Predicate Devices

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Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on real-time PCR technology and standard analytical methods. There is no mention of AI or ML in the intended use, device description, or performance evaluation sections.

Therapeutic

No

This device is intended for the qualitative detection and differentiation of HSV-1 and HSV-2 DNA to aid in diagnosis, not for treatment or therapy.

Diagnostic

Yes

The 'Intended Use / Indications for Use' section explicitly states that the device "is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS."

SaMD (Software as a Medical Device)

No

The device description explicitly states that the system consists of the Simplexa™ HSV 1 & 2 Direct (reagents), the 3M Integrated Cycler instrument, the 3M Integrated Cycler Studio Software, the Direct Amplification Disc, and associated accessories. This includes significant hardware components (the cycler and the disc) in addition to the software.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The intended use explicitly states it's for the "qualitative detection and differentiation of HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF) samples from patients suspected of Herpes Simplex Virus (HSV) infections of the central nervous system (CNS)." This is a diagnostic purpose performed on a biological sample (CSF) in vitro (outside the body).
Device Description: The description details a "real-time PCR" system that analyzes DNA from CSF samples. PCR is a common technique used in IVD tests for detecting genetic material.
Professional Use: The assay is stated to be for "professional use only," which is typical for IVD devices used in clinical laboratories.
Clinical Studies: The document includes detailed information about clinical studies comparing the device's performance to a comparator method using patient samples. This is a requirement for demonstrating the clinical validity of an IVD.
Performance Metrics: Key metrics like Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) are provided, which are standard performance indicators for IVD tests.

All these elements align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Focus Diagnostics Simplexa™ HSV 1 & 2 Direct is intended for use on the 3M Integrated Cycler instrument for the qualitative detection and differentiation of HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF) samples from patients suspected of Herpes Simplex Virus (HSV) infections of the central nervous system (CNS). This test is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS.

Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.

The assay is not intended for use as a donor screening test. The assay is for professional use only.

The Positive Control is intended to be used as a control with the Simplexa™ HSV 1 & 2 Direct. This control is not intended for use with other assays or systems.

Product codes

PGH

Device Description

The Simplexa™ HSV 1 & 2 Direct system is a real-time PCR that enables the direct amplification, detection and differentiation of HSV-2 DNA from unprocessed CSF samples without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct, the 3M Integrated Cycler (with 3M Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the SimplexaTM HSV 1 & 2 Direct, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1, HSV-2 and DNA internal control (IC) targets. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA respectively in the specimen. The IC is used to detect PCR failure and/or inhibition.

The 3M Integrated Cycler is a real-time PCR thermocycler which uses real-time fluorometric detection to identify targets within the sample wells. The instrument is controlled by an external computer running the Integrated Cycler Studio Software.

The Simplexa™ HSV 1 & 2 Direct assay reaction takes place in the DAD consumable. The DAD consumable is compartmentalized into 8 separate wedges and up to 8 separate samples or controls may be processed on each disc. Each wedge can be used only once, however, the disc may be reused until all wedges have been utilized. Each wedge contains sample and reagent input wells, microfluidic channels and laser activated valves to control the fluid flow. and a reaction chamber. The disc is specifically designed to meter the amount of reagent (Reaction Mix) and sample that are placed into specific wells in the disc. To start processing a patient sample, a foil seal is lifted and the user adds 50 µL of Reaction Mix to the reagent input well using a fixed volume pipette. Next, the user adds 50 uL of unextracted specimen to the sample input well.

The SimplexaTM HSV 1 & 2 Direct kit contains reagents for 24 reactions. Each vial contains sufficient material for a single reaction. The kit contains the Reaction Mix, the Simplexa™ HSV 1 & 2 Direct Barcode Card with assay specific information, and the Package Insert.

The Positive Control contains ten vials of Inactivated HSV-1 and HSV-2.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Cerebrospinal fluid (CSF) samples (Central Nervous System)

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Professional use only.

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

A total of 219 CSF samples were prospectively or retrospectively collected from patients with signs and symptoms of Herpes Simplex Virus (HSV) central nervous system (CNS) infection at eight external sites. All sites sent the frozen samples to Focus Diagnostics. Two aliquots were prepared from each sample and the samples were then blinded for testing. One aliquot was sent to an external testing with the SimplexaTM HSV 1 & 2 Direct. Five external sites, which included four of the collection sites, performed testing with the Simplexa™ HSV 1 & 2 Direct. The other aliquot was retained at Focus Diagnostics for the comparator PCR/sequencing method.

Results from the Simplexa™ HSV 1 & 2 Direct were compared to results from two PCR/bi-directional sequencing assays (comparator). A positive result by the comparator is defined as a positive by at least one of the two PCR/bidirectional sequencing assays. For a result to be defined as negative by the comparator, both of the two PCR/bidirectional sequencing assays should yield negative results. The samples were tested using two PCR reactions targeting two distinct regions of the HSV genome followed by analysis with two bi-directional sequencing assays. Three hundred (300) base-pair dideoxy DNA sequencing assays were validated for two different HSV gene targets. SYBR Green PCR using extracted patient sample DNA was used to test all samples. Amplicons from positive samples were used for bidirectional sequencing. Sequence requirements had a phred quality score ≥20 (for each base) and a continuous read length >200 and a 2x coverage >180. Sequence alignments were analyzed against the GenBank database using the BLAST search program to determine if the patient sample was positive for HSV-1 or HSV-2.

Neural imaging/clinical impression results documented in a case report form (CRF) as determined by the patient's attending physician and other clinical information such as chemistries, bacterial culture, MRI/CT scans and in-house PCR results were collected for all patients. The Final Diagnosis takes into consideration all of the laboratory findings and the results of a PCR test performed locally along with clinical presentation of the patient. No dual positive (both HSV-1 Positive and HSV-2 Positive) samples were found by the Simplexa HSV1&2 Direct and the comparator algorithm.

Fifty five (55) retrospective/preselected HSV positive (as determined by the collection sites) samples from four sites were collected between 2004 and 2013 and confirmed positive by the two PCR/bi-directional sequencing assays. These retrospective samples were used to supplement the prospective study to evaluate the sensitivity of the Simplexa™ HSV 1 & 2 Direct.

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

Study Type: Analytical performance: Reproducibility
Sample Size: 90 replicates per sample panel member over five non-consecutive days, across 3 sites (total 540 replicates per analyte)
Key Results: For HSV-1, HSV-2, and the DNA IC, total agreement with expected results was 100% (540/540) with a 95% CI of 99.3% to 100.0%.

