Transcript Module 06_TECHNOLOGY.

Module 6:
GeneXpert Technology and
Xpert MTB/RIF Procedures
Slides adapted from Cepheid
Global Laboratory Initiative – Xpert MTB/RIF Training Package
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Overview of the technology
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Preparation of sputum specimens (direct and processed)
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Preparation of extrapulmonary samples
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Xpert MTB/RIF assay procedures
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Monitoring the procedure
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
At the end of this module, you will be able to:
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Describe the technology underlying the GeneXpert and
Xpert MTB/RIF assay
Explain the Xpert MTB/RIF assay procedures
Use the software to monitor test results and generate
reports
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Integrated micro-fluidic based system comprised of:
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Instrument platform
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Cartridges (self-contained)
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Automated protocols
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Internal controls
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Xpert MTB/RIF Training Package
GeneXpert
Infinity-48
GeneXpert®
Module
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GX-XVI
GX-I
GX-II
GX-IV
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Xpert MTB/RIF Training Package
Plunger Motor
Motherboard
I-CORE
Valve Drive Motor
Cartridge
Inserter
Ultrasonic
Horn
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Xpert MTB/RIF Training Package
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Xpert MTB/RIF Training Package
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Fill the cartridge with prepared sample
and insert it in the instrument
Sample combines with Sample
Processing Control (SPC)
Filter captures the sample and SPC
Ultrasonically lysed cells release DNA
Eluted DNA mixes with dried-down
bead reagents
Simultaneous amplification and
detection of fluorescence
Results ready in less than 2 hours
End of hands-on work
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Xpert MTB/RIF Training Package
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Xpert MTB/RIF Training Package
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Xpert MTB/RIF Training Package
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Real-time PCR (amplification and detection at the same time)
No wet interface between instrument and cartridge to eliminate
carry-over
Total internal control of reagents system – No separate external
positive or negative controls required
Integrated ultrasonic lysis of cells for release of DNA
Software instructions to individual module motherboards to
coordinate valve movement and integral hydraulic drives
Smart fluidics - Flow of liquids directed by micro valves – Allow
using micro quantities of reaction components
Automated data analysis and results interpretation
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Movie is available from FTP: ftp://hbdc:[email protected]
(paste in Windows Explorer, not Internet Explorer)
The FTP movies are large files so should be downloaded on to a CD before the training.
Alternatively a movie can be accessed via Youtube:
http://www.youtube.com/watch?v=mIsBLmjus6Q
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
This slide may be considered optional for trainings of basic users
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Simultaneous amplification of
target DNA by PCR, and detection
Target DNA is amplified and
labelled with fluorescently tagged
probes
The accumulating product is
monitored and measured by
detecting the fluorescence
Probes used in Xpert technology are
called “Molecular Beacons”
More than one targets can be
amplified and detected (multiplex)
Each target is labelled with different
dyes
Target DNA
PCR
Fluorescence
Detection
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Xpert MTB/RIF Training Package
This slide may be considered optional for trainings of basic users
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Molecular beacon is a
short segment of
single-stranded DNA
composed of:
◦ Target specific
sequence
◦ Stem
◦ Fluorescent dye
◦ Quencher
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Xpert MTB/RIF Training Package
This slide may be considered optional for trainings of basic users
Molecular Beacon
Target
Fluorescence
No Fluorescence
No Target = No Fluorescence
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Xpert MTB/RIF Training Package
This slide may be considered optional for trainings of basic users
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Mutations in the rpoB gene encoding for β-subunit of
RNA polymerase:
◦ prevent binding of rifampicin to RNA polymerase,
synthesis of proteins and killing of bacilli
95% of all resistance to rifampicin are due to mutations in
the rpoB gene and 5% due to mutations outside the gene
> 90% of mutations in the rpoB gene are located in the 81
base pairs region (codons 507 – 533)
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Xpert MTB/RIF Training Package
This slide may be considered optional for trainings of basic users
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5 overlapping probes – bind to wild type genes and do
not bind to mutant sequences
1 probe to Sample Processing Control - SPC (Bacillus
globigii)
Total 6 fluorescent dyes detected at the same time
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Xpert MTB/RIF Training Package
This slide may be considered optional for trainings of basic users
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Probe Check Control (PCC) – testing of fluorescence
readings at different temperatures before start of thermal
