Parasitology

• Specimen collection, transport, handling and preservation

  Confirmation of a suspected parasitic condition generally depends on the result of proper laboratory examination
• Parasitology laboratory


  • Ability to generate reliable results is dependent on the proper collection, handling, and processing of specimens prior to examination, the skill of the laboratory analyst (examiner), and the quality of equipment used in the examination
• There are cases where the parasite is not demonstrable even in active infection, in light infections when parasites are still immature, recovery of parasites from infected individuals may not be possible. In such cases, immunoassays may become useful.
• Specimens available for parasitic examinations

  • Stool
  • Urine
  • Blood
  • Sputum
  • Cerebrospinal fluid
• Fecal sample examination


  The most common method of diagnosis of intestinal parasites is through the demonstration of eggs, larvae, adults, trophozoites, cysts, or oocysts in the stool
• Fecal specimen


  • Best collected in clean, wide-mouthed containers made plastic with a tight-fitting lid to ensure retention of moisture and to prevent accidental spillage
• Information to be submitted with stool specimen

  • Patient's name
  • Age
  • Sex
  • Date/time of collection
  • Requesting physician
  • Requested procedure
  • Presumptive diagnosis
  • Prior infections
  • Travel history
• Important factors to consider for stool materials to be useful in parasitic diagnosis

  • Intake of drugs/medicinal substances
  • Intake of antibiotics
  • Amount of stool to be collected
  • Contamination with toilet water, urine, or soil
  • Age of the stool sample
  • Delay in examination of specimens
• Never freeze stool samples. Never keep them in incubators.
• Stool preservatives


  • Formalin
  • Schaudinn's solution
  • Polyvinyl alcohol (PVA)
  • Merthiolate-iodine-formalin (MIF)
  • Sodium acetate-acetic acid formalin (SAF)
• Macroscopic examination


  Consistency, color, presence of blood or mucus
• Trophozoites are usually found in watery or soft stools, but almost never in formed ones. Cysts are usually found in formed specimens. Helminth eggs may be found in either watery or formed stools, but as the watery stool is usually very dilute, they may often be difficult to detect in such specimens.
• Elements observed in microscopic examination

  • White blood cells (polymorphonuclears, eosinophils)
  • Macrophages
  • Red blood cells
  • Charcot-Leyden crystals
  • Epithelial cells
  • Eggs of arthropods, plant nematodes, and other spurious parasites
  • Fungal spores
  • Plant cells/fibers, pollen grains, starch granules, vegetable spirals
  • Plant and animal hairs
• The entire slide preparation must be examined for the presence of eggs, larvae and protozoa. Low power is used to scan for large helminth eggs or larvae. High power is used to detect and identify smaller parasites and larger helminth eggs and larvae.
• Any parasites detected are reported out by their scientific name and quantity observed. If no parasites are observed report out as "No Ova or Parasite Seen or NOOPS" or "No Intestinal Parasites Seen or NIPS".
• Frequency distribution chart

  • Rare (2 to 5 organisms per 22 mm square coverslip)
  • Few (1 organism per 5 to 10 high-power fields (44x) or 1 egg/larva per 5 to 10 low-power fields (10x))
  • Moderate (1 to 2 organisms per high-power field, to as few as 1 organism per 2 to 3 high-power fields or 1 to 2 eggs/larvae per low-power field)
  • Many (Several organisms in every high-power field or Several eggs/larvae in every low-power field)
• Direct fecal smear (DFS)


  About 2 mg of stool is comminuted thoroughly with a drop of 0.85% NSS and then covered with a cover slip. A weak iodine solution can be used as a temporary stain to demonstrate nuclei.
• Kato thick smear


  About 50 to 60 mg of stool (approximately the size of two mung beans) is placed over a glass slide and covered with cut cellophane paper soaked in a mixture of glycerin and malachite green solution. The preparation is best examined within 10 to 20 minutes. It is very good in detecting eggs with thick shells (Ascaris and Trichuris) but not eggs with thin shells (hookworm).
• Kato-katz technique


  This procedure uses a measured amount of stool which has been sieved through a wire mesh and pressed under cellophane paper soaked in glycerin-malachite green solution. A uniform amount of stool is examined through the use of a template with a uniform-sized hole in the middle.
• Concentration techniques


  Can separate protozoan cysts and helminth eggs from a larger amount of stool based on differences in specific gravity. These procedures are based either on sedimentation or flotation.
• Acid ether concentration technique (AECT)


