Sputum microscopy and culture

Gram stain for bacteria and fungi; DFA for viruses (eg, RSV, influenza virus), Legionella sp, Pneumocystis jiroveci; acid fast stains for mycobacteria.

Aerobic culture on non-selective media; selective media if appropriate (eg, Legionella spp).

Mycobacterial culture; prolonged culture for fungus; virus detection, culture.

Nucleic acid detection after amplification may be used for detection of Legionella spp, Pneumocystis jiroveci, mycobacteria, mycoplasma, Chlamydia trachomatis in bronchial washes or lavage specimens.

The Gram stain may be used to screen sputum specimens before culture is performed. If squamous epithelial cells predominate the specimen is considered to be heavily contaminated with saliva and culture may not be performed as the yield is predictably low and misleading. If the specimen contains few or no epithelial cells it is cultured and the appearance of organisms on Gram stain is reported.

Interpretation of culture requires correlation with Gram stain results. For an isolate to be judged significant, it should predominate in both Gram stain and culture as well as being recognised as a respiratory pathogen.

If Streptococcus pneumoniae, Haemophilus influenzae or Moraxella catarrhalis predominate on culture with a consistent Gram stain they are likely to be significant pathogens in an appropriate clinical setting (eg, pneumonia).

Gram-negative rods (eg, E. coli, Pseudomonas aeruginosa) often colonise the airway especially in hospitalised patients receiving antibiotic therapy.
They are seldom pathogenic, but clinical judgement is required to interpret the importance of such isolates, especially in severely ill patients (eg, Klebsiella pneumoniae may be a cause of necrotising lobar pneumonia).

The isolation of Staphylococcus aureus from sputum usually reflects colonisation of the airway, but in certain circumstances it may be pathogenic (eg, post influenza pneumonia, respiratory burns, pneumonia secondary to septicaemia).

Culture is a more sensitive method than antigen detection for the diagnosis of pneumonia due to Legionella spp. PCR testing may be available in some laboratories. Positive culture for Legionella spp allows serotyping and further subtyping of the organism, which may be important in identifying the source of infection. Isolation of Legionella spp on culture indicates the presence of infection.

Infection with Pneumocystis jiroveci is diagnosed by detecting organisms with Giemsa, silver or toluidine blue stains, or by DFA or PCR.

Growth of Nocardia spp, B. pseudomallei or C. neoformans indicates the presence of infection.