module 6: genexpert technology and xpert mtb/rif procedures global laboratory initiative – xpert...

Module 6: GeneXpert Technology andXpert MTB/RIF Procedures

Global Laboratory Initiative – Xpert MTB/RIF Training Package

Slides adapted from Cepheid

Global Laboratory InitiativeXpert MTB/RIF Training Package-2-

Contents of this module

Overview of the technology

Preparation of sputum specimens (direct and processed)

Preparation of extrapulmonary samples

Xpert MTB/RIF assay procedures

Monitoring the procedure


Learning objectives

At the end of this module, you will be able to: Describe the technology underlying the GeneXpert and

Xpert MTB/RIF assay

Explain the Xpert MTB/RIF assay procedures

Use the software to monitor test results and generate reports


GeneXpert Technology

Integrated micro-fluidic based system comprised of:

Instrument platform

Cartridges (self-contained)

Automated protocols

Internal controls


GX-I

GX-XVI

GeneXpert Infinity-48

GeneXpert® Module GX-II GX-IV

GeneXpert platform


Plunger Motor

Cartridge Inserter

I-CORE

Motherboard

Valve Drive Motor

Ultrasonic Horn

GeneXpert Module


GeneXpert Module- Cartridge Bay


End of hands-on work

Automated Xpert MTB/RIF Protocol

Fill the cartridge with prepared sample and insert it in the instrument

Sample combines with Sample Processing Control (SPC)

Filter captures the sample and SPC Ultrasonically lysed cells release DNA Eluted DNA mixes with dried-down

bead reagents Simultaneous amplification and

detection of fluorescence Results ready in less than 2 hours


GeneXpert Cartridge


Components of a cartridge


Principles of GeneXpert Design

Real-time PCR (amplification and detection at the same time) No wet interface between instrument and cartridge to eliminate

carry-over Total internal control of reagents system – No separate external

positive or negative controls required Integrated ultrasonic lysis of cells for release of DNA Software instructions to individual module motherboards to

coordinate valve movement and integral hydraulic drives Smart fluidics - Flow of liquids directed by micro valves – Allow

using micro quantities of reaction components Automated data analysis and results interpretation


Movie is available from FTP: ftp://hbdc:[email protected](paste in Windows Explorer, not Internet Explorer)The FTP movies are large files so should be downloaded on to a CD before the training.Alternatively a movie can be accessed via Youtube:http://www.youtube.com/watch?v=mIsBLmjus6Q

Cartridge in Action: Movie

ftp://hbdc:[email protected]/



http://www.youtube.com/watch?v=mIsBLmjus6Q

http://www.youtube.com/watch?v=mIsBLmjus6Q


Target DNA

PCR

Fluorescence

Detection

This slide may be considered optional for trainings of basic users

Real–Time Polymerase Chain Reaction

Simultaneous amplification of target DNA by PCR, and detection

Target DNA is amplified and labelled with fluorescently tagged probes

The accumulating product is monitored and measured by detecting the fluorescence

Probes used in Xpert technology are called “Molecular Beacons”

More than one targets can be amplified and detected (multiplex)

Each target is labelled with different dyes



Molecular Beacon Technology

Molecular beacon is a short segment of single-stranded DNA composed of:

◦ Target specific

sequence

◦ Stem

◦ Fluorescent dye

◦ Quencher


Molecular Beacon

Target

Fluorescence No Fluorescence

No Target = No Fluorescence


Molecular Beacon Technology



Molecular mechanisms of resistance to rifampicin

Mutations in the rpoB gene encoding for β-subunit of RNA polymerase: ◦ prevent binding of rifampicin to RNA polymerase,

synthesis of proteins and killing of bacilli

95% of all resistance to rifampicin are due to mutations in the rpoB gene and 5% due to mutations outside the gene

> 90% of mutations in the rpoB gene are located in the 81 base pairs region (codons 507 – 533)



Detection of mutations in rpoB gene by Xpert MTB/RIF

5 overlapping probes – bind to wild type genes and do not bind to mutant sequences

