ABBOTT REALTIME HBV ASSAY

P080026 · Abbott Molecular, Inc. · MKT · Aug 13, 2010 · Microbiology

Device Facts

• Record ID: P080026
• Device Name: ABBOTT REALTIME HBV ASSAY
• Applicant: Abbott Molecular, Inc.
• Product Code: MKT · Microbiology
• Decision Date: Aug 13, 2010
• Decision: APRL
• Regulation: 21 CFR 866.3174
• Device Class: Class 2

Indications for Use

Abbott RealTime HBV assay is an in vitro polymerase chain reaction (PCR) assay for use with the Abbott m2000 System DNA reagents and with the Abbott m2000sp and m2000rt instruments for the quantitation of Hepatitis B Virus (HBV) DNA in human serum or plasma (EDTA) from chronically HBV-infected individuals. The assay is intended for use as an aid in the management of patients with chronic HBV infection undergoing antiviral therapy. The assay can be used to measure HBV DNA levels at baseline and during treatment to aid in assessing response to treatment. The results from the Abbott RealTime HBV 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 adefovir dipivoxil. This assay is not intended for use as a screening test in blood or blood products for HBV or as a diagnostic test to confirm the presence of HBV infection.

Device Story

The Abbott RealTime HBV assay is an in vitro PCR-based diagnostic test for quantifying HBV DNA in human serum or EDTA plasma. It utilizes the Abbott m2000 System, comprising the m2000sp instrument for automated sample preparation (DNA extraction using magnetic microparticles and Proteinase K) and the m2000rt instrument for real-time PCR amplification and fluorescence detection. The assay targets a highly conserved region of the HBV Surface gene. An internal control (derived from Cucurbita pepo) is processed with each sample to monitor assay validity. Results are reported in IU/mL or log IU/mL on the m2000rt workstation. Clinicians use these quantitative viral load measurements at baseline and during antiviral therapy (e.g., adefovir dipivoxil) to assess treatment response and monitor for viral rebound. The device is intended for use in clinical laboratory settings by trained personnel.

Clinical Evidence

Clinical performance was evaluated in 413 chronic HBV-infected subjects (HBeAg-positive and HBeAg-negative) undergoing adefovir dipivoxil treatment. Viral load testing was performed at baseline and weeks 12, 24, 48, and up to 240 weeks. Results were correlated with histologic, biochemical, and serological responses. Analytical studies included linearity (10 IU/mL to 10^9 IU/mL), LoD (10-15 IU/mL depending on sample volume), precision (total SD ≤ 0.42 log IU/mL), and interference testing. Clinical data demonstrated that viral load response (defined as <2000 IU/mL or ≥2 log decrease) is informative for assessing treatment response.

Technological Characteristics

In vitro PCR assay using magnetic microparticle-based DNA extraction. Targets conserved Surface gene region of HBV genome. Real-time fluorescence detection using fluorophore-quencher probes. Sample volumes: 0.2 mL or 0.5 mL. Connectivity: Networked m2000 System (m2000sp/m2000rt). Software: Assay-specific application files loaded via CD-ROM. Reagents include DNA polymerase, oligonucleotide primers, and magnesium chloride. Standardized to WHO International Standard for HBV DNA.

Indications for Use

Indicated for quantitation of HBV DNA in human serum or EDTA plasma from chronically HBV-infected individuals undergoing antiviral therapy. Used to measure baseline and on-treatment HBV DNA levels to aid in assessing treatment response. Not for blood screening or diagnostic confirmation of 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

• Abbott RealTime HIV-1 (BP060002)
• Abbott RealTime CT/NG (k080739)
• Roche Cobas® TaqMan® HBV Test

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: In vitro polymerase chain reaction (PCR) based assay for HBV viral load detection.

Device Trade Name: Abbott RealTime HBV Assay, Abbott RealTime HBV Amplification Reagent Kit, Abbott RealTime HBV Calibrator Kit, Abbott RealTime HBV Control Kit.

Applicant’s Name and Address: Abbott Molecular Inc., 1300 E. Touhy Avenue, Des Plaines, IL 60018

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P080026

Date of FDA Notice of Approval: August 13, 2010

Expedited: Not applicable

II. INDICATIONS FOR USE

Abbott RealTime HBV assay is an in vitro polymerase chain reaction (PCR) assay for use with the Abbott m2000 System DNA reagents and with the Abbott m2000sp and m2000rt instruments for the quantitation of Hepatitis B Virus (HBV) DNA in human serum or plasma (EDTA) from chronically HBV-infected individuals. The assay is intended for use as an aid in the management of patients with chronic HBV infection undergoing antiviral therapy. The assay can be used to measure HBV DNA levels at baseline and during treatment to aid in assessing response to treatment. The results from the Abbott RealTime HBV 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 adefovir dipivoxil. This assay is not intended for use as a screening test in blood or blood products for HBV or as a diagnostic test to confirm the presence of HBV infection.

III. CONTRAINDICATIONS

None known

IV. WARNINGS AND PRECAUTIONS

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For in vitro diagnostic use only

The warnings and precautions for the Abbott RealTime HBV assay are stated in the respective product labeling.

## V. DEVICE DESCRIPTION

The Abbott RealTime HBV assay is an in vitro polymerase chain reaction (PCR) assay for the quantitation of HBV DNA in human plasma (EDTA) or serum from HBV-infected individuals. The Abbott RealTime HBV assay uses PCR to generate amplified product from the DNA genome of HBV in clinical specimens. The Abbott RealTime HBV assay uses the Abbott m2000sp instrument for processing samples and the Abbott m2000rt instrument for amplification and detection. The m2000sp and m2000rt instruments, as a part of the m2000 System, were approved in the Abbott RealTime HIV-1 PMA BP060002, May 11, 2007, and subsequently cleared in the Abbott RealTime CT/NG 510(k) k080739 on 7/10/2008.

The Abbott m2000sp uses the Abbott mSample Preparation System$_{DNA}$ reagents to extract DNA for amplification and detection. The mSample Preparation reagents include lysis reagent, magnetic particle reagent for capturing nucleic acids, wash, and elution reagents. Proteinase K is included in the lysis step to digest proteins associated with the nucleic acids. The Abbott m2000sp provides automated sample eluate transfer to an Abbott 96-Deep Well Plate, and reaction assembly in the Abbott 96-Well Optical Reaction Plate. The 96-Well Optical Reaction Plate is manually sealed and transferred to the m2000rt to perform the amplification and real-time fluorescence detection reaction. Results are automatically reported on the m2000rt workstation. An internal control nucleic acid is introduced into each sample during the sample preparation process and measured on the m2000rt to demonstrate that the process was completed correctly for each sample and control. The Abbott RealTime HBV assay contains sufficient reagents to process approximately 96 tests. A total of 48 samples can be processed in each run. A negative control, a low positive control, and a high positive control must be included in each run, therefore allowing a maximum of 45 specimens to be processed in each run.

Application parameters specific to the Abbott RealTime HBV assay are contained on an assay specific application file, housed on a CD-ROM and loaded onto the m2000sp and m2000rt instruments.

The Abbott RealTime HBV assay consists of three kits:

- Abbott RealTime HBV Amplification Reagent Kit (List No. 2N40-90)
- Abbott RealTime HBV Control Kit (List No. 2N40-80)
- Abbott RealTime HBV Calibrator Kit (List No. 2N40-70)

## Sample Preparation

The purpose of sample preparation is to extract and concentrate nucleic acid, to make the target accessible for amplification, and to remove potential inhibitors of amplification from the extract. This process is accomplished by the m2000sp, an automated sample preparation system designed to use magnetic microparticle processes for the purification of nucleic acids from samples.

The Abbott mSample Preparation System$_{DNA}$ reagents lyse the virion, capture the nucleic acids and wash the particles to remove unbound sample components. Proteinase K is

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included in the lysis step to digest proteins associated with the nucleic acids. The bound nucleic acids are eluted and transferred to a 96-deep well plate. The nucleic acids are then ready for amplification. The Internal Control (IC) is introduced into the Sample Preparation Protocol and is processed along with the calibrators, controls, and specimens.

## Reagent Preparation and Reaction Plate Assembly

The Abbott m2000sp combines the Abbott RealTime HBV amplification reagent components (HBV Oligonucleotide Reagent, DNA Polymerase, and Activation Reagent). The Abbott m2000sp dispenses the resulting master mix to the Abbott 96-Well Optical Reaction Plate along with aliquots of the nucleic acid samples prepared by the Abbott m2000sp. After manual application of the Abbott Optical Adhesive Cover, the plate is ready for transfer to the Abbott m2000rt.

## Amplification

During the amplification/detection reaction on the m2000rt instrument, the target DNA is amplified by the DNA polymerase in the presence of deoxynucleotide triphosphates (dNTPs) and magnesium. First, the HBV and Internal Control primers anneal to their respective targets and are extended by the polymerase. After a denaturation step in which the temperature of the reaction is raised above the melting point of the double-stranded DNA product, the newly created DNA strand is denatured from the target DNA.

During each round of thermal cycling, amplification products dissociate to single strands at high temperature allowing primer annealing and extension as the temperature is lowered. Exponential amplification of the target is achieved through repeated cycling between high and lower temperatures. Amplification of both targets (HBV and IC) takes place simultaneously in the same reaction.

The target sequence for the Abbott RealTime HBV assay is in the Surface gene in the HBV genome. This region is specific for HBV and is highly conserved. The primers are designed to hybridize to this region with the fewest possible mismatches among HBV genotypes A through H.

The target sequence for the Internal Control is derived from the hydroxypyruvate reductase gene from the pumpkin plant *Cucurbita pepo*, and is provided as a DNA plasmid in a buffered solution.

## Detection

The presence of HBV amplification products is detected during the extension/anneal step by measuring the fluorescence of the HBV probe that binds to the target during the extension/anneal step. Similarly, the presence of IC amplification is detected during the extension/anneal step by measuring the fluorescence of the IC probe.

The HBV and IC probes are single-stranded DNA oligonucleotides consisting of a probe sequence with a fluorescent moiety that is covalently linked to the 5′ end of the probe and a quenching moiety that is covalently linked to the 3′ end of the probe.

In the absence of the HBV or IC target sequences, probe fluorescence is quenched. In the presence of the HBV or IC target, the HBV or IC probes specifically bind to their target.

During the extension/anneal step, the DNA polymerase cleaves, or nucleolytically digests, the bound probe as the polymerase moves along the template strand. This

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separates the fluorophore from the quencher, allowing fluorescent emission and detection.

The HBV and IC specific probes are each labeled with a different fluorophore, thus allowing for simultaneous detection of both amplified products at each cycle. The amplification cycle at which fluorescent signal is detected by the Abbott m2000rt is inversely related to the log of the HBV DNA concentration present in the original sample.

