Laboratory Diagnosis of GI Infections

1. Specimen Collection

• Fecal specimen (containing mucus flakes) is collected in a sterile, screw-capped, wide-mouthed container.
• In carriers, a rectal swab may be collected instead.
• Specimens must reach the laboratory within 1 hour.
• If a delay >1 hour is expected, the specimen must be placed in transport media:
  • Cary-Blair medium - standard transport medium for most enteropathogens
  • Alkaline peptone water - used specifically when cholera (Vibrio cholerae) is suspected

2. Macroscopy

3. Microscopy

a) Wet Mount Preparation (Saline or Iodine)

• Detects pus cells, RBCs, and parasitic cysts, trophozoites, eggs, or larvae
• Standard first-line microscopic examination

b) Hanging Drop Preparation

• Used for liquid specimens
• Demonstrates the characteristic darting motility of Vibrio cholerae

c) Gram Stained Smear

• Not routinely done due to the presence of normal fecal flora
• Used in special situations where morphology may give a preliminary clue:
  • Gram-negative comma-shaped bacilli - suggests Vibrio cholerae
  • Gram-positive budding oval yeast cells in an immunocompromised host or infant - suggests Candida species

d) Modified Acid-Fast Staining (0.5-1% sulfuric acid)

• Used to detect oocysts of Cryptosporidium, Isospora, and Cystoisospora

e) Electron Microscopy

• Performed on stool to detect the morphology of specific viruses causing gastroenteritis:
  • Rotaviruses - appear as spokes grouped around a hub of a wheel
  • Astroviruses - have star-like morphology
  • Coronaviruses - have cup-like depressions on the capsid surface

4. Bacterial Culture

• Identification of enteric pathogens uses conventional biochemical tests or automated methods like MALDI-TOF or VITEK, followed by serotyping with specific group/type antisera
• Antimicrobial susceptibility testing (AST) is done using the disk diffusion method on Mueller-Hinton agar to guide antibiotic therapy

5. Tissue Culture

• Used to detect enteric viruses and some diarrheagenic E. coli
• ETEC (Enterotoxigenic E. coli) - penetrates HeLa and HEp-2 cell lines
• EHEC (Enterohemorrhagic E. coli) - detected by its cytotoxic effect on Vero cell line (verocytotoxin)

6. Antigen Detection

7. Molecular Methods

• PCR assays - detect specific genes of enteric pathogens with high sensitivity and specificity
• BioFire FilmArray - a fully automated nested multiplex PCR system; its GI panel simultaneously detects common bacterial, viral, and parasitic diarrheal pathogens in a single run (one of the most comprehensive rapid diagnostic tools currently available)

8. Other Methods

Key Takeaway