1 probe to Sample Processing Control - SPC (Bacillus globigii)

Total 6 fluorescent dyes detected at the same time



Internal Quality Controls: Probe Check Control (PCC)

Probe Check Control (PCC) – testing of fluorescence readings at different temperatures before start of thermal cycling, to evaluate the response of the chemicals contained in a cartridge. 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



Internal Quality Controls: Sample Processing Control (SPC)

Sample Processing Control (SPC)

Non-infectious spores

Verifies lysis of cells occurred successfully

Detects specimen-related inhibition of amplification: 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



Algorithm for determination of Xpert MTB/RIF results


Xpert MTB/RIF procedures


1Mix

2Add 3

Insert

4Detect

Overview of the procedure


2 ml is indicated by a line on the

pipette

Sample preparation: direct sputum

Pour sample reagent (buffer) carefully into the sample to avoid production of aerosols Avoid pipetting any solid particles from the sample mix into the cartridge Avoid creating bubbles when pipetting the sample mix into the cartridge


Sample preparation: direct sputum

Carefully unscrew the lid of sputum container Pour 2 volumes of sample reagent (SR) directly into 1 volume of

sputum in the sputum container (1 ml of sputum is the minimum quantity, while 3-4 ml is the optimal quantity required)

For larger volume specimens (over 4 ml), a portion of SR from a second bottle would be needed, as each bottle contains 8 ml of SR

Replace the lid, and shake vigorously 10-20 times (one back and forth movement is a single shake), or vortex

Incubate at room temperature for 10 min After 10 min of incubation, again shake (or vortex) the specimen

vigorously 10-20 times After additional 5 min of incubation, sample should be perfectly fluid

before being tested, with no visible clumps of sputum. If still viscous, wait 5-10 more minutes before inoculating in the cartridge (2-4 ml of the final solution)


2. Add 1.5 ml of sample reagent (buffer) to

0.5 ml sediment(3:1 ratio)

Sample preparation: processed sputum sediment


Sample preparation: processed sputum sediment

Add 1.5 ml of sample reagent to 0.5 ml of suspended sediment from digested/decontaminated and concentrated sputum specimen (Note: ratio of SR to sample 3:1)

Replace the lid, and shake vigorously 10-20 times (one back and forth movement is a single shake), or vortex

Incubate at room temperature for 10 min After 10 min of incubation, again shake (or vortex) the

specimen vigorously 10-20 times After additional 5 min of incubation, sample should be perfectly

fluid before being tested with no visible clumps of sputum


Customize if samples are ever split

Sample preparation: splitting samples


WHO recommends the use of the following EPTB samples for Xpert MTB/RIF◦ Cerebral Spinal Fluid (CSF)◦ Lymph node and other tissues (samples must be prepared in a BSC)

CSF samples are typically paucibacillary and can be processed similar to sputum. However, concentration using centrifugation (sedimentation) may provide better test results. A laboratory with a BSC is needed for opening the centrifuge cups and for decanting the supernatant.

Liquid specimens which may arrive in a capped syringes should be prepared in a BSC for Xpert testing. Expressing the syringe contents into a 50 mL conical tube may also cause aerosolization of bacilli. Safety procedures for working with sharp objects should be followed.

Tissue samples require homogenization using special grinders that may create aerosols. These specimens MUST be prepared in a BSC.

SOP can be found in the WHO Xpert Implementation manual; Annex 2. www.who.int/tb/publications/xpert_implem_manual

Extrapulmonary (EPTB) samples

http://www.who.int/tb/publications/xpert_implem_manual


Lymph Node and Tissues1. Using sterile scissors and forceps cut-up tissue

sample and place into homogenizer/grinder (shown)

2. Add 2 ml of sterile phosphate buffered saline (PBS)

3. Place lid on homogenizer and grind until a finely ground homogeneous suspension

4. Allow large particles to settle (5-10 min)

5. Place 0.7 ml of supernatant into a conical screw capped tube (be sure NO tissue clumps are transferred)

6. Add 1.4 ml of Xpert Sample Reagent

7. Proceed as described for standard Xpert sample processing for sputum

8. Load cartridge with 2+ ml

9. CREATE/RUN Xpert test

Sample preparation: extrapulmonary samples

Alternative protocols are available in WHO Xpert SOP for NONSTERILE specimens. These samples require decontamination using similar protocols for indirect sputum sample processing.

