The Xpert® Carba-R Assay, performed on the GeneXpert® Instrument Systems, is a qualitative in vitro diagnostic test designed for the detection and differentiation of the blaker, blaNDM, blayIM, blackA-48, and blanny gene sequences associated with carbapenem-non-susceptibility. The test utilizes automated real-time polymerase chain reaction (PCR),

The Xpert Carba-R Assay is intended as an aid to infection of carbapenem-non- susceptible bacteria that colonize patients in healthcare settings. The Xpert Carba-R Assay is not intended to guide or monitor treatmannon-susceptible bacterial infections. A negative Xpert Carba-R Assay result does not preclude the presence of other resistance mechanisms.

The Xpert Carba-R Assay is for use with the following sample types:

Rectal Swab Specimens

The assay is performed on rectal swab specimens at risk for intestinal colonization with carbapenem-non-susceptible bacteria. Concomitant cultures are necessary to reanisms for epidemiological typing, antimicrobial susceptibility testing, and for further confirmatory bacterial identification.

Pure Colonies

The assay is performed on carbapenen-non-susceptible pure colonies of Enterobacter baumanni, or Pseudomonas aeruginosa, when grown on blood agar or MacConkey agar. For testing pure colonies, the Xpert Carba-R Assay should be used in conjunction with other laboratory tests including phenotypic antimicrobial susceptibility testing.

Device Description

The Xpert Carba-R Assay is an automated real-time polymerase chain reaction (PCR) in vitro diagnostic test for qualitative detection of the blaxpc, blaym, blaoxa-a8, and blamp gene sequences from rectal swab specimens. The Xpert Carba-R Assay is intended as an aid for infection control for monitoring the spread of carbapenem-nonsusceptible organisms in healthcare settings.

The Xpert Carba-R Assay is performed on the Cepheid GeneXpert® Instrument Systems (GeneXpert Dx, GeneXpert Infinity-48, GeneXpert Infinity-48s, and GeneXpert Infinity-80 systems). The GeneXpert Instrument System platform automates sample preparation. amplification and real-time detection.

The GeneXpert Instrument Systems require the use of single-use, disposable cartridges (the Xpert Carba-R cartridges) that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is minimized.

The Xpert Carba-R Assay cartridges contain reagents for the detection of blakec, blanDM, blaym, blaox 8-8, and blance gene sequences. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are controls utilized by the GeneXpert Instrument System platform. The SPC is present to control for adequate processing of the target bacteria and to monitor the presence of inhibitors in the real-time PCR reaction to reduce the possibility of false negative results. The PCC verifies reagent rehydration, real-time PCR tube filling in the cartridge, probe integrity, and dye stability.

The single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time PCR for the detection of the blaker, blanny, blaoxA-48. and blank gene sequences from rectal swab specimens in approximately 50 minutes. The GeneXpert Instrument Systems, comprised of the GeneXpert Dx Systems and the GeneXpert Infinity Systems, have 1 to 80 randomly accessible modules, depending upon the instrument, that are each capable of performing separate sample processing and realtime PCR and RT- PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time PCR and RT-PCR and detection.

Rectal swab specimens are placed into a sample reagent. The sample is transferred to the sample chamber of the disposable fluidic cartridge (the Xpert Carba-R cartridge). The user initiates a test from the system user interface and places the cartridge into the GeneXpert instrument platform, which performs hands-off real-time, multiplex PCR for detection of the blaker, blaypy, blaym, blaoxy-48, and blane gene sequences. The results are automatically generated at the end of the process in a report that can be viewed and printed.

AI/ML Overview

Here’s a summary of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Device: Xpert® Carba-R Assay

Indications for Use: Qualitative in vitro diagnostic test for the detection and differentiation of blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP gene sequences associated with carbapenem-non-susceptibility. Intended as an aid to infection control for detecting carbapenem-non-susceptible bacteria colonizing patients in healthcare settings (rectal swab specimens). Also for use with carbapenem-non-susceptible pure colonies of Enterobacteriaceae, Acinetobacter baumannii, or Pseudomonas aeruginosa.

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" for the clinical performance in a separate, clear table. However, the reported performance metrics in the clinical study can be considered as the evidence demonstrating the device meets an implicit acceptance level for regulatory approval. Regulatory bodies typically look for high Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). Based on the "Prospective Specimen Results" section (Table 5-11), here are the key performance metrics:

Target Gene	PPA (%) (95% CI)	NPA (%) (95% CI)
IMP	N/A (0 TP found)	99.9% (99.3-100.0)
VIM	60.0% (31.3-83.2)	98.9% (97.9-99.5)
NDM	100.0% (64.6-100.0)	99.6% (98.8-99.9)
KPC	100.0% (88.3-100.0)	99.2% (98.2-99.6)
OXA-48	96.7% (83.3-99.4)	98.6% (97.5-99.2)

Note on IMP PPA: The PPA for IMP is "N/A" with 0 True Positives (TP) among the prospective specimens, which indicates a very low prevalence of this target in the prospective cohort.

Contrived Specimen Performance (Table 5-14):

Target Gene	PPA (%) (95% CI)	NPA (%) (95% CI)
IMP	95.0% (87.8-98.0)	100.0% (98.9-100.0)
VIM	98.8% (93.4-99.8)	100.0% (98.9-100.0)
NDM	100.0% (95.4-100.0)	100.0% (98.9-100.0)
KPC	100.0% (95.4-100.0)	100.0% (98.9-100.0)
OXA-48	98.8% (93.3-99.8)	100.0% (98.9-100.0)

The reproducibility study (Table 5-15) shows high overall agreement, typically 100% for negative and moderate positive samples, and ranging from 86.1% to 97.2% for low positive samples, indicating consistent performance across sites and operators.

2. Sample Size Used for the Test Set and Data Provenance

Prospective Clinical Study:
- Initial Enrollment: 802 rectal swab specimens.
- Eligible for Inclusion: 785 specimens.
- Final Dataset: 755 specimens after exclusions (including 16 Stenotrophomonas maltophilia due to intrinsic resistance).
- Provenance: Five geographically diverse sites (three across the United States and two in Europe). Data was collected prospectively from subjects hospitalized or in long-term care facilities.
Contrived Clinical Study:
- Sample Size: 432 contrived specimens prepared in rectal swab matrix.
- Provenance: This data is laboratory-generated, not from human subjects.

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

The document refers to a "reference culture laboratory" and an "independent laboratory for reference bi-directional sequencing analysis" for establishing ground truth. It does not explicitly state the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience"). However, the methodologies employed (culture, disk diffusion, CLSI document standards, PCR, and bi-directional DNA sequencing) imply the involvement of qualified laboratory professionals and molecular biologists.

4. Adjudication Method for the Test Set

For prospective specimens:

The Xpert Carba-R Assay was evaluated against a reference method consisting of:
1. Culture: MacConkey enrichment broth.
2. Confirmation of carbapenem non-susceptibility: Disk diffusion method (per CLSI document M02 and M100) on isolated colonies.
3. Molecular Confirmation: DNA extracted from non-susceptible isolates was purified, quantified, amplified with primers specific to the 5 target genes, and confirmed for appropriate amplicon size on an Agilent 2100 Bioanalyzer.
4. Sequencing: If bands corresponded to expected amplicon sizes, the amplicon was sent to an independent laboratory for reference bi-directional sequencing analysis, which was validated for detecting the five targets.
Discordant Analysis: For specimens where the Xpert Carba-R Assay was positive but a carbapenem-non-susceptible organism was not isolated by reference culture (False Positives based on initial reference), bi-directional sequencing on DNA extracted directly from the MacConkey enrichment broth was performed to confirm the presence of the gene sequences. This acts as an adjudication step.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study was mentioned in this document. The device is an automated in vitro diagnostic test, so there is no "human reader" component in its direct output or interpretation in the way an imaging AI system would interact with a radiologist. Therefore, improvement of human readers with AI assistance is not applicable to this device.

