Investigation of an Outbreak of Cyclosporiasis in the United States: Laboratory Methods
CDC recommends the following process for confirmatory identification of Cyclospora in stool specimens:
CDC recommends laboratories obtain confirmation of cases using telediagnosis. Images may be captured from modified acid-fast stained smears or wet mounts examined by UV fluorescence. Both techniques require concentrated stool specimens. Telediagnosis will allow for rapid laboratory confirmation. Instructions for state health laboratories are found on DPDx.
It is recommended that concentration procedures be used prior to microscopic examination. The concentrate can be examined using two preferred techniques: the modified acid-fast stain or UV autofluorescence. See DPDx for procedural details.
- Modified Acid-fast Stains: In modified acid-fast stained specimens, Cyclospora oocysts retain their size, but frequently are not perfectly round. In addition, the oocyst wall is less apparent and has a wrinkled appearance, and appears collapsed or distorted on one side. The oocysts are variably stained with the modified acid-fast stain, with different oocysts in the same stained preparation ranging from colorless to deep purple. This variability in staining can lead to misidentification and is a limitation of the procedure.
- UV Fluorescence Microscopy: Cyclospora oocysts exhibit intense blue color when observed under a fluorescence microscope (UV excitation filter set at 330-365 nm). If this filter set is not available, a less intense green fluorescence can be obtained with blue excitation (450-490 nm). Because this fluorescence is very evident, UV fluorescence microscopy is a sensitive technique for rapidly examining stool sediments.
Cyclospora cannot be identified to the species level using microscopy; molecular methods must be used. At CDC a nested polymerase chain reaction (Nested-PCR) targeting the 18SrRNA gene is the method of choice for Cyclospora species identification using DNA extracted from human stool specimens. This method has been used by CDC to examine clinical specimens and food samples in different settings and during outbreak investigations. No PCR tests for Cyclospora have been approved by FDA for in vitro diagnostic use, but PCR is available at CDC and some public health laboratories.
For more information about the diagnostic procedures for cyclosporiasis, please visit DPDx.