COBAS AMPLIPREP/COBAS TAQMAN HCV TEST
Device Facts
| Record ID | P060030 |
|---|---|
| Device Name | COBAS AMPLIPREP/COBAS TAQMAN HCV TEST |
| Applicant | Roche Molecular Systems, Inc. |
| Product Code | MZP · Microbiology |
| Decision Date | Oct 30, 2008 |
| Decision | APRL |
| Regulation | 21 CFR 866.3170 |
| Device Class | Class 2 |
Intended Use
The COBAS AmpliPrep/COBAS TaqMan HCV Test is an in vitro nucleic acid amplification test for the quantitation of hepatitis C viral (HCV) RNA in human plasma or serum using the COBAS AmpliPrep Instrument for automated specimen processing and the COBAS TaqMan Analyzer or the COBAS TaqMan 48 Analyzer for automated amplification and detection. Specimens containing HCV genotypes 1 – 6 have been validated for quantitation in the assay. The COBAS AmpliPrep/COBAS TaqMan HCV Test is intended for use as an aid in the management of HCV-infected individuals undergoing anti-viral therapy. The assay measures HCV RNA levels at baseline and during treatment and can be utilized to predict sustained and non-sustained virological response to HCV therapy. The results from the COBAS AmpliPrep/COBAS TaqMan HCV Test must be interpreted within the context of all relevant clinical and laboratory findings. Assay performance characteristics have been established for individuals treated with peginterferon alfa-2a plus ribavirin. No information is available on the assay’s predictive value when other therapies are used. Assay performance for determining the state of HCV infection has not been established. The COBAS AmpliPrep/COBAS TaqMan HCV Test is not intended for use as a screening test for the presence of HCV in blood or blood products or as a diagnostic test to confirm the presence of HCV infection.
Device Story
The COBAS AmpliPrep/COBAS TaqMan HCV Test is an automated in vitro nucleic acid amplification assay for quantitating HCV RNA in human plasma or serum. The system uses the COBAS AmpliPrep instrument for automated specimen preparation and the COBAS TaqMan or TaqMan 48 Analyzer for reverse transcription, PCR amplification, and detection. The process involves adding a known concentration of HCV Quantitation Standard (QS) to each specimen to compensate for inhibition and control preparation/amplification. The system uses dual fluorescent dye-labeled oligonucleotide probes to detect HCV target and QS amplicons. The analyzer calculates HCV RNA concentration (IU/mL) by comparing target signal to QS signal. Used in clinical laboratories, the output provides viral load measurements at baseline and during treatment to help clinicians predict virological response and manage therapy. The device benefits patients by providing quantitative data to optimize antiviral treatment duration and assess response.
Clinical Evidence
Clinical utility evaluated using retrospective specimens from a Phase III randomized multicenter study (n=1281). Evaluated negative predictive value (NPV) and positive predictive value (PPV) for sustained virologic response (SVR) at Week 4 (RVR), Week 12 (EVR), and Week 24. Analytical performance: LoD 18 IU/mL (95% hit rate); linear range 43 to 6.90E+07 IU/mL. Precision evaluated across genotypes 1-6; total SD of log10 HCV RNA concentration ranged from 0.08 to 0.45 log10 IU/mL depending on genotype and concentration.
Technological Characteristics
Nucleic acid amplification test (RT-PCR). Reagents include magnetic glass particles, lysis buffer, proteinase, elution buffer, quantitation standard (Armored RNA), and master mix (Z05 DNA Polymerase, AmpErase). Automated processing on COBAS AmpliPrep; detection on COBAS TaqMan/TaqMan 48 Analyzers. Standardized against First WHO International Standard for HCV RNA (NIBSC 96/790). Results reported in IU/mL.
Indications for Use
Indicated for quantitation of HCV RNA in human plasma or serum to aid in management of HCV-infected individuals undergoing anti-viral therapy (specifically peginterferon alfa-2a plus ribavirin). Validated for HCV genotypes 1-6. Not for screening blood/blood products or confirming HCV infection.
Regulatory Classification
Identification
A nucleic acid-based hepatitis C virus (HCV) ribonucleic acid (RNA) test is identified as an in vitro diagnostic device intended for prescription use as an aid in the diagnosis of HCV infection in specified populations, and/or as an aid in the management of HCV-infected patients including guiding the selection of genotype-specific treatment in individuals with chronic HCV infection. The test is intended for use with human serum or plasma. The test is not intended for use as a donor screening test for the presence of HCV antibodies in blood, blood products, or tissue donors.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) For all nucleic acid-based HCV RNA tests, the labeling required under § 809.10(b) of this chapter must include: (i) A prominent statement that the test is not intended for use as a donor screening test for the presence of HCV RNA from human cells, tissues, and cellular and tissue-based products. (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 management. These limitations must include, but are not limited to, statements that indicate: (A) The specimen types for which the device has been cleared and that use of this test kit with specimen types other than those specifically cleared for this device may result in inaccurate test results. (B) When applicable, that assay performance characteristics have not been established in populations of immunocompromised or immunosuppressed patients or, other populations where test performance may be affected. (C) Test results are to be interpreted by qualified licensed healthcare professionals in conjunction with the individual's clinical presentation, history, and other laboratory results. (2) For all nucleic acid-based HCV RNA tests, the design verification and validation must include: (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 nature 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) Detailed documentation of analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including, but not limited to, limit of detection (LoD), upper and lower limits of quantitation (ULoQ and LLoQ, respectively), linearity, precision, endogenous and exogenous interferences, cross reactivity, carryover, matrix equivalency, and sample and reagent stability. Samples selected for use in analytical studies or used to prepare samples for use in analytical studies must be from subjects with clinically relevant circulating genotypes in the United States. Cross-reactivity studies must include samples from HCV RNA negative subjects with other causes of liver disease, including autoimmune hepatitis, alcoholic liver disease, chronic hepatitis B virus, primary biliary cirrhosis, and nonalcoholic steatohepatitis, when applicable. The effect of each claimed nucleic-acid isolation and purification procedure on detection must be evaluated. (v) Risk analysis and management strategies, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on test performance. (vi) Final release criteria to be used for manufactured test lots with appropriate evidence that lots released at the extremes of the specifications will meet the claimed analytical and clinical performance characteristics as well as the stability claims. (vii) Multisite reproducibility study that includes the testing of three independent production lots. (viii) All stability protocols, including acceptance criteria. (ix) Final release test results for each lot used in clinical studies. (x) Analytical sensitivity and specificity of the test must be the same or better than that of other cleared or approved tests. (xi) Lot-to-lot precision studies, as appropriate. (3) For devices intended for the qualitative detection of HCV RNA, in addition to the special controls listed in paragraphs (b)(1) and (2) of this section, the design verification and validation must include detailed documentation of performance from a multisite clinical study. Performance must be analyzed relative to an FDA cleared or approved qualitative HCV RNA test, or a comparator that FDA has determined is appropriate. This study must be conducted using appropriate patient samples, with appropriate numbers of HCV positive and negative samples in applicable risk categories. Additional genotypes must be validated using appropriate numbers and types of samples. The samples may be a combination of fresh and repository samples, sourced from within and outside the United States, as appropriate. The study designs, including number of samples tested, must be sufficient to meet the following criteria: (i) Clinical sensitivity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 95 percent. (ii) Clinical specificity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 96 percent. (4) For devices intended for the quantitative detection of HCV RNA, the following special controls, in addition to those listed in paragraphs (b)(1) and (2) of this section, apply: (i) Labeling required under § 809.10(b) of this chapter must include a prominent statement that the test is not intended as a diagnostic test to confirm the presence of active HCV infection, when applicable. (ii) Design verification and validation must include the following: (A) Detailed documentation of the following analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including but not limited to: LoD, ULoQ and LLoQ. LoD, LLoQ, and linearity studies must demonstrate acceptable device performance with all HCV genotypes detected by the device. (B) Detailed documentation of clinical performance testing from either: ( *1* ) A multisite clinical study with an appropriate number of clinical samples from chronically HCV infected patients in which the results are compared to an FDA-cleared or approved quantitative HCV RNA test, or a comparator that FDA has determined is appropriate. This study must include a sufficient number of HCV positive samples containing an analyte concentration near the LLoQ to describe performance at this level. Clinical samples must cover the full range of the device output and must be consistent with the distribution of these genotypes in the U.S. population. Clinical samples may be supplemented with diluted clinical samples for those viral load concentrations that are not sufficiently covered by natural clinical specimens, or( *2* ) A clinical study with prospectively collected samples demonstrating clinical validity of the device.(C) Detailed documentation of a qualitative analysis near the lower end of the measuring range demonstrating acceptable performance when used as an aid in diagnosis. (5) For devices intended for HCV RNA genotyping, in addition to the special controls listed in paragraphs (b)(1) and (2) of this section, design verification and validation must include the following: (i) Detailed documentation of an analytical performance study demonstrating the LoD for all HCV genotypes detected by the device. (ii) Detailed documentation, including results, of a multisite clinical study that assesses genotyping accuracy ( *i.e.,* the proportion of interpretable results that match with the reference method result) and the genotyping rate (*i.e.,* the proportion of results that were interpretable).(6) For any nucleic acid-based HCV RNA test intended for Point of Care (PoC) use, the following special controls, in addition to those listed in paragraphs (b)(1) and (2) of this section, apply: (i) Clinical studies must be conducted at PoC sites. (ii) Additional labeling must include a brief summary of the instructions for use that are appropriate for use in a PoC environment.
Reference Devices
- COBAS AMPLICOR HCV MONITOR Test, v2.0
- VERSANT HCV RNA 3.0
- Abbott PRISM HCV Test
- Abbott ARCHITECT Anti-HCV Reagent Kit
Related Devices
- K252484 — cobas HCV · Roche Molecular Systems, Inc. · Nov 3, 2025
- K221007 — cobas HCV · Roche Molecular Systems, Inc. · Nov 4, 2022
- P100017 — ABBOTT REALTIME HCV, ABBOTT REALTIME HCV AMPLIFICATION REAGENT KIT, ABBOTT REALTIME HVC CONTROL KIT, ABBOTT REALTIME HCV · Abbott Molecular, Inc. · May 17, 2011
Submission Summary (Full Text)
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# SUMMARY OF SAFETY AND EFFECTIVENESS
## 1. GENERAL INFORMATION
Device Generic Name: In vitro nucleic acid amplification test for the quantitation of HCV RNA in human plasma or EDTA-serum.
Device Trade Name: COBAS® AmpliPrep/COBAS® TaqMan® HCV Test
Applicant's Name and Address: Roche Molecular Systems, Inc. (RMS)
4300 Hacienda Drive
Pleasanton, CA 94588
Premarket Approval Application (PMA) Number: P060030
Date of Panel Recommendation: None
Date of Notice of Approval: October 30, 2008
## 2. INDICATIONS FOR USE
The COBAS AmpliPrep/COBAS TaqMan HCV Test is an in vitro nucleic acid amplification test for the quantitation of hepatitis C viral (HCV) RNA in human plasma or serum using the COBAS AmpliPrep Instrument for automated specimen processing and the COBAS TaqMan Analyzer or the COBAS TaqMan 48 Analyzer for automated amplification and detection. Specimens containing HCV genotypes 1 – 6 have been validated for quantitation in the assay.
The COBAS AmpliPrep/COBAS TaqMan HCV Test is intended for use as an aid in the management of HCV-infected individuals undergoing anti-viral therapy. The assay measures HCV RNA levels at baseline and during treatment and can be utilized to predict sustained and non-sustained virological response to HCV therapy. The results from the COBAS AmpliPrep/COBAS TaqMan HCV Test must be interpreted within the context of all relevant clinical and laboratory findings.
Assay performance characteristics have been established for individuals treated with peginterferon alfa-2a plus ribavirin. No information is available on the assay’s predictive value
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# SUMMARY OF SAFETY AND EFFECTIVENESS
when other therapies are used. Assay performance for determining the state of HCV infection has not been established.
The COBAS AmpliPrep/COBAS TaqMan HCV Test is not intended for use as a screening test for the presence of HCV in blood or blood products or as a diagnostic test to confirm the presence of HCV infection.
## 3. CONTRAINDICATIONS
None Known.
## 4. WARNINGS AND PRECAUTIONS:
For In-Vitro Diagnostic Use Only
The warnings and precautions for the COBAS AmpliPrep/COBAS TaqMan HCV Test are stated in the respective product labeling.
## 5. DEVICE DESCRIPTION
The COBAS AmpliPrep/COBAS TaqMan HCV Test is an in vitro nucleic acid amplification test for the quantitation of Hepatitis C Virus (HCV) RNA in human plasma or serum using the COBAS AmpliPrep (CAP) Instrument for automated specimen processing and the COBAS TaqMan (CTM) Analyzer or the COBAS TaqMan 48 Analyzer for automated amplification and detection.
