cobas® BKV is an in vitro nucleic acid amplification test for the quantitation of BKV) DNA in human EDTA plasma and urine stabilized in cobas® PCR media on the cobas® 6800/8800 Systems.

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

In urine stabilized in cobas® PCR Media, cobas® BKV is intended for use as an aid in the management of BKV in transplant patients.

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 (Figure 1) 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-OS 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).13 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.45 The fluorescent signal of the intact probes is suppressed by the quencher dye. During the PCR amplification step, hybridization of the probe to 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-OS.

AI/ML Overview

This document describes the acceptance criteria and supporting studies for the cobas® BKV device for the quantitation of BK virus (BKV) DNA in human urine. The information is extracted from a 510(k) summary.

1. Table of Acceptance Criteria and Reported Device Performance

Acceptance Criteria	Reported Device Performance (cobas® BKV in Urine)
Limit of Detection (LoD): 95% hit rate for BKV DNA.	12.2 IU/mL (WHO International Standard, 95% confidence range: 9.2-18.3 IU/mL). Achieved ≥95% hit rate for subgroups Ia, Ic, and subtypes II, III, IV at 12.2 IU/mL.
Linear Range: Accuracy within ± 0.2 log10.	7.41E+01 IU/mL to 7.41E+08 IU/mL. Maximum deviation of linear regression from better fitting non-linear regression was ≤ ± 0.2 log10 for all tested genotypes within the linear range.
Lower Limit of Quantitation (LLoQ): Mean deviation between observed and assigned log10 titer ≤ ± 0.3 log10 (based on upper 95% CI of worst performing lot). Total Analytical Error (TAE) ≤ 1 log10.	200 IU/mL. Mean deviation between observed and assigned log10 titer was ≤ 0.3 log10. TAE was ≤ 0.44 for all lots and concentrations (Table 7).
Precision (Within-Laboratory): High precision across the concentration range.	Demonstrated high precision across a concentration range of 7.41E+02 IU/mL to 7.41E+05 IU/mL (Table 8, Table 9). Total %CV ranged from 7% (highest concentration) to 23% (lowest concentration).
Analytical Specificity: No interference from listed microorganisms; mean log10 titer of positive BKV samples with interfering organisms within ± 0.5 log10 of spike control.	None of the tested non-BKV pathogens (bacteria, yeast, viruses in Table 10) interfered. Mean log10 titer of positive BKV samples was within ± 0.5 log10 of the spike control.
Interfering Substances: No interference from listed endogenous substances and drug compounds, with mean log10 titer of positive BKV samples with interfering substances within ± 0.5 log10 of spike control.	All listed endogenous interferences and drug compounds (except talcum powder at >0.05%) did not interfere. Mean log10 titer of positive BKV samples was within ± 0.5 log10 of the spike control.
Cross Contamination: Zero cross-contamination rate with a low upper 95% confidence interval.	0.0% (upper one-sided 95% CI 1.24%) with 240 replicates of negative samples.
Clinical Reproducibility: Acceptable reproducibility; 100% detection of 3 x LLoQ samples; 95% CI for difference between 2 measurements within ± 0.20 log10 copies/mL.	Acceptable clinical reproducibility. 100% of 3 x LLoQ samples detected. All estimated 95% CLs for the difference between 2 measurements from the same subject were within ± 0.20 log10 copies/mL.
Negative Percent Agreement (NPA) (Clinical): High negative percent agreement.	100% (95% Exact CI of 94.1% to 100%) for 61 valid negative samples.
Clinical Concordance with LDT: High agreement at various thresholds and strong correlation.	Concordance analysis with comparator LDT showed high agreement (e.g., 93.9% at Target Not Detected threshold, 99.5% at LLoQ threshold). Deming linear regression showed a strong correlation with CI for intercept within ±0.5 log10 IU/mL.

2. Sample Sizes Used for the Test Set and Data Provenance

The studies focused on analytical performance (Limit of Detection, Linearity, Precision, Specificity, Interference, Cross-contamination) and clinical performance (Reproducibility, Clinical Concordance).

