ARTUS CMV RGQ MDX KIT

P130027 · Qiagen, Inc. · PAB · Jun 2, 2014 · Microbiology

Device Facts

• Record ID: P130027
• Device Name: ARTUS CMV RGQ MDX KIT
• Applicant: Qiagen, Inc.
• Product Code: PAB · Microbiology
• Decision Date: Jun 2, 2014
• Decision: APRL
• Regulation: 21 CFR 866.3180
• Device Class: Class 2

Indications for Use

The artus® CMV RGQ MDx Kit is an in vitro nucleic acid amplification test for the quantitation of human cytomegalovirus (CMV) DNA in human EDTA plasma. The artus® CMV RGQ MDx Kit is intended for use as an aid in the management of solid organ transplant patients who are undergoing anti-CMV therapy. The test measures CMV DNA levels in EDTA plasma and can be used to assess CMV viral load response to antiviral drug therapy. The results from the artus® CMV RGQ MDx Kit must be interpreted within the context of all relevant clinical and laboratory findings. The artus® CMV RGQ MDx Kit is configured for use with the EZ1 DSP Virus System (EZ1 DSP Virus Kit and EZ1 Advanced instruments) for DNA extraction and the Rotor-Gene Q MDx instrument for CMV DNA amplification and quantitation. The artus® CMV RGQ MDx Kit is not intended for use as a screening test for blood or blood products.

Device Story

Device performs in vitro real-time PCR to quantitate CMV DNA in human EDTA plasma; used as aid in managing solid organ transplant patients on anti-CMV therapy. Input: 400 μL EDTA plasma. Process: Automated DNA extraction via EZ1 DSP Virus System (silica-based purification with magnetic beads); amplification of 105 bp region of CMV Major Immediate Early (MIE) gene on Rotor-Gene Q MDx instrument. Output: CMV viral load in IU/mL. Used in clinical laboratories by trained personnel. Results interpreted by clinicians alongside clinical/laboratory findings to monitor antiviral response. Benefits: Provides standardized viral load measurement to assess treatment efficacy and guide clinical management.

Clinical Evidence

Prospective multi-center study of 44 evaluable kidney transplant patients (368 specimens). Compared artus CMV RGQ MDx Kit to FDA-approved comparator. Primary endpoint: resolution of CMV DNAemia. Overall agreement 92.0% (days 7-42). PPA/NPA for LOQ threshold: 80.2%/96.8%; for 500 IU/mL: 89.7%/98.1%; for 1,000 IU/mL: 93.2%/97.5%; for 10,000 IU/mL: 90.5%/99.1%. Deming regression showed high concordance (slope 1.00).

Technological Characteristics

Real-time PCR assay; targets 105 bp region of CMV MIE gene. Automated extraction via EZ1 DSP Virus System (magnetic beads/silica). Detection via Rotor-Gene Q MDx instrument using FAM (CMV) and HEX (Internal Control) fluorophores. Quantitation via four standards (CMV QS 1-4). Internal control (282 bp synthetic DNA) monitors extraction/inhibition. Linear range: 159 to 7.94 x 10^7 IU/mL. Software-based Ct threshold analysis.

Indications for Use

Indicated for solid organ transplant patients undergoing anti-CMV therapy to quantitate CMV DNA in EDTA plasma and assess viral load response to antiviral drug therapy.

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.

Submission Summary (Full Text)

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# SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

## I. GENERAL INFORMATION

Device Generic Name: In vitro real-time polymerase chain reaction (PCR) based assay for CMV viral load measurement in human plasma

Device Trade Name: artus® CMV RGQ MDx Kit

Device Procode: PAB

Applicant’s Name and Address: QIAGEN, Inc.
1201 Clopper Road
Gaithersburg, MD 20878

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P130027

Date of FDA Notice of Approval: June 2, 2014

Expedited: Not applicable

## II. INDICATIONS FOR USE

The artus® CMV RGQ MDx Kit is an in vitro nucleic acid amplification test for the quantitation of human cytomegalovirus (CMV) DNA in human EDTA plasma. The artus® CMV RGQ MDx Kit is intended for use as an aid in the management of solid organ transplant patients who are undergoing anti-CMV therapy. The test measures CMV DNA levels in EDTA plasma and can be used to assess CMV viral load response to antiviral drug therapy. The results from the artus® CMV RGQ MDx Kit must be interpreted within the context of all relevant clinical and laboratory findings.

The artus® CMV RGQ MDx Kit is configured for use with the EZ1 DSP Virus System (EZ1 DSP Virus Kit and EZ1 Advanced instruments) for DNA extraction and the Rotor-Gene Q MDx instrument for CMV DNA amplification and quantitation.

The artus® CMV RGQ MDx Kit is not intended for use as a screening test for blood or blood products.

## III. CONTRAINDICATIONS

None

## IV. WARNINGS AND PRECAUTIONS

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The warnings and precautions can be found in the artus CMV RGQ MDx Kit labeling.

## V. DEVICE DESCRIPTION

The artus CMV RGQ MDx Kit is an in vitro nucleic acid amplification test for the quantitation of human cytomegalovirus (CMV) DNA in human EDTA plasma. Automated DNA extraction is performed using the EZ1 DSP Virus System, which consists of the EZ1 DSP Virus Kit and either the EZ1 Advanced instrument or the EZ1 Advanced XL instrument. Pathogen detection is based on the specific amplification of a fragment of the CMV genome using the artus CMV RGQ MDx Kit on the Rotor-Gene Q MDx (RGQ MDx) Instrument.

CMV DNA is isolated from human plasma using silica-based nucleic acid purification and magnetic beads on the EZ1 DSP Virus System. After DNA purification, eluates are ready to use with the artus CMV RGQ MDx Kit. The CMV RG Master provided in the kit contains reagents and enzymes for the specific amplification of a 105 bp region of the CMV Major Immediate Early (MIE) gene DNA, and for the direct detection of the amplicon on the RGQ MDx Instrument. The artus CMV RGQ MDx Kit provides four quantitation standards (CMV QS 1-4) which allow the quantitation of CMV DNA in the sample. Additionally, the artus CMV RGQ MDx Kit contains a second heterologous amplification system (Internal Control) to control the DNA extraction and to identify possible inhibition of the PCR. Oligonucleotide probes linked to fluorescence dyes specifically bind to the amplified product. The specific amplification of CMV target and the signal of the Internal Control are detected as fluorescence in different channels on the RGQ MDx Instrument allowing the detection and quantitation of CMV DNA.

## Target Selection

The CMV Major Immediate Early (MIE) gene was chosen as a target for the detection of CMV DNA. The target sequence is located specifically in the gene for the IE-1 protein, corresponding to the same region of strain AD-169 (accession number X17403). The MIE gene plays a key role in affecting activation and repression of viral and cellular genes; the region selected is specific for CMV and is highly conserved between the different CMV strains.

Alignment of sequences for three of the major gB genotypes (gB1 from Merlin strain, gB2 from AD-169 strain, and gB3 from Toledo strain) showed 100% identity for the target sequence of the artus CMV RGQ MDx Kit (105 of 105 base pairs). No sequence information was available for another gB genotype, gB4.

## Specimen Preparation

The artus CMV RGQ MDx Kit is intended to be used with plasma samples obtained with EDTA as anticoagulant. The viral DNA extraction is performed with the EZ1 DSP Virus System, which consists of the EZ1 DSP Virus Kit and an instrument from the EZ1 Advanced Instrument family (either the EZ1 Advanced or the EZ1 Advanced XL). A volume of 400 μl of EDTA plasma is used as a sample input volume. The extraction

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procedure using the EZ1 DSP Virus System comprises four steps: lyse, bind, wash and elute.

The proteolysis of viruses in plasma is performed with proteinase K and lysis buffer under highly denaturing conditions at elevated temperatures. This step ensures the digestion of virus coat proteins and the inactivation of RNases. During the binding step, binding buffer is added to the lysed samples to adjust binding conditions. Lysates are then thoroughly mixed with magnetic beads to allow the absorption of the viral DNA to the silica surface. Salt and pH conditions ensure that proteins and other contaminants, which can inhibit downstream enzymatic reactions, are not bound to the magnetic beads. Contaminants are efficiently washed away during a sequence of wash steps using first wash buffer 1, then wash buffer 2 and then ethanol. Finally during the elution step, the viral DNA is eluted in 60 μl of Elution Buffer (AVE). Extracted DNA can be stored at 4°C for up to 5 days or at -15 to -30°C for up to 6 months.

The extraction procedure is programmed on the EZ1 Advanced (or EZ1 Advanced XL) DSP Virus Card. These protocols provide both on-screen instructions for the user and operating commands for the EZ1 Advanced (or EZ1 Advanced XL) instrument.

## PCR Amplification and Detection

The CMV RG Master provided in the artus CMV RGQ MDx Kit contains reagents and enzymes for the specific amplification of a 105 bp region of the CMV Major Immediate Early Gene (MIE) on the thermocycler RGQ MDx Instrument. The initial high temperature that denaturizes the double stranded DNA of CMV permits the subsequent specific binding of the primers. DNA polymerase from Thermus aquaticus (Taq pol) catalyzes the amplification reaction of each cycle at the selected elongation temperature and the amount of amplicon increases exponentially.

The artus CMV RGQ MDx Kit also contains a second heterologous amplification system which allows the user both to control the DNA extraction and to identify a possible inhibition of the PCR. This Internal Control system is designed as a competitive PCR; a positive signal in the Internal Control channel in the absence of CMV DNA indicates that the PCR has not been inhibited.

Two different probes are used, one for the specific detection of the CMV amplicon and one for the Internal Control. The probes are flanked by a reporter fluorescence dye at one end (FAM for the CMV amplicon and HEX for the Internal Control) and a quencher at the opposite end.

In the RGQ MDx Instrument, fluorescent dyes are excited from the bottom of the sample chamber by a light-emitting diode. In the absence of a specific target sequence, the physical proximity of both dyes greatly reduces the emission of fluorescence by the reporter upon excitation. After binding of the probes to the amplicons, the exonuclease activity of the Taq polymerase permits the cleavage of the probes, liberating the fluorescence dyes and permitting the emission of fluorescence by the reporter once excited. Emitted fluorescence passes through the thin walls at the bottom of each PCR tube, through the emission filters on the side of the chamber and is detected by a

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photomultiplier tube. Detection is performed as each tube aligns with the detection optics; tubes spin pass the excitation/emission optics every 150 milliseconds.

