Cyclosporiasis laboratory findings

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1] Associate Editor(s)-in-Chief: Alejandro Lemor, M.D. [2]

Overview

The diagnosis of cyclospora infection is confirmed examining stool specimens and several stool samples are require for a more precise identification of the oocysts. Laboratory techniques used to detect the cyclospora oocytes in stool include acid-fast staining, ultraviolet (UV) fluorescence microscope, and polymerase chain reaction (PCR) analysis.

Laboratory Findings

• Cyclospora infection is diagnosed by examining stool specimens.
• Laboratory testing for cyclospora is not routinely done in most U.S. laboratories, even when stool is tested for parasites. Therefore, if indicated, health care providers should specifically request testing for cyclospora.
• Laboratory diagnosis can be difficult as the cyclospora oocysts may not be detected in stool, even if the patient is symptomatic.
• Therefore, patients might need to submit several specimens collected on different days.

Stool Examination

Wet Mount

• In bright-field microscopy using differential interference contrast (DIC), oocysts appear as refractile spheres (8 to 10 μm) with a distinct oocyst wall, but may be confused with other objects. Under UV fluorescence microscopy, the oocyst wall autofluoresces.
• An intense blue fluorescence is obtained with the preferred UV excitation filter set (330 to 365 nm).
• If this filter set is not available, a less intense green fluorescence can be obtained with blue excitation (450 to 490 nm).
• A fluorescence microscope is required and this procedure does not provide a stained slide that can be archived.
• Both DIC and UV fluorescence microscopy are efficient and reliable approaches for identification of cyclospora.

Shown below are images of oocysts of cyclospora in an unstained wet mount.

Oocyst of C. cayetanensis in an unstained wet mount. Image courtesy of the Oregon State Public Health Laboratory

Oocyst of C. cayetanensis in an unstained wet mount. Image courtesy of the Oregon State Public Health Laboratory.

Oocyst of C. cayetanensis in an unstained wet mount of stool. Image taken at 1000x magnification. Image courtesy of CDC.

Modified Acid Fast Stain

• A blue-green background, or contrasting counterstain, of fecal debris allows the oocysts to stand out.
• The oocysts are variably stained: some will stain light pink to deep purple, while others may be unstained.
• The oocysts (8 to 10 μm) may not be perfectly round; some may appear collapsed or distorted on one side.
• They may contain granules and/or have a wrinkled oocyst wall appearance (characteristics that distinguish oocysts from acid-fast artifacts).
• This staining method is the easiest and most practical, and provides a stained slide that can be archived.
• The variability in staining and confusion with artifacts is a cause of misdiagnosing this infection.

Shown below are images of oocysts of cyclospora stained with modified acid fast stain.

Image obtained from CDC

Image obtained from CDC

File:Acid fats stain 3.jpg Image obtained from CDC

Safranin Stain

• In the safranin stain, cylcospora ocysts stain uniformly, red to reddish-orange.
• This uniform staining decreases the risk of misdiagnosis.
• This technique requires heating, therefore additional equipment is necessary.

Shown below are images of oocysts of cyclospora stained with safranin stain.

Image obtained from CDC

Image obtained from CDC

Image obtained from CDC

Trichrome Stain

• Oocysts may be detected, but should not be confirmed, by this method.
• Trichrome stain is the routine staining technique for stool specimens in most laboratories and laboratorians should be familiar with the appearance of cyclospora stained with trichrome in order to detect oocysts during routine examinations.
• This staining method is inadequate for definitive diagnosis because all oocysts will appear unstained.
• Oocysts appear as clear, round, wrinkled spheres, that measure between 8 to 10 μm.

Image obtained from CDC

Image obtained from CDC

Polymerase Chain Reaction

• Molecular diagnostic methods, such as polymerase chain reaction (PCR) analysis, are used to look for the parasite's DNA in the stool.
• Nested polymerase chain reaction (nested-PCR) assay targeting the 18S rRNA gene is the method of choice for Cyclospora species identification.
• Shown below is a cyclospora positive fecal specimen. The red arrow shows the diagnostic band for cyclospora cayetanensis (size: 308 bp).

Image courtesy of CDC.