Predicate Devices

CMV Brite™ CMV Antigenemia Detection Test Kit

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a nucleic acid amplification-based qualitative assay and its performance characteristics, with no mention of AI or ML technologies.

Therapeutic

No.
The device is described as an aid in the diagnosis of active (acute or reactivated) HCMV infection, which indicates a diagnostic rather than a therapeutic purpose.

Diagnostic

Yes

The device is explicitly stated as an "aid in the diagnosis of active (acute or reactivated) HCMV infection" in both the "Intended Use / Indications for Use" and "Device Description" sections.

SaMD (Software as a Medical Device)

No

The device description explicitly states it is a "nucleic acid amplification-based qualitative assay" and is "comprised of four separate stages" involving physical processes like "Nucleic acid release," "Nucleic acid isolation," "Nucleic acid amplification," and "Nucleic acid detection." This indicates it is a laboratory assay kit with physical components, not solely software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The intended use explicitly states it's a "qualitative assay... for the detection of human cytomegalovirus (HCMV) pp67 mRNA... It is intended as an aid in the diagnosis of active (acute or reactivated) HCMV infection." This clearly indicates it's used to examine specimens from the human body to provide information for diagnostic purposes.
Device Description: The description reinforces the intended use and outlines the process of analyzing biological samples (nucleic acid release, isolation, amplification, and detection).
Anatomical Site: It specifies the use of "peripheral blood leukocytes obtained from EDTA and heparin anticoagulated human peripheral blood," which are biological specimens.
Performance Studies: The performance studies involve testing human blood samples from different patient populations (transplant and HIV-infected) and comparing the results to other diagnostic methods (cell culture and pp65 antigenemia assay).
Key Metrics: The report includes metrics like Sensitivity, Specificity, PPV, and NPV, which are standard performance indicators for diagnostic tests.
Predicate Device: The mention of a "Predicate Device" (CMV Brite™ CMV Antigenemia Detection Test Kit) further confirms its classification as a medical device, specifically an IVD, as predicate devices are used for comparison in regulatory submissions for new medical devices, including IVDs.

All these elements align with the definition of an In Vitro Diagnostic device, which is a medical device intended for use in vitro for the examination of specimens derived from the human body solely or principally for the purpose of providing information concerning a physiological state, state of health, or disease or congenital abnormality.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The NucliSens® CMV pp67 assay is a nucleic acid amplification-based qualitative assay to be used in conjunction with the NucliSens® Reader for the detection of human cytomegalovirus (HCMV) pp67 mRNA in EDTA anticoagulated whole blood from adult transplant donors and HIV infected individuals. It is intended as an aid in the diagnosis of active (acute or reactivated) HCMV infection. This product is not intended for use in screening of blood or plasma donors.

Product codes

LIN

Device Description

The NucliSens® CMV pp67 assay is a nucleic acid amplification-based qualitative assay to be used in conjunction with the NucliSens®™ Reader for the detection of human cytomegalovirus (HCMV) pp67 mRNA in EDTA anticoagulated whole blood from adult transplant donors and HIV infected individuals. It is intended as an aid in the diagnosis of active (acute or reactivated) HCMV infection. This product is not intended for use in screening of blood or plasma donors.

The NucliSens® CMV pp67 assay is comprised of four separate stages:

Nucleic acid release a.
Nucleic acid isolation b.
Nucleic acid amplification c.
Nucleic acid detection d.

a. Nucleic acid release: viral particles and cells present in the sample are disintegrated; RNases and DNases present in the sample are inactivated. Nucleic acids are released.
b. Nucleic acid isolation: Under high salt conditions, nucleic acids in NucliSens® Lysis Buffer are bound to silicon dioxide particles. These Silica particles act as a solid phase. Unbound non-nucleic acid components are removed by several washing steps. Finally, bound nucleic acid is eluted from the solid phase. Prior to the addition of Silica particles, HCMV System Control (SC) RNA molecules are added to the specimen in Lysis Buffer in a known number. SC RNA is added for validation of the test procedure.
c. Nucleic acid amplification: HCMV mRNA expressed from the UL65 open reading frame on the viral genome and encoding a phosphorylated matrix tegument protein of 67 kDa (pp67 mRNA) is converted to a double-stranded DNA molecule by the concerted action of three enzymes and two oligonucleotides. One of these oligonucleotides contains a 5'-terminal T7 RNA polymerase promoter sequence in addition to a stretch of nucleotides that is complementary to a sequence on the HCMV pp67 mRNA. The second oligonucleotide encompasses a short sequence which is identical to a segment of the mRNA and is located upstream of the region where the T7 promoter-containing oligonucleotide can anneal. Together, these oligonucleotides define a part of the HCMV pp67 mRNA which is converted to a DNA intermediate with a functional T7 promoter. This includes steps of hybridization, elongation by Avian Myeloblastosis Virus Reverse Transcriptase (AMV-RT), hydrolysis by RNase H, and re-annealing and extension by reverse transcriptase to yield a double-stranded DNA intermediate with a transcriptionally active T7 promoter. T7 RNA polymerase then produces multiple copies of RNA transcripts which are antisense to the original target RNA sequence, leading to exponential synthesis of RNA products.
d. Nucleic acid detection: Following NASBA-based amplification, single-stranded HCMV pp67 mRNA-derived amplicons can be detected by a nucleic acid hybridization procedure employing the electrochemiluminescence (ECL) principle. Amplicons are captured by hybridization to an oligonucleotide immobilized on paramagnetic beads (oligo-beads). To differentiate between amplicons derived from the HCMV pp67 mRNA (wild type or WT RNA) or from the SC RNA, aliquots of the amplification reaction are added to two hybridization solutions which, in addition to the oligo-beads contain a Ruthenium chelate-labeled oligonucleotide probe that is specific for either the WT RNA or the SC RNA. Detection is achieved by adding tripropylamine (TPA) as a substrate to the hybridization reaction mixtures followed by automated analysis in a NucliSens® Reader. Results are recorded as ECL counts by the NucliSens®Reader. The presence of HCMV pp67 mRNA in a sample is reflected by the WT RNA ECL signal. The WT RNA ECL signal and the ECL signal for SC RNA are used to determine the validity of the test.

Controls:
System Control (SC): SC RNA molecules are added to the sample during the lysis stage and serve as an internal quality control for isolation, amplification, and detection. If the combined ECL counts for WT and SC probes exceed 10,000, no inhibition is determined.
Test run controls: Additional CMV pp67 mRNA positive and negative external controls are used. The positive control contains CMV-infected cells in whole blood, which are processed similarly to test specimens. The negative control monitors contamination.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Whole blood

Indicated Patient Age Range

Adult

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Analytical specificity and sensitivity:

Interfering substances: Tested with icteric (up to 3.8 mg/dL), hemolyzed, and lipemic (up to 2,400 mg/dL) specimens, specimens with antinuclear antibodies, rheumatoid factor, and from multiparous women. 10 samples per category, spiked and unspiked. (Table b.2.1)
CMV strains and cross-reactivity: Tested with Towne, C87, Davis, ESP, and AD169 strains (infected cells). Also tested with panels of 8-10 specimens from individuals with HIV-1, HTLV-I/II, Herpes Simplex Virus Type I, Herpes Simplex Virus Type II, Epstein Barr Virus, Varicella Zoster Virus, Respiratory Syncytial Virus, Systemic Lupus Erythematosus (unspiked and spiked with HCMV RNA). Tested with 4 cell lines infected with Human Herpes Virus 6A, 6B, 7, 8. Tested with EDTA anticoagulated whole blood samples inoculated with common human bacteria flora (Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Pseudomonas fluorescens).
Whole blood donors: Evaluted 100 volunteer whole blood donors (50 HCMV seronegative, 50 HCMV seropositive by EIA). Compared to FDA cleared CMV pp65 antigenemia assay.
Analytical sensitivity: 51 clinical specimens positive for HCMV by cell culture or FDA cleared CMV pp65 antigenemia assay or both. Evaluated with NucliSens® CMV pp67 assay.
Reproducibility: 4 samples with different RNA concentrations (2500, 250, 25, 0 RNA molecules/input). Tested at 3 sites by 2 or 3 technicians, each using 3 different lots. Multiple tests (8 or 4) per condition.
Limit of detection: Data from reproducibility study (Table b.2.2.4.a.) used to estimate.

