Clinical Parasitology ( Laboratory )

• • Confirmation of a suspected parasitic condition generally depends on the result of proper laboratory examination.
• The ability of a parasitology laboratory 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.
• • Among the specimens available for parasitic examinations, the stool is most commonly utilized. Other specimens like urine, blood, sputum, cerebrospinal fluid are also used for diagnosis.
• • The most common method of diagnosis of intestinal parasites is through the demonstration of eggs, larvae, adults, trophozoites, cysts, or oocysts in the stool.
• • The fecal specimen is best collected in clean, widemouthed containers made plastic with a tight-fitting lid to ensure retention of moisture and to prevent accidental spillage.
• The stool specimen should be submitted with the following information: 1. patient’s name 2. age 3. sex 4. date/time of collection 5. requesting physician 6. requested procedure 7. presumptive diagnosis 8. prior infections 9. travel history
• For stool materials to be useful in parasitic diagnosis, there are important factors to consider: A. Intake of drugs/medicinal substances (antacids, anti-diarrheal, barium, bismuth, laxatives) • All of these drugs have been found to leave crystalline residues that can interfere with the identification of parasites. Stool samples should be collected a week after the last intake of any of these drugs.
• B. Intake of antibiotics usually decreases the number of protozoans for several weeks.
• C. Amount of stool to be collected is dictated by the techniques that will be used. A routine stool examination usually requires a thumb-sized specimen of formed stool or about 5 to 6 tablespoons of watery stool.
• D. Contamination with toilet water, urine, or soil must be prevented since these can destroy protozoan trophozoites. In addition, soil and water may contain free-living organisms that would complicate diagnosis of infections.
• E. Age of the stool sample is very important for diarrheic specimens since the trophozoites it may contain are likely to die within 30 minutes to 1 hour after passage. Therefore, stools must be examined within that period

  of time.
• F. Delay in examination of specimens may require preservation to ensure that parasites are present in the identifiable stage.
• G. Temporary storage of fecal samples in a refrigerator (3-5°C) is acceptable, but prolonged refrigeration can bring about desiccation. Trophozoites are killed by refrigeration, although helminth eggs and protozoan cysts are usually not damaged.
• NEVER FREEZE STOOL SAMPLES. NEVER KEEP THEM IN INCUBATORS.
• • Appropriate fixation of parasites in the stool will preserve protozoan morphological features and prevent possible destruction of helminth eggs and larvae.
• • Stool samples must be adequately mixed with the selected preservative in a proportion of one part stool to three parts preservative.
• 1. Formalin is an all purpose fixative. A 5% concentration is recommended for protozoan cysts, while a 10% concentration is recommended for helminth eggs and larvae. The solution may be buffered with sodium phosphate to preserve the morphological characteristics of the organisms. Preserved stool can be concentrated using formalin- ether/ethyl acetate concentration technique (FECT).
• 2. Schaudinn’s solution is used to preserve fresh stool in preparation for staining the stool smears. It contains mercuric chloride which is highly toxic to humans. Problems of mercury disposal may therefore arise.
• 3. Polyvinyl alcohol (PVA) is a plastic resin which serves to adhere a stool sample onto a slide. The main advantage of using PVA is related to the preservation of protozoan cysts and trophozoites for permanent staining.
• 4. Merthiolate-iodine-formalin (MIF) contains Merthiolate (also called thimerosal) and iodine which act as staining components, while formalin acts as the preservative. It is useful for the fixation of intestinal protozoans, helminth eggs, and larvae.
• 5. Sodium acetate-acetic acid formalin (SAF) has the advantage of not containing mercuric chloride. Images of organisms fixed in SAF, however, are not as sharp after staining compared with those fixed in PVA or Schaudinn’s solution. It is a liquid fixative with a long shelf-life.
• Stool samples are submitted to the laboratory in the fresh state or as preserved samples. If stools are fresh, the laboratory can classify the consistency of the stools as formed, semi-formed, soft, loose, or watery.
• The consistency can give an indication of the stage of the organism that may be present in the sample. Protozoan trophozoites are generally observed in soft or liquid stool, while the cysts are often found in formed or semi-formed samples.
• 1. Gross or Macroscopic Examination 1. Consistency

  2. Color

  • The surface of the entire specimen should be examined for macroscopic parasites.

  • The stool should be broken up with applicator sticks to check for helminths.

  • The presence of blood or mucus should be noted.

  • The color and the consistency of the specimen are also noted before proceeding to the microscopic

  evaluation.
• Macroscopic Examination

  ✗ The consistency of a stool specimen, whether formed, semi-formed, mushy, or watery, is of great importance, giving an indication of the types of organisms, which it may contain.

  ✗ 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.
• • 1. White blood cells:

  Polymorphonuclears (PMNs), which may indicate inflammation

  b. Eosinophils, which may indicate an immune response to a parasitic infection

  • 2. Macrophages are usually present in both bacterial and parasitic infections. In actual practice, one can mistake the active macrophages for amebic trophozoites.

  • 3. Red blood cells, which may indicate ulcerations or bleeding
• • 4. Charcot-Leyden crystals are released with the disintegration of eosinophils. They may indicate presence of hypersensitivity or parasitic infections, especially amebiasis.

  • 5. Epithelial cells from the intestinal tract can also be recovered.

  • 6. Eggs of arthropods, plant nematodes, and other spurious parasites may be mistaken for human parasites.

  • 7. Fungal spores coming from Candida spp., yeast, and yeast-like fungi may also be mistaken for parasites.