Diagnostic modalities for tb spm
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Diagnostic Modalities for Tuberculosis (SPM / Park's Textbook)
From an SPM perspective, TB diagnosis is approached through case finding - identifying infectious cases early to break the chain of transmission. The diagnostics are classified as follows:
A. MICROBIOLOGICAL (Definitive) DIAGNOSIS
1. Sputum Smear Microscopy (Primary Method)
Sputum examination by direct microscopy is the method of choice and the number one case-finding tool worldwide. It is reliable, cheap, and easy to perform. It detects cases who are excreting tubercle bacilli - the epidemiologically most important group.
Collection of specimens:
- Two sputum samples are collected (not just one - single sample catches only ~80% of smear-positive cases)
- Sample 1: On-the-spot specimen collected at first visit
- Sample 2: Early morning specimen (secretions accumulate overnight, higher bacillary load)
- A wide-mouthed, leak-proof container is used
- Collected away from others, in open-air or well-ventilated area
Staining methods:
| Method | Stain | Key Feature |
|---|---|---|
| Ziehl-Neelsen (ZN) | Carbol fuchsin | Standard, most widely used |
| Fluorescence microscopy | Auramine stain | Field of view 5-10x bigger; scan time 1-2 min per smear; used mainly in industrialized countries |
| LED Fluorescence microscopy | Auramine | Less expensive than conventional FM; comparable accuracy to conventional FM; superior to conventional ZN; WHO recommends phasing in as alternative to ZN |
Grading of smear results (NTEP):
| Grade | Bacilli seen |
|---|---|
| Negative | No AFB/100 fields |
| Scanty | 1-9 AFB/100 fields (exact count recorded) |
| 1+ | 10-99 AFB/100 fields |
| 2+ | 1-10 AFB/field in >50 fields |
| 3+ | >10 AFB/field in >20 fields |
- One positive specimen out of two is sufficient to label a patient as smear-positive
- A maximum of 20 smears per microscopist per day is recommended (visual fatigue degrades quality)
- Positivity requires at least 10,000 organisms/ml of sputum
False positives arise from: scratches on slide retaining red stain, accidental AFB transfer, environmental mycobacteria contamination, acid-fast food particles/other microorganisms.
False negatives arise from: inadequate sputum collection, improper staining technique, inadequate examination time, administrative errors.
2. Culture
- Highly sensitive and specific - the gold standard for TB diagnosis
- Requires 2-8 weeks - too slow for early diagnosis; not routine under NTEP for initial diagnosis
- Uses:
- Follow-up of DR-TB patients on treatment (to detect early recurrence)
- Long-term follow-up of DS-TB patients for relapse-free cure confirmation
Liquid Culture System - MGIT (Mycobacteria Growth Indicator Tube):
- Automated system detecting mycobacterial growth
- Culture results available within 42 days
- DST results available 14-26 days after cultures turn positive
B. RAPID MOLECULAR DIAGNOSTIC TESTS
3. CB-NAAT (Cartridge-Based Nucleic Acid Amplification Test) / GeneXpert / Xpert MTB/RIF
- Detects DNA sequences specific for MTB complex and rifampicin resistance simultaneously by PCR
- Mechanism: concentrates MTB bacilli from sputum → isolates genomic material by sonication → amplifies DNA by PCR → identifies rpoB gene mutations using fluorescent probes called molecular beacons (real-time format)
- Results from unprocessed sputum samples in 90 minutes
- Used preferentially in: key populations - children, PLHIV, extrapulmonary TB, smear-negative TB, presumptive DR-TB
- TrueNat is another NAAT device used under NTEP
4. Line Probe Assay (LPA)
Two versions used under NTEP:
| LPA Type | Targets Detected |
|---|---|
| First-line LPA (FL-LPA) | MTB complex + Rifampicin resistance (rpoB) + Isoniazid resistance (katG, inhA) |
| Second-line LPA (SL-LPA) | Fluoroquinolone resistance (gyrA, gyrB) + Second-line injectable resistance (rrs, eis) |
- DNA from smear-positive samples is extracted directly and subjected to PCR
- Smear-negative samples are first grown in liquid culture; LPA performed on culture isolate
- PCR products are reverse hybridized on nitrocellulose strips with specific probes
C. SUPPORTIVE / SUPPLEMENTARY TOOLS
5. Chest X-ray (Radiography)
- Not routinely indicated in smear-positive cases (already confirmed microbiologically)
- Indicated in:
- Smear-negative pulmonary TB (useful for diagnosis)
- TB in children
- Pleural and pericardial effusion (especially early stages when clinical signs are minimal)
- Miliary TB (essential)
- Frequent or severe haemoptysis (to exclude bronchiectasis or aspergilloma)
- Patients needing specific treatment for pneumothorax
6. Tuberculin Skin Test (TST) / Mantoux Test
- Discovered by Von Pirquet in 1907
- A positive reaction indicates past or present infection with M. tuberculosis
- The only means of estimating the prevalence of infection in a population (epidemiological utility)
Tuberculin used in India: PPD-RT 23 with Tween 80 at 1 TU dose (Note: 1 TU of PPD-RT23 = 5 TU of PPD-S)
Technique:
- 1 TU of PPD in 0.1 ml intradermally on the flexor surface of left forearm, midway between elbow and wrist
- Needle bevel facing upward; produces a pale wheal 6-10 mm in diameter
- Read at 48-96 hours (ideally 72 hours / 3rd day)
- Measure: induration only (not erythema), transverse horizontal diameter in mm
Interpretation:
| Induration | Interpretation |
|---|---|
| ≥10 mm | Positive (general population) |
| 6-9 mm | Doubtful (may be M. tuberculosis or atypical mycobacteria) |
| <6 mm | Negative |
| 0 mm | Record as '0' (no induration) |
Classification of positive TST reaction (WHO):
| Induration | Positive in Group |
|---|---|
| ≥5 mm | HIV-positive persons; recent TB contacts; CXR with fibrotic changes; immunosuppressed patients |
| ≥10 mm | Recent immigrants from high-prevalence countries; IV drug users; residents/employees of high-risk settings; mycobacteriology lab workers; children <4 years |
| ≥15 mm | All other persons with no known risk factors |
Prognostic significance: Strong reactors (≥20 mm) have greater risk of developing disease. 92% of new TB cases occur in persons already tuberculin-positive.
