What is the proper procedure for performing a Brucella culture on bone tissue?

Proper Procedure for Brucella Culture from Bone Tissue

Brucella culture from bone tissue must be performed in a biosafety level 3 (BSL-3) laboratory using aseptic technique, with specimens collected through intact skin to minimize contamination and processed with specialized culture media for optimal diagnostic yield.

Specimen Collection

Bone Biopsy Technique

• Obtain bone samples through percutaneous biopsy or during surgical intervention 1
• Use an 11-gauge bone-cutting needle (smaller for phalanges) such as:
  • Jamshidi (Perfectum Corporation/CareFusion)
  • Ostycut (Bard Products/Angiomed)
  • T-lok (Angiotech) 1
• Collect sufficient bone sample to divide for both microbiological culture and histopathological examination

Critical Safety Considerations

• Perform biopsy through intact, uninfected skin
• Maintain at least 20mm distance from any ulcer periphery when introducing the biopsy needle 1
• For plantar ulcers, use a dorsal approach to reduce contamination risk 1
• Wear appropriate personal protective equipment (PPE) including respiratory protection 1
• Process specimens in a biosafety cabinet once Brucella is suspected 1

Specimen Processing

Laboratory Handling

1. Transport specimens promptly to the laboratory in appropriate sterile containers
2. Process all specimens under BSL-3 conditions with appropriate biocontainment 1
3. Alert laboratory staff about suspected Brucella to ensure proper precautions 1
4. Divide the bone core aseptically:
   • One portion for microbiological culture
   • One portion for histopathological examination 1

Culture Methodology

1. Inoculate specimens onto specialized selective media:
   • CITA medium (containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B) offers superior isolation rates for all Brucella species 2
   • Avoid exclusive use of Farrell medium as it may inhibit growth of some Brucella species 2
2. Incubate cultures for extended periods:
   • Standard incubation for 5-7 days with automated blood culture systems 3
   • Consider extended incubation (up to 4 weeks) for optimal sensitivity 1
   • Perform blind subcultures for protracted cases 3

Diagnostic Interpretation

Culture Interpretation

• Positive culture provides definitive diagnosis of brucellosis 1
• Sensitivity of bone cultures varies (15-70%) depending on:
  • Prior antibiotic therapy
  • Laboratory practices
  • Sampling technique 1, 4
• Bone marrow culture has highest sensitivity and is considered the gold standard for diagnosis 1, 4

Complementary Testing

• Histopathological examination of bone specimens helps interpret culture results, especially with:
  • Negative cultures
  • Cultures growing only commensal skin flora 1
• Serological testing should complement culture:
  • Standard tube agglutination test (SAT)
  • ELISA for specific IgM, IgG, and IgA antibodies 4
• Consider molecular methods (PCR) for increased sensitivity 3

Common Pitfalls and Considerations

False Negatives

• Prior antibiotic therapy significantly reduces culture yield - ideally delay biopsy until patient has been off antibiotics for at least 2 weeks 1
• Sampling errors due to improper technique or insufficient sample volume
• Failure to use appropriate selective media 2
• Inadequate incubation time 1

False Positives

• Contamination from skin flora when proper aseptic technique is not followed
• Improper specimen collection through infected or ulcerated tissue 1

Safety Concerns

• Brucella is highly infectious through aerosols and direct contact
• Laboratory-acquired infections are common without proper precautions
• Respiratory protection is essential when handling potentially infected specimens 1
• Notify laboratory personnel about suspected Brucella to ensure appropriate biosafety measures 1

Treatment Considerations

If Brucella infection is confirmed, treatment typically consists of:

• Combination antibiotic therapy with doxycycline plus rifampicin or fluoroquinolones plus rifampicin 5
• Treatment duration of at least 6 weeks to prevent relapse
• Close monitoring for treatment response and potential complications 1