Title: Procedure for preparing sputum ID Number: SMH/MIC-P- 025

samples and detection of MTB/RIF

resistance using Genexpert equipment.
Revision Number: 0 Effective Date: 01/12/2013 Page 1 of 7

SEKGOMA MEMORIAL HOSPITAL LABORATORY

ALONG SEROWE/ PALAPYE ROAD

P.O BOX 120

SEROWE

TEL: 4611000

FAX: 4600664

Name Designation Signature Date

Written by W. Makhuza MSO II

Approved by: D. Kelebile PMLT

Authorized by: F. Samuel MSO 1

Revision Status Sheet

Scheduled review date: December 2015
Revision # Changes made Initial Revision
date

0 First issue W.M 01/11/2013

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 Title: Procedure for preparing sputum ID Number: SMH/MIC-P- 025
samples and detection of MTB/RIF
resistance using Genexpert equipment.
Revision Number: 0 Effective Date: 01/12/2013 Page 2 of 7

Important note: This document shall be re-issued after at most 10 changes have been
registered herein.

1. Purpose
The purpose of this procedure is to outline the steps involved in detecting
Mycobacterium Tuberculosis and resistance to rifampicin using the GeneXpert
system.

2. Scope

This procedure applies to the Microbiology Section in Sekgoma Memorial

Hospital laboratory where the equipment is used.

3. Responsibility

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 Title: Procedure for preparing sputum ID Number: SMH/MIC-P- 025
samples and detection of MTB/RIF
resistance using Genexpert equipment.
Revision Number: 0 Effective Date: 01/12/2013 Page 3 of 7

3.1 The Microbiology Section Head is responsible for effective implementation of the
procedure.
3.2 Laboratory technical staff is responsible for following the procedure when
performing this test.

4 Safety

Always use the appropriate personal equipment while handling specimens

and equipment and observe safety procedures at all times.

5 Materials

5.1 Specimen
5.1.1 Sputum(no added preservatives)

5.2 Equipment
5.2.1 GeneXpert equipment
5.2.2 Computer
5.2.3 Printer
5.2.4 Key pad
5.2.5 Mouse
5.2.6 Barcode scanner
5.2.7 Indelible labeling marker
5.2.8 Waste disposal bin (Clinical and domestic waste bins)
5.2.9 Stop watch

5.3 Reagents/material
5.3.1 Sample reagent (Buffer)
5.3.2 3ml sterile Pasteur pipettes (Graduated)
5.3.3 2ml transfer pipettes
5.3.4 Cartridges
5.3.5 Sterile sputum jars
5.3.6 Freshly prepared 0.1% sodium hypochlorite
5.3.7 Absorbent paper towel
5.3.8 70% alcohol

5.4 Glassware
N/A

6 Procedure

6.1 Principle
The Xpert MTB / RIF is a cartridge-based, automated diagnostic test for the

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 Title: Procedure for preparing sputum ID Number: SMH/MIC-P- 025
samples and detection of MTB/RIF
resistance using Genexpert equipment.
Revision Number: 0 Effective Date: 01/12/2013 Page 4 of 7

Identification of MTB and resistance to rifampicin (RIF). The GeneXpert system

Integrates and automates sample processing, nucleic acid amplification and
Detection of the target sequences in simple or complex samples using real time
PCR and reverse transcription. It purifies and concentrates MTB bacilli from
Sputum samples, isolates genomic material from the captured bacteria by
sonication and subsequently amplifies the genomic DNA by PCR and identifies
all the Rifampicin resistance. The primers in the Xpert MTB/RIF assay amplify a
portion of the rpoB gene containing 81base pair (core) region. The fluorescent
probes are able to differentiate between the conserved wild type sequence and
mutations in the core region that is associated with RIF resistance.

6.2 Procedural steps

6.2.1 Record the patient details in the Botswana District Tuberculosis

Laboratory Mycobacteriology Request Register.
6.2.2 Label each Xpert MTB/RIF cartridge with the sample ID and name
(write on the sides of the cartridge).
6.2.3 Carefully unscrew lid of the sputum container.
6.2.4 Add sample reagent 2:1 (v/v) to the sample (estimate the sputum
volume by comparing with a marked sputum container). Replace
and secure the sputum container lid.
6.2.5 Shake the diluted sample vigorously for 10-20 times using back
and forth movement.
6.2.6 Incubate for 10 minutes at room temperature.
6.2.7 After incubation mix again for 10-20 times and incubate for a
further 5 minutes.
6.2.8 Using a sterile 2 ml transfer pipette aspirate the liquefied sample
until the meniscus is above 2 ml mark.
6.2.9 Transfer into the open port of the Xpert cartridge. Dispense slowly
to minimize the risk of aerosol formation
6.2.10 Close the cartridge lid firmly.
NB: Start the test within 30 minutes of preparing the cartridge. Store
remaining prepared sample for up to 12 hours at 2-8°C in case repeat
testing may be required.

6.2.11 Log on to the GeneXpert system software using the operator user
name and password.
6.2.12 Click on Create test icon (upper left). The scan cartridge Barcode
dialog box appears.
6.2.13 Scan the barcode on the Xpert MTB/RIF cartridge. Create test
window appears.
6.2.14 Type the sample ID and patient name.
6.2.15 Click on start test. The dialog box appears. Type the operator
password.

