Xpert Xpress Strep A
Device Facts
| Record ID | K172126 |
|---|---|
| Device Name | Xpert Xpress Strep A |
| Applicant | Cepheid |
| Product Code | PGX · Microbiology |
| Decision Date | Sep 25, 2017 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.2680 |
| Device Class | Class 2 |
Indications for Use
The Xpert® Xpress Strep A Assay, performed on the GeneXpert Instrument Systems, is a rapid, qualitative in vitro diagnostic test for the detection of Streptococcus pyogenes (Group A B-hemolytic Streptococcus, Strep A) in throat swab specimens from patients with signs and symptoms of pharyngitis. The Xpert Xpress Strep A Assay utilizes an automated real-time polymerase chain reaction (PCR) to detect Streptococcus pyogenes DNA.
Device Story
Automated real-time PCR assay for qualitative detection of S. pyogenes DNA in throat swab specimens. Input: 300µL of transport medium from Copan Liquid Amies Elution Swab (ESwab) loaded into single-use disposable cartridge. Processing: GeneXpert Instrument Systems (Dx, Infinity-48s, Infinity-80) perform automated cell lysis via ultrasonic horn, DNA extraction, PCR amplification, and real-time detection. Output: Qualitative result (DETECTED, NOT DETECTED, INVALID, ERROR, NO RESULT) displayed on-screen or printed. Used in clinical laboratories by technicians. Early Termination Feature reports strongly positive results before full cycle completion. Results assist clinicians in diagnosing pharyngitis and determining appropriate antibiotic therapy.
Clinical Evidence
Two multicenter clinical studies (n=583 evaluable specimens) compared Xpert Xpress Strep A to reference culture. Sensitivity was 100% (95% CI: 97.3-100%). Specificity was 94.1% (95% CI: 91.5-95.9%). Discordant analysis using alternative PCR/sequencing suggested higher clinical sensitivity than culture. Indeterminate rate was 0.9% after retesting.
Technological Characteristics
Real-time PCR assay; automated sample prep and detection. Uses single-use, multi-chambered fluidic cartridges. Features: ultrasonic lysis, I-CORE thermocycler, syringe drive/rotary valve fluidics. Connectivity: GeneXpert Instrument Systems (standalone/networked modules). Software: automated analysis of fluorescence signals. Controls: Sample Processing Control (SPC) and Probe Check Control (PCC).
Indications for Use
Indicated for patients with signs and symptoms of pharyngitis. For prescription use only. Follow-up culture required if assay is negative and symptoms persist, or during acute rheumatic fever (ARF) outbreaks.
Regulatory Classification
Identification
A Streptococcus spp. nucleic acid-based assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify various Streptococcus spp. nucleic acids extracted directly from clinical specimens. The device detects specific nucleic acid sequences for organism identification. The identification aids in the diagnosis of diseases caused by bacteria belonging to the genus Streptococcus and provides epidemiological information on these diseases. Pathogenic streptococci are associated with infections, such as sore throat, impetigo (an infection characterized by small pustules on the skin), urinary tract infections, rheumatic fever, and kidney disease.
Special Controls
(b) Classification. Class II (special controls). The special controls for this device are: 1) Premarket notification submissions must include detailed device description documentation, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology including primer/probe sequence, design, and rationale for sequence selection. 2) Premarket notification submissions must include detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (Limit of Detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carry-over, and cross contamination. 3) Premarket notification submissions must include detailed documentation from a clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from well-accepted reference methods. 4) Premarket notification submissions must include detailed documentation for device software, including, but not limited to, software applications and hardwarebased devices that incorporate software. 5) Premarket notification submissions must include database implementation methodology, construction parameters and quality assurance protocols, as appropriate. 6) The device labeling must include limitations regarding the need for culture confirmation of negative specimens, as appropriate. 7) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling. 8) Premarket notification submissions must include details on an end user device training program that will be offered while marketing the device, as appropriate.
*Classification.* Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include detailed device description documentation, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology including primer/probe sequence, design, and rationale for sequence selection. (2) Premarket notification submissions must include detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (Limit of Detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carry-over, and cross contamination. (3) Premarket notification submissions must include detailed documentation from a clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from well-accepted reference methods. (4) Premarket notification submissions must include detailed documentation for device software, including, but not limited to, software applications and hardware-based devices that incorporate software. (5) Premarket notification submissions must include database implementation methodology, construction parameters, and quality assurance protocols, as appropriate. (6) The device labeling must include limitations regarding the need for culture confirmation of negative specimens, as appropriate. (7) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling. (8) Premarket notification submissions must include details on an end user device training program that will be offered while marketing the device, as appropriate.
Predicate Devices
- Liat Strep A Assay (K141338)
Related Devices
- K173398 — Xpert Xpress Strep A · Cepheid · Apr 26, 2018
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT COMBINATION TEMPLATE
A. 510(k) Number:
K172126
B. Purpose for Submission:
To obtain a Substantial Equivalence Determination for the Xpress Strep A Assay
C. Measurand:
Conserved regions of the *Streptococcus pyogenes* bacterial genome
D. Type of Test:
Qualitative real-time Polymerase Chain Reaction (PCR)
E. Applicant:
Cepheid
F. Proprietary and Established Names:
Xpert Xpress Strep A
G. Regulatory Information:
1. Regulation section:
21 CFR 866.2680: *Streptococcus* spp. nucleic acid-based assay
2. Classification:
Class II
3. Product code:
PGX: Groups A, C and G β-Hemolytic *Streptococcus* Nucleic Acid Amplification System
OOI: Instrumentation for clinical multiplex test systems
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4. Panel:
83-Microbiology
H. Intended Use:
1. Intended use(s):
The Xpert Xpress Strep A Assay, performed on the GeneXpert Instrument Systems, is a rapid, qualitative *in vitro* diagnostic test for the detection of *Streptococcus pyogenes* (Group A β-hemolytic *Streptococcus*, Strep A) in throat swab specimens from patients with signs and symptoms of pharyngitis.
The Xpert Xpress Strep A Assay utilizes an automated real-time polymerase chain reaction (PCR) to detect *Streptococcus pyogenes* DNA.
2. Indication(s) for use:
Same as Intended Use
3. Special conditions for use statement(s):
For prescription use only.
Additional follow-up testing by culture is required if the Xpert Xpress Strep A assay result is negative and clinical symptoms persist, or in the event of an outbreak of acute rheumatic fever (ARF).
4. Special instrument requirements:
The Xpert MRSA NxG assay is for use on the GeneXpert Dx, GeneXpert Infinity-48s and GeneXpert Infinity-80 instrument systems.
I. Device Description:
The Xpert Xpress Strep A Assay is an automated real-time polymerase chain reaction (PCR) *in vitro* diagnostic test for qualitative detection of *Streptococcus pyogenes* directly from throat swab specimens from patients with signs and symptoms of pharyngitis.
