ABBOTT REALTIME CMV
Device Facts
| Record ID | P160044 |
|---|---|
| Device Name | ABBOTT REALTIME CMV |
| Applicant | Abbott Molecular |
| Product Code | PAB · Microbiology |
| Decision Date | May 18, 2017 |
| Decision | APRL |
| Regulation | 21 CFR 866.3180 |
| Device Class | Class 2 |
Indications for Use
Abbott RealTime CMV Assay: The Abbott RealTime CMV test is an in vitro polymerase chain reaction (PCR) assay for the quantitation of cytomegalovirus (CMV) DNA in human EDTA plasma. The Abbott RealTime CMV test is intended for use as an aid in the management of Hematopoietic Stem Cell Transplant patients who are undergoing anti-cytomegalovirus therapy. In this population, serial DNA measurement can be used to assess virological response to anti-cytomegalovirus therapy. The results from the RealTime CMV test must be interpreted within the context of all relevant clinical and laboratory findings. The RealTime CMV test is not intended as a screening test for the presence of CMV DNA in blood or blood products. Abbott RealTime CMV Control Kit: The Abbott RealTime CMV controls are used to establish run validity of the Abbott RealTime CMV assay when used for the quantitation of cytomegalovirus (CMV) DNA in human plasma. Abbott RealTime CMV Calibrator Kit: The Abbott RealTime CMV calibrators are for calibration of the Abbott RealTime CMV assay when used for the quantitation of cytomegalovirus (CMV) DNA in human plasma.
Device Story
Abbott RealTime CMV is an in vitro PCR assay for quantifying CMV DNA in human EDTA plasma. System uses Abbott m2000sp for automated sample preparation (magnetic microparticle purification) and Abbott m2000rt for amplification and real-time fluorescence detection. Internal control (IC) added to each specimen to monitor process validity. Assay targets UL34 and UL80.5 genes; IC target derived from Cucurbita pepo. Results reported in IU/mL via comparison to calibration curve. Used in clinical laboratories to monitor HCT patients on anti-CMV therapy; serial measurements assess virological response. Clinicians interpret results alongside clinical/laboratory findings to guide therapy duration and assess treatment efficacy. Benefits include objective monitoring of viral load response and potential optimization of antiviral therapy duration.
Clinical Evidence
Prospective, multicenter trial of 93 CMV-seropositive HCT subjects. 64 subjects received CMV-specific antiviral therapy (AVT). Primary endpoints included viral load changes between baseline/BOT and peak/EOT. Mean viral load reduction between BOT and baseline was 2.62 log10 IU/mL (p<0.0001); reduction between peak and EOT was 2.14 log10 IU/mL (p<0.0001). Study demonstrated significant viral load differences between CMV AVT and non-CMV AVT populations. Analytical studies confirmed LoD of 31.20 IU/mL and linear range of 1.70 to 8.19 log10 IU/mL.
Technological Characteristics
In vitro PCR assay; magnetic microparticle-based nucleic acid purification. Targets CMV UL34 and UL80.5 genes. Real-time fluorescence detection using dual-labeled probes (fluorophore/quencher). Automated processing via m2000sp and m2000rt instruments. Standardized to 1st WHO International Standard for Human CMV (NIBSC 09/162). Connectivity via assay-specific application files on assay discs.
Indications for Use
Indicated for quantitation of CMV DNA in human EDTA plasma to aid management of Hematopoietic Stem Cell Transplant (HCT) patients undergoing anti-CMV therapy. Used for serial monitoring of virological response to therapy. Not for screening blood or blood products.
Regulatory Classification
Identification
A quantitative cytomegalovirus (CMV) nucleic acid test for transplant patient management is identified as a device intended for prescription use in the detection of CMV and as an aid in the management of transplant patients to measure CMV deoxyribonucleic acid (DNA) levels in human plasma and/or whole blood using specified specimen processing, amplification, and detection instrumentation. The test is intended for use as an aid in the management of transplant patients with active CMV infection or at risk for developing CMV infection. The test results are intended to be interpreted by qualified healthcare professionals in conjunction with other relevant clinical and laboratory findings.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) The labeling required under § 809.10(b) of this chapter must include: (i) A prominent statement that the device is not intended for use as a donor screening test for the presence of CMV DNA in blood or blood products. (ii) Limitations, which must be updated to reflect current clinical practice. The limitations must include, but are not limited to, statements that indicate: (A) Test results are to be interpreted by qualified licensed healthcare professionals in conjunction with clinical signs and symptoms and other relevant laboratory results; (B) Negative test results do not preclude CMV infection or tissue invasive CMV disease, and that CMV test results must not be the sole basis for patient management decisions. (iii) A detailed explanation of the interpretation of results and acceptance criteria must be provided and include specific warnings regarding the potential for variability in CMV viral load measurement when samples are measured by different devices. Warnings must include the following statement, where applicable: “Due to the potential for variability in CMV viral load measurements across different CMV assays, it is recommended that the same device be used for the quantitation of CMV viral load when managing CMV infection in individual patients.” (iv) A detailed explanation of the principles of operation and procedures for assay performance. (2) Design verification and validation must include the following: (i) Detailed documentation of the device description, including all parts that make up the device, reagents required for use with the CMV assay but not provided, an explanation of the methodology, design of the primer/probe sequences, rationale for the selected gene target, and specifications for amplicon size, guanine-cytosine content, and degree of nucleic acid sequence conservation. The design and nature of all primary, secondary, and tertiary quantitation standards used for calibration must also be described. (ii) A detailed description of the impact of any software, including software applications and hardware-based devices that incorporate software, on the device's function. (iii) Documentation and characterization of all critical reagents ( *e.g.,* determination of the identity, supplier, purity, and stability) and protocols for maintaining product integrity throughout its labeled shelf life.(iv) Stability data for reagents provided with the device and indicated specimen types, in addition to the basis for the stability acceptance criteria at all time points chosen across the spectrum of the device's indicated life cycle, which must include a time point at the end of shelf life. (v) All stability protocols, including acceptance criteria. (vi) Final lot release criteria, along with documentation of an appropriate justification that lots released at the extremes of the specifications will meet the claimed analytical and clinical performance characteristics as well as the stability claims. (vii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel CMV stains ( *e.g.,* regular review of published literature and annual in silico analysis of target sequences to detect possible primer or probe mismatches). All results of this protocol, including any findings, must be documented.(viii) Analytical performance testing that includes: (A) Detailed documentation of the following analytical performance studies: Limit of detection, upper and lower limits of quantitation, inclusivity, precision, reproducibility, interference, cross reactivity, carryover, quality control, specimen stability studies, and additional studies as applicable to specimen type and intended use for the device. (B) Identification of the CMV strains selected for use in analytical studies, which must be representative of clinically relevant circulating strains. (C) Inclusivity study results obtained with a variety of CMV genotypes as applicable to the specific assay target and supplemented by in silico analysis. (D) Reproducibility studies that include the testing of three independent production lots. (E) Documentation of calibration to a standardized reference material that FDA has determined is appropriate for the quantification of CMV DNA ( *e.g.,* a recognized consensus standard).(F) Documentation of traceability performed each time a new lot of the standardized reference material to which the device is traceable is released, or when the field transitions to a new standardized reference material. (ix) Clinical performance testing that includes: (A) Detailed documentation of device performance data from either a method comparison study with a comparator that FDA has determined is appropriate, or results from a prospective clinical study demonstrating clinical validity of the device. (B) Data from patient samples, with an acceptable number of the CMV positive samples containing an analyte concentration near the lower limit of quantitation and any clinically relevant decision points. (C) The method comparison study must include predefined maximum acceptable differences between the test and comparator method across all primary outcome measures in the clinical study protocol. (D) The final release test results for each lot used in the clinical study.