The RGQ MDx Instrument measures the fluorescence at each amplification cycle allowing the detection of both amplicons in real time. The fluorescence signals indicate the progress of the PCR reactions. Specific CMV signal is detected in the Green Channel and the signal of the Internal Control in the Yellow Channel. The RGQ MDx Instrument plots the level of fluorescence at each cycle as a curve and reports the cycle at which the fluorescence crosses a pre-defined background level (threshold). This cycle is known as the threshold cycle (Ct) and is inversely proportional to the initial amount of CMV DNA.

## CMV Quantitation

The artus CMV RGQ MDx Kit provides four quantitation standards (CMV QS 1-4) at four different concentrations, which allow the quantitation of CMV DNA in the sample. These standards are produced using primers homologous to the CMV reference strain AD 169 to amplify a 504 bp DNA fragment in the region of the MIE gene. The fragment is isolated and the concentration measured spectrophotometrically. The stock solution is used to produce QS1 (10,000 copies/μL; QS2 (1,000 copies/μL), QS3 (100 copies/μL) and QS4 (10 copies/μL) by serial dilutions. These standards are treated as purified samples, and are amplified and detected together with the samples on the RGQ MDx Instrument. A linear regression is calculated by the RGQ software at the end of the procedure with the Ct values obtained from each standard and their respective concentration.

The equation for the linear regression is then used to calculate the concentration of CMV DNA in copies/mL, using the Ct value obtained for the positive detected sample. Low Ct values indicate higher CMV DNA concentrations and high Ct values indicate lower CMV DNA concentrations. The test software provides results converted from copies/mL to International Units per mL (IU/mL) and all subsequent results are reported in IU/mL.

## Controls

- No Template Control: PCR grade nuclease free water is supplied for use with the No Template Control (NTC). The No Template Control contains water and all of the assay components except nucleic acid, and is used to detect reagent contamination with target nucleic acid or increased background in the amplification reaction. This includes potential contamination of kit components due to handling errors or contamination of the Master Mix with positive samples during PCR set-up. It also indicates contamination of the tubes or caps due to improper handling.

- High and Low Positive Control: The Low Positive Control (LPC) provided with the artus CMV RGQ MDx Kit contains a linear CMV DNA 504 bp sequence from the MIE gene at a concentration near the limit of quantitation. The Low Positive Control is used to monitor for substantial reagent failure. The quantitative result must fall within a specified range for the assay to be valid (between 315 and 1785 copies/mL). The High Positive Control (HPC) contains a linear CMV DNA 504 bp sequence from the MIE at a concentration which is in the middle of the linear range. The High Positive Control is used to verify that the calibration status of the assay is maintained

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within acceptable limits. The quantitative result must fall within a specified range for the assay to be valid (16,800-53,400 copies/mL).

- Internal Control: The Internal Control is a non-CMV, 282 bp synthetic nucleic acid sequence provided with the artus CMV RGQ MDx Kit that is co-extracted and co-amplified with the target nucleic acid. The Internal Control is added to samples prior to extraction and monitors the integrity of the reagents, equipment function, and the presence of inhibitors in the samples. A specific response must be observed with negative specimens to be accepted. Inherent in every PCR reaction is the risk of false negative results because of inhibitory substances that impair amplification. To minimize the consequences of this risk, an Internal Control was implemented into the design of the assay to detect potential inhibition. The Internal Control of the artus CMV RGQ MDx Kit contains non-CMV DNA which is amplified simultaneously with the target specific DNA in a single PCR reaction. The emitted light of the specific target is measured in the Green Channel, while the light of the Internal Control is measured in the Yellow Channel. The Master reagent of the artus CMV RGQ MDx Kit contains primers and probes for the amplification and detection of both the target specific sequence and the Internal Control sequence. Both detection systems share essential components such as enzymes and dNTPs which drive the PCR reaction. The concentrations of the Internal Control components were optimized so that the amplification of the Internal Control DNA does not impair the sensitivity of the target specific system but still creates a stable fluorescence signal for the Internal Control. If a signal is detected in the Green Channel, the result is positive for the presence of cytomegalovirus DNA. Although high amounts of target specific DNA in a sample can decrease the fluorescence signal of the Internal Control, such an outcome is irrelevant with respect to interpretation due to the positive signal in the target specific channel. If no signal is detected in the target specific Green Channel for a sample but simultaneously a signal in the Internal Control Yellow Channel is detected with an allowed CT shift of -1 up to +4 in comparison to the No Template Control ($\Delta C_{T}$), the result is negative for the presence of cytomegalovirus DNA in the sample. For a negative result to be considered valid, the $C_T$ value should be between the allowed shift (-1.0 and +0.4) compared to the NTC in the Internal Control Yellow Channel. The signal in the Internal Control channel confirms that the PCR reaction was not impaired and thereby the result is valid. If no signal is detected in the Green or Yellow Channels, the result is invalid. The PCR could be inhibited by unknown substances in the sample or the reagents could have been handled differently than prescribed

- Interpretation of Control Results:

The Rotor-Gene Q software determines whether control results are valid or invalid and therefore whether the run is valid or invalid.

Valid runs:

The conditions in Table 1 must be met for a valid run.

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Table 1: Conditions required for a valid run

| Name | Test Channel | Control Channel | Status |
| --- | --- | --- | --- |
| CMV low positive control | Valid | - | Valid |
| CMV high positive control | Valid | - | Valid |
| CMV QS 1–4 | Valid | - | Valid |
| NTC | Valid | Valid | Valid |

# Invalid runs:

If the CMV Low Positive Control, CMV High Positive Control, CMV QS 1-4, or NTC result is determined to be Invalid, the software will provide a flag/warning message. An interpretation of the possible flag/warning messages is provided in Table 2.

Table 2: Description of flag/warning messages associated with invalid control results

| Name | Flag/Warning | CMV Result | Status | Interpretation of Result |
| --- | --- | --- | --- | --- |
| QS 1-4 | Control Sample [CMV QS (1-4)] failed rule [Minimum Fluorescence] on Test Channel. Detected Fluorescence: X. Min Fluorescence: X | Invalid | Invalid | Run is invalid: signal in the CMV Test Channel is out of specification. |
| QS 1-4 | Control Sample [CMV QS (1-4)] failed rule [CT Range] on Test Channel. Detected CT: X. Min Ct: X Max CT: X | Invalid | Invalid | Run is invalid: signal in the CMV Test Channel is out of specification. |
| QS 1-4 | Failed rule [R Value] on Test Channel. Calculated R Value: X. Min R Value: X | Invalid | Invalid | Run is invalid: signal in the CMV Test Channel is out of specification. |
| LPC | Control Sample [Low Positive Control] failed rule [Concentration Range] on Test Channel. Detected Concentration: X. Min Concentration: X Max Concentration: X | Invalid | Invalid | Run is invalid: signal in the CMV Test Channel is out of specification. |
| QS 1-4 | Control Sample [CMV QS (1-4)] failed rule [Minimum Fluorescence] on Test Channel. Detected Fluorescence: X. Min Fluorescence: X | Invalid | Invalid | Run is invalid: signal in the CMV Test Channel is out of specification. |
| HPC | Control Sample [High Positive Control] failed rule [Concentration Range] on Test Channel. Detected Concentration: X. Min Concentration: X Max Concentration: X | Invalid | Invalid | Run is invalid: signal in the CMV Test Channel is out of specification. |
| NTC | Control Sample [No Template Control] failed rule [Minimum Fluorescence] on Control Channel. Detected Fluorescence: X. Min Fluorescence: X | - | Invalid | Run is invalid: signal in the Control Channel is out of specification. |

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| NTC | Control Sample [No Template Control] failed rule [CT Range] on Control Channel. Detected CT: X. Min CT: X Max CT: X | – | Invalid | Run is invalid: double intersection in Control Channel.* |
| --- | --- | --- | --- | --- |
| NTC | Control Sample [No Template Control] failed rule [Concentration Range] on Test Channel. Detected CT value: X. Expected CT value = “Not Detected” | Invalid | Invalid | Run is invalid: signal in the Test Channel is out of specification. |

## Retesting of invalid runs:

If the run is invalid due to failure of the Low Positive Control, High Positive Control, QS 1–4, or NTC, all samples in that run must be retested using remaining purified nucleic acid.

## Instrument and Software

- **EZ1 Advanced and EZ1 Advanced XL**: The EZ1 Advanced and EZ1 Advanced XL instruments perform fully automated nucleic acid purification from up to 6 or 14 samples respectively using magnetic particles in combination with the EZ1 DSP Virus Kit. The automated steps include:

- Reading reagent and sample information with a handheld bar code scanner connected to the instrument.
- Lysis of samples.
- Binding of nucleic acids to magnetic particles.
- Washing and elution of nucleic acid.
- Generating a report file that either will be transmitted to a PC or printed on an external printer after the protocol run is finished.
- Using UV radiation for decontamination.

The user inserts a card containing protocol(s) into the instrument. After starting worktable setup using the control panel and bar code reader, the user loads samples, reagent cartridges, filter-tips in tip holders, and elution tubes onto the instrument worktable. The user then closes the instrument door and starts the protocol. The door locks automatically at the start of the protocol. The protocol provides the necessary instructions for the instrument to carry out automated nucleic acid purification.

The aspiration and dispensing of samples and reagents and the separation of magnetic particles are performed by the pipettor head. The temperature of samples is regulated by a heating system.

- **Rotor-Gene Q MDx**: The Rotor-Gene Q MDx (RGQ MDx) is a real-time PCR analyzer designed for rapid thermocycling and real-time detection of amplified DNA.

The RGQ MDx Instrument incorporates a centrifugal rotary design for thermal cycling where the tubes spin in a chamber of moving air, keeping all samples at a

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uniform temperature. Samples are heated and cooled in a low-mass-air oven according to a software controlled cycle that initiates the different phases of the PCR cycle.

The RGQ MDx Instrument is capable of supporting up to six optical channels (six excitation sources and six detection filters). Only two of these channels (for FAM and HEX fluorophores) are used with the artus CMV RGQ MDx Kit.

The artus CMV Assay Package contains a template to set the PCR run parameters, assess run validity and calculate the results. The RGQ MDx instrument software supports real-time analysis procedures. The software determines Ct values and calculates the CMV concentration. A system of Flags/Warnings is embedded within the software in order to inform the user of potential problems with the assay and to indicate non-valid test runs or non-valid samples within a valid test run (inappropriate level of DNA or Internal Control failure). No results are reported for invalid runs or for non-valid samples.