The Xpert Xpress Strep A Assay is performed on the Cepheid GeneXpert Instrument Systems (GeneXpert Dx, GeneXpert Infinity-48s, and GeneXpert Infinity-80 systems), that automate sample preparation, DNA amplification and real-time detection in single-use, disposable cartridges.
The Xpert Xpress Strep A Assay cartridge contains a pair of PCR primers and a hydrolysis probe that enable detection of a conserved DNA sequence within the *S. pyogenes* genome.
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The assay also incorporates a Sample Processing Control (SPC) and a Probe Check Control (PCC) to monitor the integrity of the reagents and process workflow.
The GeneXpert Instrument Systems have between 1 and 80 randomly accessible modules, depending upon the instrument, that are each capable of performing separate sample preparation and real-time PCR assays. Each module contains a syringe drive for dispensing fluids, an ultrasonic horn for lysing cells, and a thermocycler unit for real-time PCR amplification and detection.
Once the instrument is loaded and the test initiated, all the steps associated with sample processing, PCR amplification/detection and result interpretation occur automatically. The final result report can be viewed on-screen and/or printed.
J. Substantial Equivalence Information:
1. Predicate device name(s):
Liat Strep A Assay
2. Predicate 510(k) number(s):
K141338
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3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device (K172126) | Predicate (K141338) |
| | Xpert Xpress Strep A Assay | Liat Strep A Assay |
| Regulation | 21 CFR 866.2680 | Same |
| Product Code | PGX | Same |
| Device Class | Class II | Same |
| Intended Use | The Xpert Xpress Strep A Assay, performed on the GeneXpert Instrument Systems, is a rapid, qualitative in vitro diagnostic test for the detection of Streptococcus pyogenes (Group A β-hemolytic Streptococcus, Strep A) in throat swab specimens from patients with signs and symptoms of pharyngitis. The Xpert Xpress Strep A Assay utilizes an automated real-time polymerase chain reaction (PCR) to detect Streptococcus pyogenes DNA. | The Liat Strep A Assay, performed on the Liat Analyzer, is a qualitative in vitro diagnostic test for the detection of Streptococcus pyogenes (Group A β-hemolytic Streptococcus, Strep A) in throat swab specimens from patients with signs and symptoms of pharyngitis. The Liat Strep A Assay utilizes nucleic acid purification and polymerase chain reaction (PCR) technology to detect Streptococcus pyogenes by targeting a segment of the Streptococcus pyogenes genome. |
| Analyte | Group A Streptococcus | Same |
| Measurand | Conserved region of S. pyogenes DNA | Same |
| Specimen Type | Throat swab in liquid Amies medium | Same |
| Reagent Format | Unitized ready for use | Same |
| Assay Format | Automated DNA extraction, amplification and detection | Same |
| Process Control | Cell-based | Same |
| External Controls | Available | Same |
| Result Format | Qualitative | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device (K172126) | Predicate (K141338) |
| | Xpert Xpress Strep A Assay | Liat Strep A Assay |
| Instrument System | GeneXpert Dx, GeneXpert Infinity-48s or GeneXpert Infinity-80 instrument systems | Liat Analyzer |
| Bacterial Lysis | Mechanical (sonication) | Chaotrope and enzymatic digestion |
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K. Standard/Guidance Document Referenced (if applicable):
1. Guidance for Industry, FDA Reviewers and Compliance on: Off-the-Shelf Software Use in Medical Devices; September 9, 1999.
2. General Principles of Software Validation; Final Guidance for Industry and FDA Staff; January 11, 2002.
3. Guidance for Industry: Cybersecurity for Networked Medical Devices Containing Off-the-Shelf (OTS) Software; January 14, 2005.
4. Guidance for Industry and FDA Staff: Class II Special Controls Guidance Document - Instrumentation for Clinical Multiplex Test Systems; March 10, 2005.
5. Guidance for Industry and FDA Staff: Guidance for the Content of Pre-market Submissions for Software Contained in Medical Devices; May 11, 2005.
6. Guidance for Industry and FDA Staff: Format for Traditional and Abbreviated 510(k)s; August 12, 2005.
7. Guidance for Sponsors, Institutional Review Boards, Clinical Investigators and FDA Staff: Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable; April 25, 2006.
8. Guidance for Industry and FDA Staff: Content of Premarket Submissions for Management of Cybersecurity in Medical Devices; October 2, 2014.
9. CLSI. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - 3rd Edition. CLSI document EP05-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
10. CLSI. Interference Testing in Clinical Chemistry; Approved Guideline - 2nd Edition. CLSI document EP07-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.
11. CLSI. Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline - 3rd Edition. CLSI document EP09-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2013.
12. CLSI. User Verification of Precision and Estimation of Bias; Approved Guideline - 3rd Edition. CLSI document EP15-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
13. CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - 2nd Edition. CLSI document EP17-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
14. CLSI. Molecular Diagnostic Methods for Infectious Diseases - 3rd Edition. CLSI report MM03. Wayne, PA: Clinical and Laboratory Standards Institute; 2015.
15. CLSI. Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods; Approved Guideline. CLSI document MM13-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.
16. CLSI. Verification and Validation of Multiplex Nucleic Acid Assays; Approved Guideline. CLSI document MM17-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
17. ASTM D4169-14, Standard Practice for Performance Testing of Shipping Containers and Systems, 2014.
18. Federal Communication Commission Part 15, Subparts A and B.
19. Federal Communication Commission Part 18.
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20. BS EN ISO 23640: 2011. In vitro diagnostic medical devices – Evaluation of stability of in vitro diagnostic reagents.
21. IEC CISPR-11 Industrial, Scientific, and Medical Equipment Radio Disturbance Characteristics - Limits and Methods of Measurement; 2004.
22. IEC CISPR-22 Information technology equipment - Radio Disturbance Characteristics - Limits and Methods of Measurement; 2006.
23. IEC 61010-1 Safety requirements for electrical equipment for measurement, control, and laboratory use – Part 1, General Requirements; 2001.
24. EN 61010-1 Safety requirements for electrical equipment for measurement, control, and laboratory use – Part 1, General Requirements; 2001.
25. UL 61010-1 Safety requirements for electrical equipment for measurement, control, and laboratory use – Part 1, General Requirements; 2004.
26. IEC 61010-2-101 Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use – Part 2-101: Particular requirements for In-Vitro Diagnostic (IVD) Medical Equipment; 2002.
27. EN 61010-2-101 Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use – Part 2-101: Particular requirements for In-Vitro Diagnostic (IVD) Medical Equipment; 2002.
28. EN 61326-1 Electrical Equipment for Measurement, Control, and Laboratory Use – EMC Requirements: General Requirements; 2006.
29. EN 61326-2-6 Electrical Equipment for Measurement, Control, and Laboratory Use – EMC Requirements – Part 2-6; 2006.
30. EN 55011 Industrial, Scientific and Medical (ISM) Radio-Frequency Equipment – Electromagnetic Disturbance Characteristics; 2010.
31. CAN/CSA C22.2 No. 61010-1 Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use – Part 1: General Requirements; 2004.