Limit of Detection (LoD):
- WHO International Standard: 63 replicates per concentration level (total 7 levels, x3 lots = 1323 replicates).
- Subgroups/Subtypes Verification: 63 replicates per concentration level for each genotype (total 5 genotypes, 3 levels each, x3 lots = 2835 replicates).
Linear Range:
- Main linearity: 36 replicates per panel member (10 panel members, x3 lots = 1080 replicates).
- Genotype linearity: 12 replicates per level for each genotype (8 panel members each, 5 genotypes, x3 lots = 1440 replicates).
Lower Limit of Quantitation (LLoQ): Data from the Linearity study at 100, 200, and 300 IU/mL concentrations.
Precision (Within-Laboratory): 72 replicates for each of 5 dilution levels (x3 lots = 1080 replicates).
Analytical Specificity: 3 replicates for each of the microorganisms listed in Table 10, both in BKV-negative and BKV-positive urine (number of microorganisms not explicitly totalled, but substantial).
Interfering Substances: Replicates for each substance in presence and absence of BKV DNA (number of replicates not explicitly stated, but implies multiple for each substance in Table 11).
Cross Contamination: 240 replicates of BKV-negative matrix samples, 225 replicates of high titer BKV DNA urine samples.
Clinical Reproducibility: 270 tests per concentration (5 concentrations, total 1350 tests, not including controls).
Clinical Performance / Concordance: 308 neat urine samples from 84 transplant subjects (for concordance analysis). 61 negative samples for NPA. 153 BKV positive urine samples from 55 transplant subjects (for correlation analysis).

Data Provenance: The document implies that the non-clinical studies were conducted internally or at authorized labs. The clinical performance evaluation was conducted at 3 testing sites, suggesting multi-site prospective data collection. The data samples were human EDTA plasma and urine. The origin of the samples (country) is not explicitly stated in the provided text. The clinical study used a retrospective cohort of samples from transplant patients.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

The ground truth for the analytical studies was established based on known concentrations of BKV international standards or armored DNA. For clinical performance, the comparator was a "well-established laboratory developed nucleic acid test (LDT)".

The document does not mention the use of experts to establish ground truth for the test sets in the typical sense of human readers for image-based diagnostics. The "ground truth" for this diagnostic device study is based on the highly controlled properties of the spiked samples (known concentrations, genotypes) for analytical performance, and the results from a comparator LDT for clinical concordance.

4. Adjudication Method for the Test Set

Not applicable in the context of this in vitro diagnostic device, as the "ground truth" is based on quantitative measurements and known concentrations, not subjective expert assessment requiring adjudication.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This device is an in vitro nucleic acid amplification test (NAAT) for quantitative measurement of BKV DNA. It is not an AI-assisted diagnostic device requiring human reader input or interpretation in the way an imaging diagnostic device would. Therefore, an MRMC comparative effectiveness study involving human readers and AI assistance is not relevant to this submission.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the studies described are for the standalone performance of the cobas® BKV system, which is an automated molecular diagnostic assay. The system performs "fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection." The results are "assigned... by the cobas® 6800/8800 software." While "results are intended to be read and analyzed by a qualified licensed healthcare professional in conjunction with clinical signs and symptoms and relevant laboratory findings," the primary performance metrics are based on the direct output of the automated system.

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

Analytical Studies: The ground truth for analytical studies (LoD, Linearity, Precision, Specificity, Interference) was established using known concentrations of BKV DNA, including the WHO International Standard (NIBSC 14/212), BKV armored DNA, and clinical specimens diluted to specific concentrations. Samples were spiked into BKV-negative urine.
Clinical Studies: For clinical concordance, the ground truth was based on the results from a "well-established laboratory developed nucleic acid test (LDT) (comparator BKV LDT)" on actual clinical urine samples. DNA sequencing was also used in some cases to confirm BKV presence in discordant results.

8. The Sample Size for the Training Set

The document describes performance evaluation studies (validation and verification) rather than a machine learning model development process that typically involves distinct training and test sets.
Therefore, a separate "training set" sample size for a machine learning algorithm is not applicable in the context of this in vitro diagnostic device, which is based on established molecular biological techniques (PCR).

9. How the Ground Truth for the Training Set Was Established

As this is not an AI/ML-based device with a "training set," this question is not applicable. The ground truth for the evaluation of the device was established through known concentrations of viral standards and comparison to a comparator LDT, as described in point 7.

Summary

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Image /page/0/Picture/0 description: The image shows the logos of the Department of Health & Human Services and the U.S. Food & Drug Administration (FDA). The Department of Health & Human Services logo is on the left, featuring a stylized human figure. To the right is the FDA logo, with the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG" in blue, and "ADMINISTRATION" in a smaller font size below.

January 29, 2021

Roche Molecular Systems, Inc. Raji Grewal Sr. Regulatory Specialist 4300 Hacienda Drive Pleasanton, California 94588-2722

Re: K203220

Trade/Device Name: cobas BKV Regulation Number: 21 CFR 866.3183 Regulation Name: Ouantitative Viral Nucleic Acid Test for Transplant Patient Management Regulatory Class: Class II Product Code: QLX Dated: October 30, 2020 Received: November 2, 2020

Dear Raji Grewal:

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

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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 803) 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 (OS) 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,

Maria Garcia, Ph.D. Branch Chief 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) K203220

Device Name cobas® BKV

Indications for Use (Describe)

cobas® BKV is an in vitro nucleic acid amplification test for the quantitation of BKV) DNA in human EDTA plasma and urine stabilized in cobas® PCR media on the cobas® 6800/8800 Systems.