# Interpretation of Sample Results

For each sample in a valid run, the Rotor-Gene Q software indicates the status of the analysis for CMV (Invalid, Detected, or Not Detected). Status is reported as follows:

i. "Not Detected": No CMV DNA detected in Target Specific Channel.
ii. "Detected, Below LOQ (<159 IU/mL)": CMV DNA detected in Target Specific Channel, but at a level below the Limit of Quantitation (below 159 IU/mL).
iii. "Detected": CMV DNA was detected within the linear range of the assay and the quantitated value expressed in IU/mL is provided.
iv. "Detected, Above Linear Range $(>7.97 \times 10^{7} \mathrm{IU} / \mathrm{mL})$ " : CMV DNA detected in Target Specific Channel, but at a level above the linear range (greater than $7.97 \times 10^{7} \mathrm{IU} / \mathrm{mL}$ ).
v. "Invalid": The sample result is invalid and the sample should be retested.

Negative control samples that do not meet the acceptance criteria in the Internal Control channel are flagged with a warning and no results are reported.

The calculated CMV concentration (in IU/mL) is only provided for samples within the measuring interval. A description of the sample results provided by the Rotor-Gene Q software is provided in Table 3.

Table 3: Sample results determined by the Rotor-Gene Q software

| Name | Flag/warning | CMV | Status | Interpretation of Result |
| --- | --- | --- | --- | --- |
| Sample ID | - | DNA detected | Detected | CMV DNA detected within linear range. Calculated concentration provided (IU/mL). |
| Sample ID | - | DNA not detected | Not Detected | CMV DNA not detected. |

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| Name | Flag/warning | CMV | Status | Interpretation of Result |
| --- | --- | --- | --- | --- |
| Sample ID | - | DNA detected | Detected, below LOQ | CMV DNA detected below LOQ (<159 IU/mL). |
| Sample ID | - | DNA detected | Detected, above linear range | CMV DNA detected above linear range (>7.94 x107IU/mL). |
| Sample ID | IC_LEFT_CT_SHIFT | Invalid | Invalid | Not determined: Signal in the Control Channel is out of specification range. |
| Sample ID | IC_RIGHT_CT_SHIFT | Invalid | Invalid | Not determined: Signal in the Control Channel is out of specification range. |
| Sample ID | IC_FLUORESCENCE | Invalid | Invalid | Not determined: Signal in the Control Channel is out of specification range. |
| Sample ID | IC_LEFT_CT_SHIFT IC_FLUORESCENCE | Invalid | Invalid | Not determined: Signal in the Control channel is out of specification range. |
| Sample ID | IC_RIGHT_CT_SHIFT IC_FLUORESCENCE | Invalid | Invalid | Not determined: Signal in the Control channel is out of specification range. |
| Sample ID (positive sample) | INVALID_DATA | Invalid | Invalid | Not determined: CMV test channel failed, caused by double intersection.* |
| Sample ID (negative sample) | EARLY_CT | Invalid | Invalid | Not determined: CMV test channel failed, caused by threshold intersection. |
| Sample ID (IC of negative sample) | IC_INVALID_DATA | Invalid | Invalid | Not determined: Control channel failed, caused by double intersection.* |
| Sample ID | IC_FAIL IC_FLUORESCENCE | Invalid | Invalid | No result in CMV test channel; no result in control channel. |

* Amplification curve crosses the threshold twice.

# Retesting invalid samples

Samples with invalid results must be re-extracted and retested if no eluate is left. If there is remaining eluate, it is appropriate to retest. If the retest fails, then it is necessary to resample and re-extract.

# Quality Control

The CMV Low Positive Control, High Positive Control, Quantitation Standards, and NTC (H2O) are provided with the artus CMV RGQ MDx Kit and must be included in each run of the Rotor-Gene Q MDx instrument. Control results are evaluated to determine whether the run is valid. Acceptance criteria for the controls are automatically verified by the Rotor-Gene Q software. If the run is invalid, the eluates of the samples must be retested.

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It is recommended to test a CMV negative process control, a CMV high positive process control, and a CMV low positive process control in each PCR run. The process controls should be treated as samples and subjected to the same DNA isolation procedure. Previously characterized samples may be used for this purpose. Each laboratory should ensure compliance with applicable local, state, and federal regulations, as well as the laboratory's quality control procedures.

## Kit Configuration and Components

### Materials Provided:

The contents of the artus CMV RGQ MDx Kit are sufficient for 96 reactions on the Rotor-Gene Q MDx. The Rotor-Gene Q MDx rotor holds up to 72 reaction tubes (Table 4).

Table 4: artus CMV RGQ MDx Kit content.

| Number of reactions | | 96 |
| --- | --- | --- |
| Blue | CMV RG Master | 8 x 300 μl |
| Yellow | CMV Mg-Sol | 600 μl |
| Red | CMV QS 1
1 x 10^{4} copies/μl (1.19 x 10^{6} IU/mL) | 200 μl |
| Red | CMV QS 2
1 x 10^{3} copies/μl (1.19 x 10^{5} IU/mL) | 200 μl |
| Red | CMV QS 3
1 x 10^{2} copies/μl (1.19 x 10^{4} IU/mL) | 200 μl |
| Red | CMV QS 4
1 x 10^{1} copies/μl (1.19 x 10^{3} IU/mL) | 200 μl |
| Violet | CMV Low Positive Control | 200 μl |
| Black | CMV High Positive Control | 200 μl |
| Green | CMV RG IC | 2 x 1000 μl |
| White | H_{2}O | 1 mL |
| artus CMV RGQ MDx Kit Instructions for Use (Handbook) | | 1 |

### Materials Required but Not Provided:

#### For DNA purification

**Reagents**
- EZ1 DSP Virus Kit, version 4
- Water
- 70% ethanol

**Equipment**
- EZ1 Advanced instrument or EZ1 Advanced XL instrument
- EZ1 Advanced DSP Virus Card v1.0 or higher, with firmware 1.0.1 and protocol "DSP Virus version 1.0" or higher or EZ1 Advanced XL DSP Virus Card v1.0 or higher, with firmware 1.0.1 and protocol "DSP Virus version 1.0" or higher

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- Heating block for 1.5 mL Tubes (e.g., Eppendorf® Thermomixer)
- Optional: Vortexer (if frozen samples need to be mixed)

For sample tracking, one of the following is required:

- PC and TFT Monitor, 17” (or user’s PC and monitor) with EZ1 Advanced Communicator Software (software supplied with EZ1 Advanced and EZ1 Advanced XL instruments)
- Printer and accessory package for printer Consumables
- Pipets and sterile, RNase-free pipet tips
- Soft paper tissue

## For PCR

Consumables

- Pipets (adjustable)
- Sterile pipet tips with filters
- Strip Tubes and Caps, 0.1 mL, for use with 72-well rotor

Equipment

- Vortex mixer
- Laboratory timer
- Benchtop centrifuge with rotor for 2 mL reaction tubes
- Rotor-Gene Q MDx instrument with 72-well rotor
- Rotor-Gene Q Software version 2.1.0 or higher
- Rotor-Gene Q artus CMV Assay Package 1.2.7 or higher
- Cooling block (Loading Block 72 x 0.1 mL Tubes)

## VI. ALTERNATIVE PRACTICES AND PROCEDURES

Currently, one alternative in vitro nucleic acid amplification test for the quantitative measurement of cytomegalovirus (CMV) DNA in human plasma has been FDA-approved in the United States. The pp65 antigenemia assay is another alternative to measurement of CMV viral load that has been used in transplant centers. The test is a fluorescent assay based on detection of infected cells in peripheral blood. The test is comparable in sensitivity to laboratory developed CMV amplification-based assays but has largely been supplanted by CMV PCR assays due to greater reliability and technical ease of the latter assays.

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 artus CMV RGQ MDx Kit has not been marketed in the United States or any other country.

## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

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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 artus CMV RGQ MDx Kit 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.

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 solid-organ transplant patients who are undergoing anti-CMV drug therapy, the results from the artus CMV RGQ MDx Kit must be interpreted in the context of all relevant clinical and laboratory findings.

IX. SUMMARY OF PRECLINICAL STUDIES

A. Laboratory Studies

Traceability to the 1st WHO International Standard for Human Cytomegalovirus (HCMV)

Traceability of the artus CMV Quantitation Standards (QS) to the WHO International Standard (WHO Standard) NIBSC 09/162 was carried out by testing the Quantitation Standards against the WHO Standard. This was achieved through a Quality Control test to ensure that a 5,000 IU/mL concentration of the WHO Standard was accurately quantitated. No manufactured lot of Quantitation Standards was released for inclusion in the artus CMV RGQ MDx Kit unless they had successfully passed the acceptance criteria described in the test procedure. 5,000 IU/ml (3.70 log₁₀ IU/mL) was chosen as a suitable

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concentration to be employed in the Quality Control test since this concentration is near the midpoint of the artus CMV RGQ MDx Kit calibration curve (logarithmic scale). In setting the QC release test acceptance criteria for the Quantitation Standards, a total of 113 replicates of the WHO Standard diluted to 5,000 IU/mL in negative plasma were employed. The acceptance criterion for the WHO Standard sample was set using the data obtained (logarithmic scale) from these test dilutions and it corresponded to a range of ±0.30 log₁₀ IU/ml. Preparation of the WHO Standard for use in the Quality Control test of the Quantitation Standards consisted of the following:

1. The WHO Standard was diluted to 5,000 IU/ml in human plasma confirmed to be negative for CMV. At least 14 separate dilutions were prepared.
2. Each of the 14 dilutions was extracted using the EZ1 DSP Virus Kit on the EZ1 Advanced Instrument.
3. The extracted eluates were pooled and dispensed into several aliquots.

The Quality Control test of the Quantitation Standards consisted of the following:

1. Quantitation Standards and one aliquot of the extracted WHO Standard were tested using a previously released artus CMV RGQ MDx Kit.
2. Both single test results must meet the respective QC release specifications. For the WHO Standard, the quantified value must meet the defined acceptance criteria.
3. In addition, the CT values of the Quantitation Standards were assessed against the CT values obtained with a previously released lot of Quantitation Standards to ensure consistency over time.

## Correspondence between IU/mL and copies/mL

In order to establish correspondence between U/mL to copies/mL, a serial dilution of the 1ˢᵗ WHO International Standard for Cytomegalovirus (WHO IS) for NAT based assays (NIBSC code 09/162; Merlin strain) in EDTA plasma ranging from 5.00 × 10² to 5.00 × 10⁵ IU/mL was extracted with the EZ1 DSP Virus System and analyzed with the artus CMV RGQ MDx Kit. The 1ˢᵗ WHO International Standard for Cytomegalovirus [1] represents the glycoprotein B (gB) genotype 1. Six rounds of CMV DNA extraction were conducted on 3 different days. In each run, 3 replicates of each dilution level were processed (CMV WHO Standard in EDTA plasma). All samples were subjected to single extraction using 3 different EZ1 DSP Virus Kit lots. The analysis of all eluates was performed in duplicates with 3 artus CMV RGQ MDx Kit lots on 3 Rotor-Gene Q MDx instruments.

