COBAS CMV
Device Facts
| Record ID | P160041 |
|---|---|
| Device Name | COBAS CMV |
| Applicant | Roche Molecular Systems, Inc. |
| Product Code | PAB · Microbiology |
| Decision Date | Jun 1, 2017 |
| Decision | APRL |
| Regulation | 21 CFR 866.3180 |
| Device Class | Class 2 |
Intended Use
cobas® CMV is an in vitro nucleic acid amplification test for the quantitation of cytomegalovirus (CMV) DNA in human EDTA plasma. cobas® CMV is intended for use as an aid in the management of CMV in solid organ transplant patients and in hematopoietic stem cell transplant patients. In patients receiving anti-CMV therapy, serial DNA measurements can be used to assess viral response to treatment. The results from cobas® CMV must be interpreted within the context of all relevant clinical and laboratory findings. cobas® CMV is not intended for use as a screening test for blood or blood products.
Device Story
The cobas CMV test is a quantitative PCR-based assay for CMV DNA in EDTA plasma; performed on cobas 6800/8800 Systems. Input: EDTA plasma samples. Process: Automated nucleic acid extraction/purification using magnetic glass particles; PCR amplification using primers targeting the UL54 gene; detection via fluorescent probes. Output: CMV viral load in IU/mL. Used in clinical laboratories; operated by trained personnel. Results are reviewed by clinicians to manage transplant patients, assess antiviral treatment response, and determine when to discontinue therapy. Benefits include standardized viral load monitoring to guide clinical decision-making and treatment duration.
Clinical Evidence
Clinical performance evaluated using retrospective leftover EDTA plasma from SOT (n=107) and HSCT (n=257) patients. Concordance and method comparison studies compared cobas CMV to the COBAS AmpliPrep/COBAS TaqMan CMV Test. Results showed high concordance across clinical decision thresholds. Method comparison demonstrated linear range 34.5–1.0E+07 IU/mL. Analytical studies confirmed LoD of 34.5 IU/mL, precision, and specificity. No clinical adverse events reported.
Technological Characteristics
Quantitative nucleic acid amplification assay (PCR). Uses magnetic glass particles for extraction. Targets CMV DNA polymerase (UL54) gene. Employs thermostable DNA polymerase, dUTP, and AmpErase for amplicon contamination control. Dual-reporter fluorescent probes for target and internal control (DNA-QS) detection. Automated on cobas 6800/8800 Systems. Software-based data management and result calculation.
Indications for Use
Indicated for solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) patients undergoing anti-CMV therapy to quantitate CMV DNA in EDTA plasma and assess viral 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 AmpliPrep/COBAS TaqMan CMV Test (Roche Molecular Systems)
- artus CMV RGQ MDx Kit (QIAGEN)
Related Devices
- K252481 — cobas CMV · Roche Molecular Systems, Inc. · Nov 3, 2025
- P110037 — COBAS® AMPLIPREP/COBAS® TAQMAN® CMV TEST (CAP/CTM CMV TEST) · Roche Molecular Systems, Inc. · Jul 5, 2012
- P210022 — Alinity m CMV · Abbott Molecular, Inc. · May 5, 2022
Submission Summary (Full Text)
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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)
I. GENERAL INFORMATION
Device Generic Name: Real-time polymerase chain reaction (PCR) based assay for CMV viral load measurement
Device Trade Name: cobas® CMV
Device Procode: PAB
Applicant’s Name and Address: Roche Molecular Systems, Inc. (RMS)
4300 Hacienda Drive
Pleasanton, CA 94588-2722
Date(s) of Panel Recommendation: None
Premarket Approval Application (PMA) Number: P160041
Date of FDA Notice of Approval: June 1, 2017
II. INDICATIONS FOR USE
cobas® CMV is an in vitro nucleic acid amplification test for the quantitation of cytomegalovirus (CMV) DNA in human EDTA plasma.
cobas® CMV is intended for use as an aid in the management of CMV in solid organ transplant patients and in hematopoietic stem cell transplant patients. In patients receiving anti-CMV therapy, serial DNA measurements can be used to assess viral response to treatment.
The results from cobas® CMV must be interpreted within the context of all relevant clinical and laboratory findings.
cobas® CMV is not intended for use as a screening test for blood or blood products.
III. CONTRAINDICATIONS
There are no known contraindications.
IV. WARNINGS AND PRECAUTIONS
The warnings and precautions can be found in the cobas CMV product labeling.
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V. DEVICE DESCRIPTION
The cobas CMV test is a quantitative nucleic acid amplification assay performed on either the cobas 6800 System or the cobas 8800 System. The cobas CMV test enables the detection and quantitation of CMV DNA in EDTA plasma from solid organ transplant patients (SOT) and from hematopoietic stem cell transplant (HSCT) patients. The test is intended for use as an aid in the management of SOT patients and HSCT patients who are undergoing CMV antiviral therapy.
The CMV viral load is quantified against a non-CMV DNA quantitation standard (DNA-QS), which is introduced into each specimen during sample preparation. The DNA-QS also functions as an internal control for sample preparation and the PCR amplification process. In addition, the test utilizes three external controls: a high titer positive, a low titer positive, and a negative control.
The cobas CMV test system consists of:
- cobas 6800/8800 Systems (including cobas 6800/8800 Systems Software)
- cobas CMV Assay Specific Analysis Package (ASAP) software
- cobas CMV test reagents in kit cassettes
- cobas CMV Control reagents (HPC and LPC) in kit cassettes
- cobas NHP Negative Control reagent in kit cassettes
- Specimen preparation reagents (cobas OMNI Reagents)
The cobas CMV test uses fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas 6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas 6800/8800 Systems Software which assigns test results for all tests. Results can be reviewed directly on the system screen, exported, or printed as a report.
Target Selection
Selective amplification of CMV target nucleic acid from the sample is achieved by the use of specific forward and reverse primers which are selected from highly-conserved regions of the CMV DNA polymerase (UL54) gene. A single probe is used to detect and quantify the CMV targets. Selective amplification of DNA-QS is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with the CMV genome.
Sample Preparation (Nucleic Acid Extraction and Purification)
The cobas CMV test is intended to be used with plasma (EDTA) samples. Nucleic acid from patient samples, external controls and added DNA-QS molecules are extracted simultaneously. Viral nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured proteins, cellular debris and potential PCR inhibitors are removed with subsequent wash reagent steps and
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purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature.
## Nucleic Acid Amplification and Target Detection
The cobas CMV master mix contains detection probes which are specific for the CMV target sequence and the DNA-QS nucleic acid, respectively. The specific CMV and DNA-QS detection probes are each labeled with one of two unique fluorescent dyes which act as a reporter. The two reporter dyes are measured at defined wavelengths, thus permitting simultaneous detection and discrimination of the CMV target and the DNA-QS amplification products generated by a thermostable DNA polymerase enzyme.
Each probe also has a second dye which acts as a quencher. When not bound to the target sequence, the fluorescent signals of the intact probes are suppressed by a quencher dye. During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probe is generated and the cumulative signal of the reporter dye increases concomitantly. Since the two specific reporter dyes are measured at defined wavelengths, simultaneous detection and discrimination of the amplified CMV target and the DNA-QS are possible.
The master mix also includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP); the former is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicons from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR mix, during the first thermal cycling step. However, newly formed amplicons are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.
## CMV DNA Quantitation
During the extension phase of the PCR process, the fluorescent signals of the cleaved CMV and DNA-QS probes are collected for each specimen. 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 cobas 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. CMV viral load results are reported in International Units/mL (IU/mL).
## Controls
One replicate each of the cobas CMV Negative Control, the CMV Low Positive Control, and the CMV High Positive Control must be included in each run. The validity of the results for the controls as well as for the DNA-QS is determined by the assay specific analysis software package used by the cobas 6800/8800 instrument. The run is valid if no flags appear for any of the controls. Otherwise the cobas 6800/8800 software will
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automatically invalidate the run and generate flags to provide further explanation to the user. The user should check for flags and their associated results in the cobas 6800/8800 software and/or report.
- 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 within the label of the cobas CMV Control kit. The CMV DNA IU/mL for CMV high and low positive 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.
## Instrumentation and Software
The cobas 6800/8800 platform consists of two instrument versions: the cobas 6800 System, and the cobas 8800 System. Each system is comprised of a cobas 6800 or cobas 8800 instrument, system software, Assay Specific Analysis Packages (ASAP), and a sample source unit, which can be connected to a conveyor system for automated transport of samples to and from the system. The test kits consist of assay-specific reagents and omni reagents (or common reagents) which can be used with any of the cobas assays, and on either the cobas 6800 or the cobas 8800 system.
In addition, the cobas omni (common) reagents and consumables, such as the P-plates, racks, AD-plates, waste bags, pipette tips, and secondary tubes, can be used with any of the cobas assays, and on either the cobas 6800 or the cobas 8800 systems.
Either system can be interfaced to an uninterruptible power supply (UPS), a Laboratory Information System (LIS) or middleware, and office PCs for remote monitoring functionalities.
Figure 1 below depicts the cobas 6800/8800 Platform.
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Figure 1: cobas 6800/8800 Platform
# Interpretation of Results
Results are determined automatically by the cobas software and are reported in IU/mL (scientific format). The calculated CMV concentration (in IU/mL) is only provided for samples within the linear range of the assay. Result reporting is shown in the following table:
Table 1: Result Interpretation
| Results | Interpretation |
| --- | --- |
| Target Not Detected | CMV DNA not detected. Report results as “CMV not detected.” |
| < Titer Min | Calculated titer is below the Lower Limit of Quantitation (LLoQ) of the assay. Report results as “CMV detected, less than (Titer Min).” Titer min = 34.5 IU/mL |
| Titer | Calculated titer is within the Linear Range of the assay - greater than or equal to Titer Min and less than or equal to Titer Max. Report results as “(Titer) of CMV detected”. |
| > Titer Maxa | Calculated titer is above the Upper Limit of Quantitation (ULoQ) of the assay. Report results as “CMV detected, greater than (Titer Max).” Titer max = 1.0E+07 IU/mL |
a Sample result > Titer Max refers to CMV positive samples detected with titers above the upper limit of quantitation (ULoQ). If a quantitative result is desired, the original sample should be diluted with CMV-negative human EDTA plasma and the test should be repeated. Multiply the reported result by the dilution factor.