Two-step testing: Applied in serial testing (e.g., healthcare workers) - if first test negative, repeat in 1-3 weeks; if still negative, consider uninfected.
Limitations of TST:
- Lacks specificity - false positives due to BCG vaccination, atypical mycobacteria
- High BCG coverage countries: sensitivity reduced as epidemiological tool
- Boosting effect - second dose in serial testing can produce larger reaction than first
- Hypersensitivity measured does not correlate with protective immunity
7. Interferon Gamma Release Assay (IGRA)
- Blood test measuring immune response to MTB antigens
- Detects interferon-gamma cytokine released by sensitized T-cells
- Two commercially available tests:
- QuantiFERON-TB Gold (QFT)
- T-SPOT TB test
- Results available within 24 hours
Advantages over TST:
- Single patient visit required
- Prior BCG vaccination does NOT cause false positive
- More specific than TST
Disadvantages:
- Blood sample must be processed quickly
- Requires laboratory facilities
- More expensive
- Only tests for latent TB infection (not active disease)
- Less accurate in HIV-positive individuals
- Currently used in low-prevalence, resource-rich settings in place of TST
D. TISSUE-BASED AND OTHER TESTS
8. Histopathology
- Tissue biopsy showing caseating granuloma with Langhans giant cells - characteristic of TB
- Used for extrapulmonary TB (lymph node, pleural biopsy, bone marrow biopsy, etc.)
9. Other Blood Tests
- ESR, CBC (non-specific, supportive)
- ADA (Adenosine Deaminase) - useful in pleural, pericardial, and meningeal TB
E. DIAGNOSIS IN SPECIAL POPULATIONS (NTEP Approach)
TB/HIV Co-infection:
- TB programmes should continue to prioritize sputum smear microscopy in high-HIV-burden areas
- In immunocompromised (severe): sputum smear positivity is reduced (less inflammation)
- Algorithm: Sputum smear → if positive, start treatment; if negative but TB suspected → proceed to culture/NAAT/CXR
Paediatric TB:
- Diagnosis must not be based on clinical features alone
- Sputum examination preferred; induced sputum or gastric lavage specimens used when sputum cannot be produced
- TST/IGRA, CXR, and contact history are important adjuncts
Summary Table
| Modality | Type | Key Feature (SPM Perspective) |
|---|---|---|
| Sputum ZN smear | Microbiological | Cheapest, most widely used; 2 samples; ≥10,000 org/ml needed |
| LED FM microscopy | Microbiological | Superior to ZN; cheaper than conventional FM |
| Culture (MGIT) | Microbiological | Gold standard; 2-8 weeks; used for DR-TB monitoring |
| CB-NAAT/TrueNat | Molecular rapid | 90 min; detects MTB + RIF resistance; smear-negative/HIV/children |
| LPA (FL & SL) | Molecular rapid | Detects drug resistance mutations; FL and SL versions |
| Chest X-ray | Supportive/imaging | Smear-negative, miliary, pleural/pericardial TB; not routine in smear+ |
| Mantoux/TST | Infection test | Prevalence of infection in community; 1 TU PPD-RT23; read at 72h |
| IGRA (QFT/T-SPOT) | Infection test | Latent TB; BCG does not confound; used in resource-rich settings |
| Histopathology | Tissue | Extrapulmonary TB; caseating granuloma |
Park's Textbook of Preventive and Social Medicine, pp. 215-218 (TB diagnosis, smear microscopy, CB-NAAT, LPA, CXR, Mantoux test, IGRAs - NTEP framework)
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