COBAS® AMPLIPREP/COBAS® TAQMAN® CMV TEST (CAP/CTM CMV TEST)

P110037 · Roche Molecular Systems, Inc. · PAB · Jul 5, 2012 · Microbiology

Device Facts

• Record ID: P110037
• Device Name: COBAS® AMPLIPREP/COBAS® TAQMAN® CMV TEST (CAP/CTM CMV TEST)
• Applicant: Roche Molecular Systems, Inc.
• Product Code: PAB · Microbiology
• Decision Date: Jul 5, 2012
• Decision: APRL
• Regulation: 21 CFR 866.3180
• Device Class: Class 2

Intended Use

The COBAS® AmpliPrep/COBAS® TaqMan® CMV Test is an in vitro nucleic acid amplification test for the quantitative measurement of cytomegalovirus (CMV) DNA in human EDTA plasma 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. The COBAS® AmpliPrep/COBAS® TaqMan® CMV Test is intended for use as an aid in the management of solid-organ transplant patients who are undergoing anti-CMV therapy. In this population serial DNA measurements can be used to assess virological response to antiviral treatment. The results from the COBAS® AmpliPrep/COBAS® TaqMan® CMV Test must be interpreted within the context of all relevant clinical and laboratory findings. The COBAS® AmpliPrep/COBAS® TaqMan® CMV Test is not intended for use as a screening test for the presence of CMV DNA in blood or blood products.

Device Story

The COBAS® AmpliPrep/COBAS® TaqMan® CMV Test is an automated in vitro diagnostic system for quantifying CMV DNA in human EDTA plasma. It utilizes the COBAS® AmpliPrep Instrument for silica-based automated specimen preparation and the COBAS® TaqMan® Analyzer or COBAS® TaqMan® 48 Analyzer for real-time PCR amplification and detection. The system uses a Quantitation Standard (QS) DNA construct added to each specimen to compensate for inhibition and ensure accurate quantitation. The process involves lysis, magnetic glass particle capture, washing, elution, and simultaneous PCR amplification of target CMV DNA and QS DNA. The system calculates viral load in IU/mL by comparing target and QS signals. Clinicians use serial measurements to monitor virological response to antiviral therapy in solid-organ transplant patients. The output aids in managing treatment efficacy and assessing disease resolution.

Clinical Evidence

Clinical usefulness study of 211 kidney transplant recipients with CMV disease. Primary endpoint: time to clinical resolution of CMV disease. Results showed baseline CMV viral load <18,200 IU/mL was associated with more rapid resolution (HR=1.46, p=0.015). Viral suppression (<LLoQ) by Days 7, 14, and 21 was significantly correlated with faster disease resolution (HRs 1.62, 1.83, and 1.44 respectively, all p<0.05). A 1.5 log10 decline at Day 14 was not predictive of resolution.

Technological Characteristics

In vitro nucleic acid amplification test; real-time PCR; silica-based magnetic glass particle specimen preparation; Z05 DNA polymerase; dual-labeled oligonucleotide hydrolysis probes; automated instrumentation (COBAS® AmpliPrep/TaqMan®); EDTA plasma input; quantitation range 1.37E+02 to 9.10E+06 IU/mL; results reported in IU/mL; includes AmpErase (uracil-N-glycosylase) for carryover prevention.

Indications for Use

Indicated for solid-organ transplant patients diagnosed with CMV disease undergoing anti-CMV therapy. Used for quantitative measurement of CMV DNA in human EDTA plasma to assess virological response to treatment.

Regulatory Classification

Identification

A quantitative cytomegalovirus (CMV) nucleic acid test for transplant patient management is identified as a device intended for prescription use in the detection of CMV and as an aid in the management of transplant patients to measure CMV deoxyribonucleic acid (DNA) levels in human plasma and/or whole blood using specified specimen processing, amplification, and detection instrumentation. The test is intended for use as an aid in the management of transplant patients with active CMV infection or at risk for developing CMV infection. The test results are intended to be interpreted by qualified healthcare professionals in conjunction with other relevant clinical and laboratory findings.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) The labeling required under § 809.10(b) of this chapter must include: (i) A prominent statement that the device is not intended for use as a donor screening test for the presence of CMV DNA in blood or blood products. (ii) Limitations, which must be updated to reflect current clinical practice. The limitations must include, but are not limited to, statements that indicate: (A) Test results are to be interpreted by qualified licensed healthcare professionals in conjunction with clinical signs and symptoms and other relevant laboratory results; (B) Negative test results do not preclude CMV infection or tissue invasive CMV disease, and that CMV test results must not be the sole basis for patient management decisions. (iii) A detailed explanation of the interpretation of results and acceptance criteria must be provided and include specific warnings regarding the potential for variability in CMV viral load measurement when samples are measured by different devices. Warnings must include the following statement, where applicable: “Due to the potential for variability in CMV viral load measurements across different CMV assays, it is recommended that the same device be used for the quantitation of CMV viral load when managing CMV infection in individual patients.” (iv) A detailed explanation of the principles of operation and procedures for assay performance. (2) Design verification and validation must include the following: (i) Detailed documentation of the device description, including all parts that make up the device, reagents required for use with the CMV assay but not provided, an explanation of the methodology, design of the primer/probe sequences, rationale for the selected gene target, and specifications for amplicon size, guanine-cytosine content, and degree of nucleic acid sequence conservation. The design and nature of all primary, secondary, and tertiary quantitation standards used for calibration must also be described. (ii) A detailed description of the impact of any software, including software applications and hardware-based devices that incorporate software, on the device's function. (iii) Documentation and characterization of all critical reagents ( *e.g.,* determination of the identity, supplier, purity, and stability) and protocols for maintaining product integrity throughout its labeled shelf life.(iv) Stability data for reagents provided with the device and indicated specimen types, in addition to the basis for the stability acceptance criteria at all time points chosen across the spectrum of the device's indicated life cycle, which must include a time point at the end of shelf life. (v) All stability protocols, including acceptance criteria. (vi) Final lot release criteria, along with documentation of an appropriate justification 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) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel CMV stains ( *e.g.,* regular review of published literature and annual in silico analysis of target sequences to detect possible primer or probe mismatches). All results of this protocol, including any findings, must be documented.(viii) Analytical performance testing that includes: (A) Detailed documentation of the following analytical performance studies: Limit of detection, upper and lower limits of quantitation, inclusivity, precision, reproducibility, interference, cross reactivity, carryover, quality control, specimen stability studies, and additional studies as applicable to specimen type and intended use for the device. (B) Identification of the CMV strains selected for use in analytical studies, which must be representative of clinically relevant circulating strains. (C) Inclusivity study results obtained with a variety of CMV genotypes as applicable to the specific assay target and supplemented by in silico analysis. (D) Reproducibility studies that include the testing of three independent production lots. (E) Documentation of calibration to a standardized reference material that FDA has determined is appropriate for the quantification of CMV DNA ( *e.g.,* a recognized consensus standard).(F) Documentation of traceability performed each time a new lot of the standardized reference material to which the device is traceable is released, or when the field transitions to a new standardized reference material. (ix) Clinical performance testing that includes: (A) Detailed documentation of device performance data from either a method comparison study with a comparator that FDA has determined is appropriate, or results from a prospective clinical study demonstrating clinical validity of the device. (B) Data from patient samples, with an acceptable number of the CMV positive samples containing an analyte concentration near the lower limit of quantitation and any clinically relevant decision points. (C) The method comparison study must include predefined maximum acceptable differences between the test and comparator method across all primary outcome measures in the clinical study protocol. (D) The final release test results for each lot used in the clinical study.

Reference Devices

• COBAS® AMPLICOR® CMV MONITOR Test
• COBAS® AmpliPrep / COBAS® TaqMan® HCV Test
• COBAS® TaqMan® HBV Test
• cobas® TaqScreen DPX Test

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: In vitro real-time polymerase chain reaction (PCR) based assay for CMV viral load measurement in human plasma

Device Trade Name: COBAS® AmpliPrep/COBAS® TaqMan® CMV Test

Device Procode: PAB

Applicant's Name and Address: Roche Molecular Systems, Inc.
4300 Hacienda Drive
Pleasanton, CA 94588

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P110037

Date of FDA Notice of Approval: July 5, 2012

Expedited: Not applicable

II. INDICATIONS FOR USE

The COBAS® AmpliPrep/COBAS® TaqMan® CMV Test is an in vitro nucleic acid amplification test for the quantitative measurement of cytomegalovirus (CMV) DNA in human EDTA plasma 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.

The COBAS® AmpliPrep/COBAS® TaqMan® CMV Test is intended for use as an aid in the management of solid-organ transplant patients who are undergoing anti-CMV therapy. In this population serial DNA measurements can be used to assess virological response to antiviral treatment. The results from the COBAS® AmpliPrep/COBAS® TaqMan® CMV Test must be interpreted within the context of all relevant clinical and laboratory findings.

The COBAS® AmpliPrep/COBAS® TaqMan® CMV Test is not intended for use as a screening test for the presence of CMV DNA in blood or blood products.

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# III. CONTRAINDICATIONS

None

# IV. WARNINGS AND PRECAUTIONS

The warnings and precautions can be found in the COBAS® AmpliPrep/COBAS® TaqMan® CMV Test labeling.

# V. DEVICE DESCRIPTION

The COBAS® AmpliPrep/COBAS® TaqMan® CMV Test (CAP/CTM CMV Test) is an *in vitro* nucleic acid amplification test for the quantitative measurement of cytomegalovirus (CMV) DNA in human EDTA plasma 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. The CAP/CTM CMV Test is intended for use as an aid in the management of solid-organ transplant patients who have been diagnosed with CMV disease and are undergoing antiviral therapy.

The CAP/CTM CMV Test is based on two major processes: (1) specimen preparation to isolate CMV DNA and (2) simultaneous PCR amplification of target DNA and detection of cleaved dual-labeled oligonucleotide detection probe specific to the target. The CAP/CTM CMV Test permits automated specimen preparation followed by PCR amplification and detection of CMV target DNA and CMV Quantitation Standard (QS) DNA. The Master Mix reagent contains primers and probes specific for both CMV DNA and CMV QS DNA. The detection of amplified DNA is performed using target-specific and QS-specific dual-labeled oligonucleotide probes that permit independent identification of CMV amplicon and CMV QS amplicon. The quantitation of CMV viral DNA is performed using the CMV QS. It compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation of CMV DNA in each specimen.

The CMV QS is a non-infectious DNA construct that contains CMV sequences with identical primer binding sites as the CMV target DNA and a unique probe binding region that allows CMV QS amplicon to be distinguished from CMV target amplicon. The CMV QS is added to each specimen at a known copy number and is carried through the subsequent steps of specimen preparation and simultaneous PCR amplification and detection of cleaved dual-labeled oligonucleotide detection probes. The COBAS® TaqMan® Analyzer or COBAS® TaqMan® 48 Analyzer calculates the CMV DNA concentration in the test specimens by comparing the CMV signal to the CMV QS signal for each specimen and control.

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# Target Selection

The CMV target for this test is a highly-conserved, non-drug target region of the CMV DNA polymerase (UL54) gene. Generic silica-based specimen preparation is used to capture the CMV DNA and CMV QS DNA, and defined oligonucleotides are used as primers in amplification of the CMV DNA and CMV QS DNA. A target-specific and a QS-specific dual-labeled oligonucleotide probe permit independent identification of the CMV amplicon and of the CMV QS amplicon. The CAP/CTM CMV Test uses two amplification primers for PCR. A fluorescent, signal-generating probe, modified with a 5' fluorochrome (FAM) and a 3' quencher, hybridizes to one of the two strands and is cleaved by Z05 DNA polymerase during extension of the primers.

