(150 days)
Not Found
Not Found
No
The summary describes a standard in vitro diagnostic test using PCR amplification and detection, with no mention of AI or ML in the device description, intended use, or performance studies. The "ASAP software" is likely for assay-specific analysis, not AI/ML.
No
Explanation: This device is an in vitro diagnostic test designed to quantify Epstein-Barr virus (EBV) DNA in patient samples to aid in disease management, not to treat the disease itself.
Yes.
Explanation: The device is an in vitro nucleic acid amplification test for the "quantitation of Epstein-Barr virus (EBV) DNA" and is "intended for use as an aid in the management of EBV in transplant patients." It also states that "serial DNA measurements can be used to indicate the need for potential treatment changes and to assess response to treatment," all of which are diagnostic functions.
No
The device is an in vitro diagnostic test that includes physical reagents (Proteinase Solution, DNA Quantitation Standard, Elution Buffer, Master Mix Reagent 1, EBV Master Mix Reagent 2, Positive Control Kit, Negative Control Kit) and is performed on a hardware system (cobas 6800/8800 Systems). While it utilizes software (cobas EBV Assay Specific Analysis Package (ASAP) software), it is not solely software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The "Intended Use / Indications for Use" section explicitly states that the cobas EBV is an "in vitro nucleic acid amplification test". It is designed to be used on human EDTA plasma samples, which are collected from the body but tested outside of it.
- Device Description: The description details the components of the test kit (reagents, controls) and how it is used to detect and quantify EBV DNA in plasma specimens. This process is performed in vitro.
- Performance Studies: The performance studies described (Precision, Reproducibility, Linearity, etc.) are typical evaluations conducted for IVD devices to demonstrate their analytical and clinical performance.
The definition of an In Vitro Diagnostic (IVD) is a medical device that is used to perform tests on samples such as blood, urine, or tissue, to detect diseases, conditions, or infections. The cobas EBV fits this definition perfectly.
N/A
Intended Use / Indications for Use
cobas EBV is an in vitro nucleic acid amplification test for the quantitation of Epstein-Barr virus (EBV) DNA in human EDTA plasma on the cobas 6800/8800 Systems.
cobas EBV is intended for use as an aid in the management of EBV in transplant patients. In patients undergoing monitoring of EBV, serial DNA measurements can be used to indicate the need for potential treatment changes and to assess response to treatment.
The results from cobas EBV are intended to be read and analyzed by a qualified licensed healthcare professional in conjunction with clinical signs and symptoms and relevant laboratory findings. Negative test results do not preclude EBV infection or EBV disease. Test results must not be the sole basis for patient management decisions.
cobas EBV is not intended for use as a screening test for donors of blood or blood products or human cells, tissues, and cellular and tissue-based products (HCT/Ps).
Special conditions for use statement(s)
For prescription use only.
For in vitro diagnostic use only.
Product codes (comma separated list FDA assigned to the subject device)
QLX
Device Description
cobas EBV is a quantitative test performed on the cobas 6800 System and cobas 8800 System. cobas EBV enables the detection of EBV DNA in plasma specimens. The cobas EBV assay is a dual target assay, with both targets using the same dye. The DNA Internal Control, used to monitor the entire sample preparation and PCR amplification process, is introduced into each specimen during sample processing. cobas EBV enables the detection and quantitation of EBV 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.
The cobas EBV consists of:
- Proteinase Solution
- DNA Quantitation Standard (DNA QS)
- Elution Buffer
- Master Mix Reagent 1
- EBV Master Mix Reagent 2
The EBV viral load is quantified against a non-EBV DNA quantitation standard (DNA-OS), 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 the following separately packed and sold control materials:
-
- cobas EBV Positive Control Kit:
- EBV Low Positive Control (EBV L(+)C)
- EBV High Positive Control (EBV H(+)C)
The positive control contains phage packaged EBV DNA in normal human plasma and serves as a control for the cobas EBV test.
-
- cobas Negative Control Kit:
- cobas Buffer Negative Control (BUF (-) C)
Testing with the cobas EBV test requires the following materials that are not provided:
- cobas OMNI Reagents: Including the following reagents used for specimen
processing, PCR and detection: - cobas EBV Assay Specific Analysis Package (ASAP) software .
The cobas EBV test uses sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection, all steps are fully automated by the cobas 6800/8800 platform.
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 can be used for both the cobas 6800 and the cobas 8800 systems.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Not Found
Indicated Patient Age Range
Not Found
Intended User / Care Setting
qualified licensed healthcare professional
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
Not Found
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
- Precision: Assessed for predominant genotype (EBV Genotype 1) with seven panel members ranging from the Lower Limit of Quantitation (LLoQ) up to high concentrations. Panel members prepared by spiking high titer EBV lambda phagemid into EBV negative EDTA plasma. Study involved 72 replicates per panel member over 12 days using 3 kit lots, 3 cobas 6800 systems, and 3 operators. For data analysis, only samples above LLoQ were used. Precision was determined according to CLSI guideline EP05-A3 as a multivariance analysis. Total Precision as SD of log10 titer was comparable across all kits.
- Reproducibility: Evaluated in EDTA plasma across 3 Reagent Lots, 3 Test Sites, and 3 Instruments (2 cobas 6800, 1 cobas 8800). Two runs performed per operator per day over 5 days per lot, with 3 replicates per panel member. Total of 270 test results per concentration. Test panel members prepared from EBV-VCA IgG sero-negative and RNA negative EDTA plasma spiked with EBV genotype 1 genomic material or EBV phagemid for high-positive. Panel members included Negative, 10^5 IU/mL, 5x10^3 IU/mL, 5x10^4 IU/mL, 5x10^5 IU/mL, and 5x10^7 IU/mL. Range of total lognormal coefficient of variation among positive panel members was from 13.7% to 46.16%.
- Linearity of the cobas EBV with Genotype 1: Evaluated using a 17-member test panel in EDTA-plasma, spanning 1.5E+01 IU/mL to 2.00E+08 IU/mL. Panel members included EBV phagemid DNA and a clinical specimen. Each panel member tested in 36 replicates across 3 lots and 3 instruments by 3 operators. Claimed linear measurement range is 35 IU/mL (LLoQ) to 1.0E+08 IU/mL (ULoQ).
- Verification of Linearity of cobas EBV with Genotype 2: Assessed with a cell culture derived EBV Genotype 2 panel (EBV strain Jiyoye) spanning 30 IU/mL to 1 x 10^8 IU/mL. 8 panel members prepared in EBV negative EDTA-plasma. 12 replicates across 3 lots tested per concentration. Accuracy within ± 0.12 log10 IU/mL.
- Traceability: Used WHO EBV Standard (NIBSC 09/260), Roche Molecular Systems (RMS) EBV Secondary Standard, and RMS EBV Calibration Panel. Observed quantitation values similar to expected, with deviation not more than 0.15 log10 IU/mL.