Clinical performance:

Transplant patient population: 50 bone marrow and solid organ transplant patients from Italy. Multiple serial specimens collected over an extended period. Compared to cell culture and pp65 antigenemia assay. Analysis done on per subject basis and per test basis. (Table b.2.2.7.a., b.2.2.7.b., b.2.2.7.c.)
HIV-1 Infected population: 50 HIV-1 infected individuals from the Netherlands. Multiple serial specimens collected over an extended period during routine clinic visits. Compared to cell culture and pp65 antigenemia assay. Analysis done on per subject basis and per test basis. (Table b.2.2.8.a., b.2.2.8.b., b.2.2.8.c.)

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Performance Studies:

Interfering Substances Study:
- Study Type: Interference study.
- Sample Size: 10 specimens for each interfering substance category (Icteric, Hemolyzed, Lipemic, Antinuclear Antibodies, Rheumatoid Factor, Multiparous Women), with 10 unspiked and 10 spiked (with CMV RNA) for each type. Total 60 unspiked and 60 spiked.
- Key Results: No interference observed.
  - Icteric (up to 3.8 mg/dL): Unspiked Negative: 10/10, Spiked Positive: 10/10
  - Hemolyzed: Unspiked Negative: 10/10, Spiked Positive: 10/10
  - Lipemic (up to 2,400 mg/dL): Unspiked Negative: 10/10, Spiked Positive: 10/10
  - Antinuclear Antibodies: Unspiked Negative: 10/10, Spiked Positive: 10/10
  - Rheumatoid Factor: Unspiked Negative: 10/10, Spiked Positive: 10/10
  - Multiparous Women: Unspiked Negative: 10/10, Spiked Positive: 10/10
- Conclusion: EDTA as an anticoagulant also did not interfere.
Analytical Specificity (CMV Strains & Cross-Reactivity) Study:
- Study Type: Analytical specificity.
- Sample Size: Not explicitly stated for each, but "panels of specimens" (10 per category for disease states, 4 cell lines for herpes viruses, common human bacteria flora).
- Key Results:
  - Detected Towne, C87, Davis, ESP, and AD169 CMV strains.
  - No false positive or false negative reactions with specimens from individuals exhibiting HIV-1 (10/10 unspiked negative, 10/10 spiked positive), HTLV-I/II (8/8, 8/8), Herpes Simplex Virus Type I (10/10, 10/10), Herpes Simplex Virus Type II (10/10, 10/10), Epstein Barr Virus (10/10, 10/10), Varicella Zoster Virus (10/10, 10/10), Respiratory Syncytial Virus (10/10, 10/10), Systemic Lupus Erythematosus (10/10, 10/10).
  - No cross-reactivity with Human Herpes Virus 6A, 6B, 7, 8.
  - No interference from common human bacteria flora (Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Psuedumonas fluorescens).
Analytical Specificity (Whole Blood Donors) Study:
- Study Type: Analytical specificity.
- Sample Size: 100 volunteer whole blood donors (50 HCMV seronegative, 50 HCMV seropositive).
- Key Results:
  - No specimen from 100 donors reported positive by NucliSens® CMV pp67 assay.
  - For 50 seropositive specimens, compared to an FDA cleared CMV pp65 antigenemia assay, both assays found no positive results.
  - Overall agreement relative to CMV pp65 antigenemia assay in this population was 100%.
Analytical Sensitivity Study:
- Study Type: Analytical sensitivity.
- Sample Size: 51 clinical specimens.
- Key Results:
  - NucliSens® CMV pp67 assay reported positive for 43/51 specimens.
  - CMV pp65 antigenemia assay positive for 46 specimens.
  - Culture positive for 40 specimens.
  - Overall agreement relative to antigenemia and/or culture positive was 84.3% (43/51).
Reproducibility Study:
- Study Type: Reproducibility.
- Sample Size: 4 samples (2500, 250, 25, 0 RNA molecules/input), tested multiple times (total 48, 44, or 48 tests per sample depending on site) across 3 sites, 2-3 technicians, and 3 lots.
- Key Results:
  - For 2500 RNA molecules/input, positive rates across sites/lots/techs ranged from 85% to 100%. Expected 100%.
  - For 250 RNA molecules/input, positive rates ranged from 77% to 80%. Expected 93%.
  - For 25 RNA molecules/input, positive rates ranged from 18% to 27%. Expected 13%.
  - For 0 RNA molecules/input, positive rates were 0%. Expected 0%.
  - Overall positive rates (average across sites/lots/techs regardless of RNA input) were 51%, 49%, 52%.
  - Multiple logistic regression indicated that probability of detection is not affected by lot or test site differences.
Limit of Detection Study:
- Study Type: Analytical sensitivity (using reproducibility data).
- Key Results: A 95% response rate was estimated with 700 input RNA molecules (95% fiducial limits: 480 to 1158 RNA molecules).

Clinical Performance Studies:

Transplant Patient Population Study:
- Study Type: Longitudinal clinical comparison study.
- Sample Size: 50 patients (bone marrow and solid organ transplant). Multiple specimens collected over time. Total 368 valid test result comparisons.
- Comparison: NucliSens® CMV pp67 assay vs. combined antigenemia and/or cell culture.
- Per Subject Basis (n=50 patients):
  - Sensitivity: 77% (30/39) (95% C.I.: 60.7% to 88.9%)
  - Positive Predictive Value: 100% (30/30)
  - Negative Predictive Value: 55% (11/20)
  - Overall Agreement: 82% (41/50)
  - AUC (ROC Curve): 0.902 (standard error of 0.042).
- Per Test Basis (n=368 specimens):
  - Overall Agreement: 75% ((73+203)/368)
- Time to Positivity: Median times to first positive result: Antigenemia (38 days), Culture (67 days), NucliSens® CMV pp67 (51 days).
HIV-1 Infected Population Study:
- Study Type: Longitudinal clinical comparison study.
- Sample Size: 50 individuals (HIV-1 infected). Multiple specimens collected over time. Total 450 valid test result comparisons.
- Comparison: NucliSens® CMV pp67 assay vs. combined antigenemia and/or cell culture.
- Per Subject Basis (n=50 patients):
  - Sensitivity: 78% (32/41)
  - Specificity: 89% (8/9) (95% C.I.: 51.8% to 99.7%)
  - Positive Predictive Value: 97% (32/33)
  - Negative Predictive Value: 47% (8/17)
  - Overall Agreement: 80% (40/50)
  - AUC (ROC Curve): 0.862 (standard error of 0.054).
- Per Test Basis (n=450 specimens):
  - Overall Agreement: 76% ((268+76)/450)
- Time to Positivity: Median times to first positive result: Antigenemia (405 days), Culture (519 days), NucliSens® CMV pp67 (458 days).

