GeneSTAT.MDx Coccidioides Assay

• K Number: DEN170041
• Device Name: GeneSTAT.MDx Coccidioides Assay
• Manufacturer: DxNA, LLC
• Date Cleared: 2017-11-29 ( 120 days)
• Product Code: QAA
• Regulation Number: 866.3376

Intended Use

The GeneSTAT.MDx Coccidioides Assay is a qualitative real-time polymerase chain reaction (PCR) in vitro diagnostic test for the detection of nucleic acids from Coccidioides immitis and Coccidioides posadasii. The assay does not differentiate between these two species. The assay is performed on bronchial alveolar lavage (BAL) or bronchial wash (BW) specimens from patients presenting with respiratory signs and symptoms consistent with coccidioidomycosis. The GeneSTAT.MDx Coccidioides Assay is intended to be used along with clinical findings and other laboratory results as an aid in the diagnosis of coccidioidomycosis in patients with possible or probable exposure to Coccidioides immitis or Coccidioides posadasii.

Device Description

The GeneSTAT.MDx Coccidioides Assay is a qualitative real-time PCR-based assay to detect target Coccidioides spp. DNA from bronchial alveolar lavage (BAL) or bronchial wash (BW) samples. The sample is prepared by a lysis and extraction process, with the extracted DNA then tested with the GeneSTAT.MDx Coccidioides assay on the GeneSTAT System. The GeneSTAT System is a real-time PCR nucleic acid testing platform. The GeneSTAT system consists of: - The single-use, disposable, assay-specific Test Module cartridge that contains the real-time PCR reagents, internal control, reaction wells, and test parameters. The single-use cartridge contains all the necessary reagents for amplification and detection of the Coccidioides target DNA as well as a human DNA control sequence that is used to monitor the presence of inhibitors in the PCR reaction and to ensure that the sample preparation process is adequate. All information to run the assay as well as lot number and expiration date is contained on the Radio-Frequency Identification (RFID) tag on the cartridge. The cartridge contains two PCR reaction wells. One well contains PCR reagents for amplification and detection of Coccidioides DNA and the human gene internal control (Reaction Well 1). The second well (Reaction Well 2) contains the same reagents for amplification/detection of Coccidioides DNA as used in Reaction Well 1 but does not contain the PCR reagents to detect the internal control. - The GeneSTAT Analyzer instrument that performs the mechanical, optical, and fluidic actions upon the Test Module to perform the PCR reactions. The instrument generates, analyzes, and transmits results to a connected pre-configured computer for presentation to the user. One single-use Test Module can be tested at a time in each GeneSTAT Analyzer. Up to four GeneSTAT Analyzers can be connected to single computer. - A MS Windows based laptop computer - The associated software and firmware required to run the test, analyze the results, and present the results to the user. - The Performance Verification Test Cartridges (PVT) that are used to periodically verify that the analyzer is functioning within acceptable limits. A successful PVT run is required to activate the analyzer. The PVT should be run at minimum every six months, and it is recommended that a PVT should be run monthly.

Predicate Devices

Not Found

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on standard real-time PCR technology and analysis of results, with no mention of AI or ML algorithms for data interpretation or decision making.

Therapeutic

No
The device is an in vitro diagnostic test for the detection of Coccidioides nucleic acids, intended as an aid in diagnosis, not for treating or preventing a disease.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the device is "an aid in the diagnosis of coccidioidomycosis."

SaMD (Software as a Medical Device)

No

The device description explicitly states that the GeneSTAT System includes hardware components such as the GeneSTAT Analyzer instrument and single-use Test Module cartridges, in addition to software and firmware.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

• Intended Use/Indications for Use: The description explicitly states it is an "in vitro diagnostic test" and is intended to be used "as an aid in the diagnosis of coccidioidomycosis." This directly aligns with the definition of an IVD, which is used to examine specimens from the human body to provide information for diagnosis, monitoring, or treatment.
• Device Description: The device analyzes biological specimens (BAL or BW) outside of the body using a laboratory technique (real-time PCR) to detect specific nucleic acids. This is characteristic of an in vitro diagnostic device.
• Intended User/Care Setting: The statement "For in vitro diagnostic use only" further confirms its classification as an IVD.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The GeneSTAT.MDx Coccidioides Assay is a qualitative real-time polymerase chain reaction (PCR) in vitro diagnostic test for the detection of nucleic acids from Coccidioides immitis and Coccidioides posadasii. The assay does not differentiate between these two species. The assay is performed on bronchial alveolar lavage (BAL) or bronchial wash (BW) specimens from patients presenting with respiratory signs and symptoms consistent with coccidioidomycosis. The GeneSTAT.MDx Coccidioides Assay is intended to be used along with clinical findings and other laboratory results as an aid in the diagnosis of coccidioidomycosis in patients with possible or probable exposure to Coccidioides immitis or Coccidioides posadasii.

Product codes (comma separated list FDA assigned to the subject device)

QAA

Device Description

The GeneSTAT.MDx Coccidioides Assay is a qualitative real-time PCR-based assay to detect target Coccidioides spp. DNA from bronchial alveolar lavage (BAL) or bronchial wash (BW) samples. The sample is prepared by a lysis and extraction process, with the extracted DNA then tested with the GeneSTAT.MDx Coccidioides assay on the GeneSTAT System.

The GeneSTAT System is a real-time PCR nucleic acid testing platform. The GeneSTAT system consists of:

• The single-use, disposable, assay-specific Test Module cartridge that contains the real-time PCR reagents, internal control, reaction wells, and test parameters. The single-use cartridge contains all the necessary reagents for amplification and detection of the Coccidioides target DNA as well as a human DNA control sequence that is used to monitor the presence of inhibitors in the PCR reaction and to ensure that the sample preparation process is adequate. All information to run the assay as well as lot number and expiration date is contained on the Radio-Frequency Identification (RFID) tag on the cartridge. The cartridge contains two PCR reaction wells. One well contains PCR reagents for amplification and detection of Coccidioides DNA and the human gene internal control (Reaction Well 1). The second well (Reaction Well 2) contains the same reagents for amplification/detection of Coccidioides DNA as used in Reaction Well 1 but does not contain the PCR reagents to detect the internal control.
• The GeneSTAT Analyzer instrument that performs the mechanical, optical, and fluidic actions upon the Test Module to perform the PCR reactions. The instrument generates, analyzes, and transmits results to a connected pre-configured computer for presentation to the user. One single-use Test Module can be tested at a time in each GeneSTAT Analyzer. Up to four GeneSTAT Analyzers can be connected to single computer.
• A MS Windows based laptop computer
• The associated software and firmware required to run the test, analyze the results, and present the results to the user.
• The Performance Verification Test Cartridges (PVT) that are used to periodically verify that the analyzer is functioning within acceptable limits. A successful PVT run is required to activate the analyzer. The PVT should be run at minimum every six months, and it is recommended that a PVT should be run monthly.