Study Type: Detection Limit (LoD)
Sample Size: 32 replicates per dilution for HSV-1 McIntyre and HSV-2 G strains. At least three replicates per dilution for HSV-1 HF and HSV-2 MS strains, with 32 replicates for confirmation.
Key Results:
HSV-1 McIntyre: LoD 5 TCID50/mL, 31/32 Detected Mean Ct 37.1 ± 1.17
HSV-1 HF: LoD 40 TCID50/mL, 31/32 Detected Mean Ct 36.9 ± 0.90
HSV-2 G: LoD 1.25 TCID50/mL, 31/32 Detected Mean Ct 38.3 ± 0.74
HSV-2 MS: LoD 20 TCID50/mL, 32/32 Detected Mean Ct 37.2 ± 1.03

Study Type: Analytical Reactivity
Sample Size: Triplicate testing for each viral strain.
Key Results: Simplexa™ HSV 1 & 2 Direct detected HSV-1 KOS at 20 TCID50/mL (3/3 and 2/3 detected for HSV-1, 0/3 for HSV-2), and HSV-1 F at 40 TCID50/mL (3/3 for HSV-1, 0/3 for HSV-2) and 80 TCID50/mL (3/3 for HSV-1, 0/3 for HSV-2).

Study Type: Cross-Reactivity (Analytical Specificity)
Sample Size: Triplicate testing for 51 potential cross-reactants.
Key Results: No cross-reactivity was observed for any of the tested microorganisms or substances for either HSV-1 or HSV-2 detection.

Study Type: Interfering Substances
Sample Size: Triplicate testing for 7 potentially interfering substances.
Key Results: No interference observed with Albumin, Casein, Hemoglobin, White Blood Cells, Antiviral Drug (Acyclovir), Topical Antiseptic (Betadine), or Whole Blood. All replicates detected for HSV-1, HSV-2, and DNA IC.

Study Type: Competitive Interference
Sample Size: Triplicate testing (or 5/8 replicates as noted) for various viral combinations.
Key Results: Competitive interference was observed when HSV-2 G (at 5 TCID50/mL) was tested with HSV-1 McIntyre at very high concentrations. At 20000 TCID50/mL HSV-1 McIntyre, 1/8 HSV-2 replicates were detected. At 10000 TCID50/mL HSV-1 McIntyre, 6/8 HSV-2 replicates were detected. At 5000 TCID50/mL HSV-1 McIntyre, 3/3 HSV-2 replicates were detected. No competitive interference was observed when HSV-1 McIntyre (at 20 TCID50/mL) was tested with HSV-2 G at 10000 TCID50/mL.

Study Type: Inhibition by Other Microorganisms
Sample Size: Initial triplicate testing. If any replicate was "Not Detected", five additional replicates were tested (total 8 replicates).
Key Results: None of the 51 microorganisms tested caused more than 4/8 of the replicates to be "Not Detected". One of 8 replicates of JCV (MAD-4) and Rabies were "Not Detected" for HSV-2, and 2/8 replicates of Dengue (Type1) were "Not Detected" for HSV-2. No inhibition by other organisms was observed for HSV-1.

Study Type: Sample Stability Studies (Fresh vs. Frozen)
Sample Size: 120 pairs of contrived samples (60 for HSV-1, 60 for HSV-2).
Key Results: 100% (60/60) qualitative agreement was observed with 95% CI (94.0% to 100.0%) between the fresh and frozen states for both HSV-1 and HSV-2 targets. Regression analysis showed statistically non-significant bias between Ct values for fresh and frozen samples.

Study Type: Clinical Studies (Method Comparison)
Sample Size: 164 prospective CSF samples. 55 retrospective/preselected HSV positive samples.
Key Results:
HSV-1 Prospective Samples: PPA 100.0% (3/3), NPA 98.8% (159/161).
HSV-2 Prospective Samples: PPA 85.7% (6/7), NPA 99.4% (156/157).
Retrospective/Preselected Positive Samples:
HSV-1 PPA 100.0% (13/13)
HSV-2 PPA 100.0% (42/42)

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

Prospective Samples, HSV-1:
Positive Percent Agreement (PPA): 100.0% (3/3), 95% CI: 43.8 to 100.0%
Negative Percent Agreement (NPA): 98.8% (159/161), 95% CI: 95.6 to 99.7%

Prospective Samples, HSV-2:
Positive Percent Agreement (PPA): 85.7% (6/7), 95% CI: 48.7 to 97.4%
Negative Percent Agreement (NPA): 99.4% (156/157), 95% CI: 96.5 to 99.9%

Retrospective/Preselected Positive Samples:
HSV-1 PPA: 100.0% (13/13), 95% CI: 77.2 to 100.0%
HSV-2 PPA: 100.0% (42/42), 95% CI: 91.6 to 100.0%

Predicate Device(s)

Not Found

Reference Device(s)

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Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3307 Herpes simplex virus nucleic acid-based assay for central nervous system infections.

(a)
Identification. A herpes simplex virus nucleic acid-based assay for central nervous system infections is a qualitative in vitro diagnostic device intended for the detection and differentiation of HSV-1 and HSV-2 in cerebrospinal fluid (CSF) samples from patients suspected of Herpes Simplex Virus (HSV) infections of the central nervous system (CNS). This test is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS. Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Design verification and validation must include:
(i) Detailed documentation for the device description, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including primer design and selection.
(ii) Detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (limit of detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carryover, and cross contamination. Documentation must include reagent and sample stability recommendations.
(iii) Detailed documentation from a clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to the results of two polymerase chain reaction methods followed by bidirectional sequencing.
(iv) Documentation of an appropriate end user device training program that will be offered as part of efforts to mitigate the risk of failure to correctly operate the instrument.
(v) Quality assurance protocols and detailed documentation for device software, including standalone software applications and hardware-based devices that incorporate software.
(2) The labeling required under § 809.10(b) of this chapter must include:
(i) A detailed explanation of the interpretation of results and acceptance criteria.
(ii) A limiting statement indicating that negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.

Summary

0

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Simplexa™ HSV 1 & 2 Direct

DECISION SUMMARY

A. 510(k) Number:

K133621

B. Purpose for Submission:

De novo request for evaluation of automatic class III designation for the Simplexa™ HSV 1 & 2 Direct

C. Measurand:

Target DNA sequences from conserved regions of the HSV-1 and HSV-2 DNA polymerase genes.

D. Type of Test:

A real-time Polymerase Chain Reaction (PCR) for the direct amplification, detection and differentiation of HSV-1 and HSV-2 DNA from unprocessed CSF samples without nucleic acid extraction.

E. Applicant:

Focus Diagnostics, Inc.

