GeneXpert (CBNAAT) for MTB

GeneXpert (CBNAAT) for MTB Xpert MTB/RIF Assay (Cepheid) or CBNAAT - Automated cartridge based nucleic acid amplification test (CBNAAT) - Integrated sample processing & real-time PCR - Uses 3 primers & 5 molecular probes - Minimal hands on technical time - Results in <2 hrs - Identifies MTBC & detects Rifampicin resistance simultaneously - Samples – Pulmonary (FDA approved) - Extrapulmonary (body fluids, tissues) except blood - POINT OF CARE TEST - Endorsed by WHO in 2010 - Sensitivity : 131 cfu/ml sputum (comparable to culture) Who should be tested with Xpert MTB/RIF in RNTCP Key populations tested include:  Presumptive MDR-TB patients  Children with presumptive TB  PLHIV with presumptive TB  Presumptive Extra-pulmonary TB WHO Recommendations for Xpert MTB/RIF: Xpert MTB/RIF should be used :  As the initial diagnostic test in adults & children presumed to have MDR TB or HIV associated TB  As the initial diagnostic test in testing CSF specimens from patients presumed to have TB meningitis Why GeneXpert? Why GeneXpert System for TB diagnosis?  Point of care (POC), cartridge based real time PCR system  Fully automated (extraction, amplification, detection & result interpretation)  Rapid (results available within 2 hrs)  Simple and easy to use  Less chances of carry-over or cross contamination  No separate external controls (positive or negative) required  Less exposure to infectious material  Less manual errors GeneXpert Technology GeneXpert system includes: • GeneXpert Instrument • Thermal and Optical system • Cartridge • Self contained • Disposable • Computer System • Software (GX Dx System software) • Barcode Scanner • Important Accessories • Printer • UPS Principle • GeneXpert system integrates & automates1. Sample processing 2. Nucleic acid amplification 3. Detection of target sequences • Xpert MTB/RIF assay is based on semi-nested real time PCR & molecular beacon technology for simultaneous detection of MTB & Rif resistance. GeneXpert is a software-driven cartridge processor and integrated fluorescence-based quantitative thermocycler that interfaces with single-use disposable cartridges to achieve nucleic acid isolation and amplification (rT-PCR). The microfluidics-based GeneXpert cartridge consists of multiple chambers that are designed to hold the biological sample and preloaded with the liquid buffers and lyophilized reagent beads (for sample processing, DNA/RNA extraction), and real-time PCR reagents, enzymes, and buffers and to retain all sample-processing wastes. The cartridge also has an attached PCR tube with fluidic connections to the reagents in the cartridge chambers. When inserted into the GeneXpert, the PCR tube is surrounded by heating/cooling plates and by optical blocks that enable amplification and real-time, fluorescence-based PCR product detection. • At the centre of the cartridge is a syringe barrel that has a dry interface (which nearly eliminates the potential for contamination) with the GeneXpert through a syringe plunger, thereby allowing the movement of fluids within the cartridge and between the PCR tube and the cartridge. • Fluid movement within the cartridge is controlled by a rotary valve that provides access for fluid movement between different chambers and to the PCR tube. • The valve body assembly also contains an active region fitted with a cavity containing nucleic acid purification beads. Within the cavity are inlet and outlet ports that provide for fluid movement over the purification beads. • Reagents were manually loaded into the cartridge reservoirs before the assay, and all subsequent fluid movement within the cartridge is controlled by the GeneXpert software. GeneXpert Cartridges for MTB detection Xpert MTB/RIF Xpert MTB/RIF Ultra Xpert MTB/XDR GeneXpert MTB/RIF Assay GeneXpert MTB/RIF Assay - Cartridge Based Nucleic acid Technology  The cartridge based nucleic acid amplification test (CBNAAT) or GeneXpert MTB/RIF test is a molecular test to simultaneously diagnose TB and resistance to rifampicin  More than 95% of mutations in the rpoB gene are located in the 81 base pair core region (codons 507 – 533) also known as Rifampicin Resistance Determining Region (RRDR)  The