### 5.1. Kit Configuration and Components
The COBAS AmpliPrep/COBAS TaqMan HCV Test consists of the following kits:
- COBAS AmpliPrep/COBAS TaqMan HCV Test
- COBAS AmpliPrep/COBAS TaqMan Wash Reagent
Each kit contains labeled reagents assembled according to storage temperature requirements and controlled room temperature.
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## 5.2. COBAS AmpliPrep/COBAS TaqMan HCV Test Kit
COBAS AmpliPrep/COBAS TaqMan HCV Test Kit contains reagents and controls required for sample preparation, amplification and detection. All reagents are packaged in one of four barcoded reagent cassettes which are loaded directly on to the CAP instrument along with specimens and controls. User is not required to manually add any reagent prior to amplification and detection on the CTM Analyzer. The following is a list of the reagents and controls provided in the CAP/CTM HCV Test Kit:
(1) HCV CS1 (HCV Magnetic Glass Particles Reagent Cassette) 1x48 Tests
Magnetic glass particles resuspended in a 93% (w/w) isopropanol solution
(2) HCV CS2 (HCV Lysis Reagent Cassette) 1x48 Tests
A sodium citrate buffered solution containing guanidine thiocyanate, polydocanol and ditiothreitol.
(3) HCV CS3 (HCV Multi-Reagent Cassette) 1 x 48 Tests
**Pase (Proteinase Solution)** 1 x 3.8 mL
A Tris buffered solution containing EDTA, calcium chloride, calcium acetate, proteinase and glycerol.
**EB (Elution Buffer)** 1 x 7.0 mL
A tris buffer containing methylparaben.
(4) HCV CS4 (HCV Test-Specific Reagent Cassette) 1 x 48 Tests
**HCV QS (HCV Quantitation Standard)** 1 x 3.6 mL
A Tris buffered solution containing:
EDTA,
< 0.002% Poly rA RNA (synthetic),
< 0.001% Armored HCV RNA construct containing HCV primer binding sequences and a unique probe binding region (non-infectious RNA in MS2 bacteriophage), and
0.05% sodium azide
**HCV MMX (HCV Master Mix)** 1 x 2.5 mL
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# SUMMARY OF SAFETY AND EFFECTIVENESS
A Tricine buffered solution containing:
potassium acetate,
potassium hydroxide,
< 20% dimethylsulfoxide,
Glycerol,
< 0.04% dATP, dCTP, dGTP, dUTP, dTTP,
< 0.002% Upstream and downstream primers to the UTR region of HCV,
< 0.001% Fluorescent-labeled oligonucleotide probes specific for HCV and the HCV QS,
< 0.001% Oligonucleotide aptamer,
< 0.05% Z05 DNA Polymerase (microbial)
< 0.1% AmpErase (uracil-N-glycosylase) enzyme (microbial), and
0.09% Sodium azide
**CAP/CTM Mn²⁺ (CAP/CTM Manganese Solution)** 1 x 19 mL
A solution containing:
< 0.5% manganese acetate,
glacial acetic acid, and
0.09% sodium azide.
(5) **HCV H(+ )C (HCV High Positive Control)** 4 x 1.0 mL
< 0.001% Armored HCV RNA construct containing HCV sequences (non-infectious RNA in MS2 bacteriophage)
Negative Human Plasma, non-reactive by tests for antibody to HCV, antibody to HIV-1/2, HIV p24 antigen and HBsAg; HIV-1 RNA, HCV RNA and HBV DNA not detectable by PCR methods
0.1% ProClin 300
(6) **HCV L(+ )C (HCV Low Positive Control)** 4 x 1.0 mL
< 0.001% Armored HCV RNA construct containing HCV sequences (non-infectious RNA in MS2 bacteriophage)
Negative Human Plasma, non-reactive by tests for antibody to HCV, antibody to HIV-1/2, HIV p24 antigen and HBsAg; HIV-1 RNA, HCV RNA and HBV DNA not detectable by PCR methods
0.1% ProClin 300
(7) **CTM (-) C [COBAS TaqMan Negative Control (Human Plasma)]** 4 x 1.0 mL
Negative Human Plasma, non-reactive by tests for antibody to HCV, antibody to HIV-1/2, HIV p24 antigen and HBsAg; HIV-1 RNA, HCV RNA and HBV DNA not detectable by PCR methods
0.1% ProClin 300
(8) **HCV H(+ )C Clip (HCV High Positive Control Barcode Clip)** 1 x 4 Clips
(9) **HCV L(+ )C Clip (HCV Low Positive Control Barcode Clip)** 1 x 4 Clips
(10) **HCV (-)C Clip (HCV Negative Control Barcode Clip)** 1 x 4 Clips
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## 5.3. COBAS AmpliPrep/COBAS TaqMan Wash Reagent (PG WR)
The CAP/CTM Wash Reagent (PG WR) is a generic system fluid using for various washing steps performed on the COBAS AmpliPrep instrument during sample preparation. The ingredients in PG WR are listed below:
(1) PG WR (COBAS AmpliPrep/COBAS TaqMan Wash Reagent)
1 x 5.1 L
A sodium citrate dehydrate solution containing < 0.1% Methylisothiazolone-HCl
## 5.4. Assay Principle and Format
The COBAS AmpliPrep/COBAS TaqMan HCV Test is based on three major processes: (1) specimen preparation to isolate HCV RNA; (2) reverse transcription of the target RNA to generate complementary DNA (cDNA), and (3) simultaneous PCR amplification of target cDNA and detection of amplified c-DNA (amplicon) using cleaved dual fluorescent dye-labeled oligonucleotide detection probes.
The COBAS AmpliPrep/COBAS TaqMan HCV Test permits automated specimen preparation followed by automated reverse transcription, PCR amplification and detection of HCV target RNA and HCV Quantitation Standard (QS) Armored RNA. The Master Mix reagent contains primers and probes specific for both HCV RNA and HCV QS Armored RNA. The Master Mix has been developed to ensure similar quantitation of HCV genotypes 1 through 6. The detection of amplified DNA is performed using a target-specific and a QS-specific dual-labeled oligonucleotide probe that permit independent identification of HCV amplicon and HCV QS amplicon.
The quantitation of HCV viral RNA is performed using the HCV QS. The HCV QS compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation of HCV RNA in each specimen. The HCV QS is a non-infectious Armored RNA construct that contains HCV sequences with identical primer binding sites as the
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# SUMMARY OF SAFETY AND EFFECTIVENESS
HCV target RNA and a unique probe binding region that allows HCV QS amplicon to be distinguished from HCV target amplicon.
The HCV QS is added to each specimen at a known copy number and is carried through the specimen preparation, reverse transcription, PCR amplification and detection of cleaved dual-labeled oligonucleotide detection probes. The COBAS TaqMan Analyzer or COBAS TaqMan 48 Analyzer calculates the HCV RNA concentration in the test specimens by comparing the HCV signal to the HCV QS signal for each specimen and control.
## 5.4.1. Fundamentals of COBAS TaqMan Test Quantitation:
The COBAS AmpliPrep/COBAS TaqMan HCV Test is inherently quantitative over a very wide dynamic range since the monitoring of amplicon is performed during the exponential phase of amplification. The higher the HCV titer of a specimen, the earlier the fluorescence of the reporter dye of the HCV probe rises above the baseline fluorescence level (see Figure 1). Since the amount of HCV QS RNA is constant between all specimens, the fluorescence of the reporter dye of the HCV QS probe should appear at the same cycle for all specimens (see Figure 2). In specimens, where the QS amplification and detection is affected by inhibition or poor specimen recovery, the appearance of fluorescence will be delayed, thereby enabling the calculated titer of HCV target RNA to be adjusted accordingly. The appearance of the specific fluorescent signals is reported as a critical threshold value (Ct). The Ct is defined as the fractional cycle number where reporter dye fluorescence exceeds a predetermined threshold (the Assigned Fluorescence Level), and starts the beginning of an exponential growth phase of this signal (see Figure 3). A higher Ct value indicates a lower titer of initial HCV target material. A 2-fold increase in titer correlates with a decrease of 1 Ct for target HCV RNA, while a 10-fold increase in titer correlates with a decrease of 3.3 Ct.
Figure 1 depicts the target growth curves for a dilution series spanning a $5\text{-}\log_{10}$ range. As the concentration of the virus increases, the growth curves shift to earlier cycles. Therefore, the
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# SUMMARY OF SAFETY AND EFFECTIVENESS
leftmost growth curve corresponds to the highest viral titer level, whereas, the rightmost growth curve corresponds to the lowest viral titer level.
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Figure 1: Target Growth Curves for a Dilution Series Spanning a 5-Log₁₀ Range
Figure 2 depicts the Quantitation Standard growth curves for specimens from a viral dilution series that spans a 5-log₁₀ range. The amount of Quantitation Standard added to each specimen is constant for each reaction. The Ct value of the Quantitation Standard is similar regardless of the viral titer.
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Figure 2: Quantitation Standard Growth Curves for Specimens from a Viral Dilution Series that Spans a 5-Log₁₀ Range
Figure 3 provides an example of how the fluorescence values at every cycle are normalized for each growth curve. The fractional cycle number (Ct) is calculated where the fluorescence signal crosses the Assigned Fluorescence Level.
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Figure 3: Fluorescence Values at Every Cycle are Normalized for Each Growth Curve
## 5.4.2. HCV RNA Quantitation
The COBAS AmpliPrep/COBAS TaqMan HCV Test quantitates HCV viral RNA by utilizing a second target sequence (HCV Quantitation Standard) that is added to each test specimen at a known concentration. The HCV QS is a non-infectious Armored RNA construct, containing fragments of HCV sequences with primer binding regions identical to those of the HCV target sequence. The HCV QS generates an amplification product of the same length and base composition as the HCV target RNA. The detection probe binding region of the HCV QS has been modified to differentiate HCV QS amplicon from HCV target amplicon.
During the annealing phase of the PCR on the COBAS TaqMan Analyzer or COBAS TaqMan 48 Analyzer, the specimens are illuminated and excited by filtered light and filtered emission fluorescence data are collected for each specimen. The readings from each specimen are then corrected for instrumental fluctuations. These fluorescence readings are sent by the instrument to
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# SUMMARY OF SAFETY AND EFFECTIVENESS
the AMPLILINK software and stored in a database. Pre-Checks are used to determine if the HCV RNA and HCV QS RNA data represent sets that are valid, and flags are generated when the data lie outside the preset limits. After all Pre-Checks are completed and passed, the fluorescence readings are processed to generate Ct values for the HCV RNA and the HCV QS RNA. The lot-specific calibration constants provided with the COBAS AmpliPrep/COBAS TaqMan HCV Test are used to calculate the titer value for the specimens and controls based upon the HCV RNA and HCV QS RNA Ct values. The COBAS AmpliPrep/COBAS TaqMan HCV Test is standardized against the First WHO International Standard for Hepatitis C Virus RNA for Nucleic Acid Amplification Technology Assays (NIBSC code 96/790) and titer results are reported in International Units (IU/mL) $^{1,2}$ .
## 5.5. Results
The COBAS TaqMan Analyzer or the COBAS TaqMan 48 Analyzer automatically determines the HCV RNA concentration for the specimens and controls. The HCV RNA concentration is expressed in International Units (IU)/mL.
### 5.5.1. AMPLILINK Software
- Determines the Cycle Threshold value (Ct) for the HCV RNA and the HCV QS RNA.
- Determines the HCV RNA concentration based upon the Ct values for the HCV RNA and HCV QS RNA and the lot-specific calibration coefficients provided on the cassette barcodes.
- Determines that the calculated IU/mL for HCV L(+)C and HCV H(+)C fall within the lot specific assigned ranges encoded on the COBAS AmpliPrep/COBAS TaqMan HCV Test reagent cassette barcodes supplied with the kit.
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## 5.5.2. Batch Validation
The batch is valid if no flags appear for any of the controls [HCV L(+ )C, HCV H(+ )C and CTM (–) C]. The following results are obtained for a valid batch.
| Control | Result | Interpretation |
| --- | --- | --- |
| Negative Control | Target Not Detected | Control Within Range |
| Low Postive Control | A numeric titer X.XXE+XX IU/mL | Control Within Range |
| High Positive Control | A numeric titer X.XXE+XX IU/mL | Control Within Range |
## 5.6. Interpretation of Results
For a valid batch, check each individual specimen for flags or comments on the result printout. Interpret the results as follows:
- A valid batch may include both valid and invalid specimen results depending on whether flags and/or comments are obtained for the individual specimens.