## Abbott RealTime HBV Amplification Reagent Kit Description

The Abbott RealTime HBV Amplification Reagent Kit consists of:

- Abbott RealTime HBV Internal Control (List No. 2G34Y)
- Abbott RealTime HBV Amplification Reagent Pack (List No. 2N40), which contains:
- DNA Polymerase (Part 33794)
- HBV Oligonucleotide Reagent (List No. 2G34L)
- Activation Reagent (Part 51-503200)

The Abbott RealTime HBV Amplification Reagent Kit contains four vials of Abbott RealTime HBV Internal Control and four Abbott RealTime HBV Amplification Reagent Packs.

## Abbott RealTime HBV Internal Control (List No. 2G34Y)

The Abbott RealTime HBV Internal Control consists of noninfectious linearized DNA plasmid in a buffer solution with carrier DNA and contains the preservatives sodium azide and ProClin® 950.

Prior to sample preparation, the Internal Control is introduced into the lysis buffer, which is then used during the processing of each specimen, calibrator and control and measured on the m2000rt instrument to demonstrate proper sample processing and assay validity.

## Abbott RealTime HBV Amplification Reagent Pack (List No. 2N40)

The Abbott RealTime HBV Amplification Reagent Pack consists of the DNA polymerase the HBV Oligonucleotide Reagent, and the Activation Reagent.

### DNA Polymerase (Part No. 33794)

Each vial contains DNA polymerase in a buffered solution with stabilizers. The DNA polymerase functions as an enzyme in PCR amplification.

### HBV Oligonucleotide Reagent (List No. 2G34L)

Each vial of HBV Oligonucleotide Reagent contains two pairs of oligonucleotide primers and three probes; one primer pair and two probes are specific for amplifying and detecting HBV DNA, and the other primer pair and remaining probe are specific for amplifying and detecting Internal Control DNA. The reagent also contains dNTPs and ROX™ passive reference dye. The reagent is formulated in a TRIS-potassium chloride buffer with the preservatives sodium azide and ProClin 950.

### Activation Reagent (Part No. 51-503200)

Each vial of Activation Reagent contains a 38 mM magnesium chloride solution and the preservatives sodium azide and ProClin 950.

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# Abbott RealTime HBV Control Kit (List No. 2N40-80)

The Abbott RealTime HBV Control Kit consists of:

- Abbott RealTime HBV Negative Control (List No. 2G34Z)
- Abbott RealTime HBV Low Positive Control (List No. 2G34W)
- Abbott RealTime HBV High Positive Control (List No. 2G34X)

The Abbott RealTime HBV Control Kit contains three controls (eight vials of Abbott RealTime HBV Negative Control, eight vials of Abbott RealTime HBV Low Positive Control, and eight vials of Abbott RealTime HBV High Positive Control) that are used to establish the run validity of the Abbott RealTime HBV assay.

The Abbott RealTime HBV Negative Control contains negative human plasma that is tested and found to be nonreactive by FDA licensed tests for antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material is also tested and found to be negative by FDA licensed PCR methods for HIV-1 RNA and HCV RNA, and contains the antimicrobials ProClin 300 and ProClin 950.

The Abbott RealTime HBV Low Positive Control and High Positive Control contain heat-inactivated plasma reactive for HBV DNA in negative human plasma. The negative human plasma used in the Abbott RealTime HBV Low Positive Control and High Positive Control is tested and found to be nonreactive by FDA licensed tests for antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material is also tested and found to be negative by FDA licensed PCR methods for HIV-1 RNA and HCV RNA, and contains the preservatives ProClin 300 and ProClin 950.

## 2.10.7 Abbott RealTime HBV Calibrator Kit (List No. 2N40-70)

The Abbott RealTime HBV Calibrator Kit consists of:

- Abbott RealTime HBV Calibrator A (List No. 2G34A)
- Abbott RealTime HBV Calibrator B (List No. 2G34B)

The Abbott RealTime HBV Calibrator Kit contains two calibrators (12 vials of Abbott RealTime HBV Calibrator A and 12 vials of Abbott RealTime HBV Calibrator B) that are used to generate a calibration curve for the quantitative determination of HBV in human plasma.

The Abbott RealTime HBV Calibrator A and Calibrator B contain noninfectious linearized HBV DNA plasmid in a buffer solution and contains the antimicrobials sodium azide and ProClin 950.

## Assay Calibration

A calibration curve is required to quantitate HBV DNA in the specimens and controls. Two assay calibrators are run in replicates of three to generate a calibration curve (HBV concentration [log IU/mL] versus the threshold cycle [Ct] at which a reactive level of fluorescent signal is detected). The lot specific values for Calibrator A and Calibrator B are specified on each Abbott RealTime HBV Calibrator Kit Card and must be entered into the assay test order when a run is performed. The calibration curve slope and intercept are calculated and stored on the instrument. The concentration of the HBV DNA in a sample is calculated from the calibration curve. Results are automatically reported on

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the $m2000rt$ workstation. The Low and High Positive Controls and Negative Control must be included in the calibration run.

Once an Abbott RealTime HBV calibration is accepted and stored, it may be used for six months. During this time, all subsequent samples may be tested without further calibration unless a new lot of the Abbott RealTime HBV Amplification Reagent Kit or new lot of an Abbott mSample Preparation System$_{DNA}$ is used; an Abbott RealTime HBV application specification file for a different sample volume is used; or an updated version of the Abbott RealTime HBV application specification file is installed.

## Quality Control Procedures:

### Detection of Inhibition

An IC threshold cycle [Ct] assay validity parameter is established during a calibration run. Prior to sample preparation, a defined, consistent quantity of the IC is introduced into the lysis buffer, which is then used during the processing of each specimen, calibrator, and control, and measured on the $m2000rt$ instrument to demonstrate proper sample processing and assay validity. The IC is comprised of a DNA sequence unrelated to the HBV target sequence. The median amplification cycle at which the IC target sequence fluorescent signal is detected in calibration samples establishes an IC Ct validity range to be met by all subsequent processed specimens. Specimens whose IC Ct value falls outside of the established range must be retested starting with sample preparation.

### Negative and Positive Controls

A negative control, a low positive control, and a high positive control are included in each run to evaluate run validity. The lot specific values for the low positive control and high positive control are specified on each Abbott RealTime HBV Control Kit Card and must be entered into the assay test order when a run is performed. If negative or positive controls are out of range, all of the specimens and controls from that run must be reprocessed, beginning with sample preparation. The presence of HBV must not be detected in the negative control. HBV detected in the negative control is indicative of contamination by other samples or by amplified product introduced during sample preparation or during preparation of the Abbott 96-Well Optical Reaction Plate.

### Results Calculation

The concentration of HBV DNA in a sample or control is calculated from either a stored calibration curve, or a calibration curve created by calibrators within a calibration or sample run. The Abbott $m2000rt$ instrument automatically reports the results on the $m2000rt$ workstation. Assay results are reported in IU/mL or log IU/mL. Results can also be reported in copies/mL or log copies/mL using a conversion factor of 3.41 (1 IU = 3.41 copies). Note: The assay is calibrated to the WHO International Standard for HBV. The 3.41 conversion factor is an approximation based on an average conversion factor across the assay dynamic range. The following table represents the potential $m2000rt$ outputs. Interpretation of Results:

| Sample Volume | Result | Interpretation |
| --- | --- | --- |
| 1 | 3.41 | 10 |

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| 0.5 mL | Not Detected | Target not detected |
| --- | --- | --- |
| | < 1.00 Log IU/mL^{a} | Detected^{c} |
| | 1.00 to 9.00 Log IU/mL | ^{d} |
| | > 9.00 Log IU/mL | > ULQ^{e} |
| 0.2 mL | Not Detected | Target not detected |
| | < 1.18 Log IU/mL^{b} | Detected^{c} |
| | 1.18 to 9.00 Log IU/mL | ^{d} |
| | > 9.00 Log IU/mL | > ULQ^{e} |

a 10 IU/mL
b 15 IU/mL
c Below LLQ – below lower limit of quantitation or LLoQ; HBV DNA is not quantifiable.
d Calculated results are within assay linear range. If a calculated result is obtained, the Interpretation field is left blank.
e &gt;ULQ - above upper limit of quantitation or ULoQ; if IU/mL are above the linear range of the assay, results are reported as “&gt;1,000,000,000 IU/mL HBV DNA.”

If negative or positive controls are out of range, all of the specimens and controls from that run must be reprocessed, beginning with sample preparation.

If quantitative results are desired for those specimens reported as &gt; ULQ, the original specimen should be diluted 1:50 with HBV-negative human plasma or serum (consistent with the matrix of the original specimen), and the test repeated. Multiply the reported result by the dilution factor of 50 to obtain the quantitative result.

## VI. ALTERNATIVE PRACTICES AND PROCEDURES

Currently, methods for following the progress of antiviral therapy include immunoassay (serological tests, enzyme immunoassay), biochemical (alanine aminotransferase), and histological (liver biopsy - fibrosis, inflammation). A molecular method commercially available to follow HBV DNA response to antiviral therapy during the course of treatment is the Roche Cobas® TaqMan® HBV Test.

## VII. MARKETING HISTORY

The Abbott RealTime HBV assay received CE certification and was launched in May 2007 outside of the United States, under the list number of 2G34. The following countries receive the Abbott RealTime HBV assay: Australia, Austria, Belgium, Central Africa, Croatia, Czech Republic, Finland, France, Germany, Ireland, Italy, Netherlands, Norway, West Africa, Poland, Portugal, Romania, Saudi Arabia, South Africa, Spain, Sweden, Switzerland, Turkey, and United Kingdom. In Asia, it is currently being marketed in Korea.

This product has not been withdrawn from the market from any country related to safety or effectiveness, or for any other reasons.

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# VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

To aid in the management of patients with chronic HBV infection undergoing anti-viral therapy, the results from the Abbott RealTime HBV assay must be interpreted within the context of all relevant clinical and laboratory findings. Failure of the Abbott RealTime HBV assay to perform as indicated or human error in use of the product may result in an erroneous test result that is too low or too high. An erroneous low test result may lead to inappropriate treatment, or instill a false sense of security in a patient. An erroneous high test result may contribute to unnecessary treatment or create anxiety in the patient. The assay is not intended for use as a screening test for blood or blood products for the presence of HBV or as a diagnostic test to confirm the presence of HBV infection. Assay performance characteristics have been established for individuals treated with adefovir dipivoxil. Assay performance for determining the state of HBV infection has not been established.

# IX. SUMMARY OF PRECLINICAL STUDIES

## A. Laboratory Studies

### Traceability to the WHO Standard

The figure below demonstrates the comparison of Abbott RealTime HBV Assay Calibrators to the WHO International HBV DNA Standard. Abbott RealTime HBV Calibrators trace to the World Health Organization (WHO) International Standard for Hepatitis B Virus DNA (NIBSC Code 97/746) each time a lot is manufactured. Each lot of calibrator is specifically assigned a quantitation value through testing with HBV Primary Calibrators, which are directly tested against the WHO Standard. The lot-specific quantitation values for each HBV calibrator are entered into the $m2000rt$ software when a run is being performed.

