cobas® BKV is an in vitro nucleic acid amplification test for the quantitation of BK virus (BKV) DNA in human EDTA plasma on the cobas® 6800/8800 Systems.

cobas® BKV is intended for use as an aid in the management of BKV in transplant patients. In patients undergoing monitoring of BKV, serial DNA measurements can be used to indicate the need for potential treatment changes and to assess viral response to treatment.

The results from cobas® BKV are intended to be read and analyzed by a qualified licensed healthcare professional in conjunction with clinical signs and symptoms and relevant laboratory findings. Test results must not be the sole basis for patient management decisions.

cobas® BKV is not intended for use as a screening test for blood products or human cells, tissues, and cellular and tissue-based products (HCT/Ps).

Device Description

cobas® BKV is based on 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 software which assigns test results for all tests as either target not detected, BKV DNA detected < LLoQ (lower limit of quantitation), BKV DNA detected > ULoQ (upper limit of quantitation), or a value in the linear range LLoQ < x < ULoQ. Results can be reviewed directly on the system screen, exported, or printed as a report.

Nucleic acid from patient samples and added lambda DNA-QS molecules is simultaneously extracted. In summary, 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 protein, cellular debris and potential PCR inhibitors are removed with subsequent wash reagent steps and purified nucleic acid is eluted from the glass particles with elution buffer at elevated temperature.

Selective amplification of target nucleic acid from the sample is achieved by the use of a dual target virus specific approach from highly-conserved regions of the BKV located in the BKV small t-antigen region and the BKV VP2 region. 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 BKV genome. A thermostable DNA polymerase enzyme is used for amplification. The target and DNA-QS sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicon from previous PCR runs is eliminated by the AmpErase enzyme, which is included in the PCR mix, when heated in 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.

The cobas® BKV master mix contains two detection probes specific for BKV target sequences and one for the DNA-QS. The probes are labeled with target-specific fluorescent reporter dyes allowing simultaneous detection of BKV target and DNA-QS in two different target channels. The fluorescent signal of the intact probes is suppressed by the quencher dye. During the PCR amplification step, hybridization of the specific single-stranded DNA templates results in cleavage by the 5'-to-3' nuclease 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 probes are generated and the cumulative signal of the reporter dye is concomitantly increased. Real-time detection and discrimination of PCR products are accomplished by measuring the fluorescence of the released reporter dyes for the viral targets and DNA-QS.

AI/ML Overview

The provided document is a 510(k) Summary for the cobas® BKV test for use on the cobas® 6800/8800 Systems. This document focuses on demonstrating substantial equivalence to a predicate device through non-clinical performance evaluation, rather than an AI/ML device requiring a comparative effectiveness study with human readers. Therefore, several of the requested sections (e.g., sample size for test set, number of experts, adjudication method, MRMC study, standalone performance, ground truth for test set, training set details) are not directly applicable or explicitly stated in the context of this In Vitro Diagnostic (IVD) device submission for a quantitative nucleic acid amplification test.

However, I will extract relevant information to address the applicable criteria based on the provided text.

Acceptance Criteria and Device Performance for cobas® BKV

The acceptance criteria for this diagnostic device are primarily defined by various performance characteristics required for quantitative viral nucleic acid tests. The study proves the device meets these criteria through a series of non-clinical performance evaluations.

Table of Acceptance Criteria and Reported Device Performance

Performance Characteristic	Acceptance Criteria (Implicit from Study Design/Context)	Reported Device Performance and Study Details
Limit of Detection (LoD)	Determine the concentration at which 95% hit rate is achieved for the WHO International Standard and all subgroups/subtypes, across multiple lots.	- WHO International Standard (NIBSC 14/212): Determined as 21.5 IU/mL (PROBIT, 95% hit rate) with a 95% CI of 16.3 – 32.4 IU/mL in EDTA plasma, using the least sensitive lot. - Subgroups Ia, Ic and Subtypes II, III and IV: Verified detection at 21.5 IU/mL with a ≥ 95% hit rate for all tested genotypes (e.g., Subgroup Ia at 21.5 IU/mL: 100.0% hit rate, 63/63 positives; Subgroup Ic at 21.5 IU/mL: 100.0% hit rate, 62/62 positives).
Traceability	Demonstrate proportionality and correlation to the 1st WHO International Standard for BKV DNA.	- Calibration and standardization process provides quantitation values for panels and standards similar to expected values. - Maximum deviation from expected values was not more than 0.19 log10 IU/mL. - Deming regression for BKV WHO Standard showed Y = 0.951x + 0.208 with R² = 0.973.
Linear Range	Demonstrate linearity of quantification within a specified range with acceptable deviation and accuracy.	- Demonstrated linear from 1.01E+01 to 1.97E+08 IU/mL. - Absolute deviation from non-linear regression ≤ ± 0.1 log10 in human EDTA plasma. - Accuracy within ± 0.2 log10 across the linear range. - Verified for subgroups Ia, Ic and subtypes II, III and IV: maximum deviation between linear and higher order non-linear regression ≤ ± 0.2 log10.
Lower Limit of Quantitation (LLoQ)	Determine the lowest titer meeting acceptance criteria for Total Analytical Error (TAE ≤ 1.0 log10 IU/mL) and difference between two measurements.	- Established as 21.5 IU/mL. - At 19.0 IU/mL (nominal concentration, lowest tested for LLoQ), all three lots combined showed TAE of 0.69 and difference between measurements (SD) of 0.73, both within 1.0 log10 IU/mL.
Precision – Within Laboratory	Demonstrate high precision across specified concentration ranges, instruments, operators, and days.	- High precision shown across 5.90E+01 IU/mL to 9.83E+05 IU/mL. - Total %CV ranged from 8% to 36% across the concentrations tested (e.g., 8% at 9.83E+05 IU/mL, 36% at 5.90E+01 IU/mL). - Results represent all aspects of the test procedure.
Analytical Specificity	No interference or cross-reactivity with common microorganisms and endogenous/exogenous substances.	- Microorganisms: None of 17 viruses, 13 bacteria, and 3 yeast species interfered at tested concentrations (1.00E+05 to 1.00E+06 units/mL). Negative results for BKV-negative samples, positive for BKV-spiked samples. Titer within ± 0.5 log10 of control. - Interfering Substances: Elevated triglycerides, bilirubin (conjugated/unconjugated), albumin, hemoglobin, human DNA, and 17 drug compounds (including antimicrobials and immune suppressants) did not interfere. Titer within ± 0.5 log10 of control.
Cross-Contamination	Demonstrate a low or zero cross-contamination rate.	- 0% cross-contamination rate (0/240 negative replicates) with an upper one-sided 95% CI of 1.24%.
Reproducibility	Demonstrate consistent performance across different reagent lots, test sites, batches, and testing days.	- Evaluated at 3 testing sites using 3 reagent lots per site by 2 operators over 5 days. - Total Precision Standard Deviation ranged from 0.068 to 0.304 log10 IU/mL. - Lognormal CV(%) for Total Precision ranged from 15.74% to 79.43%. - 100% detection of 3 x LLoQ samples. - Equivalence shown between cobas® 6800 and 8800 systems. - Negative percent agreement (NPA) for reproducibility study: 100% (270/270 samples negative), 95% Exact CI: 98.6% to 100%.
Clinical Concordance	Demonstrate agreement with a well-established laboratory-developed test (LDT).	- Total of 550 valid samples (217 neat, 303 diluted clinical, 30 contrived) from 129 transplant subjects were evaluable. - Agreement with Comparator BKV LDT (IU LDT): High concordance shown across different concentration ranges. - Negative Percent Agreement (NPA): 100% (43/43 samples negative) with 95% Exact CI of 91.8% to 100%. - At thresholds, percent agreement was high (e.g., ≥ threshold 2.3 Log10 IU/mL: 87.7%; < threshold 4.0 Log10 IU/mL: 100.0%). - Correlation: Deming Linear Regression Y = -0.429 + 1.019X with R-value of 0.96 (N=313 samples where both assays were BKV positive). - Mean bias was -0.357 log10 IU/mL, indicating a systematic difference but within acceptable analytical precision limits of the comparator LDT.

