GeneXpert (cartridge-based NAAT)

Principle

• GeneXpert (Cepheid) is an automated, closed cartridge system that combines sample processing, nucleic acid extraction, real-time PCR amplification and detection.
• Xpert MTB/RIF detects Mycobacterium tuberculosis complex DNA and mutations in the 81-bp rifampicin resistance-determining region (RRDR) of the rpoB gene using molecular beacon probes.
• Xpert MTB/RIF Ultra adds multicopy targets (IS6110 and IS1081) for higher sensitivity and uses melt-curve analysis for rpoB mutations.
• Xpert MTB/XDR detects resistance to isoniazid, fluoroquinolones, ethionamide and second-line injectable drugs.

Specimen

• Sputum (spot or early morning), induced sputum, bronchoalveolar lavage, gastric aspirate, and validated extrapulmonary samples (CSF, lymph node aspirate, tissue); stool for children with Ultra.

Procedure

• Add sample reagent (contains NaOH and isopropanol) to sputum at 2:1 ratio, shake, and stand about 15 minutes at room temperature with further shaking; this liquefies and inactivates bacilli.
• Transfer about 2 mL into the cartridge with the supplied pipette; load and start within about 30 minutes.
• Results are ready in about 1.5–2 hours.

Results

• MTB DETECTED (semi-quantitative: high, medium, low, very low; Ultra also "trace") with RIF resistance DETECTED, NOT DETECTED or INDETERMINATE.
• MTB NOT DETECTED.
• INVALID, ERROR or NO RESULT: repeat with a fresh sample or cartridge.
• Ultra "trace" means MTB detected at very low level; rifampicin result is indeterminate.

Quality control and pitfalls

• Built-in sample processing control and probe check control verify processing, inhibition and reagent integrity.
• Detects DNA from dead bacilli, so it is not used to monitor treatment; use smear and culture.
• RIF resistance in low-risk patients may need confirmation because of rare false-resistant results (e.g. silent mutations).
• Does not replace culture and phenotypic drug susceptibility testing.

Clinical use

• WHO-recommended initial test for TB and rifampicin-resistant TB; other cartridges detect SARS-CoV-2, HIV and HCV viral load, C. difficile, MRSA and CT/NG.