NUCLISENS CMV PP67

K983762 · Organon Teknika Corp. · LIN · Sep 15, 1999 · Microbiology

Device Facts

• Record ID: K983762
• Device Name: NUCLISENS CMV PP67
• Applicant: Organon Teknika Corp.
• Product Code: LIN · Microbiology
• Decision Date: Sep 15, 1999
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3175
• Device Class: Class 2
• Attributes: AI/ML

AI Performance

Output	Acceptance	Observed	Dev DS	Test DS
Detection of active human cytomegalovirus (HCMV) infection	Not specified	Transplant population: Sensitivity 77% (95% C.I. 60.7-88.9%), Specificity not explicitly calculated but overall agreement 82%. HIV-1 infected population: Sensitivity 78%, Specificity 89% (95% C.I. 51.8-99.7%), overall agreement 80%.	Not specified	Transplant population: 50 patients (longitudinal study in Italy). HIV-1 infected population: 50 individuals (longitudinal study in the Netherlands).

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.

Device Story

The NucliSens® CMV pp67 assay is an in vitro diagnostic test for detecting HCMV pp67 mRNA in EDTA-anticoagulated whole blood. The process involves four stages: nucleic acid release using lysis buffer (guanidine thiocyanate/Triton X-100); isolation using silica particles; isothermal nucleic acid amplification (NASBA) using AMV-RT, RNase H, and T7 RNA polymerase; and detection via electrochemiluminescence (ECL) using a NucliSens® Reader. An internal System Control (SC) RNA is added during lysis to validate the extraction, amplification, and detection process. The device is used in clinical laboratory settings by trained personnel. Results are reported as ECL counts; the presence of HCMV pp67 mRNA is indicated by the WT RNA ECL signal. The assay aids clinicians in diagnosing active HCMV infection in immunocompromised patients, potentially facilitating earlier clinical intervention compared to traditional culture or antigenemia methods.

Clinical Evidence

Clinical performance evaluated in two longitudinal studies (50 transplant patients, 50 HIV-infected individuals). In transplant patients, sensitivity was 77% (95% CI: 60.7-88.9%) and specificity 100% relative to combined antigenemia/culture. In HIV patients, sensitivity was 78% and specificity 89%. Analytical studies included 1,427 samples with 99.3% valid results. The assay demonstrated no cross-reactivity with common viruses (HSV, EBV, VZV, HHV-6/7/8) or bacterial flora. Reproducibility was confirmed across three sites and lots. Median time to positivity was 51 days (transplant) and 458 days (HIV), compared to antigenemia and culture.

Technological Characteristics

Nucleic acid amplification-based qualitative assay. Uses NASBA (Nucleic Acid Sequence-Based Amplification) technology. Components include lysis buffer (guanidine thiocyanate, Triton X-100), silica particles for isolation, and paramagnetic beads for ECL detection. Requires NucliSens® Reader for automated analysis. Employs internal System Control (SC) RNA for process validation. Sterilization/biocompatibility not applicable as it is an in vitro diagnostic reagent kit.

Indications for Use

Indicated for detection of HCMV pp67 mRNA in EDTA anticoagulated whole blood from adult transplant donors and HIV-infected individuals to aid in diagnosis of active (acute or reactivated) HCMV infection. Not for blood/plasma donor screening.

Regulatory Classification

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.

Predicate Devices

• CMV Brite™ CMV Antigenemia Detection Test Kit

Submission Summary (Full Text)

{0}------------------------------------------------

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.

