Diagnosis of Bacterial Infections

Diagnosis of Bacterial Infections

• Key Objectives:

  • Rapid submission of clinical specimens for isolation and identification.

  • Determine:

    • Infectious disease presence.

    • Specific pathogen causing the infection.

    • Antimicrobial sensitivity profile and antibiotic resistance.

    • Appropriate treatment plans.

    • Prevention measures and control plans.

    • Zoonotic potential and notification requirements.

Important Factors in Diagnosis

• Considerations:

  • Animal species affected.

  • Clinical characteristics and disease state.

  • Epidemiology and the type of biospecimen.

  • Anatomical site of infection.

  • Methodology selection for sample collection and analysis.

Phases of Diagnosis

• Phases:

  • Pre-analytical Phase:

    • Sample collection and preparation.

  • Analytical Phase:

    • Processing and analysis.

  • Post-analytical Phase:

    • Results review and reporting.

    • Interpretation and final diagnosis.

Specimen Collection and Key Steps

• Choice of Specimen Collection:

  • Based on clinical symptoms, pathogen type, infection location, and duration.

• Common Specimens:

  • Skin swabs, ear swabs, urine, wound swabs, blood, milk.

• Importance:

  • Proper specimen collection is essential for accurate diagnostic testing.

Selection of Methodology

• Detection Options:

  • Agent Detection:

    1. Direct detection of bacteria.

    2. Sample cultivation.

    3. Taxonomic identification.

  • Host Immune Response:

    1. Serology for detection of humoral immunity.

    2. Detection of cell-mediated immunity.

Direct Detection of Bacteria

• Methods:

  • Microscopy and differential staining.

  • Fluorescent antibody staining, including direct observation of stained or unstained preparations.

• Importance:

  • Assess:

    • Bacterial density.

    • Morphology.

    • Host immune response.

Microorganism Observation Techniques

• Common Staining Techniques:

  • Gram-staining.

  • Ziehl-Neelsen staining (acid-fast).

  • Fluorescent staining.

  • Example: Microscopy of milk samples indicating Gram-negative bacilli and immune response cells.

Sample Cultivation

• Isolation Process:

  • Pathogens can be isolated using simple media (liquid or solid).

  • Importance of providing optimal nutrients, atmosphere, and temperature for bacterial growth.

• Colony Formation:

  • One bacterium equals one Colony Forming Unit (CFU).

Culturing Fastidious Organisms

• Techniques:

  • Use an anaerobic chamber for strict anaerobic bacteria cultivation.

  • Employ atmospheric jars with gas converters for altering atmospheric composition.

  • Media examples: Chocolate agar, chopped-meat broth.

Identification Techniques

• Culturing:

  • Second culture techniques can yield pure pathogenic bacteria for identification.

  • Identification methods include biochemical characterization, proteomic techniques (MALDI-TOF), fatty-acid analysis (GC), and PCR.

• Antigen Detection:

  • Use of monoclonal antibodies, serotyping, ELISA, and agglutination tests for pathogen detection.

Unculturable Bacteria

• Definition:

  • Uncultivable bacteria refer to those that can't be grown on artificial media (~99%), while culturable bacteria are about 1%.

Host Immune Response Detection

• Serology Methods:

  • Detect humoral immunity and response via antibody levels in serum.

  • Importance of seroconversion indicating recent exposure to pathogens.

Techniques for Serology Testing

• Main Concepts:

  • Antibody titer measurement and its significance in diagnosing infections.

  • Seroconversion indicates development of antibodies post-exposure.

• Tests:

  • IgM and IgG levels help assess exposure over time.

Cell-Mediated Immunity

• Definition:

  • Response mechanisms involving immune cells rather than antibodies to clear intracellular pathogens (e.g., certain bacteria and viruses).

Tuberculin Skin Test

• Principle:

  • Measures cell-mediated immunity; specifically used for infection by Mycobacterium bovis.

• Procedure:

  • Conducted over 2 days; dermal response indicates prior exposure.

Interferon Gamma Test**

• Overview:

  • A whole blood test for diagnosing Mycobacterium infections based on IFN-γ levels released from cells. Results available within 24 hours after sample collection.