Sputum Microscopy and Culture

Sputum examination finds the organisms and cells in secretions from the lower respiratory tract. Gram stain and bacterial culture identify the cause of pneumonia and exacerbations of bronchiectasis and cystic fibrosis. Acid fast bacilli (AFB) smear, culture and Xpert MTB/RIF diagnose pulmonary tuberculosis and rifampicin resistance. Fungal stains, Pneumocystis tests and cytology are added in selected patients. The value of the test depends on sample quality: saliva is not sputum.

The test supports targeted treatment of community acquired and hospital acquired pneumonia, finds tuberculosis, which is infectious, and guides therapy in chronic lung disease. In mild community acquired pneumonia treated at home, sputum culture is not required.

Lower respiratory infection causes about 2.2 million deaths a year, and tuberculosis about 1.3 million deaths and 10 million new cases a year. Streptococcus pneumoniae is the commonest bacterial cause of community acquired pneumonia (30 to 50% where a pathogen is found), followed by Haemophilus influenzae, atypical organisms (Mycoplasma, Legionella, Chlamydophila) and viruses. In hospital acquired infection, Staphylococcus aureus, Pseudomonas and enteric Gram negative bacilli predominate.

Indications. Moderate or severe pneumonia, pneumonia not responding to treatment, hospital acquired or ventilator associated pneumonia, cough over 2 to 3 weeks with weight loss, fever and night sweats (tuberculosis), haemoptysis, bronchiectasis or cystic fibrosis exacerbation, HIV and immunosuppressed patients with pulmonary infiltrates, and contacts of tuberculosis patients.

Specimen. A deep cough sample taken early in the morning before eating, after rinsing the mouth with water. Collection under supervision, or induction with nebulised hypertonic saline when the patient cannot produce sputum. For tuberculosis, two or three samples (at least one early morning, spot samples 1 hour apart are acceptable) of 3 to 5 mL. Process within 2 hours (refrigerate if longer, but not for pneumococcus).

Endotracheal aspirate, bronchoscopic samples or lavage in intubated or immunosuppressed patients. Samples for Legionella need special media or urinary antigen. Sputum is not suitable for anaerobic culture.

Quality assessment (Gram stain).

• Acceptable lower respiratory sample: fewer than 10 squamous epithelial cells and more than 25 neutrophils per low power field (Murray-Washington grading). Many squamous cells indicate saliva contamination, and culture of such a sample is not reported or is interpreted with caution.
• Predominance of a single organism type with intracellular bacteria supports a pathogen: Gram positive diplococci (Streptococcus pneumoniae), Gram negative coccobacilli (Haemophilus influenzae), Gram positive cocci in clusters (Staphylococcus aureus), and Gram negative bacilli (Klebsiella, Pseudomonas).
• Sensitivity of Gram stain for pneumococcal pneumonia is about 50 to 60% and specificity about 85%, with lower sensitivity after antibiotics.

Normal flora. Viridans streptococci, Neisseria species, coagulase negative staphylococci, and diphtheroids are commensals, and growth of these does not indicate infection.

Culture. Reports a semi-quantitative count (light, moderate, heavy growth). Heavy growth of a recognised pathogen with a good quality sample supports the diagnosis, while light mixed growth does not. In ventilated patients, endotracheal aspirate counts over 10^5 to 10^6 cfu/mL, bronchoalveolar lavage over 10^4 cfu/mL and protected brush over 10^3 cfu/mL support infection.

Tuberculosis.

• AFB smear (Ziehl-Neelsen or fluorescent auramine): needs 5000 to 10 000 bacilli/mL, with sensitivity of 50 to 70% in HIV-negative and 20 to 40% in HIV-positive patients. Two to three samples increase sensitivity, with a smear positive case being highly infectious.
• Xpert MTB/RIF Ultra: sensitivity about 90% overall (about 98% in smear positive, 70 to 80% in smear negative culture positive disease), specificity 96 to 98%, result in 2 hours, and detects rifampicin resistance with high accuracy. A "trace" result needs interpretation with the clinical picture.
• Culture on liquid medium (MGIT): results in 1 to 6 weeks, the reference method, allows species identification and full drug susceptibility testing.
• Line probe assays and sequencing detect resistance to isoniazid, fluoroquinolones and second line injectables.
• Non-tuberculous mycobacteria (Mycobacterium avium complex, abscessus, kansasii) need two positive cultures or one from bronchial wash with symptoms and imaging to diagnose.