6. Standalone (Algorithm Only) Performance Study

Yes, a standalone study was performed. The clinical studies (both prospective and contrived) directly evaluated the performance of the Xpert Carba-R Assay (the algorithm/device) against the reference method without human intervention in the interpretation of the Xpert Carba-R results. The reported PPA and NPA values represent the standalone performance of the device.

7. Type of Ground Truth Used

The ground truth for the test set was established using a multi-faceted approach:

Expert Reference Method: A combination of culture (MacConkey enrichment broth followed by plating and disk diffusion based on CLSI standards) and molecular methods (PCR for specific gene amplification and bi-directional DNA sequence analysis).
Outcomes Data: Not directly applicable here; the ground truth is based on the presence or absence of specific gene sequences and organism characteristics.
Expert Consensus: While not explicitly stated as "expert consensus," the use of a multi-step reference method involving culture, phenotypic susceptibility testing, and sequencing performed by specialized laboratories implies a high standard of expert-driven verification. The discordant analysis further indicates a rigorous process to resolve discrepancies against the initial reference standard.

8. Sample Size for the Training Set

The document does not explicitly state the sample size of a "training set" for the Xpert Carba-R Assay. This is typical for in vitro diagnostic devices that are based on established molecular biology principles (like PCR) and analytical validation (LoD, reactivity, specificity) rather than machine learning models that require distinct training sets. The development process would involve extensive analytical testing to optimize primers and probes, which could be considered an iterative development phase but not a formal "training set" in the AI/ML sense.

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

As no explicit "training set" is described in the context of an AI/ML model, the establishment of ground truth for a training set is not detailed. However, for a PCR-based assay, the development process would involve establishing the analytical robustness of the assay, including:

Analytical Sensitivity (Limit of Detection - LoD): Determined by using carbapenemase-producing organisms seeded into negative human rectal swab matrix at varying concentrations and verified with known positive and negative replicates.
Analytical Reactivity (Inclusivity): Evaluated against a panel of 72 well-characterized bacterial strains with known resistance markers in rectal swab matrix, tested at concentrations (e.g., 3x LoD).
Analytical Specificity (Cross-reactivity): Evaluated against a panel of 62 carbapenem-susceptible bacteria with other resistance mechanisms, 24 commensal/enteric microorganisms, and human cells at defined concentrations.

These analytical studies use panels with known characteristics (verified through methods like individual PCR assays, DNA sequence analysis, or Check-Points array) to ensure the assay correctly identifies targets and avoids off-target detection. This rigorous analytical characterization serves a similar purpose to "ground truth establishment" for refining a diagnostic assay's performance.

Summary

{0}------------------------------------------------

Image /page/0/Picture/1 description: The image is a seal for the Department of Health & Human Services - USA. The seal is circular with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. In the center of the seal is a stylized image of three faces in profile, stacked on top of each other.

Public Health Service

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

June 29, 2016

CEPHEID JIM KELLY, Ph.D. EXECUTIVE DIRECTOR, REGULATORY AFFAIRS 904 CARIBBEAN DRIVE SUNNYVALE CA 94089-1189

Re: K160901

Trade/Device Name: Xpert® Carba-R Assay Regulation Number: 21 CFR 866.1640 Regulation Name: Antimicrobial susceptibility test powder Regulatory Class: II Product Code: POC, OOI Dated: March 31, 2016 Received: April 1, 2016

Dear Dr. Kelly:

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.

{1}------------------------------------------------

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education 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" (21 CFR 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 Industry and Consumer Education 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,

Ribhi Shawar -S

For 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

Enclosure

{2}------------------------------------------------

510(k) Number (if known) K160901

Device Name

Xpert Carba-R

Indications for Use (Describe)

The Xpert Carba-R Assay is for use with the following sample types:

Rectal Swab Specimens

Pure Colonies

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{3}------------------------------------------------

510(k) Summary

Product code:

Classification

Advisory Panel

Prescription Use

Predicate Device

Assay:

As required by 21 CFR Section 807.92(c).

Submitted by:	Cepheid904 Caribbean DriveSunnyvale, CA 90489Phone number: (847) 228-3299Fax number: (847) 890-6589
Contact:	Scott A. Campbell, PhD, MBA
Date of Preparation:	June 28, 2016
Device:
Trade name:	Xpert ® Carba-R
Common name:	Xpert Carba-R Assay
Type of Test:	Qualitative nucleic acid amplification test of the bla KPC , bla NDM ,bla VIM , bla OXA-48 , and bla IMP gene sequences associated withcarbapenem-non-susceptibility in gram-negative bacteriaobtained from rectal swab specimens
Classification:	II
Regulation number:	866.1640
Classification name:	Antimicrobial susceptibility test powder

POC, OOI

Yes

Microbiology (83)

[510(k) #K152614]

Cepheid Xpert ® Carba-R

{4}------------------------------------------------

Device Description:

{5}------------------------------------------------

Device Intended Use:

The Xpert® Carba-R Assay, performed on the GeneXpert® Instrument Systems, is a qualitative in vitro diagnostic test designed for the detection and differentiation of the blakpc, blaNDM, blaoxA-48, and blamp gene sequences associated with carbapenem-non-susceptibility. The test utilizes automated real-time polymerase chain reaction (PCR).

The Xpert Carba-R Assay is intended as an aid to infection control in the detection of carbapenem-non-susceptible bacteria that colonize patients in healthcare settings. The Xpert Carba-R Assay is not intended to guide or monitor treatment for carbapenem-nonsusceptible bacterial infections. A negative Xpert Carba-R Assay result does not preclude the presence of other resistance mechanisms.

The Xpert Carba R-Assay is for use with the following sample types:

Rectal Swab Specimens

The assay is performed on rectal swab specimens from patients at risk for intestinal colonization with carbapenem-non-susceptible bacteria. Concomitant cultures are necessary to recover organisms for epidemiological typing, antimicrobial susceptibility testing, and for further confirmatory bacterial identification.

Pure Colonies

The assay is performed on carbapenem-non-susceptible pure colonies of Enterobacteriaceae, Acinetobacter baumannii, or Pseudomonas aeruginosa, when grown on blood agar or MacConkey agar. For testing pure colonies, the Xpert Carba-R Assay should be used in conjunction with other laboratory tests including phenotypic antimicrobial susceptibility testing.

Substantial Equivalence:

The Cepheid Xpert Carba-R Assay is substantially equivalent to the Xpert® Carba-R Assay, 510(k) #K152614. Both assays utilize the same GeneXpert cartridge and detect target gene sequences (blakec, blandM, blavM, blaoxA-48, and blamp) using real-time PCR amplification and fluorogenic target-specific hybridization detection. The performance of the Xpert Carba-R Assay for an expanded indication was determined in a multi-site clinical study in which the performance of the Xpert Carba-R Assay was evaluated relative to culture and reference DNA sequence analysis. The results of the study demonstrated that the performance of the Xpert Carba-R Assay is substantially equivalent to the predicate device. Please refer to K152614 for information on the performance of the Xpert Carba-R Assay with pure colonies of carbapenem nonsusceptible Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

Table 5-1 shows the similarities and differences between the Xpert Carba-R Assay and the predicate device.