# Specimen Preparation

The CAP/CTM CMV Test utilizes automated specimen preparation on the COBAS® AmpliPrep Instrument by a generic silica-based capture technique. The procedure requires a sample input volume of 500 μL, 350 μL of which is processed by the COBAS® AmpliPrep Instrument. The CMV virus particles are lysed by incubation at elevated temperature with a protease and chaotropic lysis/binding buffer that releases nucleic acids and protects the released CMV DNA from DNAses in plasma. Protease and a known number of CMV QS DNA molecules are introduced into each specimen along with the lysis reagent and magnetic glass particles. Subsequently, the mixture is incubated and the CMV DNA and CMV QS DNA are bound to the surface of the magnetic glass particles. Unbound substances, such as salts, proteins, and other cellular impurities, are removed by washing the magnetic glass particles. After separating the magnetic glass particles and completing the washing steps, the adsorbed nucleic acids are eluted at an elevated temperature with an aqueous solution. The processed specimen, containing the released CMV DNA and CMV QS DNA, is added to the amplification mixture and transferred to the COBAS® TaqMan® Analyzer or the COBAS® TaqMan® 48 Analyzer.

# PCR Amplification

The PCR amplification reaction is performed with the thermo stable recombinant DNA Polymerase enzyme (Z05) from Thermus species Z05. In the presence of magnesium (Mg²⁺) and under the appropriate buffer conditions, Z05 has DNA polymerase activity. This allows PCR amplification to occur together with real-time detection of the amplicon. Processed specimens are added to the amplification mixture in amplification tubes (K-tubes) in which PCR amplification occurs. In the presence of Mg²⁺ and excess deoxynucleotide triphosphates (dNTPs), including deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine and deoxythymidine triphosphates (dATP, dGTP, dCTP, dUTP, and dTTP), Z05 polymerase extends the annealed primers forming double-stranded DNA.

# Target Amplification

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The Thermal Cycler in the COBAS® TaqMan® Analyzer or the COBAS® TaqMan® 48 Analyzer heats the reaction mixture to denature the double-stranded DNA and to expose the specific primer target sequences. As the mixture cools, the primers anneal to the target DNA. Z05, in the presence of $\mathrm{Mg}^{2+}$ and excess deoxynucleotide triphosphates (dNTPs), extends the annealed primers along the target template to produce double-stranded DNA molecules termed an amplicon. The COBAS® TaqMan® Analyzer or the COBAS® TaqMan® 48 Analyzer automatically repeats this process for a designated number of cycles, with each cycle intended to double the amount of amplicon DNA. The required number of cycles is preprogrammed into the COBAS® TaqMan® Analyzer or the COBAS® TaqMan® 48 Analyzer. Amplification occurs only in the region of the CMV genome between the primers; the entire CMV genome is not amplified.

## Target Detection

The CAP/CTM CMV Test utilizes real-time PCR technology. The use of fluorescently-labeled hydrolysis probes allows for real-time detection of PCR product accumulation by monitoring of the emission intensity of fluorescent reporter dyes released during the amplification process. The probes consist of CMV target and CMV QS-specific oligonucleotide probes with a reporter dye and a quencher dye. In the CAP/CTM CMV Test the CMV target and CMV QS probes are labeled with different fluorescent reporter dyes. When these probes are intact, the fluorescence of the reporter dye is suppressed by the proximity of the quencher dye due to Förster-type energy transfer effects. During PCR, the probe hybridizes to a target sequence and is cleaved by the $5' \rightarrow 3'$ nuclease activity of the thermo stable Z05 DNA polymerase during the extension phase of the PCR cycle. Once the reporter and quencher dyes are released and separated, quenching no longer occurs, and the fluorescent activity of the reporter dye is increased. The amplification of CMV DNA and CMV QS DNA are measured independently at different wavelengths. This process is repeated for a designated number of cycles, each cycle effectively increasing the emission intensity of the individual reporter dyes, permitting independent identification of CMV DNA and CMV QS DNA. The PCR cycle where a growth curve starts exponential growth is related to the amount of starting material at the beginning of the PCR.

## Selective Amplification

Selective amplification of target nucleic acid from the specimen is achieved in the CAP/CTM CMV Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP). The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine, but not DNA containing deoxythymidine. Deoxyuridine is not present in naturally occurring DNA, but is always present in amplicon due to the use of deoxyuridine triphosphate as one of the dNTPs in the Master Mix reagent; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by the AmpErase enzyme prior to amplification of the target DNA. Also, any nonspecific product formed after initial activation of the Master Mix by magnesium is destroyed by the AmpErase

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enzyme. The AmpErase enzyme, which is included in the Master Mix reagent, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme remains inactive for a prolonged period of time once exposed to temperatures above 55°C, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon formed during amplification.

## CMV DNA Quantitation

The CAP/CTM CMV Test quantifies CMV viral DNA by analyzing the difference between the CMV target and QS Ct values. The CMV QS is a non-infectious DNA construct, containing fragments of CMV sequences with primer binding regions identical to those of the CMV target sequence. The CMV QS contains CMV primer binding regions and generates an amplification product of the same length and base composition as the CMV target DNA. The detection probe binding region of the CMV QS has been modified to differentiate CMV QS amplicon from CMV target amplicon. During the extension phase of the PCR in the COBAS® TaqMan® Analyzer or the 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 the AMPLILINK software and stored in a database. Pre-Checks are used to determine if the CMV DNA target and CMV QS DNA 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 CMV DNA target and the CMV QS DNA. The lot-specific calibration constants provided with the CAP/CTM CMV Test are used to calculate the titer value for the specimens and controls based on both the CMV DNA target and CMV QS DNA Ct values. Titer results are reported in International Units/mL (IU/mL).

## Quality Control Information

One replicate each of the COBAS® TaqMan® Negative Control, the CMV Low Positive Control, and the CMV High Positive Control must be included in each test batch. The batch is valid if no flags appear for any of the controls. The user is instructed to check the run printout for flags and comments to ensure that the batch is valid.

- Negative Control: The CMV negative control must yield a “Target Not Detected” result. If the CMV negative control is flagged as invalid, then the entire batch is invalid.
- Positive Controls: The acceptable titer ranges for CMV low positive control and CMV high positive control are provided on the CAP/CTM CMV Test reagent cassette barcodes. The CMV DNA IU/mL for CMV high and low positive

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controls should fall within their acceptable titer ranges. If one or both of the positive controls are flagged as invalid, then the entire batch is invalid.

- Run Validation — AMPLILINK version 3.3 Series: The user is instructed to check AMPLILINK software results window or printout for flags and comments to ensure that the batch is valid. For control orders, a check is made to determine if the IU/mL value for the control is within its fixed range. If the IU/mL value for the control lies outside of its range, a FLAG is generated to show the control has failed.

The batch is valid if no flags appear for any of the controls. The batch is not valid if any of the following flags appear:

Negative Control

| Flag | Result | Interpretation |
| --- | --- | --- |
| NC_INVALID | Invalid | An invalid result or a “valid” result that was not negative for CMV target. |

CMV Low Positive Control

| Flag | Result | Interpretation |
| --- | --- | --- |
| LPC_INVALID | Invalid | An invalid result or a control out of range. |

CMV High Positive Control

| Flag | Result | Interpretation |
| --- | --- | --- |
| HPC_INVALID | Invalid | An invalid result or a control out of range. |

## Interpretation of Results

The "COBAS® TaqMan® Analyzer or the COBAS® TaqMan® 48 Analyzer automatically determines the CMV DNA concentration for the specimens and controls. The CMV DNA concentration is expressed in IU/mL.

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 | Report results as "CMV DNA not detected". |
| <1.37E+02 IU/mL | Calculated IU/mL results are below the Lower Limit of Quantitation (LLoQ) of the test. Report results as "CMV DNA detected, less than 137 CMV DNA IU/mL." |
| ≥1.37E+02 IU/mL and ≤9.10E+06 IU/mL | Calculated results greater than or equal to 137 CMV DNA IU/mL and less than or equal to 9.10E+06 CMV DNA IU/mL are within the Linear Range of the test. |

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| >9.10E+06 IU/mL | Calculated IU/mL results are above the Upper Limit Of Quantitation (ULoQ) of the test. Report results as "greater than 9.10E+06 CMV DNA IU/mL." Or if quantitative results are desired, the original specimen should be diluted with CMV-negative human EDTA-plasma and the test should be repeated. Multiply the obtained result by the dilution factor. |
| --- | --- |
| Failed | Specimen was not correctly processed. Test should be repeated with another aliquot of the original sample. |
| Invalid | An invalid result. Test should be repeated with another aliquot of the original sample. |

Note: Samples above the ULoQ of the test that produce an invalid result with a flag "QS_INVALID" should not be reported as &gt;9.10E+06 IU/mL. The original sample should be diluted with CMV-negative EDTA plasma and the test should be repeated. Multiply the obtained result by the dilution factor.

Note: One International Unit (IU) (as reported by the CAP/CTM CMV Test) based on the 1st WHO International Standard for Human Cytomegalovirus (HCMV) for Nucleic Acid Amplification (NAT)-based Assays (NIBSC 09/162) is equivalent to 1.1 copy (cp) CMV DNA as defined by the CAP/CTM CMV Test.

Note: The test can quantitate CMV DNA over the range of 1.37E+02 to 9.10E+06 IU/mL.

Note: The analytical measurement range of analyte values that can be directly measured for a specimen with a maximum dilution of one to one hundred using the CAP/CTM CMV Test is 1.37E+02 to 9.10E+08 IU/mL.

## Kit Configuration and Components

The CAP/CTM CMV Test utilizes two kits for the detection of CMV DNA in human plasma using automated specimen preparation on the COBAS® AmpliPrep Instrument and automated amplification/detection on the COBAS® TaqMan® Analyzer. All reagents and controls required for sample preparation, amplification and detection are provided in the CAP/CTM CMV Test kit and are packaged in one of four barcoded reagent cassettes, which are loaded directly onto the COBAS® AmpliPrep instrument along with specimens and controls.