Overall Valid Result Rate: Across analytical studies, 1427 samples were analyzed, with 99.3% (1417 samples) yielding a valid test result. 0.7% (10 samples) revealed an invalid test result. In transplant study, invalid rate was 2.3%. In HIV study, invalid rate was 1.6%.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Transplant Population (Per Subject Basis):

Sensitivity = 77% (95% C.I. using 'exact' method: 60.7 to 88.9%)
Positive Predictive Value = 100%
Negative Predictive Value = 55%
Overall Agreement = 82% (41/50)
AUC (ROC) = 0.902

Transplant Population (Per Test Basis):

Overall Agreement = 75% ((73+203)/368)

HIV-1 Infected Population (Per Subject Basis):

Sensitivity = 78%
Specificity = 89% (95% C.I. using 'exact' method: 51.8 to 99.7%)
Positive Predictive Value = 97%
Negative Predictive Value = 47%
Overall Agreement = 80% (40/50)
AUC (ROC) = 0.862

HIV-1 Infected Population (Per Test Basis):

Overall Agreement = 76% ((268+76)/450)

Predicate Device(s)

CMV Brite™ CMV Antigenemia Detection Test Kit

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3175 Cytomegalovirus serological reagents.

(a)
Identification. Cytomegalovirus serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to cytomegalovirus in serum. The identification aids in the diagnosis of diseases caused by cytomegaloviruses (principally cytomegalic inclusion disease) and provides epidemiological information on these diseases. Cytomegalic inclusion disease is a generalized infection of infants and is caused by intrauterine or early postnatal infection with the virus. The disease may cause severe congenital abnormalities, such as microcephaly (abnormal smallness of the head), motor disability, and mental retardation. Cytomegalovirus infection has also been associated with acquired hemolytic anemia, acute and chronic hepatitis, and an infectious mononucleosis-like syndrome.(b)
Classification. Class II (performance standards).

Summary

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Image /page/0/Picture/2 description: The image shows a sequence of handwritten characters, which appear to be a combination of letters and numbers. The sequence starts with the letter 'K', followed by the numbers '9', '8', '3', '7', '6', and '2'. The characters are written in a dark ink, and the handwriting style is somewhat cursive.

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A 510(k) summary of the NucliSens® CMV pp67 assay

1

510(k) Summary

NucliSens® CMV pp67

(a)(1) The submitter's name, address, telephone number, a contact person, and the date the summary was prepared;

Submitter's Name: Organon Teknika Corporation

Submitter's Address: 100 Akzo Avenue, Durham, North Carolina, 27712, USA

Submitter's Telephone: (919) 620-2373

Submitter's Contact: Ron Sanyal Ron Sanyal

Date 510(k) Summary Prepared: September 10, 1999

(a)(2) The name of the device. including the trade or proprietary name if applicable, the common or usual name, and the classification name, if known;

Trade or Proprietary Name: NucliSens® CMV pp67

Common or Usual Name: Nucleic acid amplification-based assay for CMV RNA

Classification Name: Cytomegalovirus serological reagents

(a)(3) An identification of the legally marketed device to which the submitter claims substantial equivalence;

Device Equivalent to: CMV Brite™ CMV Antigenemia Detection Test Kit

(a)(4) A description of the device.

Device Description:

The NucliSens® CMV pp67 assay is a nucleic acid amplification-based qualitative assay to be used in conjunction with the NucliSens®™ Reader for the detection of human cytomegalovirus (HCMV) pp67 mRNA in EDTA anticoagulated whole blood from adult transplant donors and HIV infected individuals. It is intended as an aid in the diagnosis of active (acute or reactivated) HCMV infection. This product is not intended for use in screening of blood or plasma donors.

2

The NucliSens® CMV pp67 assay is comprised of four separate stages:

Nucleic acid release a.
Nucleic acid isolation b.
Nucleic acid amplification c.
Nucleic acid detection d.

a. Nucleic acid release

ar Rashe and to NucliSens® Lysis Buffer containing guanidine thiocyanate and Triton X-100. Viral particles and cells present in the sample are disintegrated; RNases and DNases present in the sample are inactivated. Nucleic acids are released.

Nucleic acid isolation b.

Under high salt conditions, nucleic acids in NucliSens® Lysis Buffer are bound to silicon dioxide particles. (13-17) These Silica particles act as a solid phase. Unbound non-nucleic acid components are removed by several washing steps. Finally, bound nucleic acid is eluted from the solid phase. Prior to the addition of Silica particles, HCMV System Control (SC) RNA molecules are added to the specimen in Lysis Buffer in a known number. SC RNA is added for validation of the test procedure.

c. Nucleic acid amplification

HCMV mRNA expressed from the UL65 open reading frame on the viral genome and encoding a phosphorylated matrix tegument protein of 67 kDa (pp67 mRNA) is converted to a doublestranded DNA molecule by the concerted action of three enzymes and two oligonucleotides(18) One of these oligonucleotides contains a 5'-terminal T7 RNA polymerase promoter sequence in addition to a stretch of nucleotides that is complementary to a sequence on the HCMV pp67 mRNA. The second oligonucleotide encompasses a short sequence which is identical to a segment of the mRNA and is located upstream of the region where the T7 promoter-containing oligonucleotide can anneal. Together, these oligonucleotides define a part of the HCMV pp67 mRNA which is converted to a DNA intermediate with a functional T7 promoter. This process starts with hybridization of the oligonucleotide that contains the T7 RNA polymerase binding site to the target RNA (Primer 1; Figure 3.3). Avian Myeloblastosis Virus Reverse Transcriptase (AMV-RT) elongates the oligonucleotide, creating a cDNA copy of the RNA template and forming a RNA/DNA hybrid. The RNA portion of this hybrid is hydrolyzed by RNase H, leaving single-stranded DNA to which the second oligonucleotide can anneal (Primer 2; Figure 3.3), thereby again forming a substrate suitable for reverse transcriptase extension. This extension finally reveals a double-stranded DNA intermediate with a transcriptionally active T7 promoter portion. Recognizing the now functional promoter, T7 RNA polymerase produces multiple copies of RNA transcripts which are antisense to the original target RNA sequence. Each newly synthesized antisense RNA molecule can act as a template by itself and again be converted to a DNA intermediate with a functional T7 promoter in a way similar to the original target RNA, except that the oligonucleotide primers anneal in reverse order because the newly generated RNA molecules are opposite in orientation to the original target and the resulting DNA intermediate is only partly double-stranded. Again, many RNA copies are generated from each RNA target that reenters the reaction resulting in exponential synthesis of RNA products.

By the same process and making use of the same oligonucleotides, multiple copies of RNA can be generated from the SC RNA that was added to each sample in the nucleic acid extraction procedure. This SC RNA contains the same segment of viral RNA as defined by the oligonucleotides in addition to a unique sequence by which it can be distinguished from the viral mRNA.

3

Image /page/3/Figure/3 description: This image shows a diagram of a molecular process, possibly related to RNA amplification or transcription. The diagram includes labeled steps A through H, with each letter corresponding to a specific component or stage in the process. The legend provides definitions for each of the labeled components, such as 'ss RNA (sense),' 'Primer 1,' 'AMV-RT,' 'RNase H,' 'Primer 2,' 'T7 RNA pol,' 'ss RNA (antisense) amplificate,' and 'Isothermal amplification,' along with visual representations of sense RNA, primer 1, primer 2, antisense DNA, sense DNA, and antisense RNA.