Study Details:

Sample Size and Data Provenance:
- Test Set (Clinical Concordance): 550 valid samples (217 neat clinical, 303 diluted clinical derived from 129 transplant subjects, and 30 contrived samples).
- Provenance: Clinical samples were from BKV infected and non-infected patients. Although not explicitly stated, the context of an FDA submission for an IVD test likely implies data collected under controlled conditions at testing sites, potentially from multiple geographical locations if a multi-site study was conducted (the Reproducibility study specified 3 testing sites). The samples included both retrospective (stored clinical samples) and prospective (contrived samples) elements.
- Non-Clinical Studies: Sample sizes vary by test as detailed in the table (e.g., 63 replicates per level for LoD, 36 replicates per panel member for linear range, 72 replicates per level for precision, 240/225 replicates for cross-contamination, 270 valid tests per concentration for reproducibility).
Number of Experts and Qualifications: Not applicable. This is an IVD device for quantitative viral DNA measurement, not an AI/ML device for image interpretation or diagnosis by experts. Ground truth is established analytically (e.g., known concentrations of viral standards) or through comparison to a well-established laboratory method.
Adjudication Method: Not applicable. For IVD tests like this, objective analytical measurements are compared rather than subjective interpretations needing adjudication.
MRMC Comparative Effectiveness Study: No. This is an In Vitro Diagnostic (IVD) quantitative nucleic acid amplification test, not an AI-assisted diagnostic imaging device that involves human readers. The standard of comparison is against a well-established laboratory method (LDT) and analytical performance targets, not human reader improvement.
Standalone Performance: Yes, the entire document describes the standalone performance of the cobas® BKV assay. The device measures BKV DNA concentration directly without human-in-the-loop assistance for the measurement itself, though a qualified healthcare professional interprets the results in a clinical context.
Type of Ground Truth Used:
- Analytical/Reference Standards: For LoD, linearity, traceability, precision, and specificity, the ground truth was established using known concentrations of the 1st WHO International Standard for BK Virus DNA (NIBSC 14/212), armored DNA for specific subgroups/subtypes, and well-characterized high titer BKV DNA (subgroup Ib).
- Comparative Method: For clinical concordance, the ground truth was established by comparing results from the cobas® BKV test to those from a well-established laboratory developed nucleic acid test (LDT) from Indiana University (referred to as "comparator BKV LDT"). This LDT results served as the reference for comparison and agreement.
Sample Size for Training Set: Not applicable. This is a molecular diagnostic assay characterized through analytical and clinical performance studies, not an AI/ML model that undergoes a distinct training phase with a dedicated training set. The "training" of the assay involves its development and optimization based on scientific principles of nucleic acid amplification and detection, and then validated through the studies described.
How Ground Truth for Training Set was Established: Not applicable, as there isn't a "training set" in the AI/ML sense. The development of the assay (analogous to training) would rely on molecular biology knowledge, optimization experiments, and characterization against internally developed standards and reference materials, ultimately ensuring performance against defined analytical targets and external standards (like the WHO standard).

Summary

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which consists of the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG" in blue, and "ADMINISTRATION" in a smaller font below that.

September 2, 2020

Roche Molecular Systems, Inc. Rita Hoady Senior Manager, Regulatory Affairs 4300 Hacienda Drive Pleasanton, California 94588-2722

Re: K202215

Trade/Device Name: cobas BKV, cobas EBV/BKV Control Kit, cobas Buffer Negative Control Kit Regulation Number: 21 CFR 866.3183 Regulation Name: Quantitative Viral Nucleic Acid Test for Transplant Patient Management Regulatory Class: Class II Product Code: QMI Dated: August 5, 2020 Received: August 6, 2020

Dear Rita Hoady:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR

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for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

For:

Uwe Scherf. M.Sc., Ph.D. Director Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K202215

Device Name

cobas BKV for use on the cobas® 6800/8800 Systems

Indications for Use (Describe)

cobas® BKV is an in vitro nucleic acid amplification test for the quantitation of BK virus (BKV) DNA in human EDTA plasma on the cobas® 6800/8800 Systems.

cobas® BKV is not intended for use as a screening test for blood products or human cells, tissues, and cellular and tissue-based products (HCT/Ps).

