XPERT MTB/RIF ASSAY

DEN130032 · Cepheid · PEU · Jul 25, 2013 · Microbiology

Device Facts

• Record ID: DEN130032
• Device Name: XPERT MTB/RIF ASSAY
• Applicant: Cepheid
• Product Code: PEU · Microbiology
• Decision Date: Jul 25, 2013
• Decision: DENG
• Submission Type: Direct
• Regulation: 21 CFR 866.3373
• Device Class: Class 2

Indications for Use

The Xpert® MTB/RIF Assay, performed on the GeneXpert® Instrument Systems, is a qualitative, nested real-time polymerase chain reaction (PCR) in vitro diagnostic test for the detection of Mycobacterium tuberculosis complex DNA in raw sputum or concentrated sediments prepared from induced or expectorated sputum. In specimens where Mycobacterium tuberculosis complex (MTB-complex) is detected, the Xpert MTB/RIF Assay also detects the rifampin-resistance associated mutations of the rpoB gene. The Xpert MTB/RIF Assay is intended for use with specimens from patients for whom there is clinical suspicion of tuberculosis (TB) and who have received no antituberculosis therapy, or less than 3 days of therapy. This test is intended as an aid in the diagnosis of pulmonary tuberculosis when used in conjunction with clinical and other laboratory findings. The Xpert MTB/RIF Assay does not provide confirmation of rifampin susceptibility since mechanisms of rifampin resistance other than those detected by this device may exist that may be associated with a lack of clinical response to treatment. Specimens that have both MTB-complex DNA and rifampin-resistance associated mutations of the rpoB gene detected by the Xpert MTB/RIF Assay must have results confirmed by a reference laboratory. If the presence of rifampin-resistance associated mutations of the rpoB gene is confirmed, specimens should also be tested for the presence of genetic mutations associated with resistance to other drugs. The Xpert MTB/RIF Assay must be used in conjunction with mycobacterial culture to address the risk of false negative results and to recover the organisms for further characterization and drug susceptibility testing. The Xpert MTB/RIF Assay should only be performed in laboratories that follow safety practices in accordance with the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories publication and applicable state or local regulations.

Device Story

Automated in vitro diagnostic test for qualitative detection of M. tuberculosis complex DNA and rifampin-resistance associated mutations in raw or concentrated sputum. Input: sputum samples loaded into single-use disposable cartridges. Process: GeneXpert Instrument System performs automated sample preparation (mixing, filtration, washing, lysis via glass beads/ultrasonic horn) and real-time PCR amplification of rpoB gene core region. Detection: five molecular beacons (Probes A-E) labeled with different fluorophores differentiate wild-type from rifampin-resistance mutations. Output: tabular/graphic results displayed in ~2 hours. Used in clinical laboratories; operated by trained personnel. Benefits: rapid detection of TB and rifampin resistance; facilitates earlier treatment decisions and isolation; reduces transmission risk. Results aid clinical decision-making but require confirmation by reference laboratory for rifampin resistance and concurrent mycobacterial culture for definitive diagnosis.

Clinical Evidence

Multi-center study of 1,096 subjects with culture-confirmed active TB. Sensitivity for MTB detection: 99.7% (smear-positive), 76.1% (smear-negative). Specificity: 98.7%. Rifampin resistance detection sensitivity: 94.7%; specificity: 99.0%. Compared against culture and phenotypic drug susceptibility testing (DST).

Technological Characteristics

Nested real-time PCR assay. Materials: single-use disposable cartridges with lyophilized reagents, glass beads for lysis. Sensing: fluorogenic target-specific hybridization using five molecular beacons. Energy: electrical (GeneXpert Instrument System). Connectivity: networked/standalone PC-based software. Sterilization: N/A (disposable). Software: automated, closed-system, rule-based analysis.

Indications for Use

Indicated for patients with clinical suspicion of pulmonary tuberculosis (TB) who have received no antituberculosis therapy or less than 3 days of therapy. Used as an aid in diagnosis of pulmonary TB.

Regulatory Classification

Identification

Nucleic acid-based in vitro diagnostic devices for the detection of Mycobacterium tuberculosis complex (MTB-complex) and the genetic mutations associated with MTB-complex antibiotic resistance in respiratory specimens are qualitative nucleic acid-based devices that detect the presence of MTB-complex-associated nucleic acid sequences in respiratory samples. These devices are intended to aid in the diagnosis of pulmonary tuberculosis and the selection of an initial treatment regimen when used in conjunction with clinical findings and other laboratory results. These devices do not provide confirmation of antibiotic susceptibility since other mechanisms of resistance may exist that may be associated with a lack of clinical response to treatment other than those detected by the device.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) The FDA document entitled “Class II Special Controls Guideline: Nucleic Acid-Based In Vitro Diagnostic Devices for the Detection of *Mycobacterium tuberculosis* Complex and Genetic Mutations Associated with Antibiotic Resistance in Respiratory Specimens,” which addresses the mitigation of risks specific to the detection of MTB-complex. For availability of the document, see § 866.1(e).(2) The following items, which address the mitigation of risks specific to the detection of the genetic mutations associated with antibiotic resistance of MTB-complex: (i) The device must include an external positive assay control as appropriate. Acceptable positive assay controls include MTB-complex isolates containing one or more antibiotic-resistance associated target sequences detected by the device. (ii) The device must include internal controls as appropriate. An acceptable internal control may include human nucleic acid co-extracted with MTB-complex containing nucleic acid sequences associated with antibiotic resistance and primers amplifying human housekeeping genes (e.g., RNaseP, β-actin). (iii) The device's intended use must include a description of the scope of antibiotic resistance targeted by the assay, i.e., the specific drugs and/or drug classes. (iv) The specific performance characteristics section of the device's labeling must include information regarding the specificity of the assay oligonucleotides for detecting mutations associated with antibiotic resistance of MTB-complex, and any information indicating the potential for non-specific binding (e.g., BLAST search). (v) In demonstrating device performance you must perform: (A) Pre-analytical studies that evaluate: ( *1* )*Frozen samples.* If there is use of any frozen samples in the device performance studies, or if there is a device claim for the use of frozen samples for testing, the effect of freezing samples prior to testing and the effect of multiple freeze/thaw cycles on both antibiotic susceptible and antibiotic resistant strains of MTB-complex.( *2* )*Nucleic acid extraction methods.* Extraction methods must parallel those used in devices for the detection of MTB-complex nucleic acid and confirm that the detection of the genetic mutations associated with antibiotic resistance is not affected.(B) Analytical studies that analyze: ( *1* )*Limit of Detection.* Limit of Detection must be determined in the most challenging matrix (e.g., sputum) claimed for use with the device. The Limit of Detection must be determined using both antibiotic susceptible and antibiotic resistant strains of MTB-complex. The antibiotic resistant strains must be those with well characterized genetic mutations associated with antibiotic resistance.( *2* )*Analytical Reactivity (Inclusivity).* Testing must be conducted to evaluate the ability of the device to detect genetic mutations associated with antibiotic resistance in a diversity of MTB-complex strains. Isolates used in testing must be well characterized. Isolate strain characterization must be determined using standardized reference methods recognized by a reputable scientific body and appropriate to the strain lineage.( *3* )*Within-Laboratory (Repeatability) Precision Testing.* Within-laboratory precision studies, if appropriate, must include at least one antibiotic resistant and one antibiotic susceptible strain of MTB-complex.( *4) Between Laboratory Reproducibility Testing.* The protocol for the reproducibility study may vary slightly depending on the assay format; however, the panel must include at least one antibiotic resistant and one antibiotic susceptible strain of MTB-complex.(C) Clinical Studies. Clinical performance of the device must be established by conducting prospective clinical studies that include subjects with culture confirmed active tuberculosis. Studies must attempt to enroll subjects at risk for antibiotic-resistant MTB-complex; however, it may be necessary to include supplemental antibiotic resistant retrospective and contrived samples. Clinical studies must compare device results to both phenotypic drug susceptibility testing and genotypic reference methods. The genotypic reference method must be a polymerase chain reaction based method that uses primers different from those in the experimental device and confirmed by bidirectional sequencing.

Submission Summary (Full Text)

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### 510(k) DECISION SUMMARY

### A. 510(k) Number:

K131706

#### B. Purpose for Submission:

De novo request for evaluation of automatic class III designation for the Xpert® MTB/RIF Assay for use with the GeneXpert® Instrument Systems, including the GeneXpert® Diagnostic (Dx) Systems and the GeneXpert® Infinity Systems.