In urine stabilized in cobas® PCR Media, cobas® BKV is intended for use as an aid in the management of BKV in transplant patients.

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)
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	Raji GrewalPhone: (925) 368-0246FAX: (925) 225-0207Email: raji.grewal@roche.com
Date Prepared	October 30, 2020
Proprietary Name	cobas® BKVfor use on cobas® 6800/8800 Systems
Classification Name	Quantitative viral nucleic acid test for transplant patient management
Product Codes	QLX: 21 CFR 866.3183
Predicate Devices	cobas® BKV (K202215)
Establishment Registration	Roche Molecular Systems, Inc. (2243471)

DEVICE DESCRIPTION 1.

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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 Quantitative nucleic acid test for use on the cobas 6800/8800 Systems". The box on the right is labeled "cobas EBV/BKV Control Kit Positive control kit for use on the cobas 6800/8800 Systems". Both boxes have the Roche logo and the text "Rx Only" and "IVD".

INDICATIONS FOR USE 2.

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

In urine stabilized in cobas® PCR Media, cobas® BKV is intended for use as an aid in the management of BKV in transplant patients.

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® BKV (K202215)(Table 1).

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	Submitted Device:cobas® BKV	Predicate Device:cobas® BKV (K202215)
Regulation Number	21 CFR 866.3183	Same
Regulation Name	Quantitative viral nucleic acid test fortransplant patient management	Same
Product Code	QLX	Same
Intended Use	cobas® BKV is an in vitro nucleic acidamplification test for the quantitation of BKvirus (BKV) DNA in human EDTA plasmaand urine stabilized in cobas® PCR Mediaon the cobas® 6800/8800 Systems.In EDTA plasma, cobas® BKV is intendedfor use as an aid in the management ofBKV in transplant patients. In patientsundergoing monitoring of BKV in EDTAplasma, serial DNA measurements can beused to indicate the need for potentialtreatment changes and to assess viralresponse to treatment.In urine stabilized in cobas® PCR Media,cobas® BKV is intended for use as an aidin the management of BKV in transplantpatients.The results from cobas® BKV are intendedto be read and analyzed by a qualifiedlicensed healthcare professional inconjunction 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 blood or blood productsor human cells, tissues, and cellular andtissue-based products (HCT/Ps).	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 in EDTA plasma, serial DNAmeasurements can be used to indicate theneed for potential treatment changes andto assess viral response to treatment.The results from cobas® BKV are intendedto be read and analyzed by a qualifiedlicensed healthcare professional inconjunction 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 blood or blood productsor human cells, tissues, and cellular andtissue-based products (HCT/Ps).
Conditions for use	For prescription use	Same
Sample Types	EDTA - plasma, Urine	EDTA - plasma
Analyte Targets	BK Virus	Same
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® BKV (K202215)
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

4.1. Limit of Detection (LoD)

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 12.2 IU/mL for neat urine.

4.1.1. WHO International Standard

The limit of detection of cobas® BKV for the WHO International Standard was determined by analysis of serial dilutions of the 1* WHO BKV International Standard obtained from NIBSC (NIBSC 14/212), in BKV-negative pooled urine stabilized in cobas® PCR Media. 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 pooled urine stabilized in cobas® PCR Media 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 12.2 IU/mL with a 95% confidence range of 9.2-18.3 IU/mL in neat urine. The lowest concentration with a hit rate ≥ 95% is 10.0 IU/mL in neat urine.

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Input Titer Concentration(BKV DNA IU/mL)*	Number of Validreplicates (N)	Number of Positives(n)	Hit Rate(n/N) x100
40.0	63	63	100.0
20.0	63	63	100.0
10.0	63	60	95.2
5.0	63	47	74.6
2.5	63	25	39.7
1.25	63	26	41.3
0	63	0	0.0

Table 2: Limit of Detection in Urine, Lot 1

LoD by PROBIT at 95% hit rate: 12.2 IU/mL. 95% confidence range: 9.2–18.3 IU/mL

Urine samples tested stabilized in cobas® PCR Media. Input titer concentration used for neat urine.

Table 3: Limit of Detection in Urine, Lot 2

Input titer Concentration(BKV DNA IU/mL)*	Number of ValidReplicates (N)	Number of Positives(n)	Hit Rate(n/N) x100
40.0	63	63	100.0
20.0	63	63	100.0
10.0	63	60	95.2
5.0	63	42	66.7
2.5	63	32	50.8
1.25	63	17	27.0
0	63	0	0.0

LoD by PROBIT at 95% hit rate: 11.9 IU/mL. 95% confidence range: 9.2–17.3 IU/mL

Urine samples tested stabilized in cobas® PCR Media. Input titer concentration used for neat urine.

