The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Plague Detection Kit is a real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences of Yersinia pestis. The kit can be used to test human whole blood collected in sodium citrate or sputum collected aseptically from individuals greater than 18 years of age suspected of having septic or pneumonic plague. In addition, positive blood cultures and colonies may be tested. The JBAIDS Plague Target 2 assay is used as a supplementary test only after a positive result with the Target I Assay.

The JBAIDS Plague Target 1 and Target 2 assays are run on the JBAIDS instrument using the Diagnostic Wizard. Results are for the presumptive identification of Y. pestis in conjunction with culture and other laboratory tests. The definitive identification of Y. pestis from colony growth, liquid blood culture growth, or from blood or sputum specimens requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required.

The diagnosis of plague must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence in addition to the identification of Y. pestis from cultures or directly from whole blood or sputum specimens.

The JBAIDS Plague Detection Kit is intended for use by trained clinical laboratory personnel who have received specific training on the use of the JBAIDS Plague Detection Kit. The level of Y. pestis that would be present in blood or sputum from individuals with early systemic infection is unknown. Due to the difficulty in obtaining clinical specimens, these assays were not evaluated with blood or sputum from individuals with septic or pneumonic plague.

Device Description

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Plague Detection System is a fully integrated IVD system composed of the portable JBAIDS instrument, laptop computer and software and the JBAIDS Plague Detection Kit with two different freeze-dried PCR assays for detection of Yersinia pestis DNA. The system has been validated using four different sample preparation kits for isolating DNA from wole blood (IT 1-2-3TM Platinum Path and QFLOWdana Sample Purification Kits), sputum (IT 1-2-3TM Platinum Path and IT 1-2-3TM VIBE), positive blood cultures (IT 1-2-3TM SWIPE Sample Purification Kit), and plate cultures (IT 1-2-3™ Platinum Path and SWIPE Sample Purification Kits). Use of the JBAIDS DNA Extraction Control Kit is also recommended.

Prior to testing, specimens are processed using BioFire Diagnostic's IT 1-2-3 Sample Purification Kits. The resulting purified sample is added to Target 1 Unknown and Target 1 Inhibition Control vials, along with reconstitution buffer. Target 1 Positive Control and Negative Control vials are prepared using reconstitution buffer and water. When Y. pestis DNA is present, a fragment of Y. pestis DNA is amplified. The amplicon is detected by fluorescence using a specific hydrolysis probe. Each probe is labeled on one end with a fluorescent reporter moiety (6-carboxyfluorescein (6-FAM)) and elsewhere with a quencher moiety (carboxy tetramethylrhodamine (TAMRA)). When the probe is intact, the quencher absorbs the light emitted by the reporter moiety. During PCR, the probe hybridizes to the target sequence before the exonuclease activity of Taq polymerase hydrolyzes the probe, separating the fluorophore from the quencher and permitting detection of the fluorescent signal generated by the reporter. The fluorescent signal increases as additional templates are amplified and more probes are hydrolyzed.

JBAIDS Software analyzes the fluorescence amplification curves and reports results as positive, negative, uncertain or inhibited. A failure of the Positive or Negative Control will result in the entire run being called invalid. Retesting is required to resolve uncertain, invalid or inhibited results. The Target 2 assay is used as a supplementary test only after a positive result is obtained with the Target 1 assay.

AI/ML Overview

This document describes the validation of the JBAIDS Plague Detection Kit, specifically the modification for use with the IT 1-2-3™ Platinum Path Sample Purification Kit Accessory. The study aims to demonstrate that the new purification method provides equivalent performance to previously validated methods.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied by the "equivalence" study design, where the performance of the new extraction method (Platinum Path) is compared to the previously validated methods (QFLOWdna for whole blood, VIBE for sputum). The goal is to show that the Platinum Path method performs equivalently to the existing methods. For the clinical performance, the reported metrics are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). For Limit of Detection (LoD), the acceptance criterion is the confirmation of the previously established LoD. For reproducibility, the criteria are defined by the agreement with expected results and consistency across sites.

