Alinity m HSV 1 & 2 / VZV
Device Facts
| Record ID | K233349 |
|---|---|
| Device Name | Alinity m HSV 1 & 2 / VZV |
| Applicant | Abbott Molecular, Inc. |
| Product Code | PGI · Microbiology |
| Decision Date | May 3, 2024 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3309 |
| Device Class | Class 2 |
| Attributes | Pediatric |
Indications for Use
The Alinity m HSV 1 & 2 / VZV assay is an in vitro real-time polymerase chain reaction (PCR) assay for the qualitative detection and differentiation of Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2) and Varicella Zoster Virus (VZV) DNA from clinician-collected cutaneous or mucocutaneous lesion swab specimens from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster infection. The Alinity m HSV 1 & 2 / VZV assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster active cutaneous or mucocutaneous infections. Negative results do not preclude herpes simplex virus type 1, herpes simplex virus type 2 or varicella-zoster virus infections and should not be used as the sole basis for diagnosis, treatment or other management decisions. The Alinity m HSV 1 & 2 / VZV assay is not intended for use with cerebrospinal fluid (CSF) or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Alinity m HSV 1 & 2 / VZV assay is not intended for use in prenatal screening.
Device Story
The Alinity m HSV 1 & 2 / VZV assay is an automated, in vitro real-time PCR test performed on the Alinity m System. It processes clinician-collected cutaneous or mucocutaneous lesion swabs to detect and differentiate HSV-1, HSV-2, and VZV DNA. The system performs all steps—sample preparation, PCR assembly, amplification, detection, and result calculation—automatically without user intervention. The device is intended for use in clinical laboratories by trained personnel. Results aid clinicians in diagnosing active infections; however, they are not intended as the sole basis for management decisions. The automated workflow reduces manual handling, minimizing potential for contamination and variability compared to manual methods.
Clinical Evidence
Clinical performance was established via prospective (N=1,258) and retrospective (N=411) studies comparing the Alinity m assay to a commercially available NAAT comparator. Prospective study results: HSV-1 PPA 97.6%, NPA 98.9%; HSV-2 PPA 99.2%, NPA 99.2%; VZV PPA 97.7%, NPA 99.8%. Retrospective study results: HSV-1 PPA 100.0%, NPA 96.2%; HSV-2 PPA 98.7%, NPA 95.8%; VZV PPA 97.8%, NPA 98.4%. Discordant samples were resolved using a second NAAT method.
Technological Characteristics
Real-time PCR assay; utilizes synthetic oligonucleotides, DNA polymerase, dNTPs, and exogenous internal control (plasmid DNA). Fully automated on Alinity m System. Reagents stored at -25°C to -15°C. Qualitative detection based on fluorescent signal cycle number (CN) thresholds.
Indications for Use
Indicated for symptomatic patients of all ages (pediatric to geriatric) suspected of active HSV-1, HSV-2, or VZV cutaneous or mucocutaneous infections. Not for use with CSF, CNS infection diagnosis, or prenatal screening.
Regulatory Classification
Identification
A herpes virus nucleic acid-based cutaneous and mucocutaneous lesion panel is a qualitative in vitro diagnostic device intended for the simultaneous detection and differentiation of different herpes viruses in cutaneous and mucocutaneous lesion samples from symptomatic patients suspected of Herpetic infections. Negative results do not preclude infection and should not be used as the sole basis for treatment or other patient management decisions. The assay is not intended for use in cerebrospinal fluid samples.
Special Controls
*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 and selection. (2) Premarket notification submissions must include detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (Limit of Detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carry-over, and cross contamination. (3) Premarket notification submissions must include detailed documentation of a clinical study using lesion samples in which Herpes Simplex Virus 1, Herpes Simplex Virus 2, or Varicella Zoster Virus DNA detection was requested. The study must compare the device performance to an appropriate well established reference method. (4) 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. (5) The device labeling must include a limitation statement that reads: “The device is not intended for use with cerebrospinal fluid or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS).” (6) 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. (7) The risk management activities performed as part of the manufacturer's 21 CFR 820.10(c) design and development activities 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.
Predicate Devices
- Lyra Direct HSV 1 + 2/VZV Assay (K133448)
Related Devices
- K232286 — Savanna HSV 1+2/VZV Assay, Savanna HSV 1+2/VZV Control Set, Savanna Instrument · Quidel Corporation · Dec 20, 2023
- K140198 — IMDX HSV-1/2 FOR ABBOTT M2000 · Intelligent Medical Devices, Inc. · May 13, 2014
Submission Summary (Full Text)
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FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY
## I Background Information:
A 510(k) Number
K233349
B Applicant
Abbott Molecular Inc.
C Proprietary and Established Names
Alinity m HSV 1 & 2 / VZV
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PGI | Class II | 21 CFR 866,3309 - Herpes Virus Nucleic Acid-Based Cutaneous And Mucocutaneous Lesion Panel | MI - Microbiology |
## II Submission/Device Overview:
A Purpose for Submission:
Clearance of a new device
B Measurand:
Target DNA sequences from conserved regions of the herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) genes.
C Type of Test:
Molecular diagnostic test using real-time PCR (Polymerization Chain Reaction) technology for the direct qualitative detection and differentiation of HSV 1, HSV 2 and VZV DNA extracted and purified from cutaneous or mucocutaneous lesion samples.
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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III Intended Use/Indications for Use:
A Intended Use(s):
The Alinity m HSV 1 & 2 / VZV assay is an in vitro real-time polymerase chain reaction (PCR) assay for the qualitative detection and differentiation of Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2) and Varicella Zoster Virus (VZV) DNA from clinician-collected cutaneous or mucocutaneous lesion swab specimens from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster infection. The Alinity m HSV 1 & 2 / VZV assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster active cutaneous or mucocutaneous infections. Negative results do not preclude herpes simplex virus type 1, herpes simplex virus type 2 or varicella-zoster virus infections and should not be used as the sole basis for diagnosis, treatment or other management decisions.
The Alinity m HSV 1 & 2 / VZV assay is not intended for use with cerebrospinal fluid (CSF) or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Alinity m HSV 1 & 2 / VZV assay is not intended for use in prenatal screening.
B Indication(s) for Use:
Alinity m HSV 1 & 2 / VZV assay. See Intended Use (s)
C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
For in vitro diagnostic Use only
D Special Instrument Requirements:
Alinity m System
IV Device/System Characteristics:
A Device Description:
The Alinity m HSV 1 & 2 / VZV assay is an in vitro real-time polymerase chain reaction (PCR) assay for the qualitative detection and differentiation of Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2) and Varicella Zoster Virus (VZV) DNA from clinician-collected lesion swab specimens (including cutaneous or mucocutaneous lesion specimens).
The steps of the Alinity m HSV 1 & 2 / VZV assay consist of sample preparation, PCR assembly, amplification/detection, and result calculation and reporting. The steps involved in all stages of the Alinity HSV 1 & 2 / VZV assay procedure are executed automatically by the Alinity m System.
The Alinity m HSV 1 & 2 / VZV assay requires 2 separate assay specific kits as follows:
1) The Alinity m HSV 1 & 2 / VZV AMP Kit is comprised of 2 types of multi-well trays:
TRAY 1: Alinity m HSV 1 & 2 / VZV AMP TRAY 1
TRAY 2: Alinity m HSV 11 & 2 / V ACT TRAY 2.
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TRAY 1 - Alinity m HSV 1 & 2 / VZV AMP is individually packed in a foil pouch and contains 48 unit-dose liquid amplification reagent wells and 48 unit-dose liquid IC wells. One well of each is used per test.
- Amplification reagent wells consist of synthetic oligonucleotides, DNA Polymerase, dNTPs, and 0.15% ProClin950 in a buffered solution with a reference dye. IC wells consist of plasmid DNA with unrelated IC sequences and poly dA:dT in a TE buffer containing 0.15% ProClin 950 as a preservative. Each Alinity m HSV 1 & 2 / VZV ACT
TRAY 2 - Alinity m HSV 1 & 2 / VZV ACT is individually packed in a foil pouch and contains 48 unit-dose liquid activation reagent wells. One reagent well is used per test.