## Materials Provided:

A. COBAS® AmpliPrep/COBAS® TaqMan® CMV Test (For 72 Tests)

- CMV CS1 (CMV Magnetic Glass Particles Reagent Cassette)
- CMV CS2 (CMV Lysis Reagent Cassette)
- CMV CS3 (CMV Multi-Reagent Cassette)
- CMV CS4 (CMV Test-Specific Reagent Cassette)
- CMV H(+)C (CMV High Positive Control)

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- CMV L(+ )C (CMV Low Positive Control)
- CTM (-)C [COBAS TaqMan Negative Control (Human Plasma)]
- CMV H(+ )C Clip (High Positive Control Barcode Clip)
- CMV L(+ )C Clip (Low Positive Control Barcode Clip)
- CMV (-)C Clip (Negative Control Barcode Clip)

B. COBAS® AmpliPrep/COBAS® TaqMan® Wash Reagent

## Materials Required but Not Provided:

### Instrumentation and Software

- COBAS® AmpliPrep Instrument
- COBAS® TaqMan® Analyzer or COBAS® TaqMan® 48 Analyzer
- Optional: Docking Station
- Optional: cobas p 630 Instrument
- AMPLILINK Software, Version 3.3 Series
- Data Station for the AMPLILINK software, with printer.
- AMPLILINK Software v3.3 Series Manuals:
- COBAS® AmpliPrep Instrument Manual. For use with the COBAS® TaqMan® Analyzer, COBAS® TaqMan® 48 Analyzer, COBAS® AMPLICOR® Analyzer, or cobas p 630 Instrument, and the AMPLILINK software, Version 3.2 and 3.3 Series
- COBAS® TaqMan® Analyzer (plus optional docking station) Instrument Manual For use with the AMPLILINK software, Version 3.2 and 3.3 Series Application Manual
- COBAS® TaqMan® 48 Analyzer Instrument Manual For use with the AMPLILINK software, Version 3.2 and 3.3 series Application Manual
- AMPLILINK software Version 3.3 Series Application Manual. For use with the COBAS® AmpliPrep Instrument, COBAS® TaqMan® Analyzer, COBAS® TaqMan® 48 Analyzer, COBAS® AMPLICOR® Analyzer, and cobas p 630 Instrument.
- Optional: cobas p 630 Instrument Operator’s Manual Software Version 2.2

### Disposables

- Sample processing units (SPUs)
- Sample input tubes (S-tubes) with barcode clips
- Racks of K-tips
- Racks of K-tubes

### Other Materials Required but Not Provided:

- Sample Rack (SK 24 rack)
- Reagent Rack

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- SPU rack
- K-carrier
- K-carrier Transporter
- K-carrier rack (required for use with COBAS® TaqMan® 48 Analyzer only)
- Pipettors with aerosol barrier or positive displacement DNase-free tips (capacity 1000 µL)
- Disposable gloves, powderless
- Vortex mixer

## VI. ALTERNATIVE PRACTICES AND PROCEDURES

To date, there is no FDA cleared or approved *in vitro* nucleic acid amplification test for the quantitative measurement of Cytomegalovirus (CMV) DNA in either human blood or plasma. Current quantitative CMV DNA testing is based on non-FDA-approved laboratory developed tests and practices established by transplant centers and associated laboratories.

The pp65 antigenemia assay is an alternative to measurement of CMV viral load that has been used in transplant centers. The test is a fluorescent assay based on detection of infected cells in peripheral blood. The test is comparable in sensitivity to laboratory-developed CMV amplification-based assays but has largely been supplanted by CMV PCR assays due to greater reliability and technical ease of the latter assays.

## VII. MARKETING HISTORY

The CAP/CTM CMV Test received CE certification and was launched on March 31, 2011 outside of the United States, under the list number 4902068190.

The following countries receive the CAP/CTM CMV Test: Australia, Austria, Belgium, Croatia, Czech Republic, Denmark, Estonia, Finland, France, Germany, Hong Kong, Italy, Kuwait, Malaysia, Netherlands, New Zealand, Norway, Poland, Portugal, Republic of Korea, Saudi Arabia, Slovakia, Slovenia, South Africa, Spain, Sweden, Switzerland, Taiwan, Turkey, and the United Kingdom.

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

## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

To aid in the management of solid-organ transplant patients who are undergoing anti-CMV drug therapy, the results from the CAP/CTM CMV Test must be interpreted in the context of all relevant clinical and laboratory findings. Failure of the CAP/CTM CMV Test to perform as indicated, or human error in the use of the test or the interpretation of the test result, may result in an incorrect test result that is too low or too high. An erroneous low test result may lead to inappropriate patient management

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decisions, a delay or lack of treatment, or may instill a false sense of security in a patient or clinician. An erroneous high test result may contribute to unnecessary treatment or create anxiety in the patient.

## IX. SUMMARY OF PRECLINICAL STUDIES

### A. Laboratory Studies

#### Traceability to the 1st WHO International Standard for Human Cytomegalovirus (HCMV) for Nucleic Acid Amplification (NAA)-based Assays

Several Standards and Controls have been used during development of the CAP/CTM CMV Test to provide traceability to the 1st WHO International Standard for Human Cytomegalovirus (HCMV) for Nucleic Acid Amplification (NAT)-based Assays (NIBSC 09/162) [1] (1st CMV WHO Standard). The standards used during development of the test include the 1st CMV WHO Standard, RMS CMV Secondary Standard, RMS CMV Secondary Standard Source Material, and RMS CMV Calibration Panel (Lambda CMA1.2).

The calibration of the CAP/CTM CMV Test is traceable to the 1st WHO International Standard for HCMV through the use of the RMS CMV Secondary Standard (SS). Due to the limited amount of the available primary standard, the 1st WHO International Standard for HCMV, use of the RMS CMV SS, which is traceable to the CMV WHO International Standard, is the only practical option for use in routine manufacturing and calibration of the CAP/CTM CMV Test. Each lot of the CAP/CTM CMV Test is assigned a set of lot-specific calibration coefficients as follows:

1. The RMS CMV SS and a 5-member calibration panel, consisting of lambda packaged CMV DNA in Negative Human Plasma at the nominal concentrations of 1.00E+07 copy/mL, 3.16E+06 copy/mL, 3.16E+04 copy/mL, 1.00E+03 copy/mL, and 5.62E+02 copy/mL, are tested in multiple replicates in each of six batches.
2. A validated Internal Calibration Software (ICS) is used to pool data from all six batches and to screen out outliers (at the level of significance of 0.05) based on an Extreme Studentized Deviate (ESD) test. A dose-response curve between the log-transformed nominal titers of the calibrators and the observed delta Ct values (which are obtained by subtracting Ct value of the CMV target from the Ct value of the Quantitation Standard) is established using a second order polynomial regression. The three “a”, “b”, and “c” coefficients of the regression are the tentative calibration coefficients.
3. Based on the observed delta Ct values for the RMS CMV SS, the mean observed titer value for the RMS CMV SS for the run is calculated using the tentative calibration coefficients obtained in the step (2).

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(4) A Calibration Adjustment Factor (CAF), a ratio of the observed titer value to the assigned (fixed) titer value (in IU/mL) of the RMS CMV SS, is calculated. The resulting CAF value is then applied to adjust all titers including the expected titers of the calibrators.

(5) A new dose-response curve between the new expected titers of the calibrators and their observed delta Ct values for the calibration run is established using a second order polynomial regression. The three coefficients in the second order polynomial equation, “a”, “b”, and “c”, are the lot-specific calibration coefficients.

This process confers a lot-specific adjustment in calibration and anchors the calibration to the RMS CMV SS in a way that can be traced to the CMV WHO International Standard.

The Standards, the Calibration Panel and an independent CMV clinical specimen were tested at similar levels in the "Traceability" study. The concentration range tested for the 1st CMV WHO Standard was from 5.00E+02 IU/mL to 5.00E+05 IU/mL (2.70 – 5.70 log₁₀ IU/mL), the RMS CMV Secondary Standard Source Material was tested from 5.00E+02 IU/mL to 1.00E+07 IU/mL (2.70 – 7.00 log₁₀ IU/mL), the RMS CMV Calibration Panel was tested from 5.23E+02 to 9.30E+06 IU/mL (2.72 – 6.97 log₁₀ IU/mL), and the independent CMV clinical specimen was tested from 5.00E+02 IU/mL to 2.27E+04 IU/mL (2.70 – 4.36 log₁₀ IU/mL).

The results indicate that the calibration and standardization process of the CAP/CTM CMV Test provide quantitation values for the calibration panel, the clinical sample, the source material for the RMS CMV Secondary Standard, and the CMV WHO Standard that are similar to the expected values with deviation of no more than 0.28 log₁₀ IU/mL. The maximum deviation was obtained at the test LLoQ using the regression analyses for the Calibration Panel and the CMV WHO Standard.

Comparison of the 1st CMV WHO Standard with the RMS CMV Secondary Standard Source Material, CAP/CTM CMV Calibration Panel (Lambda CMA1.2), and an independent clinical sample:

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Linear Regression for Dilutions of CMV WHO Standard, CMV Secondary Standard Source Material AD169, Clinical Specimen #N565967-3, and CAP/CTM CMV Calibration Panel


# Limit of Detection and Lower Limit of Quantitation using the 1st WHO International Standard for Human Cytomegalovirus

The Limit of Detection (LoD) and lower limit of quantitation (LLoQ) of the CAP/CTM CMV Test were determined according to CLSI Guideline EP17-A, Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline, by analysis of six low level CMV DNA positive panels. The six independent low level CMV DNA panels were prepared using the 1st WHO International Standard for Human Cytomegalovirus (HCMV) (NIBSC 09/162, Merlin strain, genotype 1 based on the glycoprotein B gene UL55), and six independent pools of EDTA plasma as diluents. Each panel consisted of six CMV concentrations (46, 91, 137, 182, 270, and 364 IU/mL) and a CMV DNA-negative sample (blank). In addition, the limit of blank (LoB) was confirmed to be 0 IU/mL by analysis of blank samples from six unique pools of CMV DNA negative EDTA plasma, which were also used as the diluents for the low level CMV DNA positive panels.

Testing for this study was carried out using three CAP/CTM CMV Test reagent lots and three CAP/CTM systems over nine days. Two operators were employed in this study. At least 223 valid results per concentration level were obtained from 75 valid runs over nine days, which were split across three reagent lots and three CAP/CTM systems.

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# LoB Confirmation

CMV DNA-negative EDTA plasma (“blank”) samples from six different panels yielded 100% “Target Not Detected” results out of a total of 225 valid results. Since no blank replicates reported a titer value, the determined LoB is confirmed to be 0 IU/mL.

## WHO CMV Standard LoD Study — LoB Results

| Negative EDTA Plasma Panel # | Number of Positives | Number of Results | % Positive |
| --- | --- | --- | --- |
| 1 | 0 | 39 | 0% |
| 2 | 0 | 36 | 0% |
| 3 | 0 | 36 | 0% |
| 4 | 0 | 36 | 0% |
| 5 | 0 | 36 | 0% |
| 6 | 0 | 42 | 0% |
| Total | 0 | 225 | 0% |

# LoD – Hit Rate Analysis

Limit of Detection (LoD), as defined by hit rate analysis, is the lowest concentration level that has a hit rate that is greater than or equal to 95% and for which all higher concentrations tested also have hit rates greater than or equal to 95%. When analyzed lot-wise, the LoD is 46 IU/mL for lot 16578B (hit rate =100%) and 91 IU/mL for lot 16580B (hit rate =100%) and lot P11496 (hit rate = 100%). The LoD for the test when all three reagent lots are combined is 46 IU/mL (hit rate = 96.4%).

## WHO CMV Standard LoD Study — LoD Hit Rate Analysis Results by Reagent Lot

| Kit Lot | Nominal Concentration (IU/mL) | N | Total Positive | Hit Rate |
| --- | --- | --- | --- | --- |
| All 3 Lots combined | 0 | 225 | 0 | 0% |
| | 46 | 224 | 216 | 96.4% |
| | 91 | 225 | 224 | 99.6% |
| | 137 | 223 | 223 | 100% |
| | 182 | 224 | 224 | 100% |
| | 273 | 224 | 224 | 100% |
| | 364 | 224 | 224 | 100% |
| Lot 16578B | 0 | 75 | 0 | 0% |
| | 46 | 75 | 75 | 100% |
| | 91 | 75 | 74 | 98.7% |
| | 137 | 75 | 75 | 100% |
| | 182 | 75 | 75 | 100% |

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| | 273 | 74 | 74 | 100% |
| --- | --- | --- | --- | --- |
| | 364 | 75 | 75 | 100% |
| Lot 16580B | 0 | 78 | 0 | 0% |
| | 46 | 77 | 73 | 94.8% |
| | 91 | 78 | 78 | 100.0% |
| | 137 | 76 | 76 | 100% |
| | 182 | 77 | 77 | 100% |
| | 273 | 78 | 78 | 100% |
| | 364 | 78 | 78 | 100% |
| Lot P11496 | 0 | 72 | 0 | 0% |
| | 46 | 72 | 68 | 94.4% |
| | 91 | 72 | 72 | 100.0% |
| | 137 | 72 | 72 | 100% |
| | 182 | 72 | 72 | 100% |
| | 273 | 72 | 72 | 100% |
| | 364 | 71 | 71 | 100% |

## LoD — Geometric Mean Analysis

Geometric means of the observed titers obtained at the LoD level determined by the hit rate analysis were calculated. In general, the geometric mean values were in agreement with the nominal concentrations. The geometric mean titer at the hit rate based LoD level ranged from 55.6 to 111.7 IU/mL for individual lots, and was 50.8 IU/mL for all three reagent lots combined.

