Aptima HBV Quant Assay
Device Facts
| Record ID | P170025 |
|---|---|
| Device Name | Aptima HBV Quant Assay |
| Applicant | Hologic, Inc. |
| Product Code | MKT · Microbiology |
| Decision Date | Jan 23, 2018 |
| Decision | APRL |
| Regulation | 21 CFR 866.3174 |
| Device Class | Class 2 |
Indications for Use
The Aptima HBV Quant assay is an in vitro nucleic acid amplification test for the quantitation of hepatitis B virus (HBV) DNA in human plasma and serum on the fully automated Panther® system. Plasma may be prepared in ethylenediaminetetraacetic acid (EDTA), anticoagulant citrate dextrose (ACD) solution, and plasma preparation tubes (PPTs). Serum may be prepared in serum tubes and serum separator tubes (SSTs). Specimens are tested using the fully automated Panther system for sample processing, amplification, and quantitation. Specimens containing HBV genotypes A, B, C, D, E, F, G, and H are validated for quantitation in the assay. The Aptima HBV Quant assay is intended for use as an aid in the management of patients with chronic HBV infections undergoing HBV antiviral therapy. The assay can be used to measure HBV DNA levels at baseline and during treatment to aid in assessing viral response to treatment. The results from the Aptima HBV Quant assay must be interpreted within the context of all relevant clinical and laboratory findings. Assay performance for determining the clinical stage of HBV infection has not been established. Clinical performance characteristics have been established for individuals treated with tenofovir disoproxil fumarate or entecavir. The Aptima HBV Quant assay is not approved for use as a screening test for the presence of HBV DNA in blood or blood products or as a diagnostic test to confirm the presence of HBV infection.
Device Story
Aptima HBV Quant assay is an in vitro nucleic acid amplification test performed on the fully automated Panther system. It processes human plasma or serum samples to quantitate HBV DNA. The process involves three steps in a single tube: target capture using magnetic micro-particles, transcription-mediated amplification (TMA) using MMLV reverse transcriptase and T7 RNA polymerase, and real-time detection using fluorescently labeled probes (torches). The system uses a dual-target approach (polymerase and surface genes) to mitigate under-quantitation due to mutations. The Panther system software automatically calculates HBV DNA concentration by comparing fluorescent signals to calibration curves. Results are reported in IU/mL and log10 IU/mL. Clinicians use these measurements at baseline and during treatment to assess viral response, which informs clinical decision-making regarding antiviral therapy. The device benefits patients by providing accurate viral load monitoring to guide chronic HBV management.
Clinical Evidence
Pivotal study enrolled 245 evaluable subjects (126 HBeAg+, 119 HBeAg-) across 67 sites. Evaluated ability to predict virologic, biochemical, and serologic responses at Week 48 based on early responses at Weeks 12 and 24. Primary endpoint: HBV DNA <10 IU/mL. Results demonstrated clinical utility in assessing viral response to tenofovir or entecavir therapy. Analytical studies established LoD (4.8 IU/mL plasma, 5.9 IU/mL serum), linear range (1.0 to 9.0 log10 IU/mL), and precision (total SD ≤0.33 log10 IU/mL). No interference observed from endogenous substances or common HBV medications.
Technological Characteristics
Nucleic acid amplification test (TMA). Targets HBV polymerase and surface genes. Uses magnetic micro-particle target capture and fluorescent torch detection. Fully automated on Panther system. Dynamic range: 1.0–9.0 log10 IU/mL. LLoQ: 10 IU/mL. Reagents stored at 2-8°C. Software-based automated quantitation.
Indications for Use
Indicated for quantitation of HBV DNA in human plasma and serum to aid in management of patients with chronic HBV infection undergoing antiviral therapy (tenofovir disoproxil fumarate or entecavir). Validated for HBV genotypes A-H. Not for screening blood products or diagnosing HBV infection.
Regulatory Classification
Identification
A nucleic acid-based hepatitis B virus (HBV) assay is identified as an in vitro diagnostic device intended for prescription use in the detection of HBV nucleic acid in specimens from individuals with antibody evidence of HBV infection. In these devices, the detection of HBV nucleic acid is used as an aid in the management of HBV-infected individuals. The assay is intended for use with human serum or plasma (and other matrices as applicable) from individuals with HBV. The assay is not intended for use as a donor screening assay for the presence of HBV nucleic acids in blood, blood products, plasma, cells, or tissue donors, or as a diagnostic assay to confirm the presence of HBV infection.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) Labeling required under § 809.10(b) of this chapter must include: (i) A prominent statement that the assay is not intended for use as a screening assay for the presence of HBV DNA in blood or blood products, plasma, cells, or tissue donors, or as a diagnostic assay to confirm the presence of HBV infection. (ii) A detailed explanation of the principles of operation and procedures for performing the assay. (iii) A detailed explanation of the interpretation of results. (iv) Limitations, which must be updated to reflect current clinical practice and disease presentation and/or management. These limitations must include statements that indicate: (A) Management of patients undergoing HBV treatment should not be established on the basis of a single assay result but should be determined by a licensed healthcare professional in conjunction with the clinical presentation, history, and other diagnostic procedures, *e.g.,* HBV serologic testing, liver function assays, liver elastography, etc.(B) The specimen types for which the device has been cleared, and that use of this assay with specimen types other than those specifically cleared for this device may result in inaccurate assay results. (C) The results obtained with this assay may not be used interchangeably with results obtained with a different manufacturer's assay. (2) Design verification and validation must include the following: (i) Detailed device description, including the device components, ancillary reagents required but not provided, and an explanation of the device methodology. Additional information appropriate to the technology must be included such as design of primers and probes, rationale for the selected gene targets, specifications for amplicon size, and degree of nucleic acid sequence conservation. (ii) For devices with assay calibrators, the design and composition of all primary, secondary, and subsequent quantitation standards used for calibration as well as their traceability to a standardized reference material that FDA has determined is appropriate ( *e.g.,* a recognized consensus standard). In addition, analytical testing must be performed following the release of a new lot of the standard material that was used for device clearance or approval, or when there is a transition to a new calibration standard.(iii) Documentation and characterization ( *e.g.,* determination of the identity, supplier, purity, and stability) of all critical reagents (including nucleic acid sequences for primers and probes) and protocols for maintaining product integrity.(iv) Risk analysis and management strategies demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on assay performance. (v) Final release criteria to be used for manufactured assay lots with appropriate evidence that lots released at the extremes of the specification will meet the identified analytical and clinical performance characteristics as well as stability. (vi) Stability studies for reagents must include documentation of an assessment of real-time stability for multiple reagent lots using the indicated specimen types and must use acceptance criteria that ensure that analytical and clinical performance characteristics are met when stability is assigned based on the extremes of the acceptance range. (vii) All stability protocols, including acceptance criteria. (viii) Detailed documentation of analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including limit of detection (LoD), linearity, precision, endogenous and exogenous interferences, cross-reactivity, carryover, matrix equivalency, sample and reagents stability, and as applicable, upper and lower limits of quantitation (ULoQ and LLoQ, respectively). Samples selected for use must be from subjects with clinically relevant circulating genotypes in the United States. Cross-reactivity studies must include samples from HBV nucleic acid negative subjects with other viral or non-viral causes of liver disease, including autoimmune hepatitis, alcoholic liver disease, chronic hepatitis C virus, primary biliary cirrhosis, and nonalcoholic steatohepatitis, when applicable. The effect of each identified nucleic-acid isolation and purification procedure on detection must be evaluated. (ix) Analytical sensitivity of the assay that is the same or better than that of other cleared or approved assays. (x) For devices with associated software or instrumentation, documentation must include a detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review. (xi) Detailed documentation of performance from a clinical study with a design and number of clinical samples (appropriately statistically powered) that is appropriate for the intended use of the device as well as conducted in the appropriate settings by the intended users. The samples must include prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. Samples must be sourced from geographically diverse areas.
Reference Devices
- Aptima HIV-1 Quant Assay
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Submission Summary (Full Text)
SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)
I. GENERAL INFORMATION
Device Generic Name: HBV DNA quantitative test
Device Trade Name: Aptima® HBV Quant assay
Device Procode: MKT
Applicant’s Name and Address: Hologic, Inc.
10210 Genetic Center Drive
San Diego, CA 92121
Date(s) of Panel Recommendation: None
Premarket Approval Application (PMA) Number: P170025
Date of FDA Notice of Approval: January 23, 2018
II. INDICATIONS FOR USE
The Aptima HBV Quant assay is an in vitro nucleic acid amplification test for the quantitation of hepatitis B virus (HBV) DNA in human plasma and serum on the fully automated Panther® system.
Plasma may be prepared in ethylenediaminetetraacetic acid (EDTA), anticoagulant citrate dextrose (ACD) solution, and plasma preparation tubes (PPTs). Serum may be prepared in serum tubes and serum separator tubes (SSTs). Specimens are tested using the fully automated Panther system for sample processing, amplification, and quantitation. Specimens containing HBV genotypes A, B, C, D, E, F, G, and H are validated for quantitation in the assay.
The Aptima HBV Quant assay is intended for use as an aid in the management of patients with chronic HBV infections undergoing HBV antiviral drug therapy. The assay can be used to measure HBV DNA levels at baseline and during treatment to aid in assessing viral response to treatment. The results from the Aptima HBV Quant assay must be interpreted within the context of all relevant clinical and laboratory findings. Assay performance for determining the clinical stage of HBV infection has not been established. Clinical performance characteristics have been established for individuals treated with tenofovir disoproxil fumarate or entecavir.
The Aptima HBV Quant assay is not approved for use as a screening test for the presence of HBV DNA in blood or blood products or as a diagnostic test to confirm the presence of HBV infection.
PMA P170025: FDA Summary of Safety and Effectiveness Data
III. CONTRAINDICATIONS
There are no known contraindications.
IV. WARNINGS AND PRECAUTIONS
The warnings and precautions can be found in the Aptima HBV Quant assay labeling.
V. DEVICE DESCRIPTION
The Aptima HBV Quant assay involves three main steps, which all take place in a single tube on the Panther system: target capture, target amplification by transcription-mediated amplification (TMA), and detection of the amplification products (amplicon) by the fluorescent labeled probes (torches).
During target capture, viral DNA is isolated from specimens. The specimen is treated with a detergent to solubilize the viral envelope, denature proteins, and release viral genomic DNA. Capture oligonucleotides hybridize to highly conserved regions of the HBV genome, if present, in the test specimen. The hybridized target is then captured onto magnetic micro-particles that are separated from the specimen in a magnetic field. Wash steps remove extraneous components from the reaction tube.
