Introduction

The diagnosis of tuberculosis (TB) in children presents a unique challenge compared to adults. Pediatric TB is characterized by a paucibacillary nature (low bacterial load), non-specific clinical presentation, and difficulties in obtaining adequate respiratory specimens. Consequently, the traditional approach relied heavily on a "Golden Triad" of clinical history, tuberculin skin testing, and chest radiography. However, the landscape of TB diagnostics has shifted dramatically with the advent of rapid molecular diagnostics. Current guidelines (NTEP 2022) emphasize Upfront Molecular Testing (Universal Drug Susceptibility Testing or U-DST) to confirm the diagnosis microbiologically and detect drug resistance early. A systematic approach involving microbiological, radiological, immunological, and histological investigations is essential for accurate diagnosis and management.

1. Microbiological Investigations

Microbiological confirmation is the gold standard for diagnosis. It confirms the presence of Mycobacterium tuberculosis (MTB) and allows for drug susceptibility testing (DST).

A. Specimen Collection

Obtaining appropriate samples is the first critical step. Since young children often swallow sputum, alternative methods are required.

• Gastric Aspirate (GA): This is the preferred specimen for children < 6 years who cannot expectorate. Ideally, it is collected early in the morning after overnight fasting (minimum 4–6 hours) before the stomach empties. The procedure involves inserting a nasogastric tube, aspirating stomach contents, and neutralizing the acidity (if culture is delayed) to prevent killing the bacilli. Collecting samples on two consecutive days increases the yield.
• Induced Sputum (IS): In older children or those unable to expectorate spontaneously, sputum production is induced using nebulized hypertonic saline (3–5%). This irritates the airways, increasing mucus clearance and inducing a cough. It is less invasive than GA and can be performed in ambulatory settings.
• Bronchoalveolar Lavage (BAL): Reserved for difficult cases where GA/IS are negative, but suspicion remains high, or for persistent pneumonia. It is invasive but may have a higher yield in specific situations.
• Other Specimens: Depending on the site of involvement, other specimens include Cerebrospinal Fluid (CSF), Lymph Node (LN) aspirate (FNAC), pleural fluid, ascitic fluid, synovial fluid, urine, or tissue biopsies. Note: Extrapulmonary samples for culture should be sent in normal saline, never in formalin.

B. Smear Microscopy

• Ziehl-Neelsen (ZN) Staining: The traditional method to detect Acid-Fast Bacilli (AFB). It requires a high bacillary load (5,000–10,000 bacilli/ml) to be positive.
• Fluorescence Microscopy: Uses auramine-rhodamine staining. It is more sensitive than ZN staining and allows faster screening of slides.
• Limitations: Smear microscopy has low sensitivity in children (<15–20%) due to paucibacillary disease. It cannot differentiate between drug-sensitive and drug-resistant strains or between M. tuberculosis and Non-Tuberculous Mycobacteria (NTM).

C. Nucleic Acid Amplification Tests (NAAT)

Molecular diagnostic tests have revolutionized pediatric TB diagnosis by detecting MTB DNA and drug resistance mutations within hours.

• Cartridge-Based NAAT (CBNAAT) / GeneXpert MTB/RIF:
  • Principle: A semi-automated, real-time PCR assay that amplifies a specific sequence of the rpoB gene.
  • Capabilities: Detects MTB and Rifampicin resistance simultaneously in < 2 hours.
  • Sensitivity: significantly higher than smear microscopy (60–70% in culture-positive gastric aspirates). It is now the diagnostic test of choice (Upfront NAAT) for all presumptive pediatric TB cases.
• Xpert MTB/RIF Ultra: A next-generation cartridge with a larger amplification chamber and two additional molecular targets. It has a lower limit of detection (16 CFU/ml vs. 131 CFU/ml for standard Xpert), making it highly suitable for paucibacillary pediatric samples (GA, stool, CSF). It provides a semi-quantitative result, including "trace" calls for very low bacterial loads.
• TrueNat: An indigenous chip-based, battery-operated PCR test used in India (NTEP). It first detects MTB (TrueNat MTB) and then tests for Rifampicin resistance (TrueNat Rif) if the first step is positive.

