Vaccines, Blood & Biologics

May 25, 2012 Summary Basis for Regulatory Action - Procleix Ultrio Plus Assay

From: Jinjie Hu, Ph.D., Scientific Lead, BLA Efficacy Supplement Review Committee

BLA Efficacy Supplement STN#: 125113-48

Applicant Name: Gen-Probe Incorporated

Date of Submission: July 27, 2011

MDUFMA Goal Date: May 25, 2012

Trade Name: Procleix® Ultrio Plus® Assay

Proper Name: Human Immunodeficiency Virus Type 1 and/or Hepatitis C Virus and/or Hepatitis B Virus (HIV-1 and HCV and HBV/Nucleic Acid Pooled Testing/Synthetic)

Indication: To detect Human Immunodeficiency Virus Type 1 RNA and/or Hepatitis C Virus RNA and/or Hepatitis B Virus DNA in human serum and plasma specimens

Recommended Action: Approval

Office’s Signatory Authority: Jay S. Epstein, M.D.
Director, OBRR/CBER

□ I concur with the summary review.
□ I concur with the summary review and include a separate review to add further analysis.
□ I do not concur with the summary review and include a separate review.


Material Reviewed/ Consulted: Specific documentation used in developing the SBRA

Review memos from the following reviewers were used in developing the SBRA.

Discipline reviewed

Reviewer names

Clinical and non-Clinical/analytical Review

HIV: Andy Dayton, Mingjie Zhang
HCV: Deborah Taylor
HBV: Erica Silberstein

CMC Review

HIV: Pradip Akolkar
HCV: Krishnamurthy Konduru
HBV: Susan Zullo

Statistical Review

Jessica Hu

Facility Review

Daniel Kearns

Software and Instrumentation

Diane Gubernot

OCTGT review

Karoll Cortez

Bioresearch Monitoring

Christine Drabick

Lot Release Testing

Steve Kerby

Labeling and Promotional Advertizing

Jinjie Hu, Dana Jones


Intended Use

The PROCLEIX ULTRIO PLUS Assay is a qualitative in vitro nucleic acid amplification test for use on the PROCLEIX TIGRIS System to screen for human immunodeficiency virus type 1 (HIV-1) RNA, hepatitis C virus (HCV) RNA and hepatitis B virus (HBV) DNA in plasma and serum specimens from individual human donors, including donors of whole blood, blood components, and source plasma, and from other living donors. It is also intended for use in testing plasma and serum specimens to screen organ donors when specimens are obtained while the donor's heart is still beating, and in testing blood specimens from cadaveric (non-heart-beating) donors.

The assay is intended for use in testing individual samples from living donors of whole blood, blood components, and source plasma, other living donors and heart-beating organ donors, and for testing individual blood specimens from cadaveric (non-heart-beating) donors. It is also intended for use in testing pools of human plasma comprised of equal aliquots of not more than 16 individual donations from donors of whole blood, blood components, and source plasma. It is also intended for use in testing pools of human plasma comprised of equal aliquots of not more than 16 individual specimens from donors of hematopoietic stem/progenitor cells (HPCs) sourced from bone marrow, peripheral blood or cord blood, and from donors of donor lymphocytes for infusion (DLI).

This assay is intended to be used in conjunction with licensed tests for detecting antibodies to HIV-1, HCV, and hepatitis B core antigen (anti-HBc), and with licensed tests for hepatitis B surface antigen (HBsAg).

  • This assay is not intended for use as an aid in diagnosis of infection with HIV-1, HCV or HBV.
  • The assay is not intended for use on cord blood specimens.

The PROCLEIX ULTRIO PLUS Assay can be considered a supplemental test that confirms HIV-1 infection for specimens that are repeatedly reactive on a licensed donor screening test for antibodies to HIV-1, and reactive on both the PROCLEIX ULTRIO PLUS Assay and on the PROCLEIX ULTRIO PLUS HIV-1 Discriminatory Assay.

The PROCLEIX ULTRIO PLUS Assay can be considered a supplemental test that confirms HCV infection for specimens that are repeatedly reactive on a licensed donor screening test for antibodies to HCV, and reactive on both the PROCLEIX ULTRIO PLUS Assay and on the PROCLEIX ULTRIO PLUS HCV Discriminatory Assay.

The PROCLEIX ULTRIO PLUS Assay can be considered a supplemental test that confirms HBV infection for specimens that are repeatedly reactive on a licensed donor screening test for HBsAg, and reactive on both the PROCLEIX ULTRIO PLUS Assay and on the PROCLEIX ULTRIO PLUS HBV Discriminatory Assay.

Background

The PROCLEIX ULTRIO PLUS Assay (Ultrio Plus Assay) is a Nucleic Acid Test based on the licensed Procleix® Ultrio® Assay (Ultrio Assay) (STN BL 125113; approved October 03, 2006). The Ultrio Plus Assay is identical to the Ultrio Assay with minor modifications to both the assay and TIGRIS System to include an additional reagent, the Target Enhancer Reagent (TER), modifications to the TIGRIS System to allow use of the TER, and ----b(4)----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Assay but not in the Ultrio Assay). The changes to the Ultrio Plus Assay improve the detection of low copy levels of HBV DNA as compared to the Ultrio Assay, while demonstrating equivalent assay specificity and sensitivity for HIV-1 RNA or HCV RNA.
The Ultrio Plus HIV-1, HCV, and HBV Discriminatory Assays use the same procedure and reagents as the Ultrio Plus Assay, with one difference: HIV-1-specific, HCV-specific, or HBV-specific probe reagents are used in place of the Ultrio Plus Assay probe reagents.

Biological Principles of the Test

The Ultrio Plus Assay involves three main steps which take place in a single tube: Sample Preparation, HIV-1 RNA, HCV RNA and HBV DNA target amplification by Transcription-Mediated Amplification (TMA), and detection of the Amplification products (amplicons) by the Hybridization Protection Assay (HPA).

During Sample Preparation, the specimen is treated with a detergent to solubilize the viral envelope, denature protein and release viral genomic RNA and/or DNA. For the Ultrio Plus Assay, the TER is added to each reaction tube after the addition of the sample to enhance the disruption of the HBV viral particles.

Capture oligonucleotides that are homologous to highly conserved regions of HIV-1, HCV and HBV are hybridized to the HIV-1 RNA, HCV RNA and HBV DNA target, if present, in the test specimen. The hybridized target is captured onto magnetic microparticles which are then separated from the specimen in a magnetic field. Wash steps are utilized to remove extraneous components from the reaction tube. Magnetic separation and wash steps are performed with a target capture system.

Target amplification occurs via TMA, which is a transcription-based nucleic acid amplification method that utilizes two enzymes. First, MMLV reverse transcriptase generates a single stranded DNA copy of the target sequence and then T7 RNA polymerase produces multiple copies of RNA amplicons from the DNA copy template.

Detection is achieved by HPA using single-stranded nucleic acid probes with chemiluminescent labels that are complementary to the amplicon, which hybridize specifically to the amplicon. The Selection Reagent differentiates between hybridized and unhybridized probes by inactivating the label on unhybridized probes. The chemiluminescent signal produced by the hybridized probe is measured in a luminometer and is reported as Relative Light Units (RLU).

An Internal Control (IC) is added to each test specimen, control, or assay calibrator tube. The IC serves as a control for the specimen processing, Amplification, and Detection steps. IC-specific amplicons are detected using a probe with rapid emission of light (termed a “flasher signal”). Amplicons specific to HIV-1/HCV/HBV are detected using probes with relatively slower kinetics of light emission (termed a “blower signal”). The reports for the Ultrio Plus multiplex assay are a combined signal of HIV-1/HCV/HBV and are not discriminated among individual HIV-1, HCV and HBV signals.

Specimens found to be reactive in the Ultrio Plus Assay must be run in individual HIV-1, HCV and HBV Discriminatory Assays to specifically identify the source of the multiplexed reactivity.

The Ultrio Plus Assay kit consists of the following components:

  • Internal Control Reagent: A HEPES buffered solution containing detergent and an RNA transcript
  • Target Capture Reagent: A HEPES buffered solution containing detergent, capture oligonucleotides and magnetic microparticles
  • Amplification Reagent: Primers, dNTPs, NTPs and co-factors in TRIS buffered solution containing PROCLIN 300 as preservative
  • Enzyme Reagent: MMLV Reverse Transcriptase and T7 RNA Polymerase in HEPES/TRI buffered solution containing 0.05% sodium azide as preservative
  • Probe Reagent: Chemiluminescent oligonucleotide probes in succinate buffered solution containing detergent
  • Selection Reagent: Borate buffered solution containing surfactant
  • Target Enhancer Reagent: A concentrated solution of lithium hydroxide
  • Negative Calibrator: Defibrinated normal human plasma (nonreactive for HIV-1/2, HCV, and HBV when tested by FDA-licensed assays) containing gentamicin and 0.2% sodium azide as preservatives.
  • HIV-1 Positive Calibrator: Inactivated HIV-1 positive plasma in defibrinated normal human plasma (nonreactive for HIV-2, HCV, and HBV when tested by FDA-licensed assays) containing gentamicin and 0.2% sodium azide as preservatives
  • HCV Positive Calibrator: Inactivated HCV positive plasma in defibrinated normal human plasma (nonreactive for HIV-1/2 and HBV when tested by FDA-licensed assays) containing gentamicin and 0.2% sodium azide as preservatives
  • HBV Positive Calibrator: Inactivated HBV positive plasma in defibrinated normal human plasma (nonreactive for HIV-1/2 and HCV when tested by FDA-licensed assays) containing gentamicin and 0.2% sodium azide as preservatives
  • PROCLEIX Assay Fluids
    Wash Solution: --b(4)-- buffered solution
    Oil: -- b(4)------ oil
    Buffer for Deactivation Fluid: ----b(4)------------- buffered solution
  • PROCLEIX Auto Detect Reagents
    Auto Detect 1: --------b(4)-------------------------------------------------
    Auto Detect 2: --b(4)--sodium hydroxide
    The PROCLEIX HIV-1, HCV, and HBV Discriminatory Assays utilize the same procedure and reagents as the Procleix Ultrio Plus Assay with one difference: HIV-1-specific, HCV-specific, or HBV-specific probe reagents are used in place of the Ultrio Plus Assay Probe Reagent.
  • PROCLEIX HIV-1 Discriminatory Probe Reagent: Chemiluminescent oligonucleotide probe in succinate buffered solution containing detergent
  • PROCLEIX HCV Discriminatory Probe Reagent: Chemiluminescent oligonucleotide probe in succinate buffered solution containing detergent
  • PROCLEIX HBV Discriminatory Probe Reagent: Chemiluminescent oligonucleotide probe in succinate buffered solution containing detergent
     

REGULATORY REVIEW

Chemistry, Manufacturing and Controls (CMC):

  1. Primers, Probes, and Assay Controls

    -----b(4)------------ Ultrio Plus Assay oligonucleotides are identical to those of the Ultrio Assay or the licensed Procleix HIV-1/HCV Assay (HIV-1/HCV; STN 103966). ----b(4)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------and –b(4)---------- that are contained in the PROCLEIX HIV-1/HCV Assay but not in the Ultrio Assay. These oligonucleotide changes do not impact the detection of or the clinical sensitivity for HIV-1 and HCV RNA, as demonstrated in analytical and non-clinical studies.