Target amplification occurs via transcription-based nucleic acid amplification method that utilizes two enzymes, MMLV (Moloney murine leukemia virus) reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy of the target sequence which contains a promoter sequence for T7 RNA polymerase. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. The Aptima HBV Quant assay utilizes the TMA method to amplify two regions: polymerase gene and surface gene of the HBV genome. This dual target region approach mitigates the risk of under-quantitation from one of the target regions due to potential mutations. Amplifications of those regions are achieved using specific primers designed to amplify HBV genotypes A, B, C, D, E, F, G, and H. The dual target region approach with primer design targeting the most conserved regions ensure accurate quantitation of the HBV DNA.
Detection is achieved using single-stranded nucleic acid torches that are present during the amplification of the target and hybridize specifically to the amplicon as it is generated in real-time. Each torch has a fluorophore and a quencher. The quencher suppresses the signal of the fluorophore when not hybridized to the amplicon. When the torch binds to the amplicon, the quencher is moved further away from the fluorophore and it will emit a signal at a specific wavelength when excited by a light source. More torch hybridizes when more amplicon is present creating higher fluorescent signal. The time taken for the fluorescent signal to reach a threshold is proportional to the starting HBV DNA concentration. Each reaction has an internal calibrator/internal control (IC) which controls for variations in specimen processing, amplification, and detection. The concentration of a sample is determined automatically by the Panther system software
PMA P170025: FDA Summary of Safety and Effectiveness Data
using the HBV and IC signals for each reaction and comparing them to calibration information.
## Components of the Aptima HBV Quant assay Kit
The Aptima HBV Quant assay kit (100 tests) for the Panther system consists of 4 reagent kits:
Box 1: Aptima HBV Quant assay kit which contains the following reagents:
- Amplification Reagent
- Enzyme Reagent
- Promoter Reagent
- Target Capture Reagent
- Amplification Reconstitution Reagent
- Enzyme Reconstitution Reagent
- Promoter Reconstitution Reagent
Box 2: Aptima HBV Quant Controls kit which contains the following reagents:
- Negative Control
- Low Positive Control
- High Positive Control
Box 3: Aptima HBV Quant Calibrator kit which contains the following reagent:
- Positive Calibrator
Box 4: Aptima HBV Quant Target Enhancer Reagent Box
- Target Enhancer Reagent
There is one ancillary kit required to perform the assay (available separately): Aptima Assay Fluids kit (also known as Universal Fluids Kit) which contains the following reagents:
- Wash Solution
- Buffer for Deactivation Fluid
- Oil Reagent
The Aptima Specimen Diluent kit is one optional ancillary kit which can be procured separately. The Aptima Specimen Diluent reagent provided in this kit is used to dilute plasma and serum specimens that are tested with the Aptima HBV Quant assay on the Panther system.
## Quality Control Procedures
The Aptima HBV Quant assay contains three quality controls:
1. Assay Calibration
An assay calibration must be completed to generate valid results. A single positive calibrator is run in triplicate each time a reagent kit is loaded on the Panther system. Once established, the calibration is valid for up to 24 hours. Software on the Panther
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system alerts the operator when a calibration is required. The operator scans a calibration coefficient found on the Master Lot Barcode Sheet provided with each reagent kit.
During processing, criteria for acceptance of the calibrator are automatically verified by the software on the Panther system. If less than two of the calibrator replicates are valid, the software automatically invalidates the run. Samples in an invalidated run must be retested using a freshly prepared calibrator and freshly prepared controls.
## 2. Negative and Positive Controls
A set of assay controls must be tested to generate valid results. One replicate of the negative control, the low positive control, and the high positive control must be tested each time a reagent kit is loaded on the Panther system. Once established, the control measurements are valid for up to 24 hours. Software on the Panther system alerts the operator when controls measurements are required.
During processing, criteria for acceptance of controls are automatically verified by software on the Panther system. To generate valid results, the negative control must give a result of "Not Detected" and the positive controls must give results within predefined parameters. If any one of the controls has an invalid result, the software automatically invalidates the run. Samples in an invalidated run must be retested using a freshly prepared calibrator and freshly prepared controls.
## 3. Internal Calibrator/Internal Control
Each sample contains an internal calibrator/internal control (IC). During processing, IC acceptance criteria are automatically verified by the Panther system software. If an IC result is invalid, the sample result is invalidated. Every sample with an invalid IC result must be retested to obtain a valid result.
The Panther system software is designed to accurately verify processes when procedures are performed following the instructions provided in this package insert and the Panther System Operator's Manual.
## Interpretation of Results
The Panther system automatically determines the concentration of HBV DNA for specimens and controls by comparing the results to a calibration curve. HBV DNA concentrations are reported in IU/mL and $\log_{10}$ IU/mL. The interpretation of results is provided in Table 1. If the dilution option is used to dilute specimens, the Panther system automatically calculates the HBV concentration for the neat specimen by multiplying the diluted concentration by the dilution factor and diluted samples will be flagged as diluted.
For diluted specimens, results listed as "Not Detected" or "<10 detected" may be generated by diluting a specimen with a concentration above, but close to the LoD (limit of detection) or LLoQ (lower limit of quantitation). It is recommended to collect and test another neat specimen if a quantitative result is not obtained.
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Table 1: Results Interpretation
| Reported Aptima HBV Quant assay Result | | Interpretation |
| --- | --- | --- |
| IU/mL | Log10 IU/mLa | |
| Not Detected | Not Detected | HBV DNA not detected. |
| <10 detected | <1.00 | HBV DNA is detected but at a level below the LLoQ |
| 10 to 1,000,000,000 | 1.00 to 9.00 | HBV DNA concentration is within the linear range of 10 to 1,000,000,000 IU/mL |
| >1,000,000,000 | >9.00 | HBV DNA concentration is above the ULoQc |
| Invalidb | Invalidb | Error indicated in the generation of the result. Specimen should be retested |
| aValue is truncated to two decimal places. bInvalid results are displayed in blue colored font. cSerum and plasma specimens with value above the ULoQ may be diluted and retested to determine a quantitative result within the linear range Note: For diluted specimens with neat concentrations greater than the ULoQ, results will be reported using scientific notation. | | |
VI. ALTERNATIVE PRACTICES AND PROCEDURES
There are currently several FDA approved in vitro diagnostic tests for the quantitation of HBV DNA from patient samples. The patient's medical history and thorough clinical examination, in addition to serology, PCR or nucleic acid testing (NAT), determination of liver enzyme levels, and biopsy of the liver will provide further information on the status of an HBV infection. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle.
VII. MARKETING HISTORY
The Aptima HBV Quant assay, accessory kits, and Aptima Specimen Diluent are marketed in multiple countries. The device has not been withdrawn from marketing for any reasons related to its safety or effectiveness. The following is a list of countries where the product is distributed:
- Austria
- Czech Republic
- Denmark
- Estonia
- Finland
- France
- Germany
- Hungary
Lithuania
Luxembourg
Malta
The Netherlands
Norway
Poland
Romania
Switzerland
- Belgium
- Bulgaria
Croatia
Cyprus
Greece
Iceland
Latvia
Liechtenstein
Sweden
Spain
Italy
Australia
Canada
PMA P170025: FDA Summary of Safety and Effectiveness Data
- Ireland
- United Kingdom
- Slovenia
# VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH
When used according to the instructions in the package insert, there are no known potential direct adverse effects on health. The results of the Aptima HBV Quant assay must be interpreted within the context of all relevant clinical and laboratory findings. Failure of the Aptima HBV Quant assay to perform as indicated due to human error or other cause may lead to improper patient management.
An incorrect low test result or a false negative result may lead to an inappropriate treatment decision, delay in initiation of treatment, and premature discontinuation of antiviral therapy
An incorrect high test result or a false positive may contribute to a change in therapy, unnecessary treatment, prolonged duration of therapy, or may impact the psychological well being of the patient.
Inaccurate results around clinically significant HBV DNA levels such as 2,000 IU/mL and 20,000 IU/mL may impact patient management. Because medical practitioners usually obtain multiple (serial) measurements through the course of the disease, the risk of acting on a single result is lowered. In addition, other laboratory markers (e.g. ALT) may also provide additional information to the medical practitioner to guide patient management. Thus the risks are mitigated by the availability of other medical history information and other laboratory test results.
# IX. SUMMARY OF NONCLINICAL STUDIES
## A. Laboratory Studies
### Limit of Detection (LoD) Using the 3rd HBV WHO International Standard
The LoD was determined by testing dilutions of the 3rd HBV WHO International Standard (NIBSC 10/264, genotype A) in HBV negative human plasma and serum. A total of 9 panel members were tested over 3 days on 3 Panther systems with 3 reagent lots. Probit analysis was used to determine the LoD. The LoD for the Aptima HBV Quant assay using the 3rd HBV WHO International Standard standard 4.8 IU/mL for plasma and 5.9 IU/mL for serum.
### Limit of Detection by HBV Genotype
The LoD across genotypes was determined by testing dilutions of HBV positive clinical specimens for genotypes A, B, C, D, E, F, G and H in HBV negative human plasma and serum. Each panel was tested on multiple Panther systems over multiple days with multiple reagent lots. Probit analysis was used to determine the LoD. The LoD for each genotype is shown in Table 2.
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Table 2: HBV DNA Genotype LoD in Plasma and Serum
| Genotype | Plasma IU/mL | Serum IU/mL |
| --- | --- | --- |
| A | 3.3 | 4.1 |
| B | 2.9 | 3.9 |
| C | 4.9 | 5.2 |
| D | 5.7 | 5.4 |
| E | 5.8 | 5.8 |
| F | 3.0 | 4.0 |
| G | 2.8 | 7.4 |
| H | 5.5 | 6.3 |
Linear Range Genotype A: The linear range was established by testing panels of HBV genotype A virus (0.78 log10 IU/mL to 7.30 log10 IU/mL) and plasmid DNA (5.30 log10 IU/mL to 9.18 log10 IU/mL) diluted in HBV negative human plasma and serum according to CLSI EP06-A. The Aptima HBV Quant assay demonstrated linearity across the range tested with an upper limit of quantitation (ULoQ) of 9.0 log10 IU/mL as shown in Figure 1.