NASBA amplification principle Figure 3.3.

d. Nucleic acid detection

Following NASBA-based amplification, single-stranded HCMV pp67 mRNA-derived amplicons can be detected by a nucleic acid hybridization procedure employing the electrochemiluminescence (ECL) principle.(19) Amplicons are captured by hybridization to an oligonucleotide immobilized on paramagnetic beads (oligo-beads). To differentiate between

4

ORGANON TEKNIKA

@ 008

amplicons derived from the HCMV pp67 mRNA (wild type or WT RNA) or from the SC RNA, aliquots of the amplification reaction are added to two hybridization solutions which, in addition to the oligo-beads contain a Ruthenium chelate-labeled oligonucleotide probe that is specific for either the WT RNA or the SC RNA. Detection is achieved by adding tripropylamine (TPA) as a substrate to the hybridization reaction mixtures followed by automated analysis in a NucliSens® Reader. In the instrument's detection chamber, the paramagnetic beads carrying the amplicon/probe complexes are captured onto the surface of an electrode by means of a magnet. Voltage applied to this electrode triggers the ECL reaction in which light is produced by cyclical oxidation-reduction of the Ruthenium metal ion in the presence of TPA. Results are recorded as ECL counts by the NucliSens®Reader. The presence of HCMV pp67 mRNA in a sample is reflected by the WT RNA ECL signal. The WT RNA ECL signal and the ECL signal for SC RNA are used to determine the validity of the test.

NucliSens® CMV pp67 controls

System Control

The qualitative detection of HCMV pp67 RNA using the NucliSens® CMV pp67 assay is based on the co-amplification of sample RNA together with a second target RNA sequence referred to as System Control RNA (SC RNA). As the SC RNA is added to the sample during the lysis stage, the use of the system control itself functions as a significant quality control for the isolation, amplification, and detection stages of the assay to assure proper reporting of an individual specimen. Without proper isolation, amplification and detection of the system control, no or a low signal for the HCMV pp67 mRNA would be reported as an invalid test instead of a negative result.

As the SC is added to the patient specimen and competes with the WT for amplification resources, a high concentration of WT might overwhelm the SC resulting in a large WT ECL signal and a low SC ECL signal. This does not have a severe impact on the assay performance because strongly positive samples will still be called positive. If the specimen is neqative, the overwhelming does not occur and the SC signal is high and the WT signal is low. In the presence of inhibitors or interfering substances, both signals would be low resulting in an invalid assay. The use of SC RNA in the NucliSens® CMV pp87 assay allows the user to determine conditions that potentially may result in a false negative result without affecting the assay's ability to detect positives.

Since the assay makes use of an internal System Control RNA (SC RNA) which is added to each individual sample, inhibition testing is being performed on every sample(15-17). Factors inhibitory to the amplification of the CMV mRNA will also inhibit amplification of the SC-RNA which contains the same primer recognition sites as the viral target mRNA. For the interpretation of a test result, if the sum of the ECL counts for the WT probe and the ECL counts for the SC probe is greater than or equal to 10.000, no inhibition has taken place, and the ECL signal for the WT probe can be used to elucidate whether the original whole blood sample is positive or negative for the presence of CMV pp67 mRNA.

Test run controls

In addition to the System Control (provided in the kit), which functions as an internal control at the individual sample level, additional CMV pp67 mRNA positive and negative controls 1Xntrolle

control, the whole blood should additionally contain CMV-infected cells, e.g. from a spike of in vitro cultured cells infected with a strain of HCMV. Once added to NucliSens® Lysis Buffer, the external controls should be treated identically as the test specimens, including the addition of SC RNA prior to entering the nucleic acid isolation procedure.

By virtue of the external positive control lysis of cells containing CMV pp67 mRNA in whole blood specimens can be monitored, in addition to proper isolation, amplification, and detection

5

of the authentic CMV pp67 mRNA. The negative control serves to monitor contamination, both of the EDTA-anticoagulated human whole blood used for the external controls and of the reagents and materials used during the test procedure.

A statement of the intended use of the device. a)(5)

Device Intended Use: The NucliSens® CMV pp67 assay is a nucleic acid amplification-based qualitative assay to be used in conjunction with the NucliSens® Reader for the detection of human cytomegalovirus (HCMV) pp67 mRNA in EDTA anticoagulated whole blood from adult transplant donors and HIV infected individuals. It is intended as an aid in the diagnosis of active (acute or reactivated) HCMV infection. This product is not intended for use in screening of blood or plasma donors.

6

(a)(6) A summary of the technological characteristics of the new device in comparison to those of the predicate device.

The similarities and/or differences between Organon Teknika's NucliSens® CMV pp67, the Biotest Diagnostics Corporation's CMV BRITE™ Antigenemia and typical CMV cell culture are compared in Table a.6.

7

TABLE a.6

| PARAMETERS | ORGANON TEKNIKA
CORPORATION
NucliSens™ CMV pp67 | BIOTEST DIAGNOSTICS CORPORATION
CMV Brite™ Antigenemia | Typical CMV Cell Culture |
|---------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| TYPE OF ASSAY | Qualitative Assay | Qualitative Assay | Qualitative Assay |
| PANEL | Microbiology (83) | Microbiology (83) | Microbiology (83) |
| CATEGORY | Nucleic Acid Amplification-Based
In Vitro Assay | The CMV Brite test kit uses the application of a
two monoclonal antibodies (C10/C11) cocktail
in an indirect immunofluorescence staining of
cytospin preparations of peripheral blood
leukocytes. | Cell culture innoculation |
| INTENDED USE | This test is intended as an aid to
detect an active CMV infection. | This test is intended as an aid to detect an active
CMV infection. | This test is intended as an aid to detect an active
CMV infection. |
| | The NucliSens™ CMV pp67 is a
nucleic acid amplification-based in
vitro qualitative assay for detection
of the presence of human
cytomegalovirus (HCMV) pp67
mRNA in anticoagulated whole
blood. It is intended as an aid in the
diagnosis of active (acute or
reactivated) HCMV infection. This
product is not intended for use in
screening of blood or plasma donors. | The CMV Brite test kit is intended for the
qualitative detection of cytomegalovirus (CMV)
lower matrix protein pp65 by indirect
immunofluorescence using microscopy in
isolated peripheral blood leukocytes obtained
from EDTA and heparin anticoagulated human
peripheral blood. The detection of CMV pp65 in
human peripheral blood cells aids in the
diagnosis of acute or reactivated CMV infection.
This product is not FDA cleared (approved) for
use in testing (i.e. screening) of blood or plasma
donors. | Cell culture is intended for the qualitative
detection of the presence of HCMV in
leukocytes from whole blood. It aids in the
diagnosis of HCMV infection. |
| SAMPLE | Anticoagulated whole blood. | Isolated peripheral blood leukocytes obtained
from EDTA and heparin anticoagulated human
peripheral blood. | Leukocytes from whole blood |

8

(b)1) A brief discussion of the nonclinical tests submitted, referenced, or relled on in the premarket notification submission for a determination of substantial equivalency.

Not applicable.