{6}------------------------------------------------

Table 5-1: Comparison of Similarities and Differences of the Xpert Carba-R Assay with the Predicate Device

Similarities
Item	Device	Predicate Device
	Cepheid Xpert Carba-R Assay	Cepheid Xpert Carba-R AssayK152614
General Intended Use	The Xpert® Carba-R Assay, performedon the GeneXpert® Instrument Systems,is a qualitative in vitro diagnostic testdesigned for the detection anddifferentiation of the blaKPC ,blaNDM , blaVIM , blaOXA-48 , andblaIMP gene sequences associated withcarbapenem-non-susceptibility. Thetest utilizes automated real-timepolymerase chain reaction (PCR).The Xpert Carba-R Assay is intended asan aid to infection control in thedetection of carbapenem-non-susceptiblebacteria that colonize patients inhealthcare settings. The Xpert Carba-R Assay is not intended to guide ormonitor treatment for carbapenem-non-susceptible bacterial infections. Anegative Xpert Carba-R Assay resultdoes not preclude the presence of otherresistance mechanisms.The Xpert Carba-R Assay is for use withthe following sample types:Rectal Swab SpecimensThe assay is performed on rectal swabspecimens from patients at risk forintestinal colonization with carbapenem-non-susceptible bacteria. Concomitantcultures are necessary to recoverorganisms for epidemiological typing,antimicrobial susceptibility testing, andfor further confirmatory bacterialidentification.Pure ColoniesThe assay is performed on carbapenem-non-susceptible pure colonies ofEnterobacteriaceae , Acinetobacterbaumannii , or Pseudomonas aeruginosa ,when grown on blood agar orMacConkey agar. For testing purecolonies, the Xpert Carba-R Assayshould be used in conjunction with other	The Xpert® Carba-R Assay, performedon the GeneXpert® Instrument Systems,is a qualitative in vitro diagnostic test forthe detection and differentiation of theblaKPC , blaNDM , blaVIM , blaOXA -48, and blaIMP gene sequencesassociated with carbapenem-non-susceptible pure colonies ofEnterobacteriaceae , Acinetobacterbaumannii , or Pseudomonasaeruginosa grown on blood agar orMacConkey agar. The test utilizesautomated real-time polymerase chainreaction (PCR).A negative Xpert Carba-R Assayresult does not preclude the presenceof other resistance mechanisms. TheXpert Carba-R Assay should be usedin conjunction with other laboratorytests including phenotypicantimicrobial susceptibility testing.The Xpert Carba-R Assay is intendedas an aid for infection control indetecting and differentiating geneticmarkers of resistance to monitor thespread of carbapenem-non-susceptibleorganisms in healthcare settings. TheXpert Carba-R Assay is notintended to guide or monitortreatment for carbapenem-non-susceptible bacterial infections.
Similarities
Item	Device	Predicate Device
	Cepheid Xpert Carba-R Assay	Cepheid Xpert Carba-R AssayK152614
Type of test	laboratory tests including phenotypicantimicrobial susceptibility testing.	Qualitative
TechnologicalPrinciples	Same	Fully-automated nucleic acidamplification (DNA); real-timePCR
Test Cartridge	Same	Disposable single-use, multi-chamberedfluidic cartridge
Probes	Same	TaqMan® Probes
Controls	Same	Internal sample processingcontrol (SPC) and probecheck control (PCC)External controls available
Instrument System	Same	GeneXpert Instrument System(includes GeneXpert Dx, Infinity-48,Infinity-48s, and Infinity-80)
Time to obtain testresults	Same	Approximately 50 minutes to results
Interpretation of testresults	Same	Diagnostic software of the GeneXpertInstrument System
Laboratory Users	Same	Operators in CLIA Moderate or HighComplexity labs
Differences
Item	Cepheid Xpert Carba-R Assay	Cepheid Xpert Carba-R AssayK152614
Sample Types	Bacterial isolates from culture, rectalswab specimens	Bacterial isolates from culture

{7}------------------------------------------------

The Xpert Carba-R Assay has the same general intended use as the predicate device and has the same technological characteristics as the predicate device. The differences between the Xpert Carba-R Assay and the predicate device do not raise questions of safety and effectiveness. The clinical study demonstrates that the Xpert Carba-R Assay is acceptable for its intended use with inexperienced laboratory users and is substantially equivalent to the predicate device described above.

{8}------------------------------------------------

Non-Clinical Studies:

Analytical Sensitivity (Limit of Detection)

The analytical sensitivity or Limit of Detection (LoD) of the Xpert Carba-R Assay was assessed using carbapenemase-producing organisms seeded into pooled negative human rectal swab matrix. The LoD was determined for two carbapenemase-producing bacteria for each gene analyte.i.e., the genes encoding KPC, NDM, VIM, OXA-48, and IMP. Bacteria were titered by plate counts and spiked onto clean swabs. Swabs were placed into pooled negative rectal swab matrix and replicates of 20 were evaluated at a minimum of five different concentrations over four days. The LoD for each of the ten carbapenemaseproducing organisms was estimated by probit analysis. The LoD is defined as the lowest concentration of target cells (CFU/swab) that can be reproducibly distinguished from negative samples with 95% confidence. The study was performed with two different lots of Xpert Carba-R reagents and the claimed LoD is the higher of the two determinations. The estimated LoDs were verified by preparing and testing 10 replicates from two independent dilutions of each bacterium at each estimated LoD.

The claimed LoD for each pair of carbapenemase-producing organism in rectal swab matrix are shown in Table 5-2.

Target Gene and Organism	LoD Estimates (Probit)CFU /swab		LoDClaimCFU/swab	EstimatedLoDIn SampleReagentCFU/mL	Verification(Positives/20)
	Lot 1	Lot 2
IMP-1 Acinetobacter baumannii	174	141	174	35	20/20
IMP-1 Klebsiella pneumoniae	303	306	306	61	20/20
VIM-1 Klebsiella pneumoniae	247	305	305	61	20/20
VIM-4 Escherichia coli	815	468	815	163	20/20
NDM-1 Klebsiella pneumoniae ATCCBAA-2146	117	251	251	50	20/20
NDM Klebsiella pneumoniae	74	57	74	15	19/20
KPC-3 Klebsiella pneumoniae NCTC13438	373	292	373	75	20/20
KPC Enterobacter cloacae	779	537	779	156	20/20
OXA-48 Enterobacter cloacae	154	109	154	31	20/20
OXA-48 Escherichia coli	104	99	104	21	20/20

Table 5-2. LoD Estimates and Verification for Organisms Harboring Carbapenemase Genes using the Xpert Carba-R Assay in Rectal Swab Matrix

Analytical Reactivity (Inclusivity)

The analytical reactivity of the Xpert Carba-R Assay with rectal swab matrix was evaluated by testing a panel of 72 samples. This panel consisted of 11 blakec (KPC), 11 blavin (VIM), 13 blaNDM (NDM), 8 blaoxa-48 (OXA-48), 5 blands(blaoxA-18) (NDM/OXA-181), 6 blaoxa-181(OXA-181), 17 blang (IMP), and one blakec/blayM (KPC/VIM) well-characterized

{9}------------------------------------------------

bacterial strains. The strains tested in rectal swab matrix and their test concentrations are presented in Table 5-3.

For testing in rectal swab matrix, organisms were seeded into pooled negative rectal swab matrix. All bacterial strains were tested in triplicate at approximately 3x LoD for each specimen type. Xpert Carba-R Assay target genes were detected in 69 of 72 carbapenemase-producing bacterial strains although IMP-4 was detected only using a higher concentration (Table 5-3). Xpert Carba-R Assay target DNA sequences were not detected in three bacterial strains as shown in Table 5-3. In one of the three bacterial strains, the IMP-13 gene was not detected by the assay, although it was predicted to be detected by in silico analysis. In two of the other three bacterial strains, the IMP-7 and IMP-14 genes were not predicted to be detected by in silico analysis and were not detected by the assay. See Limitations in the package insert.

The variants detected, and predictions for detecting other subtypes of each resistance gene based on in silico analysis, are presented in Table 5-4.