Figure 1: Linearity in Plasma and Serum
Linearity Across HBV Genotypes: The linearity of HBV genotypes was established by testing individual clinical positive samples for genotypes A, E, F, G, and H, and PEI 1st WHO Reference panels (PEI 5086/08) for genotypes B, C, and D. Virus was used for the lower range of the assay (4 log IU/mL and below for genotypes A-G, 3 log IU/mL and below for genotype H), and plasmid DNA was used for the upper range with a 2 log overlap. Dilutions in negative human plasma were tested for all genotypes. Linearity was demonstrated across the range tested for all genotypes tested as shown in Figure 2 and Table 3 for plasma and for serum. The Aptima HBV
PMA P170025: FDA Summary of Safety and Effectiveness Data
Quant assay on the Panther system demonstrated linearity across genotypes and serum and plasma with a dynamic range of $1.0\log \mathrm{IU / mL}$ to $9.0\log \mathrm{IU / mL}$ .
Figure 2: Linear Range and Linearity (Plasma)
Table 3: Linear Fit Equations across Genotypes (Plasma)
| Genotype | Linear Equation | Maximum Non-linearity (log10 IU/mL) |
| --- | --- | --- |
| A | y = 0.9919 x + 0.0395 | 0.09 |
| B | y = 1.0238 x - 0.2043 | N/A* |
| C | y = 1.0124 x - 0.2475 | 0.10 |
| D | y = 1.0196 x - 0.2987 | 0.07 |
| E | y = 0.9872 x + 0.0832 | 0.13 |
| F | y = 0.9958 x + 0.1273 | 0.14 |
| G | y = 1.0013 x - 0.0466 | 0.11 |
| H | y = 1.0561 x - 0.323 | -0.05 |
| *No 2nd/3rdorder polynomial fit is statistically better than a linear fit at the significance level for genotype B. | | |
Lower Limit of Quantitation (LLoQ) Using the $3^{\mathrm{rd}}$ HBV WHO International Standard: The lower limit of quantitation (LLoQ) is defined as the lowest concentration at which HBV DNA is reliably quantitated within a total error, according to CLSI EP17-A2. Total error was estimated by two methods: Total Analytical Error (TAE) = |bias| + 2SD, and Total Error (TE) = SQRT(2) x 2SD. To ensure accuracy and precision of measurements, the total error of the Aptima HBV Quant assay was set at $1\log_{10}\mathrm{IU / mL}$ (i.e., at the LLoQ, the difference between two measurements of more than $1\log_{10}\mathrm{IU / mL}$ is statistically significant).
PMA P170025: FDA Summary of Safety and Effectiveness Data
The LLoQ was determined by testing panels of the 3rd WHO International Standard for Hepatitis B Virus DNA (NIBSC 10/264, genotype A) diluted in HBV negative human plasma and serum. Forty-five (45) replicates of each dilution were tested with each of three reagent lots for a minimum of 135 replicates per dilution. For a given matrix, the lowest observed result within a reagent lot that met the accuracy goal $(\mathrm{TE} \leq 1 \log_{10} \mathrm{IU/mL}$ and $\mathrm{TAE} \leq 1 \log_{10} \mathrm{IU/mL})$ with $>95\%$ detection and greater than or equal to the LOD was selected as the LLoQ for that reagent lot. The highest LLoQ observed across all reagent lots tested was selected as the final LLoQ for that matrix which is $6 \mathrm{IU/mL}$ (0.79 log $\mathrm{IU/mL}$ ) for plasma and $8 \mathrm{IU/mL}$ (0.88 log $\mathrm{IU/mL}$ ) for serum. The LLoQ was established across genotypes (see next section "Determination of the Lower Limit of Quantitation (LLoQ) Across HBV Genotypes"). This genotype data establishes the overall LLoQ for the assay as $10 \mathrm{IU/mL}$ .
Determination of LLoQ Across HBV Genotype: The LLoQ across genotypes was determined by testing dilutions of HBV positive clinical specimens for genotypes A, B, C, D, E, F, G, and H in HBV negative human plasma and serum. Assignment of the concentration of clinical specimens was determined using an FDA approved assay. Thirty-six (36) replicates of each panel member were tested with each of two reagent lots for a minimum of 72 replicates per panel member. For a given genotype and matrix, the lowest observed result within a reagent lot that met the accuracy goal $(\mathrm{TE} \leq 1 \log_{10} \mathrm{IU/mL}$ and $\mathrm{TAE} \leq 1 \log_{10} \mathrm{IU/mL})$ with $>95\%$ detection and greater than or equal to the LOD was selected as the LLoQ for that reagent lot. The highest LLoQ observed across the two reagent lots tested for a given genotype and matrix was selected as the final LLoQ for that genotype and matrix. The calculated LLoQ for genotypes A, B, C, D, E, F, G, and H in plasma and serum are summarized in Table 4: Summary of LLoQ Across Genotypes (Plasma and Serum). Genotype D in serum had the highest LLoQ at 9 IU/mL. This supports the overall LLoQ for the assay as 10 IU/mL.
Table 4: Summary of LLoQ Across Genotypes (Plasma and Serum)
| HBV Genotype | Plasma LLoQ | | Serum LLoQ | |
| --- | --- | --- | --- | --- |
| | (IU/ml) | (log10 IU/ml) | (IU/ml) | (log10 IU/ml) |
| A | 7 | 0.85 | 6 | 0.81 |
| B | 6 | 0.75 | 5 | 0.72 |
| C | 6 | 0.75 | 6 | 0.81 |
| D | 8 | 0.91 | 9 | 0.96 |
| E | 8 | 0.88 | 8 | 0.89 |
| F | 7 | 0.86 | 6 | 0.76 |
| G | 4 | 0.65 | 8 | 0.89 |
| H | 7 | 0.83 | 6 | 0.81 |
Traceability to the $3^{\mathrm{rd}}$ HBV WHO International Standard: A series of secondary standards with known concentrations were used throughout product development and product manufacturing to establish traceability to the $3^{\mathrm{rd}}$ HBV WHO International
PMA P170025: FDA Summary of Safety and Effectiveness Data
Standard. The concentrations tested for the HBV WHO standard were between 2.0 and $4.0\log_{10}\mathrm{IU / mL}$ , the secondary standards ranged in concentration from 2.4 to 8.4 $\log_{10}\mathrm{IU / mL}$ . The Aptima HBV Quant assay controls and calibrators were also tested along with the secondary standards and the WHO standard. All of the panels had similar results, and they were distributed linearly across the assay's linear range, as presented in Figure 3.
Figure 3: Traceability Between 3rd WHO Standard and Calibrators and Controls
Precision-Within Laboratory: The Aptima HBV Quant precision panel was built by diluting HBV genotype A virus and HBV plasmid DNA into HBV-negative clinical plasma and HBV-negative clinical serum (the four highest panel members in each matrix were plasmid DNA). Eleven panel members in each matrix spanned the range of the assay (target concentrations of $1.30\log_{10}\mathrm{IU / mL}$ to $8.90\log 10\mathrm{IU / mL}$ ), and were tested in three replicates per run by one operator, using three reagent lots on one Panther system over three days, two runs a day. Table 5 shows the precision of assay results (in $\log_{10}\mathrm{IU / mL}$ ) between days, between lots, between runs, within runs, and overall. Total variability was primarily due to the intra-run measurement (i.e., random error).
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 5: Precision of the Aptima HBV Quant assay
| Matrix | Sample | N | Mean Concentration | Mean Concentration | Inter-Lot | Inter-Day | Inter-Run | Intra-Run | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | (IU/mL) | (log10 IU/mL) | SD | SD | SD | SD | SD |
| Plasma | Virus | 34a | 32 | 1.21 | 0.07 | 0.07 | 0.05 | 0.28 | 0.30 |
| | Virus | 54 | 97 | 1.95 | 0.05 | 0.03 | 0.02 | 0.15 | 0.17 |
| | Virus | 54 | 1,474 | 3.16 | 0.00 | 0.02 | 0.02 | 0.07 | 0.07 |
| | Virus | 54 | 10,602 | 4.02 | 0.02 | 0.02 | 0.01 | 0.06 | 0.07 |
| | Virus | 54 | 429,428 | 5.63 | 0.03 | 0.01 | 0.01 | 0.06 | 0.07 |
| | Plasmid DNA | 54 | 652,103 | 5.80 | 0.06 | 0.01 | 0.01 | 0.06 | 0.09 |
| | Virus | 54 | 7,617,612 | 6.88 | 0.02 | 0.00 | 0.02 | 0.06 | 0.07 |
| | Plasmid DNA | 54 | 10,662,942 | 7.02 | 0.02 | 0.01 | 0.01 | 0.06 | 0.06 |
| | Virus | 54 | 89,149,358 | 7.95 | 0.01 | 0.01 | 0.01 | 0.04 | 0.05 |
| | Plasmid DNA | 54 | 103,400,000 | 8.01 | 0.04 | 0.02 | 0.01 | 0.05 | 0.07 |
| | Plasmid DNA | 54 | 612,200,000 | 8.78 | 0.03 | 0.01 | 0.01 | 0.04 | 0.05 |
| Serum | Virus | 29a | 33 | 1.27 | 0.13 | 0.08 | 0.06 | 0.29 | 0.33 |
| | Virus | 54 | 88 | 1.92 | 0.05 | 0.02 | 0.02 | 0.12 | 0.14 |
| | Virus | 54 | 1,446 | 3.15 | 0.02 | 0.01 | 0.00 | 0.08 | 0.08 |
| | Virus | 54 | 7,873 | 3.89 | 0.02 | 0.01 | 0.01 | 0.06 | 0.06 |
| | Virus | 54 | 313,518 | 5.49 | 0.01 | 0.01 | 0.01 | 0.08 | 0.08 |
| | Plasmid DNA | 54 | 599,225 | 5.77 | 0.04 | 0.01 | 0.01 | 0.06 | 0.08 |
| | Virus | 54 | 7,011,440 | 6.84 | 0.02 | 0.01 | 0.01 | 0.07 | 0.08 |
| | Plasmid DNA | 54 | 8,845,332 | 6.94 | 0.05 | 0.01 | 0.01 | 0.05 | 0.07 |
| | Virus | 54 | 70,350,774 | 7.84 | 0.03 | 0.02 | 0.02 | 0.06 | 0.07 |
| | Plasmid DNA | 53b | 122,800,000 | 8.08 | 0.04 | 0.01 | 0.01 | 0.04 | 0.06 |
| | Plasmid DNA | 54 | 678,700,000 | 8.83 | 0.02 | 0.01 | 0.01 | 0.05 | 0.05 |
| SD=standard deviationaDetected replicates with quantifiable result.bOne replicate had an invalid result. | | | | | | | | | |
Analytical Specificity-Interfering Substances: The susceptibility of the Aptima HBV Quant assay to interference by elevated levels of endogenous substances or by drugs commonly prescribed to HBV infected individuals was evaluated. HBV negative plasma samples and samples spiked with HBV to concentrations of approximately 30 IU/mL (1.48 log $_{10}$ IU/mL) and 20,000 IU/mL (4.30 log $_{10}$ IU/mL) were tested.