32. CAN/CSA C22.2 No. 61010-2-101 Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use – Part 2-101: Particular Requirements for In-Vitro Diagnostic (IVD) Medical Equipment; 2004.
33. WEEE Directive 2002/96/EC; 2002.
34. EMC (Electromagnetic Compatibility) Directive, 2004/108/EC; 2004.
35. LVD (Low Voltage Directive) 2014/35/EU; 2014.
L. Test Principle:
The Xpert Xpress Strep A Assay is performed on the GeneXpert Instrument Systems (GeneXpert Dx, GeneXpert Infinity-48s, and GeneXpert Infinity-80 systems) that automate nucleic acid extraction, amplification and detection in single-use, disposable cartridges. Each cartridge contains primers and probes for detection of the targeted region of the S. pyogenes chromosome (if present) and a Sample Processing Control (SPC). The SPC and a separate Probe Check Control (PCC) are used by the GeneXpert systems to monitor reagent and process integrity.
Throat swab specimens for testing with the Xpert Xpress Strep A Assay are collected using the Copan Liquid Amies Elution Swab (ESwab) Collection and Transport System. Upon receipt in the testing laboratory, the ESwab tube is mixed by shaking and $300\mu \mathrm{L}$ of the
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transport medium is added to an Xpert Xpress Strep A Assay cartridge using a disposable transfer pipette. The operator then initiates the test from the user interface and loads the cartridge into the GeneXpert instrument, after which all process steps are performed automatically.
Once complete, the final result report can be viewed on-screen and/or printed. Results are reported as Strep A DETECTED, Strep A NOT DETECTED, INVALID (SPC failure), ERROR (SPC and PCC failure) or NO RESULT (insufficient data collected). Instructions for retesting are provided for samples with indeterminate results.
The Xpert Xpress Strep A Assay has an Early Termination Feature whereby results for samples that are strongly positive for S. pyogenes are reported prior to completion of the full number of PCR cycles designated in the assay definition file.
## M. Performance Characteristics (if/when applicable):
### 1. Analytical performance:
#### a. Precision/Reproducibility:
To estimate the components of variance associated with the Xpert Xpress Strep A Assay a Reproducibility Study was conducted at 3 sites, with 2 operators per site. Each operator tested a 3 member panel of samples in duplicate twice per day over 6 days using 3 lots of reagents (3 sites x 2 operators/site x 2 panels/day x 2 replicates/panel member x 6 days = 144 results/panel member). Two sites conducted testing using the GeneXpert Dx and one site used the Infinity-80. Samples were prepared using simulated throat swab matrix in Liquid Amies transport medium and stored at 2-8°C until testing. Separate studies were conducted to demonstrate equivalent assay performance with simulated and natural throat swab matrix (Section M(2)(b)). The panel members used in the Reproducibility Study are summarized in Table 1.
Table 1. Summary of Reproducibility Study panel members
| S. pyogenes Strain | Panel Member | Multiple of LoD | CFU/mL^{1} |
| --- | --- | --- | --- |
| Not applicable | Negative | Not applicable | 0 |
| ATCC BAA-946 | Low Positive | 1X | ~10 |
| | Moderate Positive | 3X | ~30 |
ATCC: American Type Culture Collection; LoD: Limit of Detection
CFU per mL of ESwab transport medium
When the study was initially performed, there was an unexpectedly high rate of indeterminate results (47/432 = 10.8%), although no false positive or false negative results were observed (i.e., there was 100% agreement at all target levels among samples with valid results). An investigation showed that the high rate of indeterminate results was due to a combination of the duration of storage of the
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simulated samples prior to testing, in combination with the specific reagent lots used in the study. Even though the results of the Reproducibility Study were acceptable, the study was repeated using new panel members that were stored for a shorter duration prior to testing, as well as new reagent lots. The results of the repeat study are described below.
Eleven (11) indeterminate results were obtained over the course of the repeat study (ERROR: 5; INVALID: 4 and NO RESULT: 2) for an initial indeterminate rate of 2.5% (11/432). In all cases, the expected results were obtained upon retesting.
After retesting, percent agreement with the expected results for Negative and Moderate Positive target levels was 100% (144/144). For Low Positive samples, the percent agreement was 98.6% (142/144). ANOVA (Analysis Of Variance) was performed to assess the variance components of the Ct values for the S. pyogenes and SPC targets (Table 2). The total variation for all targets was between 0.9 and 2.3 Ct (2.5-6.6%).
The Xpert Xpress Strep A Assay demonstrated acceptable reproducibility across sites, GeneXpert systems, operators, panel members and reagent lots.
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Table 2. Summary of Ct variance components observed in the Reproducibility Study
| Sample | Target | N | Ct | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Mean | Between Site | | Between Lot | | Between Day | | Between Operator | | Within Assay | | Total | |
| | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Negative | SPC | 144 | 34.7 | 0.0 | 0.0 | 1.9 | 5.3 | 0.3 | 1.0 | 0.0 | 0.0 | 1.3 | 3.7 | 2.3 | 6.6 |
| Low Positive | GAS1 | 1422 | 37.8 | 0.2 | 0.6 | 0.0 | 0.0 | 0.1 | 0.4 | 0.1 | 0.2 | 1.0 | 2.7 | 1.1 | 2.8 |
| Moderate Positive | GAS1 | 144 | 36.5 | 0.0 | 0.0 | 0.3 | 0.8 | 0.0 | 0.0 | 0.1 | 0.3 | 0.9 | 2.3 | 0.9 | 2.5 |
GAS: Group A Streptococcus (S. pyogenes); SPC: Sample Processing Control; SD: Standard Deviation; %CV: Percent Coefficient of Variation
S. pyogenes strain ATCC BAA-946
2 Two (2) samples gave negative results for Group A Streptococcus
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b. Linearity/assay reportable range:
Not applicable.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
# Internal Controls
Each Xpert Xpress Strep A Assay includes two types of Internal Control to monitor the performance of the system:
Sample Processing Control (SPC): Designed to monitor bacterial lysis and sample inhibition. Test results are reported as INVALID if the SPC fails.
Probe Check Control (PCC): Fluorescence measurement to monitor probe rehydration and integrity, as well as filling of the reaction tube. Test results are reported as ERROR if the PCC fails.
The performance of the Internal Controls was verified by testing 29 potential failure modes due to errors in the manufacture of the Xpert Xpress Strep A Assay cartridges or the assay procedure. Under the conditions tested, no false positive results were obtained and all S. pyogenes negative samples were reported as either Negative, Error or Invalid. Two false negative results were obtained with S. pyogenes positive samples, the remainder of which were reported as Positive, Error or Invalid. The device manufacturer implemented appropriate measures by which to mitigate the risks associated with the failure modes that gave rise to false negative results, including use of a fixed volume pipette for sample transfer and $100\%$ manual inspection during cartridge assembly.
# External Controls
The device labeling includes information regarding the availability of commercially prepared External Positive and Negative Controls. These External Controls were run each day During the Clinical Study for the Xpert Xpress Strep A Assay (Section M(3)(a)) prior to testing of any clinical specimens. A summary of the results obtained is shown in Table 3.