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Input Titer Concentration(BKV DNA IU/mL)*	Number of Validreplicates (N)	Number of Positives(n)	Hit Rate(n/N) x100
40.0	63	63	100.0
20.0	63	63	100.0
10.0	63	61	96.8
5.0	63	46	73.0
2.5	63	39	61.9
1.25	63	19	30.2
0	63	0	0.0

Limit of Detection in Urine, Lot 3 Table 4:

LoD by PROBIT at 95% hit rate: 10.1 IU/mL. 95% confidence range: 7.8–14.7 IU/mL

Urine samples tested stabilized in cobas® PCR Media. Input titer concentration used for calculation based on neat urine.

Limit of Detection for subgroups la, Ic and Subtypes II, III and IV 4.2.

BKV armored DNA for subgroup Ic and clinical specimens for subgroup Ia and subtypes II, III and IV were diluted to three different concentration levels in BKV-negative urine stabilized in cobas® PCR Media. 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 12.2 IU/mL – cobas® BKV detected BKV DNA for subgroups Ia and Ic, and subtypes II, III and IV at a concentration of 12.2 IU/mL with a ≥ 95% hit rate.

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

Genotype	Test Concentration	Number of Valid Replicates (N)	Number of Positives (n)	Hit Rate(n/N)x100
Subgroup la	6.1 IU/mL	63	53	84.1%
Subgroup la	12.2 IU/mL	63	61	96.8%
Subgroup la	18.3 IU/mL	63	62	98.4%
Subgroup Ic	6.1 IU/mL	63	50	79.4%
Subgroup Ic	12.2 IU/mL	63	62	98.4%
Subgroup Ic	18.3 IU/mL	63	63	100.0%
Subtype II	6.1 IU/mL	63	56	88.9%
Subtype II	12.2 IU/mL	63	61	96.8%
Subtype II	18.3 IU/mL	63	63	100.0%

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Genotype	Test Concentration	Number of ValidReplicates (N)	Number ofPositives (n)	Hit Rate(n/N)x100
Subtype III	6.1 IU/mL	63	60	95.2%
Subtype III	12.2 IU/mL	63	62	98.4%
Subtype III	18.3 IU/mL	63	દિર	100.0%
Subtype IV	6.1 IU/mL	63	54	85.7%
Subtype IV	12.2 IU/mL	63	દિર	100.0%
Subtype IV	18.3 IU/mL	63	63	100.0%

4.3. Linear range

Linearity of cobas® BKV was evaluated using a dilution series consisting of 10 panel members using a clinical specimen (BKV subgroup Ib) spanning the assay linear range. A high titer lambda DNA stock was used to prepare 12 panel members spanning the entire 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 2.

cobas® BKV was demonstrated to be linear from 7.41E+01 IU/mL to 7.41E+08 IU/mL and shows an absolute deviation from the better fitting non-linear regression of less or equal than ± 0.1 log10 in pooled urine stabilized in cobas® PCR Media (see Figure 2). Across the linear range, the accuracy of the test was within ± 0.2 log10.

The lower limit of quantitation (LLoQ) was set to 200 IU/mL, to include the mean deviation between the observed vs. the assigned log10 titer (Accuracy) being equal or less than ±+/-0.3 log10, based on the upper 95% confidence interval of the worst performing lot using clinical specimen and calculated based on a goal for acceptable total analytical error (TAE) of < 1.0 log10. where TAE = lbias| + 2 standard deviations in alignment with the CLSI EP-17A guideline, and TAE = SQUARE 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 2.0E+02 IU/mLL21 to-1.0E+08 IU/mL. The results of calculation and claimed LLoQ are shown in Table 7.

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Figure 2: Linear Range Determination in Urine

Image /page/11/Figure/1 description: The image is a scatter plot that shows the relationship between AssignedLogTiter and ObservedLogTiter. The plot includes three trend lines: a linear fit, a polynomial fit of degree 2, and a polynomial fit of degree 3. The data points are clustered in a linear fashion, and the trend lines are all very similar, indicating a strong correlation between the two variables. The x and y axis range from 2 to 9.

Linearity for subgroups la, Ic and subtypes II, III and IV 4.4.

The dilution series used in the verification of subtype/subgroup linearity study of cobas® BK V 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 urine stabilized in cobas® PCR Media. 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.