assay identifies Mycobacterium tuberculosis complex and resistance to rifampicin by detecting mutations in 81 base pair core region of rpo B gene in <2 hours  Developed by Foundation for Innovative New Diagnostics (FIND) in partnership with Cepheid and the University of Medicine and Dentistry of New Jersey, with support from United States National Institutes of Health and the Bill & Melinda Gates Foundation  Xpert MTB/RIF assay was endorsed by WHO in 2010 for use in TB endemic countries Overview of Assay Technology The Xpert MTB/RIF assay utilizes real-time Polymerase Chain Reaction Technology, to both diagnose MTB and detect Rifampicin resistance, using clinical specimens Two key steps involved are: 1. Semi-nested PCR of MTB target DNA after its release into the solution in the cartridge 2. Detection of PCR amplified products by the instrument in real time using molecular beacons (probes) Probes bind to amplicons & generates flouorescent signals with each PCR cycle PCR Amplification of Target P 1 P 2 Semi-nested PCR using a set of three primers P1, P2 and P3 for RRDR P 3 P 1 rpoB gene containing 81 bp RRDR  1st round of amplification takes RRDR P 3 P 2 amplification of 192 bp region of place using primers P1 and P3 (19 cycles)  In the next round P2 and P3 are RRDR P 3 used to amplify the target (40 cycles) Molecular Beacon Technology MB: Oligonucleotide probe Loop: 18-30 base pair region of the molecular beacon, which is complementary to the target sequence Structure of a Molecular Beacon Probe sequence (Complementary to target) Stem: 5-7 bp complementary sequence on both the ends of the loop 5’ Fluorophore: Fluorescent dye covalently linked at the 5' end 3’ Quencher: Nonfluorescent dye covalently linked at the 3' end Stem Fluorescent dye Quencher Molecular Beacon Technology Target Absent Wild Type Target No Hybridization No Fluorescence Hybridization leads to Fluorescence Mutant Target No Hybridization No Fluorescence Molecular Beacon Technology Probe C Probe A Probe D Probe B Probe E  5 overlapping probes – bind to wild type gene and do not bind to mutant sequences.  1 probe to Sample Processing Control - SPC (Bacillus globigii) Not shown here  Each probe is labeled with a different coloured fluorophore  Total 6 fluorescent dyes detected at the same time • Xpert MTB/RIF assay has a limit of detection (minimum number of bacilli that can be detected with 95% confidence) of 131 CFU per ml of sputum. • Approximately 95% of RIF-resistant tuberculosis cases contain mutations in this 81bp region of rpoB gene. • Identify 23 common clinically occurring rpoB mutations. • The PCR assay amplifies a 192-bp segment of the M. tuberculosis rpoB gene in a Seminested real-time PCR. • M. Tuberculosis & RIF resistance is detected using five overlapping molecular beacon probes (probes A to E) that are complementary to the entire 81-bp RIF resistance-determining “core” region of the wild-type rpoB gene. • Mutations in the rpoB gene target inhibit hybridization of one or more of the rpoBspecific molecular beacons, reducing or eliminating the signal from the corresponding probes. • M. tuberculosis is identified when at least two of the five rpoB-specific molecular beacons or probes give a positive signal with cycle threshold (CT ) values that are ≤38 and that differ by no more than two cycles. • The difference in CT between the first (early CT ) and last (later CT ) M. tuberculosisspecific molecular beacon (ΔCT Max) is the basis of rpoB mutation and RIF resistance detection. • RIF resistance is identified if the ΔCT Max is >3.5 cycles. • RIF susceptibility is identified if the ΔCT Max is ≤3.5 cycles. • A sample is considered RIF indeterminate when the last probe returns a CT of >38 and the first probe has a CT value of >34.5 cycles because the assay terminates at cycle 38, and a ΔCT Max of >3.5 cannot be measured. GeneXpert Technology - Inbuilt Controls The Inbuilt Controls in GeneXpert Technology are:  System Check Control  Specimen Processing Control (SPC)  Probe Check Control (PCC) Sample Processing Control Sample Processing Control (SPC)  Sample Processing Control monitors sample processing  The SPC contains noninfectious spores of Bacillus globigii. The probes and primers are provided in the cartridge to detect its target  These spores are co-processed with MTB specimen in the cartridge by the GeneXpert machine Sample Processing Control  SPC control verifies:  Adequate sample processing  Lysis of MTB cells  Amplification  Specimen associated inhibition  Degradation of enzymes or other components of the system  SPC 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 Probe Check Control (PCC) Probe Check Control (PCC) tests the fluorescence readings of probes at different temperatures, before start of thermal cycling. 