Sputum Acid-Fast Bacilli (AFB) Smear

Sputum acid fast bacilli smear microscopy detects mycobacteria in respiratory specimens by their resistance to decolourisation with acid alcohol after staining.

Two methods are used: Ziehl-Neelsen (hot carbol fuchsin) or Kinyoun stain read under oil immersion at 1000 times magnification, and fluorescence microscopy with auramine-O or auramine rhodamine read at 200 to 400 times, which is faster and more sensitive (about 10% higher) and is read with light-emitting-diode microscopes.

The smear shows acid fast bacilli but cannot identify species or drug susceptibility. Culture and molecular tests are needed for confirmation.

Tuberculosis causes about 10 million new cases and 1.3 million deaths each year, and smear microscopy remains the first test in many settings and the main way to measure infectiousness and monitor treatment. Sensitivity is 50 to 60% for culture positive pulmonary tuberculosis in HIV-negative adults (needs about 5000 to 10 000 bacilli/mL), falling to 20 to 30% in HIV co-infection and children.

Specificity is high (over 95%) in high prevalence areas, but non-tuberculous mycobacteria also stain. Smear positive patients account for most transmission.

Indications. Cough for over 2 weeks with fever, night sweats, weight loss or haemoptysis. Abnormal chest radiograph (upper lobe infiltrate, cavity, miliary pattern). Contacts of smear positive patients with symptoms. Immunocompromised patients with pneumonia (HIV, transplant). Monitoring response to tuberculosis treatment (at 2 and 5 to 6 months). Suspected non-tuberculous mycobacterial lung disease. Other sites: pleural, CSF, lymph node, gastric aspirate, urine and tissue.

Specimen. Two to three sputum samples: one spot sample at the first visit, an early morning sample the next day (deep cough, 3 to 5 mL, mucopurulent and not saliva), and another spot sample when brought. Collection in a well ventilated area, away from others, in a wide mouthed, leak proof container.

Induced sputum with hypertonic saline, bronchoscopy washings or lavage and gastric aspirates (children) if the patient cannot produce sputum. Specimens are stored at 2 to 8 °C and processed within 48 hours.

Reference values (grading of smear).

Fluorescence smears are graded with a separate scale that depends on magnification (200 to 400 times), and the laboratory's scale is applied.

Interpretation.

• Smear positive (2+ or 3+) with compatible symptoms and chest radiograph: pulmonary tuberculosis is almost certain, and treatment is started with molecular testing (Xpert MTB/RIF) for confirmation and rifampicin resistance.
• Scanty or 1+ smear: needs confirmation by repeat smear or molecular test, culture, and clinical correlation, because of non-tuberculous mycobacteria and false positives from stain artefacts.
• Smear negative with high clinical suspicion: not exclusive of tuberculosis (up to half of cases). Molecular tests, culture, chest radiograph and empirical treatment are used as local practice allows.
• Smear grade correlates with infectiousness and bacterial load, and a smear negative, culture positive patient is less infectious but not non-infectious.
• Treatment monitoring: smear at the end of the intensive phase (2 months) should be negative. Persistent positivity at 2 months (about 10 to 20%) suggests extensive disease, poor adherence, resistance or non-tuberculous mycobacteria, and prompts drug susceptibility testing. A positive smear at 5 months or later defines treatment failure.
• Dead bacilli stain and persist for months in treated patients, so smear positivity during treatment does not always mean viable organisms, and culture is more specific.
• Non-tuberculous mycobacteria (Mycobacterium avium complex, M. kansasii, M. abscessus) give positive smears, and species identification by culture or molecular methods is needed.

Critical values and findings needing immediate action.

• Smear positive in a hospital inpatient: airborne isolation in a negative pressure room or single room, mask for staff (respirator) and patient (surgical mask), and contact tracing.
• Smear positive with HIV, severe immunosuppression, miliary disease or meningitis: urgent treatment, steroids where indicated and antiretroviral timing.
• Smear positive with rifampicin resistance on molecular testing: multidrug resistant tuberculosis regimen in a specialist centre.
• Haemoptysis, respiratory failure, pneumothorax or empyema in a smear positive patient: admission and intervention.
• Smear positive in a child, pregnant woman or healthcare worker: urgent treatment and contact screening.

Interferences and pitfalls.

• Poor specimen quality (saliva, small volume) reduces sensitivity, and good sampling (supervised collection, two to three samples) increases yield by 10 to 20%.
• Smear detects only bacilli at high density, so smear negative tuberculosis (paediatric, HIV, extrapulmonary) is missed, and molecular tests are more sensitive (Xpert MTB/RIF Ultra about 85 to 90% in smear negative, culture positive).
• Contaminants and artefacts (stain precipitate, Nocardia, Rhodococcus, Cryptosporidium, dead bacilli from previous treatment) cause false positives, and examination by trained staff with quality control is needed.
• Smear cannot differentiate Mycobacterium tuberculosis complex from non-tuberculous mycobacteria. In low prevalence settings a positive smear needs molecular confirmation before diagnosis.
• Fluorescence microscopy findings are confirmed by Ziehl-Neelsen for doubtful positives.
• Sputum smear conversion can be delayed in cavitary disease and diabetes.
• Delay in transport, heat and sunlight reduce viability for culture, so specimens are kept cool and transported promptly.
• Culture takes 2 to 6 weeks (liquid medium 1 to 3 weeks) and drug susceptibility testing adds weeks, so molecular testing for rifampicin resistance runs in parallel.

Special groups.

• Children: unable to expectorate, so gastric aspirates, induced sputum and stool molecular tests are used, and smear is rarely positive.
• Pregnancy: treat tuberculosis with first line drugs, and avoid delays.
• HIV co-infected patients: lower smear positivity (atypical radiographs, extrapulmonary disease), so molecular tests and urine lipoarabinomannan assay are used.
• Older adults: atypical presentation and lower smear yield.
• Regions: smear microscopy remains the main test in many low income settings, with LED fluorescence microscopy and molecular platforms replacing it where available. Quality assurance and prompt reporting are critical.

Follow up tests. Xpert MTB/RIF (or Ultra) on the same sample, line probe assay for drug resistance, mycobacterial culture and drug susceptibility testing, species identification, chest radiograph or CT, HIV test, CD4 count, urine lipoarabinomannan in advanced HIV, diabetes screening (HbA1c), liver function tests before treatment, sputum smear at 2 and 5 to 6 months, interferon gamma release assay in contacts, and contact tracing.