Laboratory Studies
Stool examination
Stool examination for oocysts is the standard procedure for diagnosing C cayetanensis. Other specimens that may contain oocysts include intestinal aspirates and duodenal or jejunal biopsy samples. Unusual specimens include bile and pulmonary samples (eg, induced sputum, bronchial washings, biopsy) based on the growing reports of complicated cyclosporiasis.
Three specimens should be submitted for analysis, preferably on alternate days. The number of oocysts in the stool can vary considerably, but infected individuals with symptoms continuously excrete oocysts.
Standard laboratory procedures for ova and parasites do not identify Cyclospora; therefore, the laboratory must be notified that Cyclospora is a specific consideration.
Stool specimens are submitted to the laboratory as a fresh specimen or in some type of preservative. (Many laboratories require that specimens be submitted in a preservative.)
The stool specimen is recommended to be concentrated using the formalin–ethyl acetate technique, which leads to greater recovery of oocysts.
An important detail is that the specimen be centrifuged at 500 g for 10 minutes; if the speed is reduced, recovery of Cyclospora will be compromised.
Ethyl acetate has been substituted for ether because ethyl acetate is an excellent debris extractor and a much safer agent (ie, less explosive).
Microscopy
Cyclospora oocysts are difficult to identify with microscopic examination (high dry, 400X) without special techniques: acid-fast staining (fresh or preserved specimen), safranin staining (fresh or preserved specimen), direct wet smear (no preservative, no stain) using fluorescent microscope, direct wet smear with a differential interference contrast microscope (bright field), or lacto-phenol cotton blue staining (fresh specimen). Each of these techniques is discussed below.
If an unconcentrated wet mount is prepared, the entire cover-slipped area should be scanned. Concentration of the organism on the wet mount can be effected by adding a few drops of Sheather's sucrose solution to the wet mount; the oocysts float to the surface and, reportedly, appear faint pink.
If the stool specimen has been concentrated (not wet mount concentration), the recommendation is to scan 300 fields. A single negative specimen does not rule out the diagnosis.
When viewing coccidia microscopically, calibration of the microscope's ocular micrometer for measuring is important, because size matters when differentiating Cyclospora (8-10 µm) and cryptosporidium (4-5 µm).
In circumstances in which a confirmatory step is required to verify that the oocysts are indeed those of Cyclospora, a sporulation assay can be conducted using freshly passed oocysts. See the CDC for details.
Monoclonal antibodies
In 2005, monoclonal antibodies were not yet commercially available.
Acid-fast and safranin staining
Photomicrograph of a fresh stool sample prepared with a 10% formalin solution and stained with modified acid-fast stain. This image shows four Cyclospora cayetanensis oocysts with variable staining among the four oocysts. Courtesy of CDC/DPDx - Melanie Moser [https://phil.cdc.gov/Details.aspx?pid=7827].
C cayetanensis is an acid-fast organism, but the oocyst stains very unevenly with acid-fast stains (no staining, to mixed or variable staining, to full staining), complicating identification.
A modified acid-fast stain (less intense decolorizing) may be attempted using Kinyoun acid-fast stain (cold method) or Ziehl-Neelsen acid-fast stain (hot method). With the hot method, the stained material on the slide is heated to steaming (not boiling) and allowed to stand for 5 minutes before proceeding. With the cold method, heat is not applied.
The cold method is preferred because the hot method is not thought to have a significant advantage of improved staining.
Photomicrograph of a fresh stool sample prepared using a 10% formalin solution and stained with safranin. Three homogenously stained Cyclospora cayetanensis oocysts can be seen. Courtesy of CDC/DPDx - Melanie Moser [https://phil.cdc.gov/Details.aspx?pid=7828].
Safranin staining has demonstrated superior results, producing reddish orange staining of more than 98% of oocysts on a slide, when heating the safranin application was performed using a microwave (650 W) at full power for 30 or 60 seconds. The CDC recommends heating the slide to boiling for 1 minute. The heating produces a more uniform staining.
Combining fluorescence and differential interference contrast (DIC) microscopy
According to the CDC, these two techniques provide an efficient and reliable approach to the diagnosis when used together.
Wet preparations of Cyclospora, independent of specimen age or type, exhibit an intense color when viewed with a fluorescence microscope. With a UV excitation filter set at 330-365 nanometers, the color is an intense blue. With a filter set at 450-490 nanometers, the color is a less intense green.
