XPERT MRSA/SA NASAL ASSAY

K100822 · Cepheid · NQX · Jun 1, 2010 · Microbiology

Device Facts

• Record ID: K100822
• Device Name: XPERT MRSA/SA NASAL ASSAY
• Applicant: Cepheid
• Product Code: NQX · Microbiology
• Decision Date: Jun 1, 2010
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.1640
• Device Class: Class 2

Intended Use

The Cepheid® Xpert® SA Nasal Complete Assay performed in the GeneXpert® Dx System is a qualitative in vitro diagnostic test designed for rapid detection of Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) from nasal swabs in patients at risk for nasal colonization. The test utilizes automated real-time polymerase chain reaction (PCR) to detect MRSA/SA DNA. The Xpert SA Nasal Complete Assay is intended to aid in the prevention and control of MRSA/SA infections in healthcare settings. The Xpert SA Nasal Complete Assay is not intended to diagnose, guide or monitor treatment for MRSA/SA infections, or provide results of susceptibility to methicillin. A negative result does not preclude MRSA/SA nasal colonization. Concomitant cultures are necessary to recover organisms for epidemiological typing or for further susceptibility testing.

Device Story

The Xpert SA Nasal Complete Assay is a rapid, automated, in vitro diagnostic test for simultaneous detection of SA and MRSA DNA from nasal swabs. The system uses disposable, multi-chambered fluidic cartridges processed on the GeneXpert Dx System. Input consists of nasal swab specimens; the system performs automated sample preparation, including cell lysis via ultrasonic horn and glass beads, followed by real-time multiplex PCR amplification. The system detects target sequences for staphylococcal protein A (spa), mecA gene, and SCCmec insertion. Output is a qualitative result (MRSA/SA positive/negative) provided in approximately 50 minutes. Used in healthcare settings to aid in infection prevention and control. Healthcare providers use results to manage patient colonization status; however, the device does not provide susceptibility data or clinical diagnostic confirmation, requiring concomitant cultures for epidemiological typing.

Clinical Evidence

Prospective multi-site study (n=2487) compared Xpert SA Nasal Complete Assay to enriched blood agar reference culture. MRSA sensitivity: 91.9% (95% CI: 86.8-95.5%); specificity: 97.9% (95% CI: 97.2-98.4%). SA sensitivity: 93.3% (95% CI: 91.1-95.1%); specificity: 90.5% (95% CI: 89.1-91.8%). Study included subjects with and without recent antibiotic use; no significant performance difference observed.

Technological Characteristics

Fully-automated nucleic acid amplification (real-time PCR). Materials: disposable multi-chambered plastic cartridges with integrated reaction tubes. Sensing: optical detection of fluorescent dye-labeled TaqMan probes. Energy: electrical (GeneXpert Dx System). Connectivity: standalone instrument with PC interface. Sterilization: not specified. Software: Version 2.1. Calibration: NIST-traceable thermal and optical calibration.

Indications for Use

Indicated for rapid detection of Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) from nasal swabs in patients at risk for nasal colonization. Not for diagnosing, guiding, or monitoring treatment of MRSA/SA infections or determining methicillin susceptibility.

Regulatory Classification

Identification

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

Predicate Devices

• Xpert™ MRSA/SA SSTI Assay (K080837)
• Xpert MRSA Assay (K070462)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT COMBINATION TEMPLATE

A. 510(k) Number:
k100822

B. Purpose for Submission:
To obtain a substantial equivalence determination for this 510k for MRSA/SA from nasal specimens.

C. Measurand:
Target DNA sequences for the staphylococcal protein A (spa), for methicillin/oxacillin resistance (mecA), and for the staphylococcal chromosomal cassette (SCCmec) insertion event into the staphylococcus aureus chromosomal attB site.

D. Type of Test:
Nucleic Acid Amplification Test, DNA, Methicillin-resistant Staphylococcus aureus (MRSA) / Staphylococcus aureus (SA), qualitative

E. Applicant:
Cepheid

F. Proprietary and Established Names:
Xpert™ Xpert SA Nasal Complete Assay

G. Regulatory Information:
1. Regulation section:
21 CFR 866. 1640 Antimicrobial susceptibility test powder
2. Classification:
Class II
3. Product code:
NQX- nucleic acid amplification test, DNA, methicillin resistant Staph aureus, direct specimen

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OOI- Nucleic acid amplification systems, real time

4. Panel:

83 Microbiology

## H. Intended Use:

### 1. Intended use(s):

The Cepheid® Xpert® SA Nasal Complete Assay performed in the GeneXpert® Dx System is a qualitative *in vitro* diagnostic test designed for rapid detection of *Staphylococcus aureus* (SA) and methicillin-resistant *Staphylococcus aureus* (MRSA) from nasal swabs in patients at risk for nasal colonization. The test utilizes automated real-time polymerase chain reaction (PCR) to detect MRSA/SA DNA. The Xpert SA Nasal Complete Assay is intended to aid in the prevention and control of MRSA/SA infections in healthcare settings. The Xpert SA Nasal Complete Assay is not intended to diagnose, guide or monitor treatment for MRSA/SA infections, or provide results of susceptibility to methicillin. A negative result does not preclude MRSA/SA nasal colonization. Concomitant cultures are necessary to recover organisms for epidemiological typing or for further susceptibility testing.