Specimen results are interpreted as follows:
| Titer Result | Interpretation |
| --- | --- |
| Target Not Detected | No Ct value for HCV obtained. Report results as "HCV RNA not detected". |
| < 4.3E+01 IU/mL | Below 4.3E+01 IU/mL (lower limit of quantitation, LLoQ); HCV RNA is not quantifiable. |
| ≥ 4.30E+01 IU / mL and ≤ 6.90E+07 IU / mL | Results greater than or equal to 43 IU/mL and less than or equal to 6.90E+07 IU/mL are within the Linear Range of the assay. |
| > 6.90E+07 IU / mL | Results are above the range of the assay. Report results as "greater than 6.90E+07 HCV RNA IU/mL". If quantitative results are desired, the original specimen should be diluted 1:100 with HCV-negative human serum or EDTA plasma, depending on the matrix of the original specimen, and the test repeated. Multiply the reported result by the dilution factor. |
Note: Specimens above the range of the assay may also produce an invalid result with a flag "QS_INVALID". If quantitative results are desired, the original specimen should be diluted 1:100 with HCV-negative human serum or EDTA plasma, depending on the matrix of the original specimen, and the test repeated. Multiply the reported result by the dilution factor.
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# SUMMARY OF SAFETY AND EFFECTIVENESS
## 6. ALTERNATIVE PRACTICES AND PROCEDURES
There are currently a variety of commercially available direct and indirect methods for the detection and quantitation of Hepatitis C in clinical specimens.
Some of these are listed below:
- ELISA, EIA and immunoblot procedures for measuring HCV antibody production
- Nucleic acid probe technologies for direct detection and quantitation of circulating viral particles
## 7. MARKETING HISTORY
The COBAS AmpliPrep/COBAS TaqMan HCV Test has not been withdrawn from the following markets for reasons related to safety or effectiveness. The test is currently available in the following countries:
| Argentina | Greece | Norway |
| --- | --- | --- |
| Australia | Hungary | Poland |
| Austria | Iceland | Portugal |
| Belgium | Indonesia | Romania |
| Bulgaria | Ireland | Russia |
| Canada | Italy | Slovakia |
| China | Japan | Slovenia |
| Cyprus | Korea | Spain |
| Czech Republic | Latvia | Sweden |
| Denmark | Liechtenstein | Switzerland |
| Estonia | Lithuania | Taiwan |
| Finland | Luxembourg | Thailand |
| France | Malta | Turkey |
| Germany | Netherlands | United kingdom |
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## 8. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH
Since the COBAS AmpliPrep/COBAS TaqMan HCV Test is for *in vitro* diagnostic use, there is no direct adverse effect on the health of the patient. However, failure of the product to perform as indicated or human error in use of the product may lead to a false result.
## 9. SUMMARY OF NONCLINICAL STUDIES
### 9.1. Laboratory Studies
#### 9.1.1. Traceability to the WHO Standard
Several standards and controls have been used during development of this test to provide traceability to the WHO Standard. This includes HCV WHO Standard, RMS HCV Secondary Standard, and RMS Armored HCV RNA calibrators. The HCV WHO Standard, RMS HCV Secondary Standard, RMS HCV Secondary Standard Source Material, RMS Armored HCV RNA Calibration Material and an independent HCV genotype 1a specimen were tested at similar levels. Calibrator ranged from 3.0E+02 IU/mL 2.0E+07 IU/mL (2.48 to 7.30 log₁₀), the HCV WHO Standard ranged from 3.0E+02 IU/mL to 3.6E+04 IU/mL (2.48 to 4.56 log₁₀), the HCV Secondary Standard Source Material ranged from 3.0E+02 IU/mL to 2.0E+06 IU/mL (2.48 to 6.30 log₁₀), and an HCV genotype 1a specimen ranged from 3.0E+02 IU/mL to 2.0E+06 IU/mL (2.48 to 6.30 on log₁₀).
All materials behaved similarly and demonstrated co-linear dilution performance across the linear range.
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Figure 4: Traceability of the COBAS AmpliPrep/COBAS TaqMan HCV Test to the HCV WHO Standard
# 9.1.2. Limit of Detection using the WHO International Standard
The limit of detection of the COBAS AmpliPrep/COBAS TaqMan HCV Test was determined by analysis of serial dilutions of the First WHO International Standard for Hepatitis C Virus RNA for Nucleic Acid Amplification Technology Assays (NIBSC code 96/790), genotype 1a, obtained from NIBSC, in HCV negative human EDTA plasma or serum. First WHO International Standard was freshly diluted into negative human EDTA plasma or serum on three days for each matrix. Each level of each dilution was tested with ten replicates each in three runs for each of two reagent lots for each matrix. A total of three runs were conducted over three days for each reagent lot for each matrix to give a total of 60 replicates for each level for each matrix. These studies demonstrate that the COBAS AmpliPrep/COBAS TaqMan HCV Test can detect HCV RNA in EDTA plasma and serum at concentrations as low as $18\mathrm{IU / mL}$ with a positivity rate greater than $95\%$ . The concentration of HCV RNA using the First WHO International Standard in EDTA plasma and serum that can be detected with a positivity rate of greater than $95\%$ as determined by Probit Analysis, is $13.9\mathrm{IU / mL}$ and $10.5\mathrm{IU / mL}$ , respectively (see Table 1
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# SUMMARY OF SAFETY AND EFFECTIVENESS
and Table 2 below). The difference between serum and EDTA plasma was not statistically significant.
Table 1: Limit of Detection in EDTA Plasma of the COBAS AmpliPrep/ COBAS TaqMan HCV Test using the First WHO International Standard (Genotype 1a)
| WHO Standard Based Concentration (HCV RNA IU/mL) | No. Valid Replicates | No. Positives | Positivity Rate |
| --- | --- | --- | --- |
| 0.0 | 56 | 0 | 0% |
| 2.5 | 57 | 30 | 53% |
| 5.0 | 58 | 41 | 71% |
| 7.5 | 59 | 45 | 76% |
| 10.0 | 60 | 53 | 88% |
| 15.0 | 58 | 58 | 100% |
| 25.0 | 56 | 56 | 100% |
| 50.0 | 57 | 57 | 100% |
| Probit 95% Hit Rate | 13.9 IU/mL [95% confidence limits of 11.0 – 19.8 IU/mL] | | |
Table 2: Limit of Detection in Serum of the COBAS AmpliPrep/ COBAS TaqMan HCV Test using the First WHO International Standard (Genotype 1a)
| WHO Standard Based Concentration (HCV RNA IU/mL) | No. Valid Replicates | No. Positives | Positivity Rate |
| --- | --- | --- | --- |
| 0.0 | 59 | 0 | 0% |
| 2.5 | 60 | 37 | 62% |
| 5.0 | 59 | 43 | 73% |
| 7.5 | 60 | 51 | 85% |
| 10.0 | 59 | 57 | 97% |
| 15.0 | 60 | 60 | 100% |
| 25.0 | 58 | 58 | 100% |
| 50.0 | 60 | 60 | 100% |
| Probit 95% Hit Rate | 10.5 IU/mL [95% confidence limits of 8.4 – 14.8 IU/mL] | | |
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## 9.1.3. Limit of Detection Using Clinical Specimens across HCV Genotypes
The Limit of Detection for HCV genotypes 1 to 6 was determined by obtaining eight clinical specimens representing genotypes 1, 2, 3, 4, 5, and 6. Original titers for these clinical samples were provided by the vendor and 8 member dilution panels in negative human EDTA plasma were prepared from each sample representing a genotype. The panel members were tested in two runs with 12 replicates per run for a total of 24 replicates for each member. Limit of detection for a genotype is defined as the mean concentration of the lowest panel member at which more than 95% of the results are positive. The results are presented in Table 3 below. The overall LoD for this assay is defined as 18 IU/mL.
Table 3: Limit of Detection for HCV Genotypes
| Genotype | Mean Conc. of the Panel Member with >95% Positivity Rate (IU/mL) | Number of Replicates Tested | Number of Positive Results | Positivity Rate |
| --- | --- | --- | --- | --- |
| 1 | 7.1 | 24 | 23 | 96% |
| 2 | 15.3 | 24 | 24 | 100% |
| 3 | 9.8 | 24 | 24 | 100% |
| 4 | 5.6 | 24 | 23 | 96% |
| 5 | 18.3 | 24 | 24 | 100% |
| 6 | 9.7 | 24 | 23 | 96% |
## 9.1.4. Linear Range
The linear range was evaluated in accordance with the methods defined in the CLSI (formerly NCCLS) Guideline EP6A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline.² Two linearity panels were used to evaluate the linear range of the COBAS AmpliPrep/COBAS TaqMan HCV Test. These panels consisted of dilutions in either EDTA plasma or in serum of a high titer HCV RNA positive clinical specimen for the lower and middle part of the dynamic range and, due to unavailability of very high titer clinical material, of Armored HCV RNA for the high end of the dynamic range. The study was
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performed for two lots of COBAS AmpliPrep/COBAS TaqMan HCV Test reagents. All 15 panel members for EDTA plasma and all 14 panel members for serum were tested in 104 to 111 replicates per concentration level.
The COBAS AmpliPrep/COBAS TaqMan HCV Test was found to give a linear response from 43 HCV RNA IU/mL to at least 6.90E+07 HCV RNA IU/mL with maximum observed deviation of 0.2 log₁₀ from linearity.
Figure 5 and Figure 6 are representative plots from one of the two lots tested.
The analytical measurement range of analyte values that can be directly measured on a sample with out any dilution using the COBAS AmpliPrep/COBAS TaqMan HCV Test is 43 to 6.9E+07 IU/mL.
The clinical reportable range of analyte values that can be directly measured on a sample with a maximum dilution of one to one-hundred using the COBAS AmpliPrep/COBAS TaqMan HCV Test is 43 to 6.9E+09 IU/mL.
Figure 5: Linear Range Determination for the COBAS AmpliPrep/ COBAS TaqMan HCV Test in EDTA Plasma Specimens
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Figure 6: Linear Range Determination for the COBAS AmpliPrep/ COBAS TaqMan HCV Test in Serum Specimens
# 9.1.5. Precision - Within Laboratory
Within-Run, Run-to-Run and Total Precision were evaluated in accordance with the methods defined in the NCCLS Guideline EP5-A2, Evaluation of Precision Performance of Clinical Chemistry Devices. $^{4}$ Precision of the COBAS $^{\text{®}}$ AmpliPrep/COBAS TaqMan $^{\text{®}}$ HCV Test was determined by analysis of serial dilutions of clinical HCV specimens (Genotype 1) or of Armored HCV RNA in HCV negative human EDTA plasma or in serum.
Six dilution levels of 7 replicates per level were tested in $\geq 15$ runs over $\geq 15$ days. Each sample was carried through the entire COBAS® AmpliPrep/COBAS TaqMan® HCV Test procedure, including specimen preparation, amplification and detection. Therefore, the precision reported here represents all aspects of the test procedure. The study was performed for three lots of
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# SUMMARY OF SAFETY AND EFFECTIVENESS
COBAS® AmpliPrep/COBAS TaqMan® HCV Test reagents, and the results are shown in Table 4 through Table 7.