9

(b)(2) A brief discussion of the clinical tests submitted, referenced, or relied on in the premarket notification submission for a determination of substantial equivalency.

b.2.1 Interfering substances

Elevated levels of lipids, bilirubin and hemoglobin in specimens did not interfere with the detection of CMV pp67 RNA by the NucliSens® CMV pp67 assay. The presence of antinuclear antibodies had no effect on the performance of the assay. Specimens known positive for rheumatoid factor gave no false positive or false negative results. Specimens from multiparous women gave no false positives or false negatives.

| Specimen Type | Unspiked
Negative Results/Total | Spiked with CMV RNA
Positive Results/Total |
|--------------------------------|------------------------------------|-----------------------------------------------|
| Icteric (up to 3.8 mg/dL) | 10/10 | 10/10 |
| Hemolyzed | 10/10 | 10/10 |
| Lipemic (up to 2,400
mg/dL) | 10/10 | 10/10 |
| Antinuclear Antibodies | 10/10 | 10/10 |
| Rheumatoid Factor | 10/10 | 10/10 |
| Multiparous Women | 10/10 | 10/10 |

Table b.2.1 Interfering Substances

The use of EDTA as an anticoagulant does not interfere with the performance of this assav.

Analytical specificity and sensitivity b.2.2

b.2.2.1 Analytical specificity : CMV strains and cross-reactivity

The NucliSens® CMV pp87 is capable of detecting the Towne, C87, Davis, ESP, and AD169 strains. Cells were infected with laboratory strains AD169, Towne, C87, Davis and ESP until the appearance of cytopathic effects (CPE). Nucleic acid isolated from the infected cells was analyzed with the NucliSens® CMV pp67 assay. HCMV pp67 mRNA could be amplified from all reference strains tested. In clinical and analytical studies, the NucliSens® CMV pp67 assay was performed upon patients from different geographic areas of North America and Europe. Longitudinal series of specimens were collected from these patients with episodes of HCMV infection as confirmed by conventional culture and/or high levels of antigenemia. From all of these episodes of confirmed HCMV infection, at least one or more specimen(s) tested positive in the NucliSens® CMV pp67 assay. Therefore, the NucliSens® CMV pp67 assay is capable of detecting pp67 mRNA of CMV strains encountered in patients originating from different geographical areas.

10

团 012

The specificity of the NucliSens® CMV pp67 assay was evaluated by testing a panel of specimens from individuals exhibiting the following infections or diseases, but known negative for the HCMV virus. These specimens were tested without spike to assess possible false positive type reactions and after being spiked with an aliquot of HCMV RNA to assess possible false negative type reactions.

| Specimen Type | Unspiked
Negative Results/Total | Spiked with HCMV RNA
Positive Results/Total |
|---------------------------------|------------------------------------|------------------------------------------------|
| HIV-1 | 10/10 | 10/10 |
| HTLV-I/II | 8/8 | 8/8 |
| Herpes Simplex Virus
Type I | 10/10 | 10/10 |
| Herpes Simplex Virus
Type II | 10/10 | 10/10 |
| Epstein Barr Virus | 10/10 | 10/10 |
| Varicella Zoster Virus | 10/10 | 10/10 |
| Respiratory Syncytial
Virus | 10/10 | 10/10 |
| Systemic Lupus
Erythematosus | 10/10 | 10/10 |

Table b.2.2.1 Analytical Specificity: Cross-reactivity

11

In addition, four cell lines, each infected with a related Herpes virus, were tested with the NucliSens® CMV pp67 assay to check for possible cross-reactivity. There was no cross-reactivity of the assay with these viruses:

Human Herpes Virus 6A Human Herpes Virus 6B Human Herpes Virus 7 Human Herpes Virus 8.

EDTA anticoagulated whole blood samples were inoculated with the following common human bacteria flora:

Escherichia coli Bacillus cereus Staphylococcus epidermidis Psuedumonas fluorescens.

Two 0.1 ml aliguots of each contaminated sample were added to Lysis Buffer. CMV mRNA spike was added to one of the two Lysis Buffer tubes. The specimens were then processed with the NucliSens® CMV pp67 assay. No evidence of interference by the bacteria flora with the performance of the assay was observed.

b.2.2.2.2 2 Analytical specificity : whole blood donors

The analytical specificity of the NucliSens® CMV pp67 assay was assessed in a volunteer whole blood donor population by evaluating specimens from 100 donors. No specimen was reported positive for HCMV by the NucliSens® CMV pp67 assay.

Of the one hundred (100) specimens, fifty (50) were from HCMV seronegative whole blood donors and fifty (50) were from HCMV seropositive whole blood donors. The fifty seropositive specimens were positive by a HCMV EIA assay. These fifty specimens were tested with both the NucliSens® CMV pp67 assay and an FDA cleared CMV pp65 antigenemia assay. No specimen was found positive by either assay. Relative to the CMV pp65 antigenemia assay in a whole blood donor population, the overall agreement was 100%.

12

Comparison of NucliSens® CMV pp67 to CMV pp65 Table b.2.2.2 Antigenemia in a Population of Seropositive Blood Donors

| CMV pp65

Antigenemia
	Positive	Negative	Total
NucliSens®
CMV pp67	Positive	0	0	0
	Negative	0	50	50
	Total	0	50	50

b.2.2.3 Analytical sensitivity

To assess the performance of the NucliSens® CMV pp67 assay with specimens representing active CMV infection, patients at risk for HCMV infection were evaluated at sites located in New England, in the Northeast and on the West Coast regions of the United States (Table b.2.2.3). Fifty-one (51) clinical specimens that were positive for HCMV in cell culture or in a FDA cleared CMV pp65 antigenemia assay or in both were evaluated with the NucliSens® CMV pp67 assay. The NucliSens® CMV pp67 assay reported a positive result for 43 of the specimens whereas CMV pp65 antigenernia assay tested positive for 46 specimens and culture for 40. Relative to antigeneria and/or culture positive, overall agreement was 84,3% (43/51).

Table b.2.2.3 Comparison of NucliSens® CMV pp67 to Antigenemia and/or Culture in a CMV infected population

		Positive	Negative	Total
NucliSens
®	Positive	43	0	43
CMV pp67	Negative	8	0	8
	Total	51	0	51

13

Reproducibility b,2,2.4

The following numbers of results were obtained when performing multiple tests of the same 4 samples at 3 sites with each of the same 3 lots.

Table b.2.2.4. a. Reproducibility of the NucliSens® CMV pp67 assay

	Lot A		Lot B		Lot C		Totals
Site 1	Tech.1	Tech.2	Tech.1	Tech.2	Tech.1	Tech.2	Positive %	Expected
Specimen (RNA molecules/input)	8/8	8/8	8/8	8/8	8/8	8/8	48/48 100%	100%
1 (2500)	8/8	8/8	7/8	6/8	5/8	3/8	37/48 77%	93%
2 (250)	2/8	3/8	1/8	2/8	2/8	3/8	13/48 27%	13%
3 (25)	0/8	0/8	0/8	0/8	0/8	0/8	0/48 0%	0%
4 (0)	37/64=58%		32/64=50%		29/64=45%		98/192=51%
	Lot A		Lot B		Lot C		Totals
Site 2	Tech.1	Tech.2	Tech.1	Tech.2	Tech.1	Tech.2	Positive %	Expected
Specimen(RNA molecules/input)	8/8	4/4	8/8	8/8	8/8	8/8	44/44 100%	100%
1 (2500)	8/8	3/4	6/8	7/8	5/8	6/8	35/44 80%	93%
2 (250)	0/8	0/4	2/8	0/8	2/8	4/8	8/44 18%	13%
3 (25)	0/8	0/4	0/8	0/8	0/8	0/8	0/44 0%	0%
4 (0)	23/48=48%		31/64=48%		33/64=52%		87/176=49%
	Lot A			Lot B			Lot C			Totals
Site 3	Tech.1	Tech.2	Tech.3	Tech.1	Tech.2	Tech.3	Tech.1	Tech.2	Tech.3	Positive %	Expected
Specimen (RNA molecules/input)	8/8	4/4	4/4	8/8	6/6	2/2	8/8	4/4	4/4	48/48 100%	100%
1 (2500)	7/8	2/4	4/4	7/8	6/6	2/2	6/8	3/4	4/4	41/48 85%	93%
2 (250)	0/8	0/4	1/4	2/8	2/6	0/2	2/8	2/4	1/4	10/48 21%	13%
3 (25)	0/8	0/4	0/4	0/8	0/6	0/2	0/8	0/4	0/4	0/48 0%	0%
4 (0)	30/64-47%			35/64 = 55%			34/64=53%			99/192=52%