Table 3. Summary of External Control performance in the prospective Clinical Study for the Xpert Xpress Strep A Assay
| | External Control | |
| --- | --- | --- |
| | Positive | Negative |
| Tested1 | 258 | 258 |
| Indeterminate (%) | 8/258 (3.1)2 | 6/258 (2.3)3 |
| Expected Result (%)4 | 246/250 (98.4) | 252/252 (100) |
1 Not including repeat tests
2 ERROR: 3; NO RESULT: 3; INVALID: 1
3 ERROR: 2; NO RESULT: 4
4 On initial testing
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An additional evaluation of the recommended External Controls was conducted by testing 3 lots of each control material with 3 lots of Xpert Xpress Strep A Assay reagents. All 180 pairs (100%) of the External Positive Negative Controls tested produced the expected results.
## Specimen Stability
The stability of throat swab specimens for use with the Xpert Xpress Strep A Assay was determined analytically by testing ESwabs seeded with pooled throat swab matrix in the presence and absence of *S. pyogenes* strain ATCC BAA-946 at approximately 3X LoD (26 CFU/mL). Specimens were placed at different temperatures and tested at pre-specified intervals. Results were analyzed qualitatively and by ANOVA to assess differences in Ct values for the *S. pyogenes* and SPC targets over time. No false negative or false positive results were observed during the course of the study, although a statistically significant increase in Ct values for the SPC was observed with *S. pyogenes* negative specimens after storage for 8 days at 8°C. As a result, the claimed stability of throat swab specimens at 2-8°C was restricted to ≤6 days. The study also demonstrated the stability of specimens at 15-30°C for ≤2 days.
## Reagent Stability
The shelf-life of the Xpert Xpress Strep A Assay cassettes was evaluated in a real-time stability study performed on three lots of reagents that were stored under different conditions. The results from the study to-date support assignment of an expiration date 6 months from the day of manufacture for the assay cassettes when stored under the recommended conditions.
## Cartridge Hold Time
After loading onto the GeneXpert Infinity System, assay cartridges may have to wait for a GeneXpert module to become available before being processed. A study was therefore conducted to determine the maximum permissible interval between addition of a sample to an Xpert Xpress Strep A Assay cartridge and testing on the GeneXpert Infinity System. The study was performed with *S. pyogenes* positive (3X LoD; 26 CFU/mL) and negative samples prepared with simulated throat swab matrix. The samples were added to Xpert Xpress Strep A Assay cartridges that were held for up to 5 hours under different environmental conditions prior to testing (i.e., ambient temperature/humidity, 25°C/75% relative humidity or 35°C/ambient humidity). The expected results were obtained at each time point and there were no important differences in Ct values under any of the conditions tested. The results of the study support the recommended maximum interval of 4.5 hours from sample addition to the Xpert Xpress Strep A Assay cartridges to processing on the GeneXpert Infinity System, with a maximum of 4 hours on the deck of the instrument.
## d. Detection limit:
### Determination of the Limit of Detection
The analytical sensitivity of the Xpert Xpress Strep A Assay was evaluated by testing dilutions of two strains of *S. pyogenes* in pooled throat swab matrix. The Limit of
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Detection (LoD) was determined for each strain using logistic regression according to the guidance in CLSI Document EP17-A2. Testing was performed using two lots of reagents and six different concentrations of each strain ranging from $< 2$ to 35 CFU/mL of ESwab Transport Medium, in addition to S. pyogenes negative samples. The LoD point estimates were confirmed by testing an additional 20 replicates of each strain at the claimed LoD target level. At least 19/20 replicates had to produce positive results in order for the LoD to be considered confirmed. The results of the LoD Study are shown in Table 4.
Table 4. Analytical sensitivity of the Xpert Xpress Strep A Assay
| ATCC Number | Strain | emm Sequence Type1 | LoD Point Estimate (CFU/mL of ESwab Transport Medium) |
| --- | --- | --- | --- |
| BAA-946 | GAS 10394 | 6 | 9 |
| 19615 | Bruno (CIP 104226) | 80 | 18 |
ATCC: American Type Culture Collection
1 https://www.cdc.gov/streplab/m-proteingene-typing.
# Analytical Reactivity/Inclusivity
A study was conducted to demonstrate the ability of the Xpert Xpress Strep A Assay to detect strains of S. pyogenes representing different emm sequence types, including representatives of those types most commonly isolated in cases of pharyngitis in the U.S. (Table 5). A total of 24 strains were tested at a concentration of $26\mathrm{CFU / mL}$ in simulated throat swab matrix (Section M(2)(b)), corresponding to $\sim 3\mathrm{X}$ LoD, with four replicates per strain. All 26 strains were successfully detected. These results are acceptable.
Table 5. Strains of S. pyogenes evaluated in the Analytical Reactivity Study
| ATCC Number | Strain | emm Sequence Type1 | ATCC Number | Strain | emm Sequence Type1 |
| --- | --- | --- | --- | --- | --- |
| 12202 | NCTC 8370 | 1 | 12204 | A25 | 25 |
| 12344 | T1 | 1 | 8135 | T27 | 27 |
| 700294 | SF370; M1 GAS | 1 | 12365 | C107 | 38 |
| 12383 | D58X | 3 | 12370 | C94 | 38 |
| 12384 | C203 | 3 | 700497 | CDC-SS-1147 | 75 |
| 12385 | J17A4 | 4 | 700499 | CDC-SS-1149 | 77 |
| 12203 | NCTC 8709 | 6 | 700949 | CDC-SS-1397 | 89 |
| 12352 | T11 | 11 | BAA-355 | N/A | 94 |
| BAA-1065 | MGAS 2096 | 12 | BAA-356 | N/A | 95 |
| BAA-1315 | MGAS 9429 | 12 | 14289 | C203 S | emm deficient |
| 12357 | J17C | 18 | 49399 | QC A62 | N/A |
| 10403 | T22 | 22 | 51339 | 1805 | N/A |
ATCC: American Type Culture Collection; N/A: not available
1 https://www.cdc.gov/streplab/m-proteingene-typing.
# Bioinformatic Analysis
The inclusivity of the Xpert Xpress Strep A Assay primers and probe for the targeted region of the S. pyogenes genome was analyzed in silico using the Basic Local
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Alignment Search Tool (BLAST). The assay region was shown to be well conserved, with limited evidence of polymorphisms that could affect primer or probe hybridization. The potential for false-negative Xpert Xpress Strep A Assay results due to unforeseen sequence variation is noted as a Limitation in the device labeling.
# e. Analytical specificity:
The analytical specificity of the Xpert Xpress Strep A Assay was evaluated by testing a panel of 63 potentially cross-reactive strains of bacteria, including 15 non-Group A species of Streptococcus, one strain of yeast and six viruses in simulated throat swab matrix (Table 6).