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Table 4: Precision of the COBAS AmpliPrep/COBAS TaqMan HCV Test Using Plasma (in IU/mL)
| Lot | Sample Type | Native HCV RNA | | | Armored HCV RNA | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Titer (IU/mL) | 1.42E+02 | 1.42E+03 | 1.42E+04 | 1.48E+05 | 1.48E+06 | 1.48E+07 |
| | Within Run CV (%)* | 43 | 28 | 26 | 19 | 19 | 29 |
| | Run To Run CV (%)* | 18 | 8 | 7 | 9 | 11 | 11 |
| | Total CV (%)* | 47 | 29 | 27 | 21 | 22 | 31 |
| | No. Replicates | 109 | 110 | 110 | 109 | 109 | 109 |
| 2 | Titer (IU/mL) | 1.54E+02 | 7.72E+03 | 7.72E+04 | 9.60E+04 | 9.60E+05 | 9.60E+06 |
| | Within Run CV (%)* | 43 | 29 | 28 | 35 | 32 | 26 |
| | Run To Run CV (%)* | 23 | 9 | 12 | 0 | 6 | 6 |
| | Total CV (%)* | 50 | 31 | 30 | 35 | 32 | 27 |
| | No. Replicates | 98 | 98 | 97 | 97 | 97 | 98 |
| 3 | Titer (IU/mL) | 1.42E+02 | 1.42E+03 | 1.42E+04 | 1.29E+05 | 1.29E+06 | 1.29E+07 |
| | Within Run CV (%)* | 27 | 13 | 13 | 16 | 18 | 15 |
| | Run To Run CV (%)* | 7 | 9 | 4 | 4 | 4 | 5 |
| | Total CV (%)* | 28 | 16 | 14 | 17 | 18 | 16 |
| | No. Replicates | 98 | 98 | 98 | 98 | 98 | 98 |
* %CV = 100 × $\sqrt{10^{n^2 \ln(10)} - 1}$
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Table 5: Precision of the COBAS AmpliPrep/COBAS TaqMan HCV Test using Plasma (in $\log_{10}$ IU/mL)
| Lot | Sample Type | Native HCV RNA | | | Armored HCV RNA | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Titer (log_{10} IU/mL) | 2.15 | 3.15 | 4.15 | 5.17 | 6.17 | 7.17 |
| | Within Run Standard Deviation | 0.178 | 0.118 | 0.112 | 0.083 | 0.082 | 0.122 |
| | Run To Run Standard Deviation | 0.076 | 0.036 | 0.03 | 0.038 | 0.046 | 0.047 |
| | Total Standard Deviation (log 10) | 0.194 | 0.123 | 0.116 | 0.091 | 0.094 | 0.131 |
| | No. Replicates | 109 | 110 | 110 | 109 | 109 | 109 |
| 2 | Titer (log_{10} IU/mL) | 2.19 | 3.89 | 4.89 | 4.98 | 5.98 | 6.98 |
| | Within Run Standard Deviation | 0.18 | 0.124 | 0.119 | 0.148 | 0.135 | 0.11 |
| | Run To Run Standard Deviation | 0.096 | 0.038 | 0.05 | 0 | 0.024 | 0.027 |
| | Total Standard Deviation (log 10) | 0.204 | 0.129 | 0.129 | 0.148 | 0.137 | 0.113 |
| | No. Replicates | 98 | 98 | 97 | 97 | 97 | 98 |
| 3 | Titer (log_{10} IU/mL) | 2.15 | 3.15 | 4.15 | 5.11 | 6.11 | 7.11 |
| | Within Run Standard Deviation | 0.116 | 0.057 | 0.057 | 0.069 | 0.076 | 0.063 |
| | Run To Run Standard Deviation | 0.031 | 0.039 | 0.019 | 0.017 | 0.016 | 0.023 |
| | Total Standard Deviation (log 10) | 0.12 | 0.07 | 0.06 | 0.071 | 0.077 | 0.068 |
| | No. Replicates | 98 | 98 | 98 | 98 | 98 | 98 |
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Table 6: Precision of the COBAS AmpliPrep/COBAS TaqMan HCV Test using Serum (in IU/mL)
| Lot | Sample Type | Native HCV RNA | | | Armored HCV RNA | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Titer (IU/mL) | 4.80E+02 | 4.80E+03 | 4.80E+04 | 6.90E+05 | 6.90E+06 | 6.90E+07 |
| | Within Run CV (%)* | 23 | 17 | 16 | 17 | 21 | 26 |
| | Run To Run CV (%)* | 29 | 12 | 11 | 26 | 28 | 23 |
| | Total CV (%)* | 38 | 21 | 20 | 32 | 35 | 34 |
| | No. Replicates | 104 | 104 | 104 | 104 | 104 | 104 |
| 2 | Titer (IU/mL) | 3.50E+02 | 3.50E+03 | 3.50E+04 | 1.56E+05 | 1.56E+06 | 1.56E+07 |
| | Within Run CV (%)* | 25 | 16 | 22 | 16 | 15 | 14 |
| | Run To Run CV (%)* | 10 | 4 | 10 | 12 | 18 | 13 |
| | Total CV (%)* | 27 | 17 | 24 | 20 | 23 | 20 |
| | No. Replicates | 103 | 103 | 102 | 98 | 97 | 98 |
| 3 | Titer (IU/mL) | 1.96E+02 | 1.96E+03 | 1.96E+04 | 1.69E+05 | 1.69E+06 | 1.69E+07 |
| | Within Run CV (%)* | 31 | 14 | 15 | 16 | 14 | 18 |
| | Run To Run CV (%)* | 6 | 3 | 6 | 0 | 8 | 8 |
| | Total CV (%)* | 32 | 14 | 16 | 16 | 16 | 19 |
| | No. Replicates | 90 | 91 | 90 | 90 | 90 | 90 |
* %CV = 100 × $\sqrt{10^{n^2 \ln(10)} - 1}$
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Table 7: Precision of the COBAS AmpliPrep/COBAS TaqMan HCV Test using Serum (in $\log_{10}$ IU/mL)
| Lot | Sample Type | Native HCV RNA | | | Armored HCV RNA | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Titer (log_{10} IU/mL) | 2.68 | 3.68 | 4.68 | 5.84 | 6.84 | 7.84 |
| | Within Run Standard Deviation | 0.122 | 0.052 | 0.049 | 0.112 | 0.119 | 0.114 |
| | Run To Run Standard Deviation | 0.1 | 0.072 | 0.068 | 0.075 | 0.088 | 0.106 |
| | Total Standard Deviation (log 10) | 0.158 | 0.089 | 0.084 | 0.134 | 0.148 | 0.156 |
| | No. Replicates | 104 | 104 | 104 | 104 | 104 | 105 |
| 2 | Titer (log_{10} IU/mL) | 2.54 | 3.54 | 4.54 | 5.19 | 6.19 | 7.19 |
| | Within Run Standard Deviation | 0.105 | 0.07 | 0.094 | 0.07 | 0.064 | 0.062 |
| | Run To Run Standard Deviation | 0.045 | 0.017 | 0.042 | 0.052 | 0.076 | 0.058 |
| | Total Standard Deviation (log 10) | 0.114 | 0.072 | 0.103 | 0.087 | 0.099 | 0.085 |
| | No. Replicates | 103 | 103 | 102 | 98 | 97 | 98 |
| 3 | Titer (log_{10} IU/mL) | 2.29 | 3.29 | 4.29 | 5.23 | 6.23 | 7.23 |
| | Within Run Standard Deviation | 0.131 | 0.06 | 0.064 | 0.069 | 0.06 | 0.077 |
| | Run To Run Standard Deviation | 0.024 | 0.012 | 0.024 | 0 | 0.034 | 0.033 |
| | Total Standard Deviation (log 10) | 0.133 | 0.061 | 0.068 | 0.069 | 0.068 | 0.084 |
| | No. Replicates | 90 | 91 | 90 | 90 | 90 | 90 |
## 9.1.6. Inclusivity
The performance of the COBAS AmpliPrep/COBAS TaqMan HCV Test on HCV genotypes was evaluated by (1) testing the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques, NIBSC Code 02/202 and by (2) comparing the log10 titer of clinical specimens of
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# SUMMARY OF SAFETY AND EFFECTIVENESS
HCV genotypes 1-5 to the $\log_{10}$ titer obtained for the COBAS® AMPLICOR HCV MONITOR Test, v2.0 and the VERSANT® HCV RNA 3.0.
## 9.1.6.1. HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques, NIBSC Code 02/202
The HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques, NIBSC Code 02/202 comprises 6 members of HCV genotypes 1 through 6 with a titer of 1000 IU/mL assigned by NIBSC22. The panel was tested in single determination with one lot of COBAS AmpliPrep/COBAS TaqMan HCV Test reagents. The results are presented in Table 8.
Table 8: HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques, NIBSC Code 02/202
| Panel Member | HCV Genotype | Titer (IU/mL) | Log_{10} Titer NIBSC | Log_{10} Titer CAP/CTM HCV | Difference in Titer (CAP/CTM – NIBSC) |
| --- | --- | --- | --- | --- | --- |
| NIBSC-1 | 1 | 1000 | 3.0 | 3.2 | 0.2 |
| NIBSC-2 | 2 | 1000 | 3.0 | 3.3* | 0.3 |
| NIBSC-3 | 3 | 1000 | 3.0 | 3.2 | 0.2 |
| NIBSC-4 | 4 | 1000 | 3.0 | 3.1 | 0.1 |
| NIBSC-5 | 5 | 1000 | 3.0 | 2.9** | -0.1 |
| NIBSC-6 | 6 | 1000 | 3.0 | 3.3 | 0.3 |
* Mean value of two replicates
** Repeat measurement due to volume error
## 9.1.7. Performance of the COBAS AmpliPrep/COBAS TaqMan HCV Test with HCV Negative Specimens
The performance of the COBAS AmpliPrep/COBAS TaqMan HCV Test was determined by analysis of HCV RNA-negative EDTA or serum samples from blood donors (all samples were pre-screened by either the Abbott PRISM HCV Test or by the Abbott ARCHITECT Anti-HCV Reagent Kit). A total of 808 individual EDTA plasma specimens and a total of 768 individual serum specimens were tested with two lots of COBAS AmpliPrep/COBAS TaqMan HCV Test reagents. All specimens tested negative for HCV RNA. In this panel the specificity of the
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SUMMARY OF SAFETY AND EFFECTIVENESS
COBAS AmpliPrep/COBAS TaqMan HCV Test is 100% (one-sided lower 95% confidence limit: ≥99.6%).
## 9.1.8. Cross Reactivity
The cross reactivity of the COBAS AmpliPrep/COBAS TaqMan HCV Test was evaluated by adding different pathogens (viruses, bacteria, yeast) or isolated cellular DNA (HTLV-II) into HCV negative human EDTA plasma or HCV positive human plasma (see Table 9). Stocks of non-HCV viruses as well as bacteria and yeast were diluted to a level of approximately 5E+04 particles/mL, except for HTLV-II which was available as HTLV-II infected cell DNA only. The HTLV-II infected cell DNA was used at approximately 5E+04 copies/mL. None of the non-HCV pathogens showed a false positive result in the COBAS AmpliPrep/COBAS TaqMan HCV Test.
Table 9: Cross Reactivity Specimens
| Viruses | Non-HCV Flavivirus |
| --- | --- |
| Adenovirus type 2 | West Nile Virus |
| Cytomegalovirus | St. Louis Encephalitis Virus |
| Epstein-Barr virus | Murray Valley Encephalitis Virus |
| Human Herpes Virus type 6 | Dengue Virus Type 1 |
| Herpes simplex virus type 1 | Dengue Virus Type 2 |
| Herpes simplex virus type 2 | Dengue Virus Type 3 |
| Human T-Cell Lymphotropic virus type 1 | Dengue Virus Type 4 |
| Human T-cell Lymphotronic virus type 2 | Yellow Fever Virus |
| Influenza A | Zika Virus |
| Hepatitis A virus | Banzi Virus |
| Hepatitis B virus | Ilheus |
| Human Immunodeficiency Virus Type 1B | FSME Virus |
| | Heptatis G Virus (GBV-C) |
| Bacteria | Yeast |
| Staphylococcus aureus | Candida albicans |
| Propionibacterium acnes | |
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# 9.1.9. Matrix Equivalency — Serum versus EDTA Plasma
Twenty-nine matched clinical specimen sets (each set is EDTA plasma and serum drawn from a single HCV seropositive individual) with titers ranging from 1.58 E+03 IU/mL to 1.69E+07 IU/mL (3.2 log₁₀IU/mL to 7.2 log₁₀IU/mL) were tested to demonstrate plasma—serum equivalency. Each specimen was tested in triplicate. The pooled standard deviation is calculated for each matrix (serum as well as plasma). Deming Regression is performed comparing each sample’s individual log titer for serum to the corresponding individual log titer for plasma.
The log₁₀ titer difference (mean log titer serum – mean log titer EDTA plasma) for all 29 matched sets was ≤0.3. The mean difference was -0.024 log (95% CI: -0.06 and 0.01), indicating that the results between serum and EDTA plasma were not significantly different. The result from the Deming regression analysis is shown in Figure 7. The slope is equal to 0.9855 (95% confidence interval [0.9619 to 1.0090]) with an intercept of +0.0557 (95% confidence interval is [-0.0741 to 0.1855]). The pooled standard deviation estimates for the EDTA plasma and serum samples are shown in Table 10.
Figure 7: Deming Regression Analysis of Matched Serum — EDTA Plasma Samples (n=29)
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Table 10: Pooled Standard Deviation Estimates for EDTA Plasma and Serum Specimens in the Matrix Equivalency Study
| Matrix | Mean log Titer | Pooled SD |
| --- | --- | --- |
| EDTA Plasma | 5.430 | 0.0771 |
| Serum | 5.406 | 0.0815 |
## 9.1.10. Platform Equivalency – COBAS TaqMan Analyzer & COBAS TaqMan 48 Analyzer
Comparison between the COBAS TaqMan Analyzer (CTM96) and COBAS TaqMan 48 Analyzer (CTM48) was assessed using 67 clinical specimens with titer levels ranging from $3.23\mathrm{E} + 02\mathrm{IU / mL}$ to $1.70\mathrm{E} + 07\mathrm{IU / mL}$ ($2.5\log_{10}\mathrm{IU / mL}$ to $7.2\log_{10}\mathrm{IU / mL}$). The performance was assessed using Deming regression analysis. The results of the analysis are presented in Figure 8 and Table 11. The $95\%$ confidence intervals for slope and intercept include “1” and “0” respectively, indicating that the performance of the two analyzers is equivalent when testing clinical specimens
Figure 8: Platform Equivalency – CTM96 Analyzer vs. CTM48 Analyzer
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Table 11: Results of Deming Regression
| | Coefficient | SE | 95% CI | |
| --- | --- | --- | --- | --- |
| Intercept | 0.0329 | 0.0822 | -0.1312 | to 0.1970 |
| Slope | 0.9940 | 0.0167 | 0.9607 | to 1.0274 |
## 9.1.11. Interfering Substances
Elevated levels of triglycerides, bilirubin, albumin, hemoglobin and human DNA in specimens as well as the presence of autoimmune diseases such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), and Antinuclear Antibody (ANA) were tested and did not interfere with the quantitation of HCV RNA by the COBAS AmpliPrep/COBAS TaqMan HCV Test.
