Collection, Preservation, and Processing of Samples for Parasitic Diagnosis

Ms. Neha Sharma
  1. Parasitic infections can cause various diseases in humans, affecting various body systems.
  2. Accurate diagnosis of these infections is crucial for effective treatment and control, and it begins with the appropriate collection, preservation, and processing of clinical specimens.
  3. This document outlines comprehensive laboratory procedures for handling stool, blood, body fluids, tissues, and biopsy samples for parasitic diagnosis.

 

General Principles

1.1. Importance of Proper Handling

The success of parasitic diagnosis depends on maintaining the integrity of the sample. Incorrect collection, preservation, or processing can lead to false-negative results or degradation of parasites, compromising diagnostic accuracy.

1.2. Biosafety Considerations

    • Use appropriate personal protective equipment (PPE) when handling specimens.
    • Follow biosafety guidelines for handling infectious materials (Biosafety Level 2 for most parasites).
    • Decontaminate work areas and dispose of waste according to institutional protocols.

 

Stool Samples

2.1. Collection

Stool samples are the most common specimens for diagnosing intestinal parasites such as Giardia lamblia, Entamoeba histolytica, Cryptosporidium spp., and helminths.

Procedure:

    1. Timing: Collect fresh stool samples, preferably during active diarrheal episodes or within the first three days of symptom onset.
    2. Container: Provide patients with clean, wide-mouth, leak-proof containers. Containers should not contain preservatives or disinfectants.
    3. Volume: A walnut-sized portion (solid stool) or 5–10 mL (liquid stool) is adequate.
    4. Patient Instructions:
      • Avoid contamination with urine, water, or soil.
      • Do not collect stool during or shortly after barium or antibiotic treatments.

2.2. Preservation

If immediate processing is not possible, stool samples should be preserved to prevent the degradation of parasites.

Preservation Techniques:

    • Formalin (10%): Preserves helminth eggs, larvae, protozoan cysts, and oocysts.
    • Polyvinyl Alcohol (PVA): Ideal for permanent staining of protozoa.
    • Sodium Acetate-Acetic Acid-Formalin (SAF): Suitable for concentration and staining procedures.
    • Refrigeration (4°C): For temporary storage (up to 24 hours).

2.3. Processing

Processing involves macroscopic examination, concentration methods, staining, and antigen detection.

Steps:

    1. Macroscopic Examination:
      • Check for adult worms, proglottids, blood, or mucus.
    2. Direct Wet Mount:
      • Mix a small stool sample with saline or iodine on a slide.
      • Examine under a microscope for motile trophozoites, eggs, or cysts.
    3. Concentration Methods:
      • Sedimentation (Formalin-Ether): Concentrates heavy eggs and larvae.
      • Flotation (Zinc Sulfate): Effective for lighter cysts and oocysts.
    4. Staining:
      • Trichrome Staining: For protozoan trophozoites and cysts.
      • Acid-Fast Staining: For Cryptosporidium, Cyclospora, and Isospora.
    5. Antigen Detection:
      • Use enzyme immunoassays (EIA) or rapid diagnostic tests for specific parasites like Giardia or Cryptosporidium.

 

Blood Samples

Blood samples are essential for diagnosing parasites that invade the bloodstream, such as Plasmodium spp. (malaria), Trypanosoma spp. (African and American trypanosomiasis), and Wuchereria bancrofti (lymphatic filariasis).

3.1. Collection

Procedure:

    1. Timing:
      • For malaria, collect during febrile episodes to capture parasites in circulation.
      • For filariasis, collect during nocturnal hours (depending on the parasite’s periodicity).
    2. Volume:
      • Adults: 5–10 mL.
      • Children: 2–5 mL.
    3. Anticoagulants:
      • Use EDTA for most parasitological examinations.
      • Avoid heparin, which may interfere with staining.
    4. Site and Technique:
      • Perform venipuncture under aseptic conditions.

3.2. Preservation

    • Refrigeration (4°C): Short-term storage if processing is delayed.
    • Preservatives: Not typically needed for blood films but may be required for molecular methods.

3.3. Processing

Blood processing includes preparing smears, concentration techniques, and serological or molecular tests.

Steps:

    1. Thin and Thick Smears:
      • Thin Smear: Spread a drop of blood thinly on a slide, fix it with methanol, and stain it with Giemsa.
      • Thick Smear: Place a larger drop of blood on a slide, air dry, and stain without fixing to concentrate parasites.
      • Examine under oil immersion for parasites like Plasmodium or Trypanosoma.
    2. Concentration Techniques:
      • Buffy Coat Examination: Useful for detecting Leishmania or Trypanosoma.
      • Knott’s Technique: For detecting microfilariae in peripheral blood.
    3. Serological Tests:
      • Detect antibodies or antigens for Toxoplasma gondii, Trypanosoma cruzi, or Echinococcus.
    4. Molecular Tests:
      • PCR-based methods for species-specific identification.
    1. Body Fluids (CSF, Pleural Fluid, Ascitic Fluid)

4.1. Collection

Procedure:

    1. Cerebrospinal Fluid (CSF):
      • Perform a lumbar puncture under sterile conditions.
      • Collect 1–5 mL in sterile tubes.
    2. Pleural/Ascitic Fluid:
      • Use aseptic techniques for fluid aspiration.
      • Collect in sterile containers.
    3. Timing: Process immediately to prevent degradation.

4.2. Preservation

    • Store at 4°C for short-term delays.
    • Add 1–2 drops of 10% formalin if morphological studies are required.

4.3. Processing

    1. Microscopic Examination:
      • Direct wet mount or stained smear for parasites like Naegleria fowleri (CSF) or Entamoeba histolytica (abscess fluid).
    2. Concentration:
      • Centrifuge at 2000 rpm for 5–10 minutes, and examine the sediment.
    3. Staining:
      • Use Giemsa or hematoxylin-eosin stains to identify parasites.
    4. Molecular/Serological Tests:
      • PCR for amebic meningoencephalitis or serology for hydatid cysts.

 

Tissue and Biopsy Samples

Tissue specimens are critical for diagnosing intracellular parasites like Toxoplasma gondii, Leishmania donovani, or Trichinella spiralis.

5.1. Collection

    1. Biopsy:
      • Collect tissue under sterile conditions using a scalpel or biopsy needle.
      • Preferred sites depend on clinical suspicion (e.g., lymph node, liver, or skin).
    2. Volume: A small tissue fragment (1–2 cm³) is sufficient.

5.2. Preservation

    • Formalin Fixation (10%): For histopathology.
    • Refrigeration (4°C): For molecular or culture studies.
    • Cryopreservation (-70°C): For long-term storage.

5.3. Processing

    1. Histopathology:
      • Fix tissues in formalin, embed in paraffin, and stain with hematoxylin and eosin (H&E).
      • Special stains like Giemsa or periodic acid-Schiff (PAS) may be used.
    2. Microscopy:
      • Crush preparations of fresh tissue for parasites like Leishmania.
    3. Culture:
      • Inoculate tissue samples in appropriate media (e.g., Novy-MacNeal-Nicolle [NNN] for Leishmania).
    4. Molecular Tests:
      • PCR-based methods for specific parasites.

 

Quality Assurance

To ensure reliable results, laboratories should:

    1. Standardize Procedures: Follow validated protocols for each specimen type.
    2. Training: Provide staff with regular training on parasitological techniques.
    3. Quality Control: Use positive and negative controls in staining and molecular assays.
    4. Documentation: Maintain detailed records of sample collection, storage, and processing.

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