Related Devices
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- P110037 — COBAS® AMPLIPREP/COBAS® TAQMAN® CMV TEST (CAP/CTM CMV TEST) · Roche Molecular Systems, Inc. · Jul 5, 2012
Submission Summary (Full Text)
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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)
I. GENERAL INFORMATION
Device Generic Name: CMV DNA Quantitative Assay
Device Trade Name: Abbott RealTime CMV
Device Procode: PAB
Applicant’s Name and Address: Abbott Molecular, Inc.
1300 E. Touhy Ave.
Des Plaines, IL 60018
Date of Panel Recommendation: None
Premarket Approval Application (PMA) Number: P160044
Date of FDA Notice of Approval: May, 18, 2017
II. INDICATIONS FOR USE
Abbott RealTime CMV Assay:
The Abbott RealTime CMV test is an in vitro polymerase chain reaction (PCR) assay for the quantitation of cytomegalovirus (CMV) DNA in human EDTA plasma. The Abbott RealTime CMV test is intended for use as an aid in the management of Hematopoietic Stem Cell Transplant patients who are undergoing anti-cytomegalovirus therapy. In this population, serial DNA measurement can be used to assess virological response to anti-cytomegalovirus therapy. The results from the RealTime CMV test must be interpreted within the context of all relevant clinical and laboratory findings. The RealTime CMV test is not intended as a screening test for the presence of CMV DNA in blood or blood products.
Abbott RealTime CMV Control Kit:
The Abbott RealTime CMV controls are used to establish run validity of the Abbott RealTime CMV assay when used for the quantitation of cytomegalovirus (CMV) DNA in human plasma.
Abbott RealTime CMV Calibrator Kit:
The Abbott RealTime CMV calibrators are for calibration of the Abbott RealTime CMV assay when used for the quantitation of cytomegalovirus (CMV) DNA in human plasma.
III. CONTRAINDICATIONS
There are no known contraindications.
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# IV. WARNINGS AND PRECAUTIONS
The warnings and precautions can be found in the Abbott RealTime CMV labeling.
# V. DEVICE DESCRIPTION
The Abbott RealTime CMV assay uses the Abbott m2000sp for processing samples and the Abbott m2000rt instrument for amplification and detection. Specimens for the assay are loaded on the m2000sp instrument and DNA is isolated using the sample preparation reagents (the Abbott mSample Preparation System$_{\text{DNA}}$) on board. Purified specimen nucleic acid and the CMV amplification/detection reagents are combined into a 96-well PCR tray by the m2000sp, and this tray is manually transferred to the m2000rt to perform amplification and real time fluorescence detection reaction. Patient results are automatically reported on the m2000rt workstation. A nucleic acid internal control (IC) is introduced into each specimen at the beginning of the sample preparation process and measured on the m2000rt to demonstrate that the process was completed correctly for each specimen and control and the samples are free of inhibition.
Sample preparation, extraction and concentration of nucleic acid is accomplished by the Abbott m2000sp, an automated sample preparation system which utilizes a magnetic microparticle process for the purification of nucleic acid from plasma samples. The mSample Preparation System$_{\text{DNA}}$ (4 x 24 Preps) reagents lyse the virion, capture the nucleic acids on magnetic microparticles, and wash the particles to remove unbound sample components. Proteinase K is included in the lysis step for plasma samples to digest proteins associated with the sample. The nucleic acids are eluted from the magnetic microparticles and transferred to the Abbott 96-Deep-Well Plate. The nucleic acids are then ready for amplification. The IC is introduced into the sample preparation procedure and is processed along with the calibrators, controls, and specimens.
During the amplification/detection reaction on the Abbott m2000rt instrument, the target DNA is amplified by the DNA Polymerase in the presence of deoxynucleotide triphosphates (dNTPs) and magnesium. The amplification reagent contains specific sets of amplification primers for CMV and IC. During PCR amplification, high temperature is used to separate the strands of double-stranded DNA. When the reaction is cooled to a temperature where DNA annealing can again occur, the analyte-specific, single-stranded DNA oligonucleotide primers bind to the analyte DNA. The primers are extended by DNA Polymerase, thereby making an exact copy of a short target stretch of the analyte DNA. The DNA Polymerase is a thermophilic enzyme that has been modified in its active site by a molecule that renders it inactive. When the DNA Polymerase is heated prior to the initiation of PCR, the inhibitory molecule is cleaved from the DNA Polymerase allowing it to regain its activity. In this way, the DNA Polymerase is only active at temperatures where specific DNA-DNA interactions occur. This procedure greatly reduces non-specific PCR artifacts such as primer dimers.
During each round of thermal cycling, amplification products dissociate to single strands at high temperature, allowing primer annealing and extension as the temperature is lowered. Exponential amplification of the target is achieved through repeated cycling between high and
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lower temperatures. Amplification of the CMV and IC targets takes place simultaneously in the same reaction.
The Abbott RealTime CMV assay targets 2 short sequences within the UL34 and UL80.5 genes of the CMV genome. The regions are specific for CMV and are highly conserved based on analysis of published CMV sequences. The IC target sequence is derived from the hydroxypyruvate reductase gene from the pumpkin plant Cucurbita pepo, and is provided as a linearized DNA plasmid in a buffer solution with carrier DNA.
During each round of PCR amplification, the fluorescent probes anneal to the amplified target DNA, if present. The probes are labeled with different fluorescent molecules, which allow CMV and IC to be distinguished from each other. The probes are single-stranded, linear DNA oligonucleotides modified with a fluorescent moiety covalently linked to one end of the probe and a quenching moiety to the other end. In the absence of target sequences, the probes adopt a conformation that brings the quencher close enough to the excited fluorophore to absorb its energy before it can be fluorescently emitted. When the probe binds to its complementary sequence in the target, the fluorophore and the quencher are held apart, allowing fluorescent emission and detection. Since this fluorescence occurs during every cycle, the PCR reaction can be read in real-time. The amplification cycle at which fluorescent signal is detected by the Abbott m2000rt is inversely proportional to the log of the CMV DNA concentration present in the original sample.
Application parameters specific to the Abbott RealTime CMV assay are contained in an assay specific application file, which is loaded on to an assay disc.
The optional amplification reagent extended use feature allows amplification reagent packs containing prepared master mix to be stored at $-20 \pm 5^{\circ}\mathrm{C}$, capped and protected from light, for up to 14 days before a second use. The internal control (IC) may be used again within 14 days if the vial remains capped at $-20 \pm 5^{\circ}\mathrm{C}$ until the second use.
The Abbott RealTime CMV assay is comprised of three kits which are provided separately: Abbott RealTime CMV Amplification Reagent Kit, Abbott RealTime CMV Control Kit, and Abbott RealTime CMV Calibrator Kit.
**Components of the Abbott RealTime CMV Amplification Reagent Kit:**
- Abbott RealTime CMV Internal Control
- Abbott RealTime CMV Amplification Reagent Pack
- DNA Polymerase
- CMV Oligonucleotide Reagent
- Activation Reagent
**Components of the Abbott RealTime CMV Control Kit:**
- Negative Control
- Low Positive Control
- High Positive Control
**Components of the Abbott RealTime CMV Calibrator Kit:**
- Calibrator A
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- Calibrator B
Ancillary Reagents Required:
- mSample Preparation System $_{DNA}$ reagents
$\mathrm{O}$ Abbott $m\mathrm{Lysis}_{DNA}$
$\mathrm{O}$ Abbott mWash $1_{DNA}$
$\mathrm{O}$ Abbott mWash $2_{DNA}$
$\mathrm{O}$ Abbott mElution BufferDNA
$\mathrm{O}$ Abbott mMicroparticlesDNA
Proteinase K
Interpretation of Results:
The Abbott m2000rt system automatically determines the concentration of CMV DNA for specimens and controls by comparing the results to a calibration curve. CMV DNA concentrations are reported in IU/mL. The interpretation of results is provided in the table below:
Interpretation of Results
| Result | Interpretation |
| --- | --- |
| Not detected | Target not detected |
| < 1.70 log IU/mL\( ^a \) | Detected\( ^b \) |
| 1.70 to 8.19 log IU/mL | \( ^c \) |
| > 8.19 log IU/mL | >ULQ\( ^d \) |
a 50 IU/mL
b Below LLOQ (lower limit of quantitation); CMV DNA is not quantifiable.
c Calculated results are within assay quantitation range. If calculated results are obtained, the interpretation field is left blank.