Metric (Test)	Acceptance Criteria (Implicit)	Reported Device Performance (Platinum Path)
Clinical Performance (Whole Blood)	Equivalent PPA and NPA compared to QFLOWdna method. Overall agreement ≥ 95% (lower bound of 95% CI).	PPA: 100% (49/49) with 95% CI: 92.7-100% (compared to QFLOWdna as correct result). NPA: 98% (50/51) with 95% CI: 89.6-99.9% (compared to QFLOWdna as correct result). Overall percent agreement between Platinum Path and QFLOWdna was 99% with a lower bound of the 95% CI at 94%.
Clinical Performance (Sputum)	Equivalent PPA and NPA compared to VIBE method. Overall agreement ≥ 95% (lower bound of 95% CI).	PPA: 100% (50/50) with 95% CI: 92.9-100% (compared to VIBE as correct result). NPA: 98% (49/50) with 95% CI: 89.4-100% (compared to VIBE as correct result). Overall percent agreement between Platinum Path and VIBE was 99% (99/100; 95% CI = 94.6-100%).
Limit of Detection (LoD) - Whole Blood	LoD of 50 CFU/mL to be confirmed. Detection rate of 20/20 at LoD.	20/20 independent whole blood specimens spiked at 50 CFU/mL were detected (100% positive for Target 1, 95% for Target 2). This confirmed the LoD of 50 CFU/mL.
Limit of Detection (LoD) - Sputum	LoD of 670 CFU/mL to be confirmed. Detection rate of 20/20 at LoD.	20/20 independent sputum specimens spiked at 670 CFU/mL were detected (100% positive for Target 1, 100% for Target 2). This confirmed the LoD of 670 CFU/mL.
Reproducibility (Whole Blood)	High agreement with expected results for positive and negative samples across multiple sites. Detection rate >95% for positive samples.	Medium Positive (5xLoD): Target 1 & 2: 100% Agreement (96/96) across all sites, 95% CI 96.2-100. Low Positive (1xLoD): Target 1: 100% Agreement (96/96), 95% CI 96.2-100. Target 2: 98.9% Agreement (95/96), 95% CI 94.3-99.9. Negative: Target 1: 98.9% Agreement (95/96), 95% CI 94.3-99.9. Target 2: 97.9% Agreement (94/96), 95% CI 92.7-99.7. Detection rate >99% for samples containing Y. pestis. No final false positive results for unspiked samples (both targets must be positive).
Reproducibility (Sputum)	High agreement with expected results for positive and negative samples across multiple sites. Detection rate >95% for positive samples.	Medium Positive (5xLoD): Target 1: 98.9% Agreement (89/90), 95% CI 94.0-99.9. Target 2: 100% Agreement (90/90), 95% CI 96.0-100. Low Positive (1xLoD): Target 1 & 2: 100% Agreement (90/90), 95% CI 96.0-100. Negative: Target 1: 98.9% Agreement (89/90), 95% CI 94.0-99.9. Target 2: 100% Agreement (90/90), 95% CI 96.0-100. Detection rate >99% for samples containing Y. pestis. No final false positive results for unspiked samples (both targets must be positive).
Detection of Direct Culture Samples (Platinum Path)	All Y. pestis colonies should be detected, and non-Y. pestis colonies should not be detected.	10/10 Y. pestis colonies detected for both Target 1 and Target 2. 0/10 non-Y. pestis colonies detected.

2. Sample Sizes used for the Test Set and Data Provenance

Clinical Performance (Whole Blood):
- Test Set Sample Size: 100 surrogate whole blood samples (50 spiked with inactivated Y. pestis, 50 unspiked).
- Data Provenance: Prospectively collected specimens from febrile volunteers in the USA (November 2012 - April 2013). This is surrogate data, not from actual plague patients.
Clinical Performance (Sputum):
- Test Set Sample Size: 100 surrogate sputum samples (50 spiked with inactivated Y. pestis, 50 unspiked).
- Data Provenance: Residual frozen sputum specimens (presumably from the USA). This is surrogate data.
Limit of Detection:
- Whole Blood LoD Confirmation: 20 independent whole blood specimens.
- Sputum LoD Confirmation: 20 independent sputum specimens.
Reproducibility:
- Whole Blood: A panel of 12 blood samples (4 medium positive, 4 low positive, 4 negative) tested twice a day for 4 days at 3 sites = 12 samples * 2 tests/day * 4 days * 3 sites = 288 individual tests for each target.
- Sputum: A panel of 9 sputum samples (3 medium positive, 3 low positive, 3 negative) tested twice a day for 5 days at 3 sites = 9 samples * 2 tests/day * 5 days * 3 sites = 270 individual tests for each target.
Detection of Direct Culture Samples: 10 Y. pestis colonies and 10 non-Y. pestis colonies.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The ground truth for the clinical performance and LoD studies was established by spiking samples with known quantities of inactivated Y. pestis. In the clinical performance comparison, the result obtained from the previously validated extraction method (QFLOWdna for blood, VIBE for sputum) was considered the "correct result" to which the new method (Platinum Path) was compared.
For the reproducibility study, the samples were prepared with known spike levels, making the expected outcome clear.
For the direct culture detection, the ground truth was the known identity of the bacterial colonies (Y. pestis vs. non-Y. pestis).

