Molecular Methods for Diagnosis of Tuberculosis (TB)

1. Overview & Rationale

2. Classification of Molecular Methods

3. Nucleic Acid Amplification Tests (NAATs)

3.1 Xpert MTB/RIF (GeneXpert)

• Hemi-nested real-time PCR targeting the rpoB gene (81-bp rifampicin resistance-determining region, RRDR)
• Detects both MTB complex and RIF resistance simultaneously
• Uses molecular beacon probes

• Overall sensitivity: 88% (95% CI 83–92%)
• Specificity: 98% (95% CI 97–99%)
• In smear-positive cases: sensitivity ~98%
• In smear-negative cases: sensitivity ~67–70%
• Somewhat lower sensitivity in PLHIV due to higher smear-negative disease burden; however, sensitivity increases as CD4 count falls below 500 cells/mm³ (paradoxically higher bacillary burden at very low CD4)
• Turnaround: ~2 hours
• Specimens: sputum (direct or concentrated), BAL, gastric aspirate, CSF, lymph node aspirate, stool, urine

• Closed system — minimal contamination risk
• Biosafety Level 2 lab requirements only
• Deployable at peripheral/district-level labs

• Cannot detect resistance beyond RIF (with this version)
• False RIF resistance due to non-resistance rpoB mutations
• Does not replace culture for definitive DST

3.2 Xpert MTB/RIF Ultra

• Added targets: IS6110 and IS1081 insertion sequences (high copy number) alongside rpoB
• Sensitivity increased to ~90–95% in smear-negative/culture-positive cases
• Lower limit of detection: ~16 CFU/mL vs ~112 CFU/mL for original Xpert
• Specificity slightly lower (~96%) due to detection of non-viable organisms (residual DNA)
• Particularly preferred for:
  • Smear-negative pulmonary TB
  • PLHIV
  • Extrapulmonary TB (CSF, pleural fluid)

3.3 Xpert MTB/XDR

• Targets: inhA, katG, rpoB, gyrA, gyrB, rrs, eis, fabG1
• Used as reflex test after Xpert MTB/RIF Ultra confirms RIF resistance
• WHO-recommended for detection of pre-XDR and XDR-TB

3.4 TrueNAT MTB / MTB Plus / MTB-RIF Dx

• Battery-operable, portable — designed for point-of-care use at peripheral labs
• Targets IS6110 (MTB detection) and rpoB (RIF resistance)
• WHO-endorsed for initial TB diagnosis and RIF resistance testing
• Sensitivity comparable to Xpert in smear-positive; lower in smear-negative
• Turnaround: ~1 hour

3.5 Abbott RealTime MTB / COBAS TaqMan MTB / Hologic Amplified MTD

4. Line Probe Assays (LPAs)

4.1 GenoType MTBDRplus (Hain Lifescience)

• Targets: rpoB (RIF resistance), katG + inhA promoter (INH resistance)
• Detects mutations conferring MDR-TB
• Specimens: smear-positive sputum (direct) or culture isolates
• Turnaround: ~5 hours (from DNA extraction)
• Sensitivity for RIF resistance: ~98%; INH resistance: ~85–90%
• Limitation: misses low-level INH resistance mutations in oxyR-ahpC and other regions

4.2 GenoType MTBDRsl (Hain Lifescience)

• Targets: gyrA/gyrB (fluoroquinolone resistance), rrs/eis (injectable resistance)
• Used to detect pre-XDR and XDR-TB after MDR-TB confirmed
• Version 2.0 has improved sensitivity vs v1.0

4.3 Nipro NTM+MDRTB Detection Kit

• Similar principle; detects NTM species alongside MTB
• Used in Japan and select Asian countries