Materials provided:

1. Lyticase
2. GeneSTAT Sample Vials
3. GeneSTAT.MDx Coccidioides Assay Test Module Cartridges

Materials required but not provided:

1. GeneSTAT Analyzer instrument
2. Preconfigured computer running Windows 8.1 or greater
3. Performance Verification Test (PVT) Sets

Materials required and available from other suppliers:

1. Microcentrifuge capable of reaching RCF = 20,000 x g
2. Centrifuge with rotor(s) capable of holding 15 mL or 50 mL capped centrifuge tubes
3. QIAGEN QIAamp DSP DNA Mini Kit
4. Biosafety Level 2 cabinet
5. SPUTOLYSIN Reagent

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Bronchial alveolar lavage (BAL) or bronchial wash (BW) specimens

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Reproducibility:

• Three test sites (two external, one internal).
• Each site tested three replicates of blinded and randomized samples.
• Samples contrived in negative BAL sample matrix and spiked with attenuated C. posadasii spherules (strain Acts2/Aard1/Acts3) at three levels of reactivity: Moderate Positive (3x LoD, 20.91 GEq/mL), Low Positive (1x LoD, 6.97 GEq/mL), Negative (Coccidioides-Negative BAL).
• Two operators at each site conducted the study over five non-consecutive days, resulting in 30 replicates at each site and 90 replicates overall for each sample reactivity level.
• Each operator used two GeneSTAT instruments, for a total of 12 instruments overall.
• Each site tested a different lot of Test Module cartridges.
• External positive and negative controls were run each day of testing.

Within-laboratory Repeatability:

• One internal testing site.
• Two operators using the same panel of blinded challenge samples described for the between-laboratory reproducibility study.
• The study was conducted over 12 non-consecutive days.
• Each operator tested two replicates of each panel member per day, resulting in 24 replicates per operator and 48 replicates overall.

Limit of Detection (LoD):

• Determined using a stock of attenuated C. posadasii spherules (strain Acts2/Aard1/Acts3) quantified as spherules per milliliter and genome equivalents per milliliter (GEq/mL).
• The stock sample was serially diluted in Coccidioides-Negative BAL matrix.
• Estimated by testing 20 replicates of five concentrations of C. posadasii spherules in BAL matrix by two operators on four GeneSTAT instruments over five days with two assay kit lots.
• The lowest concentration that gave a minimum 95% detection rate was then retested with an additional 60 replicates at that dilution to confirm the LoD.
• The same testing scheme was also used to determine the LoD of pre-extracted C. posadasii and C. immitis DNA diluted in a synthetic BAL matrix composed of 0.9% w/v sterile saline and 0.3 ug/mL human genomic DNA in 0.5% w/v carboxymethyl cellulose.

Analytical Reactivity (Inclusivity):

• Previously extracted DNA from different C. posadasii and C. immitis isolates representing temporal and geographical diversity of the two Coccidioides strains in the US (Table 9).
• Twenty replicates of five different DNA samples from both strains were tested at a 3x LoD concentration (30.0 GEq/mL).

Analytical Specificity (Cross reactivity/ Microbial Interference):

• A panel of 47 non-target viruses, bacteria, fungi, and DNA preparations that may be present in respiratory specimens (Table 10).
• For cross-reactivity testing, each organism was spiked into negative BAL/BW matrix at ≥ 10^6 CFU/mL for bacteria and fungi, ≥ 10^6 copies/mL for DNA preparations, and ≥ 10^5 TCID50/mL for viruses. Human Metapneumovirus, Influenza A, and Influenza B viruses were spiked at ≥ 10^4 TCID50/mL. The organisms were tested in triplicate.
• Microbial interference was evaluated with the same panel tested in triplicate in the presence of C. posadasii spherules (strain Δcts2/Δard1/Δcts3) at 3x LoD (20.91 GEq/mL).

Interfering Substances:

• A panel of 15 endogenous or exogenous substances.
• Interference challenge samples were prepared by spiking the test substances into BAL matrix to reach the test concentration shown in Table 11.
• C. posadasii spherules (strain Acts2/Aard1/Acts3) were added to each sample at 3x LoD (20.91 GEq/mL).

Carry-Over/Cross-Contamination:

• A series of high positive samples interspersed with Coccidioides-negative BAL samples.
• High positive samples contained attenuated C. posadasii spherules at 1000x LoD.
• High positive and negative samples were alternately tested on each GeneSTAT analyzer for five cycles for a total of 20 assay runs, ten high positive, and ten negative samples overall.

Specimen Stability:

• Individual or pooled Coccidioides-negative BAL/BW clinical specimens with and without C. posadasii spherules spiked at three times the LoD.
• Samples were processed and tested after storage under different conditions: neat specimens at 2-8 °C, at the Sputolysin digestion stage at 2-8 °C, and extracted DNA at 2-8 °C and -20 °C.
• Neat specimens were also tested after up to two -20 °C freeze-thaw cycles.

Prospective Sample Testing:

• A total of 236 fresh BAL/BW samples were collected prospectively from four geographically diverse regions endemic for Coccidioides (Phoenix, AZ, Albuquerque, NM, Tucson, AZ, and North Hollywood, CA).
• Samples submitted for routine fungal culture and that met the study inclusion criteria were enrolled.
• The samples were tested with the GeneSTAT.MDx Coccidioides assay prospectively at three of the sites.
• 231 prospective samples were included in the study (189 BAL and 42 BW).
• Performance compared to fungal culture followed by Coccidioides-specific DNA probe hybridization (AccuProbe, Hologic, Marlborough, MA).

Retrospective Sample Testing:

• A total of 100 frozen, de-identified BAL/BW specimens were tested at the three clinical sites.
• Samples included 51 positive (27 BAL and 24 BW) and 49 negative (38 BAL and 11 BW) samples.
• Specimens were selected from previously confirmed negative and positive samples for Coccidioides infection as determined by fungal culture; positive cultures were confirmed as Coccidioides using the AccuProbe assay.
• Prior to inclusion, samples were sent to a reference laboratory for confirmation of Coccidioides DNA status using a validated PCR assay with bi-directional sequencing. Only samples concordant by both culture and reference laboratory PCR/sequencing method were included.
• Performance compared to the original culture results.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Study Type: Analytical Performance (Reproducibility, Linearity/Assay Reportable Range, Traceability, Stability, Expected Values, Detection Limit, Analytical Reactivity (Inclusivity), Analytical Specificity (Cross reactivity/ Microbial Interference, Interfering Substances), Carry-Over/Cross-Contamination, Assay Cut-off, Specimen Stability), Clinical Studies (Prospective, Retrospective)

Reproducibility (Between-laboratory):

• Sample Size: 90 replicates per sample reactivity level (Moderate Positive, Low Positive, Negative BAL) across 3 sites. Total of 270 replicates.
• Key Results:
  • Moderate Positive: 98.9% Agreement (89/90) [95% CI: 94.0% - 99.8%]
  • Low Positive: 100% Agreement (90/90) [95% CI: 95.9% - 100%]
  • Negative BAL: 97.8% Agreement (88/90) [95% CI: 92.3% - 99.4%]

Reproducibility (Within-laboratory Repeatability):

• Sample Size: 48 replicates per sample reactivity level (Moderate Positive, Low Positive, Negative BAL).
• Key Results:
  • Moderate Positive: 100% Agreement (48/48) [95% CI: 92.6% - 100%]
  • Low Positive: 93.8% Agreement (45/48) [95% CI: 83.2% - 97.9%]
  • Negative BAL: 97.9% Agreement (47/48) [95% CI: 89.1% - 99.6%]

Detection Limit:

• Key Results: LoD of the GeneSTAT.MDx Coccidioides assay performed on the GeneSTAT System is 6.97 GEq/mL for attenuated C. posadasii spherules. Confirmed LoD for both pre-extracted C. posadasii DNA and C. immitis DNA was 10.0 GEq/mL.