Strain ID	Organism	ResistanceMarker withvariantinformation	ConcentrationTested inRectal SwabMatrix(CFU/mL)
NCTC 13438	Klebsiella pneumoniae	KPC-3	153
31551	Klebsiella pneumoniae	KPC-4	50
ATCC BAA-1705	Klebsiella pneumoniae	KPC-2	130
PA-Col	Pseudomonas aeruginosa	KPC-2	250
KBM18	Enterobacter aerogenes	KPC-2	250
BM9	Klebsiella pneumoniae	KPC-3	330
PA3	Klebsiella pneumoniae	KPC-2	100
CGNC	Serratia marcescens	KPC-2	300
CFVL	Enterobacter cloacae	KPC-2	160
COL	Escherichia coli	KPC-2	147
GR-04/KP-69	Klebsiella pneumoniae	KPC-2, VIM	80
164-3	Klebsiella oxytoca	KPC	70
NCTC 13437	Pseudomonas aeruginosa	VIM-10	500
NCTC 13439	Klebsiella pneumoniae	VIM-1	130
NCTC 13440	Klebsiella pneumoniae	VIM-1	70
758	Pseudomonas aeruginosa	VIM	250
PA-87	Klebsiella pneumoniae	VIM	200
B92A	Pseudomonas aeruginosa	VIM	2000
Col1	Pseudomonas aeruginosa	VIM-2	500
BM19	Serratia marcescens	VIM-2	250
KOW7	Escherichia coli	VIM-4	250
DIH	Klebsiella pneumoniae	VIM-19	250
MSH2014-3	Enterobacter cloacae	VIM	500
NCTC 13443	Klebsiella pneumoniae	NDM-1	80
Strain ID	Organism	ResistanceMarker withvariantinformation	ConcentrationTested inRectal SwabMatrix(CFU/mL)
ATCC BAA-2146	Klebsiella pneumoniae	NDM-1	80
34262	Klebsiella pneumoniae	NDM	80
GEN	Acinetobacter baumannii	NDM-1	130
3047	Enterobacter cloacae	NDM-1	70
7892	Proteus mirabilis	NDM-1	30
CAN	Salmonella spp.	NDM-1	70
EGY	Acinetobacter baumannii	NDM-2	40
I5	Escherichia coli	NDM-4	30
405	Escherichia coli	NDM-5	30
CF-ABE	Citrobacter freundii	NDM	30
73999	Pseudomonas aeruginosa	NDM	50
39365	Providencia rettgeri	NDM-1	70
NCTC 13442	Klebsiella pneumoniae	OXA-48	40
OM11	Klebsiella pneumoniae	OXA-48	60
501	Enterobacter cloacae	OXA-48	80
DUW	Klebsiella pneumoniae	OXA-48	120
OM22	Escherichia coli	OXA-48	80
BOU	Enterobacter cloacae	OXA-48	80
TUR	Enterobacter cloacae	OXA-48	120
11670	Escherichia coli	OXA-48	100
166643	Klebsiella pneumoniae	OXA-181	20
42194	Klebsiella pneumoniae	OXA-181	20
MSH2014-64	Klebsiella pneumoniae	OXA-181	280
MSH2014-72	Escherichia coli	OXA-181	100
74	Escherichia coli	OXA-181	100
CDC0051	Klebsiella ozaenae a	OXA-181	250
B108A	Klebsiella pneumoniae	NDM, OXA-181	10
C10192-DISCS	Enterobacter aerogenes	NDM, OXA-181	10
KP-OMA3	Klebsiella pneumoniae	NDM, OXA-181	60
1300920	Klebsiella pneumoniae	NDM, OXA-181	15
MSH2014-69	Klebsiella pneumoniae	NDM, OXA-181	20
NCTC 13476	Escherichia coli	IMP-1	250
695	Acinetobacter baumannii	IMP-1	1720
2340	Enterobacter cloacae	IMP-1	250
IMPBMI	Klebsiella pneumoniae	IMP-1	100
Yonsei 1	Acinetobacter baumannii	IMP-1	1000
Yonsei_2	Acinetobacter baumannii	IMP-1	500
6852	Klebsiella pneumoniae	IMP-1	100
MKAM	Pseudomonas aeruginosa	IMP-1	500
70450-1	Pseudomonas aeruginosa	IMP-1	250
3994	Pseudomonas spp.	IMP-10	250
Strain ID	Organism	ResistanceMarker withvariantinformation	ConcentrationTested inRectal SwabMatrix(CFU/mL)
CDC0161	Enterobacter aerogenesa	IMP-4	5.00E+04
5344	Pseudomonas aeruginosa	IMP-2	60
3985	Pseudomonas aeruginosa	IMP-11	2000
4032	Pseudomonas aeruginosa	IMP-6	80
3424	Pseudomonas aeruginosa	IMP-7b, c	1.00E+06
32443	Klebsiella pneumoniae	IMP-13c	1.00E+06
92	Pseudomonas aeruginosa	IMP-14b, c	1.00E+06

Table 5-3. Analytical Reactivity of the Xpert Carba-R Assay in Rectal Swab Matrix

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

{11}------------------------------------------------

a. These organisms were not tested as bacterial isolates.

b. IMP-7 and IMP-14 genes (Pseudomonas aeruginosa) were not detected by the assay and were not predicted to be detected by in silico analysis (see Limitations).

c. IMP-13 gene (Klebsiella pneumoniae): although predicted to be detected by in silico analysis, the IMP-13 gene was not detected by the assay (see Limitations).

	Table 5-4. Summary of Variants Detected by Wet Testing or Predicted to be Detected
Based on In Silico Analysis
Marker(or Traditional Subgroup)	No. ofSamples	Wet testing		Not tested but predicted to be detected based onin silico analysis
KPC	12	KPC-2, 3, 4	--	KPC-5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16
NDM	18	NDM-1, 2, 4, 5	--	NDM-3, 6, 7, 8, 9
VIM	12	VIM-1, 2, 4, 10, 19	--	VIM-5, 6, 7, 8, 9, 11, 12, 13, 14,15, 16, 17, 18, 20, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38
OXA-48	19	OXA-48,181(OXA-48variant)	--	OXA-162, 163, 204, 232, 244,245, 247
IMP	17	IMP-1 (9strains), IMP-2, 4, 6, 10, 11	IMP-7a, 13b,14a	IMP-3, 8, 9, 13b, 19, 20, 21, 22,24, 25, 27, 28, 30, 31, 33, 37,40, 42

a. IMP-7 and IMP-14 genes (Pseudomonas aeruginosa) were not detected by the assay and were not predicted to be detected by in silico analysis (see Limitations).

b. IMP-13 gene (Klebsiella pneumoniae) was tested: although predicted to be detected by in silico analysis, the IMP-13 gene was not detected by the assay (see Limitations).

Analytical Specificity (Cross-reactivity)

The analytical specificity of the Xpert Carba-R Assay was evaluated for bacterial isolates, organisms seeded into rectal swab matrix. For all three specimen types, a panel of 62 wellcharacterized bacterial strains of carbapenem-susceptible bacteria with carbapenem non-susceptibility due to genes or mechanisms other than the Xpert Carba-R target genes (Table 5-5 and Table 5-6) and 24 commensal bacterial strains and other enteric

{12}------------------------------------------------

microorganisms were also evaluated in the study (Table 5-7). Human cells were also tested in rectal swab matrix (Table 5-8). Resistance mechanisms were determined by individual PCR assays, DNA sequence analysis, or Check-Points array version CT102.

For rectal swab matrix samples, 62 strains were tested at concentrations >1 x 10° CFU/mL with the exception of Peptostreptococcus anaerobius that was tested at 5 x10° CFU/mL. Viruses were tested at >1 x 10 TCID50/mL or greater than 2.5 x107 RNA copies/mL. A bladder cell line (human genomic DNA) was tested at 1 x 10 cells/mL. Organisms were diluted into pooled negative rectal swab matrix and tested in triplicate. None of the 94 potentially cross-reactive organisms and nucleic acids tested was detected with the Xpert Carba-R Assay.

The Xpert Carba-R Assay did not cross react with any of the organisms tested (Table 5-6, Table 5-7, and Table 5-8). The analytical specificity of the assay was 100%.

