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Policy # MI/IC/XpertCARB-R/v01 Page 1 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual
Subject Title: Cepheid Xpert CARBA-R Assay
Issued by: LABORATORY MANAGER
Original Date: December 21, 2015
Approved by: Laboratory Director
Revision Date:
Annual Review Date:
Cepheid Xpert Carba-R Assay
I.
Introduction
The Cepheid GeneXpert® Carba-R Assay is performed on the GeneXpert® Instrument
System. It is a rapid (<1h) qualitative PCR assay for detecting KPC, NDM, VIM,
OXA48-like, and IMP1 carbapenemases that encode carbapenem-non-susceptibility in
Gram-negative bacteria. The PCR assay may be performed directly on rectal swab or
stool specimens, or it may be performed on surveillance or clinical Enterobacteriaceae
isolates suspected of producing carbapenemases.
II.
Specimen Collection and Processing
Rectal swabs are collected in the standard manner and sent in a Copan eSwab Amies
transport container to Microbiology as soon as possible. If stool is to be tested, samples
must be collected in an empty container as those collected in Enteric Transport Medium
or SAF are NOT suitable for this assay. Specimens are stable for up to 5 days at 2–8°C,
or at 20–30°C (room temperature) for up to 24h.
The Xpert CARBA-R assay has been thoroughly validated at MSH/UHN for off-label
testing of bacterial colonies isolated on a variety of commonly used culture media.
III.
Materials & Equipment
 GeneXpert Dx System

Xpert CARBA-R Assay kit

Vortex

Copan CLASSIQ Swabs Cat# 138CS01

Rainen LTS100 pipette to deliver 30uL

Aerosol-resistant tips (filtered) – long (specimens), normal (isolates)
One Xpert Carba-R Assay kit can process 10 specimens or 10 isolates (10 tests) and
contains the following items:

10 Xpert Carba-R cartridges with integrated reaction tubes and reagents

10 5mL Sample Reagent vials

10 1.7mL disposable pipettes with ridged mark to indicate transfer volume
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be
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Policy # MI/IC/Xpert CARB-R/v01 Page 2 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual
Subject Title: Cepheid Xpert CARBA-R Assay
IV.
Procedure
A. Xpert Cartridge Preparation for each new test
1. Remove one Xpert CARBA-R cartridge, one Sample Reagent vial, and the plastic
bag of graded transfer pipettes from the Cepheid box (stored at room temperature),
taking care not to touch the reaction tube at the back of the cartridge
2. Place a barcoded LIS label on the cartridge side (don’t cover the front TEST
barcode), and crack open the lid. Proceed as appropriate to 3. (Bacterial colony
testing) or to 7. (Rectal eSwab specimens)
Bacterial colonies
3. Using a sterile Copan CLASSIQ Swab, very lightly touch colonies growing closest to
the meropenem disc on the Mueller-Hinton agar plate - DO NOT OVERLOAD SWAB
4. Place swab in Sample Reagent vial and break swab at line. After discarding remnant of
swab shaft, replace lid and vortex thoroughly for 10s.
5. Using the graded transfer pipette provided in the kit, aspirate inoculated Sample
Reagent up to the marked line. Carefully transfer fluid into the “S” chamber of the
opened CARBA-R cartridge taking care not to introduce bubbles.
6. Close cartridge lid, discard Sample Reagent vial, and proceed to instrument to load
cartridge.
Rectal eSwab specimens
7. Process only one specimen for CARBA-R PCR in the Bio-Safety cabinet at one time.
8. Vortex eSwab specimen tube for 10s taking care not to wet inner lid (prevent
contamination).
9. Using a long 200
-resistant (filtered) tip, carefully pipette 30 L eSwab
fluid into the Sample reagent vial (), replace lid and vortex for 10s.
10. Using the graded transfer pipette provided in the kit, aspirate inoculated Sample
Reagent up to the marked line. Carefully transfer fluid into the “S” chamber of the
opened CARBA-R cartridge taking care not to introduce bubbles or faecal particulates.
11. Close cartridge lid, discard Sample Reagent vial, and proceed to instrument to load
cartridge.
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be
checked against the document (titled as above) on the server prior to use.
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Policy # MI/IC/Xpert CARB-R/v01 Page 3 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual
Subject Title: Cepheid Xpert CARBA-R Assay
Important: Place cartridge into GeneXpert system <15min of adding sample to cartridge
Figure 1: S-chamber of Xpert cartridge (top view)
B. Assay Method
1.
2.
3.
4.
5.
First turn on the GeneXpert computer, then the GeneXpert DX instrument
Log into Windows using password “cphd”
On the Windows desktop, double-click on the GeneXpert DX shortcut icon to open
Log using personal user name then password
Click “No” in the Database Management dialog box which appears once the system
starts up (if management tasks are required click “Yes”).
6. Similarly, click “No” if the Test Archive Reminder dialog box appears (if archiving is
required click “Yes”).
7. At the top of the GeneXpert DX System window, click “Create Test”.
8. The Scan Sample ID Barcode dialog box appears. Scan the LIS barcode using the
scanner. If the label is unreadable, click “Manual Entry” and type the LIS number into
the Patient ID Barcode Entry dialog box.
9. Next, scan the CARBA R Assay barcode on front of the cartridge - this automatically
populates the following fields in the Create Test window: Selected Assay, Reagent Lot
ID, Cartridge SN, and Expiry Date.
10. Click “Start Test” (bottom of Create Test box) and the module on the GeneXpert to
be used starts to blink green. (In the Check Status Window, the selected instrument
module progress changes to Waiting).
11. Open the module door with the blinking green light and load cartridge.
12. Click door closed. The test starts and the green light stops blinking.
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be
checked against the document (titled as above) on the server prior to use.
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Policy # MI/IC/Xpert CARB-R/v01 Page 4 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual
Subject Title: Cepheid Xpert CARBA-R Assay
13. When the test is complete the module door unlocks and the green light turns off.
Remove and appropriately discard cartridge once the door lock releases.
14. A report is automatically printed once testing is completed.
15. To view results, click “View Results” on the menu bar. In the results window now
click “View Test”. In dialog box, select test of interest and click “OK”. Results of
selected test appear in the View Results window.
16. The View Results window contains three tabs: Results, Errors, and Support. The
Results tab displays Patient ID, Sample ID, assay name and test result. The Errors
tab lists errors encountered during the test process.
17. Monthly Maintenance Procedure may be found in the GeneXpert manual (LINK)
IV.
Assay Validation