### 2. Indication(s) for use:

The Cepheid® Xpert® SA Nasal Complete Assay performed in the GeneXpert® Dx System is a qualitative *in vitro* diagnostic test designed for rapid detection of *Staphylococcus aureus* (SA) and methicillin-resistant *Staphylococcus aureus* (MRSA) from nasal swabs in patients at risk for nasal colonization. The test utilizes automated real-time polymerase chain reaction (PCR) to detect MRSA/SA DNA. The Xpert SA Nasal Complete Assay is intended to aid in the prevention and control of MRSA/SA infections in healthcare settings. The Xpert SA Nasal Complete Assay is not intended to diagnose, guide or monitor treatment for MRSA/SA infections, or provide results of susceptibility to methicillin. A negative result does not preclude MRSA/SA nasal colonization. Concomitant cultures are necessary to recover organisms for epidemiological typing or for further susceptibility testing.

### 3. Special conditions for use statement(s):

Prescription Use

### 4. Special instrument requirements:

GeneXpert® Dx System, Software version 2.1

## I. Device Description:

The Cepheid Xpert SA Nasal Complete Assay is a rapid, automated *in vitro* diagnostic test designed for rapid and simultaneous detection of *Staphylococcus aureus* (SA) and methicillin-resistant *Staphylococcus aureus* (MRSA) from nasal swabs in patients at risk for nasal colonization. The Xpert SA Nasal Complete Assay

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system performs real-time, multiplex polymerase chain reaction (PCR) for detection of DNA after an initial sample processing step. In this platform, additional sample preparation, amplification, and real-time detection are all fully-automated and completely integrated. The assay is performed on a GeneXpert® Dx System, which consists of the GeneXpert instrument, personal computer, hand-held barcode scanner, and disposable fluidic cartridges that are designed to complete sample preparation and realtime PCR for detection of MRSA and SA in approximately 50 minutes. Each instrument contains 1 to 16 randomly accessible modules that are each capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids, an ultrasonic horn for lysing cells or spores, and I-CORE® thermocycler for performing real-time PCR and detection.

The patented single-use cartridges contain: (1) eleven chambers for holding sample, reagents, or other materials, (2) a valve body composed of a plunger and syringe barrel, (3) a rotary valve system for controlling the movement of fluids between chambers, (4) an area for capturing, concentrating, washing, and lysing spores/cells, (5) dry real-time PCR reagents, and (6) an integrated PCR reaction tube that can be automatically filled by the instrument. To eliminate test-to-test contamination, all fluids including amplicons, are contained within the disposable cartridge. The instrument never comes into contact with any fluids within the cartridge. Each disposable cartridge is intended to test one sample. Cartridges are not re-usable.

The Xpert SA Nasal Complete Assay includes reagents for the simultaneous detection of the target organisms, MRSA and SA. The primers and probes in the Xpert SA Nasal Complete Assay detect nucleic acid sequences of the staphylococcal protein A (spa), the gene for methicillin/oxacillin resistance (mecA), and the staphylococcal cassette chromosome (SCCmec) inserted in the SA chromosomal attB site.

The primers and probes in the Xpert SA Nasal Complete Assay detect nucleic acid sequences of the staphylococcal protein A (spa), the gene for methicillin/oxacillin resistance (mecA), and staphylococcal cassette chromosome (SCCmec) inserted into the SA chromosomal attB site. Nasal swab specimens from the anterior nares are collected and transported to the GeneXpert® area. The swab specimen is placed in a tube containing elution reagent. Following a brief vortex, the eluted material and two other reagents are transferred to different chambers of the cartridge. The GeneXpert® performs sample preparation by mixing the eluted sample with the sample processing control (Bacillus globigii in the form of a dry spore cake within the cartridge) and treatment reagents, capturing the bacterial cells on a filter, lysing the cells using glass beads and an ultrasonic horn, then eluting the released DNA. The DNA solution is then mixed with dry PCR reagents and transferred into the PCR tube for real-time PCR and detection.

Note: Bacillus globigii spores are used as the sample preparation control because they are more difficult to lyse than Staphylococcus aureus.