### C. Measurand:

M. tuberculosis complex DNA and rifampin-resistance associated mutations of the rpoB gene.

### D. Type of Test:

Qualitative, nested real-time polymerase chain reaction (PCR)

### E. Applicant:

Cepheid®

# F. Proprietary and Established Names:

Trade Name: Xpert® MTB/RIF Assay

Common Name: Xpert MTB/RIF Assay

# G. Regulatory Information:

- 1. Regulation section: 21 CFR 866.3373
- 2. Classification: Class II (special controls)
- 3. Product code: PEU
- 4. Panel: Microbiology (83)

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# H. Intended Use:

## 1. Intended use(s):

The Xpert® MTB/RIF Assay, performed on the GeneXpert® Instrument Systems, is a qualitative, nested real-time polymerase chain reaction (PCR) in vitro diagnostic test for the detection of Mycobacterium tuberculosis complex DNA in raw sputum or concentrated sediments prepared from induced or expectorated sputum. In specimens where Mycobacterium tuberculosis complex (MTB-complex) is detected, the Xpert MTB/RIF Assay also detects the rifampin-resistance associated mutations of the rpoB gene.

The Xpert MTB/RIF Assay is intended for use with specimens from patients for whom there is clinical suspicion of tuberculosis (TB) and who have received no antituberculosis therapy, or less than 3 days of therapy. This test is intended as an aid in the diagnosis of pulmonary tuberculosis when used in conjunction with clinical and other laboratory findings.

The Xpert MTB/RIF Assay does not provide confirmation of rifampin susceptibility since mechanisms of rifampin resistance other than those detected by this device may exist that may be associated with a lack of clinical response to treatment.

Specimens that have both MTB-complex DNA and rifampin-resistance associated mutations of the rpoB gene detected by the Xpert MTB/RIF Assay must have results confirmed by a reference laboratory. If the presence of rifampin-resistance associated mutations of the rpoB gene is confirmed, specimens should also be tested for the presence of genetic mutations associated with resistance to other drugs.

The Xpert MTB/RIF Assay must be used in conjunction with mycobacterial culture to address the risk of false negative results and to recover the organisms for further characterization and drug susceptibility testing.

The Xpert MTB/RIF Assay should only be performed in laboratories that follow safety practices in accordance with the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories publication and applicable state or local regulations.

- 2. Indication(s) for use: Same as Intended Use
- 3. Special conditions for use statement(s):

The Xpert® MTB/RIF Assay is for prescription use only in accordance with 21 CFR 801.109.

- 4. Special instrument requirements:
The Xpert® MTB/RIF Assay is for use with GeneXpert® Instrument Systems, including the GeneXpert® Diagnostic (Dx) Systems and the GeneXpert® Infinity Systems.

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### I. Device Description:

The Xpert® MTB/RIF Assay is an automated in vitro diagnostic test for the qualitative detection of MTB-complex DNA and the genetic mutations associated with rifampin (Rif) resistance in raw sputum samples or concentrated sputum sediments from patients for whom there is clinical suspicion of TB and who have received no antituberculosis therapy, or less than 3 days of therapy. The primers in this test amplify a portion of the rpoB gene containing the 81 base pair core region. The probes are designed to differentiate between the conserved wild-type sequence and mutations in the core region that are associated with Rif resistance. The assay is performed on Cepheid GeneXpert® Instrument Systems.

The Xpert® MTB/RIF Assay includes single-use disposable cartridges and sample reagent for sample preparation. The Xpert® MTB/RIF Assay cartridges contain reagents for the detection of MTB-complex DNA and Rif resistance associated mutations. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included in the cartridge. The SPC is present to control for adequate processing of the target microorganism and to monitor the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability.

Sputum specimens are collected according to the institution's standard procedures and transported to the GeneXpert® Instrument System area. For raw sputum, Sample Reagent is added to the sample (2:1, v:v). Sample Reagent is added to the resuspended sputum sediment (1.5 mL Sample Reagent to 0.5 mL suspension or 3:1, v:v, for larger volumes of sediment suspension). For both specimen types, the solution is shaken vigorously to mix, and then incubated at 20-30℃ for 15 minutes. Using the transfer pipette provided, the specimen is transferred to the open port of the Xpert® MTB/RIF Assay cartridge.

The user initiates a test from the system user interface, the Xpert® MTB/RIF Assay cartridge is loaded onto the GeneXpert® Instrument System platform, which performs hands-off, automated sample processing, and real-time PCR for detection of DNA, Summary and detailed test results are obtained in approximately 2 hours and are displayed in tabular and graphic formats.

The Xpert® MTB/RIF Assay simultaneously detects MTB-complex and the genetic mutations associated with rifampin resistance by amplifying a MTB-complex specific sequence of the rpoB gene, which is probed with five molecular beacons (Probes A - E) for mutations within the rifampin-resistance determining region (RRDR). Each molecular beacon is labeled with a different fluorophore.

The valid maximum cycle threshold (Ct) of 39.0 for Probes A, B and C and 36.0 for Probes D and E are set for data analysis.

- "MTB DETECTED", is reported when at least two probes result in Ct values within the valid range and a delta Ct min (the smallest Ct difference between any pair of

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probes) of less than 2.0.

- "Rif Resistance NOT DETECTED" is reported if the delta Ct max (the Ct difference ● between the earliest and latest probe) is ≤4.0.
- "Rif Resistance DETECTED" is reported if the delta Ct max is >4.0. ●
- "Rif Resistance INDETERMINATE" is reported when the following two conditions . are met:
- 1. the Ct value of any probe exceeds the valid maximum Ct (or is zero, i.e. no threshold crossing); and
- 2. the earliest rpoB Ct value is greater than [(Valid maximum Ct of probe in condition1) - (delta Ct max cut-off of 4.0)]
- · "MTB NOT DETECTED" is reported when there is only one or no positive probe.

All assay settings are included as automatic calculations in the Xpert® MTB/RIF Assay protocol and cannot be modified by the user.

# J. Standards/Guidance Documents Referenced:

- 1. CLSI EP5-A2, Evaluation of Precision Performance of Quantitative Measurement
- 2. Methods; Approved Guideline -Second Edition. CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA, 2004
- 3. EN 13640. Stability Testing of in vitro Diagnostic Reagents. June 2002. ASTM D4169-05, Standard Practice for Performance Testing of Shipping Containers and Systems
- 4. CLSI MM3-A2, Molecular Diagnostic Methods for Infectious Disease; Approved Guideline- Second Edition. CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA, 2006
- 5. EMC (Electromagnetic Compatibility) Directive, 2004/108/EC LVD (Low Voltage Directive) 2006/95/EC
- 6. IEC 61010-1:2001 2nd Edition "Safety Requirements for electrical equipment for measurement, control, and laboratory use-Part 1: General Requirements"
- 7. EN 61010-1:2001 2nd Edition "Safety Requirements for electrical equipment for measurement, control, and laboratory use-Part 1: General Requirements"
- 8. UL 61010-1:2004 2nd Edition "Safety Requirements for electrical equipment for measurement, control, and laboratory use-Part 1: General Requirements"
- 9. EN 61010-2-101:2002 "Safety Requirements for electrical equipment for measurement, control, and laboratory use-Part 2-101: Particular Requirements for in vitro diagnostic (IVD) medical equipment"
- 10. CAN-CSA 22.2 No. 61010-1: 2004 2nd Edition "Safety Requirements for electrical equipment for measurement, control, and laboratory use-Part 1: General Requirements"
- 11. CAN-CSA 22.2 No. 61010-2-101: 2004 "Safety Requirements for electrical equipment for measurement, control, and laboratory use-Part 2-101: Particular Requirements for in vitro diagnostic (IVD) medical equipment"
- 12. WEEE Directive 2002/96/EC

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- 13. EN 55011:2007 +A1:2007 "Industrial, scientific and medical (ISM) radio-frequency equipment- Electromagnetic disturbance characteristics- Limits and methods of measurements"
- 14. EN 61326-1:2006 "Electrical Equipment for measurement, control and laboratory use-EMC Requirements"
- 15. EN 61326-2-6:2006 "Electrical Equipment for measurement, control and laboratory use- EMC requirements-Part 2-6: Particular Requirement for in vitro diagnostic (IVD) medical equipment"
- 16. FCC Part 15 Rules and Regulations for Information Technology Equipment
- 17. FCC Part 18 Rules and Regulations for Information Technology Equipment
- 18. CISPR 11:2004 "Industrial, scientific and medical equipment- Radio-frequency disturbance characteristics - Limits and methods of measurement" (Class A Radiated Emission Requirements)
- 19. CISPR 22:2006 "Information technology equipment -Radio disturbance characteristics- Limits and methods of measurement" (Class A Radiated Emission Requirements)

### K. Test Principle:

The primers and probes in the Xpert® MTB/RIF Assay detect the presence of a unique gene sequence in MTB-complex DNA by using fluorogenic target-specific hybridization for detection of the amplified DNA. Sputum specimens are collected from patients with clinical suspicion of tuberculosis. The specimen is mixed with Sample Reagent, shaken 10 to 20 times or vortexed for at least 10 seconds, and incubated for 15 minutes at 20-30℃. At 5 to 10 minutes into the incubation period, the specimen is shaken or vortexed again, and incubated for the remainder of the 15 minute incubation period prior to transferring it to the assay cartridge for testing. The GeneXpert® performs sample preparation by mixing the prepared sample with the lyophilized Sample Processing Control. The cells are filtered and washed with buffer to remove inhibitors and contaminants. and lysed using glass beads and an ultrasonic horn, eluting the released DNA. The DNA is mixed with dry real-time polymerase chain reaction (PCR) reagents and transferred into the integrated PCR tube for real-time PCR amplification and detection of chromosomal DNA gene sequences for MTBcomplex.