V.
Each cartridge includes the following internal controls:
Sample Processing Control (SPC - dry spore cake of noninfectious spores) ensures
samples are adequately extracted and detects specimen-associated PCR-inhibition; SPC
must be POSITIVE in NEGATIVE samples, but can be negative or positive in
POSITIVE samples. Tests pass if SPC meets validated acceptance criteria, but
“INVALID” test results are generated if SPC is NOT detected in NEGATIVE tests.
Probe Check Control (PCC) which must pass before start of the PCR reaction – the
GeneXpert measurements must meet assigned acceptance criteria for the fluorescence
signals from all probes (monitors bead rehydration, reaction-tube filling, probe integrity,
dye stability).
Results interpretation
Using embedded GeneXpert calculation algorithms fluorescent signals from the 5 Xpert
Carba-R assay carbapenemase gene targets are interpreted and displayed as results in the
View Results window. The following POSITIVE (one representative example only) and
NEGATIVE, INVALID, ERROR, NO RESULT are the spectrum of results that may be
expected. Repeat test for any of the following reasons:
 An INVALID result indicates that the SPC failed. The sample was not properly
processed (i.e. overloaded with specimen) resulting in PCR- inhibition
 An ERROR result indicates that the Probe Check failed and the assay aborted
possibly due to improper filling of the reaction tube, or a reagent probe integrity
problem was detected, or maximum pressure limits were exceeded.
 A NO RESULT indicates that insufficient data were collected. For example, the
operator stopped a test that was in progress. Repeat the test using a new cartridge (do
not re-use the cartridge) and new Sample Reagent vial for dilution.
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be
checked against the document (titled as above) on the server prior to use.
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Policy # MI/IC/Xpert CARB-R/v01 Page 5 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual
Subject Title: Cepheid Xpert CARBA-R Assay
POSSIBLE RESULTS
POSITIVE
IMP NOT
DETECTED
VIM DETECTED
NDM NOT
DETECTED
INTERPRETATIONS
 VIM target DNA is detected but IMP, NDM, KPC and OXA48 sequences are not
 Ct value for VIM is within the valid range and endpoint above the minimum setting
 Values for IMP, NDM, KPC, OXA are absent or below the assay detection level
 SPC is ignored as VIM target DNA can compete with control amplification
 PCC=PASS indicates all pre-PCR probe checks passed
KPC NOT
DETECTED
OXA NOT
DETECTED
NEGATIVE
IMP NOT
DETECTED
VIM NOT
DETECTED
 IMP, VIM, NDM, KPC, and OXA target DNA sequences are not detected,
indicating their complete absence or their presence below assay detection level
 SPC=PASS indicates PCR amplification of SPC DNA gave a Ct value within valid
range and a fluorescence endpoint above the minimum setting
 PCC=PASS indicates all pre-PCR probe checks passed
NDM NOT
DETECTED
KPC NOT
DETECTED
OXA NOT
DETECTED
INVALID
ERROR
NO RESULT
 IMP, VIM, NDM, KPC, and OXA target DNA sequences are not detected as target
sequences are absent or below assay detection level
 SPC=FAIL: SPC Ct value or fluorescence endpoint out of valid range
 PCC=PASS; all probe check results pass
 Presence/absence of the 5 targets cannot be determined - repeat test
 SPC=NO RESULT
 PCC=FAIL: all or one probe check result failed possibly because the reaction tube
was filled improperly or a probe integrity problem was detected; if the probe check
passed, the error is caused by a system component failure
 Presence/absence of the 5 targets cannot be determined - repeat test. Insufficient data
were collected to produce result (e.g. operator stopped a test that was in progress)
 SPC=NO RESULT, PCC=Not applicable
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be
checked against the document (titled as above) on the server prior to use.
D:\81918348.doc
Policy # MI/IC/Xpert CARB-R/v01 Page 6 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual
Subject Title: Cepheid Xpert CARBA-R Assay
VI.
Reporting
Negative:
“Negative - No carbapenemase genes detected by
Cepheid Xpert CARBA-R Assay (for research use only).
This assay is able to detect NDM, KPC, OXA48, OXA181,
OXA232, OXA244, IMP-1, and VIM carbapenemase genes.”
Test Comment Code: }CARPositive:
“______ gene DETECTED by Cepheid Xpert CARBA-R Assay
(for research use only). This assay is able to detect
NDM, KPC, OXA48, OXA181, OXA232, OXA244, IMP-1, and VIM
carbapenemase genes.”
Test Comment Code: }CAR+
Notify Infection Control Practitioner as per Isolate Notification and Freezing Table
QPCMI15003.
VII.
Quality Control
Each test includes a Sample Processing Control (SPC) and Probe Check Control (PCC).
Refer to a senior technologist if control results are outside of limits or for any other
problems with running or reporting the assay.
VIII. References
Xpert CARBA-R PCR Assay package insert v 2.0
WILLEY to provide Posters/abstracts
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be
checked against the document (titled as above) on the server prior to use.
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Policy # MI/IC/Xpert CARB-R/v01 Page 7 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual
Subject Title: Cepheid Xpert CARBA-R Assay
Record of Edited Revisions
Manual Section Name: Cepheid Xpert CARBA-R PCR Assay
Page Number / Item
Date of Revision
Signature of
Approval
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be
checked against the document (titled as above) on the server prior to use.
D:\81918348.doc