The following drug compounds tested at the Peak Plasma Level (Cmax) and at 3 times the Cmax have been shown not to interfere with the quantitation of HCV RNA by the COBAS AmpliPrep/COBAS TaqMan HCV Test:
| Nucleotide HIV / HBV DNA Polymerase Inhibitors
Adefovir dipivoxil
Tenofovir | Nucleoside HIV Reverse Transcriptase Inhibitors and DNA Polymerase Inhibitors
Lamivudine
Zidovudine
Stavudine
Abacavir
Didanosine |
| --- | --- |
| HIV Protease Inhibitors
Indinavir
Saquinavir
Ritonavir
Nelfinavir
Amprenavir
Lopinavir/Ritonavir | Non-nucleoside HIV Reverse Transcriptase Inhibitors
Nevirapine
Efavirenz |
| HIV Fusion Inhibitors
Enfurvitide |
| Immune Modulators
Interferon alfa-2a
Interferon alfa-2b
Peginterferon alfa-2a
Peginterferon alfa-2a + Ribavirin
Interferon alfa-2b+ Ribavirin | Antidepressants
Paroxetine HCl
Fluoxetine
Sertraline
Compounds for Treatment of Herpes Viruses
Ganciclovir
Valganciclovir
Acyclovir |
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## 9.2. Recommended Storage Stability
Whole kit stability was determined by a functional real time stability study conducted at 2°C – 8°C, measured over multiple time points using three kit lots of the COBAS AmpliPrep/COBAS TaqMan HCV test kits. Based on these studies, the COBAS AmpliPrep/COBAS TaqMan HCV Test Kit reagents are stable for 24 months from the date of manufacture.
## 9.3. Sample Handling and Collection
### 9.3.1. Specimen Collection
The COBAS AmpliPrep/COBAS TaqMan HCV Test is for use with serum or plasma specimens. Blood should be collected in SST® Serum Separation Tubes or in sterile tubes using EDTA (lavender top) as the anticoagulant.
Store whole blood at 2-25°C for no longer than 6 hours. Separate serum or plasma from whole blood within 6 hours of collection by centrifugation at 800-1600 x g for 20 minutes at room temperature. Transfer serum or plasma to a sterile polypropylene tube. Figure 9 and Figure 10 show specimen stability data from specimen collection studies. Studies were performed using the COBAS AmpliPrep/COBAS TaqMan HCV Test. The largest observed difference between the EDTA plasma conditions was not more than ±0.22 log₁₀ and the largest observed difference between the serum conditions was not more than ±0.14 log₁₀.
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Figure 9: HCV Stability in Whole Blood with EDTA Anticoagulant
Note: There were four replicates for each time point.
Figure 10: HCV Stability in Whole Blood without Anticoagulant
Note: There were four replicates for each time point.
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# SUMMARY OF SAFETY AND EFFECTIVENESS
## 9.3.2. Specimen Transport
Transportation of whole blood, serum or plasma must comply with country, federal, state, and local regulations for the transport of etiologic agents.⁵ Whole blood must be transported at 2-25°C and centrifuged within 6 hours of collection. Plasma or serum may be transported at 2-8°C or frozen at -70°C or colder, within the defined specimen storage period.
## 9.3.3. Specimen Stability
Serum or plasma specimens may be stored at 2-8°C for up to 3 days or frozen at -70°C or colder for up to 6 weeks. The largest observed difference between the EDTA plasma conditions was not more than ±0.13 log₁₀ and the largest observed difference between the serum conditions was not more than ±0.06 log₁₀ across the tested conditions. It is recommended that specimens be stored in 1100-1200 μL aliquots in sterile, 2.0 mL polypropylene screw-cap tubes (such as Sarstedt 72.694.006). Figure 11 and Figure 12 show specimen stability data from specimen storage studies. Studies were performed using the COBAS AmpliPrep/COBAS TaqMan HCV Test.
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Figure 11: HCV Stability in EDTA Plasma
Note: There were four replicates for each time point.
Figure 12: HCV Stability in Serum
Note: There were four replicates for each time point.
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# SUMMARY OF SAFETY AND EFFECTIVENESS
Serum and plasma specimens may be frozen and thawed up to five times without a loss of HCV RNA. The largest observed difference between the EDTA plasma conditions was not more than $\pm 0.11\log_{10}$ and the largest observed difference between the serum conditions was not more than $\pm 0.22\log_{10}$. Figure 12 and Figure 13 show the data from freeze-thaw studies performed using the COBAS AmpliPrep/COBAS TaqMan HCV Test.
Figure 13: HCV EDTA Plasma Freeze/Thaw Stability
Note: There were four replicates for each time point.
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Figure 14: HCV Serum Freeze/Thaw Stability
Note: There were four replicates for each time point.
## 10. SUMMARY OF CLINICAL STUDIES
### 10.1. Reproducibility
Note: As seen below, reproducibility studies were conducted using several levels of each of the 6 genotypes of HCV. These studies were repeated using each of the two platforms. When the results for the two platforms were compared as reported in log10 units, the results were similar for both platforms.
### 10.1.1. Reproducibility – COBAS TaqMan Analyzer (TaqMan 96)
The reproducibility of the COBAS AmpliPrep/COBAS TaqMan HCV Test was evaluated for each genotype by 2 operators at each of three clinical sites. Each operator performed 3 days of testing on each of 3 lots of reagents with each genotype panel. Each run consisted of a single genotype panel with each panel member tested in triplicate.
The results of the reproducibility study are summarized in Table 12 and Table 13 below.
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Table 12: Standard Deviation Components HCV RNA Concentration (log₁₀ IU/mL) EDTA Plasma
| Genotype | No. of Tests¹ | Lot | Site/Instrument | Operator | Day/Run | Within-Run | Total Standard Deviation of log₁₀ HCV RNA Concentration |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 163 | 0.068 | 0.085 | 0.000 | 0.052 | 0.176 | 0.214 |
| | 163 | 0.062 | 0.000 | 0.007 | 0.040 | 0.094 | 0.120 |
| | 163 | 0.027 | 0.014 | 0.002 | 0.006 | 0.075 | 0.081 |
| | 161 | 0.049 | 0.010 | 0.000 | 0.033 | 0.066 | 0.089 |
| | 163 | 0.026 | 0.024 | 0.015 | 0.022 | 0.069 | 0.082 |
| | 161 | 0.030 | 0.028 | 0.000 | 0.032 | 0.062 | 0.080 |
| 2* | 157 | 0.156 | 0.024 | 0.038 | 0.000 | 0.188 | 0.248 |
| | 154 | 0.080 | 0.036 | 0.000 | 0.000 | 0.075 | 0.115 |
| | 151 | 0.077 | 0.052 | 0.000 | 0.000 | 0.115 | 0.148 |
| | 156 | 0.068 | 0.069 | 0.000 | 0.031 | 0.056 | 0.116 |
| | 156 | 0.078 | 0.073 | 0.000 | 0.018 | 0.075 | 0.132 |
| | 157 | 0.082 | 0.058 | 0.000 | 0.033 | 0.074 | 0.129 |
| 3 | 159 | 0.130 | 0.000 | 0.000 | 0.057 | 0.121 | 0.186 |
| | 159 | 0.073 | 0.032 | 0.020 | 0.032 | 0.063 | 0.109 |
| | 158 | 0.069 | 0.079 | 0.000 | 0.036 | 0.088 | 0.141 |
| | 158 | 0.060 | 0.087 | 0.003 | 0.037 | 0.086 | 0.142 |
| | 161 | 0.112 | 0.094 | 0.000 | 0.043 | 0.066 | 0.166 |
| | 156 | 0.120 | 0.106 | 0.000 | 0.043 | 0.080 | 0.184 |
| 4 | 158 | 0.192 | 0.000 | 0.028 | 0.113 | 0.174 | 0.284 |
| | 159 | 0.179 | 0.000 | 0.000 | 0.123 | 0.077 | 0.230 |
| | 158 | 0.169 | 0.035 | 0.000 | 0.108 | 0.082 | 0.220 |
| | 153 | 0.163 | 0.044 | 0.009 | 0.132 | 0.080 | 0.230 |
| | 158 | 0.179 | 0.062 | 0.000 | 0.124 | 0.111 | 0.252 |
| | 156 | 0.204 | 0.075 | 0.040 | 0.178 | 0.122 | 0.308 |
| 5 | 154 | 0.167 | 0.037 | 0.000 | 0.049 | 0.109 | 0.209 |
| | 157 | 0.126 | 0.013 | 0.021 | 0.025 | 0.085 | 0.156 |
| | 158 | 0.096 | 0.030 | 0.000 | 0.029 | 0.066 | 0.123 |
| | 154 | 0.114 | 0.044 | 0.022 | 0.030 | 0.073 | 0.147 |
| | 157 | 0.099 | 0.078 | 0.000 | 0.033 | 0.074 | 0.149 |
| | 158 | 0.120 | 0.089 | 0.000 | 0.031 | 0.106 | 0.186 |
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{36}
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# SUMMARY OF SAFETY AND EFFECTIVENESS
| Geno-type | No. of Tests^{1} | Lot | Site/Instru-ment | Operator | Day/Run | Within-Run | Total Standard Deviation of log10 HCV RNA Concentration |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 6 | 160 | 0.044 | 0.036 | 0.000 | 0.032 | 0.155 | 0.168 |
| | 165 | 0.038 | 0.000 | 0.000 | 0.023 | 0.061 | 0.075 |
| | 168 | 0.019 | 0.034 | 0.000 | 0.000 | 0.078 | 0.087 |
| | 163 | 0.000 | 0.040 | 0.000 | 0.024 | 0.089 | 0.101 |
| | 165 | 0.012 | 0.038 | 0.000 | 0.014 | 0.092 | 0.101 |
| | 163 | 0.017 | 0.041 | 0.000 | 0.018 | 0.082 | 0.095 |
{37}
SUMMARY OF SAFETY AND EFFECTIVENESS
Table 13: Reproducibility Results Summary: Total %CV for HCV Panel Members - EDTA Plasma
| Genotype | Geometric Mean of HCV RNA Concentration (IU/mL) | Mean of HCV RNA Concentration (log10 IU/mL) | No. of Tests^{1} | Total Precision Variance of log10 HCV RNA Concentration | Total Precision Standard Deviation of log10 HCV RNA Concentration | lognormal CV (%)^{2} |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 320 | 2.45a | 163 | 0.046 | 0.21 | 52 |
| | 2,902 | 3.45 | 163 | 0.014 | 0.12 | 28 |
| | 21,795 | 4.33 | 163 | 0.007 | 0.08 | 19 |
| | 224,096 | 5.34 | 161 | 0.008 | 0.09 | 21 |
| | 2,384,233 | 6.37 | 163 | 0.007 | 0.08 | 19 |
| | 4,052,668 | 6.60 | 161 | 0.006 | 0.08 | 19 |
| 2* | 292 | 2.40b | 157 | 0.061 | 0.25 | 62 |
| | 2,713 | 3.42 | 154 | 0.013 | 0.12 | 27 |
| | 22,754 | 4.33 | 151 | 0.022 | 0.15 | 35 |
| | 110,171 | 5.03 | 156 | 0.014 | 0.12 | 27 |
| | 840,382 | 5.90 | 156 | 0.017 | 0.13 | 31 |
| | 9,696,516 | 6.97 | 157 | 0.017 | 0.13 | 30 |
| 3 | 355 | 2.51 | 159 | 0.035 | 0.19 | 45 |
| | 4,764 | 3.66 | 159 | 0.012 | 0.11 | 25 |
| | 32,876 | 4.49 | 158 | 0.020 | 0.14 | 33 |
| | 331,684 | 5.50 | 158 | 0.020 | 0.14 | 33 |
| | 1,318,936 | 6.09 | 161 | 0.028 | 0.17 | 40 |
| | 27,131,043 | 7.39 | 156 | 0.034 | 0.18 | 44 |
| 4 | 741 | 2.78 | 158 | 0.081 | 0.28 | 73 |
| | 1,847 | 3.21 | 159 | 0.053 | 0.23 | 57 |
| | 5,481 | 3.68 | 158 | 0.048 | 0.22 | 54 |
| | 13,775 | 4.08 | 153 | 0.053 | 0.23 | 57 |
| | 38,721 | 4.51 | 158 | 0.063 | 0.25 | 63 |
| | 106,098 | 4.92 | 156 | 0.095 | 0.31 | 81 |
| 5 | 254 | 2.35 | 154 | 0.044 | 0.21 | 51 |
| | 943 | 2.95 | 157 | 0.024 | 0.16 | 37 |
| | 3,164 | 3.48 | 158 | 0.015 | 0.12 | 29 |
| | 8,124 | 3.88 | 154 | 0.022 | 0.15 | 35 |
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| Genotype | Geometric Mean of HCV RNA Concentration (IU/mL) | Mean of HCV RNA Concentration (log10 IU/mL) | No. of Tests^{1} | Total Precision Variance of log10 HCV RNA Concentration | Total Precision Standard Deviation of log10 HCV RNA Concentration | lognormal CV (%)^{2} |
| --- | --- | --- | --- | --- | --- | --- |
| | 24,379 | 4.36 | 157 | 0.022 | 0.15 | 35 |
| | 253,987 | 5.37 | 158 | 0.034 | 0.19 | 45 |
| 6 | 275 | 2.41c | 160 | 0.028 | 0.17 | 40 |
| | 3,575 | 3.55 | 165 | 0.006 | 0.08 | 17 |
| | 27,274 | 4.43 | 168 | 0.008 | 0.09 | 20 |
| | 279,527 | 5.43 | 163 | 0.010 | 0.10 | 23 |
| | 1,670,245 | 6.21 | 165 | 0.010 | 0.10 | 24 |
| | 3,605,810 | 6.55 | 163 | 0.009 | 0.10 | 22 |
Note: Within assay range results are from 18 IU/mL to 6.90E+7 IU/mL (1.26 log₁₀ IU/mL to 7.84 log₁₀ IU/mL), inclusive. The limit of detection (LOD) for the assay is 18 IU/mL. Results <1.80E+1 IU/mL have been imputed as half the limit of detection, 9.0E+0 IU/mL (0.95 log₁₀ IU/mL).