	T TOTAL DECORD POLOTY OF COLUMNITY TOT SCIENCE TO PARTY LETTER POST
		Ertapenem Imipenem Meropenem
Susceptible	19	30	24
Intermediate	0	ನಿ	4
Resistant	43	24	34

Table 5-5. Number of Carbapenem-susceptible and Non-susceptible Organisms for each Antibiotic

Organism	Strain ID	Confirmed ResistanceMechanisms	Carbapenemsusceptibility (S/I/R)ª
			ETPa	IMPa	MEMª
Escherichia coli	NCTC 13441	CTX-M (-1, -type 15like); TEM	S	S	S
Klebsiella pneumoniae	NCTC 13465	CTX-M (25)	S	S	S
Enterobacter aerogenes	810	OmpC/OmpF deficient;TEM	R	R	R
Citrobacter freundii	1698	TEM (WT+164S)	S	S	S
Enterobacter cloacae	5557	AmpC (ACT/MIR)	R	R	R
Klebsiella pneumoniae	kpn5	CTX-M-2	R	S	R
Klebsiella pneumoniae	kpn12	TEM; SHV; CTX-M	R	R	R
Escherichia coli	ecol	TEM; CTX-M-2	R	R	R
Escherichia coli	eco2	CTX-M (2); TEM	R	S	S
Enterobacter cloacae	corl	CTX-M (2); TEM	R	R	R
Serratia marcescens	hpp21	CTX-M (2); TEM	S	S	S
Morganella morganii	fer29	CTX-M (2); TEM	S	R	S
Proteus mirabilis	gut25	CTX-M (2); TEM	S	R	S
Salmonella spp.	3209	CTX-M (2); TEM	S	S	S
Organism	Strain ID	Confirmed ResistanceMechanisms	Carbapenemsusceptibility (S/I/R)a
			ETPa	IMPa	MEMa
Shigella flexnerii	3331	CTX-M (2); TEM	S	S	S
Enterobacter cloacae	PA_3	AmpC; CTX-M-15; TEM	S	S	S
Klebsiella pneumoniae	32189	SHV	S	S	S
Klebsiella pneumoniae	32443	CTX-M (1, -type 15like); SHV	S	S	S
Klebsiella pneumoniae	32598	CTX-M (-1, -type 15like); SHV; TEM	R	I	R
Klebsiella pneumoniae	33560	CTX-M (15); SHV-11;TEM-1	S	S	S
Klebsiella pneumoniae	33603	SHV-2	R	I	R
Klebsiella pneumoniae	33617	SHV-27	S	S	S
Klebsiella pneumoniae	33643	SHV (-5, -55); TEM	S	S	S
Klebsiella pneumoniae	34430	SHV; TEM; CTX-M-15	S	S	S
Klebsiella pneumoniae	34680	TEM; CTX-M-2	R	S	R
Klebsiella pneumoniae	34732	CTX-M (15); SHV; TEM	R	S	S
Enterobacter cloacae	PA_174	GX-/Culture+; SHV;TEM	S	S	S
Enterobacter aerogenes	STU 645	SHV (WT+238S+240K)	R	S	R
Enterobacter aerogenes	STU 669	SHV (WT+238S+240K)	R	R	R
Escherichia coli	C3015	AmpC (CMY II); TEM	R	R	R
Enterobacter aerogenes	RI_100	AmpC (DHA); SHV	R	R	R
Klebsiella pneumoniae	B4A	SHV (WT + 238S+240K)	R	R	R
Klebsiella pneumoniae	B13A	SHV (WT + 238S+240K)	R	S	S
Enterobacter cloacae	RI_474	AmpC (ACT/MIR)	R	I	I
Enterobacter amnigenus	B71	AmpC (ACT/MIR)	R	R	R
Klebsiella pneumoniae	DD82A	SHV (WT + 238S +240K)	R	S	R
Klebsiella pneumoniae	B100	CTX-M (-1, type-15like); SHV (WT+238S);TEM	R	S	R
Enterobacter cloacae	135B	TEM	S	S	S
Klebsiella pneumoniae	B157	SHV; TEM	R	R	R
Escherichia coli	T2914280	CTX-M (-1, -15); TEM	R	S	R
Providencia stuartii	DD188	TEM (104K + 164S)	R	I	I
Enterobacter cloacae	DD189	AmpC (ACT/MIR)	R	S	S
Organism	Strain ID	Confirmed ResistanceMechanisms	Carbapenemsusceptibility (S/I/R)ª
			ETPª	IMPª	MEMª
Escherichia coli	B198B	CTX-M (-1, type -15like); TEM	R	S	R
Klebsiella pneumoniae	T3019989-1	CTX-M (-1, type-15like); SHV	R	I	R
Klebsiella pneumoniae	T3019989-2	CTX-M (-1, type-15like); SHV	R	S	R
Enterobacter cloacae	ENC-THAI14	VEB-1, TEM	S	S	S
Escherichia coli	CB154006	CTX-M (9); TEM	R	I	I
Enterobacter cloacae	S35766	AmpC(ACT/MIR)	S	S	S
Enterobacter cloacae	X1856910	AmpC (ACT/MIR); TEM	R	I	I
Klebsiella pneumoniae	W3758164	CTX-M (-1, -15 like);SHV; TEM.	R	I	R
Klebsiella pneumoniae	X2135758	CTX-M (-1, -15 like);SHV	R	S	S
Klebsiella pneumoniae	W3809535	CTX-M (-1, -15 like);SHV	R	R	R
Pseudomonas aeruginosa	CDC0064	SPM	R	R	R
Serratia marcescens	CDC0099	SME	R	R	R
Serratia marcescens	CDC0121	SME	R	R	R
Serratia marcescens	CDC0122	SME	R	R	R
Serratia marcescens	CDC0123	SME	R	R	R
Serratia marcescens	CDC0124	SME	R	R	R
Serratia marcescens	CDC0130	SME	R	R	R
Serratia marcescens	CDC0131	SME	R	R	R
Enterobacter cloacaegroup	CDC0132	IMI	R	R	R
Enterobacter cloacaecomplex	CDC0164	IMI	R	R	R

Table 5-6. Cross-reactivity Panel

{13}------------------------------------------------

{14}------------------------------------------------

a. S//R = Susceptible/Intermediate/Resistant, ETP = Ertapenem, IMP = Imipenem, MEM = Meropenem

Strain ID	Organism	Concentration Tested(CFU/mL unless otherwisespecified)
ATCC 25922	Escherichia coli	2.67E+06
ATCC 29212	Enterococcus faecalis	3.15E+06
ATCC 700603	Klebsiella pneumoniae	5.20E+06
ATCC 35218	Escherichia coli	2.47E+06
ATCC 25923	Staphylococcus aureus	4.53E+06
ATCC 27853	Pseudomonas aeruginosa	3.17E+06

{15}------------------------------------------------

Strain ID	Organism	Concentration Tested(CFU/mL unless otherwisespecified)
ATCC 9689	Clostridium difficile	1.80E+07
ATCC 700621	Enterobacter cloacae	8.95E+06
ATCC 9756	Enterococcus faecium	6.54E+06
ATCC 13182	Klebsiella oxytoca	4.76E+06
ATCC BAA-747	Acinetobacter baumannii	2.27E+06
ATCC 33128	Citrobacter freundii	2.01E+06
ATCC 49948	Morganella morganii	8.19E+06
ATCC 51331	Stenotrophomonas maltophilia	3.15E+06
ATCC 27028	Citrobacter koseri	5.05E+06
ATCC 49809	Providencia stuartii	3.01E+06
ATCC 49037	Peptostreptococcus anaerobius	5.00E+05
CCUG 29780 / ATCC 12401	Streptococcus agalactiae	5.21E+06
ATCC 15703	Bifidobacterium adolescentis	1.10E+08
ATCC 51697	Enterobacter aerogenes	3.19E+06
ATCC 43071	Proteus mirabilis	1.78E+06
CCUG 34787	Acinetobacter spp.	2.40E+06
CCUG 418	Citrobacter freundii	2.95E+06
CCUG 33629	Corynebacterium diphtheriae	4.48E+06
CCUG 17874	Helicobacter pylori	1.61E+06
CCUG 33548	Listeria monocytogenes	4.77E+06
CCUG 6325	Providencia alcalifaciens	4.91E+06
CCUG 43594 / ATCC 33560	Campylobacter jejuni	3.27E+06
MRVP/ZeptoMetrix	Adenovirus B Type 7A/NY	1.40E+05 TCID50/mL
MRVP/ZeptoMetrix	Enterovirus Type 71/NY	4.40E+05 TCID50/mL
Clinical Sample - Cepheid	Norovirus GII	2.5 x 107 RNA copies/mL

