ࡱ>  $Ebjbj ;{{;p p 8~,^C!"d}}}}}}}$.Ђ<}#CC##}4}G%G%G%#}G%#}G%G%.pzpnF$@iw@m}}0~w4 %@ zz8 {X##G%#####}}G%###~#### #########p y:  TABLE OF CONTENTS  TOC \o "1-3" \h \z \u 1. PURPOSE  PAGEREF _Toc225322812 \h 2 2. SCOPE  PAGEREF _Toc225322813 \h 2 3. RESPONSIBILITIES  PAGEREF _Toc225322814 \h 2 4. CROSS-REFERENCES  PAGEREF _Toc225322815 \h 2 5. PROCEDURES  PAGEREF _Toc225322816 \h 3 5.1. General and safety procedures  PAGEREF _Toc225322817 \h 3 5.2. Equipment & Materials  PAGEREF _Toc225322818 \h 3 5.3. Work procedure  PAGEREF _Toc225322819 \h 3 5.3.1 Expectorated Sputum Samples  PAGEREF _Toc225322820 \h 4 5.3.2. Preparing the Cartridge  PAGEREF _Toc225322821 \h 4 5.3.3. Starting the test  PAGEREF _Toc225322822 \h 5 5.4. Quality controls  PAGEREF _Toc225322823 \h 5 5.4.1. ERROR  PAGEREF _Toc225322824 \h 6 5.4.2. Reasons to repeat the assay  PAGEREF _Toc225322825 \h 6 5.5. Interpretation of Results  PAGEREF _Toc225322826 \h 6 5.5.1 MTB DETECTED  PAGEREF _Toc225322827 \h 6 5.4.2. MTB NOT DETECTED  PAGEREF _Toc225322828 \h 6 5.4.3. RIF NOT DETECTED  PAGEREF _Toc225322829 \h 7 5.4.4. INVALID  PAGEREF _Toc225322830 \h 7 5.6. Limitations of this In vitro diagnostic test (IVD)  PAGEREF _Toc225322831 \h 7 6. REFERENCES  PAGEREF _Toc225322832 \h 7 7. CHANGE HISTORY  PAGEREF _Toc225322833 \h 8  PURPOSE The purpose of SOP is to describe the stepwise procedure for the rapid detection of Mycobacteria tuberculosis (MTB) and simultaneous detection of rifampicin resistance using GeneXpert IV Dx system and the XpertMTB/RIF cartridge (Cepheid, Inc, Sunnyvale, USA). The Xpert MTB/RIF test for use with the Cepheid GeneXpert Dx system is a semi-quantitative nested real-time PCR in-vitro diagnostic test for: (1) the detection of MTB DNA in sputum samples (2) the detection of rifampicin resistance associated mutations of the rpoB gene. The GeneXpert Dx system integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences. The system requires the use of single-use disposable XpertMTB/RIF cartridges that hold the PCR reagents and host the PCR process. The Xpert MTB/Rif cartridge also contains a sample processing control (SPC) to control for adequate processing of the target bacteria and to monitor the presence of inhibitor(s) in the PCR reaction. The Probe Check control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability. The primers in the XpertMTB/RIF assay amplify a portion of the rpoB gene containing the 81 base pair core region. The probes are able to differentiate between the conserved wild-type sequence and mutations in the core region that are associated with rifampicin resistance. SCOPE This SOP relates to the testing of expectorated sputum for detection of MTB and rifampicin resistance by the GeneXpert MTB/Rif test in the _______________ TB laboratory. RESPONSIBILITIES All staff members working in the _____________________ TB Laboratory are responsible for the implementation of this operating procedure. All users of this procedure who do not understand it or are unable to carry it out as described are responsible for seeking advice from their supervisor. CROSS-REFERENCES See:Document Matrix TB 01-01_V1.0.docLocation: Refer to SOPs listed under TB 01 (General Procedures), TB 02 (Specimen Handling), TB 06 (Equipment Use and Maintenance) and TB 05 (Molecular Methods). PROCEDURES 5.1. General and safety procedures Before you start to use the GeneXpert Dx system, make sure you read the operator manual provided during the training. Using controls, making adjustments, or performing procedures other than those specified in this SOP may result in hazard or injury to person or damage to the equipment. Do not attempt to lift the instrument without proper safety training and assistance. This many damage the instrument and void the warranty. All processing of clinical specimens should be performed as per the biosafety standards. Treat all sputum specimens, including used cartridges, as potentially infectious. Wear protective gloves and laboratory coats when handling specimens and reagents. Wash hands thoroughly after handling specimens and test reagents. All benches should be decontaminated after work, or immediately after a spill, with an appropriate mycobactericidal disinfectant followed by 70% alcohol. GeneXpert system is to be handled by trained persons only. DO NOT OPERATE if you are not trained. Read and follow the instructions in the product insert of XpertMTB/Rif cartridge. Do not open Xpert MTB/Rif cartridge lid except when adding sample. Do not reuse Xpert MTB/RIF cartridges. Dispose of used Xpert MTB/RIF cartridges according to infectious waste material disposal guidelines. 