- Activation reagent wells consist of magnesium chloride, potassium chloride, and tetramethylammonium chloride. Preservative: 0.15% ProClin 950.
Table 1: Alinity m HSV 1 & 2 / VZV Assay Kit
| Alinity m HSV 1 & 2 / VZV AMP Kit | Quantity 192 Tests |
| --- | --- |
| Alinity m HSV 1 & 2 / VZV AMP TRAY 1 | 4 Trays/48 Test Each |
| Alinity m HSV 1 & 2 / VZV ACT TRAY 2 | 4 Trays/48 Test Each |
2) The Alinity m HSV 1 & 2 / VZV CTRL Kit consists of negative controls and positive controls, each supplied as liquid in single-use tubes.
Alinity m HSV 1 & 2 / VZV Negative CTRL consists of Negative Diluent / TE buffer (containing 0.085% Sodium Azide and 0.087% ProClin 950).
Alinity m HSV 1 & 2 / VZV Positive CTRL consists of linearized plasmid DNA containing HSV-1, HSV-2 and VZV DNA sequences in Negative Diluent / TE buffer (containing 0.085% Sodium Azide and 0.087% ProClin 950).
Table 2. Alinity m HSV 1 & 2 / VZV Control Kit
| Alinity m HSV 1 & 2 / VZV Negative CTRL | 12 Tube x 0.75 mL |
| --- | --- |
| Alinity m HSV 1 & 2 / VZV Positive CTRL | 12 Tube x 0.75 mL |
The Alinity m HSV 1 & 2 / VZV assay is to be run on the Alinity m system which is a fully integrated, sample to result automated system that performs real-time PCR test using the Alinity m HSV 1 & 2 / VZV AMP Kit-1 that contains 2 types of multi-well trays (TRAY 1: Alinity m HSV 1 & 2 / VZV AMP) and (TRAY 2 - Alinity m HSV 11 & 2 / V ACT) described above. The external positive and negative controls from the Alinity m HSV 1 & 2 / VZV CTRL Kit -2 are run with the Alinity m HSV 1 & 2 / VZV assay.
The Alinity m HSV 1 & 2 / VZV assay also utilizes the following items:
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| Alinity m HSV 1 & 2 / VZV Application Specification Files |
| --- |
| Alinity m Sample Prep Kit 2 |
| Alinity m Tubes and Caps |
| Alinity m System Solutions |
| Alinity m Lysis Solution |
| Alinity m Diluent Solution |
| Alinity m Vapor Barrier Solution |
| Alinity m System |
## Interpretation of Results:
The Alinity m HSV 1 & 2 / VZV assay is a qualitative assay. For each analyte (HSV-1, HSV-2 or VZV) signal, the amplification CN (Cycle Number) is determined when the fluorescent signal is detected by the Alinity m System. Each signal is either reported as “Positive” if the CN is less than or equal to a fixed assay cutoff cycle for that signal or is reported as “Negative” if the CN is not generated, or the CN is greater than the assay cutoff cycle. The Alinity m System will automatically report a result and interpretation for each specimen. If applicable a message code or flag will be displayed.
## Assay Result Interpretation
| Assay | Result | Interpretation |
| --- | --- | --- |
| HSV-1 | HSV-1 XX.XX.CN | HSV-1 Positive |
| HSV-1 | HSV-1 Target not detected (>31 CN) | HSV-1 Negative |
| HSV-2 | HSV-2 XX.XX.CN | HSV-2 Positive |
| HSV-2 | HSV-2 Target not detected (>33 CN) | HSV-2 Negative |
| VZV | VZV XX.XX.CN | VZV Positive |
| VZV | VZV Target not detected (>33 CN) | VZV Negative |
## B Principle of Operation:
Patient sample is dispensed into the Alinity m System which automates running of the assay and result reporting.
The Alinity m HSV 1 & 2 / VZV assay utilizes real-time polymerase chain reaction (PCR) to amplify and differentiate Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2) and Varicella Zoster Virus (VZV) DNA extracted from cutaneous or mucocutaneous lesion specimens. The steps of Alinity m HSV 1 & 2 / VZV assay consist of sample preparation, PCR assembly, amplification/detection, and result calculation and reporting. All stages of the Alinity m HSV 1 & 2 / VZV assay procedure are executed automatically by the Alinity m System.
Assay controls are tested at or above an established minimum frequency to help ensure that instrument and reagent performance remain satisfactory. During each control event, a negative control and a positive control are processed through sample preparation and PCR procedures that are identical to those used for specimens. Assay controls are used to demonstrate proper sample processing and assay validity.
The Alinity m HSV 1 & 2 / VZV amplification reagents include primers and probes that amplify and detect an exogenous internal control (containing plasmid DNA). Amplification and detection
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of the internal control demonstrates proper sample processing. The internal control is used to demonstrate assay validity.
Results are reported as positive or negative based on the cycle number (CN) output for each target (HSV-1, HSV-2, VZV, and IC), which is based on threshold cycle (Ct) at which the fluorescent signal surpasses a threshold to indicate the detection target nucleic acids.
The Alinity m HSV 1 & 2 / VZV assay is a dual target assay for HSV-1 and HSV-2, and a single target assay for VZV. Identification of HSV-1, HSV-2, and VZV occurs by the target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the viral genomes.
## C Instrument Description Information:
1. Instrument Name: Alinity m System
2. Specimen Identification: Specimen identification can be entered via barcode scanning.
3. Specimen Sampling and Handling: Lesion swab specimens, including cutaneous and mucocutaneous, collected in commercially available viral transport media (VTM) (Copan UTM, BD UVT, Remel M4RT) can be used with the Alinity m HSV 1+2/VZV assay on the Alinity m System.
4. Calibration: NA
5. Quality Control: There are two types of Quality Control for the Alinity m HSV 1 & 2 / VZV assay: Built-in Process Internal Control and External Controls.
a) Internal Control (IC):
The Alinity m HSV 1 & 2 / VZV assay contains an internal control (IC) for each sample. The IC DNA sequences, unrelated to the Alinity m HSV 1 & 2 / VZV assay target sequences, are amplified and detected simultaneously with the target DNAs, serving as an internal control to demonstrate that the process has proceeded correctly for each sample.
b) External Controls
The Alinity m HSV 1 & 2 / VZV CTRL Kit consists of 12 filled tubes of Negative Control and 12 filled tubes of Positive Control. Assay controls are used to demonstrate proper sample processing and assay validity. A negative control and a positive control are processed through sample preparation and PCR procedures that are identical to those used for specimens.