Figure 1 shows the linear regression analysis of IU/mL versus copies/mL.

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Figure 1: Linear Regression Analysis

The equation obtained was:

$\mathrm{Y}_{\mathrm{copies / mL}} = 0 + 1.2594212 \times \mathrm{X}_{\mathrm{IU / mL}}$

Which is equivalent to:

$\mathrm{X_{IU / mL} = Y_{copies / mL} / 1.2594212 = 0.794\times Y_{copies / mL}}$

Therefore, the equation for converting copies to international units is:

$\mathbf{X}_{\mathrm{IU / mL}} = \mathbf{0.794}\times \mathbf{Y}_{\mathrm{copies / mL}}$

(i.e.; 100 copies/mL = 79.4 IU/mL; 100 IU/mL = 125.94 copies/mL.)

# Characterization of the Different Genotypes used in the Studies

Throughout the different analytical studies, genotype gB 1 was represented by the $1^{\mathrm{st}}$ WHO International Standard for Human Cytomegalovirus for NAT Techniques (NIBSC code: 09/162); gB2 was represented by strain AD-169; gB3 by a strain Toledo provided by the Universitätsklinikum Ulm, Germany; and gB4 was represented by an EDTA-whole blood clinical isolate provided by the Groupe Hospitalier Pitié-Salpêtrière Paris, France.

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Limit of Detection (LOD)

The Limit of Detection (LOD) value of the artus CMV RGQ MDx Kit was determined to be 77 IU/mL (1.89 log $_{10}$ IU/mL) CMV in EDTA plasma.

# Limit of Detection using the $I^{st}$ WHO International Standard for Human Cytomegalovirus

The limit of detection (LOD) of the artus CMV RGQ MDx Kit was determined for the $1^{\text{st}}$ WHO International Standard for Cytomegalovirus and following the Clinical and Laboratory Standards Institute (CLSI) Guideline EP17-A2 [2]. The LOD is defined as the lowest amount of analyte in a sample that is detected with a $95\%$ probability, and it was determined by probit analysis. For this purpose, a dilution series consisting of 10 different dilutions levels of the $1^{\text{st}}$ WHO International Standard, starting with 892 IU/mL, in EDTA plasma was used. LOB was confirmed to be 0 IU/mL by analysis of blank samples.

Each dilution was determined in 6 replicates per run and day. All replicates of one dilution were tested in one PCR run. The test was performed with three different artus CMV RGQ MDx Kit lots and with each lot on four different days, by three different operators, on four different EZ1 Advanced XL and three different Rotor-Gene Q instruments, resulting in 72 overall data points per dilution.

A probit regression with SAS Software was performed and the LOD value was determined. The LODs for three different lots were all close to $54\mathrm{IU / mL}$ . The results for the combined data are shown in Table 5.

Table 5: Limit of detection using the $1^{\text{st}}$ WHO International Standard for Cytomegalovirus

| CMV target concentration (IU/mL) | Number of replicates tested (N) | Mean observed concentration (IU/mL) | Number of positives detected | Positive rate (%) |
| --- | --- | --- | --- | --- |
| 892 | 72 | 574 | 72 | 100 |
| 282 | 72 | 173 | 72 | 100 |
| 141 | 72 | 83 | 72 | 100 |
| 89 | 72 | 50 | 72 | 100 |
| 56.3 | 72 | 30 | 72 | 100 |
| 28.1 | 72 | 17 | 60 | 83 |
| 8.9 | 72 | 9 | 34 | 47 |
| 2.8 | 72 | 7 | 14 | 19 |
| 0.9 | 72 | 7 | 4 | 6 |

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| CMV target concentration (IU/mL) | Number of replicates tested (N) | Mean observed concentration (IU/mL) | Number of positives detected | Positive rate (%) |
| --- | --- | --- | --- | --- |
| 0.3 | 72 | 6 | 3 | 4 |

The LOD for the artus CMV RGQ MDx Kit using the 1st WHO International Standard for Human Cytomegalovirus (NIBBSC 09/162, Merlin strain, genotype 1 based on glycoprotein B gene UL 55) is determined to be 54 IU/mL.

## Limit of Detection Using Glycoprotein B (gB) Genotype 2

A dilution series consisting of 10 different dilution levels was used, starting with 794 IU/mL cultured CMV in CMV-negative EDTA plasma. The evaluation was performed with 3 different artus CMV RGQ MDx Kit lots, and testing was performed with each lot on 4 different days, by 4 different persons, on 3 different Rotor-Gene Q instruments. Each dilution level was tested in 6 replicates per lot and day.

The LOD values of the three lots were the same, and the final LOD for the artus CMV RGQ MDx Kit for the combined data is 77 IU/mL (using probit analysis) (Table 6).

Table 6: Limit of Detection using glycoprotein B (gB) genotype 2

| CMV target concentration (IU/mL) | Number of replicates tested (N) | Mean observed concentration (IU/mL) | Number of positives detected | Positive rate (%) |
| --- | --- | --- | --- | --- |
| 794 | 72 | 603 | 72 | 100 |
| 251 | 72 | 151 | 72 | 100 |
| 125 | 72 | 90 | 72 | 100 |
| 79 | 72 | 46 | 72 | 100 |
| 50 | 72 | 32 | 69 | 95.8 |
| 25.1 | 71 | 18 | 45 | 63.4 |
| 7.92 | 72 | 11 | 27 | 37.5 |
| 2.50 | 72 | 10 | 14 | 19.4 |
| 0.79 | 72 | 10 | 5 | 6.9 |
| 0.25 | 71 | 8 | 1 | 1.4 |

## Limit of Detection Using Glycoprotein B (gB) Genotypes 3 and 4

The claimed LOD value obtained for genotype gB2 (77 IU/mL) was verified for both CMV gB3 and gB4 genotypes following the CLSI Guideline EP17-A2 [2].

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A set of samples was prepared for each CMV gB genotype by diluting cultured virus (gB3) or clinical specimen (gB4) at the claimed LOD value concentration in 2 different EDTA plasma pools. The test was performed on 5 different days. For each CMV gB genotype, samples were analyzed in 10 replicates each day, resulting in a total number of 50 analyzed samples. Two different artus CMV RGQ MDx Kit lots, two different EZ1 Advanced (XL) instruments, and two different Rotor-Gene Q instruments were used.

The LoD value of the artus CMV RGQ MDx Kit for all lots combined was confirmed to be at least 77 IU/mL (Table 7).

Table 7: Limit of Detection using glycoprotein B (gB) genotype 3 and 4
| CMV gB genotype | CMV target concentration (IU/mL) | Number of replicates tested (N) | Number of positives detected | Positive rate (%) |
| --- | --- | --- | --- | --- |
| gB3 | 77 | 50 | 49 | 98 |
| gB4 | 77 | 50 | 47 | 94 |

Conclusion: The data were consistent with the claimed Limit of Detection (LOD) value of the artus CMV RGQ MDx Kit of 77 IU/mL (1.89 log₁₀ IU/mL) CMV in EDTA plasma.

## Limit of Blank Confirmation and Performance of Negative Samples

The limit of blank (LOB) is defined as the highest measurement result that is likely to be observed for a blank sample. In the case of the artus CMV RGQ MDx Kit, an appropriate parameter to analyze for the LOB is the end-point fluorescence intensity in the Test Channel. The fluorescence levels of negative samples should remain below a given threshold value (0.05) to generate a result "CMV DNA not detected".

The performance of negative samples determines the probability of potential false positive results. A total of 100 characterized CMV-negative EDTA plasma samples from individual donors were analyzed using two different EZ1 DSP Virus Kit manufacturing lots and two different Rotor-Gene Q instruments, over a total of four runs.

The fluorescence values at cycle 45 were measured for all samples, and the percentage of samples with fluorescence less than 0.05 was calculated. A 99% (99/100) of samples showed no result in the CMV Test Channel and fluorescence intensities below the given threshold (0.05) (95% CI: 94.6% -99.8%).

In addition, Ct values generated for each sample were analyzed. The results showed that 99 of the 100 negative samples tested yielded a negative detection result using the artus CMV RGQ MDx Kit.

To assess the missed sample, the residual eluate obtained from the discordant sample was re-tested in duplicate, obtaining negative results in both cases. Re-amplification of the sample was tested again for each duplicate, obtaining again negative results for both

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cases. These results suggest that the sample may have been contaminated with CMV during the initial PCR reaction set up. Note that the same initial PCR reaction and set of samples was used to assess performance with DNA negative samples and the same result was obtained.

# Linear Range and Limit of Quantitation

# Linear Range Using Glycoprotein B (gB) Genotype 2

The linear range of the artus CMV RGQ MDx Kit was determined following recommendations of the CLSI Guideline EP06-A [3].

A dilution series of cultured CMV ranging from $2.08\log_{10}\mathrm{IU / mL}$ to $7.90\log_{10}\mathrm{IU / mL}$ $(1.19\times 10^{2}$ to $7.94\times 10^{7}\mathrm{IU / mL})$ in EDTA plasma was prepared to determine the linear range. Samples were analyzed using the artus CMV RGQ MDx Kit with a total of one EZ1 DSP Virus Kit lot and 3 artus CMV RGQ MDx Kit lots. Each dilution level was tested in 6 replicates.

The results were analyzed to assess if the replicates at each concentration demonstrated a predefined SD (in $\log_{10}\mathrm{IU / mL}$ ) of $\leq 0.20$ ; otherwise the concentrations were considered to be outside the linear range. In addition, an assessment was performed using the polynomial evaluation of linearity to determine if the dataset was linear. None of the nonlinear coefficients in quadratic and cubic regressions were significant.

Figure 2: Linear range of the artus CMV RGQ MDx Kit for CMV gB2 genotype.

# Linear Range Using Glycoprotein B (gB) Genotypes 1, 3, and 4

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For the determination of the linear range for the other gB genotypes, dilution series from the $1^{\mathrm{st}}$ WHO International Standard (NIBSC code 09/162, gB1), cultured virus (gB3), and a clinical specimen (gB4) were used. The concentrations analyzed for gB1 and gB3 ranged from 2.08 to $5.60\log_{10}\mathrm{IU / mL}$ $(1.19\times 10^{2}$ to $3.97\times 10^{5}\mathrm{IU / mL})$ . For gB4, a dilution series ranging from 2.08 to $4.90\log_{10}\mathrm{IU / mL}$ $(1.19\times 10^{2}$ to $7.94\times 10^{4}\mathrm{IU / mL})$ was used.