#### L. Performance Characteristics (if/when applicable):

- 1. Analytical performance:
- a. Precision/Reproducibility:

# External Reproducibility (Study 154A)

The reproducibility of the Xpert® MTB/RIF Assay was evaluated at three testing sites. The test panel included five contrived panel samples, each prepared using pooled human sputa preserved with cetyl pyridinium chloride (CPC) and spiked with a cultured strain of either rifampin-resistant or rifampin-susceptible Mycobacterium tuberculosis. Strains

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were spiked at a "low positive" concentration (approximately 1X Limit of Detection -LoD) and a "moderate positive" (approximately 2 to 3X LoD). A negative sample, prepared using only CPC preserved human sputa, was also included in the test panel (see panel listing below).

- 1. Low Positive (~1 X LoD)
- 2. MTB/Rif-R Mod. Positive (~2-3 X LoD)
- 3. MTB/Rif-S Low Positive (~1 X LoD)
- 4. MTB/Rif-S Mod Positive (~2-3 X LoD)
- 5. Negative

Each sample was identified by an alpha/numeric identification code. Operators performing the testing were blinded to the expected results.

Testing of one assay kit lot was conducted over five days, by two operators, each conducting three panel runs per day. Table 1 below provides a summary of the reproducibility study results.

| Table-1. Summary of Reproducibility Results - Percent Agreement by Study Site/Instrument | | | |
|------------------------------------------------------------------------------------------|--|--|--|
| | | | |

| Sample | Site 1<br>(Infinity-80) | Site 2<br>(GeneXpert Dx) | Site 3<br>(Infinity-48) | % Total Agreement<br>by Sample |
|------------------------------------------------|-------------------------|--------------------------|-------------------------|--------------------------------|
| MTB/Rif- R<br>~2-3X LoD<br>(Moderate Positive) | 100.0%<br>(30/30) | 100.0%<br>(30/30) | 100.0%<br>(30/30) | 100.0%<br>(90/90) |
| MTB/Rif- R<br>~1X LoD<br>(Low Positive) | 93.3%<br>(28/30) | 96.7%<br>(29/30) | 96.7%<br>(29/30) | 95.6%<br>(86/90) |
| MTB/Rif- S<br>~2-3X LoD<br>(Moderate Positive) | 100.0%<br>(30/30) | 100.0%<br>(30/30) | 100.0%<br>(30/30) | 100.0%<br>(90/90) |
| MTB/Rif-S<br>~ 1X LoD<br>(Low Positive) | 96.7%<br>(29/30) | 100.0%<br>(30/30) | 100.0%<br>(30/30) | 98.9%<br>(89/90) |
| Negative | 100.0%<br>(30/30) | 100%<br>(29/29)a | 100.0%<br>(30/30) | 100%<br>(89/89) |

a One sample was non-determinate after the initial test and upon retest

- Of the MTB/Rif-R, Moderate Positive samples, 100% (90/90) were correctly . classified as "MTB DETECTED; Rif Resistance DETECTED".
- Of the MTB/Rif-R, Low Positive samples, 95.6% (86/90) were correctly . classified as "MTB DETECTED; Rif Resistance DETECTED". Four

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samples were classified as "MTB NOT DETECTED".

- Of the MTB/Rif-S. Moderate Positive samples 100% (90/90) were correctly . classified as "MTB DETECTED; Rif Resistance NOT DETECTED".
- Of the MTB/Rif-S. Low Positive samples, 98.9% (89/90) were correctly classified as ● "MTB DETECTED; Rif Resistance NOT DETECTED". One sample was classified as "MTB NOT DETECTED".
- Of the negative samples, 100% (89/89) were correctly classified as "MTB ● NOT DETECTED'. One sample was non-determinate after the initial test and upon retest.

# Comparative Precision Evaluation of the Xpert MTB/RIF Assay on the GeneXpert Dx and Infinity-80 System:

An in-house instrument precision study was conducted to compare the performance of the GeneXpert® Dx System and the Infinity-80 Instrument System. The test panel included five contrived panel samples, each prepared using pooled human sputa preserved with cetyl pyridinium chloride (CPC) and spiked with a cultured isolate of either a rifampinresistant strain of Mycobacterium tuberculosis or a rifampin susceptible strain of Mycobacterium bovis BCG. Isolates were spiked at a "low positive" concentration (approximately 1X Limit of Detection - LoD) and a "moderate positive" (approximately 2 to 3X LoD). A negative sample, prepared using only CPC preserved human sputa only, was also included in the test panel (see panel listing below).

- Low Positive (~1 X LoD) 1. MTB/Rif-R
- 2. MTB/Rif-R Mod. Positive (~2-3 X LoD)
- Low Positive (~1 X LoD) 3. MTB/Rif-S
- 4. MTB/Rif-S Mod Positive (~2-3 X LoD)
- 5. Negative

Samples were blinded with a Sample ID number which was used to define the order in which the samples were tested. Samples were tested in a different order throughout the study. Testing of one assay kit lot was conducted over 12 days, by two operators, each conducting four panel runs per day, on each instrument. Table 2 below provides a summary of the instrument precision study results.

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| Sample | GeneXpert Dx | Infinity-80 | % Total Agreement<br>by Sample |
|-----------------------------------------------|-------------------|-------------------|--------------------------------|
| MTB/Rif-R<br>~2-3X LoD<br>(Moderate Positive) | 100%<br>(94/94) | 99.0%<br>(95/96) | 99.5%<br>(189/190) |
| MTB/Rif-R<br>~1X LoD<br>(Low Positive) | 97.9%<br>(94/96) | 99.0%<br>(95/96) | 98.4%<br>(189/192) |
| MTB/Rif-S<br>~2-3X LoD<br>(Moderate Positive) | 100.0%<br>(96/96) | 100.0%<br>(96/96) | 100.0%<br>(192/192) |
| MTB/Rif-S<br>~1X LoD<br>(Low Positive) | 91.6%<br>(87/95) | 86.5%<br>(83/96) | 89.0%<br>(170/191) |
| Negative | 99.0%<br>(95/96) | 97.9%<br>(94/96) | 98.4%<br>(189/192) |

Table- 2 : Summary of Instrument Precision Results - Percent Agreement

- Of the MTB/Rif-R, Moderate Positive samples, 99.5% (189/190) were ● correctly classified as "MTB DETECTED; Rif Resistance DETECTED". Two samples tested were non-determinate after the initial test and upon repeat. One sample tested was classified "MTB NOT DETECTED".
- Of the MTB/Rif-R, Low Positive samples, 98.4% (189/192) were correctly classified as "MTB DETECTED; Rif Resistance DETECTED". Two samples were classified as "MTB NOT DETECTED". One sample was classified as "MTB DETECTED; Rif Resistance INDETERMINATE".
- Of the MTB/Rif-S, Moderate Positive samples 100% (192/192) were . correctly classified as "MTB DETECTED; Rif Resistance NOT DETECTED".
- Of the MTB/Rif-S, Low Positive samples, 89.0% (170/191) were correctly classified as "MTB DETECTED; Rif Resistance NOT DETECTED". Seventeen samples (11- Infinity 80; 6 GeneXpert DX) were classified as "MTB NOT DETECTED". Four samples (2-Infinity 80; 2-GeneXpert DX) were classified as "MTB DETECTED, Rif Resistance INDETERMINATE". One sample tested was non-determinate after the initial test and upon repeat.
- Of the negative samples, 98.4% (189/192) were correctly classified as "MTB NOT DETECTED". Three samples were classified as MTB DETECTED; Rif Resistance NOT DETECTED".

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#### b. Linearity/ assay reportable range:

Not applicable, the Xpert MTB/RIF Assay is a qualitative assay.