Multiple logistic regression indicated that the probability of detection is not affected by lot or test site differences.

14

Figure b.2.2.4.b presents the above information graphically.

Image /page/14/Figure/4 description: The image is a graph titled "CMV mRNA Transcripts Detected by the NucliSens CMV pp67 System". The graph shows the percentage of specimens positive on the y-axis and the CMV mRNA transcript on the x-axis. There are three lines on the graph, representing Lot 1, Lot 2, and Lot 3. The graph shows that as the CMV mRNA transcript increases, the percentage of specimens positive also increases for all three lots.

15

Limit of detection b.2.2.5

The data shown in Table b.2.2.4.a. were used to estimate reliable detection limits. The following figure shows the fit of a logistic regression model to the observed proportions as a function of input copy number.

Figure b.2.2.5 Logistic regression of observed detection rates for different concentrations of CMV pp67 RNA transcript using the NucliSens® CMV DD67 assay

Image /page/15/Figure/6 description: The image shows a plot of proportion detected vs. Log 10 RNA input. The x-axis is labeled "Log 10 RNA Input" and ranges from 0 to 4. The y-axis is labeled "Proportion Detected" and ranges from 0.0 to 1.0. The plot shows observed data points and a predicted curve, with the predicted curve showing a sigmoidal relationship between the two variables.

The analysis indicated a 95% response rate with 700 input RNA molecules with 95% fiducial limits ranging from 480 to 1158 RNA molecules.

b.2.2.6

In the analytical studies as presented in sections 9.1, 9.2, and 9.3, a total of 1427 samples have been analyzed. In 99.3 % of the cases (1417 samples) a valid test result was obtained. Only 10 samples (0.7%) revealed an invalid test result.

b.2.2.7 Clinical performance

b.2.2.7 .a. Transplant patient population

In a bone marrow and solid organ transplant population of 50 patients, the performance of the NucliSens® CMV pp67 assay was compared to cell culture and a pp65 antigenernia assay. Multiple specimens were collected over an extended period of time from these patients as part of a longitudinal study conducted in Italy to monitor for the presence of HCMV infection after transplantation. Only results from specimens for which all three assays revealed

16

a valid test result were considered. The percentage of invalid test results for the NucliSens® CMV pp67 assay in this study was 2.3%. In Table b.2.2.7.a., the results of this study are summarized. If either antigenermia or culture were found positive for a specimen, that particular specimen was regarded as CMV positive. If both antigenernia and culture were negative, the state of nature for that specimen was considered CMV negative. In the table, for each patient enrolled in the study, the number of CMV positive and CMV negative specimens is indicated based on the combined antigenemia/culture results and based on the NucliSens® CMV pp67 assay.

17

Table b.2.2.7.a. Numbers of Positive and Negative Tests with Antigenemia and/or Culture and with NucliSens® CMV pp67 in Serial Testing of 50 Patients in a Transplant Population

| Patient
No. | Positive
Tests
Antigenemia
or
Culture | Negative
Tests
Antigenemia
and
Culture | Positive
Tests
NucliSens® | Negative
Tests
NucliSens® | Patient
No. | Positive
Tests
Antigenemia
or
Culture | Negative
Tests
Antigenemia
and
Culture | Positive
Tests
NucliSens® | Negative
Tests
NucliSens® |
|----------------|-- | 1 | 7 | 2 | 4 | 3 | 4 | 4 | 4 | 5 | 5 | 6 | 6 | 7 | 5 | 8 | 12 | 9 | 10 | 10 | 5 | 11 | 13 | 12 | 10 | 13 | 22 | 14 | 7 | 15 | 5 | 16 | 9 | 17 | 6 | 18 | 5 | 19 | 3 | 20 | 13 | 21 | 9 | 22 | 6 | 23 | 10 | 24 | 5 | 25 | 4 -------------------------------------------------|----------------------------------------------------|---------------------------------|---------------------------------|----------------|---------------------------------------------------|----------------------------------------------------|---------------------------------|---------------------------------|
| 5 | 5 | 6 | 26 | 6 | 5 | 5 | 4 |
| 7 | 1 | 10 | 27 | 5 | 2 | 2 | 3 |
| 20 | 3 | 10 | 28 | 1 | 5 | 1 | 1 |
| 11 | 1 | 11 | 29 | 2 | 10 | 1 | 1 |
| 8 | 3 | 10 | 30 | 3 | 4 | 1 | 1 |
| 5 | 1 | 9 | 31 | 1 | 10 | 0 | 1 |
| 8 | 2 | 9 | 32 | 4 | 5 | 0 | 0 |
| 5 | 5 | 6 | 33 | 2 | 12 | 0 | 0 |
| 9 | 7 | 8 | 34 | 6 | 18 | 0 | 1 |
| 9 | 4 | 8 | 35 | 1 | 7 | 0 | 0 |
| 8 | 7 | 11 | 36 | | 20 | 0 | 0 |
| 11 | 1 | 13 | 37 | 2 | 12 | 0 | 1 |
| 3 | 14 | 8 | 38 | 2 | 14 | 0 | 1 |
| 21 | 6 | 12 | 39 | 2 | 10 | 0 | 0 |
| 7 | 3 | 7 | 40 | 0 | 12 | 0 | 0 |
| 15 | 3 | 12 | 41 | 0 | 10 | 0 | 1 |
| 8 | 6 | 7 | 42 | 0 | 11 | 0 | 0 |
| 6 | 5 | 6 | 43 | 0 | 12 | 0 | 0 |
| 6 | 2 | 6 | 44 | 0 | 17 | 0 | 1 |
| 9 | 4 | 16 | 45 | 0 | 14 | 0 | 0 |
| 11 | 5 | 7 | 46 | 0 | 11 | 0 | 0 |
| 8 | 2 | 9 | 47 | 0 | 11 | 0 | 0 |
| 6 | 8 | 7 | 48 | 0 | 15 | 0 | 0 |
| 5 | 5 | 5 | 49 | 0 | 13 | 0 | 0 |
| 5 | 5 | 4 | 50 | 0 | 18 | 0 | 1 |

Agreement between the NucliSens® CMV pp67 assay results and the combined antigenemia/culture results was calculated on a per subject basis (Table b.2.2.7.b.) and on a per test basis (Table b.2.2.7.c.). For the analysis on a per subject basis (Table b.2.2.7.b.), a similar algorithm was employed as for the individual specimens (i.e. combined antigenemia/culture versus NucliSens® CMV pp67) and a patient was considered CMV positive if any specimen for that patient was found positive during the course of the study.