- Stability:
- Clinical Specimen Stability: Evaluated in whole blood (EDTA-plasma preparation tubes) and plasma after various storage conditions and freeze-thaw cycles. Supports storage of whole blood in EDTA-plasma tubes for up to 24 hours at 2℃ to 25℃; resulting plasma stable for 24 hours at 2°C to 30°C, up to 6 days at 2℃ to 8℃, or up to 6 months at -15°C to -80°C. Plasma samples stable for up to 4 freeze/thaw cycles.
- Open kit and On-board Stability: Opened cobas EBV 192T test-specific reagent cassettes stable for up to 90 days at 2-8℃ (Open Kit) and up to 40 hours at 37°C (On-Board). Re-usable for up to 40 runs once opened.
- Reagent Stability: Supports a shelf-life stability claim of 12 months at 2-8°C.
- Detection limit (LoD):
- LoD using 1st WHO International Standard for EBV: Determined by analysis of serial dilutions in pooled EDTA-plasma. Tested with 3 lots, 3 instruments, multiple runs, and operators over 3 days. LoD by 95% hit rate was determined as the same for all tested lots. Claimed LoD value is 18.8 IU/mL.
- LoD Confirmation with EBV Genotype 2: Verified using EBV cell culture supernatants for genotype 2 strain Jiyoye (GT2-J) diluted in EBV negative EDTA plasma. Hit rate of 95% or higher observed above 18.8 IU/mL.
- LoD in Plasma vs Buffer: Evaluated comparability of LoD in Generic Specimen Diluent (GSD) and EDTA-plasma. Demonstrated comparable LoD performance.
- Lower Limit of Quantitation (LLoQ): Calculated using data from LoD study with EBV WHO International Standard. LLoQ determined to be 35 IU/mL.
- Analytical specificity:
- Cross reactivity: Tested 35 microorganisms (17 viral, 15 bacterial, 3 fungal) in 7 pools. Mean log10 titer of positive EBV samples with cross-reacting organisms was within ± 0.5 log10 of control.
- Endogenous Interference: Tested 20 individual clinical EBV-seronegative samples spiked with selected endogenous substances and EBV target. EBV-negative samples produced valid negative results. For EBV-positive samples, mean log10 titer was within ± 0.05 log10 of spike control.
- Exogenous Interference: Tested 10 individual clinical EBV-negative samples spiked with pools of 24 commercially available drugs. All produced valid results, with mean log10 titer within expected range.
- Cross Contamination: Tested 240 replicates of EBV-negative matrix and 225 replicates of high titer EBV sample (2.00E+07 IU/mL). Cross-contamination rate was 0%.
- Concordance of cobas EBV with a Comparator EBV Test: Compared to a validated, well-established comparator nucleic acid test using 464 samples (439 neat or diluted clinical samples from 72 transplant subjects and 25 contrived samples). Column percent agreement ranged from 82.5% to 100%. Negative Percent Agreement (NPA) was 95.4% (95% Exact CI: 84.2%-99.4%). Deming linear regression plot showed Y = -0.45 + 1.025X, R^2 = 0.91.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
- Precision: Standard Deviation of log10 titer (e.g., 0.04 at 5.00E+07 IU/mL nominal concentration), Lognormal Percent Coefficient of Variation (%CV) (e.g., 10% for PM01, 1.08E+06 IU/mL).
- Reproducibility: Total Precision SD and Log-Normal CV (e.g., 0.158 SD, 37.56% CV for 2.02 log10 IU/mL).
- Linearity: Accuracy within ± 0.15 log10 IU/mL (Genotype 1) and ± 0.12 log10 IU/mL (Genotype 2).
- Traceability: Maximum deviation of 0.15 log10 IU/mL. R^2 values for linear regression: 1.000 (Calibration Panel), 0.983 (WHO Standard).
- Expected values (controls): 0% invalid rate for QS and RMC, 0.02% sample failure rate.
- Limit of Detection (LoD): Highest LoD by Probit analysis of 18.8 IU/mL. 95% hit rate achieved above 18.8 IU/mL for EBV Genotype 2.
- Lower Limit of Quantitation (LLoQ): 35 IU/mL.
- Analytical specificity (Cross reactivity, Endogenous Interference, Exogenous Interference): Mean log10 titer within ± 0.5 log10 of control (cross-reactivity) or ± 0.05 log10 of spike control (endogenous interferents). Negativity Rate 100% for negative samples (exogenous interferents).
- Cross Contamination: 0% cross-contamination rate (upper one-sided 95% confidence interval: 1.24%).
- Concordance with Comparator EBV:
- Column Percent Agreement: Ranging from 82.5% to 100%. (e.g., 98.5% for "Target Not Detected" by Comparator EBV, 100% for "
§ 866.3183 Quantitative viral nucleic acid test for transplant patient management.
(a)
Identification. A quantitative viral nucleic acid test for transplant patient management is identified as a device intended for prescription use in the detection of viral pathogens by measurement of viral DNA or RNA 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 viral infection or at risk for developing viral infections. The test results are intended to be interpreted by qualified healthcare professionals in conjunction with other relevant clinical and laboratory findings.(b)
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 viral nucleic acid in blood or blood products.
(ii) Limitations which must be updated to reflect current clinical practice. These 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; and
(B) Negative test results do not preclude viral infection or tissue invasive viral disease and that 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 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 [analyte] measurements across different [analyte] assays, it is recommended that the same device be used for the quantitation of [analyte] when managing 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, ancillary reagents required for use with the 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 functions;
(iii) Documentation and characterization (
e.g., determination of the identity, supplier, purity, and stability) of all critical reagents 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 Mode 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 viral 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, carry-over, quality control, specimen stability studies, and additional studies as applicable to specimen type and intended use for the device;
(B) Identification of the viral 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 viral 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 reference standard that FDA has determined is appropriate for the quantification of viral DNA or RNA (
e.g., a recognized consensus standard); and(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 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 virus-positive samples containing an analyte concentration near the lower limit of quantitation and any clinically relevant decision points. If an acceptable number of virus-positive samples containing an analyte concentration near the lower limit of quantitation and any clinically relevant decision cannot be obtained, contrived samples may be used to supplement sample numbers when appropriate, as determined by FDA;
(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; and
(D) The final release test results for each lot used in the clinical study.
0
EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR cobas EBV DECISION SUMMARY
A. De Novo Number
B. Purpose for Submission
De Novo request for evaluation of automatic class III designation for the cobas EBV test.
C. Measurand
EBV DNA
D. Type of Test
Quantitative Polymerase Chain Reaction (PCR)
E. Applicant
Roche Molecular Systems, Inc.
F. Proprietary and Established Names
cobas EBV
G. Regulatory Information
-
- Regulation section
- 21 CFR 866.3183
-
- Classification
Class II
-
- Product code(s):
QLX
- Product code(s):
-
- Panel
1
Microbiology (83)
H. Indications For Use
-
- Indications for use:
cobas EBV is an in vitro nucleic acid amplification test for the quantitation of Epstein-Barr virus (EBV) DNA in human EDTA plasma on the cobas 6800/8800 Systems.