D. Culture Methods

Culture remains the definitive gold standard for diagnosis and is essential for phenotypic drug susceptibility testing (DST) and monitoring treatment response in drug-resistant cases.

• Liquid Culture (e.g., MGIT - Mycobacteria Growth Indicator Tube):
  • Automated systems (BACTEC MGIT 960) detect growth via fluorescence consumption of oxygen.
  • Advantages: Faster turnaround time (10–14 days) and higher recovery rate compared to solid media. It is the preferred method for second-line DST.
• Solid Culture (Lowenstein-Jensen Medium):
  • Traditional egg-based medium.
  • Disadvantage: Slow growth (takes 6–8 weeks).
  • Advantage: colonial morphology can be visualized; less expensive.

E. Line Probe Assays (LPA)

These are molecular tests used to detect specific drug resistance mutations in DNA extracted from positive cultures or direct smear-positive specimens.

• First-Line LPA (FL-LPA): Detects resistance to Isoniazid (katG, inhA genes) and Rifampicin (rpoB gene).
• Second-Line LPA (SL-LPA): Detects resistance to Fluoroquinolones (gyrA, gyrB) and Second-line Injectables (rrs, eis).

2. Radiological Investigations

Imaging supports the clinical diagnosis, especially when microbiological tests are negative.

A. Chest Radiography (CXR)

A frontal (PA/AP) view is the initial screening tool. Lateral views may help identify hilar adenopathy.

• Primary Complex: The hallmark of pediatric TB. Consists of a parenchymal focus (Ghon focus), lymphangitis, and regional lymphadenopathy.
• Lymphadenopathy: Hilar or paratracheal nodal enlargement is the most common finding. Compression of airways by nodes can lead to hyperinflation (ball-valve effect) or collapse-consolidation (segmental lesion).
• Parenchymal Disease: Consolidation (unifocal or multifocal), which may be non-specific.
• Miliary TB: Diffuse, tiny (1–2 mm) millet-seed-sized nodules distributed evenly throughout both lung fields. This is a radiological emergency.
• Pleural Effusion: Usually unilateral, exudative effusion. Common in older children/adolescents.
• Adult-type Disease: Apical infiltrates and cavitation are more common in adolescents (post-primary/reactivation TB).
• Limitations: Radiological findings are non-specific and can mimic bacterial pneumonia, viral infections, or malignancies. Up to 50% of children with confirmed TB may have a normal or equivocal CXR.

B. Computed Tomography (CT)

CT of the chest is more sensitive than CXR for detecting:

• Small hilar/mediastinal lymph nodes (especially subcarinal).
• Calcification within nodes (highly suggestive of TB).
• Necrotic nodes: Nodes with a hypodense center and peripheral rim enhancement (ring enhancement) are characteristic.
• Endobronchial complications (stenosis, bronchiectasis).
• Tree-in-bud appearance (suggests endobronchial spread).
• Indications: Persistent pneumonia, inconclusive CXR with high clinical suspicion, or to rule out other etiologies.

C. Ultrasonography (USG)

• Chest: Useful for detecting loculated pleural effusions and guiding thoracentesis.
• Abdomen: Excellent for detecting abdominal TB features like ascites (septated/loculated), omental thickening (cake-like), and necrotic mesenteric lymph nodes.
• Neck: Characterizing cervical lymphadenopathy (matted, necrotic, abscess).

D. Magnetic Resonance Imaging (MRI)

• CNS TB: The modality of choice for Tuberculous Meningitis (TBM). It detects:
  • Basal meningeal enhancement (exudates).
  • Hydrocephalus.
  • Infarcts (vasculitis).
  • Tuberculomas (ring-enhancing lesions with edema).
• Spinal TB (Pott's Spine): Superior for assessing vertebral body destruction, disc space involvement (paradiscal), pre/para-vertebral abscess, and spinal cord compression.