    Manufacture of the Ultrio Plus Assay utilizes a number of previously developed and validated formulations, processes, and container/closures used in the Ultrio Assay. The reagents that make up the Ultrio Plus Assay are identical to those in the Ultrio Assay with the following exceptions:

    • For the Ultrio Plus Assay, a new reagent (TER) has been developed to ---b(4)-----------------------. This reagent is an alkaline solution of lithium hydroxide that is added to each tube after the addition of the sample, to enhance the disruption of HBV viral particles and ------b(4)------------------------------------
    • The Ultrio Plus Assay Internal Control Reagent was reformulated to contain a ---b(4)-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
    • The formulation of the Probe Reagent and Discriminatory (HIV-1, HCV, HBV) Probe Reagents have been modified as follows:
    • ------b(4)-----------------------------------------------------------------------------------------------------------------------
    • ------b(4)----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
       
  2. Manufacturing Quality Control

    Manufacturing Overview

    The Ultrio Plus Assay is produced at the licensed -----b(4)--------------------------------------------------- and the Genetic Center Drive Facility (Registration No. 2024800) ---b(4)-------------------- as the Ultrio Assay. Establishment information can be found in the Summary Basis for Regulatory Action for the Ultrio Assay. Manufacture of the Bulk Target Enhancer Reagent is performed in -----b(4)------------------------------------------, based on demand requirements.

    The Ultrio Plus Assay components are manufactured by Gen-Probe Incorporated. The manufactured components of the assay include Oligonucleotides, Controls, Internal Control and Reagents. Acceptance criteria and specifications have been established for all kit components. Components are assembled into kits, each lot of which is subjected to a final performance test with an in-house panel of samples containing varying copy levels of HIV-1, HCV, and HBV. Meeting the established performance parameters is required for kit release.

  3. CBER Lot Release

    Three clinical lots of the Ultrio Plus Assay kit are required to be submitted to CBER for evaluation. Each master lot of commercial product, along with the lot release protocol summarizing pertinent product testing, are submitted for evaluation and approval by CBER prior to release for distribution.

Non-Clinical Studies:

Non-clinical studies were performed by the Research and Development Department at Gen-Probe per agreement with FDA. Two reagent master kit lots of the Ultrio Plus Assay and Discriminatory Assays (Pilot Lots –b(4)---) were used to perform the non-clinical studies.

  1. Determination of Limit of Detection (LOD) for the Ultrio Plus Assay and the Ultrio Plus Discriminatory Assays

    Standard panels were prepared from the World Health Organization (WHO) International Standards: HIV-1 (97/650), HCV (06/100), and HBV (97/750). A total of 6 Ultrio Plus Assay kit lots were used to test the HIV-1 and HBV panels (2 kit lots were used to test each of 3 unique HIV-1 and HBV WHO panel preparations). Each of the 6 Ultrio Plus Assay kit lots was tested in 60 replicates at each HIV-1 and HBV concentration to yield a total of 360 replicates at each level. A total of 4 Ultrio Plus Assay kit lots were used to test the HCV panel (2 kit lots were used to test each of 2 unique HCV panel preparations). Each of the 4 Ultrio Plus Assay kit lots was tested in 60 replicates at each HCV concentration for a total of 240 replicates at each level. The panels were tested with the Ultrio Plus Assay and the Ultrio Plus Discriminatory (dHIV-1, dHCV and dHBV) Assays. The SCORE method was used to calculate the 95% confidence intervals using SAS version 9.2 (Cary, NC). SAS version 9.2 was also used to perform Probit and Pearson chi-square analysis. There are no significant differences (p>0.05) in the 95% limits of detection between the Ultrio Plus Assay and the respective discriminatory assays for all three panels (see Table 1a).

    The 95% limits of detection for HIV-1 RNA by the Ultrio Plus Assay and the Ultrio Plus dHIV-1 Assay are 21.2 IU/mL and 18.9 IU/mL respectively. The 95% limits of detection for HCV RNA by the Ultrio Plus Assay and the Ultrio Plus dHCV Assay are 5.4 IU/mL and 4.4 IU/mL respectively. The 95% limits of detection for HBV DNA by the Ultrio Plus Assay and the Ultrio Plus dHBV Assay are 3.4 IU/mL and 4.1 IU/mL respectively.

    Table 1a: Limits of Detection for HIV-1 RNA, HCV RNA, and HBV DNAWHO International Standards

    Panel Tested

    PROCLEIX Assay

    95% Limit of Detection
    (95% CI),
    IU/mL

    p Values from Comparison of the Discriminatory Assays and the Ultrio Plus Assay Limits of Detection

    HIV-1 WHO (97/650)

    Ultrio Plus Assay

    21.2 (18.2 - 25.7)

    0.96

    Ultrio Plus
    dHIV-1 Assay

    18.9 (16.3 - 22.9)

    HCV WHO (06/100)

    Ultrio Plus Assay

    5.4 (4.5 - 6.7)

    0.77

    Ultrio Plus
    dHCV Assay

    4.4 (3.7 - 5.6)

    HBV WHO (97/750)

    Ultrio Plus Assay

    3.4 (3.0 - 4.1)

    0.99

    Ultrio Plus
    dHBV Assay

    4.1 (3.5 - 4.9)

    Testing of up to three replicates was performed to detect HBV WHO International Standard 97/750 at 10 copies/mL (approximately 2 IU/mL) with greater than 95% probability (see Table 1b).

    Table 1b: Number of Replicates Tested and Corresponding Detection Rate to Detect Approximately 2 IU/mL in the Ultrio Plus Discriminatory HBV Assay

    Replicates (n)

    1

    2

    3

    Probability of Detection (%)

    79.5

    95.80

    99.14

    The conversion factors for the Ultrio Plus Assay have been determined to be as follows:
    HIV-1 RNA: --b(4)----
    HCV RNA: ----b(4)------
    HBV DNA: ----b(4)------

  2. Comparison of Limit of Detection (LOD) for the Ultrio Plus Assay and the Ultrio Assay

    Analytical sensitivity panels were prepared from the WHO International Standards: HIV-1 RNA (97/650), HCV RNA (06/100) and HBV DNA (97/750). A total of six Ultrio Plus assay kit lots were used to test the HIV-1 and HBV panels and four kit lots were used to test the HCV panel. The results of Probit analysis show that the Ultrio Plus Assay has comparable sensitivity detecting HIV-1 RNAand HCV RNAand a significant increase in sensitivity for detecting HBV DNA, compared to the original PROCLEIX Ultrio Assay (see Table 2).

    Table 2: Comparison of 95% Limit of Detection of HIV-1 RNA, HCV RNAand HBV DNA WHO International Standards, Ultrio Assay vs. Ultrio Plus Assay

    Panel Tested

    95% Limit of Detection, 95% CI, (IU/mL)

    Ratio of 95% Detection Levels for Ultrio/Ultrio Plus

    p-values

    Ultrio Plus Assay

    Ultrio Assay

    HIV-1 WHO
    (97/650)

    19.6
    (12.5-32.2)

    17.4
    (13.2-30.1)

    0.9

    0.33

    HCV WHO
    (06/100)

    3.6
    (2.7-6.1)

    4.1
    (3.1-7.1)

    1.1

    0.51

    HBV WHO
    (97/750)

    4.1
    (3.0-6.6)

    10.8
    (7.9-17.0)

    2.6

    <0.0001

  3. Detection of HIV-1, HCV, and HBV in Known Positive Specimens:

    A combined total of 624 HIV-1, HCV, or HBV NAT-positive plasma specimens was obtained from a commercial vendor. Two different reagent lots were used; all testing was performed in-house. Each sample was tested neat (undiluted) and diluted 1:16 in negative donor plasma samples with the Ultrio Plus Assay. Each sample was also tested neat with the corresponding Ultrio Plus Discriminatory (dHIV-1, dHCV, or dHBV) Assay. Initially invalid tests were retested, and the valid retest results were used for the data analysis. There were 10 initially invalid reactions out of 1,248 (0.80%) for the Ultrio Plus Assay, 5 initially invalid reactions out of 200 (2.5%) for the Ultrio Plus dHIV-1 Assay, and 1 initially invalid reaction out of 200 (0.5%) for the Ultrio Plus dHCV Assay.

    Both the Ultrio Plus Assay and the Ultrio Plus dHIV-1 Assay detected 200/200 = 100% (95% CI: 98.1-100%) of neat and diluted (1:16) HIV-1 positive samples.

    Both the Ultrio Plus Assay and the Ultrio Plus dHCV Assay detected 200/200 = 100% (95% CI: 98.1-100%) of neat HCV positive samples. The Ultrio Plus Assay detected 198/200 = 99.0% (95% CI: 96.4-99.8%) of diluted (1:16) HCV positive samples.

    Both the Ultrio Plus Assay and the Ultrio Plus dHBV Assay detected 224/224 = 100% (95% CI: 98.3-100%) of neat and diluted (1:16) HBV positive samples.

    Of the 624 specimens from individuals known to be infected with HIV-1, HCV, or HBV, all were detected by the Ultrio Plus Assay and all three Ultrio Plus Discriminatory Assays. The overall detection rate for the Ultrio Plus Assay for these 624 specimens tested at a 1:16 dilution was 99.7% (622/624). Although there was variability in the detection of the diluted samples between the Ultrio Plus dHCV Assay and the Ultrio Plus dHIV-1 and Ultrio Plus dHBV Assays, the difference observed was not statistically significant, as indicated by the overlapping 95% confidence intervals (see Table 3).

    Table 3: Comparison of the Ultrio Plus Assay and the Ultrio Assay in Detection of Known Positive HIV-1, HCV, or HBV Samples

    Specimen
    Type

    N

    Ultrio Plus Reactive (95% CI)

    Ultrio Reactive (95% CI)

    Neat

    1:16

    Discrim.

    Neat

    1:16

    Discrim.

    HIV-1

    200

    100%
    (98.1 –100%)

    100%
    (98.1 –100%)

    100%
    (98.1 –100%)

    100%
    (98.1 – 100%)

    99.0%
    (96.4 –99.8%)

    100%
    (98.1 –100%)

    HCV

    200

    100%
    (98.1 –100%)

    99.0%
    (96.4 -99.8%)

    100%
    (98.1 –100%)

    99.0%
    (96.4 –99.8%)

    98.5%
    (95.7 -99.6%)

    100%
    (98.1 –100%)

    HBV

    224

    100%
    (98.3 –100%)

    100%
    (98.3 –100%)

    100%
    (98.3 –100%)

    100%
    (98.3 – 100%)

    87.9%
    (83.0 –91.7%)

    100%
    (98.3 –100%)

  4. Comparison of the Ultrio Plus Assay and the Ultrio Assay in Detection of HIV-1, HCV, or HBV Yield Specimens

    Yield specimens, defined as seronegative and previously nucleic acid test (NAT) reactive for HIV-1, HCV, or HBV by the Procleix HIV-1/HCV Assay or the Ultrio Assay were tested with the Ultrio Plus Assay and the Ultrio Assay either neat or at 1:16 dilution (see Table 4a.). Samples were obtained from a repository of positive plasma units from blood donors that is maintained by the American Red Cross (ARC) Scientific Support Office (Gaithersburg, MD).