Type of Use (Select one or both, as applicable)
-------------------------------------------------

X Prescription Use (Part 21 CFR 801 Subpart D)

| Over-The-Counter Use (21 CFR 801 Subpart C)

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cobas® BKV 510(k) Summary

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

Submitter Name	Roche Molecular Systems, Inc.
Address	4300 Hacienda DrivePleasanton, CA 94588-2722
Contact	Rita HoadyPhone: (925) 730-8397FAX: (925) 225-0207Email: rita.hoady@roche.com
Date Prepared	August 5, 2020
Proprietary Name	cobas® BKVfor use on cobas® 6800/8800 Systems
Classification Name	Quantitative viral nucleic acid test for transplant patient management
Product Codes	QMI: 21 CFR 866.3183
Predicate Devices	cobas® EBV (DEN200015)
Establishment Registration	Roche Molecular Systems, Inc. (2243471)

DEVICE DESCRIPTION 1.

Nucleic acid from patient samples and added lambda DNA-QS molecules is simultaneously extracted. In summary, viral nucleic acid is released by addition of proteinase and lysis reagent

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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 protein, cellular debris and potential PCR inhibitors are removed with subsequent wash reagent steps and purified nucleic acid is eluted from the glass particles with elution buffer at elevated temperature.

Selective amplification of target nucleic acid from the sample is achieved by the use of a dual target virus specific approach from highly-conserved regions of the BKV located in the BKV small t-antigen region and the BKV VP2 region. 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 BKV genome. A thermostable DNA polymerase enzyme is used for amplification. The target and DNA-QS sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).1-3 Any contaminating amplicon from previous PCR runs is eliminated by the AmpErase enzyme, which is included in the PCR mix, when heated in 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.

The cobas® BKV master mix contains two detection probes specific for BKV target sequences and one for the DNA-QS. The probes are labeled with target-specific fluorescent reporter dyes allowing simultaneous detection of BKV target and DNA-QS in two different target channels. 55 The fluorescent signal of the intact probes is suppressed by the quencher dye. During the PCR amplification step, hybridization of the specific single-stranded DNA templates results in cleavage by the 5'-to-3' nuclease 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 probes are generated and the cumulative signal of the reporter dye is concomitantly increased. Real-time detection and discrimination of PCR products are accomplished by measuring the fluorescence of the released reporter dyes for the viral targets and DNA-QS.

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Figure 1: cobas® BKV for use on cobas® 6800/8800 Systems

Image /page/5/Picture/1 description: The image shows two boxes of Cobas test kits. The box on the left is labeled "Cobas BKV" and is a quantitative nucleic acid test for use on the Cobas 6800/8800 systems. The box on the right is labeled "Cobas EBV/BKV Control Kit" and is a positive control kit for use on the Cobas 6800/8800 systems. Both boxes are labeled "Rx Only" and "IVD".

2. INDICATIONS FOR USE

cobas® BKV is an in vitro nucleic acid amplification test for the quantitation of BK virus (BKV) DNA in human EDTA plasma on the cobas® 6800/8800 Systems.

cobas® BKV is not intended for use as a screening test for blood or blood products or human cells, tissues, and cellular and tissue-based products (HCT/Ps).

TECHNOLOGICAL CHARACTERISTICS 3.

The primary technological characteristics and intended use of the RMS cobas® BKV for use on the cobas® 6800/8800 Systems are similar to the identified predicate device, cobas® EBV (DEN200015) (Table 1).

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	Submitted Device:cobas® BKV	Predicate Device:cobas® EBV (DEN200015)
Regulation Number	21 CFR 866.3183	Same
Regulation Name	Quantitative viral nucleic acid test fortransplant patient management	Same
Product Code	QMI	QLX
Intended Use	cobas® BKV is an in vitro nucleic acidamplification test for the quantitation of BKvirus (BKV) DNA in human EDTA plasmaon the cobas® 6800/8800 Systems.cobas® BKV is intended for use as an aidin the management of BKV in transplantpatients. In patients undergoing monitoringof BKV, serial DNA measurements can beused to indicate the need for potentialtreatment changes and to assess viralresponse to treatment.The results from cobas® BKV areintended to be read and analyzed by aqualified licensed healthcare professionalin conjunction with clinical signs andsymptoms and relevant laboratoryfindings. Test results must not be the solebasis for patient management decisions.cobas® BKV is not intended for use as ascreening test for donors of blood or bloodproducts or human cells, tissues, andcellular and tissue-based products(HCT/Ps).	cobas® EBV is an in vitro nucleic acidamplification test for the quantitation ofEpstein-Barr virus (EBV) DNA in humanEDTA plasma on the cobas® 6800/8800Systems.cobas® EBV is intended for use as an aidin the management of EBV in transplantpatients. In patients undergoing monitoringof EBV, serial DNA measurements can beused to indicate the need for potentialtreatment changes and to assessresponse to treatment.The results from cobas® EBV areintended to be read and analyzed by aqualified licensed healthcare professionalin conjunction with clinical signs andsymptoms and relevant laboratoryfindings.Negative test results do not preclude EBVinfection or EBV disease. Test resultsmust not be the sole basis for patientmanagement decisions.cobas® EBV is not intended for use as ascreening test for donors of blood or bloodproducts or human cells, tissues, andcellular and tissue-based products(HCT/Ps).
Conditions for use	For prescription use	Same
Sample Types	EDTA - plasma	Same
Analyte Targets	BK Virus	Epstein-Barr virus
Sample PreparationProcedure	Automated by cobas® 6800/8800 Systems	Same
Amplification Technology	Real-time PCR	Same
Detection Chemistry	Paired reporter and quencherfluorescence labeled probes (TaqManTechnology) using fluorescenceresonance energy transfer (FRET)	Same
	Submitted Device:cobas® BKV	Predicate Device:cobas® EBV (DEN200015)
Controls used	Sample processing control (QS)Positive and negative control	Same
Result Analysis	Based on PCR cycle threshold analysis	Same

Table 1: Comparison of the cobas® BKV for use on the cobas® 6800/8800 Systems with the Predicate Device

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4. NON-CLINICAL PERFORMANCE EVALUATION

Limit of Detection (LoD) 4.1.