cycling, to evaluate the response of the chemicals
contained in a cartridge. PCC verifies:
Re-hydration of beads
Filling of the PCR tube
Integrity of probes
Stability of a dye or the reagents/quencher
Results are automatically compared to the preestablished factory settings in the software
A test is stopped if Probe check is not PASSED
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Xpert MTB/RIF Training Package
This slide may be considered optional for trainings of basic users
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Sample Processing Control (SPC)
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Non-infectious spores
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Verifies lysis of cells occurred successfully
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Detects specimen-related inhibition of amplification:
should be positive in negative samples and can be
negative or positive in positive samples
Result is invalid if SPC is negative in a negative sample
Global Laboratory Initiative
Xpert MTB/RIF Training Package
This slide may be considered optional for trainings of basic users
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Xpert MTB/RIF Training Package
Xpert MTB/RIF procedures
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Xpert MTB/RIF Training Package
1
Mix
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2
Add
3
Insert
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Detect
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Xpert MTB/RIF Training Package
2 ml is indicated
by a line on the
pipette
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Pour sample reagent (buffer) carefully into the sample to avoid production of aerosols
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Avoid pipetting any solid particles from the sample mix into the cartridge
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Avoid creating bubbles when pipetting the sample mix into the cartridge
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Xpert MTB/RIF Training Package
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Carefully unscrew the lid of sputum container
Pour 2 volumes of sample reagent (SR) directly into 1 volume of sputum
in the sputum container (1 ml of sputum is the minimum quantity, while
3-4 ml is the optimal quantity required)
For larger volume specimens (over 4 ml), a portion of SR from a second
bottle would be needed, as each bottle contains 8 ml of SR
Replace the lid, and shake vigorously 10-20 times (one back and forth
movement is a single shake), or vortex
Incubate at room temperature for 10 min
After 10 min of incubation, again shake (or vortex) the specimen
vigorously 10-20 times
After additional 5 min of incubation, sample should be perfectly fluid
before being tested, with no visible clumps of sputum. If still viscous,
wait 5-10 more minutes before inoculating in the cartridge (2-4 ml of
the final solution)
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Xpert MTB/RIF Training Package
2. Add 1.5 ml of sample
reagent (buffer) to
0.5 ml sediment
(3:1 ratio)
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Add 1.5 ml of sample reagent to 0.5 ml of suspended sediment
from digested/decontaminated and concentrated sputum
specimen (Note: ratio of SR to sample 3:1)
Replace the lid, and shake vigorously 10-20 times (one back and
forth movement is a single shake), or vortex
Incubate at room temperature for 10 min
After 10 min of incubation, again shake (or vortex) the specimen
vigorously 10-20 times
After additional 5 min of incubation, sample should be perfectly
fluid before being tested with no visible clumps of sputum
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Customize if samples are ever split
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
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WHO recommends the use of the following EPTB samples for Xpert
MTB/RIF
◦ Cerebral Spinal Fluid (CSF)
◦ Lymph node and other tissues (samples must be prepared in a BSC)
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CSF samples are typically paucibacillary and can be processed similar to sputum.
However, concentration using centrifugation (sedimentation) may provide better
test results. A laboratory with a BSC is needed for opening the centrifuge cups
and for decanting the supernatant.
Liquid specimens which may arrive in a capped syringes should be prepared in a
BSC for Xpert testing. Expressing the syringe contents into a 50 mL conical tube
may also cause aerosolization of bacilli. Safety procedures for working with sharp
objects should be followed.
Tissue samples require homogenization using special grinders that may create
aerosols. These specimens MUST be prepared in a BSC.
SOP can be found in the WHO Xpert Implementation manual; Annex 2.
www.who.int/tb/publications/xpert_implem_manual
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
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Lymph Node and Tissues
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Using sterile scissors and forceps cut-up tissue
sample and place into homogenizer/grinder
(shown)
Add 2 ml of sterile phosphate buffered saline (PBS)
Place lid on homogenizer and grind until a finely
ground homogeneous suspension
Allow large particles to settle (5-10 min)
Place 0.7 ml of supernatant into a conical screw
capped tube (be sure NO tissue clumps are
transferred)
Add 1.4 ml of Xpert Sample Reagent
Proceed as described for standard Xpert sample
processing for sputum
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Load cartridge with 2+ ml
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CREATE/RUN Xpert test
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BSC
Required
Alternative protocols are available
in WHO Xpert SOP for NONSTERILE
specimens. These samples require
decontamination using similar protocols
for indirect sputum sample processing.