  The main reagents are 40% HCl, which can dissolve albuminous material, and ether, which can dissolve neutral fats in the stool. This technique is recommended for the recovery of Trichuris, Capillaria, and trematode eggs, especially Schistosoma.
• Formalin-ether/ethyl acetate concentration technique (FECT)


  This procedure makes use of 10% formalin which is an all purpose fixative, and ether, which can dissolve neutral fats in the stool. This is useful in the recovery of both helminth eggs and protozoan cysts.
• Zinc sulfate (ZnSO4) flotation


  The main reagent is a 33% zinc sulfate solution. Before use, the specific gravity should be checked.
• Sedimentation Procedures


  1. Acid Ether Concentration Technique (AECT)

  2. Formalin-Ether/Ethyl Acetate Concentration Technique (FECT)
• Acid Ether Concentration Technique (AECT)


  • Main reagents are 40% HCl and ether
  • Recommended for recovery of Trichuris, Capillaria, and trematode eggs, especially Schistosoma
  • Drawbacks include loss of parasite to the plug of debris and possible destruction of protozoan cysts
• Formalin-Ether/Ethyl Acetate Concentration Technique (FECT)


  • Makes use of 10% formalin and ether
  • Useful in the recovery of both helminth eggs and protozoan cysts
  • Can also be done with formalin-preserved and PVA-preserved stools
• Flotation Procedures

  1. Zinc Sulfate (ZnSO4) Flotation

  2. Brine Flotation

  3. Sheather's Sugar Flotation
• Zinc Sulfate (ZnSO4) Flotation


  • Main reagent is a 33% zinc sulfate solution
  • Specific gravity should be checked (1.18 to 1.20)
  • Parasites exposed to high specific gravity may experience distortion and shrinkage of protozoan cysts and thin-walled nematode eggs
• Brine Flotation


  • Makes use of a saturated table salt solution
  • Stools are directly mixed with the brine solution
  • No need for centrifugation as helminth eggs rise to the surface
  • Low-cost and simple but helminth eggs like hookworm and Schistosoma become badly shrunken
  • Not useful for operculated eggs like Clonorchis, Opistorchis, and heterophyids as they do not float in brine solution
• Sheather's Sugar Flotation


  • Boiled sugar solution preserved with phenol is used
  • Considered the best for the recovery of coccidian oocysts, mainly Cryptosporidium, Cyclospora, and Cystoisospora
  • Visualization of oocysts can be better appreciated through the use of a phase microscope
• Stool Culture Methods


  1. Copro Culture

  2. Harada-Mori or the Test Tube Culture Method
• Copro Culture

  • Positive stools are mixed with moistened soil or granulated charcoal to simulate environmental conditions
  • Larvae are harvested using the Baermann procedure
• Harada-Mori or the Test Tube Culture Method


  • Makes use of test tubes and filter paper strips
  • Positive stool is applied to the filter paper and placed into a test tube with water
  • Filariform larvae will generally move downwards against the upward capillary movement of water and can be recovered from the water at the bottom of the tube
• Egg Counting Procedures


  1. Kato-Katz Method

  2. Stoll Egg Count
• Stoll Egg Count


  • Makes use of 0.1 N NaOH and a stool displacement flask
  • The sodium hydroxide acts as a stool diluent, saponifying fat and freeing eggs from fecal debris
  • The constant used to multiply the total egg count depends on the amount of stool examined
• Staining of Stool Specimen


  1. Iron-Hematoxylin

  2. Trichome

  3. Periodic Acid Schiff (PAS)

  4. Chlorazol Black E

  5. Kinyouns method of acid-fast
• Perianal Swab


  Cellulose Tape or Scotch Tape Method
• Cellulose Tape or Scotch Tape Method

  • Sampling the perianal skin using a strip of cellulose tape attached onto a glass slide
  • Specimen collected early in the morning before the patient has taken a bath or washed the perineum
  • Examined under the microscope for the presence of eggs or the adult Enterobius
• Examination of Blood


  1. Wet/fresh Preparation

  2. Stained Smears (Thick and thin smear)

  3. Capillary Tube Method

  4. Knott's Concentration

  5. Membrane Filtration
• Stained Smears (Thick and thin smear)

  • Stains used include Giemsa stain, Wright's stain, and Delafield hematoxylin stain