Above ULOQ (upper limit of quantitation); if log IU/mL results are above the quantitation range of the assay, results are reported as $>8.19 \log \mathrm{IU} / \mathrm{mL}$ ; if IU/mL results are above the quantitation range of the assay, results are reported as $>156,000,000 \mathrm{IU} / \mathrm{mL}$ .
# VI. ALTERNATIVE PRACTICES AND PROCEDURES
There are currently two FDA approved in vitro diagnostic tests for the quantitation of CMV DNA. The patient's medical history and thorough clinical examination, in addition to serology, PCR or nucleic acid testing (NAT), will provide further information on the status of a CMV infection in the intended use population. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle.
# VII. MARKETING HISTORY
The Abbott RealTime CMV assay was launched March 2011 outside of the United States. The Abbott RealTime CMV assay is distributed in the following countries:
Austria
Brazil
Costa Rica
Australia
Canada
Denmark
Bahrain
Chile
Egypt
Belarus
China
France
Belgium
Colombia
Germany
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| Hungary | Norway | Spain |
| --- | --- | --- |
| India | Pakistan | Switzerland |
| Indonesia | Paraguay | Taiwan |
| Italy | Peru | Thailand |
| Kazakhstan | Philippines | Tunisia |
| Libya | Poland | Turkey |
| Luxembourg | Russia | United Arab |
| Macedonia | Saudi Arabia | Emerits |
| Mexico | Singapore | United Kingdom |
| Morocco | Slovakia | Vietnam |
| Netherlands | South Africa | |
| New Zealand | South Korea | |
This product has not been withdrawn from the market from any country related to safety or effectiveness, or for any other reasons.
## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH
Below is a list of the potential adverse effects (e.g., complications) associated with the use of the device.
The primary adverse events from use of the device are caused by an inaccurate measurement result of CMV viral load. Failure of the Abbott RealTime CMV assay to perform as indicated, or human error in the use of the test or the interpretation of the test result, may result in an incorrect test result that is too low or too high.
An erroneous low test result or a false negative result may lead to inappropriate patient management decisions, a premature discontinuation of antiviral therapy, or may instill a false sense of security in a patient or clinician. Similar assays that differ in LoD or other analytic characteristics may lead to different durations of treatment; theoretically a less sensitive assay could lead to earlier discontinuation of treatment relative to an alternative assay, with perhaps greater risk of relapse. Although clinical practice has evolved to recommend two consecutive "negative" responses as a treatment endpoint, this is based on different tests across different institutions. At the very low viral levels where differences between negative and positive assays appear, there may not be clinical repercussions from a patient 'discontinued early' from treatment.
An erroneous high test result, or a false positive result, may contribute to a change in therapy, unnecessary treatment, prolonged duration of therapy, or create anxiety in the patient. This may be mitigated by local practice; as experience with these assays evolves at local institutions, it is likely that low level positive results in the context of otherwise negative results will be recognized and that therapy would be unlikely to be unnecessarily prolonged.
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The risk of these adverse events is readily mitigated in clinical practice by serial and/or repeat measurement as well as clinical evaluation, which would likely show symptomatic improvement.
To aid in the management of Hematopoietic Stem Cell Transplant patients who are undergoing anti-CMV drug therapy, the results from the Abbott RealTime CMV assay must be interpreted in the context of all relevant clinical and laboratory findings.
## IX. SUMMARY OF NONCLINICAL STUDIES
## A. Laboratory Studies
## Limit of Detection (LoD):
The LoD was determined by testing dilutions of the WHO International Standard (IS) (genotype gB1), and strain AD169 (genotype gB2), in pooled CMV negative human plasma. Testing was performed with 4 lots of amplification reagents on 4 m2000 Systems. Testing with each lot was performed over 3 days. Each dilution was tested in replicates of 8 per day. The results obtained with the WHO IS are summarized in Table 1. Results for the AD169 strain are summarized in Table 2. The results support a claimed LoD of 31.20 IU/mL (1.49 log₁₀ IU/mL) for the Abbott RealTime CMV assay.
| IU/mL | Number Tested | Number Detected | Percent Detected |
| --- | --- | --- | --- |
| 46.80 | 96 | 96 | 100 |
| 31.20 | 96 | 95 | 99 |
| 23.40 | 96 | 93 | 97 |
| 15.60 | 96 | 91 | 95 |
| 12.48 | 96 | 91 | 95 |
| 9.36 | 96 | 83 | 86 |
| 7.80 | 96 | 86 | 90 |
| 6.24 | 96 | 66 | 69 |
| 3.90 | 96 | 52 | 54 |
| 1.56 | 96 | 24 | 25 |
| IU/mL | Number Tested | Number Detected | Percent Detected |
| --- | --- | --- | --- |
| 46.80 | 96 | 96 | 100 |
| 31.20 | 95 | 94 | 99 |
| 23.40 | 96 | 88 | 92 |
| 15.60 | 96 | 85 | 89 |
| 12.48 | 96 | 76 | 79 |
| 9.36 | 96 | 79 | 82 |
| 7.80 | 96 | 66 | 69 |
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To confirm the ability of the Abbott RealTime CMV assay to meet the claimed LoD of 31.20 IU/mL (1.49 log $_{10}$ IU/mL), the Toledo strain of CMV (genotype gB3) and a cultured clinical specimen of (genotype gB4) were each diluted to 31.20 IU/mL (1.49 log $_{10}$ IU/mL) and tested with the Abbott RealTime CMV assay. For each strain, 2 independent dilutions were made in pooled CMV negative EDTA plasma. Two amplification reagent lots were used to test 30 replicates each for a total of 60 replicates per genotype. Testing used 2 m2000 Systems and was performed over 3 days with each lot. Results demonstrated that Abbott RealTime CMV assay can detect 31.20 IU/mL of CMV DNA with strains of genotypes gB3 and gB4. Results are summarized in Table 3. The results support a claimed LoD of 31.20 IU/mL (1.49 log IU/mL) for the Abbott RealTime CMV assay.
Table 3 LoD Confirmation, Using Genotype gB3 and gB4.
| CMV Genotype | IU/mL | Number Tested | Number Detected | Percent Detected |
| --- | --- | --- | --- | --- |
| gB3 | 31.20 | 60 | 58 | 97 |
| gB4 | 31.20 | 60 | 60 | 100 |
The claimed LoD of 31.20 IU/mL was also confirmed with anti-viral resistant clinical specimens. Two anti-viral resistant clinical specimens were diluted to 31.20 IU/mL (1.49 log $_{10}$ IU/mL) and tested with the Abbott RealTime CMV assay. For each strain, 2 independent dilutions were made in pooled CMV negative EDTA plasma. Two amplification reagent lots were used to test 30 replicates each for a total of 60 replicates per specimen. Testing used 2 m2000 Systems and was performed over 3 days with each lot. Results demonstrated that Abbott RealTime CMV assay can detect 31.20 IU/mL of CMV DNA with anti-viral resistant specimens. Results are summarized in Table 4.
Table 4. LoD Confirmation Using Anti-Viral Resistant Specimens
| Anti-Viral Resistant Specimen | IU/mL | Number Tested | Number Detected | Percent Detected |
| --- | --- | --- | --- | --- |
| 1 | 31.20 | 60 | 58 | 97 |
| 2 | 31.20 | 60 | 60 | 100 |
Traceability to the $1^{\mathrm{st}}$ WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques (NIBSC code:09/162):
The Abbott RealTime CMV assay is standardized to the $1^{\mathrm{st}}$ WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques (NIBSC code: 09/162). Abbott RealTime CMV Calibrators trace to the WHO International Standard (IS)
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each time a lot is manufactured by means of value assignment using Abbott CMV Primary Calibrators.
