Chapter 18: Diagnosing Infections

Preparation for the Survey of Microbial Diseases

This chapter introduces the foundational steps and technologies used in the diagnosis of infectious diseases caused by microbes. Accurate identification of pathogens is essential for effective treatment and epidemiological control.

• Key Steps: Specimen collection, selection of diagnostic method, and interpretation of results.

• Main Categories of Diagnostic Methods: Phenotypic, Immunologic, and Genotypic.

First Steps: Specimen Collection

Proper specimen collection is critical for reliable laboratory diagnosis. The integrity of the specimen must be maintained from collection to analysis.

• Prompt Transport: Specimens should be quickly transported to the laboratory and stored appropriately, often refrigerated.

• Specialized Swabs and Transport Systems: Designed to maintain specimen stability for several hours, including nonnutritive media, buffering systems, and anaerobic environments for oxygen-sensitive bacteria.

• Sampling Sites: Common sites include blood, urine, sputum, cerebrospinal fluid, throat, wound, and genital tract.

Phenotypic Methods

Phenotypic methods rely on observable characteristics of microbes, including morphology, physiology, and biochemical properties.

• Microscopic and Macroscopic Morphology: Examination of cell shape, arrangement, and colony appearance.

• Physiology and Biochemical Properties: Growth requirements, metabolic activities, and enzyme production.

• Antimicrobial Susceptibility: Patterns of resistance or sensitivity to antibiotics can aid identification.

• Specialized Media:

  • Selective Media: Encourage growth of suspected pathogens.

  • Differential Media: Reveal identifying characteristics, such as fermentation patterns.

• Dichotomous Keys: Flowcharts used to identify specimens based on sequential tests.

• Biochemical Testing: Detects enzyme-mediated metabolic reactions, often visualized by color change.

• Phage Typing: Uses bacteriophages to identify bacterial species and trace outbreaks.

Table: Categories of Methods for Identifying Genus and Species

Immunologic Methods

Immunologic methods detect microbial antigens or patient antibodies using specific immunological reactions. These methods are highly specific and sensitive.

• Serological Testing: Uses antigen-antibody reactions to detect infection. Can be performed on serum, urine, cerebrospinal fluid, tissues, and saliva.

• Types of Immunologic Tests:

  • Agglutination: Clumping of whole cells by antibodies.

  • Precipitation: Formation of insoluble complexes with soluble antigens.

  • Lateral Flow Chromatography: Rapid tests (e.g., pregnancy, COVID-19) using prepackaged cartridges.

  • Western Blot: Electrophoresis separates proteins, detected by labeled antibodies.

  • Immunofluorescence: Direct (DFA) and indirect (IFA) methods use fluorescent-labeled antibodies.

  • ELISA: Sandwiching technique in microwells to detect antigen or antibody by color change.

  • In Vivo Tests: Introduction of antigen or antibody into patient to elicit a visible reaction (e.g., TB skin test).

• Antibody Titer: Concentration of antibodies in a sample, determined by serial dilution.

• Serotyping: Classification of bacteria into subgroups using antigen-antibody techniques.

Specificity vs. Sensitivity

• Specificity: Ability of a test to focus on only a certain antibody or antigen, minimizing false positives.

• Sensitivity: Ability to detect minute quantities of antibodies or antigens, minimizing false negatives.

Genotypic Methods

Genotypic methods analyze the genetic material of microbes, providing rapid and highly accurate identification.

• Nucleic Acid Amplification: Techniques such as PCR, real-time PCR, and multiplex PCR amplify specific DNA sequences.

• Hybridization: Use of labeled probes to detect complementary DNA or RNA sequences.

• Fluorescent In Situ Hybridization (FISH): Identifies organisms directly in patient or environmental samples without culturing.

• Whole-Genome Sequencing: High-throughput sequencing allows rapid analysis of outbreaks and drug-resistant organisms.

Table: Genotypic Diagnostic Methods

Additional Diagnostic Technologies

Emerging technologies are enhancing the speed and accuracy of microbial diagnostics, especially in point-of-care settings.

• Lab-on-a-Chip: Miniaturized devices that perform multiple diagnostic tests rapidly, useful in resource-limited settings.

• MALDI-TOF Mass Spectrometry: Analyzes protein fingerprints from pure culture isolates or patient specimens for rapid identification and antibiotic susceptibility testing.

• Imaging Technologies: CT and PET scans are used to locate deep-seated infections for targeted sampling.

Key Equations and Concepts

• Serial Dilution for Antibody Titer:

• PCR Amplification:

Where is the number of cycles.

Summary Table: Diagnostic Method Comparison

Example Applications

• COVID-19 Rapid Antigen Test: Point-of-care immunologic test for viral antigen detection.

• TB Skin Test: In vivo immunologic test for previous exposure to Mycobacterium tuberculosis.

• MALDI-TOF: Rapid identification of bacterial species from patient samples.

Additional info: These notes synthesize textbook slides and lecture content, expanding on brief points with academic context and examples for exam preparation.