Xpert® HCV Viral Load

GXHCV-VL-CE-10

In Vitro Diagnostic Medical Device 301-3019, Rev. E September 2017

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Xpert® HCV Viral Load

For In Vitro Diagnostic Use Only.

1 Proprietary Name
Xpert® HCV Viral Load
2 Common or Usual Name
HCV VL
3 Intended Use
The HCV VL assay, performed on GeneXpert® Instrument Systems, is designed for the rapid quantitation of Hepatitis C Virus
(HCV) RNA in human serum or plasma (EDTA) from HCV-infected individuals. The test utilizes automated reverse
transcriptase polymerase chain reaction (RT-PCR) using fluorescence to detect the RNA of interest for the quantitation of HCV.
The HCV VL assay quantifies HCV genotypes 1–6 over the range of 10 to 100,000,000 IU/mL. The HCV VL assay is intended
for use as an aid in the management of HCV infected patients undergoing antiviral therapy. The test measures HCV RNA levels
at baseline and during treatment and can be utilized to predict sustained and nonsustained virological responses to HCV therapy.
Results from the HCV VL assay may also be used to confirm HCV infection in anti-HCV positive individuals. In anti-HCV
positive individuals who test negative for HCV RNA, use of another HCV antibody assay may be considered for distinction
between true HCV exposure and biologic false positivity. Repeat HCV RNA testing may be indicated in cases that have had
HCV exposure in the last 6 months or have clinical evidence of HCV disease.
The assay is not intended to be used as a blood donor screening test for HCV.
4 Summary and Explanation
HCV is a member of the Flaviviridae family and has been recognized as the major causative agent of chronic liver disease,
including chronic active hepatitis, cirrhosis and hepatocellular carcinoma.1 The HCV genome is a positive-sense RNA molecule
of approximately 9500 nucleotides.1 HCV is usually transmitted through percutaneous exposure to infected blood, primarily by
intravenous drug use and receipt of unscreened donated blood products. Less frequently, HCV has been shown to be transmitted
through occupational, perinatal and sexual exposures.2
An estimated 185 million people, or roughly 3% of the world’s population, have been infected with HCV, and over 80% live in
Low and Middle Income Countries (LMICs).3 The burden of disease is greatest in developing countries; the highest reported
prevalence is in China (3.2%)4 Pakistan (4.8%)4, Nigeria (18.3%)5 and Egypt (22%).4 About 15 million European adults are
infected with HCV and most of these people are unaware of their infection.6 Each year, 350,000 to 500,000 people die from
HCV-related liver disease.7
Antiviral medicines can cure HCV, but access to diagnosis and treatment is low.7 A cure for HCV infection is now possible in
most patients with highly effective, safe and tolerable combinations of oral direct-acting antivirals (DAAs) taken for 8–24
weeks.5 Eradication of HCV is being discussed for the first time.5
Quantitation of HCV RNA has proven useful in providing a metric to evaluate the effectiveness of antiviral response to HCV
treatment. Guidelines for the management and treatment of HCV recommend quantitative testing for HCV RNA before the start
of antiviral therapy, during therapy, and after the conclusion of treatment. The primary objective of treatment is Sustained
Virologic Response (SVR), defined as undetectable HCV RNA by a sensitive test 12 or 24 weeks after the end of treatment
depending on the anti-HCV therapy.8

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5 Principle of the Procedure

The GeneXpert Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the
target sequence in simple or complex samples using RT-PCR which uses fluorescence to detect the RNA of interest. The
systems consist of an instrument, personal computer, and preloaded software for running tests and viewing the results. The
systems require the use of single-use disposable GeneXpert cartridges that hold the RT-PCR reagents and host the RT-PCR
processes. Because the cartridges are self-contained, cross-contamination between samples is minimized. For a full description
of the systems, refer to the appropriate GeneXpert Dx Operator Manual or GeneXpert Infinity Operator Manual.
The HCV VL assay includes reagents for the detection of HCV RNA in specimens as well as two internal controls used for
quantitation of HCV RNA. The internal controls monitor recovery and the presence of inhibitor(s) in the RT and PCR reactions.
The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.
6 Reagents
6.1 Materials Provided
The HCV VL assay kit contains sufficient reagents to process 10 specimens or quality control samples. The kit contains the
following:

HCV VL Assay Cartridges with Integrated Reaction Tubes 10

• Bead 1, Bead 2, and Bead 3 (freeze-dried) 1 of each per cartridge
• Lysis Reagent (Guanidinium Thiocyanate) 2.0 mL per cartridge
• Rinse Reagent 0.5 mL per cartridge
• Elution Reagent 1.5 mL per cartridge
• Binding Reagent 2.4 mL per cartridge
• Proteinase K Reagent 0.48 mL per cartridge
Disposable 1 mL Transfer Pipettes 10 per kit
CD 1 per kit
• Assay Definition File (ADF)
• Instructions to import ADF into GeneXpert software
• Instructions for Use (Package Insert)

Note Safety Data Sheets (SDS) are available at www.cepheidinternational.com under the SUPPORT tab.