				Table 5-8. Cell Line Representing Human Genomic DNA
--	--	--	--	------------------------------------------------------

Organism Name	Source
Bladder Cell Carcinoma(hgDNA)	ATCC HTB-4

Competitive Interference

A competitive interference study was performed to test whether a high titer of one or more carbapenemase-producing organisms would interfere with the detection of a second target carbapenemase-producing organism that was present at a low titer. High titered samples were formulated at concentrations of 5 x 106 CFU/swab and low titered targets were formulated at approximately 2x LoD for the respective strain in rectal swab matrix. One carbapenemase-producing bacterial strain for each gene analyte, i.e., the genes encoding

{16}------------------------------------------------

KPC, NDM, VIM, OXA-48, and IMP, was used in this study. Each carbapenemaseproducing bacterial strain type was tested at low titers in conjunction with a high titer of each of the other one or two carbapenemase-producing bacterial strain types (Table 5-9). Samples were tested in replicates of eight. An inhibitory effect was observed for three of the five targets (IMP, VIM, and OXA-48) when a low concentration of each target was present in combination with a high concentration of one or two other targets for samples tested in rectal swab matrix. The three targets (IMP, VIM, and OXA-48) were tested at a higher concentration (4x LoD) in combination with a high concentration of one of two other targets for samples in rectal swab matrix. No inhibitory effect was observed for the three targets (IMP, VIM and OXA-48) at 4x LoD in the presence of clinically relevant co-infections for the Xpert Carba-R Assay. The competitive inhibitory effect on the Carba-R targets (IMP, VIM and OXA-48) is addressed in Section 15, Limitations in the package insert.

Table 5-9. Combinations of Carbapenemase-producing Bacteria Tested with the
Xpert Carba-R Assay

Combination
High KPC/High NDM/Low VIM
High KPC/High NDM/Low OXA
High KPC/High NDM/Low IMP
High VIM/High OXA/Low KPC
High VIM/High OXA/Low NDM
High VIM/High OXA/Low IMP
High IMP/Low KPC
High IMP/Low NDM
High IMP/Low VIM
High IMP/Low OXA
High OXA/Low VIM
High VIM/Low OXA
High KPC/Low NDM
Negative

Potentially Interfering Substances

The performance of the Xpert Carba-R Assay was evaluated with 24 potentially interfering substances that may be present in rectal swab specimens. Potentially interfering substances (IS) solutions were prepared and tested at concentrations specified in Table 5-10. Positive and negative samples were included in this study. Positive samples consisted of a mix of five carbapenemase-producing organisms harboring KPC, NDM, VIM, IMP-1 and OXA-48 gene sequences seeded into pooled negative rectal swab matrix at approximately 3x LoD. Eight replicate positive samples were tested per substance. Negative samples consisted of pooled negative rectal swab matrix not seeded with carbapenemase-producing organisms. Eight replicate negative samples were tested per substance to determine the effect on the

{17}------------------------------------------------

performance of the sample processing control (SPC). Controls consisted of positive and negative samples with no interfering substances added. The effect of each potentially interfering substance on positive and negative replicates was evaluated by comparing target cycle threshold (Ct) values generated in the presence of the substance to Ct values from controls lacking the substance. The positive and negative replicate samples for 22 potentially interfering substances were correctly identified using the Xpert Carba-R Assay. Interference with the Xpert Carba-R Assay may be observed with barium sulfate at > 0.1% w/v and Pepto-Bismol at > 0.01% w/v in tests with rectal swab matrix samples. See Limitations in the package insert. Rectal swab matrix samples, positive for a mix of five carbapenemase-producing organisms harboring KPC, NDM, VIM, IMP-1 and OXA-48 gene sequences that were tested with fecal fat at 0.25% w/v, did not yield any false negative results, however, delayed cycle threshold values were observed for the VIM target. This potential interference from the presence of 0.25% w/v fecal fat is provided in the Limitations section of the package insert.

Substance/Class	Active Ingredient	Concentration Tested
Non-steroidal anti-inflammatorymedication	Naproxen	0.25% w/v
Imaging compound	Barium sulfate	0.25% and 0.1% w/v
Antibiotic (oral)	Cephalexin	0.25% w/v
Antibiotic (oral)	Ciprofloxacin	0.25% w/v
Condom withspermicidal lubricant	Nonoxynol-9	1 condoma
Creams/ointment/suppositories	Hydrocortisone	0.25% w/v
Laxative	Sennosides	0.25% w/v
Lipids	Stearic acid/Palmiticacid/Cholesterol (fecalfat)	0.25% w/v
Anti-diarrhealmedication	Loperamidehydrochloride/bismuthsubsalicylate (Imodium)	0.25% w/v
Anti-diarrhealmedication	Loperamidehydrochloride/bismuthsubsalicylate (Kaopectate)	0.25% w/v
Topical cream	K-Y Jelly	0.25% w/v
Antacids	Calciumcarbonate/aluminumhydroxide/magnesiumhydroxide/simethicone(Milk of Magnesia)	0.25% w/v
Enemas	Mineral oil	0.25% w/v
Antibiotic (topical)	Polymixin B/ Neomycin/Bacitracin (Neosporin)	0.25% w/v
Anti-fungal/anti-itch Vaginal	Nystatin	0.25% w/v
Antacid	Famotidine (Pepcid)	0.25% w/v

Table 5-10. Potentially Interfering Substances Tested

{18}------------------------------------------------

Substance/Class	Active Ingredient	ConcentrationTested
Anti-diarrhealmedication	Loperamidehydrochloride/bismuthsubsalicylate(Pepto-Bismol)	0.25%, 0.1%, 0.05%,0.025%, 0.01% w/v
Topical cream	Petroleum jelly	0.25% w/v
Anti-hemorrhoidcreams/ointments	Phenylephrine(Preparation H)	0.25% w/v
Acid reducer; antacid	Oemprazole (Prilosec)	0.25% w/v
Enemas	Saline-enema	0.25% w/v
Antacid	Cimetidine (Tagamet)	0.25% w/v
Anti-fungal/anti-itchVaginal	Benzocaine, resorcinol(Vagisil)	0.25% w/v
Moist towelettes	Benzalkonium chloride,ethanol (Wet Ones)	1 pieceb

a. One condom added to 40 mL swab matrix.

b. One piece (5 inch x 7-1/2 inch) added to 40 mL swab matrix.

Carry-Over Contamination

A study was conducted to demonstrate that single-use, self-contained GeneXpert cartridges prevent carry-over contamination in negative samples run following very high positive samples. The study consisted of a negative sample processed in the same GeneXpert module immediately following a very high positive sample. The high positive sample is composed of inactivated E. coli cells containing a plasmid with an insert consisting of a synthetic oligonucleotide of the amplicon sequences from the five Xpert Carba-R target analyte genes (KPC, NDM, VIM, IMP and OXA-48 targets). Positive cells were diluted in pooled negative rectal swab matrix to a concentration of 1 x 10 CFU/mL. The testing scheme was repeated 25 times on two GeneXpert modules for a total of 102 tests (25 high positive samples per module and 26 negative samples per module) for the rectal swab matrix. All 50 positive samples correctly reported all Xpert Carba-R targets as DETECTED. All 52 negative samples correctly reported all Xpert Carba-R targets as NOT DETECTED.

