Abstract
Cyclosporiasis is an emerging disease caused by Cyclospora cayetanensis, which induces protracting and relapsing gastroenteritis and has been linked to huge and complicated travel- and food-related outbreaks worldwide. Cyclosporiasis has become more common in both developing and developed countries as a result of increased global travel and the globalization of the human food supply. It is not just a burden on individual human health but also a worldwide public health problem. As a pathogen of interest, the molecular biological characteristics of C. cayetanensis have advanced significantly over the last few decades. However, only one FDA-approved molecular platform has been commercially used in the investigation of cyclosporiasis outbreaks. More potential molecular markers and genotyping of C. cayetanensis in samples based on the polymorphic region of the whole genomes might differentiate between separate case clusters and would be useful in tracing back investigations, especially during cyclosporiasis outbreak investigations. Considering that there is no effective vaccine for cyclosporosis, epidemiological investigation using effective tools is crucial for controlling cyclosporiasis by source tracking. Therefore, more and more epidemiological investigative studies for human cyclosporiasis should be promoted around the world to get a deeper understanding of its characteristics as well as management. This review focuses on major cyclosporiasis outbreaks and potential molecular markers for tracing back investigations into cyclosporiasis outbreaks.
Introduction
Cyclosporiasis is an emerging intestinal disease caused by a coccidian parasite, Cyclospora cayetanensis, that generally causes prolonged diarrhea, nausea, lethargy, cramps, and anorexia, among other symptoms (Giangaspero and Gasser, 2019; Ortega and Sanchez, 2010; Ramezanzadeh et al, 2022). C. cayetanensis has been found in more than 56 countries around the world, largely isolating it from tropical or subtropical countries (Li et al, 2020b). There have been many cyclosporiasis outbreaks related to travel and food infections, and it is not only a concern for individual human health but also a global public health issue (Li et al, 2020b; Ortega and Sanchez, 2010).
Cyclosporiasis is most prevalent in children with diarrhea in endemic areas (Li et al, 2020b). Cyclosporiasis is a major health concern for people in underdeveloped or developing countries where sanitation is poor and population density is high (Chacín-Bonilla, 2010; Li et al, 2020b; Pandey et al, 2011). In addition, many large cyclosporiasis outbreaks have been documented in industrialized nations (Ortega and Sanchez, 2010). The most recent large food-borne outbreaks occurred in 2018 and 2020 (Casillas et al, 2018; Hadjilouka and Tsaltas, 2020).
The fecal-oral (hand-to-mouth) route is the primary (or only) mode of transmission of C. cayetanensis infections, and it is invariably via fresh produce vehicles such as salad, basil, berries, cilantro, and some others (Dubey et al, 2022; Li et al, 2022). Furthermore, it was found that there was a marked seasonal distribution (rainy or summer season) of human C. cayetanensis infections, most notably in Nepal (Bhandari et al, 2015), China (Jiang et al, 2018; Zhou et al, 2011), Turkey (Ozdamar et al, 2010), Mexico (Orozco-Mosqueda et al, 2014), Honduras (Kaminsky et al, 2016), Columbia (Frickmann et al, 2021), Venezuela (Chacin-Bonilla et al, 2022), and elsewhere. Cyclosporiasis has varying seasonality worldwide, which may affect oocyst sporulation by precipitation, temperature, and humidity (Almeria et al, 2019).
Epidemiological tracing back investigation plays an important role in the control of epidemic cyclosporiasis. In the case of C. cayetanensis infection, ingestion of oocyst-contaminated water and food products is the main driver (Almeria et al, 2019; Li et al, 2020b). Molecular identification of the vehicles of infection, which could be fresh food, water, or soil, is critical in tracing back investigations and aids in source tracing in cyclosporiasis outbreaks (Li et al, 2020a). Several molecular methods for detecting C. cayetanensis have been developed in recent studies. This review focuses on cyclosporiasis outbreaks and potential molecular markers for their tracing-back investigations.