    Table 4a: Results of Testing Yield Specimens for HIV-1, HCV and HBV with the Ultrio Plus and Ultrio Assays

     

    Number of Specimens

    Dilution

    Total Tests*

    Ultrio Plus Reactive, 95% CI

    Ultrio Reactive, 95%CI

    HIV-1

    23

    neat

    69

    69/69
    100% (96.2-100.0%)

    69/69
    100% (96.2-100.0%)

    1:16

    69

    61/69
    88.4% (78.8-94.0%)

    64/69
    92.8% (84.1-96.9%)

    HCV

    156

    neat

    468

    466/468
    99.6% (98.5-99.9%)

    467/468
    99.8% (98.5-99.9%)

    1:16

    468

    466/468
    99.6% (98.5-99.9%)

    464/468
    99.1% (98.5-99.9%)

    HBV

    13

    neat

    39

    37/39
    94.9% (83.1-98.6%)

    36/39
    92.3% (79.7-97.4%)

    1:8

    39

    33/39
    84.6% (70.3-92.8%)

    25/39
    64.1% (48.4-77.3%)

    1:16

    39

    30/39
    76.9% (61.7-87.4%)

    22/39
    56.4% (41-70.7%)

    * each specimen was tested three times

    The Ultrio Plus and Ultrio Assays demonstrated comparable ability to detect HIV-1 yield samples and comparable ability to detect HCV yield samples.

    The results of this testing show an increased detection rate of the Ultrio Plus Assay compared to the Ultrio Assay for HBV yield specimens diluted 1:8 and 1:16.

    Testing of additional HIV-1 yield samples was performed to verify that the detection rate of the Ultrio Plus and Ultrio assays was comparable. The ARC provided two sets (one for each assay) of 17 HIV-1 yield specimens diluted 1:8 and 1:16. There were three aliquots/set, which allowed for testing of 15 replicates per diluted sample per assay.

    All 17 samples were 100% reactive (255/255) in both Ultrio Plus and Ultrio at the 1:8 dilutions. At 1:16 dilutions, Ultrio Plus was 100% reactive (255/255) and Ultrio was 98.4% reactive (251/255). All initially invalid reactions were retested and the valid retest results ere used in analysis. These data are summarized in Table 4b.

    Table 4b: Additional HIV-1 Yield Testing (Number Reactive/Number Tested)

    Sample

    Viral Load

    1:8 Dilution

    1:16 Dilution

    ULTRIO PLUS

    ULTRIO

    ULTRIO PLUS

    ULTRIO

    022GH05568

    200

    15/15

    15/15

    15/15

    15/15

    003R 43191

    260

    15/15

    15/15

    15/15

    13/15

    032FP28078

    370

    15/15

    15/15

    15/15

    15/15

    022LQ82592

    390

    15/15

    15/15

    15/15

    15/15

    004F 25254

    570

    15/15

    15/15

    15/15

    15/15

    003K 16030

    790

    15/15

    15/15

    15/15

    13/15

    035FH89864

    850

    15/15

    15/15

    15/15

    15/15

    013FY89120

    910

    15/15

    15/15

    15/15

    15/15

    036FL20959

    1,300

    15/15

    15/15

    15/15

    15/15

    003GP52279

    1,500

    15/15

    15/15

    15/15

    15/15

    029KM27572

    2,300

    15/15

    15/15

    15/15

    15/15

    006LY61030

    3,800

    15/15

    15/15

    15/15

    15/15

    013FX50837

    4,200

    15/15

    15/15

    15/15

    15/15

    053GM60877

    5,800

    15/15

    15/15

    15/15

    15/15

    013GP13645

    9,800

    15/15

    15/15

    15/15

    15/15

    011GS88902

    46,000

    15/15

    15/15

    15/15

    15/15

    054KC25532

    81,000

    15/15

    15/15

    15/15

    15/15

    Total

    255/255

    255/255

    255/255

    251/255

    % Reactive and CI

    100% reactive
    (98.9-100 CI)

    100% reactive
    (98.9-100 CI)

    100% reactive
    (98.9-100 CI)

    98.4% reactive
    (96.0-99.4 CI)

  5. Comparison of the Ultrio Plus Assay and the Ultrio Assay in Detection of HIV-1, HCV, or HBV Seropositive Specimens

    In another study, seropositive plasma units were obtained from a repository of plasma units from blood donors that is maintained by the American Red Cross (ARC) Scientific Support Office (Gaithersburg, MD).

    Seropositivity was defined as follows:

    Anti-HIV-1 confirmed positive: Donations reactive by a screening enzyme immunoassay (HIV-1/HIV-2 rDNA EIA; Abbott Laboratories, Abbott Park, IL) and positive by either HIV-1 Western blot (Calypte Biomedical, Rockville, MD) or an immunofluorescence assay (IFA, Sanochemia, Vienna, Austria).

    Anti-HCV confirmed positive: Donations reactive by a screening EIA (Ortho v3.0 ELISA; Ortho Diagnostics, Raritan, NJ) and positive by a recombinant immunoblot (RIBA 3.0, Novartis, Emeryville, CA).

    HBsAg confirmed positive: Donations reactive by a screening chemiluminescent immunoassay (ChLIA) (Abbott PRISM HBsAg, Abbott Laboratories, Abbott Park, IL) and positive by neutralization.

    Anti-HBc positive: Donations reactive by a screening ChLIA for anti-HBc (Abbott PRISM HBcore, Abbott Laboratories, Abbott Park, IL) but non-reactive for HBsAg.

    The results of testing specimens that are anti-HIV-1 confirmed positive (N=292), anti-HCV confirmed positive (N=490 or 500), HBsAg confirmed positive (N=606), or anti-HBc Reactive/HBsAg Negative (N=750) with the Ultrio Plus and Ultrio Assays are shown in Table 5.

    Table 5: Results of Testing Specimens that are Anti-HIV-1 Confirmed Positive, Anti-HCV Confirmed Positive, HBsAg Confirmed Positive, or Anti-HBc Reactive/ HBsAg Negative with the Ultrio Plus and Ultrio Assays

    Description of Specimens

    Ultrio Plus Reactive, 95% CI

    Ultrio Reactive,
    95% CI

    p-value

    Anti-HIV-1 Positive

    258/292

    88.4% (84.2-91.5%)

    254/292

    87.0% (82.6-90.4%)

    0.61

    Anti-HCV Positive

    387/490

    79.0% (75.2-82.4%)

    392/500

    78.4% (74.6-81.8%)

    0.82

    HBsAg Positive

    552/606

    91.1% (88.6-93.1%)

    489/606

    81.0% (77.4-83.6%)

    <0.0001

    Anti-HBc Reactive/ HBsAg Negative

    46/750

    6.1% (4.6-8.1%)

    27/750

    3.6% (2.5-5.2%)

    0.02

    The Ultrio Plus and Ultrio Assays demonstrated a comparable detection rate of HIV-1 antibody-positive samples and a comparable detection rate of HCV antibody-positive samples.

    Significantly better detection with the Ultrio Plus Assay was observed with samples that were initially identified as HBsAg-positive (p <0.0001) or anti-HBc-reactive/HBsAg negative (p=0.02).

    Overall, these results show that the Ultrio Plus Assay has the ability to detect HIV-1 and HCV that is comparable to Ultrio, and significantly better ability to detect HBV when compared to Ultrio.

  6. Genotype Detection

    Detection of HIV-1 Genetic Variants with the Ultrio Plus Assay and the Ultrio Plus HIV-1 Discriminatory Assay

    HIV-1 specimens and tissue culture isolates of group M (subtypes A, B, C, D, E, F, G and H), group N, and group O were quantified for HIV-1 RNA using commercially available quantitative HIV-1 RNA assays or with an in-house quantitative HIV-1 RNA test. Specimens were diluted with HIV-1/HCV/HBV NAT-negative human serum to target viral concentrations of 300, 100, and 30 copies/mL. Diluted specimens were tested in the Ultrio Plus Assay and the Ultrio Plus HIV-1 Discriminatory (dHIV-1) Assay. A total of 56 unique specimens or isolates were tested in duplicate using two pilot lots of reagents on the Procleix TIGRIS System. Three additional specimens were co-infected with HCV and/or HBV and were therefore only tested in the Ultrio Plus dHIV-1 Assay (see Table 6).

    When looking at test performance across all HIV genotypes, at 300 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHIV-1 Assay detected 100% of the replicates (216/216 and 224/224 respectively). At 100 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHIV-1 Assay detected 99.6% (223/224) and 100% (236/236) of the replicates, respectively. At 30 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHIV-1 Assay detected 94.6% (212/224) and 96.2% (227/236) of the replicates, respectively.

    Table 6: Detection of HIV-1 Genetic Variants

    Genetic
    Variant

    Copies / mL

    PROCLEIX ULTRIO PLUS Assay

    PROCLEIX ULTRIO PLUS
    HIV-1 Discriminatory Assay

    Unique Donors

    Reactive / Tested

    % Reactive

    Unique Donors

    Reactive / Tested

    % Reactive

    HIV-1 Group M Subtype A

    300

    10

    40/40

    100

    11

    44/44

    100

    100

    40/40

    100

    44/44

    100

    30

    40/40

    100

    41/44

    93.2

    HIV-1 Group M Subtype B

    300

    8

    32/32

    100

    9

    36/36

    100

    100

    32/32

    100

    36/36

    100

    30

    31/32

    96.9

    36/36

    100

    HIV-1 Group M Subtype C

    300

    8

    32/32

    100

    8

    32/32

    100

    100

    32/32

    100

    32/32

    100

    30

    31/32

    96.9

    32/32

    100

    HIV-1 Group M Subtype D

    300

    7

    28/28

    100

    7

    28/28

    100

    100

    28/28

    100

    28/28

    100

    30

    26/28

    92.9

    25/28

    89.3

    HIV-1 Group M Subtype E

    300

    9

    36/36

    100

    9

    36/36

    100

    100

    35/36

    97.2

    36/36

    100

    30

    28/36

    77.8

    34/36

    94.4

    HIV-1 Group M Subtype F

    300

    5

    20/20

    100

    5

    20/20

    100

    100

    20/20

    100

    20/20

    100

    30

    20/20

    100

    19/20

    95.0

    HIV-1 Group M Subtype G

    300

    1

    4/4

    100

    2

    4/4

    100

    100

    4/4

    100

    8/8

    100

    30

    4/4

    100

    8/8

    100

    HIV-1 Group M
    Subtype H

    300

    1

    4/4

    100

    1

    4/4

    100

    100

    4/4

    100

    4/4

    100

    30

    4/4

    100

    4/4

    100

    HIV-1 Group N

    300

    1

    4/4

    100

    1

    4/4

    100

    100

    4/4

    100

    4/4

    100

    30

    4/4

    100

    4/4

    100

    HIV-1 Group O

    300

    6

    16/16

    100

    6

    16/16

    100

    100

    24/24

    100

    24/24

    100

    30

    24/24

    100

    24/24

    100

    All Genotypes

    300

    56

    216/216

    100

    59

    224/224

    100

    100

    223/224

    99.6

    236/236

    100

    30

    212/224

    94.6

    227/236

    96.2

     

    Detection of HCV Genotypes with the Ultrio Plus Assay and the Ultrio Plus HCV Discriminatory Assay

    HCV specimens of genotypes 1, 2, 3, 4, 5, and 6 were quantified for HCV RNA using commercially available quantitative HCV RNA assays. Specimens were diluted with HIV-1/HCV/HBV NAT-negative human serum to target viral concentrations of 300, 100, and 30 copies/mL. The diluted specimens were tested with the Ultrio Plus Assay and the Ultrio Plus HCV Discriminatory (dHCV) Assay. A total of 61 unique specimens were tested in duplicate using two pilot lots of reagents on the Procleix TIGRIS System. One additional specimen was co-infected with HIV-1 and HBV and was therefore only tested in the Procleix Ultrio Plus dHCV Assay (see Table 7).

