GeneXpert MTB/RIF Assay

GeneXpert MTB/RIF assay is a rapid diagnostic test of tuberculosis (TB) and drug resistance. It is revolutionizing TB control with aids in prompt diagnosis and treatment (selection of appropriate TB regimen).

GeneXpert MTB/RIF assay is a nucleic acid amplification (NAA) test which simultaneously detects DNA of Mycobacterium tuberculosis complex (MTBC) and resistance to rifampin (RIF) (i.e. mutation of the rpoB gene) in less than 2 hours. In comparison, standard cultures can take 2 to 6 weeks for MTBC to grow and conventional drug resistance tests can add 3 more weeks.

This system integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences.

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.

The Centers for Disease Control and Prevention (CDC) recommends that NAA testing be performed on at least one respiratory specimen from patients who have a moderate or high suspicion of having pulmonary TB.

Materials/System requirement:

1. GeneXpert System
   • equipped with GX2.1 software/computer/printer/barcode wand-reader and operator manual (Cepheid Inc, Sunnyvale, USA).
   • It is available in a one, two, four, or 16-module configuration
2. GeneXpert Cartridge:
   • Single-use disposable XpertMTB/RIF cartridges
   • Sample extraction, amplification, and detection are all carried out within this self-contained cartridge.
3. Class II biological safety cabinet (BSC)  
4. Sample reagent (provided in Xpert MTB/RIF kit), 8ml volume pack per cartridge. The sample reagent solution is clear but may range from colorless to golden yellow.
5. Permanent marker pens.
6. Sterile (individually packed) disposable transfer pipettes- with a single mark for a minimum volume of sample transfer to cartridge (provided in Xpert MTB/RIF kit).
7. Sterile screw-capped specimen collection containers/cups.
8. Discard containers for pipettes and sputum containers.

For Sample Preparation and step wise guidance, please download this manual.

GeneXpert MTB RIF Manual

Basic Procedure:

1. Collect sputum sample from the patient with suspected TB.
2. The sputum is mixed with the reagent that is provided with the assay, and a cartridge containing this mixture is placed in the GeneXpert machine.
3. All processing from this point on is fully automated.

Advantages of the Xpert MTB/RIF Assay

1. Time efficient methods for detecting Mycobacterium tuberculosis bacteria and mutations isoniazid (INH) resistance.
2. Availability of quick test results leads to improved patient management and outcomes, and preventing unnecessary use of resources (avoiding unnecessary treatment, respiratory isolation).
3. Fully automated system; minimal technical training is required to run the test.
4. Prompt (quick) identification of multidrug-resistant TB (MDR TB)* cases as resistance to RIF, in most instances, co-exists with resistance to INH. Rapid diagnosis of rifampin (RIF) resistance potentially allows TB patients to start on effective treatment much sooner than waiting for results from other types of drug susceptibility testing. If rifampin resistance is detected, confirmation of resistance can be done by DNA sequencing.

*MDR TB is TB that is resistant to both isoniazid (INH) and Rifampicin (RIF).

Interpretation of GeneXpert results:

Results of GeneXpert should be interpreted along with clinical, radiographic, and other laboratory findings. The Xpert MTB/RIF assay does not replace the need for smear with microscopy for acid-fast bacilli, culture for mycobacteria, and growth-based drug susceptibility testing, in addition to genotyping for early discovery of outbreaks.

Results from the Xpert MTB/RIF assay indicate whether or not MTBC was detected in the sample. In some instances, the result is “invalid,” whereby the test should be repeated.

If MTBC was detected, the results will also state whether resistance to RIF was

• Detected: Mycobacteria have a high probability of resistance to RIF; should be confirmed by additional testing.  If RIF resistance is confirmed, rapid molecular testing for drug resistance to both first-line and second-line drugs should be performed so that an effective treatment regimen can be selected.
• Not detected: Mycobacteria are probably susceptible to RIF; All tests that are positive for MTBC should have growth-based susceptibility testing to first-line TB drugs.
• Indeterminate: the test could not accurately determine if the bacteria are resistant to RIF. Growth-based susceptibility testing to first-line TB drugs should be performed.

NOTE: Regardless of the Xpert MTB/RIF result, Center of Disease Control (CDC) strongly recommends a comprehensive testing algorithm that includes AFB smear and mycobacterial culture as well as drug susceptibility testing (DST) and genotyping.

Limitation of Nucleic Acid Amplification (GeneXpert) System

• A negative NAA test result does not exclude the possibility of a positive culture.
• A positive NAA test result does not differentiate between the species of MTBC or determine the presence of other Mycobacteria species.
• Isolates are required for all positive MTB patients in order to perform culture-based drug susceptibility testing and genotyping. 

Further Readings:

• http://www.aphl.org/AboutAPHL/publications/Documents/ID_2013Nov_Cepheid-Xpert-Fact-Sheet.pdf
• http://www.cdc.gov/tb/publications/factsheets/testing/xpert_mtb-rif.htm
• http://www.cdc.gov/tb/publications/factsheets/pdf/xpertmtb-rifassayfactsheet_final.pdf
• http://www.moleculartb.org/gb/pdf/ppt/13_SYMP_ISoundiram_GeneXperttech_2902.pdf