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# Kit Configuration and Components
## cobas CMV Reagent Cassettes:
- Proteinase Solution
- DNA Quantitation Standard (DNA-QS)
- Elution Buffer (EB)
- Master Mix Reagent 1 (MMX-R1)
- CMV Master Mix Reagent 2 (CMV MMX-R2)
## cobas CMV Control Kit:
- CMV Low Positive Control (CMV L(+)C)
- High Positive Control (CMV H(+)C)
## cobas NHP Negative Control kit:
- Normal Human Plasma (NHP) Negative Control
## cobas omni Reagents:
The cobas omni reagents are common sample preparation reagents that are used with other assays that are run on the cobas 6800/8800 Systems and are:
- cobas omni MGP Reagent (containing magnetic glass particles that bind nucleic acid)
- cobas omni Lysis Reagent
- cobas omni Specimen Diluent
- cobas omni Wash Reagent
# VI. ALTERNATIVE PRACTICES AND PROCEDURES
There are currently two other alternatives for the quantitation of CMV in the management of transplant patients.
- COBAS AmpliPrep/COBAS TaqMan CMV Test (Roche Molecular Systems)
- artus CMV RGQ MDx Kit (QIAGEN)
Each alternative has its own advantages and disadvantages.
# VII. MARKETING HISTORY
The cobas CMV test is currently available in the countries listed below. To date, there have been no adverse incidents or potentially-critical complaints reported for this test. The device has not been withdrawn from marketing for any reasons related to its safety or effectiveness.
Argentina
Brazil
Croatia
Denmark
Estonia
Austria
Bulgaria
Cyprus
Ecuador
Finland
Belgium
Columbia
Czech Republic
Egypt
France
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| Germany | Guatemala | Greece |
| --- | --- | --- |
| Hungary | Iceland | India |
| Ireland | Italy | Latvia |
| Liechtenstein | Lithuania | Luxembourg |
| Malta | Netherlands | Norway |
| Panama | Pakistan | Peru |
| Poland | Portugal | Romania |
| Russia | Saudi Arabia | Singapore |
| Slovakia | Slovenia | Spain |
| Sweden | Switzerland | Thailand |
| Turkey | United Arab Emirates | United Kingdom |
## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH
Failure of the cobas CMV test to perform as indicated, or human error in the use of the test, may result in an incorrect test result that is too low or too high. The following factors can impact the performance of the cobas CMV test:
- Mutations in the CMV UL54 gene have been observed. Mutant CMV variants may not be detectable or detectable with decreased efficiency if mutations in the primer or probe binding sites occur; such mutations consequently may result in the failure to detect CMV or in the underquantitation of CMV viral loads. The test design for the cobas CMV test mitigates, but does not entirely exclude, this risk. Additional testing should be performed if mutations are suspected.
- Invasive CMV disease may not be reflected in detectable CMV viral DNA in peripheral blood. The results from the cobas CMV test must therefore be interpreted by a qualified healthcare professional.
Failure of the device to perform as expected or failure to correctly interpret test results in the context of all clinical findings may lead to improper patient management decisions. A test result that is erroneous negative or too low may lead to 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.
To mitigate these risks in the management of transplant patients the viral load results from the cobas CMV test must be interpreted in the context of all relevant clinical and laboratory findings by a medical professional who is adequately trained in the interpretation of CMV viral load results in the context of transplant patient management.
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# IX. SUMMARY OF NONCLINICAL STUDIES
## A. Laboratory Studies
### 1. Traceability to the 1st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques Based Assays
Several standards and controls have been used during development of the cobas CMV test to provide traceability to the WHO CMV Standard [1st WHO International Standard for human Cytomegalovirus DNA for Nucleic Acid Amplification Techniques (NIBSC 09/162)]. The standards used during development of the test include the WHO CMV Standard, the Roche Molecular Systems (RMS) CMV Secondary Standard, and the RMS CMV Calibration Panel. The concentration range tested for the CMV WHO Standard was from 4.60E+01 IU/mL to 1.80E+04 IU/mL (1.66 – 4.26 log₁₀ IU/mL), the RMS CMV Secondary Standard was tested at 3.16E+04 IU/mL (4.50 log₁₀ IU/mL), and the RMS CMV Calibration Panel was tested from 1.47E+02 to 2.94E+06 IU/mL (2.17 – 6.47 log₁₀ IU/mL).
All materials demonstrated co-linear dilution performance across the linear range of cobas CMV (Figure 2) albeit the calibration panel delivers slightly higher values for the cobas CMV test. Based on these results, the calibration and standardization process of cobas CMV provides quantitation values for the calibration panel, the RMS CMV Secondary Standard, and the CMV WHO Standard that are similar to the expected values with deviation of not more than 0.23 log₁₀ IU/mL. The maximum deviation was obtained around the test LLOQ.
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Figure 2: Traceability to HCMV WHO International Standard Using cobas CMV
# 2. Limit of Detection (LOD)
# a. LOD Using the $1^{\text{st}}$ WHO International Standard for Human Cytomegalovirus
The LOD of the cobas CMV test for the $1^{\text{st}}$ WHO CMV Standard was determined by analysis of serial dilutions of the Standard in CMV-negative human EDTA plasma. Panels of eight concentration levels plus a blank were tested over three lots of cobas CMV test reagents and three instruments with multiple runs and operators over a period of 3 days. Each dilution was determined in 21 replicates per lot and day (n=63 total replicates per day).
The results from testing the WHO CMV Standard in EDTA plasma as well as the calculated LOD values are shown in Table 2. The LOD values in Table 2 were determined by Probit analysis which provided the concentration of CMV expected to give a $95\%$ hit rate. The study demonstrated that the highest LOD, namely $30.7~\mathrm{IU / mL}$ , was obtained with lot 2 as determined by Probit analysis. The lowest concentration of CMV that was experimentally observed to give a hit rate $\geq 95\%$ with lot 2 was $34.5~\mathrm{IU / mL}$ ; at this concentration, the observed hit rate was $98.4\%$ (62/63). The claimed LoD value is $34.5~\mathrm{IU / mL}$ and this concentration was used in studies for confirmation of the LoD.
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Table 2: LOD with CMV DNA 1st WHO International Standard in EDTA Plasma
| Kit Lot | Nominal Concentration (IU/mL) | Number of Valid Replicates | Number of Positive Replicates | Hit Rate [%] | LOD by Probit [95% CI] |
| --- | --- | --- | --- | --- | --- |
| Lot 1 | 92 | 63 | 63 | 100.0 | 14.7 IU/mL
[11.7 – 20.0 IU/mL] |
| | 46 | 63 | 63 | 100.0 | |
| | 34.5 | 62 | 62 | 100.0 | |
| | 23 | 63 | 62 | 98.4 | |
| | 11.5 | 63 | 57 | 90.5 | |
| | 5.8 | 63 | 45 | 71.4 | |
| | 2.9 | 63 | 26 | 41.3 | |
| | 1.4 | 63 | 11 | 17.5 | |
| | 0 | 63 | 0 | 0.0 | |
| Lot 2 | 92 | 63 | 63 | 100.0 | 30.7 IU/mL
[24.5 – 40.9 IU/mL] |
| | 46 | 63 | 62 | 98.4 | |
| | 34.5 | 62 | 62 | 98.4 | |
| | 23 | 63 | 57 | 90.5 | |
| | 11.5 | 63 | 43 | 68.3 | |
| | 5.8 | 63 | 27 | 42.9 | |
| | 2.9 | 63 | 16 | 25.4 | |
| | 1.4 | 63 | 4 | 6.4 | |
| | 0 | 63 | 0 | 0.0 | |
| Lot 3 | 92 | 63 | 63 | 100.0 | 14.6 IU/mL
[11.6 – 19.9 IU/mL] |
| | 46 | 63 | 63 | 100.0 | |
| | 34.5 | 62 | 63 | 100.0 | |
| | 23 | 63 | 62 | 98.4 | |
| | 11.5 | 63 | 58 | 92.1 | |
| | 5.8 | 63 | 45 | 71.4 | |
| | 2.9 | 63 | 24 | 38.1 | |
| | 1.4 | 63 | 13 | 20.6 | |
| | 0 | 63 | 0 | 0.0 | |
| All lots combined | 92 | 189 | 189 | 100.0 | 20.6 IU/mL
[17.9 – 24.3 IU/mL] |
| | 46 | 189 | 188 | 99.5 | |
| | 34.5 | 188 | 187 | 99.5 | |
| | 23 | 189 | 181 | 95.8 | |
| | 11.5 | 189 | 158 | 83.6 | |
| | 5.8 | 189 | 117 | 61.9 | |
| | 2.9 | 189 | 66 | 34.9 | |
| | 1.4 | 189 | 28 | 14.8 | |
| | 0 | 189 | 0 | 0.0 | |
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b. Verification of the LOD for Glycoprotein B Genotypes gB-2, gB-3 and gB-4
The Limit of Detection (34.5 IU/mL) was verified for the cobas CMV test with all relevant CMV Glycoprotein B Genotypes (gB-2, gB-3 and gB-4) following the CLSI Guideline EP17-A2. CMV cell culture supernatants for the three different Glycoprotein B genotypes were diluted to three different concentration levels in CMV negative EDTA plasma. The hit rate determination was performed with 63 replicates for each level. Testing was conducted with three lots of cobas CMV reagents across three days of testing.
The results are shown in Table 3 (below) and verify that a hit rate of 95% or higher was observed at 34.5 IU/mL for each genotype. Thus, the observed hit rates verify that the LOD for each of the three genotypes is 34.5 IU/mL or lower.
Table 3: Verification of the LOD for Different Glycoprotein B (gB) Genotypes Across Three Lots.
| gB Genotypes | Nominal Concentration (IU/mL) | Number of Valid Replicates | Number of Positive Replicates | Hit Rate [%] | LOD by Hit Rate |
| --- | --- | --- | --- | --- | --- |
| gB-2 | 17.25 | 63 | 61 | 96.8 | 17.25 IU/mL |
| | 34.5 | 63 | 63 | 100.0 | |
| | 51.75 | 63 | 63 | 100.0 | |
| gB-3 | 17.25 | 63 | 57 | 90.5 | 34.5 IU/mL |
| | 34.5 | 63 | 63 | 100.0 | |
| | 51.75 | 63 | 63 | 100.0 | |
| gB-4 | 17.25 | 63 | 55 | 87.3 | 34.5 IU/mL |
| | 34.5 | 63 | 63 | 100.0 | |
| | 51.75 | 63 | 63 | 100.0 | |
c. Verification of the LOD for Different Drug Resistant CMV Isolates
The Limit of Detection (34.5 IU/mL) was verified for the cobas CMV test with cell culture supernatants from two different drug resistant CMV isolates (one isolate resistant against foscarnet and one isolate resistant against ganciclovir, valganciclovir and cidofovir). LOD verification followed the CLSI Guideline EP17-A2. Samples of drug resistant CMV were diluted to three different concentration levels in CMV negative EDTA plasma. Testing was conducted with three lots of cobas CMV reagents. The hit rate determination was performed with 63 replicates for each level (n=21 replicates per lot).
