Introduction
First-Line Drug Susceptibility Testing (FL-DST or sometimes referred to in context as SL-DST when covering all first-line agents: Rifampicin, Isoniazid, Ethambutol, Pyrazinamide) is a crucial laboratory procedure used to determine if a patient’s Mycobacterium tuberculosis (Mtb) strain is sensitive or resistant to primary anti-TB drugs. This testing is essential to guide effective treatment, particularly in preventing the emergence of multidrug-resistant tuberculosis (MDR-TB).
- Purpose: To guide therapy by identifying effective drugs, confirming suspected drug resistance, and facilitating surveillance.
- When to Test: WHO recommends universal DST for all TB patients, particularly upon initial positive culture, or when Rifampicin resistance is found.
- First-Line Drugs (FLDs): The standard panel includes Rifampicin (RIF), Isoniazid (INH), Ethambutol (EMB), and Pyrazinamide (PZA).
- Methods: Phenotypic (culture-based) methods are the gold standard, although rapid molecular tests (e.g., GeneXpert, LPA) are increasingly used as first-line diagnostics.
Principle
The principle of phenotypic DST (such as LJ medium or MGIT) is to measure the growth of Mtb in the presence of a specific, defined Critical Concentration (CC) of an anti-TB drug.
- Critical Concentration: The lowest concentration of a drug that inhibits at least 95% of “wild-type” (drug-susceptible) Mtb strains, but does not inhibit strains from patients who have failed therapy (which are considered resistant).
- Proportion Method: Growth of the bacteria in the drug-containing medium is compared to a control medium (without drugs). If the growth in the drug tube exceeds a predefined proportion (e.g., >1% of control), the strain is reported as resistant.
Procedure (Phenotypic – Liquid/Solid)
The conventional “indirect” DST method involves cultured isolates:
- Isolate Preparation: The M. tuberculosis isolate is cultured from a patient sample, typically on Lowenstein-Jensen (LJ) or Middlebrook 7H10/7H11 media.
- Inoculum Standardization: A pure culture is suspended to a specific turbidity, often corresponding to a McFarland standard 1, to ensure a consistent bacterial load.
- Inoculation: The suspension is inoculated into tubes/plates containing the critical concentrations of the first-line drugs (RIF, INH, EMB, PZA) and control tubes (no drug).
- Incubation: Incubated at \(35-37^{\circ}\text{C}\) for up to 6 weeks for solid media, or until the liquid system (e.g., MGIT 960) flags the sample.
- Reading: The growth is interpreted.
- Note: PZA requires an acidic pH (approx. 5.5–6.0) for testing, as it is only active in acidic conditions.
Result Interpretation
- Susceptible: No growth (or minimal growth below the 1% threshold) in the tube containing the critical concentration of the drug, while the control tube shows growth.
- Resistant: Significant growth in the tube containing the critical concentration of the drug, indicating the bacteria can survive at that level.
- MDR-TB: Resistant to at least Rifampicin and Isoniazid.
- Invalid/Contaminated: Growth in the control tube is absent, or growth is contaminated with other bacteria.
Keynotes and Best Practices
- Gold Standard: Phenotypic DST on liquid media (MGIT) is faster (1-2 weeks) compared to solid media (3-6 weeks).
- Molecular Alternatives: Rapid tests like GenoType MTBDRplus or Xpert MTB/RIF can detect key RIF/INH resistance mutations in hours.
- Quality Control: It is crucial to test against known susceptible strains to ensure media accuracy and potency of drugs.
- Reporting: Results should be reported clearly, usually as “Susceptible” or “Resistant” for each drug tested.
- Limitations: Phenotypic methods can be slow. Furthermore, PZA and ETH testing can be technically difficult and less reliable compared to INH/RIF.
Further Readings
- https://pmc.ncbi.nlm.nih.gov/articles/PMC1951260/
- https://publications.ersnet.org/content/erj/25/3/564
- https://www.who.int/publications/i/item/9789241514842
- https://ntblcp.org.ng/resources/national-guidelines-for-culture-and-drug-susceptibility-testing/
- https://tbksp.who.int/en/node/3155
- https://www.acm.or.kr/2804-07/
- https://www.cdc.gov/tb/webcourses/TB101/page17050.html
- https://www.sciencedirect.com/science/article/pii/S2405579423000505
- https://www.aphl.org/programs/infectious_disease/tuberculosis/Documents/ID_2007Dec_TB-DST-Report.pdf
- https://journals.lww.com/jfmpc/fulltext/2024/13060/drug_susceptibility_testing_and_line_probe_assay.50.aspx
- https://pmc.ncbi.nlm.nih.gov/articles/PMC9570811/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC6219890/
- https://microbiologyjournal.org/tuberculosis-diagnosis-and-detection-of-drug-resistance-a-comprehensive-updated-review/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC3264138/
- https://www.sciencedirect.com/science/article/pii/S2212553112001173