Analytical Reactivity (Inclusivity):

• Sample Size: 20 replicates for each of 5 C. posadasii DNA samples and 5 C. immitis DNA samples (total 200 replicates).
• Key Results: All 20 replicate samples of the five clinical isolates of C. posadasii DNA were detected. All 20 replicate samples of the five clinical isolates of C. immitis DNA were detected.

Analytical Specificity (Cross reactivity/ Microbial Interference):

• Key Results:
  • Cross-reactivity: Initial false positive results in one out of three replicates for E. coli, Human parainfluenzavirus 2, and L. pneumophila; no false positives when retested at ten-fold higher concentrations.
  • Microbial interference: No evidence of microbial interference observed.

Interfering Substances:

• Key Results: No interference observed for any substance at the tested concentrations.

Carry-Over/Cross-Contamination:

• Sample Size: 20 assay runs (10 high positive, 10 negative).
• Key Results: No evidence of amplicon carry-over or sample cross-contamination was observed, and the expected result was obtained for all samples.

Prospective Clinical Study:

• Sample Size: 231 fresh BAL/BW samples.
• Standalone Performance: Compared to fungal culture / DNA Probe.
  • Sensitivity: 100% (3/3)
  • Specificity: 99.6% (227/228)
• Key Results: One false positive result for GeneSTAT.MDx Coccidioides assay.

Retrospective Clinical Study:

• Sample Size: 100 frozen, de-identified BAL/BW specimens (51 positive, 49 negative).
• Standalone Performance: Compared to fungal culture / DNA Probe.
  • Positive Percent Agreement (PPA): 100% (51/51)
  • Negative Percent Agreement (NPA): 95.9% (47/49)
• Key Results: Two false positive results for GeneSTAT.MDx Coccidioides assay.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Prospective Clinical Performance (All 3 Sites):

• Sensitivity (%): 100% (95% CI: 43.8% - 100%)
• Specificity (%): 99.6% (95% CI: 97.6% - 99.9%)

Retrospective Clinical Performance (All 3 Sites):

• Positive Percent Agreement (PPA) (%): 100% (95% CI: 93.0% - 100%)
• Negative Percent Agreement (NPA) (%): 95.9% (95% CI: 86.3% - 98.9%)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Not Found

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.3376 Device to detect and identify fungal nucleic acids directly in respiratory specimens.

(a)
Identification. A device to detect and identify fungal nucleic acids directly in respiratory specimens is an in vitro diagnostic device intended for the detection and identification of fungal pathogens in respiratory specimens collected from patients with signs or symptoms and suspicion of fungal infection. The device is intended to aid in the diagnosis of fungal disease in conjunction with clinical signs and symptoms and other laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The intended use must include a detailed description of what the device detects, the type of results provided to the user, the clinical indications appropriate for test use, and the specific population(s) and testing location(s) for which the device is intended.
(2) The labeling required under 21 CFR 809.10(b) must include:
(i) A detailed device description, including the parts that make up the device, instrument requirements, ancillary reagents required but not provided, and a detailed explanation of the methodology including all pre-analytical methods for processing of specimens.
(ii) Performance characteristics from analytical studies, including analytical sensitivity (Limit of Detection), inclusivity, reproducibility, interference, cross-reactivity, interfering substances, carryover/cross-contamination, and specimen stability.
(iii) A statement that the device is intended to be used in conjunction with clinical history, signs and symptoms and the results of other diagnostic tests.
(iv) A detailed explanation of the interpretation of test results and acceptance criteria for any quality control testing.
(v) A limiting statement that negative results do not preclude the possibility of infection, and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
(3) Design verification and validation activities must include:
(i) Performance characteristics from clinical studies that include prospective (sequential) samples and, if appropriate, additional characterized samples. The study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained from comparator methods. Documentation from the clinical studies must include the clinical study protocol (including predefined statistical analysis plan), clinical study report, and results of all statistical analyses.
(ii) A detailed device description of the following:
(A) Overall device design including all parts that make up the device and all control elements incorporated into the testing procedure.
(B) Thorough description of the methodology including, primer/probe sequences, primer/probe design and rationale for target sequence selection.
(C) Computational path from collected raw data to reported result (
e.g., how collected raw signals are converted into a reported signal and result, as applicable.(iii) A detailed documentation for device software, including, software applications and hardware-based devices that incorporate software.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR GeneSTAT.MDx Coccidioides Assay DECISION SUMMARY

A. DEN Number:

DEN170041

B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the GeneSTAT.MDx Coccidioides Assay

C. Measurands:

DNA sequences of Coccidioides immitis and Coccidioides posadasii

D. Type of Test:

Real-time polymerase chain reaction (PCR) assay

E. Applicant:

DxNA, LLC

F. Proprietary and Established Names:

GeneSTAT.MDx Coccidioides Assay

G. Regulatory Information:

• 1. Regulation section:
     21 CFR 866.3376
• 2. Classification:
     Class II (Special Controls)
• 3. Product code(s):
     QAA
• 4. Panel:
     83- Microbiology

1

H. Indications for Use:

1. Indications for use:

The GeneSTAT.MDx Coccidioides Assay is a qualitative real-time polymerase chain reaction (PCR) in vitro diagnostic test for the detection of nucleic acids from Coccidioides immitis and Coccidioides posadasii. The assay does not differentiate between these two species. The assay is performed on bronchial alveolar lavage (BAL) or bronchial wash (BW) specimens from patients presenting with respiratory signs and symptoms consistent with coccidioidomycosis. The GeneSTAT.MDx Coccidioides Assay is intended to be used along with clinical findings and other laboratory results as an aid in the diagnosis of coccidioidomycosis in patients with possible or probable exposure to Coccidioides immitis or Coccidioides posadasii.

2. Special conditions for use statement(s):

For in vitro diagnostic use only For prescription use only

• 3. Special instrument requirements:
     GeneSTAT System

I. Device Description:

The GeneSTAT.MDx Coccidioides Assay is a qualitative real-time PCR-based assay to detect target Coccidioides spp. DNA from bronchial alveolar lavage (BAL) or bronchial wash (BW) samples. The sample is prepared by a lysis and extraction process, with the extracted DNA then tested with the GeneSTAT.MDx Coccidioides assay on the GeneSTAT System.

The GeneSTAT System is a real-time PCR nucleic acid testing platform. The GeneSTAT system consists of:

• The single-use, disposable, assay-specific Test Module cartridge that contains the real-time PCR reagents, internal control, reaction wells, and test parameters. The single-use cartridge contains all the necessary reagents for amplification and detection of the Coccidioides target DNA as well as a human DNA control sequence that is used to monitor the presence of inhibitors in the PCR reaction and to ensure that the sample preparation process is adequate. All information to run the assay as well as lot number and expiration date is contained on the Radio-Frequency Identification (RFID) tag on the cartridge. The cartridge contains two PCR reaction wells. One well contains PCR reagents for amplification and detection of Coccidioides DNA and the human gene internal control (Reaction Well 1). The second well (Reaction Well 2) contains the same reagents for amplification/detection of Coccidioides DNA as used in Reaction Well 1 but does not contain the PCR reagents to detect the internal control.

