16. Identification of Mycobacteria

IDENTIFICATION OF MYCOBACTERIA

  1. KINYOUN ACID-FAST STAIN Rationale: Mycobacteria have lipid-rich cell walls not permeable by ordinary stains. The Kinyoun stain consists of a basic dye (fuchsin) and phenol (a lipid solvent). Phenol partially solubilizes cell wall and allows fuchsin to penetrate the wall and bind to mycolic acid. After fuchsin is incorporated, it is resistant to decolorization even after exposure to acid alcohol, a property that characterizes mycobacteria
    • can be performed on unprocessed clinical specimens, concentrated specimens, or cultures
    • Ziehl Nielsen (hot stain) : mycobacteria stain red , background is light blue
    • Kinyoun cold stain
    • Auramine fluorochrome procedure: auramine binds to mycolic acid, counterstain with potassium permanganate that reduces background fluorescence, examination under fluorescent microscope. Mycobacteria look yellow, dark background
    • In pts with minimal disease the correlation between AF stains and culture may be as low as 25-40%, but there are studies indicating a positive predictive value of 96%
  2. Digestion decontamination concentration procedure
    • for contaminated specimens (sputum, stool etc)
    • rationale: eliminate other organisms that grow faster than AFB, liquefy organic debris and mucus, so that decontaminating agents reach the bacteria and AFB are freed
    • N-acetylocysteine/NaOH
    • Centrifugation at high speed (3800xg) to concentrate bacteria (AFB are very light due to their high lipid content
  3. Blood/Bone marrow specimens
    • no digestion/ decontamination
    • addition of distilled water before the centrifugation step to lyse blood cells
  4. DNA probes for the identification of mycobacteria from cultures
    • Rationale: a chemiluminescent-labeled, single-stranded DNA probe, that is complementary to the ribosomal RNA of the mycobacteria. DNA/RNA hybrid are formed. After the selection of non-hybridized vs hybridized probes, the DNA/RNA hybrids are measured by a luminometer.
    • Sensitivity: 76%-97%, specificity:100%
  5. Biochemical identification
    • pigment production, niacin accumulation, reduction of nitrates, Tween 80 hydrolysis, catalase, arylsulfatase and urease activity, iron uptake
  6. HPLC-FL:
    • fluorescence detection of mycolic acid esters
    • rapid identification
    • sensitivity: 94.3-99%, sensitivity: 100%
  7. Direct Detection of mycobacterial DNA or rRNA in clinical specimens using PCR
    • sensitivity: 71%-94% for DNA, 97% for rRNA
    • specificity: 99%

Mycobacterium avium intracellulare

  • Characteristics :grows intracellularly in macrophages, involvement of RES, facultative, slow-growing, non-photochromogenic, Runyon group III
  • Distribution: ubiquitous organism water, soil, dust, animals, poultry
  • Mode of transmission: ingestion of contaminated water, food, inhalation of aqueous aerozols. Intestinal tract is the primary route of infection in AIDS patients. Animal to human, or human to human transmission is very rare.

In non-immunocompromized individuals:

  • usually non pathogenic
  • it can be recovered from the stool of healthy individuals
  • pulmonary infection is the main clinical condition associated with MAI predisposing factors: COPD, CF, bronchectasis, elderly women pulmonary manifestations are similar to Tb
  • lymphadenitis in children, skin, soft tissue, bone infection

In immunocompromized individuals

  • 25% of AIDS patients will develop MAI infection during the course of their disease, prevalence of 50% in autopsy specimens
  • pulmonary disease is uncommon
  • usually presents as disseminated disease:
    fever, anorexia, malaise, sweats, weight loss
    bacteremia occurs in 90% of cases
    abdominal pain, diarrhea, malabsorption are common due to gut, especially
    duodenum infiltration, pseudo-Whipple disease in contrast studies, stomach spared
    bloody stools rare: one report
    hepatomegaly, splenomegaly, lymphadenopathy
    hepatobiliary disease, the most common hepatic opportunistic infection
    other more rare manifestations: meningitis, synovitis, genitourinary tract disease
    cutaneous lesions, osteomyelitis, arthritis
  • recovery of the organisms from feces or sputum does not prove disease, although it correlates more often with or predicts disseminated disease
  • Diagnosis is made by positive blood (sensitivity:86-98%), bone marrow, tissue cultures
  • Histologic evidence of gastrointestinal or pulmonary involvement indicates disseminated disease
  • Hematologic manifestations: Anemia almost always accompanies disseminated disease Pancytopenia and especially thrombocytopenia due to hypersplenism in pts with disseminated MAI infection and splenomegaly has been proven with platelet kinetic studies and the histology of spleen specimens of pts who underwent splenectomy
  • Other lab findings:� alk phosphatase, fecal fat analysis abnormal
  • Treatment
    • Azithromycin, or clarithromycin plus ethambutol +/- rifabutin
    • gamma-interferon enhances host defense by increasing oxidative metabolism in phagocytes, enhancing antigen presentation and granuloma formation and increasing intracellular antibiotics concentration
    • Splenectomy in pts with disseminated disease and hypersplenism has been found to be beneficial, as cytopenias improved, while it has been considered as a contradication by others