Each strain was tested in triplicate at a concentration of $\geq 10^{6}$ CFU/mL for bacteria and yeast, and $\geq 10^{5}$ TCID $_{50}$ for viruses. In all cases, the expected results were obtained and there was no evidence of the potential for false-positive results due to cross-reaction with non-target organisms. These results are acceptable.
Table 6. Strains of bacteria, yeast and viruses evaluated in the Analytical Specificity Study
| Gram Negative | Strain | Gram Positive | Strain |
| --- | --- | --- | --- |
| Acinetobacter baumannii | ATCC 19606 | Bacillus cereus | ATCC 13472 |
| Acinetobacter haemolyticum | ATCC BAA-1784 | Corynebacterium diphtheriae | CCUG 33629b |
| Bordetella bronchiseptica | ATCC 4617 | Corynebacterium pseudodiphtheriticum | ATCC 10700 |
| Bordetella pertussis | ATCC 9797 | Enterococcus faecalis | ATCC 8750 |
| Burkholderia cepacia | ATCC 25416 | Enterococcus faecium | ATCC19434 |
| Campylobacter rectus | ATCC 33238 | Lactobacillus acidophilus | ATCC 314 |
| Escherichia coli | ATCC 8739 | Lactococcus lactis subsp. lactis | ATCC 9596 |
| Fusobacterium necrophorum | ATCC 25288 | Listeria monocytogenes | ATCC 15313 |
| Haemophilus parahaemolyticus | ATCC 10014 | Peptostreptococcus micros | ATCC 33270 |
| Klebsiella pneumoniae | ATCC 13883 | Staphylococcus aureus | ATCC 43300 |
| Legionella jordanis | ATCC 700762 | Staphylococcus epidermidis | NRS8c |
| Legionella micdadei | ATCC 33204 | Staphylococcus haemolyticus | NRS50c |
| Legionella pneumophila | ATCC 33152 | Streptococcus agalactiae | ATCC 13813 |
| Moraxella catarrhalis | ATCC 43628 | Streptococcus anginosus | ATCC 33397 |
| Moraxella catarrhalis | ATCC 23246 | Streptococcus bovis | ATCC 33317 |
| Moraxella lacunata | ATCC 17967 | Streptococcus canis | ATCC 43496 |
| Neisseria gonorrhoeae | ATCC 700825 | Streptococcus constellatus | ATCC 27823 |
| Neisseria lactamica | ATCC 23972 | Streptococcus dysgalactiae | F578943c |
| Neisseria meningitidis | ATCC 13102 | Streptococcus equi | ATCC 9525 |
| Neisseria mucosa | ATCC 25998 | Streptococcus gallolyticus | ATCC 9809 |
| Neisseria sicca | ATCC 29259 | Streptococcus intermedius | ATCC 27335 |
| Neisseria subflava | ATCC 49275 | Streptococcus mitis | ATTC 49456 |
| Prevotella (Bacteroides) oralis | ATCC 33269 | Streptococcus mutans | ATCC 25175 |
| Proteus mirabilis1 | ATCC 25933 | Streptococcus oralis | ATCC 9811 |
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| Gram Negative | Strain | Gram Positive | Strain |
| --- | --- | --- | --- |
| Proteus vulgaris | ATCC 29905 | Streptococcus pneumoniae | ATCC 49619 |
| Pseudomonas aeruginosa2 | ATCC 27853 | Streptococcus salivarius | ATCC 7073 |
| Pseudomonas fluorescens | ATCC 13525 | Streptococcus sanguinus | ATCC 10556 |
| Serratia marcescens | ATCC 13880 | Gram Indeterminate | Strain |
| Stenotrophomonas maltophilia | C5669c | Bordetella parapertussis | ATCC 15311 |
| Treponema denticola | ATCC 35405 | Haemophilus influenzae type A | ATCC 9007 |
| Veillonella parvula | ATCC 10790 | Haemophilus parainfluenzae | ATCC 9796 |
| Yersinia enterocolitica | ATCC 9610 | Mycoplasma pneumoniae | ATCC 15293 |
| Viruses | Strain | Yeast | Strain |
| Adenovirus Type 1 | 3292010a | Candida albicans | ATCC 14053 |
| Adenovirus Type 7 | 07142010LWa | | |
| Cytomegalovirus AD-169 | 810008a | | |
| Epstein Barr Virus 4 | 309068a | | |
| Hepatitis B Virus | NATHBV-0003b | | |
| Herpes Simplex Virus3 | 58167228a | | |
2 INVALID results obtained on initial testing; both reported as Negative on repeat
1 INVALID result obtained on initial testing; reported as Negative on repeat
3 1 ERROR on initial testing; reported as Negative on repeat
# Bioinformatic Analysis
The specificity of the Xpert Xpress Strep A primers and probes for the targeted sequence was also evaluated through extensive in silico BLAST analysis which showed that positive results with species other than S. pyogenes are unlikely to occur.
# Carry-over Contamination Study
To determine the potential for sample-to-sample contamination using the Xpert Xpress Strep A Assay, testing was performed with alternating "high positive" ( $>1 \times 10^{6}$ CFU/mL of ESwab Transport Medium) and negative samples that were prepared using simulated throat swab matrix. The study was conducted on two GeneXpert Systems (one GX-IV and one GX-XVI) with 20 high positive and 21 negative samples per instrument. No false-positive of false-negative results were obtained on either system and the results were therefore determined to be acceptable.
# f. Assay cut-off:
Thresholds and cut-offs for the Xpert Xpress Strep A Assay are embedded within the Assay Definition File that also encodes the instrument settings required to perform the test. The valid Ct ranges for the S. pyogenes target and SPC are shown in Table 7.
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Table 7. Valid Ct ranges for Xpert Xpress Strep A Assay
| Target Sequence | Valid Ct Range | |
| --- | --- | --- |
| | Minimum | Maximum |
| S. pyogenes | 10.0 | 43.0 |
| SPC | 26.0 | 43.0 |
SPC: Sample Processing Control
In addition to fixed algorithm parameters within the software Assay Definition File for the Xpert Xpress Strep A Assay, Lot Specific Parameters (LSP) are also generated for every lot of assay reagents to account for potential variations in production. The LSP are embedded within the barcode of each assay cartridge and are transferred to the GeneXpert instrument system when the barcode is scanned.
# g. Assay interference:
The potential for interference with the Xpert Xpress Strep A Assay was evaluated with endogenous and exogenous substances that may be present in throat swab specimens, as well as a representative panel of commensal or potentially pathogenic microorganisms. Testing was performed using simulated throat swab matrix (Section M(2)(b)) in the presence of S. pyogenes ATCC BAA-946 at approximately 3X LoD (26 CFU/mL). S. pyogenes negative samples were also included to assess the potential for interference with the SPC. In addition to analysis of qualitative test results, ANOVA was used to assess differences in Ct values for the S. pyogenes and SPC targets in relation to appropriate control conditions without any interfering substances or organisms present.