2

• The GeneSTAT Analyzer instrument that performs the mechanical, optical, and fluidic actions upon the Test Module to perform the PCR reactions. The instrument generates, analyzes, and transmits results to a connected pre-configured computer for presentation to the user. One single-use Test Module can be tested at a time in each GeneSTAT Analyzer. Up to four GeneSTAT Analyzers can be connected to single computer.
• A MS Windows based laptop computer
• The associated software and firmware required to run the test, analyze the results, and ● present the results to the user.
• The Performance Verification Test Cartridges (PVT) that are used to periodically verify that the analyzer is functioning within acceptable limits. A successful PVT run is required to activate the analyzer. The PVT should be run at minimum every six months, and it is recommended that a PVT should be run monthly.

Materials provided:

• 1. Lyticase
• 2. GeneSTAT Sample Vials
• 3. GeneSTAT.MDx Coccidioides Assay Test Module Cartridges

Materials required but not provided:

• 1. GeneSTAT Analyzer instrument
• 2. Preconfigured computer running Windows 8.1 or greater
• 3. Performance Verification Test (PVT) Sets

Materials required and available from other suppliers:

• Microcentrifuge capable of reaching RCF = 20,000 x g 1.
• 2. Centrifuge with rotor(s) capable of holding 15 mL or 50 mL capped centrifuge tubes
• 3. QIAGEN QIAamp DSP DNA Mini Kit
• 4. Biosafety Level 2 cabinet
• 5. SPUTOLYSIN Reagent

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

Not applicable

K. Test Principle:

The GeneSTAT.MDx Coccidioides assay is a qualitative real-time PCR-based assay that detects but does not differentiate between DNA sequences of Coccidioides immitis and Coccidioides posadasii, the causative agents of Valley Fever. The assay detects target DNA that has been previously extracted from bronchial alveolar lavage (BAL) or bronchial wash (BW) samples. The BAL/BW sample preparation process includes the lysis of Coccidioides organisms within the sample with sputolysin, and the subsequent extraction and purification of DNA using the QIAGEN QIAamp DSP DNA Mini Kit.

3

The extracted DNA is placed inside the sample vial, which is then attached to the Coccidioides assay cartridge. The cartridge is placed in the GeneSTAT instrument, the RFID tag is read by the instrument, and the user follows a series of data-entry steps, including adding sample ID information, and the run is started. All subsequent steps of the assay process are performed by the GeneSTAT instrument without user intervention, including transfer of sample from the sample vial to the reaction wells in the cartridge, hydration of PCR reagents. PCR amplification and real-time detection of target sequences, and results analysis. The GeneSTAT assay process, from loading the extracted sample into the cartridge to assay result, takes approximately 1.5 hours.

L. Performance Characteristics:

1. Analytical performance:

a. Reproducibility

Between-laboratory reproducibility of the GeneSTAT.MDx Coccidioides assay was assessed at three test sites (two external, one internal). Each of the three sites tested three replicates of blinded and randomized samples contrived in negative BAL sample matrix and spiked with attenuated C. posadasii spherules (strain Acts2/Aard1/Acts3) at three levels of reactivity, expressed in genome equivalents per milliliter (GEq/mL):

(1) Moderate Positive: 3x LoD (20.91 GEq/mL)

(2) Low Positive: 1x LoD (6.97 GEq/mL)

(3) Negative: Coccidioides-Negative BAL

Two operators at each site conducted the study over five non-consecutive days, resulting in 30 replicates at each site and 90 replicates overall for each sample reactivity level. Each operator used two GeneSTAT instruments, for a total of 12 instruments overall. Each site tested a different lot of Test Module cartridges. External positive and negative controls were run each day of testing. One Moderate Positive sample and two Negative samples produced Invalid results and were not repeated. Qualitative results are summarized in Table 1, and average Ct and %CV values for the positive samples are summarized in Table 2 for individual sites and across all sites. Qualitative and quantitative analyses conducted by day and by operator are summarized in Table 3, Table 4, Table 5, and Table 6. The reproducibility results are acceptable.

4

| Sample

ID	Site 1	Site 2	Site 3	All 3 Sites
	%
Agreement	%
Agreement	%
Agreement	% Agreement
[95% CI]
Moderate
Positive	96.7%
(29/30)+	100%
(30/30)	100%
(30/30)	98.9% (89/90)
[94.0% - 99.8%]
Low
Positive	100%
(30/30)	100%
(30/30)	100%
(30/30)	100% (90/90)
[95.9% - 100%]
Negative
BAL	96.7%
(29/30)+	100%
(30/30)	96.7%
(29/30)+	97.8% (88/90)
[92.3% - 99.4%]

Table 1. Between-Site Reproducibility - Qualitative Analysis

1 One Moderate Positive sample and two Negative samples produced Invalid results and were not repeated

Table 2. Between-Site Reproducibility - Quantitative Analysis

	Site 1		Site 2		Site 3		All 3 Sites
Sample
ID	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)
Moderate	36.8	33.8	34.3	32.9	36.3	33.5	35.8	33.4
Positive	(6.7%)	(3.2%)	(3.7%)	(2.2%)	(8.7%)	(4.3%)	(6.4%)	(3.2%)
Low	39.1	35.3	37.8	34.8	38.0	35.6	38.3	35.3
Positive	(9.3%)	(4.4%)	(7.1%)	(3.6%)	(9.7%)	(6.4%)	(8.7%)	(4.8%)

Table 3. Reproducibility by Day - Qualitative Analysis

| Sample ID | Day 1
% Agreement | Day 2
% Agreement | Day 3
% Agreement | Day 4
% Agreement | Day 5
% Agreement |
|----------------------|----------------------|----------------------|----------------------|----------------------|----------------------|
| Moderate
Positive | | | (b) (4) | | |
| Low
Positive | | | | | |
| Negative
BAL | | | | | |

Table 4. Reproducibility by Day - Quantitative Analysis

Sample ID	Day 1		Day 2		Day 3		Day 4		Day 5
	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)
Mod.
Positive					(b) (4)
Low
Positive

5

	Site 1	Site 2	Site 3
Sample ID
(b) (4)
Moderate
Positive
Low
Positive
Negative
BAL

Table 5. Reproducibility by Operator - Qualitative Analysis

Table 6. Reproducibility by Operator - Quantitative Analysis

| Sample

ID	Site 1				Site 2				Site 3
	Operator 1		Operator 2		Operator 1		Operator 2		Operator 1		Operator 2
	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)
Mod.
Positive	(b) (4)
Low
Positive

Within-laboratory Repeatability

Within-laboratory repeatability testing was performed at one internal testing site by two operators using the same panel of blinded challenge samples described for the between-laboratory reproducibility study. The study was conducted over 12 nonconsecutive days. Each operator tested two replicates of each panel member per day, resulting in 24 replicates per operator and 48 replicates overall. Qualitative results are summarized in Table 7, and average Ct values and %CV values are shown in Table 8. The results are acceptable.

