GeneXpert Xpert MTB RIF User manual

BCR-ABL say
GXMTB/RIF-10GXMTB/RIF-10
301-0191 Rev. C, May 2012
In Vitro Diagnostic Medical Device
Xpert MTB/RIF
R

Copyright © Cepheid. Cepheid®, the Cepheid logo, GeneXpert®, and Xpert® are trademarks of Cepheid.
This product is licensed under US Patent No. 5,851,767 and corresponding claims of any non-US counterpart(s) thereof.
This product is sold under license from the Public Health Research Institute and may be used under PHRI patent rights only for human in vitro
diagnostics.
NO OTHER RIGHTS ARE CONVEYED EXPRESSLY, BY IMPLICATION OR BY ESTOPPEL TO ANY OTHER PATENTS. FURTHERMORE, NO RIGHTS FOR RESALE
ARE CONFERRED WITH THE PURCHASE OF THIS PRODUCT.

301-0191 Rev. C, May 2012 1
English
In Vitro Diagnostic Medical Device
PROPRIETARY NAME
Xpert® MTB/RIF Assay
COMMON OR USUAL NAME
Xpert MTB/RIF Assay
A. INTENDED USE
The Xpert MTB/RIF Assay for use with the Cepheid GeneXpert® system is a semi-quantitative,
nested real-time PCR in-vitro diagnostic test for the detection of:
•Mycobacterium tuberculosis complex DNA in sputum samples or concentrated sediments prepared
from induced or expectorated sputa that are either acid-fast bacilli (AFB) smear positive or
negative
• Rifampin-resistance associated mutations of the rpoB gene in samples from patients at risk for
rifampin resistance
The Xpert MTB/RIF Assay is intended for use with specimens from untreated patients for whom
there is clinical suspicion of tuberculosis (TB). Use of the Xpert MTB/RIF Assay for the detection of
M. tuberculosis (MTB) or determination of rifampin susceptibility has not been validated for patients
who are receiving treatment for tuberculosis.
B. SUMMARY AND EXPLANATION
Globally, about 2 billion people are infected with MTB.1Every year almost 9 million people develop
active disease, and 2 million people die of the illness. Active MTB, which is predominantly
pulmonary in nature, is a highly infectious airborne disease. Given the infectious nature of MTB, fast
and accurate diagnosis is an important element of MTB treatment and control.
Treatment involves prolonged administration of multiple drugs and is usually highly effective.
However, MTB strains can become resistant to one or more of the drugs, which makes cure difficult
to achieve. Four common first-line drugs used in anti-tuberculosis therapy are:
• Isoniazid (INH)
• Rifampin (RIF or Rifampicin)
• Ethambutol (EMB)
• Pyrazinimide (PZA)
RIF resistance rarely occurs in isolation and usually indicates resistance to a number of other anti-TB
drugs.2RIF resistance is most commonly seen in multi-drug resistant (MDR-TB) strains and has a
reported frequency of greater than 95% in such isolates.3, 4, 5 MDR-TB is defined as a tuberculous
disease caused by a bacterial strain that is resistant to at least INH and RIF. Resistance to RIF or
other first-line drugs usually indicates the need for full susceptibility testing, including testing against
second-line agents.

