What methods are used to identify Nocardia species in a clinical specimen and culture?

Identification of Nocardia Species in Clinical Specimens and Culture

Direct Specimen Examination

Modified acid-fast staining is the primary method for identifying Nocardia in clinical specimens, revealing characteristic branching, beaded, filamentous bacteria that are partially acid-fast. 1, 2

Microscopic Methods

• Modified acid-fast stain (modified carbol-fuchsin stain) should be performed on all respiratory specimens when Nocardia is suspected, as this reveals the characteristic partially acid-fast branching filaments 1, 2
• Gram stain shows gram-positive, branching, beaded filamentous organisms, though this is less specific than modified acid-fast staining 1
• Calcofluor-KOH staining can be used as an alternative fluorescent method to visualize the branching filamentous structure 1

Specimen Types and Collection

• Corneal scrapings should be collected using a Kimura spatula and inoculated directly onto culture plates at bedside for ocular infections 1
• Vitreous aspirate or biopsy is optimal for endophthalmitis cases, with direct bedside inoculation of plates 1
• Sputum, bronchoalveolar lavage fluid, and tissue biopsies are appropriate for pulmonary nocardiosis 3, 4

Culture Methods

The laboratory must be notified when Nocardia is suspected so that culture plates can be incubated for extended periods (up to 7 days) and special media can be used, as Nocardia are slow-growing organisms. 1

Culture Media and Conditions

• Buffered charcoal yeast extract (BCYE) agar should be added to enhance recovery of Nocardia species 1
• Standard sheep blood agar and chocolate agar plates should be inoculated directly at bedside when possible 1
• Fungal media (such as Sabouraud dextrose agar) support optimal growth of Nocardia species 1
• Extended incubation for 7 days or longer is essential, as Nocardia are slow-growing organisms that may not be visible within the standard 48-72 hour incubation period 1
• Aerobic conditions are required, as Nocardia are strictly aerobic actinomycetes 1

Culture Characteristics

• Nocardia colonies typically appear as chalky white, orange, or tan colonies with aerial hyphae that may fragment into bacillary forms 5
• Growth is usually visible after 3-7 days but may take up to 2-4 weeks in some cases 1

Species-Level Identification

Molecular methods, particularly multilocus sequence analysis (MLSA), are the gold standard for accurate species identification of Nocardia, as biochemical methods and 16S rRNA sequencing alone cannot reliably discriminate between closely related species. 6, 7

Molecular Methods (Preferred)

• Multilocus sequence analysis (MLSA) using gyrB, 16S rRNA, secA1, and hsp65 genes provides the most accurate species identification, correctly identifying 98.5-99.5% of isolates 6, 7
• 16S rRNA gene sequencing alone is insufficient for discriminating closely related species but can be used as part of MLSA 6, 7, 4
• Mass spectrometry (MALDI-TOF) can identify Nocardia to species level when adequate reference databases are available 3

Biochemical Methods (Supplementary)

• API 20C AUX yeast identification system can provide supplementary identification data, with unique patterns for N. transvalensis, N. asteroides IV, and N. brevicatena 5
• Specific biochemical tests include:
  • Arylsulfatase positivity (characteristic of N. nova) 5
  • Opacification of Middlebrook 7H11 agar (characteristic of N. farcinica) 5
  • Gelatin liquefaction (characteristic of N. brasiliensis and N. pseudobrasiliensis) 5
  • Citrate and acetamide utilization 5

Antimicrobial Susceptibility Patterns

• Kirby-Bauer susceptibility testing to gentamicin, tobramycin, amikacin, and erythromycin can provide species-specific patterns for N. nova, N. farcinica, and N. brevicatena 5
• Broth microdilution method is the standard for determining antimicrobial susceptibility 3

Practical Algorithm for Identification

1. Perform modified acid-fast stain on direct specimen to visualize partially acid-fast branching filaments 1, 2
2. Notify laboratory of suspected Nocardia to ensure extended incubation and appropriate media 1
3. Inoculate BCYE agar, blood agar, and chocolate agar at bedside when possible 1
4. Incubate aerobically for 7 days minimum (up to 2-4 weeks if necessary) 1
5. Perform MLSA (gyrB-16S-secA1-hsp65) for definitive species identification 6, 7
6. Conduct antimicrobial susceptibility testing by broth microdilution to guide therapy 3

Common Pitfalls

• Premature discarding of cultures due to standard 48-72 hour incubation periods will miss Nocardia, which requires 7+ days 1
• Relying solely on 16S rRNA sequencing fails to discriminate between closely related species in 22.1% of cases due to foreign alleles 7
• Misidentification as fungi can occur due to branching filamentous morphology; modified acid-fast staining differentiates Nocardia from true fungi 1
• False-negative cultures may occur in patients already receiving antifungal or antibacterial therapy 1