# Interfering Substances Study
All S. pyogenes positive and negative samples (n = 8 per condition) were correctly identified in the presence of each of the potentially inhibitory substances shown in Table 8 and there were no statistically significant differences in Ct values between the test and control conditions for either the SPC or S. pyogenes targets. Nevertheless, a significant reduction in fluorescence intensity was observed for the S. pyogenes target in the presence of antiseptic mouthwash at $6.5\%$ w/v. These results are noted in the device labeling.
Table 8. Potentially interfering substances testing with the Xpert Xpress Strep A Assay
| Substance Group | Description/Active Ingredient | Concentration Tested |
| --- | --- | --- |
| Saliva | Human Saliva, 100% | 6.5% (v/v) |
| Mucin | Porcine mucin Type III Bound Sialic Acid, 0.5-1.5% | 2.5% (w/v) |
| Blood | Whole Human Blood EDTA anticoagulant | 5.0% (v/v) |
| Antiseptic | Rite Aid Oral Care Tartar Control Plus Antiseptic Mouth Rinse, Ice Mint: Eucalyptol, 0.92% Menthol, 0.042% Methyl Salicylate, 0.060% | 6.5% (v/v)1 |
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| Substance Group | Description/Active Ingredient | Concentration Tested |
| --- | --- | --- |
| | Thymol, 0.64% | |
| Cough Medicine | Wal-Tussin DM Max Cough and Chest Congestion Liquid Berry: Dextromethorphan HBr USP, 10mg Guaifenesin USP, 200mg | 5mg/mL2 |
| Sugar-containing Cold & Flu Remedies | Vicks NyQuil Severe Cold & Flu Nighttime Relief Berry: Acetaminophen, 650mg Dextromethorphan HBr, 20mg Doxylamine Succinate, 12.5mg Phenylephrine HCl 10mg | 6.5% (v/v) |
| Salt-modifying Remedies | Saline Nasal Spray: Sodium Chloride, 0.65% | 6.5% (v/v) |
| Foods/drinks That Increase Salivary Viscosity | Organic Grade A Whole Milk with Vitamin D3 | 6.5% (v/v) |
| pH Modifying Remedies | Orange Juice, 100% | 6.5% (v/v) |
| Antacids | Equate Liquid Maximum Strength Original Classic Antacid/Anti Gas: Aluminum Hydroxide, 400mg Magnesium Hydroxide, 400mg Simethicone, 40mg | 6.5% (v/v) |
1 Although all samples were reported appropriately as positive, reduced fluorescent signal was observed for the S. pyogenes target in the presence of antiseptic mouthwash at $6.5\%$ v/v
2 1 S. pyogenes negative specimen produced an ERROR on initial testing; reported as Negative on repeat
# Microbial Interference Study
The list of microorganisms tested is shown in Table 9. Each of the species was tested at $\geq 10^{6}$ CFU/mL of ESwab Transport Medium. The Xpert Xpress Strep A Assay correctly identified all the S. pyogenes positive and negative specimens in the study (n = 4 per condition) and there were no statistically significant differences in Ct values between the test samples and controls. However, a reduction in fluorescence intensity was observed for both the S. pyogenes target and SPC in the presence of a high concentration of N. lactamica. The results of the study are noted in the device labeling.
Table 9. Species of commensal organisms tested with the Xpert Xpress Strep A Assay in the Microbial Interference Study
| Species | Strain | Species | Strain |
| --- | --- | --- | --- |
| Acinetobacter baumannii | ATCC 19606 | Streptococcus constellatus | ATCC 27823 |
| Candida albicans | ATCC 14053 | Streptococcus dysgalactiae | F578943 |
| Enterococcus faecalis | ATCC 8750 | Streptococcus equi | ATCC 9525 |
| Fusobacterium necrophorum | ATCC 25288 | Streptococcus gallolyticus | ATCC 9809 |
| Haemophilus influenzae type A | ATCC 9007 | Streptococcus intermedius | ATCC 27335 |
| Lactobacillus acidophilus | ATCC 314 | Streptococcus mitis | ATCC 49456 |
| Neisseria lactamica1 | ATCC 23972 | Streptococcus mutans | ATCC 25175 |
| Peptostreptococcus micros | ATCC 33270 | Streptococcus oralis | ATCC 9811 |
| Prevotella (Bacteroides) oralis | ATCC 33269 | Streptococcus pneumoniae | ATCC 49619 |
| Staphylococcus epidermidis | NRS8 | Streptococcus salivarius | ATCC 7073 |
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| Species | Strain | Species | Strain |
| --- | --- | --- | --- |
| Streptococcus agalactiae | ATCC 13813 | Streptococcus sanguinis | ATCC 10556 |
| Streptococcus anguinosus | ATCC 33397 | Treponema denticola | ATCC 35405 |
| Streptococcus bovis | ATCC 33317 | Veillonella parvula | ATCC 10790 |
| Streptococcus canis | ATCC 43496 | | |
1 Although all samples were reported appropriately as positive or negative, reduced fluorescent signal was observed for both the S. pyogenes target and SPC in the presence of a high concentration of N. lactamica
# 2. Comparison studies:
a. Method comparison with predicate device:
Not applicable.
b. Matrix comparison:
Comparison of Fresh and Frozen Cell Stocks
Specimens for analytical evaluation of assay performance were prepared using S. pyogenes cell stocks that were grown on Tryptic Soy Agar plates for 18-24 hours, suspended in Typtic Soy Broth containing $15\%$ (v/v) glycerol and frozen at $-80^{\circ}\mathrm{C}$ . The concentration of the stocks was determined by performing viable counts. Testing of samples prepared from fresh cell stocks and cell stocks that were frozen at $-80^{\circ}\mathrm{C}$ for 10 days showed negligible difference in qualitative assay performance or Ct values. Use of frozen cells stocks in preparation of samples for analytical testing was therefore deemed acceptable.
Comparison of Performance with Natural and Simulated Matrices
To provide a sufficient quantity of material for testing, a simulated throat matrix was used for the majority of Analytical Studies. To represent a worst case scenario, for each sample, the ESwab Transport Medium was seeded with $150\mu \mathrm{L}$ of simulated matrix (equal to the absorptive capacity of the swab head). Studies were performed in accordance with the standard assay procedure by transferring $300\mu \mathrm{L}$ of the simulated matrix to the Xpert Xpress Strep A Assay cartridge.
To assess the suitability of the simulated matrix for use in analytical testing, a comparison study was conducted to confirm that the Xpert Xpress Strep A Assay performed similarly in the presence of natural and simulated throat swab matrix. Testing was performed with S. pyogenes ATCC BAA-946 at levels close to the LoD of the assay. The results are summarized in Table 10 and show that the pre-defined acceptance criteria were met at each target level and therefore that the assay exhibited similar analytical sensitivity when performed in the presence of either natural or simulated matrix. Ct values for the S. pyogenes and SPC targets were also similar in both matrices. The results of this study support the use of simulated matrix in the Analytical Studies to characterize the performance of the Xpert Xpress Strep A Assay.
