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Laboratory Diagnosis of Cyclospora Infection

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Published: September 2013

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Cyclospora infection is a diarrheal disease that occurs in foodborne outbreaks or can occur in travelers to endemic areas. Dr. Rosenblatt describes the clinical aspects of Cyclospora infection and discusses the appropriate laboratory tests for diagnosis.

Presenter: Jon Rosenblatt, MD

Presenters: Bobbi S. Pritt, MD

  • Associate Professor of Laboratory Medicine and Pathology, College of Medicine
  • Director of the Initial Processing and Media Laboratory and the Parasitology Laboratory at Mayo Clinic in Rochester, Minnesota
  • Consultant in the Division of Clinical Microbiology

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Thank you…..I have no disclosures. By the way, in case you were wondering, I am the more mature person in the pictures on the previous slide and was formerly director of the Mayo Clinic Parasitology Lab. The youthful Dr. Pritt is currently director of that lab.

The optimum test utilization messages in this presentation are first, that cyclosporiasis is a diarrheal disease which occurs in foodborne outbreaks or is seen in travelers to endemic areas;  second, Cyclospora oocysts are best detected using modified acid-fast or modified safranin stains;  and thirdly, that use of the Parasitic Investigation of Stool Specimens Algorithm can aid in optimum test utilization.  The algorithm can be found on the MML website.

Cyclospora cayetanensis is a single-celled coccidian parasite of the subphylum Apicomplexa, and is genetically related to Cryptosporidium and Isospora, which actually is now known as Cystoisospora. Cyclospora causes the diarrheal disease cyclosporiasis, which is endemic in tropical and subtropical regions but also occurs as sporadic foodborne outbreaks in temperate climates such as the United States. The infection is transmitted by ingestion of sporulated oocysts in food or water contaminated by feces.

This life cycle slide is from the CDC dpd website and illustrates the fecal-oral pattern of transmission. It should be noted that unsporulated oocysts excreted in the stool must undergo sporulation in the environment in order for the infection to be transmitted by oral ingestion of contaminated food or water.

Cyclospora was first recognized as a significant cause of gastroenteritis in Lima Peru in 1989 by microbiologists at the University Peruana Cayetano and ultimately the species name cayetanensis was assigned in recognition of that work. The morphology and staining characteristics were similar to those of Cryptosporidium but the new organism was larger (8-10 microns in diameter vs 4-5 microns for Cryptosporidium). The new organism was initially thought to possibly be a larger form of species of Cryptosporidium and the term “Crypto Grand” was coined. Subsequently, it was described as a CLB, an acronym for coccidian- or cyano-like body. However, finally in 1992, Ortega et al characterized the organism as a new species of Cyclospora.

Cyclosporiasis is endemic in tropical and subtropical countries in Latin America and Asia and occurs in travelers to those areas. Since 1990, foodborne outbreaks have occurred in the United States related to fruits (esp raspberries) and vegetables produced in Latin America and the United States. The recent outbreak in the United States which began in June of 2013, has been traced to commercially packaged salad mix from Mexico. As of 8/21/2013, the CDC has reported 601 ill persons from 22 different states.  In actuality, not all the cases have been linked to the salad mix, but the outbreak is waning since the product has been withdrawn from the market.

The incubation period averages 7 days with a range of 2 to 14 days. The predominant symptom is watery diarrhea which may be persistent or have a remitting/relapsing course. Other symptoms include anorexia, abdominal pain, nausea and vomiting, myalgias, low-grade fever, and fatigue.  Sulfamethoxazole–trimethoprim is effective treatment – but symptoms may persist for months, especially in the elderly or those with comorbidities.

For the laboratory detection of Cyclospora oocysts, stools should be processed as soon after collection as possible. If specimens cannot be processed within 1 to 2 hours after collection, they should be placed in an appropriate fixative such as formalin or Ecofix. Specimens should be concentrated before being studied and at least 3 specimens should be examined before a negative report is given.

Oocysts may be seen on wet-mount microscopy as part of the routine ova and parasite exam. However, this is not the most sensitive method for their detection. If Cyclospora oocysts are suspected on the wet mount, their identification must be confirmed using specific stains.

This slide illustrates the appearance of Cyclospora oocysts using wet-mount microscopy. The size, consistent oval or round shape, and visible outer wall suggest the identification.

The Parasitic Investigation of Stool Specimens Algorithm, which can be found on the Mayo Medical Laboratories website , may help users determine the most appropriate testing for intestinal parasites. I will not review the entire algorithm here, but I do want to point out the pale blue box in the lower left hand corner which indicates the special stains needed for identification of Cyclospora, as well as for Microsporidia.

Cyclospora oocysts are best detected using a modified acid-fast or modified safranin stain. Using the modified acid-fast stain, oocysts are round, 8 to 10 microns in diameter, and pink to purple in color. Alternatively, the oocyst interior may not stain well, resulting in a “ghost cell” appearance. Using the modified safranin stain, the color is red to orange. However, depending on the exact method used, there may be many “ghost cells” here as well. Staining methods are described at the CDC dpd website which is indicated at the bottom of this slide.

This slide illustrates Cyclospora oocysts stained by the modified acid-fast method. Both well-stained and ghost cells are seen. The consistent round shape is apparent and the approximate 10-micron size can be determined from the bottom right hand scale.

The next slide also illustrates well-stained oocysts and ghost cells with the modified acid-fast method.

The next slide shows the reddish-orange color obtained with a well done modified safranin stain. The ocular micrometer is useful in determining the size of the oocysts.

Cyclospora oocysts may also be detected using differential interference contrast microscopy or UV fluorescence microscopy. With wet-mount/UV microscopy, oocysts appear round and refractile and fluoresce either blue or green depending on the exact method used. The methods for staining oocysts are described in A Practical Guidance Document to the Detection of Cyclospora, which has been prepared by the American Society for Microbiology Committee on Laboratory Practices, and which can be viewed at the ASM website, asm.org.

This slide illustrates the appearance of a Cyclospora oocyst using wet-mount/UV fluorescence microscopy and is from the CDC dpd website.

The key points from this presentation are that: Cyclosporiasis should be suspected in patients with diarrhea related to foodborne outbreaks and in those who have traveled to endemic areas; that the routine ova and parasite examination is not the most sensitive method for detecting Cyclospora oocysts and the orders process should indicate this to physicians; and, finally that Cyclospora oocysts  are best detected by microscopy of stool concentrates using the modified acid-fast or modified safranin stains.

The final slide lists a few references which may be of some interest to those wishing further information on this topic.

Thank you for your attention.


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