	Operator 1	Operator 2	Combined
Sample ID	% Agreement
[95% CI]	% Agreement
[95% CI]	% Agreement
[95% CI]
Moderate
Positive	100% (24/24)
[86.2% - 100%]	100% (24/24)
[86.2% - 100%]	100% (48/48)
[92.6% - 100%]
Low
Positive	87.5% (21/24)†
[69.0% - 95.7%]	100% (24/24)
[86.2% - 100%]	93.8% (45/48)†
[83.2% - 97.9%]
Negative
BAL	95.8% (23/24)*
[79.8% - 99.3%]	100% (24/24)
[86.2% - 100%]	97.9% (47/48)*
[89.1% - 99.6%]

Table 7. Within-Site Repeatability - Qualitative Analysis

Three Low Positive samples produced Not Detected results and were not repeated. One Negative sample produced an Invalid result and was not repeated.

6

Sample ID	Operator 1		Operator 2		Combined
	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)	Well 1
Avg. Ct
(%CV)	Well 2
Avg. Ct
(%CV)
Moderate
Positive	34.3
(5.4%)	33.2
(4.2%)	34.9
(5.3%)	33.4
(3.2%)	34.6
(5.4%)	33.3
(3.7%)
Low
Positive	36.7
(6.8%)	35.5
(4.6%)	37.0
(4.5%)	36.0
(6.2%)	36.9
(5.5%)	35.8
(5.5%)

Table 8. Within-Site Repeatability - Quantitative Analysis

b. Linearity/assay Reportable Range:

Not Applicable

• Traceability, Stability, Expected Values (controls, calibrators, or methods): C.

Internal Control:

One of the reaction wells in the test cartridge contains two PCR primer/probe sets: a set for the detection of Coccidioides spp. DNA and an internal control set to detect human DNA (RNase P gene) that is present in the clinical sample and co-extracted with the Coccidioides organism. The internal control is used to verify that the sample processing and DNA purification process occurred efficiently, that the assay reagents and equipment are functioning correctly, and to monitor for the presence of PCR inhibitors in the sample.

External Controls:

External negative and positive quality control samples should be run periodically, and in conformance with local, state and/or federal regulations or requirements. Negative and positive controls should be tested using the same procedure used for patient samples, starting from the first step of the sample extraction process.

Previously characterized BAL/BW samples that have tested negative in the GeneSTAT.MDx Coccidioides Assay or commercially available human DNA dissolved in molecular biology grade water or sterile saline should be used as the external negative control. Note: Human DNA must be present in the negative control to avoid generation of an invalid result in the GeneSTAT.MDx Coccidioides Assay.

Previously characterized BAL/BW samples that have tested positive in the GeneSTAT.MDx Coccidioides Assay or commercially available Coccidioides materials suitable for external positive controls (e.g., BEI Resources, Cat. No. NR-166).

Positive and negative controls were run each day of testing during the analytical and clinical testing. Of the total 94 external controls that were run across all sites in the clinical study, one sample produced an initial invalid result (1.1%) which resolved to the expected valid result upon re-test. One positive control out of 47 (2.1%) produced an initial false negative result which resolved to the expected positive result upon re

7

test.

d. Detection Limit:

The Limit of Detection (LoD) for the GeneSTAT.MDx assay was determined using a stock of attenuated C. posadasii spherules (strain Acts2/Aard1/Acts3) quantified as spherules per milliliter and genome equivalents per milliliter (GEq/mL). The stock sample was serially diluted in Coccidioides-Negative BAL matrix. The LoD of the assay was estimated by testing 20 replicates of five concentrations of C. posadasii spherules in BAL matrix by two operators on four GeneSTAT instruments over five days with two assay kit lots. The lowest concentration of C. posadasii spherules that gave a minimum 95% detection rate was then retested with an additional 60 replicates at that dilution to confirm the LoD. The LoD of the GeneSTAT.MDx Coccidioides assay performed on the GeneSTAT System is 6.97 GEq/mL. An attenuated strain of C. immitis was not available for testing. The same testing scheme was also used to determine the LoD of pre-extracted C. posadasii and C. immitis DNA diluted in a synthetic BAL matrix composed of 0.9% w/v sterile saline and 0.3 ug/mL human genomic DNA in 0.5% w/v carboxymethyl cellulose. The confirmed LoD for both C. posadasii DNA and C. immitis DNA was 10.0 GEq/mL.

e. Analytical Reactivity (Inclusivity):

The inclusivity of the GeneSTAT.MDx Coccidioides assay was assessed by testing previously extracted DNA from different C. posadasii and C. immitis isolates representing temporal and geographical diversity of the two Coccidioides strains in the US (Table 9). Twenty replicates of five different DNA samples from both strains were tested at a 3x LoD concentration (30.0 GEq/mL).

All 20 replicate samples of the five clinical isolates of C. posadasii DNA were detected in the assay. Likewise, all 20 replicate samples of the five clinical isolates of C. immitis DNA were detected in the assav. An invalid result was observed for one strain replicate: however, the expected positive result was produced when the sample was re-tested per the assay instructions. The results are acceptable.

Strain	Collection Site
C. posadasii	Yuma, AZ
C. posadasii	San Joaquin, CA
C. posadasii	Maricopa County, AZ
C. posadasii	Tucson, AZ
C. posadasii	San Antonio, TX
C. immitis	Monterey, CA
C. immitis	Barstow, CA
C. immitis	San Joaquin, CA
C. immitis	San Joaquin, CA
C. immitis	Tucson, AZ

Table 9. Inclusivity Panel

8

f. Analytical Specificity:

Cross reactivity/ Microbial Interference

The GeneSTAT.MDx Coccidioides assay was used to test a panel of 47 non-target viruses, bacteria, fungi, and DNA preparations that may be present in respiratory specimens (Table 10). For cross-reactivity testing, each organism was spiked into negative BAL/BW matrix at ≥ 106 CFU/mL for bacteria and fungi, ≥ 106 copies/mL for DNA preparations, and ≥ 105 TCID50/mL for viruses. Human Metapneumovirus, Influenza A, and Influenza B viruses were spiked at ≥ 104 TCID≤0/mL. The organisms were tested with the GeneSTAT.MDx Coccidioides assay in triplicate. Initial false positive results were observed in one out of three replicates of E. coli, Human parainfluenzavirus 2, and L. pneumophila; however, no false positive results were observed for any of these panel members when an additional three replicates were tested at ten-fold higher concentrations. Three initial invalid results were observed in the study, and all three samples resolved to the expected negative result upon re-test.

Microbial interference was evaluated with the same panel tested in triplicate in the presence of C. posadasii spherules (strain Δcts2/Δard1/Δcts3) at 3x LoD (20.91 GEq/mL). No evidence microbial interference was observed. The results are acceptable.