The limit of detection (LoD) of cobas® BKV was determined by analysis of serial dilutions of the WHO International Standard (subgroup Ib) and verified for subgroups Ia, Ic and subtypes II, III and IV. The overall concentration for which 95% hit rate is expected by PROBIT is 21.5 IU/mL for EDTA plasma.

WHO International Standard 4.1.1.

The limit of detection of cobas® BKV for the WHO International Standard was determined by analysis of serial dilutions of the 1st WHO BKV International Standard obtained from NIBSC (NIBSC 14/212) in BKV-negative human EDTA plasma. Panels of six concentration levels plus a blank were tested over three lots of cobas® BKV reagents, multiple runs, days, operators, and instruments.

The results for EDTA plasma are shown in Table 2 through Table 4. The study demonstrates that with the least sensitive lot, the concentration for which 95% hit rate is expected by PROBIT is 21.5 IU/mL with a 95% confidence range of 16.3 – 32.4 IU/mL in EDTA plasma. The lowest concentration with a hit rate ≥ 95% is 19.0 IU/mL in EDTA plasma.

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Input titer concentration(BKV DNA IU/mL)	Number of validreplicates	Number of positives	Hit rate in %
80.0	63	63	100.0
38.0	63	63	100.0
19.0	63	60	95.2
9.5	63	46	73.0
4.75	63	36	57.1
2.38	63	23	36.5
0	62	0	0.0

Limit of Detection in EDTA Plasma, Lot 1 Table 2:

LoD by PROBIT at 95% hit rate: 21.5 IU/mL, 95% confidence range: 16.3 – 32.4 IU/mL

Table 3: Limit of Detection in EDTA Plasma, Lot 2

Input titer concentration(BKV DNA IU/mL)	Number of validreplicates	Number of positives	Hit rate in %
80.0	62	62	100.0
38.0	63	63	100.0
19.0	63	61	96.8
9.5	63	48	76.2
4.75	63	34	54.0
2.38	63	23	37.1
0	62	0	0.0

LoD by PROBIT at 95% hit rate: 19.7 IU/mL, 95% confidence range: 15.0 – 29.2 IU/mL

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Input titer concentration(BKV DNA IU/mL)	Number of valid replicates	Number of positives	Hit rate
80.0	63	63	100.0
38.0	63	63	100.0
19.0	63	60	95.2
9.5	63	50	79.4
4.75	63	35	55.6
2.38	63	22	35.0
0	63	0	0.0

Table 4: Limit of Detection in EDTA Plasma, Lot 3

LoD by PROBIT at 95% hit rate: 19.3 IU/mL, 95% confidence range: 14.8 – 28.5 IU/mL

Limit of Detection for Subgroups Ia, Ic and Subtypes II, III and IV 4.2.

BKV armored DNA for subgroup Ic and subtype III, and clinical specimens for subgroup Ia and subtypes II and IV were diluted to three different concentration levels in BKV-negative EDTA plasma. The hit rate determination was performed with 63 replicates for each level. Testing was conducted with three lots of cobas® BKV reagents.

The combined results from three lots shown in Table 5 verify that – consistent with an LoD of 21.5 IU/mL – cobas® BKV detected BKV DNA for subgroups Ia and Ic, and subtypes II, III and IV at a concentration of 21.5 IU/mL with a ≥ 95% hit rate.

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Genotype	Test concentration	Number of validreplicates (N)	Number ofpositives (n)	Hit rate(n/N)x100
Subgroup la	5.4 IU/mL	Not Tested	Not Tested	Not Tested
Subgroup la	10.8 IU/mL	63	54	85.7%
Subgroup la	21.5 IU/mL	63	63	100.0%
Subgroup Ic	5.4 IU/mL	62	57	91.9%
Subgroup Ic	10.8 IU/mL	63	61	96.8%
Subgroup Ic	21.5 IU/mL	62	62	100.0%
Subtype II	5.4 IU/mL	63	54	85.7%
Subtype II	10.8 IU/mL	63	63	100.0%
Subtype II	21.5 IU/mL	63	63	100.0%
Subtype III	5.4 IU/mL	63	49	77.8%
Subtype III	10.8 IU/mL	63	63	100.0%
Subtype III	21.5 IU/mL	63	63	100.0%
Subtype IV	5.4 IU/mL	63	57	90.5%
Subtype IV	10.8 IU/mL	63	63	100.0%
Subtype IV	21.5 IU/mL	63	63	100.0%

Table 5: BKV DNA Subgroups Ia, Ic and Subtypes II, III and IV Verification of Limit of Detection in EDTA Plasma

4.3. Traceability to the 1st WHO International Standard for BK Virus for Nucleic Acid Amplification Techniques (NAT)-based Assays

Several standards and controls have been used during development of this test to provide traceability to the WHO standard [the 1st WHO International Standard for BK Virus DNA (NIBSC 14/212)"]. The standards used during development of the test include the BKV WHO Standard, the RMS BKV Secondary Standard, and the RMS BKV Calibration Panel. The Standards and the Calibration Panel were tested. The concentration range tested for the BKV WHO Standard was from 1.90E+01 IU/mL to 2.00E+04 IU/mL (1.28-4.30 log10 IU/mL), the RMS BKV Secondary Standard was tested at 1.86E+04 IU/mL (4.27 log10 IU/mL), and the RMS BKV Calibration Panel was tested from 1.00E+03 IU/mL to 5.00E+08 IU/mL (3.00-8.70 log10 IU/mL).