Note: Three extra panels (one Genotype 1 panel and two Genotype 6 panels) than the number of panels/genotype planned were additionally tested in the study.
* For genotype 2 the results from 3 mis-positioned aliquots are excluded.
¹ Number of tests with detectable viral load. In total, 165 tests/panel member were performed for Genotype 1, 162 tests/panel member were performed for Genotypes 2, 3, 4, and 5, and 168 tests/panel member were performed for Genotype 6. Invalid tests were not repeated.
$$
2 \% CV_{\log} = 100 \times \sqrt{10^{\sigma^2 \ln(10)} - 1}
$$
ᵃ Two <1.80E+1 IU/mL results were observed for this panel member.
ᵇ Two <1.80E+1 IU/mL results were observed for this panel member.
ᶜ One <1.80E+1 IU/mL result was observed for this panel member.
Table 14 summarizes the results for the HCV negative panel members from the reproducibility study. There were 2 false positive results in 961 tests. Specificity was 99.8% [95% CI = (0.99, 1.00)].
Table 14: HCV Negative Panel Member Summary
| Expected HCV RNA Concentration | Total Valid Results | Target Not Detected | Target Detected but Below LOD^{1} | >=18 and <43 IU/mL^{2} | Within Linear Range^{3} |
| --- | --- | --- | --- | --- | --- |
| Negative | 961 | 959 | 1 | 0 | 1 |
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SUMMARY OF SAFETY AND EFFECTIVENESS
| Expected HCV RNA Concentration | Total Valid Results | Target Not Detected | Target Detected but Below LOD^{1} | >=18 and <43 IU/mL^{2} | Within Linear Range^{3} |
| --- | --- | --- | --- | --- | --- |
1 The limit of detection (LOD) for the assay is 18 IU/mL. Results <1.80E+1 IU/mL are below the LOD.
2 Results 18 IU/mL to <43 IU/ml are above the LOD, but below the linear range.
3 Within linear range results are from 43 IU/mL to 6.90E+7 IU/mL, inclusive.
## 10.2. Reproducibility - COBAS TaqMan 48 Analyzer
The reproducibility of the COBAS AmpliPrep/COBAS TaqMan HCV Test was evaluated for each genotype by two operators at each of three clinical sites. Each operator performed three days of testing on each of three lots of reagents with each genotype panel. Each run comprised a single genotype panel with each panel member tested in triplicate.
The results of the reproducibility study are summarized in Table 15 and Table 16 below.
Table 15: Standard Deviation Components HCV RNA Concentration (log10 IU/mL) EDTA Plasma
| Genotype | Geometric Mean of HCV RNA Concentration (IU/mL) | Mean of HCV RNA Concentration (log_{10} IU/mL) | No. of Tests^{1} | Lot | Site/Instrument | Operator | Day/Run | Within-Run | Total Standard Deviation of log_{10} HCV RNA Concentration |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 133 | 2.01^{a} | 159 | 0.000 | 0.058 | 0.000 | 0.052 | 0.306 | 0.316 |
| | 1,488 | 3.16 | 159 | 0.063 | 0.000 | 0.010 | 0.000 | 0.104 | 0.122 |
| | 10,145 | 3.99 | 161 | 0.056 | 0.000 | 0.000 | 0.028 | 0.090 | 0.109 |
| | 76,432 | 4.87 | 154 | 0.043 | 0.000 | 0.000 | 0.024 | 0.082 | 0.096 |
| | 726,306 | 5.85 | 159 | 0.013 | 0.000 | 0.000 | 0.037 | 0.095 | 0.103 |
| | 1,264,581 | 6.05 | 159 | 0.060 | 0.012 | 0.000 | 0.118 | 0.175 | 0.220 |
| 2 | 108 | 1.93^{b} | 132 | 0.067 | 0.000 | 0.000 | 0.000 | 0.283 | 0.291 |
| | 1,428 | 3.11 | 131 | 0.078 | 0.000 | 0.000 | 0.074 | 0.165 | 0.196 |
| | 9,839 | 3.95 | 131 | 0.047 | 0.000 | 0.000 | 0.000 | 0.178 | 0.184 |
| | 72,711 | 4.84 | 132 | 0.018 | 0.000 | 0.017 | 0.062 | 0.101 | 0.121 |
| | 477,320 | 5.66 | 134 | 0.028 | 0.040 | 0.000 | 0.001 | 0.117 | 0.127 |
| | 5,583,514 | 6.71 | 133 | 0.056 | 0.000 | 0.000 | 0.150 | 0.083 | 0.180 |
| 3 | 105 | 1.88^{c} | 157 | 0.275 | 0.094 | 0.000 | 0.070 | 0.182 | 0.349 |
| | 8,253 | 3.86 | 160 | 0.186 | 0.087 | 0.000 | 0.024 | 0.097 | 0.228 |
{40}
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# SUMMARY OF SAFETY AND EFFECTIVENESS
| Geno-type | Geometric Mean of HCV RNA Concentration (IU/mL) | Mean of HCV RNA Concentration (log_{10} IU/mL) | No. of Tests^{1} | Lot | Site/Instrument | Operator | Day/Run | Within-Run | Total Standard Deviation of log_{10} HCV RNA Concentration |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | 774,131 | 5.79 | 160 | 0.187 | 0.111 | 0.036 | 0.132 | 0.124 | 0.286 |
| | 12,215,944 | 7.02 | 160 | 0.158 | 0.095 | 0.000 | 0.096 | 0.110 | 0.235 |
| 4 | 713 | 2.62 | 163 | 0.373 | 0.104 | 0.000 | 0.107 | 0.199 | 0.448 |
| | 4,428 | 3.49 | 162 | 0.332 | 0.102 | 0.000 | 0.066 | 0.112 | 0.371 |
| | 20,340 | 4.21 | 165 | 0.244 | 0.075 | 0.025 | 0.032 | 0.134 | 0.291 |
| | 213,356 | 5.23 | 164 | 0.246 | 0.110 | 0.029 | 0.068 | 0.106 | 0.299 |
{41}
SUMMARY OF SAFETY AND EFFECTIVENESS
| Genotype | Geometric Mean of HCV RNA Concentration (IU/mL) | Mean of HCV RNA Concentration (log_{10} IU/mL) | No. of Tests^{1} | Lot | Site/Instrument | Operator | Day/Run | Within-Run | Total Standard Deviation of log_{10} HCV RNA Concentration |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 219 | 2.21^{d} | 156 | 0.243 | 0.088 | 0.022 | 0.091 | 0.194 | 0.337 |
| | 1,570 | 3.08 | 157 | 0.231 | 0.111 | 0.021 | 0.077 | 0.164 | 0.315 |
| | 13,760 | 4.07 | 159 | 0.186 | 0.071 | 0.029 | 0.063 | 0.109 | 0.238 |
| | 98,283 | 4.94 | 158 | 0.157 | 0.088 | 0.033 | 0.028 | 0.116 | 0.218 |
| 6 | 106 | 1.96 | 158 | 0.103 | 0.044 | 0.000 | 0.000 | 0.212 | 0.240 |
| | 5,271 | 3.67 | 159 | 0.165 | 0.026 | 0.040 | 0.073 | 0.101 | 0.212 |
| | 39,349 | 4.55 | 161 | 0.114 | 0.049 | 0.040 | 0.000 | 0.143 | 0.194 |
| | 495,696 | 5.65 | 160 | 0.110 | 0.087 | 0.039 | 0.053 | 0.135 | 0.206 |
Note: Within assay range results are from 18 IU/mL to 6.90E+7 IU/mL (1.26 log<sub>10</sub> IU/mL to 7.84 log<sub>10</sub> IU/mL), inclusive. The limit of detection (LOD) for the assay is 18 IU/mL. Results <1.80E+1 IU/mL have been imputed as half the limit of detection, 9.0E+0 IU/mL (0.95 log<sub>10</sub> IU/mL).
Note: Three extra panels (one Genotype 1 panel and two Genotype 6 panels) were additionally tested in the study. Seven fewer Genotype 2 panels than the number of panels/genotype planned were tested.
Note: One result above the linear range (>6.90E+7 IU/mL) from a Genotype 1 panel was excluded from this analysis.
1 Number of tests with detectable viral load. In total, 162 tests/panel member were performed for Genotypes 1, 3, and 5, 141 tests/panel member were performed for Genotype 2, and 165 tests/panel member were performed for Genotypes 4 and 6. Invalid tests were not repeated.
a One <1.80E+1 IU/mL result was observed for this panel member.
b Four <1.80E+1 IU/mL results were observed for this panel member.
c Five <1.80E+1 IU/mL results were observed for this panel member.
d One <1.80E+1 IU/mL result was observed for this panel member.
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Table 16: Reproducibility Results Summary: Total %CV for HCV Panel Members — EDTA Plasma
| Genotype | Geometric Mean of HCV RNA Concentration (IU/mL) | Mean of HCV RNA Concentration (log_{10} IU/mL) | No. of Tests^{1} | Total Precision Variance of log_{10} HCV RNA Concentration | Total Precision Standard Deviation of log_{10} HCV RNA Concentration | lognormal CV (%)^{2} |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 133 | 2.01^{a} | 159 | 0.100 | 0.32 | 84 |
| | 1,488 | 3.16 | 159 | 0.015 | 0.12 | 29 |
| | 10,145 | 3.99 | 161 | 0.012 | 0.11 | 26 |
| | 76,432 | 4.87 | 154 | 0.009 | 0.10 | 22 |
| | 726,306 | 5.85 | 159 | 0.011 | 0.10 | 24 |
| | 1,264,581 | 6.05 | 159 | 0.048 | 0.22 | 54 |
| 2 | 108 | 1.93^{b} | 132 | 0.085 | 0.29 | 75 |
| | 1,428 | 3.11 | 131 | 0.039 | 0.20 | 48 |
| | 9,839 | 3.95 | 131 | 0.034 | 0.18 | 44 |
| | 72,711 | 4.84 | 132 | 0.015 | 0.12 | 28 |
| | 477,320 | 5.66 | 134 | 0.016 | 0.13 | 30 |
| | 5,583,514 | 6.71 | 133 | 0.032 | 0.18 | 43 |
| 3 | 105 | 1.88^{c} | 157 | 0.122 | 0.35 | 95 |
| | 8,253 | 3.86 | 160 | 0.052 | 0.23 | 56 |
| | 774,131 | 5.79 | 160 | 0.082 | 0.29 | 74 |
| | 12,215,944 | 7.02 | 160 | 0.055 | 0.23 | 58 |
| 4 | 713 | 2.62 | 163 | 0.201 | 0.45 | 138 |
| | 4,428 | 3.49 | 162 | 0.138 | 0.37 | 104 |
| | 20,340 | 4.21 | 165 | 0.085 | 0.29 | 75 |
| | 213,356 | 5.23 | 164 | 0.089 | 0.30 | 78 |
| 5 | 219 | 2.21^{d} | 156 | 0.113 | 0.34 | 91 |
| | 1,570 | 3.08 | 157 | 0.099 | 0.31 | 83 |
| | 13,760 | 4.07 | 159 | 0.056 | 0.24 | 59 |
| | 98,283 | 4.94 | 158 | 0.048 | 0.22 | 54 |
{43}
SUMMARY OF SAFETY AND EFFECTIVENESS
| Genotype | Geometric Mean of HCV RNA Concentration (IU/mL) | Mean of HCV RNA Concentration (log_{10} IU/mL) | No. of Tests^{1} | Total Precision Variance of log_{10} HCV RNA Concentration | Total Precision Standard Deviation of log_{10} HCV RNA Concentration | lognormal CV (%)^{2} |
| --- | --- | --- | --- | --- | --- | --- |
| 6 | 106 | 1.96 | 158 | 0.058 | 0.24 | 60 |
| | 5,271 | 3.67 | 159 | 0.045 | 0.21 | 52 |
| | 39,349 | 4.55 | 161 | 0.038 | 0.19 | 47 |
| | 495,696 | 5.65 | 160 | 0.042 | 0.21 | 50 |
Note: Within assay range results are from 18 IU/mL to 6.90E+7 IU/mL (1.26 log<sub>10</sub> IU/mL to 7.84 log<sub>10</sub> IU/mL), inclusive. The limit of detection (LOD) for the assay is 18 IU/mL. Results <1.80E+1 IU/mL have been imputed as half the limit of detection, 9.0E+0 IU/mL (0.95 log<sub>10</sub> IU/mL).
