B57: BAL, sputum, associated specimens

Type of specimen

• Sputum - is prone to contamination; an early morning sample could be better—A minimum of 1 ml is needed.
• BAL - can be done using a bronchoscope or without one (non-directed BAL). The sample is from the lower respiratory tract.
• Bronchial aspirate - direct aspiration of material from the large airways of the respiratory tract using a flexible bronchoscope.
• Bronchial brushing - uses a protected brush catheter in the bronchoscope to tease material from the airways. A pure bacterial count greater than 10^3 cfu/mL in a brush specimen obtained bronchoscopically has been found to correlate with a histological diagnosis of pneumonia.
• Bronchial washings - similar to aspiration, but a small amount of saline is instilled and then aspirated.
• Protected catheter specimens - similar to bronchial brushing.
• Transthoracic aspirate - obtained through the chest wall via a needle passed between the ribs. This procedure may be undertaken to sample, for instance, an aspergilloma, abscess or any focal lung lesion that is accessible.
• Tracheal aspirate - taken via ET tube. Quality is similar to sputum.
• Induced sputum - it is taken by physiotherapists.
  
  

Target organism

CAP

• Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis,
• Staph aureus - in the context of recent flu or complication of blood culture
• Kleb pneumoniae (Friedländer’s pneumonia) - in the context of alcoholism and homelessness.
• Mycoplasma pneumonia (20% of CAP; epidemic every 4 -5 years; younger patients)
• Chlamydophila pneumoniae
• Chlamydophila psittaci and Coxiella burnetii (bird and farm exposure)
• Legionella pneumophila (exposure to water features/AC)
• RSV, flu, adenovirus
  

HAP

• Enterobacterales - Klebsiella, Enterobacter (often multi-resistant)
• Pseudomonas
• Acinetobacter
• Staph aureus, including MRSA
• Legionella (consider using a chain of evidence form for possible medicolegal action)

Lung abscess

• S aureus, K pneumonia - multifocal necrotising pneumonia
• Nocardia - immunosuppressed patients
• Burkholderia pseudomallei (diabetic and travel to SE Asia, Northern Australia)
• Fusobacterium necrophorum (Complication of Lemierre's syndrome or necrobacillosis)

Cystic fibrosis

• S. aureus,
• H. influenzae (usually non-encapsulated in CF patients),
• S. pneumoniae and
• Pseudomonads - mucoid P aeruginosa
• Burkholderia cepacia
• complex
• Aspergillus spp
• Mycobacteria other than Mycobacterium tuberculosis (MOTT) - M. abscessus
• Ralstonia, Achromobacter and Pandoraea are emerging pathogens
• Anaerobe

Mycobacteria disease

• TB
• Other Mycobacteria - Mycobacterium avium-intracellulare, Mycobacterium abscessus, Mycobacterium kansasi, Mycobacterium malmoense, Mycobacterium xenopi, Mycobacterium fortuitum and Mycobacterium haemophilum.
• All BAL should be tested for Mycobacteria unless specifically suggested not to.

Parasites

• Syndrome Tropical Pulmonary Eosinophilia (pulmonary infiltrate, cough, SOB, eosinophilia) - Larval forms of Ascaris lumbricoides, hookworms and Strongyloides stercoralis
• Lung fluke - Paragonimus westermanii ( Far East, Indian subcontinent and West Africa; uncooked fish/crayfish)

Fungal infection

• Candida species are extremely rare causes of LRTI. Airway could be colonised with Candida, which may contaminate the specimen.
• Invasive aspergillosis - patients receiving corticosteroids, individuals with haematological malignancies and those with previous pulmonary infections. If risk is present, a portion of BAL should be tested for Aspergillus galactomannan.
• Pneumocystis jirovecii
• Dimorphic fungi - Histoplasma, Coccidioidomycosis, Paracoccidioidomycosis, Blastomyces, Talaromyces
• Cryptococcosis

Consider bioterrorism for these organisms if there is no travel history

• Bacillus anthracis (Anthrax)
• Brucella species (Brucella)
• Francisella tularensis (Tularemia)
• Burkholderia mallei (Glanders)
• Burkholderia pseudomallei (Melioidosis)
• Clostridium botulinum (Botulism)
• Coxiella burnetii (Q fever)
• Yersinia pestis (Plague)