## V Substantial Equivalence Information:
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A Predicate Device Name(s):
Lyra Direct HSV 1 + 2 / VZV Assay
B Predicate 510(k) Number(s):
K133448
Comparison with Predicate(s):
| Device & Predicate Device(s): | New Device
K233349 | Predicate
K133448 |
| --- | --- | --- |
| Device Trade Name | Alinity m HSV 1 & 2 / VZV | Lyra Direct HSV 1 + 2/VZV Assay |
| General Device Characteristic Similarities | | |
| Intended Use/Indication For Use | The Alinity m HSV 1 & 2 / VZV assay is an in vitro real-time polymerase chain reaction (PCR) assay for the qualitative detection and differentiation of Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2) and Varicella Zoster Virus (VZV) DNA from clinician-collected cutaneous or mucocutaneous lesion swab specimens from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 a and/or varicella-zoster infection. The Alinity m HSV 1 & 2 / VZV assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster active cutaneous or mucocutaneous infections. Negative results do not preclude herpes simplex virus type 1, herpes simplex virus type 2 or varicella-zoster virus infections and should not be used as the sole basis for diagnosis, treatment or other management decisions. | The Lyra Direct HSV 1 + 2/VZV Assay is an in vitro multiplex Real-Time PCR test for qualitative detection and differentiation of herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus DNA isolated and purified from cutaneous or mucocutaneous lesion samples obtained from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster infection. The Lyra Direct HSV 1 + 2/VZV Assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and varicella-zoster virus active cutaneous or mucocutaneous infections. Negative results do not preclude herpes simplex virus type 1, herpes simplex virus type 2 or varicella-zoster virus infections and should not be used as the sole basis for diagnosis, treatment or other management decisions. The Lyra Direct HSV 1 + 2/VZV Assay is not intended for use with cerebrospinal fluid or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Lyra Direct HSV 1 + 2/VZV Assay is not intended for use in prenatal screening. The device is not intended for point-of-care use. |
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| | The Alinity m HSV 1 & 2 / VZV assay is not intended for use with cerebrospinal fluid (CSF) or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Alinity m HSV 1 & 2 / VZV assay is not intended for use in prenatal screening. | |
| --- | --- | --- |
| Amplification Technology | Real-Time Polymerase Chain Reaction (PCR) | Same |
| Qualitative | Yes | Same |
| Analyte | Viral DNA from HSV 1, HSV 2 and VZV | Same |
| Specimen Types | Cutaneous or mucocutaneous lesion swabs in transport medium | Same |
| Automated Analysis | Yes | Same |
| External Assay Controls | Positive and Negative Control | Same |
| General Device Characteristic Differences | | |
| Assays Steps | All steps of the Alinity m HSV 1 & 2 / VZV assay procedure are executed automatically by the Alinity m System. No intermediate processing or transfer steps are performed by the user. | Assay processing steps are executed manually until placed on instrument for signal evaluation:
• Life Technologies Quant Studio Dx
• Applied Biosystems 7500 Fast Dx
• Cepheid Smart Cycler II System |
| Sample Preparation Instrument Components | Automated liquid handling and robotic manipulation platform. Automated process for sample processing using Internal control (IC) | Mechanical lysis and addition of Process Buffer. Manual process for sample processing using processing control (PRC) |
| Reagent Kit Storage (Unopened) until expiration date | -25°C to -15°C | 2°C to 8°C |
VI Standards/Guidance Documents Referenced:
Not Applicable
VII Performance Characteristics (if/when applicable):
A Analytical Performance:
1. Precision/Reproducibility:
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# 1a. Within-Laboratory Precision:
The Alinity m HSV 1 & 2 / VZV assay within-laboratory Precision was evaluated by testing a 4-member precision panel consisting of 4 target levels (Moderate Positive, Low Positive, Sub-LoD, and Negative) for each assay analyte. The positive panel members were prepared by spiking HSV-1, HSV-2, and VZV stocks into negative matrix to achieve the targeted level of Moderate Positive (3X LoD), Low Positive (1X to 2X LoD), and High Negative (<1X LoD). Each panel member was tested with 3 replicates in a run, 2 runs on each of 5 days, on 3 Alinity m Systems operated by 3 operators (one operator per system), using 3 Alinity m HSV 1 & 2 / VZV AMP Kit lots (one lot per system) for a total of 90 replicates of each panel member. The precision results for HSV-1, HSV-2, and VZV are summarized below in Table 3.
Table 3. Within-laboratory Precision
| Analyte | Sample | Agreement with Expected Results (Na/Nb) | Mean CN | Within Run | | Between Run | | Between Day | | Within laboratoryc | | Between instrument Lot/Operator | | Totald | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | % CV | SD | % CV | SD | %CV | SD | % CV | SD | % CV | SD | % CV |
| HSV-1 | 3x LoD | 100% (90/90) | 26.31 | 0.233 | 0.9 | 0.000 | 0.0 | 0.000 | 0.0 | 0.233 | 0.9 | 0.178 | 0.7 | 0.287 | 1.1 |
| | 1 - 2x LoD | 100% (90/90) | 26.82 | 0.181 | 0.7 | 0.060 | 0.2 | 0.089 | 0.3 | 0.210 | 0.8 | 0.245 | 0.9 | 0.323 | 1.2 |
| | <1x LoD | 78.9% (71/90) | 30.26 | 0.418 | 1.4 | 0.095 | 0.3 | 0.000 | 0.0 | 0.428 | 1.4 | 0.125 | 0.4 | 0.446 | 1.5 |
| | Negative | 100% (90/90) | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| HSV-2 | 3x LoD | 100% (90/90) | 29.03 | 0.265 | 0.9 | 0.000 | 0.0 | 0.024 | 0.1 | 0.266 | 0.9 | 0.233 | 0.8 | 0.354 | 1.2 |
| | 1 - 2x LoD | 100% (90/90) | 29.60 | 0.270 | 0.9 | 0.156 | 0.5 | 0.000 | 0.0 | 0.311 | 1.1 | 0.243 | 0.8 | 0.395 | 1.3 |
| | <1x LoD | 90% (81/90) | 32.07 | 0.474 | 1.5 | 0.219 | 0.7 | 0.000 | 0.0 | 0.522 | 1.6 | 0.063 | 0.2 | 0.526 | 1.6 |
| | Negative | 100% (90/90) | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| VZV | 3x LoD | 100% (90/90) | 27.38 | 0.257 | 0.9 | 0.118 | 0.4 | 0.000 | 0.0 | 0.283 | 1.0 | 0.104 | 0.4 | 0.301 | 1.1 |
| | 1 - 2x LoD | 100% (90/90) | 28.77 | 0.305 | 1.1 | 0.204 | 0.7 | 0.000 | 0.0 | 0.387 | 1.3 | 0.125 | 0.4 | 0.388 | 1.3 |
| | <1x LoD | 88.9% (80/90) | 31.92 | 0.586 | 1.8 | 0.353 | 1.1 | 0.000 | 0.0 | 0.684 | 2.4 | 0.315 | 1.0 | 0.753 | 2.4 |
| | Negative | 100% (90/90) | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
a Number of replicates with expected results used in the Mean and SD calculation.
b Total Number of replicates.
c Within-Laboratory includes Within-Run, Between-Run and Between-Day Components.
Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument/Lot/Operator Components.
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# 1b. Reproducibility Study (multi-site precision):
The reproducibility of the Alinity m HSV 1 & 2 / VZV assay was evaluated by testing a 4 - member panel, including a 5X LoD (Positive), 1-2X LoD (Low Positive), $< 1\mathrm{X}$ LoD (High Negative), and Negative panel member. The positive panel members were prepared by spiking cultured viral particles HSV-1, HSV-2 and VZV into the negative matrix. A total of 3 Alinity m HSV 1 & 2 / VZV kit lots were used for the reproducibility study. Each of external sites used 2 different lots of reagent kits and a different Alinity m system. Six replicates of each panel member were tested with 3 reagent lots at 3 external sites on 5 non-consecutive days (6 replicates x 2 kit lots/site x 3 sites x 5 days = 180). The reproducibility results of the Alinity m HSV 1 & 2 / VZV assay for HSV-1, HSV-2, and VZV are presented below in Table 4.