It is recommended to also perform a culture from the ground tissue

supernatant.

BSC Require

d


CSF samples (Note: blood stained samples may interfere with the Xpert test)

Sample preparation: extrapulmonary samples (2)

1. > 5 ml CSF sample

a) Transfer into conical tube and centrifuge (3000g:15 min)

b) Decant supernatant into disinfectant solution (BSC required)

c) Resuspend sediment to a volume of 2 ml using Xpert MTB/RIF Sample Reagent

d) Add concentrated specimen to cartridge and proceed with Xpert testing

2. 1-5 ml CSF sample (including blood stained)

a) Add equal volume of Xpert MTB/RIF Sample Reagent

b) Add 2 ml into the cartridge and proceed with Xpert testing

3. 0.1-1 ml CSF sample

a) Add 2 ml Xpert MTB/RIF Sample Reagent

b) Add 2 ml into the cartridge and proceed with Xpert testing

4. <0.1 ml CSF sample

c) Insufficient sample for Xpert MTB/RIF testing

A BSC must be used

whenever there is a risk

of aerosolizatio

n, e.g., decanting


Sample preparation: work organization

Prepare at a time only as many samples as the number of available (i.e. functioning) modules

Start sample preparation no more than 4 hours before a module is available

Do not open a cartridge until you are ready to insert the sample; the lid should be closed as soon as the sample is added.


Do not use the cartridge if:

The expiration date has passed It appears wet Its lid seal appears broken or (accidentally) opened It has been dropped or shaken after you added the

treated sample The reaction tube on the back side appears to be

damaged It has already been processed: each cartridge is for

single-use only, and can not be reused once scanned Its package (pouch of 10 cartridges) has been open for

more than 6 weeks


Preparation of Cartridge: Labelling

Pick up the cartridge only by the right and left side. Do not touch the lid, the barcode on the front side, or the reaction tube on the back side

Label the cartridge with the sample ID by writing on the left or right side of the cartridge or affix ID label

Do not put the label on the lid of the cartridge or obstruct the existing 2D barcode on the cartridge


Preparation of cartridge: Inoculation

Open the cartridge and pipette 2-4 ml of prepared sample using the plastic transfer pipette

Pipette sample carefully to avoid aerosols and bubbles

Do not transfer solid particles into the cartridge

Close lid firmly Start the test


Storage of samples and inoculated cartridges Storage of specimen: Direct or processed (decontaminated/concentrated) sputum Refrigerate at 2–8°C, for 10 days maximum If necessary, at room temperature (up to 35°C) for up to 3 days,

and then refrigerated at 2–8°C, for a combined maximum of 10 days

Storage of specimen in presence of sample reagent: Direct or processed (decontaminated/concentrated) sputum Process within 12 hours, kept at 2-8°C degrees. If refrigeration

is not possible, process within 5 hours Storage of inoculated cartridge (eg, in case of power failure): Run test within 4 hours from the addition of the sample If more than 4 hours has elapsed, inoculate a new cartridge


1. Switch on GeneXpert instrument(a small blue light will appear at the front panel)

2. Switch on the computer

3. Log on to Windows as Cepheid user : User name: CepheidPassword : cphd

4. Double click on “GeneXpert Dx” icon on the desktop

5. Log-in with the user account

Windows 7

Windows XP

Getting Started


6. Click on “No” in the “Database management” dialogue box to begin your work session

Start Software


7. Click on Check Status to confirm all modules available- If not, proceed to troubleshooting (Module 9)

Start Software: Check Status


1.Click on “CREATE TEST”

2. A window opens requesting to scan the cartridge barcode

Starting a test


3. Take the barcode scanner and scan the cartridge barcode by holding the yellow button pressed.

NOTE: If the barcode scanner is not working you can enter the cartridge barcode manually by typing the 2 line number on the cartridges

After the ‘BEEP’ sound, move the scanner away from cartridge to avoid scanning the barcode twice

In case of barcode reader failure while using a new lot, contact Cepheid technical support to collect the Lot Specific Parameter

Starting a test: scan the cartridge


4. After you have scanned the barcode, this window will appear

5. Enter Patient ID = Name

6. Enter Sample ID = Lab Serial Nr.

By default, the software displaystest type as Specimen.