The results are shown in Table 4 (below) and verify that the observed LOD for each of the two drug resistant strains of CMV was 34.5 IU/mL or lower.
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Table 4: Verification of the LOD for Drug Resistant CMV Isolates
| gB Genotypes | Nominal Concentration (IU/mL) | Number of Valid Replicates | Number of Positive Replicates | Hit Rate [%] | LOD by Hit Rate |
| --- | --- | --- | --- | --- | --- |
| Forscarnet (E756Q) | 17.25 | 63 | 58 | 92.1 | 34.5 IU/mL |
| | 34.5 | 63 | 63 | 100.0 | |
| | 51.75 | 63 | 63 | 100.0 | |
| Ganciclovir, Valganciclovi, Cidofovir (L545S) | 17.25 | 63 | 59 | 93.7 | 34.5 IU/mL |
| | 34.5 | 63 | 63 | 100.0 | |
| | 51.75 | 63 | 63 | 100.0 | |
The Limit of Detection (LOD) for the cobas CMV test across the tested genotypes and phenotypes (i.e., drug sensitive and drug resistant) is 34.5 IU/mL (1.54 log₁₀ IU/mL).
## 3. Lower Limit of Quantitation (LLOQ)
The LLOQ was determined using data from the LOD study with the WHO CMV Standard (Section 2 above) for each of the lots using all concentration levels with a hit rate ≥ 95%.
The LLOQ is defined as the lowest level of CMV that can be reliably detected and at which the total analytical error (TAE) meets both of the following two criteria:
- The TAE, when calculated as |Bias| + 2SD, is ≤1.0 log₁₀ IU/mL, and
- The TAE has to be such that the standard deviation for the difference between two measurements calculated as SQRT(2) × 2 × SD is ≤ 1.0 log₁₀ IU/mL
Meeting the |Bias| + 2SD ≤ 1.0 log₁₀ IU/mL criterion ensures that, for samples with assay values equal to the LLOQ, there is 95% or greater probability that the measured value will be within 1.0 log₁₀ IU/mL of the true value. Meeting the SQRT(2) × 2 × SD ≤ 1.0 log₁₀ IU/mL criterion ensures that, for samples with assay values equal to the LLOQ, a difference of more than 1.0 log₁₀ IU/mL between two measurements is statistically significant (a true change is detected).
Table 5 below shows both criteria for the total analytical error (TAE = |Bias| + 2 × SD ≤ 1.0 log₁₀ IU/mL and SQRT(2) × 2 × SD ≤ 1.0 log₁₀ IU/mL).
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Table 5: LLOQ - TAE and Difference between Measurements
| Lot | Nominal Concentration (IU/mL) | Nominal Concentration (log10 IU/mL) | Mean Observed Concentration (log10 IU/mL) | SD (log10 IU/mL) | Absolute Bias (log10 IU/mL) | TAE (log10 IU/mL) | Difference Between Measurements (log10 IU/mL) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 23 | 1.36 | 1.04 | 0.32 | 0.32 | 0.96 | 0.91 |
| | 34.5 | 1.54 | 1.28 | 0.29 | 0.26 | 0.83 | 0.81 |
| | 46 | 1.66 | 1.43 | 0.17 | 0.23 | 0.57 | 0.48 |
| | 92 | 1.96 | 1.76 | 0.16 | 0.2 | 0.53 | 0.46 |
| 2* | 34.5 | 1.54 | 1.42 | 0.19 | 0.11 | 0.5 | 0.55 |
| | 46 | 1.66 | 1.63 | 0.22 | 0.03 | 0.47 | 0.61 |
| | 92 | 1.96 | 1.84 | 0.16 | 0.12 | 0.44 | 0.46 |
| 3 | 23 | 1.36 | 1.15 | 0.25 | 0.21 | 0.72 | 0.72 |
| | 34.5 | 1.54 | 1.32 | 0.25 | 0.22 | 0.71 | 0.70 |
| | 46 | 1.66 | 1.48 | 0.24 | 0.18 | 0.66 | 0.67 |
| | 92 | 1.96 | 1.8 | 0.18 | 0.16 | 0.52 | 0.51 |
| Across lots | 23 | 1.36 | 1.17 | 0.27 | 0.2 | 0.74 | 0.77 |
| | 34.5 | 1.54 | 1.34 | 0.25 | 0.19 | 0.69 | 0.70 |
| | 46 | 1.66 | 1.51 | 0.21 | 0.15 | 0.57 | 0.59 |
| | 92 | 1.96 | 1.8 | 0.17 | 0.16 | 0.5 | 0.47 |
* Concentration 23 IU/mL for Lot 2 did not meet 95% hit rate in the LOD study above and was therefore not included in this analysis.
The LLOQ was determined to be 23 IU/mL for lots 1 and 3 and 34.5 IU/mL for lot 2, calculated based on the calculation of the Total Analytical Error (TAE) and the difference between two measurements. The claimed LLOQ for the cobas CMV test is 34.5 IU/mL.
# 4. Linear Range
# a. Linear Range for Glycoprotein B Genotype gB-1
Linearity of the cobas CMV test was evaluated using a dilution series consisting of 10 panel members with CMV genotype gB-1 DNA concentrations spanning the range of $2.45\mathrm{E} + 01$ IU/mL to $1.34\mathrm{E} + 07$ IU/mL. Each panel member was tested in 48 replicates across three lots of cobas CMV test reagents and the results of the study are presented in Table 6 and Figure 3 below.
The cobas CMV test was demonstrated to be linear from $2.45\mathrm{E} + 01$ IU/mL to $1.34\mathrm{E} + 07$ IU/mL and shows an absolute deviation from the better fitting non-linear regression of less than $\pm 0.2\log_{10}\mathrm{IU / mL}$ . Across the linear range, the accuracy of the test was within $\pm 0.1\log_{10}\mathrm{IU / mL}$ .
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Based on the LLOQ (34.5 IU/mL) and the determined linear range, the claimed linear measurement range of the cobas CMV test is 34.5 – 1.0E+07 IU/mL.
Table 6: cobas CMV Linearity with CMV Genotype gB-1
| GT | Linear Equation HCV Genotype Linearity Study (Intercept and Slope in log10 IU/mL) | Maximum Difference Between 1st Order Model and Higher Order Model (log10 IU/mL) |
| --- | --- | --- |
| gB-1 | y= 1.0169x + -0.089 | n.a. |
| Note: The linear model (1storder) was observed to be the best fit model after analysis of the combined three lot data as no higher order model is significant. Hence there is no deviation to be shown between 1st order model and any higher order model. | | |
Figure 3: Linear Range Determination in EDTA Plasma with Genotype gB-1*
*Units on Axis in $\log_{10}$ IU/mL
# b. Linear Range for Glycoprotein B Genotypes gB-2, gB-3, and gB-4
The dilution series used in the verification of linearity for CMV genotypes gB-2, gB-3, and gB-4 consisted of seven panel members for each genotype spanning the intended linear range of $34.5 - 1.0\mathrm{E} + 07$ IU/mL. Panel members were prepared in EDTA plasma. Sixteen replicates were tested across two lots of cobas CMV reagents for each level.
All relevant genotypes (gB-2, gB-3 and gB-4) were detected within the linear range for EDTA plasma established for the predominant genotype gB-1. The linearity within the linear range of cobas CMV was verified for all three CMV Glycoprotein B genotypes
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(gB-2, gB-3 and gB-4. The maximum deviation between the linear regression and the better fitting non-linear regression was equal to or less than $\pm 0.1\log_{10}$ .
Table 7: cobas CMV Linearity Using CMV Genotype gB-2, 3 and 4
| GT | Linear Equation HCV Genotype Linearity Study (Intercept and Slope in log10 IU/mL) | Maximum Difference Between 1st Order Model and Higher Order Model (log10 IU/mL) |
| --- | --- | --- |
| gB-2 | y= 1.0225 x + -0.0566 | n.a.* |
| gB-3 | y= 1.0221 x + -0.0705 | n.a.* |
| gB-4 | y= 1.0361 x + -0.1099 | -0.11 |
| * n.a.: The linear model (1storder) was observed to be the best fit model after analysis of the combined three lot data as no higher order model is significant. Hence there is no deviation to be shown between 1storder model and any higher order model. | | |
# c. Linear Range for CMV Drug Resistant Isolates
The dilution series used in the verification of linearity for CMV drug resistant isolates for the cobas CMV test consisted of seven panel members spanning the intended linear range of $34.5 - 1.0\mathrm{E} + 07$ IU/mL. Sixteen replicates were tested across two lots of cobas CMV reagent for each level in EDTA plasma.
The linearity within the linear range of cobas CMV was verified for two CMV drug resistant isolates (one isolate resistant against foscarnet and one isolate resistant against ganciclovir, valganciclovir and cidofovir). Table 8 shows that the maximum deviation between the linear regression and the better fitting non-linear regression was equal to or less than $\pm 0.1\log_{10}\mathrm{IU / mL}$
Table 8: cobas CMV Linearity Using Drug Resistant CMV
| Resistant Phenotype | Linear Equation HCV Genotype Linearity Study (Intercept and Slope in log10 IU/mL) | Maximum Difference Between 1st Order Model and Higher Order Model (log10 IU/mL) |
| --- | --- | --- |
| Ganciclovir /Valganciclovir /Cidofovir | y= 1.0182 x + -0.0259 | n.a.* |
| Foscarnet | y= 1.0285 x + -0.111 | -0.09 |
| * The linear model (1storder) was observed to be the best fit model after analysis of the combined two lot data as no higher order model is significant. Hence there is no deviation to be shown between 1st order model and any higher order model. | | |
The claimed linear range of the cobas CMV assay, based on the linearity results, was determined to be between 34.5 and $1 \times 10^{7} \mathrm{IU/mL}$ , or $1.54 \log_{10} \mathrm{IU/mL}$ to $7.00 \log_{10} \mathrm{IU/mL}$ , for CMV in EDTA plasma, with maximum deviation from linearity of less than or equal to $0.1 \log_{10} \mathrm{IU/mL}$ .
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# 5. Precision - Within Laboratory
The precision of the cobas CMV test was determined by analysis of serial dilutions of high titer cultured virus (Merlin strain, gB-1 genotype) in CMV negative EDTA plasma. Ten dilution levels were tested in 48 replicates for each level across three lots of cobas CMV test reagents using three instruments and three operators over 12 days. Each sample was carried through the entire cobas CMV test procedure. Therefore, the reported precision represents all aspects of the test procedure.