Note: Three extra panels (one Genotype 1 panel and two Genotype 6 panels) were additionally tested in the study. Seven fewer Genotype 2 panels than the number of panels/genotype planned were tested.
Note: One result above the linear range (>6.90E+7 IU/mL) from a Genotype 1 panel was excluded from this analysis.
1 Number of tests with detectable viral load. In total, 162 tests/panel member were performed for Genotypes 1, 3, and 5, 141 tests/panel member were performed for Genotype 2, and 165 tests/panel member were performed for Genotypes 4 and 6. Invalid tests were not repeated.
$$
2 \% CV_{\log} = 100 \times \sqrt{10^{\sigma^{2} \ln(10)} - 1}
$$
a One <1.80E+1 IU/mL result was observed for this panel member.
b Four <1.80E+1 IU/mL results were observed for this panel member.
c Five <1.80E+1 IU/mL results were observed for this panel member.
d One <1.80E+1 IU/mL result was observed for this panel member.
Table 17 summarizes the results for the HCV negative panel members from the reproducibility study. There were no false positive results in 932 tests. Specificity was 100% [95% CI = (0.996, 1.000)].
Table 17: HCV Negative Panel Member Summary
| Expected HCV RNA Concentration | Total Valid Results | Target Not Detected | Target Detected but Below LOD^{1} | >=18 and <43 IU/mL^{2} | Within Linear Range^{3} |
| --- | --- | --- | --- | --- | --- |
| Negative | 932 | 932 | 0 | 0 | 0 |
1 The limit of detection (LOD) for the assay is 18 IU/mL. Results <1.80E+1 IU/mL are below the LOD.
2 Results 18 IU/mL to <43 IU/ml are above the LOD, but below the linear range.
3 Within linear range results are from 43 IU/mL to 6.90E+7 IU/mL, inclusive.
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## 10.3. Clinical Utility
The use of HCV RNA for the on-treatment assessment of HCV antiviral therapy has become an increasingly important tool for individualizing treatment and optimizing patient outcomes. The critical on-treatment time points for evaluating therapy for customization or discontinuation are at Week 4, Week 12, and Week 24.
The primary objective of this study was to evaluate the clinical utility of the COBAS AmpliPrep/COBAS TaqMan HCV Test, for the clinical management of patients infected with chronic hepatitis C (CHC) by estimating the negative predictive value (NPV) and positive predictive value (PPV) for achieving an sustained virologic response (SVR) at established clinically relevant time points during antiviral treatment (Week 4/Rapid virologic response (RVR), Week 12/early virologic response (EVR), and Week 24).
## 10.3.1. Study Population
Retrospectively collected specimens from patients enrolled in a Phase III, randomized, multicenter study comparing 48 weeks with 24 weeks of treatment with peginterferon alfa-2a given in combination with either a standard dose or a low dose of ribavirin were studied.⁷
The patient population included subjects with serologically proven CHC who had not been previously treated with an interferon or ribavirin. A total of 1311 patients were enrolled in the original study, 1284 of whom received treatment. Specimens from a total of 1281 subjects were available for testing, for at least one time point, which was performed at 5 US sites, 3 sites used the COBAS TaqMan 48 Analyzer and 2 used the COBAS TaqMan Analyzer 96.
Determination of HCV RNA viral levels at Screening/Baseline, Week 4, Week 12, and Week 24 were performed using the COBAS AmpliPrep/COBAS TaqMan HCV Test. End of Treatment (EOT) and End of Follow-up (EOF) results were determined using the FDA-approved the COBAS® AMPLICOR HCV Test, v2.0.
Three predictability analysis subsets were established from the cohort based on the availability of serum samples at the key established clinically relevant time points as follows: Week 4/RVR
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# SUMMARY OF SAFETY AND EFFECTIVENESS
Analysis was performed for the subset of patients with viral load results available for Screening/Baseline, Week 4 and EOF time points. This subset contained 984 patients. Week 12/EVR Analysis was performed for the subset of patients with viral load results available for Screening/Baseline, Week 12 and EOF time points. This subset contained 991 patients. Week 24 Analysis was performed for the subset of patients with viral load results available for Screening/Baseline, Week 24 and EOF time points. This subset contained 982 patients. Baseline demographics of the study population are presented in Table 18.
Table 18: Description of the Study Population at Baseline
| Characteristic | Category | Summary Statistics | Combined Over All Four Treatment Arms |
| --- | --- | --- | --- |
| Total Number of Subjects | | N | 1281 |
| Age | < 40 | N (%) | 503 (39.3) |
| | ≥40 | N (%) | 778 (60.7) |
| Gender | Male | N (%) | 837 (65.3) |
| | Female | N (%) | 444 (34.7) |
| Genotype | 1 | N (%) | 739 (57.7) |
| | 2 | N (%) | 202 (15.8) |
| | 3 | N (%) | 288 (22.5) |
| | 4 | N (%) | 36 (2.8) |
| | 5 | N (%) | 7 (0.5) |
| | 6 | N (%) | 9 (0.7) |
| Week 0 HCV RNA | ≤ 7.40E+5 IU/mL^{1} | N (%) | 306 (23.9) |
| | > 7.40E+5 IU/mL | N (%) | 910 (71.0) |
| | Missing | N (%) | 65 (5.1) |
| Baseline Biopsy Result | Cirrhotic | N (%) | 91 (7.1) |
| | Non-Cirrhotic | N (%) | 959 (74.9) |
| | Transition to Cirrhotic | N (%) | 231 (18.0) |
| Baseline SGPT^{3} | ≤ 3 * ULN^{2} | N (%) | 880 (68.7) |
| | > 3 * ULN | N (%) | 401 (31.3) |
| Baseline Serum Creatinine (mg/dL) | | Mean | 0.9 |
| | | SD | 0.2 |
| Baseline Creatinine Clearance (mL/min) | | Mean | 97.5 |
| | | SD | 25.3 |
{46}
SUMMARY OF SAFETY AND EFFECTIVENESS
| Characteristic | Category | Summary Statistics | Combined Over All Four Treatment Arms |
| --- | --- | --- | --- |
$^{1}$ 2,000,000 copies/mL = 7.40E+5 IU/mL = 5.87 log10 IU/mL.
$^{2}$ ULN = Upper Limit of Normal Range.
$^{3}$ SGPT, serum glutamic pyruvic transaminase.
## 10.3.2. Predictability Analysis
### 10.3.2.1. Association between Baseline Covariates and Sustained Virologic Response
Established host-, viral-, and treatment-related baseline covariates predictive of SVR with peginterferon/ribavirin therapy were analyzed using the unadjusted odds ratios (univariate) shown in Table 19. The data subset used for this analysis comprises 1017 patients who have baseline and End of Follow-up responses. Distribution of subjects for various characteristics in this subset is similar to that in Table 21. These results demonstrate that genotype non-1 and low baseline viral load for genotype 1 (defined as $<7.40\mathrm{E}^{+5}$) are the two most significant positive predictors of SVR.
Table 19: Predictors of Sustained Virological Response at Baseline
| Characteristic^{1} | Category | N (%) | Percent with SVR | Odds Ratio (95% CI) Using Univariate Analysis |
| --- | --- | --- | --- | --- |
| Age | ≥ 40 | 612(60.2) | 60.3 | |
| | < 40 | 405(39.8) | 71.9 | 1.7 (1.3, 2.2) |
| Gender | Male | 663(65.2) | 63.2 | |
| | Female | 354(34.8) | 68.1 | 1.2 (0.9, 1.7) |
| Treatment^{1} | A: 24-W LD RBV^{2,3} | 177(17.4) | 54.8 | |
| | B: 24-W HD RBV | 244(24.0) | 68.9 | 1.8 (1.2, 2.8) |
| | C: 48-W LD RBV | 261(25.7) | 59.0 | 1.2 (0.8, 1.8) |
| | D: 48-W HD RBV | 335(32.9) | 71.9 | 2.1 (1.4, 3.1) |
| Genotype | 1 | 575(56.5) | 49.0 | |
| | Non-1 | 442(43.5) | 85.5 | 6.1 (4.5, 8.5) |
| Week 0 HCV RNA for Genotype 1 | > 7.40E+5 IU/mL^{4} | 434(42.7) | 43.3 | |
| | ≤ 7.40E+5 IU/mL | 141(13.9) | 66.7 | 2.6 (1.7, 4.0) |
| Week 0 HCV RNA for Genotype non-1 | > 7.40E+5 IU/mL | 335(32.9) | 84.5 | |
| | ≤7.40E+5 IU/mL | 107(10.5) | 88.8 | 1.5 (0.7, 3.1) |
{47}
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# SUMMARY OF SAFETY AND EFFECTIVENESS
| Characteristic^{1} | Category | N (%) | Percent with SVR | Odds Ratio (95% CI)
Using Univariate Analysis |
| --- | --- | --- | --- | --- |
| Baseline Biopsy Result | Cirrhotic/ Transition to Cirrhotic | 250(24.6) | 58.4 | |
| | Non-Cirrhotic | 767(75.4) | 67.0 | 1.4 (1.1, 2.0) |
| Baseline SGPT^{5} | ≤ 3*ULN^{5} | 706(69.4) | 60.5 | |
| | > 3*ULN | 311(30.6) | 74.9 | 2.0 (1.4, 2.7) |
1 Treatment is 180 mcg/wk PEG-IFN + RBV.
2 24-W = 24-week therapy ; 48-W = 48-week therapy.
3 LD = low dose of RBV, 800 mg/day ; HD = high dose of RBV, 1,000 or 1,200 mg/day.
4 2,000,000 copies/mL = 7.40E+5 IU/mL = 5.87 log10 IU/mL, based on the AASLD Practice Guideline.
5 SGPT, serum glutamic pyruvic transaminase; ULN, upper limit of normal range.
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# SUMMARY OF SAFETY AND EFFECTIVENESS
## 10.3.2.2. Definitions of Prediction Rules, NPV, PPV, and Odds Ratios
- Rapid Virologic Response Analysis = HCV-RNA < LOD at Week 4 of antiviral therapy
- Early Virologic Response = achievement of either a 2-log₁₀ drop or absence of HCV RNA at Week 12 of antiviral therapy
- Week 24 Virologic Response = HCV-RNA < LOD at Week 24 of antiviral therapy
- Positive Predictive Value = the probability of SVR given an on-treatment virologic response at Week 4, Week 12, or Week 24
- Negative Predictive Value = the probability of NO SVR given no on-treatment virologic response at Week 4, Week 12, or Week 24
Odds ratio (OR) describes the measure of association between virologic response and SVR and is equal to:
$$
OR = \frac{NPV * PPV}{(1 - NPV) * (1 - PPV)}
$$
The relationship between SVR and RVR, EVR, or Week 24 results was studied after adjusting for baseline covariates and treatment arm. Factors such as HCV genotype, baseline viral load, cirrhosis, age, ethnicity, and body weight are cited in the literature as predictors for SVR.
Each of the 3 study subsets were initially analyzed for both PPV and NPV as pooled data for all treatment arms and further stratified by individual treatment arms, genotype and predictive rule cut-off (where appropriate).
