xpert mtb rif - ghdonline · 2010. 11. 11. · • xpert® mtb/rif should be used as the initial...

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Xpert® MTB/RIF

• 2013

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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

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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.

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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

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Automated Xpert® MTB/RIF Protocol

in less than 2h

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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

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Genetics of Rifampin Resistance in M. tuberculosis

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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

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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

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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

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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

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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

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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

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Description- Packaging

Kit box

CD

Xpert ® MTB/RIF

cartridge SR (Sample Reagent) Disposable

transfer pipette

Sample reagent

pouch

• 10 or 50 cartridges per kit

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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

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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.

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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

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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

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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

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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

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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.

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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

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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*

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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

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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

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Semi Quantitative Results

0

10

20

NegativeVery Low

Low

MediumHigh

Nu

mb

er

of

sam

ple

s

Xpert MTB/RIF results

AFB Smear Results

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Xpert® MTB/RIF Positive Result

MTB DETECTED - RIF NOT DETECTED

Internal Control

5 targets amplified

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Xpert® MTB/RIF Positive Result

MTB DETECTED - RIF DETECTED

Internal Control 4 targets amplified

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Xpert® MTB/RIF Positive Result

MTB DETECTED - RIF INDETERMINATE

Internal Control

5 targets amplified

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Xpert® MTB/RIF Negative Result

Internal Control

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PAGE | 37

Xpert® MTB/RIF Invalid Result

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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