LAB DIAGNOSIS FOR MYCOBACTERIA

• BSC of M. tuberculosis

  BSC 2; but Biosafety level 3 practice are recommended
• BSC 3 practice

  1. restricted lab access
  2. negative pressure airflow
  3. special PPE
• Effective disinfectants for Mycobacterium spp.

  1. Amphyl
  2. 0.05% to 0.5% sodium hypochlorite
  3. Phenol soap mixtures
• Recommended container for spx collection

  • sterile
  • wide-mouth cup
  • tightly-fitted lid
• Sputum specimen collection

  • early-morning specimen
• Collection of sputum (days)

  • 3 consecutive days
• If atleast two of the first three sputum, direct smears are positive, then three specimens are needed for culture confirmation
• Amount of sputum needed

  • 5-10mL
  • produced: deep cough of expectorated sputum
  • or induced by inhalation of an aerosol of hypertonic saline
• Bronchial washing

  • specimen of choice for detecting NTM and other opportunistic pathogens in patients with immune dysfunction
• Brushings

  • diagnostic than washing or biopsy
  • possibly due to inhibitory effect on mycobacteria by lidocaine used in adults during bronchoscopy or of dilution of the specimen with saline
• Children with difficulty producing sputum, gastric aspiration is recommended
• Gastric Aspirates specimen collection

  • morning after an overnight fast
• Gastric lavage


  used to collect sputum from patients who may have swallowed sputum during the night
• Gastric aspirates: three specimens should be collected within 3 days
• Sterile water: 30-60mL, instilled orally or via nasogastric tube aspiration
• Gastric aspirates: should be neutralized with sodium carbonate or another buffer to pH 7.0 ASAP
• Gastric aspirates: processed within 4 hours
• Gastric aspirates container

  • top: tight-screwed
  • tube: held upright
• Specimen collection of Urine

  • First midstream specimen is preferred
  • amount: 15mL
  • Early morning urine: voided
  • amount: 40mL minimum
• Clinical manifestations of Urinary TB

  • frequency of urination
  • dysuria
  • hematuria
  • flank pain
• Urine: sterile containers should be submitted daily for atleast 3 days
• Specimen choice for patients with AIDS: stool
• Test for stool specimen

  • AFB staining
  • Culture
• Specimen container for stool

  • dry
  • clean
  • wax-free container
  • without preservative or diluent
• Blood: for mycobacteremia
• Anticoagulant for recovery of Mycobacterium spp.

  • SPS
  • heparin
  • citrate
• Specialized automated systems and culture media for Mycobacterium spp. growth

  • Myco/F lytic bottles
  • BacT/ALERT MB blood medium
• Tissue or body fluids specimen collection

  • 10-15mL of sterile saline is added to prevent dehydration
  • at least 10mL for CSF
  • 3-5mL for exudates and pericardial and synovial fluids
  • 10-15mL for abdominal and chest
• Container for pleural, peritoneal and pericardial fluids

  • syringe with Luer-tip cap
  • sterile container
• Best type of specimen collection for culturing of skin lesion or wound

  aspirate
• If volume is insufficient for aspiration, pus and exudates may be obtained on a swab, and then placed in a transport medium such as Amie’s or Stuart’s medium.
• These media inactivates or neutralize fatty acid. They contain charcoal which inhibits fatty acids.
• Purpose of digestion-decontamination process

  1. To liquefy the sample through digestion of the proteinaceous material
  2. To allow the chemical decontaminating agent to contact and kill the nonmycobacterial organisms
• Specimens that require digestion-decontamination

  • sputum
  • gastric washing
  • BAL
  • bronchial washing
  • transtracheal aspirate
• Specimen that requires decontamination

  • voided urine
  • autopsy tissue
  • abdominal fluid
• Decontaminating agents

  1. Sodium Hydroxide
  2. N-acetyl-L-cysteine
  3. Benzalkonium Chloride
  4. Oxalic acid
• Sodium Hydroxide

  • Usual concentration: 2%, 3%, or 4%
  • Digestant and decontaminating agent
• N-Acetyl-L-Cysteine

  • Combination of N-acetyl-L-cysteine (digestant) and Sodium Hydroxide (decontaminating agent)
  • Treatment with mucolytic agents such as NALC splits mucoprotein, allowing greater sedimentation
• Benzalkonium Chloride

  • Another digestant-decontamination
  • Benzalkonium combined (decontamination) with trisodium phosphate (digestant)
  • Trisodium phosphate - liquefies sputum rapidly
  • Benzalkonium chloride - shortens exposure time and effectively destroys many contaminants, with little bactericidal effect on the tubercle bacilli.
• Oxalic Acid

  • amount: 5%
  • used to decontaminate specimens contaminated with Pseudomonas aeruginosa, such as sputum specimens from patients with Cystic fibrosis.