping Kits gave the expected positive result ≥ 98% of the time for samples containing influenza virus ≥ LoD. As expected, samples spiked below LoD had variable results. Interfering Substances Substances present in NPS or NPW specimens or introduced during sample preparation were evaluated for their potential to interfere with assay performance. The following substances were found to be potential interfering substances with the JBAIDS Influenza kits. See 510(k) submission for complete data. • Snuff (nasal tobacco) • MagNA Pure Proteinase K Solution (well 1) • MagNA Pure Lysis Solution (well 2) • MagNA Pure Magnetic Glass Particles (well 3) • MagNA Pure Wash Buffer I (wells 4,5) • Ethanol • Bleach • DNAZap™ In addition, the JBAIDS influenza A, B, A/H1, A/H3 reacted with the viral material contained in the 2009-2010 version of the FluMist ® intranasal influenza vaccine (MedImmune). In the presence of vaccine material (e.g. a recent vaccination), inaccurate positive results may be obtained for the influenza assays. Prospective Clinical Evaluation Respiratory specimens (NPS or NPW) were analyzed from over 800 volunteers (adults and children) suspected of influenza infections or presenting with influenza-like illness (ILI) at five geographically distinct U.S. sites during the 2010/2011 influenza season (December through April). Study Site Principal Investigator Enrolled Population Valid Specimens Tested Study Started - Completed Site 1 Womack Army Medical Center; Fayetteville, NC CPT Brook Danboise, Ph.D. Department of Emergency Medicine, Pediatrics, and Acute and Minor Illness Clinics (AMIC). NPS: 51 NPW: 325 December 14, 2010 – April 11, 2011 Site 2 Naval Health Research Center; San Diego, CA Dennis Faixs, Ph.D. Naval Hospital Camp Pendleton, Naval Medical Center San Diego, and TriCare Clinic Clairemont Mesa. NPS: 207 January 20, 2011- April 13, 2011 Site 3 David Grant Medical Center; Fairfield, CA Maj Jason Lane, M.D. Pediatrics (Acute Care), Primary Care, Family Medicine, Flight Medicine and Emergency Room. NPS: 56 January 31, 2011- April 14, 2011 Site 4 Wright-Patterson Air Force Base; Dayton, OH Maj Vidhya Prakash, M.D. Wright Patterson Medical Center: Primary Care, Pediatrics, and Emergency Room. NPW: 119 January 21, 2011- March 29, 2011 Site 5 Brooke Army Medical Center; San Antonio, TX MAJ Jennifer Russell, M.D. Troop Medical Clinic (adults only). NPW: 46 February 1, 2011-February 28, 2011 The specimens were first purified using either the IT 1-2-3 Platinum Path Sample Purification kit or Roche MagNA Pure Compact Nucleic Acid Isolation kit I and then tested with the JBAIDS assays. For comparator testing to determine clinical truth, each specimen was also analyzed using the Centers for Disease Control and Prevention (CDC) Human Influenza Virus Real-time RT-PCR Detection and Characterization Panel (rRT-PCR Flu Panel) and influenza A positives were further analyzed with subtype-specific PCR followed by bi-directional sequencing. Due to similar performance, data for each purification kit were combined. Influenza A H1 was not circulating during the prospective evaluation. Influenza Strain Sample Matrix PPA NPA TP/(TP+FN) Percent 95% CI TN/(TN+FP) Percent 95% CI Flu A NPW 104/104 100% 96.5-100% 383/386 99.2% 97.8-99.8% NPS 62/62 100% 94.2-100% 251/252 99.6% 97.8-100% Flu B NPW 68/72 94.4% 86.4-98.5% 414/418 99.0% 97.6-99.7% NPS 24/25 96.0% 79.7-99.9% 289/289 100% 98.7-100% Seasonal H1 a NPW 0/0 - - 482/482 100% 99.3-100% NPS 0/0 - - 312/312 100% 98.8-100% 2009 H1N1 NPW 66/66 100% 94.6-100% 414/416 99.5% 98.3-99.9% NPS 34/34 100% 89.7-100% 277/278 99.6% 98.0-100% Seasonal H3 NPW 33/33 100% 89.4-100% 449/449 100% 99.2-100% NPS 26/26 100% 86.8-100% 286/286 100% 98.7-100% a Seasonal influenza A H1 was not circulating during the prospective evaluation. Surrogate Clinical Specimens Seasonal influenza A H1 was not circulating during the prospective evaluation and there were insufficient archived specimens containing this analyte for analysis. Therefore, surrogate clinical