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

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) Xpert MTB/RIF assay procedures Monitoring the procedure 2

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

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

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

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

GeneXpert Module- Cartridge Bay 7

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

GeneXpert Cartridge 9

Components of a cartridge 10

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

Cartridge in Action: Movie Movie is available from FTP: ftp://hbdc:Crasa7Uc@ftp.caplaser.net (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

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

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

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

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)

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

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

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

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

Xpert MTB/RIF procedures 21

Overview of the procedure 1 Mix 2 Add 4 Detect 3 Insert 22

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

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 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)

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

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 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

Sample preparation: splitting samples Customize if samples are ever split

Sample preparation: work organization Prepare at a time only as many samples as the number of available (i.e. functioning) modules Start sample preparation within 30 minutes before a module is available Do not open a cartridge until you are ready to perform testing Use the cartridge within 30 minutes after opening the cartridge lid

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

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

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

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

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

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

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

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

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

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”

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

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

Summary of test processes: Movie Movie is available from FTP: ftp://hbdc:Crasa7Uc@ftp.caplaser.net (paste in Windows Explorer, not Internet Explorer) The FTP movies are large files so should be downloaded on to a CD before the training.

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)

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

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”

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

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

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) or Processed Sputum Sediment (3:1) – 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

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?