1. GeneXpert Technology and Xpert MTB/RIF Procedures
Module 6:
GeneXpert Technology and
Xpert MTB/RIF Procedures
Slides adapted from Cepheid
Global Laboratory Initiative – Xpert MTB/RIF Training Package

2. Contents of this module
World Health Organization
08/12/13
Contents of this module
Overview of the technology
Preparation of sputum specimens (direct and processed)
Preparation of extrapulmonary samples
Xpert MTB/RIF assay procedures
Monitoring the procedure 2

3. Learning objectives At the end of this module, you will be able to:
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

4. GeneXpert Technology
Integrated micro-fluidic based system comprised of:
Instrument platform
Cartridges (self-contained)
Automated protocols
Internal controls 4

5. GeneXpert platform GeneXpert Infinity-48 GX-XVI GeneXpert® Module GX-I
GX-II
GX-IV 5 5

6. GeneXpert Module Plunger Motor Motherboard I-CORE Valve Drive Motor
Cartridge Inserter
Valve Drive Motor
Ultrasonic Horn 6

7. GeneXpert Module- Cartridge Bay7

8. Automated Xpert MTB/RIF Protocol
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

9. GeneXpert Cartridge 9

10. Components of a cartridge10

11. Principles of GeneXpert Design
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 11

12. Cartridge in Action: Movie
Movie is available from FTP: (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:

13. Real–Time Polymerase Chain Reaction
This slide may be considered optional for trainings of basic users
Target DNA
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
PCR
Fluorescence
Detection

14. Molecular Beacon Technology
This slide may be considered optional for trainings of basic users
Molecular beacon is a short segment of single-stranded DNA composed of:
Target specific sequence
Stem
Fluorescent dye
Quencher

15. Molecular Beacon Technology
This slide may be considered optional for trainings of basic users
Molecular Beacon
Target
Fluorescence
No Fluorescence
No Target = No Fluorescence

16. Molecular mechanisms of resistance to rifampicin
This slide may be considered optional for trainings of basic users
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)

17. Detection of mutations in rpoB gene by Xpert MTB/RIF
This slide may be considered optional for trainings of basic users
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

18. Internal Quality Controls: Probe Check Control (PCC)
This slide may be considered optional for trainings of basic users
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 pre- established factory settings in the software
A test is stopped if Probe check is not PASSED

19. Internal Quality Controls: Sample Processing Control (SPC)
This slide may be considered optional for trainings of basic users
Sample Processing Control (SPC)
Non-infectious spores
Verifies lysis of cells occurred successfully
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 19

20. Algorithm for determination of Xpert MTB/RIF results
This slide may be considered optional for trainings of basic users 20

21. Xpert MTB/RIF procedures21

22. Overview of the procedure1
Mix 2
Add 4
Detect 3
Insert 22

23. Sample preparation: direct sputum
2 ml is indicated by a line on the pipette
Pour sample reagent (buffer) carefully into the sample to avoid production of aerosols
Avoid pipetting any solid particles from the sample mix into the cartridge
Avoid creating bubbles when pipetting the sample mix into the cartridge

24. Sample preparation: direct sputum
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 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 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)

25. Sample preparation: processed sputum sediment
2. Add 1.5 ml of sample reagent (buffer) to 0.5 ml sediment (3:1 ratio)

26. Sample preparation: processed sputum sediment
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 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 times
After additional 5 min of incubation, sample should be perfectly fluid before being tested with no visible clumps of sputum

27. Sample preparation: splitting samples
Customize if samples are ever split

28. Extrapulmonary (EPTB) samples
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)
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.

29. Sample preparation: extrapulmonary samples
Lymph Node and Tissues
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
Load cartridge with 2+ ml
CREATE/RUN Xpert test
Alternative protocols are available
in WHO Xpert SOP for NONSTERILE
specimens. These samples require decontamination using similar protocols for indirect sputum sample processing.
BSC Required
It is recommended to also perform a culture from the ground tissue supernatant.

30. Sample preparation: extrapulmonary samples (2)
CSF samples (Note: blood stained samples may interfere with the Xpert test)
> 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
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
ml CSF sample
Add 2 ml Xpert MTB/RIF Sample Reagent
Add 2 ml into the cartridge and proceed with Xpert testing
4. <0.1 ml CSF sample
Insufficient sample for Xpert MTB/RIF testing
A BSC must be used whenever there is a risk of aerosolization, e.g., decanting

31. Sample preparation: work organization
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.

32. Do not use the cartridge if:
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

33. Preparation of Cartridge: Labelling
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

34. Preparation of cartridge: Inoculation
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

35. Storage of samples and inoculated cartridges
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:
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

36. Getting Started 1. Switch on GeneXpert instrument
(a small blue light will appear at the front panel)
2. Switch on the computer
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
Windows XP
Windows 7

37. Start Software
6. Click on “No” in the “Database management” dialogue box to begin your work session

38. Start Software: Check Status
7. Click on Check Status to confirm all modules available
- If not, proceed to troubleshooting (Module 9)

39. Starting a test Click on “CREATE TEST”
2. A window opens requesting to scan the cartridge barcode

40. Starting a test: scan the cartridge
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
In case of barcode reader failure while using a new lot, contact Cepheid technical support to collect the Lot Specific Parameter

41. Starting a test
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”

42. Always hold cartridge upright: avoid shaking, tilting or dropping
Load the cartridge
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

43. Manual entry of the cartridge barcode
If the barcode scanner is not working you can enter the cartridge barcode manually
Scan cartridge barcode and click on «Manual Entry»
Click on «Create Test»
Type manually the 2 line numbers of the cartridges

44. Summary of test processes: Movie
Movie is available from FTP: (paste in Windows Explorer, not Internet Explorer)
The FTP movies are large files so should be downloaded on to a CD before the training.

45. Monitor the test status
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)

46. Monitor the test status
Click on
“CHECK STATUS”
2. Progress, status,
and remaining time
are indicated
3. Messages
with more details

47. How to stop a test and why
In case your cartridge(s) was(were) not properly prepared, you might have to stop the test to avoid waste of time:
Click on “STOP TEST”
Select the module(s) that need to be stopped, by marking the tickbox
After selecting, click “Stop”

48. How to stop a test and why
Confirm your choice by clicking “Yes”
You will see the stopped test’s details in “Check Status” section

49. Visualize, generate and manage test reports
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

50. Summary
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

51. Assessment
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?

52. 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.