specimens were used to demonstrate performance for this analyte. Residual, influenza-negative NPS and NPW specimens from the prospective clinical evaluation were spiked with a representative influenza A H1 strain at and above the system LoD or unspiked. Specimens were randomized and assigned study code numbers such that laboratory personnel were blinded to the analyte contents. Specimens were then evaluated using the JBAIDS system. Positive Percent Agreement (PPA) for all JBAIDS influenza assays in both sample types using both purification methods was greater than 99%. Negative Percent Agreement (NPA) was at least 93% for all assays in both matrices using both purification methods. See 510(k) submission for complete data regarding PPA and NPA. Influenza Strain Sample Matrix PPA NPA TP/ (TP+FN) Percent 95% CI TN/ (TN+FP) Percent 95% CI Flu A NPW 53/54 98.1% 90.1- 100% 8/8 100% 63.1- 100% NPS 59/59 100% 93.9- 100% 7/7 100% 59.0- 100% Flu B NPW 0/0 - - 66/67 98.5% 92.0- 100% NPS 0/0 - - 65/65 100% 94.5- 100% Seasonal H1 NPW 54/54 100% 93.4- 100% 8/8 100% 63.1- 100% NPS 59/59 100% 93.9- 100% 7/7 100% 59.0- 100% 2009 H1N1 NPW 0/0 - - 62/62 100% 94.2- 100% NPS 0/0 - - 66/66 100% 94.6- 100% Seasonal H3 NPW 0/0 - - 62/62 100% 94.2- 100% NPS 0/0 - - 66/66 100% 94.6- 100% Range of Analyte Levels relative to Assay LoD Data collected during the clinical evaluation were analyzed to look at the distribution of analyte levels in specimens for the influenza A and B assays. The LoD for each assay is indicated with a green line in the histograms below. Cp values for NPS and NPW specimens were combined. Samples processed with the Roche MagNA Pure (MP) are indicated in red; those processed with Platinum Path (PP) are shown in purple. This data shows that the majority of clinical specimens contained analyte levels well above the system LoD, and that influenza was still detected in patients with analyte levels below the system LoD. A similar analyte distribution with respect to LoD was seen for all of the subtyping assays as well (not shown). CONCLUSION These studies demonstrated that the JBAIDS Influenza A & B Detection Kit and JBAIDS Influenza A Subtyping Kit are robust, highly sensitive, and highly specific in vitro diagnostic devices for the detection of influenza viruses. The studies presented here, and others not shown, were used to support two 510(k) applications to the FDA. Clearance was granted on September 13, 2011 for both the JBAIDS Influenza A&B Detection Kit (K111775) and the JBAIDS Influenza A Subtyping Kit (K111778). Wizard-based software and Auto Results Rehydrate Freeze-Dried PCR Reagents Sample Purification
Transcript of Clinical Evaluation of the JBAIDS Influenza A & B - BioFire Defense
Presented at the Society of Armed Forces Medical Laboratory Scientists, April 2012
CONTACT INFORMATIONLou BanksIdaho Technology, [email protected]
See all of ITI’s scientific posters by scanning the QR code to access the Scientific Poster Page
Kevin Bourzac1, Sean Phipps1, Cynthia Phillips1, Andrew Hemmert1, Cynthia Andjelic1, Brook Danboise2, Dennis Faixs3, Jason Lane4, Vidhya Prakash5, Jennifer Russell6, Patricia Beverly7, Jennifer C. Dabisch8, Gary Uzzell1, and Beth Lingenfelter1
1 Idaho Technology, Inc., Salt Lake City, UT, 2 Womack Army Medical Center, Fayetteville, NC, 3 Naval Health Research Center, San Diego, CA, 4 David Grant Medical Center, Farfield, CA, 5 Wright-Patterson Air Force Base, Dayton, OH, 6 Brooke Army Medical Center, San Antonio, TX, 7 United States Army Medical Materiel Development Activity, Fort Detrick, MD, 8 Joint Project Management Office for Chemical, Biological Medical Systems - Biosurveillance, Frederick, MD
Clinical Evaluation of the JBAIDS Influenza A & B Detection Kit and JBAIDS Influenza A Subtyping Kit
DISCLAIMERThe views, opinions, and findings contained in this report are those of the author(s) and should not be construed as an official Department of Defense position, policy, or decision, unless so designated by other official documentation.