    For all genotypes taken together, at 300 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHCV Assay detected 99.6% (243/244) and 100% (248/248) of the replicates, respectively. At 100 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHCV Assay detected 98.8% (241/244) and 99.2% (246/248) of the replicates, respectively. At 30 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHCV Assay detected 93.9% (229/244) and 94.4% (234/248) of the replicates, respectively.

    Table 7: Detection of HCV Genotypes


    Genotype

    Copies / mL

    PROCLEIX ULTRIO PLUS Assay

    PROCLEIX ULTRIO PLUS
    HCV Discriminatory Assay

    Unique Donors

    Reactive / Tested

    % Reactive

    Unique Donors

    Reactive / Tested

    % Reactive

    HCV
    Genotype 1

    300

    11

    44/44

    100

    11

    44/44

    100

    100

    44/44

    100

    44/44

    100

    30

    44/44

    100

    43/44

    97.7

    HCV
    Genotype 2

    300

    13

    51/52

    98.1

    14

    56/56

    100

    100

    49/52

    94.2

    54/56

    96.4

    30

    42/52

    80.8

    44/56

    78.6

    HCV
    Genotype 3

    300

    12

    48/48

    100

    12

    48/48

    100

    100

    48/48

    100

    48/48

    100

    30

    45/48

    93.8

    48/48

    100

    HCV
    Genotype 4

    300

    14

    56/56

    100

    14

    56/56

    100

    100

    56/56

    100

    56/56

    100

    30

    55/56

    98.2

    55/56

    98.2

    HCV
    Genotype 5

    300

    6

    24/24

    100

    6

    24/24

    100

    100

    24/24

    100

    24/24

    100

    30

    24/24

    100

    24/24

    100

    HCV
    Genotype 6

    300

    5

    20/20

    100

    5

    20/20

    100

    100

    20/20

    100

    20/20

    100

    30

    19/20

    95.0

    20/20

    100

    All Genotypes

    300

    61

    243/244

    99.6

    62

    248/248

    100

    100

    241/244

    98.8

    246/248

    99.2

    30

    229/244

    93.9

    234/248

    94.4

    Detection of HBV Genotypes with the Ultrio Plus Assay and the Ultrio Plus HBV Discriminatory Assay

    HBV specimens of genotypes A, B, C, D, E, F, and G were quantified for HBV DNA using commercially available quantitative HBV DNA assays. Specimens were diluted with HIV-1/HCV/HBV NAT-negative human serum to target viral concentrations of 300, 100, and 30 copies/mL. Diluted specimens were tested with the Ultrio Plus Assay and the Ultrio Plus HBV Discriminatory (dHBV) Assay. A total of 58 unique specimens were tested in duplicate using two pilot lots of reagents on the Procleix TIGRIS System (see Table 8).
    For all genotypes taken together, at 300 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHBV Assay detected 100% (228/228) and 99.6% (227/228) of the replicates, respectively. AT 100 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHBV Assay detected 99.1% (230/232) and 99.6% (231/232) of the replicates, respectively. At 30 copies/mL, the Ultrio Plus Assay and the Ultrio Plus dHBV Assay detected 94.8% (220/232) and 98.3% (228/232) of the replicates, respectively.

    Table 8: Detection of HBV Genotypes

    Genotype

    Copies / mL

    PROCLEIX ULTRIO PLUS Assay

    PROCLEIX ULTRIO PLUS
    HBV Discriminatory Assay

    Unique Donors

    Reactive / Tested

    % Reactive

    Unique Donors

    Reactive / Tested

    % Reactive

    HBV
    Genotype A

    300

    12

    48/48

    100

    12

    47/48

    97.9

    100

    46/48

    95.8

    47/48

    97.9

    30

    38/48

    79.2

    44/48

    91.7

    HBV
    Genotype B

    300

    10

    40/40

    100

    10

    40/40

    100

    100

    40/40

    100

    40/40

    100

    30

    40/40

    100

    40/40

    100

    HBV
    Genotype C

    300

    10

    40/40

    100

    10

    40/40

    100

    100

    40/40

    100

    40/40

    100

    30

    40/40

    100

    40/40

    100

    HBV
    Genotype D

    300

    9

    36/36

    100

    9

    36/36

    100

    100

    36/36

    100

    36/36

    100

    30

    36/36

    100

    36/36

    100

    HBV
    Genotype E

    300

    8

    28/28

    100

    8

    28/28

    100

    100

    32/32

    100

    32/32

    100

    30

    31/32

    96.9

    32/32

    100

    HBV
    Genotype F

    300

    8

    32/32

    100

    8

    32/32

    100

    100

    32/32

    100

    32/32

    100

    30

    31/32

    96.9

    32/32

    100

    HBV
    Genotype G

    300

    1

    4/4

    100

    1

    4/4

    100

    100

    4/4

    100

    4/4

    100

    30

    4/4

    100

    4/4

    100

    All Genotypes

    300

    58

    228/228

    100

    58

    227/228

    99.6

    100

    230/232

    99.1

    231/232

    99.6

    30

    220/232

    94.8

    228/232

    98.3

  7. Detection of HIV-1, HCV, and HBV in Low Titer Samples

    The low titer samples for this study were samples with the lowest titers that were commercially available and had sufficient volume for testing multiple replicates. Each sample was tested in triplicate neat and diluted 1:16 in pools of negative plasma, using both the Ultrio and Ultrio Plus Assays on TIGRIS. The samples tested were as follows:

    HIV-1, 60 samples, titer range from below the level of quantitation to 1,760 copies/mL
    HCV, 34 samples, titer range from below the level of quantitation to 9,421 copies/mL
    HBV, 50 samples, titer range from below the level of quantitation to 231 IU/mL

    The results showed comparable detection by the Ultrio and Ultrio Plus Assays of low titer HIV-1 and HCV specimens tested neat and diluted 1:16. The Ultrio Plus Assay showed significantly better detection than the Ultrio Assay for the low titer HBV specimens. For HBV, the Ultrio Plus Assay detected 98.7% of neat specimens compared to 75.3% detected with the Ultrio Assay. When tested at a 1:16 dilution, the Ultrio Plus Assay detected 78.0% of the specimens compared to 42.0% detected with the Ultrio Assay (see Table 9).

    Table 9: Results of Testing Low Titer Specimens

    Specimens

    Dilution

    Assay

    # Reactive

    # Valid
    Replicates

    % Reactive

    p-value

    HIV-1

    Neat

    Ultrio

    170

    180

    94.4%

    0.81

    Ultrio Plus

    172*

    180

    95.6%

    1:16

    Ultrio

    128

    180

    71.1%

    0.40

    Ultrio Plus

    136 †

    180

    75.6%

    HCV

    Neat

    Ultrio

    74

    102

    72.5%

    0.42

    Ultrio Plus

    80

    102

    78.4%

    1:16

    Ultrio

    64

    102

    62.7%

    1.00

    Ultrio Plus

    63

    102

    61.8%

    HBV

    Neat

    Ultrio

    113

    150

    75.3%

    <0.0001

    Ultrio Plus

    148**

    150

    98.7%

    1:16

    Ultrio

    63

    150

    42.0%

    <0.0001

    Ultrio Plus

    117 ††

    150

    78.0%

    * One neat HIV-1 specimen with an HIV-1 titer of 100 copies/mL was not detected by Ultrio Plus in all 3 replicates tested.
    ** One neat HBV specimen with an HBV titer of 59 IU/mL was not detected by Ultrio Plus in 2 of 3 replicates tested.
    † Four HIV-1 specimens diluted 1:16 with HIV-1 titers of 88, 100, 512 and 678 copies/mL were not detected by Ultrio Plus in at least 1 of the 3 replicates tested.
    †† Four HBV specimens diluted 1:16 with HBV titers of 25, 30, 54 and 59 IU/mL were not detected by Ultrio Plus in at least 1 of the 3 replicates tested.
    Note: All other neat and diluted 1:16 samples that were not detected by Ultrio Plus were below the level of quantitation.

  8. Detection of HIV-1, HCV, and HBV in Seroconversion Panels

    Commercial seroconversion panels were tested to determine the ability of the Ultrio Plus Assay and the three Discriminatory Assays to reduce the pre-seroconversion window period of HIV-1, HCV, and HBV detection. The results are shown as the following:

    • HIV-1 Detection in seroconversion panels

    Compared to an anti-HIV-1/2 Ab test and an HIV-1 p24 Ag test, respectively, the Ultrio Plus Assay was able to detect HIV-1 RNA an average of 14.5 and 8.6 days earlier in neat samples, 11.7 and 5.8 days earlier in 1:8 dilutions, and 12.5 and 6.6 days earlier in 1:16 dilutions. The Ultrio Plus dHIV-1 Assay was able to detect HIV-1 RNA an average of 14.1 and 8.2 days earlier than the Anti-HIV-1/2 Ab test and the HIV-1 p24 Ag test, respectively (see Table 10).

    Table 10: Detection of HIV-1 RNA in HIV-1 Seroconversion Panels

    Panel ID

    Days Earlier Detection Than HIV-1/2 Antibody

    Days Earlier Detection than HIV-1 p24 Ag

    Procleix Ultrio Plus Assay

    dHIV-1
    Assay

    Procleix Ultrio Plus Assay

    dHIV-1
    Assay

    Neat

    1:8

    1:16

    Neat

    Neat

    1:8

    1:16

    Neat

    6244

    11

    8

    8

    11

    6

    3

    3

    6

    6247

    16

    14

    14

    14

    7

    5

    5

    5

    9020

    17

    14

    14

    14

    7

    4

    4

    4

    9021

    14

    14

    14

    14

    4

    4

    4

    4

    9030

    14

    14

    14

    14

    7

    7

    7

    7

    9031

    15

    12

    15

    15

    22

    19

    22

    22

    9032

    14

    10

    10

    17

    14

    10

    10

    17

    9076

    14

    8

    8

    14

    8

    2

    2

    8

    9077

    16

    16

    16

    16

    4

    4

    4

    4

    9079

    14

    7

    12

    12

    7

    0

    5

    5

    Mean

    14.5

    11.7

    12.5

    14.1

    8.6

    5.8

    6.6

    8.2

    • HCV Detection in seroconversion panels

    When compared to the Anti-HCV 3.0 antibody tests the Ultrio Plus Assay was able to detect HCV RNA an average of 32.6 days earlier in neat samples, 31.8 days earlier in 1:8 dilutions, and 32.1 days earlier in 1:16 dilutions. The Ultrio Plus dHCV Assay was able to detect HCV RNA an average of 32.6 days earlier than the Anti-HCV antibody tests.
    Note: In 5 of the 12 seroconversion panels (6214, 6226, 6228, 9045, and 9047), the first available bleed in the series was already reactive with both the Ultrio Plus Assay and Ultrio Plus dHCV Assay, so the number of days of window closure may underestimate the true window closure period for the assays (see Table 11).