The precision data were subjected to multivariate analysis accounting for: reagent lots, operators/instruments, days, runs and within-run replicates. No outliers were removed for analysis of results. The results are shown in Table 9.
Table 9: Within-Laboratory Precision of the cobas CMV test - Lot Specific Standard Deviation (SD*)
| Nominal Concentration | | Assigned Concentration | | Lot 1 | Lot 2 | Lot 3 | All Lots |
| --- | --- | --- | --- | --- | --- | --- | --- |
| (IU/mL) | Log10(IU/mL) | (IU/mL) | Log10(IU/mL) | SD | SD | SD | Pooled SD |
| 2.00E+07 | 7.30 | 1.34E+07 | 7.13 | 0.03 | 0.06 | 0.02 | 0.04 |
| 9.11E+06 | 6.96 | 6.11E+06 | 6.79 | 0.04 | 0.04 | 0.03 | 0.04 |
| 1.00E+06 | 6.00 | 6.71E+05 | 5.83 | 0.05 | 0.03 | 0.06 | 0.05 |
| 1.00E+05 | 5.00 | 6.71E+04 | 4.83 | 0.06 | 0.05 | 0.03 | 0.05 |
| 1.80E+04 | 4.26 | 1.21E+04 | 4.08 | 0.06 | 0.04 | 0.05 | 0.05 |
| 1.80E+03 | 3.26 | 1.21E+03 | 3.08 | 0.04 | 0.03 | 0.04 | 0.04 |
| 2.00E+02 | 2.30 | 1.34E+02 | 2.13 | 0.13 | 0.10 | 0.11 | 0.12 |
| 1.00E+02 | 2.00 | 6.71E+01 | 1.83 | 0.14 | 0.11 | 0.09 | 0.12 |
| 4.60E+01 | 1.66 | 3.09E+01 | 1.49 | 0.20 | 0.23 | 0.17 | 0.20 |
| 3.65E+01 | 1.56 | 2.45E+01 | 1.39 | 0.22 | 0.20 | 0.23 | 0.22 |
* Standard deviations (SD) was calculated based on $\log_{10}$ -transformed results from the cobas CMV test.
# 6. Reproducibility
The reproducibility of the cobas CMV test was evaluated with the Merlin strain (gB-1) diluted into EDTA plasma on the cobas 6800 System. Reproducibility and lot-to-lot variability testing was performed at 3 sites (2 external clinical sites and one internal site), using 3 reagent lots. Two operators at each site tested each reagent lot for 6 days (3 days for Operator 1 and 3 days for Operator 2). Two runs were performed each day; 3 replicates of each panel member were performed for each run. Data were analyzed using a mixed model to estimate total variance. The evaluation results are summarized in Table 10 through Table 14 below.
Table 10 below shows the clinical reproducibility of the assay at points across the linear range. The relative contributions of different factors to the observed variance are shown.
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Table 10: Attributable Percentage of Total Variance, Total Precision Standard Deviation, and Lognormal CV(%) of CMV DNA Concentrations (log₁₀ IU/mL) by Positive Panel Member
| CMV DNA Concentration (log₁₀ IU/mL) | | | Percent Contribution to Total Variance (Lognormal CV(%)) Standard Deviationc | | | | | Total Precision | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Expected | Observed Meana | Number of Testsb | Lot | Site | Operator /Day | Run | Within -Run | SDd | Log-normal CV(%)e |
| 2.01 | 2.07 | 324 | 1% (2.97) | 6% (6.49) | 0% (0.00) | 3% (4.47) | 90% (25.15) | 0.114 | 26.61 |
| | | | 0.0129 | 0.0282 | 0.0000 | 0.0194 | 0.1076 | | |
| 3.26 | 3.27 | 322 | 10% (4.29) | 13% (4.85) | 3% (2.50) | 0% (0.00) | 74% (11.71) | 0.059 | 13.64 |
| | | | 0.0186 | 0.0210 | 0.0109 | 0.0000 | 0.0507 | | |
| 3.86 | 3.90 | 324 | 23% (7.26) | 0% (0.00) | 0% (0.22) | 0% (0.00) | 77% (13.50) | 0.066 | 15.36 |
| | | | 0.0315 | 0.0000 | 0.0010 | 0.0000 | 0.0584 | | |
| 6.70 | 6.74 | 324 | 15% (5.16) | 3% (2.31) | 1% (1.52) | 0% (0.00) | 81% (11.98) | 0.058 | 13.35 |
| | | | 0.0224 | 0.0100 | 0.0066 | 0.0000 | 0.0518 | | |
| Note: The table only includes results with detectable viral load..aCalculated using SAS MIXED procedure.bNumber of valid tests with detectable viral load.cCalculated using the variance component from the SAS MIXED procedure.dCalculated using the total variability from the SAS MIXED procedure.eLognormal CV(%) = sqrt(10^√[SD^2 * ln(10)] - 1) * 100. | | | | | | | | | |
Table 11 below shows the estimated detectable viral load difference for each positive panel member. The detectable fold difference can be used to assess statistically significant changes in a patient's viral load when measured serially.
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Table 11: Detectable Viral Load Difference by Positive Panel Member
| CMV DNA Concentration (log10 IU/mL) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Expected | Observed Mean | No. of Testsa | Total Precision Standard Deviation (log10 IU/mL) | Standard Deviation of Difference Between Two Measurementsb | 95% Confidence Limitc (± log10 IU/mL) | Detectable Fold Differenced |
| 2.01 | 2.07 | 324 | 0.11 | 0.16 | 0.31 | 2.06 |
| 3.26 | 3.27 | 322 | 0.06 | 0.08 | 0.16 | 1.46 |
| 3.86 | 3.90 | 324 | 0.07 | 0.09 | 0.18 | 1.53 |
| 6.70 | 6.74 | 324 | 0.06 | 0.08 | 0.16 | 1.45 |
| Note: The table only includes results with detectable viral load. The lower limit of quantitation (LLOQ) for the assay is 3.45E+01 IU/mL, and the upper limit of quantitation (ULoQ) is 1.0E+07 IU/mL. a Number of valid tests with detectable viral load. b Standard deviation of difference between two measurements = sqrt[2 * (total precision standard deviation)^2]. c 95% CL = 1.96 * standard deviation of difference between two measurements. d Detectable Fold Difference = 10^(1.96 * sqrt(2 * (total standard deviation)^2)). CL = confidence limit. | | | | | | |
Table 12 below presents the reproducibility results for the cobas 6800 System using negative panel member. $100\%$ of test results were negative.
Table 12: Reproducibility Results With the Negative Panel Member
| Expected CMV DNA Concentration | Number of Valid Tests | Positive Results | Negative Results | Negative Percent Agreementa | 95% Exact CIb |
| --- | --- | --- | --- | --- | --- |
| Negative | 323 | 0 | 323 | 100.00 | (98.86, 100.00) |
| a Negative Percent Agreement = (number of negative results / total valid tests in negative panel member)*100%.b Calculated using the Clopper-Pearson exact binomial confidence interval method.CI = confidence interval; CMV = cytomegalovirus. | | | | | |
# 7. Analytical Specificity - Cross-Reactivity
The analytical specificity of cobas CMV was evaluated by diluting a panel of microorganisms to a concentration of $1.00\mathrm{E} + 06$ particles, copies, IU, genome equivalents or $\mathrm{CFU / mL}$ . Organisms were spiked into negative human EDTA plasma and into human EDTA plasma containing $230~\mathrm{IU / mL}$ CMV DNA (Table 13). Each sample was tested in replicates of three. None of the non-CMV pathogens interfered with test performance. Negative results were obtained with cobas CMV for all microorganism samples without CMV target and positive results were obtained for all of the microorganism samples with CMV target. Furthermore, the mean $\log_{10}\mathrm{IU / mL}$ of each of the positive CMV samples containing potentially cross-reacting organisms was within $\pm 0.5\log_{10}\mathrm{IU / mL}$ of the mean $\log_{10}\mathrm{IU / mL}$ of the respective positive spike control.
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Table 13: Microorganisms Tested for Cross-Reactivity
| Viruses | Bacteria | Yeast and Fungi |
| --- | --- | --- |
| Adenovirus type 5 | Propionibacterium acnes | Aspergillus niger |
| BK Polyomavirus | Staphylococcus aureus | Candida albicans |
| Epstein-Barr Virus | Chlamydia trachomatis | Cryptococcus neoformans |
| Hepatitis B Virus | Clostridium perfringens | |
| Hepatitis C Virus | Enterococcus faecalis | |
| Herpes Simplex Virus type1 | Escherichia coli | |
| Herpes Simplex Virus type 2 | Klebsiella pneumonia | |
| Human Herpes Virus type-6 | Listeria monocytogenes | |
| Human Herpes Virus type-7 | Mycobacterium avium | |
| Human Herpes Virus type-8 | Neisseria gonorrhoeae | |
| Human Immunodeficiency Virus-1 | Staphylococcus epidermidis | |
| Human Immunodeficiency Virus-2 | Streptococcus pyrogenes | |
| Human Papillomavirus | Mycoplasma pneumonia | |
| JC virus | Salmonella typhimurium | |
| Parvovirus B19 | Streptococcus pneumonia | |
| Varicella-Zoster Virus | | |
## 8. Analytical Specificity – Interfering Substances
Elevated levels of triglycerides (34.5 g/L), conjugated bilirubin (0.25 g/L), unconjugated bilirubin (0.25 g/L), albumin (58.7 g/L), hemoglobin (2.9 g/L) and human DNA (2 mg/L) were tested in samples in the presence and absence of CMV DNA (230 IU/mL). The tested endogenous interfering substances were shown not to interfere with the performance of the cobas CMV test.
Specimens from patients with autoimmune diseases such as systemic lupus erythematosus (SLE, n=6 specimens), rheumatoid arthritis (RA; n=7 specimens) and antinuclear antibody (ANA; n=7 specimens) were tested with CMV DNA spiked to 230 IU/mL (3 replicates per specimen) and without spiked CMV DNA (one replicate per specimen).
In addition, drug compounds listed in Table 14 were tested at three times the $C_{\mathrm{max}}$ in the presence (230 IU/mL) and absence of CMV DNA.