INTRODUCTIONThe Joint Biological Agent Identification and Diagnostic System (JBAIDS), a real-time PCR-based system, is the United States Department of Defense’s Program of Record for diagnostic testing of infectious diseases of operational concern. The JBAIDS Influenza A & B Detection Kit is designed to detect influenza viruses A and B, and the JBAIDS Influenza A Subtyping Kit is designed to differentiate influenza A hemagglutinin subtypes seasonal influenza A H1, seasonal influenza A H3, and 2009 influenza A H1N1 when testing nasopharyngeal swabs (NPS) or nasopharyngeal washes (NPW). Here, we present selected analytical and clinical studies that were performed in support of 510(k) pre-market applications for JBAIDS Influenza A & B Detection kits and the Influenza A Subtyping kit to the US FDA.
Two nucleic acid purification kits were also evaluated, both of which use magnetic bead-based technology. The IT 1-2-3™ Platinum Path™ Sample Purification kit, manufactured by Idaho Technology, Inc, is a manual extraction method and the Roche MagNA Pure Compact Nucleic Acid Isolation kit I is an automated system.
PERFORMANCE TESTING
FOOTNOTESThis work was funded by the Armed Forces Health Surveillance Center (AFHSC) as the Requesting Agency under the Memorandum of Agreement (MOA) between AFHSC and the Chemical Biological Medical Systems Joint Project Management Office (CBMS JPMO).
Limit of Detection
The LoD for each target influenza assay was determined using both NPS and NPW samples spiked with quantified live virus. The LoD was the lowest concentration for which greater than 95% of samples yielded positive results for both sample matrices and both nucleic acid purification kits.
Kit Assay(s) Influenza Type Strain LoD (EID50/mL)
Influ
enza
A &
BD
etec
tion
Kit
Flu A
Influenza A H1N1 A/New Caledonia/20/1999 50Influenza A H3N2 A/New York/55/2004 5
Influenza A H1N1 2009 A/New York/18/2009 50
Flu BInfluenza B B/Ohio/1/2005 5Influenza B B/Florida/7/2004 10
Influ
enza
A
Subt
ypin
g K
it Flu A H1 Influenza A H1N1A/New Caledonia/20/1999 50a
A/Hawaii/15/2001 5,000
Flu A H3 Influenza A H3N2A/New York/55/2004 5A/Wisconsin/67/2005 10
Flu A H1 2009/ Flu A Sw Influenza A H1N1 2009
A/New York/18/2009 1,500A/California/7/2009 5,000
a The aliquot of A/New Caledonia/20/1999 tested contains 17-45 times more PCR target copies per EID50 than the aliquot of A/Hawaii/15/2001 tested.
Assay DesignThe JBAIDS Influenza assays are based on influenza assays from the ‘CDC Human Influenza Virus Real-time RT-PCR Detection and Characterization Panel (CDC rRT-PCR Flu Panel)’ and the ‘CDC Influenza 2009 A(H1N1)pdm RT-PCR Panel’. The JBAIDS assays were re-optimized to work with Idaho Technology’s proprietary freeze-dried PCR reagent formulation. Testing demonstrated that the assays detected a wide range of influenza strains, as shown in the tables below. JBAIDS Influenza A & B Detection Kit
Assay Name Organisms Identified
Flu A 28/28 Influenza A strains
Flu B 11/11 Influenza B strains
JBAIDS Influenza A Subtyping Kit
Assay Name Organisms Identified
Flu A H1 9/10a Influenza A H1 strains
Flu A H3 9/12b Influenza A H3 strainsFlu A H1 2009
Detected 13/13 pandemic influenza A H1 2009 strainsFlu A SW
a The influenza A/1/ Denver/1/57 strain was not detected at a final concentration of 5,000 TCID50/mL. b Flu A H3 assay, the following strains were not detected at the following concentration: A/ Aichi/2/68 at 114,000 TCID50/mL, A/Hong Kong/8/68 at 137,000 TCID50/mL, and A/MRC-2 recomb at 7,350 TCID50/mL.