The calibration and standardization process of cobas® BKV provides quantitation values for the calibration panel, the RMS BKV Secondary Standard, and the BKV WHO Standard that are

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similar to the expected values with deviation of not more than 0.19 log10 IU/mL (Figure 2). The maximum deviation was obtained at 19.0 IU/mL (approximately LLoQ).

Figure 2: Traceability to WHO International Standard [Bivariate Fit of Observed BKV DNA Concentration (log10 IU/mL) by Expected BKV DNA Concentration (log10 IU/mL)] using cobas® BKV

Image /page/11/Figure/2 description: The image shows a scatter plot comparing observed BKV DNA concentration to expected BKV DNA concentration, both in Log10 IU/mL. Three testing materials are plotted: BKV Calibration Panel, BKV Secondary Standard, and BKV WHO Standard. Linear fit equations and R-squared values are provided for the BKV Calibration Panel (y = 1.001x - 0.011, R^2 = 0.999) and the BKV WHO Standard (y = 0.951x + 0.208, R^2 = 0.973).

4.4. Linear Range

Linearity of cobas® BKV was evaluated using a dilution series consisting of 18 panel members with BKV subgroup Ib DNA spanning the assay linear range. A high titer lambda DNA stock was used to prepare 11 panel members spanning the entire linear range. A clinical specimen was used to prepare seven panel members covering the intermediate - and lower levels of the linear range.

Each panel member was tested in 36 replicates across three lots of cobas® BKV reagents and the results of the study are presented in Figure 3.

cobas® BKV was demonstrated to be linear from 1.01E+01 to 1.97E+08 IU/mL and shows an absolute deviation from the better fitting non-linear regression of less or equal than ± 0.1 log10 in human EDTA plasma (see Figure 3). Across the linear range, the accuracy of the test was within ± 0.2 log10.

The lower limit of quantitation (LLoQ) is 21.5 IU/mL, calculated based on a goal for acceptable total analytical error (TAE) of ≤ 1.0 log10, where TAE = |bias| + 2 standard

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deviations in alignment with the CLSI EP-17A guideline, and TAE = SOUARE ROOT(2) x 2 standard deviations based on the "difference between 2 measurements" approach.

Based on the LLoQ and the determined linear range, as well as the medical value the linear measurement range of the test was set to 21.5-1.0E+08 IU/mL. The results of calculation and claimed LLoO are shown in Table 6.

Image /page/12/Figure/2 description: The image is a scatter plot that compares AssignedLogTiter and ObservedLogTiter. The x-axis represents AssignedLogTiter, and the y-axis represents ObservedLogTiter. The plot includes a linear fit line and a polynomial fit line with a degree of 3. Most of the data points are clustered between 1 and 2 on the x-axis, with a few points scattered up to 8.

Image /page/12/Figure/3 description: The image is a title for a figure. The title reads "Figure 3: Linear Range Determination in EDTA Plasma". The title is written in a bold, sans-serif font. The figure number is 3.

Linearity for Subgroups la, Ic and subtypes II, III and IV 4.5.

The dilution series used in the verification of subgroup/subtype linearity study of cobas® BKV consisted of eight panel members spanning the linear range of the assay. Testing was conducted with three lots of cobas® BKV reagent, 12 replicates per level were tested in EDTA plasma. The results of the study are presented in Table 6.

The linearity within the linear range of cobas® BKV was verified for subgroups Ia, Ic and subtypes II, III and IV. The maximum deviation between the linear regression and the better fitting non-linear regression was equal to or less than ± 0.2 log10.

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Genotype	Linear regression	Better fitting higher ordermodel regression	Maximum difference betweenlinear regression and thebetter fitting higher ordermodel (log10 IU/mL)
Subgroup la	y = 0.9794912x +0.2792632	y = -0.0120248x3 + 0.1792256x2 +0.2014722x +1.1614041	0.21
Subgroup Ic	y = 0.9820273x +0.1365877	y = -0.0024169x3 + 0.0403425x2 +0.7853471x + 0.3881234	0.06
Subtype II	y = 0.9856895x +0.1313346	y = -0.0063337x3 + 0.0966686x2 +0.5547977x + 0.6352469	0.12
Subtype III	y = 0.9742446x +0.1747927	y = -0.0039425x3 + 0.0693297x2 +0.6211286x + 0.6415592	0.12
Subtype IV	y = 0.9802729x +0.1452696	y = -0.0054353x3 + 0.0880830x2 +0.5657132x + 0.6484089	0.14

Table 6: Linearity Verification on Subgroups Ia, Ic and Subtypes II, III and IV

4.6. Lower Limit of Quantitation

The analysis for LLoQ was performed with data obtained from the LoD study at concentration levels of 19.0 IU/mL, 38.0 IU/mL and 80.0 IU/mL. The LLoQ is the lowest titer within the linear range that is not lower than the LoD and meets the acceptance criteria for the Total Analytical Error (|Bias| + 2x SD) (TAE) and the difference between two measurements. The acceptance criteria for both is ≤1.0 log10 IU/mL.

The results of calculation and claimed LLoQ are shown in Table 7 the lower limit of quantitation (LLoQ) is 21.5 IU/mL.

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Lot	Nominalconcentration(IU/mL)	log10titernominal	Mean log10titerobserved	SD(log10)	AbsoluteBias	TAE( Bias +2x SD)	Difference betweenMeasurements inSD (= SQRT(2) x2x SD)
1	19.0	1.28	1.39	0.25	0.11	0.61	0.71
1	38.0	1.58	1.62	0.25	0.04	0.53	0.69
1	80.0	1.90	1.89	0.26	0.01	0.52	0.73
2	19.0	1.28	1.50	0.26	0.22	0.74	0.74
2	38.0	1.58	1.76	0.21	0.18	0.60	0.59
2	80.0	1.90	2.02	0.27	0.11	0.65	0.76
3	19.0	1.28	1.47	0.27	0.19	0.72	0.75
3	38.0	1.58	1.66	0.26	0.08	0.59	0.72
3	80.0	1.90	1.91	0.19	0.00	0.38	0.53
3 Lotscombined	19.0	1.28	1.45	0.26	0.18	0.69	0.73
3 Lotscombined	38.0	1.58	1.68	0.24	0.10	0.57	0.67
3 Lotscombined	80.0	1.90	1.94	0.24	0.04	0.52	0.68

Table 7: Lower Limit of Quantitation (LLoQ) of cobas® BKV using the 1st WHO International Standard for BK Virus (BKV) (NIBSC 14/212)

Precision – Within Laboratory 4.7.

