Drug susceptible test
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Drug Susceptibility Testing (DST)
Drug susceptibility testing determines whether a microorganism - most commonly Mycobacterium tuberculosis - is susceptible or resistant to specific antimicrobial agents. It is the cornerstone of guiding targeted therapy and preventing treatment failure.
Why DST Matters
- WHO considers universal DST the current standard of care for all TB patients
- DST to rifampin resistance serves as an excellent proxy for MDR-TB (multidrug-resistant TB) diagnosis
- Recommended for all initial M. tuberculosis isolates
- Essential when RR-TB (rifampin-resistant TB) is detected, to guide selection of appropriate regimens for second-line drugs
Two Main Categories of DST
1. Phenotypic Methods (Culture-Based)
Based on actual growth of M. tuberculosis in the presence of drugs.
Two sub-methods:
- Absolute method - growth observed in drug-containing media vs. control
- Proportional method (reference standard) - resistance is present when growth on drug-containing medium is ≥1% of control growth on drug-free media
Media options:
- Liquid media - faster (average ~3 weeks), preferred; automated systems recommended
- Solid media - slower (results may take ≥8 weeks)
Growth detection approaches:
| Method | Principle | Notes |
|---|---|---|
| Colorimetric | Reduction of oxidation-reduction indicator (alamarBlue, resazurin, tetrazolium) | Color change signals viable organisms; 97-98% sensitive for RIF & INH resistance |
| Nitrate Reductase Assay | M. tuberculosis reduces nitrate to nitrite (detected by color reaction) | 97% sensitive, 100% specific for rifampin resistance |
| MODS (Microscopic Observation of Drug Susceptibility) | Identifies M. tuberculosis AND determines drug susceptibility in the same test | Detects RIF & INH susceptibility simultaneously |
2. Genotypic Methods (Molecular)
Faster than culture-based tests - detect drug resistance-associated gene mutations.
Key mutations targeted:
| Drug | Gene Mutation | Detection Rate |
|---|---|---|
| Rifampin | rpoB (rifampin resistance-determining region) | ~96% of resistant strains |
| Isoniazid | katG or inhA | 65-75% of resistant strains |
| Fluoroquinolones | gyrA and gyrB | 42-85% of resistant strains |
Key Molecular DST Platforms
Line Probe Assays (LPAs)
- DNA strip-based tests detecting bacterial DNA + resistance-associated mutations
- Process: DNA extraction → PCR amplification → labeled probe hybridization → colorimetric detection
- WHO recommends LPAs for detecting INH and RIF resistance in smear-positive sputum or cultured isolates
- Second-line LPAs now recommended for rapid detection of fluoroquinolone and second-line injectable drug resistance in MDR-TB
Xpert MTB/RIF / Xpert MTB/RIF Ultra / Truenat MTB-Rif Dx
- Rapid, cartridge-based molecular tests
- Simultaneously detect M. tuberculosis AND rifampin resistance
- WHO-recommended as the initial diagnostic test in HIV-associated TB suspects (preferred over conventional microscopy + culture)
Whole Genome Sequencing (WGS)
- Provides comprehensive information on all resistance-conferring mutations
- Historically required culture, but targeted next-generation sequencing (tNGS) can now be performed directly from sputum
- WHO now recommends tNGS to detect drug resistance after TB diagnosis to guide treatment decisions
- Limitation: suboptimal sensitivity for some newer/repurposed drugs; confirmation by phenotypic DST still needed
Which Drugs Are Tested?
| Priority | Drugs | Notes |
|---|---|---|
| First-line (mandatory) | Isoniazid (INH), Rifampin (RIF) | Reliable; RIF resistance = MDR-TB proxy |
| First-line (less reliable) | Streptomycin, Ethambutol, Pyrazinamide | Less reproducible |
| WHO Group A second-line | Moxifloxacin, Levofloxacin, Bedaquiline, Linezolid | Automated liquid culture preferred |
| WHO Group B | Clofazimine, Cycloserine/Terizidone | Clofazimine via automated liquid culture |
| WHO Group C | Amikacin, Delamanid, Pyrazinamide | PZA testing: high false-positive rate without QA |
| Not standardized | EMB, Ethionamide, PAS, Imipenem, Meropenem | Not currently recommended |
Direct vs. Indirect Testing
- Direct DST: performed on the clinical specimen (sputum) - faster
- Indirect DST: performed on cultured isolates - standard approach when molecular methods aren't available
Clinical Application Summary
- Collect sputum → send for smear microscopy + culture + DST
- Use rapid molecular test (Xpert MTB/RIF) at least once as baseline
- If RIF-resistant → immediately expand to second-line DST (fluoroquinolones, injectables)
- If MDR-TB confirmed → tNGS or LPA for comprehensive resistance profiling
- Repeat DST if patient remains culture-positive during treatment
Sources: Murray & Nadel's Textbook of Respiratory Medicine; Harrison's Principles of Internal Medicine 22E (2025)
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