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Laboratory Diagnosis of Tuberculosis

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Key Principle

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1. Specimens

• Pulmonary TB: Fresh sputum (3 consecutive morning samples preferred), gastric washings (especially in children who cannot produce sputum)
• Extrapulmonary TB: Pleural fluid, CSF, urine, joint fluid, biopsy material, blood
• Disseminated/miliary TB: Blood cultures

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2. Decontamination and Concentration

1. Liquefied with N-acetyl-L-cysteine (NALC)
2. Decontaminated with NaOH (kills contaminating bacteria and fungi)
3. Neutralized with buffer
4. Concentrated by centrifugation

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3. Smear Microscopy (Acid-Fast Staining)

A. Ziehl-Neelsen (ZN) Stain

• Classic carbolfuchsin stain - mycobacteria appear red against a blue background
• Based on the acid-fast property: once stained, organisms resist decolorization with acid-alcohol due to high mycolic acid content in cell wall
• Sensitivity ~30-40% for smear-positive TB

B. Fluorochrome Stain (Preferred)

• Auramine-rhodamine stain - mycobacteria fluoresce yellow-orange
• More sensitive than ZN stain and is the preferred method for clinical material
• LED fluorescence microscopes (some battery-powered) have extended this method to resource-limited settings

Note: Gastric washings and urine smears are generally not recommended because saprophytic mycobacteria may be present and give false positives.

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4. Culture

A. Media

• Both selective (with antibiotics to prevent overgrowth) and nonselective media should be used
• Incubation at 35-37°C in 5-10% CO2 for up to 8 weeks
• If results are negative with a positive smear, or if slowly-growing NTM is suspected: incubate a set at a lower temperature (24-33°C) for 12 weeks

B. Colony Characteristics of M. tuberculosis

• Slow-growing: doubling time ~18 hours; colonies appear in 2-8 weeks
• Buff/cream-coloured, rough, wrinkled ("breadcrumb" or "cauliflower" colonies) on LJ
• Non-pigmented (nonchromogen)
• Produces niacin and catalase (heat-labile catalase) - important biochemical tests

C. Runyon Classification (Traditional)

D. Identification Methods

• Rate of growth, colony morphology, pigmentation, biochemical profiles (niacin, nitrate reduction, catalase, urease)
• Requires 6-8 weeks

• DNA probes - species-level ID within days of a positive culture
• HPLC (High-Performance Liquid Chromatography): mycolic acid profiling - available in reference labs
• MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry): species-level ID
• Pyrosequencing

E. Blood Cultures (for MAC/disseminated)

• Two methods: lysis centrifugation system OR commercially available broth media for blood cultures

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5. Susceptibility Testing

• Standardized broth culture technique for first-line drugs
• Agar-based technique (reference labs) for first- and second-line drugs
• MODS assay (Microscopic Observation Drug Susceptibility): multi-well plate, examines for M. tuberculosis "cording" - used widely outside the US
• First-line drugs: INH, rifampicin, pyrazinamide, ethambutol
• Second-line drugs: kanamycin, capreomycin, ethionamide, cycloserine, fluoroquinolones

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6. Nucleic Acid Amplification Tests (NAATs)

• Permit rapid, direct detection of M. tuberculosis in clinical specimens
• GeneXpert MTB/RIF (Cepheid): Real-time multiplex PCR
  • Detects M. tuberculosis complex AND rifampin resistance mutations simultaneously
  • Sensitivity: 98.2% for smear-positive specimens; 72.5% for smear-negative
  • Specificity: 99.2%
• Faster than culture (results in ~2 hours vs. weeks)
• Earlier FDA-cleared commercial assays (Amplified MTD test, Amplicor) also available

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7. Tuberculin Skin Test (TST / Mantoux)

Material

• PPD (Purified Protein Derivative) - chemically fractionated from old tuberculin
• Standardized in Tuberculin Units (TU): 1 TU (first strength), 5 TU (standard/intermediate), 250 TU (second strength)

Technique

• 0.1 mL injected intracutaneously (Mantoux method), volar aspect of forearm
• Read at 48-72 hours - measure induration (not erythema)

Interpretation (CDC cut-points)

• Becomes positive within 4-6 weeks after infection
• BCG vaccination may cause false-positive TST

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8. Interferon-Gamma Release Assays (IGRAs)

Mechanism

• Detect interferon-γ released by sensitized CD4 T cells in response to M. tuberculosis-specific antigens:
  • ESAT-6 (Early Secretory Antigenic Target-6)
  • CFP-10 (Culture Filtrate Protein-10)
  • TB7.7
• These antigens are absent from BCG and most NTM - hence higher specificity

Available Assays

• Results reported as positive, negative, or indeterminate
• Not recommended in severely immunocompromised hosts or children < 5 years
• Comparable to TST for detecting latent TB infection
• Advantage over TST: Not affected by BCG vaccination

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9. Molecular Typing / Genotyping (Epidemiological)

• IS6110 RFLP (Restriction Fragment Length Polymorphism): Gold standard historically; based on variable positions of insertion sequence IS6110 in M. tuberculosis chromosome
• Spoligotyping: PCR-based, targets direct repeat locus
• MIRU-VNTR (Mycobacterial Interspersed Repetitive Units - Variable Number Tandem Repeats): Replacing IS6110 typing; faster
• Done at CDC, state health labs, and research labs

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Summary Algorithm

Suspect TB
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Collect appropriate specimen
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    ├── Smear (ZN / Auramine-rhodamine) → Rapid, cheap, but ~50-80% sensitive
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    ├── Culture (LJ / BACTEC MGIT) → Gold standard; 2-8 weeks
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    ├── NAAT (GeneXpert MTB/RIF) → Rapid (<2h), detects rifampin resistance
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    ├── Tuberculin skin test (TST/PPD) → Latent TB screening
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    └── IGRA (QFT-GIT / T-SPOT.TB) → Latent TB; better in BCG-vaccinated

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