DIAGNOSIS OF BACTERIAL INFECTIONS

• Diagnosis of Bacterial Infections

  Rapid, Accurate
• Rapid submission of clinical specimens for isolation, identification and susceptibility testing will improve and update this knowledge
• Accurate diagnosis questions


  • Is it an infectious disease?
  • Which pathogen is?
  • What is the antimicrobial sensitivity profile?
  • Does it have any antibiotic resistance?
  • Which treatment plan should I recommend?
  • Any prevention measurements?
  • How about control plan?
  • Is it a zoonosis?
  • Is it notifiable disease?
• Diagnosis of bacterial infections phases

  • Pre-analytical
  • Analytical
  • Post-analytical
• Pre-analytical phase


  • Animal species
  • Clinical characteristics
  • Disease state
  • Epidemiology
  • Biospecimen type
  • Anatomical site
• Analytical phase


  • Selection of methodology
  • Sample collection
  • Processing
  • Preparation
  • Analysis
• Post-analytical phase


  • Result & review
  • Reporting
  • Interpretation
  • Diagnosis
  • Treatment
• Choice for specimen collection

  Depends on clinical symptoms, type of pathogen, location of infection, duration of infection, diagnostic tests to be performed
• Common specimens for bacterial infections

  • skin
  • ear swab
  • urine
  • wound swabs
  • blood
  • milk
• Proper specimen collection is the FIRST and KEY step for accurate diagnostic testing!
• Specimen types


  • Blood
  • Tissues
  • Scraps/Swabs/Impression
  • Transudate/Exudates
  • Urine/Misc. fluids
  • Feces
  • Vomitus/sputum
• Selection of methodology


  • Detection of the agent
  • Detection of host immune response
• Detection of the agent

  1. Direct detection of the bacteria

  2. Sample cultivation

  3. Taxonomic identification of pathogen
• Detection of host immune response

  1. Serology: detection of humoral immunity

  2. Detection of cell-mediated immunity
• Direct detection of the bacteria

  Microscopy and differential staining

  Fluorescent antibody staining
• Examination of stained or unstained preparations ('smears') is the single most important and cost-effective procedure
• Direct microscopy observation


  • Gives an idea of bacterial density (estimation of bacterial numbers), bacterial morphology, host immune response (cellular reaction)
• Common staining


  • Gram-staining
  • Ziehl-Neelsen staining (acid-fast staining)
  • Fluorescent antibody staining
  • Electron microscopy (generally used only for research)
• Sample cultivation


  Many pathogens can be isolated from clinical samples in the laboratory, using simple media
• Colony Forming Unit (CFU)


  1 bacterium = 1 CFU = 1 bacterial colony
• Types of microbial solid growth media

  • General-purpose media

  Selective media

  Differential media
• MacConkey Agar


  • Selective & Differential medium

  Energy source: Lactose or Peptones

  Inhibitory compounds: Bile salts and crystal violet

  Differentiation: pH indicator
• Media can be both selective and differential
• Blood agar


  General purpose, enriched medium often used to grow fastidious organisms and to differentiate bacteria based on their hemolytic properties
• Routine sample culture


  Cultured on three different solid media:

  1. General-purpose or enriched medium

  2. Differential and selective medium for Gram-negative

  3. Differential and/or selective medium for Gram-positive

  Incubation: Most pathogens growth at 37°C (mesophiles), 18h-48h

  Primary culture: Mixed culture, with pathogen and microbiota

  2nd culture: May have the pure pathogenic bacteria
• Culture of fastidious organisms

  Anaerobic chamber or Atmospheric jar with gas converter

  Chocolate agar, Chopped-meat broth, Incubator with controlled CO2 %
• Identification


  • Biochemical characterization

  Proteomic characterization (MALDI-TOF)

  Fatty-acid characterization (GC)

  PCR

  Real-time PCR
• Monoclonal antibodies


  For antigen-recognition for detection and/or identification

  Goals: SEROTYPING & Antigen detection

  Fluorescent antibody staining, Enzyme-linked immunosorbent assay (ELISA), Agglutination tests
• The term 'unculturable' is used to describe bacteria that are not grown on artificial media till date
• Culturable vs Unculturable bacteria

  Culturable (~1%)

  Unculturable (~99%)

  All bacteria in nature
• Serology: detection of humoral immunity

  Diagnostic identification of antibodies in serum

  Evidence of exposure to an infectious agent does not necessarily confirm an etiological role

  Collection of 2 serum samples with 2 weeks apart and a four-fold rise of antibody titer are indicative for recent exposure (SEROCONVERSION)
• Antibody titer


  Measurement of antibody level (quantity). Expressed as the reciprocal of the highest dilution of serum that positively reacts in a specific test.
• Seroconversion


  A four-fold rise of antibody titer indicates antibody development after exposure to a pathogen or antigen
• Cell-mediated immunity


  Adaptive immune response that is independent of antibodies but involves immune cells that specifically recognize, target, and clear infected host cells
• Delayed-type hypersensitivity


  Cell-mediated immunity based method
• Tuberculin skin test


  Testing for bovine tuberculosis (Mycobacterium bovis)

  Measure of cell-mediated immunity (delayed hypersensitivity)

  Obligatory in the EU
• Tuberculin skin test procedure

  Day 1: two sites are clipped, and tuberculin injected (M. bovis and M. avium antigens)

  Day 4: measure skin reaction ➔ if bovine site > 4 mm = animal is reactor
• Interferon gamma test (IFN-ϒ)


  Whole blood test that can aid in diagnosis of Mycobacterium infections

  Based on the amount of IFN-ϒ that is released or on the number of cells releasing IFN-ϒ

  One visit: results within 24h