Genotype	Linear Regression	Better Fitting Higher OrderModel Regression	Maximum Difference BetweenLinear Regression and theBetter Fitting Higher OrderModel(log10 IU/mL)
Subgroup la	y = 0.9756016x +0.2367214	y = -0.0081339x3 + 0.1373308x2 +0.2602376x + 1.3507964	0.12
Subgroup Ic	y = 0.9773177x +0.2051674	y = -0.005087x3 + 0.0902742x2 +0.4865276x + 0.9928655	0.10
Subtype II	y = 0.9762885x +0.2271355	y = -0.0072558x3 + 0.1259492x2 +0.3032666x + 1.2971372	0.12

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

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Genotype	Linear Regression	Better Fitting Higher OrderModel Regression	Maximum Difference BetweenLinear Regression and theBetter Fitting Higher OrderModel(log10 IU/mL)
Subtype III	v = 0.9762129x +0.2270439	y = -0.0081255x3 + 0.1370235x2 +0.2645895x + 1.327922	0.12
Subtype IV	y = 0.9758502x +0.2028958	y = -0.0086141x3 + 0.1427957x2 +0.2499516x + 1.2930774	0.13

Lower Limit of Quantitation 4.5.

The analysis for LLoQ of cobas® BKV in urine was performed with data obtained from the Linearity study at concentration levels of 100 IU/mL and 300 IU/mL and 300 IU/mL. The LLoQ was set at the concentration level of 200 IU/mL to include the mean deviation between the observed vs. the assigned log10 titer (Accuracy) being equal or less than ± 0.3 log10, based on the upper 95% confidence interval of the worst performing lot using clinical specimen. The LLoQ within the linear range meets the acceptance criterion for the Total Analytical Error (JBias| + 2x SD) (TAE). The TAE criterion is ≤≤ 1 log10.

The results of calculation and claimed LLoQ are shown in Table 7. The LLoQ is 200 IU/mL.

Lot	NominalConcentration(IU/mL)	log10 TiterAssigned	Mean log10TiterObserved	SD(log10)	AbsoluteBias	TAE( Bias + 2xSD)	Difference BetweenMeasurements inSD (= SQRT(2) x 2xSD)
1	3.00E+02	2.44	2.50	0.05	0.06	0.15	0.13
1	2.00E+02	2.27	2.37	0.07	0.11	0.25	0.21
1	1.00E+02	1.96	2.06	0.11	0.10	0.32	0.32
2	3.00E+02	2.44	2.63	0.05	0.19	0.29	0.14
2	2.00E+02	2.27	2.49	0.06	0.22	0.34	0.17
2	1.00E+02	1.96	2.22	0.09	0.26	0.44	0.25
3	3.00E+02	2.44	2.58	0.07	0.13	0.27	0.20
3	2.00E+02	2.27	2.41	0.07	0.15	0.28	0.19
3	1.00E+02	1.96	2.14	0.09	0.17	0.36	0.26
3 Lotscombined	3.00E+02	2.44	2.57	0.08	0.13	0.28	0.22

LLoQ of cobas® BKV using Clinical Sample (Urine) Table 7:

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Lot	NominalConcentration(IU/mL)	log10 TiterAssigned	Mean log10TiterObserved	SD(log10)	AbsoluteBias	TAE( Bias + 2xSD)	Difference BetweenMeasurements inSD (= SQRT(2) x 2xSD)
3 Lotscombined	2.00E+02	2.27	2.42	0.08	0.16	0.32	0.23
3 Lotscombined	1.00E+02	1.96	2.14	0.12	0.18	0.41	0.33

4.6. Precision - within laboratory

Precision of cobas® BKV was determined by analysis of serial dilutions of high titer BKV DNA (subgroup Ib) in BKV-negative pooled urine stabilized in cobas® PCR Media. Five dilution levels were tested in 72 replicates for each level across three lots of cobas® BKV reagents using two 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 7.41E+02 IU/mL to 7.41E+05 IU/mL.

NominalConcentration(IU/mL)	AssignedConcentration[IU/mL]	Urine Stabilizedin cobas® PCRMediaLot 1 SD	Urine Stabilized incobas® PCR MediaLot 2 SD	Urine Stabilizedin cobas® PCRMediaLot 3 SD	Urine Stabilizedin cobas® PCRMedia all LotsPooled SD
1.00E+08	7.41E+07	0.02	0.01	0.02	0.02
1.00E+06	7.41E+05	0.02	0.02	0.02	0.02
1.00E+05	7.41E+04	0.02	0.03	0.02	0.03
1.00E+04	7.41E+03	0.03	0.03	0.03	0.03
6.00E+03	4.44E+03	0.04	0.03	0.04	0.03
1.00E+03	7.41E+02	0.05	0.05	0.04	0.05
3.00E+02	2.22E+02	0.08	0.07	0.05	0.07

Within-laboratory Precision of cobas® BKV* (in Stabilized Urine) Table 8:

Titer data are considered to be log-normally distributed and are analyzed following log10 transformation. Standard deviations (SD) columns present the total of the log-transformed titer for each of the three reagent lots.

