Cyclosporiasis

[Cyclospora cayetanensis]

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Causal Agents

Cyclospora cayetanensis is a coccidian protozoan. It appears that all human cases are caused by this species; no animal reservoirs for C. cayetanensis have been identified.

Life Cycle

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When freshly passed in stools, the oocyst is not infective (thus, direct fecal-oral transmission cannot occur; this differentiates Cyclospora from another important coccidian parasite, Cryptosporidium). In the environment , sporulation occurs after days or weeks at temperatures between 22°C to 32°C, resulting in division of the sporont into two sporocysts, each containing two elongate sporozoites . The sporulated oocysts can contaminate fresh produce and water which are then ingested . The oocysts excyst in the gastrointestinal tract, freeing the sporozoites, which invade the epithelial cells of the small intestine . Inside the cells they undergo asexual multiplication into type I and type II meronts. Merozoites from type I meronts likely remain in the asexual cycle, while merozoites from type II meronts undergo sexual development into macrogametocytes and microgametocytes upon invasion of another host cell. Fertilization occurs, and the zygote develops to an oocyst which is released from the host cell and shed in the stool . Several aspects of intracellular replication and development are still unknown, and the potential mechanisms of contamination of food and water are still under investigation.

Hosts

Humans appear to be the only major host for C. cayetanensis. Occasionally, cysts are recovered from animal feces, but it is likely that this represents spurious passage following coprophagy.

Geographic Distribution

Cyclosporiasis has been reported in many countries, but is most common in tropical and subtropical areas. In the United States, the majority of cases are reported during the spring and summer months. Outbreaks have been identified nearly every year since the mid-1990s.

Clinical Presentation

After an average incubation period of one week, symptomatic infections typically manifest as watery diarrhea of varying severity. Other manifestations include complications of dysentery, further abdominal symptoms, and sometimes non-specific systemic symptoms (e.g. headache, low-grade fever). Untreated infections typically last for 10–12 weeks and may follow a relapsing course. The duration of symptoms and associated weight loss are greater in individuals with HIV or possibly other immunosuppressive conditions. Infections can be asymptomatic in disease-endemic regions.

Cyclospora cayetanensis oocysts in wet mounts.

Cyclospora cayetanensis oocysts are spherical, 7.5-10 µm in diameter and unsporulated when passed in feces. Sporulation in the environment is temperature-dependent and may take one to several weeks for an infective oocyst to contain two sporocysts, each containing two sporozoites. Oocysts of C. cayetanensis will autofluoresce under ultraviolet (UV) microscopy.

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

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

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

Figure D: Oocyst of C. cayetanensis in an unstained wet mount of stool. Image taken at 1000x magnification.

C. cayetanensis oocysts stained with trichrome.

Figure A: Oocyst of C. cayetanensis? stained with trichrome; while the oocyst is visible, the staining characteristics are inadequate for a reliable diagnosis.

Figure B: Oocysts of C. cayetanensis stained with trichrome; while the oocyst is visible, the staining characteristics are inadequate for a reliable diagnosis.

C. cayetanensis oocysts viewed under ultraviolet (UV) microscopy.

Figure A: Oocyst of C. cayetanensis viewed under UV microscopy.

Figure E: Oocyst of C. cayetanensis viewed under UV microscopy.

Figure B: Oocyst of C. cayetanensis viewed under UV microscopy.

Figure F: Oocyst of C. cayetanensis viewed under UV microscopy.

Figure C: Oocyst of C. cayetanensis viewed under UV microscopy.

Figure D: Oocyst of C. cayetanensis viewed under UV microscopy.

C. cayetanensis oocysts stained with modified acid-fast.

Figure A: Oocysts of C. cayetanensis stained with modified acid-fast stain. Note the variability of staining in the four oocysts.

Figure B: Two oocysts of C. cayetanensis stained with modified acid-fast stain. Both oocysts failed to take up the carbol fuschin stain. Image courtesy of the Arizona State Public Health Laboratory.