The evaluation was conducted with the WHO 1st International HBV DNA Standard, and one lot of HBV Calibrators, and was performed on one run. The WHO International HBV DNA Standard was reconstituted to a concentration of $1 \times 10^{5}$ IU/mL and then diluted to $1 \times 10^{4}$, $1 \times 10^{3}$, and $1 \times 10^{2}$ IU/mL in negative human plasma. The highest assay calibrator, Calibrator B, which is lot-assigned at 6.42 log IU/mL, was diluted to $1 \times 10^{5}$, $1 \times 10^{4}$, $1 \times 10^{3}$, and $1 \times 10^{2}$ IU/mL in Tris-EDTA (TE) buffer. The data for Calibrator B and its dilution series are presented in comparison to the WHO International HBV DNA Standard dilution series in the figure below. The results indicate that the assay standardization process provides quantitation values for the RealTime HBV Calibrators and the WHO International HBV DNA Standard that are similar to the expected values with deviation of not more than 0.33 log IU/mL. The maximum deviation was obtained at the assay ULQ.

Comparison of WHO 1999 $1^{\text{st}}$ International Standard with Abbott RealTime HBV Calibrators:

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Abbott RealTime HBV Calibrators
WHO 1999 1st International Standard Dilution series

## Linear Range

In one study, a 13-member panel prepared by diluting an HBV-positive specimen targeted from 9.13 log IU/mL to 0.29 log IU/mL in HBV negative human plasma was tested and evaluated in accordance with methods defined in the CLSI EP6-A, using the 0.5 mL sample preparation protocol. The Abbott RealTime HBV assay was shown to be linear in plasma across the range of HBV DNA concentrations tested (shown in the figure below) with deviation from linearity of not more than 0.20 log IU/mL.

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Abbott RealTime HBV Linear Range Least Squares Linear Regression Analysis:

In a second study, one panel consisting of Genotype A and one panel consisting of Genotype C were tested. The two 10-member panels were prepared by diluting to concentrations targeted from 1.27 log IU/mL to 8.47 log IU/mL for Genotype A and 1.59 log IU/mL to 8.79 log IU/mL for Genotype C. The two panels were prepared with high copy HBV-positive specimens diluted in HBV serologically-negative human plasma. Least squares linear regression analysis was performed for Genotypes A and C separately. Analysis for Genotype A and Genotype C is shown in the two figures below. The Abbott RealTime HBV assay was shown to be linear in plasma across the range of HBV DNA concentrations tested for HBV Genotype A and HBV Genotype C.

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Abbott RealTime HBV linear range least squares linear regression analysis Genotype A:

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Abbott RealTime HBV linear range least squares linear regression analysis Genotype C:


In addition, a 14-member serum panel and a 10-member plasma panel targeting the linear range of the assay was tested as part of the reproducibility studies and evaluated.

The Abbott RealTime HBV assay was shown to give a linear response from 10 IU/mL (1.00 log IU/mL) HBV DNA to $10^{9}$ IU/mL (9.00 log IU/mL) HBV DNA in both EDTA plasma and serum with deviation from linearity not more than 0.20 log IU/mL for the 0.5 mL sample preparation protocol.

The Abbott RealTime HBV assay was shown to give a linear response from 15 IU/mL (1.18 log IU/mL) HBV DNA to $10^{9}$ IU/mL (9.00 log IU/mL) HBV DNA in both EDTA plasma and serum with deviation from linearity not more than 0.20 log IU/mL for the 0.2 mL sample preparation protocol.

## Limit of Detection (LoD) using the WHO international Standard

The LoD, defined as the HBV DNA concentration detected with a probability of 95%, was determined by testing dilutions of the WHO International Standard for Hepatitis B Virus DNA (NIBSC 97/746) which were prepared in HBV negative human plasma and serum. Probit analysis of the data was used to determine the concentration of the WHO Standard detected with 95% probability.

The results of the LoD in plasma and serum at both sample volumes are summarized in the table below.

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| Sample Volume | Sample Matrix | Concentration Detected with 95% Probability | 95% Confidence Interval | Concentration Detected with 95% Probability | 95% Confidence Interval |
| --- | --- | --- | --- | --- | --- |
| | | Log IU/mL | | IU/mL | |
| 0.5 mL | Plasma | 0.81 | (0.60, 1.12) | 6.40 | (3.97, 13.03) |
| 0.2 mL | Plasma | 1.03 | (0.85, 1.29) | 10.66 | (7.11, 19.38) |
| 0.5 mL | Serum | 0.58 | (0.19, 1.84) | 3.82 | (1.55, 69.76) |
| 0.2 mL | Serum | 0.75 | (0.56, 1.04) | 5.61 | (3.62, 10.94) |

Limit of detection (LoD) in plasma using WHO international standard; 0.5 mL sample preparation protocol:

| IU/mL | Number Tested | Number Detected | Percent Detected |
| --- | --- | --- | --- |
| 20.00 | 26 | 26 | 100 |
| 10.00 | 26 | 25 | 96 |
| 5.00 | 26 | 26 | 100 |
| 2.50 | 26 | 23 | 88 |
| 1.00 | 26 | 12 | 46 |
| 0.50 | 26 | 7 | 27 |
| 0.25 | 26 | 7 | 27 |
| 0.10 | 26 | 4 | 15 |

Probit analysis of the data determined that the concentration of HBV DNA detected with 95% probability was 6.40 IU/mL (95% CI 3.97-13.03 IU/mL).

Limit of detection (LoD) in serum using WHO International Standard 0.5 mL sample preparation protocol:

| IU/mL | Number Tested | Number Detected | Percent Detected |
| --- | --- | --- | --- |
| 20.00 | 30 | 30 | 100 |
| 10.00 | 30 | 30 | 100 |
| 5.00 | 30 | 30 | 100 |
| 2.50 | 30 | 29 | 97 |
| 1.00 | 30 | 17 | 57 |
| 0.50 | 30 | 16 | 53 |
| 0.25 | 30 | 1 | 3 |
| 0.10 | 30 | 8 | 27 |

Probit analysis of the data determined that the concentration of HBV DNA detected with 95% probability was 3.82 IU/mL (95% CI 1.55-69.76 IU/mL).

Limit of detection (LoD) in plasma using WHO International Standard 0.2 mL sample preparation protocol:

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| IU/mL | Number Tested | Number Detected | Percent Detected |
| --- | --- | --- | --- |
| 40.00 | 27 | 27 | 100 |
| 20.00 | 27 | 27 | 100 |
| 10.00 | 27 | 26 | 96 |
| 5.00 | 27 | 23 | 85 |
| 2.50 | 27 | 12 | 44 |
| 1.00 | 27 | 11 | 41 |
| 0.50 | 27 | 6 | 22 |
| 0.20 | 27 | 0 | 0 |

Probit analysis of the data determined that the concentration of HBV DNA detected with 95% probability was 10.66 IU/mL (95% CI 7.11-19.38 IU/mL).

Limit of detection (LoD) in serum using WHO International Standard 0.2 mL sample preparation protocol:

| IU/mL | Number Tested | Number Detected | Percent Detected |
| --- | --- | --- | --- |
| 40.00 | 30 | 30 | 100 |
| 20.00 | 30 | 30 | 100 |
| 10.00 | 30 | 30 | 100 |
| 5.00 | 29* | 25 | 86 |
| 2.50 | 30 | 27 | 90 |
| 1.00 | 30 | 17 | 57 |
| 0.50 | 30 | 17 | 57 |
| 0.20 | 30 | 4 | 13 |

* One replicate was deleted due to instrument error.
Probit analysis of the data determined that the concentration of HBV DNA detected with 95% probability was 5.61 IU/mL (95% CI 3.62-10.94 IU/mL).

The LoD of the Abbott RealTime HBV assay is determined as 10 IU/mL for the 0.5 mL sample preparation protocol and 15 IU/mL for the 0.2 mL sample preparation protocol.

## Limit of Detection (LoD) by Genotype Using Clinical Specimens

The LoD was determined by analysis of a dilution series of patient samples representing HBV Genotypes A, B, C, D, E, F, G, H and of the WHO (World Health Organization) International Standard. One patient sample for each HBV genotype was tested. Serial dilutions were made in HBV serologically negative human plasma and serum to create an eight-member panel with the target concentrations 0.10 IU/mL, 0.25 IU/mL, 0.50 IU/mL, 1.00 IU/mL, 2.50 IU/mL, 5.00 IU/mL, 10.0 IU/mL, and 20.0 IU/mL for the 0.5 mL sample preparation protocol; and target concentrations of 0.20 IU/mL, 0.50 IU/mL, 1.00 IU/mL, 2.50 IU/mL, 5.00 IU/mL, 10.00 IU/mL, 20.00 IU/mL, and 40.00 IU/mL for the 0.2 mL sample preparation protocol.

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Probit analysis of the data was used to determine the concentration of each HBV genotype detected with 95% probability.

Summaries of the results of LoD by genotype at both volumes are shown in tables below.

Summary of LoD by genotype for 0.5 mL sample preparation protocol:

| Genotype Tested | Plasma | Serum |
| --- | --- | --- |
| | Concentration Detected (IU/mL) (95% Confidence Interval) | Concentration Detected (IU/mL) (95% Confidence Intervals) |
| WHO | 3.69 (2.59, 6.19) | * |
| A | 2.31 (1.59, 4.08) | 5.49 (2.86, 19.05) |
| B | 2.96 (2.12, 4.90) | ** |
| C | 4.53 (3.18, 7.58) | 3.92 (2.09, 14.50) |
| D | 3.23 (2.23, 5.62) | ** |
| E | 4.73 (2.11, 39.36) | 3.72 (2.55, 6.47) |
| F | 4.22 (2.80, 7.73) | ** |
| G | 2.51 (1.80, 4.21) | 1.94 (1.43, 3.14) |
| H | 8.11 (4.18, 27.97) | ** |

* WHO standard was not tested in serum in this study.
** Genotypes B, D, F, and H were tested in serum with 0.2 mL volume only.

The LoD for the assay to detect HBV in clinical specimens using 0.5 mL sample preparation protocol volume, detecting any of the eight genotypes tested, considering that assay does not differentiate between HBV genotypes, is determined to be 10 IU/mL.

Summary of LoD by genotype for 0.2 mL sample preparation protocol:

| Genotype Tested | Plasma | Serum |
| --- | --- | --- |
| | Concentration Detected (IU/mL) (95% Confidence Interval) | Concentration Detected (IU/mL) (95% Confidence Intervals) |
| WHO | 8.16 (5.63, 13.93) | * |
| A | 5.86 (4.00, 10.22) | ** |
| B | 5.37 (3.72, 9.23) | 2.40 (1.61, 4.65) |
| C | 8.61 (5.95, 14.68) | ** |
| D | 5.34 (3.54, 9.93) | 2.26 (1.55, 4.21) |
| E | 14.57 (9.63, 26.28) | ** |
| F | 6.60 (4.41, 11.98) | 7.18 (4.75, 13.20) |

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| G | 3.84 (2.61, 6.94) | ** |
| --- | --- | --- |
| H | 10.86 (7.34, 19.10) | 7.65 (5.01, 14.26) |

* WHO standard was not tested in serum in this study.
** Genotypes A, C, E, and G were tested in serum with 0.5 mL volume only.