This is a molecular diagnostic test, so there isn't an "expert" interpreting images or clinical signs to establish ground truth. The ground truth is determined by the controlled experimental design (spiking, known bacterial cultures) and the performance of the predicate device.

4. Adjudication Method for the Test Set

Not applicable in the traditional sense for a molecular diagnostic device measuring defined targets in spiked samples or known cultures. The "adjudication" in the clinical equivalency studies was implicitly done by comparing the new method's results to those of the previously validated predicate method. JBAIDS operators were blinded to the analyte content of the samples.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This is a molecular diagnostic test, not an AI-powered diagnostic imaging device requiring human interpretation of results. The device provides a qualitative "positive," "negative," "uncertain," or "inhibited" result.

6. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done

Yes, the device operates autonomously to detect target DNA sequences and report results (positive, negative, uncertain, or inhibited) based on its pre-programmed algorithms. The performance data presented (PPA, NPA, LoD confirmation, reproducibility, direct culture detection) are all standalone performance metrics of the device with the new extraction method. Human operators perform the sample preparation and initiate the test, but the interpretation of the PCR amplification curves and the final result determination are automated by the JBAIDS software.

7. The Type of Ground Truth Used

Spiking studies (Clinical Performance, LoD, Reproducibility): Ground truth was established by spiking clinically relevant matrices (whole blood, sputum) with known quantities of inactivated Y. pestis. For the clinical performance comparison, the results from the previously cleared extraction methods served as the comparator "ground truth" to determine equivalency.
Direct Culture Detection: Ground truth was established by using known bacterial colonies (Y. pestis vs. non-Y. pestis).

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of developing the JBAIDS Plague Detection Kit or its modification. This is a PCR-based assay, not a machine learning algorithm that typically requires a large training dataset. The device utilizes pre-defined molecular targets and amplification curves for detection. Any 'training' would refer to the initial development and optimization of the PCR primers/probes and reaction conditions, rather than a data-driven machine learning training set described here.

9. How the Ground Truth for the Training Set Was Established

As noted above, a "training set" in the machine learning sense is not applicable here. The development of the JBAIDS Plague Detection Kit and its components (like the PCR assays and sample purification methods) would involve:

Design and optimization: Identifying specific DNA targets for Y. pestis.
Analytical validation: Testing the assay with characterized strains of Y. pestis and related/unrelated organisms at various concentrations.
Limit of Detection (LoD) determination: Empirically determining the lowest concentration consistently detected.
Specificity and Sensitivity testing: Challenging the assay with known positive and negative samples.

These processes would utilize known samples and cultures where the presence/absence and concentration of Y. pestis are precisely controlled.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

February 20, 2019

CYNTHIA PHILLIPS, Ph.D. VICE PRESIDENT OF REGULATORY AND CLINICAL AFFAIRS BIOFIRE DEFENSE, LLC 79 W. 4500 SOUTH, SUITE 14 SALT LAKE CITY UT 84107

Re: K131729 Trade/Device Name: JBAIDS Plague Detection Kit Regulatory Class: Unclassified Product Code: OIH Dated: June 10, 2013

Dear Dr. Phillips:

Received: June 12, 2013

This letter corrects our substantially equivalent letter of July 31, 2013.

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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Page 2-Dr. Phillips

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Uwe Scherf -S

Uwe Scherf, M.Sc., Ph.D. Director, Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

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Indications for Use

510(k) Number: K131729 Device Name: JBAIDS Plague Detection System

Prescription Use X (Part 21 CFR 801 Subpart D) AND/OR Over-The-Counter Use (21 CFR 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostics and Radiological Health (OIR)

Image /page/2/Picture/10 description: The image shows the text "John-Hobson-S 2013.07.31 10:04:30 -04'00'". The text is overlaid on top of a logo. The date and time are clearly visible in the center of the image.