Each dilution was analyzed in 8 replicates. All replicates of one dilution were tested in one Rotor-Gene Q run. The linear range was determined using one artus CMV RGQ MDx Kit lot.

An assessment was performed using the polynomial evaluation of linearity to determine if the dataset was linear. None of the nonlinear coefficients in quadratic and cubic regressions were significant.

Linear regression analyses of the mean $\log_{10}$ observed titer vs. the nominal $\log_{10}$ titer for each of the gB genotype linearity panels is shown in Figure 3. Table 8 summarizes the results obtained for all CMV gB genotypes analyzed.

Figure 1: Linear range of the artus CMV RGQ MDx Kit for all CMV gB genotypes analyzed.

Table 8: Linear range of the artus CMV RG PCR Test for all CMV gB genotypes analyzed and maximum difference between regression equations

| gB genotype | Linear range |
| --- | --- |
| gB1 | 159 IU/mL to 3.97 x 105IU/mL(2.20 log10 IU/mL to 5.60 log10 IU/mL) |

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Table 9: CMV gB Genotypes Linearity Study — Observed Titer Summary for CMV Glycoprotein B Genotype 1. Best fitted $1^{\text{st}}$ -order line is $y = 1.0357x - 0.2949$

| Nominal concentration (log10 IU/mL) | N | Mean Observed (log10 IU/mL) | SD log10 IU/mL | Bias | Predicted 1st-order | Deviation from linearity |
| --- | --- | --- | --- | --- | --- | --- |
| 5.60 | 8 | 5.52 | 0.02 | -0.08 | 5.51 | 0.01 |
| 4.60 | 8 | 4.49 | 0.01 | -0.11 | 4.47 | 0.02 |
| 3.60 | 8 | 3.39 | 0.04 | -0.21 | 3.43 | -0.04 |
| 2.90 | 8 | 2.65 | 0.08 | -0.25 | 2.71 | -0.06 |
| 2.60 | 8 | 2.38 | 0.10 | -0.31 | 2.40 | -0.02 |
| 2.38 | 8 | 2.19 | 0.15 | -0.22 | 2.17 | 0.02 |
| 2.20 | 8 | 2.05 | 0.15 | -0.15 | 1.98 | 0.07 |
| 2.08 | 8 | 1.83 | 0.24 | -0.25 | 1.86 | -0.03 |

For gB1, the assay was linear at interval $[2.10\log_{10}\mathrm{IU / mL} - 5.60\log_{10}\mathrm{IU / mL}]$ , with deviation from linearity $\leq 0.07\log_{10}\mathrm{IU / mL}$ ; bias was $\leq 0.31\log_{10}\mathrm{IU / mL}$ .

Table 10: CMV gB Genotypes Linearity Study — Observed Titer Summary for CMV Glycoprotein B Genotype 2. Best fitted $1^{\text{st}}$ -order line is $y = 1.0467x - 0.2792$

| Nominal concentration (log10 IU/mL) | N | Mean Observed (log10 IU/mL) | SD log10 IU/mL | Bias | Predicted 1st-order | Deviation from linearity |
| --- | --- | --- | --- | --- | --- | --- |
| 7.90 | 18 | 8.02 | 0.06 | 0.12 | 7.99 | 0.03 |
| 6.90 | 18 | 6.95 | 0.05 | 0.05 | 6.94 | 0.01 |
| 5.90 | 18 | 5.86 | 0.05 | -0.04 | 5.90 | -0.04 |
| 4.90 | 18 | 4.83 | 0.05 | -0.07 | 4.85 | -0.02 |
| 3.90 | 18 | 3.79 | 0.04 | -0.11 | 3.80 | -0.01 |
| 2.90 | 18 | 2.73 | 0.11 | -0.17 | 2.76 | -0.03 |
| 2.60 | 18 | 2.46 | 0.09 | -0.14 | 2.44 | 0.02 |
| 2.38 | 18 | 2.21 | 0.17 | -0.17 | 2.21 | 0.00 |
| 2.20 | 18 | 2.02 | 0.13 | -0.18 | 2.02 | 0.00 |
| 2.08 | 18 | 1.93 | 0.13 | -0.15 | 1.89 | 0.04 |

For gB2, the assay was linear at interval $[2.10\log_{10}\mathrm{IU / mL} - 7.90\log_{10}\mathrm{IU / mL}]$ , with deviation from linearity $\leq 0.04\log_{10}\mathrm{IU / mL}$ ; bias was $\leq 0.18\log_{10}\mathrm{IU / mL}$ .

Table 11: CMV gB Genotypes Linearity Study — Observed Titer Summary for CMV Glycoprotein B Genotype 3. Best fitted $1^{\text{st}}$ -order line is $y = 1.0621x - 0.3809$

| Nominal concentration (log10 IU/mL) | N | Mean Observed (log10 IU/mL) | SD log10 IU/mL | Bias | Predicted 1st-order | Deviation from linearity |
| --- | --- | --- | --- | --- | --- | --- |
| 5.60 | 8 | 5.56 | 0.02 | -0.04 | 5.57 | -0.01 |
| 4.60 | 8 | 4.50 | 0.01 | -0.10 | 4.50 | 0.00 |
| 3.60 | 8 | 3.45 | 0.03 | -0.15 | 3.44 | 0.01 |
| 2.90 | 8 | 2.73 | 0.06 | -0.17 | 2.70 | 0.03 |
| 2.60 | 8 | 2.37 | 0.06 | -0.22 | 2.38 | -0.01 |
| 2.38 | 8 | 2.16 | 0.18 | -0.22 | 2.15 | 0.01 |

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For gB3, the assay was linear at interval $[2.10\log_{10}\mathrm{IU / mL} - 5.60\log_{10}\mathrm{IU / mL}]$ , with deviation from linearity $\leq 0.15\log_{10}\mathrm{IU / mL}$ ; bias was $\leq 0.40\log_{10}\mathrm{IU / mL}$ .

Table 12: CMV gB Genotypes Linearity Study — Observed Titer Summary for CMV Glycoprotein B Genotype 4. Best fitted $1^{\text{st}}$ -order line is $y = 1.0956x - 0.5596$

| Nominal concentration (log10 IU/mL) | N | Mean Observed (log10 IU/mL) | SD log10 IU/mL | Bias | Predicted 1st-order | Deviation from linearity |
| --- | --- | --- | --- | --- | --- | --- |
| 4.90 | 8* | 4.80 | 0.03 | -0.10 | 4.81 | -0.01 |
| 4.60 | 8 | 4.46 | 0.04 | -0.14 | 4.48 | -0.02 |
| 3.60 | 8 | 3.43 | 0.04 | -0.17 | 3.38 | 0.05 |
| 2.90 | 8 | 2.63 | 0.09 | -0.27 | 2.62 | 0.01 |
| 2.60 | 8 | 2.29 | 0.08 | -0.31 | 2.29 | 0.00 |
| 2.38 | 8 | 2.00 | 0.17 | -0.38 | 2.05 | -0.05 |
| 2.20 | 8 | 1.86 | 0.12 | -0.34 | 1.85 | 0.01 |
| 2.08 | 8 | 1.71 | 0.14 | -0.37 | 1.79 | -0.01 |

*1 outlier value of 5.09 log $_{10}$ IU/mL was excluded

For gB4, the assay was linear at interval $[2.10\log_{10}\mathrm{IU / mL} - 4.90\log_{10}\mathrm{IU / mL}]$ , with deviation from linearity $\leq 0.05\log_{10}\mathrm{IU / mL}$ ; bias was $\leq 0.38\log_{10}\mathrm{IU / mL}$ .

Table 13 contains the linear equation obtained for each genotype and the maximum difference between the gB1 (1 $^{\text{st}}$ WHO International Standard) and the other genotypes based on the linear fit.

Table 13: Linear equations obtained for all CMV gB genotypes analyzed

| CMV gB genotype | Linear equation in gB genotype linearity study | Maximum difference between gB1 and corresponding gB genotype (log10 IU/mL) |
| --- | --- | --- |
| 1 | y = 1.0357x - 0.2949 | n.a.* |
| 2 | y = 1.0467x - 0.2792 | 0.08 |
| 3 | y = 1.0621x - 0.3809 | 0.06 |
| 4 | y = 1.0956x - 0.5596 | 0.13 |

* n.a.: not applicable.

The claimed linear range of the assay, based on the linearity results, the homology of sequences in the target region among the genotypes, the single primers/probe used, and the tight overlap in the curves, was considered to be between 159 and $7.94 \times 10^{7}$ IU/mL, or $2.20 \log_{10} \mathrm{IU} / \mathrm{mL}$ to $7.90 \log_{10} \mathrm{IU} / \mathrm{mL}$ CMV in EDTA plasma, with maximum deviation from linearity of less or equal to $0.15 \log_{10} \mathrm{IU} / \mathrm{mL}$ .

# Lower Limit of Quantitation (LOQ)

The lower limit of quantitation (LOQ) was determined by spiking the different CMV genotypes into 5 unique EDTA plasma pools at a concentration equal to the lower limit of the linear range for each genotype. Data was generated on 5 different days with 12

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replicates per genotype each day (e.g., day 1, pool 1, 12 replicates). A total of 60 data points for each genotype was obtained. Two different artus CMV RGQ MDx Kit lots were used.

The LOQ was defined as the lowest level of CMV that can be reliably detected and at which the total error is $\leq 1.0\log_{10}\mathrm{IU / mL}$ , where total error is calculated as $|\mathrm{Bias}| + 2\mathrm{SD}$ . Meeting the $|\mathrm{Bias}| + 2\mathrm{SD} \leq 1.0\log_{10}\mathrm{IU / mL}$ criterion ensures that, for samples with assay values equal to the LOQ, there is $95\%$ or greater probability that the measured value will be within $1.0\log_{10}\mathrm{IU / mL}$ of the true value. In addition, total error is such that difference between two measurements $&gt;1.0\log_{10}\mathrm{IU / mL}$ is statistically significant (a true change is detected). The standard deviation for the difference between two measurements is $\sqrt{2}\times$ SD, and meeting the $\sqrt{2}\times 2\times \mathrm{SD} \leq 1.0\log_{10}\mathrm{IU / mL}$ criterion ensures that, for samples with assay values equal to the LOQ, the difference between two measurements of more than $1.0\log 10\mathrm{IU / mL}$ is statistically significant.