    Table 11: Detection of HCV RNA in HCV Seroconversion Panels

     

    Panel ID

    Days Earlier Detection Than HCV Antibody

     

    PROCLEIX ULTRIO Plus Assay

     

    dHCV Assay

     

    Neat

     

    1:8

     

    1:16

     

    Neat

    6213

    26

    26

    26

    26

    6214

    30

    30

    30

    30

    6222

    23

    23

    23

    23

    6225

    39

    33

    33

    39

    6226

    39

    39

    39

    39

    6227

    32

    32

    32

    32

    6228

    31

    31

    31

    31

    9041

    38

    38

    38

    38

    9045

    37

    37

    37

    37

    9047

    28

    28

    28

    28

    9054

    30

    30

    30

    30

    9055

    38

    34

    38

    38

    Mean

    32.6

    31.8

    32.1

    32.6

    • HBV Detection in seroconversion panels

    Compared to two licensed HBsAg tests, the Ultrio Plus Assay was able to detect HBV DNA an average of 23.6 and 27.0 days earlier in neat samples, 13.5 and 16.8 days earlier in 1:8 dilutions, and 10.5 and 13.9 days earlier in 1:16 dilutions. The Ultrio Plus dHBV Assay was able to detect HBV DNA an average of 23.7 and 27.1 days earlier than the two licensed HBsAg tests (see Table 12).

    Table 12: Detection of HBV DNA in HBV Seroconversion Panels

    Panel
    ID

    Days Earlier Detection Than HBV Surface Antigen, Abbott PRISM HBsAg Test

    Days Earlier Detection Than HBV Surface Antigen, Ortho HBsAg ELISA Test System 3

    Procleix Ultrio Plus Assay

    dHBV Assay

    Procleix Ultrio Plus Assay

    dHBV Assay

    Neat

    1:8

    1:16

    Neat

    Neat

    1:8

    1:16

    Neat

    6283

    18

    10

    10

    21

    18

    10

    10

    21

    6289

    20

    13

    0

    15

    20

    13

    0

    15

    6290

    21

    14

    7

    21

    23

    16

    9

    23

    6292

    27

    8

    8

    29

    27

    8

    8

    29

    9073

    21

    10

    10

    21

    25

    14

    14

    25

    9074

    14

    14

    11

    18

    17

    17

    14

    21

    11006

    28

    14

    14

    28

    30

    16

    16

    30

    11007

    21

    5

    7

    14

    28

    12

    14

    21

    11008

    21

    11

    7

    21

    31

    21

    17

    31

    11015

    36

    34

    27

    44

    36

    34

    27

    44

    11024

    33

    15

    15

    29

    42

    24

    24

    38

    Mean

    23.6

    13.5

    10.5

    23.7

    27.0

    16.8

    13.9

    27.1

  9. Potentially Interfering Substances and Unrelated Medical Conditions

    Cross-reactivity and interference studies were done on Ultrio Plus and the Ultrio Plus HIV-1, HCV, and HBV Discriminatory Assays with various donor and donation specimens with potential interference materials or medical conditions. HIV-1, HCV, and HBV positive specimens were created by individually spiking to a final concentration of 150 copies/mL of HIV-1, 150 copies/mL of HCV, or 15 IU/mL of HBV. Cross-reactivity and interference are defined as greater than 5% unexpected results.

    The following potential interfering substances were tested:

    • icteric, hemolyzed, or lipemic specimens
    • plasma containing the following exogenously added substances:
      albumin (60 g/L)
      hemoglobin (5,000 mg/L)
      bilirubin (200 mg/L)
      lipids (30,000 mg/L)
    • Specimens from individuals with the following medical conditions, specimens with bacterial contamination, and specimens from subjects infected with other blood-borne pathogens or who had received HBV and flu vaccines:

      Antinuclear antibody
      Alcoholic cirrhosis
      Elevated alanine aminotransferase
      Hyperglobulinemia (elevated IgG and/or IgM)
      Systemic lupus erythematosis
      Multiple myeloma
      Multiple sclerosis
      Rheumatoid arthritis
      Rheumatoid factor

      Candida albicans
      Corynebacterium diphtheriae
      Micrococcus luteus
      Propionibacterium acnes
      Pneumocystis carinii
      Staphylococcus aureus
      Staphylococcus epidermidis

      Cytomegalovirus (CMV)
      Dengue virus (serotypes 1-4)
      Epstein-Barr virus (EBV)
      Flu vaccinees
      Hepatitis A virus
      HBV vaccinees
      HIV-2
      Herpes simplex virus 1 or 2 (HSV I/II)
      Human T-cell Lymphotrophic Virus Type I and II (HTLV I/II)
      Parvovirus B19
      Rubella virus
      West Nile Virus (WNV)

      No Cross-reactivity or interference was observed for any of the conditions tested in any of the assays.

  10. Reproducibility (Precision Study)

    Reproducibility of the Ultrio Plus Assay and the Ultrio Plus HIV-1, HCV, and HBV Discriminatory Assays on the Procleix TIGRIS System was evaluated. For determination of the reproducibility of each assay, 7 quality control panel members were tested as individual specimens. Six of the panel members were positive for HIV-1 RNA (100 and 30 copies/mL), HCV RNA (100 and 30 copies/mL), or HBV DNA (32 and 11 IU/mL), and 1 panel member was HIV-1, HCV and HBV negative.

    The reproducibility panels were tested by a total of three operators with three different pilot lots and three Procleix TIGRIS instruments over multiple days. Nine valid runs were generated with the Ultrio Plus Assay and each Ultrio Plus Discriminatory Assay. For the Ultrio Plus Assay, each of the seven panel members was tested in 360 replicates (120 replicates for each lot, and 40 replicates on each day). In each of the Ultrio Plus Discriminatory Assays, each of the six positive panel members was tested in 360 replicates, and the negative panel member was tested in 108 replicates.

    Reproducibility analyses included evaluation of percent agreement and mean Signal to Cutoff (S/CO) ratios for panel members, evaluation of mean Relative Light Unit (RLU) values for the Negative, HIV-1 Positive, HCV Positive, and HBV Positive Calibrators, and evaluation of standard deviation (SD) and percent coefficient of variation (%CV) of the S/CO ratios and RLU values for each of the five variance factors (see Tables 13, 14, 15, and 16). The mean analyte S/CO ratios were analyzed for the positive and negative panel members and the Internal Control S/CO ratios were analyzed for the negative panel members. The mean analyte RLU values were analyzed for the Positive and Negative Calibrators and the Internal Control RLU values were analyzed for the Negative Calibrators. The percent agreement between the assay results and the true status of each panel member was calculated using the analyte S/CO for all panel members. For the Ultrio Plus and the Ultrio Plus Discriminatory Assays, results for each panel member are shown individually.

    For the Ultrio Plus Assay and the three Ultrio Plus Discriminatory Assays, the overall percent agreement of test results was 93.1 to 100% for positive panel members and 100% for the negative panel member. With regard to signal variability, intra-assay (or random error) and inter-instrument factors, in most cases, were the largest and second largest contributors to total variance (according to SD values) in the Ultrio Plus Assay and the Ultrio Plus HIV-1, HCV, and HBV Discriminatory Assays. It should be noted that while these factors were responsible for the majority of the variance in the assays, the total %CV did not exceed 6.8% for any positive panel members at 100 copies/mL (HIV-1 and HCV) or 32 IU/mL (HBV), and did not exceed 25.5% for any positive panel members at 30 copies/mL (HIV-1 and HCV) or 11 IU/mL (HBV), in any assay.

    Table 13: Result of Reproducibility Study of the Ultrio PLUS Assay

    Sample

    Titer*

    N

    Agreement
    (%)

    Mean S/CO

    Inter-
    Instrument

    Inter-
    Operator

    Inter-
    Lot

    Inter-
    Day

    Intra-
    Run

    SD

    % CV

    SD

    %CV

    SD

    %CV

    SD

    %CV

    SD

    %CV

    Negative,
    IC**

    0

    360

    100%

    2.11

    0.00

    0.0

    0.05

    2.4

    0.00

    0.0

    0.04

    1.9

    0.10

    4.7

    Negative,
    Analyte

    0.09

    0.02

    22.2

    0.00

    0.0

    0.00

    0.0

    0.00

    0.0

    0.04

    44.4

    HIV-1

    100

    360

    100%

    11.90

    0.40

    3.4

    0.00

    0.0

    0.13

    1.1

    0.32

    2.7

    0.81

    6.8

    30

    360

    98.1%

    10.32

    0.38

    3.7

    0.21

    2.0

    0.00

    0.0

    0.00

    0.0

    2.27

    22.0

    HCV-1a

    100

    360

    100%

    8.70

    0.25

    2.9

    0.15

    1.7

    0.06

    0.7

    0.13

    1.5

    0.28

    3.2

    30

    360

    95.8%

    8.60

    0.32

    3.7

    0.20

    2.3

    0.00

    0.0

    0.14

    1.6

    0.50

    5.8

    HBV A

    32

    360

    100%

    14.63

    0.64

    4.4

    0.30

    2.1

    0.55

    3.8

    0.29

    2.0

    0.37

    2.5

    11

    360

    99.7%

    14.56

    0.60

    4.1

    0.29

    2.0

    0.56

    3.9

    0.31

    2.1

    0.38

    2.6

    Sample

    N

    Agreement
    (%)

    Mean RLU

    Inter-
    Instrument

    Inter-
    Operator

    Inter-
    Lot

    Inter-
    Day

    Intra-
    Run

    SD

    % CV

    SD

    %CV

    SD

    %CV

    SD

    %CV

    SD

    %CV

    Negative Calibrator,
    IC**

    27

    N/A

    151,240

    2,950

    2.0

    2,180

    1.4

    13,300

    8.8

    2,680

    1.8

    5,340

    3.5

    Negative Calibrator,
    Analyte

    10,490

    3,240

    30.9

    170

    1.6

    0

    0.0

    0

    0.0

    5,070

    48.3

    HIV-1 Positive Calibrator

    18

    N/A

    1,070,200

    57,340

    5.4

    21,480

    2.0

    4,330

    0.4

    31,910

    3.0

    29,910

    2.8

    HCV Positive Calibrator

    18

    N/A

    764,310

    41,400

    5.4

    8,260

    1.1

    8,700

    1.1

    0

    0.0

    25,200

    3.3

    HBV Positive Calibrator

    18

    N/A

    1,299,600

    60,590

    4.7

    0

    0.0

    50,450

    3.9

    0

    0.0

    41,560

    3.2

    N = Number of panel members combined for this analysis; S/CO = Signal to Cutoff ratio; IC = Internal Control
    * Concentration = copies/mL for HIV-1 and HCV, IU/mL for HBV.
    ** Analysis of Internal Control signal

    Table 14: Result of Reproducibility Study of the ULTRIO PLUS HIV-1 Discriminatory Assay

    Sample

    Titer*

    N

    Agreement
    (%)