All potentially interfering substances tested were shown to not interfere with the test performance. Negative results were obtained with cobas CMV for all samples without CMV target and positive results were obtained with all of the samples with CMV target. Furthermore, the mean $\log_{10}$ IU/mL of each of the positive CMV samples containing potentially interfering substances was within $\pm 0.5$ $\log_{10}$ IU/mL of the mean $\log_{10}$ IU/mL of the respective positive spike control.
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Table 14: Drug Compounds Tested for Interference with the Quantitation of CMV DNA by cobas CMV
| Class of Drug | Generic Drug Name | |
| --- | --- | --- |
| Antibimicrobial | Cefotetan
Clavulanate potassium
Fluconazole
Piperacillin
Tazobactam sodium | Sulfamethoxazole
Ticarcillin disodium
Trimethoprim
Vancomycin |
| Compounds for Treatment of Herpes Viruses | Ganciclovir
Valganciclovir | Cidofovir
Foscarnet |
| Immune Suppressant | Azathioprine
Cyclosporine
Everolimus
Mycophenolate mofetil
Mycophenolic acid | Prednisone
Sirolimus
Tacrolimus |
## 9. Cross-Contamination
The cross-contamination rate for the cobas CMV test was determined by testing 240 replicates of a normal, CMV DNA negative human EDTA-plasma sample and 225 replicates of a high titer CMV sample at 1.00E+06 IU/mL. In total, five runs were performed with positive and negative samples arranged in a checkerboard configuration. All 240 replicates of the negative sample were negative, resulting in a cross-contamination rate of 0% (95% confidence interval 0%-1.5%).
## B. Animal Studies
Not applicable
## C. Additional Studies
None
## X. SUMMARY OF PRIMARY CLINICAL STUDIES
To establish a reasonable assurance of safety and effectiveness of viral load testing with the cobas CMV test for use as an aid in the management of CMV in solid organ transplant (SOT) recipients and hematopoietic stem cell transplant (HSCT) recipients, clinical performance evaluation of the cobas CMV test carried out with EDTA plasma specimens left over from clinical studies that tested investigational prophylactic anti-CMV treatment regimens. The use of left-over specimens in the clinical performance evaluation of the cobas CMV test did not require an IDE for either study. Data from these clinical performance studies were the basis for the PMA approval decision.
The objective for the studies listed below was to evaluate the cobas CMV test with respect to clinical decision making (i.e., Clinical Concordance study) and numeric viral
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load results (i.e., Method Comparison study) by comparing the cobas CMV test results to the test results obtained with the previously approved COBAS AmpliPrep/COBAS TaqMan CMV Test. EDTA plasma specimens from SOT and Hematopoetic Stem Cell Transplant (HSCT) patient populations were used in all studies. A summary of the clinical studies is presented in the sections below.
A. Clinical Concordance in SOT Patients
B. Method Comparison in SOT Patients
C. Clinical Concordance in HSCT Patients
D. Method Comparison in HSCT Patients
## A. Clinical Concordance in SOT Patients
### 1. Study Design
EDTA plasma samples were obtained from kidney transplant recipients at high risk for CMV viremia (CMV D+/R-) and who participated in a phase 2a international, multicenter, randomized, placebo-controlled (two-armed), double-blind clinical trial of a prophylactic treatment. Samples were prospectively collected for the drug trial and stored frozen; leftover. De-identified samples were then used for performance validation of the cobas CMV test.
Subjects in the drug trial were provided with either the prophylactic treatment (treatment arm) or a placebo (control arm). Patients were managed with serial plasma CMV viral load testing as per the institutions standard of care.
Blood was drawn and tested from the study subjects at least weekly for the first 12 weeks after transplant and then biweekly for a subsequent 12 weeks. Participants who developed CMV viremia had anti-CMV treatment initiated and underwent more frequent blood draws for viral load testing (at least once per week until 2 sequential negative test results were obtained). The key timepoints as defined by the cobas CMV testing protocol were Baseline, Day 7, 14, 21, 28, 35, 42 and 49.
SOT patient samples in the parent drug study were collected between December 2012 and October 2014. The database for this PMA included n=107 SOT patients.
EDTA plasma samples were tested with the cobas CMV test and with an FDA approved comparator test (cobas AmpliPrep/cobas TaqMan CMV Test abbreviated as TaqMan CMV Test for this document) and concordance of CMV viral load measurements was assessed (see below under "Analysis"). For the cobas CMV device study testing was performed at 3 external clinical sites and one manufacturer's site. The sponsor internal site performed comparator testing only. The external clinical sites performed cobas testing of the specimens. For further discussion of the analysis methods, see Section 4, Analysis, below.
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# Inclusion and Exclusion Criteria
Enrollment in the parent drug study was limited to patients who met the following clinical inclusion criteria:
- ≥ 18 years of age
- Signed Informed Consent Form
- Seronegative for CMV (based on CMV immunoglobulin G) before transplantation
- Recipient of primary or secondary renal allograft from a living or cadaveric CMVseropositive donor
Enrollment in the cobas CMV testing study was limited to patients who met the following inclusion criteria:
- Patients in the parent study with more than 5 samples with sufficient volume to permit testing with the cobas CMV test and the TaqMan CMV Test
Patients were not permitted to enroll in the studies if the inclusion criteria were not met.
## Follow-up schedule and endpoints
See Study Design (above).
## 2. Accountability of SOT Subjects
A total of 107 subjects participated in the parent drug study and provided a total of 1,913 specimens. All specimens were assessed for entry into the clinical performance evaluation of the cobas CMV test.
For additional accountability with respect to the different analysis performed on the study cohort, please refer to the analysis sections below (section 4).
## 3. SOT Study Population Demographics and Baseline Parameters
The demographics of the study population are representative for a study performed in the US that assesses CMV in SOT transplant patients.
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Table 15: Demographics and Baseline Clinical Characteristics of SOT Subjects
| Characteristics | Statistic |
| --- | --- |
| Total, N | 107 |
| Age (years) | |
| Mean ± Standard Deviation | 49 ± 13.6 |
| Median | 50 |
| Range | 18 - 76 |
| Gender, n(%) | |
| Male | 74 (69.2%) |
| Female | 33 (30.8%) |
| Ethnicity, n(%) | |
| Hispanic / Latino | 10 (9.3%) |
| Not Hispanic / Not Latino | 91 (85.0%) |
| Unknown | 6 (5.6%) |
| Race, n(%) | |
| Asian | 1 (0.9%) |
| Black / African-American | 16 (15.0%) |
| White | 88 (82.2%) |
| Other | 2 (1.9%) |
| Immunosuppression Induction, n(%) | |
| Yes | 26 (24.3%) |
| No | 81 (75.7%) |
| Study Arm, n(%) | |
| Anti-CMV Prophylaxis Regimen | 53 (49.5%) |
| Placebo | 54 (50.5%) |
| CMV Serology Status, n(%) | |
| Donor Positive, Recipient Negative | 107 (100.0%) |
## 4. Analysis
The concordance of CMV measurements between the cobas CMV test and the TaqMan CMV Test with specimens from SOT patients was assessed with respect to the following clinical assessments:
### a. Agreement of SOT Patient Viral Load Results Using Different Threshold Values
Viral load guided initiation of anti-CMV medication in the management of transplant patients depends on multiple patient risk factors as well as the experience of the treating physicians and the institution's standard of care. Hence, no universally applicable treatment threshold can be validated. Consequently, result concordance between the
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cobas CMV test and the TaqMan CMV Test was analyzed using four different arbitrary thresholds: Target Not Detected [LOD], 137 IU/mL [2.137 log₁₀ IU/mL], 500 IU/mL [2.699 log₁₀ IU/mL] and 1,800 IU/mL [3.255 log₁₀ IU/mL]. The analysis included all evaluable samples of the clinical study independent of treatment and independent of the specific time elapsed post treatment initiation.
In addition, results from the cobas CMV and the TaqMan CMV tests were analyzed in a 6x6 table that takes into account that the SD for the reproducibility of the TaqMan CMV Test was less than ± 0.3 log₁₀ IU/mL. The categories in the table derived from the threshold value (in log₁₀ IU/mL) ± 0.6 log₁₀ IU/mL (2x the SD of the reproducibility) with the log-transformed nominal threshold values in log₁₀ rounded to one significant figure. The analysis is based on the assumption that samples with values above a threshold of 2.7 log₁₀ IU/mL IU/mL are considered unlikely to be below the threshold of 2.1 log₁₀ IU/mL because of random measurement error. Similarly, samples with values above a threshold of 3.3 log₁₀ IU/mL IU/mL are considered unlikely to be below the threshold of 2.7 log₁₀ IU/mL and samples with values above a threshold of 3.9 log₁₀ IU/mL IU/mL are considered unlikely to be below the threshold of 3.3 log₁₀ IU/mL because of random measurement error. Therefore samples were considered discordant only if they were discrepant across more than the immediately adjacent categories.
## Analysis Specific Accountability:
A total of 1,898 samples from the 107 subjects had paired results (i.e., valid results with both, the cobas CMV test and the TaqMan CMV Test) and were included in the analysis.
## b. Agreement of SOT Patient Viral Load Results at Baseline in Order to Assess Initiation of Anti-CMV Therapy
The cobas CMV test and TaqMan CMV test results were analyzed as described under 4.a. (above) but analysis was performed only for paired evaluable samples collected at baseline. For the purpose of this analysis measurements prior to initiation of anti-CMV therapy were considered in the baseline analysis as well because they were used to guide the decision to initiate anti-CMV treatment.
## Analysis Specific Accountability:
Of the 107 subjects in the clinical study, 71 subjects initiated therapy; 125 of their samples with paired results were evaluable and collected at baseline or prior to anti-CMV treatment initiation and were included in the analysis.
## c. Agreement of SOT Patient Viral Load Results in Order to Assess Response to Anti-CMV Therapy
The cobas CMV and TaqMan CMV test results were analyzed as described under 4.a. (above), but analysis was performed for all paired evaluable samples collected at the protocol defined timepoints post anti-CMV therapy initiation (Day 14, Day 21, Day 28, Day 35, and Day 49).
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# Analysis Specific Accountability:
Of the 107 total subjects in the clinical study, 71 subjects initiated therapy; 272 of their samples with paired results were evaluable and collected at protocol defined time points post anti-CMV therapy initiation and were included in the analysis.
## d. Agreement of SOT Patient Viral Load Results in Order to Determine When to Stop Anti-CMV Therapy
An analysis was carried out to determine the concordance between the cobas CMV test and the TaqMan CMV Test when used to aid in determining whether or not to stop anti-CMV treatment at visit times of Day 14, Day 21, Day 28, Day 35, and Day 49 (post anti-CMV therapy initiation). The analysis considered all viremic patients who started anti-CMV medication (n=71).