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2 301-0191 Rev. C, May 2012
Molecular detection of MTB and rpoB gene mutations associated with RIF resistance speeds the
diagnosis of both drug-susceptible and MDR-TB. With the Xpert MTB/RIF Assay, this can be
accomplished in fresh sputum samples and in prepared sediments in less than 2.5 hours. The rapid
detection of MTB and RIF resistance allows the physician to make critical patient management
decisions regarding therapy during the same medical encounter.
C. PRINCIPLE OF THE PROCEDURE
The GeneXpert Dx system integrates and automates sample processing, nucleic acid amplification,
and detection of the target sequences in simple or complex samples using real-time PCR and reverse
transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and
preloaded software for running tests on collected samples and viewing the results. The system
requires the use of single-use disposable GeneXpert cartridges that hold the PCR reagents and host
the PCR process. Because the cartridges are self-contained, cross-contamination between samples is
eliminated. For a full description of the system, see the GeneXpert Dx System Operator Manual or
GeneXpert Infinity System Operator Manual.
Xpert MTB/RIF includes reagents for the detection of MTB and RIF resistance and a Sample
Processing Control (SPC) to control for adequate processing of the target bacteria and to monitor
the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent
rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.
The primers in the Xpert MTB/RIF Assay amplify a portion of the rpoB gene containing the 81 base
pair “core” region. The probes are able to differentiate between the conserved wild-type sequence and
mutations in the core region that are associated with RIF resistance.
D. REAGENTS AND INSTRUMENTS
D.1 MATERIAL PROVIDED
The Xpert MTB/RIF kit (GXMTB/RIF-10) contains sufficient reagents to process 10
patient or quality-control specimens.
The kit contains the following items:
•CD
• Assay definition file (ADF)
• Instructions to import ADF into GX software
•PackageInsert
• Xpert MTB/RIF cartridges with integrated reaction tubes 10
• Bead 1 (freeze-dried) 2 per cartridge
-Primers
-Probes
-KCl
-MgCl
2
- HEPES, pH 8.0
- Bovine serum albumin (BSA)

D. Reagents and Instruments
301-0191 Rev. C, May 2012 3
• Bead 2 (freeze-dried) 2 per cartridge
-Probe
- Polymerase
-KCl
-MgCl
2
-dNTPs
- HEPES, pH 7.2
-BSA
• Bead 3 (freeze-dried)1 per cartridge
- Approximately 6000 non-infectious sample preparation
control spores
• Reagent 1 (tris buffer, EDTA, and surfactants) 4 mL per cartridge
• Reagent 2 (tris buffer, EDTA, and surfactants) 4 mL per cartridge
• Sample Reagent (SR) – sodium hydroxide and isopropanol 10 x 8 mL bottles
• Disposable transfer pipettes 12
Note: The Sample Reagent (SR) solution is clear, ranging from colorless to golden yellow.
Note: Safety Data Sheets (SDS) are available at www.cepheid.com/tests-and-reagents/literature/msds or
www.cepheidinternational.com/tests-and-reagents/literature/msds.
Note: The bovine serum albumin (BSA) in the beads within this product was produced exclusively from bovine
plasma sourced in the United States. The manufacturing of the BSA is also performed 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 commingling of the material with other animal
materials.
Note: The transfer pipettes have a single mark representing the minimum volume of sample necessary to transfer
to the GX cartridge. Use only for this purpose. All other pipettes must be provided by the laboratory.
D.2 STORAGE AND HANDLING
• Store the Xpert MTB/RIF cartridges and reagents at 2 to 28 °C.
• Do not use reagents or cartridges that have passed the expiration date.
• Do not open a cartridge lid until you are ready to perform testing.
• Process the cartridge within 4 hours after adding the sample to the cartridge.
• The cartridges are stable up to 2 weeks at 2 to 48°C after opening the pouch.