    Mean
    S/CO

    Inter-
    Instrument

    Inter-
    Operator

    Inter-
    Lot

    Inter-
    Day

    Intra-
    Run

    SD

    % CV

    SD

    % CV

    SD

    % CV

    SD

    %CV

    SD

    % CV

    Negative,
    IC**

    0

    108

    100%

    2.07

    0.00

    0.0

    0.05

    2.4

    0.00

    0.0

    0.03

    1.5

    0.09

    4.4

    Negative,
    Analyte

    0.14

    0.03

    21.4

    0.00

    0.0

    0.00

    0.0

    0.00

    0.0

    0.07

    50.0

    HIV-1

    100

    360

    100%

    21.30

    1.13

    5.3

    0.55

    2.6

    0.00

    0.0

    0.26

    1.2

    0.93

    4.4

    30

    360

    98.9%

    18.02

    0.94

    5.2

    0.00

    0.0

    0.00

    0.0

    0.84

    4.7

    4.61

    25.5

    Sample

    N

    Agreement
    (%)

    Mean RLU

    Inter-
    Instrument

    Inter-
    Operator

    Inter-
    Lot

    Inter-
    Day

    Intra-
    Run

    SD

    % CV

    SD

    % CV

    SD

    % CV

    SD

    %CV

    SD

    % CV

    Negative Calibrator,
    IC**

    27

    N/A

    149,300

    0

    0.0

    2,300

    1.5

    8,370

    5.6

    4,000

    2.7

    4,890

    3.3

    Negative Calibrator,
    Analyte

    7,570

    3,080

    40.6

    1,590

    21.0

    1,120

    14.7

    0

    0.0

    2,530

    33.4

    HIV-1 Positive Calibrator

    27

    N/A

    1,103,000

    39,370

    3.6

    14,310

    1.3

    22,360

    2.0

    0

    0.0

    31,900

    2.9

    N = Number of panel members combined for this analysis; S/CO = Signal to Cutoff ratio; IC = Internal Control
    * Concentration = copies/mL for HIV-1 and HCV, IU/mL for HBV.
    ** Analysis of Internal Control signal

    Table 15: Result of Reproducibility Study of the ULTRIO PLUS HCV Discriminatory Assa

    Sample

    Titer*

    N

    Agreement
    (%)

    Mean
    S/CO

    Inter-
    Instrument

    Inter-
    Operator

    Inter-
    Lot

    Inter-
    Day

    Intra-
    Run

    SD

    % CV

    SD

    %
    CV

    SD

    % CV

    SD

    % CV

    SD

    % CV

    Negative,
    IC**

    0

    108

    100

    2.04

    0.00

    0.0

    0.02

    1.0

    0.01

    0.5

    0.04

    2.0

    0.11

    5.4

    Negative,
    Analyte

    0.1

    0.03

    30.0

    0.01

    10.0

    0.02

    20.0

    0.01

    10.0

    0.06

    60.0

    HCV-1a

    100

    360

    100

    23.46

    1.03

    4.4

    0.00

    0.0

    0.69

    2.9

    0.44

    1.9

    0.79

    3.4

    30

    360

    93.1

    22.39

    1.26

    5.6

    0.19

    0.9

    0.84

    3.8

    0.00

    0.0

    2.93

    13.0

    Sample

    N

    Agreement
    (%)

    Mean
    RLU

    Inter-
    Instrument

    Inter-
    Operator

    Inter-
    Lot

    Inter-
    Day

    Intra-
    Run

    SD

    % CV

    SD

    %
    CV

    SD

    % CV

    SD

    % CV

    SD

    % CV

    Negative Calibrator,
    IC**

    27

    N/A

    147,200

    3,750

    2.6

    2,120

    1.4

    7,030

    4.8

    2,560

    1.7

    6,470

    4.4

    Negative Calibrator,
    Analyte

    4,370

    2,460

    56.3

    0

    0.0

    1,190

    27.2

    850

    19.4

    1,530

    35.0

    HCV Positive Calibrator

    27

    N/A

    1,223,000

    51,940

    4.3

    0

    0.0

    47,460

    3.9

    8,570

    0.7

    24,620

    2.0

    N = Number of panel members combined for this analysis; S/CO = Signal to Cutoff ratio; IC = Internal Control
    * Concentration = copies/mL for HIV-1 and HCV, IU/mL for HBV.
    ** Analysis of Internal Control signal

    Table 16: Result of Reproducibility Study of the ULTRIO PLUS HBV Discriminatory Assay

    Sample

    Titer*

    N

    Agreement
    (%)

    Mean S/CO

    Inter-
    Instrument

    Inter-
    Operator

    Inter-
    Lot

    Inter-
    Day

    Intra-
    Run

    SD

    % CV

    SD

    %CV

    SD

    %CV

    SD

    %CV

    SD

    %CV

    Negative,
    IC**

    0

    108

    100

    2.06

    0.04

    1.9

    0.03

    1.5

    0.00

    0.0

    0.00

    0.0

    0.12

    5.8

    Negative,
    Analyte

    0.07

    0.02

    28.6

    0.00

    0.0

    0.00

    0.0

    0.00

    0.0

    0.05

    71.4

    HBV A

    32

    360

    100

    23.15

    1.05

    4.5

    0.39

    1.7

    0.00

    0.0

    0.28

    1.2

    0.51

    2.2

    11

    355

    100

    23.01

    1.03

    4.5

    0.37

    1.6

    0.00

    0.0

    0.30

    1.3

    0.59

    2.6

    Sample

    N

    Agreement
    (%)

    Mean RLU

    Inter-
    Instrument

    Inter-
    Operator

    Inter-
    Lot

    Inter-
    Day

    Intra-
    Run

    SD

    %CV

    SD

    %CV

    SD

    %CV

    SD

    %CV

    SD

    %CV

    Negative Calibrator,
    IC**

    27

    N/A

    149,600

    5,400

    3.6

    1,540

    1.0

    0

    0.0

    0

    0.0

    6,090

    4.1

    Negative Calibrator,
    Analyte

    5,220

    3,100

    59.4

    320

    6.1

    0

    0.0

    0

    0.0

    3,180

    60.9

    HBV Positive Calibrator

    27

    N/A

    1,436,000

    42,020

    2.9

    0

    0.0

    8,580

    0.6

    13,250

    0.9

    39,900

    2.8

    N = Number of panel members combined for this analysis; S/CO = Signal to Cutoff ratio; IC = Internal Control
    * Concentration = copies/mL for HIV-1 and HCV, IU/mL for HBV.
    ** Analysis of Internal Control signal

  11. Data to Support Limited Supplemental Test Claims for HIV-1, HCV, and HBV

    Samples with confirmed positive serological status were tested to demonstrate that the Ultrio Plus Assay and a Discriminatory assay, when reactive, can be used as a supplemental test that confirms infection for individual donor specimens that are repeatedly reactive on a licensed donor screening serological test. All of the samples included in this analysis were EIA repeatedly reactive and confirmed positive by the following criteria:

    • Anti-HIV-1 confirmed positive: Samples reactive by a screening enzyme immunoassay (HIV-1/HIV-2 rDNA EIA; Abbott Laboratories, Abbott Park IL) and positive by either HIV-1 western blot (Calypte Biomedical, Rockville MD) or an immunofluorescent assay (IFA, Sanochemia, Vienna Austria).
    • Anti-HCV confirmed positive: Samples reactive by a screening EIA (Ortho v3.0 ELISA; Ortho Diagnostics, Raritan NJ) and positive by a recombinant immunoblot (RIBA 3.0, Novartis Emeryville, CA).
    • HBsAg confirmed positive: Samples reactive by a screening ChLIA (chemiluminescent immunoassay; Abbott PRISM) and positive by neutralization.

    The samples were tested with the Ultrio Plus Assay in-house. Samples had been previously tested with the licensed Ultrio Assay at Creative Testing Solutions (CTS, St. Petersburg, FL).

    Results

    HIV-1
    There were 292 confirmed anti-HIV-1/-2 positive specimens: 258 were Ultrio Plus Assay reactive and 34 were nonreactive. Positive agreement between Ultrio Plus Assay results and confirmatory assay results in this study was 258/292 = 88.4% (95% CI: 84.2% to 91.5%). In comparison, positive agreement between the licensed Ultrio Assay and confirmatory assay results was 254/292 = 87.0% (95% CI: 82.6% to 90.4%) (see Table 17).

    HCV
    There were 490 confirmed anti-HCV positive specimens tested with the Ultrio Plus Assay and 500 confirmed anti-HCV positive specimens tested with the Ultrio Assay. Of these, 387 were Ultrio Plus Assay reactive and 103 were nonreactive. Positive agreement between Ultrio Plus Assay results and confirmatory assay results in this study was 387/490 = 79.0% (95% CI: 75.2% to 82.4%). In comparison, positive agreement between the licensed Ultrio Assay and confirmatory assay results was 392/500 = 78.4% (95% CI: 74.6% to 81.8%). Approximately 20% of RIBA positive samples will have undetectable HCV RNA due to a resolved HCV infection (see Table 17).

    HBV
    There were 606 confirmed HBsAg positive specimens: 552 were Ultrio Plus Assay reactive and 54 were nonreactive. Positive agreement between Ultrio Plus Assay results and confirmatory assay results in this study was 552/606 = 91.1% (95% CI: 88.6% to 93.1%). In comparison, positive agreement between the licensed Ultrio Assay and confirmatory assay results was 489/606 = 80.6% (95% CI: 77.4% to 83.6%). The results for the specimens that were HBsAg neutralization reactive and Ultrio and Ultrio Plus nonreactive are not unexpected, as HBsAg may be present in particles that do not contain nucleic acids or after vaccination with a vaccine derived from HBsAg (see Table 17).

    Table 17: Ultrio and Ultrio Plus Performance with Known Serological Results

    Specimen Type, Serology Test

    N

    Ultrio Plus Assay

    Ultrio Assay

    Reactive

    Nonreactive

    Reactive

    Nonreactive

    HIV-1 screening test repeatedly reactive and WB or IFA positive*

    292

    258 (88.4%)

    34
    (11.6%)

    254
    (87.0%)

    38
    (13.0%)

    HCV Screening Test repeatedly reactive and RIBA positive*

    490
    or 500

    387/490 (79.0%)

    103/490
    (21.0%)

    392/500
    (78.4%)

    108/500
    (21.6%)

    HBsAg screening test repeatedly reactive and neutralization test positive*

    606

    552 (91.1%)

    54
    (8.9%)

    489
    (80.6%)

    117
    (19.4%)

    * Only WB, IFA, RIBA, and Neutralization test positives have been tested in this study.

    Testing of repeatedly reactive samples that are indeterminate or negative has not been performed.