The following Definitions were used:
- **Baseline**: Baseline was defined as the date the anti-CMV medication was started.
- **Days of Interest**: Testing days of interest were Day 14, Day 21, Day 28, Day 35, and Day 49 and were defined relative to the Baseline with a window of ±3 days.
- **Viremic Episode**: A viral load of ≥137 IU/mL (or ≥ 2.137 log₁₀ IU/mL) for both tests was used to define a viremic episode.
- **Resolution of Viremia**: Consistent with current guideline recommendations resolution of the viremic episode is defined by two consecutive measurements below the LLOQ of the comparator test (i.e., <137 IU/mL or < 2.137 log₁₀ IU/mL) and/or Target Not Detected (TND) with the samples taken one week apart.
For subjects in the cohort who had multiple episodes of viremia, once the subject met the criterion for resolution of their initial CMV episode at a given timepoint, that resolution status was carried forward for the corresponding assay and recurrent episodes of viremia were not analyzed anymore.
Not all subjects have all samples available at each of the time points. Missing data points were imputed through linear regression if surrounding datapoints with viral loads > 137 IU/mL were available. Imputation was performed across a maximum of two missing data points (2 weeks).
A missing data point can be considered as "not resolved" for the purpose of resolution analysis if the previous data point was viremic because two consecutive viral load measurements of < 137 IU/mL (or undetectable CMV DNA) are required to define a resolution of the viremic episode.
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# Analysis Specific Accountability:
Of the 107 subjects that were included in the Clinical Concordance analysis, the following number of subjects were excluded for the purpose of the resolution analysis:
- Thirty-nine (39) subjects with 609 samples were excluded for the resolution analysis because they did not initiate therapy during the course of the study or they did not have viral load measurements at any of the required time points so that neither the occurrence of a viremic episode nor the resolution of a viremic episode could be determined.
- Six (6) subjects who initiated therapy but were not viremic at any of the collected time points were excluded because neither a viremic episode nor its resolution could be determined.
- One (1) subject was excluded because the subject became viremic only at Day 42 so that resolution of viremia could not be analyzed.
A total of 61 subjects started anti-CMV therapy during the course of the parent drug study; 1,184 samples from these subjects provided paired viral load measurements from the cobas CMV and the TaqMan CMV tests at any of the following time points and were included in the resolution analysis: Baseline, Day 7, 14, 21, 28, 35, 42 and 49.
# 5. Safety and Effectiveness Results
The key safety and effectiveness (performance) outcomes for the Clinical Concordance study of Solid Organs Transplant (SOT) patients are presented below in Tables 16 to 29.
Performance evaluation was based on 107 patients evaluable for Clinical Concordance. Adverse effect reporting is not applicable to this study as it is a retrospective study with leftover specimens and no patient management was based on the results of this device during the study.
# a. Agreement of SOT Patient Viral Load Results Using Different Threshold Values
Table 16 below considers all 1,898 specimens for which a result was available for both the cobas CMV and TaqMan CMV tests (so called "paired results"). These specimens were obtained from 107 subjects (viremic and non-viremic). Concordance for the viral load results of the specimens is provided in Table 17.
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Table 16: Concordance Analysis of Samples From SOT Patients Using Different Thresholds (All Paired Samples)
| | TaqMan CMV | | Total | |
| --- | --- | --- | --- | --- |
| | | Target Not Detected | | Detected |
| cobas CMV | Target Not Detected | 1,022 | 8 | 1,030 |
| | Detected | 1721 | 696 | 868 |
| Total | | 1,194 | 704 | 1,898 |
| | | < 137 IU/mL (< 2.1 log10 IU/mL*) | ≥ 137 IU/mL (≥ 2.1 log10 IU/mL*) | Total |
| cobas CMV | < 137 IU/mL (< 2.1 log10 IU/mL*) | 1,391 | 6 | 1397 |
| | ≥ 137 IU/mL (≥ 2.1 log10 IU/mL*) | 972 | 404 | 501 |
| Total | | 1,488 | 410 | 1,898 |
| | | < 500 IU/mL (< 2.7 log10 IU/mL**) | ≥ 500 IU/mL (≥ 2.7 log10 IU/mL**) | Total |
| cobas CMV | < 500 IU/mL (< 2.7 log10 IU/mL**) | 1,537 | 8 | 1,545 |
| | ≥ 500 IU/mL (≥ 2.7 log10 IU/mL**) | 1023 | 251 | 353 |
| Total | | 1,639 | 259 | 1,898 |
| | | < 1,800 IU/mL (< 3.3 log10 IU/mL***) | ≥ 1,800 IU/mL (≥ 3.3 log10 IU/mL***) | Total |
| cobas CMV | < 1,800 IU/mL (< 3.3 log10 IU/mL***) | 1,693 | 1 | 1,694 |
| | ≥ 1,800 IU/mL (≥ 3.3 log10 IU/mL***) | 624 | 142 | 204 |
| Total | | 1,755 | 143 | 1,898 |
| All paired samples evaluable for Clinical Concordance analysis were included in this table. Note: All paired samples evaluable for Clinical Concordance analysis were included in this table. Samples with a “Target Not Detected” results were categorized as “< Threshold in IU/mL”.128 of the 172 discordant samples were sequenced and showed impactful sequenced mismatch225 of the 97 discordant samples were sequenced and showed impactful sequenced mismatch327 of the 102 discordant samples were sequenced and showed impactful sequenced mismatch419 of the 62 discordant samples were sequenced and showed impactful sequenced mismatch* Threshold Log10 of 2.137 abbreviated as 2.1 log10 IU/mL** Threshold Log10 of 2.699 abbreviated as 2.2log10 IU/mL*** Threshold Log10 of 3.255 abbreviated as 3.3 log10 IU/mL | | | | |
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Table 17: Summary Concordance of Viral Load Results from SOT Patients Using Different Thresholds
| | Percent Agreement < Threshold 95% CI (n/N) | Percent Agreement ≥ Threshold (n/N) 95% CI (n/N) | Overall Percent Agreement 95% CI (n/N) |
| --- | --- | --- | --- |
| Target Not Detected | 85.6
83.5%, 87.5%
(1,022/1,194) | 98.9
97.8%, 99.5%
(696/704) | 90.5
89.1%, 91.8%
(1,718/1,898) |
| 137 IU/mL
(2.1 log10 IU/mL*) | 93.5
92.1%, 94.7
(1,391/1,488) | 98.5
96.8%, 99.5%
(404/410) | 94.6
93.5%, 95.5%
(1,795/1,898) |
| 500 IU/mL
(2.7 log10 IU/mL**) | 93.8
92.5%, 94.9%
(1,537/1,639) | 96.9
94.0%, 98.7%
(251/259) | 94.2
93.1%, 95.2%
(1,788/1,898) |
| 1,800 IU/mL
(3.3 log10 IU/mL***) | 96.5
95.5%, 97.3%
(1,693/1,755) | 99.3
96.2%, 100%
(142/143) | 96.7
95.8%, 97.4%
(1,835/1,898) |
| Note: All paired samples evaluable for Clinical Concordance analysis were included in this table.
Samples with a “Target Not Detected” results were categorized as “< Threshold in IU/mL”.
* Log10 of 2.137 abbreviated as 2.1 log10 IU/mL
** Log10 of 2.699 abbreviated as 2.2 log10 IU/mL
*** Log10 of 3.255 abbreviated as 3.3 log10 IU/mL.
95% confidence interval (CI) calculated by exact method assuming independence between all samples.
1 IU/mL = 1.1 copy/mL. | | | |
The concordance analysis of viral load results as determined by the cobas CMV and TaqMan CMV tests was further analyzed in the 6x6 Table (Table 18) below as described in section A.4.a. (above). Samples were considered discordant if they were discrepant across more than the immediately adjacent categories; this analysis considers the reproducibility of the cobas CMV test.
Most samples were not discrepant by more than 1 category from the diagonal. The 4 samples with wider discrepancies (see footnotes a to d in Table 18) were sequenced and a total of 12 out of 13 were found to contain a CMV variant with a significant mismatch mutation in one of the TaqMan CMV Test primer binding sites.
Agreements were found to be as follows:
Agreement for TND: 99.7% (1,190/1,194)
Agreement for Detected < 2.1 log10 IU/mL: 94.2% (277/294)
Agreement for 2.1 to < 2.7 log10 IU/mL: 92.75% (140/151)
Agreement for 2.7 to < 3.3 log10 IU/mL: 99.1% (115/116)
Agreement for 3.3 to < 3.9 log10 IU/mL: 100% (104/104)
Agreement for ≥ 3.9 log10 IU/mL: 100% (39/39)
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Table 18: Concordance Analysis for Samples From SOT Patients (All Paired Samples)
| cobas CMV (log10 IU/mL) | Target Not Detected | TaqMan CMV Test (log10 IU/mL) | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | < 2.1 | 2.1 to < 2.7 | 2.7 to < 3.3 | 3.3 to < 3.9 | ≥ 3.9 | |
| Target Not Detected | 1,022 | 8 | 0 | 0 | 0 | 0 | 1,030 |
| < 2.1* | 168 | 193 | 6 | 0 | 0 | 0 | 367 |
| 2.1 to < 2.7 | 3a | 76 | 61 | 8 | 0 | 0 | 148 |
| 2.7 to < 3.3 | 0 | 12c | 73 | 63 | 1 | 0 | 149 |
| 3.3 to < 3.9 | 1b | 5d | 8e | 44 | 58 | 0 | 116 |
| ≥ 3.9 | 0 | 0 | 3f | 1g | 45 | 39 | 88 |
| Total | 1,194 | 294 | 151 | 116 | 104 | 39 | 1,898 |
| Note: All 1898 paired samples evaluable for Clinical Concordance analysis from all 71 subjects were included in this table. *The LLOQ is 34.5 IU/mL for the cobas CMV and 137 IU/mL for TaqMan CMV Test. Therefore for the cobas CMV test the category of <137 IU/mL includes non-quantifiable results < the LLOQ and quantifiable results ≥ 34.5 IU/mL but < 137 IU/mL. log10(137) = 2.137 (abbreviated above as 2.1); log10(1,800) = 3.255 (abbreviated above as 3.3); log10(7,943) = 3.900 (abbreviated above as 3.9). a These samples were sequenced and 2 out of 3 were found to contain a significant impact mutation in the forward primer binding site of the Taqman CMV test. b This sample was sequenced and was found to contain a significant impact mutation in the forward primer binding site of the Taqman CMV test. c 8 of the 12 discordant samples derived from 5 subjects and all 8 samples were sequenced and found to have significant impact mutations. d These 5 samples derived from 3 subjects; they were sequenced and all 5 were found to contain a significant impact mutation in the forward primer binding site of the Taqman CMV test. e 7 of the 8 discordant samples derived from 3 subjects and all 7 samples were sequenced and found to have a significant impact mutation. f These 3 samples derived from 2 subjects; they were sequenced and all 3 were found to contain a significant impact mutation in the forward primer binding site of the Taqman CMV test. g The one discrepant sample was found to have significant impact mutation | | | | | | | |
# b. Agreement of SOT Patient Viral Load Results at Baseline Using Different Threshold Values
Table 19 below considers all 71 subjects that had paired baseline viral load results. If the baseline sample was missing the sample immediately prior to treatment initiation was used instead (if available). Concordance for the viral load result of these 71 samples is provided in Table 19.
