Table 1.

Common Cyclospora cayetanensis detection methods.

Methods	Characteristics	LoD (limit of detection)	References
Wet smears using light microscopy	Smears from fresh or concentrated feces; oocysts identified as 8- to 10-μm, spherical, refractile, with a central morula, and resembling wrinkled cellophane	Low, usually detection with other methods	Zhou et al., 2011; Becker et al., 2013
Modified acid-fast stain	A series of modified acid-fast staining methods, such as: ziehl-neelsen, Kinyoun's, carbolfuchsin, etc. Some oocysts stain in deep red, whereas others stain pink, or remain unstained against the blue-green background	Recommended for diagnosis of clinical samples	Brennan et al., 1996; Alakpa et al., 2002; Gonçalves et al., 2005; Dillingham et al., 2009
Modified safranin stain	Oocysts reddish orange stained, and the cyst wall more clearly with background	It is reported being superior to acid-fast stain, with fast, reliable, and easy to perform	Visvesvara et al., 1997
Lacto-phenol cotton blue (LPCB) stain	Oocysts stained in blue, and internal structures is clear	Recommended, if the acid-fast stain is not performed	Parija et al., 2003
Fluorescence microscopy	Oocysts autofluorescence; they appear blue when exposed to 365 nm UV light and looks green under 450–490 nm excitation	At least 2-fold over the direct wet mount	Ortega and Sterling, 1996; Berlin et al., 1998; McHardy et al., 2014
Flow cytometry	Oocysts morphology and auto fluorescence features, with higher automation	No differences with qPCR assay for oocyst detection and counts	Dixon et al., 2005; Li et al., 2014
Serological test- ELISA	No commercial serological assays are available	Specific IgG and IgM antibodies needed for oocysts	Wang et al., 2002
Nested PCR molecular detection	Specific PCR primers for small subunit rRNA or ITS regions	One to 10 oocysts	Relman et al., 1996; Olivier et al., 2001; Li et al., 2007*
PCR-RFLP molecular detection	Use of restriction enzyme AluI	As few as one oocyst in 10 liters water	Shields and Olson, 2003b
Quantitative PCR	Specific primers and probe	Estimate the DNA of 0.5 oocysts	Verweij et al., 2003
Quantitative PCR	Using the inherent genetic uniqueness of the 18S ribosomal gene sequence	As few as one oocyst per 5 μL reaction volume	Varma et al., 2003
Multiplex PCR	Simultaneous detection of Cyclospora cayetanensis, Cystoisospora belli, Enterocytozoon bieneusi, and Encephalitozoon intestinalis; detection of the amplicon is through specific probes coupled to Luminex beads	103 plasmid copies	Taniuchi et al., 2011
Multiplex PCR	Commercially available DNA-based technologies for stool specimens	Simultaneous detection of 22 different enteric pathogens	Buss et al., 2015; Hitchcock et al., 2019
PCR assays and qPCR	Polymorphic junction region in the mitochondrial genome for human stool samples	As few as one oocyst	Guo et al., 2019; Nascimento et al., 2019
Multiplex qPCR and T4 phage internal control	Simultaneous detection of Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis in human stool samples	20 copies: equivalently: 103 of oocysts	Shin et al., 2018
FDA validated qPCR technique	FDA validated technique used in fresh produce matrices and prepared dishes	As few as five oocysts	Murphy et al., 2017, 2018b; Almeria et al., 2018
Multiplex qPCR	Highly specific, precise, and robust method that has potential for application in food-testing on berries	~10 oocysts	Temesgen et al., 2019a
PCR assay targeting the ITS	Potential for standard use in food testing, particularly berry fruits	~6.4 pg: equivalent to DNA of one oocyst	Temesgen et al., 2019b
Multiplex PCR	Simultaneous detection of protozoan (oo)cysts (Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii) in leafy greens	1–10 oocysts/g spinach in 10 g samples processed	Shapiro et al., 2019

^*It was initially developed to analyze cattle samples, now it is widely used to analyze human samples.