Mod 8

• Gram-positive bacilli


  Bacilli that are Gram-positive, unlike most bacilli which are Gram-negative
• Gram-positive bacilli exceptions


  • Mycobacteria
  • Clostridia
  • Corynebacteria

  
• Gram-positive bacilli exceptions


  • Bacillus
  • Listeria
  • Erysipelothrix
  • Lactobacillus
  • Actinomyces
• Gram-positive bacilli exceptions


  • Propionibacterium
  • Eubacterium
  • Mobiluncus
  • Bifidobacterium

  
• Mycobacteria


  • Strictly aerobic
  • Beaded appearance (Much granules)
  • Palisades or snapping (X,V,Y,L) formation
  • Acid fast!
  • Slow growing

  
• Slow growth of mycobacteria

  Due to hydrophobic cell surface, which causes clumping and reduces nutrient intake, resulting in growth taking 2 to 60 days
• Gram staining of mycobacteria

  Gram-positive, but actual stain shows gram ghost, neutral bacilli<|>High lipid content (60%), including mycolic acid and cord factor wax D

  
• Acid-fastness of mycobacteria


  Due to mycolic acid or hydroxymethoxy acid, fatty acids with 50-90 carbon atoms<|>Peptidoglycan layer has N-glycolylmuramic acid instead of N-acetylmuramic acid, and very high lipid content
• Other acid-fast organisms


  • Nocardia
  • Rhodococcus
  • Leigonella micdadae
  • Isospora
  • Cryptosporidium
• Pappenheim's method


  1. Differentiates M. smegmatis (blue) from MTB (red)

  2. Decolorizer: mixture of Rosalic acid & alcohol
• Baumgarten's method


  1. Stain: dilute alcoholic fuchsin

  2. Differentiates MTB (blue) from M. leprae (red)
• Tuberculosis (TB)


  Common, lethal infectious disease caused by Mycobacterium tuberculosis<|>Worldwide, the second greatest contributor among infectious diseases to adult mortality

  
• Risk of active pulmonary TB

  Low after one exposure, but increases under conditions of stress or in a confined environment with repeated exposures
• Primary tuberculosis


  Typically a disease of the respiratory tract
• Symptoms of tuberculosis


  Low-grade fever, night sweats, fatigue, anorexia (loss of appetite), and weight loss
• Pathophysiology of tuberculosis in the lungs

  1. Inflammatory response causing pneumonia-like symptoms

  2. Bacteria form lesions in the lungs and solidify into nodules called tubercles

  3. Tubercles near blood vessels can perforate the vessels, leading to hemorrhage, blood-tinged spit, hemoptysis (coughing out of blood)
• Tuberculosis pathophysiology


  1. Bacteria in the lungs are phagocytized by T cells and macrophages, leading to intracellular multiplication and not being eliminated by the host

  2. This results in granuloma/hard tubercle formation

  3. In cases of large bacterial load, no granuloma formation occurs, instead leading to caseous necrosis

  
• Spread of tuberculosis


  1. Via the lymph system or hematogenously, leading to meningeal or miliary (disseminated) tuberculosis, often in patients with depressed or ineffective cellular immunity

  2. Common sites of dissemination: spleen, liver, bone marrow, kidneys, adrenal gland, eyes

  
• Pott's disease


  Tuberculosis spondylitis or skeletal tuberculosis of the spine
• Latent tuberculosis


  No apparent signs, symptoms, or pathologic condition, although the organism is present in granulomas<|>Can progress to active disease (reactivation tuberculosis) at any time, especially in HIV patients

  
• First-line anti-tuberculosis drugs


  • Ethambutol
  • Isoniazid
  • Pyrazinamide
  • Rifamycin
• Antibiotic resistance in tuberculosis

  Occurs through spontaneous mutation in several chromosomal genes, leading to vertical gene transfer

  
• Multidrug-resistant TB (MDR-TB)


  Does not respond to the standard 6-month treatment regimen, requires up to 2 years of treatment with more toxic and costly drugs<|>Caused by tubercle bacilli resistant to rifampin and isoniazid
• Extensively drug-resistant tuberculosis (XDR-TB)


  MDR-TB plus resistance to any fluoroquinolone and any second-line injectables (e.g., amikacin, kanamycin, capreomycin)

  
• Second-line anti-tuberculosis drugs


  • Aminoglycosides
  • Aminosalysicylic acid
  • Capreomycin
  • Cycloserine
  • Ethionamide
  • Fluoroquinolones
  • Macrolides
• Mycobacterium tuberculosis


  Also known as Koch's bacillus or human tubercle bacilli<|>Slow growing, requires 5-10% CO2 at 37°C, growth enhanced by glycerol

  
• Cultural characteristics of M. tuberculosis

  Rough/dry/warty/granular resembling "cauliflower", tan to bough colonies (nonpigmented)

  
• Biochemical characteristics of M. tuberculosis

  Positive for niacin, nitrate, and pyrazinamidase

  
• Specimen types for M. tuberculosis

  • Sputum/urine for 3 consecutive days
  • Blood
  • Peritoneal/pericardial fluid
  • CSF (pellicle/web-like clot formation suggestive of tubercular meningitis)

  
• Staining methods for M. tuberculosis

  • Kinyoun's (cold)
  • Ziehl-Neelsen (hot)
  • Fite-Faraco (hematoxylin as counterstain)
  • Auramine-rhodamine fluorochrome stain (fluorescent yellow organisms on black background)

  
• For fuchsin staining, the smear should be examined carefully by scanning at least 300 oil immersion fields (magnification x1000), equivalent to three full horizontal sweeps of a smear that is 2 cm long and 1 cm wide before reporting as negative

  
• For fluorochrome staining, the equivalent number of fields (30) at lower magnification (LPO or HPO) is required, thus less time is needed compared to fuchsin staining
• Virulence tests for M. tuberculosis


  Serpentine cord formation (seen on direct observation of colonies on cord medium)<|>Neutral red dye test (virulent strains bind the dye and stain pink to red, avirulent strains remain colorless)

  
• Skin tests for M. tuberculosis

  Primary means of identification, identifies TB infection (recent/past, with/without disease)<|>Principle: persons infected develop hypersensitivity to the proteins of the organisms

  
• Types of TB skin test reagents

  • Old tuberculin (boiled broth cultures of MTB)
  • PPD (culture extract of M. tuberculosis, i.e., PPD of tuberculin/ammonium sulphate precipitated MTB)

  
• The PPD test is not 100% sensitive or specific, can't differentiate latent and acute infection, and a positive reaction does not necessarily signify the presence of disease
• With HIV, it is difficult to diagnose using skin tests because they are frequently anergic (lack biologic response)
• Methods of PPD administration

  • Mantoux (intracutaneous, >15mm induration is positive)
  • Von Pirquet (scratch, raised area is positive)
  • Vollmer patch (piece of cloth on skin)
  • Moro percutaneous (rubbed)
  • Tuberculin tine (multiple puncture)
• 1. Spot TB test


  Measures T cells activated by M. tuberculosis antigens, >8 spots formation is positive
• QuantiFERON-TB Gold (ELISA)


  Uses a mixture of synthetic peptides to detect M. tuberculosis infection