It is recommended to also perform a
culture from the ground tissue
supernatant.
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Xpert MTB/RIF Training Package
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CSF samples (Note: blood stained samples may interfere with the Xpert test)
1. > 5 ml CSF sample
a) Transfer into conical tube and centrifuge (3000g:15 min)
b) Decant supernatant into disinfectant solution (BSC required)
c) Resuspend sediment to a volume of 2 ml using Xpert MTB/RIF Sample Reagent
d) Add concentrated specimen to cartridge and proceed with Xpert testing
2. 1-5 ml CSF sample (including blood stained)
a) Add equal volume of Xpert MTB/RIF Sample Reagent
b) Add 2 ml into the cartridge and proceed with Xpert testing
3. 0.1-1 ml CSF sample
a) Add 2 ml Xpert MTB/RIF Sample Reagent
A BSC must be
used whenever
there is a risk of
aerosolization,
e.g., decanting
b) Add 2 ml into the cartridge and proceed with Xpert testing
4. <0.1 ml CSF sample
a) Insufficient sample for Xpert MTB/RIF testing
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Xpert MTB/RIF Training Package
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Prepare at a time only as many samples as the number of
available (i.e. functioning) modules
Start sample preparation no more than 4 hours before a
module is available
Do not open a cartridge until you are ready to insert the
sample; the lid should be closed as soon as the sample is
added.
Global Laboratory Initiative
Xpert MTB/RIF Training Package
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The expiration date has passed
It appears wet
Its lid seal appears broken or (accidentally) opened
It has been dropped or shaken after you added the
treated sample
The reaction tube on the back side appears to be
damaged
It has already been processed: each cartridge is for
single-use only, and can not be reused once scanned
Its package (pouch of 10 cartridges) has been open for
more than 6 weeks
Global Laboratory Initiative
Xpert MTB/RIF Training Package
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Pick up the cartridge only by
the right and left side. Do not
touch the lid, the barcode on
the front side, or the reaction
tube on the back side
Label the cartridge with the
sample ID by writing on the left
or right side of the cartridge or
affix ID label
Do not put the label on the lid
of the cartridge or obstruct the
existing 2D barcode on the
cartridge
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Xpert MTB/RIF Training Package
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Open the cartridge and pipette
2-4 ml of prepared sample
using the plastic transfer
pipette
Pipette sample carefully to
avoid aerosols and bubbles
Do not transfer solid particles
into the cartridge
Close lid firmly
Start the test
Global Laboratory Initiative
Xpert MTB/RIF Training Package
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Storage of specimen:
Direct or processed (decontaminated/concentrated) sputum
Refrigerate at 2–8°C, for 10 days maximum
If necessary, at room temperature (up to 35°C) for up to 3 days,
and then refrigerated at 2–8°C, for a combined maximum of 10
days
Storage of specimen in presence of sample reagent:
Direct or processed (decontaminated/concentrated) sputum
Process within 12 hours, kept at 2-8°C degrees. If refrigeration
is not possible, process within 5 hours
Storage of inoculated cartridge (eg, in case of power failure):
Run test within 4 hours from the addition of the sample
If more than 4 hours has elapsed, inoculate a new cartridge
Global Laboratory Initiative
Xpert MTB/RIF Training Package
1. Switch on GeneXpert instrument
(a small blue light will appear at the front
panel)
Windows XP
2. Switch on the computer
Windows 7
3. Log on to Windows as Cepheid user :
User name: Cepheid
Password : cphd
4. Double click on “GeneXpert Dx” icon on the
desktop
5. Log-in with the user account
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Xpert MTB/RIF Training Package
6. Click on “No” in
the “Database
management”
dialogue box to
begin your work
session
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Xpert MTB/RIF Training Package
7. Click on Check Status to confirm all modules available
- If not, proceed to troubleshooting (Module 9)
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Xpert MTB/RIF Training Package
1.Click on “CREATE TEST”
2. A window opens requesting to scan the cartridge barcode
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Xpert MTB/RIF Training Package
3. Take the barcode scanner and scan the cartridge barcode by holding
the yellow button pressed.
