XPERT MTB/RIF ASSAY by CEPHEID | FDA 510(k) Summary

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 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 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
- 1. 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.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study proving the device meets them, based on the provided text:

Acceptance Criteria and Device Performance for Xpert® MTB/RIF Assay

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally demonstrated through the sensitivity and specificity values against a reference method (culture and DST).

Acceptance Criteria Category	Specific Criteria (Derived from text)	Reported Device Performance
MTB-Complex Detection (vs. Culture)
- Overall Sensitivity	High sensitivity and specificity for MTB-complex detection (implicitly required by regulatory guidelines for diagnostic tests aid in diagnosis).	Overall Sensitivity (US prospective study): 93.8%
- Overall Specificity		Overall Specificity: 98.7% (95% CI: 97.5% - 99.4%)
- Sensitivity (AFB Smear-Positive)	High sensitivity for smear-positive specimens.	99.7% (350/351) with 95% CI: 98.4% - 99.9% (Table 19)
- Specificity (AFB Smear-Positive)	High specificity for smear-positive specimens.	98.5% (65/66) with 95% CI: 91.9% – 99.7% (Table 19)
- Sensitivity (AFB Smear-Negative)	Acceptable sensitivity for smear-negative specimens.	76.1% (89/117) with 95% CI: 67.6% - 82.9% (Table 20)
- Specificity (AFB Smear-Negative)	High specificity for smear-negative specimens.	98.8% (555/562) with 95% CI: 97.5% – 99.4% (Table 20)
Rifampin Resistance Detection (vs. DST)
- Sensitivity (Rif Res vs. DST)	High sensitivity for detecting rifampin resistance.	94.7% (18/19) with 95% CI: 75.4% - 99.1% (Table 26)
- Specificity (Rif Susc vs. DST)	High specificity for detecting rifampin susceptibility.	99.0% (404/408) with 95% CI: 97.5% - 99.6% (Table 26)
LoD for MTB-Complex	LoD must be determined in the most challenging matrix (e.g., sputum) claimed for use with the device, using both antibiotic susceptible and resistant strains. This is a general regulatory requirement (Q. Risks to Health and Mitigations, P. Proposed Labeling, S. Conclusion).	Demonstrated via probit analysis (Table 8) with claimed LoD values for different M.bovis BCG and M. tuberculosis strains in sputum and sediment, ranging from 525 to 4000 CFU/mL.
Reproducibility (Overall Agreement %)	Reproducibility studies must include at least one antibiotic resistant and one antibiotic susceptible strain of Mycobacterium tuberculosis complex (Q. Risks to Health and Mitigations, P. Proposed Labeling, S. Conclusion). Implicitly looking for high percentage agreement.	External Reproducibility (Table 1):
Cross-Reactivity (Analytical Exclusivity)	Absence of cross-reactivity with common respiratory pathogens and non-tuberculosis mycobacteria. Identification of any potential for cross-reactivity through in silico analysis. Implicitly required to prevent false positives.	Wet-tested microorganisms: No cross-reactivity observed with 131/132 microorganisms. One replicate of M. scrofulaceum showed cross-reactivity at 10^0 CFU/mL but not at 10^7 CFU/mL (presumably a false positive at lower concentration). (Table 14) In silico analysis: 13/18 predicted non-cross-reactive. 5/18 (M. kumamontonense, M. leprae, M. mucogenicum, Tsukamurella spp., Nocardia otitidiscaviarum) showed slight potential for cross-reactivity (Table 15, 16).
Analytical Reactivity (Inclusivity)	Ability of the device to detect genetic mutations associated with antibiotic resistance in a diversity of Mycobacterium tuberculosis complex strains; isolates used must be well characterized. (Q. Risks to Health and Mitigations, P. Proposed Labeling, S. Conclusion).	Wet-tested strains: 62 well-characterized M. tuberculosis strains (26 Rif-S, 36 Rif-R) tested at near LoD. Rif-S detection: 87% (67/77) accurate. 3 DST-susceptible strains showed Rif-resistance mutations by sequence analysis and Xpert. 1 replicate of one Rif-S strain showed "INDETERMINATE." Rif-R detection: 100% (107/107) accurate. (Table 11) In silico analysis: High likelihood of amplification and detection for M. africanum, M. bovis, M. canettii, M. caprae, and M. microti based on sequence alignment (Table 13).
Interfering Substances	Performance should not be significantly impacted by common interfering substances found in sputum. Implicitly required.	Inhibition observed for Lidocaine (30%), mucin (5% and 2.5%), Ethambutol (50, 25, 10 ug/mL), Guaifenesin (5 mg/mL), Phenylephrine (100% and 50%), and tea tree oil (0.5% to 0.015%), leading to false negative or indeterminate results (Table 17).
Carry-over Contamination	No significant carry-over/cross-contamination in single-use cartridges. Implicitly required for device safety and effectiveness.	100% correct results for both high positive samples (20/20 "MTB DETECTED; Rif Resistance NOT DETECTED") and negative samples (22/22 "MTB NOT DETECTED") when negative samples followed high positive samples. No carry-over or cross-contamination observed.
Internal Controls	Device must include internal controls (Sample Processing Control, Probe Check Control) to ensure adequate processing, monitor inhibition, and verify reagent/probe integrity. (Q. Risks to Health and Mitigations, P. Proposed Labeling, S. Conclusion).	SPC: Present in each cartridge, verifies adequate processing and monitors inhibitors. Passes if valid Ct in negative sample. PCC: Verifies reagent rehydration, PCR tube filling, probe integrity, and dye stability. Passes if fluorescence meets acceptance criteria.
External Controls	Device must include an external positive assay control (and implicitly a negative control) as appropriate. (Q. Risks to Health and Mitigations, P. Proposed Labeling, S. Conclusion).	Commercially available INTROL TB Controls evaluated; showed 100% expected results for negative, wild type, and two mutant controls (Tables 4-7). During clinical trial, 97% (453/467) of external controls gave expected results on first attempt, with 8/14 failed controls giving expected results on retest.
Specimen Stability	Claims for transport and storage of sputum and sputum sediment must be supported. Implicitly required.	Sputum: Storage at 3-5°C for up to 3 days, then 2-8°C for additional 7 days supported. Sputum Sediment: Storage at 2-8°C for up to 7 days supported.
Cartridge Hold Time	Claims for cartridge hold time (from adding sample to start of processing) must be supported. Implicitly required.	Sputum & Sputum Sediment: 4-hour hold time supported.
Shelf-Life	Product shelf-life must be demonstrated. Implicitly required.	18 months at 2-28°C (unopened). 6 weeks at 2-45°C (open package).
Failure Mode Testing	Evaluation of effect of operator errors, manufacturing errors, and instrument malfunction. Implicitly required to ensure device robustness.	Operator Errors: Reduced sample volumes (70-90%) and "no sample added" all resulted in correct positive/negative results, except for "no sample" resulting in negative. (Table 29) Manufacturing Errors: Most simulated errors (missing reagents, improper filter loading) resulted in "Error" or "Invalid" results, indicating the system detects internal failures and prevents erroneous results. Some (e.g., missing pre-filter) did not impede correct results. (Table 30)