F. Proprietary and Established Names:

Simplexa™ HSV 1 & 2 Direct

G. Regulatory Information:

1. Regulation: 21 CFR 866.3307
1. Classification: Class II (special controls)
1. Product code: PGH
1. Panel: Microbiology (83)

1

H. Intended Use:

1. Intended use(s):

Simplexa™ HSV 1 & 2 Direct

The Focus Diagnostics Simplexa™ HSV 1 & 2 Direct is intended for use on the 3M Integrated Cycler instrument for the qualitative detection and differentiation of HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF) samples from patients suspected of Herpes Simplex Virus (HSV) infections of the central nervous system (CNS). This test is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS.

Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.

The assay is not intended for use as a donor screening test. The assay is for professional use only.

The Positive Control is intended to be used as a control with the Simplexa™ HSV 1 & 2 Direct. This control is not intended for use with other assays or systems.

1. Indication(s) for use:
  Same as intended use
1. Special conditions for use statement(s):
  The Simplexa" HSV 1 & 2 Direct is for prescription use only in accordance with 21 CFR 801.109.
1. Special instrument requirements:
  To be used with the 3MTM Integrated Cycler with Integrated Cycler Studio Software version 5.0 or higher

I. Device Description:

Simplexa™ HSV 1 & 2 Direct

The Simplexa™ HSV 1 & 2 Direct system is a real-time PCR that enables the direct amplification, detection and differentiation of HSV-2 DNA from unprocessed CSF samples without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct, the 3M Integrated Cycler (with 3M Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the SimplexaTM HSV 1 & 2 Direct, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1, HSV-2 and DNA internal control (IC) targets. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA respectively in the specimen. The IC

2

is used to detect PCR failure and/or inhibition.

The 3M Integrated Cycler is a real-time PCR thermocycler which uses real-time fluorometric detection to identify targets within the sample wells. The instrument is controlled by an external computer running the Integrated Cycler Studio Software.

The Simplexa™ HSV 1 & 2 Direct assay reaction takes place in the DAD consumable. The DAD consumable is compartmentalized into 8 separate wedges and up to 8 separate samples or controls may be processed on each disc. Each wedge can be used only once, however, the disc may be reused until all wedges have been utilized. Each wedge contains sample and reagent input wells, microfluidic channels and laser activated valves to control the fluid flow. and a reaction chamber. The disc is specifically designed to meter the amount of reagent (Reaction Mix) and sample that are placed into specific wells in the disc. To start processing a patient sample, a foil seal is lifted and the user adds 50 µL of Reaction Mix to the reagent input well using a fixed volume pipette. Next, the user adds 50 uL of unextracted specimen to the sample input well.

The SimplexaTM HSV 1 & 2 Direct kit contains reagents for 24 reactions. Each vial contains sufficient material for a single reaction. The kit contains the Reaction Mix, the Simplexa™ HSV 1 & 2 Direct Barcode Card with assay specific information, and the Package Insert.

| Kit

Component	Contents
Simplexa™
HSV 1 & 2
Direct Reaction
Mix	DNA polymerase, buffer, dNTPs, template DNA (IC), dye-labeled
fluorescent probe-primers specific for detection of HSV-1, HSV-2, and the
DNA IC.
	Target	Probe
Fluorophore
(Dye)	Excitation
(nm)	Emission
(nm)	Targeted Gene
	HSV-1	CFR610	590	610	HSV-1 DNA
polymerase
	HSV-2	FAM	495	520	HSV-2 DNA
polymerase
	DNA
IC	Q670	644	670	NA
Simplexa™
HSV 1 & 2 Kit
Barcode Card	Assay specific parameters.

Component Description

The Positive Control contains ten vials of Inactivated HSV-1 and HSV-2.

3

J. Standard/Guidance Documents Referenced:

Guidance for Industry and FDA Staff: Administrative Procedures for CLIA Categorization, May 7, 2008

Guidance for Industry and FDA Staff: Format for Traditional and Abbreviated 510(k), August 12, 2005

Guidance for Industry, FDA Reviewers and Compliance on Off-The-Shelf Software Use in Medical Devices, September 9, 1999

Guidance for Industry: Cybersecurity for Networked Medical Devices Containing Off-The-Shelf (OTS) Software, July 18, 2007

Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices, May 11, 2005

General Principles of Software Validation: Final Guidance for Industry and FDA Staff, January 11, 2002

K. Test Principle:

The Simplexa™ HSV 1 & 2 Direct system is a real-time PCR for the direct amplification, detection and differentiation of HSV-1 and HSV-2 DNA from unprocessed CSF specimens without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct, the 3M Integrated Cycler (with 3M Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ HSV 1 & 2 Direct assay reaction, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1. HSV-2 and IC targets. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA, respectively, in the specimen. An internal control is used to detect PCR failure and/or inhibition. The principle of the test and the procedural steps are summarized below.

Specimen Collection:

The specimen type is CSF. Specimens should be transported on ice and stored at 2 to 8°C for up to 7 days post collection. If there is delay for more than 7 days before processing, the specimens should be stored at -70°C.

Real-Time PCR Instrument Setup:

The 3M Integrated Cycler Operator Manual provides details on how to configure the 3M Integrated Cycler Studio Software, to add an assay definition, and to set up and analyze runs on the 3M Integrated Cycler.

Direct Amplification Disc Loading and Real-Time PCR Amplification:

1. Select samples that need to be tested.
1. Thaw Reaction Mix vials at room temperature (approximate range 18 to 25°C). Thaw one Reaction Mix vial for each sample or control to be tested.

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1. Scan the barcode on the Simplexa™ HSV 1 & 2 Direct Reaction Mix vial or barcode card.
1. Scan the disc barcode on the Direct Amplification Disc (DAD).
1. Scan or type in each sample identifier.
1. For one wedge at a time, peel the adhesive foil back to expose the Sample (SAMPLE) and Reaction (R) wells without completely removing the adhesive foil cover (Figures 1 & 2). A void touching the under side of the foil that will be in contact with the wells and disc surface.
1. Ensure that the Reaction Mix is completely thawed. Briefly spin down the tubes as needed. (Do not vortex the Reaction Mix).
Use the fixed volume pipette to transfer 50 uL of the Reaction Mix into the Reaction (R) 8. well.
1. Use the fixed volume pipette to transfer 50 µL of sample or control; pipette sample or control into the Sample well (SAMPLE).
1. Cover the wedge sealing the wells with the peeled adhesive foil, pressing down firmly near the edge of the wedge. If the original foil is torn, do not load the wells in the wedge. Instead load another wedge.
1. Tear off the tab portion of the foil cover along the perforation.
1. Repeat steps 6 to 11 for the next sample(s).
1. Load the sealed Direct Amplification Disc into the 3M Integrated Cycler and start the run.

Image /page/4/Figure/11 description: The image contains two figures showing a disc with pre-use foil lifted from sample and reaction wells. Figure 1 shows the disc with the foil lifted from wedge #3, revealing the numbers 2, 3, and 4 on the disc. Figure 2 shows a close-up of the sample and reaction wells, labeled as "SAMPLE" and "R" respectively, with the number 4 visible on the foil.