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510(k) Summary BioFire Diagnostics, Inc.

Modification of the JBAIDS Plague Detection Kit for use with the IT 1-2-3TM Platinum Path Sample Purification Kit Accessory

Introduction: According to the requirements of 21 CFR 807.92, the following information provides sufficient detail to understand the basis for a determination of substantial equivalence.

Submitted by:

BioFire Diagnostics, Inc. 390 Wakara Way Salt Lake City, UT 84108

Telephone: 801-736-6354 Facsimile: 801-588-0507

Contact: Cynthia Phillips, ext. 370

Date Submitted: June 10, 2013

Device Name and Classification:

Trade Name: JBAIDS Plague Detection Kit Classification: Unclassified Product Code: OIH

Predicate Device:

JBAIDS Plague Detection Kit (K072631)

Intended Use:

The JBAIDS Plague Target 1 and Target 2 assays are run on the JBAIDS instrument using the Diagnostic Wizard. Results are for the presumptive identification of Y, nestis in

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conjunction with culture and other laboratory tests. The definitive identification of Y. pestis from colony growth, liquid blood culture growth, or from blood or sputum specimens requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required.

Device Description:

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invalid or inhibited results. The Target 2 assay is used as a supplementary test only after a positive result is obtained with the Target 1 assay.

Substantial Equivalence:

The JBAIDS Plague Detection Kit is substantially equivalent to the previously cleared JBAIDS Plague Detection Kit. The following tables compare the modified JBAIDS Plague Detection Kit to the previously cleared JBAIDS Plague Detection Kits (K072631). The first table outlines the similarities between the two systems and the second table outlines the differences.

Element	New Device:JBAIDS Plague Detection Kit	Predicate:JBAIDS Plague Detection Kit(K072631)
Intended Use	Identification of Plague infectionthrough the detection of two DNAsequence targets unique to Yersiniapestis. Results are used in conjunctionwith clinical information, culture, andother laboratory tests as an aid in thediagnosis of systemic Plague infectionin individuals suspected of having thedisease.	Same
Technology	Real-time PCR using hydrolysis probes	Same
OrganismDetected	Qualitative in vitro detection ofYersinia pestis DNA	Same
SpecimenTypes	Whole blood (collected in 3.2% sodiumcitrate), sputum collected asepticallyfrom individuals greater than 18 yearsof age suspected of having septic orpneumonic plague, blood culture(grown in soybean-casein digest broth)or bacterial culture (grown on bloodagar)	Same
Platform	JBAIDS Instrument	Same
Time Requiredfor Analysis ofSpecimen	Less than 3 hours	Same

Table 1. Similarities between the New Device and the Predicate

Table 2. Differences between the New Device and the Predicate

Element	New Device:JBAIDS Plague Detection Kit	Predicate:JBAIDS Plague Detection Kit(K072631)
---------	--------------------------------------------	--------------------------------------------------------

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DNAExtractionMethods	Whole blood purified with IT 1-2-3 TMPlatinum Path or IT 1-2-3 TM QFLOWdnaSample Purification Kits (or validatedequivalent).	Whole blood purified with IT 1-2-3 TMQFLOWdna Sample Purification Kit (orvalidated equivalent).
	Sputum purified with IT 1-2-3 TMPlatinum Path or IT 1-2-3 TM VIBESample Purification Kits (or validatedequivalent).	Sputum purified with IT 1-2-3 TM VIBESample Purification Kits (or validatedequivalent).
	Blood culture purified with IT 1-2-3 TMSWIPE Sample Purification Kit (orvalidated equivalent).	Same
	Direct bacterial culture purified with IT1-2-3 TM Platinum Path or IT 1-2-3 TMSWIPE Sample Purification Kit (orvalidated equivalent).	Direct bacterial culture purified with IT1-2-3 TM SWIPE Sample Purification Kit(or validated equivalent).