Major Cyclosporiasis Outbreaks
Travel-related outbreaks
C. cayetanensis has been implicated as a cause of traveler's diarrhea (Ortega and Sanchez, 2010). The first documented outbreak of cyclosporiasis (the causal agent known as an “alga-like organism” at that time) was recorded among 55 British expatriates in Nepal who suffered from chronic diarrhea between June and November 1989 (Shlim et al, 1991). Since then, there have been some outbreaks in developed countries, with travel to developing countries (areas) being one of the key risk factors for infections (Table 1).
Travel-Related Outbreaks of Cyclosporiasis
Thus far, the most significant and well-documented cyclosporiasis outbreak has been recorded in Australia, which occurred on a cruise ship in 2010 (Gibbs et al, 2013). A total of 314 persons were sick, including 266 travelers and 48 crew members, although the number of infected persons was likely much higher because no thorough examination was performed. During the tracing back investigations, it was revealed that fresh vegetables bought in Singapore or Malaysia were a possible source of C. cayetanensis in this outbreak (Gibbs et al, 2013).
Another large-scale travel-related cyclosporiasis outbreak occurred almost concurrently in the United Kingdom and Canada (Nichols et al, 2015). A total of 176 cases (79 in the United Kingdom and 97 in Canada) occurred from June to September 2015 after returning from Mexico. Epidemiological investigations revealed that the outbreak was caused by a contaminated foodstuff that was marketed throughout Mexico's Riviera Maya area (Nichols et al, 2015). Early communication and international collaboration are critical for gaining a better understanding of the source and scale of this recurring issue (Marques et al, 2017).
The risk of travel-related cyclosporiasis outbreaks is mostly associated with people traveling to or returning from developing countries (areas) such as Nepal, Indonesia, Guatemala, Peru, Mexico, and so on, which could be considered as cyclosporiasis endemic countries (areas) (Table 1).
Food-borne outbreaks
Cyclosporiasis outbreaks have been reported in the United States, Canada, and Europe since the mid-1990s, with fresh produce, including raspberries, lettuce, basil, mesclun, cilantro, green onions, and snow peas, being the most common sources (Table 2). So far, the largest cyclosporiasis outbreaks were observed in the United States and Canada, with over 1400 cases of cyclosporiasis reported between May and August 1996 (Herwaldt et al, 1997).
Food-Borne Outbreaks of Cyclosporiasis
The most recent large food-borne cyclosporiasis outbreaks have been documented in 2018 and 2020, respectively (Casillas et al, 2018; Hadjilouka and Tsaltas, 2020). The outbreak season (June to July) was notable for large outbreaks in the United States in 2018, resulting in 761 laboratory-confirmed cases (Casillas et al, 2018). Tracing back investigations indicated that the outbreak was associated with prepackaged fresh vegetable trays and salads sold at convenience store chain (Casillas et al, 2018). In the case of the 2020 cyclosporiasis outbreak in the United States, 690 people from 13 states had Cyclospora infection confirmed in the laboratory, and fresh express salad items were shown to be the most probable source of contamination (Hadjilouka and Tsaltas, 2020).
Until now, human infections with C. cayetanensis have been documented in more than 56 countries around the world, including all five human-inhabited continents (Li et al, 2020b). Despite the fact that some water-borne cyclosporiasis outbreaks have also been documented (Huang et al, 1995; Rabold et al, 1994), C. cayetanensis is the protozoan thought to be responsible for the majority of transmission via the food-borne pathway (Beshearse et al, 2021) (Table 2).
Tracing Back Investigations
Many reports showed that epidemiological and tracing back investigations were used during cyclosporiasis outbreaks to identify potential sources of infection and modes of transmission. Case-control studies demonstrated that intake of raspberries was substantially linked with sickness in cyclosporiasis outbreaks in the United States and Canada during 1996–1997 (Herwaldt and Ackers, 1997).