Table 14 shows both criteria for the total analytical error (TAE = |Bias| + 2 x SD ≤1.0 log10 IU/mL and $\sqrt{2}$ x 2 x SD ≤1.0 log10 IU/mL) for the log10 IU/mL (Tables 14 and 15) following the recommendations of the CLSI Guideline EP17-A2 [2].

The LoQs for two individual lots were similar. The LoQ results for the combined lots are presented in Table 14.

Table 14: Lower limit of quantitation for the different CMV gB genotypes (log ${}_{10}\mathrm{{IU}}/\mathrm{{mL}}$ )

| CMV gB genotype | Nominal Concentration (IU/mL) | Nominal Concentration (log10 IU/mL) | Average Measured Concentration (log10 IU/mL) | Bias (log10 IU/mL) | SD (log10 IU/mL) | TAE = |Bias| + 2 x SD (log10 IU/mL) | √2 x 2 x SD (log10 IU/mL) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| gB1 | 159 | 2.20 | 1.87 | -0.33 | 0.25 | 0.84 | 0.72 |
| gB2 | 159 | 2.20 | 2.03 | -0.17 | 0.23 | 0.62 | 0.65 |
| gB3 | 159 | 2.20 | 1.79 | -0.41 | 0.26 | 0.93 | 0.73 |
| gB4 | 159 | 2.20 | 1.89 | -0.31 | 0.18 | 0.66 | 0.50 |

The claimed LOQ for the artus CMV RGQ MDx Kit considering all CMV gB genotypes is 159 IU/mL (2.20 log $_{10}$ IU/mL), with the geometric mean of the observed titer value of 107 IU/mL (2.03 log $_{10}$ IU/mL).

Table 15 shows the linear range for the different gB genotypes according to the results obtained for the LOQ.

Table 15: LOQ obtained for all CMV gB genotypes analyzed

| gB genotype | LOQ |
| --- | --- |
| gB1 | 159 IU/mL to 3.97 x 105IU/mL(2.20 log10 IU/mL to 5.60 log10 IU/mL) |
| gB2 | 159 IU/mL to 7.94 x 107IU/mL(2.20 log10 IU/mL to 7.90 log10 IU/mL) |
| gB3 | 159 IU/mL to 3.97 x 105IU/mL(2.20 log10 IU/mL to 5.60 log10 IU/mL) |

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# Analytical Specificity (Cross-reactivity)

The analytical specificity of the artus CMV RGQ MDx Kit was evaluated by testing the cross-reactivity of a panel of different pathogens consisting of 21 viruses, 3 fungi, and 1 protozoan parasite. The pathogens were tested at the highest concentration available. Samples were prepared by diluting the organisms or DNA/RNA either in CMV negative EDTA plasma or in CMV-spiked EDTA plasma at 2 concentrations (near the LOD value, and within the linear range). Each sample was extracted and tested in four replicates. There were no false-positive or invalid results among the 25 pathogens tested (Table 16).

Table 16: Analytical Specificity

| Pathogen | Concentration* | CMV |
| --- | --- | --- |
| Viruses | | |
| Adenovirus type 2 | 1.26 x 109TCID50/mL | - |
| Adenovirus type 4 | 4.77 x 105TCID50/mL | - |
| Adenovirus type 5 | 2.75 x 1012TCID50/mL | - |
| BK polyomavirus deposited as BK virus | 1.41 x 104TCID50/mL | - |
| EBV B95-8 strain (type 1) purified virus | 1.50 x 108copies/mL | - |
| Enterovirus type 71 | 3.62 x 104TCID50/mL | - |
| Hepatitis A virus RNA NAT assays | 5.00 x 103IU/mL | - |
| Hepatitis B virus DNA | 5.00 x 104IU/mL | - |
| Hepatitis C virus (HCV) RNA | 7.75 x 103IU/mL | - |
| HSV-1 MacIntyre strain purified virus | 3.30 x 105TCID50/mL | - |
| Herpes simplex virus type 2 (HSV-2) | 6.15 x 106TCID50/mL | - |
| Human herpesvirus 3 deposited as varicella-zoster | 1.41 x 104TCID50/mL | - |
| HHV-6A GS strain purified viral lysate | 2.50 x 109VP/mL | - |
| Human herpesvirus 6B strain Z-29 | 1.41 x 102TCID50/mL | - |
| HHV-7 H7-4 strain quantitated DNA control | 6.00 x 105copies/mL | - |
| KSHV/HHV-8 KS-1 strain quantitated viral DNA | 6.00 x 105copies/mL | - |
| HIV-1 RNA, 2nd International Standard | 1.82 x 104IU/mL | - |
| HTLV-I MT-2 strain purified virus | 2.30 x 107VP/mL | - |
| Human T lymphotropic virus type II (HTLV-II) | 3.25 x 107VP/mL | - |
| Human T lymphotropic virus type III (HTLV-III) | 3.25 x 107VP/mL | - |
| Human T lymphotropic virus type IV (HTLV-IV) | 3.25 x 107VP/mL | - |

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| Pathogen | Concentration* | CMV |
| --- | --- | --- |
| Parvo B19 DNA | 5.00 x 104IU/mL | - |
| WNV | 5.45 x 108copies/mL | - |
| Fungi | | |
| Aspergillus fumigatus Z014 | 1.09 x 107CFU/mL | - |
| Candida albicans Z006 | 1.05 x 107CFU/mL | - |
| Pneumocystis jirovecii | 4.15 x 103copies/mL | - |
| Protozoan Parasite | | |
| Plasmodium falciparum | 5.00 x 107IU/mL | - |

* TCID50: Tissue culture infective dose 50%; VP: Viral particles; CFU: Colony forming units; IU: International Unit.

All negative CMV EDTA plasma samples tested negative. Moreover, all samples with a CMV concentration near the LOD value were detected positive in the Test Channel in the presence of the tested organisms. Samples with a nominal concentration around the middle of the linear range (3.08 log $_{10}$ IU/mL, 1.19 x 10 $^{3}$ IU/mL CMV) were quantified within $\pm 0.25$ log $_{10}$ IU/mL.

The results showed no cross-reactivity of the respective pathogens with the specific CMV detection in terms of sensitivity and quantitation.

# Interference - Endogenous Interfering Substances

Potentially interfering endogenous substances that may be found in patient specimens were spiked into CMV-negative EDTA plasma in the presence of different concentrations of CMV (3 x LOD, and 1,191 IU/mL). Samples were then tested using the artus CMV RGQ MDx Kit. Samples containing potentially interfering substances were compared to control EDTA plasma samples containing no spiked interfering substance. Each CMV concentration level for each interfering substance was tested in four replicates.

Qualitatively, samples were considered positive if a $\mathrm{CT}$ value was detected in the target specific channel. Samples were considered negative if no $\mathrm{CT}$ value was detected in the target specific channel and the sample validity criteria for the internal control were met.

The samples were also analyzed for potential inhibition by assessing the validity criteria of the Internal Control for negative specimens which is an allowed $\mathrm{CT}$ shift of -1 up to +4 in comparison to the No Template Control ( $\Delta \mathrm{CT}$ ).

For all tested samples, the IU/mL values were transformed into $\log_{10}$ IU/mL. On the basis of the logarithmic values, the average for the control (CMV in EDTA-plasma without interferent) was calculated. Subsequently, the difference between a single value and the average control value was calculated for each sample to evaluate whether all samples were quantified within the pre-specified acceptance criteria (based on International

{24}

Consensus Guidelines on the Management of Cytomegalovirus in Solid Organ Transplantation [7]).

The test concentrations for each interfering substance (Table 17) were selected based on available literature references and guidance provided by the CLSI Guideline EP07-A2 [3].

Table 17: Endogenous Interfering Substances

| Potential interfering substance | Concentration |
| --- | --- |
| Bilirubin (conjugated) | 30.3 mg/dL |
| Bilirubin (unconjugated) | 20.3 mg/dL |
| Hemoglobin | 2 g/dL |
| Human genomic DNA | 10 μg/dL |
| Total protein (albumin) | 11 g/dL |
| Triglyceride (intralipid) | 1.1 g/dL |

All tested interfering substance concentrations showed no influence on the performance of the artus CMV RGQ MDx Kit with regards to analytical specificity, sensitivity, and quantitation.

## Interference - Exogenous Interfering Substances

Potentially interfering exogenous substances were spiked into EDTA plasma in the absence or presence of different concentrations of CMV. Samples were then tested with the artus CMV RGQ MDx Kit. Samples containing potentially interfering substances were compared to control EDTA plasma samples containing no spiked interfering substance. Each concentration level for each interfering substance was tested in four replicates.

Qualitatively, samples were considered positive if a Ct value was detected in the target specific channel. Samples were considered negative if no Ct value was detected in the target specific channel and the sample validity criteria for the internal control was met. The samples were also analyzed for potential inhibition by assessing the validity criteria of the Internal Control for negative specimens which is an allowed Ct shift of -1 up to +4 in comparison to the No Template Control (ΔCt).

For all tested samples the copies/mL values were transformed into $\log_{10}$ copies/mL. On the basis of the logarithmic values, the average for the control (CMV in EDTA-plasma without interferent) was calculated. Subsequently, the difference between a single value and the average control value was calculated for each sample to evaluate whether all samples were quantified within the specified acceptance criteria.

The test concentrations for each interfering substance were selected based on available literature references and guidance provided by the CLSI Guideline EP07-A2 [3]. The potentially interfering substances and the test concentrations are presented in Table 18.

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Table 28: Exogenous Interfering Substances

| Potential interfering substance | Concentration |
| --- | --- |
| Amoxicillin | 125 mg/liter |
| Azathioprine-sodium | 4 mg/liter |
| Cefotaxim | 1 g/liter |
| Cidofovir | 81 mg/liter |
| Clavulanic acid | 25 mg/liter |
| Cyclosporine | 1.125 g/liter |
| Di-sodium EDTA | 1.5 mg/liter |
| Fluconazole | 1 mg/liter |
| Foscarnet (phosphonoformic acid trisodium hexahydrate) | 700 mg/liter |
| Ganciclovir | 32 mg/liter |
| Heparin-sodium | 3000 U/liter |
| Mycophenolate sodium | 80 mg/liter |
| Piperacillin | 1 g/liter |
| Prednisolone-21-hydrogensuccinate, sodium salt | 4 mg/liter |
| Prednisone | 0.5 mg/liter |
| Rapamycin | 100 mg/liter |
| Sulfamethoxazole | 200 mg/liter |
| Tazobactam | 125 mg/liter |
| Ticarcillin | 1 g/liter |
| Trimethoprim | 5.2 mg/liter |
| Valganciclovir hydrochloride | 22 mg/liter |
| Vancomycin | 125 mg/liter |

All tested interfering substance concentrations showed no influence on the performance of the artus CMV RGQ MDx Kit at the CMV concentrations evaluated with regards to analytical specificity, sensitivity, and quantitation.

