Presentation on theme: "GeneXpert Technology and Xpert MTB/RIF Procedures"— Presentation transcript:
1 GeneXpert Technology and Xpert MTB/RIF Procedures Module 6:GeneXpert Technology andXpert MTB/RIF ProceduresSlides adapted from CepheidGlobal Laboratory Initiative – Xpert MTB/RIF Training Package
2 Contents of this module World Health Organization08/12/13Contents of this moduleOverview of the technologyPreparation of sputum specimens (direct and processed)Preparation of extrapulmonary samplesXpert MTB/RIF assay proceduresMonitoring the procedure2
3 Learning objectives At the end of this module, you will be able to: Describe the technology underlying the GeneXpert and Xpert MTB/RIF assayExplain the Xpert MTB/RIF assay proceduresUse the software to monitor test results and generate reports
4 GeneXpert TechnologyIntegrated micro-fluidic based system comprised of:Instrument platformCartridges (self-contained)Automated protocolsInternal controls4
8 Automated Xpert MTB/RIF Protocol Fill the cartridge with prepared sample and insert it in the instrumentSample combines with Sample Processing Control (SPC)Filter captures the sample and SPCUltrasonically lysed cells release DNAEluted DNA mixes with dried-down bead reagentsSimultaneous amplification and detection of fluorescenceResults ready in less than 2 hoursEnd of hands-on work
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-overTotal internal control of reagents system – No separate external positive or negative controls requiredIntegrated ultrasonic lysis of cells for release of DNASoftware instructions to individual module motherboards to coordinate valve movement and integral hydraulic drivesSmart fluidics - Flow of liquids directed by micro valves – Allow using micro quantities of reaction componentsAutomated data analysis and results interpretation11
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 usersTarget DNASimultaneous amplification of target DNA by PCR, and detectionTarget DNA is amplified and labelled with fluorescently tagged probesThe accumulating product is monitored and measured by detecting the fluorescenceProbes used in Xpert technology are called “Molecular Beacons”More than one targets can be amplified and detected (multiplex)Each target is labelled with different dyesPCRFluorescenceDetection
14 Molecular Beacon Technology This slide may be considered optional for trainings of basic usersMolecular beacon is a short segment of single-stranded DNA composed of:Target specific sequenceStemFluorescent dyeQuencher
15 Molecular Beacon Technology This slide may be considered optional for trainings of basic usersMolecular BeaconTargetFluorescenceNo FluorescenceNo Target = No Fluorescence
16 Molecular mechanisms of resistance to rifampicin This slide may be considered optional for trainings of basic usersMutations in the rpoB gene encoding for β-subunit of RNA polymerase:prevent binding of rifampicin to RNA polymerase, synthesis of proteins and killing of bacilli95% 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 users5 overlapping probes – bind to wild type genes and do not bind to mutant sequences1 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 usersProbe 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 beadsFilling of the PCR tubeIntegrity of probesStability of a dye or the reagents/quencherResults are automatically compared to the pre- established factory settings in the softwareA 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 usersSample Processing Control (SPC)Non-infectious sporesVerifies lysis of cells occurred successfullyDetects specimen-related inhibition of amplification: should be positive in negative samples and can be negative or positive in positive samplesResult is invalid if SPC is negative in a negative sample19
20 Algorithm for determination of Xpert MTB/RIF results This slide may be considered optional for trainings of basic users20
22 Overview of the procedure 1Mix2Add4Detect3Insert22
23 Sample preparation: direct sputum 2 ml is indicated by a line on the pipettePour sample reagent (buffer) carefully into the sample to avoid production of aerosolsAvoid pipetting any solid particles from the sample mix into the cartridgeAvoid creating bubbles when pipetting the sample mix into the cartridge