Conversion factors were determined by diluting the CMV WHO IS to approximately 5 log₁₀ IU/mL in pooled, normal EDTA plasma and testing against the Abbott CMV Primary Calibrators. The conversion factors were confirmed using the CMV WHO IS diluted to approximately 3.5 log₁₀ IU/mL.
The mathematical relationship between log₁₀ copies/mL and log₁₀ IU/mL is log₁₀ copies/mL + 0.19 = log₁₀ IU/mL. The mathematical relationship between copies/mL and IU/mL is copies/mL × 1.56 = IU/mL.
## Limit of Blank and Performance With Negative Specimens:
A total of 100 anti-CMV IgG negative specimens were tested with the Abbott RealTime CMV assay. Two amplification reagent lots were used to test 50 specimens each. Testing was performed on 2 Abbott RealTime m2000 Systems and each lot was used in 2 independent runs testing 25 specimens. All specimens had reported results of "Not detected". The specificity was 100% (100/100; 95% CI of 96.3 to 100.0%). The percent of false positives was 0% (0/100). The Limit of Blank (LoB) for the Abbott RealTime CMV assay is confirmed to be 0 IU/mL.
## Linear Range:
The upper limit of linearity of the Abbott RealTime CMV assay for plasma specimens is 8.19 log₁₀ IU/mL (156 million IU/mL) and the lower limit of linearity is 1.70 log₁₀ IU/mL (50 IU/mL).
The linear range for genotypes gB1 to gB4 was determined using 2 panels per genotype. The 9-member plasmid panels were prepared by diluting CMV DNA to concentrations ranging from 1.49 to 8.49 log₁₀ IU/mL in pooled CMV negative human plasma. The 5-member virus panels were made using cultured virus strains of genotypes gB1 to gB4 diluted in pooled CMV negative human plasma. The concentrations ranged from 1.49 to 5.19 log₁₀ IU/mL for genotypes gB1, gB2, and gB4 and 1.46 to 5.00 log₁₀ IU/mL for gB3. Testing was performed with a single lot of amplification reagents for 3 days with 4 replicates per day for a total of 12 replicates per panel member.
The deviation from linearity, defined as the difference between values predicted using a linear model vs the best fit polynomial model, was ≤ 0.10 log₁₀ IU/mL for all panel members of genotypes gB1 to gB4.
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Figure 1 Abbott RealTime CMV Assay Linearity using CMV genotype gB2 strain AD169.
Note: Error Bars represent $\pm 1$ SD of the mean
Sample Size (n) 107
Correlation Coefficient (r) 0.999
Slope 0.93
95% CI for slope (0.92, 0.93)
Intercept 0.36
95% CI for intercept (0.31, 0.41)
Target Concentration (Log IU/mL) Min 1.49 Max 8.49
Abbott Real Time CMV (Log IU/mL) Min 1.47 Max 8.30
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Figure 2
Abbott RealTime CMV Assay Linearity by Genotype
The Abbott RealTime CMV assay was shown to be linear across the range of CMV concentrations tested. See Table 5 for linear equations.
Table 5. Linear Equations for CMV Genotypes gB1 to gB4
| CMV Genotype | Plasmid Panel | Virus Panel |
| --- | --- | --- |
| gB1 | y = 0.9821x + 0.1991 | y = 0.9485x + 0.3143 |
| gB2 | y = 0.9257x + 0.3627 | y = 0.9356x + 0.2144 |
| gB3 | y = 0.9872x+0.0833 | y = 1.0367x - 0.1767 |
| gB4 | y = 0.9689x + 0.2178 | y = 0.9305x + 0.2382 |
# Lower Limit of Quantitation:
The claimed lower limit of quantitation (LLoQ) for the Abbott RealTime CMV assay is $50~\mathrm{IU / mL}$ $(1.70\log_{10}\mathrm{IU / mL})$
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The total analytical error (TAE) was calculated using estimates determined through analysis of data from limit of detection (LoD) and precision studies. These studies included all four genotypes of CMV.
TAE was estimated by 2 different methods: $|\mathrm{Bias}| + (2 \times \mathrm{SD})$ and SQRT $(2) \times 2 \times \mathrm{SD}$ . The TAE estimates for panel members that had an observed concentration at or near the claimed assay limit of detection $(1.49\log_{10}\mathrm{IU / mL})$ were evaluated.
For CMV genotypes gB1 to gB4, the TAE analyses demonstrated that the Abbott RealTime CMV assay can determine CMV DNA concentration of 31.20 IU/mL (1.49 $\log_{10}$ IU/mL) in plasma with an acceptable level of accuracy (TAE $\leq 1.00\log_{10}$ IU/mL). The absolute value of the bias plus two SDs (TAE) $\leq 1.00\log_{10}$ IU/mL ensures that, for samples with a true value equal to the LLoQ, there is $95\%$ or greater probability that the measured value will be within $1\log_{10}$ IU/mL of the true value. The square root of two times two SDs $\leq 1.00\log_{10}$ IU/mL ensures that, for samples with a true value equal to the LLoQ, the difference between two measurements of more than $1\log_{10}$ IU/mL is statistically significant. Table 6 shows the TAE analyses for the lowest concentration that meets both TAE acceptance criteria for genotypes gB1 to gB4, anti-viral resistant, and clinical strains. Across all strains, the lowest concentration that met TAE acceptance criteria across all CMV genotypes and clinical strains was 46.80 IU/mL (1.67 $\log_{10}$ IU/mL). The results support a claimed LLOQ of 50 IU/mL (1.70 $\log_{10}$ IU/mL).
| Table 6. Lower Limit of Quantitation for CMV Genotypes gB1 to gB4, anti-viral resistant, and clinical strains | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| CMV strain | Nominal Concentration IU/mL | Nominal Concentration Log10IU/mL | n | Mean (Log10 IU/mL) | SD (Log10 IU/mL) | Bias (Log10 IU/mL) | TAE = |Bias| + (2×SD) (Log10 IU/mL) | TAE = SQRT(2) ×2×SD (Log10 IU/mL) |
| gB1 | 31.20 | 1.49 | 95 | 1.81 | 0.20 | 0.32 | 0.72 | 0.57 |
| gB2 | 31.20 | 1.49 | 94 | 1.52 | 0.28 | 0.03 | 0.59 | 0.79 |
| gB3 | 31.20 | 1.49 | 58 | 1.60 | 0.33 | 0.11 | 0.77 | 0.93 |
| gB4 | 31.20 | 1.49 | 60 | 1.72 | 0.24 | 0.23 | 0.71 | 0.68 |
| CMV Anti-Viral Resistant Strain 1 | 46.80 | 1.67 | 60 | 1.79 | 0.16 | 0.12 | 0.44 | 0.45 |
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| Table 6. Lower Limit of Quantitation for CMV Genotypes gB1 to gB4, anti-viral resistant, and clinical strains | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| CMV strain | Nominal Concentration IU/mL | Nominal Concentration Log_{10} IU/mL | n | Mean (Log_{10} IU/mL) | SD (Log_{10} IU/mL) | Bias (Log_{10} IU/mL) | TAE = |Bias| + (2×SD) (Log_{10} IU/mL) | TAE = SQRT(2) ×2×SD (Log_{10} IU/mL) | | |
| CMV Anti-Viral Resistant Strain 2 | 31.20 | 1.49 | 60 | 1.77 | 0.24 | 0.28 | 0.76 | 0.68 | | |
| Clinical Strain | 31 | 1.49 | 78 | 1.43 | 0.33 | -0.06 | 0.72 | 0.93 | | |
| Clinical Strain | 31 | 1.49 | 119 | 1.68 | 0.24 | 0.19 | 0.67 | 0.68 | | |
In addition, the claimed LLoD of 31.20 IU/mL was verified using a CMV anti-viral resistant strain.