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Table 19: Concordance Analysis for Baseline Samples From SOT Patients Using Different Thresholds
| | TaqMan CMV | | Total | |
| --- | --- | --- | --- | --- |
| | | Target Not Detected | | Detected |
| cobas CMV | Target Not Detected | 9 | 0 | 9 |
| | Detected | 21 | 60 | 62 |
| Total | | 11 | 60 | 71 |
| | | < 137 IU/mL (< 2.1 log10 IU/mL*) | ≥ 137 IU/mL (≥ 2.1 log10 IU/mL*) | Total |
| cobas CMV | < 137 IU/mL (< 2.1 log10 IU/mL*) | 24 | 1 | 25 |
| | ≥ 137 IU/mL (≥ 2.1 log10 IU/mL*) | 52 | 41 | 46 |
| Total | | 29 | 42 | 71 |
| | | < 500 IU/mL (< 2.7 log10 IU/mL**) | ≥ 500 IU/mL (≥ 2.7 log10 IU/mL**) | Total |
| cobas CMV | < 500 IU/mL (< 2.7 log10 IU/mL**) | 33 | 2 | 35 |
| | ≥ 500 IU/mL (≥ 2.7 log10 IU/mL**) | 73 | 29 | 36 |
| Total | | 40 | 31 | 71 |
| | | < 1,800 IU/mL (< 3.3 log10 IU/mL***) | ≥ 1,800 IU/mL (≥ 3.3 log10 IU/mL***) | Total |
| cobas CMV | < 1,800 IU/mL (< 3.3 log10 IU/mL***) | 48 | 0 | 48 |
| | ≥ 1,800 IU/mL (≥ 3.3 log10 IU/mL***) | 44 | 19 | 23 |
| Total | | 52 | 19 | 71 |
| Only paired samples at or before baseline and evaluable for Clinical Concordance analysis were included in this table. Samples with a “Target Not Detected” results were categorized as “< threshold value in IU/mL”. 10 of the 2 discordant samples derived from subjects with an impactful sequence mismatch; discrepancy of results was <0.5 log10 IU/mL for both discrepant samples. 22 of the 5 discordant samples derived from subjects with an impactful sequence mismatch and for three samples the discrepancy of results was >0.5 log10 IU/mL including the two samples with impactfull sequence mismatch. 33 of the 7 discordant samples derived from subjects with an impactful sequence mismatch and all three had discrepancies >0.5log10 IU/mL. For the remaining 4 discrepant samples the discrepancy was <0.5 log IU/mL. | | | | |
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$^{4}$ 1 of the 4 discordant samples derived from subject with impactful sequence mismatch. Two of the discordant samples had discrepancies of $>0.5 \log_{10} \mathrm{IU/mL}$ (including the sample with the sequence mismatch); the other two samples had discrepancies $<0.5 \log_{10} \mathrm{IU/mL}$ .
* Threshold $\mathrm{Log}_{10}$ of 2.137 abbreviated as $2.1\log_{10}\mathrm{IU / mL}$
** Threshold $\mathrm{Log}_{10}$ of 2.699 abbreviated as $2.2\log_{10}\mathrm{IU / mL}$
*** Threshold $\mathrm{Log}_{10}$ of 3.255 abbreviated as $3.3\log_{10}\mathrm{IU / mL}$ .
Table 20: Summary Concordance of Viral Load Results for Baseline Samples From SOT Using Different Thresholds
| | Percent Agreement < Threshold 95% CI (n/N) | Percent Agreement ≥ Threshold (n/N) 95% CI (n/N) | Overall Percent Agreement 95% CI (n/N) |
| --- | --- | --- | --- |
| Target Not Detected | 81.8%
48.2%, 97.7%
(9/11) | 100.0%
94.0%, 100.0%
(60/60) | 97.2%
90.2%, 99.7%
(69/71) |
| 137 IU/mL
(2.1 log10 IU/mL*) | 82.8%
64.2%, 94.2%
(24/29) | 97.6%
87.4%, 99.9%
(41/42) | 91.5%
82.5%, 96.8%
(65/71) |
| 500 IU/mL
(2.7 log10 IU/mL**) | 82.5%
67.2%, 92.7%
(33/40) | 93.5%
78.6%, 99.2%
(29/31) | 87.3%
77.3%, 94.0%
(62/71) |
| 1,800 IU/mL
(3.3 log10 IU/mL***) | 92.3%
81.5%, 97.9%
(48/52) | 100.0%
82.4%, 100.0%
(19/19) | 94.4%
86.2%, 98.4%
(67/71) |
| Note: Only paired samples at or before baseline and evaluable for Clinical Concordance analysis were included in this table. Samples with a “Target Not Detected” results were categorized as “< threshold value in IU/mL”.
* Log10 of 2.137 abbreviated as 2.1 log10 IU/mL
** Log10 of 2.699 abbreviated as 2.2 log10 IU/mL
*** Log10 of 3.255 abbreviated as 3.3 log10 IU/mL.
CI: 2-sided 95% confidence interval calculated by exact method assuming independence between all samples. | | | |
The concordance analysis of viral load results as determined by the cobas CMV and TaqMan CMV tests was further analyzed in the 6x6 Table (Table 21) below as described in section A.4.a. (above). Samples were considered discordant if they were discrepant across more than the immediately adjacent categories; this analysis considers the reproducibility of the cobas CMV test. Most samples were not discrepant by more than 1 category from the diagonal. The two samples with wider discrepancy (see footnote a in Table 21) were sequenced and found to contain a CMV variant with a significant mismatch mutation in one of the TaqMan CMV Test primer binding sites.
Agreements were found to be as follows:
Agreement for TND: 100% (11/11)
Agreement for $< 2.1\log_{10}\mathrm{IU / mL}$ 88.9% (16/18)
Agreement for 2.1 to $< 2.7\log_{10}\mathrm{IU / mL}$ 100% (11/11)
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Agreement for 2.7 to < 3.3 log₁₀ IU/mL: 100% (12/12)
Agreement for 3.3 to < 3.9 log₁₀ IU/mL: 100% (15/15)
Agreement for ≥ 3.9 log₁₀ IU/mL: 100% (4/4)
Table 21: Concordance Analysis for Baseline Samples From SOT Patients
| cobas CMV (log₁₀ IU/mL) | Target Not Detected | TaqMan CMV Test (log₁₀ IU/mL) | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | < 2.1 | 2.1 to < 2.7 | 2.7 to < 3.3 | 3.3 to <3.9 | ≥ 3.9 | |
| Target Not Detected | 9 | 0 | 0 | 0 | 0 | 0 | 9 |
| < 2.1* | 2 | 13 | 1 | 0 | 0 | 0 | 16 |
| 2.1 to < 2.7 | 0 | 3 | 5 | 2 | 0 | 0 | 10 |
| 2.7 to < 3.3 | 0 | 1^{a} | 5 | 7 | 0 | 0 | 13 |
| 3.3 to <3.9 | 0 | 1^{a} | 0 | 3 | 15 | 0 | 19 |
| ≥ 3.9 | 0 | 0 | 0 | 0 | 0 | 4 | 4 |
| Total | 11 | 18 | 11 | 12 | 15 | 4 | 71 |
| Note: Only paired samples at or before baseline and evaluable for Clinical Concordance analysis were included in this table. Samples with a “Target Not Detected” results were categorized as “< threshold value in IU/mL”.
*The LLOQ is 34.5 IU/mL for the cobas CMV and 137 IU/mL for TaqMan CMV Test. Therefore for the cobas CMV test the category of <137 IU/mL includes non-quantifiable results < the LLOQ and quantifiable results ≥ 34.5 IU/mL but < 137 IU/mL.
Log₁₀ of 2.137 abbreviated as 2.1 log₁₀ IU/mL
Log₁₀ of 2.699 abbreviated as 2.2 log₁₀ IU/mL
Log₁₀ of 3.255 abbreviated as 3.3 log₁₀ IU/mL
Log₁₀ of 7,943 abbreviated as 3.9 log₁₀ IU/mL
a This sample was sequenced and found to contain a significant impact mutation in the forward primer binding site of the Taqman CMV test. | | | | | | | |
## c. Agreement of SOT Patients Viral Load Results in Order to Assess Response to Anti-CMV Therapy
Tables 22 and 23 provide an analysis of concordance with respect to viral load results for the 272 paired results from the 71 subjects who started anti-CMV therapy and had samples collected at any of the protocol defined days post therapy initiation (i.e., Days 7, 14, 21, 28, 35, and 49 post treatment).
The concordance analysis of viral load results as determined by the cobas CMV and TaqMan CMV tests was further analyzed in the 6x6 Table (Table 24) below as described in section 4. a. (above). Samples were considered discordant if they were discrepant across more than the immediately adjacent categories; this analysis considers the reproducibility of the cobas CMV test.
Except for the categories < 2.1 log₁₀ IU/mL and 2.1 to < 2.7 log₁₀ IU/mL all categories had 100% agreement between the cobas CMV and the TaqMan CMV Test. Agreements for the categories < 2.1 log₁₀ IU/mL and 2.1 to < 2.7 log₁₀ IU/mL was 92.3% (72/78) and 96.1% (74/77), respectively. However, 7 out of 9 discrepant samples were samples that
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contained a significant impact mutation in the primer binding site of the TaqMan CMV test.