The LoD for the assay to detect HBV in clinical specimens using 0.2 mL sample preparation protocol volume, detecting any of the eight genotypes tested, considering that assay does not differentiate between HBV genotypes, is determined to be 15 IU/mL.

## Limit of Quantitation

The total analytical error (TAE) was calculated using estimates determined from the reproducibility studies that were conducted at three sites: two external sites and one internal site. Genotypes A and C were tested at both sample volumes and in both plasma and serum. Presented in the table below are the TAE estimates for the plasma panel members that had an observed concentration at or near the assay limit of detection, for each sample input volume.

| Plasma: Total Analytical Error Estimates (Log IU/mL) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample Protocol Volume (mL) | n | HBV Genotype (Panel Member) | Expected Conc. | Observed Conc. | Absolute Bias | SD^{b} | TAE^{c} Absolute Bias + (2 x SD) | TAE^{d} SQRT(2) x 2 x SD |
| 0.5 | 110 | A (5) | 1.04 | 0.90^{a} | 0.14 | 0.32 | 0.78^{a} | 0.91^{a} |
| 0.5 | 119 | C (10) | 1.14 | 1.13 | 0.01 | 0.29 | 0.59 | 0.82 |
| 0.2 | 37 | A (5) | 1.04 | 1.03^{a} | 0.01 | 0.40 | 0.81^{a} | 1.13^{a} |
| 0.2 | 46 | C (10) | 1.14 | 1.24 | 0.10 | 0.30 | 0.70 | 0.85 |

a Panel Member is below the assay Limit of Detection (1.00 log IU/ml for 0.5 ml and 1.18 log IU/ml for 0.2 ml). TAE is provided for information only.
b SD = within-run component variability + between-run component variability.
c Per Section 5.1 of EP17-A CLSI guideline.
d Based on difference between two measurements approach.

Presented in the table below are the TAE estimates for the serum panel study. TAE was estimated by two different methods (see table footnotes).

| Serum: Total Analytical Error Estimates (Log IU/mL) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample Protocol Volume (mL) | n | HBV Genotype (Panel Member) | Expected Conc. | Observed Conc. | Absolute Bias | SD^{b} | TAE^{c} Absolute Bias + (2 x SD) | TAE^{d} SQRT(2) x 2 x SD |
| 0.5 | 88 | A (6) | 1.36 | 1.04 | 0.32 | 0.20 | 0.72 | 0.57 |
| 0.5 | 90 | C (12) | 1.48 | 1.29 | 0.19 | 0.20 | 0.59 | 0.57 |
| 0.5 | 88 | C (13) | 1.27 | 0.95^{a} | 0.32 | 0.25 | 0.82^{a} | 0.71^{a} |
| 0.2 | 88 | A (5) | 1.56 | 1.10^{a} | 0.46 | 0.24 | 0.94^{a} | 0.68^{a} |
| 0.2 | 89 | C (12) | 1.48 | 1.14 | 0.34 | 0.24 | 0.82 | 0.68 |

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a Panel Member is below the assay Limit of Detection (1.00 log IU/ml for 0.5 ml and 1.18 log IU/ml for 0.2 ml). TAE is provided for information only.
b SD = within-run component variability ÷ between-run component variability.
c Per Section 5.1 of EP17-A CLSI guideline.
d Based on difference between two measurements approach.

These studies demonstrated that the Abbott RealTime HBV assay can determine with an acceptable level of accuracy the concentration of HBV DNA in EDTA plasma and serum at concentrations of 10 IU/mL (1.00 log IU/mL) for the 0.5 mL sample protocol volume and 15 IU/mL (1.18 log IU/mL) for the 0.2 mL sample protocol volume. At these concentrations, the difference between two measurements of more than 1.0 log IU/mL is statistically significant.

## Linearity of assay by HBV genotype

The ability of the RealTime HBV assay to detect and quantitate HBV genotypes was evaluated through linearity studies by diluting eight specimens, one of each genotype A through H, to target concentrations of 4.47 log IU/mL, 3.47 log IU/mL, 2.47 log IU/mL, and 1.17 log IU/mL in HBV serologically negative human plasma. Three replicates were tested at each concentration for each genotype, using the 0.5 mL sample preparation protocol. The results are summarized in the table and figure below.

Abbott RealTime HBV linearity of assay by HBV Genotype

| HBV Genotype | Linear Equation in Linearity Study | Maximum Difference^{a} Between Genotype A and Corresponding Genotype (log IU/mL) |
| --- | --- | --- |
| A | y = 0.95x + 0.19 | n/a |
| B | y = 0.89x + 0.40 | 0.35 |
| C | y = 0.93x + 0.32 | 0.11 |
| D | y = 0.89x + 0.43 | 0.32 |
| E | y = 0.86x + 0.58 | 0.44 |
| F | y = 0.90x + 0.42 | 0.23 |
| G | y = 0.85x + 0.61 | 0.51 |
| H | y = 0.86x + 0.60 | 0.42 |

a The maximum difference was obtained at the assay ULoQ or LLoQ.

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21

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Abbott RealTime HBV linearity of assay by HBV genotypes (dilutional linearity):


The data from the studies demonstrate that the Abbott RealTime HBV assay is capable to quantitate different HBV genotypes across the linear range with deviation of not more 0.51 log IU/mL.

## Precision

### Within-Laboratory Precision (Lot-to-Lot)

The between instrument and lot precision of the assay was evaluated within a laboratory using an 8-member panel. Panel members 2, 3, 5, and 7 were prepared by diluting a high copy HBV patient sample in HBV serologically negative human serum. Panel members 1, 4, 6, and 8 were prepared by diluting the same high copy HBV patient sample into HBV serologically negative human plasma.

A total of three reagent lots were used and each lot was assigned an $m2000sp$ and $m2000rt$ instrument pair. A total of 45 replicates were tested for each panel member across the three pairs of $m2000sp$ and $m2000rt$ instruments. One run was performed per day on each instrument pair for five days for a total of 15 runs. Panel members 1 through 8 were run in replicates of three. The $0.5\mathrm{mL}$ sample preparation protocol was used. Results are summarized in the table below.

Abbott RealTime HBV Within-Laboratory Precision for the $0.5\mathrm{mL}$ Sample Preparation Protocol:

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| Panel | Specimen Type^{a} | n | Mean Conc. (Log IU/mL) | Within-Run Component SD^{b} | Between-Run/Day Component SD^{b} | Between-Lot/Instrument Component SD^{b} | Total SD^{b,c} |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | P | 45 | 1.41 | 0.19 | 0.00 | 0.08 | 0.21 |
| 2 | S | 45 | 2.32 | 0.07 | 0.04 | 0.07 | 0.11 |
| 3 | S | 45 | 3.48 | 0.06 | 0.05 | 0.08 | 0.11 |
| 4 | P | 45 | 4.38 | 0.06 | 0.06 | 0.08 | 0.12 |
| 5 | S | 45 | 5.47 | 0.09 | 0.00 | 0.10 | 0.13 |
| 6 | P | 45 | 6.38 | 0.06 | 0.07 | 0.11 | 0.15 |
| 7 | S | 45 | 7.54 | 0.05 | 0.08 | 0.14 | 0.17 |
| 8 | P | 45 | 8.44 | 0.04 | 0.05 | 0.13 | 0.14 |

a P = Plasma; S = Serum
b Standard deviations (SD) are in log IU/mL.
c Total precision includes within-run, between-run and between-lot components of precision

The between-lot component of precision was less or equal to 0.14 log IU/mL.

## Within-Laboratory Precision (Operator-to-Operator)

The within-run, between-run, and between-technician (operator) precision of the Abbott RealTime HBV assay was evaluated by testing 84 replicates of each HBV panel member that span the dynamic range of the assay from approximately 1.0 log IU/mL to approximately 9.00 log IU/mL, for HBV Genotypes A and C. Panel members 1 through 5 were HBV Genotype A, and panel members 6 through 10 were HBV Genotype C. The 0.5 mL sample preparation protocol was used for this study. This same panel was also used as a part of the site-to-site reproducibility study. One lot of amplification reagents was run on one m2000sp and m2000rt instrument pair by three technicians. Each technician completed one run per day for seven days, for a total of 21 runs. Four replicates were run for each panel member. The SD for the between-technician component and the total SD for the Abbott RealTime HBV assay were found to be less than or equal to 0.06 log IU/mL and 0.11 log IU/mL, respectively, for all panel members greater than the assay limit of detection (1.00 log IU/mL). The between-technician precision component was lower than the within-run component for all panel members. The results are summarized in the table below.

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Abbott RealTime HBV Within-Laboratory Precision (Operator-to-Operator):

| Panel Member | n | Mean Concentration (log IU/mL) | Within-Run Component SD^{a} | Between-Run/Day Component SD^{a} | Between-Technician Component SD^{a} | Total SD^{a,d} |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 84 | 8.87 | 0.06 | 0.03 | 0.05 | 0.08 |
| 2 | 84 | 6.77 | 0.05 | 0.03 | 0.01 | 0.06 |
| 3 | 84 | 4.53 | 0.10 | 0.00 | 0.05 | 0.11 |
| 4 | 84 | 2.72 | 0.06 | 0.02 | 0.02 | 0.07 |
| 5 | 73^{b,c} | 0.49 | 0.24 | 0.00 | 0.07 | 0.25 |
| 6 | 84 | 8.57 | 0.08 | 0.02 | 0.06 | 0.10 |
| 7 | 84 | 6.72 | 0.07 | 0.00 | 0.04 | 0.08 |
| 8 | 84 | 4.66 | 0.09 | 0.03 | 0.04 | 0.10 |
| 9 | 83^{c} | 2.69 | 0.07 | 0.03 | 0.05 | 0.09 |
| 10 | 84 | 0.78 | 0.19 | 0.00 | 0.04 | 0.19 |

a Standard Deviations (SD) are in log IU/mL
b Target not detected for 10 samples
c Error code 4457 “Internal Control Failed” for one sample
d Total precision includes within-run, between-run and between-technician components of precision

## Reproducibility in Plasma

The plasma reproducibility panel was tested at three different sites by one technologist and one instrument pair at each site. Panels tested at each site consisted of a 40-member panel (10 unique panel members) that included five concentration levels of one prevalent HBV genotype and five concentration levels of a second prevalent HBV genotype, repeated four times within the panel. The concentration levels targeted for the reproducibility panels spanned the linear quantitation range of the assay. The HBV genotypes selected for the reproducibility panels were genotype A and genotype C, recognized as prevalent in the US population. Each 5-member panel was prepared from a high copy source sample, which was comprised of at least two individual patient specimens that had a common genotype. A total of three reagent lots were used. For the 0.5 mL reproducibility, each of the three clinical sites tested two of the three lots for five days each. Site 1 used lots A and B, Site 2 used lots B and C, and Site 3 used lots A and C. The 0.2 mL reproducibility was tested at each of the three clinical sites using two lots for two days each. The SD for the between-site component was less than or equal to 0.10 log IU/mL. The results are summarized in tables below.