Summary of Performance Data

Clinical Performance

True clinical specimens from patients infected with Yersinia pestis (plague), are not available for testing due to the extreme rarity of natural infection with these organisms. Therefore, two clinical evaluations using surrogate specimens were performed to validate the use of the IT 1-2-3TM Platinum Path Sample Purification Kit for use with the JBAIDS Plague Detection Kit. For the evaluation using blood samples, whole blood specimens were prospectively collected from patients with fever, after which the samples were spiked with inactivated Y. pestis, purified by both the new and old extraction methods, and then tested. For the evaluation using sputum samples, residual frozen sputum specimens were spiked with inactivated Y. pestis, purified by both the new and old extraction methods, and then tested.

Testing of Surrogate Whole Blood Clinical Specimens

One hundred (100) surrogate whole blood samples were prepared using prospectively collected specimens that were collected from febrile volunteers from November of 2012 into April of 2013. Fifty (50) of the specimens were spiked with inactivated Y. pestis at concentrations near and above the system LoD, while the remaining 50 specimens were not spiked with Y. pestis. Samples were then processed using both the new nucleic acid extraction method (Platinum Path) and the original nucleic acid extraction method (IT I-2-3TM QFLOWdana Sample Purification Kit; QFLOWdad by testing with the JBAIDS Plague Detection Kit. JBAIDS operators were blinded to the analyte content of the samples. Table 3 presents the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for the surrogate whole blood specimen testing. In this study, a success was defined as a surrogate sample having identical JBAIDS results for both the Platinum Path and QFLOWana processed samples, where the JBAIDS result for a sample purified using from the QFLOWda® kit was considered the correct result. Overall percent agreement between the two purification kits was 99% with a lower bound of the 95%

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confidence interval at 94%. The IT 1-2-3 QFLOWdar and Platinum Path Sample Purification Kits performed equivalently with respect to detection of Y. pestis in surrogate whole blood specimens tested with the JBAIDS Plague Detection Kit.

Positive Agreement				Negative Agreement
QFLOWdna +PlatinumPath +	QFLOWdna +PlatinumPath -	PPA	95%CIa	QFLOWdna -PlatinumPath -	QFLOWdna -PlatinumPath +	NPA	95% CI
49	0	100%(49/49)	92.7-100%	50	1b	98%(50/51)	89.6-99.9%

Table 3. JBAIDS Plague Detection Kit Performance on Spiked Whole Blood Samples Processed with the IT 1-2-3 Platinum Path and OFLOWdna Sample Purification Kits

a C.J. Clopper and E.S. Pearson. 1934. The use of confidence or fiducial limits illustrated in the case of the binomial. Biometrika 26:404-413.

b One false positive result was obtained for a specimen spiked at the 1×LoD level for which the result was positive for the Platinum Path purified sample, but negative for the QFLOW®® purified sample. When a sample is spiked at the 1×LoD level, ≥ 95% of results are expected to be positive. Occasional negative results are therefore expected (approximately 1 out of 20), with the consequence in this case of a false positive result for a specimen spiked at the 1×LoD level.

Testing of Surrogate Sputum Clinical Specimens

One hundred (100) surrogate sputum samples were prepared using frozen residual sputum specimens. Fifty (50) of the samples were spiked with inactivated Y. pestis at concentrations near and above the system LoD, while the remaining 50 specimens were not spiked with Y. pestis. Samples were then processed using both the new nucleic acid extraction method (Platinum Path) and the original nucleic acid extraction method (IT /-2-3TM VIBE Sample Purification Kit) followed by testing with the JBAIDS Plague Detection Kit. JBAIDS operators were blinded to the analyte content of the samples. Table 4 presents the PPA and NPA for the surrogate sputum clinical specimen testing. Overall percent agreement between the two purification kits was 99% (99/100; 95% CI = 94.6-100%). The IT 1-2-3 VIBE and Platinum Path Sample Purification Kits performed equivalently with respect to detection of Y. pestis in surrogate sputum samples tested with the JBAIDS Plague Detection Kit.

Table 4. JBAIDS Plague Detection Kit Performance on Spiked Sputum Samples Processed with the IT 1-2-3 Platinum Path and VIBE Sample Purification Kits

Positive Agreement			Negative Agreement
VIBE +Plat Path+	VIBE +Plat Path-	PPA	VIBE -Plat Path-	VIBE -Plat Path+	NPA	95% CIa

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50	0	100%(50/50)	92.9-100%	49	1b	98%(49/50)	89.4-100%
----	---	-----------------	---------------	----	----	----------------	---------------

ª C.J. Clopper and E.S. Pearson. 1934. The use of confidence or fiducial limits illustrated in the case of the binomial. Biometrika 26:404-413.

b A single Platinum Path-purified sputum sample not spiked with Y. pestis gave a false positive result. It may have been contaminated with Y. pestis during processing or testing, as PCR amplification of that sample was delayed relative to samples spiked at the 1×LoD level, and no other false positive results were obtained during the study.