The bovine serum albumin (BSA) in the beads within this product was produced and manufactured exclusively from bovine plasma
Note sourced in the United States. No ruminant protein or other animal protein was fed to the animals; the animals passed ante- and post-
mortem testing. During processing, there was no mixing of the material with other animal materials.

7 Storage and Handling

+28
+2 • Store the HCV VL assay cartridges and reagents at 2–28 °C.
• Do not open the cartridge until you are ready to perform the assay.
• Do not use a cartridge that has leaked.
• Do not use HCV VL assay cartridges and reagents that were previously frozen.
• Do not use reagents or cartridges that have passed the expiration date.
8 Materials Required but Not Provided
• GeneXpert Dx System or GeneXpert Infinity Systems (catalog number varies by configuration): GeneXpert Instrument,
computer with proprietary GeneXpert Dx Software Version 4.7b or higher (GeneXpert Dx systems); or Xpertise 6.4b or
higher (Infinity-80/Infinity-48s), barcode scanner, and operator manual.
• Printer: If a printer is needed, contact Cepheid Technical Support to arrange for the purchase of a recommended printer.
• Bleach or sodium hypochlorite

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9 Warnings and Precautions

• Treat all biological specimens, including used cartridges, as if capable of transmitting infectious agents. Because it is often
impossible to know which might be infectious, all biological specimens should be treated with standard precautions.
Guidelines for specimen handling are available from the U.S. Centers for Disease Control and Prevention9 and the Clinical
and Laboratory Standards Institute.10
• Good laboratory practices and changing gloves between handling specimens are recommended to avoid contamination of
specimens or reagents.
• Follow your institution’s safety procedures for working with chemicals and handling biological samples.
• Do not substitute HCV VL assay reagents with other reagents.
• Do not open the HCV VL assay cartridge lid except when adding sample.
• Do not use a cartridge that has been dropped after removing it from the packaging.
• Do not shake the cartridge. Shaking or dropping the cartridge after opening the lid may yield invalid results.
• Do not use a cartridge that has a damaged reaction tube.
• Do not use a cartridge that has leaked.
• Each single-use HCV VL assay cartridge is used to process one test. Do not reuse cartridges.
• The single-use disposable pipette is used to transfer one specimen. Do not reuse spent disposable pipettes.
• Wear clean lab coats and gloves. Change gloves between processing each sample.
• In the event of contamination of the work area or equipment with samples or controls, thoroughly clean the contaminated
area with a solution of 1:10 dilution of household chlorine bleach or sodium hypochlorite and then 70% ethanol or 70%
denatured ethanol. Wipe work surfaces dry completely before proceeding.
• Consult your institution’s environmental waste personnel on proper disposal of used cartridges and unused reagents. Check
state, territorial, or local regulations as they may differ from national disposal regulations. The material may exhibit
characteristics of hazardous waste requiring specific disposal requirements. Institutions should check their hazardous waste
disposal requirements.
• Biological specimens, transfer devices, and used cartridges should be considered capable of transmitting infectious agents
requiring standard precautions. Follow your institution’s environmental waste procedures for proper disposal of used
cartridges and unused reagents. These materials may exhibit characteristics of chemical hazardous waste requiring specific
disposal. If country or regional regulations do not provide clear direction on proper disposal, biological specimens and used
cartridges should be disposed per WHO [World Health Organization] medical waste handling and disposal guidelines.
10 Chemical Hazards11,12
• Signal Word: WARNING
• UN GHS Hazard Statements:
• May be harmful if swallowed
• Causes mild skin irritation
• Causes eye irritation
• UN GHS Precautionary Statements:
• Prevention:
• Wash thoroughly after handling.
• Response:
• Call a POISON CENTER of doctor/physician if you feel unwell.
• If skin irritation occurs: Get medical advice/attention.
• IF IN EYES: rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do.
Continue rinsing.
• If eye irritation persists: Get medical advice/attention.

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11 Specimen Collection, Storage, and Transport

Whole blood should be collected in K2-EDTA tubes, EDTA-PPT or serum collection tubes and centrifuged to separate the
plasma/serum and red blood cells per the manufacturer’s instructions.
• A minimum of 1 mL plasma or serum is required for the HCV VL assay. If using the transfer pipette included in the kit, a
minimum of 1.2 mL plasma or serum is required. Alternatively, if using a precision pipette, a minimum of 1 mL plasma or
serum is required.
+30
+15 • Whole blood may be held at 15–30 °C for up to 24 hours or at 2–8 °C for up to 3 days prior to plasma/serum preparation.
+8 Centrifugation should be performed according to the manufacturer instructions.
+2
°C
+35 +8 • After centrifugation and separation, plasma and serum may be held at 15–35 °C for up to 24 hours or at 2–8 °C for up to
+15 +2
°C
3 days prior to testing.
-18 • Plasma and serum specimens are stable frozen (-70 to -18 °C) for 6 weeks.
-70
°C
• Plasma and serum specimens are stable for up to three freeze/thaw cycles.
• Plasma and serum specimens must be thawed and equilibrated to room temperature prior to transfer to the cartridge.
+2
+8
• Ship whole blood, plasma or serum specimens at 2–8 °C.
• Transportation of whole blood, plasma or serum specimens must comply with country, federal, state and local regulations for
the transportation of etiologic agents.
12 Procedure
12.1 Preparing the Specimen
1. Following centrifugation of whole blood samples, 1 mL of plasma can be pipetted directly into the cartridge. Sufficient
volume is critical to obtaining valid test results (see instructions in Section 12.2, Preparing the Cartridge Option 1 below).
+35 2. If using frozen specimens, place the specimens at room temperature (20–35 °C) until completely thawed and equilibrated to
+20
room temperature before use.
+8 3. Plasma and serum samples stored at 2–8 °C should be removed from the refrigerator and equilibrated to room temperature
+2
°C
before use.