WHO CMV Standard LoD Study — Geometric Mean Titers at the Hit Rate Based LoD Level

| Kit Lot | LoD (Nominal Concentration in IU/mL) | N Positive | Geometric Mean Titer (IU/mL) |
| --- | --- | --- | --- |
| ALL LOTS | 46 | 216 | 50.8 |
| LOT 16578B | 46 | 75 | 55.6 |
| LOT 16580B | 91 | 78 | 84.1 |
| LOT P11496 | 91 | 72 | 111.7 |

Based on the analyses above, the LoD of the CAP/CTM CMV Test using the 1st WHO International Standard for Human Cytomegalovirus (HCMV) (NIBSC 09/162, Merlin strain, genotype 1 based on the glycoprotein B gene UL55) is determined to be 91 IU/mL, with the geometric mean of the observed titer at the LoD level of 111.7 IU/mL.

## LLoQ Analysis

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LLoQ is defined as the lowest level of CMV DNA that can be reliably detected (i.e., percent of detected greater than 95%) and at which the total error for accuracy is less than or equal to 1.0 log₁₀, where total error (TAE) is calculated as |bias| + 2 x standard deviations (SDs) per CLSI EP-17A guideline, and TAE is also calculated as SQRT(2) x 2 x SDs based on the “difference between two measurements” approach.

## WHO CMV Standard LoD Study — LLoQ Summary by Reagent Lot

| Kit Lot | Nominal CMV Concentration (IU/mL) | log₁₀ Nominal (IU/mL) | N | AVG log₁₀ Titer (IU/mL) | SD log₁₀ Titer (IU/mL) | Bias | TAE = |Bias| + 2 x SD | TAE = SQRT(2) x 2 x SD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| All 3 Lots combined | 46 | 1.66 | 224 | 1.71 | 0.37 | 0.05 | 0.79 | 1.05 |
| | 91 | 1.96 | 225 | 1.99 | 0.31 | 0.04 | 0.66 | 0.88 |
| | 137 | 2.14 | 223 | 2.20 | 0.29 | 0.06 | 0.64 | 0.82 |
| | 182 | 2.26 | 224 | 2.34 | 0.27 | 0.08 | 0.62 | 0.76 |
| | 273 | 2.44 | 224 | 2.55 | 0.26 | 0.11 | 0.63 | 0.74 |
| | 364 | 2.56 | 224 | 2.60 | 0.27 | 0.04 | 0.58 | 0.76 |
| Lot 16578B | 46 | 1.66 | 75 | 1.74 | 0.39 | 0.09 | 0.87 | 1.10 |
| | 91 | 1.96 | 75 | 2.02 | 0.32 | 0.06 | 0.70 | 0.90 |
| | 137 | 2.14 | 75 | 2.23 | 0.26 | 0.10 | 0.62 | 0.74 |
| | 182 | 2.26 | 75 | 2.37 | 0.24 | 0.11 | 0.59 | 0.68 |
| | 273 | 2.44 | 74 | 2.58 | 0.25 | 0.14 | 0.64 | 0.71 |
| | 364 | 2.56 | 75 | 2.63 | 0.29 | 0.07 | 0.65 | 0.82 |
| Lot 16580B | 46 | 1.66 | 77 | 1.63 | 0.34 | -0.03 | 0.71 | 0.96 |
| | 91 | 1.96 | 78 | 1.92 | 0.33 | -0.03 | 0.69 | 0.93 |
| | 137 | 2.14 | 76 | 2.13 | 0.33 | 0.00 | 0.66 | 0.93 |
| | 182 | 2.26 | 77 | 2.28 | 0.29 | 0.02 | 0.60 | 0.82 |
| | 273 | 2.44 | 78 | 2.49 | 0.25 | 0.05 | 0.55 | 0.71 |
| | 364 | 2.56 | 78 | 2.55 | 0.27 | -0.01 | 0.55 | 0.76 |
| Lot P11496 | 46 | 1.66 | 72 | 1.75 | 0.36 | 0.09 | 0.81 | 1.02 |
| | 91 | 1.96 | 72 | 2.05 | 0.28 | 0.09 | 0.65 | 0.79 |
| | 137 | 2.14 | 72 | 2.23 | 0.27 | 0.09 | 0.63 | 0.76 |
| | 182 | 2.26 | 72 | 2.37 | 0.25 | 0.11 | 0.61 | 0.71 |
| | 273 | 2.44 | 72 | 2.58 | 0.28 | 0.14 | 0.70 | 0.79 |
| | 364 | 2.56 | 71 | 2.64 | 0.24 | 0.08 | 0.56 | 0.68 |

Based on the analyses above, the LLoQ of the CAP/CTM CMV Test using the 1st WHO International Standard for Human Cytomegalovirus (HCMV) (NIBSC 09/162, Merlin strain, genotype 1 based on the glycoprotein B gene UL55) is determined to be 91 IU/mL, with the mean of the observed titer of 111.7 IU/mL. The results of this study also support the claimed LLoQ of 137 IU/mL.

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# Limit of Detection and Lower Limit of Quantitation Using CMV Glycoprotein B (gB) Genotypes 2, 3, and 4 Specimens

The Limit of Detection (LoD) and the Lower Limit of Quantitation (LLoQ) for the CAP/CTM CMV Test using CMV glycoprotein B (gB) genotypes 2-4 clinical specimens were evaluated by testing at least 49 valid replicates at 137 IU/mL, 91 IU/mL, and 27 IU/mL, respectively, for each gB genotype 2-4. The testing was completed with 25 valid runs tested across six days using two kit lots of reagents and two CAP/CTM instrument systems. Two operators participated in the execution of this study.

CMV clinical specimens of three different glycoprotein-B (gB) genotypes, genotype 2 (IMPACT Accession Number P915569), genotype 3 (IMPACT Accession Number M554408, and genotype 4 (IMPACT Accession Number P722647), were collected as part of the IMPACT clinical trial for the Roche drug valganciclovir (protocol NT18435/A). The gB genotype of the clinical specimens was determined by DNA sequencing analysis. The gB genotype specimen titers were assigned by normalizing the mean measured titer of a prepared dilution of each clinical specimen to the mean measured titer of the CMV Secondary Standard (Lot TRLOT03, RMD Pleasanton, CA.) tested at two levels (2.18E+04 IU/mL (neat) and 4.55E+02 IU/mL).

## LoD – Hit Rate Analysis

Limit of Detection, as defined by hit rate analysis, is the lowest concentration level that has a hit rate that is greater than or equal to 95% and for which all higher concentrations tested also have hit rates greater than or equal to 95%. For all three genotypes, both the 137 IU/mL and 91 IU/mL levels gave 100% hit rates. At the level of 27 IU/mL, observed hit rates were less than 95% for all three genotypes (Genotype 2 = 88.0%, Genotype 3 = 78.0%, and Genotype 4 = 94.0%).

CMV gB Genotypes 2-4 LoD Study — Summary Results

| Sample ID | Nominal Concentration in IU/mL | Number of Observed Positive Hits | Number of Valid Replicates | Hit Rate |
| --- | --- | --- | --- | --- |
| gB2 - 1 | 27 IU/mL | 44 | 50 | 88% |
| gB2 - 2 | 91 IU/mL | 50 | 50 | 100% |
| gB2 - 3 | 137 IU/mL | 49 | 49 | 100% |
| | | | | |
| gB3 - 1 | 27 IU/mL | 39 | 50 | 78% |
| gB3 - 2 | 91 IU/mL | 50 | 50 | 100% |
| gB3 - 3 | 137 IU/mL | 49 | 49 | 100% |
| | | | | |
| gB4 - 1 | 27 IU/mL | 47 | 50 | 94% |
| gB4 - 2 | 91 IU/mL | 50 | 50 | 100% |

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| gB4 - 3 | 137 IU/mL | 49 | 49 | 100% |
| --- | --- | --- | --- | --- |

The observed Limit of Detection for each of the three genotypes in this study is 91 IU/mL, which is similar to the observed LoD performance of the predominant gB genotype 1 in the LoD study using the 1st WHO International Standard for Human Cytomegalovirus (HCMV) (NIBSC 09/162, Merlin strain, genotype 1 based on the glycoprotein B gene UL55).

Based on the analyses above, the LoD of the CAP/CTM CMV Test using CMV Glycoprotein B (gB) Genotypes 2-4 clinical specimens is determined to be 91 IU/mL.

## LLoQ Analysis

LLoQ is defined as the lowest level of CMV DNA that can be reliably detected (i.e., percent of detected greater than 95%) and at which the total error for accuracy is less than or equal to 1.0 log₁₀, where total error (TAE) is calculated as |bias| + 2 x standard deviations (SDs) per CLSI EP-17A guideline, and TAE is also calculated as SQRT(2) x 2 x SDs based on the “difference between two measurements” approach.

CMV gB Genotypes 2-4 LoD Study — LLoQ Summary Results

| Sample ID | Nominal Concentration in (IU/mL) | log_{10} Nominal (IU/mL) | N | AVG log_{10} Titer (IU/mL) | SD log_{10} Titer (IU/mL) | Bias | TAE = |Bias| + 2 x SD | TAE = SQRT(2) x 2 x SD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| gB2 - 1 | 27 | 1.43 | 50 | 1.61 | 0.30 | 0.17 | 0.77 | 0.85 |
| gB2 - 2 | 91 | 1.96 | 50 | 2.04 | 0.22 | 0.08 | 0.52 | 0.62 |
| gB2 - 3 | 137 | 2.14 | 49 | 2.21 | 0.20 | 0.08 | 0.48 | 0.57 |
| | | | | | | | | |
| gB3 - 1 | 27 | 1.43 | 50 | 1.51 | 0.39 | 0.08 | 0.86 | 1.10 |
| gB3 - 2 | 91 | 1.96 | 50 | 1.93 | 0.27 | -0.03 | 0.57 | 0.62 |
| gB3 - 3 | 137 | 2.14 | 49 | 2.22 | 0.13 | 0.08 | 0.34 | 0.37 |
| | | | | | | | | |
| gB4 - 1 | 27 | 1.43 | 50 | 1.52 | 0.32 | 0.09 | 0.73 | 0.90 |
| gB4 - 2 | 91 | 1.96 | 50 | 2.03 | 0.22 | 0.07 | 0.51 | 0.62 |
| gB4 - 3 | 137 | 2.14 | 49 | 2.24 | 0.21 | 0.11 | 0.53 | 0.59 |

Based on the analyses above, the LLoQ of the CAP/CTM CMV Test using CMV Glycoprotein B (gB) Genotypes 2-4 clinical specimens is determined to be 91 IU/mL, with the mean of the observed titer of 111.7 IU/mL. The results of this study also support the claimed LLoQ of 137 IU/mL.