Wet preparations of Cyclospora viewed with a DIC microscope (bright field) appear as refractile spheres (8-10 µm in size) with a distinct oocyst wall.
Lactol-phenol cotton blue (LPCB) staining
In laboratories where acid-fast stains are not routinely used, such as in rural areas and in developing countries, the LPCB wet mount is a suitable and practicable substitute with many advantages. It is a single stain that can be used to detect both coccidian and noncoccidian parasites, and it is simpler and less expensive than the acid-fast stain. [27, 28]
Polymerase chain reaction
PCR of Cyclospora stages isolated from stool or other specimens has been used for diagnosis. The protocol originally developed in 1996 by Relman, who was unable to differentiate Cyclospora species and Eimeria species.
This protocol was modified using a restriction enzyme Mn/I to distinguish the two species, and was designated a nested-PCR restriction length fragment polymorphism (RLFP).
The PCR-RLFP protocol is reliable for clinical purposes in detecting C cayetanensis in stool specimens but is unreliable when applied to environmental specimens because of the presence of genetically similar microorganisms. This has led to the development of PCR protocols that can reliably detect C cayetanensis in large numbers of environmental and clinical specimens (see below). The BioFire GI panel is a multiplex PCR and can detect a high percentage of GI infections, with one study noting a detection rate of 95%. [29]
Environmental and outbreak sampling
C cayetanensis produces an environmentally resistant oocyst that is infective in humans (see Pathophysiology). The US Environmental Protection Agency's Interim Enhanced Surface Water Treatment Rule (EPA-IESWTR) sets zero as the maximum contaminant level goal for Cryptosporidium and other pathogens in water.
In the late 1990s, several outbreaks of cyclosporiasis were attributed to food imported into the United States. These outbreaks generated the need to have techniques for sampling large numbers of environmental specimens, as well as large numbers of clinical specimens in order to prevent outbreaks before they occur and to better and more rapidly manage them when they do occur.
Emerging techniques
Three techniques that meet these criteria and are evolving include the new PCR protocols, flow cytometry, and electrorotation.
PCR
Newer PCR assays have been reported that can reliably screen large numbers of clinical and environmental specimens for C cayetanensis. New primers have been identified for C cayetanensis and used to modify existing protocols (an alternative nested-primer PCR-RFLP) or to develop new protocols. Such new protocols include single-nucleotide polymorphisms (SNP-PCR), which amplifies allelic sequences that differ by at least a single base pair; and real-time PCR, which uses the 5 prime-exonuclease activity of AmpliTaq Gold DNA polymerase to cleave C cayetanensis. Despite this, better environment detection methods are needed. [30]
PCR techniques are emerging that can enable detection of C cayetanensis on fresh produce (eg, raspberry, basil, cucumbers) via usage of various DNA isolation kits and having more than three different detection targets. [31] Detection methods for detecting C cayetanensis in soil and water are scant, with the FDA approving a validated detection method called Dead-End ultrafiltration (DEUF) in 2019 for water samples. [32] PCR sensitivity and specificty can also possibly decrease with natural PCR inhibitors found in foods such polyphenols in berries. [33] Commercial DNA cleanup kits can help decrease this via removing PCR inhibitors and eveb augmenting the process; one study that examined five different kits noted some kits had more efficacy than others, but this often depends on the substrate examined. [34]
A modified Relman-PCR protocol was used to determine the sensitivity of PCR in detecting C cayetanensis in experimentally spiked raspberries, basil, and mesclun lettuce. The protocol detected 40 or fewer oocysts per 100 g of raspberries or basil and 1,000 per 100 g of mesclun lettuce. This experiment demonstrated the importance of testing different the various PCR protocols on food to determine the detection sensitivity.
Flow cytometry
Flow cytometry has been used to accurately detect and enumerate various organisms in a prepared suspension (0.5 mL), including Cyclospora, by recording characteristic forward (size) and side (complexity) scattering of incident light emitted by an argon-ion laser operating at 488 nm. Scatter patterns are specific to the organism.
Electrorotation This technique (which is not commercially available) takes advantage of the unique patterns of rotation around their axis of biological organisms, including Cyclospora, secondary to the application of uniform rotating alternating current fields. These real-time and noninvasive measurements are performed on a microscale (ie, one cell at a time) and allow the identification of the organism and the determination of its viability. Electrorotation could potentially be used for rapid assessment of food and water.
Other procedures
Other procedures for the diagnosis of cyclosporiasis include demonstration of oocyst sporulation and polymerase chain reaction (PCR) of Cyclospora stages isolated from stool or duodenal or jejunal aspirates or biopsies.