+2
+8 4. Plasma samples stored at 2–8 °C or frozen and thawed should be vortexed for 15 seconds before use, if the specimen is
cloudy, clarify by a quick centrifugation.

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12.2 Preparing the Cartridge

1. Wear protective disposable gloves.
2. Inspect the test cartridge for damage. If damaged, do not use it.
3. Open the lid of the test cartridge.
• Option 1: If using the transfer pipette included in the kit (Figure 1), fill to just below the bulb but above the line to transfer
at least 1 mL plasma or serum from the collection tube into the sample chamber of the test cartridge (Figure 2). Do NOT
pour the specimen into the chamber!
• Option 2: If using an automatic pipette, transfer at least 1 mL of plasma or serum into the sample chamber of the test
cartridge (Figure 2). Do NOT pour the specimen into the chamber!

Bulb

Fill the specimen to just below

the bulb and above the mark on
the pipette

Figure 1. HCV VL Assay Transfer Pipette

4. Close the cartridge lid.
5. Load the cartridge into the GeneXpert Dx instrument or Infinity system.

Sample Chamber
(Large Opening)

Figure 2. HCV VL Assay Cartridge (Top View)

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12.3 Starting the Test

Important Before you start the test, make sure the HCV VL Assay Definition File (ADF) is imported into the software.

Note The steps you follow can be different if the system administrator changed the default workflow of the system.

This section lists the basic steps for running the test. For detailed instructions, see the GeneXpert Dx System Operator Manual or
the GeneXpert Infinity System Operator Manual, depending on the model that is being used.
1. Turn on the GeneXpert instrument:
• If using the GeneXpert Dx instrument, first turn on the instrument and then turn on the computer. The GeneXpert
software will launch automatically. If it doesn't, double-click the GeneXpert Dx software shortcut icon on the
Windows® desktop.
or
• If using the GeneXpert Infinity instrument, power up the instrument. The GeneXpert software will launch
automatically. If it doesn't, double-click the Xpertise software shortcut icon on the Windows® desktop.
2. Log on to the GeneXpert Instrument System software using your user name and password.
3. In the GeneXpert System window, click Create Test (GeneXpert Dx) or Orders and Order Test (Infinity).
4. Scan in the Patient ID (optional). If typing the Patient ID, make sure the Patient ID is typed correctly. The Patient ID is
associated with the test results and is shown in the View Results window.
5. Scan or type in the Sample ID. If typing the Sample ID, make sure the Sample ID is typed correctly. The Sample ID is
associated with the test results and is shown in the View Results window and all reports. The Scan Cartridge dialog box
appears.
6. Scan the barcode on the HCV VL assay cartridge. The Create Test window appears. Using the barcode information, the
software automatically fills the boxes for the following fields: Select Assay, Reagent Lot ID, Cartridge SN, and Expiration
Date.
7. Click Start Test (GeneXpert Dx) or Submit (Infinity). Enter your password, if requested.
8. For the GeneXpert Infinity System, place the cartridge on the conveyor belt. The cartridge will be automatically loaded, the
test will run, and the used cartridge will be placed into the waste container.
or
For the GeneXpert Dx Instrument:
A. Open the instrument module door with the blinking green light and load the cartridge.
B. Close the door. The test starts and the green light stops blinking. When the test is finished, the light turns off.
C. Wait until the system releases the door lock before opening the module door and removing the cartridge.
D. The used cartridges should be disposed in the appropriate specimen waste containers according to your institution’s
standard practices.
13 Viewing and Printing Results
This section lists the basic steps for viewing and printing results. For more detailed instructions on how to view and print the
results, see the GeneXpert Dx System Operator Manual or the GeneXpert Infinity System Operator Manual, depending on the
instrument used.
1. Click the View Results icon to view results.
2. Upon completion of the test, click the Report button of the View Results window to view and/or generate a PDF report file.