## J. Substantial Equivalence Information:

1. Predicate device name(s):

Xpert™ MRSA/SA SSTI Assay

Xpert MRSA Assay

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2. Predicate 510(k) number(s):

K080837

K070462

3. Comparison with predicate:

Similarities

| | Device | Predicate | |
| --- | --- | --- | --- |
| Item | Xpert SA Nasal Complete Assay | Xpert MRSA/SA SSTI Assay (K080837) | Xpert MRSA Assay (K070462) |
| Intended Use | Rapid detection of MRSA and SA | Same | MRSA only |
| Indication for Use | Identification of MRSA and SA | Same | MRSA only |
| Technological Principles | Fully-automated nucleic acid amplification (DNA); real-time PCR | Same | Same |
| Test Cartridge | Disposable single-use, multi-chambered, fluidic cartridge. | Same | Same |
| Instrument System | Cepheid GeneXpert Dx System | Same | Same |
| Fluidics | Self-contained and automated after specimen preparation | Same | Same |
| Probes | TaqMan® Probes | Same | Same |
| Internal Controls | Sample processing control (SPC) and probe check control (PCC). | Same | Same |
| DNA Target Sequence | Sequence incorporating the insertion site (attBssc) of Staphylococcal Cassette Chromosome mec (SCCmec) for detection of MRSA. | Same | Same |
| | Sequence specific to methicillin/oxacillin resistance (mecA gene) | Same | N/A, assay does not detect SA |
| Rapid test results | Approximately 50 minutes to results. | Same | Approximately 75 minutes. |

Differences

| Intended Use | Simultaneous rapid detection of SA and MRSA. | Same | Only detects MRSA |
| --- | --- | --- | --- |
| Specimen Type | Direct from nasal swabs. | Direct from skin and soft tissue infection swabs. | Direct from nasal swabs. |
| DNA Target Sequence | Sequence specific to Staphylococcus aureus species (spa gene) | Same | NA |
| Software version | 2.1 | 1.6b | 1.6 |

K. Standard/Guidance Document Referenced (if applicable):

Not applicable

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# L. Test Principle:

The primers and probes in the Xpert SA Nasal Complete Assay detect the presence of proprietary sequences for the staphylococcal protein A (Spa), the gene for methicillin/oxacillin resistance (mecA), and the staphylococcal cassette chromosome (SCCmec) inserted into the SA chromosomal attB site. Nasal swabs from the anterior nares are collected and transported to the GeneXpert® area. The swab is placed in a tube containing elution reagent. Following a brief vortex, the eluted material and two other liquid reagents are transferred to different chambers of the cartridge. The GeneXpert® Dx System performs sample preparation by mixing the eluted sample with the sample preparation control (Bacillus globigii in the form of a dry spore cake within the cartridge) and treatment reagents, capturing the bacterial cells on a filter, lysing the cells using glass beads and an ultrasonic horn, then eluting the released DNA. The DNA solution is then mixed with dry PCR reagents and transferred into the PCR tube for real-time PCR and detection.

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

# 1. Analytical performance:

# a. Precision/Reproducibility:

A Reproducibility study was performed using a panel of 10 specimens with varying concentrations of SA, MRSA added to a mixture of human blood and porcine mucin in a background of methicillin-susceptible Staphylococcus epidermidis (MSSE) were tested in duplicate on 10 different days at each of the three sites (10 specimens x 2 times/ day x 10 days x 3 sites). One lot of Xpert SA Nasal Complete kit was used at each of the 3 testing sites.

Summary of Reproducibility Results a

| Specimen ID | Site 1 | Site 2 | Site 3 | Total Agreement |
| --- | --- | --- | --- | --- |
| Negative (MSSE) | 100.0% (20/20) | 100.0% (20/20) | 100.0% (20/20) | 100.0% (60/60) |
| MSSA – High Neg | 95.0% (19/20) | 95.0% (19/20) | 95.0% (19/20) | 95.0% (57/60) |
| MSSA – Low Pos | 85.0% (17/20) | 95.0% (19/20) | 100.0% (20/20) | 93.3% (56/60) |
| MSSA – Mod Pos | 100.0% (20/20) | 100.0% (20/20) | 100.0% (20/20) | 100.0% (60/60) |
| MRSA1 – High Neg | 100.0% (20/20) | 95.0% (19/20) | 85.0% (17/20) | 93.3% (56/60) |
| MRSA1 – Low Pos | 95.0% (19/20) | 95.0% (19/20) | 100.0% (20/20) | 96.7% (58/60) |
| MRSA1 – Mod Pos | 95.0% (19/20) | 100.0% (20/20) | 100.0% (20/20) | 98.3% (59/60) |
| MRSA2 – High Neg | 60.0% (12/20) | 60.0% (12/20) | 50.0% (10/20) | 56.7% (34/60) |
| MRSA2 – Low Pos | 95.0% (19/20) | 95.0% (19/20) | 95.0% (19/20) | 95.0% (57/60) |
| MRSA2 – Mod Pos | 100.0% (20/20) | 100.0% (20/20) | 100.0% (20/20) | 100.0% (60/60) |
| % Total Agreement by Site | 92.5% (185/200) | 93.5% (187/200) | 92.5% (185/200) | 92.8% (557/600) |

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a For negative and high negative samples, %Agreement = (# negative results/total samples run); for low and moderate positive samples, %Agreement = (# positive results/total samples run).