No reliable immunological techniques are available to detect Cyclospora.
Histologic Findings
Duodenal and jejunal overall microscopic architecture is altered with mild to moderately severe villous atrophy (villous-to-crypt ratio reported 0.6-1.5:1 versus normal 3-4:1).
Total length of the villi is reduced. Villi are widened secondary to infiltration of both the surface epithelium and villous mucosa.
The surface epithelium is less organized, with loss of normal polarity, and is infiltrated extensively with lymphocytes, which displace the nuclei.
Neutrophils are often present.
Vessel congestion and dilatation are increased.
Focal vacuolization is present.
Loss of the brush border and altered cell shape from columnar to cuboidal is observed (more at tips of villi).
The villous mucosa is diffusely edematous and infiltrated with a mixed inflammatory cell infiltrate of plasma cells, lymphocytes, and, in some cases, eosinophils.
Mitotic activity in the crypts is increased.
The lamina propria is mildly to intensely inflamed.
All stages of the known life cycle of C cayetanensis have been observed in the enterocytes.
Electron microscopy (EM) demonstrates pronounced enterocyte vacuolization and abundant intraepithelial reactive cells. Predominantly in the apical areas, numerous intraenterocytic coccidial organisms are within parasitophorous vacuoles. The concentration of cells with organisms decreases progressively to zero in the crypts.
Follow-up studies of a few patients indicate that mild inflammation may persist (duration not known), manifested on light microscopy as focal vacuolization and gapping of enterocytes and mild increase in intraepithelial lymphocytes or other reactive cells. On EM, myelinlike material was uncommon. Its significance is unknown. Scarce and moderate amounts have been noted in protozoal infections of the upper gastrointestinal tract. It may be a marker of cell injury, but that is not substantiated.
The above discussion of histology is adapted from Connor (1993 [35] , 1999 [36] ) and Ortega (1997 [37] ).
-
Photomicrograph of a fresh stool sample prepared with a 10% formalin solution and stained with modified acid-fast stain. This image shows four Cyclospora cayetanensis oocysts with variable staining among the four oocysts. Courtesy of CDC/DPDx - Melanie Moser [https://phil.cdc.gov/Details.aspx?pid=7827].
-
Photomicrograph of a fresh stool sample prepared using a 10% formalin solution and stained with safranin. Three homogenously stained Cyclospora cayetanensis oocysts can be seen. Courtesy of CDC/DPDx - Melanie Moser [https://phil.cdc.gov/Details.aspx?pid=7828].
-
Life cycle of Cyclospora cayetanensis in pictorial form. Courtesy of CDC/DPDx [https://www.cdc.gov/parasites/cyclosporiasis/biology.html].
Tables
Pattern of Spread |
Countries |
Comments |
Endemic |
Bangladesh, Brazil, Chile, China, Cuba, Dominican Republic, Egypt, Guatemala, Haiti, India, Indonesia, Jordan, Mexico, Morocco, Nepal, Nigeria, Pakistan, Peru, Puerto Rico, Romania, Saudi Arabia, Tanzania, Thailand, Turkey, Venezuela, Viet Nam, Zimbabwe |
Prevalence (1-15%†) varies significantly with the season and from year to year; children (≤ 9 y, most studies) account for 70-80% cases, which are typically asymptomatic (72-94%); asymptomatic disease is higher in older children (10-18 y) and adults (>18 y); infection rate in those with HIV is significantly higher than overall prevalence |
International travel-related |
Australia, Belgium, Czech Republic, Germany, Greece, Ireland, Italy, Japan, The Netherlands, Spain, Switzerland, United Kingdom, United States |
≤4% returning travelers with diarrhea |
Foodborne outbreaks |
Canada, United States, Germany, Mexico |
Canada/United States: raspberries, blackberries, mesclun, basil‡; Germany: lettuce imported from Southern France/Southern Italy; Mexico: watercress |
Waterborne outbreaks |
United States (Chicago), Nepal |
14 cases of cyclosporiasis; tap water in medical dormitory, suspected source was contaminated water storage tank; 12 of 14 developed cyclosporiasis |
* Community-based studies † Highest in spring and early summer ‡ Fresh produce. Raspberries, peas, or blackberries from Guatemala; mesclun (young salad greens, eg, spring mix, field greens, baby greens, gourmet salad mix) or lettuce from Peru or United States; basil from Mexico or United States. |
||
