How to Run Gene Expert Machine for MTB

Assay Technical Training Xpert® MTB/RIF Ultra For CE-IVD Use Only 1 © Cepheid 301-7915 Rev. B July 2017 Training Agenda • Xpert® MTB/RIF Ultra ‒ Clinical utility ‒ Reagents ‒ Sample collection ‒ Kit storage and handling ‒ Preparing the cartridge ‒ Quality Controls ‒ Results analysis • Discussion 2 © Cepheid Training Objectives • At the end of the training, user will be able to: ‒ Properly store and handle the Xpert® MTB/RIF Ultra cartridge kit ‒ Follow proper laboratory safety precautions ‒ Collect and transport appropriate specimen ‒ Prepare a cartridge and run the assay ‒ Report the various software generated results ‒ Understand the assay control strategy 3 © Cepheid The Cepheid Solution • Simultaneous detection ‒ MTB Complex and Rifampicin resistance associated mutations • On-board internal controls for each sample ‒ Probe Check Control (PCC) ‒ Specimen Processing Control (SPC) • Results in approximately: ‒ 65 minutes for a NEGATIVE ‒ 77 minutes for a POSITIVE • Closed cartridge system minimizes risk of contamination • On-demand results • Random access 4 © Cepheid Intended Use The Xpert MTB/RIF Ultra Assay, is a semi-quantitative, nested real-time polymerase chain reaction (PCR) in vitro diagnostic test for the detection of Mycobacterium tuberculosis (MTB) complex DNA in unprocessed sputum samples or concentrated sediments prepared from induced or expectorated sputum. In MTB positive specimens, it also detects rifampicin-resistance associated mutations of the rpoB gene. The Xpert MTB/RIF Ultra Assay is intended for use with specimens from untreated patients for whom there is clinical suspicion of tuberculosis (TB) and who have received no anti-tuberculosis therapy, or less than 3 days of therapy in the last 6 months. This test is intended as an aid in the diagnosis of pulmonary tuberculosis when used in conjunction with clinical and other laboratory findings. 5 © Cepheid Targets and Probes Targets ‒ IS1081 and IS6110 (multi-copy insertion element target present in most clinical TB strains) ‒ Rifampicin resistance determining region (RRDR) of rpoB gene (81 base pair) Probes ‒ 1 probe for SPC (Sample Processing Control) ‒ 2 probes bind to IS1081-IS6110 ‒ 4 probes bind to Rifampicin-R mutations in rpoB A melt analysis with the 4 rpoB probes differentiates between the conserved wild-type sequence and mutations associated with RIF resistant. 6 © Cepheid Assay Requirements GeneXpert Systems • GeneXpert Dx Software v4.7b or higher • Xpertise Software v6.4b or higher Test Kits (CE-IVD) • GXMTB/RIF-Ultra-10 and GXMTB/RIF-Ultra-50 Sample Collection • Leak proof, sterile screw-capped collection containers Other materials • Personal Protective Equipment (PPE) • 1:10 Bleach • 70% ethanol or denatured ethanol Optional • Uninterruptible Power Supply /Surge Protector • Printer • Vortex 7 © Cepheid Good Laboratory Practice Personnel Protective Equipment (PPE) Lab Bench area Specimens, Samples, and Kits Storage • Wear clean lab coats and gloves • Change gloves between processing samples • Clean work surfaces routinely with:  1:10 dilution of household bleach  70% Ethanol Solution • After cleaning, ensure work surfaces are dry • Store specimens and sample away from kit to prevent contamination • Use filtered tips when recommended Equipment(s) 8 © Cepheid • Follow the manufacturer’s requirements for calibration and maintenance of equipment(s) Kit Handling 9 © Cepheid Xpert MTB/RIF Ultra Kit Contents Catalog Number GXMTB/RIF-Ultra-10 GXMTB/RIF-Ultra-50 Cartridges Per Kit 10 or 50 Reagent Vials 10 or 50 Transfer Pipettes 12 or 60 Assay Definition File (ADF) Kit CD Kit of 10 cartridges Assay Import Instructions Package Insert (PDF) Storage 2-28 °C Cartridges contain chemically hazardous substances-please see Package Insert and Safety Data Sheet for more detailed information. 