Abbott RealTime HBV Reproducibility in Plasma - 0.5 mL Sample Preparation Protocol:

| Panel | Genotype | n | Mean Concentration (Log | Mean Concentration (IU/mL) | Within-Run Component SD^{c} | Between-Run Component SD^{c} | Between-Lot Component SD^{c} | Between-Site Component SD^{c} | Total SD^{c,d} |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

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| | | | IU/mL) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | A | 120 | 8.93 | 872,502,276 | 0.07 | 0.00 | 0.03 | 0.08 | 0.11 |
| 2 | A | 119^{a} | 6.84 | 7,087,010 | 0.04 | 0.03 | 0.06 | 0.05 | 0.09 |
| 3 | A | 120 | 4.70 | 52,574 | 0.09 | 0.02 | 0.06 | 0.10 | 0.15 |
| 4 | A | 120 | 2.81 | 665 | 0.05 | 0.02 | 0.06 | 0.08 | 0.12 |
| 5 | A | 110^{b} | 0.90^{c} | 27^{c} | 0.31 | 0.07 | 0.26 | 0.10 | 0.42 |
| 6 | C | 119^{a} | 8.64 | 446,037,175 | 0.07 | 0.01 | 0.04 | 0.07 | 0.11 |
| 7 | C | 120 | 6.83 | 6,922,148 | 0.06 | 0.01 | 0.06 | 0.05 | 0.10 |
| 8 | C | 119^{a} | 4.84 | 72,954 | 0.08 | 0.00 | 0.08 | 0.09 | 0.15 |
| 9 | C | 120 | 2.84 | 722 | 0.06 | 0.02 | 0.09 | 0.08 | 0.13 |
| 10 | C | 119^{b} | 1.13 | 26 | 0.29 | 0.00 | 0.22 | 0.00 | 0.37 |

a Invalid replicate not included.
b Target not detected not included.
c Standard deviations (SD) are in log IU/mL.
d The total precision includes within-run, between-run, between-lot, and between-site components of precision.
e Concentration is below the assay LoD.

Abbott RealTime HBV Reproducibility in Plasma (0.2 mL Sample Preparation Protocol):

| Panel | Genotype | n | Mean Concentration (Log IU/mL) | Mean Concentration (IU/mL) | Within-Run Component t SD^{b} | Between-Run Component t SD^{b} | Between-Lot Component SD^{b} | Between-Site Component t SD^{b} | Total SD^{b,c} |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | A | 48 | 8.99 | 1,019,710,342 | 0.07 | 0.00 | 0.13 | 0.00 | 0.15 |
| 2 | A | 48 | 6.87 | 7,526,185 | 0.04 | 0.05 | 0.09 | 0.00 | 0.12 |
| 3 | A | 48 | 4.70 | 52,678 | 0.06 | 0.08 | 0.14 | 0.00 | 0.17 |
| 4 | A | 48 | 2.83 | 716 | 0.06 | 0.06 | 0.13 | 0.00 | 0.16 |
| 5 | A | 37^{a} | 1.03^{d} | 21 | 0.40 | 0.00 | 0.18 | 0.02 | 0.44 |
| 6 | C | 48 | 8.64 | 451,101,262 | 0.05 | 0.05 | 0.12 | 0.00 | 0.14 |
| 7 | C | 48 | 6.85 | 7,255,246 | 0.05 | 0.05 | 0.11 | 0.00 | 0.13 |
| 8 | C | 48 | 4.83 | 71,717 | 0.08 | 0.07 | 0.14 | 0.00 | 0.18 |
| 9 | C | 48 | 2.84 | 738 | 0.08 | 0.04 | 0.15 | 0.00 | 0.18 |
| 10 | C | 46^{a} | 1.24 | 26 | 0.30 | 0.00 | 0.27 | 0.00 | 0.41 |

a Target not detected not included.
b Standard deviations (SD) are in log IU/mL.
c The total precision includes within-run, between-run, between-lot, and between-site components of precision.
d Concentration is below the assay LoD.

## Reproducibility in Serum

The serum reproducibility panel tested at each site consisted of a 42-member panel (14 unique panel members) that included seven concentration levels of one prevalent HBV genotype and seven concentration levels of a second prevalent HBV genotype, repeated three times within the panel. The concentration levels targeted for the reproducibility panels spanned the linear

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quantitation range of the assay and also included some members below the lower limit of quantitation. The HBV genotypes selected for the serum reproducibility panels were genotypes that were recognized as prevalent in the US population. Each seven-member panel was prepared from a high copy source sample. A total of three reagent lots were used. Each of the three clinical sites tested two of the three amplification reagent lots for five days each. Site 1 used lots A and B, Site 2 used lots B and C, and Site 3 used lots A and C. Each site conducted the five day reproducibility at both the 0.2 mL volume and 0.5 mL volume for two lots of amplification reagents.

The results are summarized in tables below.

Abbott RealTime HBV Reproducibility in Serum (0.5 mL Sample Preparation Protocol):

| Panel | Genotype | n | Mean Concentration (Log IU/mL) | Mean Concentration (IU/mL) | Within-Run Component SD^{c} | Between-Run Component SD^{c} | Between-Lot Component SD^{c} | Between-Site Component SD^{c} | Total SD^{c,d} |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | A | 89^{a} | 8.24 | 184,508,108 | 0.13 | 0.07 | 0.07 | 0.06 | 0.17 |
| 2 | A | 90 | 6.19 | 1,613,319 | 0.04 | 0.07 | 0.07 | 0.03 | 0.11 |
| 3 | A | 90 | 3.94 | 9,725 | 0.07 | 0.08 | 0.11 | 0.19 | 0.24 |
| 4 | A | 89^{a} | 1.96 | 105 | 0.12 | 0.07 | 0.07 | 0.20 | 0.25 |
| 5 | A | 90 | 1.25 | 22 | 0.20 | 0.10 | 0.12 | 0.20 | 0.32 |
| 6 | A | 88^{b} | 1.04 | 13 | 0.17 | 0.10 | 0.08 | 0.18 | 0.28 |
| 7 | A | 84^{b} | 0.74^{e} | 7^{e} | 0.22 | 0.12 | 0.21 | 0.12 | 0.35 |
| 8 | C | 90 | 7.22 | 17,211,265 | 0.05 | 0.05 | 0.08 | 0.05 | 0.12 |
| 9 | C | 90 | 6.23 | 1,741,264 | 0.05 | 0.06 | 0.08 | 0.02 | 0.12 |
| 10 | C | 89^{a} | 3.89 | 9,068 | 0.11 | 0.07 | 0.10 | 0.21 | 0.27 |
| 11 | C | 89^{a} | 1.64 | 55 | 0.18 | 0.12 | 0.06 | 0.28 | 0.36 |
| 12 | C | 90 | 1.29 | 23 | 0.18 | 0.09 | 0.12 | 0.15 | 0.27 |
| 13 | C | 88^{b} | 0.95^{e} | 12^{e} | 0.24 | 0.08 | 0.15 | 0.24 | 0.38 |
| 14 | C | 87^{b} | 0.88^{e} | 9^{e} | 0.23 | 0.06 | 0.10 | 0.10 | 0.28 |

a Invalid replicate not included.
b Target not detected not included.
c Standard deviations (SD) are in log IU/mL.
d The total precision includes within-run, between-run, between-lot, and between-site components of precision.
e Concentration is below the assay LoD.

Abbott RealTime HBV Reproducibility in Serum (0.2 mL Sample Preparation Protocol):

| Panel | Genotype | n | Mean Concentration (Log IU/mL) | Mean Concentration (IU/mL) | Within-Run Component SD^{c} | Between-Run Component SD^{c} | Between-Lot Component SD^{c} | Between-Site Component SD^{c} | Total SD^{c,d} |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | A | 90 | 8.28 | 205,545,691 | 0.16 | 0.04 | 0.03 | 0.00 | 0.17 |

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| 2 | A | 88^{a} | 6.21 | 1,638,140 | 0.04 | 0.04 | 0.03 | 0.00 | 0.06 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 3 | A | 89^{a} | 3.89 | 8,306 | 0.10 | 0.07 | 0.07 | 0.13 | 0.19 |
| 4 | A | 89^{a} | 1.90 | 95 | 0.22 | 0.04 | 0.16 | 0.16 | 0.32 |
| 5 | A | 88^{b} | 1.10^{e} | 16^{e} | 0.21 | 0.12 | 0.07 | 0.18 | 0.31 |
| 6 | A | 86^{a,b} | 0.89^{e} | 10^{e} | 0.27 | 0.03 | 0.10 | 0.10 | 0.31 |
| 7 | A | 82^{b} | 0.62^{e} | 7^{e} | 0.33 | 0.00 | 0.06 | 0.16 | 0.37 |
| 8 | C | 90 | 7.22 | 16,648,045 | 0.05 | 0.02 | 0.04 | 0.00 | 0.07 |
| 9 | C | 90 | 6.25 | 1,776,403 | 0.04 | 0.04 | 0.04 | 0.00 | 0.07 |
| 10 | C | 90 | 3.84 | 7,550 | 0.12 | 0.07 | 0.07 | 0.13 | 0.20 |
| 11 | C | 90 | 1.57 | 48 | 0.18 | 0.10 | 0.02 | 0.31 | 0.37 |
| 12 | C | 89^{b} | 1.14^{e} | 17^{e} | 0.22 | 0.10 | 0.11 | 0.12 | 0.29 |
| 13 | C | 87^{a,b} | 0.80^{e} | 8^{e} | 0.26 | 0.13 | 0.15 | 0.08 | 0.34 |
| 14 | C | 82^{b} | 0.73^{e} | 9^{e} | 0.35 | 0.06 | 0.17 | 0.00 | 0.39 |

a Invalid replicate not included.
b Target not detected not included.
c Standard deviations (SD) are in log IU/mL.
d The total precision includes within-run, between-run, between-lot, and between-site components of precision.
e Concentration is below the assay LoD.

## Analytical Specificity

## Potentially Interfering Substances

The susceptibility of the Abbott RealTime HBV assay to interference by elevated levels of potentially interfering substances was evaluated. HBV-negative samples and HBV-positive samples containing 2,933 IU/mL (3.47 log IU/mL) of HBV DNA were tested. Potential interference at HBV DNA concentrations close to the assay LLoQ was not assessed. HBV-negative and positive samples were tested in a plasma matrix and were not tested in serum.

No interference in the performance of the Abbott RealTime HBV assay was observed in the presence of high levels of hemoglobin (500 mg/dL), triglycerides (3,000 mg/dL), bilirubin (20 mg/dL), and protein (9 g/dL). For hemoglobin and protein, there was a slight trend towards lowering of the values of the high level HBV specimens in the presence of interfering substances. The mean differences of the test and control conditions for hemoglobin and protein are small (-0.058 and -0.112 log IU/mL, respectively) compared to the clinically significant difference between two samples (1 log); as such, these values are not expected to be clinically significant.