No interference in the performance of the assay was observed in the presence of albumin (90 mg/mL), hemoglobin (5 mg/mL), triglycerides (30 mg/mL), or unconjugated bilirubin (0.2 mg/mL).
HBV negative clinical plasma specimens from patients with elevated levels of defined substances or from patients with the indicated diseases (ten samples for each substance) listed in Table 6 were tested with the Aptima HBV Quant assay. No interference in the performance of the assay was observed.
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 6: Clinical Specimens Tested for Interference
| Antinuclear antibody (ANA) | Systemic lupus erythematosus (SLE) |
| --- | --- |
| Rheumatoid factor (RF) | Hyperglobulinemia |
| Alcoholic cirrhosis (AC) | Rheumatoid arthritis (RA) |
| Alcoholic hepatitis | Anti-Jo1 antibody (JO-1) |
| Non-alcoholic hepatitis | Multiple myeloma (MM) |
| Autoimmune hepatitis | Hemolyzed (elevated hemoglobin) |
| Elevated alanine aminotransferase (ALT) | Icteric (elevated bilirubin) |
| Hepatocellular carcinoma (HCC) | Lipemic (elevated lipid) |
| Multiple sclerosis (MS) | Elevated protein |
No interference in the performance of the assay was observed in the presence of the exogenous substances listed in Table 7 at concentrations at least three times the $C_{\mathrm{max}}$ (human plasma).
Table 7: Exogenous Substances Tested for Interference
| Exogenous Substance Pool | Exogenous Substances Tested |
| --- | --- |
| 1 | Saquinavir, ritonavir, amprenavir, indinavir, lopinavir, nelfinavir mesylate |
| 2 | Clarithromycin, valganciclovir hydrochloride, efavirenz, nevirapine |
| 3 | Paroxetine HCI, enfuvirtide, zidovudine, didanosine, abacavir sulfate |
| 4 | Ribavirin, entecavir, adefovir dipivoxil, tenofovir disoproxil fumarate, lamivudine, ganciclovir, acyclovir |
| 5 | Stavudine, ciprofloxacin, fluoxetine, azithromycin, valacyclovir, sertraline, zalcitabine |
| 6 | Interferon alpha -2a, interferon alpha -2b, pegylated interferon alpha-2b |
Analytical Specificity – Cross Reactivity: Potential cross-reactivity to the pathogens listed in Table 8 was evaluated in HBV negative human plasma in the presence or absence of $30~\mathrm{IU / mL}$ ( $1.5\log_{10}\mathrm{IU / mL}$ ) and $20,000~\mathrm{IU / mL}$ ( $4.3\log_{10}\mathrm{IU / mL}$ ) HBV DNA. No cross-reactivity or interference was observed.
Table 8: Pathogens Tested for Cross-Reactivity
| Microorganism/ Pathogen | Concentration | Microorganism/ Pathogen | Concentration |
| --- | --- | --- | --- |
| Adenovirus type 5 | 100,000 TCID50/mL1 | Parvo B19 | 100,000 IU/mL |
| BK human polyomavirus | 1,000 TCID50/mL | Rubella virus | 10,000 TCID50/mL |
| Cytomegalovirus | 100,000 TCID50/mL | St. Louis Encephalitis virus | 100,000 TCID50/mL |
| Dengue virus 1 | 10,000 TCID50/mL | vaccinia virus | 1,000 TCID50/mL |
| Dengue virus 2 | 10,000 TCID50/mL | West Nile Virus | 100,000 TCID50/mL |
| Dengue virus 3 | 10,000 TCID50/mL | Yellow Fever Virus | 100,000 TCID50/mL |
PMA P170025: FDA Summary of Safety and Effectiveness Data
| Microorganism/ Pathogen | Concentration | | Microorganism/ Pathogen | Concentration | |
| --- | --- | --- | --- | --- | --- |
| Dengue virus 4 | 100,000 | TCID50/mL | Candida albicans | 1,000,000 | CFU/mL5 |
| Epstein-Barr virus | 100,000 | copies/mL | Chlamydia trachomatis | 1,000,000 | IFU/mL6 |
| Flu H1N1 | 100,000 | TCID50/mL | Corynebacterium diphtheriae | 1,000,000 | CFU/mL |
| Hepatitis A | 100,000 | TCID50/mL | Neisseria gonorrhoeae | 1,000,000 | CFU/mL |
| Hepatitis C | 100,000 | IU/mL2 | Propionibacterium acnes | 1,000,000 | CFU/mL |
| Hepatitis G virus | 100,000 | copies/mL | Staphylococcus aureus | 1,000,000 | CFU/mL |
| Human Herpesvirus Type 6B | 100,000 | copies/mL | Staphylococcus epidermidis | 1,000,000 | CFU/mL |
| Human Herpesvirus Type 8 | 100,000 | copies/mL | Streptococcus pneumoniae | 1,000,000 | CFU/mL |
| HIV-1 | 100,000 | IU/mL | Trichomonas vaginalis | 1,000,000 | CFU/mL |
| HIV-2 | 10,000 | TCID50/mL | | | |
| Human Palillomavirus | 100,000 | copies/mL | | | |
| Herpes Simplex Virus Type 1 | 100,000 | TCID50/mL | | | |
| Herpes Simplex Virus Type 2 | 100,000 | TCID50/mL | | | |
| Human T-lymphotropic virus 1 | 100,000 | vp/mL3 | | | |
| Human T-lymphotropic virus 2 | 100,000 | vp/mL | | | |
| Japanese encephalitis virus | N/A | | | | |
| Murray Valley Encephalitis virus | 2,000 | LD50/mL4 | | | |
| 1TCID50/mL = Tissue culture Infectious dose units per mL2IU/mL = International units per mL3vp/mL = Viral particles per mL4LD50/mL = Lethal dose per mL5CFU/mL = Colony forming units per mL6IFU/mL = Inclusion forming units per mL | | | | | |
Matrix Equivalency: To demonstrate equivalent performance between serum and plasma collection tube types, one hundred eighteen sample sets of matched blood collection tubes (serum tube, ACD, K2 EDTA, K3 EDTA, PPT, SST) were assessed. Of these, 44 sets were naturally infected HBV-positive, and 74 sets were HBV-negative spiked with HBV virus. Correlation was measured by using the serum collection tube as a comparator. Deming regression analysis between serum and each of the collection tubes (SST, K2 EDTA, K3 EDTA, ACD and PPT) demonstrated that the Aptima HBV Quant assay has equivalent performance in reporting quantitative results with the 6 different collection tube types as shown in Table 9.
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 9: Matrix Equivalency Study
| Blood Collection Tube | Deming Regression | 95% CI of Slope | | 95% CI of Intercept | | R² | Mean Difference (log₁₀) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Lower Limit | Upper Limit | Lower Limit | Upper Limit | | |
| ACD | y = 1.01x - 0.04 | 1.00 | 1.02 | -0.10 | 0.01 | 0.998 | -0.01 |
| K2 EDTA | y = 1.02x - 0.14 | 1.00 | 1.03 | -0.20 | -0.07 | 0.997 | -0.07 |
| K3 EDTA | y = 1.01x - 0.12 | 1.00 | 1.03 | -0.18 | -0.06 | 0.997 | -0.06 |
| PPT | y = 1.02x - 0.14 | 1.00 | 1.03 | -0.21 | -0.07 | 0.996 | -0.06 |
| SST | y = 1.00x - 0.03 | 0.99 | 1.01 | -0.07 | 0.03 | 0.999 | -0.01 |
Sample Dilution Using Aptima Specimen Diluent (1:3): To assess the quantitation accuracy of HBV DNA in samples diluted with Aptima Specimen Diluent, samples that spanned the linear range (1.20 log10 IU/mL to 9.39 log10 IU/mL) were diluted 1:3 with Aptima Specimen Diluent (such as 240μL of sample combined with 480μL of Aptima Specimen Diluent). Each sample was tested neat and diluted (1:3) in triplicate. Testing was performed using one lot of assay reagents on two Panther systems with two Aptima Specimen Diluent lots. The difference between the average reported concentration in native matrix (dilution factor applied to the diluted sample result) and the average concentration in Aptima Specimen Diluent are shown in Table 10 for plasma and Table 11 for serum. The sample concentrations were accurately quantitated in the diluted samples.
Table 10: Plasma Specimen 1:3 Dilution Matrix Comparison Summary
| Plasma Matrix Average Reported Concentration (log10 IU/mL) n = 9 | Diluent Average Reported Concentration (log10 IU/mL) n = 18 | Difference of Diluent from Plasma Matrix (log IU10/mL) |
| --- | --- | --- |
| 1.20a | 1.11b | -0.09 |
| 1.56a | 1.36b | -0.20 |
| 2.15 | 2.04 | -0.11 |
| 3.10 | 2.97 | -0.13 |
| 3.92 | 3.89 | -0.03 |
| 4.82 | 4.79 | -0.03 |
| 5.70 | 5.70 | 0.00 |
| 7.07 | 6.98 | -0.09 |
| 7.74 | 7.60 | -0.14 |
| 8.74 | 8.62 | -0.12 |
| 9.29 | 9.19 | -0.10 |
| 9.39 | 9.29 | -0.10 |
| a n=21 b n=42 | | |
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 11: Serum Specimen 1:3 Dilution Matrix Comparison Summary
| Serum Matrix
Average Reported
Concentration
(log_{10} IU/mL) n = 9 | Diluent
Average Reported
concentration
(log_{10} IU/mL) n = 18 | Difference
of Diluent from
Plasma Matrix
(log IU_{10}/mL) |
| --- | --- | --- |
| 1.21^{a} | 1.11^{b} | -0.10 |
| 1.54^{a} | 1.36^{b} | -0.18 |
| 2.21 | 2.03 | -0.18 |
| 3.06 | 2.98 | -0.08 |
| 3.90 | 3.83 | -0.07 |
| 4.77 | 4.76 | -0.01 |
| 5.77 | 5.74 | -0.03 |
| 7.03 | 7.00 | -0.03 |
| 7.85 | 7.71 | -0.14 |
| 8.87 | 8.76 | -0.11 |
| 9.37 | 9.30 | -0.07 |
| 9.46 | 9.36 | -0.10 |
| a n=21 b n=42 | | |
Sample Dilution Using Aptima Specimen Diluent (1:100): To assess the quantitation accuracy of HBV DNA in samples diluted with Aptima Specimen Diluent, plasma or serum, eight individual plasma specimens and eight individual serum specimens spiked with HBV virus targeting between 6 to 8 log $_{10}$ IU/mL, along with eight individual plasma specimens and eight individual serum specimens spiked with HBV plasmid DNA targeting 9.16 log $_{10}$ IU/mL, were tested in 5 replicates. A 1:100 dilution was performed with one part sample and 99 parts Aptima Specimen Diluent just prior to testing. Testing was performed using one lot of assay reagents on two Panther systems with two Aptima Specimen Diluent lots. The difference between the average reported concentration in native matrix (dilution factor applied to the diluted sample result) and the average concentration in Aptima Specimen Diluent was calculated for each sample set as shown in Table 12 for plasma and Table 13 for serum.