Other pathogens.

• Legionella pneumophila: urinary antigen (detects serogroup 1) and PCR or culture on buffered charcoal yeast extract agar.
• Pneumocystis jirovecii: induced sputum or lavage with immunofluorescence or PCR, in HIV patients with hypoxia.
• Fungi: Aspergillus (galactomannan in lavage and serum), Cryptococcus, histoplasmosis, and Candida (usually colonisation).
• Viral PCR panels (influenza, respiratory syncytial virus, SARS-CoV-2) from nasopharyngeal or lower tract samples.
• Pseudomonas aeruginosa and Burkholderia in cystic fibrosis: mucoid colonies indicate chronic infection.

Cytology. Eosinophils in asthma and eosinophilic bronchitis (sputum eosinophils over 3% suggest eosinophilic airway inflammation and steroid responsiveness), malignant cells (sensitivity 30 to 60% for lung cancer, higher for central tumours with repeated samples), and haemosiderin laden macrophages in pulmonary haemorrhage.

Critical values and findings needing immediate action.

• AFB smear positive or Xpert positive: respiratory isolation, notification, and start of antituberculous treatment. Rifampicin resistance needs a drug resistant regimen and specialist care.
• Gram stain showing Gram positive diplococci with severe pneumonia: appropriate cover without delay, and blood cultures.
• Sputum Gram stain with Gram negative bacilli in a ventilated or neutropenic patient: broad spectrum cover.
• Legionella urinary antigen positive: macrolide or fluoroquinolone therapy and public health notification.
• Haemoptysis with a positive smear or cavity: massive haemoptysis is an emergency.
• Malignant cells on cytology: oncology referral.

Interferences and pitfalls.

• Salivary contamination is the commonest problem, and a poor quality sample can mislead treatment.
• Antibiotics before sampling reduce the yield of culture and Gram stain.
• Colonisation of the airways (chronic obstructive pulmonary disease, bronchiectasis, intubation) gives positive cultures without infection.
• A negative culture does not exclude pneumonia, since many cases (up to 50%) have no pathogen identified, and atypical organisms and viruses need other tests.
• Delay in processing reduces pneumococcus and Haemophilus survival.
• A negative smear does not exclude tuberculosis, and a positive smear may be non-tuberculous mycobacteria.
• Xpert may remain positive in treated patients (dead bacilli), and is not used to monitor response or test for cure.
• Single sputum samples miss infection, and repeat samples help.
• Blood cultures and urinary antigens (pneumococcus, Legionella) complement sputum.

Special groups.

• Children: usually cannot expectorate, and gastric aspirate, induced sputum or nasopharyngeal aspirate is used for tuberculosis.
• Older adults: pneumonia may present without cough or fever, and aspiration is frequent.
• HIV patients: smear sensitivity is lower, and Xpert and urine lipoarabinomannan add yield. Pneumocystis, tuberculosis, bacterial pneumonia and fungi are all possible.
• Cystic fibrosis: sputum culture every visit to track Pseudomonas, Staphylococcus aureus, Burkholderia, Achromobacter, and fungi.
• Regions: tuberculosis testing capacity varies, and Xpert is the preferred first test where available.

Follow up tests. Blood cultures, urinary pneumococcal and Legionella antigens, chest radiograph or CT, CRP and procalcitonin, HIV test, full blood count, pleural fluid analysis if an effusion is present, bronchoscopy with bronchoalveolar lavage, interferon gamma release assay, drug susceptibility testing, viral PCR panel, galactomannan, and cytology or biopsy for suspected malignancy.