- Indications for use:
cobas EBV is intended for use as an aid in the management of EBV in transplant patients. In patients undergoing monitoring of EBV, serial DNA measurements can be used to indicate the need for potential treatment changes and to assess response to treatment.
The results from cobas EBV are intended to be read and analyzed by a qualified licensed healthcare professional in conjunction with clinical signs and symptoms and relevant laboratory findings. Negative test results do not preclude EBV infection or EBV disease. Test results must not be the sole basis for patient management decisions.
cobas EBV is not intended for use as a screening test for donors of blood or blood products or human cells, tissues, and cellular and tissue-based products (HCT/Ps).
Special conditions for use statement(s)
For prescription use only.
For in vitro diagnostic use only.
-
- Special instrument requirements
The test is run on the cobas 6800/8800 instrument systems.
- Special instrument requirements
I. Device Description
cobas EBV is a quantitative test performed on the cobas 6800 System and cobas 8800 System. cobas EBV enables the detection of EBV DNA in plasma specimens. The cobas EBV assay is a dual target assay, with both targets using the same dye. The DNA Internal Control, used to monitor the entire sample preparation and PCR amplification process, is introduced into each specimen during sample processing. cobas EBV enables the detection and quantitation of EBV 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.
The cobas EBV consists of:
- Proteinase Solution ●
- DNA Quantitation Standard (DNA QS) ●
- Elution Buffer ●
2
- Master Mix Reagent 1
- . EBV Master Mix Reagent 2
The EBV viral load is quantified against a non-EBV DNA quantitation standard (DNA-OS), 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 the following separately packed and sold control materials:
-
- cobas EBV Positive Control Kit:
- . EBV Low Positive Control (EBV L(+)C)
- EBV High Positive Control (EBV H(+)C) ●
The positive control contains phage packaged EBV DNA in normal human plasma and serves as a control for the cobas EBV test.
-
- cobas Negative Control Kit:
- cobas Buffer Negative Control (BUF (-) C) ●
Testing with the cobas EBV test requires the following materials that are not provided:
- cobas OMNI Reagents: Including the following reagents used for specimen ● processing, PCR and detection:
- cobas EBV Assay Specific Analysis Package (ASAP) software .
The cobas EBV test uses sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection, all steps are fully automated by the cobas 6800/8800 platform.
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 can be used for both the cobas 6800 and the cobas 8800 systems.
J. Standard/Guidance Document Referenced
EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline-Third Edition
3
EP6-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline.
EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline-Second Edition
EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline -Second Edition
K. Test Principle
The cobas EBV test is a quantitative PCR test performed on the fully automated cobas 6800/ 8800 Systems that detects and quantifies EBV DNA from (EDTA) plasma specimens of transplant patients as follows:
Target Selection
Selective amplification of EBV target nucleic acid from the sample is achieved by the use of specific forward and reverse primers which are selected to amplify highly-conserved regions of the EBV DNA EBNA-1 and BMRF-2 gene. Specific probes for each amplicon are used to detect and quantify the EBV targets. Selective amplification of DNA-OS is achieved by the use of DNA-QS specific forward and reverse primers, selected to have no homology with the EBV genome, detected through a DNA-QS specific probe.
Sample Preparation (Nucleic Acid Extraction and Purification)
Nucleic acid from patient samples and external controls are extracted upon addition of a DNA Quantitation standard (DNA-QS). The DNA-QS molecules are extracted simultaneously with the samples/controls serving as an extraction control. Viral nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid, along with the added DNA-QS binds to magnetic glass particles. Unbound substances and impurities are removed with subsequent wash reagent steps and purified nucleic acid is then eluted from the magnetic glass particles with elution buffer.
Nucleic Acid Amplification and Target Detection
The cobas EBV master mix contains detection probes which are specific for the two EBV target sequences and the DNA-QS nucleic acid, respectively. The two EBV specific detection probes are labeled with the same fluorescent dye while the DNA-OS detection probe is labeled with a second fluorescent dye both acting as reporter dyes. Each probe also has a second dye which acts as a quencher that suppresses the fluorescent signals of the intact probes when they are not bound to their respective target sequence. Target bound probes however, emit fluorescence of the two reporter dyes. This fluorescence is measured at defined wavelengths, thus permitting simultaneous detection and discrimination of the EBV targets and the DNA-QS amplification products generated by a thermostable DNA polymerase enzyme.
EBV DNA Quantitation
During the extension phase of the PCR process fluorescence readings are processed to generate Ct values for the EBV DNA target and the EBV QS DNA. The lot-specific
4
calibration constants provided with the cobas EBV test are used to calculate the titer value for the specimens and controls based on both the EBV DNA target and the EBV QS DNA Ct values. EBV viral load results are reported in International Units/mL (IU/mL).
L. Performance Characteristics
1. Analytical performance
a. Precision
The Precision was assessed for the predominant genotype (EBV Genotype 1) with seven panel members ranging from (b) (4) Panel members were prepared by spiking a high titer EBV lambda phagemid into EBV negative EDTA plasma.
Precision was calculated on results generated over twelve days using three kit lots and three cobas 6800 systems by three operators. Per test day, two (2) runs were performed containing three (3) within-run replicates per panel member. The study design accounts for a total of 72 replicates per panel member.
For data analysis only samples with titers above the Lower Limit of Ouantification (LLoO). (i.e., (b) (4)), were used. Precision was determined according to the CLSI guideline EP05-A3 as a multivariance analysis accounting for reagent lots, operators/instruments, days, runs and within-run replicates.
| Nominal
Concentration | Assigned
concentration
(IU/mL) | Standard Deviation | | | |
|--------------------------|--------------------------------------|--------------------|-------|-------|----------|
| | | Lot 1 | Lot 2 | Lot 3 | All Lots |
| 5.00E+07 | 5.40E+07 | 0.03 | 0.04 | 0.04 | 0.04 |
| 1.00E+06 | 1.08E+06 | 0.02 | 0.03 | 0.02 | 0.02 |
| 1.00E+05 | 1.08E+05 | 0.02 | 0.02 | 0.03 | 0.02 |
| 1.00E+04 | 1.08E+04 | 0.04 | 0.02 | 0.03 | 0.03 |
| 1.00E+03 | 1.08E+03 | 0.05 | 0.05 | 0.05 | 0.05 |
| 1.00E+02 | 1.08E+02 | 0.17 | 0.18 | 0.15 | 0.17 |
| 6.00E+01 | 6.48E+01 | 0.17 | 0.17 | 0.13 | 0.16 |
Table 1: Precision - Standard Deviation
- Titer data are considered to be log-normally distributed and are analyzed following logio transformation. Standard deviations (SD) columns present the total of the log-transformed titer for each of the three reagent lots.
