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1 © Cepheid – Proprietary & Confidential © 2012 Cepheid 1
Xpert® MTB/RIF
• 2013
2 © Cepheid – Proprietary & Confidential
TB transmission
http://www.medicinenet.com/tuberculosis/article.htm
Exposure to TB
Latent TB (90%) - Never develop TB
- Not infectious
Active TB (10%) ill - 5% develop TB within 2 years
- 5% develop TB within many years later
Untreated Treated
50% die within 5 years
25% remain sick
25% recover
No infection
(70-90%)
Infection
(10-30%)
Cavities open into the bronchi allowing spread of M.tuberculosis
trough coughing
5 © Cepheid – Proprietary & Confidential
Intended Use
Detection of Mycobacterium tuberculosis (MTB) complex DNA and
Rifampicin resistant Mycobacterium tuberculosis.
• Semi Quantitative in vitro Diagnostic test
• Specimen :
- Sputum sample
- induced sputum
• Detects
- rpoB gene responsible for the resistance determining region to
Rifampicin for the wild type of Mycobacterium tuberculosis
The Xpert® MTB/RIF test is intended for use with specimens from untreated
patients for whom there is clinical suspicion of tuberculosis.
6 © Cepheid – Proprietary & Confidential
The WHO STAG Committee Recommendations *
• Xpert ® MTB/RIF should be used as the initial diagnostic test in individuals
suspected of having MDR-TB or HIV-associates TB (Strong recommendation)
• Xpert ® MTB/RIF may be used as a follow-on test to microscopy where MDR and
HIV is a lesser concern, especially in smear-negative specimens (conditional
recommendation, recognising major resource implications)
* From the presentation to the FIND Symposium by Mario Raviglione, Head of WHO Stop TB-11/11/2010
7 © Cepheid – Proprietary & Confidential
Automated Xpert® MTB/RIF Protocol
in less than 2h
8 © Cepheid – Proprietary & Confidential
The Xpert ® MTB/RIF Molecular Beacon Assay
5 - Probes bind to wild type (do not bind to mutant
sequence)
1 - Probe for SPC (Sample Processing Control)
6 - fluorescent dyes detected simultaneously
rpoB gene
target
Hybridization
Molecular beacon
reporter quencher
Increase of
temperature
9 © Cepheid – Proprietary & Confidential
Genetics of Rifampin Resistance in M. tuberculosis
10 © Cepheid – Proprietary & Confidential
Algorithm
Probe Check Control
Pass Fail
Error
Yes
SPC
Pos No Value
MTB Not Detected Invalid
Test
Stopped
At least 2 probes positive?
Number of positive probes
No
All positives
within a Delta Ct of 4
1 neg. or
more neg.
No Result
11 © Cepheid – Proprietary & Confidential
All Common rpoB mutations detected
Representative selection of known
mutations
Representative selection of known
mutations
Representative selection of
wild-type strains
Representative selection of
wild-type strains
Wild-type strains - all probe
signals tightly grouped
Wild-type strains - all probe
signals tightly grouped
No signal from Probe B No signal from Probe B
Delayed signal from Probe A Delayed signal from Probe A
Xpert ® MTB/RIF detects
the most prevalent rpoB
mutations
12 © Cepheid – Proprietary & Confidential
Exclusivity and Interference
Selected Mycobacteria Other
Than Tuberculosis group – do not
meet interpretation criteria
Selected Mycobacteria Other
Than Tuberculosis group – do not
meet interpretation criteria
Mycobacterium tuberculosis
complex– all probes in tight
formation
Mycobacterium tuberculosis
complex– all probes in tight
formation
Mycobacterium tuberculosis
detected
Mycobacterium tuberculosis
detected
Xpert MTB/RIF is specific
for Mycobacterium
tuberculosis complex
The presence of MOTTs in
positive samples does not
interfere with the reaction
Specimen collection, transport, and storage
14 © Cepheid – Proprietary & Confidential
Sputum Samples
• Subject must be seated or standing
• Rinse the patient’s mouth twice with water
• Explain the patient how to: - open and close the collection device
- produce good sputum (collecting real sputum, not saliva;
deep inhalation and exhalation of breath followed by cough
from as deep inside the chest as possible)
- avoid contamination of the exterior of the container
(carefully spitting and closing the container);
- collect and safely deliver the sputum to the laboratory
Sputum is usually thick and mucous. It may be fluid and contain pieces of purulent material.