## Precision – Within Laboratory:
The precision of the Abbott RealTime CMV assay was evaluated with a 10-member panel with CMV DNA concentrations that spanned 12.48 IU/mL to 312,000,000 IU/mL. Four replicates of each panel member were tested per run with 1 run per day on each lot/instrument pair for 5 days, for a total of 15 runs and 60 replicates per panel member. Table 7 presents the precision assay results, between runs, within runs, inter-assay, between lot, and overall.
Table 7: RealTime CMV Precision (Log IU/mL)
| | | | | Within-run component | | Between-run component | | Inter-assay | | Between-lot/instrument component | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| panel | n | Target Concentration (Log IU/mL) | Mean Concentration (Log IU/mL) | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 | 40^{b} | 1.11^{e} | 1.23 | 0.232 | 18.9 | 0.055 | 4.5 | 0.239 | 19.4 | 0.00 | 0.0 | 0.239 | 19.4 |
| 2 | 53^{c} | 1.41^{e} | 1.41 | 0.269 | 19.1 | 0.173 | 12.3 | 0.320 | 22.7 | 0.00 | 0.0 | 0.320 | 22.7 |
| 3 | 60 | 2.11 | 1.99 | 0.134 | 6.7 | 0.020 | 1.0 | 0.136 | 6.8 | 0.024 | 1.2 | 0.138 | 6.9 |
| 4 | 60 | 3.11 | 2.92 | 0.058 | 2.0 | 0.067 | 2.3 | 0.088 | 3.0 | 0.024 | 0.8 | 0.092 | 3.1 |
| 5 | 60 | 4.11 | 3.83 | 0.053 | 1.4 | 0.035 | 0.9 | 0.063 | 1.7 | 0.011 | 0.3 | 0.064 | 1.7 |
| 6 | 60 | 5.11 | 4.83 | 0.046 | 1.0 | 0.042 | 0.9 | 0.062 | 1.3 | 0.065 | 1.4 | 0.090 | 1.9 |
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| 7 | 60 | 6.11 | 6.07 | 0.036 | 0.6 | 0.017 | 0.3 | 0.039 | 0.6 | 0.052 | 0.9 | 0.065 | 1.1 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 8 | 60 | 7.19 | 7.08 | 0.050 | 0.7 | 0.028 | 0.4 | 0.057 | 0.8 | 0.054 | 0.8 | 0.079 | 1.1 |
| 9 | 59d | 7.89d | 7.77 | 0.036 | 0.5 | 0.026 | 0.3 | 0.044 | 0.6 | 0.079 | 1.0 | 0.091 | 1.2 |
| 10 | 59d | 8.49d | 8.26 | 0.042 | 0.5 | 0.020 | 0.2 | 0.047 | 0.6 | 0.079 | 0.9 | 0.091 | 1.1 |
a Between-run (total inter-assay SD) contains the within-run and between-run components
b CMV DNA was not detected in 20 replicates
c CMV DNA was not detected in 6 replicates. One replicate was identified as an outlier and was excluded.
One replicate did not generate a result due to instrument error.
e The target value for the panel members falls below the claimed LOD, which is 50 IU/mL (1.70 log IU/mL).
## Reproducibility (Multi-Site):
Clinical reproducibility and precision were evaluated in a multi-center study that included 3 external sites that used the Abbott RealTime CMV assay to test an 8 member panel spanning a targeted range from 1.19 to 8.49 log₁₀ IU/mL. The panel was made with CMV positive clinical specimens, cultured strain AD169 or plasmid DNA diluted in pooled plasma. Each panel member was repeated 4 times within the panel. Testing was performed with 3 lots of the Abbott RealTime CMV Amplification Reagent Kit and Sample Preparation DNA System Kit. Each of the 3 clinical sites tested 2 of the 3 amplification reagent lots and sample preparation reagent lots for 5 non-consecutive days each, resulting in a total of 10 runs at each site. Results are summarized in Table 8.
| Table 8. Clinical Reproducibility Precision | | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Within-run Component | | Between-run Component | | Between-lot Component | | Between-site Component | | Totalg | |
| Panel Member | Source | n | Mean Log₁₀ IU/mL | SDᵃ | % CV | SDᵃ | % CV | SDᵃ | % CV | SDᵃ | % CV | SDᵃ | % CV |
| 1 | Plasmid | 120 | 8.34 | 0.17 | 2.0 | 0.07 | 0.8 | 0.16 | 1.9 | 0.10 | 1.1 | 0.26 | 3.1 |
| 2 | Plasmid | 120 | 6.70 | 0.13 | 1.9 | 0.04 | 0.6 | 0.08 | 1.2 | 0.17 | 2.5 | 0.23 | 3.4 |
| 3 | Cultured Virus | 120 | 5.29 | 0.12 | 2.3 | 0.04 | 0.7 | 0.05 | 1.0 | 0.19 | 3.6 | 0.23 | 4.4 |
| 4 | Cultured Virus | 120 | 3.94 | 0.14 | 3.5 | 0.05 | 1.4 | 0.06 | 1.5 | 0.19 | 4.7 | 0.24 | 6.2 |
| 5 | Cultured Virus | 119ᵇ | 3.06 | 0.15 | 4.9 | 0.05 | 1.6 | 0.04 | 1.2 | 0.15 | 4.9 | 0.22 | 7.2 |
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| Table 8. Clinical Reproducibility Precision | | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Within-run Component | | Between-run Component | | Between-lot Component | | Between-site Component | | Totalg | |
| Panel Member | Source | n | Mean Log10 IU/mL | SD^{a} | % CV | SD^{a} | % CV | SD^{a} | % CV | SD^{a} | % CV | SD^{a} | % CV |
| 6 | Clinical Specimen | 119^{c} | 2.26 | 0.12 | 5.4 | 0.05 | 2.3 | 0.03 | 1.2 | 0.13 | 5.8 | 0.19 | 8.3 |
| 7 | Clinical Specimen | 119^{d} | 1.68^{f} | 0.21 | 12.8 | 0.10 | 6.0 | 0.04 | 2.5 | 0.13 | 8.0 | 0.27 | 16.4 |
| 8 | Clinical Specimen | 101^{e} | 1.43^{f} | 0.26 | 18.5 | 0.00 | 0.0 | 0.06 | 4.3 | 0.14 | 9.5 | 0.30 | 21.2 |
a Standard deviations (SD) are in $\log_{10}$ IU/mL.
b A result was not generated for one replicate due to a missing sample.
c One replicate was excluded from the analysis due to technician error.
d CMV DNA was not detected in one replicate.
e CMV DNA was not detected in 19 replicates.
f The mean concentration is below the claimed assay LLoQ (1.70 log$_{10}$ IU/mL).
g Total variability includes within-run, between-run, between-lot, and between-site variability.