Interpretation of Results:

Upon completion of the run, the Integrated Cycler Studio Software automatically calculates and displays results. The display presents the user with a separate report box for each of the analytes as well as the IC. The IC can be reported as "valid" or "invalid".

The software reports one of four possible outcomes for each of HSV-1 and HSV-2 after a run

5

is completed for each sample ID entered: "Detected". "Not Detected". "Invalid" or "EC500".

"Detected" result indicates that HSV-1 and/or HSV-2 DNA were detected in the patient a. sample tested (whether the DNA IC was detected or not detected)
b. "Not Detected" result indicates that HSV-1 and/or HSV-2 DNA were not detected in the patient sample tested whereas the DNA IC was detected.
c. "Invalid" result points to the inability to detect presence or absence of HSV-1 and/or HSV-2 DNA in the patient sample. This result may be due to:
- 1. DNA Internal Control (DNA IC) failure, or
- 1. Failure to detect sufficient specimen.

If an invalid result occurs, the sample needs to be re-tested per the instructions provided in the device Package Insert.

d. "EC500" result points to a data quality error for the particular viral analyte(s). The software was unable to determine a valid amplification for that analyte(s). The sample should be re-tested.

L. Performance Characteristics (if/when applicable):

1. Analytical performance:
- a. Reproducibility

The reproducibility study evaluated the device's inter-laboratory, inter-assay, and intra-assay reproducibility for high negative, low positive (approximately equal to the Limit of Detection (LoD)) and moderately positive (approximately 4 times LoD) samples for HSV-1 (McIntyre strain) and HSV-2 (G strain) and a positive control. The testing panel consisted of a total of 6 samples: a high negative pool containing both HSV-1 and HSV-2 targets, a low positive and a moderately positive sample separately for each of HSV-1 and HSV-2, and a positive control containing both HSV-1 and HSV-2 targets. The samples (except for the positive control) were generated by spiking viral stock into CSF that was screened to be negative for both analytes. For each sample panel member, two different operators performed two runs per day with 3 replicates per run, for five days at each of the three sites. This provided a total of 90 (3 sites x 5 days x 2 runs (each by different operator) x 3 replicates) replicates per sample panel member over five non-consecutive days. Three sites assessed the device's inter-laboratory reproducibility and inter/intra-assay reproducibility. Combined results for all sites and results stratified by site are presented in the tables below.

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	Site - 1			Site - 2	Site - 3
Sample	%
Agreement
with
Expected
Results	Avg.
Ct	Total
%CV	%
Agreement
with
Expected
Results	Avg.
Ct	Total
%CV	%
Agreement
with
Expected
Results	Avg.
Ct	Total
%CV	Total %
Agreement
with
Expected
Results	95% CI
HSV-1 Low
Positive	100.0%
(30/30)	36.9	2.0	100.0%
(30/30)	35.4	2.2	100.0%
(30/30)	36.6	2.7	100.0%
(90/90)	95.9%
to
100.0%
HSV-1
Medium
Positive	100.0%
(30/30)	34.6	1.6	100.0%
(30/30)	33.2	1.2	100.0%
(30/30)	34.3	1.8	100.0%
(90/90)	95.9%
to
100.0%
HSV-2 Low
Positive	100.0%
(30/30)a	NA	NA	100.0%
(30/30)a	NA	NA	100.0%
(30/30)a	NA	NA	100.0%
(90/90)a	95.9%
to
100.0%
HSV-2
Medium
Positive	100.0%
(30/30)a	NA	NA	100.0%
(30/30)a	NA	NA	100.0%
(30/30)a	NA	NA	100.0%
(90/90)a	95.9%
to
100.0%
High
Negative	100.0%
(30/30)a	NA	NA	100.0%
(30/30)a	NA	NA	100.0%
(30/30)a	NA	NA	100.0%
(90/90)a	95.9%
to
100.0%
Positive
Control	100.0%
(30/30)	30.7	1.5	100.0%
(30/30)	30.6	1.2	100.0%
(30/30)	30.2	1.4	100.0%
(90/90)	95.9%
to
100.0%
Total
Agreement	100.0% (180/180)			100.0% (180/180)	100.0% (180/180)	100.0%
(540/540)	99.3%
to
100.0%

ª Expected Results for HSV-2 Low Positive, HSV-2 Medium Positive and High Negative samples are "Negative" for
HSV-1.

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	Site - 1			Site - 2	Site - 3
Sample	%
Agreement
with
Expected
Results	Avg.
Ct	Total
%CV	%
Agreement
with
Expected
Results	Avg.
Ct	Total
%CV	%
Agreement
with
Expected
Results	Avg.
Ct	Total
%CV	Total %
Agreement
with
Expected
Results	95% CI
HSV-1 Low
Positive	100.0%
(30/30)b	NA	NA	100.0%
(30/30)b	NA	NA	100.0%
(30/30)b	NA	NA	100.0%
(90/90)b	95.9%
to
100.0%
HSV-1
Medium
Positive	100.0%
(30/30)b	NA	NA	100.0%
(30/30)b	NA	NA	100.0%
(30/30)b	NA	NA	100.0%
(90/90)b	95.9%
to
100.0%
HSV-2 Low
Positive	96.7%
(29/30)	38.1	2.9	90.0%
(27/30)	38.4	3.2	83.3%
(25/30)	38.3	2.4	90.0%
(81/90)b	82.1%
to
94.6%
HSV-2
Medium
Positive	100.0%
(30/30)	35.0	1.3	100.0%
(30/30)	34.6	1.8	100.0%
(30/30)	35.0	1.4	100.0%
(90/90)	95.9%
to
100.0%
High
Negative	93.3%
(28/30)b	39.7	0.2	96.7%
(29/30)b	38.4	NA	96.7%
(29/30)b	39.1	NA	95.6%
(86/90)b	89.1%
to
98.3%
Positive
Control	100.0%
(30/30)	30.1	0.9	100.0%
(30/30)	30.6	1.1	100.0%
(30/30)	30.0	1.2	100.0%
(90/90)	95.9%
to
100.0%
Total
Agreement	98.3% (177/180)			97.8% (176/180)	96.7% (174/180)	97.6%
(527/540)	95.9%
to
98.6%

b Expected Results for HSV-1 Low Positive, HSV-1 Medium Positive and High Negative samples are "Negative" for HSV-2.