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14 Quality Control
Each test includes a Sample Volume Adequacy (SVA), Internal Quantitative Standard High and Low (IQS-H and IQS-L, also
acts a specimen processing control [SPC]) and Probe Check Control (PCC).
• Sample Volume Adequacy (SVA) – Ensures the sample was correctly added to the cartridge. The SVA verifies that the
correct volume of sample has been added in the sample chamber. The SVA passes if it meets the validated acceptance
criteria. If the SVA does not pass, an ERROR 2096 will be displayed if there is no sample or an ERROR 2097 will be
displayed if there is not enough sample. The system will prevent the user from resuming the test.
• Internal Quantitative Standard High and Low (IQS-H and IQS-L) – IQS-H and IQS-L are two Armored RNA®
constructs in the form of a dry bead that goes through the whole assay process. The IQS-H and IQS-L are standards
calibrated against the WHO 4th International standard for HCV. They are used for quantification by using lot specific
parameters for the calculation of HCV RNA concentration in the sample. Additionally IQS-H and IQS-L detect specimen-
associated inhibition of the RT-PCR reaction. The IQS-H and IQS-L pass if they meet the validated acceptance criteria.
• Probe Check Control (PCC) – Before the start of the PCR reaction, the GeneXpert Instrument System measures the
fluorescence signal from the probes to monitor bead rehydration, reaction tube filling, probe integrity and dye stability. The
PCC passes if it meets the validated acceptance criteria.
• External Controls – Following good laboratory practices, external controls, not available in the kit, should be used in
accordance with local, state, and federal accrediting organizations’ requirements as applicable.
15 Interpretation of Results
The results are interpreted automatically by the GeneXpert Instrument System from measured fluorescent signals and embedded
calculation algorithms and are clearly shown in the View Results window (Figure 3 and Figure 5). Possible results are shown in
Table 1.

Table 1. HCV VL Assay Results and Interpretation

Result Interpretation
HCV DETECTED The HCV RNA is detected at XX IU/mL.
XX IU/mL (log X.XX) • The HCV RNA has a titer within the linear range setting of the assay and the endpoint above the
See Figure 3. minimum.
• IQS-H and IQS-L: PASS.
• Probe Check: PASS; all probe check results pass.
HCV DETECTED The HCV RNA is detected above the quantitative range of the assay.
> 1.00E08 IU/mL • IQS-H and IQS-L: PASS.
See Figure 4. • Probe Check: PASS; all probe check results pass.
HCV DETECTED The HCV RNA is detected below the quantitative range of the assay.
< 10 IU/mL • IQS-H and IQS-L: PASS.
See Figure 5. • Probe Check: PASS; all probe check results pass.
HCV NOT DETECTED The HCV RNA is not detected.
See Figure 6. • HCV RNA is not detected.
• IQS-H and IQS-L: PASS.
• Probe Check: PASS; all probe check results pass.
INVALID Presence or absence of the HCV RNA cannot be determined. Repeat test according to the
See Figure 7. instructions in Section 16.2, Retest Procedure.
• IQS-H and/or IQS-L: FAIL; Cycle thresholds (Cts) are not within valid range and the endpoint is
below the minimum setting.
• Probe Check: PASS; all probe check results pass.

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Table 1. HCV VL Assay Results and Interpretation (Continued)

Result Interpretation
ERROR Presence or absence of HCV RNA cannot be determined. Repeat test according to the instructions
See Figure 8. in Section 16.2, Retest Procedure.
• Probe Check: FAIL*; all or one of the probe check results fail.
* If the probe check passed, the error is caused by the maximum pressure limit exceeding the acceptable range
or by a system component failure.
NO RESULT Presence or absence of HCV RNA cannot be determined. Repeat test according to the instructions
in Section 16.2, Retest Procedure. A NO RESULT indicates that insufficient data were collected. For
example, the operator stopped a test that was in progress.

Assay screenshots are for example only. Assay name and version number may vary from the screenshots shown in this package
Note
insert.

Figure 3. HCV Detected and Quantified

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Figure 4. HCV Detected

Figure 5. HCV Detected

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Figure 6. HCV Not Detected

Figure 7. Invalid

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Figure 8. Error
16 Retests
16.1 Reasons to Repeat the Assay
If any of the test results mentioned below occur, repeat the test according to the instructions in Section 16.2, Retest Procedure.
• An INVALID result indicates one or more of the following:
• The IQS-H and/or IQS-L Cts are not within valid range.
• The sample was not properly processed or PCR was inhibited.
• An ERROR result indicates that the assay was aborted. Possible causes include: insufficient volume of sample was added, the
reaction tube was filled improperly, a reagent probe integrity problem was detected, or the maximum pressure limit was
exceeded.
• A NO RESULT indicates that insufficient data were collected. For example, the operator stopped a test that was in progress,
or a power failure occurred.
16.2 Retest Procedure
For retest of a NO RESULT, INVALID, or ERROR result, use a new cartridge (do not re-use the cartridge) and new reagents.
1. Remove a new cartridge from the kit.
2. See Section 12, Procedure, including Section 12.1, Preparing the Specimen, Section 12.2, Preparing the Cartridge, and
Section 12.3, Starting the Test.
17 Limitations
Good laboratory practices and changing gloves between handling specimens are recommended to avoid contamination of
reagents.
Mutations or polymorphisms in primer or probe binding regions may affect detection of new or unknown HCV variants
resulting in a false negative result.