Antivirals and antibiotics at concentrations equal to or in excess of peak plasma or serum levels were tested in five pools. No interference in the performance of the Abbott RealTime HBV assay was observed in the presence of the following drug pools for all HBV-positive and negative samples tested:

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| Drug Pool | Drugs Tested |
| --- | --- |
| 1 | Zidovudine, Saquinavir, Ritonavir, Clarithromycin, Interferon 2a, Interferon 2b, Didanosine |
| 2 | Abacavir sulfate, Amprenavir, Peginterferon 2a, Peginterferon 2b, Ribavirin,Entecavir, Adefovir |
| 3 | Tenofovir, Lamivudine, Indinavir, Ganciclovir, Valganciclovir, Acyclovir, Paroxetine |
| 4 | Stavudine, Efavirenz, Lopinavir, Enfuvirtide, Ciprofloxacin, Fluoxetine |
| 5 | Zalcitabine, Nevirapine, Nelfinavir, Azithromycin, Valacyclovir, Sertraline |

A consideration was made to avoid combining specific drugs within a pool that would not be used together in a clinical setting. However, because the listed drugs were tested only as pools, individual drug effects were not assessed.

## Cross-reactivity studies with clinical specimens

The specificity of the assay was evaluated by testing 60 clinical specimens that were positive for at least one of the following DNA virus markers, RNA viruses, non-viral hepatitis, or autoimmune disease states (summarized in table below). Clinical specimens that were tested for DNA virus markers were serum. Clinical specimens that were tested for RNA virus markers were plasma or serum. HBV DNA was not detected in any of the 60 specimens tested.

| DNA and RNA Viruses | Non-viral Hepatitis and Autoimmune States |
| --- | --- |
| Epstein Barr Virus (EBV) | Anti-nuclear Antibody (ANA) |
| Herpes Simplex Virus 1 (HSV-1) | Rheumatoid Factor (RF) |
| Herpes Simplex Virus 2 (HSV-2) | Cirrhosis |
| Cytomegalovirus (CMV) | Alcoholic Hepatitis |
| Human Immunodeficiency Virus (HIV-1) | Non-alcoholic Steatohepatitis (NASH) |
| Hepatitis C Virus (HCV) | Autoimmune Hepatitis (AUH) |
| Hepatitis A Virus (HAV) | Hepatocellular Carcinoma |

## Performance of the assay with HBV-Negative Specimens

Performance of the Abbott RealTime HBV assay was evaluated by testing 124 HBV seronegative serum and 125 HBV seronegative plasma specimens from blood donors. The specimens were tested on one $m2000$ instrument system with one lot of amplification reagents. HBV DNA was not detected for all 249 specimens, resulting in $100\%$ correct

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results: 100% (124/124) with 95% CI: 97.0% to 100% for serum samples and 100% (125/125) with 95% CI: 97.0% to 100% for plasma samples.

## Cross-Reactivity studies using nucleic acid or viral lysate

The viruses and microorganisms in the table below were evaluated for potential cross-reactivity in the Abbott RealTime HBV assay. Purified nucleic acid or viral lysate from each microorganism or virus was added at a concentration of 100,000 copies/mL to HBV DNA negative samples and HBV DNA positive samples that contained 2,933 IU/mL HBV DNA (3.47 log IU/mL). No interference in the Abbott RealTime HBV assay was observed in the presence of nucleic acids from potentially cross-reactant microorganisms or viruses for all the HBV-positive and negative samples tested.

| Microorganism/Virus | Source |
| --- | --- |
| Human immunodeficiency virus 1 (HIV-1) | Viral lysate, cell culture |
| Human immunodeficiency virus 2 (HIV-2) | Viral lysate, cell culture |
| Human T-lymphotropic virus I (HTLV-I) | Viral lysate, cell culture |
| Hepatitis C virus (HCV) | Viral lysate, human specimen |
| Hepatitis A virus (HAV) | Purified nucleic acid |
| Epstein-Barr virus (EBV) | Purified nucleic acid |
| Herpes simplex virus 1 (HSV-1) | Purified nucleic acid |
| Herpes simplex virus 2 (HSV-2) | Purified nucleic acid |
| Cytomegalovirus (CMV) | Purified nucleic acid |
| Human herpesvirus 6B (HHV-6B) | Purified nucleic acid |
| Human herpesvirus 8 (HHV-8) | Purified nucleic acid |
| Varicella-zoster virus (VZV) | Purified nucleic acid |
| Vaccinia virus (VACV) | Purified nucleic acid |
| BK human polyomavirus | Purified nucleic acid |
| Human papilloma virus 16 (HPV-16) | Purified nucleic acid |
| Human papilloma virus 18 (HPV-18) | Purified nucleic acid |
| Neisseria gonorrhoeae | Purified nucleic acid |
| Chlamydia trachomatis | Purified nucleic acid |
| Candida albicans | Purified nucleic acid |
| Staphylococcus aureus | Purified nucleic acid |
| Staphylococcus epidermidis | Purified nucleic acid |
| Mycobacterium gordonae | Purified nucleic acid |
| Mycobacterium smegmatis | Purified nucleic acid |

## Analytical Carryover

Potential carryover was determined by performing three studies in which high copy HBV-positive samples were interspersed with negative samples in a checkerboard pattern. For these studies, the targeted level for the high copy HBV-positive samples was greater than 8 log IU/ml. The carryover rate is defined as the number of HBV-negative samples that have a

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concentration reported greater than assay LoD over the total number of HBV-negative samples tested. The carryover rate in these representative studies ranged from 0% to 2% with an overall rate of 0.63% (95% CI 0.08%-2.24%). Results from the three studies are summarized in the table below.

| Study | Number of Runs | Number of Negatives Tested | Number Detected | Number Detected (> LoD) | Percent Detected (> LoD) | 95% CI of Percent Detected |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 5 | 100 | 1 | 0 | 0.00 | (0.00, 3.62) |
| 2 | 5 | 100 | 2 | 2 | 2.00 | (0.24, 7.04) |
| 3 | 6 | 120 | 2 | 0 | 0.00 | (0.00, 3.03) |
| Overall | 16 | 320 | 5 | 2 | 0.63 | (0.08, 2.24) |

## Comparison of 0.2 mL vs. 0.5 mL Sample Preparation Protocols

This study used the Abbott RealTime HBV assay to quantitate HBV-positive patient specimens. Sixty HBV-positive EDTA plasma specimens were tested in duplicate with both the 0.2 mL and 0.5 mL sample preparation protocols. Each duplicate pair was tested in the same run. The study was designed to cover the dynamic range of the Abbott RealTime HBV assay with actual patient samples representing genotypes (A, B, C, D) commonly encountered within the US. The data showed a slope of 0.99 and an intercept of 0.09. The results are summarized in the figure below.

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# Abbott RealTime HBV
Correlation of 0.2 mL vs. 0.5 mL Sample Preparation Protocols
Least Squares Linear Regression


| Sample Size (n) | | | | 60 |
| --- | --- | --- | --- | --- |
| Correlation Coefficient (r) | | | | 0.999 |
| Slope | | | | 0.99 |
| 95% CI for Slope | | | | (0.98, 1.00) |
| Intercept | | | | 0.09 |
| 95% CI for Intercept | | | | (0.03, 0.14) |
| 0.5 mL Mean conc. (log IU/mL) | Min | 1.46 | Max | 8.75 |
| 0.2 mL Mean conc. (log IU/mL) | Min | 1.51 | Max | 8.71 |

The observed lowest value in the specimen population for the 0.2 mL sample volume was 1.33 log IU/mL (mean value of the duplicate pair was 1.51 log IU/mL). For the 0.5 mL sample volume, the same specimen had an observed lowest value of 1.40 log IU/mL (mean value of duplicate pair was 1.46 log IU/mL).

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# Sample Handling and Collection

The assay is for use with serum or EDTA plasma specimens only.

# Serum vs. Plasma Across the Linear Range

Specimens from 30 individual HBV serologically-negative donors were tested. The specimens from each donor were collected as matched sets in serum and in EDTA- plasma tubes. Each pair of serum and plasma specimens was spiked with HBV-positive material at two targeted concentration levels throughout the dynamic range of the Abbott RealTime HBV assay. Specimen types at each targeted concentration were tested once using the 0.5 mL sample preparation protocol. Two plasma-serum pairs had quantitation values below the assay dynamic range and were excluded from the analysis.

Using a sample size of 58, linear regression analysis demonstrated a slope of 1.00 (95% CI 0.98 to 1.01) and an intercept of 0.03 (95% CI -0.04 to 0.10). The mean difference between serum and plasma specimens was -0.02 log IU/mL (95% CI -0.05 to 0.01). The results are summarized in the figure below.

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# Abbott RealTime HBV Specimen Type - Serum vs. Plasma Least Squares Linear Regression


| Sample Size (n) | | | | 58 |
| --- | --- | --- | --- | --- |
| Correlation Coefficient (r) | | | | 0.998 |
| Slope | | | | 1.00 |
| 95% CI for Slope | | | | (0.98, 1.01) |
| Intercept | | | | 0.03 |
| 95% CI for Intercept | | | | (-0.04, 0.10) |
| Plasma (log IU/mL) | Min | 1.26 | Max | 7.95 |
| Serum (log IU/mL) | Min | 1.32 | Max | 7.89 |

## Recommended storage stability

The stability study data supports 18 month dating for the Abbott RealTime HBV Amplification Reagent Kits.

The stability study data supports 18 month dating for the Abbott RealTime HBV Control and Calibrator Kit components and the Internal Control from the Amplification Reagent Kit.

## Recommended sample stability

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Human serum or plasma specimens may be stored at 15 to 30°C for up to 24 hours or at 2 to 8°C for up to three days. Freshly drawn whole blood (plasma or serum) specimens may be held for up to 6 hours at 2 to 30°C prior to centrifugation. Freeze/thaw effect was tested in both serum and plasma for up to eight cycles. Frozen specimens may be thawed at 15 to 30°C or 2 to 8°C. Thawed specimens may be stored at 2 to 8°C for up to 6 hours, if not processed immediately. Serum and plasma specimens may be stored at -20°C or colder for longer term storage. Stability testing results are summarized in the table below.