Reproducibility – DNA IC

| Sample | %
Agreement
with
Expected
Results | Avg.
Ct | Total
%CV | Site - 1 | | | Site - 2 | | | Site - 3 | | | Total %
Agreement
with
Expected
Results | 95% CI |
|-----------------------------|-----------------------------------------------|------------|--------------|-----------------------------------------------|------------|--------------|-----------------------------------------------|------------|--------------|-----------------------------------------------|-----------------------|--------------|-----------------------------------------------------|-----------------------|
| | | | | %
Agreement
with
Expected
Results | Avg.
Ct | Total
%CV | %
Agreement
with
Expected
Results | Avg.
Ct | Total
%CV | %
Agreement
with
Expected
Results | Avg.
Ct | Total
%CV | | |
| HSV-1 Low
Positive | 100.0%
(30/30) | 29.6 | 0.7 | 100.0%
(30/30) | 30.0 | 1.7 | 100.0%
(30/30) | 29.1 | 0.6 | 100.0%
(90/90) | 95.9%
to
100.0% | | | |
| HSV-1
Medium
Positive | 100.0%
(30/30) | 29.6 | 0.8 | 100.0%
(30/30) | 30.1 | 1.2 | 100.0%
(30/30) | 29.1 | 0.6 | 100.0%
(90/90) | 95.9%
to
100.0% | | | |
| HSV-2 Low
Positive | 100.0%
(30/30) | 29.4 | 0.7 | 100.0%
(30/30) | 30.1 | 1.0 | 100.0%
(30/30) | 29.1 | 0.7 | 100.0%
(90/90) | 95.9%
to
100.0% | | | |
| HSV-2
Medium
Positive | 100.0%
(30/30) | 29.4 | 0.9 | 100.0%
(30/30) | 30.0 | 1.3 | 100.0%
(30/30) | 29.2 | 1.6 | 100.0%
(90/90) | 95.9%
to
100.0% | | | |
| High
Negative | 100.0%
(30/30) | 29.4 | 0.8 | 100.0%
(30/30) | 30.1 | 1.3 | 100.0%
(30/30) | 29.2 | 1.3 | 100.0%
(90/90) | 95.9%
to
100.0% | | | |
| Positive
Control | 100.0%
(30/30) | 29.6 | 1.0 | 100.0%
(30/30) | 30.0 | 1.1 | 100.0%
(30/30) | 29.2 | 0.8 | 100.0%
(90/90) | 95.9%
to
100.0% | | | |
| Total
Agreement | | | | 100.0% (180/180) | | | 100.0% (180/180) | | | 100.0% (180/180) | | | 100.0%
(540/540) | 99.3%
to
100.0% |

The positive control Inter-Lot precision was evaluated to assess if there was lot to lot

8

variability of the positive control. The study assessed the reproducibility of 3 different lots of positive control Pack by testing each lot of control with one lot of Reaction Mix, using one instrument and one operator over 3 consecutive days. Each lot of positive control was run with 2 replicates per run, 2 runs per day for 3 days, thereby providing a total 12 replicates per lot. The testing was performed at Focus Diagnostics. Results of the study were analyzed for overall mean, overall (total) variability, variability between day (inter-day), variability between run (inter-run), variability between lot (inter-lot) and the residual. The variability is presented in terms of standard deviation (SD) and/or percent coefficient of variation (%CV) of Ct. The inter-lot variability component is summarized in the table below.

Quantitative Summary of PC Inter-Lot Reproducibility
			Inter-Day		Inter-Run		Inter-Lot		Intra-Run/Lot		Total
Analyte	N	Mean Ct	SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
HSV-1	36	29.4	0.00	0.0	0.00	0.0	0.11	0.4	0.11	0.4	0.16	0.5
HSV-2	36	29.8	0.00	0.0	0.00	0.0	0.12	0.4	0.13	0.5	0.18	0.6
IC	36	29.6	0.00	0.0	0.08	0.3	0.08	0.3	0.19	0.7	0.22	0.8

b. Linearity/assay reportable range:

Not applicable.

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

Positive Control Stability:

The positive control for this product is a blend of inactivated HSV-1 and HSV-2 provided in single use aliquots frozen at -20°C. The positive control has been assigned an expiration date of 12 months at -20℃.

To assess the room temperature stability of positive control, aliquots of the positive control were thawed and kept at room temperature for up to 24 hours, and were then evaluated.

Room-temperature stability of Positive Control

9

	HSV-2 (FAM)		HSV-1 (CFR610)		IC (Q670)
Time (hrs) at room temperature	Mean Ct	Change from time '0'	Mean Ct	Change from time '0'	Mean Ct	Change from time '0'
0	30.6		30.7		30.1
8 hours	30.5	-0.33%	30.9	0.65%	30.3	0.66%
0	30.8		31.3		30.2
24 hours	30.8	0.00%	31.0	-0.96%	30.2	0.00%

A negative percent difference indicates earlier Ct detection after room temperature storage. As shown in the table above, no significant change in detection of each target analyte was observed when positive control was stored on the bench top at room temperature for 24 hours. Based on these results, the positive control is stable for at least 24 hours at room temperature.

d. Assay cut-off:

The Simplexa™ HSV 1 & 2 Direct cut-off was determined during feasibility and verification studies. During verification, all studies were performed to a maximum of 45 cycles of amplification for informational use, but the data were calculated with a cut-off of Ct=40 for HSV-1 and HSV-2 targets. When the verification data were analyzed, there were some results that prompted the Ct cut-off for HSV-2 to be re-set from Ct=40 to Ct=42. The LoD verification study detected HSV-2 between a Ct of 40 and 41 for four replicates at HSV-2 concentrations below the established HSV-2 LoD concentration. No HSV-2 LoD replicates (at or below LoD concentration) were detected beyond Ct=41. The HSV-1 cut-off was not adjusted from Ct=40 because no HSV-1 LoD replicates from the LoD verification study were detected at a Ct>40. Based on the results of the verification studies, the final cut-offs were set at Ct=40 for HSV-1 and at Ct=42 for HSV-2. The PCR cycling protocol was reduced to 42 cycles because all target cut-offs are at Ct4/8) replicates were "Not Detected", then an inhibitory effect would be determined. None of the microorganisms caused >4/8 of the replicates to be "Not Detected". One of 8 replicates of JCV (MAD-4) and Rabies were "Not Detected" for HSV-2 and 2/8 replicates of Dengue (Type1) were "Not Detected" for HSV-2. No inhibition by other organisms was observed for either HSV-1 or HSV-2.