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18 Performance Characteristics
18.1 Limit of Detection
The limit of detection (LOD) of the HCV VL assay was determined by testing eight different dilutions prepared from a HCV
genotype 1 reference standard in HCV negative EDTA plasma and serum. The HCV genotype 1 material used in the LOD study
was the WHO 4th International standard, NIBSC code 06/102. The limit of detection was determined for three reagent lots and
a total of 72 or 73 replicates per concentration level were tested. One additional low concentration level was included for both
sample types after the first day of testing. The number of tested replicates for this level was thus smaller (49 in plasma and 53 in
serum). The evaluation was performed according to CLSI guideline E17-A2. The HCV RNA concentration that can be detected
with a positivity rate of greater than 95% was determined by Probit regression analysis and the results for the individual lots and
specimens are shown in Table 2. The maximum observed LOD with Probit analysis for HCV genotype 1 in EDTA plasma is 4.0
IU/mL (95% CI 2.8 – 5.2). The maximum observed LOD with Probit analysis for HCV genotype 1 in serum is 6.1 IU/mL (95%
CI 4.2 – 7.9).

Table 2. HCV VL LOD Estimates with Probit Regression and 95% Upper and Lower Confidence Intervals for
HCV Genotype 1 Specimens in Plasma and Serum per Kit Lot

LOD 95% 95% CI

Specimen Lot (IU/mL) (IU/mL)
1 3.3 2.4 - 4.2
WHO
2 4.0 2.7 - 5.2
(Plasma)
3 4.0 2.8 - 5.2
1 6.1 4.2 - 7.9
WHO
2 2.6 1.9 - 3.3
(Serum)
3 2.3 1.8 - 2.9

Hit rate analysis shows a positivity of > 95% at 6 IU/mL for the HCV genotype 1 material tested as shown in Table 3.

Table 3. HCV VL LOD for HCV Genotype 1 in EDTA Plasma and Serum

Concentration No. Positivity

Specimen (IU/mL) Replicates No. Positives Rate (%)
0.5a 49 24 49
1 72 47 65
2 72 61 85
WHO 3 72 69 96
(Plasma) 4 72 67 93
6 72 71 99
8 73 73 100
10 72 72 100
a
0.5 53 21 40
1 73 47 64
2 73 64 88
WHO 3 72 69 96
(Serum) 4 73 71 97
6 72 71 99
8 72 70 97
10 72 72 100
a. 0.5 IU/mL was added day 2 due to the high positivity rate observed at 1 IU/mL after day 1

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In addition, dilutions of clinical specimens representing HCV genotype 1a, 2b, 3a, 4a, 5a and 6a in negative human EDTA
plasma were analyzed with one reagent lot and 24 replicates per concentration level. The assignment of the nominal
concentration of clinical specimens was determined by Abbott RealTime HCVTM assay. Hit rate analysis shows a positivity of
>95% for all genotypes at 10 IU/mL as shown in Table 4.

Table 4. HCV VL LOD Hit Rate Analysis for HCV Genotype 1 – 6 Specimens in EDTA Plasma

Lowest
Concentration
Level > 95% Hit
Genotype Rate (IU/mL) Hit Rate (%)
1a 10 100
2b 4 100
3a 6 100
4a 4 100
5a 2 96
6a 4 96

18.2 Limit of Quantitation

The total analytical error (TAE) was calculated using estimates determined through analysis of data from LOD study (WHO
standard) and the Precision/Reproducibility study according to CLSI guideline E17-A2. The TAE for the dilutions that had an
observed concentration at or near the assay limit of detection 10 IU/mL (1.0 log10) are presented in Table 5. TAE was estimated
by two different methods.

Table 5. HCV VL TAE Analysis for Determination of LOQ

TAEa
Absolute TAEb
Specimen Concentration Bias + 2xSQRT
(Study) DL Lot N (Log10 IU/mL) Bias Total SD 2xSD (2)xSD
Expected Observed
DL1 72 1.40 1.31 0.09 0.15 0.38 0.41
Acrometrix
DL2 72 1.40 1.29 0.11 0.14 0.40 0.41
(Precision)
DL3 72 1.40 1.24 0.16 0.12 0.41 0.35
DL1 72 1.00 0.92 0.08 0.22 0.52 0.62
Acrometrix
DL2 72 1.00 0.82 0.18 0.18 0.54 0.51
(Precision)
DL3 72 1.00 0.75 0.25 0.19 0.63 0.54
DL1 24 1.00 0.91 0.09 0.21 0.51 0.59
WHO, Plasma
DL2 24 1.00 0.82 0.18 0.30 0.78 0.86
(LOD)
DL3 24 1.00 0.86 0.14 0.17 0.48 0.48
DL1 24 1.00 0.96 0.04 0.13 0.30 0.37
WHO, Serum
DL2 24 1.00 0.88 0.12 0.23 0.58 0.66
(LOD)
DL3 24 1.00 0.80 0.20 0.18 0.57 0.52
a. TAE calculated according to the Westgard model in CLSI EP17-A2 (Section 6.2)
b. TAE based upon the difference between two measurements approach

The results of the TAE analysis demonstrate that the HCV VL assay can determine 10 IU/mL (1.0 log10) with an acceptable
trueness and precision.