3. Immunological Investigations

These tests indicate infection (Latent TB Infection - TBI) but do not differentiate between infection and active disease. They are supportive tools in high-burden settings.

A. Tuberculin Skin Test (TST) / Mantoux Test

• Method: Intradermal injection of 2 Tuberculin Units (TU) of PPD RT23 (or 5 TU PPD-S).
• Reading: The transverse diameter of induration (not erythema) is measured after 48–72 hours.
• Interpretation:
  • $\ge$ 10 mm: Considered Positive in immunocompetent children (including those with BCG vaccination).
  • $\ge$ 5 mm: Considered Positive in high-risk groups: HIV-infected, severely malnourished (SAM), or close contacts of infectious cases.
• False Negatives (Anergy): Severe TB (miliary/meningitis), viral infections (measles, varicella), malnutrition, immunosuppression (steroids), improper storage/technique.
• False Positives: NTM infection, recent BCG vaccination (effect wanes after 2–3 years).

B. Interferon-Gamma Release Assays (IGRA)

Blood tests that measure the release of interferon-gamma by T-cells in response to specific MTB antigens (ESAT-6, CFP-10).

• Types: QuantiFERON-TB Gold Plus (ELISA-based) and T-SPOT.TB (ELISPOT-based).
• Advantages: Single visit, objective result, no cross-reactivity with BCG (highly specific).
• Disadvantages: Expensive, requires sophisticated lab, indeterminate results in young/immunocompromised children. Like TST, it cannot distinguish latent infection from active disease.

4. Histopathological Investigations

Tissue diagnosis is crucial for extrapulmonary TB (EPTB).

• Fine Needle Aspiration Cytology (FNAC): Useful for accessible lymph nodes (cervical).
  • Findings: Epithelioid cell granulomas with Caseous Necrosis.
  • Microbiology: The aspirate should also be sent for CBNAAT/culture.
• Biopsy: Required for pleural tissue (pleural biopsy has higher yield than fluid), liver, bone marrow, or deep-seated nodes. Demonstrates caseating granulomas.

5. Ancillary and Supportive Investigations

These help in assessing the severity and ruling out comorbidities.

• Complete Blood Count (CBC): Anemia (chronic disease), leukocytosis (or leukopenia in severe disease), lymphocytosis/monocytosis.
• ESR: Usually elevated, but non-specific.
• HIV Screening: Mandatory for all children diagnosed with TB. HIV co-infection alters the presentation (more extrapulmonary/disseminated) and management.
• Liver Function Tests (LFT) / Renal Function Tests (RFT): Baseline assessment before starting hepatotoxic drugs (INH, Rifampicin, PZA) and to adjust doses in renal failure.
• Adenosine Deaminase (ADA):
  • An enzyme marker of T-cell activation.
  • Utility: High levels in fluids (Pleural, Ascitic, Pericardial, CSF) support a diagnosis of TB.
  • Cut-off: Typically > 40 U/L in pleural fluid; > 10 U/L in CSF (varies by lab).
  • Caution: Guidelines (NTEP) consider it supportive, not confirmatory, due to false positives in empyema/malignancy.

6. Specific Investigations for Extrapulmonary TB (EPTB)

A. TB Meningitis (TBM)

• CSF Analysis:
  • Appearance: Clear or "cobweb" formation on standing.
  • Cytology: Pleocytosis (10–500 cells/mm³), predominantly lymphocytes (early stage may show neutrophils).
  • Biochemistry: Elevated Protein (100–500 mg/dL), Low Glucose (< 40 mg/dL or CSF:Blood ratio < 0.5).
  • Microbiology: Smear yield is very low. CSF CBNAAT is recommended (though sensitivity is ~50–60%, specificity is high). Culture is gold standard.
• Neuroimaging: Contrast-enhanced MRI or CT (basal exudates, hydrocephalus, tuberculomas).

B. Pleural TB

• Pleural Fluid Analysis: Exudative (Protein > 3g/dL), Lymphocytic predominant. Glucose may be low.
• Microbiology: Fluid smear/CBNAAT yield is low.
• Biopsy: Pleural biopsy is the most sensitive diagnostic tool for pleural TB.