Table 12: Plasma Specimen 1:100 Dilution Matrix Comparison Summary
| Plasma Matrix
Average Reported
Concentration
(log_{10} IU/mL) n = 5 | Diluent
Average Reported
Concentration
(log_{10} IU/mL) n = 10 | Difference
of Diluent from
Plasma Matrix
(log IU_{10}/mL) |
| --- | --- | --- |
| 7.86 | 7.85 | -0.01 |
| 7.84 | 7.83 | -0.01 |
| 7.78 | 7.75 | -0.03 |
| 7.80 | 7.80 | 0.00 |
| 6.58 | 6.53 | -0.05 |
| 6.58 | 6.52 | -0.06 |
| 6.58 | 6.53 | -0.05 |
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 13: Serum Specimen 1:100 Dilution Matrix Comparison Study
| Serum Matrix Average Reported Concentration (log10 IU/mL) n = 5 | Diluent Average Reported concentration (log10 IU/mL) n = 10 | Difference of Diluent from Plasma Matrix (log IU10/mL) |
| --- | --- | --- |
| 7.70 | 7.85 | 0.15 |
| 7.84 | 7.85 | 0.01 |
| 7.79 | 7.82 | 0.03 |
| 7.75 | 7.79 | 0.04 |
| 6.77 | 6.77 | 0.00 |
| 6.75 | 6.80 | 0.05 |
| 6.75 | 6.71 | -0.04 |
| 6.70 | 6.73 | 0.03 |
| 9.27a | 9.08a | -0.19 |
| 9.24a | 9.06a | -0.18 |
| 9.29a | 9.08a | -0.21 |
| 9.31a | 9.11a | -0.20 |
| 9.14a | 8.91a | -0.23 |
| 9.18a | 8.92a | -0.26 |
| 9.19a | 8.90a | -0.29 |
| 9.08a | 8.84a | -0.24 |
| aSpiked using plasmid DNA | | |
# Confirmation of the LLoQ in Specimens Diluted in Aptima Specimen Diluent
The LLoQ of the Aptima HBV Quant assay was confirmed with HBV genotype A clinical specimens diluted into Aptima Specimen Diluent. Specimens were prepared in HBV negative human plasma and serum at 21, 30, and $45\mathrm{IU / mL}$ . Each panel was diluted 1:3 into Aptima Specimen Diluent just prior to testing to give final concentrations of approximately 7, 10, and $15\mathrm{IU / mL}$ . Thirty-six (36) replicates of each panel member were tested with one reagent lot across three days. An LLoQ $\leq 10$ IU/mL for HBV plasma and serum diluted into Aptima Specimen Diluent was confirmed as shown in Table 14.
Table 14: Confirmation of LLoQ - Samples in Aptima Specimen Diluent
PMA P170025: FDA Summary of Safety and Effectiveness Data
| Matrix | % Detected | Aptima HBV Quant | Aptima HBV Quant | SD | Bias | Calculated TE | Calculated TAE |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | (IU/mL) | (log10 IU/mL) | (log10 IU/mL) | (log10 IU/mL) | (log10 IU/mL) | (log10 IU/mL) |
| Plasma | 100% | 3 | 0.50 | 0.19 | 0.10 | 0.54 | 0.48 |
| Serum | 100% | 2 | 0.38 | 0.12 | 0.46 | 0.33 | 0.70 |
Precision of Diluted Samples: The Aptima HBV Quant precision panel was built by diluting HBV-positive plasma and HBV plasmid DNA into HBV-negative clinical plasma and serum. Positive panels were diluted into Aptima Specimen Diluent. These were tested in five replicates per run by one operator, using three lots of Aptima Specimen Diluent on one Panther system over three test days, two runs a day. Table 15 shows the precision of assay results (in SD $\log_{10}$ IU/mL) for three lots of Aptima Specimen Diluent. Total variability was $\leq 0.15$ SD across all panel members and Diluent lots.
Table 15: Precision of Panels Diluted in Aptima Specimen Diluent
| Matrix | Target Concentration log10 IU/mL | Dilution | Lot 1 Specimen Diluent (n=10) | | Lot 2 Specimen Diluent (n=10) | | Lot 3 Specimen Diluent (n=10) | | Combined Lots (n=30) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Avg. log10 IU/mL | SD | Avg. log10 IU/mL | SD | Avg. log10 IU/mL | SD | Avg log10 IU/mL | SD |
| Plasma | 3.30 | Neat | 3.46 | 0.07 | 3.43 | 0.08 | 3.46 | 0.06 | 3.45 | 0.07 |
| | | 1:3 | 3.36 | 0.09 | 3.35 | 0.07 | 3.39 | 0.09 | 3.37 | 0.08 |
| | 4.30 | Neat | 4.33 | 0.06 | 4.27 | 0.03 | 4.41 | 0.05 | 4.34 | 0.08 |
| | | 1:3 | 4.34 | 0.05 | 4.35 | 0.05 | 4.38 | 0.10 | 4.35 | 0.07 |
| | 9.18 | Neat | 9.13 | 0.05 | 9.10 | 0.03 | 9.26a | 0.15 | 9.16a | 0.11 |
| | | 1:100 | 9.18 | 0.03 | 9.14 | 0.04 | 9.33 | 0.10 | 9.21 | 0.10 |
| Serum | 3.30 | Neat | 3.52 | 0.05 | 3.48 | 0.06 | 3.50 | 0.07 | 3.50 | 0.06 |
| | | 1:3 | 3.45 | 0.08 | 3.40 | 0.06 | 3.39 | 0.08 | 3.41 | 0.07 |
| | 4.30 | Neat | 4.35 | 0.05 | 4.37 | 0.06 | 4.43 | 0.06 | 4.38 | 0.06 |
| | | 1:3 | 4.35 | 0.05 | 4.37 | 0.05 | 4.41 | 0.04 | 4.37 | 0.05 |
| | 9.18 | Neat | 9.08 | 0.03 | 9.14 | 0.05 | 9.31b | 0.12 | 9.17b | 0.12 |
| SD = standard deviation.; a1 replicate excluded (above calculable range). b2 replicates excluded (above calculable range). | | | | | | | | | | |
Carryover: To establish that the Panther system minimizes the risk of false positive results arising from carryover contamination, a study was conducted using spiked panels on three Panther systems. Carryover was assessed using high titer HBV DNA spiked plasma samples (8 log IU/mL) interspersed between HBV negative samples in a checkerboard pattern. Testing was carried out over fifteen runs. The overall carryover rate was $0.14\%$ (1/705).
Specimen Stability: Specimen stability studies demonstrated that, for the Aptima HBV Quant assay, specimens should be stored as follows:
PMA P170025: FDA Summary of Safety and Effectiveness Data
Whole blood can be stored at $2^{\mathrm{0}}\mathrm{C}$ to $30^{\mathrm{0}}\mathrm{C}$ and must be centrifuged within 24 hours of specimen collection. Plasma or serum may then be stored under one of the following conditions:
- In the primary collection tube or specimen aliquot tube (SAT) at $2^{\mathrm{O}}\mathrm{C}$ to $30^{\mathrm{O}}\mathrm{C}$ for up to 24 hours.
- In the primary collection tube or SAT at $2^{\mathrm{O}}\mathrm{C}$ to $8^{\mathrm{O}}\mathrm{C}$ for up to 5 days, or
- In the SAT at $-20^{\circ}\mathrm{C}$ for up to 60 days.
Real-Time Reagent (including Controls) Stability: Expiration dating for the Aptima HBV Quant assay has been established and approved as shown in Table 16:
Table 16: Real-Time Reagent Stability
| Kit Description | Shelf Life |
| --- | --- |
| Aptima HBV Quant assay Kit | 22 months at 2°C to 8°C |
| Aptima HBV Quant Target Enhancer Reagent Kit | 22 months at 15°C to 30°C |
| Aptima HBV Quant Calibrator Kit | 24 months at -15°C to -35°C |
| Aptima HBV Quant Controls Kit | 24 months at -15°C to -35°C |
Antimicrobial Effectiveness (AET): Testing was performed and approved for the Aptima HIV-1 Quant Assay. The Aptima HBV Quant assay uses similar base formulations with minor differences in concentrations. Because the formulations are so similar and the preservatives used are identical, the AET established for the Aptima HIV-1 Quant assay verifies the preservative effectiveness for the Aptima HBV Quant assay. Results of the study were compared to the requirements of USP51. All reagents met the USP requirements for AET. Unlike Aptima HIV-1 Quant Assay, Aptima HBV Quant assay also contains Target Enhancer Reagent which is a concentrated solution of lithium hydroxide. No microbial control is needed since this reagent is caustic and self-preserving.
PMA P170025: FDA Summary of Safety and Effectiveness Data
PMA P170025: FDA Summary of Safety and Effectiveness Data
Page 19
# X. SUMMARY OF PRIMARY CLINICAL STUDY(IES)
## A. Study Design
The study was designed to assess the ability of the Aptima HBV Quant assay to predict virologic, biochemical, and serologic responses to HBV treatment at clinically relevant time points during 12-, 24-, or 48-week antiviral treatment.
