LAB DIAGNOSIS FOR MYCOBACTERIA

• Specimen Collection


  • Sterile, wide-mouth cup with tightly fitted lid
  • Sputum and other respiratory secretions
  • Bronchial washing, bronchoalveolar lavage (BAL), or transbronchial biopsy specimens
  • Brushings
  • Gastric aspiration
  • Urine
  • Stool
  • Blood for mycobacteremia
  • Tissue or body fluids
• Sputum and other respiratory secretions collection

  1. Early-morning specimen collected on three consecutive days

  2. If at least two of the first three sputum direct smears are positive, then three specimens are often sufficient to confirm a diagnosis

  3. When none, or only one, of the first three sputum smears is positive, additional specimens are needed for culture confirmation

  4. 5 to 10 mL of sputum produced by a deep cough of expectorated sputum or induced by inhalation of an aerosol of hypertonic saline should be used
• Gastric aspiration


  1. Gastric aspirates should be obtained in the morning after an overnight fast

  2. Three specimens should be collected within 3 days

  3. Sterile water, 30 to 60 mL, is instilled orally or via nasogastric tube aspiration
• Urine collection


  1. First morning midstream specimen is preferred

  2. A minimum of 15 mL, is collected in a sterile container
• Tissue or body fluids collection

  1. 10 to 15 mL of sterile saline should be added to prevent dehydration

  2. 2 mL for CSF, 3 to 5 mL for exudates and pericardial and synovial fluids, and 10 to 15 mL for abdominal and chest fluids
• Digestion and Decontamination of Specimens

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

  • Sputum
  • Gastric washing
  • BAL
  • Bronchial washing
  • Transtracheal aspirate
  • Voided urine
  • Autopsy tissue
  • Abdominal fluid
  • Any contaminated fluid
• Sodium Hydroxide


  Usual concentration 2%, 3%, or 4%—serves as a digestant and decontaminating agent
• 1. Acetyl-L-cysteine (NALC)


  A combination of a liquefying agent, plus NaOH, is commonly used
• Benzalkonium Chloride (Zephiran)


  Another digestant-decontamination agent, combined with trisodium phosphate (Z-TSP)
• Oxalic Acid


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

  
• Staining for Acid-Fast Bacilli

  1. Ziehl-Neelsen and Kinyoun stains use carbolfuchsin as the primary stain, acid-alcohol as a decolorizing agent, and a methylene blue counterstain

  2. Ziehl-Neelsen staining involves the application of heat with the carbolfuchsin stain, whereas the Kinyoun acid-fast stain is a cold stain

  3. Slides are examined using a ×100 oil immersion objective on a light microscope for 15 minutes, viewing a minimum of 300 fields before a slide is called negative

  
• Mycobacteria


  • Strictly aerobic and grow more slowly
  • The generation time of mycobacteria is longer than 12 hours; M. tuberculosis has the longest replication time, at 20 to 22 hours
  • Most pathogenic mycobacteria require 2 to 6 weeks of incubation
  • Growth of M. tuberculosis is enhanced by an atmosphere of 5% to 10% CO2
  • Mycobacteria require a pH between 6.5 and 6.8
• M. genavense


  Does not grow on media used routinely to isolate mycobacteria and requires extended incubation (6 to 8 weeks)
• M. leprae


  Fails to grow on artificial media
• Egg-Based Media


  • Löwenstein-Jensen (LJ)
  • Petragnani
  • American Thoracic Society (ATS) media
• Egg-Based Media


  • Contain fresh whole eggs, potato flour, and glycerol, with slight variations in defined salts, milk, and potato flour
  • Each contains malachite green to suppress the growth of gram-positive bacteria
• Selective media


  Gruft modification of LJ and Mycobactosel, used in combination with nonselective media to increase the isolation of mycobacteria from contaminated specimens
• Agar-Based Media


  • Middlebrook 7H10
  • Middlebrook 7H11
• Middlebrook 7H11 medium


  Contains 0.1% casein hydrolysate, which improves the recovery of isoniazid-resistant strains of M. tuberculosis
• Agar-Based Media


  Drug susceptibility tests may be performed on agar-based media without altering drug concentrations, which occurs with egg-based media
• Liquid Media


  • Middlebrook 7H9 broth and Dubos Tween albumin are nonselective liquid media used for subculturing stock strains
  • Mycobacterium spp. grow more rapidly in liquid medium

  
• Colony Morphology


  • Smooth and soft or rough and friable appearance
  • Colonies of M. tuberculosis that are rough often exhibit a prominent patterned texture referred to as cording (curved strands of bacilli)
  • Colonies of Mycobacterium avium complex have a variable appearance, with glossy whitish colonies often occurring with smaller translucent colonies
• Growth Rate