Specimen Stability (Log IU/mL)

| Sample Type | Test Condition | Test Condition Mean | Baseline Condition Mean | Mean Difference |
| --- | --- | --- | --- | --- |
| Plasma | 24-26h at 28-32°C | 3.781 | 3.722 | 0.059 |
| | 72-74h at 2-8°C | 3.777 | 3.722 | 0.055 |
| Serum | 24-26h at 28-32°C | 3.871 | 3.844 | 0.027 |
| | 72-74h at 2-8°C | 3.870 | 3.844 | 0.026 |
| Plasma (Whole Blood) | 6-8h at 28-32°C | 3.863 | 3.866 | -0.003 |
| | 6-8h at 2-8°C | 3.862 | 3.866 | -0.004 |
| Serum (Whole Blood) | 6-8h at 28-32°C | 3.823 | 3.628 | 0.195 |
| | 6-8h at 2-8°C | 3.730 | 3.628 | 0.102 |
| Plasma Freeze/Thaw | 8 freeze/thaw cycles (frozen at -20°C or colder for a minimum of 8 hours; thawed at 15°C to 30°C for a maximum of 24 hours) | 2.704 | 2.693 | 0.011 |
| Serum Freeze/Thaw | | 2.722 | 2.714 | 0.007 |

## X. SUMMARY OF PRIMARY CLINICAL STUDIES

A summary of the clinical study is presented below.

## B. Study Population and Baseline Parameters

The clinical performance of the Abbott RealTime HBV Assay for use with the m2000 System was evaluated by assessing the antiviral therapy response in chronic HBV-infected subjects undergoing treatment with adefovir dipivoxil. The HBV DNA data were obtained from testing patient samples previously collected under two study protocols, one of which evaluated patients with chronic HBeAg-positive HBV

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infection and compensated liver function⁹ and one that evaluated patients with HBeAg-negative HBV infection with compensated liver function.¹⁰ The relationship between HBV DNA viral levels at various time points to histologic, biochemical, and serological responses to treatment was determined in this study.

The study population consisted of chronic HBV-infected subjects enrolled in double-blind, randomized, placebo-controlled studies of adefovir dipivoxil that spanned 240 weeks. In the HBeAg-positive protocol, patients were randomized to 10 mg adefovir dipivoxil, 30 mg adefovir dipivoxil, or placebo for the first 48 weeks. Only the 10 mg adefovir dipivoxil treated patients (169) and placebo patients (60) were included in this study. Viral load testing was performed at baseline and at weeks 12, 24, and 48. The viral load results were evaluated against histologic, biochemical, and serological response at 48 weeks. In addition, patients that remained on the 10 mg adefovir dipivoxil treatment were also tested at weeks 144, 192, 240, as available.

In the HBeAg-negative protocol, patients were randomized to either 10 mg adefovir dipivoxil or placebo for the first 48 weeks. The adefovir dipivoxil treated patients (123) and placebo patients (61) were tested at baseline and at weeks 12, 24, and 48. The viral load results were evaluated against histologic and biochemical response at 48 weeks. In addition, patients that remained on the 10 mg adefovir dipivoxil treatment were also tested at weeks 96, 144, 192, and 240, as available.

Demographic data, HBV genotype, HBeAg, anti-HBe, and HBsAg seroconversion results, and baseline (pretreatment) and post-treatment liver biopsy results were available.

The table below summarizes the subject demographics.

| Subject Demographics Characteristic | Category | Summary Statistics | HBeAg+ | HBeAg- | Total |
| --- | --- | --- | --- | --- | --- |
| Total Number of Subjects | - | n | 229 | 184 | 413 |
| Placebo | - | n (%) | 60^{a} (26.20) | 61 (33.15) | 121 |
| 10 mg adefovir dipivoxil | - | n (%) | 169^{a} (73.80) | 123 (66.85) | 292 |
| Total Number of Subjects with Demographic Information | - | n | 220 | 184 | 404 |
| Age (yr) | - | Median (Min, Max) | 34 (16, 65) | 46 (18, 65) | 40 (16, 65) |
| Weight (kg) | - | Median (Min, Max) | 71 (43, 117.73) | 74.55 (46, 135) | 72.5 (43, 135) |
| Sex | Male | n (%) | 164 (74.55) | 152 (82.61) | 316 (78.22) |
| | Female | n (%) | 56 (25.45) | 32 (17.39) | 88 (21.78) |
| Race | White | n (%) | 80 (36.36) | 122 (66.30) | 202 (50.00) |
| | Asian | n (%) | 129 (58.64) | 56 (30.43) | 185 (45.79) |
| | Other | n (%) | 11 (5.00) | 6 (3.26) | 17 (4.21) |
| Genotype | A | n (%) | 64 (29.09) | 11 (5.98) | 75 (18.56) |
| | B | n (%) | 41 (18.64) | 31 (16.85) | 72 (17.82) |

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| | C | n (%) | 82 (37.27) | 24 (13.04) | 106 (26.24) |
| --- | --- | --- | --- | --- | --- |
| | D | n (%) | 27 (12.27) | 114 (61.96) | 141 (34.90) |
| | Other | n (%) | 6 (2.73) | 4 (2.17) | 10 (2.48) |
| Knodeill Score | - | n | 210 | 175 | 385 |
| Total | - | Mean (SD) | 9.38 (3.29) | 9.35 (3.34) | 9.37 (3.31) |
| Necroinflammatory | - | Mean (SD) | 7.70 (2.71) | 7.50 (2.75) | 7.61 (2.73) |
| Fibrosis | - | Mean (SD) | 1.67 (1.09) | 1.86 (1.15) | 1.76 (1.12) |

a Clinical response data was not provided for three placebo and six treatment subjects

The HBeAg-positive subjects were primarily Asian and HBV Genotypes A and C, while the HBeAg-negative subjects were primarily White and HBV Genotype D. Patients included in the clinical performance analysis received either the standard 10 mg adefovir dipivoxil dosing or placebo.

The table below summarizes available subjects by treatment arm.

| Summary of Available Subjects by Treatment Arm Population | Number of Subjects - Placebo | Number of Subjects -10 mg Adefovir | No. of Specimens per Subject^a | Total No. of Specimens Tested |
| --- | --- | --- | --- | --- |
| Chronic HBeAg+ | 60 | 169 | 2 to 7 | 1,036 |
| Chronic HBeAg- | 61 | 123 | 2 to 8 | 939 |
| Total subjects tested | 121 | 292 | 2 to 8 | 1,975 |
<a>^a</a> This number is reported as a range because the number of specimens varied for each subject.

## Within-Subject Variability in Absence of Treatment

The objective of this analysis was to assess the change in viral load (in log IU/mL units) between two successive measurements of placebo patients. There were 55 patients in the placebo arm of the HBeAg-positive group and 57 patients in the HBeAg-negative group that had available results for both Weeks 0 and 12. These results were used to estimate within-subject variability, which includes biological variability as well as total assay variability.

The within-subject variability (SD) based on these results was estimated to be 0.79 log IU/mL for HBeAg-positive patients and 0.86 log IU/mL for HBeAg-negative patients. Biological variability was similar to the within-subject variability since the assay variability was negligible. The median change (Week 12 - Week 0) of viral load within a subject was estimated to be 0.00 log IU/mL for HBeAg-positive patients and -0.28 log IU/mL for HBeAg-negative patients. Approximately 89% of the HBeAg-positive patient’s and 81% of HBeAg-negative patient’s change of viral load was less than 2.0 log IU/mL.

## C. Safety and Effectiveness Results

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Clinical Study Results and Statistical Analyses

Statistical analysis of clinical data was used to assess whether viral response to treatment measured with Abbott RealTime HBV Assay for use with the m2000 System is informative for assessing the response to treatment in HBeAg+ and HBeAg- patients with chronic hepatitis B. Observing changes in viral load in individual patients over time may help the clinician in the assessment of a patient’s response to therapy.

## HBeAg-Positive Patients

The table and figure below illustrate the efficacy of treating HBeAg-positive patients with 10 mg adefovir dipivoxil compared to placebo, based on HBV viral load testing results using Abbott RealTime HBV Assay for use with the m2000 System. At Week 48, 22.92% (33/144) of HBeAg-positive patients on treatment vs. 0% (0/55) on placebo had achieved very low viral loads below 100 IU/mL. In addition, only 23.61% (34/144) of patients on treatment vs. 81.82% (45/55) on placebo had viral loads greater than or equal to 10⁶ IU/mL.

Distribution of HBV Viral Load at Week 48 for HBeAg-Positive Patients:

| Viral Load (IU/mL) | Adefovir Dipivoxil | | | Placebo | | |
| --- | --- | --- | --- | --- | --- | --- |
| | n | % | Cumulative % | n | % | Cumulative % |
| TND^{a} | 4 | 2.78 | 2.78 | 0 | 0.0 | 0.0 |
| < 15 | 13 | 9.03 | 11.81 | 0 | 0.0 | 0.0 |
| 15 - < 100 | 16 | 11.11 | 22.92 | 0 | 0.0 | 0.0 |
| 100 - < 10^{3} | 21 | 14.58 | 37.50 | 2 | 3.64 | 3.64 |
| 10^{3} - < 10^{4} | 18 | 12.50 | 50.00 | 1 | 1.82 | 5.46 |
| 10^{4} - < 10^{5} | 14 | 9.72 | 59.72 | 4 | 7.27 | 12.73 |
| 10^{5} - < 10^{6} | 24 | 16.67 | 76.39 | 3 | 5.45 | 18.18 |
| 10^{6} - < 10^{9} | 32 | 22.22 | 98.61 | 43 | 78.18 | 96.36 |
| ≥ 10^{9} | 2 | 1.39 | 100.00 | 2 | 3.64 | 100.00 |
| Total | 144 | 100.00 | | 55 | 100.00 | |

a Target not detected.

The figure below demonstrates the median viral load change and inter-quartile range of change from baseline for HBeAg-positive subjects on treatment compared to placebo. This shows the impact of treatment with adefovir dipivoxil on the viral load of the HBeAg-positive patients with chronic hepatitis B.

Median and inter-quartile range of change in HBV DNA from baseline (HBeAg-positive subjects):

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The goal of therapy for patients with chronic HBV infection is to reduce the HBV DNA to low or undetectable levels, and monitor for viral rebound that could be associated with resistance. Results in the table below show that 70.41% (119/169) of the treated subjects achieved a nadir, or lowest concentration, viral load level by Week 48. Of the 49 subjects that achieved a nadir by Week 24, 13.79% (4/29) subjects had a greater than or equal to one log IU/mL increase by Week 48 (20 of these subjects did not have a Week 48 result).