### 1. Clinical Inclusion and Exclusion Criteria
Enrollment in the study was limited to patients who met the following inclusion criteria:
- The subject is chronically infected with HBV (historical record of persistence of HBsAg for greater than 6 months).
- The subject is initiating HBV antiviral therapy with either entecavir or tenofovir as indicated in the US FDA approved label (0.5 mg once daily of entecavir or 300 mg once daily for tenofovir), and has been treated for less than 12 weeks (±7 days).
- Treatment-naïve subjects and certain treatment-experienced subjects are eligible.
- Treatment-experienced subjects who discontinued a previous nucleotide/nucleoside treatment regimen (eg, tenofovir, entecavir, adefovir, lamivudine, telbivudine) due to reasons other than treatment failure or resistance (ie, breakthrough, rebound, or non-response) or treatment success (eg, seroconversion) are eligible to participate (eg, discontinued due to pregnancy, cost). Previous treatment must have ended ≥6 months before the initiation of current treatment with entecavir or tenofovir.
- For subjects who have taken their first dose of current entecavir or tenofovir treatment, a standard of care HBV quantitative assay result is available for a sample that was collected ≤180 days before the start of therapy.
- The subject is at least 18 years of age at the time of enrollment
- Adequate medical records are available for collection of protocol-defined demographics, baseline patient characteristics, medical history, virology, and specific laboratory results, and other information to verify enrollment criteria
- The subject and/or legally authorized representative is willing and able to provide consent prior to providing a specimen(s)
Patients were not permitted to enroll in the study if they met any of the following exclusion criteria:
- Treatment experienced subjects who discontinued an HBV treatment regimen due to treatment failure or resistance (ie breakthrough, rebound, nonresponse) or treatment success (eg., seroconversion)
- Subject is in one of the following patient populations:
PMA P170025: FDA Summary of Safety and Effectiveness Data
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- Acute HBV infection
- Patients who are HBV immune tolerant
- Human immunodeficiency virus (HIV) and/or hepatitis c virus (HCV) co-infection
- Solid organ or bone marrow transplant recipients
- Renal failure or dialysis
- Evidence or history of hepatic decompensation
- Evidence of history of hepatocellular carcinoma
- Underlying liver disease other than HBV
- Receiving chemotherapy, immunosuppressive agents
- Subject is unsuitable for study participation based on the Investigator’s decision (eg., unlikely to comply with study visit schedule, significant medical complications)
- Participating in another investigational study that the Investigator believes might interfere with the subject’s participation in this study.
2. Response to Antiviral Therapy Definitions
Aptima HBV Quant assay clinical utility was assessed for individuals treated with tenofovir and entecavir. No information is available on the assay's clinical utility when other HBV antiviral therapies are used.
Definitions:
Early virologic response outcomes
- Week 12 and Week 24 virologic response = HBV DNA <10 IU/mL (<LLoQ) as assessed by the Aptima HBV Quant assay on the Panther system
- Week 12 alternative virologic response = HBV DNA ≥2 log10 decrease from baseline Week 24 alternative virologic response = HBV DNA <2000 IU/mL (for HBeAg+) or <50 IU/mL (for HBeAg-)
Clinical utility endpoints
- Week 48 virologic response = HBV DNA <10 IU/mL (<LLoQ) as assessed by an FDA-approved HBV quantitative assay
- Alternative Week 48 virologic response = HBV DNA <50 IU/mL as assessed by an FDA-approved HBV quantitative assay
- Biochemical response = Normalization of ALT test results at Week 48 (ALT <30 U/L for males and <19 U/L for females)
- Serologic response = Loss of HBeAg (HBeAg-negative results) at Week 48
Measures of association and predictive value
- Positive Predictive Value (PPV) = True Positive / (True Positive + False Positive) or the probability of response at Week 48 (for the clinical utility endpoint being assessed) in subjects with virologic response at the early time point
- Negative Predictive Value (NPV) = True Negative / (False Negative + True Negative) or the probability of non-response at Week 48 (for the clinical utility
endpoint being assessed) in subjects with virologic non-response at the early time point
- Odds Ratio (OR) = (True Positive × True Negative) / (False Positive × False Negative)
# 3. Demographics
The study enrolled 331 subjects from a total of 67 clinical sites. These were composed of 37 U.S. and 30 outside of U.S. clinical sites (Australia, Canada, Germany, Hungary, New Zealand, Romania, and Turkey). Of the 331 enrolled subjects, 86 subjects were not evaluable due to withdrawal, discontinuation, early treatment halt, missing Week 48 results, or missing baseline HBV DNA viral load, or low baseline HBV DNA viral load. The remaining 245 subjects were evaluable for at least one of the clinical utility endpoints and included 126 HBeAg positive and 119 HBeAg negative subjects (Table 17).
Table 17: Demographics and Baseline Clinical Characteristics of Evaluable Subjects
| Characteristics | | Total |
| --- | --- | --- |
| Total, N | N | 245 |
| Entecavir | n (%) | 94 (38.4) |
| Tenofovir | n (%) | 151 (61.6) |
| Sex, n (%) | Male | 154 (62.9) |
| | Female | 91 (37.1) |
| Age (years) | Mean ± SD | 43.5 ± 13.63 |
| | Median | 44.0 |
| | Range | 18 – 83 |
| Age category (years), n (%) | 18–29 | 40 (16.3) |
| | 30-49, n (%) | 120 (49.0) |
| | 50-70, n (%) | 80 (32.7) |
| | >70, n (%) | 5 (2.0) |
| Ethnicity, n (%) | Hispanic or Latino | 7 (2.9) |
| | Not Hispanic or Latino | 236 (96.3) |
| | Unknown/Refused | 2 (0.8) |
| Racea, n, (%) | White | 89 (36.3) |
| | Black or African American | 16 (6.5) |
| | Asian | 132 (53.9) |
| | American Indian/Alaska | 0 (0.0) |
| | Native Hawaiian/Pacific | 6 (2.4) |
| | Other | 1 (0.4) |
| | Unknown/Refused | 1 (0.4) |
| Genotype, n (%) | A | 28 (11.4) |
| | B | 64 (26.1) |
| | C | 36 (14.7) |
| | D | 47 (19.2) |
| | E | 2 (0.8) |
| | F | 0 (0.0) |
| | G | 0 (0.0) |
| | H | 3 (1.2) |
| | Unknown | 65 (26.5) |
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| Characteristics | | Total |
| --- | --- | --- |
| HBV treatment status, n (%) | Experienced | 27 (11.0) |
| | Naïve | 218 (89.0) |
| Previous drug treatment, n (%) | Tenofovir | 5 (18.5) |
| | Entecavir | 4 (14.8) |
| | Adefovir | 2 (7.4) |
| | Lamivudine | 1 (3.7) |
| | Telbivudine | 0 (0.0) |
| | Interferon | 10 (37.0) |
| | Other^{b} | 5 (18.5) |
| Previous treatment outcome, n (%) | Failure | 1 (3.7) |
| | Success | 0 (0.0) |
| | Discontinued for other reasons | 26 (96.3) |
| HBsAg serostatus, n (%) | Positive/reactive | 210 (85.7) |
| | Not Tested | 35 (14.3) |
| Cirrhotic status, n (%) | Cirrhotic | 26 (10.6) |
| | Non-cirrhotic | 201 (82.0) |
| | Not Tested | 18 (7.3) |
| HBV viral load (log10 IU/mL), n | Mean ± SD | 6.3 ± 1.93 |
| | Median | 6.4 |
| | Range | 3 – 9 |
| ALT (U/L) | Mean ± SD | 102.4 ± 175.97 |
| | Number above ULN^{c}, n (%) | 177 (85.9) |
| ^{a}Subjects may report multiple races
^{b}Various combinations of the specific drugs listed
^{c}The upper limit of normal range (ULN) for alanine aminotransferase (ALT) was 30 U/L for males and 19 U/L for females | | |
## 4. Results
Predicting Week 48 Virologic Response, Defined as HBV DNA < 10 IU/mL
The primary definition of virologic response being HBV DNA < 10 IU/mL was used for both early virologic response at Weeks 12 and 24, as well as the virologic response at Week 48. The association between early virologic responses at Weeks 12 and 24 and Week 48 clinical utility endpoints (virologic response, biochemical response, and serologic response) were assessed.
Predicting Virologic Response at Week 48
Associations between virologic response at Week 48 and virologic response at Week 12 and Week 24 are summarized in Table 18.
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 18: PPV, NPV, and Odds Ratio for Virologic Response Predicated by Early Virologic Response During Treatment: Week 48 Virologic Response Defined as <10 IU/mL
| HBeAg Status | Week of Early Virologic Response | Treatment | PPV (%) | | NPV (%) | | OR |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Estimate (95% CI) | n/N | Estimate (95% CI) | n/N | Estimate (95% CI)a,b |
| HBeAg(+) | 12 | Entecavir | 0.0 (0.0, 93.2) | 0/1 | 82.5 (80.0, 88.4) | 33/40 | 1.49 (<0.01, 30.95) |
| | | Tenofovir | 100 (27.3, 100) | 2/2 | 74.4 (72.6, 78.7) | 61/82 | 14.30 (1.11, >999.99) |
| | | All | 66.7 (15.4, 98.2) | 2/3 | 77.0 (75.7, 79.9) | 94/122 | 6.71 (0.62, 147.55) |
| | 24 | Entecavir | 50.0 (6.4, 93.2) | 2/4 | 88.2 (83.5, 95.4) | 30/34 | 7.50 (0.74, 79.76) |
| | | Tenofovir | 75.0 (52.7, 92.3) | 12/16 | 84.1 (78.5, 90.0) | 58/69 | 15.81 (4.62, 65.54) |
| | | All | 70.0 (50.3, 88.1) | 14/20 | 85.4 (81.3, 90.1) | 88/103 | 13.69 (4.74, 44.16) |
| HBeAg(-) | 12 | Entecavir | 94.1 (87.1, 99.0) | 32/34 | 22.2 (7.6, 36.0) | 4/18 | 4.57 (0.80, 35.85) |
| | | Tenofovir | 83.3 (70.5, 93.2) | 25/30 | 46.9 (35.0, 58.9) | 15/32 | 4.41 (1.42, 15.71) |
| | | All | 89.1 (82.1, 94.7) | 57/64 | 38.0 (29.0, 47.1) | 19/50 | 4.99 (1.96, 14.00) |
| | 24 | Entecavir | 93.0 (88.0, 98.1) | 40/43 | 37.5 (6.4, 67.2) | 3/8 | 8.00 (1.21, 55.54) |
| | | Tenofovir | 82.6 (74.3, 90.4) | 38/46 | 75.0 (54.1, 92.0) | 12/16 | 14.25 (3.92, 62.71) |
| | | All | 87.6 (82.7, 92.6) | 78/89 | 62.5 (44.8, 78.0) | 15/24 | 11.82 (4.30, 34.94) |
| CI=95% profile-likelihood confidence intervala Shading indicates statistical significance of odds ratios.b For calculation of odds ratios and their confidence intervals, 0.5 was added to all cells whenever at least one cell was zero | | | | | | | |
Predicting Biochemical Response at Week 48
Associations between biochemical response at Week 48 and virologic response at Week 12 and Week 24 are summarized in Table 19.