  The range in recovery time is wide, from 3 to 60 days
• Niacin Accumulation


  • Accumulation of niacin, detected as nicotinic acid, is the most commonly used biochemical test for the identification of MTB
  • Nicotinic acid reacts with cyanogen bromide in the presence of an amine to form a yellow-pigmented compound
  • It is recommended that the test be done on egg agar cultures 3 to 4 weeks old and with at least 50 colonies

  
• Nitrate Reduction


  • The production of nitroreductase, which catalyzes the reduction of nitrate to nitrite, is relatively uncommon among Mycobacterium spp., but a positive result may be seen in M. kansasii, M. szulgai, M. fortuitum, and M. tuberculosis
  • Bacteria are incubated in 2 mL of sodium nitrate at 37° C for 2 hours. Hydrochloric acid, sulfanilamide, and N-naphthylenediamine dihydrochloride are then added; if the bacteria reduce the nitrate to nitrite, a red color forms
  • The nitrate reduction test differentiates M. tuberculosis from the scotochromogens and MAC
• Catalase


  • Mycobacteria are catalase positive
  • Most M. tuberculosis complex organisms do not produce heat-stable catalase; exceptions are certain strains resistant to isoniazid
  • Other heat-stable, catalase-negative species include M. gastri, M. haemophilum, and M. marinum
  • Semiquantitation of catalase production is based on the addition of Tween 80 and hydrogen peroxide to a 2-week-old culture grown in an agar deep, and the resulting column of bubbles is measured
• Hydrolysis of Tween 80

  • Some mycobacteria possess a lipase that can split the detergent Tween 80 into oleic acid and polyoxyethylated sorbitol
  • The pH indicator, neutral red, is initially bound to Tween 80 and has an amber color. After hydrolysis of Tween 80, neutral red can no longer bind, and it is released, causing a pink color to form

  
• Iron Uptake


  • Some mycobacteria can convert ferric ammonium citrate to an iron oxide, resulting in rusty brown colonies in a positive reaction on the addition of 20% aqueous solution of ferric ammonium citrate
  • The test is most useful in distinguishing M. chelonae, which is generally negative
• Arylsulfatase


  • Most members of the genus Mycobacterium possess the enzyme arylsulfatase, which liberates phenolphthalein, causing a pH change in the presence of sodium bicarbonate, indicated by the formation of a pink color change
  • The M. fortuitum complex, M. chelonae, M. xenopi, and M. triviale have rapid arylsulfatase activity that can be detected in 3 days
  • M. marinum and M. szulgai exhibit activity with 14 days of incubation

  
• Pyrazinamidase


  Hydrolyzes pyrazinamide to pyrazinoic acid and ammonia in 4 days
• Tellurite Reduction


  Reduction of colorless potassium tellurite to black metallic tellurium in 3 to 4 days is a characteristic of MAC
• Urease


  • Detection of urease activity can be used to distinguish M. scrofulaceum, urease-positive, from M. gordonae, urease-negative
  • A pink to red color is indicative of a positive reaction
• NAP


  NAP (p-nitroacetylamino-β-hydroxypropiophenone) selectively inhibits the M. tuberculosis complex
• Thiophene-2-Carboxylic Acid Hydrazide (T2H)


  M. bovis is susceptible to lower concentrations of T2H than MTB
• Sodium Chloride Tolerance


  • High salt concentration (5% NaCl) in egg-based media (e.g., LJ) inhibits the growth of most mycobacteria
  • M. flavescens, M. triviale, and most rapidly growing Mycobacterium spp. are exceptions that do grow in the presence of 5% NaCl
• Growth on MacConkey Agar

  The Mycobacterium fortuitum-chelonae complex can grow on MacConkey agar without crystal violet, whereas most other mycobacteria cannot
• The CDC currently recommends that when isolated, M. tuberculosis be tested for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin
• Skin Testing


  1. The tuberculin skin test uses purified protein derivative (PPD) extracted and purified from the cell wall of culture-grown M. tuberculosis as the antigen

  2. A standardized amount of antigen is injected intradermally into the patient's forearm

  3. Reactivity is read at 48 hours; in immunocompetent individuals, the presence of a raised firm area (induration) 10 mm or larger is considered reactive

  4. A reactive tuberculin skin test indicates past exposure to M. tuberculosis; other Mycobacterium spp. generally result in an induration smaller than 10 mm. Immunocompromised patients with previous