Precision of cobas® BKV was determined by analysis of serial dilutions of high titer BKV DNA (subgroup Ib) in BKV-negative EDTA plasma. Six dilution levels were tested in 72 replicates for each level across three lots of cobas® BKV reagents using four instruments and two operators over 12 days. Each sample was carried through the entire cobas® BKV procedure on fully automated cobas® 6800/8800 Systems. Therefore, the precision reported here represents all aspects of the test procedure. The results are shown in Table 8. The results of the variance component estimation are shown in Table 9.

cobas® BKV showed high precision for three lots of reagents tested across a concentration range of 5.90E+01 IU/mL to 9.83E+05 IU/mL.

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NominalConcentration[IU/mL]	AssignedConcentration[IU/mL]	EDTA plasmaLot 1SD	EDTA plasmaLot 2SD	EDTA plasmaLot 3SD	EDTA plasmaAll LotsSD
1.00E+06	9.83E+05	0.02	0.02	0.04	0.03
1.00E+05	9.83E+04	0.03	0.04	0.04	0.04
1.00E+04	9.83E+03	0.04	0.05	0.03	0.04
6.00E+03	5.90E+03	0.03	0.05	0.03	0.04
1.00E+02	9.83E+01	0.09	0.11	0.11	0.11
6.00E+01	5.90E+01	0.14	0.11	0.13	0.13

Table 8: Within-laboratory Precision of cobas® BKV*

Titer data are considered to be log-normally distributed and are analyzed following logo transformation. Standard deviations (SD) columns present the total of the log-transformed titer for each of the three reagent lots.

Table 9: Lognormal Percent Coefficient of Variation (%CV) of cobas® BKV by Positive Panel and Contributing Components of Variance*

Nominalconcen-trationTiter(IU/mL)	Nominalconcen-trationLog10 titer(IU/mL)	Assignedconcen-trationTiter(IU/mL)	Assignedconcen-trationLog10 titer(IU/mL)	N	Instrument/Operator%CV	Lot%CV	Day%CV	Run%CV	WithinRun%CV	Total%CV
1.00E+06	6.00	9.83E+05	5.99	72	2%	5%	3%	2%	5%	8%
1.00E+05	5.00	9.83E+04	4.99	71	3%	6%	3%	0%	8%	11%
1.00E+04	4.00	9.83E+03	3.99	70	3%	7%	5%	3%	9%	13%
6.00E+03	3.78	5.90E+03	3.77	72	2%	8%	2%	1%	8%	12%
1.00E+02	2.00	9.83E+01	1.99	72	5%	8%	6%	4%	24%	26%
6.00E+01	1.78	5.90E+01	1.77	71	4%	14%	7%	15%	29%	36%

Titer data are considered to be log-normally distributed and the %CV values are analyzed as Lognormal CV(%) = sqrt(10^[SD^2 * In(10)] - 1) * 100%

4.8. Analytical Specificity

The analytical specificity of cobas® BKV was evaluated by testing a panel of microorganisms at a concentration of 1.00E+06 units/mL (CFU/mL, cells/mL, CCU/mL, IFU/mL) for bacteria and yeast and between 1.00E+05 units/mL and 1.00E+06 units/mL (copies/mL, TCIDso/mL, IU/mL, cells/mL) for viruses. Microorganisms were diluted into BKV DNA negative human EDTA plasma as well as human EDTA plasma containing (100 IU/mL) BKV DNA. The specific organisms tested are listed in Table 10. Each sample was tested in replicates of three. None of the non-BKV pathogens interfered with test performance at the concentrations tested. Negative results were obtained with cobas® BKV for all microorganism samples without BKV target and positive results were obtained for all of the microorganism samples with BKV target.

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Furthermore, the mean logio titer of each of the positive BKV samples containing potentially cross-reacting organisms was within ± 0.5 log10 of the mean log10 titer of the respective positive spike control.

Viruses	Bacteria	Yeast
Adenovirus Type 5	Propionibacterium acnes	Aspergillus niger
Cytomegalovirus	Staphylococcus aureus	Candida albicans
Epstein-Barr Virus	Chlamydia trachomatis	Cryptococcus neoformans
Hepatitis B Virus	Clostridium perfringens	-
Hepatitis C Virus	Enterococcus faecalis	-
Herpes Simplex Virus Type 1	Escherichia coli	-
Herpes Simplex Virus Type 2	Klebsiella pneumoniae	-
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 pyogenes	-
Human Papillomavirus	Mycoplasma pneumoniae	-
JC virus	Salmonella enterica	-
Parvovirus B19	Streptococcus pneumoniae	-
Simian Virus 40	-	-
Varicella-Zoster Virus	-	-

Table 10: Microorganisms Tested for Cross-Reactivity

Interfering Substances 4.9.

Elevated levels of triglycerides (37 mmol/L), conjugated bilirubin (0.2 g/L), unconjugated bilirubin (0.2 g/L), albumin (60 g/L), hemoglobin (2 g/L) and human DNA (2 mg/L) in samples were tested in the presence (100 IU/mL) and absence of BKV DNA. The tested endogenous interferences were shown not to interfere with the test performance of cobas® BKV.

In addition, drug compounds listed in Table 11 were tested at three times the Cmax in presence (100 IU/mL) and absence of BKV DNA.

All potentially interfering substances have been shown to not interfere with the test performance. Negative results were obtained with cobas® BKV for all samples without BKV target and positive results were obtained on all of the samples with BKV target. Furthermore, the mean

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logio titer of each of the positive BKV samples containing potentially interfering substances was within ± 0.5 log10 of the mean log10 titer of the respective positive spike control.