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 19: PPV, NPV, and Odds Ratio for Biochemical Response Predicted by Early Virologic Response During Treatment: Week 48 Virologic Response Defined as <10 IU/mL
| HBeAg Status | Week of Early Virologic Response | Treatment | PPV (%) | | NPV (%) | | OR |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Estimate (95% CI) | n/N | Estimate (95% CI) | n/N | Estimate (95% CI)a |
| HBeAg(+) | 12 | Entecavir | NC (NC) | 0/0 | 67.9 (63.4, 76.1) | 19/28 | 2.05 (0.01, 393.52) |
| | | Tenofovir | 100 (27.6, 100) | 2/2 | 58.5 (55.1, 63.9) | 31/53 | 7.00 (0.54, 983.90) |
| | | All | 100 (27.3, 100) | 2/2 | 61.7 (59.7, 65.5) | 50/81 | 8.02 (0.63, >999.99) |
| | 24 | Entecavir | 66.7 (16.4, 98.2) | 2/3 | 68.2 (60.1, 80.0) | 15/22 | 4.29 (0.35, 101.82) |
| | | Tenofovir | 58.3 (33.2, 81.0) | 7/12 | 61.4 (54.3, 69.7) | 27/44 | 2.22 (0.61, 8.61) |
| | | All | 60.0 (36.6, 80.9) | 9/15 | 63.6 (58.2, 70.0) | 42/66 | 2.62 (0.84, 8.70) |
| HBeAg(-) | 12 | Entecavir | 43.8 (25.1, 61.2) | 7/16 | 50.0 (25.1, 74.9) | 6/12 | 0.78 (0.17, 3.53) |
| | | Tenofovir | 52.9 (35.1, 72.2) | 9/17 | 76.2 (61.2, 89.8) | 16/21 | 3.60 (0.93, 15.39) |
| | | All | 48.5 (36.0, 60.9) | 16/33 | 66.7 (54.5, 78.6) | 22/33 | 1.88 (0.70, 5.20) |
| | 24 | Entecavir | 45.8 (36.0, 56.0) | 11/24 | 75.0 (25.2, 98.7) | 3/4 | 2.54 (0.28, 55.45) |
| | | Tenofovir | 42.9 (32.8, 53.1) | 12/28 | 80.0 (53.0, 97.1) | 8/10 | 3.00 (0.61, 22.34) |
| | | All | 44.2 (37.8, 50.8) | 23/52 | 78.6 (55.1, 95.0) | 11/14 | 2.91 (0.80, 13.98) |
| CI=95% profile-likelihood confidence interval, NC=not calculable
a For calculation of odds ratios and their confidence intervals, 0.5 was added to all cells whenever at least one cell was zero, unless there were either no Week 48 responses or no Week 48 non-responses which resulted in reporting the odds ratio as NC. | | | | | | | |
Predicting Serologic Response at Week 48
Associations between serologic response at Week 48 and virologic response at Week 12 and Week 24 are summarized in Table 20. The value of early virologic responses at Weeks 12 and 24 as a predictor of Week 48 serologic response varied by week and treatment.
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 20: PPV, NPV, and Odds Ratio for Serologic Response PRedicted by Early Virologic Response During Treatment: Week 48 Virologic Response Defined as $< {10}\mathrm{{IU}}/\mathrm{{mL}}$
| | | | PPV (%) | | NPV (%) | | OR |
| --- | --- | --- | --- | --- | --- | --- | --- |
| HBeAg Status | Week of Early Virologic Response | Treatment | Estimate (95% CI) | n/N | Estimate (95% CI) | n/N | Estimate (95% CI)a |
| HBeAg(+) | 12 | Entecavir | 100 (6.5, 100) | 1/1 | 86.8 (84.2, 93.9) | 33/38 | 18.27 (0.86, >999.99) |
| | | Tenofovir | 0.0 (0.0, 72.0) | 0/2 | 82.9 (81.7, 86.0) | 68/82 | 0.95 (<0.01, 12.45) |
| | | All | 33.3 (1.8, 84.3) | 1/3 | 84.2 (83.1, 86.8) | 101/120 | 2.66 (0.12, 29.11) |
| | 24 | Entecavir | 50.0 (6.4, 93.2) | 2/4 | 88.2 (83.5, 95.4) | 30/34 | 7.50 (0.74, 79.76) |
| | | Tenofovir | 18.8 (3.1, 39.1) | 3/16 | 84.1 (80.6, 89.1) | 58/69 | 1.22 (0.25, 4.59) |
| | | All | 25.0 (8.5, 43.6) | 5/20 | 85.4 (82.5, 89.4) | 88/103 | 1.96 (0.57, 5.94) |
| CI=95% profile-likelihood confidence intervala For calculation of odds ratios and their confidence intervals, 0.5 was added to all cells whenever at least one cell was zero. | | | | | | | |
Predicting Week 48 Virologic Response, Defined as HBV DNA $< 50$ IU/mL (Alternative Definition)
Alternative definitions of early (Weeks 12 and 24) and Week 48 virologic responses also were assessed (see section X.A.2 Response to Antiviral Therapy Definitions). Associations between clinical utility endpoints and virologic response at Week 12 and Week 24, using these alternate definitions of virologic response are summarized in Table 21 (virologic response), Table 22 (biochemical response), and Table 23 (serologic response).
Table 21: PPV, NPV, and Odds Ratio for Virologic Response Predicted by Early Virologic Response during Treatment: Week 48 Virologic Response Defined as $< {50}\mathrm{{IU}}/\mathrm{{mL}}$
| HBeAg Status | Week of Early Virologic Response | Treatment | PPV (%) | | NPV (%) | | OR |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Estimate (95% CI) | n/N | Estimate (95% CI) | n/N | Estimate (95% CI)a,b |
| HBeAg(+) | 12 | Entecavir | 34.2 (26.9, 39.2) | 13/38 | 66.7 (16.1, 98.2) | 2/3 | 1.04 (0.09, 23.60) |
| | | Tenofovir | 55.1 (51.9, 59.4) | 43/78 | 83.3 (43.5, 99.2) | 5/6 | 6.14 (0.93, 120.54) |
| | | All | 48.3 (45.6, 51.3) | 56/116 | 77.8 (45.3, 97.1) | 7/9 | 3.27 (0.75, 22.54) |
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 22: PPV, NPV, and Odds Ratio for Biochemical Response Predicted by Early Virologic Response During Treatment: Week 48 Virologic Response Defined as <50 IU/mL
| | | | PPV (%) | | NPV (%) | | OR |
| --- | --- | --- | --- | --- | --- | --- | --- |
| HBeAg Status | Week of Early Virologic Response | Treatment | Estimate (95% CI) | n/N | Estimate (95% CI) | n/N | Estimate (95% CI)a,b |
| HBeAg(+) | 12 | Entecavir | 33.3 (25.1, 39.0) | 9/27 | 100 (6.7, 100) | 1/1 | 1.54 (0.07, 233.77) |
| | | Tenofovir | 44.2 (39.6, 48.6) | 23/52 | 66.7 (15.7, 98.2) | 2/3 | 1.59 (0.14, 35.36) |
| | | All | 40.5 (37.1, 43.7) | 32/79 | 75.0 (23.9, 98.7) | 3/4 | 2.04 (0.25, 42.30) |
| | 24 | Entecavir | 50.0 (31.2, 69.5) | 7/14 | 81.8 (58.9, 97.1) | 9/11 | 4.50 (0.79, 37.15) |
| | | Tenofovir | 52.5 (44.0, 61.8) | 21/40 | 81.3 (60.1, 96.7) | 13/16 | 4.79 (1.30, 23.28) |
| | | All | 51.9 (44.1, 60.2) | 28/54 | 81.5 (66.4, 93.1) | 22/27 | 4.74 (1.66, 15.82) |
| HBeAg(-) | 12 | Entecavir | 46.4 (39.5, 52.6) | 13/28 | NC (NC) | 0/0 | 0.87 (<0.01, 166.17) |
PMA P170025: FDA Summary of Safety and Effectiveness Data
Table 23: PPV, NPV, and Odds Ratio for Serologic Response Predicted by Early Virologic Response During Treatment: Week 48 Virologic Response Defined as $< {50}\mathrm{{IU}}/\mathrm{{mL}}$
| | | | PPV (%) | | NPV (%) | | OR |
| --- | --- | --- | --- | --- | --- | --- | --- |
| HBeAg Status | Week of Early Virologic Response | Treatment | Estimate (95% CI) | n/N | Estimate (95% CI) | n/N | Estimate (95% CI)a,b |
| HBeAg(+) | 12 | Entecavir | 16.7 (10.4, 19.6) | 6/36 | 100 (44.1, 100) | 3/3 | 1.49 (0.12, 209.89) |
| | | Tenofovir | 16.7 (12.9, 18.6) | 13/78 | 83.3 (44.2, 99.2) | 5/6 | 1.00 (0.14, 19.98) |
| | | All | 16.7 (13.9, 18.1) | 19/114 | 88.9 (58.8, 99.5) | 8/9 | 1.60 (0.27, 30.56) |
| | 24 | Entecavir | 30.0 (16.5, 41.6) | 6/20 | 100 (86.3, 100) | 18/18 | 16.59 (1.72, >999.99) |
| | | Tenofovir | 22.4 (16.9, 26.8) | 13/58 | 96.3 (84.7, 99.9) | 26/27 | 7.51 (1.37, 140.31) |
| | | All | 24.4 (20.0, 28.4) | 19/78 | 97.8 (90.4, 99.9) | 44/45 | 14.17 (2.77, 259.25) |
| CI=95% profile-likelihood confidence interval aShading indicates statistical significance of odds ratios. bFor calculation of odds ratios and their confidence intervals, 0.5 was added to all cells whenever at least one cell was zero. | | | | | | | |
# Conclusion
Overall, the results demonstrated that the Aptima HBV Quant assay can be used to assess HBV DNA viral load in subjects with chronic HBV infection at the start of and during antiviral treatment. This study demonstrated that HBV DNA levels measured at baseline, and decreases in HBV DNA levels after 12 or 24 varied by week and treatment. The Aptima HBV Quant assay can be used to quantitate HBV DNA levels to aid in the management of chronic HBV-infected patients undergoing HBV antiviral therapy.