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# Linear Range Using Cultured CMV AD169 Virus

A 10-member panel was used to evaluate the linear range of the CAP/CTM CMV Test in accordance with CLSI Guideline EP06-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline. The panel was prepared by diluting cultured CMV virus (strain AD169, genotype 2 based on the glycoprotein B gene UL55) using a pool of CMV DNA negative EDTA plasma as the diluent. Each panel member was tested with two replicates per run, with three runs per day, for a minimum of six days for a total of 36 replicates per panel member evenly distributed across three lots of CAP/CTM CMV Test kit reagents, and three CAP/CTM systems.

Cultured human CMV stock material (Advanced Biotechnology Inc.; Columbia, MD; M/N: 10-103-100, Lot: 7E0006-PV, strain AD169, genotype 2 based on the glycoprotein B gene UL55) was diluted into a pool of CMV DNA negative EDTA plasma to prepare the 10-level linearity panel. The cultured human CMV stock material (source material for the RMS CMV Secondary Standard, Lot TRLOT03) was value assigned as $7.28\mathrm{E} + 10$ IU/mL based on the CMV Secondary Standard lot TRLOT03 $(2.184\mathrm{E} + 04$ IU/mL, n=62, COBAS® AMPLICOR® CMV MONITOR Test).

## CMV AD169 Linearity Study — Test Panel Levels

| Level / Panel Member # | Nominal Titer (copy/mL) | Nominal Titer (IU/mL) | Log_{10} Nominal Titer (IU/mL) | Description |
| --- | --- | --- | --- | --- |
| 1 | 2.0E+07 | 1.82E+07 | 7.26 | > ULoQ |
| 2 | 1.0E+07 | 9.10E+06 | 6.96 | ULoQ |
| 3 | 1.0E+06 | 9.10E+05 | 5.96 | < ULoQ |
| 4 | 1.0E+05 | 9.10E+04 | 4.96 | Intermediate Level |
| 5 | 1.0E+04 | 9.10E+03 | 3.96 | Intermediate Level |
| 6 | 1.0E+03 | 9.10E+02 | 2.96 | Intermediate Level |
| 7 | 5.0E+02 | 4.55E+02 | 2.66 | > LLoQ |
| 8 | 2.5E+02 | 2.28E+02 | 2.36 | > LLoQ |
| 9 | 1.5E+02 | 1.37E+02 | 2.14 | LLoQ |
| 10 | 1.0E+02 | 9.10E+01 | 1.96 | < LLoQ |

Deviation from linearity and bias were assessed by evaluating mean $\log_{10}$ observed values and the nominal values.

Acceptance criteria for the CMV AD169 Linearity Study were the following:

- Linearity range: Maximum deviation between linear regression and better fitting non-linear regression (i.e., deviation from linearity) should be $\leq 0.3$ $\log_{10}$ for all tested concentration levels.

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- Repeatability: $\leq 0.3 \log_{10}$ for all concentration levels tested pooled.

Out of the 377 valid results, two outliers were identified by the Extreme Studentized Deviate Test (alpha = 0.01). One outlier (4.38 $\log_{10}$ titer IU/mL) was identified from level 9.10E+01 IU/mL (expected titer = 1.96 $\log_{10}$) with ESD score of 4.1773, exceeding the lambda cutoff of 3.3561. A second outlier (3.91 $\log_{10}$ titer IU/mL) was identified from level 1.37E+02 IU/mL (expected titer = 2.14 $\log_{10}$) with ESD score of 3.8050, exceeding the lambda cutoff of 3.3561. Data was analyzed with and without the outliers.

Linear ($1^{\text{st}}$ order), quadratic ($2^{\text{nd}}$ order), and cubic ($3^{\text{rd}}$ order) polynomial models were fitted to the total data set (with and without the two outliers). It was determined that the linear ($1^{\text{st}}$ order) model was the best fit for each of the two data sets (with and without the two outliers). Linear regression analyses of the mean $\log_{10}$ observed titer vs. the nominal $\log_{10}$ titer with and without the two outliers are presented below.

CMV AD169 Linearity Study — Linearity of the CAP/CTM CMV Test (All Data Combined, Including the Two Outliers)

CMV AD169 Linearity Study — Observed Titer Summary (All Data Combined, Including the Two Outliers)

| Nominal (IU/mL) | Log_{10} Nominal (IU/mL) | N | Mean log_{10} Titer (IU/mL) | SD log_{10} Titer (IU/mL) | Bias | Predicted 1^{st}-order | Deviation |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9.1E+01 | 1.96 | 38 | 1.99 | 0.57 | 0.03 | 2.02 | -0.030 |
| 1.4E+02 | 2.14 | 38 | 2.20 | 0.45 | 0.07 | 2.19 | 0.000 |
| 2.3E+02 | 2.36 | 38 | 2.41 | 0.25 | 0.05 | 2.42 | -0.010 |
| 4.6E+02 | 2.66 | 37 | 2.75 | 0.23 | 0.09 | 2.72 | 0.031 |
| 9.1E+02 | 2.96 | 38 | 3.02 | 0.25 | 0.06 | 3.02 | 0.002 |
| 9.1E+03 | 3.96 | 37 | 4.01 | 0.19 | 0.05 | 4.01 | -0.006 |
| 9.1E+04 | 4.96 | 37 | 5.01 | 0.20 | 0.05 | 5.01 | -0.004 |
| 9.1E+05 | 5.96 | 38 | 6.01 | 0.17 | 0.05 | 6.01 | -0.002 |
| 9.1E+06 | 6.96 | 38 | 7.01 | 0.13 | 0.05 | 7.01 | 0.001 |
| 1.8E+07 | 7.26 | 38 | 7.30 | 0.19 | 0.04 | 7.31 | -0.009 |

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The bias of the observed mean $\log_{10}$ titer was within $0.03\log_{10}$ and $0.09\log_{10}$ of the $\log_{10}$ nominal titer for all the concentration levels tested, including the two outliers. The maximum deviation between the observed mean $\log_{10}$ titer and the best fitted $1^{\text{st}}$-order model (deviation from linearity) was $&lt; 0.04\log_{10}$ for each concentration level tested, including the two outliers.

CMV AD169 Linearity Study — Linearity of the CAP/CTM CMV Test (All Data Combined, Both Outliers Removed)

CMV AD169 Linearity Study — Observed Titer Summary (All Data Combined, Outliers Removed)

| Nominal (IU/mL) | Log_{10} Nominal (IU/mL) | N | Mean log_{10} Titer (IU/mL) | SD log_{10} Titer (IU/mL) | Bias | Predicted 1^{st}-order | Deviation |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9.1E+01 | 1.96 | 37 | 1.92 | 0.42 | -0.04 | 2.00 | -0.076 |
| 1.4E+02 | 2.14 | 37 | 2.15 | 0.35 | 0.02 | 2.17 | -0.027 |
| 2.3E+02 | 2.36 | 38 | 2.41 | 0.25 | 0.05 | 2.39 | 0.013 |
| 4.6E+02 | 2.66 | 37 | 2.75 | 0.23 | 0.09 | 2.70 | 0.051 |
| 9.1E+02 | 2.96 | 38 | 3.02 | 0.25 | 0.06 | 3.00 | 0.020 |
| 9.1E+03 | 3.96 | 37 | 4.01 | 0.19 | 0.05 | 4.00 | 0.006 |
| 9.1E+04 | 4.96 | 37 | 5.01 | 0.20 | 0.05 | 5.01 | 0.002 |
| 9.1E+05 | 5.96 | 38 | 6.01 | 0.17 | 0.05 | 6.01 | -0.002 |
| 9.1E+06 | 6.96 | 38 | 7.01 | 0.13 | 0.05 | 7.01 | -0.006 |
| 1.8E+07 | 7.26 | 38 | 7.30 | 0.19 | 0.04 | 7.32 | -0.017 |

The bias of the observed mean $\log_{10}$ titer was within $-0.04\log_{10}$ and $0.09\log_{10}$ of the $\log_{10}$ nominal titer for all the concentration levels tested, with the two outliers removed. The maximum deviation between the observed mean $\log_{10}$ titer and the best fitted $1^{\text{st}}$-order model (deviation from linearity) was $&lt; 0.08\log_{10}$ for each concentration level tested, with the two outliers removed.

Based on the analyses above, the CAP/CTM CMV Test was found in the CMV AD169 Linearity Study to give a linear response from $9.1\mathrm{E} + 01$ ($\log_{10} = 1.96$) CMV DNA IU/mL to at least $9.1\mathrm{E} + 06$ ($\log_{10} = 6.96$) CMV DNA IU/mL, with maximum deviation from linearity of less than or equal to $0.3\log_{10}$ in this interval. The results of this study support the claimed LLoQ of $1.37\mathrm{E} + 02$ IU/mL and the claimed linear range of $1.37\mathrm{E} + 02$ to $9.1\mathrm{E} + 06$ IU/mL.

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Linear Range Using CMV Glycoprotein B (gB) Genotypes 1 - 4 Specimens

Four 5-member panels were used to verify the linear range of the CAP/CTM CMV Test for CMV glycoprotein B (gB) genotypes 1, 2, 3, and 4 in accordance with CLSI Guideline EP6-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline. Each of the four panels was prepared by diluting the four gB plasmids to $9.10\mathrm{E} + 06$, $3.35\mathrm{E} + 05$, $1.24\mathrm{E} + 04$, $4.55\mathrm{E} + 02$, and $1.37\mathrm{E} + 02$ IU/mL using a pool of CMV DNA negative EDTA plasma as the diluent.

Each of the gB genotype samples used in this study were full-length UL54 gene plasmid clones. Plasmid clones for CMV gB genotypes 1 and 2 were prepared using CMV Merlin and CMV AD169, respectively. Plasmid clones for CMV gB genotypes 3 and 4 were prepared using clinical specimens. Each of the four glycoprotein B genotype plasmid stocks was assigned a concentration by the Calibrator Bracketing Method using CMV Secondary Standard (Lot TRLOT03) which is traceable to the $1^{\text{st}}$ WHO International Standard for Human Cytomegalovirus (NIBSC code 09/162). Four 5-member panels were then prepared (one panel for each glycoprotein B genotype) by serial dilution using a pool of negative CMV DNA EDTA plasma (see the table below).

CMV gB Genotypes Linearity Study — Panel Members

| Nominal Titer (IU/mL) | Nominal Titer (copy/mL) | Log_{10} Nominal Titer (IU/mL) | Description |
| --- | --- | --- | --- |
| 9.10E+06 | 1.000E+07 | 6.96 | ULoQ |
| 3.35E+05 | 3.684E+05 | 5.53 | Intermediate level |
| 1.24E+04 | 1.360E+04 | 4.09 | Intermediate level |
| 4.55E+02 | 5.000E+02 | 2.66 | 3.3x LLoQ |
| 1.37E+02 | 1.500E+02 | 2.14 | 1x LLoQ |

One run was comprised of one replicate of each genotype panel member. A total of 16 runs were completed in four days for a total of 16 replicates per genotype panel member evenly distributed among two lots of CAP/CTM CMV Test kit reagents and two CAP/CTM systems. Eight replicates were run for each level for each lot of CAP/CTM CMV Test kit reagents; a minimum of seven valid replicates per lot for each level were required for data analysis.

In addition, bias was assessed by evaluation of mean $\log_{10}$ differences between the observed and nominal value.

Acceptance criteria for the linearity study are the following:

Linearity range: Maximum deviation between linear regression and better fitting nonlinear regression (i.e., deviation from linearity) should be $\leq 0.3\log_{10}$ for each concentration level tested.

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Repeatability: $\leq 0.3 \log_{10}$ for each genotype tested (all concentration levels pooled).