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NominalConcentrationTiter (IU/mL)	NominalConcentrationLog10 Titer(IU/mL)	AssignedConcentrationTiter (IU/mL)	AssignedconcentrationLog10 titer(IU/mL)	N	Instrument/ Operator%CV	Lot%CV	Day%CV	Run%CV	WithinRun%CV	Total%CV
1.00E+08	8.00	7.41E+07	7.87	57**	3%	5%	1%	1%	4%	7%
1.00E+06	6.00	7.41E+05	5.87	72	2%	6%	1%	2%	4%	8%
1.00E+05	5.00	7.41E+04	4.87	72	4%	7%	1%	2%	5%	10%
1.00E+04	4.00	7.41E+03	3.87	71	6%	9%	2%	1%	6%	12%
6.00E+03	3.78	4.44E+03	3.65	72	6%	7%	0%	1%	7%	11%
1.00E+03	3.00	7.41E+02	2.87	72	3%	11%	2%	2%	11%	16%
3.00E+02	2.48	2.22E+02	2.35	70	5%	15%	5%	6%	15%	23%

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

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%.

**15/72 replicates had results above the Upper Limit of Quantification and were excluded from the analysis.

4.7. Analytical specificity

The analytical specificity of cobas® BKV was evaluated by testing a panel of microorganisms to a concentration between 1.00E+06 units/mL and 2.00E+06 units/mL (CFU/mL, cells/mL, CCU/mL, IFU/mL) for bacteria and yeast and at 1.00E+05 units/mL (copies/mL, TCID50/mL, IU/mL, cells/mL) for viruses. Microorganisms were diluted into BKV DNA negative urine as well as urine containing (600 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. Furthermore, the mean log10 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.

Table 10: Microorganisms tested for cross-reactivity in urine

Bacteria	Yeast	Viruses
Bacillus cereus	Candida albicans	Herpes Simplex Virus-2
Bacillus subtilis	Candida glabrata	Human Papillomavirus 16
Chlamydia trachomatis	Candida parapsilosis	-
Corynebacterium diphteriae	Candida tropicalis	-

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Bacteria	Yeast	Viruses
Enterobacter cloacae	-	-
Enterococcus faecalis	-	-
Enterococcus faecium	-	-
Escherichia coli	-	-
Klebsiella pneumoniae	-	-
Lactobacillus acidophilus	-	-
Lactobacillus crispatus	-	-
Lactobacillus jensenii	-	-
Lactobacillus vaginalis	-	-
Morganella morganii	-	-
Mycoplasma genitalium	-	-
Neisseria gonorrhoeae	-	-
Proteus mirabilis	-	-
Pseudomonas aeruginosa	-	-
Staphylococcus aureus	-	-
Staphylococcus epidermidis	-	-
Staphylococcus saphrophyticus	-	-
Streptococcus agalactiae	-	-
Streptococcus bovis	-	-
Streptococcus oralis/viridans	-	-
Streptococcus pneumoniae	-	-
Treponema pallidum	-	-
Trichomonas vaginalis	-	-
Ureaplasma urealyticum	-	-

4.8. Interfering substances

Elevated levels of albumin (0.5% w/v), conjugated bilirubin (1% w/v), glucose (1% w/v), peripheral blood mononuclear cells (1.00E+06 cells/mL), mucus (in presence of 1 mucus swab per 4.3 mL of specimen), acidic pH (pH 4), alkaline pH (pH 9), semen (1 swab dipped into semen per 4.3mL of specimen) , sodium (300 mEq/L) and whole blood (10% v/v) in urine samples were tested in the presence (600 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 in presence and absence of BKV DNA.

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All potentially interfering substances, with the exception of talcum powder, have been shown to not interfere with the test performance. Talcum powder at ≤ 0.05% showed no interference with cobas® BKV. 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 log10 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 spike control.

Class of Drug	Active Ingredient	Concentration	Generic Drug Name
Antimicrobial	Clotrimazole	100 µg/mL	Gyne-Lotrimin 7
Antimicrobial	Metronidazole	701 µmol/L	Arilin rapid,Vaginal suppositories,Vagi Metro Cream,Nidazea Gel
Estrogen steroid hormone	Estradiol	4.41 nmol/L	Estrace
Analgesics	PhenazopyridineHydrochloride	200 µg/mL	Azo Standard
Analgesics	Acetaminophen	1324 µmol/L	Acetaminophen
Lubricant	Propylene Glycol	1000 µg/mL	K-Y UltraGel
Nonsteroidal anti-inflammatorydrug	Acetylsalicylic Acid	3.62 mmol/L	Acetylsalicylic Acid
Nonsteroidal anti-inflammatorydrug	Naproxen	2170 µmol/L	Naproxen
Nonsteroidal anti-inflammatorydrug	Ibuprofen	2425 µmol/L	Ibuprofen
Not applicable	Talc	0.05% (w/v)	Talcum powder

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

4.9. 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 urine sample stabilized in cobas® PCR Media at approximately 1.00E+09 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 0.0% (upper one-sided 95% confidence interval 1.24%).