10 © Cepheid Kit of 50 cartridges Xpert MTB/RIF Ultra Assay Kit Storage and Handling • Store the Xpert MTB/RIF Ultra Assay cartridges and reagents at 2–28°C • Follow your institution’s safety procedures for working with chemicals and handling biological samples • Do not use Collection Reagent tubes that have not been validated by Cepheid • Open the Assay cartridge lid only when adding the Sample, close the lid and proceed with processing 11 © Cepheid Warnings and Precautions • Do not shake the cartridge • Do not use a cartridge that… : ‒ appears wet, has leaked or if the lid seal appears to have been broken ‒ appears damaged ‒ has been dropped after removing it from packaging ‒ has been dropped or shaken after adding the sample to it ‒ has a damaged reaction tube ‒ has been used: each cartridge is single-use to process one test ‒ is expired ‒ Do not reuse spent disposable pipettes 12 © Cepheid Warnings and Precautions • Biological specimens, transfer devices, and used cartridges should be considered capable of transmitting infectious agents requiring standard precautions. • Follow your institution’s environmental waste procedures for proper disposal of used cartridges and unused reagents. These materials may exhibit characteristics of chemical hazardous waste requiring specific national or regional disposal procedures. • If national or regional regulations do not provide clear direction on proper disposal, biological specimens and used cartridges should be disposed per WHO [World Health Organization] medical waste handling and disposal guidelines. 13 © Cepheid Xpert MTB/RIF Ultra Assay Limitations • The Xpert MTB/RIF Ultra Assay has only been validated with Sputum and Sputum sediment using Cepheid’s recommended procedure as per Package Insert. • A positive result does not necessarily indicate the presence of viable organisms. It is, however, presumptive for the presence of MTB and Rifampicin resistance. • Mutations or polymorphism in primer or probe binding regions may affect detection of new or unknown MDR MTB or Rifampicin resistant strains resulting in a false negative result. • The Xpert MTB/RIF Ultra Assay has only been evaluated in Patients > 18 year old. For detailed information, refer to the current Package Insert 14 © Cepheid Specimen Collection, Storage and Transport 15 © Cepheid Sputum Sample Collection • Subject must be seated or standing • Rinse the patient’s mouth twice with water • Explain to the patient how to: - open and close the collection device produce good sputum (collecting real sputum, not saliva) inhale deeply 2/3 times, breath out hard each time, cough deeply from the chest avoid contamination of the exterior of the container (carefully spitting and closing the container) - collect and safely deliver the sputum to the laboratory - keep the best sample Sputum is usually thick and mucous. It may be fluid and contain pieces of purulent material (pus). Color varies from opaque white to green. Bloody specimens will appear reddish or brown. Clear saliva or nasal discharge is not suitable as a TB specimen. Source: http://www.stoptb.org/wg/gli/assets/documents/TBLabDiagnosisSputum%20Microscopy_Handbook.pdf-(Images courtesy of A. Van Deun) 16 © Cepheid Specimen Collection, Transport and Storage Sample type Volume Transportation Storage Conditions up to 3 days Unprocessed Sputum 1-4 mL from day 4 to 10 Sputum sediment 17 0.5-2.5 mL up to 7 days • Collect sputum or induced sputum following your institution’s standard procedures. • Do not accept specimens with obvious food particles or other solid particles © Cepheid Cartridge Preparation 18 © Cepheid Cartridge Preparation – Unprocessed Sputum 2 1 3 • Shake* vigorously 10 to 20 times or vortex for at least 10 seconds. • Incubate at room temperature for 10 minutes. • Shake or vortex again vigorously 10 to 20 times. • Incubate for another 5 minutes **See Note below. Estimate volume of sputum. Add 2 volumes of SR to 1 volume of Sputum. Replace container lids. Obtain one Xpert cartridge, sample reagent (SR), and sputum collection container for each sample. *One back and forth motion equals one shake ** Shake and