9

Table 10: Cross Reactivity Panel
Acinetobacter baumanii	Legionella pneumophila #
Adenovirus	Moraxella catarrhalis
Aspergillus fumigatus	Mycobacterium tuberculosis
Blastomyces dermatitidis†	Mycoplasma pneumoniae
Bordetella parapertussis	Neisseria meningitidis
Bordetella pertussis	Neisseria sicca
Burkholderia cepacia	Porphyromonas gingivalis†
Candida albicans	Proteus vulgaris
Candida glabrata	Pseudomonas aeruginosa
Chlamydophila pneumoniae†	Respiratory Syncytial Virus
Corynebacterium diphtheriae	Scedosporium prolificans
Escherichia coli #	Serratia marcescens
Haemophilus influenzae	Staphylococcus aureus (MRSA)
Histoplasma capsulatum†	Staphylococcus aureus (MSSA)
Human Influenza A	Staphylococcus epidermidis (MRSE)
Human Influenza B	Staphylococcus epidermidis (MSSE)
Human Metapneumovirus	Staphylococcus haemolyticus
Human parainfluenzavirus 1	Streptococcus agalactiae
Human parainfluenzavirus 2#	Streptococcus dysgalactiae
Human parainfluenzavirus 3	Streptococcus mitis
Human parainfluenzavirus 4	Streptococcus pneumoniae
Klebsiella pneumoniae	Streptococcus pyogenes
Lactobacillus acidophilus	Streptococcus salivarius
Lactobacillus plantarum

Table 10. Cross Reactivity Panel

Tested as purified DNA

Initial false positive results were observed in one out of three replicates of these organisms; however, no false positive results were observed for any of these organisms when an additional three replicates were tested at ten-fold higher concentrations.

Interfering Substances

The susceptibility of the GeneSTAT.MDx Coccidioides assay to interference by a panel of 15 endogenous or exogenous substances was assessed. Interference challenge samples were prepared by spiking the test substances into BAL matrix to reach the test concentration shown in Table 11 below. C. posadasii spherules (strain Acts2/Aard1/Acts3) were added to each sample at 3x LoD (20.91 GEq/mL). No interference was observed for any substance at the tested concentrations. The results are acceptable.

| Substance | Test
Concentration | Substance | Test
Concentration |
|--------------------------|-----------------------|----------------------|-----------------------|
| Lidocaine HCl,
4% w/v | 20% v/v | Human Genomic
DNA | 50 ng/mL |
| Levofloxacin | 5 mg/mL | Albuterol Sulfate | 50 $μ$ g/mL |
| Tobramycin | 5% w/v | Mucin | 1% w/v |
| Zanamivir | 500 ng/mL | Phenylephrine | 1.5% v/v |
| Oseltamivir | 100 ng/mL | Fluticasone | 5 $μ$ g/mL |

Table 11. Interfering Substances Panel

10

| Substance | Test
Concentration | Substance | Test
Concentration |
|-------------|-----------------------|--------------|-----------------------|
| Whole blood | 2% v/v | NaCl 5% | 5% w/v |
| Epinephrine | 1 mg/mL | Nicogel, 40% | 0.5% w/v |
| FluMist | 1% v/v | | |

Carry-Over/ Cross-Contamination

Carry-over and cross-contamination on the GeneSTAT.MDx Coccidioides assay was evaluated by testing a series of high positive samples interspersed with Coccidioides-negative BAL samples on two GeneSTAT instruments. The high positive samples contained attenuated C. posadasii spherules at 1000x LoD. High positive and negative samples were alternately tested on each GeneSTAT analyzer for five cycles for a total of 20 assay runs, ten high positive, and ten negative samples overall. No evidence of amplicon carry-over or sample cross-contamination was observed, and the expected result was obtained for all samples. The results are acceptable.

• g. Assay Cut-off:
  The cut-off values for the assay Coccidioides result and the internal control were established and verified by analyzing contrived samples and clinical BAL/BW samples known to be positive and negative for Coccidioides. The cut-offs were validated by the results of the samples tested in the clinical and analytical studies.

The following decision table is used by the assay data analysis software to generate the reported assay result. This table is not displayed:

Reaction Well 1		Reaction Well 2	Reported Assay Result
Coccidioides
PCR	Internal
Control
PCR	Coccidioides
PCR
Negative	Positive	Negative	Coccidioides DNA Not Detected
Positive	Positive	Negative	Coccidioides DNA Detected
Positive	Positive	Positive	Coccidioides DNA Detected
Positive	Negative	Positive	Coccidioides DNA Detected
Negative	Positive	Positive	Coccidioides DNA Detected
Positive	Negative	Negative	Coccidioides DNA Detected
Negative	Negative	Positive	INVALID
Negative	Negative	Negative	INVALID

h. Specimen Stability

BAL/BW sample stability was demonstrated by testing individual or pooled Coccidioides-negative BAL/BW clinical specimens with and without C. posadasii spherules spiked at three times the LoD. Samples were processed and tested with the GeneSTAT.MDx Coccidioides assay per the labeled instructions after storage under different conditions that included storage of neat specimens at 2-8 ℃, storage at the Sputolysin digestion stage at 2-8 ℃, and storage of extracted DNA at 2-8 ℃ and -20 °C. To support the use of frozen samples in analytical and clinical studies, neat

11

specimens were also tested after up to two -20 ℃ freeze-thaw cycles. The results of the analytical studies and the results of the prospective and retrospective clinical studies support the specimen collection and storage recommendations outlined in the labeling:

BAL/BW specimens should be maintained at 2-8 °C and must be processed at least through the Sputolysin digestion stage within seven days or less from the time of collection. Samples that have undergone Sputolysin digestion should be maintained at 2-8 °C and must be processed through the DNA extraction procedure within three days or less from Sputolysin treatment. Extracted DNA can be stored for up to eight hours at 2-8 ℃ or can be frozen at -20 ℃ or lower for up to 30 days prior to testing.

• 2. Comparison Studies:
  • a. Clinical Comparison:

See section L3

• b. Matrix Equivalence Study
  Not applicable

• 3. Clinical Studies:
     The clinical performance of the GeneSTAT.MDx Coccidioides assay performed on the GeneSTAT System was determined by testing prospective and retrospective samples. Due to the low prevalence of coccidioidomycosis, retrospective samples were tested to demonstrate product performance with additional positive clinical samples. All samples were tested by fungal culture followed by Coccidioides-specific DNA probe hybridization to confirm recovery of Coccidioides in positive fungal cultures (AccuProbe, Hologic, Marlborough, MA).

Prospective Sample Testing

A total of 236 fresh BAL/BW samples were collected prospectively from four geographically diverse regions endemic for Coccidioides (Phoenix, AZ, Albuquerque, NM, Tucson, AZ, and North Hollywood, CA). Samples submitted for routine fungal culture and that met the study inclusion criteria were enrolled. The samples were tested with the GeneSTAT.MDx Coccidioides assay prospectively at three of the sites (several samples collected from CA were sent to the NM site for testing).

Five samples were excluded from the study due to protocol violations, sample handling violations, incomplete culture results, or insufficient specimen volume. Of the remaining 231 prospective samples that were included in the study, 189 were BAL and 42 were BW. Performance compared to fungal culture was evaluated based on results from testing BAL and BW samples combined. In comparison to fungal culture with fresh prospective specimens, the GeneSTAT.MDx Coccidioides assay sensitivity was 100% (3/3) and specificity was 99.6% (227/228). The GeneSTAT .MDx Coccidioides assay produced a false positive result in one sample. The prospective sample testing results are summarized

12

below for all sites combined (Table 12) and for each site individually (Table 13, Table 14, and Table 15).