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

Clinical Study (MTB-Complex & Rifampin Resistance):
- Eligible Subjects: 1,126
- Analyzed Subjects (MTB-Complex Detection): 1,096 (after excluding non-determinate Xpert results and culture contamination)
- Analyzed Subjects (Rifampin Resistance Detection): 1,082 (after further exclusions for indeterminate Rif resistance and missing DST results)
- Data Provenance: Multi-center study, includes both US (49% from California, New York, Florida) and non-US (51% from Vietnam, Peru, South Africa, Mexico, Bangladesh) populations. Data included both prospectively collected specimens (450 US, 23 non-US) and archived specimens (92 US, 531 non-US).
Reproducibility Studies:
- External Reproducibility (Study 154A): 5 contrived panel samples (1 negative, 4 positive at low/moderate concentrations) x 3 sites x 2 operators x 3 panel runs/day x 5 days = 90 tests per sample type over 5 types of samples (450 total tests).
- Instrument Precision: 5 contrived panel samples (1 negative, 4 positive at low/moderate concentrations) x 2 instruments x 2 operators x 4 panel runs/day x 12 days = 192 tests per sample type over 5 types of samples (960 total tests). One sample type (MTB/Rif-R ~2-3X LoD) was 190 total tests.
Rifampin Resistance Study (Analytical): 185 isolates (87 Rif-R, 98 Rif-S) spiked into sputum.
Analytical Reactivity (Inclusivity): 62 M. tuberculosis strains (26 Rif-S, 36 Rif-R) tested in triplicate (186 total tests).
Exclusivity/Cross-Reactivity: 132 microorganisms (24 NTM, 87 bacteria, 7 fungi, 14 viruses) tested in triplicate (396 total tests for wet testing).