## Summary of Ct value results by sample level and probe

| SPC | | | |
| --- | --- | --- | --- |
| Level | Mean | Std Dev | %CV |
| Neg (MSSE) | 34.3 | 0.72 | 2.1% |
| MSSA High Neg | 34.3 | 0.75 | 2.2% |
| MRSA1 High Neg | 34.6 | 0.86 | 2.5% |
| MRSA2 High Neg | 34.6 | 0.75 | 2.2% |
| Level | mean | Std Dev | %CV |
| --- | --- | --- | --- |
| MSSA Low Pos | 33.7 | 0.91 | 2.7% |
| MSSA Moderate Pos | 31.6 | 0.71 | 2.2% |
| MRSA1 Low Pos | 32.6 | 1.53 | 4.7% |
| MRSA1 Moderate Pos | 31.7 | 0.79 | 2.5% |
| MRSA2 Low Pos | 32.7 | 0.97 | 3.0% |
| MRSA2 Moderate Pos | 30.6 | 0.85 | 2.8% |
| Level | mean | Std Dev | %CV |
| --- | --- | --- | --- |
| MRSA1 Low Pos | 33.3 | 0.88 | 2.6% |
| MRSA1 Moderate Pos | 32.2 | 0.82 | 2.5% |
| MRSA2 Low Pos | 33.4 | 1.02 | 3.1% |
| MRSA2 Moderate Pos | 31.1 | 0.75 | 2.4% |
| SCCmec | | | |
| --- | --- | --- | --- |
| Level | Mean | Std Dev | %CV |
| MRSA1 Low Pos | 34.1 | 0.86 | 2.5% |
| MRSA1 Moderate Pos | 32.9 | 0.79 | 2.4% |
| MRSA2 Low Pos | 34.6 | 1.19 | 3.4% |
| MRSA2 Moderate Pos | 32.5 | 0.80 | 2.5% |

## b. Linearity/assay reportable range:

Not applicable

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

The Xpert SA Nasal Complete Assay includes a Sample Processing Control (SPC) and a Probe Check Control (PCC). The success rate was >95% on the first attempt and was acceptable.

Recommended external quality control organisms were also tested at the sites. External Controls may be used in accordance with accrediting institutions and government regulations. External Controls are not provided in the test kit; however, the outside source and the catalog numbers are provided in the "Materials Available but Not Provided" section of the Xpert SA Nasal Complete Assay Package Insert.

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d. Detection limit:

# Limit of Detection Study

The Limit of Detection study was determined using Staphylococcus aureus (SA) cells and methicillin-resistant Staphylococcus aureus (MRSA) cells diluted into a simulated nasal matrix. The nasal matrix consisted of mucin and $1\%$ of blood in PBS with $15\%$ glycerol. The limit of detection is defined as the lowest number of colony forming units (CFU) per sample that can be reproducibly distinguished from negative samples with $95\%$ confidence or the lowest concentration at which 19 of 20 replicates were positive.

For SA, replicates of 20 were evaluated at various concentrations for three (3) individual isolates. USA types USA900 and USA1200 were represented.

For MRSA, replicates of 20 were evaluated at various concentrations for ten (10) individual isolates representing SCCmec types I, II, III, IVa, IVd, V, VI, VII, and VIII. When characterized by pulsed-field gel electrophoresis (PFGE), USA100 and USA400 are represented. Isolates of heterogeneous oxacillin resistant subpopulations were included.

The estimate and confidence intervals were determined using logistic regression with data (number of positive results per number of replicates at each level) over the range of CFU/swab tested. The confidence intervals were determined using maximum likelihood estimates on the logistic model parameters using the large sample variance-covariance matrix. The LoD point estimates and $95\%$ upper and lower confidence intervals for each SA and each MRSA SCCmec type tested are summarized in tables below:

95% Confidence Intervals for Analytical LoD - SA

| SA Strain ID | PFGE | LoD (CFU/swab) | Lower 95% CI (CFU/swab) | Upper 95% CI (CFU/swab) |
| --- | --- | --- | --- | --- |
| N7129 | USA900 | 154 | 132 | 197 |
| 102-04 | USA1200 | 128 | 109 | 177 |
| 29213 | unknown | 94 | 76 | 138 |

95% Confidence Intervals for Analytical LoD - MRSA

| MRSA Strain ID | SCCmec Type | PFGE | LoD (CFU/swab) | Lower 95% CI (CFU/swab) | Upper 95% CI (CFU/swab) |
| --- | --- | --- | --- | --- | --- |
| 64/4176 | I | USA500 | 79 | 64 | 119 |
| N315 | II | USA100 | 94 | 76 | 131 |
| BK2464 | II | USA100 | 143 | 116 | 212 |
| 11373 | III | unknown | 52 | 42 | 77 |
| MW2 | IVa | USA400 | 85 | 69 | 130 |
| BK2529* | IVd | USA500 | 256 | 216 | 334 |
| ST59-MRSA-V | V | USA1000 | 127 | 105 | 170 |
| HDE288* | VI | USA800 | 97 | 78 | 141 |
| JCSC6082 | VII | unknown | 214 | 182 | 276 |
| WA MRSA16 | VIII | unknown | 292 | 259 | 384 |