All 3 Sites		Fungal Culture / DNA Probe
		POS	NEG	TOTAL
GeneSTAT.MDx
Coccidioides
Assay	POS	3	1†	4
	NEG	0	227	227
	TOTAL	3	228	231
All 3 Sites		95% CI
Sensitivity (%)		100%	43.8%	100%
Specificity (%)		99.6%	97.6%	99.9%

Table 12. Prospective Clinical Performance - All Sites

I One false positive result was obtained from the GeneSTAT.MDx Coccidioides assay at Site #2. This sample was negative for Coccidioides DNA when tested by an independent real-time PCR and bi-directional sequencing method.

Site #1		Fungal Culture / DNA Probe
		POS	NEG	TOTAL
GeneSTAT.MDx
Coccidioides
Assay	POS	0	0	0
	NEG	0	92	92
	TOTAL	0	92	92
Site #1		95% CI
Sensitivity (%)		*	*	*
Specificity (%)		100%	96.0%	100%

Table 13. Prospective Clinical Performance - Site #1

*There were no positive samples collected at Site #1.

Table 14. Prospective Clinical Performance - Site #2
------------------------------------------------------	--	--

Site #2	Fungal Culture / DNA Probe
	POS	NEG	TOTAL
GeneSTAT.MDx
Coccidioides
Assay	POS	2	1	3
	NEG	0	50	50
	TOTAL	2	51	53
Site #2			95% CI
Sensitivity (%)		100%	34.2%	100%
Specificity (%)		98.0%	89.7%	99.7%

13

| Site #3 | | Fungal Culture / DNA
Probe | | |
|---------------------------------------|-------|-------------------------------|--------|-------|
| | | POS | NEG | TOTAL |
| GeneSTAT.MDx
Coccidioides
Assay | POS | 1 | 0 | 1 |
| | NEG | 0 | 85 | 85 |
| | TOTAL | 1 | 85 | 86 |
| Site #3 | | | 95% CI | |
| Sensitivity (%) | | 100% | 20.7% | 100% |
| Specificity (%) | | 100% | 95.7% | 100% |

Table 15. Prospective Clinical Performance - Site #3

Retrospective Sample Testing

A total of 100 frozen, de-identified BAL/BW specimens were tested at the three clinical sites. The enrolled samples included 51 positive (27 BAL and 24 BW) and 49 negative (38 BAL and 11 BW) samples. Specimens were selected from previously confirmed negative and positive samples for Coccidioides infection as determined by fungal culture; positive cultures were confirmed as Coccidioides using the AccuProbe assay. Prior to inclusion in the study, the retrospective samples were sent to a reference laboratory for confirmation of Coccidioides DNA status using a validated PCR assay with bi-directional sequencing. Only samples that were concordant by both culture and the reference laboratory PCR/sequencing method were included in the retrospective sample set.

The performance of the GeneSTAT.MDx Coccidioides assay was compared to the original culture results for this sample set. Overall positive percent agreement (PPA) was 100% (51/51) and negative percent agreement (NPA) was 95.9% (47/49). The retrospective sample testing results are summarized below for all sites combined (Table 16) and for each site individually (Table 17, Table 18, and Table 19).

| All 3 Sites | | Fungal Culture / DNA
Probe | | |
|--------------|-------|-------------------------------|--------|-------|
| | | POS | NEG | TOTAL |
| GeneSTAT.MDx | POS | 51 | 2† | 53 |
| Coccidioides | NEG | 0 | 47 | 47 |
| Assay | TOTAL | 51 | 49 | 100 |
| All 3 Sites | | | 95% CI | |
| PPA (%) | | 100% | 93.0% | 100% |
| NPA (%) | | 95.9% | 86.3% | 98.9% |

Table 16. Retrospective Clinical Performance - All Sites

T Two false positive results were obtained from the GeneSTAT.MDx Coccidioides assay at Site #2. These samples were negative for Coccidioides DNA when tested by an independent real-time PCR and bi-directional sequencing method.

14

| Site #1 | | Fungal Culture / DNA
Probe | | |
|---------------------------------------|-------|-------------------------------|--------|-------|
| | | POS | NEG | TOTAL |
| GeneSTAT.MDx
Coccidioides
Assay | POS | 36 | 0 | 36 |
| | NEG | 0 | 17 | 17 |
| | TOTAL | 36 | 17 | 53 |
| Site #1 | | | 95% CI | |
| PPA (%) | | 100% | 90.4% | 100% |
| NPA (%) | | 100% | 81.6% | 100% |

Table 17. Retrospective Clinical Performance - Site #1

Table 18. Retrospective Clinical Performance - Site #2

| Site #2 | | Fungal Culture / DNA
Probe | | |
|--------------|-------|-------------------------------|--------|-------|
| | | POS | NEG | TOTAL |
| GeneSTAT.MDx | POS | 13 | 2† | 15 |
| Coccidioides | NEG | 0 | 30 | 30 |
| Assay | TOTAL | 13 | 32 | 45 |
| Site #2 | | | 95% CI | |
| PPA (%) | | 100% | 77.2% | 100% |
| NPA (%) | | 93.8% | 79.9% | 98.3% |

Table 19. Retrospective Clinical Performance - Site #3

| Site #3 | | Fungal Culture / DNA
Probe | | |
|---------------------------------------|-------|-------------------------------|--------|-------|
| | | POS | NEG | TOTAL |
| GeneSTAT.MDx
Coccidioides
Assay | POS | 2 | 0 | 2 |
| | NEG | 0 | 0 | 0 |
| | TOTAL | 2 | 0 | 2 |
| Site #3 | | | 95% CI | |
| PPA (%) | | 100% | 34.2% | 100% |
| NPA (%) | | * | * | * |

*There were no negative samples tested at Site #3.

• 4. Clinical cut-off:
     Not applicable

5. Expected values/Reference range:

The prospective study included 189 BAL and 42 BW samples that were submitted for routine fungal culture from four regions endemic for coccidioidomycosis (Phoenix, AZ, Albuquerque, NM, Tucson, AZ, and North Hollywood, CA). Among the total 231 samples, 124 were collected from males (53.7%), 102 were collected from females (44.2%), and the gender was not recorded for five samples (2.2%). Three of the samples (1.3%) were positive for Coccidioides by Accuprobe confirmed fungal culture, and four of the samples (1.7%) were positive by the GeneSTAT.MDx Coccidioides assay.

15

M. Instrument Name

GeneSTAT System

N. 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 __________________________________________________________________________________________________________________________________________________________________________

Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission?