Exclusivity testing demonstrated that the assays were specific for influenza viruses and did not detect 36 non-influenza Viruses and Organisms (e.g. Coronaviruses, Rhinovirus, Parainfluenza viruses, Haemophilus influenzae, Mycoplasma pneumoniae, etc.). Additionally, with the following exceptions, we found that subtyping assays were not cross-reactive with incorrect subtypes. Influenza A H1N1 strains (A/Maryland/12/1991, A/Iowa/1/2006, and A/swine/ Wisconsin/125/1997) were detected by the Flu A Sw assay. These results may be explained because the first two of these strains were isolated from humans but have an origin of swine lineage and the third was isolated from swine. The Influenza A H3N2 virus strain A/SW/IA/1/99 (swine origin) was detected by both the Flu A H3 and Flu A Sw assays. Detection of swine Influenza A H3 viruses by the Flu A H3 assay may be attributed to the similarity between the hemagglutinin sequences for swine and human isolates.
Reproducibility
A multicenter reproducibility study was performed to determine the overall system reproducibility. Multiple sample panels were prepared by spiking NPS and NPW samples with representative influenza viruses at three designated concentrations (~3×LoD, ~1×LoD, ~1/20×LoD). Simulated NPS and NPW samples (HeLa cells in VTM) were used for influenza A H3N2 and 2009 influenza a H1N1 spiking. At each testing site, each panel was tested twice daily for five days. On each testing day, two users at each site purified and tested one aliquot of each sample, nine total samples per operator per day. One operator used the Platinum Path purification kit and the other used the Roche MagNA Pure purification kit prior to JBAIDS influenza testing. The Flu SC was also tested with each sample. Each concentration level was tested a total of 90 times. The JBAIDS Influenza A & B Detection and Influenza A Subtyping Kits gave the expected positive result ≥ 98% of the time for samples containing influenza virus ≥ LoD. As expected, samples spiked below LoD had variable results.
Interfering Substances
Substances present in NPS or NPW specimens or introduced during sample preparation were evaluated for their potential to interfere with assay performance. The following substances were found to be potential interfering substances with the JBAIDS Influenza kits. See 510(k) submission for complete data.
• Snuff (nasal tobacco)
• MagNA Pure Proteinase K Solution (well 1)
• MagNA Pure Lysis Solution (well 2)
• MagNA Pure Magnetic Glass Particles (well 3)
• MagNA Pure Wash Buffer I (wells 4,5)
• Ethanol
• Bleach
• DNAZap™
In addition, the JBAIDS influenza A, B, A/H1, A/H3 reacted with the viral material contained in the 2009-2010 version of the FluMist® intranasal influenza vaccine (MedImmune). In the presence of vaccine material (e.g. a recent vaccination), inaccurate positive results may be obtained for the influenza assays.
Prospective Clinical Evaluation
Respiratory specimens (NPS or NPW) were analyzed from over 800 volunteers (adults and children) suspected of influenza infections or presenting with influenza-like illness (ILI) at five geographically distinct U.S. sites during the 2010/2011 influenza season (December through April).
Study SitePrincipal Investigator Enrolled Population Valid Specimens
TestedStudy Started -
Completed
Site 1Womack Army Medical Center;
Fayetteville, NC CPT Brook Danboise, Ph.D.
Department of Emergency Medicine, Pediatrics, and Acute and
Minor Illness Clinics (AMIC).
NPS: 51NPW: 325
December 14, 2010 – April 11, 2011
Site 2Naval Health Research Center;
San Diego, CADennis Faixs, Ph.D.
Naval Hospital Camp Pendleton, Naval Medical Center San Diego,
and TriCare Clinic Clairemont Mesa. NPS: 207 January 20, 2011-
April 13, 2011
Site 3David Grant Medical Center;
Fairfield, CAMaj Jason Lane, M.D.
Pediatrics (Acute Care), Primary Care, Family Medicine, Flight
Medicine and Emergency Room. NPS: 56 January 31, 2011-
April 14, 2011
Site 4Wright-Patterson Air Force Base;
Dayton, OHMaj Vidhya Prakash, M.D.
Wright Patterson Medical Center: Primary Care, Pediatrics, and
Emergency Room. NPW: 119 January 21, 2011-
March 29, 2011
Site 5Brooke Army Medical Center;
San Antonio, TXMAJ Jennifer Russell, M.D.
Troop Medical Clinic (adults only). NPW: 46
February 1, 2011-February 28,
2011
The specimens were first purified using either the IT 1-2-3 Platinum Path Sample Purification kit or Roche MagNA Pure Compact Nucleic Acid Isolation kit I and then tested with the JBAIDS assays. For comparator testing to determine clinical truth, each specimen was also analyzed using the Centers for Disease Control and Prevention (CDC) Human Influenza Virus Real-time RT-PCR Detection and Characterization Panel (rRT-PCR Flu Panel) and influenza A positives were further analyzed with subtype-specific PCR followed by bi-directional sequencing. Due to similar performance, data for each purification kit were combined. Influenza A H1 was not circulating during the prospective evaluation.