Table 4. Reproducibility
| Analyte | Panel Member | Agreement with Expected Results (Na/Nb) | Mean CN | Within Run/Day | | Between Run/Day | | Between Lot | | Between Site/Instrument | | Totala | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| HSV-1 | 5x LoD | 100% (180/180) | 27.21 | 0.322 | 1.2 | 0.140 | 0.5 | 0.097 | 0.4 | 0.000 | 0.0 | 0.364 | 1.3 |
| | 1 - 2x LoD | 98.9% (178/180) | 28.96 | 0.413 | 1.4 | 0.000 | 0.0 | 0.041 | 0.1 | 0.066 | 0.2 | 0.421 | 1.5 |
| | < 1x LoD | 49.4% (89/180) | 30.52 | 0.406 | 1.3 | 0.000 | 0.0 | 0.000 | 0.0 | 0.037 | 0.1 | 0.408 | 1.3 |
| | Negative | 100% (180/180) | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| HSV-2 | 5x LoD | 100% (180/180) | 29.53 | 0.311 | 1.1 | 0.142 | 0.5 | 0.054 | 0.2 | 0.105 | 0.4 | 0.362 | 1.2 |
| | 1 - 2x LoD | 100% (180/180) | 30.56 | 0.391 | 1.3 | 0.102 | 0.3 | 0.192 | 0.6 | 0.084 | 0.3 | 0.455 | 1.5 |
| | < 1x LoD | 35.6% (64/180) | 32.38 | 0.421 | 1.3 | 0.000 | 0.0 | 0.115 | 0.4 | 0.000 | 0.0 | 0.437 | 1.3 |
| | Negative | 100% (180/180) | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| VZV | 5x LoD | 100% (180/180) | 27.38 | 0.409 | 1.5 | 0.154 | 0.6 | 0.000 | 0.0 | 0.184 | 0.7 | 0.474 | 1.7 |
| | 1 - 2x LoD | 100% (180/180) | 29.22 | 0.438 | 1.5 | 0.152 | 0.5 | 0.057 | 0.2 | 0.133 | 0.5 | 0.486 | 1.7 |
| | < 1x LoD | 62.2% (112/180) | 35.21 | 0.577 | 1.8 | 0.000 | 0.0 | 0.000 | 0.0 | 0.097 | 0.3 | 0.585 | 1.8 |
| | Negative | 100% (180/180) | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
${}^{a}$ Number of replicates with expected results used in the Mean and SD calculation.
bTotal Number of replicates.
Total includes Within-Run/Day, Between-Run/Day, Between-Lot, and Between-Site/Instrument Components.
# 2. Linearity:
Not applicable
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# 3. Analytical Specificity/Interference:
# a. Cross-Reactivity:
A study was performed to evaluate the performance of the Alinity m HSV 1 & 2 / VZV assay in the presence of 55 potential cross-reactivity microorganisms that could be found in lesion swab specimens. The microorganisms were tested at $10^{5}$ Units/mL for viruses and fungi, and $10^{6}$ Units/mL for bacteria or the highest titer available. There was no cross reactivity observed with the 55 microorganisms tested with the Alinity m HSV 1 & 2 / VZV assay tested at the concentrations indicated below in Table 5.
Table 5. Cross-Reactivity
| Organism | Organism Type | Concentration of the Microorganism | Unit of Concentration |
| --- | --- | --- | --- |
| Acinetobacter calcoaceticus | Bacteria | 1.10E+06 | cfu/mL |
| Acinetobacter lwoffii | Bacteria | 1.10E+06 | cfu/mL |
| Actinomyces israelii | Bacteria | 1.10E+06 | cfu/mL |
| Adenovirus type 1 | Virus | 1.10E+05 | TCID50/mL |
| Adenovirus type 7 | Virus | 1.10E+05 | TCID50/mL |
| Bacteroides fragilis | Bacteria | 1.10E+06 | cfu/mL |
| BK virus | Virus | 1.10E+05 | copies/mL |
| Bordetella pertussis | Bacteria | 1.10E+06 | cfu/mL |
| Campylobacter jejuni | Bacteria | 1.10E+06 | cfu/mL |
| Candida albicans | Fungi | 1.10E+05 | cfu/mL |
| Chlamydophila pneumoniae | Bacteria | 1.10E+06 | IFU/mL |
| Chlamydia trachomatis serovar D | Bacteria | 1.10E+06 | IFU/mL |
| Chlamydia trachomatis serovar I | Bacteria | 1.10E+06 | IFU/mL |
| Clostridium difficile | Bacteria | 1.00E+06 | cfu/mL |
| Clostridium perfringens | Bacteria | 1.10E+06 | cfu/mL |
| Cryptococcus neoformans | Fungi | 1.10E+05 | cfu/mL |
| Cytomegalovirus | Virus | 1.10E+05 | copies/mL |
| Enterobacter cloacae | Bacteria | 1.10E+06 | cfu/mL |
| Enterococcus faecium | Bacteria | 1.10E+06 | cfu/mL |
| Enterococcus faecalis | Bacteria | 1.10E+06 | cfu/mL |
| Epstein-Barr virus | Virus | 1.10E+05 | copies/mL |
| Escherichia coli | Bacteria | 1.10E+06 | cfu/mL |
| Fusobacterium nucleatum | Bacteria | 1.10E+06 | cfu/mL |
K233349 - Page 10 of 25
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| Haemophilus ducreyi | Bacteria | 1.10E+06 | copies/mL |
| --- | --- | --- | --- |
| Haemophilus influenzae | Bacteria | 1.10E+06 | cfu/mL |
| Hepatitis B virus | Virus | 1.10E+05 | IU/mL |
| Hepatitis C virus | Virus | 1.10E+05 | IU/mL |
| Herpes Virus 6A strain GS | Virus | 1.10E+05 | copies/mL |
| Herpes Virus 6B strain Z29 | Virus | 1.10E+05 | copies/mL |
| HIV-1 | Virus | 1.10E+05 | IU/mL |
| HPV 16 | Virus | 1.10E+05 | copies/mL |
| HPV 18 | Virus | 1.10E+05 | copies/mL |
| Kingella kingae | Bacteria | 1.00E+06 | cfu/mL |
| Klebsiella pneumoniae | Bacteria | 1.10E+06 | cfu/mL |
| Listeria monocytogenes | Bacteria | 1.10E+06 | cfu/mL |
| Mobiluncus mulieris | Bacteria | 1.10E+06 | cfu/mL |
| Moraxella catarrhalis | Bacteria | 1.10E+06 | cfu/mL |
| Mycobacterium tuberculosisa | Bacteria | 1.10E+06 | copies/mL |
| Neisseria gonorrhoeae | Bacteria | 1.10E+06 | cfu/mL |
| Neisseria meningitidis | Bacteria | 1.10E+06 | cfu/mL |
| Propionibacterium acnes | Bacteria | 1.10E+06 | cfu/mL |
| Proteus mirabilis | Bacteria | 1.10E+06 | cfu/mL |
| Proteus vulgaris | Bacteria | 1.10E+06 | cfu/mL |
| Pseudomonas aeruginosa | Bacteria | 1.10E+06 | cfu/mL |
| Staphylococcus aureus | Bacteria | 1.10E+06 | cfu/mL |
| Staphylococcus epidermidis | Bacteria | 1.10E+06 | cfu/mL |
| Staphylococcus saprophyticus | Bacteria | 1.10E+06 | cfu/mL |
| Streptococcus agalactiae | Bacteria | 1.10E+06 | cfu/mL |
| Streptococcus mitis | Bacteria | 1.10E+06 | cfu/mL |
| Streptococcus mutans | Bacteria | 1.10E+06 | cfu/mL |
| Streptococcus pneumoniae | Bacteria | 1.10E+06 | cfu/mL |
| Streptococcus pyogenes | Bacteria | 1.10E+06 | cfu/mL |
| Streptococcus salivarius | Bacteria | 1.10E+06 | cfu/mL |
| Treponema pallidum | Bacteria | 1.00E+06 | cells/mL |
| Toxoplasma gondii | Bacteria | 1.10E+06 | copies/mL |
aMycobacterium tuberculosis genomic DNA was used as target.
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b. Microbial Interference Study:
The microbial interference study was performed with a list of microorganisms listed above in the Table 5 for the Cross Reactivity Study.
Each potentially interfering microorganism was tested in the presence of 3X LoD levels for each analyte e.g., HSV-1, HSV-2 and VZV viruses spiked into negative matrix. All positive samples reported positive results for HSV-1, HSV-2, and VZV in the presence of these microorganisms. There was no interference observed with the 55 microorganisms tested with the Alinity m HSV 1 & 2 / VZV Assay.
c. Interfering Substances:
The performance of the Alinity m HSV 1 & 2 / VZV assay was evaluated with potentially interfering endogenous and exogenous substances that may be present in cutaneous or mucocutaneous lesion specimens. A panel composed of twenty-four (24) substances listed in Table 6 was tested in the absence or presence of HSV-1, HSV-2, or VZV (Isolate 2, G strain, Ellen strain, respectively) viruses at 3X LOD in the Alinity m HSV 1 & 2 / VZV assay. There was no evidence of interference (false positive or false negative results) caused by the substances tested at the concentrations shown below in Table 6.
