Microbio

• Mycobacterium tuberculosis Complex (MTBC)

  General Characteristics

  • Acid fast, long, slender, straight rods
  • High lipid content in the cell wall
  • In X, Y, V and L arrangement (Chinese letter arrangement)
  • Serpentine cord formation (due to cord factor)
  • Obligate aerobe predilection with organs with high oxygen tension bone
  • marrow, lungs, kidneys and brain
  • Slow growth rate generation time 15 to 2 hours
  • Habitat human lungs
  • Transmitted via respiratory droplets (almost always transmitted via active respiratory droplets)
• Virulence factors of Mycobacterium tuberculosis

  1. Cord factor
  2. Wax D
• Cord Factor

  • Composed of trehalose dimycolate
  • Associated with the serpentine cord like pattern of virulent strains
• Granulomas and caseation by cell mediated immunity primarily due to host

  tissue reaction
• Wax D

  • Enhancement of immune response in the host
• Resistance to acids and dehydration

  • Droplet nuclei drying of the respiratory droplets ->bacilli remains suspended in the air for indefinite periods of time
• Pathogenesis of Mycobacterium tuberculosis

  Classification:

  • Pulmonary tuberculosis
  • Extrapulmonary tuberculosis- tuberculosis in the brain, kidney or bone marrow
• Primary Tuberculosis

  • Pulmonary TB
  • Lesions usually occur in the LOWER LOBES of the lungs
  • Organisms are engulfed by alveolar macrophages
  • Organisms survive and multiply inside alveolar macrophages due to the sulfatides that enable the survival (prevention of the fusion of lysosomes)
• Types of Lesions

  1. Exudative Lesions

  • Consists of inflammatory response, with edema fluid,
  • polymorphonuclear cells and later mononuclear cells
  • May heal in healthy individuals or may develop into granulomatous type (central area of giant cells containing the bacilli surrounded by epitheloid tissue— Tubercle Granuloma
• 2. Granulomatous Lesions

  • Consist of central are of giant cells (Langhan’s giant cells) containing the tubercle bacilli surrounded by a zone of epitheloid cells
• Tubercle


  - a granuloma surrounded by fibrous tissue that has undergone central caseation necrosis

  • In healthy individuals, tubercles may heal spontaneously by fibrosis and calcification and persist as such for lifetime
  • Appear as radio opaque nodules in chest x rays
• Secondary Tuberculosis

  • Lesions occur most commonly in the apex of the lungs
  • Dormant mycobacteria has been reactivated (viable non proliferating bacilli within the healed lesions)
  • Especially seen in immunocompromised hosts
• Disseminated Tuberculosis

  • When tubercle lesion liquefies, its caseous contents may drain into the bronchus
  • Facilitates spread of the organism to other parts of the lungs
  • New tubercles are formed
  • Dissemination of the tubercle into the bloodstream forms MILIARY TUBERCULOSIS
  • The blood carries the organism to many organs of the body

  Disseminated Tuberculosis
• Clinical Findings

  • Fever
  • Fatigue
  • Night Sweats
  • Weight Loss
• Pulmonary TB

  cough, chest pain, dyspnea and hemoptysis
• Scrofula


  mycobacterial cervical adenitis; seen in children with tuberculosis, caused by M. scrofulaceum
• Gastrointestinal TB

  • abdominal pain, diarrhea, fever and weight loss; may cause intestinal obstruction and/or hemorrhage


  Intestinal Obstruction
• Renal TB


  fever, dysuria, hematuria (blood in the urine), flank pain, sterile pyuria (excretion of urine filled with pus cells with negative bacterial culture)
• TB of the bone or joints

  • most frequently affected is the spine, leading to the collapse of the vertebrae; paralysis may occur due to nerve compression
• TB meningitis


  mental deterioration, retardation, blindness and

  deafness
• Treatment

  • INH (isonicotinic acid hydrazine or ISONIAZID), rifampin pyrazinamide, ethambutomal
  • Protracted therapylong duration of treatment (3 months)
  • Multiple Drug Therapy-done to prevent the emergence of drug resistance mutants during the long duration of treatment
• Bacille Calmette et Guerin (BCG) Vaccine

  • Vaccine containing live, attenuated strain of Mycobacterium bovis, administered intracutaneously
  • Vaccine limits the extent of the disease but not prevent the diseases
• Laboratory Diagnosis

  • Microscopy- sputum microscopy
  • Staining- Ziehl Neelsen Stain
  • Fluorescent dyes- Rhodamine auramine
• Microscopy

  1. Sputum specimen

  • Number

  1. Case findings 2 specimen
  2. On the same day with several hours interval (for spot collection); or
  3. On two consecutive days
• 2. Quality

  • Macroscopic- yellowish, mucopurulent
  • Microscopic - >25 WBC per LPO or 5 WBC per OIO; presence of alveolar macrophages or dust cells
• Culture


  Decontamination, Digestion and Concentration
• Components

  • Decontamination
  • Digestion
  • Concentration
• Decontamination


  kill other contaminants
• Digestion


  mucoid material of the sputum is liquefied
• Concentration


  high speed centrifugation
• 4% NaOH


  Traditional decontamination and concentration solution
• 4% NaOH

  • Time exposure must be carefully controlled to no more than 15 minutes
  • Effects mucolytic action to promote concentration by centrifugation
• N acetyl L cysteine (NALC) + 2% NaOH

  • Mild decontamination olution with mucolytic agent frees —> mycobacteria entrapped in mucus
  • Limit exposure to NaOH to 15 minutes
• Dithiothreitol + 2% NaOH

  • Very effective mucolytic agent used with 2% NaOH
  • Reagent is more expensive than NALC
  • Limit exposure to NaOH to 15 minutes
• 13% Trisodium phosphate + Benzalkonium chloride (Zephiran)

  • Zephiran should be neutralized with lecithin
  • Not inoculated to egg based culture medium
• 13% Trisodium phosphate


  Not as effective as TSP Zephiran mixture
• 5% Oxalic acid


  Most useful in processing specimen that contains Pseudomonas Aeruginosa as contaminant or present as coinfection with MTB
• 1% Cetylpyridinium chloride + 2% NaCl


  Tubercle bacilli have survived 8 day transit without significant loss
• Culture media


  incubated at 35°C to 37°C with 5 to 10% CO2 tension;

  examined weekly for growth up to 8 weeks; colonies appear as small, buff in color, dry, scaly or warty (cauliflower like)
• Media

  1. Egg-based Media

  • Lowenstein Jensen
  • Petragnani
  • American Thoracic
  • Society