(150 days)
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).
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:
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- 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.
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- 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.
Here's an analysis of the acceptance criteria and the studies that prove the device meets these criteria, based on the provided text:
Acceptance Criteria and Device Performance
| Criteria | Reported Device Performance |
|---|---|
| Precision | Standard Deviation (log10 transformed titer): Ranged from 0.02 to 0.17 across various concentrations (Table 1). |
| Lognormal Percent Coefficient of Variation (%CV): Ranged from 7% to 43% for positive panel members (Table 2). | |
| Reproducibility | Total Lognormal Coefficient of Variation (% CV): Ranged from 13.7% to 46.16% among positive panel members when tested across three reagent lots, three test sites, and three instruments (Table 3). |
| Linearity (Genotype 1) | Linear Range: 35 IU/mL (LLoQ) to 1.0E+08 IU/mL (ULoQ). |
| Accuracy: Within ± 0.15 log10 IU/mL (Mean Square Error) across the linear range. | |
| Best-fitting model: 1st order for individual lots/panel types, 2nd order for combined data (minor difference to 1st order: ≤ ± 0.01 log10 IU/mL). (Table 4, Figure 1) | |
| Linearity (Genotype 2) | Accuracy: Within ± 0.12 log10 IU/mL (Mean Square Error) across the linear range, with minor differences (–0.06 log IU/mL to +0.08 log IU/mL) between 1st and 3rd order regression models. (Figure 2) |
| Traceability | Quantitation values for the cobas EBV calibration panel and RMS EBV Secondary Standard showed deviations not more than 0.15 log10 IU/mL from expected values, demonstrating traceability to the 1st WHO International Standard for EBV (NIBSC 09/260). (Figure 3) |
| Regression equations: EBV Calibration Panel: y = 1.000x - 0.002; R2 = 1.000. | |
| EBV 1st WHO Standard: y = 0.975x + 0.159; R2 = 0.983. | |
| Clinical Specimen Stability | Whole blood (EDTA-plasma tubes) stable for up to 24 hours at 2-25°C. Plasma stable for 24 hours at 2-30°C, then up to 6 days at 2-8°C, or up to 6 months at -15 to -80°C. Plasma stable for up to four freeze/thaw cycles. Mean log10 titers at different time points/conditions were within ±0.5 log10 of the reference (T0). |
| Open Kit and On-board Stability | Open 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). Reusable for up to 40 runs. |
| Reagent Stability | Shelf-life stability claim of 12 months when stored at 2-8°C (supported by real-time stability data). |
| Limit of Detection (LoD) | WHO EBV Standard (Genotype 1): LoD = 18.8 IU/mL by Probit analysis (highest LoD across lots). LoD by 95% hit rate was (b) (4) for all lots. (Table 5) |
| EBV Genotype 2: LoD verified at 18.8 IU/mL with a hit rate of 95% or higher. (Table 6) | |
| Plasma vs Buffer (GSD): Comparable LoD performance in Plasma and GSD (Hit rate 98.4% at 1.5xLoD, 92.1% at 1.0xLoD). (Table 7) | |
| Lower Limit of Quantitation (LLoQ) | LLoQ: 35 IU/mL. Determined based on Total Analytical Error (TAE) and difference between two measurements. (Table 8) |
| Cross-reactivity | EBV-negative samples: Negativity rate was high (not explicitly stated as 100% but implied by lack of reported positives). |
| EBV-positive samples: Mean log10 titer of positive EBV samples with 35 potential cross-reacting microorganisms was within ±0.5 log10 of the positive spike control. (Table 9) | |
| Endogenous Interference | EBV-negative samples: All produced valid negative results. |
| EBV-positive samples: Mean log10 titer of positive EBV samples with interfering substances (NaOH2, Albumin, Bilirubin, Human DNA, Hemoglobin, Triglycerides) was within ±0.05 log10 of the spike control. (Table 10) | |
| Exogenous Interference | EBV-negative samples: 100% negativity rate. |
| EBV-positive samples: Mean log10 titer of positive EBV samples with 24 common drugs was observed to be within acceptable range (implied by table values, e.g., Max Mean Difference in log10 Titer of -0.03 for Ethanol SC). (Table 11) | |
| Cross-Contamination | 0% cross-contamination rate (upper one-sided 95% CI: 1.24%) from testing 240 replicates of EBV-negative matrix sample in checkerboard configuration with high-titer samples. |
| Concordance with Comparator EBV Test | Overall Column Percent Agreement: Ranged from 82.5% to 100% depending on analyte concentration (for valid samples on both assays, n=464). (Table 12) |
| Negative Percent Agreement (NPA): 95.4% (95% CI: 84.2%-99.4%) with comparator EBV negative samples (n=43). | |
| Agreement at clinical thresholds: High agreement, e.g., 98.0% ( |
§ 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.