    Review Commentm

    When a sample is repeatedly reactive on a licensed screening serology test and confirmed positive by an additional, more specific serology test, and the Ultrio Plus Assay is reactive and Discriminatory test reactive, the Ultrio Plus Assay also acts as a supplemental test that confirms infection. However, specimens that are repeatedly reactive for antibodies to HIV-1, HCV, or HBV on the screening assay and negative or indeterminate on the standard supplemental assays (HIV-1 Western blot, HCV RIBA, and HBsAg neutralization test, respectively), were not tested in these studies. Considering that the specificity of Ultrio Plus Assay is comparable to the approved Ultrio Assay, the review committee decided that the limited supplemental claim can be granted. ----b(4)-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

  12. Stability

    Stability studies were conducted to support dating of the Ultrio Plus Assay kit. The shelf-life stability study is composed of the following types of testing:
     

    • Real-time Stability – Monitors the master kit and individual kit reagents using QC panels to support expiration dating under recommended storage conditions.
    • Open kit stability – Monitors stability after a kit is opened, thawed and exposed to conditions of use and then stored as recommended (e.g. thawed, with some reagents diluted).
    • On-board TIGRIS – Monitors performance of the kit according to use conditions on the TIGRIS System.

    Performance results show acceptable kit performance out to –b(4)----- for ----b(4)--------------------------------------- in Real-time Stability studies, and acceptable kit performance under open kit 30-day, open kit 60-day, and on-board TIGRIS conditions. Testing of positive QC panels resulted in acceptable signal/reactivity rates and the negative QC panel results in acceptable background signal at the time points tested. Stability studies for -----b(4)--------------- are currently ongoing. At present, all performance acceptance criteria have been met with no degradation of performance in the Real-time Stability study beyond 24 months, which is the approved expiration dating for the kit.

  13. Serum and plasma specimens collected in various anticoagulants

    The performance of Ultrio Plus and Ultrio Plus Discriminatory Assays for serum and samples collected in various anticoagulants was evaluated. The anticoagulants tested were: ACD (Acid Citrate Dextrose), CPD (citrate phosphate dextrose), CP2D (citrate phosphate double dextrose), CPDA (citrate phosphate dextrose adenine), K2EDTA (ethylene diamine tetraacetic acid), K2EDTA Sep (K2 EDTA separation tube), K3 EDTA, LiH (lithium heparin), NaC (sodium citrate), PPT (K2 EDTA Plasma Preparation Tube), and a serum collection tube.

    HIV-1, HCV, and HBV positive specimens were created by individually spiking specimens collected in various anticoagulants and control specimens to a final concentration of 150 copies/mL of HIV-1, 150 copies /mL HCV, or 15 IU/mL of HBV. For all anticoagulants and serum, 100% detection of the spiked HIV-1, HCV and HBV was observed.


CLINICAL STUDIES

  1. Clinical Specificity - U.S. Blood Donor Specimens and Source Plasma Donor Specimens

    Study Design

    A prospective multicenter clinical trial was conducted in the United States. Blood donations were collected at blood collection facilities in accordance with their respective SOPs. Samples were also collected from Source Plasma donors. Samples from all donors were sent to testing laboratories. Three external sites were used for this study. At the testing sites, samples were combined to create 16-sample pools, and the pools were tested with the Ultrio Assay on the TIGRIS System and with the Ultrio Plus Assay on the TIGRIS System. Three lots of the Ultrio Plus Assay were used over the course of the study at each testing site. Samples were also tested with serologic tests in accordance with site SOPs.

    Samples from pools with discordant results between the Ultrio Plus Assay and the Ultrio Assay were tested with additional NAT assays. Results from testing of pools with the Ultrio Plus Assay were compared to Ultrio Assay results to estimate clinical specificity in 16-member pools with a 2-sided 95% confidence interval (CI). Results from additional testing were used to determine the true status of the pools.

    Sample size

    • 3043 fresh and frozen normal blood donor plasma specimens
    • 2104 16-sample pools consisting of donations from volunteer whole blood donors and 1025 16-member pools consisting of donations from Source Plasma donors.

    Statistical methods of analysis

    • Ultrio Assay nonreactive pools containing Ultrio Plus Assay nonreactive Individual Donor Samples (IDS) are considered nonreactive for HIV-1 RNA, HCV RNA and HBV DNA.
    • Ultrio Plus Assay reactive pools containing at least 1 Ultrio Plus Assay reactive, discriminated IDS are considered reactive.
    • All other combinations are indeterminate.
    • Pools with indeterminate status are listed in the clinical study report along with the available licensed and investigational assay results for each pool constituent; truth is defined by available licensed and investigational assay results?.
    • Tests that were invalid due to instrument hardware error were not retested, and are excluded from the data analysis.
    • The clinical specificity of the Ultrio Plus Assay in pools of 16 individual donor samples is calculated as: Specificity = TN/(TN+FP) x 100%

    Results

    1. Clinical specificity in 16-sample pools of donations from volunteer blood donors

      Of the 2104 pools, 2090 had nonreactive results on the Ultrio Plus Assay. There were 14 pools that had reactive results on the Ultrio Plus Assay. Of these 14 pools, 12 pools were determined to be true positive pools (see Table 18).

      Table 18.

       

      Number of Pools

      Total pools tested

      2104

      Nonreactive pools

      2090

      Initially reactive pools

      14

      True positive pools

      12

      False positive pools

      2

      Two of the pools were determined to be false positives as neither pool contained samples that were reactive when tested as individual donor samples with the Ultrio Plus Assay (see Table 19). In this study the specificity of the Ultrio Plus Assay in 16-sample pools of donations from volunteer blood donors was 2090/2092 = 99.90% (95% CI: 99.77% - 100%).

      Table 19. Clinical Specificity Study: Ultrio Plus Assay Reactivity in 16-Sample Pools From Voluntary Blood Donations

      Results

      n

      Percentage (95% CI)

      Total pools tested

      2,104

      100%

      Nonreactive pools

      2,090

      99.3% (98.9 - 99.6%)

      Initially reactive pools

      14

      0.7% (0.4 - 1.1%)

      Pool, individual constituent(s), and discriminatory assay reactive;
      Ultrio Assay reactive; True Positive

      12

      0.6% (0.3 - 1.0%)

      Pool reactive, individual constituent(s) non-reactive; Ultrio
      Assay non-reactive; Alternate NAT not performed; False Positive

      2

      0.1% (0.0 - 0.3%)

    2. Clinical specificity in 16-sample pools of Source Plasma donations

      Of the 1025 pools, 1013 had non-reactive results on the Ultrio Plus Assay. There were 12 pools that had reactive results on the Ultrio Plus Assay, and all of them were determined to be true positive pools (see Table 20).

      Table 20. Clinical Specificity Study: PROCLEIX ULTRIO PLUS Assay Reactivity in 16-Sample Pools From Source Plasma Donations

      Results

      n

      Percentage (95% CI)

      Total pools tested

      1,025

      100%

      Nonreactive pools

      1,013

      98.8% (98.0 - 99.3%)

      Initially reactive pools

      12

      1.2% (0.7 - 2.0%)

      Pool, individual constituent(s), and discriminatory assay reactive; Ultrio Assay reactive; True Positive

      11

      1.1% (0.6 - 1.9%)

      Pool, individual constituent(s), and discriminatory assay reactive; Ultrio Assay non-reactive; Alternate NAT confirmed reactive; True Positive

      1

      0.1% (0.0 - 0.6%)

      n = number of pools

      Twelve of the pools were determined to be true positives as all 12 pools contained samples that were reactive when tested as individual donor samples (see Table 19). In this study the specificity of the Ultrio Plus Assay in 16-sample pools of Source Plasma donations was 1013/1013 = 100% (95% CI: 99.6% - 100%).

      Presumptive HBV yield case

      One specimen from the Source Plasma pool study was identified as a possible HBV yield case. The test results for this specimen are listed below:

      Ultrio Plus Assay on pool of 16: Reactive
      Ultrio Plus Assay IDS: Reactive
      Ultrio Plus Assay dHBV: Reactive
      Ultrio Assay on pool of 16: Non-reactive
      Alternate NAT: Reactive
      HBsAg: Negative

      In addition, plasma specimens from 3 donations from this donor (donation 10 days previous, the presumptive yield donation, and a subsequent donation) were tested in 10 replicates with the Ultrio Plus Assay and the Ultrio Assay. All replicates were reactive on the Ultrio Plus Assay (10/10, 10/10, 10/10); 1 replicate from the presumptive yield case donation was nonreactive on the Ultrio Assay (10/10, 9/10, 10/10). All of these specimens were negative for anti-HBc.

      No follow-up samples from this donor were tested to demonstrate seroconversion and to prove HBV infection in this presumptive yield case.

    3. Clinical specificity – in individual donations from volunteer blood donors

      Of 3043 individual blood donations tested, 3042 had nonreactive results on the Ultrio Plus Assay. One false positive specimen was initially reactive on Ultrio Plus Assay, and non-reactive upon retesting in the Ultrio Plus Assay and the Ultrio Plus Discriminatory Assays. In this study the specificity of the Ultrio Plus Assay in individual volunteer blood donations was 3042/3043 = 99.97% (95% CI = 99.90-100%).

      For the Ultrio Plus HIV-1 Discriminatory Assay, the specificity was 578/578 = 100.00% (95% CI: 99.5-100%) in this study.
      For the Ultrio Plus HCV Discriminatory Assay, the specificity was 717/717 = 100.00% (95% CI: 99.6-100%) in this study.
      For the Ultrio Plus HBV Discriminatory Assay, the specificity was 712/714 = 99.72% (95% CI: 99.0-99.9%) in this study. Two false positive specimens were initially reactive on the Ultrio Plus HBV Discriminatory Assay, and nonreactive upon retesting on the Ultrio Plus Assay and the Ultrio Plus HBV Discriminatory Assay.

      Performance of the Ultrio Plus Assay and Ultrio Plus Discriminatory Assays in Cadaveric Blood Specimens:

      Specificity

      HIV-1, HCV and HBV seronegative cadaveric blood specimens were tested to determine the specificity of the Ultrio Plus Assay and the Discriminatory Assays for HIV-1, HCV and HBV. 32 cadaveric specimens were tested using two reagent lots. The specificity of the Ultrio Plus Assay and of each of the Ultrio Plus Discriminatory Assays for cadaveric specimens in this study was 32/32 = 100% (95% CI = 89.3% - 100%) (see Table 21) .

      Table 21: Specificity of Ultrio Plus Assay and Discriminatory Assays in Cadaveric Blood Specimens

      ULTRIO
      PLUS Assay

      Mean IC S/CO

      2.10

      Mean Analyte S/CO

      0.09

      Specificity

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/0

      ULTRIO
      PLUS dHIV-1 Assay

      Mean IC S/CO

      2.01

      Mean Analyte S/CO

      0.13

      Specificity

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/0

      ULTRIO
      PLUS dHCV Assay

      Mean IC S/CO

      2.02

      Mean Analyte S/CO

      0.12

      Specificity

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/0

      ULTRIO
      PLUS dHBV Assay

      Mean IC S/CO

      2.03

      Mean Analyte S/CO

      0.10

      Specificity

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/0

      IC = Internal Control
      S/CO = Signal to Cutoff ratio
      * Sixteen unique cadaveric plasma specimens and 16 unique cadaveric serum specimens were each tested in singlet.

      Sensitivity

      For HIV-1:

      Cadaveric blood specimens that were seronegative for HIV-1, HCV, and HBV were spiked with a low level of HIV-1 and tested to determine the sensitivity of the Ultrio Plus Assay and the Ultrio Plus HIV-1 Discriminatory (dHIV-1) Assay. Sixteen cadaveric specimens were tested in duplicate using two reagent lots after spiking each specimen with approximately 150 copies/mL of HIV-1. The results are shown in Table 22.