The results for cobas EBV Precision are shown in Table 1. The Variance Component Analysis demonstrated the contribution of components of variance to the total precision
5
variance (Table 2). Overall, the total Precision as SD of log10 titer is comparable across all kits.
| Panel | Assigned
Concentration
(IU/mL) | | N | Instrument/
Operator | Kit
Lot | Day | Run | Within
Run | Total |
|--------|--------------------------------------|---------------------------|----|-------------------------|------------|-----|-----|---------------|-------|
| Member | Titer
(IU/mL) | Log10
Titer
(IU/mL) | | %CV | %CV | %CV | %CV | %CV | %CV |
| PM01 | 1.08E+06 | 7.73 | 72 | 5 | 1 | 2 | 2 | 8 | 10 |
| PM02 | 1.08E+05 | 6.03 | 72 | 1 | 4 | 1 | 3 | 4 | 7 |
| PM03 | 5.94E+03 | 5.03 | 72 | 2 | 2 | 3 | 2 | 5 | 7 |
| PM04 | 1.08E+04 | 4.03 | 72 | 2 | 1 | 3 | 3 | 7 | 8 |
| PM05 | 1.08E+03 | 3.03 | 72 | 4 | 1 | 4 | 4 | 10 | 12 |
| PM06 | 1.08E+02 | 2.03 | 72 | 3 | 5 | 8 | 14 | 42 | 43 |
| PM07 | 6.48E+01 | 1.81 | 68 | 7 | 3 | 6 | 12 | 39 | 40 |
Table 2: Lognormal Percent Coefficient of Variation (% CV) *
- Titer data are considered to be log-normally distributed and the %CV values are analyzed as Lognormal CV (%) = sqrt (10^ [SD^2 * ln (10)] - 1) * 100%
b. Reproducibility
Reproducibility of cobas EBV was evaluated in EDTA plasma across three Reagent Lots, three Test Sites, three Instruments (two cobas 6800 Systems and one cobas 8800 System). Two runs were performed per operator per day (1 run = 1 batch; 1 batch = 1 panel + 3 controls) over five days per reagent lot and each run had three replicates per panel member. The total number of tests (not including controls) was as follows: 3 lots × 3 sites × 5 days/lot × 2 runs × 3 replicates/concentration = 270 test results/concentration.
Test panel members were prepared from EBV-VCA IgG sero-negative and RNA negative EDTA plasma spiked with EBV genotype 1 genomic material in the form of EBV cell culture supernatant. EBV phagemid was used for preparing the high-positive panel member (5x107 IU/mL) due to the lack of adequate volumes of high concentration sample. Test panel members had the following concentrations: Negative, 105 IU/mL, 5x103 IU/mL, 5x104 IU/mL, 5x105 IU/mL, and 5x107 IU/mL. Two invalid results were excluded. The results are summarized in Table 3 below:
6
Table 3: Reproducibility Study
| EBV DNA
Concentration
(log10 IU/mL) | | Number
of
Testsb | Percent of Total Variance
[Log-Normal CV (%)] | | | | | Total
Precision | |
|-------------------------------------------|-------------------|------------------------|--------------------------------------------------|----------------|------------------|----------------|------------------|--------------------|------------------------------|
| Expected | Observed
Meana | | Lot | Site | Day/
Operator | Batch | Within
-Batch | SDc | Log-
Normal
CV
(%)d |
| 2.02 | 2.09 | 270 | 11%
(11.97) | 2%
(5.30) | 0%
(0.00) | 3%
(6.34) | 84%
(34.25) | 0.158 | 37.56 |
| 3.70 | 3.68 | 270 | 43%
(10.07) | 15%
(5.92) | 0%
(0.00) | 16%
(6.23) | 26%
(7.81) | 0.067 | 15.43 |
| 4.70 | 4.68 | 270 | 39%
(8.54) | 10%
(4.24) | 0%
(0.00) | 24%
(6.63) | 28%
(7.18) | 0.059 | 13.70 |
| 5.70 | 5.50 | 268 | 7%
(11.39) | 58%
(34.36) | 0%
(0.00) | 21%
(20.18) | 15%
(17.08) | 0.191 | 46.16 |
| 7.70 | 7.76 | 270 | 27%
(8.63) | 15%
(6.52) | 0%
(0.88) | 13%
(6.01) | 45%
(11.26) | 0.073 | 16.83 |
a Calculated using SAS MIXED procedure.
b Number of valid tests with detectable DNA level.
C Calculated using the total variability from the SAS MIXED procedure.
d Lognormal CV (%) = sqrt (10^ [SD^2 * In (10)] - 1) * 100
Note: The table only includes results with detectable DNA level. CV (%) = percent coefficient of variation; SD = standard deviation.
Analysis of variance and a mixed model that included lot, site, day/operator, batch and within-batch (random error) as random effects was performed. The variance contribution of each component to the total variance was estimated. The range of the total lognormal coefficient of variation, among positive panel members, was from 13.7% to 46.16%. The largest total lognormal coefficient of variation was observed in the expected 5x10 -panel member and most of that variability (58% of the total variance) was attributed to site. The largest total lognormal coefficient of variation observed in the lowest panel member (37.56%) was explained by the within-batch component.
7
c. Linearity
Linearity of the cobas EBV with Genotype 1
Linearity of the cobas EBV test was evaluated for the predominant EBV genotype (GT 1) in EDTA-plasma using a 17-member test panel. Eleven panel members were generated using an EBV phagemid DNA and covered the entire linear range. Six panel members were generated using an EBV GT1-positive clinical specimen. Due to the lack of sufficient volume of high positive EBV-positive transplant patient samples the clinical sample panel was designed to cover the range from 13.6 IU/mL to 4 log IU/mL (i.e., only the low and intermediate part of the measuring range) to overlap with the phagemid-based higher concentration panel members.
Panel member concentrations spanned the range of 1.5E+01 IU/mL to 2.00E+08 IU/mL (nominal concentration). Each panel member was tested in 36 replicates across three lots of cobas EBV test reagents (12 replicates/lot) and the results of the study are presented in Table 4 and Figure 1 below. The Linearity panel was tested across three instruments by three operators. Resulting data were analyzed to identify the linear range according to CLSI guideline EP6-A and the best fitting polynomial regression fit.
| Lot | Equation 1st Order
$Y=b_0+b_1x$ | Equation 2nd Order
$Y=b_0+b_1x+b_2x^2$ | Maximum
Difference
(log10 IU/mL) |
|---------------------------------------|------------------------------------|-------------------------------------------|----------------------------------------|
| Clinical Sample | | | |
| 1 | (b) (4) | | |
| 2 | (b) (4) | | N/A |
| 3 | (b) (4) | | N/A |
| Phagemid | | | |
| 1* | (b) (4) | | |
| 2 | (b) (4) | | N/A |
| 3 | (b) (4) | | N/A |
| Clinical Sample and Phagemid Combined | | | |
| 1 | (b) (4) | | N/A |
| 2 | (b) (4) | | N/A |
| 3 | (b) (4) | | N/A |
| ALL | (b) (4) | | |
Table 4: cobas EBV Linearity with EBV Genotype 1
bo-Intercept; bj=slope; all coefficients are provided in log10 IU/mL; best fitting model is bolded N/A=not applicable; if linearity is described best by the 1st order model, there is no deviation to be shown between the 1st order model and any higher order model
- For this lot the 3d order was the best fitting model, however, the difference to the 1st order regression model was so minor that it not shown here.