| No. | Microorganism (#Detected/#Total) | |
|-----|----------------------- | | | 1 | Baseline | 2 | Adenovirus (type 1) | 3 | Adenovirus (type 7A) | 4 | BKV | 5 | Citrobacter freundii | 6 | Citrobacter koseri | 7 | Cryptococcus neoformans | 8 | Cytomegalovirus (AD169) | 9 | Dengue (Type1) | 10 | Encephalomyocarditis virus | 11 | Enterobacter aerogenes | 12 | Enterovirus 71 | 13 | Epstein Barr virus (B95-8) | 14 | Escherichia coli | 15 | Haemophilus influenzae | 16 | Haemophilus influenzae (MinnA) | 1.00 X 106 cfu/mL | 17 | Hepatitis B | 18 | Hepatitis C | 19 | HIV1 (type IIIB) | 20 | HIV2 (type NIHZ) | 21 | Human herpesvirus 6 | 22 | Human herpesvirus 7 (Type SB) | 23 | Human herpesvirus 8 | 24 | Influenza A/California NYMC x-179-A | 1.00 X 105 TCID50/mL | 25 | Influenza B/Florida/02/2006 | 26 | JCV (MAD-4) | 27 | Klebsiella pneumoniae | No. | Microorganism (#Detected/#Total) | |
| | | 28 | LA Crosse encephalitis | 29 | Listeria monocytogenes | 30 | Measles | 31 | Mumps | 32 | Mycobacterium tuberculosis
(Genomic DNA) | 1.00 X 106 cfu/mL | 33 | Naegleria fowleri | 34 | Neisseria meningitides
(serogroup A) | 1.00 X 106 cfu/mL | 35 | Parainfluenza Virus 1 | 36 | Parainfluenza Virus 2 | 37 | Parainfluenza Virus 3 | 38 | Parvovirus B19 | 39 | Poliovirus (Type 3) | 40 | Proteus mirabilis (Z050) | 41 | Pseudomonas aeruginosa | 42 | Rabies | 43 | Rhinovirus (Type 1A) | 44 | Rotavirus (Type Wa) | 45 | Rubella | 46 | St. Louis Encephalitis | 47 | Staphylococcus aureus COL | 48 | Streptococcus agalactiae | 49 | Streptococcus pneumoniae
Z022; 19F | 1.00 X 106 cfu/mL | 50 | Toxoplasma gondii | 51 | Varicella zoster virus | 52 | West Nile Virus | Tested Concentration | Qualitative Result
------------------------|---------------------------|------------------------------------------|--------|
| | HSV-1 | HSV-2 |
| Not Applicable | 20/20 | 20/20 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 copies/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 8/8 | *6/8 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 copies/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
type b
| 3/3 | 3/3 |
| 1.00 X 105 IU/mL | 3/3 | 3/3 |
| 1.00 X 105 IU/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| Not Available | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 copies/mL | 3/3 | 3/3 |
/7/2009
| 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 8/8 | ***7/8 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| Tested Concentration | Qualitative Result
| | HSV-1 | HSV-2 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 3/3 | 3/3 |
| 1.00 X 104 cells/mL | 3/3 | 3/3 |
| 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 IU/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 1.00 X 106 copies/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 8/8 | ***7/8 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 1.00 X 106 cfu/mL | 3/3 | 3/3 |
| 3/3 | 3/3 |
| 1.00 X 106 tachyzoites/mL | 3/3 | 3/3 |
| 1.00 X 105 copies/mL | 3/3 | 3/3 |
| 1.00 X 105 TCID50/mL | 3/3 | 3/3 |

Simplexa™ HSV 1 & 2: Inhibition by Other Microorganisms

16

*Quantified material was not available to test; instead the vendor provided a culture fluid with a known Ct value. The site was directed to dilute the stock to a relevant Ct value; 1:200 dilution factor. ** 1/3 and 1/5 replicates were "Not Detected" for HSV-2 during initial and confirmatory testing respectively.

***1/3 replicate was "Not Detected" for HSV-2 during initial testing.

Sample stability studies i.

The objective of this study was to evaluate the difference between results from the Simplexa™ HSV 1 & 2 Direct testing of fresh (never frozen, unextracted and tested within 72 hours of preparation) specimens versus frozen (unextracted and tested after

17

2 freeze-thaw steps) specimens. A panel of contrived samples consisting of 120 pairs (fresh and frozen) of spiked specimens prepared in negative human CSF matrix was used for this study; 60 of the pairs spanned the analytical range for HSV-1 and the other 60 pairs spanned the range for HSV-2. Sixty percent of the samples (36/60) were spiked to be close to LoD so that 18 of the samples were 1-1.5 times LoD and 18 samples were spiked at 4 times LoD. The remaining 40% of the samples (24/60) were spiked across the assay range. One lot of Simplexa ™ HSV 1 & 2 Direct was used for the study. The frozen samples were stored at -20°C for a minimum of 5 days, after which they were thawed for at least 1 hour and refrozen for >24 hours and then thawed again for testing. The HSV-1 (McIntyre) and HSV-2 G strains were used in the study.

No significant storage effect on the samples stored fresh was detected when compared to the results from the same samples frozen. Below is the summary of results from the Fresh vs. Frozen Study of the Simplexa™ HSV 1 & 2 Direct along with the concentration [TCID50/mL] of the virus strains. In the SimplexaTM HSV 1 & 2 Direct Fresh vs. Frozen study, 100% (60/60) qualitative agreement was observed with 95% CI (94.0% to 100.0%) between the fresh and frozen states for both HSV-1 and HSV-2 targets. Regression analysis was performed to estimate quantitative bias (in terms of Ct) between Ct values from the paired fresh and frozen samples. The regression plots are shown below. The 95% confidence intervals for intercept and slope of both targets indicate statistically non-significant bias between Ct values for fresh and frozen samples. These results support the testing of frozen samples to establish performance characteristics of the assay.

HSV-1 Sample Distribution
HSV-1 (McIntyre)
Spiked Concentration
[TCID50/mL]	Number of
Replicates
(% of Total)
5 (LoD)	18 (30%)
20 (4XLoD)	18 (30%)
78.125	4 (6.7%)
156.25	4 (6.7%)
312.5	4 (6.7%)
625	4 (6.7%)
1250	4 (6.7%)
2500	4 (6.7%)
Total	60 (100%)

18

Image /page/18/Figure/0 description: The image contains a Passing-Bablok regression fit with a confidence bound, showing the relationship between fresh and frozen HSV1 Ct values. The regression line equation is P-B Reg Line = -1.54 + 1.05 * X, with a 95% confidence interval for the intercept of (-3.1146, 0.1000) and for the slope of (1.0000, 1.0976), based on a sample size of n=60. Additionally, there is a table showing the HSV-2 sample distribution, detailing the number of replicates and their percentage of the total for various spiked concentrations of HSV-2 (G strain), ranging from 1.875 (1.5XLoD) to 2500 [TCID50/mL].