#### c. Traceability, Stability, Expected values (controls, calibrators, or methods)

#### Internal Controls

Sample Processing Control (SPC): The SPC verifies that the specimen processing is adequate for cartridge tested. In addition, the SPC detects specimen-associated inhibition of the real-time PCR reactions. The SPC, which is included in each cartridge, contains non-infectious spores in the form of a dry spore cake. The SPC PASSES if a valid cycle threshold (Ct) is generated in a negative sample. A SPC result is not required in a positive sample because MTB amplification can compete with this control. If the SPC fails, the test should be repeated.

Probe Check Control (PCC): Before the start of the first and second nested real-time PCR reactions, the GeneXpert Instrument System measures the fluorescent signal from the probes to monitor bead rehydration, reaction-tube filling, probe integrity, and dye stability. These readings are then compared to default settings established by Cepheid. The PCC PASSES if the fluorescence generated meets the validated acceptance criteria. If the PCC fails, the test should be repeated.

#### External Controls:

A study was conducted to evaluate commercially available ready-to-use control materials for the Xpert® MTB/RIF Assay. The control material was evaluated using the GeneXpert® DX instrument in combination with the Xpert® MTB/RIF Assay. INTROL TB Controls are custom made by Main Molecular Quality Controls Inc. (MMQCI; Scarborough Maine). The controls are non-infectious and can be shipped and stored at 2-8°C. The INTROL TB controls were tested exactly as a raw sputum sample. The INTROL TB control panel included a wild-type control. (2) mutant controls and a negative control. Replicates of 20 were run per control type. Table 3 provides a detailed description of the external controls evaluated in the study.

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| External Control ID | rpoB<br>Genotype | Probes Affected by<br>rpoB Gene Mutations<br>of Control | Concentration<br>(CFU/mL) | Expected Result |
|---------------------|-----------------------------------------------------|---------------------------------------------------------|---------------------------|---------------------------------------------------------|
| INTROL TBWT-04 | rpoB: wild type<br>(H37Rv) | None | $2X10^4$ | "MTB Detected,<br>Rifampin Resistance<br>Not Detected " |
| INTROL TBMDR1-04 | rpoB: Mutants<br>(F505L,<br>L511P, D516V,<br>H526Y) | A, B, and D | $2X10^4$ | "MTB Detected,<br>Rifampin Resistance<br>Detected" |
| INTROL TBMDR2-04 | rpoB: Mutants<br>(S522L,<br>H526D, S531L) | C, D, and E | $2X10^4$ | "MTB Detected,<br>Rifampin Resistance<br>Detected" |
| INTROL TBNEG-04 | rpoB: NEG | None | No Cells | "MTB Not Detected" |

# Table 3. External Controls

The results of the study are described in the following summary tables – Table 4 through Table 7.

# Table 4: INTROL TBNEG-04

| Analyte | Ct Range | Mean Ct Value | Result |
|--------------------------------------|-----------|---------------|-----------------------------------------------------------------------------------------|
| Specimen Processing<br>Control (SPC) | 23.1-27.9 | 24.6 | All replicates tested were<br>correctly reported as<br>expected - “MTB Not<br>Detected” |

# Table 5: INTROL TBWT-04

| Analyte | Ct Range | Mean Ct Value | Result |
|--------------------------------------|-----------|---------------|--------------------------------------------------------------------------------------------------------------------------|
| Specimen Processing<br>Control (SPC) | 23.1-27.9 | 24.6 | All replicates tested were<br>correctly reported as<br>expected - “MTB<br>Detected; Rifampin<br>Resistance Not Detected” |
| Probe E | 21.2-24.7 | 22.5 | |
| Probe D | 21.2-25.4 | 22.8 | |
| Probe C | 19.9-23.8 | 21.0 | |
| Probe B | 21.2-25.3 | 23.0 | |
| Probe A | 19.4-23.3 | 20.9 | |

#### Table 6: INTROL TBMDR1-04

| Analyte | Ct Range | Mean Ct Value | Result |
|--------------------------------------|-----------|---------------|---------------------------------------------------------------------------------------------------|
| Specimen Processing<br>Control (SPC) | 22.3-28.9 | 24.1 | All replicates tested were<br>correctly reported as<br>expected - "MTB |
| Probe E | 20.8-23.5 | 22.1 | Detected; Rifampin<br>Resistance Detected" |
| Probe D | 0-0 | 0 | |
| Probe C | 19.8-22.9 | 21.5 | No Ct values were<br>obtained for Probes A, B,<br>and D as expected using<br>this mutant control. |
| Probe B | 0-0 | 0 | |
| Probe A | 0-0 | 0 | |

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| Analyte | Ct Range | Mean Ct Value | Result |
|--------------------------------------|-----------|---------------|---------------------------------------------------------------------------------------------------|
| Specimen Processing<br>Control (SPC) | 21.6-34.7 | 24.2 | All replicates tested were<br>correctly reported as<br>expected - “MTB |
| Probe E | 0-0 | 0 | Detected; Rifampin<br>Resistance Detected” |
| Probe D | 0-0 | 0 | |
| Probe C | 0-0 | 0 | |
| Probe B | 19.4-28.7 | 22.9 | |
| Probe A | 18.5-27.3 | 21.5 | No Ct values were<br>obtained for Probes C, D,<br>and E as expected using<br>this mutant control. |

### Table 7: INTROL TBMDR2-04

External controls may be used in accordance with accrediting institutions and government regulations. External controls are not provided in the assay kit; however they are available for purchase from outside sources. The outside source and the catalog numbers are provided in the Materials Available but Not Provided section of the Xpert® MTB/RIF Assay Package Insert.

During the clinical trial, three external controls (MTB NEG, MTB Positive Rifsusceptible, and MTB Positive Rif-resistant) were run each day that study specimens were tested. Study specimens were not run until correct results were obtained for both the negative and positive controls. Of the 467 external control samples that were run, 97% (453/467) gave expected results on the first attempt. Of the 14 external controls that failed to give expected results on the first attempt (i.e., a nondeterminate result of INVALID, ERROR, or NO RESULT), eight gave expected results when retested. Six external control specimens which failed to give expected results on the first attempt were not repeated; no study specimens were tested on these occasions.

- d. Limit of Detection:
Studies were performed to determine the limit of detection (LoD) of isolates of Mycobacterium tuberculosis and Mycobacterium bovis BCG (Bacille Calmette-Guerin) diluted in human sputum and human sputum sediment. The LoD is the lowest concentration reported in CFU/mL that can be reproducibly distinguished from negative samples with 95% confidence. Replicates of 20 were evaluated at five to eight concentrations and LoD was determined using probit analysis with the exception of testing performed with M. tuberculosis mutant rifampin-resistant strain TDR125 cells in sputum sediment which was performed at one concentration only, in replicates of 40. See Table 8 below.

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

| Microorganism | Specimen Type | LoD Estimate | Claimed LoD<br>(CFU/mL) |
|-----------------------------|---------------|--------------|-------------------------|
| M. bovis BCG | Sputum | 486 | 525 |
| | Sediment | 703 | 700 |
| M. tuberculosis<br>(H37Rv) | Sputum | 414 | 600 |
| | Sediment | 2046 | 3000 |
| M. tuberculosis<br>(TDR125) | Sputum | 872 | 1000 |
| | Sediment | NDa | 4000 |

Table 8: Probit Analysis Data and Claimed LoD in CFU/mL

4Not determined (ND) by probit analysis.

#### Rifampin Resistance Study e.

Due to the low prevalence of rifampin resistance, an additional study was conducted to evaluate the performance of the Xpert MTB/RIF Assay in detecting genetic mutations associated with rifampin resistance in well characterized rifampinsusceptible and rifampin-resistant clinical isolates spiked into pooled sputum confirmed negative for MTB-complex and mycobacteria other than MTB-complex. Fifty aliquots of pooled negative sputa were randomly intermixed for testing among the positives. Results of Xpert MTB/RIF Assay were compared to prior MTB identification, phenotypic drug susceptibility testing (agar proportion method), and bi-directional sequencing results. The results of the study are shown in the Table 9 below.

| | | DST | | |
|---------------------------|--------------------------------------------|------------------------------------------------------|------|-------|
| | | Res | Susc | Total |
| Xpert<br>MTB/RIF<br>Assay | MTB DETECTED<br>Rif Resistance<br>DETECTED | 85 | 9 | 94 |
| | MTB DETECTED<br>Rif Resistance<br>DETECTED | 2 | 89 | 91 |
| | Total | 87 | 98 | 185 |
| | Sensitivity:<br>Specificity: | 97.7% (95% CI 92.0-99.4)<br>90.8% (95% CI 83.5-95.1) | | |

Table 9: Xpert MTB/RIF Performance vs. DST

Of the 11 samples with rifampin discordant results, bi-directional sequencing results were concordant with the Xpert MTB/RIF Assay for 10 of 11 samples and discordant with one sample. See Table 10 below.