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

The document does not explicitly state the number or qualifications of experts used to establish the ground truth for the clinical study's test set.

Ground truth for MTB-complex detection: Established by "culture (solid and/or liquid)". This implies standard microbiological laboratory procedures performed by qualified laboratory personnel, but no specific number or qualification of "experts" is given beyond this.
Ground truth for Rifampin resistance: Established by "phenotypic drug susceptibility testing (DST) using agar proportions methods with Middlebrook or Lowenstein-Jensen media or the BD BACTECTM MGITTM 960 SIRE assay." This also implies standard microbiological laboratory procedures.
Discordant analysis: "Bi-directional sequencing of the rpoB region of the MTB genome" was used to resolve discordant results between Xpert and culture/DST. This is a molecular method, likely performed by molecular biologists/scientists, but again, no specific number or detailed qualifications are provided.

4. Adjudication Method (e.g., 2+1, 3+1, none) for the Test Set

The document does not explicitly describe a formal adjudication method like "2+1" or "3+1" for the clinical study test set's primary ground truth (culture/DST).

However, it does describe a process for handling discordant results:

For MTB-complex detection, "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."
For Rifampin resistance, "Discordant results were further evaluated using bi-directional sequencing of the rpoB region of the MTB genome."
This implies that sequencing served as an adjudicator for specific discrepancies, effectively acting as an additional, confirmatory ground truth method in those cases. The primary ground truth for clinical performance remained culture and DST.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study focusing on human readers' improvement with AI vs. without AI assistance was not done.

This device is an in vitro diagnostic test (a PCR-based assay), not an AI-powered image analysis tool for human readers. Therefore, the concept of "human readers improve with AI vs. without AI assistance" does not apply to this type of device.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

Yes, this entire study is a standalone (algorithm only) performance assessment.

The Xpert® MTB/RIF Assay is an automated PCR system that processes samples and provides quantitative results (Ct values) and qualitative interpretations ("MTB DETECTED", "Rif Resistance DETECTED/NOT DETECTED/INDETERMINATE") directly. The results are "displayed in tabular and graphic formats." There is no "human-in-the-loop" interpretation step where a human reviews the algorithm's output and modifies it, or where a human's performance is compared before and after using the device's output. The performance metrics (sensitivity, specificity, reproducibility, etc.) directly reflect the device's algorithmic output compared to the established ground truth.

7. The Type of Ground Truth Used

For Mycobacterium tuberculosis complex DNA detection:
- Primary Ground Truth: Mycobacterial culture (solid and/or liquid).
- Confirmatory (for discordant results): Bi-directional sequencing of the rpoB region of the MTB genome.
For Rifampin-resistance associated mutations:
- Primary Ground Truth: Phenotypic drug susceptibility testing (DST) using agar proportions methods or BD BACTECTM MGITTM 960 SIRE assay.
- Confirmatory (for discordant results): Bi-directional sequencing of the rpoB region of the MTB genome.

8. The Sample Size for the Training Set

The document does not explicitly describe a separate "training set" in the context of machine learning or AI algorithm development. This is expected, as the Xpert® MTB/RIF Assay is a PCR-based diagnostic, not an AI/ML algorithm that is "trained" in the typical sense. Its development would involve analytical verification and validation based on known strains and biological principles, rather than iterative learning from a large dataset.

The term "training set" is generally not applicable to traditional in vitro diagnostic assays like this one. If an analogy were to be drawn, the internal "assay settings" (like valid Ct values) and the design of primers/probes were established during product development using various analytical studies and possibly earlier R&D experiments, which could be considered akin to "training" or "calibration" data, but not a formally delineated machine learning training set.

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

As noted above, a formal "training set" in the AI/ML sense is not described. Therefore, the method for establishing ground truth for such a set is also not applicable here.

The analytical performance characteristics (like LoD, inclusivity, exclusivity) were established using well-characterized strains (e.g., ATCC Strain H37Rv, specific mutant strains) and clinical isolates, with ground truth determined by:

Prior MTB identification
Phenotypic drug susceptibility testing (DST)
Bi-directional sequencing

These methods serve to define the characteristics of these samples, which are then used to challenge and validate the assay's performance.

Summary

Regulation Number and Section

§ 866.3373 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.

(a)
Identification. Nucleic acid-based in vitro diagnostic devices for the detection ofMycobacterium 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.(b)
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.