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 (ERROR message) Test continues if the Probe check PASSED. Steps involved in Probe Check Control Biosafety Requirements for Xpert MTB/RIF  The Xpert MTB/RIF test is a low-risk procedure and requires the same level of precautions, as those used for direct AFB sputum-smear microscopy:  Carry out the procedure in a well ventilated area  Wear gloves and a laboratory coat at all times when handling patient samples  Minimize generation of aerosols during sample processing and handle specimens carefully and responsibly  Based on risk assessment, additional biosafety precautions may be required in Xpert MTB/RIF laboratories  These precautions may include the use of N95 respirators or biosafety cabinets Personal Protective Equipment (PPE) Personal Protective Equipment (PPE) are important, but they do not replace good microbiological and good laboratory practices Gloves: Essential (Disposable and Powder-free) Laboratory Coats: Essential Respirators: Usually not required but:  May be needed, based on risk assessment  Must always be included in a spill kit  Recommended respirators are N95 or chosen based on program guidelines Sample Processing-Sputum Storage and Handling  Store the Xpert cartridges at 2-28°C  Ensure cartridges have reached room temperature before using them (wait 10 min)  Do not use cartridges that have passed the expiry date  Do not open the cartridge lid until you are ready to perform the test  Cartridges should be used for testing within 4 hours of sample loading Content of cartridges are non-hazardous Sample Processing- Lymph Node and Tissue Lymph Node and Tissue sample processing : 1. Using sterile scissors and forceps cut-up tissue sample and homogenize (special grinders) 2. Add 2ml of sterile phosphate buffered saline (PBS) 3. Decontaminate using NaOH/NALC method 4. Resuspend in 2ml Phosphate Buffer 5. Proceed as described for standard Xpert sample processing for sputum Each laboratory must optimize this SOP to minimize the error rate Biosafety cabinet and safety centrifuge required Sample Processing-CSF Sample preparation: Cerebrospinal Fluid (CSF)  5 ml CSF sample i. ii. iii. iv. Transfer into conical tube and centrifuge (3000g:15 min) Decant supernatant into disinfectant solution (BSC required) Resuspend sediment to a volume of 2 ml using Xpert MTB/RIF Sample Reagent Add concentrated specimen to cartridge and proceed with Xpert testing  1-5 ml CSF sample i. ii. Add equal volume of Xpert MTB/RIF Sample Reagent Add 2 ml into the cartridge and proceed with Xpert testing  0.1-1 ml CSF sample Makeup the volume to 2 ml by adding sample reagent and proceed with Xpert testing  <0.1 ml CSF sample Insufficient sample for Xpert MTB/RIF testing Each laboratory must optimize this SOP to minimize the error rate Biosafety cabinet and centrifuge must be used Precautions for Processing Specimen and Cartridge Handling Preparing Samples for Xpert MTB/RIF Testing When preparing samples for Xpert MTB/RIF testing:  The sample reagent should be added to the specimen in such a manner that the procedure minimizes the aerosol generation  Although sample reagent inactivates M. tuberculosis bacilli, it reduces, but does not eliminate, the biosafety risk to the laboratory technician  There is an increased risk of generating aerosols when splitting and handling concentrated and extrapulmonary TB specimens. These procedures must be performed in a certified biosafety cabinet  When transferring cartridges to the machine for loading, an appropriate size enclosed container should be used. Care should be taken, as sample may leak if the