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

| DST | Xpert MTB/RIF | n | Bi-directional<br>sequencing |
|-----------------|-----------------------------|---|-----------------------------------------------------------------|
| Rif-resistant | Rif Resistance NOT DETECTED | 2 | 2 of 2 Wild Type |
| Rif-susceptible | Rif Resistance DETECTED | 9 | 8 of 9 Rif resistance mutations<br>present.<br>1 of 9 Wild Type |

# Table 10: Discrepant Testing Results

#### Analytical Reactivity (Inclusivity): f.

A total of 62 well characterized strains of Mycobacterium tuberculosis representing geographic and phenotypic diversity were evaluated with the Xpert MTB/RIF Assay. Whole organism was used for testing the majority of the strains. For 15 strains, due to lack of availability or viability of whole organism, DNA was used for testing (see footnote "a" in Table 11). All strains were tested in triplicate near the analytical limit of detection.

Table 11 below includes 26 rifampin susceptible and 36 rifampin resistant strains, as determined by phenotypic drug susceptibility testing (DST).

Xpert MTB/RIF Assay results of "MTB DETECTED; Rif Resistance NOT DETECTED" compared to DST was 87% (67/77) accurate in replicate tests using 26 strains sensitive to rifampin. One replicate in 78 tests was reported as "INVALID" and not repeated. Three of the DST susceptible strains (see footnote "c" in Table 11) that tested as "MTB DETECTED; Rif Resistance DETECTED" by the Xpert MTB/RIF Assay were found to have rifampin resistance mutations according to DNA sequence analysis. One of three replicates of the wild type strain TDR 33 was reported "MTB DETECTED; Rif Resistance INDETERMINATE".

Xpert MTB/RIF Assay results of "MTB DETECTED; Rif Resistance DETECTED" compared to DST was 100% (107/107) accurate in replicate tests using 36 strains resistant to rifampin. One replicate in 108 tests was reported as "INVALID" and not repeated. Results by strain are shown in Table 11.

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

| Strain ID | Origin | Rifampin<br>Susceptibility<br>by DSTd | Rifampin<br>Susceptibility<br>by Xperte |
|------------|------------|---------------------------------------|-----------------------------------------|
| TDR 116 | S. Korea | R | R |
| TDR 21 | RD Congo | R | R |
| TDR 28ª | Bangladesh | R | R |
| TDR 191 ª | Peru | R | R |
| TDR 125 | Brazil | R | ાર |
| TDR 34 | Bangladesh | R | ાર |
| TDR 73 | Peru | R | ાર |
| TDR 35 | Bangladesh | R | R |
| TDR 190 ª | Spain | R | R |
| TDR 117 | S. Korea | R | R |
| TDR 129 | Brazil | R | R |
| TDR 186 ª | Morocco | R | ાર |
| TDR 59 ª | Burundi | R | R |
| TDR 185 ª | Nigeria | R | R |
| TDR 6 | Bangladesh | R | R |
| TDR 19 | Azerbaijan | R | R |
| TDR 148 | Nepal | R | R |
| TDR 13 ª | Bangladesh | R | R |
| TDR 12 | Bangladesh | R | ાર |
| H37Rvb | Lab strain | S | S |
| TDR 22 | RD Congo | S | S |
| TDR 29 | Azerbaijan | S | S |
| TDR 33 | Belgium | S | S |
| CDC 1551 ª | USA | S | S |
| TDR 146 ª | Nepal | S | S |
| TDR 78 ª | S. Korea | S | S |
| TDR 54 ª | Bangladesh | S | S |
| TDR 215 ª | Peru | S | S |
| TDR 158 ª | Peru | S | S |
| TDR 178 ª | Guinea | S | S |
| TDR 64 ª | S. Africa | S | S |
| 97-05193 | Peru | R | R |
| 97-05201 | Peru | R | ાર |
| 97-06877 | Peru | R | ાર |
| 97-08341 | Peru | R | R |
| 97-12004 | Peru | R | R |
| 97-17582 | Peru | R | R |
| 97-18875 | Peru | R | R |
| 97-20784 | Peru | R | R |
| 97-20985 | Peru | R | R |
| Strain ID | Origin | Rifampin<br>Susceptibility<br>by DSTd | Rifampin<br>Susceptibility<br>by Xperte |
| 99-09120 | Peru | R | R |
| 99-R396 | Peru | R | R |
| 01-R0612 | Beijing | R | R |
| 02-R1141 | Beijing | R | R |
| 02-R1794 | Beijing | R | R |
| 02-R1840 | Beijing | R | R |
| 03-R1517 | Beijing | R | R |
| TDR 0116 | S. Korea | R | R |
| 01-R1403c | Peru | S | R |
| 97-15246c | Peru | S | R |
| 98-R839c | Peru | S | R |
| 99-R460 | Peru | S | S |
| 99-R485 | Peru | S | S |
| 00-06461 | US | S | S |
| 00-R0222 | Peru | S | S |
| 00-R0454 | US | S | S |
| 00-R0460 | Peru | S | S |
| 01-10979 | US | S | S |
| 01-R1118 | Peru | S | S |
| 02-02880 | US | S | S |
| 02-03222 | Peru | S | S |
| 02-R0040 | Peru | S | S |

Table 11: Analytical Reactivity (Inclusivity) of the Xpert MTB/RIF Assay

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

4DNA used for testing; quantified cultured strains was not available..

bATCC Strain H37Rv tested as both whole organism and DNA.

·Isolate rifampin susceptible by DST, but bi-directional sequencing and

Xpert MTB/RIF Assay revealed the presence of rifampin resistance mutations.
ªR=Rif resistant; S=Rif susceptible.

°R=Rif resistance mutations detected, S=Rif resistance mutations not detected.

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

An additional five Mycobacterium tuberculosis complex strains, i.e., M. africanum M. bovis, M. canettii, M. caprae, and M. microti, were not wet tested but were evaluated in silico to assess the analytical reactivity. The results of the in silico analyses predict a very high likelihood of amplification and detection using the Xpert MTB/RIF Assay. See Table 13.

| Table 13: Alignments of Primers and Probes to the rpoB sequences of M. africanum, M. |
|--------------------------------------------------------------------------------------|
| bovis, M. canettii, M. caprae and M. microti |

| MTB complex organism | Sequence Alignment (number of identical residues) of Xpert MTB/RIF Assay Primer/Probe to MTB-complex Strains | | | | | | | |
|----------------------------------------|--------------------------------------------------------------------------------------------------------------|-----------|----------|---------------|---------------|---------------|---------------|---------------|
| | RpoB For1 | RpoB For2 | RpoB Rev | RpoB Probe Ab | RpoB Probe Bb | RpoB Probe Cb | RpoB Probe Db | RpoB Probe Eb |
| Mycobacterium africanum (taxid:33894)a | 24/24 | 24/24 | 23/24 | 17/17 | 24/24 | 17/17 | 18/18 | 18/18 |
| Mycobacterium bovis (taxid:1765)a | 24/24 | 24/24 | 23/24 | 17/17 | 24/24 | 17/17 | 18/18 | 18/18 |
| Mycobacterium canetti (taxid:78331)a | 24/24 | 24/24 | 23/24 | 17/17 | 24/24 | 17/17 | 18/18 | 18/18 |
| Mycobacterium caprae (taxid:115862)a | 24/24 | 24/24 | 23/24 | 17/17 | 24/24 | 17/17 | 18/18 | 18/18 |
| Mycobacterium microti (taxid:1806)a | 24/24 | 24/24 | 23/24 | 17/17 | 24/24 | 17/17 | 18/18 | 18/18 |

4 taxid - unique identifier for an organism in the NCBI taxonomy database. b Sequence alignment of probes done with no stem sequences.