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5. CLINICAL PERFORMANCE EVALUATION

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, of a positive and a negative sample panel with a total number 270 tests per concentration (not including controls). The panels were made from urine stabilized with cobas® PCR Media that was confirmed negative for BKV DNA using a urine nucleic acid test (NAT) release protocol and spiked with a BKV WHO international standard, or BKV genotype Ia cultured virus DNA. Two operators at each site tested each reagent lot for 5 days. Two runs (1 run = 1 batch = 1 panel + 3 controls) were performed each day and 3 replicates of each panel member were performed for each run. The evaluation results are summarized in Table 12.

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

Expected BKVDNAConcentration	ObservedMeana BKVDNAConcentration	NumberofTestsb	Lot%TVc(CV%)d	Site%TVc(CV%)d	Day/Operator%TVc(CV%)d	Batch%TVc(CV%)d	Within-Batch%TVc(CV%)d	TotalPrecisionSDe	TotalPrecisionCV(%)d
2.78	2.92	270	59%(12.64)	0%(1.15)	0%(0.00)	0%(0.00)	40%(10.41)	0.071	16.47
3.70	3.78	270	47%(8.14)	2%(1.62)	8%(3.31)	0%(0.00)	43%(7.72)	0.051	11.83
4.70	4.80	270	38%(5.02)	2%(1.28)	6%(2.07)	0%(0.00)	53%(5.96)	0.035	8.17
5.70	5.70	270	21%(3.12)	0%(0.00)	0%(0.00)	0%(0.00)	79%(6.12)	0.030	6.87
7.70	7.69	270	2%(1.51)	19%(4.84)	6%(2.79)	0%(0.00)	73%(9.53)	0.048	11.17

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

a Calculated using SAS MIXED procedure.

b Number of valid tests with detectable DNA level.

° %TV = Percent contribution to Total Variance.

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

e Calculated using the total variability from the SAS MIXED procedure.

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cobas® BKV showed acceptable 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 ± 0.20 log10 copies/mL, indicating that the assay can assess changes in BKV DNA levels that are thought to be clinically significant.

The system showed a 99.26% negative percent agreement with a CI of 97.3% to 99.9%. Of the 270 valid tests for the negative panel members, two samples (0.74%) showed a DNA level of < LLoQ positivity. Further investigation of these results showed that they were not associated with a particular instrument/site or reagent lot. Additional DNA sequencing confirmed the presence of BKV. The identified BKV sequences were different from those of the positive control and the BKV strain used for panel preparation, excluding contamination during panel preparation and suggesting trace viruria in one of the 25 urine specimens of the pooled urine sample that was used for the negative panel preparation.

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 urine samples of BKV infected and non-infected patients that were stabilized in cobas® PCR Media. 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 308 neat urine samples stabilized in cobas® PCR Media from 84 transplant subjects 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 (log10 IU/mL) Results for all Samples (Stabilized Urine)

cobas® BKV(log10 IU/mL)	Comparator BKVLDTTarget NotDetected	Comparator BKV LDT< LLoQ(<3.0)	Comparator BKV LDT3.0 to < 3.3	r BKV LDT> 3.9	Total
Target NotDetected	62	6	0	0	୧୫
< LLoQ (<3.0)	ব	22	0	1	27
3.0 to < 3.3	0	2	O	0	2

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cobas® BKV(log10 IU/mL)	Comparator BKVLDTTarget NotDetected	Comparator BKV LDT< LLoQ(<3.0)	Comparator BKV LDT3.0 to < 3.3	Comparator BKV LDT3.3 to < 3.6	Comparator BKV LDT3.6 to 3.9	Comparator BKV LDT> 3.9	Total
3.3 to < 3.6	0	0	6	3	0	0	9
3.6 to 3.9	0	0	2	11	10	0	23
> 3.9	0	0	0	2	8	169	179
Total	66	30	8	16	18	170	308
Column Agreement(%)	(66/66)100.0%	(30/30)100.0%	(6/8)75.0%	(14/16)87.5%	(18/18)100.0%	(169/170)99.4%	-
(95% Score CI)a	(94.5%,100.0%)	(88.6%,100.0%)	(40.9%,92.9%)	(64.0%,96.5%)	(82.4%,100.0%)	(96.7%,99.9%)	-

Note: CI = Confidence Interval; LLoQ = lower limit of quantitation; LDT = laboratory developed test; BKV = BK virus.

Standard Deviation of Comparator BKV LDT estimated at 0.15 log10 U/mL (comparator BKV LDT validation study). Analyte concentration of 3.3 log10 IU/mL represented LLoQ + 2σ, 3.6 log10 IU/mL represented LLoQ + 4σ and 3.9 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.