Table 6. Interference
| Interfering Substance | Test Concentration |
| --- | --- |
| Blood (human) | 5% v/v |
| Leukocytes | 4.0×10^{5} cells/mL |
| Mucin | 0.3% v/v |
| Urine | 5% v/v |
| Feces | 0.1% w/v |
| Human Serum Albumin | 1% w/v |
| Saliva | 4% v/v |
| Seminal Fluid | 5% v/v |
| K-Y Jelly (Personal Lubricant) | 2.5% w/v |
| Vaginal Contraceptive Gel | 2.5% w/v |
| Monistat (Miconazole Nitrate Vaginal Cream) | 3% w/v |
| Preparation H (Hemorrhoidal Ointment) | 2.5% w/v |
| Abreva (Cold Sore Cream) | 2.5% w/v |
| Acyclovir | 2.5% w/v |
| Vagisil (Anti-Itch Cream) | 0.25% w/v |
| Vagicaine (Anti-Itch Cream) | 2.5% w/v |
| Feminine Wash (Douche) | 2.5% w/v |
| Denavir (Anti-retro viral cream) | 2.5% w/v |
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| Feminine Deodorant Spray | 2.5% w/v |
| --- | --- |
| Lip Balm | 3% w/v |
| Summer’s Eve (Body Powder) | 2.5% w/v |
| Toothpaste | 2.5% w/v |
| Casein protein | 0.7% w/v |
| Mouthwash | 3% v/v |
d. Competitive Interference: A competitive interference study was performed to evaluate the performance of the Alinity m HSV 1 & 2 / VZV assay in the presence of the 3 target analytes. Each sample was prepared with 2 of the analytes (HSV-1, HSV-2 or VZV viruses) at 3X LoD and the third analyte at 1000X LoD in negative simulated swab matrix.
The three Panel Members (PM) were evaluated as follow:
- PM 1: HSV-1 and HSV-2 at 3X LoD and VZV at 1000X LoD
- PM 2: HSV-1 and VZV at 3X LoD and HSV-2 at 1000X LoD
- PM 3: HSV-2 and VZV at 3X LoD and HSV-1 at 1000X LoD
Across panel members, all replicates at the low concentration were detected for each of the 3 analytes. None of the analyte targets present at the high concentration interfered with the detection of the other 2 analyte targets at low levels. Results are presented below in Table7.
Table 7. Competitive Interference Results
| Panel Number | High Analyte | HSV-1 Positivity | HSV-2 Positivity | VZV Positivity |
| --- | --- | --- | --- | --- |
| PM 1 | HSV-1 & 2 at 3X LoD and VZV at 1000X LoD | 100.0% (24/24) | 100.0% (24/24) | 100.0% (24/24) |
| PM 2 | HSV-1 and VZV at 3X LoD and HSV-2 at 1000X LoD | 100.0% (23/23) | 100.0% (23/23) | 100.0% (23/23) |
| PM 3 | HSV-2 and VZV at 3X LoD and HSV-1 at 1000X LoD | 100.0% (24/24) | 100.0% (24/24) | 100.0% (24/24) |
4. Assay Reportable Range:
Not applicable
5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
Not applicable
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# 6. Limit of Detection:
The limit of detection (LoD) for Alinity m HSV 1 & 2 / VZV assay for each analyte were determined by quantified (TCID50/mL) cultures of HSV-1 (MacIntyre), HSV-2 (MS) or VZV (Ellen) viral strains diluted into pooled negative clinical swab specimens. The LoD was established in 2 phases as follows:
**Phase 1: LoD Range Finding** - A dilution series was made from viral stocks of HSV-1, HSV-2 and VZV into negative clinical matrix (CM). For each panel member (PM), a total of 72 replicates of each dilution (panel member) were tested across 3 Alinity m HSV 1 & 2 / VZV AMP Kit lots and 3 Alinity m Systems (one lot per system) over 3 days (8 replicates x 3 days x 3 instruments/lot = 72 replicates per each panel member).
**Phase 2: LoD Confirmation** - The LoD determined for each analyte from Phase I Study was further confirmed in negative clinical matrix. For each target analyte, a total of 32 replicates of each PM in clinical negative matrix were tested across 2 Alinity m HSV 1 & 2 / VZV AMP kit lots and 2 Alinity m Systems, with each lot of reagents assigned to a specific instrument. A total of 16 replicates were tested for each PM on each of the 2 instrument/lot pair, resulting in a total of 32 replicates per PM (16 replicates × 2 instrument/lot = 32 replicates per PM).
Probit analysis of the data determined that the concentration detected with 95% probability (LoD by Probit) for HSV-1, HSV-2, and VZV was 5.90 TCID50/mL, 2.07 TCID50/mL, and 0.055 TCID50/mL respectively shown below in Table 8.
Table 8. Limit of Detection
| Analyte | Analyte Strain | LoD Concentration (TCID_{50}/mL) |
| --- | --- | --- |
| HSV-1 | MacIntyre | 5.90 |
| HSV-2 | MS | 2.07 |
| VZV | Ellen | 0.055 |
# 7. Analytical Reactivity (Inclusivity):
Analytical reactivity or inclusivity of the Alinity m HSV 1 & 2 / VZV assay was further evaluated by functional testing of clinically relevant strains/isolates, in addition to those strains used in the LoD study. The inclusivity for the Alinity m HSV 1 & 2 / VZV assay was determined by testing 5 HSV-1 strains/isolates, 3 HSV-2 strains/isolates and 5 VZV strains/isolates. The strains whose reported units of measure were in TCID<sub>50</sub>/mL, were diluted to a concentration ≤3X LoD. For strains where concentration in TCID<sub>50</sub>/mL was not available, a dilution series based on copies/mL was prepared and tested. The dilution series consisted of at least one concentration that resulted in positive results 100% of the time, and at least one concentration that resulted in positive results <100% of the time. The Alinity m HSV 1 & 2 / VZV assay detected all strains listed at a level near LoD in Table 9.
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Table 9: Inclusivity Test Results
| Analyte | Strain/Isolate | Analyte Concentration | Inclusive (Yes/No) |
| --- | --- | --- | --- |
| HSV-1 | Macintyre | 17.7 TCID_{50}/mL | Yes |
| | HF | 17.7 TCID_{50}/mL | Yes |
| | F | 17.7 TCID_{50}/mL | Yes |
| | KOS | 300 copies/mL^{a} | Yes |
| | Vero 2 Isolate | 17.7 TCID_{50}/mL | Yes |
| HSV-2 | MS | 6.21 TCID_{50}/mL | Yes |
| | G | 6.21 TCID_{50}/mL | Yes |
| | Vero 2 Isolate | 6.21 TCID_{50}/mL | Yes |
| VZV | Ellen | 0.093 TCID_{50}/mL | Yes |
| | 82 | 100 copies/mL | Yes |
| | Oka | 0.093 TCID_{50}/mL | Yes |
| | Webster | 0.093 TCID_{50}/mL | Yes |
| | AV92-3 L | 0.093 TCID_{50}/mL | Yes |
aLowest level that has 100% detection.
8. Carry-Over:
The carry-over rate for Alinity m HSV 1 & 2 / VZV assay was determined by testing 360 alternating replicates of negative and high-positive samples. High positive samples consisted of HSV-2 DNA targeted to a CN value of 10.00. The testing was performed across 3 different Alinity m Systems. All negative samples reported negative interpretations, resulting in an overall carry-over rate of 0.0% (0/360, 95% CI: 0.0%, 1.1%).