During the cyclosporiasis outbreaks in the United States (Pennsylvania) in 2000, stool examinations revealed the presence of C. cayetanensis oocysts, and dietary items were investigated to determine the source of infection (Ho et al, 2002). The wedding cake, which had a creamy raspberry filling, was substantially linked to the sickness, according to multivariate analysis. The remnants were then tested for C. cayetanensis DNA using a polymerase chain reaction (PCR). Finally, it was found that raspberries imported from Guatemala were associated with this cyclosporiasis outbreak (Hadjilouka and Tsaltas, 2020). In the United States in 2013, a mixed salad of romaine lettuce, iceberg lettuce, carrots, and red cabbage served in restaurants was linked to the cyclosporiasis outbreak in Iowa and Nebraska. The tracing back investigation showed that fresh cilantro originated from Puebla, Mexico, was related to the disease in case-control studies (Abanyie et al, 2015).
During the 2017 cyclosporiasis outbreaks in the United States, case-control study revealed that green onions were the only dietary item significantly linked to the disease. A tracing back investigation was conducted in the outbreak-related restaurant to identify the source of the concerned food; however, the inspections were unable to determine the source of the green onions (Keaton et al, 2018).
According to another tracing back investigation, the cyclosporiasis outbreak in the United States during the 2018 breakout season (June to July) was linked to packed vegetable baskets and salads sold at a supermarket chain (Casillas et al, 2018). During the 2019 cyclosporiasis outbreaks in the United States, the CDC determined that consumption of contaminated fresh basil was the most probable cause of the outbreak, while the FDA determined that the fresh basil was exported to the United States by Morelos, Mexico (CDC, 2019).
Thus, a routine workflow for the extraction and recovery of molecular sequences from contaminated clinical, food, and even environmental samples is required, which would facilitate identification for source-tracking and case linking (Gopinath et al, 2018). During the cyclosporiasis outbreaks, questionnaires to interview sick people are necessary for the tracing back investigations. For the laboratory confirmed, it could be identified by microscopical observations (light microscopy/ultraviolet [UV] epifluorescence microscopy) or by several molecular tools that are being continuously developed (e.g., PCR, quantitative PCR [qPCR] and multilocus sequence typing [MLST]) (Hadjilouka and Tsaltas, 2020). So far, the only commercially available molecular multiplex panel that includes a Cyclospora target is the FDA-approved BioFire FilmArray Gastrointestinal Panel (FilmArray GI). Because of the sensitivity and accuracy of PCR, the use of FilmArray GI likely increases the identification of Cyclospora infections, as demonstrated in a large Cyclospora outbreak in Wisconsin in 2018 (Bateman et al, 2020).
Potential Molecular Markers
Single locus molecular makers
A traditional Ova and Parasite (O&P) examination was the standard laboratory method for diagnosing Cyclospora infection. Even today, modified acid fast staining and UV fluorescence microscopy are useful diagnostic methods (Li et al, 2020a). The first FDA-approved gastrointestinal PCR panel with a Cyclospora target became commercially available in 2014, which is more sensitive than traditional O&P examination and has the potential to improve case detection (Casillas et al, 2018).
The lack of reliable and accurate C. cayetanensis typing tools has hindered outbreak investigations. Despite this, several possible C. cayetanensis detection and genotyping markers have been established and effectively used in cyclosporiasis epidemiological trace-back investigations (Table 3). The availability of sequence data might help researchers better understand C. cayetanensis biology and develop diagnostic markers for cyclosporiasis outbreak investigations (Ogedengbe et al, 2015; Tang et al, 2015). When epidemiological data for case linkage and source tracking are lacking, targeted amplicon sequencing and molecular genotyping are useful techniques for outbreak investigations (Li et al, 2020b).
Cyclospora cayetanensis Genotyping and Source Tracking Markers
cnPCR, conventional PCR; HSP70, 70 kda heat shock protein; ITS, internal transcribed spacer; MLST, multilocus sequence typing; mt, mitochondrial; nPCR, nested-PCR; PCR, polymerase chain reaction; qPCR, quantitative PCR; SNP, single nucleotide polymorphism; SSU rRNA, small subunit ribosomal RNA.
Over the last few decades, several molecular markers for C. cayetanensis have been developed. The small subunit ribosomal RNA (seven small subunit [SSU] rRNA) gene has been frequently used for molecular characterization of C. cayetanensis. For the identification of C. cayetanensis, many conventional, nested, and qPCR tests as well as multiplex PCR assays (along with other parasites) based on targeting the SSU rRNA gene have been established (Almeria et al, 2019; Li et al, 2020b; Relman et al, 1996; Taniuchi et al, 2011; Varma et al, 2003). The SSU rRNA gene sequences revealed that C. cayetanensis isolates from all around the world have very little genetic variability compared with other molecular loci (Sulaiman et al, 2014).