(*) Heterogeneous oxacillin-resistant isolates

The results of this study indicate that the Xpert SA Nasal Complete Assay will

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produce a positive SA result 95% of the time with 95% confidence for a nasal swab containing 175 CFU and a positive MRSA result 95% of the time with 95% confidence for a nasal swab containing 300 CFU.

## Effect of Competing Amounts of SA on the Limit of Detection of MRSA

An additional study was conducted to determine the effect of increasing amounts of methicillin-susceptible Staphylococcus aureus (MSSA) cells on the Limit of Detection (LoD) of methicillin-resistant Staphylococcus aureus (MRSA) cells established for the Xpert SA Nasal Complete Assay.

The competitive inhibitory effect of increasing amounts of SA relative to MRSA at LoD was evaluated for each SCCmec type I, II, III, IVa, V, VI, VII, and VIII.

For MRSA SCCmec types I, II, III, IVa, V, VI, and VII, the effect of competing MSSA is inhibitory at the claimed LoD at MRSA: MSSA ratios of 1:1×10⁶.

For MRSA SCCmec type VIII, the effect of competing MSSA is inhibitory at the claimed LoD at a MRSA: MSSA ratio of 1:1×10³.

## Analytical Inclusivity

A total of 248 isolates (199 MRSA strains and 49 methicillin sensitive S. aureus strains) were tested using the Xpert SA Nasal Complete Assay. All strains were tested in triplicate using bacterial cell stocks diluted to concentrations at or near the assay cut-off. Known USA300 isolates (39 in total), including USA300-0114, are represented. Other PFGE types included in the study are USA100 (54), USA200 (5), USA400 (1), USA500 (7), USA600 (2), USA700 (2), USA800 (9), USA1000 (8), USA1100 (2), IBERIAN isolates (2), and strains designated PGFE types A, B, and F. All results are as expected.

## Evaluation of Empty Cassette Variants:

In another study, 22 Staphylococcus aureus isolates identified as "empty cassette variants" received from a hospital were tested using the Xpert SA Nasal Complete Assay. All strains were tested at cell concentrations near the analytical LoD, at ~300 CFU/test and at ~3×10⁵ CFU/test.

All 22 isolates were identified as MRSA negative and SA positive. No mecA signals were reported.

## e. Analytical specificity:

### Cross Reactivity

A total of 114 strains (83 Gram positive, 28 Gram negative, and three yeasts) phylogenetically related to Staphylococcus aureus or those potentially present in nasopharyngeal flora were tested. There were 103 strains from the American Type Culture Collection (ATCC), seven strains from the Network

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on Antimicrobial Resistance in Staphylococcus aureus (NARSA); the remaining four isolates were from Sweden, Germany and Japan. The isolates included 34 methicillin-sensitive coagulase negative staphylococci (MSCoNS), and 12 methicillin-resistant coagulase negative staphylococci (MRCoNS).

Each strain was tested using 100 μL of culture adjusted to 4.5 to 9.5×10⁸ CFU/mL. Three replicates of each strain were tested. All isolates were reported MRSA Negative; SA Negative by the Xpert SA Nasal Complete Assay.

## Evaluation of BORSA Strains:

All seven BORSA isolates (including the apparent "empty cassette" isolate) were reported MRSA negative; SA positive at both high and low cell concentrations using the Xpert SA Nasal Complete Assay. No mecA signals were reported.

## Interference Study

In another analytical study, potential interfering substances that may be present in clinical nasal specimen were tested with the following results:

Summary of Test Results per Substance and Concentration Tested

| Substance | Conc Tested | Accurate Tests per # of Replicates Tested | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Negs* | MSSA | | MRSA^ | |
| | | | N7129 | 102-04 | type II | type IVa |
| Buffer (Control) | 100% (v/v) | 24/24 | 16/16 | 8/8 | 16/16 | 8/8 |
| Anefrin Nasal Spray | 100% (v/v) | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Blood | 100% (v/v) | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Flonase | 100% (v/v) | 2/8 | 8/8 | - | - | - |
| | 50% (v/v) | 3/8 | 8/8 | 8/8 | - | - |
| | 25% (v/v) | 4/8 | 8/8 | 8/8 | - | - |
| | 10% (v/v) | 7/8 | - | - | 6/8 | 7/8 |
| | 5% (v/v) | 8/8 | - | - | 7/8 | 8/8 |
| | 1% (v/v) | 8/8 | - | - | 8/8 | - |
| Mucin | 5% (w/v) | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| NasalCrom | 100% (v/v) | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Nasonex | 100% (v/v) | 2/8 | 8/8 | - | - | - |
| | 50% (v/v) | 4/8 | 8/8 | 8/8 | - | - |
| | 25% (v/v) | 6/8 | - | 8/8 | - | - |
| | 10% (v/v) | 8/8 | - | - | 8/8 | 7/8 |
| | 5% (v/v) | 8/8 | - | - | - | 8/8 |
| Neo-Synephrine | 100% (v/v) | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Rhinolast (Astelin) | 100% (v/v) | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Saline Nasal Spray | 100% (v/v) | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Zicam Nasal Gel | 100% (v/v) | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |

(*) In negatives per substance (excluding buffer control), if not 8/8, then Invalid or Error test result(s) reported

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(^) In MRSA positives per substance (excluding buffer control), if not 8/8, then false negative test result(s) reported
(-) Level of substance not tested

Inhibitory effects resulting in invalid test results were observed in the presence of Flonase or Nasonex in negative samples at concentrations greater than 5% (v/v) and 10% (v/v), respectively.

Inhibitory effects resulting in MRSA false negative test results were observed in the presence of Flonase at greater than 1% (v/v) and in the presence of Nasonex greater than 5% (v/v).

**Carry-Over Contamination** study consisted of a negative sample (no MRSA cells spiked into the elution buffer) processed within the same GeneXpert module immediately following a very high positive sample (1×10⁷ MRSA cells spiked into the elution buffer). This was repeated 20 times between two GeneXpert modules for a total of 42 runs (10 positive samples per module and 11 negative samples per module). The study demonstrated no evidence of carry-over contamination.

f. Assay cut-off:

The valid minimum cycle threshold setting for spa, mecA and SCCmec is 10 because in practice, a Ct cannot be calculated before the end of the minimum background subtraction range, which is 10 cycles. The earliest spa Ct reported in true SA and MRSA Positive results during pre-clinical testing was 16.5.

The valid maximum cycle threshold (Ct) setting for spa, mecA, and SCCmec is 35.0, 36.0, and 38.0 respectively.

To obtain a valid SA Positive test result, the spa Ct must be reported within the valid cycle range. To obtain a valid MRSA Positive test result, all spa, mecA, and SCCmec Cts must be reported within the valid cycle range. To obtain a valid MRSA or SA Negative test result, the spa, mecA, and SCCmec Cts must not be reported within the valid cycle range and the SPC Ct must be reported within its valid cycle range. If the SPC falls outside the valid cycle range, the test result is Invalid and must be repeated.

The valid maximum cycle settings (assay cut-offs) for the Xpert SA Nasal Complete targets (spa, mecA, and SCCmec) were derived from pre-clinical data (n=736) collected using clinical specimens to maximize percent sensitivity and percent specificity relative to enriched culture. The SA specificity was poor if the spa Ct cut-off was moved from 35 (91.5%) to 36 (88.6%) and poor SA sensitivity was calculated if the spa Ct cut-off was moved from 35 (92.7%) to 34 (90.0%). These assay cut-offs were subsequently validated in the pivotal clinical study, Protocol 128. The clinical study supports and validates the Ct cut-offs chosen for spa, mecA, and SCCmec targets. From the analysis it is demonstrated that the established assay settings maximize sensitivity (highest priority) and specificity.

2. Comparison studies:

a. Method comparison with predicate device:

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Not applicable

b. Matrix comparison:

Not applicable

3. Clinical studies:

Clinical Sensitivity/Specificity:

Performance characteristics were determined in a multi-site prospective investigation study at eight US institutions by comparing the Xpert SA Nasal Complete Assay with Reference Culture. Double swabs were collected from each subject. One swab was tested by the Xpert SA Nasal Complete Assay at the enrolling center and the other swab was sent to the central laboratory for Reference Culture testing.

At the centralized laboratory, the specimen was enriched overnight in trypticase soy broth with 6.5% NaCl. The trypticase soy broth was then streaked onto a sheep blood agar plate. Confirmation of presumptive positive colonies was performed with catalase, tube coagulase, and Gram stain. MecA-Mediated Oxacillin resistance was tested by disk diffusion test using a 30μg cefoxitin disk and cutoff of ≤21 mm (R), ≥22 mm (S).

Performance of the Xpert SA Nasal Complete Assay was calculated relative to the Reference Culture (enriched blood agar culture) results. The overall performance of the Xpert SA Nasal Complete Assay relative to direct CHROMagar, and enriched CHROMagar methods was for information only.

Overall Results

A total of 2487 specimens were tested for MRSA and SA by Xpert the SA Nasal Complete Assay and enriched blood agar culture. Among the eligible 2487 cases, there were 141 subjects who had antibiotic use within 7 to 21 days prior to sample collection, 2323 subjects with confirmed no antibiotic use, and 23 cases of unknown antibiotic status. There was no statistically significant difference in the culture positivity rate or the Xpert SA Nasal Complete Assay performance based on antibiotic status.