Media

• Blood agar
• Chocolate agar with bacitracin
• Burkholderia cepacia selective agar - if testing CF patients
• BMPAα- Legionella
• Sabouraud dextrose agar

Safety

• If suspecting a category 3 pathogen process in a CL3 room, inside a cabinet. Otherwise, cat 2 and process inside a cabinet.
• Take appropriate precautions for aerosol-generating procedures (process inside a cabinet).
• Centrifugation must be carried out in sealed buckets, which are subsequently opened in a microbiological safety cabinet.
  
  Where to process depends upon a local risk assessment. We do all sputum handling in CL3.

• For staining mycobacteria heat-fix inside a safety cabinet (remember heat-fix may not kill all Mycobacteria - take precaution)

Processing specimen

Review the sputum: Is it salivary, mucosalivary, mucoid, mucopurulent, purulent and/or bloodstained? Put in on the report. If salivary, reject (unless the patient is immunocompromised or you intend to test for Mycobacteria - in that case process all types of sputum) but retain specimens at 4°C for at least 48hr. No such grading is needed for BAL.

Prepare the sample -

• Sputum - Add 0.1% solution of dithiothreitol or N-acetyl L-cysteine (NALC) to sputum. Dilute 10µL of homogenised sputum in 5mL of sterile distilled water.
• BAL - Centrifuge BAL at 1200 xg for 10 mins. Tip off all but 0.5mL of supernatant and re-suspend the centrifuged deposit in the remaining fluid.

Gram & other stain

Although it can be used to assess the quality of the sputum or to predict the culture from BAL, it is usually not done unless a microbiologist specifically requests it (for example, a microbiologist may want to have an idea from a BAL for starting an antibiotic therapy in a very unwell patient, but it is rare in my experience).

Heat treatment for Legionella - is not recommended in SMI.
If you need microscopy for fungus - use KOH - Calcofluor white preparation.
If Legionella staining is needed, use the Fluorescent staining technique

Inoculation

• Put 1µL loopful of the final solution on each culture plate. Put another 1µL of more concentrated sputasol on the same plate (use half plate) if the patient has CF or immunocompromised. For patients with cystic fibrosis who have no previous B. cepacia colonisation, inoculate 100µL of the liquefied sputum onto a B. cepacia plate.
• BAL - serial dilutions are made or calibrated loops are used to inoculate 3 dilutions - 1:10, 1:1000 and 1:100,000. Diagnostic thresholds are - Diagnostic thresholds are 10^5-10^6cfu/mL for bronchoscopic aspirates, 10^3cfu/mL for protected brush specimens and 10^4cfu/mL for BAL

Media

BAL

• All specimens: Chocolate agar with bacitracin disc or incorporated, Blood agar (if choco agar with bacitracin incorporated used), CLED, SAB
• Bronchiectasis or CF: Above media+ Mannitol Salt/Chromogen ic Agar
• CF: B. cepacia selective agar (target - B cepacia, M abscessus)
• CF - Media for Mycobacteria - RGM, LJ or an automated liquid culture
• CAP with flu-like symptoms: Legionella selective agar

Sputum

• All specimens - Chocolate agar with bacitracin + blood agar
• Bronchiectasis, CF, immunocompromised, ITU- above + SAB, CLED, mannitol Salt/chromogenic agar
• CF: B cepacia selective agar
• CF - Media for Mycobacteria - RGM, LJ or an automated liquid culture
• Mycological investigation requested - SAB
• Legionella selective agar - if Legionella suspected.

Level of identification

• Species-level - H influenza, M catarrhalis, N meningitidis, S pneumoniae, Legionella, S aureus, Pasteurella, Stenotrophomonas, Burkholderia and P aeruginosa.
  For P aerugonosa - report mucoid or non mucoid.
• Genus level - Moulds
• Yeast - yeast level
• Enterobacterales - report coliform if from the community (except K pneumoniae), if from the hospital - species level