There were no invalid results in this study. No outlier was identified by the Extreme Studentized Deviate Test (alpha = 0.01). The linear ($1^{\text{st}}$ order), quadratic ($2^{\text{nd}}$ order), and cubic ($3^{\text{rd}}$ order) polynomial models were fitted to the dataset for each gB genotype 1-4. It was determined that for each of the four gB genotypes, the linear ($1^{\text{st}}$ order) model was the best fit.

Linear regression analyses of the mean $\log_{10}$ observed titer vs. the nominal $\log_{10}$ titer for each of the gB genotype linearity panels is shown for gB genotypes 1 - 4 in the following figure: .

CMV gB Genotypes Linearity Study — Linearity of the CAP/CTM CMV Test for CMV Glycoprotein B Genotypes 1 to 4


The accuracy of the observed mean $\log_{10}$ titer was within $0.08\log_{10}, 0.18\log_{10}, 0.12\log_{10}$ and $0.09\log_{10}$ of the nominal $\log_{10}$ titer for all the concentration levels tested with gB genotypes 1 to 4, respectively. The maximum deviation between the observed mean $\log_{10}$ titer and the best fitted $1^{\text{st}}$-order model (deviation from linearity) was $\leq 0.03\log_{10}, \leq 0.08\log_{10}, \leq 0.08\log_{10}$, and $\leq 0.03\log_{10}$ for each concentration level tested with gB genotypes 1 to 4, respectively.

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CMV gB Genotypes Linearity Study — Observed Titer Summary for CMV Glycoprotein B Genotype 1

| Nominal Concentration (IU/mL) | Log10 Nominal (IU/mL) | N | AVG Titer (IU/mL) | SD Titer (IU/mL) | AVG log_{10} Titer (IU/mL) | SD log_{10} Titer (IU/mL) | Bias | Predicted 1^{st}-order | Deviation |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.37E+02 | 2.14 | 16 | 1.71E+02 | 6.87E+01 | 2.21 | 0.15 | 0.07 | 2.20 | 0.01 |
| 4.55E+02 | 2.66 | 16 | 5.70E+02 | 2.12E+02 | 2.73 | 0.16 | 0.07 | 2.73 | 0.00 |
| 1.24E+04 | 4.09 | 16 | 1.48E+04 | 2.30E+03 | 4.16 | 0.07 | 0.07 | 4.16 | 0.00 |
| 3.35E+05 | 5.53 | 16 | 3.70E+05 | 5.68E+04 | 5.56 | 0.07 | 0.04 | 5.59 | -0.03 |
| 9.10E+06 | 6.96 | 16 | 1.17E+07 | 4.93E+06 | 7.04 | 0.16 | 0.08 | 7.02 | 0.02 |

CMV gB Genotypes Linearity Study — Observed Titer Summary for CMV Glycoprotein B Genotype 2

| Nominal Concentration (IU/mL) | Log10 Nominal (IU/mL) | N | AVG Titer (IU/mL) | SD Titer (IU/mL) | AVG log_{10} Titer (IU/mL) | SD log_{10} Titer (IU/mL) | Bias | Predicted 1^{st}-order | Deviation |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.37E+02 | 2.14 | 16 | 1.71E+02 | 7.48E+01 | 2.19 | 0.19 | 0.06 | 2.25 | -0.06 |
| 4.55E+02 | 2.66 | 16 | 7.00E+02 | 1.29E+02 | 2.84 | 0.08 | 0.18 | 2.76 | 0.08 |
| 1.24E+04 | 4.09 | 16 | 1.36E+04 | 1.66E+03 | 4.13 | 0.05 | 0.04 | 4.16 | -0.03 |
| 3.35E+05 | 5.53 | 16 | 3.61E+05 | 6.39E+04 | 5.55 | 0.08 | 0.03 | 5.56 | -0.01 |
| 9.10E+06 | 6.96 | 16 | 9.35E+06 | 1.55E+06 | 6.96 | 0.07 | 0.01 | 6.96 | 0.00 |

CMV gB Genotypes Linearity Study — Observed Titer Summary for CMV Glycoprotein B Genotype 3

| Nominal Concentration (IU/mL) | Log10 Nominal (IU/mL) | N | AVG Titer (IU/mL) | SD Titer (IU/mL) | AVG log_{10} Titer (IU/mL) | SD log_{10} Titer (IU/mL) | Bias | Predicted 1^{st}-order | Deviation |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.37E+02 | 2.14 | 16 | 1.23E+02 | 6.71E+01 | 2.02 | 0.29 | -0.12 | 2.10 | -0.08 |
| 4.55E+02 | 2.66 | 16 | 5.29E+02 | 1.45E+02 | 2.71 | 0.11 | 0.05 | 2.63 | 0.08 |
| 1.24E+04 | 4.09 | 16 | 1.30E+04 | 9.54E+02 | 4.11 | 0.03 | 0.02 | 4.08 | 0.03 |
| 3.35E+05 | 5.53 | 16 | 3.11E+05 | 3.40E+04 | 5.49 | 0.05 | -0.03 | 5.53 | -0.04 |
| 9.10E+06 | 6.96 | 16 | 9.81E+06 | 1.77E+06 | 6.99 | 0.07 | 0.03 | 6.98 | 0.01 |

CMV gB Genotypes Linearity Study — Observed Titer Summary for CMV Glycoprotein B Genotype 4

| Nominal Concentration (IU/mL) | Log10 Nominal (IU/mL) | N | AVG Titer (IU/mL) | SD Titer (IU/mL) | AVG log_{10} Titer (IU/mL) | SD log_{10} Titer (IU/mL) | Bias | Predicted 1^{st}-order | Deviation |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.37E+02 | 2.14 | 16 | 1.58E+02 | 5.64E+01 | 2.17 | 0.18 | 0.03 | 2.14 | 0.03 |
| 4.55E+02 | 2.66 | 16 | 4.64E+02 | 1.50E+02 | 2.65 | 0.14 | -0.01 | 2.67 | -0.02 |
| 1.24E+04 | 4.09 | 16 | 1.36E+04 | 1.44E+03 | 4.13 | 0.05 | 0.04 | 4.12 | 0.01 |
| 3.35E+05 | 5.53 | 16 | 3.45E+05 | 3.13E+04 | 5.54 | 0.04 | 0.01 | 5.57 | -0.03 |
| 9.10E+06 | 6.96 | 16 | 1.22E+07 | 7.19E+06 | 7.05 | 0.16 | 0.09 | 7.02 | 0.03 |

Based on these analyses above, the CAP/CTM CMV Test was found in the CMV gB Genotypes Linearity Study to give a linear response from $1.37\mathrm{E} + 02$ ($\log_{10} = 2.14$) CMV DNA IU/mL to $9.1\mathrm{E} + 06$ ($\log_{10} = 6.96$) CMV DNA IU/mL, with maximum deviation from linearity of less than or equal to $0.3\log_{10}$ in this interval. The results of this study support the claimed LLoQ of $1.37\mathrm{E} + 02$ IU/mL and the claimed linear range of $1.37\mathrm{E} + 02$ to $9.1\mathrm{E} + 06$ IU/mL.

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In addition, the linearity performance of the CAP/CTM CMV Test detecting CMV gB genotypes 1 to 4 was also assessed by determining the maximum difference between gB1 and the other three gB genotypes (i.e., gB 2, 3, and 4) as the following:

CAP/CTM CMV Test linearity by CMV gB Genotype

| CMV gB Genotype | Linear Equation in gB Genotype Linearity Study | Maximum Difference* Between gB1 and Corresponding gB Genotype (log IU/mL) |
| --- | --- | --- |
| 1 | y = 0.9988x + 0.071 | n/a |
| 2 | y = 0.9767x + 0.161 | 0.06 |
| 3 | y = 1.0125x - 0.0642 | 0.11 |
| 4 | y = 1.0126x -0.0227 | 0.06 |

* The maximum difference was obtained at the assay ULoQ or LLoQ

This analysis demonstrated that the CAP/CTM CMV Test is able to quantitate different CMV genotypes across the linear range with deviation of not more 0.11 $\log_{10}$ IU/mL.

## Precision

The precision of the CAP/CTM CMV Test was determined according to CLSI guideline EP05-A2, Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline — Second Edition by analysis of an 8-member panel. The panel was prepared using a CMV DNA-positive clinical specimen for the lower end of the dynamic range and by diluting cultured CMV (strain AD169) for the mid and high end of the dynamic range. Both source materials were diluted in CMV-negative EDTA plasma. The 8-member panel covered a range from $1.82\mathrm{E} + 02$ CMV DNA IU/mL to $9.10\mathrm{E} + 06$ CMV DNA IU/mL.

Cultured human CMV stock material (Advanced Biotechnology Inc.; Columbia, MD; M/N: 10-103-100, Lot: 7E0006-PV, strain AD169, genotype 2 based on the glycoprotein B gene UL55) was diluted into a pool of CMV DNA negative EDTA plasma to prepare a part of the 8-level linearity panel. The cultured human CMV stock material (source material for the RMS CMV Secondary Standard, Lot TRLOT03) was value assigned at $7.28\mathrm{E} + 10$ IU/mL based on the CMV Secondary Standard lot TRLOT03 ($2.184\mathrm{E} + 04$ IU/mL, n=62, COBAS® AMPLICOR® CMV MONITOR Test).

A CMV DNA positive clinical specimen (Roche IMPAACT study, Accession #: T439708-3) was diluted into a pool of CMV DNA negative EDTA plasma to prepare the rest of the 8-level linearity panel. The clinical specimen was value assigned at $7.30\mathrm{E} + 05$ IU/mL by the CAP/CTM CMV Test by normalizing the mean measured titer $(n = 12)$ of a prepared dilution of the clinical specimen to the mean measured titer $(n = 12)$ of the CMV Secondary Standard (Lot TRLOT03) tested at two levels $(2.184\mathrm{E} + 04$

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IU/mL (neat) and diluted to 4.55E+02 IU/mL), which bracketed the level of the prepared clinical specimen.

Precision Panel Levels

| Level # | Nominal Titer (IU/mL) | Nominal Log_{10} Titer (Log_{10} IU/mL) | Description | CMV DNA Source |
| --- | --- | --- | --- | --- |
| 8 | 9.10E+06 | 6.96 | ULoQ | Cultured Virus |
| 7 | 9.10E+05 | 5.96 | <ULoQ | Cultured Virus |
| 6 | 9.10E+04 | 4.96 | Intermediate Level | Cultured Virus |
| 5 | 1.82E+04 | 4.26 | Intermediate Level | Cultured Virus |
| 4 | 4.55E+02 | 2.66 | Intermediate Level | Cultured Virus |
| 3 | 9.10E+02 | 2.96 | LLoQ minimum product requirement | Clinical Specimen |
| 2 | 2.91E+02 | 2.46 | ~LLoQ | Clinical Specimen |
| 1 | 1.82E+02 | 2.26 | LLoQ target product requirement | Clinical Specimen |

Each panel member was tested with two replicates per run, with two runs per day, for 12 days, and for each of the two workflows (COBAS® AmpliPrep docked to a COBAS® TaqMan® and COBAS® AmpliPrep linked to a COBAS® TaqMan® 48) for a total 96 replicates per panel member, with replicates evenly distributed across three reagent kit lots, four COBAS® AmpliPrep/COBAS® TaqMan® systems, and at least two operators.

All results, without any exclusion of outliers, were included in the analysis. Each sample was carried through the entire CAP/CTM CMV Test procedure, including specimen preparation, amplification, and detection. Therefore, the precision reported below represents all aspects of the test procedure.