9. Fresh/Frozen Specimen Stability
Study 1: Fresh/frozen specimen stability was evaluated by testing a panel member (PM) targeted to 2X LoD for HSV-1 and HSV-2 and 1.13X LoD for VZV prepared in pooled negative clinical specimens using Alinity m HSV 1 & 2 / VZV assay reagents and the Alinity m System. The PM was tested immediately (control condition) and after 1 freeze-thaw (FT1) and 3 freeze-thaw (FT2) test conditions. The PM were frozen at -70°C and thawed at 5°C± 3°C for at least 1 hr before testing. A total of 40 replicates were tested for each PM at each storage condition. The study results support that HSV-1/HSV-2/VZV positive panel member are stable undergoing 3 freeze/thaw cycles prior to testing shown below in Table 10.
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Table 10: Freeze Thaw Stability (Study 1)
| Analyte | Condition | Positive Rate | Mean | SD |
| --- | --- | --- | --- | --- |
| HSV-1
(2X LoD) | No Freeze/Thaw | 40/40 (100%) | 27.04 | 0.332 |
| | 1 Freez/Thaw (FT1) | 40/40 (100%) | 27.09 | 0.271 |
| | 3 Freez/Thaw (FT2) | 40/40 (100%) | 27.07 | 0.182 |
| HSV-2
(2X LoD) | No Freeze/Thaw | 40/40 (100%) | 29.81 | 0.323 |
| | 1 Freez/Thaw (FT1) | 40/40 (100%) | 29.74 | 0.293 |
| | 3 Freez/Thaw (FT2) | 40/40 (100%) | 29.69 | 0.316 |
| VZV
(1X LoD) | No Freeze/Thaw | 40/40 (100%) | 28.62 | 0,262 |
| | 1 Freez/Thaw (FT1) | 40/40 (100%) | 28.58 | 0.410 |
| | 3 Freez/Thaw (FT2) | 40/40 (100%) | 28.52 | 0.251 |
**Study 2**: Fresh/frozen specimen stability was evaluated by testing panel members (PMs) prepared in pooled negative clinical specimens using Alinity m System. The PMs were prepared by spiking HSV-1, HSV-2, and VZV virus stock into pooled negative clinical specimens to achieve the targeted final concentration of 4X Limit of Detection (LoD) for the respective analytes for Panel Member 1, 100X LoD for Panel Member 2, and 1000X LoD for Panel Member 3. All 3 targets (HSV-1, HSV-2, VZV) were spiked together in a given PM. For PM1, a total of 20 replicates were tested at each condition. For PM2 and PM3, a total of 10 replicates were tested at each condition. Negative clinical matrix was prepared by pooling HSV-1/HSV-2/VZV negative clinical swab specimens. The concentrations of PM for each analyte are listed below in Table 11.
Table 11. Target Concentration of the Panel Member
| Panel Member | Target concentration | Analyte | Target concentration (TCID_{50}/mL) |
| --- | --- | --- | --- |
| 1 | 4X LoD | HSV-1 | 23.60 |
| | | HSV-2 | 8.28 |
| | | VZV | 0.22 |
| 2 | 100X LoD | HSV-1 | 590 |
| | | HSV-2 | 207 |
| | | VZV | 5.5 |
| 3 | 1000X LoD | HSV-1 | 5900 |
| | | HSV-2 | 2070 |
| | | VZV | 55 |
The panel members were tested at the following storage conditions:
- Control Conditions: Not frozen or thawed before testing (Fresh)
- Test Conditions: 3 freeze/thaws (frozen at -70°C or colder for 24 hours, thawed at 5°C + 3°C for a minimum of 1 hour, repeat 3X before testing.
Results are presented below in Table 12.
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Table 12: Freeze Thaw Stability (Study 2)
| Analyte | Target Concentration | Condition | Positive Rate | Mean (CN) | SD |
| --- | --- | --- | --- | --- | --- |
| HSV-1 | 3-4X LoD | No FreezeThaw | 20/20 (100%) | 26.56 | 0.170 |
| | | 3 Freeze/Thaw | 20/20 (100%) | 26.91 | 0.161 |
| | 100X LoD | No Freeze/Thaw | 10/10 (100%) | 22.93 | 0.161 |
| | | 3 Freeze/Thaw | 10/10 (100%) | 22.90 | 0.160 |
| | 1000X LoD | No Freeze/Thaw | 10/10 (100%) | 19.49 | 0.190 |
| | | 3 Freeze/Thaw | 10/10 (100%) | 19.33 | 0.307 |
| HSV-2 | 3-4X LoD | No Freeze/Thaw | 20/20 (100%) | 29.16 | 0.208 |
| | | 3 Freeze/Thaw | 20/20 (100%) | 29.43 | 0.236 |
| | 100X LoD | No Freeze/Thaw | 10/10 (100%) | 25.44 | 0.157 |
| | | 3 Freeze/Thaw | 10/10 (100%) | 25.47 | 0.121 |
| | 1000X LoD | No Freeze/Thaw | 10/10 (100%) | 21.96 | 0.193 |
| | | 3 Freeze/Thaw | 10/10 (100%) | 21.91 | 0.152 |
| VZV | 3-4X LoD | No Freeze/Thaw | 20/20 (100%) | 27.28 | 0.454 |
| | | 3 Freeze/Thaw | 20/20 (100%) | 27.62 | 0.408 |
| | 100X LoD | No Freeze/Thaw | 10/10 (100%) | 23.28 | 0.261 |
| | | 3 Freeze/Thaw | 10/10 (100%) | 23.56 | 0.147 |
| | 1000X LoD | No Freeze/Thaw | 10/10 (100%) | 19.65 | 0.217 |
| | | 3 Freeze/Thaw | 10/10 (100%) | 19.84 | 0.261 |
10. Specimen Storage/Onboard Stability Study: Storage conditions and on-board stability of HSV-1, HSV-2 and VZV clinical swab specimens were evaluated by 2 different studies:
Study 1 – Specimen Stability Study with Pooled Negative Clinical Matrix: The study was executed by spiking HSV-1, HSV-2 and VZV in clinical swab matrix that was prepared by pooling HSV-1, HSV-2 and VZV negative specimens followed by testing with the Alinity m HSV 1 & 2 / VZV assay at the following storage conditions.
Specimen Storage/Onboard Stability conditions evaluated:
- 20°C ±5°C for 35, 61, 91, and 99 days
- 2°C to 8°C for 4.4, 5.5, 8.8, and 15.5 days
- 26°C for 1.1, 2.2, and 4.4 days
- 30°C for 2.2, 3.3 and 4.4 days
The positive specimens were prepared by spiking all three targets at a concentration of 3X LoD in pooled negative clinical matrix. A minimum of 5 replicates for each specimen were evaluated for each control condition (baseline), test conditions, and at different timepoints and temperature. The study was conducted using 1 lot of reagent kit and 1 lot of control kit reagent.
Data supported specimen stability storage at -20°C ±5°C for 99 days, and at 2°C to 8°C for up to 15 days and at 26°C for 1 day.
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Study 2 - Specimen Stability Study with individual clinical swab samples: The study was executed by spiking HSV-1, HSV-2 and VZV in individual negative clinical swab matrix rather than a pooled HSV-1, HSV-2 and VZV negative specimens followed by testing with the Alinity m HSV 1 & 2 / VZV assay at the storage conditions indicated below. This study represents the actual use of the test.
Ten positive samples were prepared by spiking each analyte from positive patient specimen to a target concentration of 3X LoD in individual negative clinical swab specimens. Replicates of spiked specimens were tested immediately (control condition or baseline) and specimen stability conditions (test conditions) described as follows:
- Baseline (Control Condition)
- Specimens stored for 2.2 days at 26°C + storage-on board for 4 hrs.
- Specimens stored 4.4 days at 26°C + storage-on board for 4 hrs.
Ten replicates for each condition were tested. For each analyte (HSV-1, HSV-2 and VZV) a total of 34 results were generated in the study. There were 4 valid control results and 30 valid sample results included in the analyses for each analyte. All positive samples for each analyte were tested 100% positive. The results support that individual specimens are stable at 26°C±1°C for 4 days and Onboard for 4 hours.