incubate an additional 5 minutes if the sample is not completely liquefied Note: Minimum Sputum volume for one test is 1 ml 5 4 Bring the cartridge to room temperature. Write on the side of the cartridge or affix an ID label. Open the cartridge lid. 19 © Cepheid 6 Aspirate the liquefied sample just above the line on the pipette. Adapted from 301-7000 7 Slowly empty the sample into the sample chamber of the cartridge. Close the lid firmly. Start the test within the time frame specified in the package insert. Cartridge Preparation – Sputum Sediment Sputum sediment prepared according to the Kent and Kubica method* is re-suspended in 67mM Phosphate/H2O buffer. 2 1 3 • Shake** vigorously 10 to 20 times or vortex for at least 10 seconds. • Incubate at room temperature for 10 minutes. • Shake or vortex again vigorously 10 to 20 times. • Incubate for another 5 minutes **See Note below. Collect at least 0.5mL of sediment*. For volumes ≥ 0.7mL add 2 volumes of reagent. Total volume must be at least 2 mL. Obtain one Xpert cartridge, sample reagent (SR), and sediment collection container for each sample. * For volume ~0.5-0.7mL, add 3 volumes of sample reagent to the sediment. 4 5 Bring the cartridge to room temperature. Write on the side of the cartridge or affix an ID label. Open the cartridge. 20 © Cepheid ** Shake and incubate an additional 5 minutes if the sample is not completely liquefied 7 6 Aspirate the liquefied sample just above the line on the pipette. Adapted from 301-7001 Slowly empty the sample into the sample chamber of the cartridge. Close the lid firmly. Start the test within the time frame specified in the package insert. Run a Test 1 Create Test 2 Scan barcode messages: 3 Scan the cartridge Cartridge/ Patient and/or Sample ID GeneXpert Start the test within 4 hours after adding the sample to the cartridge By default, do not click on Manual Entry or Cancel GeneXpert Infinity Place the cartridge on the conveyor within 30 minutes of adding the sample. "For complete details on how to run a test, refer to the Package Insert and the GeneXpert Dx or Xpertise Dx Operator Manuals. 21 © Cepheid Create a Test on GeneXpert Dx Software 4 Complete the fields as required 5 The Assay Protocol is selected automatically 6 The module is selected automatically DO NOT CHANGE IT!!! 7 Click on Start Test 8 A green light will flash on the module Load the cartridge into module and close the door 22 © Cepheid Xpert MTB/RIF Ultra Create a Test on Xpertise Dx Software 4 Complete the fields as required 5 The Assay Protocol is selected automatically 23 6 Click on SUBMIT 7 Place the cartridge into the conveyor belt © Cepheid Xpert MTB/RIF Ultra Automated Xpert Protocol 3 2 1 24 © Cepheid The cartridge is loaded into the System Sample is added to the cartridge Nucleic acids are purified Eluted DNA Purified mixes nucleicwith acids dried-down mix with the reagent PCR reagents beads 4 Simultaneous amplification and detection occurs 5 Results are ready to view 6 Quality Controls 25 © Cepheid Cepheid Control Strategy • System System Control – Check Status ‒ System control checks the optics, temperature of the module and mechanical integrity of each cartridge. ‒ If the system controls fail, an ERROR test result will be reported. • Assay Quality Controls • Each Xpert cartridge is a self-contained test device • Cepheid designed specific molecular methods to include internal controls that enable the system to detect specific failure modes within each cartridge. ‒ Specimen Processing Control (SPC) ‒ Probe Check Controls (PCC) 26 © Cepheid Internal Quality Controls • Probe Check Controls (PCC) • Before the PCR step, fluorescence signal is measured on all probes and compared with default factory settings to monitor ‒ bead rehydration ‒ probe integrity ‒ reaction tube filling ‒ dye stability • Sample Processing Controls (SPC) ‒ non-infectious spore in each cartridge ‒ Verifies adequate sample processing ‒ Verifies lysis, presence of the organism and detects PCR inhibition ‒ Should be positive in a negative sample ‒ Can be positive or negative in a positive sample 27 © Cepheid Commercially Available External Controls Catalog Number Description Configuration Storage MTB/RIF Ultra External Control manufactured by SmartSpots DCS-5 SmartSpots Positive Control (HBDC specific) 4 dried spots Room Temperature MTB/RIF Ultra Control Bundle External Control manufactured by MMQCI for Cepheid TBWT-04 (No mutation/ WT H37v) CTRL-M114-5 (MMQCI Cat. number: M114-5) TBMDR1-04 (mutations in: rpoB, inhA, katG) TB NEG (Negative control: No TB DNA, no cells) 5 x 1 mL 2-8°C 5 x 1 mL 2-8°C 5 x 1 mL 2-8°C For DCS-5 Controls, follow the instruction from manufacturer SmartSpot : http://www.tbgxmonitor.com/ For CRTL-M114-5 Controls, follow the instructions from manufacturer MMQCI : http://www.mmqci.com/qc-m114.php - Other vendors for quality control material are also available than the one outlined above. - External controls should be used in accordance with local, state accrediting organizations, as applicable 28 © Cepheid http://www.mmqci.com/qc-m114.php Result Interpretation 29 © Cepheid Result Interpretation Algorithm IS1081/IS6110 Pass Positive 0 or 1 probe Positive RpoB Probe Check Control (PCC) Fail ERROR Negative SPC 2 to 4 probes Positives Too low bacilli concentration Negative INVALID Positive MTB DETECTION RIFAMPICIN All 4 Wild Type peaks detected At least One MUT melt peak detected RESISTANCE < 4 valid melt peaks detected DETERMINATION Test stopped anytime 30 © Cepheid NO RESULT Xpert MTB/RIF Ultra – All Possible Results 31 IS1081 / IS6110 rpoB 1/2/3/4 Melt Analysis performed TB Results RIF Results + 2 and more + Yes MTB DETECTED HIGH RIF Resistance DETECTED + All + Yes MTB DETECTED HIGH RIF Resistance NOT DETECTED + 2 and more + Yes MTB DETECTED HIGH RIF Resistance INDETERMINATE + 2 and more + Yes MTB DETECTED MEDIUM RIF Resistance DETECTED + All + Yes MTB DETECTED MEDIUM RIF Resistance NOT DETECTED + 2 and more + Yes MTB DETECTED MEDIUM RIF Resistance INDERMINATE + 2 and more + Yes MTB DETECTED LOW RIF Resistance DETECTED + All + Yes MTB DETECTED LOW RIF Resistance NOT DETECTED + 2 and more + Yes MTB DETECTED LOW RIF Resistance INDERTERMINATE + 2 and more + Yes MTB DETECTED VERY LOW RIF Resistance DETECTED + All + Yes MTB DETECTED VERY LOW RIF Resistance NOT DETECTED + 2 and more + Yes MTB DETECTED VERY LOW RIF Resistance INDETERMINATE RIF Resistance INDETERMINATE © Cepheid + 0 or 1 + No MTB TRACE DETECTED FAIL 0,1 or 2 + No MTB NOT DETECTED FAIL FAIL No INVALID FAIL FAIL No INVALID MTB DETECTED MEDIUM; RIF Resistance DETECTED The MTB target is present within the sample: – At least one rpoB mutation has been detected. – SPC: NA (not applicable). An SPC signal is not required because MTB amplification can compete with this control. – Probe Check: PASS 32 © Cepheid MTB DETECTED MEDIUM; RIF Resistance NOT DETECTED The MTB target is present within the sample: – No mutation in the rpoB gene target sequence has been detected. – SPC: NA (not applicable) An SPC is not required because MTB amplification can compete with this control – Probe Check: PASS 33 © Cepheid MTB DETECTED LOW; RIF Resistance INDETERMINATE The MTB target is present within the sample: – IS1081/IS6110 signal is detected – RIF resistance could not be determined due to insufficient signal detection – SPC: NA (not applicable). An SPC signal is not required because MTB amplification can compete with this control. – Probe Check: PASS 34 © Cepheid MTB Trace DETECTED The MTB target is present within the sample: – IS1081-IS6110 signal is detected. – RIF resistance cannot be determined due to insufficient signal detection. – SPC: NA (not applicable). An SPC signal is not required because MTB amplification can compete with this control. – Probe Check: PASS A TRACE result call means that low levels of MTB are detected but no RIF resistant result is detected. A TRACE result is always RIF Resistance INDETERMINATE 35 © Cepheid MTB NOT DETECTED The MTB target is NOT detected