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# SUMMARY OF SAFETY AND EFFECTIVENESS
## 10.3.2.3. Predictive Values at Week 4 of Antiviral Therapy (RVR Analysis)
The RVR analysis in the current study has been performed using the prediction rule of HCV RNA <18 IU/mL, the established LOD for the test. These results demonstrate a high PPV for all patients at 4 weeks (greater than 0.87) independent of genotype. The NPV for not achieving SVR is less than 0.63 for all subgroups and is less useful for predicting NO SVR, especially in the non-1 genotype population due to the high response rate in this population. The analysis was also performed using a prediction rule of <50 IU/mL$^6$. No significant differences were noted in either PPV or NPV when comparing the two prediction rules.
Table 20 presents the performance statistics by treatment arm for RVR evaluation. This table shows that the positive predictive value for all patients at Week 4 generally remains high when the analysis is done by individual treatment arm (A through D) compared to the pooled results of all groups, regardless of genotype. The NPV for not achieving an SVR also remains low, particularly in the non-1 genotype patients due to the high response rate in this population.
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Table 20: NPV and PPV at Week 4 and Corresponding Odds Ratios: Treatment Arms A through D
| Treatment Arm^{1} | Genotype | Prediction Rule | Negative Predictive Value (NPV) | | Positive Predictive Value (PPV) | | Odds Ratio (95% CI) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Estimate (95% CI) | N | Estimate (95% CI) | N | Unadjusted | Adjusted^{2} |
| A | 1 | <18 IU/mL^{3} | 0.88 (0.78, 0.95) | 61/69 | 0.87 (0.60, 0.98) | 13/15 | 49.6 (8.3, 487.5) | 47.5 (8.0, 282.4) |
| | Non-1 | <18 IU/mL^{3} | 0.47 (0.23, 0.72) | 8/17 | 0.90 (0.80, 0.96) | 63/70 | 8.0 (1.9, 32.5) | 6.8 (1.7, 26.7) |
| B | 1 | <18 IU/mL^{3} | 0.81 (0.69, 0.89) | 54/67 | 0.94 (0.80, 0.99) | 31/33 | 64.4 (12.9, 587.4) | 89.9 (14.9, 542.5) |
| | Non-1 | <18 IU/mL^{3} | 0.43 (0.24, 0.63) | 12/28 | 0.95 (0.89, 0.98) | 100/105 | 15.0 (4.1, 60.1) | 13.2 (3.9, 44.3) |
| C | 1 | <18 IU/mL^{3} | 0.61 (0.52, 0.69) | 86/142 | 0.85 (0.68, 0.95) | 28/33 | 8.6 (3.0, 29.9) | 7.9 (2.6, 23.7) |
| | Non-1 | <18 IU/mL^{3} | 0.29 (0.13, 0.51) | 7/24 | 0.86 (0.74, 0.94) | 49/57 | 2.5 (0.7, 9.3) | 2.4 (0.7, 8.6) |
| D | 1 | <18 IU/mL^{3} | 0.46 (0.38, 0.54) | 68/148 | 0.84 (0.71, 0.93) | 42/50 | 4.5 (1.9, 11.7) | 3.2 (1.3, 7.7) |
| | Non-1 | <18 IU/mL^{3} | 0.07 (0.01, 0.24) | 2/27 | 0.87 (0.79, 0.93) | 86/99 | 0.5 (0.1, 2.6) | 0.4 (<0.1, 2.0) |
NPV: The denominator is the number of patients with no RVR at 4 weeks; the numerator is the number of patients who did not achieve SVR among patients with no RVR at 4 weeks.
PPV: The denominator is the number of patients with RVR at 4 weeks; the numerator is the number of patients who achieved SVR among patients with RVR.
1. Treatment Arm A = 24-week PEG-IFN + low-dose RBV;
Treatment Arm B = 24-week PEG-IFN + high-dose RBV
Treatment Arm C = 48-week PEG-IFN + low-dose RBV;
Treatment Arm D = 48-week PEG-IFN + high-dose RBV
2. Based on the logistic regression model including covariates for treatment arm, genotype (non-1 vs 1), baseline viral load (<= 7.40 E+5 IU/mL vs > 7.40 E+5 IU/mL), liver disease (non-cirrhotic vs cirrhotic), baseline SGPT (> 3*ULN vs <= 3*ULN) and age (<40 vs >=40). Genotype and/or treatment arm covariates were excluded if the analysis was by genotype and/or treatment arm.
3. Limit of detection for CAP/CTM HCV Test is 18 IU/mL
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# SUMMARY OF SAFETY AND EFFECTIVENESS
## 10.3.2.4. Predictive Values at Week 12 of Antiviral Therapy (EVR Analysis)
The NPV is higher for genotype 1 patients than non-1 patients. Additionally, the PPV is less predictive and significantly different for genotype 1 and non-1 patient groups.
Table 21 presents the NPV and PPV for all 4 treatment arms at 12 weeks stratified by genotype. Note that the sample sizes for non-1 patients are too small due to high response rate in this subgroup and insufficient to make meaningful conclusions.
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Table 21: NPV and PPV at Week 12 and Corresponding Odds Ratios: Treatment Arms A through D
| Treatment Arm^{1} | Genotype | Prediction Rule | Negative Predictive Value (NPV) | | Positive Predictive Value (PPV) | | Odds Ratio (95% CI) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Estimate (95% CI) | N | Estimate (95% CI) | N | Unadjusted | Adjusted^{3} |
| A | 1 | 2 Log Drop or No HCV | 1.00 (0.81, 1.00) | 18/18 | 0.34 (0.23, 0.46) | 23/68 | >8.7 (1.2, 378.6)^{3} | >13.7 (1.4, 132.0)^{3} |
| | Non-1 | 2 Log Drop or No HCV | 0.50 (0.01, 0.99) | 1/2 | 0.85 (0.75, 0.92) | 72/85 | NA | NA |
| B | 1 | 2 Log Drop or No HCV | 1.00 (0.75, 1.00) | 13/13 | 0.51 (0.40, 0.61) | 47/93 | >12.3 (1.7, 535.0)^{3} | >10.2 (1.1, 97.8)^{3} |
| | Non-1 | 2 Log Drop or No HCV | 1.00 (0.29, 1.00) | 3/3 | 0.90 (0.84, 0.95) | 117/130 | NA | NA |
| C | 1 | 2 Log Drop or No HCV | 0.94 (0.71, 1.00) | 16/17 | 0.54 (0.45, 0.62) | 83/155 | 18.4 (2.7, 782.6) | 17.0 (2.2, 134.1) |
| | Non-1 | 2 Log Drop or No HCV | 1.00 (0.03, 1.00) | 1/1 | 0.82 (0.72, 0.90) | 65/79 | NA | NA |
| D | 1 | 2 Log Drop or No HCV | 0.76 (0.50, 0.93) | 13/17 | 0.65 (0.58, 0.72) | 119/183 | 6.0 (1.8, 26.3) | 6.5 (1.9, 21.8) |
| | Non-1 | 2 Log Drop or No HCV | 1.00 (0.03, 1.00) | 1/1 | 0.90 (0.83, 0.94) | 113/126 | NA | NA |
NPV: The denominator is the number of patients with no EVR at 12 weeks; the numerator is the number of patients who did not achieve SVR among patients with no EVR at 12 weeks.
PPV: The denominator is the number of patients with EVR at 12 weeks; the numerator is the number of patients who achieved SVR among patients with EVR.
1. Treatment Arm A = 24-week PEG-IFN + low-dose RBV;
2. Treatment Arm B = 24-week PEG-IFN + high-dose RBV
3. Treatment Arm C = 48-week PEG-IFN + low-dose RBV;
4. Treatment Arm D = 48-week PEG-IFN + high-dose RBV
Based on the logistic regression model including covariates for treatment arm, genotype (non-1 vs 1), baseline viral load (<= 7.40 E+5 IU/mL vs > 7.40 E+5 IU/mL), liver disease (non-cirrhotic vs cirrhotic), baseline SGPT (>3*ULN vs <= 3*ULN) and age (<40 vs >=40). Genotype and/or treatment arm covariates were excluded if the analysis was by genotype and/or treatment arm.
Since NPV = 1.0 odds ratio estimate is not available. Conservative estimates of unadjusted and adjusted odds ratio are obtained by artificially subtracting one (1) from the numerator.
NA: with ≤ 3 in the denominator, the performance of the device for EVR in Non-1 cannot be determined.
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# SUMMARY OF SAFETY AND EFFECTIVENESS
## 10.3.2.5. Predictive Values at Week 24 of Antiviral Therapy
The Week 24 analysis is performed using the prediction rule of defining $<18 \, \text{IU/mL}$ (the LOD of the test) as response at Week 24. The NPV at Week 24 for all patients is extremely high ($>0.98$), regardless of genotype. However, the PPV at Week 24 is less predictive of SVR and varies by genotype. The analysis was also performed using a prediction rule of $<50 \, \text{IU/mL}^2$. No significant differences were noted in either PPV or NPV when comparing the two prediction rules.
Table 22 presents performance characteristics at Week 24 classified by treatment arm. This table shows that the NPV for 24 weeks for all subgroups are at least 0.96, independent of treatment duration and genotype. The numbers of patients in non-1 genotype subsets are too small due to high response rate in this subgroup and are insufficient to draw conclusions. Once again, the PPV is less predictive of SVR and varies by genotype.
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Table 22: NPV and PPV at Week 24 and Corresponding Odds Ratios: Treatment Arms A through D
| Treatment Arm^{1} | Genotype | Prediction Rule | Negative Predictive Value (NPV) | | Positive Predictive Value (PPV) | | Odds Ratio (95% CI) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Estimate (95% CI) | N | Estimate (95% CI) | N | Unadjusted | Adjusted^{2} |
| A | 1 | <18 IU/mL^{3} | 1.00 (0.85, 1.00) | 22/22 | 0.35 (0.24, 0.49) | 22/62 | >11.6 (1.6, 499.0)^{4} | >18.7 (2.0, 176.4)^{4} |
| | Non-1 | <18 IU/mL^{3} | 1.00 (0.16, 1.00) | 2/2 | 0.84 (0.74, 0.91) | 69/82 | NA | NA |
| B | 1 | <18 IU/mL^{3} | 1.00 (0.81, 1.00) | 18/18 | 0.53 (0.42, 0.64) | 45/85 | >19.1 (2.7, 816.6)^{4} | >13.5 (1.6, 111.2)^{4} |
| | Non-1 | <18 IU/mL^{3} | 1.00 (0.29, 1.00) | 3/3 | 0.90 (0.84, 0.95) | 120/133 | NA | NA |
| C | 1 | <18 IU/mL^{3} | 0.97 (0.84, 1.00) | 31/32 | 0.60 (0.51, 0.68) | 83/139 | 45.9 (7.1, 1894.5) | 36.0 (4.7, 274.7) |
| | Non-1 | <18 IU/mL^{3} | 1.00 (0.16, 1.00) | 2/2 | 0.82 (0.72, 0.90) | 64/78 | NA | NA |
| D | 1 | <18 IU/mL^{3} | 0.96 (0.80, 1.00) | 25/26 | 0.70 (0.63, 0.77) | 121/172 | NA | NA |
| | Non-1 | <18 IU/mL^{3} | 1.00 (0.16, 1.00) | 2/2 | 0.89 (0.82, 0.94) | 110/124 | >19.1 (2.7, 816.6)^{4} | >13.5 (1.6, 111.2)^{4} |
NPV: The denominator is the number of patients with no response at 24 weeks; the numerator is the number of patients who did not achieve SVR among patients with no response at 24 weeks.
PPV: The denominator is the number of patients with response at 24 weeks; the numerator is the number of patients who achieved SVR among patients with a response at 24 weeks.
1. Treatment Arm A = 24-week PEG-IFN + low-dose RBV;
2. Treatment Arm B = 24-week PEG-IFN + high-dose RBV
3. Treatment Arm C = 48-week PEG-IFN + low-dose RBV;
4. Treatment Arm D = 48-week PEG-IFN + high-dose RBV
Based on the logistic regression model including covariates for treatment arm, genotype (non-1 vs 1), baseline viral load (<= 7.40 E+5 IU/mL vs > 7.40 E+5 IU/mL), liver disease (non-cirrhotic vs cirrhotic), baseline SGPT (>3*ULN vs <= 3*ULN) and age (<40 vs >=40).
Genotype and/or treatment arm covariate were excluded if the analysis was by genotype and/or treatment arm.
3. Limit of detection for CAP/CTM HCV Test
4. Since NPV = 1.0 odds ratio estimate is not available. Conservative estimates of unadjusted and adjusted odds ratio are obtained by artificially subtracting one (1) from the numerator.
NA: with ≤ 3 in the denominator, the performance of the device for Week 24 in Non-1 cannot be calculated.
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## 10.3.3. Within-Subject Variability in Absence of Treatment
The objective of this analysis is to estimate the change in viral load (in log units) between two successive measurements of patients not receiving anti-viral therapy.
Baseline and screening serum sample results were available from 196 subjects enrolled in the clinical study to evaluate the effect of pegylated-interferon 2b treatment duration and Ribavir…