The performance of the Xpert SA Nasal Complete Assay is summarized in the tables below:

Xpert

Reference Culture

| | MRSA+ | SA+/MRSA- | Neg/No Growth | Total |
| --- | --- | --- | --- | --- |
| MRSA+ | 159 | 24 | 25 | 208 |
| SA+/MRSA- | 9 | 393 | 152 | 554 |
| SA- | 5 | 37 | 1683 | 1725 |
| Total | 173 | 454 | 1860 | 2487 |
| MRSA: Sensitivity: 91.9% (159/173) (95% CI: 86.8-95.5%) | | | | |

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| Specificity: | 97.9% (2265/2314) (95% CI: 97.2-98.4%) |
| --- | --- |
| PPV: | 76.4% (159/208) (95% CI: 70.1-82.0%) |
| NPV: | 99.4% (2265/2279) (95% CI: 99.0-99.7%) |
| SA: | |
| Sensitivity: | 93.3% (585/627) (95% CI: 91.1-95.1%) |
| Specificity: | 90.5% (1683/1860) (95% CI: 89.1-91.8%) |
| PPV: | 76.8% (585/762) (95% CI: 73.6-79.7%) |
| NPV: | 97.6% (1683/1725) (95% CI: 96.7-98.2%) |

Of the Xpert Assays run on eligible specimens, $96.5\%$ (2487/2578) of these specimens were successful on the first attempt. The remaining 91 gave indeterminate results on the first attempt (31 "INVALID", 51 "ERROR" and 9 "NO RESULT"). The study design did not allow for repeat testing.

# Empty Cassette Variants

There were 14 isolates that fit the empty cassette profile (i.e. spa positive, SCCmec positive, mecA negative) in the clinical studies. All of the 14 specimens were verified true negative MRSA isolates, and true positive SA isolates relative to Reference Culture.

c. Other clinical supportive data (when a. and b. are not applicable):

Not applicable

# 4. Clinical cut-off:

Not applicable

# 5. Expected values/Reference range:

A total of 2487 nasal specimens were included from eight institutions across the United States. The number and percentage of positive cases by the reference culture method, calculated by age group, are presented in the tables below.

Observed Prevalence of MRSA and SA by Reference Culture

| Age Group | Total N | MRSA By Culture | | SA By Culture | |
| --- | --- | --- | --- | --- | --- |
| | | Number Positive | Observed Prevalence | Number Positive | Observed Prevalence |
| Ages 22 to 30 | 325 | 10 | 3.1% | 71 | 21.8% |
| Ages 31 to 40 | 359 | 17 | 4.7% | 84 | 23.4% |
| Ages 41 to 50 | 459 | 28 | 6.1% | 118 | 25.7% |
| Ages 51 to 60 | 487 | 36 | 7.4% | 141 | 29.0% |
| Ages 61 to 70 | 315 | 25 | 7.9% | 75 | 23.8% |
| Ages >70 | 542 | 57 | 10.5% | 138 | 25.5% |

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N. Instrument Name: The GeneXpert® Dx Instrument

O. System Descriptions:

1. Modes of Operation:

The GeneXpert Dx System operates in random access mode. The instrument is available in configurations with one module base, four module bases, and 16 module bases. The multiple module bases allow one (1), up to four (4) and up to 16 single use cartridges, respectively, to be run simultaneously. Up to four of the four module base GeneXpert Dx Systems may be linked together.

2. Software:

FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:

☐ Yes ☑ or No

The software that controls the operation of the sample processing and the I-CORE module, and collects, analyzes and interprets the acquired optical data is the GeneXpert Dx Version 2.1 software.

3. Specimen Identification: Barcode

3. Specimen Sampling and Handling: Automated

GeneXpert Dx System Hardware Components for Automated Sample Processing

| Module Hardware Components | Function |
| --- | --- |
| Valve Drive | Rotates the cartridge valve body to address the different cartridge chambers. |
| Syringe Pump drive | Dispenses fluids to and from the different cartridge chambers. |
| Ultrasonic horn | Lyses the bacterial cells and sample prep control. |

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| I-CORE® module | Performs PCR amplification and detection. As the user inserts the cartridge into the system, the reaction tube component of the cartridge is inserted into the I-CORE module. After sample preparation within the cartridge, the sample and reagent mixture is transferred from the cartridge chamber into the reaction tube. During the amplification process, the I-CORE heater heats up and the fan cools down the reaction tube contents. Two optical blocks positioned within the ICORE excite the dye molecules that make up the probes and detect the fluorescence emitted. The system uses calibration and data analysis algorithms to determine a relative fluorescence value for each reporter dye after each thermal cycle. |
| --- | --- |
| Hand-held Barcode Scanner | Scans cartridge barcode and optional Patient or Sample ID barcode into the GeneXpert Dx System. |

5. Calibration:

Optical and thermal calibration of the GeneXpert Dx System is performed by Cepheid at the time of manufacture prior to installation and once yearly or after 1000 runs per module. The user does not calibrate or perform any serviceable functions on the instrument. The normalization function compensates for any optical degradation between calibrations.