5. Loop-Mediated Isothermal Amplification (TB-LAMP)

• Target: IS6110, devR, 16S rRNA (depending on kit)
• WHO-endorsed as replacement for smear microscopy in symptomatic TB suspects
• Sensitivity: ~80% (smear-positive), ~50–60% (smear-negative) — better than smear, inferior to Xpert
• Turnaround: ~1 hour
• Visual readout with naked eye using fluorescent dye (no complex reader needed)
• Suitable for peripheral/low-resource settings
• Does not detect drug resistance

6. Whole Genome Sequencing (WGS)

Clinical Applications:

Platforms:

• Short-read: Illumina MiSeq/NextSeq (most common; high accuracy, slower)
• Long-read: Oxford Nanopore (faster, deployable; higher error rate)
• Direct WGS from sputum: Emerging — bypasses culture; currently limited by sensitivity and host DNA contamination

WHO Position:

Turnaround: 1–7 days (from culture positive isolate); 2–5 days in optimized pipelines

7. Molecular Typing Methods (Epidemiology)

7.1 MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit – Variable Number Tandem Repeat)

• Gold-standard molecular typing method pre-WGS
• 24-locus MIRU-VNTR panel provides high discriminatory power
• PCR-based; faster than IS6110 RFLP
• Standardized global database (SITVIT/MIRU-VNTRplus) for strain comparison
• Still widely used where WGS is not available

7.2 Spoligotyping (Spacer Oligonucleotide Typing)

• Targets the direct repeat (DR) region of MTB genome
• Identifies lineages and families (e.g., Beijing strain, Haarlem, LAM, T-family)
• Lower discriminatory power than MIRU-VNTR; often used in combination
• Global database: SPOTCLUST / SpolDB4

7.3 IS6110 RFLP (Restriction Fragment Length Polymorphism)

• Historical gold standard for typing
• High discriminatory power for strains with ≥6 IS6110 copies
• Labor-intensive, requires large amounts of DNA, slow (~6 weeks from culture)
• Largely replaced by MIRU-VNTR and WGS

8. Extrapulmonary TB: Molecular Diagnosis

9. Pediatric TB

• Gastric aspirate and nasopharyngeal aspirate: validated specimens for Xpert
• Stool Xpert Ultra: WHO-endorsed — sensitivity ~67–70% vs culture
• LPAs generally not useful in children (low bacillary load)
• Stool and urine Xpert expanding access in low-resource pediatric settings

10. Drug Resistance Detection: Summary

11. WHO mWRD Recommendations Summary

1. Xpert MTB/RIF Ultra — recommended as initial diagnostic test for all individuals with signs/symptoms of TB (pulmonary and extrapulmonary), replacing smear microscopy as the first-line test where feasible
2. TrueNAT — alternative to Xpert at peripheral levels
3. TB-LAMP — replacement for smear microscopy in symptomatic individuals (not for resistance detection)
4. LPA (MTBDRplus) — for rapid MDR-TB detection from smear-positive specimens or culture isolates
5. LPA (MTBDRsl) — for second-line DST after MDR-TB confirmed
6. Xpert MTB/XDR — for detection of pre-XDR and XDR-TB
7. WGS — for comprehensive resistance profiling at reference laboratory level

12. Advantages vs. Limitations Summary

Key Points

• Xpert MTB/RIF Ultra is the WHO-recommended initial diagnostic test for TB across all settings (pulmonary and extrapulmonary), combining TB detection with RIF resistance in ~2 hours.
• LPAs (MTBDRplus/sl) provide rapid MDR/XDR-TB detection, critical for timely initiation of appropriate regimens.
• WGS is the comprehensive reference standard, increasingly used for resistance profiling and outbreak control.
• All molecular tests complement but do not replace culture, which remains necessary for complete phenotypic DST, growth confirmation, and species identification.
• In PLHIV, Xpert sensitivity correlates inversely with CD4 count — patients with CD4 <100 cells/mm³ often have higher bacillary burden (Prevention and Treatment of Opportunistic Infections, p. 480).
• Pediatric and extrapulmonary TB remain diagnostic challenges; stool Xpert Ultra and tissue biopsies are key advances.