24 Sample preparation: direct sputum Carefully unscrew the lid of sputum containerPour 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 SRReplace the lid, and shake vigorously times (one back and forth movement is a single shake), or vortexIncubate at room temperature for 10 minAfter 10 min of incubation, again shake (or vortex) the specimen vigorously timesAfter 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 vortexIncubate at room temperature for 10 minAfter 10 min of incubation, again shake (or vortex) the specimen vigorously timesAfter 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/RIFCerebral 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 TissuesUsing 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 suspensionAllow 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 ReagentProceed as described for standard Xpert sample processing for sputumLoad cartridge with 2+ mlCREATE/RUN Xpert testAlternative protocols are availablein WHO Xpert SOP for NONSTERILEspecimens. These samples require decontamination using similar protocols for indirect sputum sample processing.BSC RequiredIt 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 samplea) 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 Reagentd) Add concentrated specimen to cartridge and proceed with Xpert testingml CSF sample (including blood stained)a) Add equal volume of Xpert MTB/RIF Sample Reagentb) Add 2 ml into the cartridge and proceed with Xpert testingml CSF sampleAdd 2 ml Xpert MTB/RIF Sample ReagentAdd 2 ml into the cartridge and proceed with Xpert testing4. <0.1 ml CSF sampleInsufficient sample for Xpert MTB/RIF testingA 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) modulesStart sample preparation no more than 4 hours before a module is availableDo 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 passedIt appears wetIts lid seal appears broken or (accidentally) openedIt has been dropped or shaken after you added the treated sampleThe reaction tube on the back side appears to be damagedIt has already been processed: each cartridge is for single-use only, and can not be reused once scannedIts 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 sideLabel the cartridge with the sample ID by writing on the left or right side of the cartridge or affix ID labelDo 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 pipettePipette sample carefully to avoid aerosols and bubblesDo not transfer solid particles into the cartridgeClose lid firmlyStart the test
35 Storage of samples and inoculated cartridges Storage of specimen:Direct or processed (decontaminated/concentrated) sputumRefrigerate at 2–8°C, for 10 days maximumIf 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 daysStorage 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 hoursStorage of inoculated cartridge (eg, in case of power failure):Run test within 4 hours from the addition of the sampleIf 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 computer3. Log on to Windows as Cepheid user :User name: CepheidPassword : cphd4. Double click on “GeneXpert Dx” icon on the desktop5. Log-in with the user accountWindows XPWindows 7
37 Start Software6. 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 twiceNOTE: If the barcode scanner is not working you can enter the cartridge barcode manually by typing the 2 line number on the cartridgesIn case of barcode reader failure while using a new lot, contact Cepheid technical support to collect the Lot Specific Parameter
41 Starting a test4. After you have scanned the barcode, this window will appear5. Enter Patient ID = Name6. Enter Sample ID = Lab Serial Nr.By default, the software displaystest type as Specimen.Software automatically assigns amodule to be used(Note: the least used module isselected; a different module canbe manually selected)7. Click on “Start test”
42 Always hold cartridge upright: avoid shaking, tilting or dropping Load the cartridgeCompletely open the cartridge bay door of the assigned module indicated by the blinking green light above the selected moduleLoad the cartridge carefully with the barcode in frontClosing cartridge bay door automatically starts the testAlways 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 manuallyScan 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 startedIf 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 teststatus 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 timeare indicated3. Messageswith 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 tickboxAfter 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 resultsEdit test-related informationGenerate a result report (specimen and patient)How to print automatically test reportDifference between archive/backup: how to archive, retrieve, backup and restore data from a backupHow to copy/paste data to Excel
50 SummaryThe 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 accordinglyCheck for cartridge integrity before use, properly label, and inoculate with sufficient amount (2-4 ml) of final solutionAfter loading a cartridge into the GeneXpert, the accompanying software allows for test progress to be monitored
51 AssessmentList and describe the different components of the GeneXpert technologyList and describe the different controls and their mechanismDescribe the sample preparation procedure (according to the sample examined) and the subsequent steps necessary to create and run a testHow do you monitor the status of the test?Why might you want to stop a test? How would you do that?
52 AcknowledgementsThe 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.