#### Analytical Exclusivity (Cross Reactivity): g.

The Xpert MTB/RIF Assay was evaluated by testing a panel of 132 microorganisms (24 non-tuberculosis mycobacteria, 87 bacteria, 7 fungi and 14 viruses) representing common respiratory pathogens potentially encountered in the oral/respiratory tract. See Table 14. Nucleic acid (DNA or RNA) was used in place of whole organisms for 9 bacteria, 1 fungus, and 6 viral strains due to lack of availability or due to biosafety restrictions. All microorganisms were tested in triplicate, at a concentration of at least:

- 10° CFU/mL (or DNA at 1x107 copies/mL) for bacteria and fungi; ●
- 103 TCID50/mL (or nucleic acid at 2x10 copies/mL) for viruses; ●
- 106 elementary bodies (EB) per mL for Chlamydia; and ●
- 106 CFU/mL for two nontuberculous mycobacteria (M. genavense and M. ● smegmatis)

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

There was no cross-reactivity observed with 131 of the 132 microorganisms tested. Cross-reactivity was observed in one of three replicates of M. scrofulaceum at a concentration of 10° CFU/mL, however, no cross-reactivity was observed when tested at a concentration of 107 CFU/mL.

| Acinetobacter baumannii | Human influenza virus Bc | Neisseria lactamica |
|----------------------------------------------|--------------------------------------------------------|------------------------------------------------------------|
| Acinetobacter calcoaceticus | Human Metapneumovirus | Neisseria meningitidis |
| Actinomyces israeliia | Human parainfluenzae Type 1 | Neisseria mucosa |
| Actinomyces odontolyticus | Human parainfluenzae Type 2 | Neisseria sicca |
| Adenovirus | Human parainfluenzae Type 3 | Nocardia asteroidesa |
| Aspergillus fumigatusb | Human respiratory syncytial virus<br>Ac | Nocardia cyriacigeorgicaa |
| Bacillus cereus | Human respiratory syncytial virusBc | Pasteurella multocida subsp.<br>tigris |
| Bacillus subtilis subsp. subtilis | Kingella kingae | Pediococcus pentosaceusa |
| Bacteroides fragilis | Klebsiella oxytoca | Peptostreptococcus anaerobius |
| Bordetella parapertussisa | Klebsiella pneumoniae producing<br>KPC-3 carbapenemase | Porphyromonas<br>asaccharolytica |
| Bordetella pertussis | Klebsiella pneumoniae subsp.<br>pneumoniae | Prevotella melaninogenica |
| Burkholderia cepacia | Lactobacillus acidophilus | Propionibacterium acnes |
| Campylobacter jejuni subsp. jejunia | Lactobacillus casei | Proteus mirabilis |
| Candida albicans | Legionella pneumophila subsp.<br>pneumophila | Proteus vulgaris |
| Candida glabrata | Leuconostoc mesenteroides subsp.<br>mesenteroides | Providencia stuartii |
| Candida krusei | Listeria monocytogenes | Pseudomonas aeruginosa |
| Candida parapsilosis | Moraxella catarrhalis | Rhinovirus Strain 1A |
| Candida tropicalis | Morganella morganii subsp.<br>morganii | Rhodococcus equi |
| Chlamydia trachomatis | Mycobacterium abscessus | Salmonella enterica subsp.<br>enterica serovar Dublin |
| Chlamydophila pneumoniaea | Mycobacterium asiaticum | Salmonella enterica subsp.<br>enterica serovar typhimurium |
| Citrobacter freundii | Mycobacterium avium subsp. avium | Serratia marcescens subsp.<br>marcescens |
| Clostridium perfringens | Mycobacterium celatum | Shigella flexneri |
| Corynebacterium diphtheriaea | Mycobacterium chelonae | Shigella sonnei |
| Corynebacterium jeikeium | Mycobacterium flavescens | Staphylococcus aureus subsp.<br>aureus |
| Corynebacterium<br>pseudodiphtheriticum | Mycobacterium fortuitum subsp.<br>fortuitum | Staphylococcus capitis subsp.<br>capitis |
| Corynebacterium xerosis | Mycobacterium gastri | Staphylococcus epidermidis |
| Cryptococcus neoformans | Mycobacterium genavense | Staphylococcus haemolyticus |
| | | |
| Cytomegalovirus | Mycobacterium gordonae | Staphylococcus hominis subsp.<br>hominis |
| Eikenella corrodens | Mycobacterium haemophilum | Staphylococcus lugdunensis |
| Enterobacter aerogenes | Mycobacterium intracellulare | Stenotrophomonas maltophilia |
| Enterobacter cloacae subsp.<br>cloacae | Mycobacterium kansasii | Streptococcus agalactiae |
| Enterococcus avium | Mycobacterium malmoense | Streptococcus constellatus<br>subsp. constellatus |
| Enterococcus faecalis | Mycobacterium marinum | Streptococcus equi subsp. equi |
| Enterococcus faecium | Mycobacterium scrofulaceum | Streptococcus mitis |
| Enterovirus Type 71/NY | Mycobacterium simiae | Streptococcus mutans |
| Escherichia coli | Mycobacterium smegmatis | Streptococcus parasanguinis |
| Escherichia coli producing CTX-<br>M-15 ESBL | Mycobacterium szulgai | Streptococcus pneumoniae |
| Fusobacterium nucleatum subsp.<br>nucleatum | Mycobacterium terrae | Streptococcus pyogenes |
| Haemophilus influenzae | Mycobacterium thermoresistibile | Streptococcus salivarius subsp.<br>salivarius |
| Haemophilus parahaemolyticus | Mycobacterium triviale | Streptococcus sanguinis |
| Haemophilus parainfluenzae | Mycobacterium vaccae | Streptococcus uberis |
| Herpes simplex virus Type 1° | Mycobacterium xenopi | Veillonella parvula |
| Herpes simplex virus Type 2° | Mycoplasma pneumoniaeᵃ | Weissella paramesenteroides |
| Human influenza virus A° | Neisseria gonorrhoeae | Yersinia enterocolitica subsp.<br>enterocolitica |

Table 14: Microorganisms Tested for Analytical Specificity

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

4 Genomic DNA used; concentrations tested ranged from 1x107 to 1x101º copies/mL.

b Genomic DNA used; concentration tested at 3.2 x 10° copies/mL.

& Genomic DNA or RNA used; concentrations tested ranged from 3.1 x 10 to 1.2 x 10" copies/mL.

Potential cross-reactivity of 18 microorganisms that could not be wet tested using whole organisms or nucleic acid was evaluated by in silico analysis. Thirteen of the 18 microorganisms tested revealed no potential for cross-reactivity. See Table 15. Five isolates demonstrated a slight potential for cross reactivity which may result in false positive results with the Xpert MTB/RIF Assay. See Table 16.

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

| Bacteria | Fungi | Virus |
|--------------------------|-----------------------------------------------------------|---------------------------|
| Kingella oralis | Blastomyces dermatitidis<br>(Ajellomyces<br>dermatitidis) | Rubella virus |
| Legionella micdadei | Penicillium spp. | Rubeola virus |
| Nocardia brasiliensis | Rhizopus spp. | Rubulavirus |
| Streptomyces<br>anulatus | Scedosporium spp. | Varicella Zoster<br>Virus |
| | Histoplasma capsulatum | |

Table 15: Microorganisms Predicted to be Non-cross Reactive by in silico Analysis

#### Table 16. Microorganisms Predicted to be Potentially Cross Reactive by in silico Analysis

Image /page/18/Figure/3 description: The image shows a table with five rows. Each row contains the name of a different type of bacteria. The bacteria listed are Mycobacterium kumamontonense, Mycobacterium leprae, Mycobacterium mucogenicum, Tsukamurella spp., and Nocardia otitidiscaviarum.

#### h. Interfering Substances:

Performance of the Xpert MTB/RIF Assay was evaluated in the presence of 32 potentially interfering substances. These substances are listed in Table 17 along with the active ingredients and concentrations tested. Positive and negative samples vere included in this study. Positive samples were tested near the analytical limit of detection using one inactivated rifampin-susceptible strain of M. tuberculosis (H37Rv) and one inactivated rifampin-resistant strain of M. tuberculosis TDR6 (probe E mutant). Both strains were tested in replicates of eight. Negative samples, which consisted of the interfering substance without the MTB strain, were tested in replicates of eight to determine the effect on the performance of the sample processing control (SPC). Inhibition of the Xpert MTB/RIF Assay was observed in the presence of Lidocaine at 30%; mucin at 5% and 2.5%; Ethambutol at 50 µg/mL, 25 ug/mL, and 10 ug/mL; Guaifenesin at 5 mg/mL; Phenylephrine at 100% and 50%; and tea tree oil at 0.5% to 0.015%, resulting in a false negative result "MTB NOT DETECTED" or a "Rif Resistance INDETERMINATE" result.