团 0 0 0 2

# 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: [phone]

**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

{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<br>CORPORATION<br>NucliSens™ CMV pp67 | BIOTEST DIAGNOSTICS CORPORATION<br>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<br>In Vitro Assay | The CMV Brite test kit uses the application of a<br>two monoclonal antibodies (C10/C11) cocktail<br>in an indirect immunofluorescence staining of<br>cytospin preparations of peripheral blood<br>leukocytes. | Cell culture innoculation |
| INTENDED USE | This test is intended as an aid to<br>detect an active CMV infection. | This test is intended as an aid to detect an active<br>CMV infection. | This test is intended as an aid to detect an active<br>CMV infection. |
| | The NucliSens™ CMV pp67 is a<br>nucleic acid amplification-based in<br>vitro qualitative assay for detection<br>of the presence of human<br>cytomegalovirus (HCMV) pp67<br>mRNA in anticoagulated whole<br>blood. It is intended as an aid in the<br>diagnosis of active (acute or<br>reactivated) HCMV infection. This<br>product is not intended for use in<br>screening of blood or plasma donors. | The CMV Brite test kit is intended for the<br>qualitative detection of cytomegalovirus (CMV)<br>lower matrix protein pp65 by indirect<br>immunofluorescence using microscopy in<br>isolated peripheral blood leukocytes obtained<br>from EDTA and heparin anticoagulated human<br>peripheral blood. The detection of CMV pp65 in<br>human peripheral blood cells aids in the<br>diagnosis of acute or reactivated CMV infection.<br>This product is not FDA cleared (approved) for<br>use in testing (i.e. screening) of blood or plasma<br>donors. | Cell culture is intended for the qualitative<br>detection of the presence of HCMV in<br>leukocytes from whole blood. It aids in the<br>diagnosis of HCMV infection. |
| SAMPLE | Anticoagulated whole blood. | Isolated peripheral blood leukocytes obtained<br>from EDTA and heparin anticoagulated human<br>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<br>Negative Results/Total | Spiked with CMV RNA<br>Positive Results/Total |
|--------------------------------|------------------------------------|-----------------------------------------------|
| Icteric (up to 3.8 mg/dL) | 10/10 | 10/10 |
| Hemolyzed | 10/10 | 10/10 |
| Lipemic (up to 2,400<br>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<br>Negative Results/Total | Spiked with HCMV RNA<br>Positive Results/Total |
|---------------------------------|------------------------------------|------------------------------------------------|
| HIV-1 | 10/10 | 10/10 |
| HTLV-I/II | 8/8 | 8/8 |
| Herpes Simplex Virus<br>Type I | 10/10 | 10/10 |
| Herpes Simplex Virus<br>Type II | 10/10 | 10/10 |
| Epstein Barr Virus | 10/10 | 10/10 |
| Varicella Zoster Virus | 10/10 | 10/10 |
| Respiratory Syncytial<br>Virus | 10/10 | 10/10 |
| Systemic Lupus<br>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<br>Antigenemia | | | | |
|-------------------------|----------|----------|-------|----|
| | Positive | Negative | Total | |
| NucliSens®<br>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<br>® | 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<br>No. | Positive<br>Tests<br>Antigenemia<br>or<br>Culture | Negative<br>Tests<br>Antigenemia<br>and<br>Culture | Positive<br>Tests<br>NucliSens® | Negative<br>Tests<br>NucliSens® | Patient<br>No. | Positive<br>Tests<br>Antigenemia<br>or<br>Culture | Negative<br>Tests<br>Antigenemia<br>and<br>Culture | Positive<br>Tests<br>NucliSens® | Negative<br>Tests<br>NucliSens® |
|----------------|---------------------------------------------------|----------------------------------------------------|---------------------------------|---------------------------------|----------------|---------------------------------------------------|----------------------------------------------------|---------------------------------|---------------------------------|
| 1 | 7 | 5 | 5 | 6 | 26 | 6 | 5 | 5 | 4 |
| 2 | 4 | 7 | 1 | 10 | 27 | 5 | 2 | 2 | 3 |
| 3 | 4 | 20 | 3 | 10 | 28 | 1 | 5 | 1 | 1 |
| 4 | 4 | 11 | 1 | 11 | 29 | 2 | 10 | 1 | 1 |
| 5 | 5 | 8 | 3 | 10 | 30 | 3 | 4 | 1 | 1 |
| 6 | 6 | 5 | 1 | 9 | 31 | 1 | 10 | 0 | 1 |
| 7 | 5 | 8 | 2 | 9 | 32 | 4 | 5 | 0 | 0 |
| 8 | 12 | 5 | 5 | 6 | 33 | 2 | 12 | 0 | 0 |
| 9 | 10 | 9 | 7 | 8 | 34 | 6 | 18 | 0 | 1 |
| 10 | 5 | 9 | 4 | 8 | 35 | 1 | 7 | 0 | 0 |
| 11 | 13 | 8 | 7 | 11 | 36 | | 20 | 0 | 0 |
| 12 | 10 | 11 | 1 | 13 | 37 | 2 | 12 | 0 | 1 |
| 13 | 22 | 3 | 14 | 8 | 38 | 2 | 14 | 0 | 1 |
| 14 | 7 | 21 | 6 | 12 | 39 | 2 | 10 | 0 | 0 |
| 15 | 5 | 7 | 3 | 7 | 40 | 0 | 12 | 0 | 0 |
| 16 | 9 | 15 | 3 | 12 | 41 | 0 | 10 | 0 | 1 |
| 17 | 6 | 8 | 6 | 7 | 42 | 0 | 11 | 0 | 0 |
| 18 | 5 | 6 | 5 | 6 | 43 | 0 | 12 | 0 | 0 |
| 19 | 3 | 6 | 2 | 6 | 44 | 0 | 17 | 0 | 1 |
| 20 | 13 | 9 | 4 | 16 | 45 | 0 | 14 | 0 | 0 |
| 21 | 9 | 11 | 5 | 7 | 46 | 0 | 11 | 0 | 0 |
| 22 | 6 | 8 | 2 | 9 | 47 | 0 | 11 | 0 | 0 |
| 23 | 10 | 6 | 8 | 7 | 48 | 0 | 15 | 0 | 0 |
| 24 | 5 | 5 | 5 | 5 | 49 | 0 | 13 | 0 | 0 |
| 25 | 4 | 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®<br>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®<br>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<br>Tests<br>Antigenemia<br>or<br>Culture | Negative<br>Tests<br>Antigenemia<br>and<br>Culture | Positive<br>Tests<br>NucliSens® | Negative<br>Tests<br>NucliSens® | Subject | Positive<br>Tests<br>Antigenemia<br>or<br>Culture | Negative<br>Tests<br>Antigenemia<br>and<br>Culture | Positive<br>Tests<br>NucliSens® | Negative<br>Tests<br>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}------------------------------------------------

| ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | | Any Sep 2007 | - A4 -- A Mich Allehouse | A LAND AND L A . A . A . L<br>488 | Comments of the control of the consisted on the consisted on the consisted on the control of the contribution of the contribution of the contribution of the contribution of t | | | -------------------------------------- |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|---------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---|----------------------------------------|
| A Acres and<br>t | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | because and the distribution of the collection of the collection and the contract and the consisted to the consisted to the consisted to the consisted to the consisted to the | | . Accomments of the consideration and the defense<br>And Control Control Concession Company of Concession Company of Concession | ------------------------------------------------------------------------------------------------------------------------<br>1 | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------<br>------------------------------------------------------------------------------------------------------------<br>. | . | |

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®<br>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®<br>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.

## References

- 1. Stagno, S. (1990). Cytomegalovirus. In: Infectious Diseases of the Fetus and Newborn Infant. Remington, J.S. and Klein, J., eds. Saunders, W.B., Philadelphia, pp 241-281.
- 2. Drew, W.L. (1992). Cytomegalovirus infection in patients with AIDS. Clin. Infect. Dis. 14, 608-615.
- 3. Gozlan, J., Caburet, F., Tancrede, C., and Petit, J.-C. (1992). A reverse polymerase chain reaction method for detection of human cytomegalovirus late transcripts in cells infected in vitro, J. Virol. Methods 40, 1-10.
- Bitsch, A., Kirchner, H., Dupke, R., and Bein, G. (1993). Cytomegalovirus ব transcripts in peripheral blood leukocytes of actively infected transplant patients detected by reverse transcription-polymerase chain reaction. J. Infect. Dis. 167, 740-743.
- Gozlan, J., Salord, J.-M., Chouaïd, C., Duvivier, C., Picard, O., Meyohas, M.-5. C., and Petit, J.-C. (1993). Human cytomegalovirus (HCMV) late-mRNA detection in peripheral blood of AIDS patients: diagnostic value for HCMV disease compared with those of viral culture and HCMV DNA detection. J. Clin. Microbiol. 31, 1943-1945.
- Meyer, T., Reischl, U., Wolf, H., and Arndt, R. (1994). Identification of active o cytomegalovirus infection by analysis of immediate-early, early and late transcripts in peripheral blood cells of immunodeficient patients. Mol. Cell. Probes 8, 261-271.
- 7. Meyer-König, U., Serr, A., von Laer, D., Kirste, G., Wolff, C., Haller, O., Neumann-Haefelin, D., and Hufert, F. T. (1995). Human cvtomegalovirus immediate early and late transcripts in peripheral blood leukocytes; diagnostic value in renal transplant recipients. J. Infect. Dis. 171, 705-709.
- Velzing, J., Rothbarth, P. H., Kroes, A. C. M., and Quint, W. G. V. (1994). 8. Detection of cytomegalovirus mRNA and DNA encoding the immediate early gene in peripheral blood leukocytes from immunocompromised patients. J. Med. Virol. 42. 164-169.

{26}------------------------------------------------

- ல் Nelson, P. N., Rawal, B. K., Boriskin, Y. S., Mathers, K. E., Powles, R. L., Steel, H. M., Tryhorn, Y. S., Butcher, P. D., and Booth, J. C. (1996). A polymerase chain reaction to detect a spliced late transcript of human cytomegalovirus in the blood of bone marrow transplant recipients. J. Virol. Methods 56, 139-148.
- 10. Gaeta, A., Nazzari, C., Angeletti, S., Lazzarini, M., Mazzei, E., and Mancini, C. (1997). Monitoring for cytomegalovirus infection in organ transplant recipients: analysis of op65 antigen. DNA and late mRNA in peripheral blood leukocytes. J. Med. Virol. 53, 189-195.
- 11. Blok, M. J., Goossens, V. J., Vanherle, S. J. V., Top, B., Tacken, N., Middeldorp, J., Christiaans, M. H. L., van Hooff, J. P., and Bruggeman, C. A. (1998). The diagnostic value of monitoring human cytomegalovirus (CMV) late pp67 mRNA expression using nucleic acid sequence-based amplification (NASBA) in renal allograft recipients. J. Clin. Microbiol. J. Clin. Microbiol. May, 1998, p. 1341 - 1346.
- 12 Davis, M. G., and Huang, E .- S. (1985). Nucleotide sequence of a human cytomegalovirus DNA fragment encoding a 67-kilodalton phosphorylated viral protein. J. Virol. 56, 1-11.
- 13. Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M., and van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495-503.
- 14. 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.
- 15. 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.
- 19. 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.

{27}------------------------------------------------

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.

{28}------------------------------------------------

Page 2

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 [phone].

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 [phone]. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at [phone]. 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 [phone] or at [phone] 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

{29}------------------------------------------------

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. T…