Enhanced concentration and isolation of Cyclospora cayetanensis oocysts from human fecal samples

Riner, Diana K.; Mullin, Andrew S.; Lucas, Sasha; Cross, John H.; Lindquist, Heather

doi:10.1016/j.mimet.2007.06.021

Cited by 11 publications

(9 citation statements)

References 12 publications

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“…A modified aqueous detachment solution containing 0.563 mM H 2 Na 2 P 2 O 7 and 42.8 mM NaCl followed by a discontinuous sucrose gradient with diatrizoate meglumine (Renocal) in 0.25 M sucrose yielded cleaner oocysts than did the use of sucrose gradients (160). In the event that the samples were preserved in SAF fixative, it is recommended that equal volumes of SAF-fixed samples and 10% KOH be subjected to vortex homogenization and centrifugation with 0.85% saline solution (12).…”

Section: Molecular Methodsmentioning

confidence: 99%

Update on Cyclospora cayetanensis , a Food-Borne and Waterborne Parasite

2010

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SUMMARY The coccidian parasite Cyclospora cayetanensis is recognized as an emerging pathogen that causes protracted diarrhea in humans. The first cases of Cyclospora infection were reported in the late 1970s and were observed among expatriates and travelers in regions where infections are endemic. Since then, Cyclospora has been considered a cause of traveler's diarrhea. Epidemiological investigations were reported and examined in areas of endemicity even before the true identity of Cyclospora was elucidated. Cyclospora was fully characterized in the early 1990s, but it was not until the 1995 Cyclospora outbreak in the United States and Canada that it caught the attention of the public and physicians. The biology, clinical presentation, epidemiology, diagnosis, treatment, and control of cyclosporiasis are reviewed, with a focus on diagnostic assays currently being used for clinical and environmental samples. Challenges and limitations in working with Cyclospora are also discussed.

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Section: Molecular Methodsmentioning

confidence: 99%

Update on Cyclospora cayetanensis , a Food-Borne and Waterborne Parasite

2010

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“…Purified oocysts from fecal samples are required for experimental seeding for the validation of detection techniques in produce, or for setting up other molecular sequencing and typing techniques directly from oocysts. Discontinuous percoll gradients [255] as well as a detachment solution, renocal-sucrose gradient sedimentation, and flow cytometry [256] have been used in the isolation of C. cayetanensis oocysts. A recent study used discontinuous density gradients in the presence of a detergent and flow cytometry to obtain highly purified oocysts for use in genomic studies [257].…”

Section: Diagnosismentioning

confidence: 99%

Cyclospora cayetanensis and Cyclosporiasis: An Update

2019

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Cyclospora cayetanensis is a coccidian parasite of humans, with a direct fecal–oral transmission cycle. It is globally distributed and an important cause of foodborne outbreaks of enteric disease in many developed countries, mostly associated with the consumption of contaminated fresh produce. Because oocysts are excreted unsporulated and need to sporulate in the environment, direct person-to-person transmission is unlikely. Infection by C. cayetanensis is remarkably seasonal worldwide, although it varies by geographical regions. Most susceptible populations are children, foreigners, and immunocompromised patients in endemic countries, while in industrialized countries, C. cayetanensis affects people of any age. The risk of infection in developed countries is associated with travel to endemic areas and the domestic consumption of contaminated food, mainly fresh produce imported from endemic regions. Water and soil contaminated with fecal matter may act as a vehicle of transmission for C. cayetanensis infection. The disease is self-limiting in most immunocompetent patients, but it may present as a severe, protracted or chronic diarrhea in some cases, and may colonize extra-intestinal organs in immunocompromised patients. Trimetoprim-sulfamethoxazole is the antibiotic of choice for the treatment of cyclosporiasis, but relapses may occur. Further research is needed to understand many unknown epidemiological aspects of this parasitic disease. Here, we summarize the biology, epidemiology, outbreaks, clinical symptoms, diagnosis, treatment, control and prevention of C. cayetanensis; additionally, we outline future research needs for this parasite.

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“…A previous study has concluded that a detachment solution (containing disodium pyrophosphate) can improve the recovery of C. cayetanensis from stool during density gradient purification compared to 0.01% Tween 20 [ 15 ]. Moreover, the addition of 0.1% of detergent Alconox in the wash solution can improve oocyst recovery from environmental samples [ 16 ].…”

Section: Discussionmentioning

confidence: 99%

Purification of Cyclospora cayetanensis oocysts obtained from human stool specimens for whole genome sequencing

et al. 2018

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BackgroundCyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. There is a need for efficient laboratory methods for strain-level characterization to assist in outbreak investigations. By using next generation sequencing, genomic sequences can be obtained and compared to identify potential genotyping markers. However, there is no method available to propagate this parasite in the laboratory. Therefore, genomic DNA must be extracted from oocysts purified from human stool. The objective of this study was to apply optimized methods to purify C. cayetanensis oocysts and extract DNA in order to obtain high-quality whole genome sequences with minimum contamination of DNA from other organisms.ResultsOocysts from 21 human stool specimens were separated from other stool components using discontinuous density gradient centrifugation and purified further by flow cytometry. Genomic DNA was used to construct Ovation Ultralow libraries for Illumina sequencing. MiSeq sequencing reads were taxonomically profiled for contamination, de novo assembled, and mapped to a draft genome available in GenBank to assess the quality of the resulting genomic sequences. Following all purification steps, the majority (81–99%) of sequencing reads were from C. cayetanensis. They could be assembled into draft genomes of around 45 MB in length with GC-content of 52%.ConclusionsDensity gradients performed in the presence of a detergent followed by flow cytometry sorting of oocysts yielded sufficient genomic DNA largely free from contamination and suitable for whole genome sequencing of C. cayetanensis. The methods described here will facilitate the accumulation of genomic sequences from various samples, which is a prerequisite for the development of typing tools to aid in outbreak investigations.

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Enhanced concentration and isolation of Cyclospora cayetanensis oocysts from human fecal samples

Cited by 11 publications

References 12 publications

Update on Cyclospora cayetanensis , a Food-Borne and Waterborne Parasite

Update on Cyclospora cayetanensis , a Food-Borne and Waterborne Parasite

Cyclospora cayetanensis and Cyclosporiasis: An Update

Purification of Cyclospora cayetanensis oocysts obtained from human stool specimens for whole genome sequencing

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