Clinical Studies

Clinical Performance

Performance characteristics of the Xpert Carba-R Assay with rectal swab specimens were determined in a multi-site investigational study. The positive percent agreement (PPA) and negative percent agreement (NPA) of the Xpert Carba-R Assay was evaluated relative to a reference method of culture (MacConkey enrichment broth) and PCR/bi-directional DNA sequence analysis.

Five geographically diverse sites (three across the United States and two in Europe) prospectively collected paired rectal swab specimens from subjects who were hospitalized or in a long-term care facility. Highly soiled rectal swab specimens, were excluded from the study. Due to low prevalence of each of the Xpert Carba-R Assay target genes in the absence of an outbreak, contrived specimens were also included in the study.

{19}------------------------------------------------

One rectal swab of the pair was used for Xpert Carba-R Assay testing. The second rectal swab was inoculated into MacConkey enrichment broth and used for reference method testing. A reference culture laboratory determined the presence of carbapenem nonsusceptible organisms by culturing the MacConkey enrichment broth from each of the specimens. The MacConkev enrichment broth was screened for the presence of carbapenem-non-susceptible organisms initially by plating the broth on MacConkey agar plates with a meropenem disk. For specimens that exhibited growth of gramnegative bacteria around the meropenem disk, confirmation of carbapenem nonsusceptibility was determined on isolated colonies by using the disk diffusion method (per CLSI document M02) as well as CLSI document M100. DNA extracted from the carbapenem non-susceptible isolates was purified, quantified, and amplified using primers specific to all 5 target genes; amplified regions included more bases than the regions amplified by the Xpert Carba-R Assay. The production of the appropriate size amplification product was confirmed on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

If bands shown on the Bioanalyzer corresponded to the expected size of the amplicon from any of the five target genes detected by the Xpert Carba-R Assay, the amplicon for the isolate was sent to an independent laboratory for reference bi-directional sequencing analysis, which was validated for detection of the five targets in the Xpert Carba-R Assay. If no bands were shown on the Bioanalyzer for any of the five target genes, the isolate was not sent for sequence analysis and the reference method result was considered negative for the five target genes.

Prospective Specimen Results Obtained with the Xpert Carba-R Assay in Comparison to the Reference Method

A total of 802 prospective rectal swab specimens were initially enrolled in this clinical study, of which 785 were eligible for inclusion. From the 785 eligible specimens. 755 specimens were included in the final dataset after exclusions based on protocol deviations (including 16 Stenotrophomonas maltophilia organisms that were excluded due to their intrinsic resistance to the carbapenems tested).

When tested with prospective rectal swab specimens, the Xpert Carba-R Assay demonstrated a PPA range from 60.0% to 100% for the four assay targets (blakec, blaNDM, blaym, and blaoxA-48) relative to the reference method (Table 5-11). The NPA for the blakpc, blann, blanx 4-48, and blansp gene sequences ranged from 98.6%-99.9% relative to the reference method (Table 5-11).

For specimens with discordant results (the Xpert Carba-R Assay was positive for a target gene but a carbapenem-non-susceptible organism was not isolated by reference culture), discordant analysis was performed using bi-directional sequencing on DNA extracted directly from the MacConkey enrichment broth. Discrepant testing results are footnoted in Table 5-11.

{20}------------------------------------------------

SpecimenType	Target	N	TP	FP	TN	FN	PPA%(95 CI)	NPA%(95 CI)
Prospectiveg	IMP	755	0	1a	754	0	N/A	99.9%(99.3-100.0)
	VIM	755	6	8b	737	4	60.0%(31.3-83.2)	98.9%(97.9-99.5)
	NDM	755	7	3c	745	0	100.0%(64.6-100.0)	99.6%(98.8-99.9)
	KPC	755	29	6d,e	720	0	100.0%(88.3-100.0)	99.2%(98.2-99.6)
	OXA-48	755	29	10f	715	1	96.7%(83.3-99.4)	98.6%(97.5-99.2)

Table 5-11. Xpert Carba-R Performance vs. Reference Culture + Sequencing -Prospective Specimens

N = Number, TP = True Positive, FP = False Positive, TN = True Negative, FN = False Negative

a. 1 discordant specimen was confirmed as FP after discordant analysis.

b.2 of the 8 FPs were determined to be TPs after discordant analysis.

c. 1 of the 3 FPs was determined to be TP after discordant analysis.

d. 1of the 6 FPs was determined to be TP after discordant analysis.

e. Site reported that subject was on ertapenem during time of specimen collection.

f. 3 of the 10 FPs were determined to be TPs after discordant analysis.

g. Of the 755 prospective rectal swab specimens evaluated in the study, 636 specimens did not yield a culture isolate by Reference Culture. From the remaining 119 specimens, 112 carbapenem-non-susceptible organisms were recovered by Reference Culture in addition to 7 carbapenem susceptible organisms [Pseudomonas aeruginosa (5); Escherichia coli (1), and Enterobacter cloacae (1)].

Performance of the Xpert Carba-R Assay on the prospective specimens is shown in Table 5-12 by species. Only organisms for which at least one positive specimen was collected are included in Table 5-12.

Speciesa	Target	N	TP	TN	PPA(95% CI)	NPA(95% CI)
Enterobacter cloacae	IMP	4	0	4	NA	100%(51.0-100.0)
	VIM	4	1	3	100%(20.7-100.0)	100%(43.9-100.0)
	NDM	4	0	4	NA	100%(51.0-100.0)
	KPC	4	0	4	NA	100%(51.0-100.0)
	OXA-48	4	1	3	100%(20.7-100.0)	100%(43.9-100.0)
E. coli	IMP	10	0	10	NA	100%(72.3-100.0)
	VIM	10	0	10	NA	100%(72.3-100.0)
	NDM	10	3	7	100%(43.9-100.0)	100%(64.6-100.0)
	KPC	10	2	8	100%(34.2-100.0)	100%(64.6-100.0)
	OXA-48	10	3	7	100%(43.9-100.0)	100%(64.6-100.0)

Table 5-12. Xpert Carba-R Performance vs. Reference Culture + Sequencing by Organism type - Prospective Specimens

{21}------------------------------------------------

Speciesa	Target	N	TP	FP	TN	FN	PPA(95% CI)	NPA(95% CI)
Klebsiellaoxytoca	IMP	1	0	0	1	0	NA	100%(20.7-100.0)
	VIM	1	0	0	1	0	NA	100%(20.7-100.0)
	NDM	1	0	0	1	0	NA	100%(20.7-100.0)
	KPC	1	0	0	1	0	NA	100%(20.7-100.0)
	OXA-48	1	1	0	0	0	100%(20.7-100.0)	NA
Klebsiellapneumoniae	IMP	60	0	1	59	0	NA	98.3%(94.0-100.0)
	VIM	60	0	1	59	0	NA	98.3%(94.0-100.0)
	NDM	60	4	1	55	0	100%(51.0-100.0)	98.2%(90.6-99.7)
	KPC	60	27	1	32	0	100%(87.5-100.0)	97.0%(89.6-100.0)
	OXA-48	60	24	3	32	1	96.0%(80.5-99.3)	91.4%(77.6-97.0)
	Pseudomonasaeruginosa	IMP	30	0	0	30	0	NA
VIM		30	5	0	21	4	55.6%(26.7-81.1)	100%(84.5-100.0)
NDM		30	0	1	29	0	NA	96.7%(83.3-99.4)
KPC		30	0	1	29	0	NA	96.7%(83.3-99.4)
OXA-48		30	0	0	30	0	NA	100%(88.7-100.0)

a. Acinetobacter baumannii (13) and Enterobacter amnigensus (1) were recovered but did not contain target sequences by the Reference Method.

Multiple targets were detected by the Xpert Carba-R Assay in eight prospective specimens. The details are provided in Table 5-13, along with the discrepant sequencing result.