Precision of the CAP/CTM CMV Test (EDTA-Plasma in IU/mL)

| Level | Average Observed CMV DNA Titer (IU/mL) | Total No. Replicates | Within-Run %CV | Between-Run/Operator %CV | Between-Lot %CV | Between-Workflow %CV | Between Instrument %CV | Between Day %CV | Total %CV |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 8.29E+01 | 96 | 50% | 0% | 18% | 0% | 6% | 0% | 55% |
| 2 | 1.66E+02 | 96 | 33% | 12% | 14% | 0% | 14% | 0% | 41% |
| 3 | 2.83E+02 | 96 | 54% | 0% | 0% | 0% | 15% | 18% | 60% |
| 4 | 5.20E+02 | 96 | 19% | 16% | 14% | 3% | 12% | 0% | 32% |
| 5 | 1.49E+04 | 96 | 29% | 10% | 4% | 3% | 3% | 0% | 31% |
| 6 | 8.00E+04 | 96 | 17% | 13% | 10% | 0% | 7% | 5% | 25% |
| 7 | 8.05E+05 | 96 | 15% | 0% | 11% | 0% | 12% | 6% | 23% |
| 8 | 7.62E+06 | 96 | 20% | 0% | 18% | 3% | 15% | 7% | 32% |

*Between-Run is confounded with Between-Operator and therefore, presented as Between Run/Operator.

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Precision of the CAP/CTM CMV Test (EDTA-Plasma in $\log_{10}$ IU/mL)

| Level | Average Observed CMV DNA Titer (log_{10} IU/mL) | Total No. Replicates | Within-Run SD | Between-Run/Operator* SD | Between-Lot SD | Between-Workflow SD | Between Instrument SD | Between Day SD | Total SD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 1.92 | 96 | 0.21 | 0.00 | 0.08 | 0.00 | 0.03 | 0.00 | 0.22 |
| 2 | 2.22 | 96 | 0.14 | 0.05 | 0.06 | 0.00 | 0.06 | 0.00 | 0.17 |
| 3 | 2.45 | 96 | 0.22 | 0.00 | 0.00 | 0.00 | 0.06 | 0.08 | 0.24 |
| 4 | 2.72 | 96 | 0.08 | 0.07 | 0.06 | 0.01 | 0.05 | 0.00 | 0.14 |
| 5 | 4.17 | 96 | 0.12 | 0.04 | 0.02 | 0.01 | 0.01 | 0.00 | 0.13 |
| 6 | 4.90 | 96 | 0.07 | 0.06 | 0.04 | 0.00 | 0.03 | 0.02 | 0.11 |
| 7 | 5.91 | 96 | 0.07 | 0.00 | 0.05 | 0.00 | 0.05 | 0.03 | 0.10 |
| 8 | 6.88 | 96 | 0.08 | 0.00 | 0.08 | 0.01 | 0.06 | 0.03 | 0.13 |

*Between-Run is confounded with Between-Operator and therefore, presented as Between Run/Operator.

## Performance with CMV DNA-Negative Samples

The performance of the CAP/CTM CMV Test with CMV DNA-negative samples was determined by testing 227 anti-CMV IgG seronegative EDTA plasma specimens obtained from an FDA-registered Donor Testing Laboratory. The 227 anti-CMV IgG seronegative EDTA plasma specimens were from de-identified EDTA plasma specimens from patients under routine diagnostic care.

For CMV IgG seronegative specimens, all 227 specimens tested negative for CMV DNA by the CAP/CTM CMV Test, yielding a 100% negativity rate with 95% CI: 98.3% to 100%.

## Analytical Specificity (Cross-reactivity)

Various pathogenic organisms that may be present in patient specimens were evaluated for cross-reactivity with the CAP/CTM CMV Test by adding cultured organisms (viruses, bacteria, fungi) or positive clinical specimens at $1.0\mathrm{E} + 06$ particles/mL input concentration into CMV DNA-negative human EDTA plasma and into CMV DNA-positive EDTA plasma at $6.82\mathrm{E} + 02$ IU/mL CMV. Each sample was tested in triplicates using the CAP/CTM CMV Test.

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# Analytical Specificity Specimens

| **Human Herpesviruses**
Herpes simplex virus types 1 and 2
Varicella-Zoster virus
Epstein-Barr virus
Human herpesvirus 6, 7, and 8 | **Other Viruses**
BK Polyomavirus
JC Polyomavirus
Hepatitis virus A, B, and C
HIV type 1
Adenovirus 5
Parvovirus B19 |
| --- | --- |
| **Bacteria**
Mycoplasma pneumoniae
Propionibacterium acnes
Salmonella typhimurium
Staphylococcus aureus
Streptococcus pneumoniae | **Fungi**
Aspergillus niger
Candida albicans
Cryptococcus neoformans |

# Analytical Specificity Specimens – Method of Quantitation

| Specimen # | Description | Unit | Method of Quantitation | Specimen # | Description | Unit | Method of Quantitation |
| --- | --- | --- | --- | --- | --- | --- | --- |
| S1. | Adenovirus 5 | PFU/mL | Plaque Assay | S13. | Human immunodeficiency virus type 1 | PFU/mL | Plaque Assay |
| S2. | Aspergillus niger | CFU/mL | Plate Count | S14. | Herpes simplex virus type 1 | PFU/mL | Plaque Assay |
| S3. | BK Polyomavirus | copies/mL | Real-Time PCR using Roche LightCycler® | S15. | Herpes simplex virus type 2 | PFU/mL | Plaque Assay |
| S4. | Candida albicans | CFU/mL | Plate Count | S16. | JC Polyomavirus | copies/mL | Real-Time PCR using Roche LightCycler® |
| S5. | Cryptococcus neoformans | CFU/mL | Plate Count | S17. | Mycoplasma pneumoniae | CFU/mL | Plate Count |
| S6. | Epstein-Barr virus | copies/mL | Real-Time PCR using Roche LightCycler® | S18. | Parvovirus B19 | IU/mL | cobas® TaqScreen DPX Test |
| S7. | Hepatitis A virus | copies/mL | RT-qPCR | S19. | Propionibacterium acnes | CFU/mL | Plate Count |

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| S8. | Hepatitis B virus | IU/mL | High Pure System / COBAS® TaqMan® HBV Test, US-IVD | S20. | Salmonella typhimurium | CFU/mL | Plate Count |
| --- | --- | --- | --- | --- | --- | --- | --- |
| S9. | Hepatitis C virus | IU/mL | COBAS® AmpliPrep / COBAS® TaqMan® HCV Test, US-IVD | S21. | Staphylococcus aureus | CFU/mL | Plate Count |
| S10. | Human Herpesvirus type 6 | vp/mL | Transmission Electron Microscopy (TEM) | S22. | Streptococcus pneumoniae | CFU/mL | Plate Count |
| S11. | Human Herpesvirus type 7 | copies/mL | Real-Time PCR using Roche LightCycler® | S23. | Varicella-Zoster Virus | PFU/mL | Plaque Assay |
| S12. | Human Herpesvirus type 8 | copies/mL | Real-Time PCR using Roche LightCycler® | | | | |

None of the organisms at the concentrations tested showed cross-reactivity with the CAP/CTM CMV Test. CMV-positive specimens returned titer results that were within $\pm 0.3\log_{10}$ from a CMV-positive control without the potentially cross-reactive organism spiked.

## Interfering Substances (Exogenous)

A total of 21 exogenous substances (i.e., pharmaceutical drugs) that may be present in patient specimens were evaluated for potential interference with the CAP/CTM CMV Test by adding the drugs at levels recommended in CLSI guideline EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline – 2nd Edition, or three times the peak plasma drug concentration level ($C_{\mathrm{max}}$) (see footnote in the table below), whichever was greater, into CMV DNA-negative human EDTA plasma, and into CMV DNA-positive EDTA plasma at $6.82\mathrm{E} + 02\mathrm{IU/mL}$ CMV. Each sample was tested in triplicate using the CAP/CTM CMV Test.

These 21 exogenous substances, described in the table below, were shown not to interfere with the CAP/CTM CMV Test when tested at the levels mentioned above.

The mean $\log_{10}$ titer of the positive CMV specimens containing a potential interfering substance was within $\pm 0.3\log_{10}$ of the mean $\log_{10}$ titer of the positive CMV specimens without the potential interfering substance. In addition, specimens negative for CMV DNA, regardless of containing potential interfering substance, had “Target Not Detected” as a result.

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Potentially Interfering Exogenous Substance

| ID | Drug | | Function | CLSI Test Concentration (ug/mL) | 1 x C_{max} (ug/mL) | 3 x C_{max} (ug/mL) |
| --- | --- | --- | --- | --- | --- | --- |
| 1. | Azathioprine (Imuran)† | | Immunosuppressant | 3.0 | 1.0 | 3.0 |
| 2. | Sulfamethoxazole† | | Antibiotic | 399.8 | 68 | 204 |
| 3. | Trimethoprim† | | Antibiotic | 40.1 | 2 | 6 |
| 4. | Cefotetan (Cefotan)† | | Antibiotic | NA | 237 | 711 |
| 5. | Cidofovir (Vistide)† | | Anti-CMV | NA | 19.6 | 58.8 |
| 6. | Cyclosporine (Genraf, Neoral, Sandimmune)† | | Immunosuppressant | NA | 1.8 | 5.4 |
| 7. | Everolimus (Afinitor)‡ | | Immunosuppressant | NA | 4.0 | 12.0 |
| 8. | Fluconazole (Diflucan or Trican)† | | Anti-fungal | 75.0 | 14.1 | 42.3 |
| 9. | Foscarnet (Foscavir, Sodium phosphonoformate tribasic hexahydrate)† | | Anti-CMV | NA | 187 | 561 |
| 10. | Ganciclovir (Cytovene)† | | Anti-CMV | NA | 9.0 | 27.0 |
| 11. | Mycophenolate mofetil (CellCept)† | | Immunosuppressant | NA | 26.0 | 78.0 |
| 12. | Mycophenolate sodium (Myfortic)† | | Immunosuppressant | NA | 37 | 111 |
| 13. | Zosyn | Piperacillin† | Antibiotic | NA | 298 | 894 |
| 14. | | Tazobactam sodium† | Antibiotic | NA | 34 | 102 |
| 15. | Prednisone‡ | | Immunosuppressant | 0.3 | 12.0 | 36.0 |
| 16. | Sirolimus (Rapamune or rapamycin)† | | Immunosuppressant | NA | 0.035 | 0.105 |
| 17. | Tacrolimus (FK506 or Prograf)† | | Immunosuppressant | 0.040 | 0.069 | 0.207 |
| 18. | Timentin | Clavulanate potassium† | Antibiotic | 7.0 | 8.0 | 24.0 |
| 19. | | Ticarcillin disodium† | Antibiotic | NA | 330.0 | 990.0 |
| 20. | Valganciclovir (Valcyte)† | | Anti-CMV | NA | 9.5 | 28.5 |
| 21. | Vancomycin† | | Antibiotic | 100.0 | 63 | 189 |

* Test levels recommended in the CLSI Guideline EP07-A2 (Interference Testing in Clinical Chemistry; Approved Guideline-Second Edition)
† Levels of ~1xCmax found at www.drugs.com/pro/
‡ Levels calculated according to CLSI guidelines as 1 and 3 times the maximum dose in 5L of blood. Doses used for calculations were 20 mg and 60 mg for Everolimus and Prednisone, respectively (ref. www.drugs.com/pro/ for dose information)

## Interfering Substances (Endogenous)

A list of physiologically occurring substances that may be present in patient specimens were evaluated for potential interference with the CAP/CTM CMV Test by adding the endogenous substances at levels recommended in CLSI guideline EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline – 2nd Edition, into CMV DNA-negative human EDTA plasma, and into CMV DNA-positive EDTA

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plasma at 6.82E+02 IU/mL CMV. Each sample was tested in duplicate using the CAP/CTM CMV Test.