## Carryover/Cross-Contamination

The artus CMV RGQ MDx Kit showed no evidence of carryover or cross-contamination when 30 high positive CMV samples with 2.38 x 10⁶ IU/mL were extracted and tested by alternating positive samples with 30 CMV-negative samples in five extraction runs and one PCR run. The CMV tested concentration represents the highest viral load observed

26

{26}

within a diagnostic evaluation study analyzing 203 retrospectively and prospectively collected patient specimens.

All CMV negative samples were analyzed regarding the presence of a Ct value in the target specific channel to check for a possible carryover event. The samples were also analyzed regarding the validity criteria for the Internal Control for negative specimens (Ct shift of -1 up to +4 in comparison to the No Template Control). On the basis of these results the percentage of valid negative results for all negative samples tested was calculated.

No Ct was observed in the target specific channel for any negative sample. Since the Internal Control of all samples showed a Ct value within the predefined acceptance range, 100% of the negative samples were considered to be truly negative. No cross-contamination occurred throughout the test system, including nucleic acid purification and real-time PCR detection.

## Precision

The precision of the artus CMV RGQ MDx Kit was determined following the recommendations of the CLSI Guideline EP05-A2 [4] by testing a 4-member panel (a negative sample, a sample with a concentration near the LOD, and 2 concentrations in the linear range of the assay; all samples were in EDTA plasma). Each panel member was evaluated for 20 days, with two runs per day, and two replicates per run. A total of two different EZ1 Advanced and two different EZ1 Advanced XL instruments, as well as three different Rotor-Gene Q instruments were used for the testing. Three different EZ1 DSP Virus Kit lots and three different artus CMV RGQ MDx Kit lots were used for the study. A total of three different operators performed the test. The results are summarized in Table 19 and Table 20.

Table 19: Precision of the artus CMV RGQ MDx Kit (in log₁₀ IU/mL)

| Nominal Values IU/mL, (log₁₀ IU/mL) | Average Observed CMV DNA Titer (log₁₀ IU/mL) | N of tests | Within-Run SD | Between-Run SD | Between-EZ1 Advanced Instrument SD* | Between-EZ1 DSP Virus Kit Lot SD* | Between-RGQ Instrument SD* | Between-artus CMV RGQ MDx Kit Lot SD* | Between-Operator SD* | Between-Day SD* | Total SD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 230 (2.362) | 2.110 | 80 | 0.136 | 0.052 | 0.000 | 0.000 | 0.000 | 0.042 | 0.000 | 0.043 | 0.158 |
| 1,191 (3.076) | 2.901 | 80 | 0.068 | 0.052 | 0.003 | 0.009 | 0.000 | 0.034 | 0.019 | 0.000 | 0.095 |
| 79,400 (4.900) | 4.764 | 80 | 0.025 | 0.019 | 0.013 | 0.050 | 0.000 | 0.000 | 0.018 | 0.004 | 0.063 |
| Negative | | 80 | 100% (80/80) “Not Detected” Results | | | | | | | | |

* Estimates of some components of variance have large uncertainty due to only 80 measurements.

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Table 20: Precision of the artus CMV RGQ MDx Kit (in IU/mL)

| Nominal Values (IU/mL) | Geometric Mean CMV DNA Titer (IU/mL) | N of tests | Within-Run %CV | Between-Run % | Between-EZ1 Advanced Instrument %CV* | Between-EZ1 DSP Virus Kit Lot %CV* | Between-RGQ Instrument %CV* | Between-artus CMV RGQ MDx Kit Lot %CV* | Between-Operator %CV* | Between-Day %CV* | Total %CV |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 230 | 129 | 80 | 32.1% | 12.0% | 0% | 0% | 0% | 9.7% | 0% | 9.9% | 37.6% |
| 1,191 | 796 | 80 | 15.8% | 12.0% | 0.7% | 2.1% | 0% | 7.8% | 4.4% | 0% | 22.1% |
| 79,400 | 58,076 | 80 | 5.8% | 4.4% | 3.0% | 11.6% | 0% | 0% | 4.2% | 0.9% | 14.6% |
| Negative | | 80 | 100% (80/80) “Not Detected” Results | | | | | | | | |

* Estimates of some components of variance have large uncertainty due to only 80 measurements.

# Specimen Stability

The stability of CMV DNA in EDTA Whole Blood, in EDTA Plasma, and after extraction under various storage conditions when using the artus CMV RGQ MDx Kit was evaluated using CMV from cultured virus stocks.

The stability testing was conducted in four separate parts:

- Stability of CMV in EDTA Whole Blood
- Stability of CMV in EDTA Plasma
- Stability of purified CMV DNA
- Stability of CMV covering the complete storage process.

A contrived sample stability study was performed for all parts, in which CMV whole virus was added to either CMV-negative whole blood or plasma samples from five randomly selected individuals pre-screened to ensure they did not contain endogenous CMV. Samples were tested with the artus CMV RGQ MDx Kit after storage at defined conditions. Samples subjected to the various storage conditions were compared to a baseline value $(\mathrm{T_0})$ obtained at the start of the incubation period. Whole blood or plasma samples not spiked with CMV DNA were also subjected to the various storage conditions and served as a negative control.

Negative samples should show no detectable signal in the target specific channel. Samples prepared near the LOD (3x LOD) should be detected positive throughout the course of the study. The acceptance criteria for the sample with 3 x LOD and the sample with concentration $3.08\log_{10}\mathrm{IU / mL}$ (1,191 IU/mL), within the linear range, were based on information derived from the International Consensus Guidelines on the Management of Cytomegalovirus in Solid Organ Transplantation (Kotton et al, Transplantation 2010; 89: 779-795) [7].

The contrived sample specimen stability is defined in Table 21 for the different specimen types.

Table 21: Contrived Sample Specimen Stability

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| Specimen Type | Storage Temperature | Duration |
| --- | --- | --- |
| EDTA Whole Blood | 20-25°C | 48 hours |
| | 2-8°C | 72 hours |
| EDTA Plasma | 20-25°C | 48 hours |
| | 2-8°C | 120 hours |
| | -15 to -30°C | 12 months |
| Eluted DNA | 2-8°C | 120 hours |
| | -15 to -30°C | 6 months |

In addition to the specimen stability study using CMV from viral culture, a limited specimen stability study using actual clinical specimens was started to demonstrate the stability of CMV DNA in EDTA Whole Blood, in EDTA Plasma, and after extraction under various storage conditions when using the artus CMV RGQ MDx Kit. The results generated to date are presented here.

Whole blood from a clinical sample containing CMV DNA was added to CMV negative whole blood from 5 randomly selected donors pre-screened to ensure they did not contain endogenous CMV. Samples subjected to various storage conditions were compared to a baseline value $(\mathrm{T_0})$ obtained at the start of the incubation period. The testing schedules for each storage condition were extended beyond the desired storage claim so that stability was supported by test data and not based on an extrapolation. Whole blood not spiked with a clinical sample containing CMV DNA was also subjected to the various storage conditions and served as a negative control.

Whole blood samples containing 3x LOD and $3.08\log_{10}\mathrm{IU / mL}$ (1,191 IU/mL) of CMV DNA were maintained at 20 to $25^{\circ}\mathrm{C}$ for 26 hours. EDTA plasma was then prepared from the whole blood and the resulting plasma maintained at -15 to $-30^{\circ}\mathrm{C}$ for a total of 13 months or 20 to $25^{\circ}\mathrm{C}$ for 26 hours. After the plasma was stored at -15 to $-30^{\circ}\mathrm{C}$ for 13 months, nucleic acid was extracted from EDTA plasma samples using the EZ1 DSP Virus System. The resulting eluates were maintained at -15 to $-30^{\circ}\mathrm{C}$ for 7 months or at 2 to $8^{\circ}\mathrm{C}$ for 144 hours (6 days). After designated storage conditions, the samples were tested with the artus CMV RGQ MDx Kit. Samples stored at -15 to $-30^{\circ}\mathrm{C}$ included two freeze thaw cycles one month apart during the incubation period. This is represented in Figure 4 as “ $1/1/x\mathrm{m}$ ”, where “x” is the number of months the samples were stored after the two freeze-thaw cycles. Time points that have been completed are indicated with a checkmark, and time points yet to be tested are indicated with a grey arrow. Time points $\mathrm{T}_{3}$ and $\mathrm{T}_{6}$ are tested after two months of the 13 month incubation at -15 to $-30^{\circ}\mathrm{C}$ . The last of the samples are scheduled to be tested in January 2015.