In addition, operator-to-operator precision of the Abbott RealTime CMV assay was evaluated by testing the same 8 member panel. One lot of amplification reagents was run on 1 m2000sp and m2000rt instrument pair by 3 operators. Each operator completed 1 run per day for 7 days, for a total of 21 runs. Four replicates were tested for each panel member in each run.
| Table 9. Operator-to-Operator Precision | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Within-run Component | | Between-run Component | | Between-operator Component | | Total^{f} | | |
| Panel Member | Source | n | Mean Log_{10} IU/mL | SD^{a} | % CV | SD^{a} | % CV | SD^{a} | % CV | SD^{a} | % CV | |
| 1 | Plasmid | 84 | 8.24 | 0.13 | 1.6 | 0.06 | 0.7 | 0.12 | 1.4 | 0.18 | 2.2 | |
| 2 | Plasmid | 84 | 6.62 | 0.14 | 2.1 | 0.03 | 0.5 | 0.07 | 1.0 | 0.16 | 2.4 | |
| 3 | Cultured Virus | 83^{b} | 5.17 | 0.12 | 2.4 | 0.08 | 1.6 | 0.10 | 1.9 | 0.18 | 3.5 | |
| 4 | Cultured Virus | 84 | 3.83 | 0.11 | 2.9 | 0.09 | 2.4 | 0.10 | 2.7 | 0.18 | 4.6 | |
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| Table 9. Operator-to-Operator Precision | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Within-run Component t | | Between-run Component t | | Between-operator Component t | | Totalf | |
| Panel Member | Source | n | Mean Log10 IU/mL | SD^{a} | % CV | SD^{a} | % CV | SD^{a} | % CV | SD^{a} | % CV |
| 5 | Cultured Virus | 83^{b} | 2.96 | 0.10 | 3.4 | 0.10 | 3.3 | 0.11 | 3.7 | 0.18 | 6.0 |
| 6 | Clinical Specimen | 84 | 2.10 | 0.11 | 5.4 | 0.05 | 2.5 | 0.11 | 5.4 | 0.17 | 8.0 |
| 7 | Clinical Specimen | 78^{c} | 1.43^{e} | 0.33 | 23.2 | 0.00 | 0.0 | 0.11 | 7.7 | 0.35 | 24.4 |
| 8 | Clinical Specimen | 70^{d} | 1.28^{e} | 0.32 | 24.7 | 0.05 | 3.7 | 0.04 | 2.8 | 0.32 | 25.1 |
aStandard deviations (SD) are in log¹⁰ IU/mL.
b A result was not generated for one replicate due to an instrument error.
c CMV DNA was not detected in 6 replicates.
d CMV DNA was not detected in 14 replicates.
eThe mean concentration is below the claimed assay LLoQ (1.70 log¹⁰ IU/mL).
f Total variability includes within-run, between-run, and between-operator variability.
## Analytical Specificity – Cross Reactivity:
The following microorganisms (viruses, bacteria and fungi) were evaluated for potential cross-reactivity in the Abbott RealTime CMV assay. Each microorganism was added to CMV DNA negative plasma samples and plasma samples containing approximately 100 IU/mL and 3,120 IU/mL of CMV DNA. Microorganisms were tested at 10⁵ - 10⁶ copies/mL, IU/mL, viral particles/mL, cells/mL, TCID₅₀/mL, IFU/mL, or CFU/mL. No interference in the performance of the Abbott RealTime CMV assay was observed in the presence of the potential cross-reactants for all positive and negative samples tested.
| Microorganisms Used for Potential Cross-reactivity Study | |
| --- | --- |
| Human Immunodeficiency Virus 1 | Human Papillomavirus 18 |
| Human Immunodeficiency Virus 2 | Adenovirus |
| Human T lymphotropic virus type I | Parvovirus B19 |
| Hepatitis A Virus | JC Polyomavirus |
| Hepatitis B virus | Neisseria gonorrhoeae |
| Hepatitis C Virus | Chlamydia trachomatis |
| Epstein-Barr Virus | Staphylococcus aureus |
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Microorganisms Used for Potential Cross-reactivity Study
| Herpes simplex virus type 1 | Staphylococcus epidermidis |
| --- | --- |
| Herpes simplex virus type 2 | Mycobacterium gordonae |
| Human herpesvirus 6 | Mycobacterium smegmatis |
| Human herpesvirus 7 | Propionibacterium acnes |
| Human herpesvirus 8 | Streptococcus pneumonia |
| Varicella-Zoster virus | Salmonella typhi |
| Vaccinia Virus | Aspergillus niger |
| BK Polyomavirus | Candida albicans |
| Human Papillomavirus 16 | Cryptococcus neoformans |
## Analytical Specificity - Interfering Substances:
The susceptibility of the Abbott RealTime CMV assay to interference by elevated levels of potentially interfering substances was evaluated. Ten Anti-CMV IgG negative plasma samples and plasma samples containing approximately 100 IU/mL and 3,120 IU/mL of CMV DNA were spiked with high levels of hemoglobin, bilirubin, protein, lipids, or genomic DNA and tested. No interference in the performance of the Abbott RealTime CMV assay was observed in the presence of the following endogenous substances for all CMV positive and negative samples tested:
- Hemoglobin 2 g/L
- Bilirubin 342 µM
- Protein 120 g/L
- Lipid 37 mM
- Genomic DNA 350 µg/dL
Forty-four therapeutic drugs were tested in 9 pools, and individually for 13 drugs. CMV DNA negative plasma samples and plasma samples containing approximately 100 IU/mL and 3,120 IU/mL of CMV DNA were spiked with the drugs. No interference in the performance of the Abbott RealTime CMV assay was observed in the presence of the following drugs and drug pools in excess of peak plasma or serum levels, or in excess of therapeutic dose when peak plasma or serum levels were not available.
| Drug Pool | Drugs Tested |
| --- | --- |
| 1 | zidovudine, saquinavir, ritonavir, clarithromycin, interferon 2b |
| 2 | abacavir sulfate, amprenavir, peginterferon 2a, peginterferon 2b, ^{a} ribavirin |
| 3 | tenofovir disoproxil fumarate, lamivudine, indinavir sulfate, ganciclovir, valganciclovir hydrochloride, acyclovir |
| 4 | stavudine, efavirenz, lopinavir, enfuvirtide, ciprofloxacin |
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| Drug Pool | Drugs Tested |
| --- | --- |
| 5 | nevirapine, nelfinavir, azithromycin, valacyclovir |
| 6 | adefovir, didanosine, entecavir, cidofovir, mycophenolate mofetil |
| 7 | famotidine, cyclosporine |
| 8 | prednisone, sirolimus, tacrolimus, azathioprine |
| 9 | atenolol, amlodipine besylate, lisinopril, rabeprazole, valsartan |
a Peginterferon 2b was not tested with the 100 IU/mL sample.
Note: A consideration was made to avoid combining specific drugs within a pool that would not be used together in a clinical setting. For drug interference evaluated using drug pools, effects of individual drug were not assessed with the exception of the 13 drugs that were tested individually.
## Drugs Tested Individually
- lymphocyte immune globulin
- cyclosporine
- tacrolimus
- mycophenolate mofetil
- azathioprine
- ganciclovir
- valganciclovir
- foscarnet
- everolimus
- adefovir*
- didanosine*
- entecavir*
- cidofovir*
*These drugs were tested individually with CMV positive samples at 100 IU/mL.
The susceptibility of the Abbott RealTime CMV assay to interference by autoimmune disease states was evaluated. Plasma from 10 patients each with Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), and Anti-nuclear antibodies (ANA) were tested. Each sample was tested unspiked and spiked with CMV virus to approximately 100 IU/mL and 3,120 IU/mL. Results showed that these autoimmune disease states do not interfere with the Abbott RealTime CMV assay.
## Specimen Stability:
The stability of CMV DNA in specimens processed from whole blood and stored under various conditions was evaluated. Whole blood samples from 10 unique donors were spiked with CMV from clinical specimens at a target concentration of 3.49 log₁₀ IU/mL (3.3 log₁₀ copies/mL).
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The storage conditions are shown in the following flow diagram. A portion of each whole blood sample was processed to obtain EDTA plasma and tested the same day as the blood was collected for a baseline quantitation (Test 1). The remainder of the spiked whole blood was stored at $30^{\circ}\mathrm{C}$ for 25 hours before being processed to plasma. After plasma processing, one aliquot for each of the ten donors was tested the same day as processing (Test 2); the remainder was stored at $30^{\circ}\mathrm{C}$ for 25 hours at which time one aliquot from each donor was tested (Test 3). The remaining aliquots of plasma were stored either at $2 - 8^{\circ}\mathrm{C}$ for $137\mathrm{h}$ (approximately 5.7 days, Test 4) or $-70^{\circ}\mathrm{C}$ or colder for a total of 1,319 hours (approximately 55 days, Test 5), including 5 rounds of freeze / thaw prior to testing.