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 Title: Procedure for preparing sputum ID Number: SMH/MIC-P- 025
samples and detection of MTB/RIF
resistance using Genexpert equipment.
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6.2.16 Open the instrument module door with the blinking green light and
load the cartridge.
6.2.17 Close the door. The test starts automatically.
6.2.18 When the testing is finished the light turns off and the module door
is released. Remove cartridges and dispose them in the waste
disposal bin.

6.3 Quality control

6.3.1 Use cartridges within the expiry dates
6.3.2 Store cartridges and reagent (buffer) at 2-28°C
6.3.3 Do not open a cartridge lid until ready to perform the testing.
6.3.4 Equipment operation temperature should not exceed 30°C.
6.3.5 The cartridges are stable for up to 2 weeks after opening the
pouch.
6.3.6 Ensure the sample processing control (SPC) has passed before
reporting negative results. The SPC should be positive in a
negative sample and can be negative/positive in a positive
sample.
6.3.7 At least 1ml of sputum sample should be collected.
6.3.8 Store samples at room temperature for 3 days and 2-8°C for 10
days

6.4 Reporting results

6.4.1 Check the table below for interpretation of results.

RESULT INTERPRETATION
MTB DETECTED The MTB target is present within the sample:
Rif Resistance DETECTED ·A mutation in the rpoB gene has been detected that falls
within the valid delta Ct setting.
·SPC: NA (not applicable). An SPC signal is not required
because MTB amplification can compete with this control.
·Probe check: PASS. All probe check results passed.
MTB DETECTED The MTB target is present within the sample:
Rif resistance NOT DETECTED ·A mutation in the rpoB gene has been detected that falls

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 Title: Procedure for preparing sputum ID Number: SMH/MIC-P- 025
samples and detection of MTB/RIF
resistance using Genexpert equipment.
Revision Number: 0 Effective Date: 01/12/2013 Page 6 of 7

within the valid delta Ct setting.

·SPC: NA (not applicable). An SPC signal is not required
because MTB amplification can compete with this control
·Probe check: PASS. All probe check results passed

MTB DETECTED The MTB target is present within the sample

Rif resistance ·RIF resistance could not be determined due to insufficient
INDETERMINANT signal detection.
·SPC: NA (not applicable). An SPC signal is not required
because MTB amplification can compete with this control.
·Probe check: PASS. All probe check results passed.

MTB Not Detected The MTB target is not detected within the sample:
·SPC: PASS. The SPC met the acceptance criteria.
·Probe check: PASS. All probe check results passed

INVALID The presence or absence of MTB cannot be determined. The

SPC does not meet the acceptance criteria, the sample was
not properly processed or PCR was inhibited. Repeat the test.
·MTB INVALID: The presence/absence of MTB DNA cannot be
determined.
·SPC: FAIL. The MTB target result is negative and SPC Ct is
not within valid range.
·Probe check: PASS. All probe check results passed.
ERROR The presence or absence of MTB cannot be determined.
Repeat the test.
·MTB: NO RESULT
·SPC:NO RESULT
·Probe Check: FAIL. All or one of the probe check results
failed.
NB: if the probe check passed, the error is caused by a system
component failure.
NO RESULT The presence or absence of MTB cannot be determined.
Repeat the test. A NO RESULT indicates that insufficient data
was collected. E.g. the operator stopped a test that was in
progress.
·MTB NO RESULT
·SPC: NO RESULT
·Probe Check: NA (not applicable)

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 Title: Procedure for preparing sputum ID Number: SMH/MIC-P- 025
samples and detection of MTB/RIF
resistance using Genexpert equipment.
Revision Number: 0 Effective Date: 01/12/2013 Page 7 of 7

6.4.2 Attach the equipment printout results to the Mycobacteriology

request and report form (MH2011). Put the analysts’ initials, date
and dispatch.
6.4.3 Transcribe results into the Botswana District Tuberculosis
Laboratory Mycobacteriology Request Register.
6.4.4 If possible alert concerned wards/clinics for positive MTB/RIF
results.
6.4.5 Repeat the test using a new cartridge if one of the following test
results occurs: INVALID, ERROR/NO RESULT.

6.5 Limitations
6.5.1 The equipment only detects the MTB/Rifampicin resistance.
6.5.2 Bloody samples interfere with the PCR.
6.5.3 The maximum operating temperature for the equipment is 30°C
6.5.4 Erroneous test results might occur from improper specimen
collection, handling/storage or technical error.
6.5.5 Test results might be affected by antecedent or concurrent
antibiotic therapy. Therefore, therapeutic success or failure cannot
be assessed using this test because DNA might persist following
antimicrobial therapy.
6.5.6 A positive test result does not necessarily indicate the presence of
viable organisms. Therefore therapeutic success/failure cannot be
assessed using this technique.
6.5.7 Mutations/polymorphism in primer/probe binding regions may
affect detection of new or unknown MDR-TB or RIF resistant
strains resulting in false negative results.

7 Records and Related Forms

7.1 Botswana District Tuberculosis Laboratory Mycobacteriology Request Register.

7.2 GeneXpert result printout.
7.3 Mycobacteriology request and report form (MH2011).

8 Supporting documents

8.1 General Laboratory Safety Practices- SMH/SAF-002.

8.2 Procedure for preparing effervescent chlorine tablets disinfectant. SMH/SAF-P-
019.
8.3 Procedure for Waste Management. SMH/SAF-014.

9 References
9.1 MTB/RIF GeneXpert user manual.

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