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Figure 4: Specimen Stability Plan

| | Donor #2 | Negative | | | | 3x LOD | | 1,191 IU/mL | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Storage Condition | Sample ID | Target Specific Channel CT | Internal Control Channel CT | Internal Control Channel ΔCT | Sample ID | Result | Sample ID | CMV IU/mL | CMV log10 IU/mL | Δlog10 IU/mL compared to baseline |
| T0 | Baseline (0 hr) | 0240004 | 0.00 | 26.26 | 1.07 | 0240025 | Detected | 0240046 | 1,232 | 3.09 | 3.04 (Avg)** |
| | | 0240005 | 0.00 | 26.48 | 1.29 | 0240026 | Detected | 0240047 | 1,011 | 3.01 | |
| | | 0240005 | 0.00 | 26.32 | 1.13 | 0240027 | Detected | 0240048 | 1,052 | 3.02 | |
| T1 | Whole Blood RT/26 hr* | 0240064 | 0.00 | 27.50 | 2.01 | 0240069 | Detected | 0240074 | 823 | 2.92 | -0.12 |
| T2 | Whole Blood RT/26 hr + Plasma RT/26 hr | 0240079 | 0.00 | 28.03 | 2.36 | 0240084 | Detected | 0240089 | 504 | 2.70 | -0.34 |
| T3 | Whole Blood RT/26 hr + Plasma - 20°C*/2 mo | 0240094 | 0.00 | 27.32 | 1.86 | 0240099 | Detected | 0240104 | 600 | 2.78 | -0.26 |
| | | | | | | | | | | | |
| | Donor #3 | Negative | | | | 3x LOD | | 1,191 IU/mL | | | |
| | Storage Condition | Sample ID | Target Specific Channel CT | Internal Control Channel CT | Internal Control Channel ΔCT | Sample ID | Result | Sample ID | CMV IU/mL | CMV log10 IU/mL | Δlog10 IU/mL compared to baseline |

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| T0 | Baseline (0 hr) | 0240007
0240008
0240009 | 0.00
0.00
0.00 | 25.92
25.93
25.98 | 0.73
0.74
0.79 | 0240028
0240029
0240030 | Detected
Detected
Detected | 0240049
0240050
0240051 | 1,404
1,550
1,864 | 3.15
3.19
3.27 | 3.20 (Avg) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| T1 | Whole Blood RT/26 hr | 0240065 | 0.00 | 26.51 | 1.02 | 0240070 | Detected | 0240075 | 1,279 | 3.11 | -0.10 |
| T2 | Whole Blood RT/26 hr
+ Plasma RT/26 hr | 0240080 | 0.00 | 26.87 | 1.20 | 0240085 | Detected | 0240090 | 1,253 | 3.10 | -0.10 |
| T3 | Whole Blood RT/26 hr
+ Plasma - 20°C/2 mo | 0240095 | 0.00 | 26.52 | 1.06 | 0240100 | Detected | 0240105 | 1,181 | 3.07 | -0.13 |
| | | | | | | | | | | | |
| | Donor #4 | Negative | | | | 3x LOD | | 1,191 IU/mL | | | |
| | Storage Condition | Sample ID | Target Specific Channel | Internal Control Channel CT | Internal Control Channel ΔCT | Sample ID | Result | Sample ID | CMV IU/mL | CMV log10 IU/mL | Δlog10 IU/mL compared to baseline |
| T0 | Baseline (0 hr) | 024001
0
024001
1 | 0.00
0.00
0.00 | 26.28
26.43
26.37 | 1.09
1.24
1.18 | 0240031
0240032
0240033 | Detected
Detected
Detected | 0240052
0240053
0240054 | 1,067
1,038
1,349 | 3.03
3.02
3.13 | 3.06 (Avg) |
| T1 | Whole Blood RT/26 hr | 024006
6 | 0.00 | 27.57 | 2.08 | 0240071 | Detected | 0240076 | 1,850 | 2.93 | -0.13 |
| T2 | Whole Blood RT/26 hr
+ Plasma RT/26 hr | 024008
1 | 0.00 | 27.85 | 2.18 | 0240086 | Detected | 0240091 | 590 | 2.77 | -0.29 |
| T3 | Whole Blood RT/26 hr
+ Plasma - 20°C/2 mo | 024009
6 | 0.00 | 27.28 | 1.82 | 0240101 | Detected | 0240106 | 687 | 2.84 | -0.22 |
| | | | | | | | | | | | |
| | Donor #5 | Negative | | | | 3x LOD | | 1,191 IU/mL | | | |
| | Storage Condition | Sample ID | Target Specific Channel | Internal Control Channel CT | Internal Control Channel ΔCT | Sample ID | Result | Sample ID | CMV IU/mL | CMV log10 IU/mL | Δlog10 IU/mL compared to baseline |
| T0 | Baseline (0 hr) | 024001
3
024001
4 | 0.00
0.00
0.00 | 25.96
25.97
25.91 | 0.77
0.78
0.72 | 0240034
0240035
0240036 | Detected
Detected
Detected | 0240055
0240056
0240057 | 1,026
1,173
924 | 3.01
3.07
2.97 | 3.02 (Avg) |
| T1 | Whole Blood RT/26 hr | 024006
7 | 0.00 | 26.24 | 0.75 | 0240072 | Detected | 0240077 | 977 | 2.99 | -0.03 |
| T2 | Whole Blood RT/26 hr
+ Plasma RT/26 hr | 024008
2 | 0.00 | 26.59 | 0.92 | 0240087 | Detected | 0240092 | 619 | 2.79 | -0.22 |
| T3 | Whole Blood RT/26 hr
+ Plasma - 20°C/2 mo | 024009
7 | 0.00 | 26.08 | 0.62 | 0240102 | Detected | 0240107 | 530 | 2.72 | -0.29 |

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| | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Donor #6 | Negative | | | | 3x LOD | | 1,191 IU/mL | | | |
| | Storage Condition | Sample ID | Target Specific Channel CT | Internal Control Channel CT | Internal Control Channel ΔCT | Sample ID | Result | Sample ID | CMV IU/mL | CMV log10 IU/mL | Δlog10 IU/mL compared to baseline |
| T0 | Baseline (0 hr) | 024001 | 0.00 | 26.10 | 0.91 | 0240037 | Detected | 0240058 | 2,095 | 3.32 | 3.28 (Avg) |
| | | 6 | 0.00 | 26.85 | 1.66 | 0240038 | Detected | 0240059 | 1,970 | 3.29 | |
| | | 024001 | 0.00 | 26.73 | 1.54 | 0240039 | Detected | 0240060 | 1,671 | 3.22 | |
| T1 | Whole Blood RT/26 hr | 024006 | 0.00 | 26.86 | 1.37 | 0240073 | Detected | 0240078 | 1,081 | 3.03 | -0.25 |
| T2 | Whole Blood RT/26 hr + Plasma RT/26 hr | 024008 | 0.00 | 27.22 | 1.55 | 0240088 | Detected | 0240093 | 1,191 | 3.08 | -0.20 |
| T3 | Whole Blood RT/26 hr + Plasma - 20°C/2 mo | 024009 | 0.00 | 26.90 | 1.44 | 0240103 | Detected | 0240108 | 785 | 2.90 | -0.38 |

The data for all samples tested to date demonstrated the following:

- Negative Samples: No detectable signal observed in the target specific channel.
- 3x LOD: All samples were detected in the target specific channel.
- The 1,191 IU/mL sample values remained between $\pm 0.38$ log10 IU/mL of the baseline control average at time (T0).

The data collected supported a clinical specimen stability of whole blood at room temperature for 24 hr, and plasma at room temperature for 24 hr and at -20C for two months.

## Kit Stability

The stability of the artus CMV RGQ MDx Kit and kit reagents under possible usage conditions was determined. Five different aspects of kit stability were examined:

- Closed Bottle Stability: Demonstrates stability of the kit under intended storage conditions.
- Component In Use Stability: Demonstrates stability of reagents under simulated usage conditions.
- Reaction Mix In Use Stability: Demonstrates stability of prepared Reaction Mix under simulated usage conditions.
- Open Bottle Stability: Demonstrates stability of reagents that have been opened and returned to storage conditions.
- Transport Simulation Stability: Demonstrates stability of reagents that have been handled under simulated shipping conditions.

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For each part, reagents were stored under prescribed conditions, and then used to perform the artus CMV RGQ MDx Kit assay. To demonstrate stability, the assay acceptance criteria must have been met, as well as the criteria established for a panel of samples.

The samples used in the stability testing include kit controls, derivatives of kit controls, and samples intended to simulate clinical specimens at two different concentrations (Table 22). For the purposes of this study, the medical decision points were defined as those within the linear range of the assay and near the limit of detection. For the artus CMV RG PCR kits to be considered stable, these test samples should produce results that fall within predefined acceptance ranges throughout the course of the study. These ranges were the same at both baseline (i.e., time zero) and each subsequent time point.

For the assay run controls, functional testing at each time point should yield results that fall within the pre-defined acceptance criteria necessary to validate the run. The acceptance criteria for these run controls were established during the development of the assay, and are applicable both at baseline and at each subsequent time point.

The QS4 1:10 dilution sample was prepared to resemble a CMV DNA concentration near the limit of detection. A stable product should detect this sensitivity-challenging sample throughout its shelf life. The negative, and the 397 and 397,000 IU/mL samples reflect clinical samples, they consist of CMV particles spiked into negative EDTA-plasma. The 397 IU/mL sample (near LoD), should produce positive results throughout the course of the stability study. The acceptance criteria for the 397,000 IU/mL sample were based on an internal evaluation which accounted for the viral load variability of this particular sample under non-stressed test conditions. The viral load measurement of this sample should fall within this acceptable range, both at baseline and at each subsequent time point (Table 22).

Table 22: Samples Included in Kit Stability Testing

| Sample | Acceptance Criteria | Description |
| --- | --- | --- |
| NTC | Not detected | Assay Acceptance Criteria |
| QS4 | CT: 27-34 | Assay Acceptance Criteria |
| QS3 | CT: 24-30 | Assay Acceptance Criteria |
| QS2 | CT: 21-27 | Assay Acceptance Criteria |
| QS1 | CT: 18-24 | Assay Acceptance Criteria |
| Negative Sample | Not detected | CMV negative plasma sample |
| 397 copies/mL Sample | Detected | CMV Virus spiked into CMV- negative plasma near LOD |
| QS 4 (1:10)- 4 replicates | Detected | CMV DNA sample near LOD (QS4 diluted 1:10) |
| CMV RG LPC | 250-1,418 IU/mL | Assay Acceptance Criteria; CMV DNA sample in linear range |
| CMV RG HPC | 13,353-42,400 IU/mL | Assay Acceptance Criteria; CMVDNA sample in linear range |

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The kit stability of the artus CMV RGQ MDx Kit is defined in Table 23.

Table 23: Kit Stability

| Stability Type | Storage Condition | Duration |
| --- | --- | --- |
| Closed Bottle Stability | -15°C to -30°C | 24 months |
| Component In Use Stability | 2°C to 8°C | 5hr |
| | Room Temperature | 2 hr |
| Master Mix In Use Stability | 2°C to 8°C | 5 hr |
| | Room Temperature | 2 hr |
| Open Bottle Stability | -15°C to -30°C after closed bottle | 24 months |
| Transport Simulation Stability | -15°C to -30°C or dry ice | 24 months |

# Reproducibility Study

The reproducibility of the artus CMV RGQ MDx Kit was evaluated using 3 sites. A 10 member panel with 5 simulated specimens (2 of each in the panel) including negatives, high negatives, low positives, moderate positives and high positives was provided for testing. The 10 member panel was tested in duplicate by two different technologists each day for 6 days at each site with 3 reagent kit lots. A total of 144 measurements was analyzed for each pair of simulated specimens.

The percentage of variance due to each component and SD of the $\log_{10}$ transformed CMV DNA concentration were calculated. The detectable difference in viral load between two test results for each expected $\log_{10}$ CMV DNA concentration was estimated by using the total variance and was calculated as the antilog of the $95\%$…