5.2. Equipment & Materials GeneXpert Dx system equipped with GX2.1 software/computer/printer/barcode wand-reader and operator manual (Cepheid Inc, Sunnyvale, USA) (Cat Item No GXIV-4N1-6). Class II biological safety cabinet (BSC) Xpert MTB/RIF kit- contains 10 individually packed cartridges: Store the Xpert MTB/RIF cartridges and reagents at 2-28C. Do not open the cartridge until you are ready to perform testing. Use cartridge within 30 minutes after opening the cartridge lid. Cartridge is stable up to 7 days after opening the package. Sample reagent (provided in Xpert MTB/RIF kit), 8ml volume pack per each cartridge. The sample reagent solution is clear, but may range from colorless to golden yellow. Permanent marker pens. Sterile (individually packed) disposable transfer pipettes- with single mark for minimum volume of sample transfer to cartridge (provided in Xpert MTB/RIF kit). Sterile screw-capped specimen collection containers/cups. Discard containers for pipettes and sputum containers. 5.3. Work procedure Before proceeding, determine how many samples to be processed. Batch the samples to a maximum of four per run. Enter the patient/suspect details on the Xpert MTB/Rif worksheet. 5.3.1 Expectorated Sputum Samples To each specimen in sputum container with screw cap (ensure prior that the cup is leak proof): Unscrew lid of sputum collection container. Using a plastic disposable pipette, measure and note the sputum volume (Note: the maximum volume on the graduated disposable pipette is 3ml). Record the volume on the Xpert/Rif worksheet. Carefully discard the pipette. Using separate plastic disposable pipette, add the sample reagent at 2:1 (v/v) ratio to the sputum sample. Replace the lid of sputum cup (Note: avoid leakages on the cup). Shake the sputum cup vigorously 10-20 times using back and forth movements in a single shake. Incubate the sample in the sputum cup for 15 minutes at room temperature. During the incubation period, shake the sputum cup at least once, as described above. The sputum sample should be liquefied with no visible clumps of sputum after incubation. Particulate matter may exist that is not part of the sample. Label each Xpert MTB/RIF cartridge with the Laboratory number, as the same given on the sputum cup. Write with marker pen on the front side bottom of the cartridge, never on the lid of the cartridge or on barcode. 5.3.2. Preparing the Cartridge Note: Start the test within 30 minutes of adding the sample to the cartridge. Using the sterile transfer pipette provided in the Xpert/Rif kit, draw the liquefied sample into the transfer pipette until the meniscus of pipette is above the minimum mark. Do not process the sample further if there is insufficient volume. Avoid touching the sterile transfer pipette. Open the front of the pipette wrapper at the bulb-end of pipette. Take the pipette out carefully. Retain the paper cover. Open the cartridge lid. Transfer sample into the open port of the Xpert MTB/RIF cartridge. See Figure 1 below. Dispense slowly to minimize the risk of aerosol formation.  Figure 1: Xpert MTB/RIF cartridge (top view) Carefully put the pipette back into the paper/plastic cover. Discard the transfer pipette into bio-hazard waste bin. Close the cartridge lid. Make sure the lid snaps firmly into place. Remaining liquefied sample may be kept for up to 12 hours at 2-8C (for repeat testing). Important: Be sure to load the cartridge into the GeneXpert Dx instrument and start the test within 30 minutes of preparing the cartridge. IMPORTANT: The minimum required amount to be loaded into the cartridge is 2ml (as marked on the Pasteur pipette). 5.3.3. Starting the test Important: Before you start the test, ensure the system attached to a working uninterrupted power source (UPS). Turn on the computer, Password is cphd. Turn on the GeneXpert Dx instrument. On the Windows desktop of computer, double-click the GeneXpert Dx shortcut Icon. In the GeneXpert Dx system window, click Create Test. The Scan Cartridge Barcode dialog box appears. Skip or cancel the dialog. In the Sample ID box, type the Laboratory number. Insure the correct Laboratory number is recorded. The Laboratory number must match the number on the cartridge and on sputum cup. Click Start Test. Open the instrument module door which displays the blinking green light, and load the cartridge Close the door of module firmly (an audible click sound should be heard). The test starts and the green light stops blinking. When the test is finished, the light turns off. Continue with loading next cartridge following the steps described above. Once the run is completed- results are printed automatically. It takes around 1 hour 55 minutes to complete run. Once run ends, the result print-out is generated automatically. Wait until the system releases the door lock at the end of run, then open the module door and remove the cartridge. Dispose the used cartridge in the biohazard waste container. Using either the result print-out, or transcribing from the monitor; enter the results on the Xpert MTB/RIF worksheet. Sign, date and initial the result-print out. Use the Ink-pad and