After the ‘BEEP’ sound, move the
scanner away from cartridge to avoid
scanning the barcode twice
NOTE: If the barcode scanner is not
working you can enter the cartridge
barcode manually by typing the 2
line number on the cartridges
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In case of barcode reader failure
while using a new lot, contact
Cepheid technical support to
collect the Lot Specific Parameter
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Xpert MTB/RIF Training Package
4. After you have scanned the barcode,
this window will appear
5. Enter Patient ID = Name
6. Enter Sample ID = Lab Serial Nr.
By default, the software displays
test type as Specimen.
Software automatically assigns a
module to be used
(Note: the least used module is
selected; a different module can
be manually selected)
7. Click on “Start test”
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Xpert MTB/RIF Training Package
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Completely open the cartridge
bay door of the assigned module
indicated by the blinking green
light above the selected module
Load the cartridge carefully with
the barcode in front
Closing cartridge bay door
automatically starts the test
Always hold cartridge upright: avoid shaking, tilting or dropping
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Xpert MTB/RIF Training Package
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If the barcode scanner is not working you can enter the
cartridge barcode manually
Click on «Create Test»
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Scan cartridge barcode and
click on «Manual Entry»
Type manually the 2
line numbers of the
cartridges
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Movie is available from FTP: ftp://hbdc:[email protected]
(paste in Windows Explorer, not Internet Explorer)
The FTP movies are large files so should be downloaded on to a CD before the training.
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
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Under “Check status” (see next slide), ensure that the status has
been updated from “loading” to “run”, meaning that the testing
process has started
If you want to see the progress of the test run, you can check:
progress of the test (for example, 3/45 means the test is on the
third PCR cycle out of 45 cycles)
amount of time remaining until the end of the test
status of the test (for example: “OK”)
If the Status displays Error or Warning, look at the Messages for a
description of the problem (see next slide)
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Xpert MTB/RIF Training Package
1.Click on
“CHECK STATUS”
2. Progress, status,
and remaining time
are indicated
3. Messages
with more details
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Xpert MTB/RIF Training Package
In case your cartridge(s)
was(were) not properly
prepared, you might have to
stop the test to avoid waste
of time:
1. Click on “STOP TEST”
2. Select the module(s) that
need to be stopped, by
marking the tickbox
3. After selecting, click
“Stop”
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Xpert MTB/RIF Training Package
4. Confirm your choice by
clicking “Yes”
5. You will see the
stopped test’s details in
“Check Status” section
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
See Module 7 on Results Interpretation for the following
topics:
 Visualizing the test results
 Edit test-related information
 Generate a result report (specimen and patient)
 How to print automatically test report
 Difference between archive/backup: how to archive,
retrieve, backup and restore data from a backup
 How to copy/paste data to Excel
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
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The Xpert MTB/RIF assay is an integrated micro-fluidic based
system comprised of a GeneXpert instrument, Xpert MTB/RIF test
cartridges, an automated protocol (simultaneous DNA
amplification and detection of fluorescence) with in-built controls
(SPC and PCC)
Sample preparation protocols should be followed for direct
sputum (2:1), processed sputum sediment (3:1), and
extrapulmonary samples – adjust biosafety requirements
accordingly
Check for cartridge integrity before use, properly label, and
inoculate with sufficient amount (2-4 ml) of final solution
After loading a cartridge into the GeneXpert, the accompanying
software allows for test progress to be monitored
Global Laboratory Initiative
Xpert MTB/RIF Training Package
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List and describe the different components of the
GeneXpert technology
List and describe the different controls and their
mechanism
Describe the sample preparation procedure (according to
the sample examined) and the subsequent steps
necessary to create and run a test
How do you monitor the status of the test?
Why might you want to stop a test? How would you do
that?
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Acknowledgements
The Xpert MTB/RIF Training Package has been developed by a consortium of
GLI partners, including FIND, KNCV, US CDC, USAID, TB CARE I and WHO, with
funding from USAID.
The modules are based on materials originally developed by FIND, KNCV and
Cepheid.