The physiologically occurring substances shown in the table below, when tested at the test levels recommended in CLSI guideline EP07-A2, were shown not to interfere with the CAP/CTM CMV Test.

Potentially Interfering Endogenous Substances

| Potential Interfering Substance | Reference Interval | Test Level |
| --- | --- | --- |
| Conjugated bilirubin (Ditaurobilirubin) | 0 – 0.2 mg/dL | ≥ 20 mg/dL* |
| Un-conjugated Bilirubin | 0.3 – 1.2 mg/dL | ≥ 20 mg/dL* |
| Hemoglobin | 100 - 200 mg/dL | ≥ 200 mg/dL* |
| Human DNA | N/A | 0.4 mg/dL |
| Human albumin | 3,900 – 5,100 mg/dL | ≥ 6,000 mg/dL* |
| Triglycerides | 30 – 330 mg/dL | ≥ 3,300 mg/dL* |
| Systemic Lupus Erythematosus | Natural specimens obtained from 10 different patients (per disease) diagnosed with one of the three auto-immune diseases. | |
| Rheumatoid Factor | | |
| Antinuclear antibody | | |

* Test levels recommended in the CLSI Guideline EP07-A2

The mean log₁₀ titer of the positive CMV specimens containing a potential endogenous interfering substance were all within ± 0.3 log₁₀ of the mean log₁₀ titer of the CMV positive control specimens without the potential interfering substance. In addition, specimens negative for CMV DNA and containing elevated levels of potential interfering substance all tested as “Target Not Detected.”

## Sample to Sample Cross Contamination (Carryover)

The potential for sample to sample carryover contamination was evaluated for the CAP/CTM CMV Test by performing five runs, with each run comprised of three full SK-24 racks of alternating high positive samples (at a concentration of 9.10E+06 IU/mL) and negative specimens. Out of 165 CMV negative specimens tested, 0% were positive for CMV DNA when tested with the CAP/CTM CMV Test (95% CI: 0% to 2.3%).

## Robustness — Whole System Failure

A study was conducted to assess the whole system failure rate when the entire process is performed as prescribed by the manufacturer. The whole system failure rate is defined as the number of false negative results observed in a minimum of 100 replicates at a titer level less than or equal to approximately three times the LoD.

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For this study, a low CMV DNA positive sample was prepared by diluting a CMV DNA positive clinical specimen (IMPACT Study Accession No: T439708-3) with a pool of CMV DNA negative EDTA plasma to a final concentration of $2.72\mathrm{E} + 02$ IU/mL, which is approximately $3\times$ LoD. Five SK24 racks were run on an individual COBAS® AmpliPrep/COBAS® TaqMan® system and another five SK24 racks were run on an individual COBAS® AmpliPrep/COBAS® TaqMan® 48 system. One kit lot of reagents was used for this study. Each SK24 rack contained three controls (one Negative Control, one Low Positive Control, and one High Positive Control) and 20 replicates of the CMV specimen at $2.72\mathrm{E} + 02$ IU/mL (3x LoD).

The results from this study showed that 100 replicates of a CMV specimen at a concentration of $3\mathrm{x}$ LoD reported $100\%$ positivity rate for both the CAP/CTM and the CAP/CTM 48 workflows (95% CI: $96.3\%$ to $100\%$), meeting the minimum requirement for Whole System Failure.

## Unprocessed Specimen Stability

Unprocessed specimen stability was determined by examining the $\log_{10}$ titers results of 10 unique CMV DNA negative clinical specimens spiked with a CMV DNA positive clinical specimen to $5\mathrm{x}$ LLoQ and two CMV DNA negative clinical specimens. All 12 specimens were stored at conditions and lengths of times that simulated the maximum intended limits for specimen handling (e.g., plasma separated from whole blood within six hours), transportation (no more than six hours at $25^{\circ}\mathrm{C}$), storage ($2 - 8^{\circ}\mathrm{C}$ or frozen), and specimen testing (on the COBAS® AmpliPrep instrument for six hours prior to processing).

For the unprocessed specimens to be considered stable, the following criteria must be met:

- For CMV (+) specimens at $5 \times$ LLoQ, the difference between the mean measured and Day 0 mean measured $\log_{10}$ titer results must be within $\pm 0.3 \log_{10}$ for each condition at each time point to be considered stable. In addition, all replicates must have a positive result for each condition at each time point to be considered stable.
- For CMV (-) specimens, all replicates must have a "CMV Target Not Detected" result for each condition at each time point to be considered stable.

The mean $\log_{10}$ titer was within $\pm 0.3\log_{10}$ of the Day 0 mean $\log_{10}$ titer for each time point up through seven days at $2 - 8^{\circ}\mathrm{C}$ and through six weeks at $-20^{\circ}\mathrm{C}$. The observed $\log_{10}$ differences in titer across the time points ranged from -0.28 to 0.36.

Unprocessed Specimen Stability Summary

| Time Point | Mean Log10 Titer | |
| --- | --- | --- |
| | 4°C | -20°C |
| D0 | 2.66 | 2.66 |
| D3 | 2.67* | N/A |

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| D7 | 2.73* | N/A |
| --- | --- | --- |
| W6 | N/A | 2.77#* |

* Includes 6 hours On-board Stability (38°C)
# Includes 1, 2, and 3 Freeze/Thaw Cycles

The average $\log_{10}$ titer was also within $\pm 0.3\log_{10}$ of the Day 0 average $\log_{10}$ titer for the 6 week time point for 1, 2, and 3 freeze/thaw cycles. All 12 CMV specimens tested met the criteria describe above after storage at $2 - 8^{\circ}C$ for seven days and at $-20^{\circ}C$ for six weeks. The figure below shows the $\log_{10}$ titer result for each of the 10 low positive specimens over time.

CMV Stability in Unprocessed EDTA-Plasma Specimens

In addition, all 12 CMV specimens stored frozen at $-20^{\circ}\mathrm{C}$ for six weeks also met the criteria described above after three freeze/thaw cycles. The figure below shows the $\log_{10}$ titer result for each of the 10 low positive specimens over multiple freeze/thaw cycles. The observed $\log_{10}$ differences in titer across the freeze/thaw conditions ranged from -0.33 to 0.32.

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CMV Results after up to Three Freeze/Thaw Cycles
(Unprocessed EDTA-Plasma Specimens)

Based on the results of this functional stability testing, the following unprocessed specimen stability statements in the product package insert are substantiated:

- EDTA whole blood may be stored or transported at room temperature (25°C) for up to six hours. Plasma should be separated from whole blood within six hours of collection.
- Unprocessed EDTA-plasma CMV specimens are stable for up to seven days at 2-8°C and up to six weeks at -20°C.
- Unprocessed EDTA-plasma CMV specimens are stable for up to three freeze/thaw cycles.
- EDTA-plasma CMV specimens are stable on-board the COBAS® AmpliPrep Instrument (38°C) for up to six hours.

## Prepared Specimen Stability (PCR Mixture)

To determine the stability of the PCR mixture (35μL CMV MMx, 15μL MgCl₂, and 50μL processed sample) on-board the COBAS® AmpliPrep Instrument (CAP) prior to amplification/ detection on the COBAS® TaqMan® Analyzer (CTM), CAP/CTM CMV Test kit controls (HPC, LPC, and NC) and CMV samples at four concentration levels (1.37E+02 IU/mL, 4.55E+03 IU/mL, 2.18E+04 IU/mL, and 9.10E+06 IU/mL) were processed on the CAP instrument and the resulting PCR Mixtures were amplified/detected on the CTM after 0, 120, 180, and 240 minutes incubation at 40°C in the dark. (Note: 38°C is defined as the upper temperature limit for the CAP environment area).

For the prepared specimens (PCR mixtures) to be considered stable, the following criteria must be met:

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- For 9.10E+06 IU/mL, 2.18E+04 IU/mL, and 4.55E+03 IU/mL samples, the mean log₁₀ titer of the replicates must be within ± 0.3 log₁₀ titer units of the mean log₁₀ titer for Time Point 0.
- For 1.37E+02 IU/mL samples, the upper bound of 95% confidence interval for the observed positive hit rate should be greater than or equal to 99% (based on Clopper-Pearson confidence intervals). For example, 9 out of 10 results must be positive (90% hit rate with upper bound of 95% confidence interval of 99.8). If repeat testing occurs, 19 out of 20 trials must be positive (95% hit rate with upper 95% confidence interval of 99.9%).
- All heat stressed CAP/CTM CMV Controls must be valid.

All kit controls evaluated for PCR mixture stability were valid up to 240 minutes.

For 4.55E+03 IU/mL, 2.18E+04 IU/mL, and 9.10E+06 IU/mL PCR mixtures, the difference in mean log₁₀ titers at each time point and Time 0 were within ±0.12 log₁₀ at all time points. For 1.37E+02 IU/mL PCR mixtures, the upper bound of 95% confidence interval was 100% (10 positives/10 replicates) at 0, 120, and 180 minutes and was 99.8% (9 positives/10 replicates) at 240 minutes. Based on this data, the PCR mixtures remained within specification when stored at 40°C in the dark for up to 240 minutes.

After the eluate is mixed with activated MMx for the first sample of the batch, each subsequent sample in the batch takes approximately 4 minutes to complete. With a maximum batch size of 24, the PCR mixture for the first sample would be sitting onboard for ~92 minutes at the completion of the batch. When calculating the allowable time between the completion of the specimen and control preparation and the start of the amplification/detection, at least 92 minutes should be subtracted from the total stability time of 240 minutes. To be conservative, the COBAS® TaqMan® Analyzer or COBAS® TaqMan® 48 Analyzer run must be started within 120 minutes following completion of specimen and control preparation.

Based on the results of this functional stability testing, the following processed specimen stability (PCR mixture) claim in the product package insert is substantiated:

- The COBAS® TaqMan® Analyzer or COBAS® TaqMan® 48 Analyzer run must be started within 120 minutes following completion of specimen and control preparation.

## Open Vial Reagent Stability

Onboard Reagent and Open Vial Stability for the CAP/CTM CMV Test was verified at CMV DNA concentrations of 1.37E+02 IU/mL, 9.10E+04 IU/mL, and 9.10E+06 IU/mL. Stability of the test was verified by simulating onboard storage of cap-punctured bottles for up to six runs (Note: 1 kit, consisting of 72 tests, is configured to

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allow for six runs of 12 tests). In between runs, opened reagents were stored at 2-8°C for two weeks.

Test samples were prepared using CMV cultured strain AD169 diluted in a pool of CMV DNA negative EDTA plasma at concentrations of 1.37E+02 IU/mL, 9.10E+04 IU/mL and 9.10E+06 IU/mL. Twenty-seven (27) replicates of each of the three concentrations were tested at time 0 to establish a baseline. Nine replicates of each of the three concentrations were tested at each subsequent time point for a total of six time points (including baseline).

Each on-board reagent time point consisted of one and a half hours run on the CAP, followed by incubation at 28°C (except MGPs that were kept at 38°C) for 18.5 hours for a total cumulative time of 20 hours. Following 28°C incubation (or 38°C for MGPs), open vials were stored at 2-8°C uncovered until the next time point. Refer to the tables below for the testing schedule for each time point.

Open Vial / On-board Reagent Stability Study — Study Time Points

| Time Point | Onboard Stability (Hours) | Open Vial Stability (Days) |
| --- | --- | --…