Applied Bacteriology

• Importance of specimen collection in laboratory diagnosis
• Types of specimen collection
• Different laboratory methods for the identification of etiologic diagnosis
• Clinical question precedes and guides approach to etiologic diagnosis
• Purpose of testing:

  • Surveillance
  • Validation of syndromic management
  • Diagnosis
  • Screening
  • Determination of antimicrobial susceptibility
• Test-specific considerations:

  • Test performance
  • Specimen collection
  • Infection prevalence
  • Associated morbidity and complications
  • Costs of tests
  • Resources
  • Infrastructure
• Ancillary Tests
• Quality of specimen is crucial
• Specimens for Diagnosis:

  1. Direct Tissue or Fluid Samples
  2. Indirect Samples
  3. Samples from Microbiota Sites
• Specimen Collection and Transport:

  • Take the appropriate specimen
  • Collect the specimen at the appropriate time
  • Collect enough material and an adequate number of samples
  • Avoid contamination
• Specimen Collection and Transport:

  • Use the correct containers and appropriate transport media
  • Label specimens properly
  • Complete request form with enough clinical information and a statement of possible etiology
  • Inform the laboratory if special tests are required
  • Transport specimens rapidly to the laboratory
• Laboratory Methods:

  1. Direct detection

  a. Microscopy

  b. Culture methods

  c. Nucleic acid-based assays

  d. Immunologic systems: Antigen detection

  2. Indirect detection

  a. Immunologic systems: Antibody
• Direct Examination:

  • Light (bright-field) microscopy: Simple stain
  • Light (bright-field) microscopy: Differential Stain
  • Light (bright-field) microscopy: Gram Staining
  • Light (bright-field) microscopy: Acid-fast Staining
  • Light (bright-field) microscopy: Special stains
  • Dark-field and Fluorescence microscopy
  • Electron microscopy
• Direct Examination:

  • Culture methods
  • Nutrient media
  • Selective media
  • Indicator media
• Non-amplification Techniques: Hybridization
• Polymerase Chain Reaction
• Quantitative or Real-time PCR
• Multiplex PCR
• Isothermal Nucleic Acid Amplification
• Sequencing
• Modifications of Monoclonal Antibodies
• Immunodiffusion
• Agglutination
• Complement Fixation Assays
• ELISA
• Immunochromatographic Assays
• Western Blotting
• Immunohistochemistry
• Immunofluorescence
• Pointers on Immunoassays:

  • Paired specimens are compared (acute vs convalescent)
  • Titer is the highest serum dilution demonstrating activity
  • Seroconversion or fourfold rise in titer most conclusive
  • IgM responses indicate acute infection
• Emerging Technology: MALDI-TOF MS

  • Matrix assisted laser desorption ionization-time of flight mass spectrometry
  • Peptide mass fingerprint
  • Generate analytes from pure cultures
• Parting Words:

  • Conventional microbiological, histological and radiological techniques remain the cornerstone of diagnosis but are insensitive and have a limited impact on clinical decision-making
  • Molecular-based assays in infectious diseases must be correlated with the clinical picture or risk factors that the patient has for particular infections or drug resistance
• Questions?
• Thank you for your kind attention
• Psalm 16:8-11