18

Table b.2.2.7.b.Agreement of NucliSens® CMV pp67 Assay with Antigenemia and/or Culture on a Per Subject Basis in a Transplant Population

	Antigenemia and/or Culture*
NucliSens®
CMV pp67	+	-	Total
+	30	0	30
-	9	11	20
Total	39	11	50

Represents the result of any positivity over course of study of serial specimens from 50 patients

Overall Agreement =100 (41/50) = 82%

Overali Agreement = 100 (41/30) = 82%
Sensitivity = 100(30/39) = 77% (95% C.I. using 'exact' method: 60.7 to 88.9%) Positive Predictive Value = 100(30/30/30) = 100% Negative Predictive Value = 100(11/20) = 55%

Table b.2.2.7.c.Agreement of NucliSens® CMV pp67 Assay with Antigenernia and/or Culture on a Per Test Basis in a Transplant Population

	Antigenemia and/or Culture*
NucliSens®
CMV pp67	+	-	Total
+	73	19	92
-	73	203	276
Total	146	222	368

Represents multiple determinations from serial specimens from 50 patients where all tests had valid responses

Overall Agreement =100 (73+203)/368 = 75%

Using the combined antigenemia/culture results as the true state of nature, a Receiver Operator Characteristic (ROC) curve was constructed for the NucliSens® CMV pp67 assay on a per subject basis (Figure b.2.2.7.d.). The estimated area under the curves as a diagnostic measure for the discriminatory capacity of the assay for specimens catagorized as CMV positive or CMV negative in this way, was 0.902 with a standard error of 0.042 for the per subject analysis.

19

Figure b.2.2.7.d. ROC Curve of NucliSens® CMV pp67 Assay with Antigenemia and/or Culture as Truth on a Per Subject Basis in a Transplant Population.

Image /page/19/Figure/4 description: The image is a graph with "1 - specificity" on the x-axis and "Sensitivity" on the y-axis. The x-axis ranges from 0.0 to 1.0, while the y-axis ranges from 0 to 1. The graph shows a curve that starts at approximately (0, 0.8), gradually increasing to (1, 1). The curve indicates the relationship between sensitivity and specificity.

ー NucliSens® CMV pp67

..

20

Time to Positivity in a Transplant Population

To investigate which test first revealed a positive result in the course of time, a time to positivity analysis was carried out. For this analysis, results for antigenemia and culture were considered separately. The Kaplan-Meier curve shown in Figure b.2.2.7.e plots the probability that a test remains negative versus time. Median times to first positive result for antigenermia, culture, and NucliSens® CMV pp67 were 38, 67, and 51 days, respectively.

Image /page/20/Figure/5 description: The image is a graph titled "Time to Positivity - Transplant Population". The graph shows the probability of remaining negative over time, measured in days. There are four lines on the graph, representing different tests: Antigenemia, Culture, HCMV pp67, and Test. The x-axis represents the time to first positive result in days, ranging from 0 to 110, and the y-axis represents the probability of remaining negative, ranging from 0.2 to 1.0.

Figure b.2.2.7.e.'

21

2023

b.2.2.8 HIV-1 Infected population

In a HIV-1 infected population of 50 individuals, the performance of the NucliSens® CMV pp67 assay was compared to cell culture and a pp65 antigenemia assay. Multiple specimens were collected over an extended period of time from these individuals as part of a longitudinal study conducted in the Netherlands to monitor for the presence of HCMV infection or disease during routine clinic visits. Only results from specimens for which all three assays revealed a valid test result were considered. The percentage of invalid test results for the NucliSens® CMV pp67 assay in this study was 1.6%. In Table b.2.2.8.a. the results of this study are summarized. If either antigenemia or culture were found positive for a specimen, that particular specimen was regarded as CMV positive. If both antigenemia and culture were negative, the state of nature for that specimen was considered CMV negative. In the table, for each patient enrolled in the study, the number of CMV positive and CMV negative specimens is indicated based on the combined antigenemia/culture results and based on the NucliSens® CMV pp67 assay.

Table b.2.2.8.a Numbers of Positive and Negative Tests with Antigenemia and/or Culture and with NuclISens® CMV pp67 In Serial Testing of 50 Patients in a HIV Infected Population

| | Positive
Tests
Antigenemia
or
Culture | Negative
Tests
Antigenemia
and
Culture | Positive
Tests
NucliSens® | Negative
Tests
NucliSens® | Subject | Positive
Tests
Antigenemia
or
Culture | Negative
Tests
Antigenemia
and
Culture | Positive
Tests
NucliSens® | Negative
Tests
NucliSens® |
|----|---------------------------------------------------|----------------------------------------------------|---------------------------------|---------------------------------|---------|---------------------------------------------------|----------------------------------------------------|---------------------------------|---------------------------------|
| 1 | 3 | 5 | 0 | 8 | 26 | 2 | 4 | 0 | 5 |
| 2 | 7 | 3 | 7 | 3 | 27 | 7 | 3 | 1 | 5 |
| 3 | 8 | 5 | 6 | 5 | 28 | 4 | 1 | 1 | 4 |
| 4 | 10 | 2 | 2 | 10 | 29 | 3 | 21 | 1 | 22 |
| 5 | 3 | 8 | 0 | 11 | 30 | 2 | 7 | 3 | 11 |
| 6 | 0 | 9 | 0 | 9 | 31 | 0 | 8 | 0 | 5 |
| 7 | 10 | 12 | 6 | 16 | 32 | 0 | 6 | 0 | 6 |
| 8 | 3 | 21 | 1 | 22 | 33 | 3 | 6 | 1 | 8 |
| 9 | 4 | 5 | 2 | 6 | 34 | 5 | 0 | 1 | 4 |
| 10 | 2 | 6 | 0 | 8 | 35 | 6 | 2 | 5 | 3 |
| 11 | 6 | 9 | 5 | 10 | 36 | 3 | 4 | 0 | 7 |
| 12 | 0 | 11 | 0 | 10 | 37 | 5 | 4 | 3 | 6 |
| 13 | 1 | 6 | 0 | 7 | 38 | 0 | 6 | 0 | 6 |
| 14 | 1 | 2 | 0 | 3 | 39 | 1 | 2 | 1 | 2 |
| 15 | 0 | 20 | 0 | 19 | 40 | 4 | 3 | 4 | 3 |
| 16 | 0 | 6 | 0 | 6 | 41 | 1 | 13 | 1 | 12 |
| 17 | 9 | 6 | 4 | 11 | 42 | 5 | 7 | 2 | 10 |
| 18 | 3 | 2 | 2 | 3 | 43 | 1 | 18 | 1 | 18 |
| 19 | 2 | 6 | 1 | 6 | 44 | 4 | 3 | 1 | 6 |
| 20 | 3 | 2 | 1 | 4 | 45 | 2 | 7 | 1 | 8 |
| 21 | 2 | 6 | 1 | 7 | 46 | 7 | 3 | 2 | 8 |
| 22 | 7 | 16 | 5 | 18 | 47 | 7 | 1 | 6 | 2 |
| 23 | 3 | 3 | 0 | 5 | 48 | 0 | 16 | 1 | 15 |

22

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Agreement between the NucliSens® CMV pp67 assay results and the combined antigenemia/culture results was calculated on a per subject basis (Table b.2.2.8.b) and on a per test basis (Table b.2.2.8.c). For the analysis on a per subject basis (Table b.2.2.8.b), a similar algorithm was employed as for the individual specimens (i.e. combined antigenemia/culture versus NucliSens® CMV pp67) and a patient was considered CMV positive if any specimen of the patient was found positive during the course of the study.