Spiked Virus-HSV1 Passing-Bablok Regression Fit with Confidence Bound

19

Spiked Virus-HSV2 Passing-Bablok Regression Fit with Confidence Bound

Image /page/19/Figure/1 description: The image is a scatter plot that shows the correlation between fresh and frozen HSV2 Ct values. The x-axis represents fresh HSV2 Ct, and the y-axis represents frozen HSV2 Ct. The plot includes a Passing-Bablok regression line with the equation P-B Reg Line = -0.91 + 1.04 * X, along with 95% confidence intervals for the intercept (-2.6000, 0.2000) and slope (1.0000, 1.0889), with a sample size of n=60. Different symbols represent varying concentrations of TCID50/mL.

j. Stability studies

A real-time stability was completed over a 14 month time period supporting a shelflife claim of 12 months for the Simplexa™ HSV 1 & 2 Direct and the positive control.

k. Carry-over Contamination

The amplification carry-over for the Simplexa™ assays including the DirectSimplexa™ HSV 1 & 2 Direct Simplexa™ HSV 1 & 2 Direct was assessed from the Simplexa™ Flu A/B & RSV Direct (K120413). The carry-over study in K120413 can be applied to the Simplexa™ HSV 1 & 2 Direct as carry-over contamination is not expected to be analyte specific and there have been no hardware changes to the instrument since the K120413 clearance. The carry-over study in K120413 searched for the presence of contamination in negative samples that were adjacent to high positive samples. The study was designed by alternately placing high positive and negative samples on each disc. A total of 60 negative samples and 60 high positive samples were tested across 17 runs using 4 different instruments. The

20

sample size of 60 was chosen to provide an acceptance criterion of > 90% for the lower bound of the two-sided 95% Confidence Interval for the observed negative rate of the negative samples. This acceptance criterion would be met if no more than one of the 60 negative samples gave a "Detected" result indicative of carry-over contamination. All 60 negative samples gave a result of "Not Detected" for all three target viruses in the K120413 study with a negative rate of 100% and a lower bound of the two-sided 95% CI of 94.0%. Therefore, no evidence of carryover was observed.

2. Comparison studies:

a. Method comparison with predicate device:
Not applicable. Refer to the Clinical Studies section of this document.
b. Matrix comparison:
Not applicable.
1. Clinical studies:
  A total of 219 CSF samples were prospectively or retrospectively collected from patients with signs and symptoms of Herpes Simplex Virus (HSV) central nervous system (CNS) infection at eight external sites. All sites sent the frozen samples to Focus Diagnostics. Two aliquots were prepared from each sample and the samples were then blinded for testing. One aliquot was sent to an external testing with the SimplexaTM HSV 1 & 2 Direct. Five external sites, which included four of the collection sites, performed testing with the Simplexa™ HSV 1 & 2 Direct. The other aliquot was retained at Focus Diagnostics for the comparator PCR/sequencing method.

Results from the Simplexa™ HSV 1 & 2 Direct were compared to results from two PCR/bi-directional sequencing assays (comparator). A positive result by the comparator is defined as a positive by at least one of the two PCR/bidirectional sequencing assays. For a result to be defined as negative by the comparator, both of the two PCR/bidirectional sequencing assays should yield negative results. The samples were tested using two PCR reactions targeting two distinct regions of the HSV genome followed by analysis with two bi-directional sequencing assays. Three hundred (300) base-pair dideoxy DNA sequencing assays were validated for two different HSV gene targets. SYBR Green PCR using extracted patient sample DNA was used to test all samples. Amplicons from positive samples were used for bidirectional sequencing. Sequence requirements had a phred quality score ≥20 (for each base) and a continuous read length >200 and a 2x coverage >180. Sequence alignments were analyzed against the GenBank database using the BLAST search program to determine if the patient sample was positive for HSV-1 or HSV-2.

Neural imaging/clinical impression results documented in a case report form (CRF) as

21

determined by the patient's attending physician and other clinical information such as chemistries, bacterial culture, MRI/CT scans and in-house PCR results were collected for all patients. The Final Diagnosis takes into consideration all of the laboratory findings and the results of a PCR test performed locally along with clinical presentation of the patient. No dual positive (both HSV-1 Positive and HSV-2 Positive) samples were found by the Simplexa HSV1&2 Direct and the comparator algorithm. The table below shows the distribution of the different clinical parameters collected from patients who were confirmed positive and negative by the results from the two PCR/bi-directional sequencing assays.

| Parameter | Patients which
are positive by
the comparator
for HSV
(Number and %) | Patients which
are negative by
the comparator
for HSV
(Number and %) |
|---------------------------------------------|----------------------------------------------------------------------------------|----------------------------------------------------------------------------------|
| MRI results suggestive of HSV infection | 18/65 (27.7%) | 9/154 (5.8%) |
| MRI results not suggestive of HSV infection | 43/65 (66.2%) | 121/154 (78.6%) |
| MRI results not available | 4/65 (6.2%) | 24/154 (15.6%) |
| Final Diagnosis positive for HSV infection. | 61/65 (93.8%) | 33/154 (21.4%) |
| Final Diagnosis negative for HSV infection | 4/65 (6.2%) | 121/154 (78.6%) |
| Bacteria culture positive | 2/65 (3.1%) | 4/154 (2.6%) |
| Bacteria culture negative | 63/65 (96.9%) | 145/154 (94.2%) |
| Bacteria culture not performed | 0/65 (0.0%) | 5/154 (3.2%) |

22

The following tables summarize the results of the Simplexa™ HSV 1 & 2 Direct assay's
agreement with the comparator (results of the 2 PCR/bidirectional sequencing assays).

HSV-1: Prospective Samples				HSV-2: Prospective Samples
Simplexa™
Results	Comparator: Two PCR/SEQ			Simplexa™
Results	Comparator: Two PCR/SEQ
	Detected	Not
Detected	Total		Detected	Not
Detected	Total
Detected	3	2*	5	Detected	6	1##	7
Not Detected	0	159	159	Not Detected	1#	156	157
Total	3	161	164	Total	7	157	164
PPA	100.0% (3/3)
95% CI: 43.8 to 100.0%			PPA	85.7% (6/7)
95% CI: 48.7 to 97.4%
NPA	98.8% (159/161)
95% CI: 95.6 to 99.7%			NPA	99.4% (156/157)
95% CI: 96.5 to 99.9%
*1/2 sample had a final diagnosis of meningitis
from the chart information.				#Sample was collected from patient with a final
diagnosis of meningitis from the chart
information.
##Sample was collected from patient with a final
diagnosis of meningitis from the chart
information.

Note: PPA: Positive Percent Agreement; NPA: Negative Percent Agreement

Fifty five (55) retrospective/preselected HSV positive (as determined by the collection sites) samples from four sites were collected between 2004 and 2013 and confirmed positive by the two PCR/bi-directional sequencing assays. These retrospective samples were used to supplement the prospective study to evaluate the sensitivity of the Simplexa™ HSV 1 & 2 Direct. The results are shown in the table below.