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: CDC wwwn.cdc.gov/dls/ila/.../ParticipantModule3.doc
15 © Cepheid – Proprietary & Confidential
Storage and handling of the sample
• Do not collect less than 1 mL of sputum per specimen
• Do not accept specimens with obvious food particles or other solid particles
• Do not leave the specimen at 35 0С more than 3 days (stable 4 to 10 days at 40C)
• Specimens should be held at 2–80C whenever possible including during
transportation to the laboratory
16 © Cepheid – Proprietary & Confidential
Storage of Xpert ® MTB/RIF
• Store the Xpert® MTB/RIF kit at 2-28 °C.
• Cartridges are stable up to 2 weeks at 2-28 °C once pouch opened.
• The Xpert® MTB/RIF kit CANNOT be used once it passes the expiration date
Kit handling
18 © Cepheid – Proprietary & Confidential
Description- Packaging
Kit box
CD
Xpert ® MTB/RIF
cartridge SR (Sample Reagent) Disposable
transfer pipette
Sample reagent
pouch
• 10 or 50 cartridges per kit
19 © Cepheid – Proprietary & Confidential
50 Test Xpert MTB/RIF kit
• Available
• Item Reference CGXMTB/RIF-50
• Reduced packaging and storage volume
• Open pouch Shelf life 6 weeks 2 - 45 °C / 75rh
Composed of
5 x 10-pack pouched cartridges
One box of 50 SR bottles
5 packs of 12 2ml pipettes
One CD-ROM
Expected to replace the 10 test kit format
20 © Cepheid – Proprietary & Confidential
Warnings and Precautions
• The performance of Xpert ® MTB/RIF for the detection of MTB complex has not
been demonstrated from non-respiratory specimens such as blood, CSF, stool or
urine.
• The performance of the Xpert ® MTB/RIF test has not been evaluated with
specimens processed by methods other than those described in this package insert.
• Open the Xpert ® MTB/RIF cartridge lid ONLY when adding decontaminated sample.
• The cartridges should be processed within 4 hours once sample added to the
cartridge.
21 © Cepheid – Proprietary & Confidential
Warnings and Precautions
• Do not use a cartridge:
- if it appears wet or if the lid seal appears to have been broken.
- that has been dropped or shaken after you have added the treated sample.
- that has a damaged reaction tube.
- that have already been processed: each cartridge is for a single-use and
cannot be reused once scanned.
• Dispose of used Xpert ® MTB/RIF cartridges according to your institution’s and
country’s safety guidelines for hazardous material
22 © Cepheid – Proprietary & Confidential
Materials not Provided
Materials Required but Not Provided
o Use clean, translucent, screw-capped specimen collection containers
(Single use disposable plastic containers (50 ml capacity are preferred)
o Disposable Gloves/ Coat/ Respiratory mask
o Pen
o Timer
Optional
o UPS
o Printer
o Additional 2 ml sterile transfer pipettes
23 © Cepheid – Proprietary & Confidential
Xpert ® MTB/RIF –> Low Risk Biosafety level
References:
1-http://www.stoptb.org/wg/gli/assets/documents/Mtg2pres/01%20-%20Current%20draft%20WHO-
CDC%20Biosafety%20recommendations.pdf
2-Containment of Bioaerosol Infection Risk by the Xpert MTB/RIF, Banada et Al.; JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3551–3557
Results suggest that the Xpert ® MTB/RIF assay poses equivalent
biohazard risk than the performance of a direct AFB smear 2
Direct AFB Smear Microscopy 1 Limited risk of generating infectious aerosols
- Work can be done on an open bench
- Restricted access to the lab
- Separate bench for smear-microscopy preparation
- Adequately ventilated lab (natural/mechanical ventilation)
- Proper disposal of infectious material
24 © Cepheid – Proprietary & Confidential
Biosafety requirements for Xpert ® MTB/RIF
- well ventilated area.
- wear gloves and lab coat.
- use BSL2 with laminar flow hood
General Laboratory
Smear preparation area Cartridge preparation
Xpert is a low risk procedure and requires the same level of precaution as for performing direct AFB sputum smear microscopy.
Concentrated sediment (pellet)
10-20x
Direct Sputum
10-20x
Procedure
26 © Cepheid – Proprietary & Confidential
Global Procedure
Handling Time = 2 MINUTES
Mix “Sample Reagent” with the
Sample.
Incubate for 15 minutes at room
temperature.
Transfer sample into the open port of
the Xpert® MTB/RIF cartridge Insert the cartridge and start the test
Cartridge contains all necessary reagents: Just add the sample treated with the
Sample Reagent
Once cartridge is ready, test should be loaded in the system within 30 minutes.