The quantitation of CMV in the stored samples was compared to the baseline (Test 1) as an indicator of sample storage stability.
Sample Storage and Processing Conditions
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| Table 10. Specimen Stability Study Results | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Baseline Condition (Test 1) | | | Storage Condition | | | | | |
| Condition | n | Mean Log10 IU/mL | SD | n | Mean Log10 IU/mL | SD | Mean Difference (Storage-Baseline) Log10 IU/mL | Pooled SD | 95% CI Mean Difference |
| Test 2 | 40 | 3.31 | 0.078 | 40 | 3.27 | 0.102 | -0.04 | 0.091 | (-0.09, -0.00) |
| Test 3 | 40 | 3.31 | 0.078 | 40 | 3.23 | 0.113 | -0.08 | 0.097 | (-0.12, -0.04) |
| Test 4 | 40 | 3.31 | 0.078 | 40 | 3.26 | 0.102 | -0.05 | 0.091 | (-0.09, -0.01) |
| Test 5 | 40 | 3.31 | 0.078 | 39 | 3.28 | 0.113 | -0.03 | 0.097 | (-0.07, 0.01) |
The results support the specimen storage claims stated in the assay package insert: Prior to preparing plasma specimens through centrifugation, freshly drawn whole blood specimens may be held at 2 to $30^{\circ}C$ for up to 24 hours. After centrifugation, remove plasma from cells. Plasma specimens may be stored:
- At 15 to $30^{\circ}C$ for up to 24 hours
- At 2 to $8^{\circ}C$ for up to 5 days
- At - 70°C or colder for longer term
- Multiple freeze/thaw cycles should be avoided and should not exceed 3 freeze/thaw cycles. Thaw plasma specimens at 15 to $30^{\circ}C$ or at 2 to $8^{\circ}C$ . Once thawed, if plasma specimens are not being processed immediately, they can be stored at 2 to $8^{\circ}C$ for up to 24 hours.
## Analytical Carryover:
Potential sample carryover in the automated Abbott m2000 instrument was determined by testing 218 high concentration CMV positive plasma samples interspersed with 220 negative samples arranged in a checkerboard pattern. The positive samples were spiked with CMV DNA at a target concentration of 15.6 million IU/mL. The carryover rate is defined as the number of CMV negative samples that report a value greater than the assay LoD over the total number of CMV negative samples tested. A total of 5 runs were evaluated. The carryover rate was $0.0\%$ (0/220).
## X. SUMMARY OF PRIMARY CLINICAL STUDY:
The clinical utility of the Abbott RealTime CMV assay was evaluated in a prospective, multicenter trial of subjects undergoing allogenic, hematopoietic stem cell transplantation (HCT).
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# Study Design
All enrolled study subjects were CMV-seropositive. The Abbott RealTime CMV assay was used for CMV plasma viral load measurement by the central laboratories in the study. Plasma CMV viral load levels were monitored in subjects according to the following schedule: weekly during days 0 through 100 post-transplant, every other week during days 101 through 180, every 30 days during days 181 through 365. Once a subject commenced CMV-specific antiviral treatment (CMV AVT), plasma viral load testing occurred on a weekly basis until CMV AVT was discontinued; at which point regularly scheduled viral load assessments resumed.
The date and time of initiation and discontinuation of CMV AVT was recorded and CMV viral level in close proximity was used to establish beginning of therapy (BOT) and end of therapy (EOT) viral load levels, respectively. Viral load levels to define baseline were obtained within 0 to +14 days of transplantation, while BOT viral load levels were collected -7 to +2 days of initiation of CMV AVT. EOT was defined as immediately following discontinuation of CMV AVT. If there were multiple viral load measurements within the window, the one closest to the baseline, BOT, or EOT date was chosen.
Data from 93 subjects were analyzed. Of the 93 subjects, 64 were treated with CMV AVT. The remaining 29 subjects received AVT that was not specific for CMV infection (non-CMV AVT). The demographics and ages of the CMV AVT and non-CMV AVT subjects included in the analyses are shown in Table 11 and Table 12, respectively. The mean age for the 93 subjects included in the analysis was 52 years with a range of 21 to 72 years.
| Table 11. Summary of Demographics | | | | |
| --- | --- | --- | --- | --- |
| | | CMV AVT Subjects (n=64) | Non-CMV AVT Subjects (n=29) | Total Subjects (n=93) |
| | Category | n (%) | n (%) | n (%) |
| Gender | Female | 29 (45.3%) | 13 (44.8%) | 42 (45.2%) |
| | Male | 35 (54.7%) | 16 (55.2%) | 51 (54.8%) |
| Race | Asian | 10 (15.6%) | 2 (6.9%) | 12 (12.9%) |
| | Black or African American | 0 (0.0%) | 1 (3.4%) | 1 (1.1%) |
| | White | 54 (84.4%) | 26 (89.7%) | 80 (86.0%) |
| | Other | 1 (1.6%) | 0 (0.0%) | 1 (1.1%) |
| Race | Asian | 10 (15.6%) | 2 (6.9%) | 12 (12.9%) |
| | Black or African American | 0 (0.0%) | 1 (3.4%) | 1 (1.1%) |
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| Ethnicity | Hispanic or Latino | 2 (3.1%) | 1 (3.4%) | 3 (3.2%) |
| --- | --- | --- | --- | --- |
| | Not Hispanic or Latino | 62 (96.9%) | 28 (96.6%) | 90 (96.8%) |
| Table 12. Summary of Age (Years) | | | | |
| --- | --- | --- | --- | --- |
| | | CMV AVT Subjects | Non-CMV AVT Subjects | Total Subjects |
| | Category | (n=64) | (n=29) | (n=93) |
| Age | Mean Years (SD) | 53 (12) | 51 (13) | 52 (13) |
| | Range (min, max) | (21, 72) | (25, 72) | (21, 72) |
The mean number and range of CMV viral load measurements for the 93 subjects included in the analyses are shown in Table 13.
| Table 13 Mean and Range of the Number of Viral Load Measurements | | | |
| --- | --- | --- | --- |
| Status | Subjects | Mean | Range |
| CMV AVT | 64 | 25.0 | 6, 56 |
| Non-CMV AVT | 29 | 23.6 | 3, 38 |
| All Subjects | 93 | 24.6 | 3, 56 |
The mean and duration of the first course of therapy for 63 of the CMV AVT subjects is shown in Table 14. The first course of CMV AVT is defined as the earliest CMV AVT start date and end date for CMV AVT subjects.
| Table14. Mean and Range of the Duration of First Course of CMV AVT (Days) | | |
| --- | --- | --- |
| Subjectsa | Mean (Days) | Range |
| 63 | 28.6 | 1, 123 |
${}^{a}$ One subject was excluded; the CMV AVT end date was not provided.
