GeneXpert test

What it is. Xpert MTB/RIF is an automated, cartridge-based, closed-system, real-time PCR (nucleic acid amplification test) run on the GeneXpert platform. In a single step it simultaneously detects Mycobacterium tuberculosis complex DNA and rifampicin resistance, the latter by using five molecular beacon probes to detect mutations in the 81-base-pair "hot-spot" (core) region of the rpoB gene, which harbours >95% of rifampicin-resistance mutations. Result in about 2 hours, with minimal training and biosafety requirements — hence its Indian programme name, CBNAAT (cartridge-based nucleic acid amplification test).

Xpert MTB/RIF Ultra. The improved-generation assay adds two multi-copy amplification targets (IS6110 and IS1081), a larger reaction chamber and melting-curve analysis for rifampicin resistance. This lowers the limit of detection from about 114 to 16 colony-forming units/mL, giving markedly better sensitivity in smear-negative, paucibacillary, HIV-associated, paediatric and extrapulmonary disease. Ultra reports an additional semi-quantitative "trace" category, which is treated as positive in high-risk groups but may require clinical correlation elsewhere.

Performance. Against culture, pooled sensitivity ≈85% and specificity ≈98% for pulmonary TB; sensitivity is about 98% in smear-positive but only ≈67% in smear-negative disease (Ultra improves this). For rifampicin resistance, sensitivity ≈95% and specificity ≈98%. It therefore vastly outperforms Ziehl–Neelsen smear microscopy, which needs about 10,000 bacilli/mL.

Indications. WHO recommends Xpert MTB/RIF or Ultra as the initial diagnostic test in all people with signs or symptoms of TB, replacing smear microscopy. It is particularly indicated in presumptive MDR-TB, HIV-associated TB, paediatric TB, and extrapulmonary specimens — and it is the preferred initial test on cerebrospinal fluid for TB meningitis (where a rapid answer changes outcome), as well as on lymph node aspirate, pleural and other body fluids, gastric aspirate and tissue.

Limitations — the marks are here. It cannot distinguish live from dead bacilli, so it must never be used for treatment monitoring or as a test of cure (remains positive for months). It does not detect isoniazid or fluoroquinolone resistance — that needs a line probe assay, Xpert MTB/XDR or sequencing. False rifampicin resistance can arise from silent or borderline rpoB mutations, so a positive result in a low-prevalence setting should be confirmed by a second method. Sensitivity is lower in pleural fluid and in paucibacillary extrapulmonary disease, so a negative result never excludes TB. Practical constraints: stable electricity, ambient temperature control, annual module calibration, and cartridge cost.

Indian context. Under NTEP, universal drug susceptibility testing mandates a rapid molecular test with rifampicin resistance detection for every diagnosed TB patient, and CBNAAT machines have been placed down to district and sub-district level. India also uses the indigenous Truenat MTB / MTB Plus and MTB-RIF Dx — a chip-based, battery-operated, portable real-time PCR system (Molbio, Goa), WHO-endorsed in 2020, which can be deployed at primary health centre level without air conditioning and has brought molecular diagnosis to peripheral settings.

The WHO rapid-diagnostics menu has broadened well beyond Xpert alone: targeted next-generation sequencing (tNGS) directly on sputum was recommended in 2023 for comprehensive drug-resistance profiling in one test; low-complexity automated assays (Xpert MTB/XDR) now detect isoniazid, fluoroquinolone, ethionamide and amikacin resistance as a reflex test after a positive Xpert MTB/RIF; and computer-aided detection (CAD) software on digital chest radiographs is recommended for TB triage in people aged 15 and over. Tongue-swab sampling with Xpert Ultra — which removes the need for sputum production, a real advantage in frail and cognitively impaired older adults — is under active evaluation for programmatic use.