27 © Cepheid – Proprietary & Confidential
Cartridge Preparation – Expectorated Sputum
Sample
Open the cartridge
Unscrew the Sample
Reagent (SR)‘s and
the sputum collection
container ‘s lids.
Add 2 volumes of SR to 1
volume of sputum. Replace
the lid
Shake vigorously 10-20 times. Incubate at room temperature. After 10
min. of incubation, shake the specimen vigorously 10-20 times.
After 5 min of incubation, Sample should be perfectly fluid before being
processed, with no visible clumps of sputum. If still viscous, wait 5-10
further minutes.
Open the cartridge pouch
and label the cartridge
with the sample ID by
writing on the left or right
side or affix ID label.
Pipette at least 2 ml with the
plastic transfer pipette from the
collection container to the
cartridge.
Close the cartridge lid. Release these 2 ml to the
cartridge
28 © Cepheid – Proprietary & Confidential
Cartridge Preparation– Induced Sputum
Shake vigorously 10-20 times.
(Note: one back and forth movement is a single shake.)
Incubate at room temperature. After 10 min. of
incubation, shake (or vortex) the specimen vigorously 10-
20 times
Unscrew the
Sample Reagent
(SR)‘s lid
Collect 0.5 ml of prepared
sediment and add 1.5 ml of
the sample reagent.
Replace the lid.
Open the cartridge. Pipette at least 2 ml with the
plastic transfer pipette from the
collection container to the
cartridge.
Release these 2 ml to the
cartridge
Close the cartridge lid.
5 minutes open the cartridge
pouch and label the cartridge
with the sample ID by writing
on the left or right side or
affix ID label.
Induced sputum prepared according to the Kent and Kubica method* is re-suspended in 67mM Phosphate/H2O buffer.
Kent and Kubica method is available upon request*
29 © Cepheid – Proprietary & Confidential
Control Materials
• ATCC strains
• External Positive Controls :
o QC organisms such as ATCC 27290 as BCG Copenhagen,
• External Negative Controls :
o ATCC SmT Mc2 2500 as Mycobacterium avium
o ATCC 35790 or 35771 as Mycobacterium intracellular
o ATCC 2278 as Mycobacterium xenopii
o ATCC 12478 as Mycobacterium kansasii
External controls should be used in accordance with local, state accrediting organizations, as applicable.
Result Interpretation
31 © Cepheid – Proprietary & Confidential
Algorithm
Probe Check Control
Pass Fail
Error
Yes
SPC
Pos No Value
MTB Not Detected Invalid
Test
Stopped
At least 2 probes positive?
Number of positive probes
No
All positives
within a Delta Ct of 4
1 neg. or
more neg.
No Result
32 © Cepheid – Proprietary & Confidential
Semi Quantitative Results
0
10
20
NegativeVery Low
Low
MediumHigh
Nu
mb
er
of
sam
ple
s
Xpert MTB/RIF results
AFB Smear Results
33 © Cepheid – Proprietary & Confidential
Xpert® MTB/RIF Positive Result
MTB DETECTED - RIF NOT DETECTED
Internal Control
5 targets amplified
34 © Cepheid – Proprietary & Confidential
Xpert® MTB/RIF Positive Result
MTB DETECTED - RIF DETECTED
Internal Control 4 targets amplified
35 © Cepheid – Proprietary & Confidential
Xpert® MTB/RIF Positive Result
MTB DETECTED - RIF INDETERMINATE
Internal Control
5 targets amplified
36 © Cepheid – Proprietary & Confidential
Xpert® MTB/RIF Negative Result
Internal Control
37 © Cepheid – Proprietary & Confidential
PAGE | 37
Xpert® MTB/RIF Invalid Result
38 © Cepheid – Proprietary & Confidential
PAGE | 38
Xpert ® MTB/RIF Analytical Studies
• Analytical Sensitivity is 131 CFU/mL with a 95% confidence interval ranging from 106.2
CFU to 176.4 CFU.
• Analytical Specificity (Exclusivity) Cultures of 18 mycobacteria other than tuberculosis
(MOTT) strains were tested and all were reported MTB negative.
• Analytical Inclusivity: Xpert ® MTB/RIF correctly detected all MTB strains and correctly
identified the rifampicin resistant isolates
• Analytical Inactivation of Mycobacteria in sputum samples : The viability of the M. bovis
organisms from the treated sputum was reduced by at least 6 logs relative to the
untreated control.
Please refer to the Package insert for all information regarding sensitivity and analytical
performances