Distribution of the HBeAg-positive subjects by week on treatment and the viral load at which the nadir was reached:

| Nadir Viral Load (IU/mL) | Number (%) of Patients With the Nadir Viral Load Achieved By Week | | | | | | Total By Viral Load | Cumulative By Viral Load |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | 12 | 24 | 48 | 144 | 192 | 240 | | |
| TND^{4} | 0 (0.00) | 0 (0.00) | 4 (2.37) | 1 (0.59) | 1 (0.59) | 4 (2.37) | 10 (5.92) | 10 (5.92) |
| < 15 | 0 (0.00) | 1 (0.59) | 13 (7.69) | 1 (0.59) | 6 (3.55) | 1 (0.59) | 22 (13.02) | 32 (18.94) |
| 15 -< 100 | 0 (0.00) | 0 (0.00) | 12 (7.10) | 3 (1.78) | 4 (2.37) | 2 (1.18) | 21 (12.43) | 53 (31.37) |
| 100 -< 10^{3} | 1 (0.59) | 3 (1.78) | 15 (8.88) | 0 (0.00) | 1 (0.59) | 7 (4.14) | 27 (15.98) | 80 (47.35) |

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| 10^{3}–<10^{4} | 3
(1.78) | 8
(4.73) | 5
(2.96) | 1
(0.59) | 1
(0.59) | 2
(1.18) | 20
(11.83) | 100
(59.18) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 10^{4}–<10^{5} | 3
(1.78) | 5
(2.96) | 7
(4.14) | 3
(1.78) | 1
(0.59) | 2
(1.18) | 21
(12.43) | 121
(71.61) |
| 10^{5}–<10^{6} | 3
(1.78) | 5
(2.96) | 8
(4.73) | 1
(0.59) | 3
(1.78) | 2
(1.18) | 22
(13.02) | 143
(84.63) |
| 10^{6}–<10^{9} | 8
(4.73) | 9
(5.33) | 6
(3.55) | 1
(0.59) | 0
(0.00) | 2
(1.18) | 26
(15.38) | 169
(100.00) |
| Total by Week | 18
(10.65) | 31
(18.34) | 70
(41.42) | 11
(6.51) | 17
(10.06) | 22
(13.02) | | |
| Cumulative By Week | 18
(10.65) | 49
(28.99) | 119
(70.41) | 130
(76.92) | 147
(86.98) | 169
(100.00) | | |

*Target Not Detected

Two patients out of 169 achieved HBsAg seroconversion. One patient had results showing HBsAg seroconversion at both Weeks 192 and 240. The other patient achieved seroconversion at Week 240. These two patients were white males, HBV genotype A, and &gt;30 years of age. A summary of these results is provided in the table below.

HBeAg-Positive Subjects with HBsAg Seroconversion:

| | Concentration (Log IU/mL) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Week 0 | Week 12 | Week 24 | Week 48 | Week 144 | Week 192 | Week 240 |
| Subject 1 | 6.99 | 4.98 | 2.08 | 1.50 | TND* | TND* | TND* |
| Subject 2 | 8.59 | 6.57 | 6.85 | 6.72 | 5.68 | 1.45 | ** |

* TND = Target Not Detected
** The Abbott RealTime HBV result for the Week 240 time point was excluded due to technician error.

Summaries of the effect of baseline covariates for the HBeAg-negative population are provided in the three tables that follow.

Association between responses to treatment at week 48 and baseline covariates for HBeAg-positive patients association:

| Response to Treatment | Covariate | Category | n | No. of Patients with Response | Proportion (%) of Patients with Response | Unadjusted Odds Ratio (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| Histological | Race | Asian | 75 | 47 | 62.67 | 1.44 (0.66, 3.14) |
| | | Other | 52 | 28 | 53.85 | |
| | Sex | Male | 97 | 58 | 59.79 | 1.14 (0.45, 2.81) |
| | | Female | 30 | 17 | 56.67 | |
| | Age | ≤30 | 52 | 34 | 65.38 | 1.57 (0.71, 3.49) |
| | | >30 | 75 | 41 | 54.67 | |
| | Genotype | B,C | 72 | 47 | 65.28 | 1.81 (0.83, 3.95) |

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| | | Non-B,C | 55 | 28 | 50.91 | |
| --- | --- | --- | --- | --- | --- | --- |
| Biochemical | Race | Asian | 82 | 47 | 57.32 | 1.77 (0.82, 3.82) |
| | | Other | 51 | 22 | 43.14 | |
| | Sex | Male | 102 | 53 | 51.96 | 1.01 (0.42, 2.45) |
| | | Female | 31 | 16 | 51.61 | |
| | Age | ≤30 | 56 | 33 | 58.93 | 1.63 (0.77, 3.48) |
| | | >30 | 77 | 36 | 46.75 | |
| | Genotype | B,C | 79 | 45 | 56.96 | 1.65 (0.78, 3.53) |
| | | Non-B,C | 54 | 24 | 44.44 | |
| Response to Treatment | Covariate | Category | n | No. of Patients with Response | Proportion (%) of Patients with Response | Unadjusted Odds Ratio (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| HBeAg Loss | Race | Asian | 84 | 21 | 25.00 | 0.89 (0.38, 2.09) |
| | | Other | 55 | 15 | 27.27 | |
| | Sex | Male | 105 | 26 | 24.76 | 0.79 (0.31, 2.11) |
| | | Female | 34 | 10 | 29.41 | |
| | Age | ≤30 | 59 | 13 | 22.03 | 0.70 (0.29, 1.63) |
| | | >30 | 80 | 23 | 28.75 | |
| | Genotype | B,C | 80 | 19 | 23.75 | 0.77 (0.34, 1.78) |
| | | Non-B,C | 59 | 17 | 28.81 | |
| HBeAg Sero-conversion | Race | Asian | 84 | 8 | 9.52 | 0.47 (0.15, 1.45) |
| | | Other | 55 | 10 | 18.18 | |
| | Sex | Male | 105 | 14 | 13.33 | 1.15 (0.33, 5.18) |
| | | Female | 34 | 4 | 11.76 | |
| | Age | ≤30 | 59 | 7 | 11.86 | 0.84 (0.26, 2.58) |
| | | >30 | 80 | 11 | 13.75 | |
| | Genotype | B,C | 80 | 6 | 7.50 | 0.32 (0.09, 1.00) |
| | | Non-B,C | 59 | 12 | 20.34 | |

The statistical significance of the associations of the Race, Sex, Age and Genotype covariates with viral response was studied and the results are summarized in the two tables below. All lower limits of the 95% confidence intervals in these two tables are smaller than 1, except for Race and Genotype at Weeks 12 and 24 (when response is defined as &lt; 2000 IU/mL). This is in agreement with logistic regression analyses resulting in no statistically significant associations between the four covariates and viral load. All lower limits of the 95% confidence intervals in Table 25 are smaller than 1

PMA P080026: FDA Summary of Safety and Effectiveness Data

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(when viral response is defined as $\geq 2$ log decrease). When response is defined as $\geq 2$ log decrease, logistic regression analyses resulted in only gender at Week 12 showing a borderline statistically significant association ($p = 0.043$) with viral load. Generally, the virological responses at Weeks 12, 24 and 48 do not appear to be correlated with Race, Sex, Age and HBV Genotype.

Odds ratios for the association between viral response ($&lt; 2000 \mathrm{IU} / \mathrm{mL}$) and covariates, by week, for an HBeAg-positive population:

| Covariate | Category | Week | n | No. Below 2000 IU/mL | Proportion (%) Below 2000 IU/mL | Unadjusted Odds Ratio (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| Race | Asian | 12 | 87 | 21 | 24.14 | 2.92 (1.04, 9.41) |
| | Other | | 61 | 6 | 9.84 | |
| | Asian | 24 | 81 | 30 | 37.04 | 3.20 (1.30, 8.42) |
| | Other | | 58 | 9 | 15.52 | |
| | Asian | 48 | 79 | 34 | 43.04 | 1.56 (0.71, 3.47) |
| | Other | | 52 | 17 | 32.69 | |
| Sex | Male | 12 | 112 | 20 | 17.86 | 0.90 (0.32, 2.78) |
| | Female | | 36 | 7 | 19.44 | |
| | Male | 24 | 104 | 27 | 25.96 | 0.67 (0.28, 1.70) |
| | Female | | 35 | 12 | 34.29 | |
| | Male | 48 | 100 | 37 | 37.00 | 0.71 (0.29, 1.76) |
| | Female | | 31 | 14 | 45.16 | |
| Age | ≤30 | 12 | 63 | 10 | 15.87 | 0.75 (0.28, 1.92) |
| | >30 | | 85 | 17 | 20.00 | |
| | ≤30 | 24 | 61 | 18 | 29.51 | 1.14 (0.50, 2.55) |
| | >30 | | 78 | 21 | 26.92 | |
| | ≤30 | 48 | 55 | 21 | 38.18 | 0.95 (0.44, 2.05) |
| | >30 | | 76 | 30 | 39.47 | |
| Genotype | B,C | 12 | 81 | 20 | 24.69 | 2.81 (1.04, 8.41) |
| | Non-B,C | | 67 | 7 | 10.45 | |
| | B,C | 24 | 76 | 29 | 38.16 | 3.27 (1.36, 8.29) |
| | Non-B,C | | 63 | 10 | 15.87 | |
| | B,C | 48 | 74 | 32 | 43.24 | 1.52 (0.70, 3.34) |
| | Non-B,C | | 57 | 19 | 33.33 | |

Odds ratios for the association between viral response ($\geq 2$ log decrease from baseline result) and covariates, by week, for an HBeAg-positive population:

PMA P080026: FDA Summary of Safety and Effectiveness Data
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41

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| Covariate | Category | Week | n | No. with ≥ 2 Log Decrease | Proportion (%) with ≥ 2 Log Decrease | Unadjusted Odds Ratio (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| Race | Asian | 12 | 87 | 54 | 62.07 | 0.86 (0.41, 1.79) |
| | Other | | 61 | 40 | 65.57 | |
| | Asian | 24 | 81 | 62 | 76.54 | 1.24 (0.53, 2.88) |
| | Other | | 58 | 42 | 72.41 | |
| | Asian | 48 | 79 | 56 | 70.89 | 0.81 (0.33, 1.91) |
| | Other | | 52 | 39 | 75.00 | |
| Sex | Male | 12 | 112 | 66 | 58.93 | 0.41 (0.15, 1.03) |
| | Female | | 36 | 28 | 77.78 | |
| | Male | 24 | 104 | 74 | 71.15 | 0.41 (0.11, 1.22) |
| | Female | | 35 | 30 | 85.71 | |
| | Male | 48 | 100 | 71 | 71.00 | 0.71 (0.23, 1.96) |
| | Female | | 31 | 24 | 77.42 | |
| Age | ≤ 30 | 12 | 63 | 41 | 65.08 | 1.13 (0.54, 2.36) |
| | > 30 | | 85 | 53 | 62.35 | |
| | ≤ 30 | 24 | 61 | 44 | 72.13 | 0.78 (0.34, 1.81) |
| | > 30 | | 78 | 60 | 76.92 | |
| | ≤ 30 | 48 | 55 | 40 | 72.73 | 1.02 (0.44, 2.41) |
| | > 30 | | 76 | 55 | 72.37 | |
| Genotype | B,C | 12 | 81 | 51 | 62.96 | 0.95 (0.46, 1.96) |
| | Non-B,C | | 67 | 43 | 64.18 | |
| | B,C | 24 | 76 | 58 | 76.32 | 1.19 (0.51, 2.75) |
| | Non-B,C | | 63 | 46 | 73.02 | |
| | B,C | 48 | 74 | 52 | 70.27 | 0.77 (0.32, 1.80) |
| | Non-B,C | | 57 | 43 | 75.44 | |

## Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Odds Ratio (OR) Analysis in an HBeAg-Positive Population

For each patient, the clin…