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

| Substance | Description/Active Ingredient | Concentration Tested |
|--------------------------------------------|--------------------------------------------------------------------|-----------------------------------------------|
| Blood (human) | | 5% (v/v) |
| Germicidal Mouthwash | Chlorhexidine gluconate (0.12%),<br>20% | 20% (v/v) |
| Specimen Processing<br>Reagents | Cetylpyridinium chloride, 1% in 2%<br>NaCl | 0.5% (v/v) in 1% NaCl |
| Specimen Processing<br>Reagents | Cetylpyridinium chloride, 1% in 2%<br>NALC | 0.5% (v/v) in 1% NALC |
| Specimen Processing<br>Reagents | Cetylpyridinium chloride, 1% in 2%<br>NALC plus 25 mM Citrate | 0.5% (v/v) in 1% NALC plus<br>12.5 mM Citrate |
| Gastric Acid | pH 3 to 4 solution in water,<br>neutralized | 100% (v/v) |
| Human DNA/Cells | HELA 229 | 106 cells/mL |
| Antimycotic; Antibiotic | Nystatin oral suspension, 20% | 20% (v/v) |
| White Blood Cells (human) | WBC/Pus matrix (30% buffy coat;<br>30% plasma; 40% PBS) | 100% (v/v) |
| Anesthetics (endotracheal<br>intubation) | Lidocaine HCl 4% | 20% to 30% (v/v) |
| Nebulizing solutions | NaCl 5% (w/v) | 5% (w/v) |
| Mucin | Mucin 5% (w/v) | 1.5% to 5% (w/v) |
| Antibacterial, systemic | Levofloxacin 25 mg/mL | 5 mg/mL |
| Nasal corticosteroids | Fluticasone<br>500 mcg/spray | 5 µg/mL |
| Inhaled bronchodilators | Albuterol Sulfate 2.5 mg/3mL | 50 µg/mL; 100 µg/mL |
| Oral anesthetics | Orajel (20% Benzocaine) | 5% (w/v) |
| Anti-viral drugs | Acyclovir, IV 50 mg/mL | 50 µg/mL |
| Antibiotic, nasal ointment | Neosporin (400U Bacitracin, 3.5 mg<br>Neomycin, 5000U Polymyxin B) | 5% (w/v) |
| Tobacco | Nicogel (40% tobacco extract) | 0.5% (w/v) |
| Anti-tuberculosis drugs | Streptomycin 1mg/mL | 25 µg/mL |
| Anti-tuberculosis drugs | Ethambutol 1 mg/mL | 5 µg/mL to 50 µg/mL |
| Anti-tuberculosis drugs | Isoniazid 1 mg/mL | 50 µg/mL |
| Oral expectorants | Guaifenesin (400mg/tablet) | 2.5 mg/mL; 5 mg/mL |
| Anti-tuberculosis drugs | Pyrazinamide 10 mg/mL | 100 µg/mL |
| Nasal gel (Homeopathic) | Zicam gel | 50% (w/v) |
| Nasal spray | Phenylephrine 0.5% | 25% to 100% (v/v) |
| Anti-tuberculosis drugs | Rifampicin 1mg/mL | 25 µg/mL |
| Allergy relief medicine<br>(Homeopathic) | Tea tree oil (<5% Cineole, >35%<br>Terpinen-4-01) | 0.008% to 0.5% (v/v) |
| Live intranasal influenza<br>virus vaccine | Live influenza virus vaccine | 5% (v/v) |
| Pneumocystis jiroveci<br>medication | Pentamidine | 300 ng/mL |
| Bronchodilator | Epinephrine (injectable formulation) | 1mg/mL |
| Neutralizing buffer | YPP plus Neutralizing Buffer | >67 mM phosphate |

Table 17. Potential Interfering Substances in Xpert MTB/RIF Assay

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

#### i. Carry-over Contamination:

A study was conducted to demonstrate whether potential carry-over and crosscontamination occurs when using the single-use, self-contained Xpert MTB/RIF Assay cartridges. In the study, a negative sample (TET buffer) was processed in the same GeneXpert module immediately following a very high positive sample containing M. bovis BCG at a concentration of approximately 1x106 CFU/mL spiked into TET buffer. This testing scheme was repeated 20 times on two GeneXpert modules for a total of 42 runs resulting in 20 positive and 22 negative specimens. All 20 positive samples were correctly reported as "MTB DETECTED; Rif Resistance NOT DETECTED" and all 22 negative samples were correctly reported as "MTB NOT DETECTED".

#### j. Assay cut-off:

The valid minimum cycle threshold (Ct) setting for all rpoB probes A through E in the MTB/RIF Assay is 3 because in practice, a Ct cannot be calculated before the end of the minimum background subtraction range, which is two cycles. A minimum Ct setting of 3 is well below the earliest Cts reported for all targets.

The valid maximum cycle threshold (Ct) setting for probes A, B, and C is 39. The valid maximum cycle threshold setting for probes D and E is 36. These settings were established to mitigate potential false rifampin resistant results due to very late probe D and E Cts. By evaluating three sets of pre-clinical data in which all maximum valid rpoB Cts were set to 39, three of six false "Rif Resistance DETECTED" results were reported as "Rif Resistance INDETERMINATE" by setting the valid maximum cycle for Probe D and Probe E to 36. To further justify these setting, analytical testing with M. bovis BCG demonstrated that in a total of 55 positive results, two false "Rif Resistance DETECTED" results were reported as "Rif Resistance INDETERMINATE" when the probe D and E maximum valid Ct settings were 36 relative to 39.

- 2. Comparison studies:
- a. Method comparison with predicate device:

Not Applicable. Refer to Clinical Studies section.

- b. Matrix comparison:
Not applicable.

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

### 3. Clinical studies:

Performance characteristics of the Xpert MTB/RIF Assay for detection of MTB-complex DNA and for detection of Rif resistance associated mutations in sputum samples relative to results from culture (solid and/or liquid) followed by phenotypic drug susceptibility testing (DST) were determined in a multi-center study using prospective and archived sputum specimens collected from both US and non-US populations. Leftover standard of care sputum specimens or concentrated sediment prepared from induced or expectorated sputa were tested by the Xpert MTB/RIF Assay from study subjects suspected of tuberculosis (TB). All AFB smears were performed on concentrated sediments.

Specimens from subjects 18 years or older were eligible for the multi-center study if they were suspected of pulmonary tuberculosis, on no TB treatment or with less than 3 days of TB treatment, had sufficient volume for testing on the Xpert MTB/RIF Assay, and had AFB smear, MTB culture and phenotypic drug susceptibility testing (DST) results. There were 1,126 subjects eligible and tested by the Xpert MTB/RIF Assay (Table 18).

| Table 18. Accountability of Eligible Study Subjects for Evaluation of Xpert MTB/RIF |
|--------------------------------------------------------------------------------------|
| Assay for the Detection of MTB-complex vs. MTB Culture |

| | | Culture | | | |
|------------------|---------------------|---------|-----|----------------|-------|
| | | + | - | Not Determined | Total |
| Xpert | MTB<br>DETECTED | 439 | 8 | 2 | 449 |
| MTB/RIF<br>Assay | MTB NOT<br>DETECTED | 29 | 620 | 15 | 664 |
| | Non-<br>Determinate | 9 | 4 | 0 | 13 |
| | Total | 477 | 632 | 17 | 1126 |

Thirteen subjects (1.2%) had Xpert MTB/RIF Assay non-determinate results (i.e., INVALID, ERROR or NO RESULT) and were excluded from the analysis. Seventeen (1.5%) subjects had MTB culture contamination. Analysis of missing values with regard to potential biases was performed (i.e., analysis of covariate distributions and logistic regression) and the 17 subjects were excluded from the analysis. One thousand ninety six (1,096) subjects were used in the analysis of the performance of the Xpert MTB/RIF Assay relative to the detection of MTB-complex. The specimens came from study subjects who were >18 years old, 62% (n=679) male, 36% female (n=396); and for 1.9% (n=21) gender was unknown. Subjects were from geographically diverse regions: 49% (n=542) were from the United States (California, New York and Florida) and 51% (n=554) were from outside the United States (Vietnam, Peru, South Africa, Mexico and Bangladesh). Of the 542 US specimens, 450 were prospectively collected and 92 were from an archived specimen bank. Of the 554 non-US specimens, 23 were prospectively collected and 531 were archived from a specimen bank.

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

## Xpert MTB/RIF Assay Performance vs. MTB Culture

One to three sputum specimens were collected from each study subject for use in the clinical study (33.9% of study subjects had 1 sputum specimen collected, 44.2% had 2 sputum specimens, and 22% had 3 sputum specimens). If more than one specimen was collected from a subject, the first sample with sufficient volume was tested by the Xpert MTB/RIF Assay. If the assay result was non-determinate (ERROR, INVALID or NO RESULT), the same specimen was retested if there was sufficient volume. Overall, 1.2% of tested samples (13/1,126; 95% CI: 0.7% to 2.0%) were non-determinate. Among 1,096 subjects with MTB culture results, an Xpert MTB/RIF Assay result was obtained with the first specimen for 85.5% of subjects, with the second specimen for 11.2% of subjects, and with the third specimen for 0.3% of subjects. AFB smear status for a subject was determined by the AFB smear result from the specimen with a corresponding Xpert MTB/RIF Assay result. The MTB culture status for a subject was defined based on the evaluation of the MTB culture results of all specimens collected for use in the study for this subject.