C. Abdominal TB

• Ascitic Fluid: Exudative, lymphocytic, high ADA.
• Imaging: USG/CT showing characteristic lymphadenopathy (necrotic), bowel wall thickening, or loculated ascites.
• Biopsy: Laparoscopic biopsy of peritoneum or lymph nodes.

7. Integrated Diagnostic Algorithm (NTEP 2022)

The current diagnostic approach integrates clinical suspicion with upfront molecular testing.

1. Screening: Identify Presumptive TB (Fever/Cough > 2 weeks, Weight loss, Contact history).
2. Chest X-ray:
   • Highly Suggestive: Proceed to sample collection.
   • Non-specific: Trial of antibiotics for 7–10 days. If symptoms persist -> Sample collection.
3. Sample Collection: Collect Sputum/Induced Sputum/Gastric Aspirate.
4. Upfront Molecular Test (CBNAAT/TrueNat):
   • MTB Positive, Rif Sensitive: Treat as Drug-Sensitive TB.
   • MTB Positive, Rif Resistant: Refer for DR-TB management (LPA/Liquid Culture).
   • MTB Negative: If clinical suspicion remains high (High-grade fever, contact history, suggestive CXR), treat as "Clinically Diagnosed TB" (Probable TB) after ruling out other causes.

Conclusion

The investigation of TB in children has evolved from a reliance on surrogate markers (TST, X-ray) to precise microbiological confirmation. While the "Golden Triad" remains relevant for clinical decision-making in resource-limited or culture-negative scenarios, Upfront NAAT (CBNAAT) is now the standard of care. This ensures not only the diagnosis of TB but also the early detection of drug resistance, enabling the initiation of appropriate therapy and improving outcomes in pediatric tuberculosis.

Newer Modalities in the Diagnosis of Tuberculosis

1. Molecular Diagnostics (Nucleic Acid Amplification Tests - NAAT)

These tests amplify specific DNA sequences (e.g., IS6110, rpoB) to detect M. tuberculosis (MTB) and drug resistance mutations. They are now the diagnostic tests of choice (Upfront NAAT) for all presumptive pediatric TB cases.

A. Cartridge-Based NAAT (CBNAAT) / GeneXpert MTB/RIF

• Principle: Automated, real-time PCR assay integrated into a self-contained cartridge.
• Targets: Amplifies the rpoB gene to detect MTB and mutations conferring Rifampicin resistance.
• Turnaround Time: Results are available in approximately 2 hours.
• Sensitivity: Significantly higher than smear microscopy (60–70% in culture-positive gastric aspirates). It requires a lower bacillary load (~131 CFU/mL) compared to smear (~10,000 CFU/mL).
• Application: Recommended for use with sputum, gastric aspirate, CSF, and lymph node aspirates.

B. Xpert MTB/RIF Ultra (Next Generation)

• Advantage: Uses a larger amplification chamber and two additional multi-copy amplification targets (IS6110 and IS1081).
• Limit of Detection: It has a much lower limit of detection (16 CFU/mL) compared to standard Xpert.
• Utility in Children: Because of its high sensitivity, it is particularly useful for paucibacillary pediatric samples (e.g., gastric aspirate, stool, nasopharyngeal aspirate). It provides semi-quantitative results, including "trace" calls for very low bacterial loads, which should be interpreted in the clinical context.

C. Truenat

• Features: An indigenous, chip-based, battery-operated, micro-PCR device developed in India. It is portable and suitable for peripheral health centers.
• Process: It involves a two-step process:
  1. Truenat MTB: Detects the presence of MTB.
  2. Truenat MTB-Rif Dx: If MTB is positive, a second chip is used to detect Rifampicin resistance.
• Status: Endorsed by the WHO and NTEP as a point-of-care rapid molecular test.