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Table 10. Comparison of Xpert Xpress Strep A Assay performance with natural and simulated throat swab matrix
| Matrix | Target Level1 | N | Xpert Xpress Strep A Positive (%) | Ct Value | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | S. pyogenes2 | | SPC3 | |
| | | | Expected | Observed | Mean | SD | Mean | SD |
| Natural | Negative | 10 | 0(0) | 0(0) | NA | NA | 35.1 | 0.8 |
| | <1X | 10 | 1-9(10-90) | 6(60) | 39.8 | 1.3 | 34.9 | 0.5 |
| | 2X | 30 | ≥27(≥95) | 30(100) | 37.9 | 0.6 | NA | NA |
| | 5X | 10 | 10(100) | 10(100) | 36.4 | 0.5 | NA | NA |
| | 10X | 5 | 5(100) | 5(100) | 35.5 | 0.3 | NA | NA |
| Simulated | Negative | 10 | 0(0) | 0(0) | NA | NA | 34.4 | 0.4 |
| | <1X | 10 | 1-9(10-90) | 7(70) | 40.1 | 0.6 | 34.3 | 0.3 |
| | 2X | 30 | ≥27(≥95) | 30(100) | 38.2 | 0.9 | NA | NA |
| | 5X | 10 | 10(100) | 10(100) | 36.7 | 0.6 | NA | NA |
| | 10X | 5 | 5(100) | 5(100) | 35.8 | 0.3 | NA | NA |
NA: Not Applicable; SD: Standard Deviation; SPC: Sample Processing Control
1 Multiple of Limit of Detection (LoD = 9 CFU/mL ESwab Transport Medium)
Actual concentrations tested (CFU/150μL matrix): <1X: 3; 2X: 17; 5X: 42; 10X: 84
2 Values shown are for samples that were S. pyogenes-positive by the Xpert Xpress Strep A Assay
3 Values shown are for samples that were S. pyogenes-negative by the Xpert Xpress Strep A Assay
# 3. Clinical studies:
# a. Clinical Sensitivity:
The performance of the Xpert Xpress Strep A Assay was evaluated in two multicenter studies that were conducted at a total of nine (9) clinical sites in the U.S. Specimens were collected from subjects who presented with a sore throat and at least one other symptom that was characteristic of pharyngitis (e.g., tonsillar exudate, tender cervical lymphadenopathy or fever). All the specimens were collected using the Copan Liquid Amies Elution Swab (ESwab) Collection and Transport System and were used for both Xpert Xpress Strep A testing and the reference culture method. One study evaluated performance of the Xpert Xpress Strep A Assay using residual, de-identified specimens that were left over from standard of care testing $(1^{\mathrm{st}}$ swab), while the second study evaluated specimens that were collected prospectively under informed consent following the collection of swabs for use in standard of care patient management $(2^{\mathrm{nd}}$ swab).
Testing with the Xpert Xpress Strep A Assay was conducted at each of the clinical sites. Residual sample after Xpert testing was shipped at $2 - 8^{\circ}\mathrm{C}$ to a central laboratory for reference culture (inoculation of $10\mu \mathrm{L}$ transport medium onto Trypticase Soy Agar with $5\%$ Sheep Blood and incubation at $35^{\circ}\mathrm{C}$ in $5\%$ $\mathrm{CO}_{2}$ ). All reference cultures were inoculated within 48 hours of specimen collection. Isolated colonies
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that exhibited $\beta$ -hemolysis were typed by latex agglutination (Remel Streptex). Culture plates that did not exhibit $\beta$ -hemolytic colonies after 48 hours were recorded as negative for Group A Streptococcus.
Eight hundred and forty-four (844) specimens were initially enrolled in the two studies. Of these, 261 were excluded from the analysis of performance due to failure to comply with the inclusion criteria (19), reference culture procedural error (184), delay in reference culture inoculation (31), delay in shipment (26) or labeling error (1). A total of 583 specimens was therefore included in the analysis. On initial testing, 18/583 specimens $(3.1\%)$ produced indeterminate results (Error [9], Invalid [6], No Result [3]). Sixteen of the 18 were retested, of which 12 produced valid results that were included in the analysis of performance for a final indeterminate rate of 6/583 $(0.9\%)$ (Table 11).
Table 11. Expert Xpress Strep A Assay Clinical Performance vs Reference Culture (1 $^{\text{st}}$ and $2^{\text{nd}}$ swab data combined)
| | Reference Culture | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| Xpert Xpress Strep A | Positive | 138 | 261,2 | 164 |
| | Negative | 0 | 413 | 413 |
| | Total | 138 | 439 | 5773 |
| Sensitivity | | 138/138 = 100% (95% CI: 97.3-100%) | | |
| Specificity | | 413/439 = 94.1% (95% CI: 91.5-95.9%) | | |
| Positive Predictive Value | | 138/164 = 84.1% (95% CI: 77.8-88.9%) | | |
| Negative Predictive Value | | 413/413 = 100% (95% CI: 99.1-100%) | | |
95% CI: Two-sided 95% score confidence interval
25/26 specimens were tested by an alternative PCR assay with bi-directional sequencing of the amplified products; 21/25 were positive for S. pyogenes by this method
10/26 subjects were positive by a rapid antigen test method used as standard of care
3 On initial testing, 18/583 specimens $(3.1\%)$ produced indeterminate results (Error (9), Invalid (6), No Result (3)); 16/18 were retested, of which 12 produced valid results that were included in the analysis of performance for a final indeterminate rate of 6/583 $(0.9\%)$
The results of the two Clinical Studies were also analyzed separately to determine the performance of the Xpert Xpress Strep A Assay with residual clinical specimens from standard of care testing (1 $^{\text{st}}$ swab) and specimens that were collected prospectively (2 $^{\text{nd}}$ swab) (Table 12). Although both residual and prospectively collected specimen types yielded $100\%$ sensitivity, higher specificity was observed with the residual specimens (96.4% vs 90.9%).