The internal transcribed spacer (ITS) sequences of C. cayetanensis are extremely variable both within and across isolates (Olivier et al, 2001). These sequences might be numerous clones from a single clinical source, or they could be variations in the ITS region across various copies of the rRNA gene unit (Adam et al, 2000). There was no genetic variation found in the 70 kda heat shock protein (HSP70) locus (Sulaiman et al, 2013), confirming the presence of a genetically homogenous population of C. cayetanensis at this genetic locus (Sulaiman et al, 2014). Recently, a diagnostic technique based on multiplex real-time PCR and a T4 phage internal control has been developed that can concurrently identify Giardia lamblia, Cryptosporidium parvum, and C. cayetanensis (targeting the ITS-1) in human stool samples (Shin et al, 2018).
The resolution of single molecular marker-based technique for case linkage and source tracing of cyclosporiasis is relative low. More recently, PCR or qPCR based on polymorphism in the mitochondrial (mt), apicoplast, and nuclear genomes has been established for C. cayetanensis identification (Bednarska et al, 2015; Cinar et al, 2020; Guo et al, 2019; Houghton et al, 2020).
mt molecular markers
C. cayetanensis mt genome was found to be 6274 bp long by the Illumina MiSeq platform, and the mt genome structure was validated by PCR amplification and sequencing (Cinar et al, 2015). The mt genome of C. cayetanensis contains three protein-coding genes, including cytb, cox1, and cox3, as well as 12 large subunit and SSU fragmented rRNA genes, all of which are highly conserved with those found in Eimera spp. (Cinar et al, 2015; Ogedengbe et al, 2015). The mt genome of C. cayetanensis appears to exist as a linear monomeric genome (Cinar et al, 2015; Ogedengbe et al, 2015).
Many molecular markers targeting polymorphic regions of the mt genome of C. cayetanensis has been developed to assess the genetic heterogeneity. A qPCR targeting the mt polymorphic link region (∼357 bp) between copies of C. cayetanensis has been developed and tested on 36 specimens from six countries, yielding nine genotypes (Guo et al, 2019). Among the investigated samples, geographical segregation of genotypes was identified, which might be beneficial for tracking geographic sources. In another study, the mt junction area (≈200 bp) was tested as a possible genotyping marker in stool samples from 134 laboratory-confirmed cases in the United States, which were classified into 14 sequence types (Nascimento et al, 2019).
DNA sequencing of the PCR products might offer genotyping, case clustering, and geographical source-tracking of the isolates, which could be useful during cyclosporiasis outbreak investigations (Guo et al, 2019; Nascimento et al, 2019). A large number of epidemiological linkages have confirmed the relationships, implying that the technique could be useful in a number of cyclosporiasis outbreak investigations such as trace-back, case-linkage, source-tracking, and case-clustering investigations (Barratt et al, 2019; Guo et al, 2019; Nascimento et al, 2019).
For genotyping of C. cayetanensis, a method was developed to acquire full mt genome (6.3-kb) sequences by amplifying four overlapping amplicons. According to the findings, genomic study of mt genome sequences might aid in tracing epidemic cases back to their source (Cinar et al, 2020). Meanwhile, a hybrid reference-guided de novo assembly technique for C. cayetanensis mt genomes was developed, which proved beneficial in expanding the number of C. cayetanensis mt genomes available from dietary or environmental materials (Gopinath et al, 2018).
MLST tools
MLST analysis provides a higher resolution technique for linking cases and finding clusters of cyclosporiasis outbreaks compared to single-locus genotyping. A MLST tool for C. cayetanensis involving five microsatellite loci (CYC3, CYC13, CYC15, CYC21, CYC22) has been developed by searching the whole genome (Guo et al, 2016). The MLST analysis revealed significant geographic clustering in C. cayetanensis isolates from humans worldwide, with a total of 54 different multilocus sequence types (Li et al, 2017).