8
Image /page/8/Figure/0 description: The image shows the title of a figure. The title is "Figure 1: Linearity Across Both Panel Types (all lots)". The title is written in a bold, sans-serif font. The figure number is 1.
Image /page/8/Figure/1 description: The image is a scatter plot that shows the relationship between AssignedLogTiter and ObservedLogTiter. The x-axis represents AssignedLogTiter, ranging from 1 to 8, while the y-axis represents ObservedLogTiter, ranging from 0 to 9. The plot shows a cluster of data points between 1 and 2 on the x-axis, with a linear trend between the two variables as x increases. A dashed line is plotted through the data, showing a positive correlation between the two variables.
Except for Lot 1 (clinical sample), the 1st order model is the best fitting model for all lots and all panel members (clinical sample and phagemid) when analyzed separately. However, for all panel member and lots combined the 2nd order model (62) is significant at a 5% level. Therefore, the 2nd order polynomial was chosen as the best-fitting regression model for the analysis. However, the absolute difference between the 1st order and the better fitting 2nd order regression is minimal (i.e., equal or less than ± 0.01 log10 IU/mL). Across the linear range, the accuracy of the test was within ± 0.15 log10 IU/mL (Mean Square Error).
Based on the LLoQ (35 IU/mL) and the determined linear range, the claimed linear measurement range of the cobas EBV test is from 35 IU/mL (LLoQ) to 1.0E+08 IU/mL (ULoQ).
Verification of Linearity of cobas EBV with Genotype 2
Linearity was assessed with a cell culture derived EBV Genotype 2 panel (EBV strain Jiyoye) spanning the expected linear range from the 1 x 10° IU/mL (at or near expected ULoQ) to 30 IU/mL (at or near expected LLoQ). The following 8 panel members were prepared in EBV negative EDTA-plasma: 30 IU/mL, 150 IU/mL, 3, 4, 5, 6, 7, and 8 log IU/mL. Twelve replicates across three test-specific reagent lots were tested per concentration level (i.e., 4 replicates per kit lot). Data were analyzed according to CLSI guideline EP6-A and the 3td order polynomial regression was the best fitting modes using a 5% significance level. The following equations were obtained.
| 1st order: | y = 0.2124 + 0.9713x | |
|---|---|---|
| 2nd order: | y = 0.2800 + 0.9346x + 0.0039x2 | |
| 3rd order: | y = 0.7323 + 0.5420x + 0.0980x2 + -0.0066x3 |
9
The differences in logio values calculated from the 1st order and the 3th order regression were minor, ranging from -0.06 log IU/mL to +0.08 log IU/mL. Linearity for EBV genotype 2 is shown in
Figure 2. Across the linear range, the accuracy of the test was within ± 0.12 log10 IU/mL (Mean Square Error).
Image /page/9/Figure/2 description: The image shows the label (b) (4) in the upper left corner. The rest of the image is a gray rectangle. The gray rectangle takes up most of the image.
Figure 2: Verification of Linearity for EBV Genotype 2
Figure 1 Regression plot of EBV Genotype 2 - including outliers
d. Traceability, Stability, Expected values (controls, calibrators, or methods)
i) Traceability
Several standards and controls were used during development of the cobas EBV test to provide traceability to the WHO EBV Standard [1st WHO International Standard for Epstein-Barr Virus for Nucleic Acid Amplification Techniques (NIBSC 09/260)]. The standards used during development of the test include the WHO EBV Standard, the Roche Molecular Systems (RMS) EBV Secondary Standard, and the RMS EBV Calibration Panel.
Traceability of the calibration panel and the RMS EBV Secondary Standard to the 1st EBV WHO Standard was verified as shown in Figure 3.
| The concentration range tested for the EBV WHO Standard was from (b) (4) | 10 | |
|---|---|---|
| (b) (4) | . the RMS EBV Secondary Standard was tested at | |
| (b) (4) | and the RMS EBV Calibration Panel was tested from | |
| (b) (4) |
Figure 3: Traceability to the 1st WHO International Standard for EBV (NIBSC 09/260) using cobas EBV
10
Image /page/10/Figure/0 description: The image is a scatter plot comparing observed EBV DNA concentration to expected EBV DNA concentration. The x-axis represents the expected EBV DNA concentration (Log 10 IU/mL) ranging from 1 to 9, while the y-axis represents the observed EBV DNA concentration (Log 10 IU/mL) ranging from 1 to 9. The plot includes data points for EBV Calibration Panel, EBV Secondary Standard, and EBV WHO Standard, with trend lines showing a positive correlation between expected and observed concentrations.
Figure 3 shows the bivariate fit of observed EBV DNA concentration (log10 IU/mL) by expected EBV DNA concentration (log10 IU/mL). Observed quantitation values are similar to the expected values with deviation of not more than 0.15 log10 IU/mL. All materials demonstrated co-linear dilution performance across the linear range of cobas EBV (Figure 2) The maximum deviation was observed at 200 IU/mL (approximately 6x LLoQ). The following linear regression equations were obtained:
EBV Calibration Panel: y = 1.000x - 0.002; R2 = 1.000 EBV 1st WHO Standard: y = 0.975x + 0.159; R2 = 0.983
Based on these results, the calibration and standardization process of cobas EBV provides quantitation values for the cobas EBV calibration panel and the RMS EBV Secondary Standard provide traceability to the 1st WHO international Standard for EBV.
ii) Expected values
To monitor the assay performance, reagent performance, and procedural errors, positive and negative external controls must be run in accordance with the guidelines or requirements of local, state, and/or federal regulations or accrediting organizations.
External controls are provided separately from the cobas EBV test kit in the cobas EBV Positive Control Kit and the cobas Negative Control. The cobas EBV Positive Control includes an EBV High Positive control, and a Low Positive control containing EBV phagemid-DNA diluted into negative human plasma. The cobas Negative Control includes Tris buffer. The cobas Negative Control, the EBV Low Positive Control, and the EBV 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 amount of EBV DNA for EBV high and low positive controls must fall within their acceptable titer ranges.
11
The failure rates of the kit controls and samples were assessed by evaluating outcomes of the nine analytical studies (LoD, LoD verification for Genotype, Linearity, Linearity verification for Genotype, Precision, Cross contamination, Lot interchangeability, Accuracy, and LoD in Plasma vs Buffer). Results demonstrated an overall invalid rate of 0% for QS and RMC and a sample failure rate of 0.02%.
iii) Stability
iii.a. Clinical Specimen Stability
The clinical specimen stability of EBV viral target in whole blood specimens collected in EDTA-plasma preparation tubes and/or plasma samples after various storage conditions with or without freeze-thawing cycles was evaluated using cobas EBV for use on the cobas 6800/8800 Systems.
