Diagnosing Infectious Diseases — Laboratory Methods, ELISA, PCR, and Antibiotic Testing Explained | Chapter 29 from Brock Biology of Microorganisms

How do clinicians accurately detect and identify infectious agents to guide treatment? Chapter 29 of Brock Biology of Microorganisms provides a comprehensive overview of clinical microbiology diagnostics, including traditional culture methods, advanced immunological assays, and rapid molecular techniques. This summary covers laboratory safety, specimen handling, immunoassays like ELISA, nucleic acid testing such as PCR, and how labs determine antibiotic susceptibility.

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Clinical Safety and Healthcare Environments

To protect laboratory workers and patients, strict protocols and personal protective equipment (PPE) are standard. Healthcare-associated infections (HAIs) occur in about 10% of patients, often caused by pathogens such as MRSA, E. coli, Klebsiella, and C. difficile. Clinical labs operate under biosafety levels (BSL-1 to BSL-4) depending on pathogen risk, with BSL-2 being standard and BSL-4 reserved for the most dangerous agents like Ebola virus.

Specimen Collection and Pathogen Culture

Diagnostic accuracy begins with proper specimen collection, transport, and timely processing. Techniques include:

• Microscopy (e.g., Gram stain) and direct antigen detection.
• Culture on selective, differential, or enrichment media (e.g., MacConkey, EMB).
• Automated systems that detect growth via turbidity, gas, or fluorescence.

Sample types and approaches:

• Blood/CSF: Automated cultures; CSF must be sterile.
• Urine: Cultured for UTIs using enteric media.
• Feces: Examined for pathogens like Campylobacter.
• Wounds/Abscesses: Aerobic/anaerobic culture; MRSA detected on chromogenic agar.
• Genital samples: N. gonorrhoeae on MTM agar, Gram staining for rapid ID.

Antibiotic Susceptibility Testing

Choosing effective antibiotics relies on susceptibility testing:

• Minimum Inhibitory Concentration (MIC): Determined by broth dilution or Etest.
• Disk diffusion (Kirby-Bauer): Measures inhibition zones on agar plates.
• Antibiograms: Summarize resistance trends for specific pathogens, guiding treatment and monitoring emerging resistance.

Immunological Methods and Rapid Testing

Immunoassays detect pathogens or antibodies in patient samples via antigen–antibody reactions:

• Serology: Measures antibody titers, revealing infection history or stage.
• Monoclonal antibodies (mAbs): Offer highly specific detection.
• Precipitation and agglutination: Visible clumping or reactions (e.g., latex beads for S. aureus).
• Immunofluorescence: Uses labeled antibodies for direct or indirect visualization.
• Skin tests: (e.g., tuberculin) assess delayed-type hypersensitivity.

ELISAs (enzyme immunoassays) are versatile:

• Direct ELISA: Detects antigen.
• Indirect ELISA: Detects antibody.
• Sandwich ELISA: Uses two antibodies for greater sensitivity.
• Combo tests: Detect both antigen and antibody.

Rapid tests (such as for strep throat or HIV) deliver results at the point of care. Immunoblots (Western blots) confirm protein presence after gel separation, providing diagnostic confirmation.

Nucleic Acid-Based Diagnostics: PCR and Beyond

Molecular diagnostics offer rapid, sensitive, and specific detection:

• PCR (Polymerase Chain Reaction): Amplifies pathogen DNA.
• qPCR: Quantifies DNA in real time.
• RT-PCR: Detects RNA viruses (e.g., SARS-CoV-2).
• Nucleic acid hybridization: Labeled probes detect specific genes.
• Melting curve analysis: Differentiates closely related pathogens (e.g., HSV-1 vs HSV-2).

Molecular diagnostics are rapid, culture-independent, and highly specific, but cannot always confirm if organisms are alive or if they are antibiotic resistant.

Glossary: Key Terms from Chapter 29

• HAI: Healthcare-associated infection.
• MIC: Lowest drug concentration that inhibits pathogen growth.
• ELISA/EIA: Enzyme-linked immunoassays for antigen or antibody detection.
• qPCR/RT-PCR: Real-time PCR and reverse-transcriptase PCR for nucleic acid detection.
• Titer: Concentration of antibody or antigen in a sample.
• Agglutination/Precipitation: Visible antigen–antibody reactions.
• Antibiogram: Summary chart of antimicrobial resistance patterns.

Conclusion: The Power of Modern Microbial Diagnostics

Chapter 29 highlights the critical role of laboratory diagnostics in identifying infectious diseases and guiding therapy. By combining traditional culture methods, immunological assays, and cutting-edge molecular tools, clinical microbiology empowers modern medicine to improve patient outcomes and combat antimicrobial resistance.

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