Laboratory Diagnosis of Infections

Laboratory Diagnosis of Infections

Laboratory Diagnosis of Infections

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Specimen Handling - Get It Right!

  • Aseptic technique is crucial. Collect before antimicrobials.
  • 📌 5 Rights: Right Patient, Right Site, Right Time, Right Specimen, Right Container.
  • Adequate quantity essential. Label meticulously: Name, ID, Date, Time, Site.
  • Transport ASAP. Use transport media (e.g., Stuart's, Amies) if delay >2h.
  • Special handling: Anaerobes (anaerobic vial), Viruses (VTM). Viral transport medium and swabs

⭐ For suspected bacteremia, collect 2-3 blood culture sets (aerobic & anaerobic bottles each) from different sites before antibiotics administration to maximize yield and guide therapy effectively.

Microscopy & Stains - Stain & See

  • Foundation of rapid diagnosis; initial pathogen clues.
  • Gram Stain: Differentiates bacteria based on cell wall.
    • Gram +ve: Purple/Violet (e.g., Staphylococcus, Streptococcus)
    • Gram -ve: Pink/Red (e.g., E. coli, Klebsiella)
    • 📌 Positive Purple, Negative piNk.
  • Ziehl-Neelsen (ZN) Stain: For acid-fast bacilli (AFB).
    • Mycobacterium tuberculosis (red bacilli, blue background).
    • Modified ZN: Nocardia, Cryptosporidium.
  • KOH Mount ($10-20%$): Visualizes fungal elements (hyphae, yeasts) by dissolving host cells.
  • India Ink: Negative stain for capsules, classically Cryptococcus neoformans.
  • Giemsa Stain: For blood parasites (Malaria, Leishmania), Chlamydia inclusions, Borrelia.
  • Albert's Stain: Detects metachromatic granules in Corynebacterium diphtheriae. India ink stain of Cryptococcus neoformans

⭐ Ziehl-Neelsen stain uses carbolfuchsin (primary stain), heat (mordant), acid-alcohol (decolorizer), and methylene blue (counterstain).

Culture Methods - Growth & ID

  • Goal: Isolate, ID microbes, & enable Antimicrobial Susceptibility Testing (AST). Gold standard for many infections.
  • Culture Media Types:
    • Basal: e.g., Nutrient agar, Peptone water.
    • Enriched: Blood Agar (BA), Chocolate Agar (CA for Haemophilus, Neisseria).
    • Selective: MacConkey (Gram-neg), Lowenstein-Jensen (LJ for TB), TCBS (Vibrio).
    • Differential: MacConkey (Lactose Fermenter/Non-Lactose Fermenter), CLED (urine pathogens).
    • Transport: Stuart's, Amies, Cary-Blair (fecal).
  • Incubation: Standard 35-37°C; O₂ needs (aerobic; anaerobic - Robertson Cooked Meat (RCM); microaerophilic - Campylobacter; capnophilic - 5-10% CO₂ for Neisseria, Haemophilus).
  • Identification: Colony morphology, Gram stain, biochemical tests (catalase, oxidase, IMViC), automated systems (e.g., VITEK). Bacterial colonies on culture media

⭐ For blood cultures, the optimal blood-to-broth ratio is 1:5 to 1:10 to dilute antibacterial substances in blood & improve recovery of organisms.

Immunological Dx - Immune Scope

  • Detects host immune response (antibodies, CMI) or microbial antigens.
  • Antibody Detection:
    • IgM: Acute infection indicator.
    • IgG: Past/chronic infection, immunity.
    • Seroconversion: Appearance of antibodies.
    • Significant rise: ≥4-fold increase in titre (acute vs. convalescent sera).
  • Antigen Detection:
    • Directly identifies microbial components (e.g., proteins, polysaccharides).
    • Enables rapid diagnosis.
  • Cell-Mediated Immunity (CMI) Tests:
    • E.g., Tuberculin skin test (TST), Interferon-Gamma Release Assays (IGRAs) for TB.
  • Techniques: ELISA, Rapid tests (immunochromatography), Immunofluorescence (IF), Agglutination. Antibody response curve IgM and IgG over time

⭐ ELISA is a versatile, highly sensitive, and specific technique widely used for detecting either antigens or antibodies, crucial for HIV and Hepatitis diagnosis.

Molecular Techniques - Gene Detectives

Directly detect microbial genetic material (DNA/RNA). They offer high sensitivity, specificity, and rapid results, vital for unculturable or slow-growing pathogens.

  • PCR (Polymerase Chain Reaction): Core method for DNA amplification.
    • RT-PCR: Detects RNA (e.g., HIV, Influenza).
    • qPCR (Real-time PCR): Allows quantification of pathogen load.
  • Nucleic Acid Hybridization: Uses labeled probes to find specific DNA/RNA sequences (e.g., FISH).
  • Sequencing (e.g., NGS): Determines exact nucleotide order for precise ID, genotyping, resistance.

Traditional vs. Molecular Diagnostic Approaches

⭐ PCR can detect as few as 1-10 microbial genomes, revolutionizing diagnosis of infections like TB from paucibacillary samples.

High‑Yield Points - ⚡ Biggest Takeaways

  • Gram stain (cell wall) & ZN stain (acid-fast bacilli) are key microscopy tests.
  • Culture is gold standard for ID & antibiotic susceptibility testing (AST).
  • Serology (antibodies/antigens) indicates infection stage; IgM suggests acute infection.
  • Molecular tests (e.g., PCR) offer rapid, sensitive microbial nucleic acid detection.
  • India Ink for Cryptococcus (capsule); KOH mount for rapid fungal element detection.
  • Optimal blood culture collection (volume, timing, multiple sets) is crucial for sepsis diagnosis.
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Practice Questions: Laboratory Diagnosis of Infections

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Arrange the following in sequential order with regards to the steps of collection of samples for pap smear testing: Use posterior vaginal wall retractor Take the sample Make smear on a slide Fix the smear

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Flashcards: Laboratory Diagnosis of Infections

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Which EBV pathology other than Hodgkin's lymphoma may show Reed-Sternberg cells?_____

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Which EBV pathology other than Hodgkin's lymphoma may show Reed-Sternberg cells?_____

Infectious mononucleosis

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