Freshly drawn whole blood from ten unique individual EBV-negative donors collected in EDTA-plasma tubes (five donor samples collected in PPT and five in lavender top tubes) were spiked with EBV to a concentration of 150 IU/mL (approximately 5xLLoQ). The reference time point (T0) was processed directly after spiking of the target by separating the blood into EDTA-plasma; all other samples were stored at indicated conditions until further processing. All samples used for this study in each PPT/ lavender top tube type were tested unspiked at time point 0 (T0).
All EBV-positive samples tested generated positive results for EBV and the mean log10 titers including the two-sided 95 confidence interval of each of the tested time points/conditions and tube types was within ±0.5 logio of the mean logio titer of the respective reference condition (T0 = reference).
The results support the following storage conditions for whole blood collected in BD Vacutainer PPT or BD Vacutainer EDTA tubes (lavender top) and the respective separated plasma:
- Whole blood collected in EDTA-plasma tubes (lavender top and PPT) may be ● stored or transported for up to 24 hours at 2℃ to 25℃ before further processing and plasma separation.
- . Afterwards, whole blood samples should be centrifuged, and the resulting plasma samples are additionally stable for:
- o 24 hours at 2°C to 30°C in primary or secondary tubes, followed by
- o up to 6 days at 2℃ to 8℃ in primary or secondary tubes, or
- o up to 6 months at -15°C to -80°C in secondary tubes.
- Plasma samples are stable in secondary tubes for up to four freeze/thaw cycles . when frozen between -15°C to -80°C.
12
iii.b. Open kit and On-board Stability
The data submitted support the claim that, once opened, the cobas EBV 192T test-specific reagent cassettes are stable for up to 90 days at 2-8℃ (Open Kit Stability) and remain stable for up to 40 hours at 37°C (On-Board Stability). Furthermore. 192T test-specific cassettes once opened are re-usable for up to 40 runs.
iii.c. Reagent Stability
Three lots of the cobas EBV were tested to demonstrate stability of the test-specific reagents of cobas EBV and cobas EBV Control Kit when stored at stressed temperature conditions (accelerated stability) and at the targeted storage temperature of 2°C to 8°C (real-time stability).
Real-time stability: The data submitted supports a shelf-life stability claim of 12 months. when stored at 2-8°C.
e. Detection limit
i) Limit of Detection (LoD) using the 1st WHO International Standard for EBV
The LoD of the cobas EBV test for the 1st WHO EBV Standard (Genotype 1) was determined by analysis of serial dilutions of the Standard diluted into a pooled EDTA-plasma derived from EBV IgG/IgM negative individuals following the recommendations in CLSI Guideline EP17-A2. Panels of six concentration levels plus a blank were tested with three lots of cobas EBV test reagents and three instruments with multiple runs and operators over a period of three days. Each dilution was determined in " replicates per lot and day (n=10)(4) total replicates per day). The results from testing the WHO EBV Standard in EDTA plasma as well as the calculated LoD values are shown in Table 5. The LoD values in Table 5 were determined by Probit analysis and by 95% hit rate.
| Kit Lot | Nominal
Concentration
(IU/mL) | Number of
Positive
Replicates | Number of
Valid
Replicates | Hit Rate
[%] | LoD by
Probit
[95% CI] |
|---------|-------------------------------------|-------------------------------------|----------------------------------|-----------------|------------------------------|
| Lot 1 | (b) (4) | | | | |
Table 5: LoD with EBV DNA 1st WHO International Standard in EDTA Plasma
13
| Kit Lot | Nominal
Concentration
(IU/mL) | Number of
Positive
Replicates | Number of
Valid
Replicates | Hit Rate
[%] | LoD by
Probit
[95% CI] |
|----------------------|-------------------------------------|-------------------------------------|----------------------------------|-----------------|------------------------------|
| Lot 2 | (b) (4) | | | | |
| Lot 3 | | | | | |
| All lots
combined | | | | | |
When determined by Probit analysis, the different lots have similar LoD for all tested lots; the highest LoD of 18.8 IU/mL was obtained with lot 1, which is only slightly lower than the LoD determined by 95% hit rate. The LoD by 95% hit rate was (b) (4) and was the same for all tested lots. The claimed LoD value is (b) (4) determined by the least sensitive kit lot and this concentration was used in studies for confirmation of the LoD.
ii) Limit of Detection (LoD) Confirmation with EBV Genotype 2
The Limit of Detection (18.8 IU/mL) was verified for the cobas EBV test with EBV genotype 2 following the CLSI Guideline EP17-A2. EBV cell culture supernatants for genotype 2 strain Jiyoye (GT2-J) were diluted to three different concentration levels in EBV negative EDTA plasma. The hit rate determination was performed with 63 replicates for each level. Testing was conducted with three lots of cobas EBV reagents across three days of testing.
The results are shown in Table 6 and verify that a hit rate of 95% or higher was observed above 18.8 IU/mL for EBV genotype 2. Thus, the observed hit rates verify the LoD for EBV Genotype 2 at 18.8 IU/mL.
14
| Lot | Nominal
Concentration
(IU/mL) | Number of
Positive
Replicates | Number of
Valid
Replicates | Hit Rate
[%] | LOD by Hit Rate |
|----------|-------------------------------------|-------------------------------------|----------------------------------|-----------------|-----------------|
| Lot 1 | (b) (4) | | | | |
| Lot 2 | | | | | |
| Lot 3 | | | | | |
| All Lots | | | | | |
Table 6: Verification of the LOD for EBV Genotype 2
iii) Limit of Detection (LoD) in Plasma vs Buffer
This study evaluated whether the Limit of Detection in Generic Specimen Diluent (GSD) is equivalent to the LoD in EDTA-plasma in the cobas EBV assay, so that GSD can be used as a negative control for cobas EBV. Three independent dilution series (0.5x, 1.0x, and 1.5x LoD of cobas EBV) were prepared on three consecutive days using EBV WHO International Standard in GSD and tested.
The results (Table 7) demonstrated that the hit rate was 98.4% and 92.1% at 1.5xLoD and 1.0xLoD, respectively. The study demonstrated a comparable LoD performance of cobas EBV in Plasma and GSD.
Table 7: Hit Rates for all Dilution Series combined
| Conc. Level | Conc. IU/mL | Hit Rate % | Two-sided 95% CI |
|---|---|---|---|
| (b) (4) |
Lower Limit of Quantitation (LLoQ) f.