Software automatically assigns amodule to be used(Note: the least used module isselected; a different module canbe manually selected)

7. Click on “Start test”

Starting a test


Always hold cartridge upright: avoid shaking, tilting or dropping

Load the cartridge

Completely open the cartridge bay door of the assigned module indicated by the blinking green light above the selected module

Load the cartridge carefully with the barcode in front

Closing cartridge bay door automatically starts the test


Click on «Create Test» Scan cartridge barcode and click on «Manual Entry»

Type manually the 2 line numbers of the

cartridges

Manual entry of the cartridge barcode

If the barcode scanner is not working you can enter the cartridge barcode manually


Movie is available from FTP: ftp://hbdc:[email protected](paste in Windows Explorer, not Internet Explorer)The FTP movies are large files so should be downloaded on to a CD before the training.

Summary of test processes: Movie





Monitor the test status

Under “Check status” (see next slide), ensure that the status has been updated from “loading” to “run”, meaning that the testing process has started

If you want to see the progress of the test run, you can check: progress of the test (for example, 3/45 means the test is on the

third PCR cycle out of 45 cycles) amount of time remaining until the end of the test status of the test (for example: “OK”) If the Status displays Error or Warning, look at the Messages for

a description of the problem (see next slide)


1.Click on “CHECK STATUS”

2. Progress, status,and remaining timeare indicated

3. Messageswith more details

Monitor the test status


In case your cartridge(s) was(were) not properly prepared, you might have to stop the test to avoid waste of time:

1. Click on “STOP TEST”

2. Select the module(s) that need to be stopped, by marking the tickbox

3. After selecting, click “Stop”

How to stop a test and why


4.Confirm your choice by clicking “Yes”

5.You will see the stopped test’s details in “Check Status” section

How to stop a test and why


Visualize, generate and manage test reports

See Module 7 on Results Interpretation for the following topics: Visualizing the test results Edit test-related information Generate a result report (specimen and patient) How to print automatically test report Difference between archive/backup: how to archive,

retrieve, backup and restore data from a backup How to copy/paste data to Excel


Summary

The Xpert MTB/RIF assay is an integrated micro-fluidic based system comprised of a GeneXpert instrument, Xpert MTB/RIF test cartridges, an automated protocol (simultaneous DNA amplification and detection of fluorescence) with in-built controls (SPC and PCC)

Sample preparation protocols should be followed for direct sputum (2:1), processed sputum sediment (3:1), and extrapulmonary samples – adjust biosafety requirements accordingly

Check for cartridge integrity before use, properly label, and inoculate with sufficient amount (2-4 ml) of final solution

After loading a cartridge into the GeneXpert, the accompanying software allows for test progress to be monitored


Assessment

List and describe the different components of the GeneXpert technology

List and describe the different controls and their mechanism

Describe the sample preparation procedure (according to the sample examined) and the subsequent steps necessary to create and run a test

How do you monitor the status of the test? Why might you want to stop a test? How would you do

that?

Acknowledgements

The Xpert MTB/RIF Training Package has been developed by a consortium of GLI partners, including FIND, KNCV, US CDC, USAID, TB CARE I and WHO, with funding from USAID.

The modules are based on materials originally developed by FIND, KNCV and Cepheid.

module 6: genexpert technology and xpert mtb/rif procedures global laboratory initiative – xpert...

Documents

cepheid slide

procedure slide

results interpretation

ftp movies

internet explorer ftp

instrument sample

genexpert technology

windows explorer