specimen collection

• In areas in which parasitic infections are not considered a major cause of human disease, it can be difficult for health care professionals to recognize that these agents may be a cause of the patient's clinical condition
• With the increased number of populations at risk for contracting parasitic infections, it is critical for clinicians to obtain knowledge of the clinical manifestations of parasitic diseases and understand the appropriate laboratory test(s) to order
• Laboratory technicians must have an understanding of these diseases to guide the physician in selecting the appropriate tests
• The diagnosis of these diseases can be challenging and is not always straightforward, so it is imperative that strong communication exist between the physician and clinical laboratory
• This chapter is designed to introduce readers to representative testing methods available for the diagnosis of parasitic infections
• Parasites that may be determined using these testing methods are identified, but the lists are not intended to be exhaustive in nature
• Appropriate testing methods are mentioned in the specific parasite laboratory diagnosis sections, which may or may not be noted in this chapter
• Successful laboratory identification of parasites

  1. Preanalytic steps

  2. Analytic steps

  3. Postanalytic steps
• In the preanalytic phase, a specimen received in the laboratory that is compromised because of improper collection, labeling, or transport should be rejected and a new specimen requested
• Laboratory techniques performed in the analytic phase of testing of these samples should be completed with care to ensure that accurate results are obtained
• Interpretation and reporting of results obtained, completed in the postanalytic phase of testing, should be accurately reported in a timely manner
• The most common procedure performed in the area of parasitology is the examination of a stool specimen for ova and parasites (abbreviated as O&P), where ova refers to the egg stage of select parasites and parasites encompasses the other morphologic forms that may be present
• There are two general components associated with this routine parasitology procedure - macroscopic and microscopic examination
• Morphologic forms of protozoa and helminths may be detected from a properly collected and prepared stool specimen
• Protozoan forms known as trophozoites and cysts may be recovered from these samples, and helminth stages such as eggs, larvae, proglottids, and adult worms may also be found
• Parasites are often shed (i.e., enter and subsequently passed in the stool) intermittently, so multiple specimens are recommended for adequate detection
• Typical stool collection protocol

  Three specimens, one specimen collected every other day or a total of three collected in 10 days
• One exception is in the diagnosis of amebiasis, in which up to six specimens in 14 days is acceptable
• Certain medications and substances may interfere with the detection of parasites, so stool samples from patients whose therapy includes barium, bismuth, or mineral oil should be collected prior to therapy or not until 5 to 7 days after the completion of therapy
• Collection of specimens from patients who have taken antibiotics or antimalarial medications should be delayed for 2 weeks following therapy
• Stool specimens should be collected in a clean, watertight container with a tight-fitting lid, and the acceptable amount of stool required for parasite study is 2 to 5 g, often referred to as the size of a walnut
• Urine should not be allowed to contaminate the stool specimen, and stool should not be retrieved from toilet bowl water
• The specimen container should be labeled with the patient's name and identification number, the physician's name, and the date and time of sample collection
• Some form of requisition, paper or computer-based, should accompany the specimen indicating the test(s) requested
• When handling all specimens, gloves and a protective coat should be worn at all times, and biohazard hoods should also be used in laboratories, when present
• To demonstrate the motility of protozoan trophozoites, a fresh specimen is required, as the trophozoite stage is sensitive to environmental changes and disintegrates rapidly
• Other parasite stages (e.g., protozoan cysts, helminth eggs and larvae) are not as sensitive and can survive for longer periods outside the host
• Liquid specimens should be examined within 30 minutes of passage, semiformed specimens within 1 hour of passage, and formed stool specimens can be held for 24 hours following collection
• If the guidelines for timely examination cannot be met, the specimen should be placed into a preservative
• The ratio of fixative to stool is important for the successful recovery of parasites, and the recommended ratio is three parts fixative to one part stool
• The choice of fixative(s) for O&P use depends on the preference of the laboratory performing the test, and the laboratory technician must be familiar with and understand the uses and limitations of each fixative
• Fixatives used for O&P testing

  • Formalin
  • Polyvinyl Alcohol (PVA)
  • Sodium Acetate Formalin (SAF)
  • Modified Polyvinyl Alcohol
• Formalin


  An all-purpose fixative for the recovery of protozoa and helminths, with advantages of being easy to prepare, preserving specimens for up to several years, and having a long shelf life, but disadvantages of not preserving parasite morphology adequately for permanent smears and not allowing recovery of trophozoites
• Polyvinyl Alcohol (PVA)


  A fixative that can be used for preparation of a permanent stained smear, with the advantage of having a long shelf life when stored at room temperature, but the disadvantage of containing mercuric chloride which has potential health problems
• Sodium Acetate Formalin (SAF)


  A viable alternative to PVA and Schaudinn fixative that is mercury-free, easy to prepare, and has a long shelf life, but the disadvantages of not having good adhesive properties and not providing as clear protozoa morphology in permanent stains as mercury-containing preservatives
• Modified Polyvinyl Alcohol


  Alternatives to mercury-based PVA that contain copper sulfate or zinc sulfate, with the advantage of being able to be used for concentration methods and permanent stained smears
• Preparing smears for staining

  With the modified acid-fast stain to detect coccidian oocysts
• SAF (sodium acetate-acetic acid-formalin)

  • Disadvantages: Adhesive properties are not good, so addition of albumin to the microscope slide may be necessary to ensure adhesion of the specimen
  • Protozoa morphology from SAF-preserved specimens is not as clear in permanent stains as when mercury-containing preservatives are used
  • Limiting factor in the choice of permanent stains made from this fixative
• Many experts believe that permanent stained smears with iron hematoxylin staining provide better results than staining SAF-preserved material using the Wheatley trichrome stain
• Modified Polyvinyl Alcohol (PVA)

  Alternatives to mercury-based PVA using substitute compounds containing copper sulfate or zinc sulfate