D. Xpert MTB/XDR

• Scope: A newer cartridge compatible with GeneXpert platforms (requires 10-color modules).
• Targets: Detects mutations associated with resistance to Isoniazid (H), Fluoroquinolones (FQ), Second-line Injectables (SLI), and Ethionamide.
• Utility: Allows for rapid molecular drug susceptibility testing (DST) for second-line drugs, essential for initiating appropriate regimens for drug-resistant TB (e.g., shorter oral Bedaquiline regimens). Processing time is ~90 minutes.

E. Line Probe Assays (LPA)

• Principle: DNA strip assays that use PCR and reverse hybridization to detect specific drug resistance mutations.
• Types:
  • First-Line LPA (FL-LPA): Detects resistance to Rifampicin (rpoB) and Isoniazid (katG for high-level, inhA for low-level resistance).
  • Second-Line LPA (SL-LPA): Detects resistance to Fluoroquinolones (gyrA, gyrB) and Second-line Injectables (rrs, eis).
• Constraint: Requires a high bacillary load; thus, it is performed directly on smear-positive specimens or indirectly on culture isolates. Turnaround time is 48–72 hours.

2. Newer Approaches to Specimen Collection

To overcome the difficulty of obtaining sputum in young children, newer diagnostic modalities are being validated on non-invasive specimens.

• Stool Testing: MTB DNA can be detected in stool (swallowed sputum) using Xpert MTB/RIF or Ultra. Processing involves simple filtration or centrifugation. It is non-invasive and useful in children who cannot expectorate or tolerate gastric lavage.
• Nasopharyngeal Aspirate (NPA): Easier to collect than gastric aspirate; useful for Xpert Ultra testing, though sensitivity may be lower than gastric aspirate.

3. Newer Tests for TB Infection (Latent TB)

These tests identify immune sensitization to MTB but do not distinguish between latent infection and active disease.

A. Interferon-Gamma Release Assays (IGRA)

• Principle: Measures interferon-gamma released by T-lymphocytes after stimulation with MTB-specific antigens (ESAT-6, CFP-10, and TB7.7).
• Types: QuantiFERON-TB Gold Plus (ELISA-based) and T-SPOT.TB (ELISPOT-based).
• Advantages: No cross-reactivity with BCG vaccination (highly specific); single patient visit required.
• Disadvantage: Expensive; reduced sensitivity in young children (<2 years) and immunocompromised hosts due to indeterminate results.

B. C-Tb (Next Generation Skin Test)

• Description: A novel skin test developed (e.g., by Statens Serum Institut) that combines the operational ease of the tuberculin skin test (TST) with the high specificity of IGRAs.
• Antigens: Uses recombinant ESAT-6 and CFP-10.
• Key Feature: Like IGRAs, it is unaffected by prior BCG vaccination.
• Reading: Induration is measured after 48–72 hours; a universal cutoff of $\ge$ 5 mm is proposed. It is expected to be adopted in programs once commercially available.

4. Advances in Culture and Phenotypic DST

While molecular tests are rapid, culture remains the gold standard for monitoring treatment response and detecting resistance to newer drugs (e.g., Bedaquiline).

• Liquid Culture Systems (e.g., MGIT 960):
  • Mycobacteria Growth Indicator Tube (MGIT) uses fluorescence to detect oxygen consumption by growing bacteria.
  • Advantage: Faster detection (5–14 days) compared to solid Lowenstein-Jensen media (6–8 weeks) and higher recovery rates.
  • Application: Essential for testing susceptibility to Bedaquiline, Clofazimine, and Linezolid.

5. Other Emerging Modalities

• Lipoarabinomannan (LAM) Assay: A lateral flow urine test detecting the mycobacterial cell wall antigen LAM. Currently recommended by WHO primarily for HIV-positive patients with low CD4 counts or those who are seriously ill, as sensitivity is low in immunocompetent children.
• Next-Generation Sequencing (NGS): Whole Genome Sequencing (WGS) or Targeted NGS can rapidly provide a complete drug resistance profile for all anti-TB drugs. Currently restricted to reference laboratories and research settings but holds promise for comprehensive U-DST in the future.

Summary Table