Yes __________________________________________________________________________________________________________________________________________________________________________

• 2. Software:
     FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes X or No __________________________________________________________________________________________________________________________________________________________________

The sponsor provided adequate verification and validation testing covering the following software/instrument components of the GeneSTAT System:

| Component of

GeneSTAT System	Descriptions of System Element
DxNA Test Module	Assay specific cartridge containing reagents, reaction wells, and
associated test parameters. The analyzer reads assay specific
information contained on the identification tag of Test Modules.
GeneSTAT Analyzer	Benchtop analyzer that executes mechanical, optical, fluidic
actions upon the Test Module to perform PCR reactions, generate
results, and transmit results to PCReports.
Firmware	Control and analysis firmware on the analyzer that runs
mechanical, thermal, mixing, and optical detection functions
required to execute assay protocols. The firmware processes the
fluorescence signals generated and compares these values to assay
specific thresholds to determine if the target has been detected.
PC	MS Windows based laptop computer locally connected via USB to
the analyzer
PCReports	Software resident on the PC providing reports, user interface, and
related functions.
Performance
Verification Test
Cartridges (PVT)	Test Modules containing functional chemistry used to periodically
verify that the analyzer is functioning within acceptable limits.

Table 20. GeneSTAT System

16

3. Specimen Identification:

The sample ID is manually entered into the GeneSTAT System during the assay run process.

• 4. Specimen Sampling and Handling:
     Specimens are manually prepared and transferred to the assay Test Module. Briefly, the user is required to transfer the extracted sample to the Sample Vial, attach the Sample Vial to the assay cartridge, and load the attached Sample Vial and cartridge (referred to as the Test Module) onto the GeneSTAT analyzer when prompted by the software. Subsequent specimen handling is automated.
• 5. Calibration: Calibration is not required by the user.
• 6. Quality Control: See section L1c.

O. Other Supportive Instrument Performance Characteristics Data Not Covered in the "Performance Characteristics" Section above:

Not applicable

P. Proposed Labeling:

The labeling is sufficient and satisfies the requirements of 21 CFR parts 801 and 809 as well as the Special Controls for this type of device.

17

Q. Identified Risks to Health and Mitigation Measures:

Identified Risk	Mitigation Measures
Incorrect identification or lack of
identification of a pathogenic microorganism
by the device can lead to improper patient
management	General Controls and Special Controls
(1), (2)(i), (2)(ii), (2)(iii), (2)(iv), (2)(v),
(3)(i), (3)(ii), and (3)(iii)
Failure to correctly interpret test results	General Controls and Special Controls
(1), (2)(iii), (2)(iv), and (2)(v)
Failure to correctly operate the instrument	General Controls and Special Controls
(1), (2)(i), (3)(ii), and (3)(iii)

R. Benefit/Risk Analysis:

Summary
Summary of
the Benefit(s)	• The GeneSTAT.MDx Coccidioides Assay is the first assay to use a qualitative real- time polymerase chain reaction (PCR) to detect and identify Coccidioides spp. nucleic acids directly from patient bronchial alveolar lavage specimens and bronchial wash specimens.
	• The GeneSTAT.MDx Coccidioides Assay can provide results from an extracted sample in approximately 1.5 hours, compared to traditional fungal culture which may take up to three weeks to return results. The quicker time to result may allow for more rapid initiation of antifungal therapy, which may result in improved patient outcomes with decreases in morbidity or mortality.
	• Compared to traditional fungal culture, the GeneSTAT.MDx Coccidioides Assay demonstrated sensitivity/PPA of 100% in both prospective and retrospective studies. Specificity/NPA was 99.6% and 95.9% in the prospective and retrospective studies respectively. The performance in the prospective and retrospective clinical studies suggests that the GeneSTAT.MDx Coccidioides Assay will be highly sensitive and specific for the diagnosis of coccidioidomycosis.
Summary of
the Risk(s)	• False positive results, and false negative results are the primary risks associated with use of the GeneSTAT.MDx Coccidioides Assay.
	• A false positive result may lead to unnecessary antifungal therapy, with associated adverse events, such as potential allergic reactions.
	• A false negative result may result in a delay of antifungal therapy, with subsequent worsening of infection and associated increase in morbidity or mortality.
Summary of
Other
Factors	None.

18

| Conclusions
Do the
probable
benefits
outweigh the
probable
risks? | The probable benefits of the GeneSTAT.MDx Coccidioides Assay outweigh the potential
risks in light of the listed special controls and applicable general controls. The
GeneSTAT.MDx Coccidioides Assay is the first assay to use a qualitative real-time
polymerase chain reaction to detect and identify Coccidioides spp. nucleic acids directly
from bronchial alveolar lavage specimens and bronchial wash specimens and is likely to
benefit patients by more rapidly diagnosing coccidioidomycosis. The excellent
performance observed during the clinical trials in comparison to traditional fungal culture
and the proposed special controls suggest that errors will be uncommon and will be well
mitigated by current laboratory practices, which includes standard of care fungal culture
and product labelling. The GeneSTAT.MDx Coccidioides Assay could provide substantial
benefits as an aid in the diagnosis of coccidioidomycosis. |

-------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

S. Patient Perspectives

This submission did not include specific information on patient perspectives for this device.

T. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.3376. FDA believes that the stated special controls, and applicable general controls, including design controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Product Code:	QAA
Device Type:	Device to detect and identify fungal nucleic acids directly in respiratory
specimens
Class:	II (special controls)
Regulation:	21 CFR 866.3376

• (a) Identification. A device to detect and identify fungal nucleic acids directly in respiratory specimens is an in vitro diagnostic device intended for the detection and identification of fungal pathogens in respiratory specimens collected from patients with signs or symptoms and suspicion of fungal infection. The device is intended to aid in the diagnosis of fungal disease in conjunction with clinical signs and symptoms and other laboratory findings.
• (b) Classification. Class II (special controls). A device to detect and identify fungal nucleic acids directly in respiratory specimens must comply with the following special controls:
• The intended use for the 21 CFR 809.10 compliant labeling must include a detailed (1) description of what the device detects, the type of results provided to the user, the clinical indications appropriate for test use, and the specific population(s) and testing location(s) for which the device is intended.
• (2) The 21 CFR 809.10 compliant labeling must include:

19

• (i) A detailed device description, including the device components, instrument requirements, ancillary reagents required but not provided, and a detailed explanation of the methodology including all pre-analytical methods for processing of specimens.
• (ii) Performance characteristics from analytical studies, including but not limited to analytical sensitivity (Limit of Detection), inclusivity, reproducibility, interference, cross reactivity, interfering substances, carryover/cross contamination, and specimen stability.
• (iii) A statement that the device is intended to be used in conjunction with clinical history, signs and symptoms and the results of other diagnostic tests.
• (iv) A detailed explanation of the interpretation of test results and acceptance criteria for any quality control testing.
• (v) A limiting statement that negative results do not preclude the possibility of infection, and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
• (3) The compliant design controls must include:
  • (i) Performance characteristics from clinical studies that include prospective (sequential) samples and, if appropriate, additional characterized samples. The study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained from wellaccepted comparator methods. Documentation from the clinical studies must include the clinical study protocol (including predefined statistical analysis plan), clinical study report, and results of all statistical analyses.
  • (ii) A detailed device description of the following:
    • (A)Overall device design including all device components and all control elements incorporated into the testing procedure
    • (B) Thorough description of the methodology including, but not limited to. primer/probe sequences, primer/probe design and rationale for target sequence selection
    • (C) Computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported signal and result), as applicable
  • (iii) A detailed documentation for device software, including, but not limited to, software applications and hardware-based devices that incorporate software.