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4 301-0191 Rev. C, May 2012
E. MATERIALS REQUIRED BUT NOT PROVIDED
• GeneXpert Dx system equipped with GX4.0 software or higher (catalog number varies by
configuration): GeneXpert instrument, computer, barcode reader, and operator manual
• Printer (See GeneXpert Dx System Operator Manual or GeneXpert Infinity Operator Manual for
compatibility guidelines)
• Leak-proof, sterile, screw-capped specimen collection containers
• Disposable gloves and eye protection
• Labels or permanent marking pen
• Transfer pipettes for sample processing
F. 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 Prevention6 and the Clinical and Laboratory Standards
Institute (formerly National Committee for Clinical Laboratory Standards)7.
• Wear protective disposable gloves, laboratory coats and eye protection when handling specimens
and reagents. Wash hands thoroughly after handling specimens and test reagents.
• Follow your institution’s safety procedures for working with chemicals and handling biological
samples.
• The performance of Xpert MTB/RIF Assay for the detection of MTB complex has not been
demonstrated from non-respiratory specimens, such as blood, CSF, stool or urine. The
performance of the Xpert MTB/RIF Assay has not been evaluated with specimens processed by
methods other than those described in this package insert.
• When processing more than one sample at a time, open only one cartridge, add the Sample
Reagent-treated sample (or decontaminated, liquefied sample), and close the cartridge before
adding Sample Reagent-treated sample to the next cartridge.
• Do not open the Xpert MTB/RIF cartridge lid except when adding the treated sample.
• Do not use a cartridge that has been dropped or shaken after you have added the treated sample.
• Do not use a cartridge if it appears wet or if the lid seal appears to have been broken.
• Do not use a cartridge that has a damaged reaction tube.
• Each Xpert MTB/RIF cartridge is used to process one test. Do not reuse processed cartridges.
• Check your regional/country hazardous and medical waste disposal requirements. If regulations
(or lack thereof) do not provide clear direction on proper disposal, biological specimens, including
used cartridges, should be treated as capable of transmitting infectious agents. Dispose used
cartridges as hazardous health-care waste in durable waste containers per WHO [World Health
Organization} medical waste handling and disposal guidelines.
• Sample Reagent contains sodium hydroxide (pH > 12.5) and isopropanol. Harmful if swallowed
(H302), causes severe skin burns and eye damage (H314). Highly flammable liquid and vapor
(H225).

G. Specimen Collection and Transport
301-0191 Rev. C, May 2012 5
G. SPECIMEN COLLECTION AND TRANSPORT
You can process resuspended sediment or fresh sputum with this assay. See Table 1 to determine
adequate specimen volume.
Note: To obtain an adequate fresh sputum specimen, follow the instructions in this section. The patient should
be seated or standing.
G.1 STORAGE AND TRANSPORT
Store and transport specimens at 2 to 8 °C prior to processing whenever possible. However, if
necessary the specimens can be stored at a maximum of 35 °C for 3 days and at 2 to 8 °C for
4 to 10 days.
G.2 SPECIMEN COLLECTION PROCEDURE
1. Have the patient rinse his or her mouth twice with water.
2. Open the lid on the sputum collection container.
3. Have the patient inhale deeply, cough vigorously, and expectorate the material into the
container. Avoid spills or soiling the outside of the container.
4. Secure the lid on the collection device, and then send the container to the processing area.
H. ASSAY PROCEDURE(S)
H.1 SPUTUM SEDIMENTS PROCEDURE
Note: Reject specimens with obvious food particles or other solid particulates.
Volume Requirements: Sputum sediments prepared according to the method of Kent and
Kubica8and re-suspended in 67mM Phosphate/H2O buffer) can be tested using Xpert MTB/
RIF Assay. After resuspension, keep at least 0.5 mL of the resuspended sediment for the Xpert
MTB/RIF Assay.
1. Label each Xpert MTB/RIF cartridge with the sample ID.
Note: Write on the sides of the cartridge or affix an ID label. Do not put the label on the lid of the cartridge or
cover the existing 2D barcode on the cartridge.
2. Transfer at least 0.5 mL of the total resuspended pellet to a conical, screw-capped tube for
the Xpert MTB/RIF using a transfer pipette. Alternatively, the entire sample can be
processed in the original tube.
Note: Store re-suspended sediments at 2 to 8 °C if they are not immediately processed. Do not store for more
than 12 hours.
3. Transfer 1.5 mL of Xpert MTB/RIF Sample Reagent (SR) to 0.5 mL of resuspended
sediment using a transfer pipette.
Table 1. Required Specimen Volume
Specimen Type Minimum Volume for One Test
Minimum Total Volume for Test and
Retest
Sputum sediment 0.5 mL 1 mL
Fresh sputum 1 mL 2 mL