A summary of the CMV specific drugs used for treating the subjects for their first course of CMV AVT are shown in Table 15. Ganciclovir, Valganciclovir Hydrochloride, and
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Valganciclovir were the most commonly used CMV AVT drugs for the subjects' first CMV AVT.
| Table 15. Summary of Drugs Used for First CMV AVT | | | |
| --- | --- | --- | --- |
| Drug Name | Total Number of Subjects with CMV AVT | Number of Subjects Receiving the Drug | Percentage of Subjects Receiving the Drug (%) |
| Ganciclovir | 64 | 31 | 48.4 (31/64) |
| Ganciclovir Sodium | 64 | 2 | 3.1 (2/64) |
| Valganciclovir Hydrochloride | 64 | 29 | 45.3 (29/64) |
| Valganciclovir | 64 | 17 | 26.6 (17/64) |
| Foscarnet Sodium | 64 | 3 | 4.7 (3/64) |
| Foscarnet | 64 | 2 | 3.1 (2/64) |
| Immunoglobulin Cytomegalovirus | 64 | 3 | 4.7 (3/64) |
Comparison of viral loads at BOT versus baseline and peak versus EOT are shown in Table 16 for CMV AVT subjects. 62 CMV AVT subjects were included in this analysis. Measurements for 58 CMV AVT subjects fell within the time window for baseline and BOT. Measurements for 61 CMV AVT subjects fell within the time window for EOT. The mean difference in viral load between the BOT and baseline for 58 subjects for whom results were available at both time points was $2.62\log_{10}\mathrm{IU / mL}$ with a $95\%$ confidence interval of 2.16 to 3.08. The p-value is less than 0.0001 which shows the mean difference is significantly different than zero. The mean difference in viral load between peak and EOT for the 61 subjects for whom results were available at both time points was $2.14\log_{10}\mathrm{IU / mL}$ , with a $95\%$ confidence interval of 1.81 to 2.47. The p-value is less than 0.0001 which shows the mean difference is significantly different than zero.
| Table 16. Comparison of CMV Viral Load Between Different Time Points (Baseline vs BOT, Peak vs EOT) for the First Course of CMV AVT | | | | |
| --- | --- | --- | --- | --- |
| | na | Mean Difference (SD) | 95% Confidence Interval | p-value |
| Difference in VL Between BOT and Baseline (log10 IU/mL) | 58b | 2.62 (1.733) | 2.16, 3.08 | < 0.0001d |
| Difference in VL Between Peak and EOT (log10 IU/mL) | 61c | 2.14 (1.293) | 1.81, 2.47 | < 0.0001e |
a A total of 2 subjects were excluded from the original 64 CMV AVT subjects. One subject was excluded because no CMV AVT end date was provided; 1 subject was excluded because there was no viral load measurement during the first course of CMV AVT.
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b The viral load measurements of 2 subjects fell outside of the time window of BOT; the viral load measurements of 2 subjects fell outside of the time window of baseline.
c The viral load measurement of 1 subject fell outside of the time window of EOT.
$^{\mathrm{d}}$ p-value comparing VL at BOT vs VL at baseline (using paired t-test).
e p-value comparing VL at peak vs VL at EOT (using paired t-test).
Viral loads for CMV AVT subjects during the first course of CMV AVT versus viral loads for non-CMV AVT subjects during 1 year post-transplant were also analyzed and are shown in Table 17.
This analysis demonstrates that, during the first course of CMV AVT, the CMV viral loads at BOT and peak are statistically significantly higher than the highest CMV viral loads for subjects with non-CMV AVT.
| Table 17. Viral Load Analysis for the First Course of CMV AVT vs non-CMV AVT Populations | | | | |
| --- | --- | --- | --- | --- |
| | na | Mean (SD) | Range | p-value |
| VL at BOT (log10IU/mL) for CMV | | 3.48 | 0, | |
| AVT Subjects | 60b | (1.205) | 5.93 | 0.0001c |
| VL at Peak (log10IU/mL) for CMV | | 3.66 | 0, | |
| AVT Subjects | 62 | (1.138) | 5.93 | < 0.001d |
| VL at Peak (log10IU/mL) for non-CMV AVT Subjects | 29 | 2.46 | 0, | |
| | | (0.976) | 4.04 | |
a A total of 2 subjects were excluded from the original 64 CMV AVT subjects. One subject was excluded because no CMV AVT end date was provided; 1 subject was excluded because there was no viral load measurement during the first course of CMV AVT.
b The viral load measurements of 2 subjects fell outside of the time window of BOT.
${}^{c}$ p-value comparing VL at BOT for first CMV AVT subjects vs VL at peak for non-CMV AVT subjects using a t-test.
$^{\mathrm{d}}$ p-value comparing VL at peak for first CMV AVT subjects vs VL at peak for non-CMV AVT subjects using a t-test.
## Safety and Effectiveness Results
### 1. Safety Results
There were no adverse effects of the device reported while the study was conducted.
### 2. Effectiveness Results
The results of the clinical study support the use of the Abbott RealTime CMV assay as an aid in the management of HCT patients who are undergoing anti-cytomegalovirus antiviral therapy (see Tables 16 and 17). In this population, serial DNA measurement can be used to assess virological response to anti-cytomegalovirus antiviral therapy.
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3. Subgroup Analyses
Not applicable.
4. Pediatric Extrapolation
In this premarket application, existing clinical data was not leveraged to support approval of a pediatric patient population.
**Financial Disclosure**
The Financial Disclosure by Clinical Investigators regulation (21 CFR 54) requires applicants who submit a marketing application to include certain information concerning the compensation to, and financial interests and arrangement of, any clinical investigator conducting clinical studies covered by the regulation. The pivotal clinical study included 4 investigators. None of the clinical investigators had disclosable financial interests/arrangements as defined in sections 54.2(a), (b), (c), and (f). The information provided does not raise any questions about the reliability of the data.
XI. PANEL MEETING RECOMMENDATION AND FDA'S POST-PANEL ACTION
In accordance with the provisions of section 515(c)(3) of the act as amended by the Safe Medical Devices Act of 1990, this PMA was not referred to the Microbiology Devices Advisory Panel, an FDA advisory committee, for review and recommendation because the information in the PMA substantially duplicates information previously reviewed by this panel.
XII. CONCLUSIONS DRAWN FROM PRECLINICAL AND CLINICAL STUDIES
A. Effectiveness Conclusions
- The effectiveness of the Abbott RealTime CMV assay has been demonstrated when used for the quantitation of cytomegalovirus (CMV) DNA in human plasma for the management of Hematopoietic Stem Cell Transplant patients who are undergoing anti-cytomegalovirus antiviral therapy.
- There are no issues with endogenous interferents at physiological levels or with commonly administered medications.
- Whole blood can be stored at 2 to 30°C for up to 24 hours. After centrifugation and removal from cells, EDTA plasma may be stored:
- At 15 to 30°C for up to 24 hours
- At 2 to 8°C for up to 5 days
- At –70°C or colder for longer term
- No more than 3freeze/thaw cycles
B. Safety Conclusions
Based on the results of the analytical and clinical laboratory studies, the Abbott RealTime CMV assay, when used according to the provided directions and in
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conjunction with other laboratory results and clinical information, should be safe and pose minimal risk to the patient due to false test results.
## C. Benefit-Risk Determination
The benefits outweigh the risks at the level of performance observed in the pivotal clinical study. Complementary analytical studies strengthen this conclusion. When used for the proposed intended use, benefits to both the clinician and patient include confirmation that CMV viral load is responding to treatment as anticipated, and an approximation of the time that therapy can be discontinued.
The risk from a falsely high result is the misinterpretation that a patient is not responding to treatment. This is mitigated by the known likelihood that most patients respond to treatment, understanding of the time course of response, and monitoring of the patient’s clinical symptoms. The risks of false negative results may be greater, and the need for the sponsor to monitor MDR and literature suggesting the emergence or recognition of CMV viral mutations is essential. This risk is mitigated, however, by the recognition in transplant centers that CMV disease can occur absent detectable viremia.
Patient Perspectives
This submission did not include specific information on patient perspectives for this device.
In conclusion, given the available information above, the data support that for the management of HCT patients who are undergoing anti-cytomegalovirus antiviral therapy, the probable benefits outweigh the probable risks.
## D. Overall Conclusions
The results of the nonclinical and clinical laboratory studies support the use of the Abbott RealTime CMV assay as an aid in the management of HCT patients who are undergoing anti-cytomegalovirus antiviral therapy. In this population, serial DNA measurement can be used to assess virological response to anti-cytomegalovirus antiviral therapy.
## XIII. CDRH DECISION
CDRH issued an approval order on May 18, 2017.
The applicant’s manufacturing facilities have been inspected and found to be in compliance with the device Quality System (QS) regulation (21 CFR 820).
## XIV. APPROVAL SPECIFICATIONS
Directions for use: See device labeling.
Hazards to Health from Use of the Device: See Indications, in the device labeling.
Post-approval Requirements and Restrictions: See approval order.
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