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Table 12. Expert Xpress Strep A Assay Clinical Performance vs Reference Culture
| 1stswab: Residual Specimens | | | | |
| --- | --- | --- | --- | --- |
| | Reference Culture | | | |
| | | Positive | Negative | Total |
| Xpert Xpress Strep A | Positive | 65 | 91,2 | 74 |
| | Negative | 0 | 244 | 244 |
| | Total | 65 | 253 | 3183 |
| Sensitivity | | 65/65 = 100% (95% CI: 94.4-100%) | | |
| Specificity | | 244/253 = 96.4% (95% CI: 93.4-98.1%) | | |
| Positive Predictive Value | | 65/74 = 87.8% (95% CI: 78.5-93.5%) | | |
| Negative Predictive Value | | 244/244 = 100% (95% CI: 98.5-100%) | | |
| 2ndswab: Prospective Specimens | | | | |
| | Reference Culture | | | |
| | | Positive | Negative | Total |
| Xpert Xpress Strep A | Positive | 73 | 174,5 | 90 |
| | Negative | 0 | 169 | 169 |
| | Total | 73 | 186 | 2596 |
| Sensitivity | | 73/73 = 100% (95% CI: 95.0-100%) | | |
| Specificity | | 169/186 = 90.9% (95% CI: 85.9-94.2%) | | |
| Positive Predictive Value | | 73/90 = 81.1% (95% CI: 71.8-87.9%) | | |
| Negative Predictive Value | | 169/169 = 100% (95% CI: 97.8-100%) | | |
95% CI: Two-sided 95% score confidence interval
1 8/9 specimens were tested by an alternative PCR assay with bi-directional sequencing of the amplified products; 7/8 were positive for S. pyogenes by this method
2 All 9 subjects who had Xpert Xpress Strep A false positive results were also evaluated with a rapid antigen test method used as standard of care (SOC); 3/9 subjects were positive by the SOC test method
3 On initial testing, 9/321 specimens $(2.8\%)$ produced indeterminate results (Error (4), Invalid (4), No Result (1)); 7/9 were retested, of which 6 produced valid results that were included in the analysis of performance for a final indeterminate rate of 3/321 $(0.9\%)$
4 All 17 specimens were tested by an alternative PCR assay with bi-directional sequencing of the amplified products; 14/17 were positive for S. pyogenes by this method
5 All 17 subjects who had Xpert Xpress Strep A false positive results were also evaluated with an SOC rapid antigen test method; 7/17 subjects produced a positive result by the SOC test method
6 On initial testing, 9/262 specimens $(3.4\%)$ produced indeterminate results (Error (5), Invalid (2), No Result (2)); all 9 were retested, of which 6 produced valid results that were included in the analysis of performance for a final indeterminate rate of 3/262 $(1.1\%)$
The performance of the Xpert Xpress Strep A Assay stratified by clinical site and $1^{\text{st}}$ or $2^{\text{nd}}$ swab (residual or prospectively collected) is shown in Table 13. The Xpert Xpress Strep A Assay exhibited low specificity at Sites 5 and 6, although 7/16 subjects at these sites with false positive Xpress Strep A Assay results were positive for S. pyogenes by a
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standard of care rapid antigen test and 13/16 were positive by PCR/bidirectional sequencing. These results are acceptable.
Table 13. Xpert Xpress Strep A performance stratified by site and $1^{\text{st}}$ or $2^{\text{nd}}$ swab (residual or prospectively collected)
| Specimen Source | Site | Culture Positive (%) | Xpert Xpress Strep A (%; 95% Confidence Interval) |
| --- | --- | --- | --- |
| Sensitivity | Specificity1,2 |
| 1st Swab (Residual) | 1 | 28/157(17.8) | 28/28(100; 87.9-100) | 125/129(96.9; 92.3-98.8) |
| 3 | 37/161(23.0) | 37/37(100; 90.6-100) | 119/124(96.0; 90.9-98.3) |
| Total | 65/318(20.4) | 65/65(100; 94.4-100) | 244/253(96.4 93.4-98.1) |
| 2nd Swab (Prospective) | 2 | 11/37(29.7) | 11/11(100; 74.1-100) | 26/26(100; 87.1-100) |
| 4 | 3/23(13.0) | 3/3(100; 43.9-100) | 19/20(95.0; 76.4-99.1) |
| 5 | 4/17(23.5) | 4/17(100; 51.0-100) | 10/13(76.9; 49.7-91.8) |
| 6 | 38/111(34.2) | 38/38(100; 90.8-100) | 60/73(82.2; 71.9-89.3) |
| 7 | 9/39(23.1) | 9/9(100; 70.1-100) | 30/30(100; 88.7-100) |
| 8 | 6/17(35.3) | 6/17(100; 61.0-100) | 11/11(100; 74.1-100) |
| 9 | 2/15(13.3) | 2/15(100; 34.2-100) | 13/13(100; 77.2-100) |
| Total | 73/259(28.2) | 73/73(100; 95.0-100) | 169/186(90.9; 85.9-94.2) |
| Overall | 138/577(23.9) | 138/138(100; 97.3-100) | 413/439(94.1; 91.5-95.9) |
Standard of care rapid antigen test results on subjects with discordant Xpert Xpress Strep A and reference culture results: Site 1: 0/4 positive; Site 3: 3/5 positive; Site 4: 0/1 positive; Site 5: 2/3/ positive; Site 6: 5/13 positive
2 PCR/bidirectional sequencing results on subjects with discordant Xpert Xpress Strep A and reference culture results: Site 1: 3/4 positive (1 not tested); Site 3: 4/5 positive; Site 4: 1/1 positive; Site 5: 3/3/ positive; Site 6: 10/13 positive
b. Clinical specificity:
Refer to Section M(3)(a), above.
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable.
4. Clinical cut-off:
Not applicable.
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# 5. Expected values/Reference range:
The performance of the Xpert Xpress Strep A Assay was evaluated in a Clinical Study conducted at multiple sites in the US (Section M(3)(a)). The overall prevalence of S. pyogenes (Group A Streptococcus) in throat swab specimens was $28.4\%$ as determined by the Xpert assay and $23.9\%$ as determined by culture. The prevalence of Group A Streptococcus as determined by the Xpert assay is shown in Table 14, stratified by the age and gender of the subjects.
Table 14. Prevalence of S. pyogenes positive subjects by age and gender
| Age/Gender | Number | Xpert Xpress Strep A Positive | % Prevalence1 |
| --- | --- | --- | --- |
| 0 to 1 years | 3 | 0 | 0 |
| 2-5 years | 76 | 28 | 36.8 |
| 6-12 years | 189 | 80 | 42.3 |
| 13-21 years | 129 | 25 | 19.4 |
| >22-65 years | 170 | 30 | 17.6 |
| >65 years | 10 | 1 | 10.0 |
| Male | 251 | 74 | 29.5 |
| Female | 326 | 90 | 27.6 |
| Total | 577 | 164 | 28.4 |
As determined by the Xpert Xpress Strep A Assay
# N. Instrument Name:
GeneXpert Instrument Systems (GeneXpert Dx, GeneXpert Infinity-48s and GeneXpert Infinity-80).
# O. System Descriptions:
# 1. Modes of Operation:
Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device?
Yes X or No
Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission?
Yes or No X
# 2. Software:
FDA has reviewed applicant's Hazard Analysis and software development processes for
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this line of product types:
Yes ☐ X or No ☐
3. Specimen Identification:
Barcode scan or manual entry.
4. Specimen Sampling and Handling:
Throat swab samples are collected using the Copan Liquid Amies Elution Swab (ESwab) and transported to the testing laboratory. The test operator briefly shakes the ESwab tube containing the swab to elute the sample and adds 300μL of the transport medium to an Xpert Xpress Strep A Assay cartridge using a transfer pipette. The operator then loads the cartridge into the GeneXpert System and initiates the run from the user interface.
5. Calibration:
No calibration by the user is required.
6. Quality Control:
Quality control is addressed for each separately cleared assay for use on the GeneXpert systems. Refer to Section M(1)(c) for information on internal and external controls.
P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:
Not applicable.
Q. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
R. Conclusion:
The submitted information in this premarket notification is complete and supports substantial equivalence decision.