The performance of the Xpert MTB/RIF Assay for detection of MTB-complex relative to MTB culture, stratified by AFB smear status is shown in Table 19 and Table 20. Discordant results for MTB culture positive and Xpert MTB/RIF Assay "MTB NOT DETECTED" were further evaluated using bi-directional sequencing of the rpoB region of the MTB genome. No discordant analysis was performed on MTB culture negative specimens.

| AFB Smear-Positive Subjects | | | | |
|-------------------------------------------------------------------------------------------------------------------|---------------------|---------|----|-------|
| | | Culture | | |
| | | + | - | Total |
| Xpert<br>MTB/RIF<br>Assay | MTB<br>DETECTED | 350 | 1b | 351 |
| | MTB NOT<br>DETECTED | 1a | 65 | 66 |
| | Total | 351 | 66 | 417 |
| Sensitivity = 99.7% (350/351) with 95% CI: 98.4% - 99.9%<br>Specificity =98.5% (65/66) with 95% CI: 91.9% – 99.7% | | | | |

Table 19: Xpert MTB/RIF Assay Performance vs. MTB Culture for

4 One MTB culture positive specimen was not detected by the Xpert MTB/RIF Assay. This culture isolate was determined to be MTB by bi-directional sequencing analysis. 6The Xpert MTB/RIF Assay detected MTB in one specimen that was MTB culture negative. The culture result was based on one sputum specimen for this subject.

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| | | Culture | | |
|---------------------------------------------------------------------------------------------------------------------|---------------------|---------|-----|-------|
| | | + | - | Total |
| Xpert<br>MTB/RIF<br>Assay | MTB<br>DETECTED | 89 | 7b | 96 |
| | MTB NOT<br>DETECTED | 28a | 555 | 583 |
| | Total | 117 | 562 | 679 |
| Sensitivity = 76.1% (89/117) with 95% CI: 67.6% - 82.9%<br>Specificity = 98.8% (555/562) with 95% CI: 97.5% – 99.4% | | | | |

Table 20: Xpert MTB/RIF Assay Performance vs. MTB Culture for AFB Smear-Negative Subjects

4Twenty-eight MTB culture positive specimens were not detected by the Xpert MTB/RIF Assay. These culture isolates were determined to be MTB by bi-directional sequencing analysis.

bThe Xpert MTB/RIF Assay detected MTB in seven specimens that were MTB culture negative. The culture results were based on one sputum specimen for three subjects, two sputum specimens for two subjects, and three sputum specimens for two subjects.

Overall sensitivity depends on the percent of AFB smear positive subjects among the subjects with positive MTB culture. In the US prospective study, this percent was 75.5% and the overall sensitivity was 93.8%. The overall specificity was 98.7% (95% CI: 97.5% - 99.4%).

In clinical use, overall sensitivity will vary depending on the percentage of patients with AFB-smear positive tuberculosis in the population being tested; overall sensitivity will be lower in a population where the probability of having AFB-smear positive tuberculosis is lower, e.g., a patient population with a higher prevalence of HIV co-infection.

#### Xpert MTB/RIF Assay Performance vs. MTB Culture by Collection Method

The performance of the Xpert MTB/RIF Assay for detection of MTB-complex was determined relative to MTB culture in expectorated and induced sputum specimens. Results are shown in Table 21 and Table 22. Of the 1,096 specimens, 535 were expectorated specimens, 234 were induced specimens, and 327 were of unknown collection method.

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| | AFB Smear-Positive<br>Subjects | AFB Smear-Negative<br>Subjects |
|--------------------|------------------------------------------|----------------------------------------|
| <b>Sensitivity</b> | 99.6% (271/272)<br>95% CI: 97.9% - 99.9% | 79.0% (75/95)<br>95% CI: 69.7% - 85.9% |
| <b>Specificity</b> | 97.6% (164/168)<br>95% CI: 94.0% - 99.1% | |

Table 21: Xpert MTB/RIF Assay Performance vs. MTB Culture (Expectorated)

# Table 22: Xpert MTB/RIF Assay Performance vs. MTB Culture (Induced)

| | AFB Smear-Positive<br>Subjects | AFB Smear-Negative<br>Subjects |
|-------------|------------------------------------------|---------------------------------------|
| Sensitivity | 100% (15/15)<br>95% CI: 79.6% - 100% | 40.0% (4/10)<br>95% CI: 16.8% - 68.7% |
| Specificity | 99.0% (207/209)<br>95% CI: 96.6% - 99.7% | |

# Xpert MTB/RIF Assay Performance vs. Culture by Specimen Type

The performance of the Xpert MTB/RIF Assay for detection of MTB-complex was determined relative to MTB culture in raw sputum and concentrated sputum sediment. Results are shown in Table 23 and Table 24. Among 1,096 specimens, there were 606 raw sputum specimens and 490 concentrated sputum sediment specimens.

### Table 23: Xpert MTB/RIF Assay Performance vs. MTB Culture (Raw Sputum)

| | AFB Smear Positive<br>Subjects | AFB Smear Negative<br>Subjects |
|-------------|------------------------------------------|----------------------------------------|
| Sensitivity | 99.7% (285/286)<br>95% CI: 98.0% - 99.9% | 79.4% (77/97)<br>95% CI: 70.3% - 86.2% |
| Specificity | 97.8% (218/223)<br>95% CI: 94.9% -99.0% | |

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| (Concentrated Sediment) | | | | |
|-------------------------|-----------------------------|-----------------------|--|--|
| | AFB Smear Positive Subjects | AFB Smear Negative | | |
| | | Subjects | | |
| Sensitivity | 100% (65/65) | 60.0% (12/20) | | |
| | 95% CI: 94.4% - 100% | 95% CI: 38.7% - 78.1% | | |
| | | | | |
| Specificity | 99.3% (402/405) | | | |
| | 95% CI: 97.8% - 99.7% | | | |

# Table 24: Xpert MTB/RIF Assay Performance vs. MTB Culture

# Xpert MTB/RIF Assay Performance vs. Drug Susceptibility Testing for Rifampin (Rif)

MTB positive culture isolates underwent phenotypic drug susceptibility testing (DST) to Rif using agar proportions methods with Middlebrook or Lowenstein-Jensen media or the BD BACTECTM MGITTM 960 SIRE assay. The performance of the Xpert MTB/RIF Assay for the detection of genetic mutations associated with Rif resistance was determined relative to the DST results of the MTB culture isolates. There were 1,096 subjects eligible, with both Xpert MTB/RIF Assay and MTB culture results.

| | DST | | | | | |
|---------------------------|--------------------------------------------------|---------|----------|-----------------------------------------|-----------------------------------------|-------|
| | | Rif Res | Rif Susc | DST Not Done,<br>TB Culture<br>Positive | DST Not Done,<br>TB Culture<br>Negative | Total |
| | MTB DETECTED,<br>Rif Resistance<br>DETECTED | 18 | 4 | 0 | 0 | 22 |
| Xpert<br>MTB/RIF<br>Assay | MTB DETECTED,<br>Rif Resistance NOT<br>DETECTED | 1 | 404 | 7 | 7 | 419 |
| | MTB DETECTED,<br>Rif Resistance<br>INDETERMINATE | 0 | 4 | 1 | 1 | 6 |
| | MTB NOT<br>DETECTED | 2 | 26 | 1 | 620 | 649 |
| | Total | 21 | 447 | 9 | 628 | 1096 |

Table 25: Accountability of Eligible Study Subjects for Evaluation of Xpert MTB/RIF Assay for the Detection of Rif Resistance vs. DST

There were 447 subjects with an Xpert MTB/RIF Assay result "MTB DETECTED": 6 out of 447 (1.3%) subjects had an Xpert MTB/RIF Assay result of "MTB DETECTED, Rif Resistance INDETERMINATE" and were excluded from the analysis of the performance of the Xpert MTB/RIF Assay with regard to Rif resistance. Eight subjects with MTB positive cultures did not have DST results. Analysis of missing values with regard to potential biases was performed (i.e., analysis of covariate distributions and logistic regression) and the eight subjects were excluded from the analysis. In the analysis of the performance of the Xpert MTB/RIF Assay with regard to the detection of Rif resistance associated mutations, 1,082 subjects were used.

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Results for the detection of Rif resistance associated mutations are reported by the Xpert MTB/RIF Assay only when MTB-complex is detected by the device. Discordant results were further evaluated using bi-directional sequencing of the rpoB region of the MTB genome. Overall results are reported in Table 26.

| | DST | | | | |
|---------------------------|--------------------------------------------------…