LLoQ using the 1st WHO International Standard for EBV
The LLoQ for cobas EBV was calculated using data generated from the Limit of Detection study using EBV WHO International Standard. The LLoQ was determined, per CLSI document EP17-A2, as the lowest titer within the linear range with a hit rate of at least 95% and at which the total analytical error (TAE) meets both of the following two criteria:
- The TAE, when calculated as (b) (4) and
15
- The TAE has to be such that the standard deviation for the difference between two measurements calculated as (b) (4)
The LLoQ was determined for each kit lot; all calculations are based on non-rounded values but the results shown in here are rounded.
| Lot | Nominal
Concentrati
on (IU/mL) | Log10
titer
Nominal | Mean log10
titer
Observed | SD
(log10) | Absolute
Bias | TAE
([Bias] +
2SD) | Difference
Between
Measurements
(SD) |
|-------------|--------------------------------------|---------------------------|---------------------------------|---------------|------------------|--------------------------|-----------------------------------------------|
| 1 | 20 | 1.30 | 1.34 | 0.38 | 0.04 | 0.80 | 1.07 |
| 1 | 35 | 1.54 | 1.60 | 0.25 | 0.06 | 0.56 | 0.71 |
| 1 | 50 | 1.70 | 1.74 | 0.22 | 0.04 | 0.49 | 0.63 |
| 2 | 20 | 1.30 | 1.29 | 0.37 | 0.01 | 0.75 | 1.05 |
| 2 | 35 | 1.54 | 1.58 | 0.27 | 0.04 | 0.58 | 0.76 |
| 2 | 50 | 1.70 | 1.77 | 0.23 | 0.07 | 0.53 | 0.65 |
| 3 | 20 | 1.30 | 1.33 | 0.31 | 0.03 | 0.65 | 0.88 |
| 3 | 35 | 1.54 | 1.58 | 0.32 | 0.04 | 0.67 | 0.89 |
| 3 | 50 | 1.70 | 1.76 | 0.21 | 0.06 | 0.48 | 0.60 |
| All
lots | 20 | 1.30 | 1.32 | 0.35 | 0.02 | 0.73 | 1.00 |
| | 35 | 1.54 | 1.59 | 0.28 | 0.05 | 0.60 | 0.79 |
| | 50 | 1.70 | 1.76 | 0.22 | 0.06 | 0.50 | 0.63 |
Table 8: LLoQ - TAE and Difference between Measurements
The LLoQ was determined to be 35 IU/mL for lots 1 and 20 IU/mL for lot 3, calculated based on the calculation of the Total Analytical Error (TAE) and the difference between two measurements. The LLoQ for the cobas EBV test is 35 IU/mL.
g. Analytical specificity
i) Cross reactivity
For potential cross reactants 35 microorganisms, including 17 viral isolates, 15 bacterial strains and three fungal isolates were used and divided into seven pools with 4 to 5 microorganisms per cross reactant pool and HCV as single interferent. Potential cross reactants in EBV-seronegative EDTA plasma were tested in the absence and presence of EBV DNA at a concentration of 5xLLoQ. Potential cross reactants were tested at bl(4)
Results are shown in Table 9. For EBV-negative samples the negativity rate was determined. For EBV-positive samples the positivity rate was determined together with the correct
16
quantitation of EBV DNA by computing the Mean concentration detected across the replicates, the SD, and the difference between the control condition (no cross reactant) and the test condition containing the potential cross reactant organism. The mean log10 titer of each of the positive EBV samples containing potentially cross-reacting organisms was within ± 0.5 log10 of the mean log10 titer of the respective positive spike control.
17
| | Organisms | Test
Concentrat
ion | No
EBV | | EBV | | |
|------|-----------------------------------------------------------------------------------------------------------------------------------------|---------------------------|------------------------|------------------------|-----------------|---------------|-----------------------------------------|
| Pool | | | Negati
vity
Rate | Positi
vity
Rate | Mean
[Log10] | SD
[Log10] | Mean
Difference
in log10
Titer |
| 1 | HSV 1
HSV 2
HSV 6
HSV 7
HSV 8 | (b) (4) | | | | | |
| 2 | Adenovirus
Type 5
Candida
albicans
Chlamydia
trachomatis
Clostridium
perfringens
CMV | | | | | | |
| 3 | Enterococcus
faecalis
Escherichia coli
HBV
HIV-1
HIV-2 | | | | | | |
| 4 | Klebsiella
pneumoniae
Listeria
monocytogenes
Mycobacterium
avium
Mycoplasma
pneumoniae
Neisseria
gonorrhoeae | | | | | | |
Table 9: Cross Reactivity
18
| (b) (4) | ||
|---|---|---|
| 5 | Parvovirus B19 | |
| Propionibacterium acnes | ||
| Salmonella | ||
| enterica | ||
| Simian Virus 40 | ||
| Staphylococcus | ||
| aureus | ||
| 6 | Staphylococcus | |
| epidermis | ||
| Streptococcus | ||
| pyogenes | ||
| Streptococcus | ||
| pneumoniae | ||
| VZV | ||
| 7 | Aspergillus | |
| niger | ||
| Cryptococcus | ||
| neoformans | ||
| Human | ||
| Papilloma Virus | ||
| (HPV) | ||
| JC Virus | ||
| BK Virus | ||
| Hepatitis C | ||
| Virus (HCV) | ||
| Control (EBV | ||
| negative) | ||
| Control (EBV | ||
| positive) – for | ||
| Pools 1-6 and | ||
| HCV | ||
| Control (EBV | ||
| positive) – for | ||
| Pool 7 |
*cp/mL= copies /mL
19
ii) Endogenous Interference
The effect of potentially interfering endogenous substances on the sensitivity/quantitation of cobas EBV was determined by testing 20 individual clinical EBV-seronegative samples spiked with selected endogenous substances and EBV target at 150 IU/mL (5xLLoQ). The negative sample spiked solely with EBV target was used as a Positive Spike Control (PSC). To analyze specificity, the same 20 individual clinical negative samples were individually spiked with potentially interfering endogenous substances and tested in the absence of EBV target DNA. The un-spiked samples were used as Negative Spiked Controls (NSC). Interferent concentrations were used as recommended by the CLSI guideline EP7-A2. Human DNA levels were tested at 2mg/mL. Control conditions were tested with one replicate per specimen, and test conditions were tested with 3 replicates per specimen. Results are summarized in Table 10.
| Interferent | C1 | No EBV | EBV [150 IU/mL] | ||||
|---|---|---|---|---|---|---|---|
| Negativity | |||||||
| Rate | Positivity | ||||||
| Rate | Mean Ct | Mean | |||||
| [Log 10] | SD | ||||||
| [Log 10] | Mean | ||||||
| Difference | |||||||
| in log10 | |||||||
| Titer | |||||||
| Control | (b) (4) | ||||||
| NaOH2 | |||||||
| Albumin | |||||||
| Bilirubin | |||||||
| (conj.) | |||||||
| Bilirubin | |||||||
| (unconj.) | |||||||
| Human DNA | |||||||
| Hemoglobin | |||||||
| Triglycerides |
Table 10: Endogenous Interference
1C= Test Concentration; 2 solvent control; 3 0.2 g/L = 342 umol/L
EBV-negative samples with endogenous interferents all produced valid negative results (target not detected) in the presence of endogenous interferents.
