Laboratory Diagnosis

Direct Microscopy (Stool Examination):

- Sample Collection:
- Collect three stool samples for examination.
- In rare cases (especially in HIV-positive patients), sputum, bronchial washings, or duodenal/jejunal aspirates may be collected.

- Direct Wet Mount:
- Perform direct wet mounting from the mucus plug of the stool sample.
- Demonstrates highly refractile, double-walled oocysts.

- Concentration Techniques:
- Used when the oocyst load is low.
- Flotation Techniques:
- Sheather’s sugar flotation technique (widely used for coccidian parasites).
- Saturated salt flotation technique.
- Sedimentation Techniques:
- Formalin-ether sedimentation technique.

- Acid-Fast Staining:
- Oocysts are acid-fast to 0.5–1% sulfuric acid or acid alcohol.
- Appear as red-colored oocysts against a blue background.
- Kinyoun’s Method (Cold Acid-Fast Staining):
- Method of choice for staining.
- Sensitivity:
- Low sensitivity; requires a minimum of 50,000 oocysts/mL in liquid stool and 500,000 oocysts/mL in formed stool.

- Direct Fluorescent Antibody Staining:
- Available for detecting Cryptosporidium oocysts.
- Uses fluorescent-labeled monoclonal antibodies against cyst wall antigens.
- Advantages:
- Ten times more sensitive and more specific than acid-fast staining.
- Useful for detecting oocysts in water and environmental samples.
- Considered the gold standard test for cryptosporidiosis.

- Autofluorescence:
- Cyclospora oocysts fluoresce under ultraviolet epifluorescence microscopy.
- Rapid and sensitive method, though not highly specific.
- Cystoisospora oocysts may also show autofluorescence, but less consistently than Cyclospora.

- Phase-Contrast Microscopy:
- Useful for visualizing oocysts and other developmental stages.

Histopathology:

- Demonstrates various developmental stages of coccidian parasites from intestinal (jejunum) biopsy specimens.
- Hematoxylin and Eosin (H&E) Staining:
- Cryptosporidium appears as 1–3 μm basophilic round bodies within the cell membrane of enterocytes.
- These are referred to as "blue beads" under the microscope.

Antigen Detection from Stool:

- Enzyme-Linked Immunosorbent Assay (ELISA):
- Detects Cryptosporidium parvum specific coproantigen (oocyst antigen) in stool.
- Sensitivity ranges from 66% to 100% with excellent specificity.

- Immunochromatographic Tests (ICT):
- Examples include the Triage parasite panel.
- Simultaneously detects antigens of Cryptosporidium (protein disulfide isomerase antigen), Giardia, and Entamoeba histolytica.
- Shows sensitivity of 83–96% and specificity of 99–100%.

Antibody Detection:

- Methods like ELISA detect antibodies in the patient's serum against oocyst antigens of Cryptosporidium or Cyclospora.
- Primarily used for sero-epidemiological studies, not for routine diagnosis.

Molecular Diagnosis:

- Polymerase Chain Reaction (PCR) and Real-Time PCR:
- Detect specific genes (e.g., 18S rRNA or small subunit rRNA) from clinical and environmental samples.
- Advantages:
- More sensitive and faster than traditional methods.
- Can differentiate between Cryptosporidium, Cyclospora, and Cystoisospora.
- Identifies various genotypes of Cryptosporidium, important in outbreak investigations.

- BioFire FilmArray:
- Fully automated commercial PCR system (by bioMérieux).
- The gastrointestinal panel detects common bacterial, viral, and parasitic diarrheal pathogens, including Cryptosporidium, Cyclospora, E. histolytica, and Giardia.

Other Methods:

- CD4+ T Lymphocyte Count:
- Low counts (especially in HIV-positive patients) can be indicative of infection severity.

- Fecal Leukocyte Marker—Lactoferrin:
- Increased lactoferrin levels in 75% of cases indicate the presence of pus cells in feces.

IN points