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4. Shake vigorously 10 to 20 times or vortex for at least 10 seconds.
Note: One back-and-forth movement is a single shake.
5. Incubate for 10 minutes at room temperature, and then shake the specimen vigorously 10
to 20 times or vortex for at least 10 seconds.
6. Incubate the sample at room temperature for an additional 5 minutes.
H.2 EXPECTORATED SPUTUM SAMPLE PROCEDURE
Note: Reject specimens with obvious food particles or other solid particulates.
1. Label each Xpert MTB/RIF cartridge with the sample ID.
Note: Write on the sides of the cartridge or affix an ID label. Do not put the label on the lid of the cartridge or
cover the existing 2D barcode on the cartridge.
Figure 1. Writing on the cartridge with a permanent marking pen
2. In a leak-proof sputum collection container:
A. Carefully open the lid of the sputum collection container.
Figure 2. Opening the sample container
B. Pour approximately 2 times the volume of the SR to the sputum (2:1 dilution,
SR:sputum).
Figure 3. Examples of 2:1 dilutions
Example 1
8 mL SR:4 mL sputum
Example 2
2 mL SR:1 mL sputum
Note: Discard the leftover SR
and the bottle in a chemical
waste container.
3 mL line
1 mL sputum

H. Assay Procedure(s)
301-0191 Rev. C, May 2012 7
C. Replace and secure the lid.
D. Shake vigorously 10 to 20 times or vortex for at least 10 seconds.
Note: One back-and-forth movement is a single shake.
3. Incubate the sample for 10 minutes at room temperature, and then shake the specimen
vigorously 10 to 20 times or vortex for at least 10 seconds.
4. Incubate the sample at room temperature for an additional 5 minutes.
H.3 PREPARING THE CARTRIDGE
Note: Start the test within 4 hours of adding the sample to the cartridge.
1. Open the cartridge lid, and then open the sample container.
2. Using the provided transfer pipette, aspirate the liquefied sample to the line on the pipette.
Do not process the sample further if there is insufficient volume. See Figure 4.
Figure 4. Aspirating to the line on the pipette
3. Transfer the sample into the sample chamber of the Xpert MTB/RIF cartridge. Dispense
the sample slowly to minimize the risk of aerosol formation. See Figure 5.
Figure 5. Dispensing decontaminated liquefied sample into the sample chamber of the cartridge
4. Close the cartridge lid firmly.
Important: Be sure to load the cartridge into the GeneXpert Dx instrument and start the
test within 5 hours of preparing the cartridge.

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H.4 STARTING THE TEST
Important: Before you start the test, make sure that the system is running GX 4.0 software
or higher and that the Xpert MTB/RIF assay definition file is imported into the
software.
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.
Note: The steps you follow can be different if the system administrator changed the default workflow of the
system.
1. Turn on the GeneXpert instrument:
• If using the GeneXpert Dx instrument, first turn on the GX Dx instrument, and then
turn on the computer. The GeneXpert software will launch automatically.
or
• If using the GeneXpert Infinity instrument, power up the instrument. On the
Windows® desktop, double-click the Software shortcut icon.
2. Log on to the GeneXpert Dx System software using your user name and password.
3. In the GeneXpert Dx System window, click Create Test. The Scan Sample ID dialog box
appears.
4. In the Sample ID box, scan or type the sample ID. Make sure you type the correct sample
ID (sample ID is associated with the test results and is shown in the View Results window
and all the reports). The Scan Cartridge Barcode dialog box appears.
5. Scan the barcode on the Xpert MTB/RIF 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.
6. Click Start Test. Enter your password if requested.
7. Open the instrument module door with the blinking green light and load the cartridge.
8. Close the door. The test starts and the green light stops blinking. When the test is finished,
the light turns off.
9. Wait until the system releases the door lock at the end of the run, then open the module
door and remove the cartridge.