Based on Study 1 and Study 2, the data supports the following specimen storage conditions:
- 15°C to 25°C (4 days)
- 2°C to 8°C (14 days)
- -25°C to -15°C (90 days)
- -70°C or colder (90 days)
10. Comparison Studies:
a. Method Comparison with Predicate Device:
See Section B Clinical Studies 3 below.
b. Matrix Equivalency Study:
i). Equivalency between Clinical Swab Matrix and Simulated Swab Matrix:
The matrix equivalency was established between clinical swab matrix (CM) and simulated swab matrix (SM) for the Alinity m HSV 1 & 2 / VZV assay. Four panel members for each analyte were prepared by spiking with HSV-1, HSV-2 and VZV stock to achieve the targeted level at 3X LoD, 1X LoD, <1X LoD, <0.1X LoD. For each target analyte, a total of 32 replicates of each PM in each matrix were tested across 2 Alinity m HSV 1 & 2 / VZV AMP kit lots and 2 Alinity m Systems, with each lot of reagents assigned to a specific instrument. For each matrix, a total of 16 replicates were tested for each PM on each of the 2 instrument/lot pair, resulting in a total of 32 replicates per PM (16 replicates × 2 instrument/lot = 32 replicates per PM).
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The data supports that the simulated swab matrix (SM) is found to be equivalent to the clinical swab matrix (CM).
ii). Compatibility between Transport Media (Swab) and Simulated swab matrix:
The study was conducted for transport media compatibility and simulated swab matrix equivalency. The samples were prepared as follow:
1) Remel M4RT: Thirty (30) samples were tested in duplicate for a total of 60 replicates. All 30 samples were prepared by pooling 2 or 3 individual clinical specimens. Pooling was performed to ensure there was enough volume per sample for duplicate testing and for any potential repeat testing needed due to invalid results. Each of the 30 samples was spiked with HSV-1, HSV-2 and VZV stock to achieve the targeted final concentration of 3X LOD.
2) COPAN UTM/BD UTV media: Thirty (30) samples were tested in duplicate for a total of 60 replicates. Out of these 30 samples, 8 samples were individual clinical specimens, and the remaining 22 samples were prepared by pooling 2 individual specimens. Pooling was performed to ensure that there was enough volume per sample for duplicate testing and for any potential repeat testing needed due to invalid results. Each of the 30 samples was spiked with HSV-1, HSV-2 and VZV stock to achieve the approximate targeted final concentration of 3X LOD.
3) Simulated Swab Matrix (SM) was spiked with HSV-1, HSV-2 and VZV stock to achieve the approximate targeted final concentration of 3X LOD. For each matrix, a total of 60 replicates were tested. The results demonstrated that the performance of Alinity m HSV 1 & 2 / VZV assay is equivalent in Remel M4RT, COPAN UTM/BD UTV and Simulated Swab Matrix. The results demonstrated that the Alinity m HSV 1 & 2 / VZV assay is equivalent in Remel M4RT, COPAN UTM/BD UTV, and simulated swab matrix. Results are presented below in Table 13.
Table 13. Matrix Equivalency Results
| Analyte | Matrix | Positive Rate | Mean CN | SD |
| --- | --- | --- | --- | --- |
| HSV-1 | Remel M4RT | 100% (60/60) | 26.89 | 0.465 |
| | COPAN/BD | 100% (60/60) | 26.44 | 0.389 |
| | Simulated Swab matrix (SM) | 100% (60/60) | 26.30 | 0.186 |
| HSV-2 | Remel M4RT | 100% (60/60) | 29.82 | 0.544 |
| | COPAN/BD | 100% (60/60) | 29.43 | 0.407 |
| | Simulated Swab matrix (SM) | 100% (60/60) | 29.34 | 0.245 |
| VZV | Remel M4RT | 100% (60/60) | 28.97 | 0.669 |
| | COPAN/BD | 100% (60/60) | 28.06 | 0.472 |
| | Simulated Swab matrix (SM) | 100% (60/60) | 27.96 | 0.238 |
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B. Clinical Studies:
1. Clinical Sensitivity:
Not applicable
2. Clinical Specificity:
Not applicable
3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
Performance characteristics of the Alinity m HSV 1 & 2 / VZV assay were established in prospective and retrospective clinical studies conducted in the United States.
The swab specimens for prospective and retrospective studies were categorized as cutaneous (eg, skin lesion), mucocutaneous (eg, anorectal, vaginal/cervical, and oral lesion), and un categorized (lesion type could not be determined).
The clinical performance of the Alinity m HSV 1 & 2 / VZV assay was established by comparing to an FDA-cleared nucleic acid amplification test (NAAT 1). For specimens with discordant results between Alinity m and NAAT 1, testing with another FDA-cleared nucleic acid test (NAAT 2) was performed. Results of testing on discordant samples were not included in the analysis of device performance and are considered for information purposes only.
Prospective Clinical Study #1
The multicenter, prospective clinical study was conducted using swab specimens from lesions (including cutaneous and mucocutaneous) of symptomatic individuals suspected of HSV-1, HSV-2, and/or VZV infection at geographically diverse locations in the US. All lesion swab specimens were prospectively collected in BD UVT, COPAN UTM, or Remel M4RT collection media. The subjects were male and female individuals of all ages, including pediatric and geriatric populations ages >1 to <90 years.
A total of 1,257 prospective specimens (1017 fresh + 240 frozen) were included for each analyte (HSV-1, HSV-2, and VZV), for clinical performance analysis, excluding 1 sample that did not have complete history information.
The gender and age demographics for each category are listed below in Table 14.
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Table 14. Prospective Study - Age and Gender Distribution by Sample Type
| Number of Specimens from | | | | |
| --- | --- | --- | --- | --- |
| Lesion Type | Age | Female Subjects (# Lesions) | Male Subjects (# Lesions) | Total (# Lesions) |
| Cutaneous | ≤ 5 Years | 10 (11) | 15 (16) | 25 (27) |
| | 6 to 21 Years | 24 (28) | 27 (28) | 51 (56) |
| | 22 to 59 Years | 176 (195) | 163 (183) | 339 (378) |
| | ≥ 60 Years | 82 (95) | 78 (86) | 160 (181) |
| | Total | 292 (329) | 283 (313) | 575 (642) |
| Mucocutaneous | ≤ 5 Years | 13 (20) | 14 (29) | 27 (49) |
| | 6 to 21 Years | 33 (34) | 12 (13) | 45 (47) |
| | 22 to 59 Years | 219 (240) | 120 (170) | 339 (410) |
| | ≥ 60 Years | 62 (80) | 21 (27) | 83 (107) |
| | Total | 327 (374) | 167 (239) | 494 (613) |
| Uncategorized | ≤ 5 Years | 0 (0) | 0 (0) | 0 (0) |
| | 6 to 21 Years | 0 (0) | 0 (0) | 0 (0) |
| | 22 to 59 Years | 0 (0) | 0 (0) | 0 (0) |
| | ≥ 60 Years | 3 (3) | 0 (0) | 3 (3) |
| | Total | 3 (3) | 0 (0) | 3 (3) |
| Total | Total | 622 (706) | 450 (552) | 1072 (1258) |
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The Clinical Study #1 performance is summarized in Table 15.