Class of drug	Generic drug name
Antimicrobial	Cefotetan
Antimicrobial	Clavulanate potassium
Antimicrobial	Fluconazole
Antimicrobial	Piperacillin
Antimicrobial	Tazobactam sodium
Antimicrobial	Sulfamethoxazole
Antimicrobial	Ticarcillin disodium
Antimicrobial	Trimethoprim
Antimicrobial	Vancomycin
Antimicrobial	Micafungin
Compounds for Treatment of Herpes Viruses	Ganciclovir
Compounds for Treatment of Herpes Viruses	Valganciclovir
Compounds for Treatment of Herpes Viruses	Acyclovir
Compounds for Treatment of Herpes Viruses	Cidofovir
Compounds for Treatment of Herpes Viruses	Foscarnet
Compounds for Treatment of Herpes Viruses	Letermovir
Immune suppressant	Azathioprine
Immune suppressant	Cyclosporine
Immune suppressant	Everolimus
Immune suppressant	Mycophenolate mofetil
Immune suppressant	Prednisone
Immune suppressant	Sirolimus
Immune suppressant	Tacrolimus
Immune suppressant	Mycophenolic acid

Table 11: Drug Compounds Tested for Interference with the Quantitation of BKV DNA by cobas® BKV

4.10. Cross Contamination

The cross-contamination rate for cobas® BKV was determined by testing 240 replicates of a BKV-negative matrix sample and 225 replicates of a high titer BKV DNA sample at approximately 2.00E+07 IU/mL. In total, five runs were performed with positive and negative samples in a checkerboard configuration.

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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%).

CLINICAL PERFORMANCE EVALUATION 5.

Reproducibility of cobas® BKV 5.1.

The reproducibility of cobas® BKV was evaluated across factors (reagent lot, test site, batch and testing days) that could affect reported results in routine clinical testing. The evaluation was conducted at 3 testing sites, using 3 reagent lots per site, of a positive and a negative sample panel with a total number 270 tests per concentration (not including controls). The panels were made from EDTA plasma that was BKV VCA IgG negative and were tested for BKV with a plasma NAT release protocol, and spiked with a BKV WHO international standard, or BKV genotype Ib (most common genotype) cultured virus DNA. Two operators at each site tested each of three reagent lots for 5 days. One run per operator (1 run = 1 batch = 1 panel + 3 controls) was performed each day and 3 replicates of each panel member were included in each run. The evaluation results are summarized in Table 12.

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Table 12: Attributable Percentage of Total Variance, Total Precision Standard Deviation, and lognormal CV(%) of BKV DNA Concentration (log10 IU/mL) bv Positive Panel Member

Expected BKV DNAConcentration(log10 IU/mL)	Observed Meana BKV DNAConcentration(log10 IU/mL)	NumberofTestsb	Lot%TVc(CV%)d	Site%TVc(CV%)d	Day/Operator%TVc(CV%)d	Batch%TVc(CV%)d	Within-Batch%TVc(CV%)d	TotalPrecisionSDe	TotalPrecisionLog-normalCV(%)d
1.81	1.74	270	9%(20.63)	6%(17.69)	0%(0.00)	7%(19.15)	78%(68.05)	0.304	79.43
3.70	3.52	270	10%(9.79)	10%(9.57)	14%(11.44)	25%(15.16)	40%(19.38)	0.131	30.91
4.70	4.51	270	3%(4.42)	24%(13.46)	0%(0.00)	56%(20.58)	17%(11.27)	0.118	27.71
5.70	5.54	270	7%(5.66)	28%(11.50)	0%(0.00)	40%(13.85)	25%(10.84)	0.094	21.94
7.70	7.62	269	4%(3.27)	49%(11.00)	0%(0.00)	13%(5.60)	34%(9.10)	0.068	15.74

a Calculated using SAS MIXED procedure.

b Number of valid tests with detectable DNA level.

c %TV = Percent contribution to Total Variance.

d CV% = Lognormal percent coefficient of variation = sqrt(10^[SD^2 * ln(10)] - 1) * 100

e Calculated using the total variability from the SAS MIXED procedure

Note: The table only includes results with detectable DNA level. SD = standard deviation. cv = coefficient of variation; and BKV = BK Virus

cobas® BKV showed excellent clinical reproducibility at concentrations throughout the linear range. In addition, the system detected 100% of the 3 x LLoQ samples. The cobas® 6800 and cobas® 8800 Systems share a modular design and they showed equivalency when using cobas® BKV. All of the estimated 95% confidence limits (CLs) for the difference between 2 measurements from the same subject were within ± 0.84 log10 copies/mL, indicating that the assay can assess changes in BKV DNA levels that are thought to be clinically significant.

Of the 270 valid tests for the negative panel members performed on the cobas® 6800/8800 Systems, all samples showed a "Target Not Detected" result, therefore the negative percent agreement (NPA) was 100% with the 95% Exact CI of 98.6% to 100%.

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Clinical Performance of cobas® BKV 5.2.

The clinical performance of cobas® BKV was further evaluated at three testing sites by measuring BKV DNA levels in clinical samples (neat and diluted) of BKV infected and non-infected patients and contrived EDTA plasma samples spiked with cultured BKV virus, compared with a well-established laboratory developed nucleic acid test (LDT) (comparator BKV LDT).

From all samples tested with cobas® BKV and the comparator BKV test, there were a total of 550 samples (217 neat and 303 diluted clinical samples from 129 transplant subjects and 30 contrived samples) that were valid on both assays and evaluable for the clinical concordance analysis (Table 13).