Lab Stamp/seal. 5.4. Quality controls Each Xpert MTB/RIF cartridge includes a Sample processing control (SPC) and Probe Check control (PCC). Print out of the test result indicates the validation of controls. Sample Processing Control (SPC): Ensures the sample was correctly processed. The SPC contains non-infectious spores in the form of a dry spore cake that is included in each cartridge to verify adequate processing of MTB. SPC should be positive in a negative sample and can be negative or positive in a positive sample. The SPC passes if it meets the validated acceptance criteria. The test result will be Invalid if the SPC is not detected in a negative test. Probe Check Control (PCC): Before the start of the PCR reaction, the GeneXpert Dx system measures the fluorescence signal from the probes to monitor bead hydration, reaction-tube filling, probe integrity and dye stability. Probe Check passes if it meets the assigned acceptance criteria. 5.4.1. ERROR MTB-NO RESULT SPC-NO RESULT Probe Check-NA (not applicable) 5.4.2. Reasons to repeat the assay Repeat the test using a new cartridge or initiate alternate procedures if one of the following test results occurs: An INVALID result indicate that the SPC failed. The sample was not properly processed or PCR was inhibited. An ERROR result indicates that the probe check control failed and the assay was aborted possibly due to the reaction tube being filled improperly, a reagent probe integrity problem was detected, or because the maximum pressure limits were exceeded or there was a GeneXpert module failure. A NO RESULT indicates that insufficient data was collected. For example, the operator stopped a test that was in progress. 5.5. Interpretation of Results This is DNA based test, meant for New TB suspect, ensure dont enrol follow-up patient. The results are interpreted by the GeneXpert Dx system from measured fluorescent signals and embedded calculation algorithms and will be displayed in the View Results window. Lower Ct values represent a higher starting concentration of DNA template; higher Ct values represent a lower concentration of DNA template. 5.5.1 MTB DETECTED If MTB target DNA is detected- the MTB result will be displayed at High, Medium, Low or Very Low depending on the Ct value of the MTB target present in the sputum sample. Below table lists the Ct value ranges for the displayed MTB results: MTB resultCt rangeHigh<16Medium16-22Low22-28Very Low>28 Rifampicin resistance result types, when MTB is detected: Rifampicin resistance DETECTED: a mutation in the rpoB gene has been detected that falls within the valid delta Ct setting. Rifampicin resistance NOT DETECTED: no mutation in the rpoB has been detected. Rifampicin resistance INDETERMINATE: the MTB concentration was very low and resistance could not be detected. 5.4.2. MTB NOT DETECTED MTB target DNA is not detected. SPC meets acceptance criteria. MTB NOT DETECTED-MTB target DNA is not detected SPC- Pass; SPC has a Ct value range and endpoint above the endpoint minimum setting. Probe Check- PASS: all probe check results pass. 5.4.3. RIF NOT DETECTED Rifampicin target DNA is not detected, SPC meets acceptance criteria. RIF NOT DETECTED- Rifampicin target DNA is not detected. SPC- Pass; SPC has a Ct value range and endpoint above the endpoint minimum setting. Probe Check- PASS: all probe check results pass. 5.4.4. INVALID Presence or absence of MTB cannot be determined, repeat test with extra specimen. SPC does not meet acceptance criteria, the sample was not properly processed, or PCR is inhibited. MTB INVALID- Presence or absence of MTB DNA cannot be determined SPC-FAIL; MTB target results is negative and the SPC Ct is not within valid range. Probe Check- PASS; all probe check results pass. 5.6. Limitations of this In vitro diagnostic test (IVD) Perform the test and validate results as per this SOP and details of test package insert Reliable results depend on proper specimen collection, handling, and storage. A positive test result does not necessarily indicate the presence of viable organisms. It is however, presumptive for the presence of MTB and rifampicin resistance. The results might be affected by antecedent or concurrent anti-TB drug therapy. Fill in results on the Xpert MTB/Rif worksheet. Use:Xpert MTB/Rif worksheet_form.docLocation:  REFERENCES Xpert MTB/Rif product insert Policy statement: automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF system. World Health Organization. 2011. WHO/HTM/TB/2011.4 Boehme CC, Nicol MP, Nabeta P, Michael JS, Gotuzzo E, Tahirli R, Gler MT, Blakemore R, Worodria W, Gray C, Huang L, Caceres T, Mehdiyev R, Raymond L, Whitelaw A, Sagadevan K, Alexander H, Albert H, Cobelens F, Cox H, Alland D, Perkins MD. Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study. Lancet. 2011 Apr 30;377(9776):1495-505. 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