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18.3 Precision/Reproducibility
The precision/reproducibility of the HCV VL assay was determined by analysis of parallel dilutions of HCV reference materials
in HCV negative EDTA plasma. The nominal concentration of the reference material used was calibrated to the WHO 4th HCV
International Standard (06/102). The study was a two site, blinded, comparative study using a seven-member panel of HCV
reference material in HCV negative EDTA plasma with RNA concentrations that span the HCV VL assay quantitation range.
Two operators at each of the two study sites tested one panel of twenty-one samples once per day over six testing days per lot.
One site used an Infinity-80 instrument and the other site used GeneXpert Dx instruments. Three lots of HCV VL assay reagents
were used for the study. Precision/reproducibility was evaluated in accordance with “Evaluation of Precision Performance of
Clinical Chemistry Devices; Approved Guideline” CLSI document EP5-A2. The precision results for each regent lot are shown
in Table 6.

Table 6. HCV VL Precision per Lot

Expected HCV Total Precision per Lot

RNA Lot 1 Lot 2 Lot 3
Concentration
log10 IU/mL SD CVa SD CVa SD CVa
1.0 0.23 55.8% 0.18 44.2% 0.20 48.1%
1.4 0.15 35.1% 0.15 35.8% 0.13 29.6%
2.7 0.09 20.7% 0.09 20.6% 0.09 20.2%
4.2 0.07 16.4% 0.08 18.9% 0.07 15.3%
5.4 0.12 28.3% 0.09 19.9% 0.07 16.2%
6.9 0.13 31.8% 0.09 20.9% 0.07 17.0%
8.2 0.10 22.7% 0.10 23.7% 0.08 17.8%
a. “CV” is lognormal CV, as obtained using the formula:

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The reproducibility and precision of the HCV VL assay was evaluated by using nested ANOVA with terms for Site/Instrument,
Lot, Day, Operator/Run and Within-Run. The standard deviation and the percentage of variability due to each component of the
log10 HCV transformed concentrations were calculated, see Table 7.

Table 7. Standard Deviation and Contributable Percentage of Variability for Each Term and Total Precision

HCV RNA
Concentration Contribution to Total Variance SD (CV%) Total Precision
log10 IU/mL
Operator/ Within-
Site/Inst Lot Day Total
Run Run
Lower Upper
Expected Actual N SD (%)a SD (%)a SD (%)a SD (%)a SD (%)a SD CI CI CVb
1.0 0.83 216 0.03 1.8% 0.08 13.2% 0.04 3.5% 0.00 0.0% 0.19 81.6% 0.21 0.18 0.25 51.7%
1.4 1.28 216 0.00 0.0% 0.04 7.1% 0.00 0.0% 0.00 0.0% 0.14 92.9% 0.14 0.13 0.16 34.1%
2.7 2.66 216 0.00 0.0% 0.04 17.2% 0.00 0.0% 0.02 3.2% 0.08 79.5% 0.09 0.08 0.11 22.1%
4.2 4.18 215 0.00 0.0% 0.05 30.9% 0.01 2.6% 0.00 0.0% 0.07 66.5% 0.09 0.07 0.12 20.6%
5.4 5.44 216 0.00 0.0% 0.06 26.5% 0.00 0.0% 0.01 1.3% 0.09 72.2% 0.11 0.09 0.14 25.8%
6.9 6.86 216 0.00 0.0% 0.07 34.0% 0.02 3.4% 0.00 0.0% 0.10 62.5% 0.13 0.10 0.17 29.8%
8.2 8.11 216 0.00 0.0% 0.09 47.9% 0.00 0.0% 0.02 2.6% 0.09 49.5% 0.13 0.10 0.19 30.5%

a. (%) is contribution of variance component to overall lognormal CV

b. “CV” is lognormal CV, as obtained using the formula:

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18.4 Linear Range and Inclusivity

The linear range of the HCV VL assay was determined by analysis of a twelve member panel covering a range from ~5 (0.75
log10) to ~1 x 108 (8 log10) IU/mL. Panels were prepared by parallel dilutions of HCV reference material (Armored RNA®
genotype 1 and clinical specimen genotype 1) in HCV negative EDTA plasma and serum. The nominal concentration of the
reference material used was calibrated to the WHO 4th HCV International Standard (06/102). Each panel member was tested in
replicates of four on each of three testing days using two kit lots. Totally, 24 replicates per panel member and sample type were
tested. The linearity analysis was performed according to CLSI guideline EP06-A. The combined results for both lots are shown
in Figure 9 and Figure 10. The HCV VL assay is linear within a range 0.8–8.0 log10 IU/mL with a R2 value of >0.997.