Table 22: Concordance Analysis of cobas CMV and TaqMan CMV Test Results for Samples from SOT Patients Collected at Protocol Defined Days Post Treatment Initiation Using Different Thresholds (n=272 Samples)
| | TaqMan CMV | | Total | |
| --- | --- | --- | --- | --- |
| | | Target Not Detected | | Detected |
| cobasCMV | Target Not Detected | 24 | 3 | 27 |
| | Detected | 361 | 209 | 245 |
| Total | | 60 | 212 | 272 |
| | | < 137 IU/mL (< 2.1 log10 IU/mL*) | ≥ 137 IU/mL (≥ 2.1 log10 IU/mL*) | Total |
| cobasCMV | < 137 IU/mL (< 2.1 log10 IU/mL*) | 105 | 1 | 106 |
| | ≥ 137 IU/mL (≥ 2.1 log10 IU/mL*) | 332 | 133 | 166 |
| Total | | 138 | 134 | 272 |
| | | < 500 IU/mL (< 2.7 log10 IU/mL**) | ≥ 500 IU/mL (≥ 2.7 log10 IU/mL**) | Total |
| cobasCMV | < 500 IU/mL (< 2.7 log10 IU/mL**) | 151 | 0 | 151 |
| | ≥ 500 IU/mL (≥ 2.7 log10 IU/mL**) | 343 | 87 | 121 |
| Total | | 185 | 87 | 272 |
| | | < 1,800 IU/mL (< 3.3 log10 IU/mL***) | ≥ 1,800 IU/mL (≥ 3.3 log10 IU/mL***) | Total |
| cobasCMV | < 1,800 IU/mL (< 3.3 log10 IU/mL***) | 196 | 0 | 196 |
| | ≥ 1,800 IU/mL (≥ 3.3 log10 IU/mL***) | 264 | 50 | 76 |
| Total | | 222 | 50 | 272 |
| Note: Samples with a “Target Not Detected” or a detectable viral load below the threshold result were categorized as “< Threshold IU/mL. Note: LLOD cobas CMV = 34.5 IU/mL; LLOQ TaqMan CMV = 137 IU/mL. 1 IU/mL = 1.1 copy/mL.”14 of the 36 discordant samples were sequenced and showed impactful sequenced mismatch | | | | |
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Table 23: Summary of Concordance of Viral Load Results by Different Baseline Threshold
| | Percent Agreement < Threshold 95% CI (n/N) | Percent Agreement ≥ Threshold (n/N) 95% CI (n/N) | Overall Percent Agreement 95% CI (n/N) |
| --- | --- | --- | --- |
| Target Not Detected | 40.0
27.6%, 53.5%
(24/60) | 98.6
95.9%, 99.7%
(209/212) | 85.7
80.9%, 89.6%
(233/272) |
| 137 IU/mL
(2.1 log10 IU/mL*) | 76.1
68.2%, 82.9
(105/138) | 99.3
95.9%, 100%
(133/134) | 87.5
83.0%, 91.2%
(238/272) |
| 500 IU/mL
(2.7 log10 IU/mL**) | 81.6
75.3%, 86.9%
(151/185) | 100
95.8%, 100%
(87/87) | 87.5
83.0%, 91.2%
(238/272) |
| 1,800 IU/mL
(3.3 log10 IU/mL***) | 88.3
83.3%, 92.2%
(196/222) | 100
92.9%, 100%
(50/50) | 90.4
86.3%, 93.7%
(246/272) |
| Note: Samples with a “Target Not Detected” or a detectable viral load below the threshold result were categorized as “< Threshold IU/mL.
* Log10 of 2.137 abbreviated as 2.1 log10 IU/mL
** Log10 of 2.699 abbreviated as 2.2log10 IU/mL
*** Log10 of 3.255 abbreviated as 3.3 log10 IU/mL.
CI: 2-sided 95% confidence interval calculated by exact method assuming independence between all samples. | | | |
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Table 24: Concordance Analysis for Samples From SOT Patients Collected at Protocol Defined Time Points Post Treatment Initiation
| cobas CMV (log10 IU/mL) | TaqMan CMV Test (log10 IU/mL) | | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Target Not Detected | < 2.1 | 2.1 to < 2.7 | 2.7 to < 3.3 | 3.3 to < 3.9 | ≥ 3.9 | |
| Target Not Detected | 24 | 3 | 0 | 0 | 0 | 0 | 27 |
| < 2.1* | 36 | 42 | 1 | 0 | 0 | 0 | 79 |
| 2.1 to < 2.7 | 0 | 27 | 18 | 0 | 0 | 0 | 45 |
| 2.7 to < 3.3 | 0 | 4a | 25 | 16 | 0 | 0 | 45 |
| 3.3 to < 3.9 | 0 | 2b | 1c | 21 | 12 | 0 | 36 |
| ≥ 3.9 | 0 | 0 | 2b | 0 | 26 | 12 | 40 |
| Total | 60 | 78 | 47 | 37 | 38 | 12 | 272 |
| Note: A total of 272 paired samples evaluable for Clinical Concordance analysis from 68 viremic subjects at protocol defined time points (Day 14, Day 21, Day 28, Day 35 or Day 49 post anti-CMV therapy initiation) were included in this table. *The LLOQ is 34.5 IU/mL for the cobas CMV and 137 IU/mL for TaqMan CMV Test. Therefore for the cobas CMV test the category of <137 IU/mL includes non-quantifiable results < the LLOQ and quantifiable results ≥ 34.5 IU/mL but < 137 IU/mL. log10(137) = 2.137 (abbreviated above as 2.1); log10(1,800) = 3.255 (abbreviated above as 3.3); log10(7,943) = 3.900 (abbreviated above as 3.9). a two of the 4 discrepant samples were sequenced and found to contain a significant impact mutation in the forward primer binding site of the Taqman CMV test. b These 2 samples were sequenced and both were found to contain a significant impact mutation in the forward primer binding site of the Taqman CMV test. c The discrepant sample was sequenced and found to contain a significant impact mutation in the forward primer binding site of the Taqman CMV test. | | | | | | | |
# d. Agreement of SOT Patients Viral Load Results in Order to Determine When to Stop Anti-CMV Therapy
The concordance analysis between the cobas CMV test and the TaqMan CMV Test when used to aid in determining whether or not to stop anti-CMV treatment at visit times of Day 14, Day 21, Day 28, Day 35, and Day 49 (post anti-CMV therapy initiation) considered all viremic patients who started anti-CMV medication $(n = 71)$ .
From the total of $n = 71$ SOT recipients who initiated therapy $n = 4$ subjects were excluded because they did not have any viral load measurements at any of the protocol define timepoints so that a viremic episode could not be defined and a resolution thereof could not be determined. From the remaining $n = 67$ subjects who started therapy $n = 7$ only had negative viral load results up to $\geq$ Day 42 and hence resolution of viremia could therefore not be assessed within the protocol defined time points (up to Day 49). Therefore the resolution analysis below contains samples from $n = 60$ subjects.
Viremia was defined as a viral load of $\geq 137\mathrm{IU / mL}$ with both assays, the cobas CMV and the FDA approved comparator test. Resolution of viremia is defined as two consecutive samples with a "Target Not Detected" result or a detectable viral load below $137\mathrm{IU / mL}$ .
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Table 25: Concordance Analysis of cobas CMV and TaqMan CMV Test Results When Used to Determining Resolution of Viremia for SOT Patients
| Day 14 Post Anti-CMV Therapy Initiation | | | |
| --- | --- | --- | --- |
| | TaqMan CMV | | |
| cobas CMV | Resolution of CMV Episode | No Resolution of CMV Episode | Total |
| Resolution of CMV Episode | 0 | 0 | 0 |
| No Resolution of CMV Episode | 0 | 40 | 40 |
| Total | 0 | 40 | 40 |
| Column Agreement (95% Exact CI)a | NC | 100.0% (91.2%, 100.0%) | |
| Overall Percent Agreement (95% CI)a | 100.0% (91.2%, 100.0%) | | |
| Day 21 Post Anti-CMV Therapy Initiation | | | |
| | TaqMan CMV | | |
| cobas CMV | Resolution of CMV Episode | No Resolution of CMV Episode | Total |
| Resolution of CMV Episode | 0 | 0 | 0 |
| No Resolution of CMV Episode | 1 | 50 | 51 |
| Total | 1 | 50 | 51 |
| Column Agreement (95% CI)a | 0.0% (0.0%, 95%) | 100.0% (92.9%, 100.0%) | |
| Overall Percent Agreement (95% CI)a | 98.0% (89.6%, 100%) | | |
| Day 28 Post Anti-CMV Therapy Initiation | | | |
| | TaqMan CMV | | |
| cobas CMV | Resolution of CMV Episode | No Resolution of CMV Episode | Total |
| Resolution of CMV Episode | 6 | 0 | 6 |
| No Resolution of CMV Episode | 4 | 46 | 50 |
| Total | 10 | 46 | 56 |
| Column Agreement (95% CI)a | 60.0% (26.2%, 87.8%) | 100.0% (92.3%, 100.0%) | |
| Overall Percent Agreement (95% CI)a | 92.9% (82.7%, 98.0%) | | |
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| Day 35 Post Anti-CMV Therapy Initiation | | | |
| --- | --- | --- | --- |
| | TaqMan CMV | | |
| cobas CMV | Resolution of CMV Episode | No Resolution of CMV Episode | Total |
| Resolution of CMV Episode | 16 | 1 | 17 |
| No Resolution of CMV Episode | 8 | 31 | 39 |
| Total | 24 | 32 | 56 |
| Column Agreement (95% CI)a | 66.7% (44.7%, 84.4%) | 96.9% (83.8%, 99.9%) | |
| Overall Percent Agreement (95% CI)a | 83.9% (71.7%, 92.4%) | | |
| Day 49 Post Anti-CMV Therapy Initiation | | | |
| --- | --- | --- | --- |
| | TaqMan CMV | | |
| cobas CMV | Resolution of CMV Episode | No Resolution of CMV Episode | Total |
| Resolution of CMV Episode | 38 | 0 | 38 |
| No Resolution of CMV Episode | 7 | 12 | 19 |
| Total | 45 | 12 | 57 |
| Column Agreement (95% CI)a | 84.4% (70.5%, 93.5%) | 100.0% (73.5%, 100.0%) | |
| Overall Percent Agreement (95% CI)a | 87.7% (76.3%, 94.9%) | | |
Only paired samples evaluable for Clinical Concordance analysis from Non-ViremicSubjects at each of the indicated Days Post Anti-CMV Therapy Initiation were included in this table. Sample with a "Target Not Detected" or a detectable viral load below 137 IU/mL result was categorized as "< 2.1 log10 IU/mL (< 137 IU/mL)."
a CI calculated with exact method
NC = Not calculable
## 6. Subgroup Analysis
No Subgroup analysis was performed
## 7. Pediatric Extrapolation
In this premarket application, existing clinical data was not leveraged to support approval of a pediatric patient population.
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## B. Method Comparison in SOT Patients
### 1. Study Design
The objective of this study was to compar…