Selected Analytic Studies

Limit of Detection

Twenty out of 20 independent whole blood specimens spiked with Y. pestis at the previously established LoD level and processed with the IT 1-2-3 Platinum Path Sample Purification Kit were detected with the JBAIDS Plague Detection Kit. This confirmed the LoD of 50 CFU/mL in whole blood that was previously established for whole blood samples processed using the IT 1-2-3 OFLOWd10 Sample Purification Kit.

Twenty out of 20 independent sputum specimens spiked with Y. pestis at the LoD level and processed with the IT 1-2-3 Platinum Path Sample Purification Kit were detected with the JBAIDS Plague Detection Kit. This confirmed the LoD of 670 CFU/mL in sputum previously established for sputum samples processed using the IT 1-2-3 VIBE Sample Purification Kit.

Table 5. Confirmation of the Y. pestis LoDs for Platinum Path-Purified Whole Blood and Sputum Samples Tested with the JBAIDS Plague Detection Kit

SampleMatrix	Spiked Y.pestisConcentration(CFU/mL)	PlagueAssay	#Positive	%Positive	Plague TargetAssay Mean Cp+/- Std Dev
WholeBlood	50	Target 1	20/20	100.0%	34.25 ± 1.18
WholeBlood		Target 2	19/20	95.0%	34.67 ± 1.01
Sputum	670	Target 1	20/20	100.0%	31.17 ± 0.93
Sputum		Target 2	20/20	100.0%	31.91 ± 1.68

Reproducibility

A multicenter study was performed to determine the overall system reproducibility when whole blood and sputum samples were processed with the IT 1-2-3 Platinum Path Sample Purification Kit prior to testing with the JBAIDS Plague Detection Kit.

A panel of 12 blood samples was tested twice each day for four days at each of three testing sites. The panel contained four samples spiked with inactivated Y. pestis CO92 strain at a medium positive (5×LoD) level, four samples spiked at a low positive level (1×LoD), and four samples that were not spiked. Results for whole blood testing are summarized in Table 6.

A panel of nine sputum samples was similarly tested twice each day for five days at each of three testing sites. This panel contained three samples spiked with inactivated Y. pestis CO92 strain at a medium positive (5×LoD) level, three samples spiked at a low positive

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level (1×LoD), and three samples that were not spiked. Results for sputum testing are summarized in Table 7. The detection rate was >99% for both whole blood and sputum samples containing Y. pestis spiked near or above the LoD for the respective sample matrix. True negative whole blood and sputum samples did not have 100% negative results for each individual target assay, but since both targets must be detected for a final positive result for Plague, there were no final false positive results for unspiked samples. The JBAIDS Plague Detection System is reproducible when used to test whole blood and sputum samples processed with the IT 1-2-3 Platinum Path Sample Purification Kit.

		Plague Target 1						Plague Target 2
Blood Spike Level	Test Location	Number Positive	Number Negative	% Agreement with Expected Result	95% CI	Mean Cp	Std Dev	%CV	Number Positive	Number Negative	% Agreement with Expected Result	95% CI	Mean Cp	Std Dev	%CV
MediumPositive(5xLoD)	Site 1	32/32	0/32	100%		29.66	0.95	3.20	32/32	0/32	100%		32.52	0.86	2.64
	Site 2	32/32	0/32	100%		28.75	0.87	3.03	32/32	0/32	100%		31.80	0.83	2.61
	Site 3	32/32	0/32	100%		28.51	1.40	4.91	32/32	0/32	100%		31.04	0.93	3.00
	AllSites	96/96	0/96	100%	96.2-100	28.98	1.20	4.14	96/96	0/96	100%	96.2-100	31.79	1.06	3.33
LowPositive(1xLoD)	Site 1	32/32	0/32	100%		31.16	0.98	3.15	31/32a	0/32	96.9%		33.88	1.16	3.42
	Site 2	32/32	0/32	100%		30.08	1.27	4.22	32/32	0/32	100%		32.67	1.01	3.09
	Site 3	32/32	0/32	100%		29.55	0.96	3.25	32/32	0/32	100%		32.09	0.65	2.03
	AllSites	96/96	0/96	100%	96.2-100	30.26	1.27	4.20	95/96	0/96	98.9%	94.3-99.9	32.88	1.21	3.68
Negative	Site 1	0/32	32/32	100%					1/32	31/32	96.9%		36.40d
	Site 2	0/32	31/32b	96.9%					0/32	31/32c	96.9%
	Site 3	0/32	32/32	100%					0/32	32/32	100%
	AllSites	0/96	95/96	98.9%	94.3-99.9				1/96	94/96	97.9%	92.7-99.7	36.40d