Table 15. Clinical Agreement by analyte and Lesion Type (Prospective Clinical study #1)
| Analyte | Specimen Type | Total | True Positive | False Negative | True Negative | False Positive | PPA % with 95% CI | NPA % with 95% CI |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| HSV1 | Cutaneous Lesion | 641 | 36 | 0 | 600 | 5 | 100% (36/36) (90.4% -100%) | 99.2% (600/605) (98.1% -99.6%) |
| | Mucocutaneous Lesion | 613 | 87 | 2 | 517 | 7 | 97.8% (87/89) (92.2% -99.4%) | 98.7% (517/524) (97.3% -99.4%) |
| | Uncategorized^{a} | 3 | 0 | 1 | 2 | 0 | 0.0% (0/1) (0.0% - 79.3%) | 100% (2/2) (34.2% - 100%) |
| | All | 1257 | 123 | 3^{b} | 1119 | 12^{c} | 97.6% (123/126) (93.2% - 99.2%) | 98.9% (1119/1131) (98.2% - 99.4%) |
| HSV2 | Cutaneous Lesion | 641 | 61 | 1 | 575 | 4 | 98.4% (61/62) (91.4% - 99.7%) | 99.2% (600/605) (98.1% -99.6%) |
| | Mucocutaneous Lesion | 613 | 60 | 0 | 548 | 5 | 100% (60/60) (94.0% -100%) | 99.1% (548/553) (98.1% -99.6%) |
| | Uncategorized^{a} | 3 | 1 | 0 | 2 | 0 | 100% (1/1) (20.7% - 100%) | 100% (2/2) (34.2% - 100%) |
| | All | 1257 | 122 | 1^{d} | 1125 | 9^{e} | 99.2% (122/123) (95.5% - 99.9%) | 99.2% (1125/1134) (98.5% - 99.6%) |
| VZV | Cutaneous Lesion | 641 | 38 | 0 | 601 | 2 | 100% (38/38) (90.8% -100%) | 99.7% (601/603) (98.8% -99.9%) |
| | Mucocutaneous Lesion | 613 | 5 | 1 | 607 | 0 | 83.3% (5/6) (43.6% - 97.0%) | 100% (607/607) (99.4% - 100%) |
| | Uncategorized^{a} | 3 | 0 | 0 | 3 | 0 | 0/0 | 100% (3/3) (43.9% - 100%) |
| | All | 1257 | 43 | 1^{f} | 1211 | 2^{g} | 97.7% (43/44) (88.2% - 99.6%) | 99.8% 91211/1213) (99.4% - 100%) |
The lesion type for these swabs could not be determined or categorized.
b 3 out of 3 NAAT 1+/Alinity m HSV-1 negatives were negatives by NAAT 2.
c 2 out of 12 NAAT 1- /Alinity m HSV-1 positives results were positives by NAAT 2.
d 1 out of 1 NAAT 1+/Alinity m HSV-2 negative were negative by NAAT 2.
e 3 out of 9 NAAT 1-/Alinity m HSV-2 positive results were positive by NAAT 2.
f 1 out of 2 NAAT 1+Alinity m VZV negative results were negative by NAAT 2.
g 0 out of 2 NAAT 1-/Alinity m VZV positive were positive by NAAT 2.
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# Retrospective Clinical Study #2
The retrospective study was conducted using archived, leftover lesion swab specimens from routine clinical testing, collected from male and female individuals of all ages, including pediatric and geriatric populations ages $>1$ to $<90$ years. All lesion swab specimens were previously collected in BD UVT, COPAN UTM, or Remel M4RT collection media per standard of care. One lesion swab specimen was obtained from each individual.
A total of 411 specimens were included in the clinical performance analysis that had results for at least one of the analytes (410 results for HSV-1, 410 results for HSV-2, and 411 results for VZV).
The gender and age demographics for each category are listed below in Table 16.
Table 16. Retrospective Study - Age and Gender Distribution by Sample Type
| Number of Samples from | | | | |
| --- | --- | --- | --- | --- |
| Lesion Type | Age | Female Subjects (# Lesions) | Male Subjects (# Lesions) | Total (# Lesions) |
| Cutaneous | ≤ 5 Years | 8 (8) | 17 (17) | 25 (25) |
| | 6 to 21 Years | 9 (9) | 9 (9) | 18 (18) |
| | 22 to 59 Years | 60 (60) | 25 (25) | 85 (85) |
| | ≥ 60 Years | 9 (9) | 10 (10) | 19 (19) |
| | Total | 86 (86) | 61 (61) | 147 (147) |
| Mucocutaneous | ≤ 5 Years | 31 (31) | 28 (28) | 59 (59) |
| | 6 to 21 Years | 18 (18) | 13 (13) | 31 (31) |
| | 22 to 59 Years | 78 (78) | 33 (33) | 111 (111) |
| | ≥ 60 Years | 20 (20) | 13 (13) | 33 (33) |
| | Total | 147 (147) | 87 (87) | 234 (234) |
| Uncategorized | ≤ 5 Years | 5 (5) | 4 (4) | 9 (9) |
| | 6 to 21 Years | 2 (2) | 1 (1) | 3 (3) |
| | 22 to 59 Years | 8 (8) | 8 (8) | 16 (16) |
| | ≥ 60 Years | 0 (0) | 2 (2) | 2 (2) |
| | Total | 15 (15) | 15 (15) | 30 (30) |
| Total | Total | 248 (248) | 163 (163) | 411 (411) |
The Clinical Study #2 performance is summarized in Table 17.
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Table 17. Clinical Agreement by analyte and Lesion Type (Retrospective Clinical study #2)
| Analyte | Specimen Type | Total | True Positive | False Negative | True Negative | False Positive | PPA % with 95% CI | NPA % with 95% CI |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| HSV1 | Cutaneous Lesion | 147 | 16 | 0 | 124 | 7 | 100% (16/16)
(80.6% - 100%) | 94.7% (124/131)
(89.4% - 97.4%) |
| | Mucocutaneous Lesion | 234 | 51 | 0 | 178 | 5 | 100% (51/51)
(93.0% - 100%) | 97.3% (178/183)
93.8% - 98.8%) |
| | Uncategorized^{a} | 29 | 5 | 0 | 23 | 1 | 100% (5/5)
(56.6% - 100%) | 95.8% (23/24)
(79.8% - 99.3%) |
| | All | 410 | 72 | 0 | 325 | 13^{b} | 100% (72/72)
(94.9% - 100%) | 96.2% (325/338)
(93.5% - 97.7%) |
| HSV2 | Cutaneous Lesion | 147 | 23 | 1 | 115 | 8 | 95.8% (23/24)
(79.8% - 99.3%) | 93.5% (115/123)
(87.7% - 96.7%) |
| | Mucocutaneous Lesion | 234 | 44 | 0 | 186 | 4 | 100% (44/44)
(92.0% - 100%) | 97.9% (186/190)
(2094.7% - 99.2%) |
| | Uncategorized^{a} | 29 | 7 | 0 | 20 | 2 | 100% (7/7)
(64.6% - 100%) | 90.9% (20/22)
(72.2% - 97.5%) |
| | All | 410 | 74 | 1^{c} | 321 | 14^{d} | 98.7% (74/75)
(92.8% - 99.8%) | 95.8% (321/335)
(93.1% - 97.5%) |
| VZV | Cutaneous Lesion | 147 | 20 | 0 | 125 | 2 | 100% (20/20)
(83.9% - 100%) | 98.4% (125/217)
(94.4% - 99.6%) |
| | Mucocutaneous Lesion | 234 | 17 | 0 | 214 | 3 | 100% (17/17)
(81.6% - 100%) | 98.6% (214/127)
(96.6% - 99.5%) |
| | Uncategorized^{a} | 30 | 7 | 1 | 21 | 1 | 87.5% (7/8)
(52.9% - 97.8) | 95.5% (21/22)
(78.2% - 99.2%) |
| | All | 411 | 44 | 1^{e} | 360 | 6^{f} | 97.8% (44/45)
(88.4% - 99.6%) | 98.4% (360/366)
(96.5% - 99.2%) |
The lesion type for these swabs could not be determined or categorized.
b 0 out of 13 NAAT 1-/Alinity m HSV-1 positives were positive by NAAT 2.
c 1 out of 1 NAAT 1+/Alinity m HSV-2 negative results were negative by NAAT 2.
d 0 out of 14 NAAT 1-/Alinity m HSV-2 positives were positive by NAAT 2.
e 1 out of 1 NAAT 1+/Alinity m VZV negative results were negative by NAAT 2.
f 0 out of 6 NAAT 1-/Alinity m VZV positives were positive by NAAT 2.
B Clinical Cut-Off:
Not applicable
C Expected Values/Reference Range:
Not applicable
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VIII Proposed Labeling:
The labeling is sufficient, and it satisfies the requirements of 21 CFR Part 809.10.
IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