Figure 9. Linearity Genotype 1 in EDTA Plasma for the HCV VL Assay

Figure 10. Linearity Genotype 1 in Serum for the HCV VL Assay

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To confirm the linear range and evaluate the inclusivity of the HCV VL assay, panels consisting of clinical specimens
representing HCV genotype 2 – 6 and Armored RNA® when available (genotypes 2 and 3 only) were prepared in negative
human EDTA plasma. 7 – 13 panel members per genotype covering as wide a range as possible, varying from ~ 0.9 – 6 log10
IU/mL for genotype 5 to ~ 0.9 – 8.3 log10 for genotype 3, were prepared and analyzed in replicates of four on each of three
testing days using two kit lots. For each genotype, 24 replicates per panel member were tested. The nominal concentrations of
the reference materials used were calibrated to the WHO 4th HCV International Standard (06/102). All genotypes responded
linearly with R2 values ranging from 0.994 – 0.998.
18.5 Analytical Specificity (Exclusivity)
The analytical specificity of the HCV VL assay was evaluated by adding potentially cross-reacting organisms at 1 x 105 CFU/
mL, copies/mL or TCID50/mL input concentration into HCV negative EDTA plasma and in plasma that contained ~25 IU/mL
HCV reference material (clinical specimen genotype 1). Tested organisms are listed in Table 8.

Table 8. Analytical Specificity Organisms

Human Immunodeficiency virus 1

Human Immunodeficiency virus 2
Human T-cell lymphotropic virus I
Human T-cell lymphotropic virus II
Candida albicans
Cytomegalovirus
Epstein-Barr virus
Hepatitis A virus
Hepatitis B virus
Herpes simplex virus 1
Herpes simplex virus 2
Human herpes virus 6
Human herpes virus 8
Varicella Zoster virus
BK Human polyoma virus
Banzi virus
Ilheus virus
West Nile virus
Zika virus
Human papilloma virus 16
Human papilloma virus 18
Staphylococcus epidermidis
Staphylococcus aureus

None of the tested organisms showed cross reactivity and all positive replicates resulted in concentrations of HCV RNA within
± 0.5 log from a HCV positive control when tested using the HCV VL assay. In addition to the species listed in Table 8, Dengue
virus and vaccinia virus were analyzed in silico since material representing the viruses could not be obtained for testing. No
practical significant sequence similarity was found between the analyzed viruses and the primers and probes of the
Xpert HCV VL assay.

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18.6 Potentially Interfering Substances

The susceptibility of the HCV VL assay to interference by elevated levels of endogenous substances, by drugs prescribed to
HCV infected patients and by autoimmune disease markers was evaluated. HCV negative EDTA plasma and plasma that
contained ~25 IU/mL HCV reference material (clinical specimen genotype 1) were tested.
Elevated levels of the endogenous substances listed in Table 9 were shown not to interfere with the quantification of
the HCV VL assay or impact the assay specificity.

Table 9. Endogenous Substances and Concentration Tested

Substance Tested Concentration

Albumin 9 g/dL
Bilirubin 20 mg/dL
Hemoglobin 500 mg/dL
Human DNA 0.4 mg/dL
Triglycerides 3000 mg/dL

The drug components as presented in Table 10 were shown not to interfere with the quantification of the HCV VL assay or
impact the assay specificity when tested at three times peak level concentration in five drug pools.

Table 10. Drug Pools Tested

Pool Drugs
Control N/A
1 Zidovudine, Saquinavir, Ritonavir, Interferon alfa-2b, Clarithromycin
2 Abacavir sulfate, Fosamperavir Calcium, Peginterferon 2b, Ribavirin
3 Tenofovir disoproxil fumarate, Lamivudine (3TC), Indinavir sulfate, Ganciclovir,
Valganciclovir HCl, Acyclovir
4 Stavudine (d4T), Efavirenz, Lopinavir, Enfuvirtide (T-20), Ciprofloxacin
5 Nevirapine, Nelfinavir mesylate, Azithromycin, Valacyclovir HCl

Testing of specimens from ten individuals per autoimmune disease marker shows no interference with the autoimmune disease
markers systemic lupus erythematosus (SLE), anti-nuclear antibody (ANA), or rheumatoid factor (RF) using the HCV VL assay.

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18.7 Sample Collection Media Equivalence (EDTA, PPT-EDTA and Serum)

For each sample collection media (EDTA, PPT-EDTA and serum) specimens from 50 matched HCV positive individuals and 25
matched HCV negative specimens were collected and tested using one kit lot of the HCV VL assay.
As shown in Figure 11 and Figure 12 equivalent performance of the HCV VL assay was shown for EDTA plasma versus serum
samples and EDTA plasma versus PPT-EDTA plasma samples. All HCV positive specimens collected in serum or PPT-EDTA
plasma produced concentrations of HCV RNA within ±0.5 log10 IU/mL of the HCV positive specimen collected in EDTA
plasma when tested using the HCV VL assay.

Figure 11. Scatterplot of Log IU/mL PPT-EDTA versus Log IU/mL EDTA

Figure 12. Scatterplot of Log IU/mL Serum versus Log IU/mL EDTA Plasma

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19 Performance Characteristics – Clinical Performance

Specificity
The specificity of the HCV VL assay was evaluated using 100 EDTA plasma specimens from HCV negative blood donors.
None of the 100 specimens tested were detected by the HCV VL assay equating to 100% specificity (95% CI = 96.1-100).
Method Correlation
A multi-site study was conducted to evaluate the performance of the HCV VL assay relative to a comparator method using fresh
and frozen human plasma or serum specimens collected from HCV infected individuals. Of the 607 eligible specimens, each
from unique individuals, 408 (67.2%) were collected from male subjects. The average age was 50.2 ± 13.2 years with an age
range of 21 to 86 years.
Of the 607 specimens, 389 were within the quantitation range of both assays including 23 specimens that were HCV non-1
genotypes (2, 2a, 2b, 2c, 3, 3a, 4 & 6) and one mixed genotype (HCV 1 & 6). The Deming regression shows very good
correlation between the HCV VL and the comparator method with a slope of 1.022 and intercept of 0.082. The R2 was 0.986.