The thermal reaction chamber thermistors are calibrated to $\pm 0.50^{\circ}\mathrm{C}$ using National Institute of Standards and Technology (NIST)-traceable standards. During the manufacturing process, the temperature of the heating system is measured at two temperatures: $60^{\circ}\mathrm{C}$ and $95^{\circ}\mathrm{C}$. Calibration coefficients that correct for small errors in the raw thermistor readings of the heaters are stored in the memory of each I-CORE module.

The optical system is calibrated using standard concentrations of individual unquenched fluorescent dye-oligos. For each optical channel, the signal produced by a tube alone (the blank signal) is subtracted from the raw signal produced by the dye-oligo standard to determine the spectral characteristics. Using the individual spectral characteristics of the pure dye-oligos, signals from an unknown mixture of dye-oligos can be resolved into corrected signals for the individual dye-oligos in the mixture.

6. Quality Control:

The Xpert SA Nasal Complete Assay test includes a Sample Processing Control (SPC) and Probe Check Control (PCC) pre-loaded in the cartridge and provided with the assay.

The SPC verifies that conditions adequate for the processing of the target SA and MRSA bacteria have occurred. It consists of Bacillus globigii spores formulated into a dry reagent bead included in each Xpert SA Nasal Complete cartridge. Furthermore the SPC verifies (1) the effectiveness of each sample preparation step, (2) reaction tube filling, (3) that all reaction components are present and functioning, and monitors the presence of potential inhibitor(s) in the PCR assay.

The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability. The PCC is considered to PASS

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if the fluorescence generated meets the validated acceptance criteria. If the PCC fails for any SA or MRSA target or SPC, a probe check error is reported and the test will not continue. For these conditions, the test will need to be repeated using a new specimen, a new cartridge and new reagents.

## P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

### Shelf-life

The Xpert MRSA/SA Assay shelf-life was demonstrated in stability studies using real-time stability results and linear regression analysis to support a shelf-life of 12 months when the reagents and cartridge are stored at 2-28°C; open package for up to 24 hours at 2-28°C. Currently there are three lots that have 9 months of real time data, and one lot has 12 months of real time data.

The stability of the product is being evaluated at four temperatures (5° ± 3°C, 25° ± 3°C, 35° ± 3°C and 45° ± 3°C) at predefined time point intervals up to 36 months following the study plan described in the 510k submission. One lot has an extra time point for both 4°C and 25°C storage conditions.

A Specimen Stability study was conducted to establish specimen transport and storage claims for the Xpert SA Nasal Complete Assay. The following collection device types were tested:

- LSD: Liquid Stuart-Dry (Cepheid Sample Collection Device)
- LSW: Liquid Stuart-Wet (swab pre-moistened with sterile 0.9% NaCl solution)
- LAW: Liquid Amies-Wet (swab pre-moistened with the provided transport medium)

The recommended storage conditions are room temperature (15 - 28°C) of up to 24 hours or refrigerated 2-8°C for up to five days. The study supported this claim.

Failure Modes Testing was performed to determine the effect of failure modes that might occur with the Xpert SA Nasal Complete Assay. Failures may be due to operator errors, manufacturing errors or instrument malfunction.

Operator error might include adding an insufficient amount of reagent to the cartridge or omitting a liquid reagent or adding a liquid reagent to the wrong chamber.

A manufacturing error might result in beads being loaded incorrectly into the cartridge before packaging (missing beads or double beads), or a cartridge being assembled incorrectly (missing the filter required for cell capture).

An instrument malfunction examples include an ultrasonic horn failure, motion of the syringe drive not detected, syringe pressure reading exceeds protocol limit, the system failed to find the plunger home position, a valve positioning error was detected, digital temperature reading of thermistor(s) not within acceptable range, and the optical signal from the detector(s) did not reach the expected value. Because the software performs self-check procedures prior to the start of each

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test, if any of the instrument malfunctions described above occur the test is aborted; no assay results are reported.

Replicates of eight (8) MRSA negative and eight (8) MRSA positive samples were tested per condition. Positive samples contained MRSA cells at ~600 CFU/test. Results are compared to positive and negative controls in which all cartridges are assembled correctly (1 EZR- enzyme bead, 1 TSR-primer/probe bead, 1 SPC- sample processing bead, and a capture filter) and all liquid reagents (reagent 1, reagent 2, and elution buffer) are dispensed correctly and completely. Of the 354 runs, 3 provided uninformative GeneXpert results (Error). Errors consisted of 1 pressure abort, 1 probe check control error, and 1 valve positioning error.

**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 a substantial equivalence decision.