In another recent study, four potential molecular genotyping markers (CDS-1, CDS-2, CDS-3, and CDS-4) from C. cayetanensis whole genomes were chosen for PCR and Sanger sequencing to assess their genotyping usefulness (Houghton et al, 2020). These four markers encompassed 13 single nucleotide polymorphisms (SNPs) and were effectively amplified in 57 of the 93 positive stool samples from humans, resulting in 19 distinct genotypes (Houghton et al, 2020). This might offer an extra resolution needed for an effective technique to help in epidemic investigations.
MLST analysis of C. cayetanensis results in mostly unreadable sequences due to repeated sequences containing a large number of small satellites and microsatellites (Hofstetter et al, 2019). It may be difficult to substantially improve the performance of the MLST method because of the nucleotide repeat features of the markers along with the frequent occurrence of mixed genotypes in Cyclospora infections (Hofstetter et al, 2019).
Three SNP-rich loci from C. cayetanensis genomes, one mt locus and two nu loci, with amplicon sizes smaller than 1 kb and capturing between 4 and 20 SNPs, were chosen for genotyping assessment, which grouped the 88 genotyped specimens into 16 genetic clusters using the ensemble method (Barratt et al, 2019).
To offer greater resolution for C. cayetanensis genotyping, a small panel of markers has been proposed to be utilized in combination with previously reported panels. This might be more useful and have a greater resolution for case linkage and source monitoring of cyclosporiasis outbreaks in the laboratory detection. Unfortunately, no substantiated molecular typing techniques are currently available to help effectively link cases to one another, food vehicles, or sources. As a result, many of the cases could not be connected to the cyclosporiasis outbreak directly. Great efforts should be done for developing more reliable and accurate C. cayetanensis typing tools for tracing back investigations.
Treatment
Trimethoprim sulfamethoxazole (TMP-SMX, also known as cotrimoxazole) chemotherapy twice daily for seven days was reported to cure human cyclosporiasis (Escobedo et al, 2009; Hoge et al, 1995). In some cases where TMP-SMX causes intolerance and allergy, ciprofloxacin antibiotic with having less effectivity than TMP-SMX is a suitable treatment option for human cyclosporiasis (Verdier et al, 2000). When sulfonamide intolerance and ciprofloxacin resistance occur, another drug called nitazoxanide can be used to treat cyclosporiasis (Cohen, 2005; Diaz et al, 2003; Zimmer et al, 2007). More research into treatment methods is being conducted; in a recent study in mice, silver nanoparticles were effective against Cyclospora infection (Gaafar et al, 2019). These have the potential to be used as an alternative to standard cyclosporiasis treatment.
Conclusions
There have been many human cyclosporiasis outbreaks related to travel and food infections, and cyclosporiasis is not only a concern for individual human health but also a global public health issue. Epidemiological source tracking investigations are important for controlling epidemic diseases such as cyclosporiasis. Several promising molecular markers have been developed and are being used in cyclosporiasis outbreak investigations, case linkage, and source tracking. Single-locus makers, mt markers, and MLST markers derived from mt, apicoplast, and nuclear genomes are among the molecular markers that are used to analyze genetic variability in C. cayetanensis isolates, as well as case linking and source-tracking. When an appropriate typing approach for C. cayetanensis is available, epidemiological case linking and source monitoring of cyclosporiasis outbreaks may be triggered, allowing for early response, which is important for managing any disease outbreak.
Footnotes
Authors' Contributions
L.Z.: Conceptualization and reviewing and editing. J.L.: Writing—original draft, formal analysis, and reviewing and editing. F.X.: Methodology; software, writing, and reviewing and editing. M.R.K.: Writing—original draft (partial) and reviewing and editing. The published version of the article has been reviewed and approved by all authors.
Disclosure Statement
No competing financial interests exist.
Funding Information
This research was funded by the National Natural Science Foundation of China (32102689), the Henan Postdoctoral Scientific Research Initiation Project (282851), the Outstanding Talents of Henan Agricultural University (30501055), and the Leading talents of the Thousand Talents Program of Central China (19CZ0122).