within the sample: – RIF resistance cannot be determined due to insufficient signal detection – SPC: PASS. The SPC met the acceptance criteria. – Probe Check: PASS 36 © Cepheid Troubleshooting 37 © Cepheid Factors That Negatively Affect Results • Improper specimen collection ‒ The bacterial load in the specimen is below the detection limit of the test ‒ Performance with extra-pulmonary specimen types has not been assessed • Improper transport or storage of collected specimen ‒ Storage and transport conditions are specimen specific ‒ Refer to the Package Insert for the appropriate handling instructions • Improper testing procedure ‒ Modification to the testing procedures may alter the performance of the test ‒ Careful compliance with the package insert is necessary to avoid erroneous results 38 © Cepheid INVALID Result The presence or absence of MTB target cannot be determined – SPC: FAIL. The SPC does not meet the acceptance criteria. – Probe Check: PASS ‒ Possible Cause ‒ Improper sample collection ‒ Incorrect sample preparation ‒ Improper storage of the cartridges ‒ Inefficient sample processing in cartridge ‒ Missing primer/probe or enzyme beads ‒ Presence of interfering substances in the sample ‒ Solution ‒ 39 © Cepheid Repeat the test with a new cartridge and new sample INVALID Result – The presence or absence of MTB cannot be determined ‒ Cause ‒ Defect in reaction tube ‒ Poor thermal contact of the reaction tube with the instrument thermal plates ‒ Solution ‒ 41 © Cepheid Repeat the test with a new cartridge ERROR – The presence or absence of MTB cannot be determined – MTB: NO RESULT. – SPC: NO RESULT. – If Probe Check: FAIL Possible Cause – Improper Sample collection – Incorrect Sample volume added to the cartridge – If Probe Check: PASS Cause – Check the GeneXpert System module ‒ Solution ‒ 42 © Cepheid Repeat the test with a new cartridge NO RESULT Xpert MTB-RIF Ultra 1 – The presence or absence of MTB cannot be determined. – MTB: NO RESULT – SPC: NO RESULT – Probe Check: NA (not applicable) ‒ Possible Cause ‒ A NO RESULT indicates that insufficient data were collected. ‒ Test was stopped with stop test button ‒ Electrical failure ‒ Solution 43 © Cepheid ‒ Secure the power ‒ Repeat the test with a new cartridge Re-test Procedure 1 Discard used cartridge Follow your institution’s safety guidelines for disposal of cartridges 2 Obtain the residual sample, mix according to Package Insert If the leftover sample volume is insufficient, or the retest continues to return an INVALID, ERROR, or NO RESULT, collect a new sample 44 © Cepheid 3 Obtain a new cartridge Label appropriately as retest on the new cartridge Process the sample per the package insert 4 Run the test on the System Technical Assistance • Before contacting Cepheid Technical Support, collect the following information: ‒ ‒ ‒ ‒ ‒ Product name Lot number Serial number of the System Error messages (if any) Software version and, if applicable, Computer Service Tag number • Log your complaint online using the following link http://www.cepheid.com/us/support : Create a Support Case Region US Australia and New Zealand Brazil and Latin America China France Germany India, Bangladesh, Bhutan, Nepal, and Sri Lanka Italy Japan South Africa United Kingdom Belgium and Netherlands Other European, Middle East, and African countries 45 © Cepheid Telephone Technical Support Email + 1 888 838 3222 techsupport@cepheid.com + 1800 107 884 (AU) + 0800 001 028 (NZ) + 55 11 3524 8373 + 86 021 5406 5387 + 33 563 825 319 + 49 69 710 480 480 + 91 11 48353010 + 39 800 902 567 + 0120 95 4886 + 27 861 22 76 35 + 44 3303 332 533 +33 563 825 3319 + 33 563 825 319 + 971 4 253 3218 techsupportANZ@cepheid.com latamsupport@cepheid.com techsupportchina@cepheid.com support@cepheideurope.com support@cepheideurope.com techsupportindia@cepheid.com support@cepheideurope.com support@japan.cepheid.com support@cepheideurope.com support@cepheideurope.com support@cepheideurope.com support@cepheideurope.com 46 © Cepheid