PMA P170025: FDA Summary of Safety and Effectiveness Data
B. Reproducibility
Reproducibility was evaluated on the Panther system at three external U.S. sites. Two operators performed testing at each site. Each operator performed two runs per day over three days, using three reagent lots over the course of testing. Each run had three replicates of each panel member. Overall, 108 replicates of each panel member were tested. Reproducibility was tested using panel members prepared with HBV-negative plasma. The positive panel members were positive for HBV genotypes A or C. HBV DNA concentrations spanned the linear range of the assay. Table 24 shows the reproducibility and precision of assay results for each positive panel member between sites, between operators/days, between lots, between runs, within runs, and overall. The coefficient of variation was calculated using the following equation where $\sigma^2$ is the sample variance of the data after $\log_{10}$ transformation:
$$
\%CV = 100 \times \sqrt{(10^{\sigma^2 \ln(10)} - 1)}
$$
Table 24: Reproducibility of Aptima HBV Quant assay HBV DNA Levels on the Panther System in Positive Panel Members
| | | | | Between Sites | Between Operators/ Daysa | Between Lots | Between Runs | Within Runs | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| GT | N | IU/mL | Log10 IU/mL | SD (%CV) | SD (%CV) | SD (%CV) | SD (%CV) | SD (%CV) | SD (%CV) |
| A | 108 | 17.6 | 1.85 | 0.059 (13.578) | <0.001 (<0.001) | 0.138 (32.693) | 0.090 (20.869) | 0.178 (42.883) | 0.250 (62.666) |
| 108 | 129.4 | 2.092 | 0.009 (2.162) | 0 (0) | 0.074 (17.109) | 0.051 (11.869) | 0.106 (24.736) | 0.139 (32.886) |
| 107 | 1056.0 | 3.012 | 0.035 (7.994) | 0.032 (7.432) | 0.014 (3.246) | 0.032 (7.356) | 0.085 (19.666) | 0.103 (24.060) |
| 108 | 7663.0 | 3.875 | 0 (0) | 0.027 (6.262) | 0.040 (9.235) | 0.044 (10.088) | 0.066 (15.194) | 0.092 (21.540) |
| 108 | 188172.1 | 5.263 | 0.027 (6.281) | 0.042 (9.707) | 0.042 (9.787) | 0.030 (6.829) | 0.072 (16.689) | 0.102 (23.772) |
| 108 | 9389094.1 | 6.961 | 0.038 (8.846) | 0 (0) | 0.031 (7.237) | 0.064 (14.791) | 0.068 (15.756) | 0.106 (24.692) |
| 107 | 86664677.2 | 7.931 | 0.038 (8.692) | 0.029 (6.753) | 0.020 (4.584) | 0.037 (8.635) | 0.049 (11.375) | 0.081 (18.725) |
| 107 | 753726183.2 | 8.868 | 0.024 (5.476) | 0.052 (11.997) | 0.015 (3.499) | 0.045 (10.304) | 0.053 (12.187) | 0.091 (21.163) |
| C | 107 | 17.0 | 1.174 | 0.041 (9.521) | 0.041 (9.392) | 0.074 (17.147) | 0.092 (21.438) | 0.189 (45.704) | 0.230 (57.010) |
| 108 | 152.9 | 2.151 | 0.035 (8.127) | 0 (0) | 0.055 (12.706) | 0.064 (14.925) | 0.131 (30.748) | 0.160 (38.013) |
| 108 | 1363.8 | 3.125 | 0.042 (9.583) | 0.023 (5.316) | 0 (0) | 0.061 (14.033) | 0.055 (12.623) | 0.094 (22.002) |
| 108 | 9871.9 | 3.988 | 0.011 | 0.014 | 0.040 | 0.038 | 0.059 | 0.083 |
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| | | | | Between Sites | Between Operators/ Daysa | Between Lots | Between Runs | Within Runs | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| GT | N | IU/mL | Log10 IU/mL | SD (%CV) | SD (%CV) | SD (%CV) | SD (%CV) | SD (%CV) | SD (%CV) |
| | | | | (2.472) | (32.70) | (9.337) | (8.801) | (13.651) | (19.291) |
| | 108 | 217400.5 | 5.329 | 0.031 (7.255) | 0.047 (10.843) | 0.016 (3.791) | 0.026 (6.023) | 0.063 (14.685) | 0.090 (21.044) |
| | 108 | 12087179.5 | 7.069 | 0.046 (10.543) | 0 (0) | 0.02 (4.652) | 0.064 (14.762) | 0.073 (16.922) | 0.109 (25.501) |
| | 108 | 577434712.8 | 7.754 | 0.044 (10.232) | 0.028 (6.472) | 0.013 (2.944) | 0.043 (10.026) | 0.052 (12.010) | 0.087 (20.146) |
| | 108 | 572184754.9 | 8.749 | 0.042 (9.711) | 0.048 11.160 | 0.028 6.374 | 0.034 7.740 | 0.048 11.081 | 0.091 (21.208) |
| | %CV=log-normal coefficient of variation, GT=genotype, SD=standard deviation
Note: Variability from some factors may be numerically negative. This can occur if the variability due to those factors is very small. In these cases, SD and CV are shown as 0.
a Between Operators may be confounded with Between Days, therefore, Between Operators and Between Days estimates are combined in Between Operators/Days. | | | | | | | | |
## C. Safety and Effectiveness Results
1. **Safety Results**
There were no adverse effects of the device reported while the study was conducted.
2. **Effectiveness Results**
The analysis of effectiveness of the Aptima HBV Quant assay was assessed by determining the ability of the test to measure HBV DNA levels at baseline, during treatment, and after treatment. See results in Section X. A and B, above. Overall the clinical studies demonstrate the effectiveness of the Aptima HBV Quant assay in accurately measuring HBV DNA levels in patients undergoing treatment.
3. **Subgroup Analyses**
Not Applicable.
4. **Pediatric Extrapolation**
In this premarket application, existing clinical data was not leveraged to support approval of a pediatric patient population.
## D. Financial Disclosure
The Financial Disclosure by Clinical Investigators regulation (21 CFR 54) requires applicants who submit a marketing application to include certain information concerning the compensation to, and financial interests and arrangement of, any clinical investigator conducting clinical studies covered by the regulation. The pivotal clinical study included 70 investigators. None of the clinical investigators had
PMA P170025: FDA Summary of Safety and Effectiveness Data
disclosable financial interests/arrangements as defined in sections 54.2(a), (b), (c), and (f). The information provided does not raise any questions about the reliability of the data.
## XI. PANEL MEETING RECOMMENDATION AND FDA'S POST-PANEL ACTION
In accordance with the provisions of section 515(c)(3) of the act as amended by the Safe Medical Devices Act of 1990, this PMA was not referred to the FDA Microbiology Devices Advisory Panel, an FDA advisory committee, for review and recommendation because the information in the PMA substantially duplicates information previously reviewed by this panel.
## XII. CONCLUSIONS DRAWN FROM PRECLINICAL AND CLINICAL STUDIES
### A. Effectiveness Conclusions
- The effectiveness of the Aptima HBV Quant assay has been demonstrated when used for the quantitation of HBV DNA in human plasma [EDTA, ACD, and PPTs] and serum and serum separation tubes (SST) for the management of patients undergoing treatment.
- There are no issues with endogenous interferents at physiological levels or with commonly administered medications.
- Whole blood can be stored at 2°C to 30°C and must be centrifuged within 24 hours of specimen collection. Plasma or serum may then be stored under one of the following conditions:
- In the primary collection tube or specimen aliquot tube (SAT) at 2°C to 25°C for up to 24 hours.
- In the primary collection tube or SAT at 2°C to 8°C for up to 5 days, or
- In the SAT at -20°C for up to 60 days.
- The preservatives that the Aptima HCV Quant Dx Assay reagents and controls contain have been shown to meet USP Chapter 51 criteria.
- The Aptima Quant Dx assay reagents can be stored at 2-8°C for 22 months.
- The clinical performance was evaluated in an ethnically diverse population representative of the intended use population (patients undergoing HBV therapy (see clinical performance studies, above).
### B. Safety Conclusions
Based on the results of the analytical and clinical laboratory studies, the Aptima HBV Quant assay, when used according to the provided directions and in conjunction with other laboratory results and clinical information, should be safe and pose minimal risk to the patient due to incorrect test results.
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C. Benefit-Risk Determination
The benefits outweigh the risks at the level of performance observed in the pivotal clinical study. Complementary analytical studies strengthen this conclusion. Accurate detection and quantitation of HBV DNA is an essential component in the management of chronic HBV infected patients undergoing antiviral therapy. Risks include inaccurate quantification. This risk is substantially mitigated by device design (i.e., use of controls), the likelihood of additional testing for incorrect results, and the overall clinical assessment.
1. Patient Perspectives
This submission did not include specific information on patient perspectives for this device.
In conclusion, given the available information above, the data support that for the Aptima HBV Quant assay as an aid in the management of chronic HBV infected patients undergoing antiviral therapy the probable benefits outweigh the probable risks.
D. Overall Conclusions
The data in this application support the reasonable assurance of safety and effectiveness of this device when used in accordance with the indications for use. The device accurately measures the viral load in the patient at baseline and at intervals during antiviral therapy. Therefore, this device should benefit the physician and the patients in the management of chronic HBV infected individuals undergoing antiviral therapy when used according to the directions for use in the labeling.
XIII. CDRH DECISION
CDRH issued an approval order on January 23, 2018.
The applicant’s manufacturing facilities have been inspected and found to be in compliance with the device Quality System (QS) regulation (21 CFR 820).
XIV. APPROVAL SPECIFICATIONS
Directions for use: See device labeling.
Hazards to Health from Use of the Device: See Indications, Contraindications, Warnings, Precautions, and Adverse Events in the device labeling.
Post-approval Requirements and Restrictions: See approval order.
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