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Tuberculosis (TB) - Investigation Tests

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1. Microbiological Tests

A. Sputum Smear Microscopy (ZN Stain / Auramine-Rhodamine)

• Ziehl-Neelsen (ZN) stain - identifies Acid-Fast Bacilli (AFB); red bacilli on blue background
• Auramine-Rhodamine (fluorescence microscopy) - more sensitive; glowing bacilli under UV
• Grades: 0 (no AFB), Scanty (1-9 AFB/100 fields), 1+ (10-99/100 fields), 2+ (1-10/field), 3+ (>10/field)
• Minimum 3 sputum samples (spot-morning-spot) recommended
• Sensitivity ~40-60%; Specificity ~98%
• Limitation: cannot distinguish live vs. dead bacilli; negative in paucibacillary disease

B. CBNAAT / GeneXpert MTB/RIF (Cartridge-Based Nucleic Acid Amplification Test)

• First-line molecular test (preferred over smear microscopy per Indian RNTCP/WHO guidelines)
• Detects M. tuberculosis DNA and rifampicin resistance simultaneously in ~2 hours
• High sensitivity and specificity; useful even in smear-negative cases
• Can be used on sputum, BAL, CSF, lymph node aspirate, gastric lavage, urine, etc.
• Result categories: MTB detected/not detected; RIF resistance detected/not detected

C. Culture

• Gold standard for definitive TB diagnosis
• Solid media: Lowenstein-Jensen (LJ) medium - takes 4-8 weeks
• Liquid media: MGIT (Mycobacteria Growth Indicator Tube) - faster (1-3 weeks)
• Allows full Drug Sensitivity Testing (DST) including first-line and second-line drugs
• More sensitive than smear (detects as few as 10-100 bacilli/mL vs. 5,000-10,000 for smear)

D. Drug Sensitivity Testing (DST)

• Line Probe Assay (LPA): First-line (FL-LPA) and second-line (SL-LPA)
  • FL-LPA: detects resistance to H (isoniazid) and R (rifampicin)
  • SL-LPA: detects resistance to fluoroquinolones and second-line injectables
• Liquid Culture DST (LC-DST): full phenotypic sensitivity panel
• Performed when CBNAAT shows RIF resistance or on treatment failure

E. Other Molecular Tests

• TrueNat MTB / MTB Plus: point-of-care NAAT; comparable to GeneXpert
• Whole Genome Sequencing (WGS): emerging gold standard for complete resistance mapping
• PCR: conventional or real-time PCR for M. tuberculosis

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2. Immunological Tests

A. Tuberculin Skin Test (TST) - Mantoux Test

• Technique: 0.1 mL of PPD (Purified Protein Derivative) RT-23 (2 TU) injected intradermally on volar aspect of forearm
• Read at 48-72 hours by measuring induration (not erythema) in mm
• Interpretation:

• Limitations: False positive with BCG vaccination and NTM infection; false negative in immunosuppression, malnutrition, miliary TB, recent viral infection

B. Interferon-Gamma Release Assays (IGRA)

• Types: QuantiFERON-TB Gold Plus (QFT-Plus), T-SPOT.TB
• Measures IFN-γ released by T-cells in response to M. tuberculosis-specific antigens (ESAT-6, CFP-10)
• Advantages over TST: Not affected by BCG vaccination; requires only 1 visit; more specific
• Limitations: Expensive; cannot distinguish latent from active TB; may be indeterminate in immunosuppressed patients
• Used mainly for latent TB infection (LTBI) diagnosis
• As per Goldman-Cecil: TST and IGRA are both positive in latent and active TB; culture and smear distinguish the two

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3. Radiological Investigations

A. Chest X-Ray (CXR)

• Most widely used initial investigation
• Classical findings:
  • Primary TB: Ghon focus (mid/lower lobe opacity) + hilar lymphadenopathy = Ghon complex; calcified = Ranke complex
  • Post-primary/Reactivation TB: Cavitary lesions in apical and posterior segments of upper lobes (classically right upper lobe), fibronodular shadows, calcifications
  • Miliary TB: Diffuse miliary mottling (1-2 mm nodules) throughout both lung fields
  • Pleural effusion, collapse/consolidation

B. CT Chest (HRCT/CT)

• More sensitive than CXR, especially for early disease, miliary TB, mediastinal nodes
• Identifies cavitation, tree-in-bud pattern (endobronchial spread), pleural disease
• Used when CXR is inconclusive

C. CT/MRI Brain

• For TB meningitis or tuberculoma - ring-enhancing lesions, hydrocephalus, basal exudate

D. CT Abdomen/Pelvis

• For abdominal TB - lymphadenopathy, ascites, ileocaecal involvement

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4. Histopathological Investigation

• Tissue biopsy (lymph node, pleura, lung, bowel, liver, bone marrow)
• Classic finding: Caseating granuloma - central caseous necrosis surrounded by epithelioid macrophages, Langhans giant cells, lymphocytes
• AFB stain on tissue sections (ZN stain)
• Also send for culture and GeneXpert

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5. Other Investigations

Bronchoscopy

• For smear-negative pulmonary TB; BAL (bronchoalveolar lavage) sent for smear, culture, CBNAAT
• Transbronchial lung biopsy (TBLB) or EBUS (endobronchial ultrasound-guided biopsy) for lymph nodes

CSF Analysis (for TB Meningitis)

Adenosine Deaminase (ADA)

• Elevated in pleural fluid (>40 U/L), CSF, ascitic fluid
• Highly suggestive of TB in pleural effusion; quick, cheap, useful test

Pleural Fluid Analysis

• Exudative, lymphocytic effusion; ADA >40 U/L strongly suggests TB

Blood Tests

• ESR elevated (non-specific)
• CBC: anemia, lymphocytosis
• LFT (baseline before treatment and monitoring)
• Serum creatinine (especially if injectable drugs used in MDR-TB)

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6. Summary Table

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