Table b.2.2.8.b Agreement of NucliSens® CMV pp67 Assay with Antigenemia and/or Culture on a Per Subject Basis in a HIV-1 Infected Population

	Antigenemia and/or Culture*
NucliSens®
CMV pp67	+	-	Total
+	32	1	33
-	9	8	17
Total	41	9	50

Represents the result of any positivity over course of study of serial specimens from 50 patients

Overall Agreement =100 (40/50) = 80% Sensitivity = 100(32/41) = 78% Specificity = 100(8/9) = 89% (95% C.I. using 'exact' method: 51.8 to 99.7%) Positive Predictive Value = 100(32/33) = 97% Negative Predictive Value = 100(8/17) = 47%

Table b.2.2.8.c Agreement of NucliSens® CMV pp67 Assay with Antigenemia and/or Culture on a Per Test Basis in a HIV-1 Infected Population

	Antigenemia and/or Culture*
NucliSens®
CMV pp67	+	-	Total
+	76	11	87
-	95	268	363
Total	171	279	450

Represents multiple determinations from serial speciments where all tests had valid responses

Overall Agreement =100 (268+76)/450 = 76%

Using the combined antigenemia/culture results as the true state of nature, a ROC curve were constructed for the NucliSens® CMV pp67 assay on a per subject basis (Figure b.2.2.8.d.). The estimated area under the curve as a diagnostic measure for the discriminatory capacity of the assay for specimens

23

团 025

catagorized as CMV positive or CMV negative in this way, was 0.862 with a standard error of 0.054 for the per subject analysis.

Figure b.2.2.8.d. ROC Curve of NucllSens® CMV pp67 Assay with Antigenemia and/or Culture as Truth on a Per Subject Basis in a HIV-1 Infected Population is shown in the following figure.

Image /page/23/Figure/5 description: This image is a plot of sensitivity versus 1-specificity. The x-axis is labeled "1 - specificity" and ranges from 0.0 to 1.0. The y-axis is labeled "Sensitivity" and ranges from 0 to 1. The plot shows a curve that starts at (0,0), rises sharply to around (0, 0.6), then increases in steps to around (0.3, 0.85) before gradually increasing to (1,1).

24

Time to Positivity in a HIV Infected Population

To investigate which test first revealed a positive result in the course of time, a time to positivity analysis was carried out. For this analysis, results for antigenemia and culture were considered separately. The Kaplan-Meier curve shown in Figure b.2.2.8.e. plots the probability that a test remains negative versus time. Median times to first positive result for antigenemia, culture, and NucliSens® CMV pp67 were 405, 519, and 458 days, respectively. Figure b.2.2.8.e

Time to Positivity - HIV Population

Image /page/24/Figure/4 description: The image is a graph that shows the probability of remaining negative over time for different tests. The x-axis represents the time to first positive result in days, ranging from 0 to 1200. The y-axis represents the probability of remaining negative, ranging from 0.0 to 1.0. The graph compares four different tests: Antigenemia, Culture, and NucliSens.

25

(b)3) The conclusions drawn from the nonclinical and clinical tests that demonstrate that the device is as safe, as effective, and performed as well or better than the legally marketed device identified in (a)(3).

The conclusions drawn from the clinical tests demonstrate that the NucliSens™ CMV pp67 assay is as safe, as effective, and performed as well as the legally marketed CMV Brite™ CMV Antigenemia Detection Test Kit and typical cell culure in the diagnosis of active HCMV infection.

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1. Verhofstede, C., Fransen, K., Marissens, D., Verhelst, R., van der Groen, G., Lauwers, S., Zissis, G., and Plum, J. (1996). Isolation of HIV-1 RNA from plasma: Evaluation of eight different extraction methods. J. Virol. Methods 60. 155-159.
1. Vernazza, P. L., Dyer, J. R., Fiscus, S. A., Eron, J. J., and Cohen, M. S. (1997). HIV-1 viral load in blood, semen and saliva. AIDS 11, 1058.
16 Gupta, P., Mellors, J., Kingsley, L., Riddler, S., Singh, M. K., Schreiber, S., Cronin, M., and Rinaldo, C. R. (1997). High viral load in semen of human immunodeficiency virus type 1-infected men at all stages of disease and its reduction by therapy with protease and nonnucleoside reverse transcriptase inhibitors. J. Virol. 71, 6271-6275.
17 Dyer, J. R., Gilliam, B. L., Eron, J. J., Grosso, L., Cohen, M. S., and Fiscus, S. A. (1996). Quantitation of human immunodeficiency virus type 1 RNA in cell free seminal plasma: Comparison of NASBA™ with Amplicor™ reverse transcription-PCR amplification and correlation with quantitative culture. J. Virol. Methods 60, 161-170.
18 Compton, J. (1991). Nucleic acid sequence-based amplification. Nature 350, 91-92.
1. Kenten, J.H., Gudibande, S., Link, J., Willey, J.J., Curfman, B., Major, E.O., and Massey, R.J. (1992). Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV-1 polymerase chain reaction products. Clin. Chem. 38, 873-879.

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Image /page/27/Picture/1 description: The image shows the seal of the Department of Health & Human Services - USA. The seal features a stylized eagle with three stripes representing the department's mission. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" is arranged in a circular pattern around the eagle.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

SEP 1 5 1999

Mr. Ron Sanyal, M. Pharm., CQE, RAC Regulatory Affairs Administrator Organon Teknika Corporation 100 Akzo Avenue Durham, North Carolina 27712

Re: K983762 Trade Name: NucliSens® CMV pp67 Regulatory Class: II Product Code: LIN Dated: July 1, 1999 Received: July 2, 1999

Dear Mr. Sanyal:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we 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). You may, therefore, market the device, subject to the general controls provisions of the Act. 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.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 895. A substantially equivalent determination assumes compliance with the Current Good Manufacturing Practice requirements, as set forth in the Quality System Regulation (QS) for Medical Devices: General regulation (21 CFR Part 820) and that, through periodic QS inspections, the Food and Drug Administration (FDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory action. In addition, FDA may publish further announcements concerning your device in the Federal Register. Please note: this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal laws or regulations.

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Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at (770)488-7655.

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll free number (800) 638-2041 or at (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsmamain.html"

Sincerely yours,

Steven Sutman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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Page 1 of 1

510(k) NUMBER (IF KNOWN): K983762

DEVICE NAME: NucliSens® CMV pp67

INDICATIONS FOR USE:

Then NucliSens® CMV pp67 assay is a nucleic acid amplification-based qualitative assay to be used in conjunction with the Nuclisens® Reader for the detection of human cytomegalovirus (HCMV) pp67 mRNA in EDTA anticoagulated whole blood from adult transplant donors and HIV infected individuals. It is intended as an aid in the diagnosis of active (acute or reactivated) HCMV infection. This product is not intended for use in screening of blood or plasma donors.

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

	Concurrence of CDRH, Office of Device Evaluation (ODE)

	(Division Sign-Off)
	Division of Clinical Laboratory Devices
	510(k) Number
	K923762
Prescription Use
(Per 21 CFR 801.109)	OR	Over-The Counter- Use
(Optional Format 1-2-96)