*HCV non-1 genotypes are represented as triangles. A single outlier was not included in the analysis.
Figure 13. Xpert v. Comparator Method

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20 References
1. Di Bisceglie AM. Natural history of Hepatitis C: it impact on clinical management. Hepatology 2000; 31:1014-1018.
2. EASL Clinical Practice Guidelines: Management of Hepatitis C. Consensus Statement. J. Hepatology 2011; vol. 55:245-
264.
3. Mohd Hanafiah K., Groeger J., Flaxman AD., Wiersma S.T Global epidemiology of hepatitis C virus infection: new
estimates of age-specific antibody to HCV seroprevalence. Hepatology 2013; 57(4): 1333-1342.
4. Shepard CW, Finelli L, Alter MJ. Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 2005; 5:558-67.
doi:10.1016/S1473-3099(05)70216-4PMID: 16122679.
5. Graham CS., Swan T. A Path to Eradication of Hepatitis C in Low-and-Middle-Income Countries. Antiviral Res. 2015 Jan
20; pii: S0166-3542(15)00005-4. doi: 10.1016/j.antiviral.215.01.004. [Epub ahead of print].
6. Region H Press Release. The number of people living with HIV and hepatitis is on the rise in Europe, Oct 2014.
http://newsite.hiveurope.edu/
7. Hepatitis C Fact Sheet No 164 Updated April 2014, accessed January 28, 2015 at
http://www.who.int/mediacentre/factsheets/fs164/en/
8. Ghany MG, Strader DB, Thomas DL, et al. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology
2009;49 (4):1335-1374.
9. Centers for Disease Control and Prevention. Biosafety in Microbiological and Biomedical Laboratories (refer to latest
edition. http:..www.cdc.gov/biosafety/publications/
10. Clinical and Laboratory Standards Institute. Protection of Laboratory Workers from Occupationally Acquired Infections,
Approved Guideline. Document M29 (refer to latest edition).
11. REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 16 December
2008 on the classification labeling and packaging of substances and mixtures amending and repealing, List of Precautionary
Statements, Directives 67/548/EEC and 1999/45/EC (amending Regulation (EC).
12. Occupational Safety and Health Standards, Hazard Communication, Toxic and Hazard Substances (March 26, 2012) (29
C.F.R., pt. 1910, subpt. Z).

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21 Cepheid Headquarters Locations

Corporate Headquarters European Headquarters

Cepheid Cepheid Europe SAS
904 Caribbean Drive Vira Solelh
Sunnyvale, CA 94089 81470 Maurens-Scopont
USA France
Telephone: +1 408.541.4191 Telephone: +33 563 825 300
Fax: +1 408.541.4192 Fax: +33 563 825 301
www.cepheid.com www.cepheidinternational.com

22 Technical Assistance
Before contacting Cepheid Technical Support, collect the following information:
• Product name
• Lot number
• Serial number of the instrument
• Error messages (if any)
• Software version and, if applicable, Computer Service Tag number

Region Telephone Email

US +1 888.838.3222 TechSupport@cepheid.com
Brazil and Latin America + 55 11 3524 8373 latamsupport@cepheid.com
France +33 563 825 319 Support@cepheideurope.com
Germany +49 69 710 480 480 Support@cepheideurope.com
India, Bangladesh, Bhutan, Nepal
+91 11 48353010 techsupportindia@cepheid.com
and Sri Lanka
Italy +39 800 902 567 Support@cepheideurope.com
United Kingdom +44 3303 332 533 Support@cepheideurope.com
South Africa +27 861 22 76 35 Support@cepheideurope.com
Other European, Middle East and +33 563 825 319
Support@cepheideurope.com
African countries +971 4 253 3218
+1800 107 884
Australia, New Zealand techsupportanz@cepheid.com
+0800 001 028
China +86 021 5406 5387 techsupportchina@cepheid.com
Japan 0120 95 4886 support@japan.cepheid.com
Other countries not listed above +1 408.400.8495 TechSupport@cepheid.com

Contact information for other Cepheid offices is available on our website at www.cepheidinternational.com under the
SUPPORT tab. Select the Contact Us option.

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23 Table of Symbols

Symbol Meaning

Catalog number

In vitro diagnostic medical device

Do not reuse

Batch code

Caution

Manufacturer

Contains sufficient for <n> tests

Control

Expiration date

CE marking – European Conformity

Temperature limitation

Biological risks

Warning

Cepheid AB
Röntgenvägen 5
SE-171 54 Solna
Sweden

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