Table 6. Reproducibility of the Plague Target 1 and Target 2 Assays in the JBAIDS Plague Detection Kit for Whole Blood Samples Purified with the IT 1-2-3 Platinum Path Purification Kit

ª One initial Uncertain result; retest also Uncertain.

b One initial Inhibited result; retest Inhibited for undiluted sample and negative for 1:10 dilution; no further testing.

· One initial Inhibited result; inadequate volume for retesting.

d Mean Cp for single false positive sample

Table 7. Reproducibility of the Plague Target 2 Assays in the JBAIDS Plague Detection Kit for Sputum Samples Purified with the IT 1-2-3 Platinum Path Purification Kit

F	S	p.i.k	e	o	1	o	5	4	Plague Target 1	Plague Target 2
---	---	-------	---	---	---	---	---	---	-----------------	-----------------

{10}------------------------------------------------

		Number Positive	Number Negative	% Agreement withExpected Result	95% CI	Mean Cp	Std Dev	%CV	Number Positive	Number Negative	% Agreement withExpected Result	95% CI	Mean Cp	Std Dev	%CV
Medium Positive(5xLoD)	Site 1	29/30	1/30	96.7%		29.82	0.70	2.35	30/30	0/30	100%		32.70	0.70	2.14
	Site 2	30/30	0/30	100%		28.60	0.59	1.88	30/30	0/30	100%		31.61	0.57	1.80
	Site 3	30/30	0/30	100%		27.59	0.58	2.10	30/30	0/30	100%		30.80	0.53	1.72
	AllSites	89/90	1/90	98.9%	94.0-99.9	28.65	1.10	3.84	90/90	0/90	100%	96.0-100	31.70	0.99	3.12
Low Positive(1xLoD)	Site 1	30/30	0/30	100%		32.60	0.77	2.36	30/30	0/30	100%		35.62	1.19	3.34
	Site 2	30/30	0/30	100%		31.31	0.58	2.03	30/30	0/30	100%		34.40	0.59	1.72
	Site 3	30/30	0/30	100%		30.36	0.67	2.21	30/30	0/30	100%		33.48	0.72	2.15
	AllSites	90/90	0/90	100%	96.0-100	31.42	1.14	3.63	90/90	0/90	100%	96.0-100	34.50	1.23	3.57
Negative	Site 1	0/30	30/30	100%					0/30	30/30	100%
	Site 2	0/30	29/30a	96.7%					0/30	30/30	100%
	Site 3	0/30	30/30	100%					0/30	30/30	100%
	AllSites	0/90	89/90	98.9%	94.0-99.9				0/90	90/90	100%	96.0-100

ª One initial Uncertain result; retest also Uncertain.

Detection of Direct Culture Samples Processed with the IT 1-2-3 Platinum Path Sample Purification Kit

Y. pestis colonies can be detected using a modified Platinum Path protocol to process the colonies followed by testing with the JBAIDS Plague Detection Kit. Ten Yersinia pestis colonies were purified alongside ten non- Y. pestis colonies. All ten Y. pestis colonies were detected with the JBAIDS Plague Detection Kit, while the non- Y. pestis colonies were not detected.

Table 8. Plague Target 1 and Target 2 Detection from Colonies Purified with Platinum Path
Plague Target 1	Plague Target 2

Colony Type	Plague Target 1			Plague Target 2
	Positive Results/Total	Cp (cycles) Mean	SD	Positive Results/Total	Cp (cycles) Mean	SD
Y. pestis	10/10	19.25	0.74	10/10	20.70	0.67
Non- Y. pestis	0/10	-	-	0/10	-	-

Regulation Number and Section

N/A