Influenza Strain
Sample Matrix
PPA NPATP/(TP+FN) Percent 95% CI TN/(TN+FP) Percent 95% CI
Flu A NPW 104/104 100% 96.5-100% 383/386 99.2% 97.8-99.8%NPS 62/62 100% 94.2-100% 251/252 99.6% 97.8-100%
Flu B NPW 68/72 94.4% 86.4-98.5% 414/418 99.0% 97.6-99.7%NPS 24/25 96.0% 79.7-99.9% 289/289 100% 98.7-100%
Seasonal H1a
NPW 0/0 - - 482/482 100% 99.3-100%NPS 0/0 - - 312/312 100% 98.8-100%
2009 H1N1
NPW 66/66 100% 94.6-100% 414/416 99.5% 98.3-99.9%NPS 34/34 100% 89.7-100% 277/278 99.6% 98.0-100%
Seasonal H3
NPW 33/33 100% 89.4-100% 449/449 100% 99.2-100%NPS 26/26 100% 86.8-100% 286/286 100% 98.7-100%
a Seasonal influenza A H1 was not circulating during the prospective evaluation.
Surrogate Clinical Specimens
Seasonal influenza A H1 was not circulating during the prospective evaluation and there were insufficient archived specimens containing this analyte for analysis. Therefore, surrogate clinical specimens were used to demonstrate performance for this analyte. Residual, influenza-negative NPS and NPW specimens from the prospective clinical evaluation were spiked with a representative influenza A H1 strain at and above the system LoD or unspiked. Specimens were randomized and assigned study code numbers such that laboratory personnel were blinded to the analyte contents. Specimens were then evaluated using the JBAIDS system. Positive Percent Agreement (PPA) for all JBAIDS influenza assays in both sample types using both purification methods was greater than 99%. Negative Percent Agreement (NPA) was at least 93% for all assays in both matrices using both purification methods. See 510(k) submission for complete data regarding PPA and NPA.
Influenza Strain
Sample Matrix
PPA NPATP/
(TP+FN)Percent 95% CI
TN/(TN+FP)
Percent 95% CI
Flu ANPW 53/54 98.1%
90.1-100%
8/8 100%63.1-100%
NPS 59/59 100% 93.9-100%
7/7 100%59.0-100%
Flu BNPW 0/0 - - 66/67 98.5%
92.0-100%
NPS 0/0 - - 65/65 100% 94.5-100%
Seasonal H1
NPW 54/54 100%93.4-100%
8/8 100%63.1-100%
NPS 59/59 100%93.9-100%
7/7 100%59.0-100%
2009 H1N1
NPW 0/0 - - 62/62 100%94.2-100%
NPS 0/0 - - 66/66 100%94.6-100%
Seasonal H3
NPW 0/0 - - 62/62 100%94.2-100%
NPS 0/0 - - 66/66 100%94.6-100%
Range of Analyte Levels relative to Assay LoD
Data collected during the clinical evaluation were analyzed to look at the distribution of analyte levels in specimens for the influenza A and B assays. The LoD for each assay is indicated with a green line in the histograms below. Cp values for NPS and NPW specimens were combined. Samples processed with the Roche MagNA Pure (MP) are indicated in red; those processed with Platinum Path (PP) are shown in purple. This data shows that the majority of clinical specimens contained analyte levels well above the system LoD, and that influenza was still detected in patients with analyte levels below the system LoD. A similar analyte distribution with respect to LoD was seen for all of the subtyping assays as well (not shown).
CONCLUSIONThese studies demonstrated that the JBAIDS Influenza A & B Detection Kit and JBAIDS Influenza A Subtyping Kit are robust, highly sensitive, and highly specific in vitro diagnostic devices for the detection of influenza viruses.
The studies presented here, and others not shown, were used to support two 510(k) applications to the FDA. Clearance was granted on September 13, 2011 for both the JBAIDS Influenza A&B Detection Kit (K111775) and the JBAIDS Influenza A Subtyping Kit (K111778).
Wizard-based software and Auto Results
Rehydrate Freeze-Dried
PCR Reagents
Sample Purification