Figure C: Oocysts of C. cayetanensis stained with modified acid-fast stain. Note the wrinkled edge and the lack of stain in the two oocysts. Image courtesy of the Arizona State Public Health Laboratory.

Figure D: Oocyst of C. cayetanensis stained with modified acid-fast stain.

Figure E: Oocysts of C. cayetanensis stained with modified acid-fast stain.

Figure F: Oocysts of C. cayetanensis stained with modified acid-fast stain.

C. cayetanensis oocysts stained with safranin (SAF).

Figure A: Oocyst of C. cayetanensis stained with safranin (SAF).

Figure E: A pair of oocysts of C. cayetanensis stained with safranin (SAF).

Figure B: Oocyst of C. cayetanensis stained with safranin (SAF).

Figure F: Oocyst of C. cayetanensis (yellow arrow) along with an oocyst of Cryptosporidium parvum (red arrow), stained with safranin (SAF). Cryptosporidium spp. also stain with the safranin and modified acid-fast stains.

Figure C: Oocyst of C. cayetanensis stained with safranin (SAF).

Figure D: Oocyst of C. cayetanensis stained with safranin (SAF).

C. cayetanensis oocysts viewed under differential interference contrast (DIC) microscopy.

Figure A: Oocyst of C. cayetanensis viewed under differential interference contrast (DIC) microscopy. The refractile globules are easily visible under DIC.

Figure B: Oocyst of C. cayetanensis viewed under differential interference contrast (DIC) microscopy. The refractile globules are easily visible under DIC.

Figure C: Oocyst of C. cayetanensis viewed under DIC microscopy. There are two sporocysts are visible in this image.

Figure D: Oocyst of C. cayetanensis viewed under DIC microscopy.

Figure E: A pair of oocysts of C. cayetanensis viewed under DIC microscopy.

Figure F: Rupturing oocyst of C. cayetanensis viewed under DIC microscopy. One sporocyst has has been released from the mature oocyst; the second sporocyst is still contained within the oocyst wall.

Laboratory Diagnosis

Unpreserved stool collected in enteric transport media (e.g., Cary-Blair) is commonly used for culture independent diagnostic tests (CIDTs) and can be used for confirmatory testing by microscopy and/or PCR if needed. Unpreserved specimens should be refrigerated and sent to the diagnostic laboratory as rapidly as possible.

Cyclospora oocysts can be excreted intermittently and in small numbers. Thus:

A single negative stool specimen does not rule out the diagnosis; three or more specimens at 2- or 3-day intervals may be required.
Concentration procedures should be used to maximize recovery of oocysts. The method most familiar to laboratorians is the formalin-ethyl acetate sedimentation technique (centrifuge for 10 minutes at 500 × g). Other methods can also be used (such as the Sheather’s flotation procedure).

Microscopic Examination

The sediment can be examined microscopically with the following techniques:

Wet mounts (by conventional light microscopy, which can be enhanced by UV fluorescence microscopy or differential interference contrast [DIC, Nomarsky])
Stained smears (using modified acid-fast stain or a modified safranin stain). Both modified acid-fast stains and modified safranin staining will stain Cyclospora cysts from a pink to brilliant red. Staining may be more variable using modified acid-fast.
Other common stains including Giemsa and trichrome are not adequate for the reliable detection of Cyclospora.

Molecular Diagnosis

Several conventional and real-time PCR protocols have been developed to specifically detect Cyclospora cayetanensis in stool. Commercial, FDA-approved panels (that include other enteric pathogens) are available and are highly sensitive.

Laboratory Safety

Standard precautions for the processing of stool samples apply.

Cyclospora oocysts are not immediately infectious when passed, however, precautions should still be taken to avoid exposure to other pathogens that may be present in stools.

Key points for laboratory diagnosis of Cyclospora, Cryptosporidium, and Cystoisospora belli [PDF, 157 KB, 1 Page]