23

| Retrospective/Preselected Positive Samples –

Positive Percent Agreement (PPA)
Simplexa™
Results	Comparator: Two PCR/SEQ
	HSV-1	HSV-2
HSV-1	13	0
HSV-2	0	42
Not Detected	0	0
Total	13	42
	HSV-1	HSV-2
PPA	100.0% (13/13)
95% CI: 77.2 to
100.0%	100.0% (42/42)
95% CI: 91.6 to
100.0%

4. Expected Values

The observed expected values of HSV-1 and HSV-2 in the prospective population of the Simplexa™ HSV 1 & 2 Direct clinical study varied between institutions, and are shown in the tables below.

| Sample
Population | Sample
Collection
Site | No. of
Samples | Expected Values Based on
Simplexa™ HSV1/2 Direct | | |
|----------------------|------------------------------|-------------------|-----------------------------------------------------|-----------------|--------------------|
| | | | HSV-1 | HSV-2 | Not
Detected |
| Prospective | 1 | 31 | - | 6.5%
(2/31) | 93.5%
(29/31) |
| | 7 | 24 | 4.2%
(1/24) | - | 95.8%
(23/24) |
| | 8 | 94 | 4.3%
(4/94) | 5.3%
(5/94) | 90.4%
(85/94) |
| | 9 | 15 | - | - | 100.0%
(15/15) |
| | All | 164 | 3.0%
(5/164) | 4.3%
(7/164) | 92.7%
(152/164) |

24

| Age

Category	Prospective - Number of Samples Positive by Simplexa™ HSV1/2 Direct
	Combined				Female				Male
	HSV-1	HSV-2	Not
Detected	All	HSV-1	HSV-2	Not
Detected	All	HSV-1	HSV-2	Not
Detected	All
From birth
to 1 month	1	1	9	10			6	6		1	3	4
>1 month to
2 years	1	1	25	26			8	8		1	17	18
>2 years to
12 years			9	9			6	6			3	3
>12 years to
21 years	1	1	11	12	1		4	5			7	7
>21 years to
60 years	4	4	65	73	3	1	35	39	1	3	30	34
>60 years	1		33	34	1		20	21			13	13
All	5	7	152	164	4	2	79	85	1	5	73	79

25

M. Instrument Names:

Integrated Cycler with Integrated Cycler Studio Software version 5.0 or higher (3M)

N. System Description:

1. Modes of Operation: Batch
1. Specimen Identification: Entered manually by user
1. Specimen Sampling and Handling: Samples are processed according to assay instructions.
1. Calibration:

In-field calibration for the Integrated Cycler is not necessary. Calibration of the optical modules (excitation and emission gain settings) is performed during the manufacturing process and the values are stored in the instrument firmware.

1. Quality Control:
  Quality control is addressed for each separately cleared specific assay to be run on the instrument.

The positive control may be used as an external control for Quality Control (QC) testing, training or proficiency testing. Synthetic CSF is recommended as a No Template Control (NTC). Ouality control ranges have been established as indicated in the table below. If the controls are not within these parameters, patient results should be considered invalid and the assay repeated. Focus Diagnostics recommends testing controls once per day. Each laboratory should establish its own QC ranges and frequency of QC testing based on applicable local laws, regulations and standard good laboratory practice. Refer to the Simplexa" HSV 1 & 2 Direct package insert for instructions on testing the positive control.

| Control Type | HSV-1 | HSV-2 | DNA Internal
Control (DNA IC) |
|---------------------------------------------|--------------|--------------|----------------------------------|
| Simplexa™ HSV 1
& 2 Positive
Control1 | Detected | Detected | Not applicable2 |
| No Template
Control (NTC) | Not Detected | Not Detected | Valid |

Expected Control Results

1 Typical Ct values for the Positive Control range between 25 to ≤40.

2 Detection of the Simplexa™ DNA Internal Control (DNA IC) is not required for a valid result when HSV is detected.

26

1. Software:
  FDA has reviewed applicant's Hazard Analysis and Software Development processes for this line of product types:

Yes___________ or No__________________________________________________________________________________________________________________________________________________________

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, 21 CFR 801.109, and the special controls.

Q. Identified Potential Risks and Required Mitigation Measures

Identified Potential Risks	Required Mitigation Measures
Risk of false results	Special controls (1), (2), and (3)
Failure to correctly interpret test results	Special controls (4) and (5)
Failure to correctly operate the
instrument	Special controls (6) and (7)

R. Benefit/Risk Analysis

Summary
Summary of
the Benefit(s)	When used for the proposed intended use, the benefits to the clinician and the patient
include: (1) establishment of the device performance in a manner that demonstrates
consistent accurate test results; and 2) ability to use a well validated device to diagnose
HSV in the CNS, which will allow prompt initiation of disease specific treatment.

27

| Summary of
the Risk(s) | The risks associated with the device, when used as intended, are those related to the
risk of false results, failure to correctly interpret the test results and failure to correctly
operate the instrument.
They may lead to error or delay in the diagnosis of HSV-1 and HSV-2 infections,
error or delay in treatment of these infections, unnecessary use of anti-viral therapy,
and delay in establishing the patient's true diagnosis. |
|----------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Conclusions
Do the
probable
benefits
outweigh the
probable risks? | The probable benefits of this device outweigh the probable risks associated with its
use. There are no substantial clinical concerns with the classification of this device in
Class II given the combination of general and special controls. |

S. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.3307 with special controls. FDA believes that special controls, along with the applicable general controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Device Type: Herpes simplex virus nucleic acid-based assay for central nervous system infections.

Class: II (special controls)

Regulation: 21 CFR 866.3307

(a) Identification. A herpes simplex virus nucleic acid-based assay for central nervous system infections is a qualitative in vitro diagnostic device intended for detection and differentiation of HSV-1 and HSV-2 in cerebrospinal fluid (CSF) samples from patients suspected of Herpes Simplex Virus (HSV) infections of the central nervous system (CNS). This test is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS. Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.

(b) Classification. Class II (special controls). The special controls for this device are:

1. Premarket notification submissions must include detailed documentation for the device description, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology including primer design

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and selection.

1. Premarket notification submissions must include detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (Limit of Detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carry-over, and cross contamination. Premarket notification submissions must also document reagent and sample stability recommendations.
1. Premarket notification submissions must include detailed documentation from a clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to the results of two PCR methods followed by bidirectional sequencing.
1. A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling.
1. The device labeling must include a limitation statement that reads: "Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions."
1. Premarket notification submissions must include quality assurance protocols and a detailed documentation for device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software.
1. The risk management activities performed as part of the manufacturer's 21 CFR 820.30 design controls must document an appropriate end user device training program that will be offered as part of efforts to mitigate the risk of failure to correctly operate the instrument.