Table 13: Concordance Analysis Between cobas® BKV and the Comparator LDT on
BKV DNA Level Results For all Samples

cobas® BKV(log₁₀ IU/mL)	ComparatorBKV LDT(log₁₀ IU/mL)Target NotDetected	ComparatorBKV LDT(log₁₀ IU/mL)< LLoQ(< 2.3)	ComparatorBKV LDT(log₁₀ IU/mL)2.3 to < 3.0	ComparatorBKV LDT(log₁₀ IU/mL)3.0 to < 3.7	ComparatorBKV LDT(log₁₀ IU/mL)3.7 to 4.4	ComparatorBKV LDT(log₁₀ IU/mL)> 4.4	Total
Target Not Detected	107	7	5	0	0	0	119
< LLoQ (< 2.3)	23	51	39	0	0	0	113
2.3 to < 3.0	0	3	40	62	1	0	106
3.0 to < 3.7	0	0	1	71	42	0	114
3.8 to 4.4	0	0	0	0	26	26	52
> 4.4	0	0	0	0	1	45	46
Total	130	61	85	133	70	71	550
Column Agreement(%)	(130/130)100.0%	(61/61)100.0%	(80/85)94.1%	(133/133)100.0%	(69/70)98.6%	(71/71)100.0%
(95% Score CI)a	(97.1%,100%)	(94.1%,100.0%)	(87.0%,97.5%)	(97.2%,100.0%)	(92.3%,99.7%)	(94.9%,100.0%)

Note: CI = Confidence Interval; LLoQ = lower limit of quantitation of Comparator BKV LDT (200 IU/mL). Standard Deviation of Comparator BKV LDT estimated at 0.37 log10 IU/mL ffrom Indiana University BKV LDT analytical precision study).

Analyte concentration of 3.0 log10 IU/mL represented LLoQ + 20, 3.7 log10 IU/mL represented LLoQ + 40 and 4.4 log10 IU/mL represented LLoQ + 60 with a range interval of 20.

Paired samples evaluable for clinical concordance analysis were included in this table. aAssumed independence between all samples.

Discordant results were defined as those that are more than one box away from the diagonal

(indicated by shading). For Target Not Detected (TND) by LDT Column Agreement the cobase

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BKV Target Not Detected and < LLoQ (< 2.3) cells were combined. The rationale for adding the adjacent <LLoO and TND cells for the TND column is that the difference between a TND and <LLoQ is not clinically meaningful and that these are analytically at the lower end of the measuring range, which may be impacted by random error.

Of the 43 BKV DNA-negative samples collected for the estimation of the NPA with the cobas® BKV, all 43 samples were negative by cobas® BKV, therefore the NPA was 100% with the 95% Exact CI of 91.8% to 100%.

Concordance between cobas® BKV and the comparator BKV LDT was also evaluated using different clinical thresholds (Table 14).

Table 14: Summary of Concordance of cobas® BKV and Comparator BKV LDT using Different Thresholds for All Samples

Thresholds*	Percent Agreement< Threshold95% Cl(n/N)	Percent Agreement≥ Threshold95% Cl(n/N)
Target Not Detected	82.3% (107/130)(74.8%, 87.9%)	97.1% (408/420)(95.1%, 98.4%)
LLoQ (2.3 Log10 IU/mL)	98.4% (188/191)(95.5%, 99.5%)	87.7% (315/359)(83.9%, 90.7%)
3.0 Log10 IU/mL	99.6% (275/276)(98.0%, 99.9%)	77.0% (211/274)(71.7%, 81.6%)
4.0 Log10 IU/mL	100.0% (447/447)(99.1%, 100.0%)	67.0% (69/103)(57.4%, 75.3%)

Note: Samples with a Target Not Detected results were categorised as <threshold value in IU/mL. LLoQ = lower limit of quantitation of Comparator BKV LDT (200 IU/mL = 2.3 log10 IU/mL). 95% confidence interval (CI) calculated by Score method assuming independence between all samples.

Thresholds of 1000 IU/ml = 3.0 Log10 IU/ml and 10,000 IU/ml = 4.0 Log10 IU/ml.

From all samples tested with cobas® BKV that were BKV positive with the comparator BKV test, there were a total of 313 (133 neat and 159 diluted clinical samples from 68 transplant subjects and 21 contrived samples), which were evaluable for the correlation analysis at the three testing sites (Figure 4).

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Correlation between cobas® BKV and Comparator BKV LDT for All Samples: Figure 4: Deming Linear Regression Plot of DNA Levels (log10 IU/mL)

Image /page/22/Figure/1 description: This scatter plot shows the correlation between cobas BKV and Comparator BKV LDT, both measured in log10 IU/mL. The data points are clustered around a regression line, indicating a positive correlation. The Deming Regression analysis (N=313) yields the equation Y = -0.429 + 1.019X, with an R-value of 0.96. The 95% confidence intervals for the intercept are (-0.592, -0.266), and for the slope are (0.975, 1.063).

Additional bias plot analysis of DNA level differences indicated a systematic difference between both assays that is constant across the overlapping linear range. The 95% CI of the intercept of the fitted line in the bias plots was (-0.404 to -0.168), which is within ±0.74 log10 IU/mL (± 2 times analytical precision standard deviation of comparator BKV LDT). Furthermore, the mean bias was estimated at -0.357 log10 IU/mL and using the equation of the fitted line in the bias plots, the systematic difference between both assays was -0.343 log10 IU/mL and -0.362 log10 IU/mL for samples with DNA levels at 3 and 4 log10 IU/mL, respectively.

CONCLUSIONS 6.

The conclusions drawn from the nonclinical and clinical studies demonstrate that the device is as safe, as effective, and performs as well as the predicate device.

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7. REFERENCES

Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over 1. contamination in polymerase chain reactions. Gene. 1990;93:125-8. PubMed PMID: 227421.
Savva R. McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-2. excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-93. PubMed PMID: 7845459.
1. Mol CD, Arvai AS, Slupphau G, et al. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell. 1995;80:869-78. PubMed PMID: 7697717.
Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of 4. specific DNA sequences. Biotechnology (NY). 1992;10:413-7. PubMed PMID: 1368485.
1. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. doi: 10.1101/gr.6.10.986. PubMed PMID: 8908518.
National Institute for Biological Standards and Control. 1st WHO International Standard for 6. BK Virus DNA (NIBSC 14/212) version 2.0: Hertfordshire, England; NIBSC, 2016.

Regulation Number and Section

§ 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.