      Table 22: Reactivity of the Ultrio Plus Assay and the Ultrio Plus HIV-1 Discriminatory Assay in Cadaveric Blood Specimens Spiked with HIV-1

      ULTRIO
      PLUS Assay

      Mean Analyte S/CO

      9.45

      Reactive Rate

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/32

      ULTRIO
      PLUS dHIV-1 Assay

      Mean Analyte S/CO

      16.38

      Reactive Rate

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/32

      S/CO = Signal to Cutoff ratio
      * Eight unique cadaveric plasma specimens and 8 unique cadaveric serum specimens were each tested in duplicate.

      For HCV:

      Cadaveric blood specimens that were seronegative for HIV-1, HCV, and HBV were spiked with a low level of HCV and tested to determine the sensitivity of the Ultrio Plus Assay and the Ultrio Plus HCV Discriminatory (dHCV) Assay. Sixteen cadaveric specimens were tested in duplicate using two reagent lots after spiking each specimen with approximately 150 copies/mL of HCV. The results are shown in Table 23.

      Table 23: Reactivity of the Ultrio Plus Assay and the Ultrio Plus HCV Discriminatory Assay in Cadaveric Blood Specimens Spiked with HCV

      ULTRIO
      PLUS Assay

      Mean Analyte S/CO

      8.31

      Reactive Rate

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/32

      ULTRIO
      PLUS dHCV Assay

      Mean Analyte S/CO

      22.21

      Reactive Rate

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/32

      For HBV:

      Cadaveric blood specimens that were seronegative for HIV-1, HCV, and HBV were spiked with a low level of HBV and tested to determine the sensitivity of the Ultrio Plus Assay and the Ultrio Plus HBV Discriminatory (dHBV) Assay. Sixteen cadaveric specimens were tested in duplicate using two reagent lots after spiking each specimen with approximately 15 IU/mL of HBV. The results are shown in Table 24.

      Table 24: Reactivity of the Ultrio Plus Assay and the Ultrio Plus HBV Discriminatory Assay in Cadaveric Blood Specimens Spiked with HBV

      ULTRIO
      PLUS Assay

      Mean Analyte S/CO

      14.10

      Reactive Rate

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/32

      ULTRIO
      PLUS dHBV Assay

      Mean Analyte S/CO

      22.82

      Reactive Rate

      100%

      95% CI

      89.3-100%

      Number Tested*/Reactive

      32/32


REVIEW ISSUES

During the review of the BLA Efficacy Supplement, the review committee identified the following issues:

CMC:

  • Clarification of the rationale for the modifications to the purity specification for the ---b(4)---- used in the clinical lots.
  • Verification of –b(4)---- for purity and shelf life.
  • Reagents’ on-board stability.
  • Stability of specimens collected in various anticoagulants, and stability of cadaveric specimens.

Non-clinical:

  • HCV and HBV genotype detection for low copy number panels.

Clinical:

  • Need for additional data to support the Limited Supplemental Test claims for the Ultrio Plus Assay.
  • Comparison of results for Ultrio Plus and Ultrio for Clinical Sensitivity Panels (Known Positives).

Software:

  • Clarification for several software issues.

Labeling:

  • Conversion factors for copies/mL to IU/mL.

These issues were communicated to Gen-Probe on February 2, 2012. Gen-Probe addressed all of the issues in their response submitted on February 24, 2012 and in their further communications with the reviewers. The review committee agreed that all of the review issues are adequately resolved. There are no additional issues that may prevent the approval of this supplement.

Bioresearch Monitoring Inspection (BIMO Inspection)

The inspections were conducted in accordance with FDA’s Compliance Program Guidance Manual (CPGM) 7348.811. BIMO inspections were conducted at all three clinical study sites, as follows:

Site ID

Study Site

Location

Form FDA 483 Issued

Inspection Final Classification

ACHA

American Red Cross Charlotte National Testing Laboratory

Charlotte, North Carolina

No

NAI

ADET

American Red Cross Detroit National Testing Laboratory

Detroit, Michigan

No

NAI

QLTX

QualTex Laboratories

San Antonio, Texas

Yes

VAI

The inspection final classification for two sites was “No Action Indicated” and for one site was “Voluntary Action Indicated”.

Software and Instrumentation

Minor modifications to hardware (i.e., a larger syringe) and software (i.e., to accommodate the larger syringe) were necessary because of the addition of a new reagent, TER, used with the Ultrio Plus Assay. This modified system will also be used to perform the WNV Assay and the Ultrio Assay. Even though these changes to the TIGRIS System were made to specifically accommodate the Ultrio Plus Assay, modifications to the software for the WNV Assay and the Ultrio Assay to accommodate the larger syringe were also necessary because a single TIGRIS System is desired to perform all Procleix assays.

The hardware modifications to the TIGRIS System include:

  • Using a 1.25 mL amplification syringe instead of a 0.5 mL amplification syringe
  • Modified reagent holder (reagent “wedge”), used only with the Ultrio Plus Assay
  • Changes to the Assay Reagent Carousel lid to accommodate TER, which will be used only with the Ultrio Plus Assay.

The hardware modifications to the TIGRIS System occur at the customer site, not at the time of manufacture. A qualified Field Service Engineer installs the parts as well as the associated software.

Additional modifications to the TIGRIS System include:

  • Maintenance procedures were updated to aspirate a different volume with the 1.25 mL amplification syringe when compared to the 0.5 mL amplification syringe.
  • The Prime Operation (used for priming fluid lines before starting assay processing) was updated to aspirate a different volume with the 1.25 mL amplification syringe compared to the 0.5 mL amplification syringe.
  • User Interface and Assay Reagent Inventory was modified to allow for the loading of TER (specific for the Ultrio Plus Assay only).
  • Assay Processing and Assay Processing Verifier changes were made to support addition of TER (specific for the Ultrio Plus Assay only).
  • The TIGRIS Interface and Assay Reagent Inventory modules were modified to allow for the coexistence of the WNV and Ultrio Plus Assays on the modified TIGRIS (however the WNV and Ultrio Plus reagents cannot be loaded on the TIGRIS at the same time.)

These modifications are either assay independent or are specific to the system.

The following is a description of the assay processing parameter and process differences in the Assay Software (ADMs) for the Ultrio Plus Assay compared to the Ultrio Assay:

  • Addition of an aspirate airgap after sample addition for the Ultrio Plus Assay to improve sample liquid level sensing (LLS) accuracy by reducing the risk of hanging droplets affecting LLS readings.
  • Updated parameter settings that are used for aspirating and dispensing reagents to accommodate the 1.25 mL amplification syringe.
  • One liquid level sense check, instead of two, is performed during the aspiration of the following reagents: TER, Amplification, Enzyme, Probe and Selection.
  • Aspirating and dispensing TER into MTUs only occurs for the Ultrio Plus Assay. As a result, it takes longer for Assay Processing to be completed for the Ultrio Plus Assay due to the addition of TER.
  • Amplification and HPA pipettor volumes used for rinsing the pipettors after dispensing the following reagents have been updated to allow extra system fluid needed for the TER rinse: Amplification, Enzyme, Probe and Selection.
  • Reagent Dispense Verification (RDV) acceptable ranges for Amplification, Enzyme, Probe and Selection reagent dispenses have been updated as a result of the modified dispense parameters for the 1.25mL Amplification syringe.
  • MTU dwell times that are performed for various steps throughout Assay Processing were modified to accommodate the assay sequence differences that resulted from the TER addition in the Ultrio Plus Assay.

Gen-Probe correctly identified the software as Major Level of Concern and provided the following documents: hazard and risk analyses, traceability analyses, validation and verification documentation, software life cycle, configuration management, various software tests, revision level history and release version, unresolved anomalies, comparability testing with larger syringe, and other documents. All the documents provided by Gen-Probe were satisfactory.

Establishment Description:

  • Establishment Inspection

As there is not a substantive change in manufacture, procedures, or facilities, a pre-approval inspection would not enhance understanding of the change, procedures, validation exercise, or data submitted to support the change. In addition, the Gen-Probe facility was inspected from August 9 to 18, 2011 and was classified voluntary action indicated (VAI). Therefore, the DMPQ reviewer recommended that an inspection not be conducted in conjunction with this supplement.

  • Environmental Impact Analysis, Claims for Categorical Exclusion

Gen-Probe requested that the requirement for an Environmental Assessment be waived for this BLA Efficacy Supplement. Under 21 CFR 25.31(c), the requirement for an environmental impact assessment is categorically waived for an application to market a biologic product if the substances associated with that product occur naturally in the environment and the action of the product does not significantly alter the concentration or distribution of those substances, their metabolites, or degradation products in the environment. Gen-Probe, in accordance with current Good Manufacturing Practices, performs all production activities in compliance with applicable environmental regulations. The Ultrio Plus Assay does not have significant environment impact. This claim for categorical exclusion was found to be justified.

Lot Release Testing Result:

Three consecutive clinical lots of the Ultrio Plus Assay were received and tested in the Division of Biological Standards and Quality Control (DBSQC). Three lots met the current Expected Reactivity for the FDA/CBER Reference Panels for HIV-1 RNA, HCV RNA, and HBV DNA used to evaluate kit function.

Note: Lot Release Testing of the Ultrio Plus Assay is performed on the eSAS instrument, not on the TIGRIS instrument. FDA lot release cannot be done on the TIGRIS platform on which the test is intended to be used because FDA does not have a TIGRIS instrument at this time. A TIGRIS instrument will not be available to FDA in the near future ---b(4)---------------------------------------------. The original Ultrio and Ultrio Plus Assays are similar, and since each lot of Ultrio kits is currently being released based on FDA testing using only eSAS, lot release testing for Ultrio Plus is likewise being done using the eSAS instrument at FDA. It is highly unlikely that a lot that would not pass when tested on TIGRIS would pass using eSAS. At the same time FDA will request Gen-Probe to perform the testing of a coded lot release panel on the TIGRIS instrument and to submit the results to FDA.

The following is a list of the kit lots submitted in support of approval:

Master Lot NumberExpiration DateLot Release Assay Determination
--b(4)---2013-08-15PASS
---b(4)---2013-09-15PASS
--b(4)-----2013-10-15PASS

Recommendations

Based on the review of the information submitted in the application and additional information submitted in response to all review issues raised by the review committee members, all reviewers agreed that the Ultrio Plus Assay may be approved contingent upon the fulfillment of a Postmarket Commitment (PMC). The PMC will allow the agency to update the labeling for the HIV-1, HCV, and HBV limited supplemental claims.

The requirements and conditions in the PMC are:
In order to further update the Ultrio Plus Assay labeling for the HIV-1, HCV, and HBV limited supplemental claims, Gen-Probe will provide the results of Ultrio Plus Assay testing of the following:

  1. -----b(4)----------------------------------------------------------------------------------------------------------------------------------------------------
  2. -----b(4)---------------------------------------------------------------------------------------------------------------------------------------------------------
  3. ------b(4)----------------------------------------------------------------------------------------------------------------------------------------------------------------
  4. ------b(4)------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  5. ------------------------------b(4)-------------------------------------------------------------------------------------------------------------------------------------

Gen-Probe agreed that they will provide the results of this testing as a supplement to the BLA within six months of approval of the BLA efficacy supplement for the Ultrio Plus Assay.

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