For EBV-positive samples with endogenous interferents the mean log10 titer of each of the positive EBV samples containing endogenous interferents was within ± 0.05 logio of the mean log10 titer of the spike control.
20
iii) Exogenous Interference
The effect of potentially interfering exogenous substances on the sensitivity/quantitation of cobas EBV was determined by testing 10 individual clinical EBV-negative samples spiked with pools of 24 commercially available drugs at three times the plasma peak level per CLSI EP7-A2. The same samples were also tested in the presence of EBV target at 150 IU/mL (5x LLoQ). The negative sample spiked solely with EBV target was used as a Positive Spike Control (PSC). The un-spiked samples were used as Negative Spiked Controls (NSC). Conditions were tested with 3 replicates per specimen. The following drugs were tested, and the results are summarized in Table 11 below.
- Pool 1: Azathioprine, Sulfamethoxzole, Trimethoprim. Cefotan, Cidofovir .
- Pool 2: Foscarnet, Piperacillin, Tazobactam, Prednisone, Vancomycin .
- Pool 3: Cyclosporine, Everolimus, Fluconazole, Ganciclovir .
- Pool 4: Mycophenolate mofetil, Mycophenolic acid, Valganciclovir .
- Pool 5: Sirolimus, Tacrolimus .
- Pool 6: Letermovir, Micafungin, Acyclovir Clavulanate potassium .
- Pool 7: Ticarcillin disodium .
| No EBV | EBV [150 IU/mL] | |||||
|---|---|---|---|---|---|---|
| Pool | Negativity | |||||
| Rate | Positivity | |||||
| Rate | Mean | |||||
| Ct | Mean | |||||
| [Log 10] | SD | |||||
| [Log 10] | Mean | |||||
| Difference | ||||||
| in log10 | ||||||
| Titer | ||||||
| Pool 11 | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.5 | 2.22 | 0.18 | -0.18 | ||
| Pool 21 | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.7 | 2.19 | 0.15 | -0.21 | ||
| Pool 32 | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.7 | 2.23 | 0.18 | -0.17 | ||
| Pool 42 | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.7 | 2.23 | 0.15 | -0.17 | ||
| Pool 53 | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.6 | 2.24 | 0.19 | -0.16 | ||
| Pool 61 | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.7 | 2.20 | 0.16 | -0.20 | ||
| Pool 71 | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.6 | 2.21 | 0.17 | -0.19 | ||
| PBS SC | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.4 | 2.30 | 0.19 | -0.10 | ||
| DMSO SC | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.5 | 2.26 | 0.15 | -0.13 | ||
| Ethanol SC | 10/10 | |||||
| 100% | 30/30 | |||||
| 100% | 34.4 | 2.37 | 0.23 | -0.03 |
Table 11: Exogenous Interference
21
| Negative
Control | 10/10
100% | - | N/A | N/A | N/A | N/A |
|---------------------|---------------|---------------|------|-----|------|-----|
| Positive
Control | - | 30/30
100% | 34.2 | 2.4 | 0.22 | N/A |
The superscripts in Pools 1-7 indicate the solvent that was used for constituting the interferents (i.e., 1 = PBS; 2 = DMSO; and 3 = Ethanol); SC = solvent control.
- h. Cross Contamination
The cross-contamination rate for cobas EBV was determined by testing 240 replicates of an EBV-negative matrix sample and 225 replicates of a high titer EBV sample at approximately 2.00E+07 IU/mL. In total, five runs were performed with positive and negative samples in a checkerboard configuration.
All 240 replicates of the negative sample were negative, resulting in a cross-contamination rate of 0% (upper one-sided 95% confidence interval: 1.24%).
-
Assay cut-off i.
Not applicable -
- Comparison studies
-
a. Method comparison with predicate device
Not applicable
-
b. Matrix comparison
Not applicable -
- Clinical studies
Concordance of cobas EBV with a Comparator EBV Test
The clinical performance of cobas EBV was compared to a validated well-established comparator nucleic acid test(comparator EBV) by measuring EBV DNA levels in longitudinal clinical samples (neat and diluted) of EBV-infected and non-infected patients. Contrived EDTA plasma samples spiked with cultured EBV virus were used to cover the linear range.
The comparator EBV is well described, currently used in clinical practice at a major transplant center in the United States, is traceable to the WHO standard and its use is acceptable. Due to different methods of measuring EBV viral load at each institution, EBV viral load quantitation may vary between laboratories and hence should not be compared to make clinical management decisions.
A total of 464 samples (439 neat or diluted clinical samples of 72 transplant subjects and 25 contrived samples) were valid on both assays and evaluable for the clinical concordance
22
analysis. Results presented in Table 12 demonstrate a column percent agreement between the cobas EBV and comparator EBV ranging from between 82.5% to 100% depending on the analyte concentration in the samples. DNA sequencing on representative samples from subjects with results consistently offset by more than 1 log10 IU/mL DNA level did not reveal any sequence mismatches for any primer or probe targets for the cobas EBV assay.
| Comparator EBV (log10 IU/mL) | |||||||
|---|---|---|---|---|---|---|---|
| cobas® EBV | |||||||
| (log10 IU/mL) | Target | ||||||
| Not | |||||||
| Detected | 3.8 | Total | |||||
| Target Not | |||||||
| Detected | 95 | 17 | 17 | 0 | 0 | 0 | 129 |
| 3.8 | 0 | 0 | 0 | 0 | 1 | 29 | 30 |
| Total | 136 | 65 | 113 | 63 | 46 | 41 | 464 |
| Column | (134/136) | (65/65) | (96/113) | (52/63) | (40/46) | (40/41) | |
| Agreement (%) | 98.5% | 100% | 85.0% | 82.5% | 87.0% | 97.6% | |
| (95% Score CI)a | (94.8, | ||||||
| 99.6%) | (94.4%, | ||||||
| 100%) | (77.2%, | ||||||
| 90.4%) | (71.4%, | ||||||
| 90.0%) | (74.3%, | ||||||
| 93.9%) | (87.4%, | ||||||
| 99.6%) |
Table 12: Concordance analysis between cobas EBV and the comparator EBV DNA level results for all samples
Note: LLoO = lower limit of quantitation of comparator EBV (100 IU/mL). Standard Deviation of comparator EBV estimated at 0.3 logio IU/mL (comparator EBV analytical precision study).
Paired samples evaluable for clinical concordance analysis were included in this table.
a Assumed independence between all samples: CI =Confidence Interval by Score Method.
Discordant results were defined as those that are more than one box away from the diagonal (indicated by shading). For Target Not Detected by comparator EBV Column Agreement the cobas EBV Target Not Detected and