cartridges are toppled during transport Interpretation of Test Results GeneXpert: Result Interpretation • Genexpert software generates results from measured fluorescence signals & embedded calculation algorithms. • In addition to qualitative results, the software provides semi-quantitative results which tells about bacterial load in specimen. • Semi-quantitative result interpretation is based on Ct values of MTB specific targets. Semiquantitative Results Semi-Quantitative Result MTB Result Ct Range High Less than 16 Medium 16-22 Low 22-28 Very Low Greater than 28 BACTERIAL LOAD is inversely proportional to Ct VALUE Troubleshooting Important reasons for repeat test • • • In case of problem, look for an error message on the screen (pop-up windows, message box at the bottom of the screen or in the "Error" tab in the menu "View Results") Look for the error code in the GeneXpert user manual and follow the recommended corrective actions. If the problem remains, contact your local service provider (if applicable) or Cepheid assistance. Maintenance of GeneXpert Instrument Frequency Daily Weekly Monthly Annual or after 2000 tests per module Task  Remove and properly dispose of cartridges  Clean and disinfect work area  Put on dust cover when instrument not in use  Disinfect the instrument’s surface  Disinfect cartridge bay interior and plunger rod  Filter cleaning  Disinfect cartridge bay interior and plunger rod  Clean instrument filter  Archive and back-up test results  Filter cleaning  Module Xpert Check (calibration) and maintenance of the module USES OF GENE XPERT • Xpert® Xpress SARS-CoV-2 • Xpert® Xpress SARS-CoV2/Flu/RSV • Xpert® Xpress /Flu/RSV • Xpert® Xpress Strept A • Xpert® Ebola • Xpert® EBV • Xpert® HPV • Xpert® GBS • Xpert® MRSA • Xpert® Carba R • • • • • • • • • Xpert® C.difficile BT Xpert® Van A/ VanB Xpert® Norovirus Xpert® HBV Viral Load Xpert® HCV Viral Load Xpert® HIV Viral Load Xpert® BCR-ABL Ultra Xpert® Bladder cancer Xpert® Breast cancer Xpert® Xpress SARS-CoV-2 • The specimen is briefly mixed by rapidly inverting the collection tube 5 times. • Using the supplied transfer pipette, transfer one draw of the external control sample (300 µL) into the large opening (Sample Chamber) in the cartridge. • After filling pipette, excess sample will be seen in the overflow reservoir bulb of the pipette. Check that the pipette does not contain bubbles. • Shaking or dropping the cartridge after opening the cartridge lid may yield nondeterminate results. • Do not place the sample ID label on the cartridge lid or on the barcode label on the cartridge. • Do not use a cartridge with a damaged barcode label. • Do not use a cartridge if it appears wet or if the lid seal appears to have been broken. • Do not open the Xpert Xpress SARS-CoV-2 cartridge lid except when adding specimen. Xpert Xpress SARS-CoV-2 Possible Results Result Text N2 E SPC SARS-CoV-2 POS + +/- +/- SARS-CoV-2 PRESUMPTIVE POS - + +/- SARS-CoV-2 NEGATIVE - - + INVALID - - - Xpert MTB/RIF :  Adv. - Automated, Quick results - Highly sensitive for both smear positive & negative samples (esp. HIV) - High specificity - Easy preparation; little technical training required (~1day)  Disadv. – Costly; shelf life of cartridge only 18mths - Cannot detect INH resistance - Can only identify MTBC, not MTB species - Require uninterrupted power supply - Cannot differs b/w live & dead bacilli (not be used for treatment monitoring) - Machine & Cartridge are temperature sensitive - Blood & blood tinged samples cannot be used Xpert MTB/RIF Ultra Assay (A next generation Xpert MTB/RIF assay)  Approval by WHO in 2017  Ultra Cartridge (Cepheid) : - Higher sensitivity for paucibacillary specimens & RIF resistance detection - Includes : - A large chamber for DNA amplification - Two additional molecular targets (IS6110 & IS1081)  Detection limit- 16 bacilli/ml sputum  Can be used on same Gene Xpert equipment with faster results (around an hour)  Low specificity Xpert Omni & Xpert XDR Xpert Omni • • • • Same test platform as Xpert MTB/Rif Single module Battery powered Robust, for use in remote settings Xpert XDR • An Xpert assay for detection of resistance to INH, FQ & SLIDs (second line injectable drugs)