Table 5-13. Prospective Specimens with Multiple Targets Detected
------------------------------------------------------------------	--	--	--	--	--

Specimen	Targets Detectedby Xpert Carba-RAssay	TargetsDetected byReferenceSequencing	Discrepant TestingResults - TargetsDetected byReferenceSequencing
1	KPC, OXA-48	NEG	NEG
2	VIM, KPC	NEGa	NEGa
3	VIM, OXA-48	OXA-48	OXA-48
4	KPC, OXA-48	KPC	KPC, OXA-48
5	NDM, OXA-48	NDM	NDM, OXA-48
6	VIM, NDM	NEGa	NEG
7	NDM, KPC	KPC	NDM, KPC
8	VIM, KPC	VIM	VIM, KPC

a. An organism was not isolated from reference culture, therefore, reference sequencing was not performed.

{22}------------------------------------------------

Contrived Specimen Results Obtained with the Xpert Carba-R Assay in Comparison to the Reference Method

A total of 432 contrived specimens prepared in rectal swab matrix were also tested as part of the clinical study.

In addition to Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii groups tested in the contrived study, 5 other non-Enterobacteriaceae strains were also evaluated: Pseudomonas stutzeri (1), Pseudomonas oryzihabitans (1), Pseudomonas putida (2), and Empedobacter brevis (1).

When tested with contrived specimens, the Xpert Carba-R Assay demonstrated a PPA range from 95% to 100% across the assay targets (blakp., blandM. blagsA.48. and blamp). The NPA for the blakec, blaNDM, blaoxA-48, and blamp gene sequences was 100% relative to the reference method (Table 5-14).

Table 5-14. Xpert Carba-R Performance vs. Reference Method-Contrived Specimens

Contrived Specimens
Target	N	TP	FP	TN	FN	PPA%(95 CI)	NPA%(95 CI)
IMP	432	76	0	352	4	95.0%(87.8-98.0)	100.0%(98.9-100.0)
VIM	432	81	0	350	1	98.8%(93.4-99.8)	100.0%(98.9-100.0)
NDM	432	80	0	352	0	100.0%(95.4-100.0)	100.0%(98.9-100.0)
KPC	432	80	0	352	0	100.0%(95.4-100.0)	100.0%(98.9-100.0)
OXA-48	432	79	0	352	1	98.8%(93.3-99.8)	100.0%(98.9-100.0)

Reproducibility Study

Reproducibility of the Xpert Carba-R Assay was evaluated using two panels of 11 samples, prepared in pooled negative rectal swab matrix. Two operators at each of the three study sites tested one panel of 11 samples in replicates of four per day over six testing days (11 samples x 2 replicates x 2 times/day x 6 days x 2 operators x 3 sites). Three lots of Xpert Carba-R Assay cartridges were used at each of the 3 testing sites. The Xpert Carba-R Assay was performed according to the Xpert Carba-R Assay procedure. Results are summarized in Table 5-15.

{23}------------------------------------------------

Sample	Matrixª	Site 1			Site 2			Site 3			% TotalAgreementby Sample
		Op 1	Op 2	Site	Op 1	Op 2	Site	Op 1	Op 2	Site
Neg	R	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(144/144)
IMPMod Pos	R	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(144/144)
IMPLow Pos	R	91.7%(22/24)	87.5%(21/24)	89.5%(43/48)	83.3%(20/24)	87.5%(21/24)	85.4%(41/48)	87.5%(21/24)	79.2%(19/24)	83.3%(40/48)	86.1%(124/144)
VIMMod Pos	R	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(144/144)
VIMLow Pos	R	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(144/144)
NDMMod Pos	R	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(144/144)
NDMLow Pos	R	91.7%(22/24)	95.8%(23/24)	93.8%(45/48)	95.8%(23/24)	95.8%(23/24)	95.8%(46/48)	100%(24/24)	91.7%(22/24)	95.8%(46/48)	95.1%(137/144)
KPCMod Pos	R	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(144/144)
KPCLow Pos	R	95.8%(23/24)	100%(24/24)	97.9%(47/48)	100%(24/24)	91.7%(22/24)	95.8%(46/48)	95.8%(23/24)	95.8%(23/24)	95.8%(46/48)	96.5%(139/144)
OXA-48Mod Pos	R	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(24/24)	100%(24/24)	100%(48/48)	100%(144/144)
OXA-48Low Pos	R	95.8%(23/24)	100%(24/24)	97.9%(47/48)	95.8%(23/24)	100%(24/24)	97.9%(47/48)	91.7%(22/24)	100%(24/24)	95.8%(46/48)	97.2%(140/144)

Table 5-15. Summary of Reproducibility Results - % Agreement, Rectal Swab Matrix

a. R=rectal

The reproducibility of the Xpert Carba-R Assay was also evaluated in terms of the fluorescence signal expressed in Ct values for each target detected. The mean, standard deviation (SD), and coefficient of variation (CV) between-sites, between-lots, betweendays, between-operators, and within-assays for each panel member are presented in Table 5-16.

Between-Within-Between-Site Between-Lot Between-Day Total Assay Operator Assay Mean Nº Matrixª Channel Sample Ct CV CV CV CV CV CV (Analyte) SD SD SD SD SD SD (%) (%) (%) (%) (%) (%) 0.2 2.0 Neg R SPC 144 32.9 0.2 0.5 0.7 0.0 0.1 0.0 0 0.6 18 0.7 IMP Mod Pos 34.5 0.0 0.2 0.2 R IMP 144 0.0 0:5 0.0 0.1 0.7 2.0 0.7 2.1 0 IMP Low Pos R IMP 140 36.4 0.0 0.0 0.0 0.0 0.2 0.5 0.0 0 1.2 3.3 1.2 3.4 VIM Mod Pos VIM 144 31.0 0.5 R 0.0 0.0 0.3 0.9 0 0.0 0.2 0.5 1.6 0.6 1.9 VIM Low Pos R VIM 144 33.8 0.0 0.0 0.6 1.8 0.3 0.9 1.0 1.4 4.0 4.6 0.3 1.6 NDM Mod Pos 144 33.7 0.0 R NDM 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.6 1.7 0.6 1.7 NDM Low Pos R NDM 143 36.2 0.2 0.7 0.0 0.0 0.3 0.7 0.0 0.0 0.8 2.3 0.9 2.5 KPC Mod Pos R КРС 144 34.2 0.0 0.0 0.3 0.8 0.2 0.6 0.0 0.0 0.4 1.2 0.6 1.6 35.8 0.5 1.9 KPC Low Pos R КРС 141 0.0 0.0 1.5 0.0 0.0 0.3 0.9 0.7 0.9 2.6 OXA-48 Mod Pos R OXA-48 144 34.3 0.0 0.0 0.2 0.5 0.2 0.5 0.1 0.3 0.5 1.6 0.6 1.7 OXA-48 OXA-48 Low Pos R 143 36.1 0.0 0.0 0.0 0.0 0.2 0.6 0.0 0.0 0.8 2.3 0.9 2.4

Table 5-16. Summary of Reproducibility Data, Rectal Swab Matrix

a. R=rectal

b. Results with non-zero Ct values out of 144.

{24}------------------------------------------------

Conclusions

The results of the nonclinical analytical and clinical performance studies summarized above demonstrate that the Xpert Carba-R Assay is safe and effective for its intended use and is substantially equivalent to the predicate device.

Regulation Number and Section

§ 866.1640 Antimicrobial susceptibility test powder.

(a)
Identification. An antimicrobial susceptibility test powder is a device that consists of an antimicrobial drug powder packaged in vials in specified amounts and intended for use in clinical laboratories for determining in vitro susceptibility of bacterial pathogens to these therapeutic agents. Test results are used to determine the antimicrobial agent of choice in the treatment of bacterial diseases.(b)
Classification. Class II (performance standards).