DNA sequencing performed on representative samples from subjects with results consistently offset by more than 1 logio IU/mL DNA level did not reveal any sequence mismatches for any primer or probe targets for cobas® BKV.

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 cobas® BKV Target Not Detected and < LLoQ (< 3.0) cells were combined. The rationale for adding the adjacent <LLoQ 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 66 BKV DNA-negative samples collected for the estimation of the NPA with the cobas® BKV 61 provided valid results, all 61 samples were negative by cobas® BKV, therefore the NPA was 100% with the 95% Exact CI of 94.1% to 100%.

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

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Table 14: Summary of Concordance of cobas® BKV and Comparator BKV LDT Using Different Thresholds for all Samples (Stabilized Urine)

Threshold*	Percent Agreement< Threshold95% CI (n/N)	Percent Agreement≥ Threshold95% CI (n/N)
Target Not Detected	93.9% (62/66)(85.4%, 97.6%)	97.5% (236/242)(94.7%, 98.9%)
LLoQ (3.0 log10 IU/mL)	97.9% (94/96)(92.7%, 99.4%)	99.5% (211/212)(97.4%, 99.9%)
4.0 log10 IU/mL	90.9% (130/143)(85.1%, 94.6%)	99.4% (164/165)(96.6%, 99.9%)
7.0 log10 IU/mL	97.2% (242/249)(94.3%, 98.6%)	94.9% (56/59)(86.1%, 98.3%)

Note: Samples with Target Not Detected results were categorized as < threshold value in IU/mL.

LLoQ = lower limit of quantitation of Comparator BKV LDT (1000 IU/mL = 3.0 log10 IU/mL).

95% confidence interval (CI) calculated by Score method assuming independence between all samples.

Thresholds of 10,000 IU/mL = 4.0 log10 IU/mL and 10,000,000 IU/mL = 7.0 log10 IU/mL.

From all samples tested with cobas® BKV that were BKV positive with the comparator BKV test, there were a total of 153 neat urine samples stabilized in cobas® PCR Media from 55 transplant subjects evaluable for the correlation analysis at the three testing sites (Figure 3).

Figure 3: Correlation Between cobas® BKV and Comparator BKV LDT for All Samples: Deming Linear Regression Plot of DNA Levels (log10 IU/mL) (Stabilized Urine)

Image /page/20/Figure/8 description: The image is a scatter plot with a linear regression line. The data points are clustered around the regression line, indicating a strong positive correlation. The 95% confidence intervals for the intercept are (0.080, 0.381), and for the slope are (0.968, 1.032). A dashed line is also plotted, showing a 1:1 relationship.

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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 (0.168 to 0.488), which is within ±0.5 log10 IU/mL. Furthermore, the mean bias was estimated at 0.231 log10 IU/mL and using the equation of the fitted line in the bias plots, the systematic difference between both assays was -0.248 log10 IU/mL and 0.188 log10 IU/mL for samples with DNA levels at 4 and 7 log10 IU/mL, respectively.

CONCLUSIONS 6.

The results of non-clinical and clinical performance studies demonstrate that cobas® BKV for use on the cobas® 6800/8800 Systems is as effective, and performs as well as the predicate device .

REFERENCES 7.

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: 2227421.
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.
Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 5. 1996;6:986-94. doi: 10.1101/gr.6.10.986. PubMed PMID: 8908518.

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.

Indications for Use

Indications for Use (Describe)

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cobas® BKV 510(k) Summary

DEVICE DESCRIPTION 1.

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

INDICATIONS FOR USE 2.

TECHNOLOGICAL CHARACTERISTICS 3.

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

4. NON-CLINICAL PERFORMANCE EVALUATION

4.1. Limit of Detection (LoD)

4.1.1. WHO International Standard

Table 2: Limit of Detection in Urine, Lot 1

Table 3: Limit of Detection in Urine, Lot 2

Limit of Detection for subgroups la, Ic and Subtypes II, III and IV 4.2.

4.3. Linear range

Figure 2: Linear Range Determination in Urine

Linearity for subgroups la, Ic and subtypes II, III and IV 4.4.

Lower Limit of Quantitation 4.5.

4.6. Precision - within laboratory

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

4.7. Analytical specificity

4.8. Interfering substances

4.9. Cross contamination

5. CLINICAL PERFORMANCE EVALUATION

Reproducibility of cobas® BKV 5.1.

Clinical Performance of cobas® BKV 5.2.

Figure 3: Correlation Between cobas® BKV and Comparator BKV LDT for All Samples: Deming Linear Regression Plot of DNA Levels (log10 IU/mL) (Stabilized Urine)

CONCLUSIONS 6.

REFERENCES 7.

§ 866.3183 Quantitative viral nucleic acid test for transplant patient management.

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