Cyclospora and Cyclosporiasis: The Nepalese Perspective

Cyclosporiasis is an emerging disease that is ubiquitous in humans and other animals worldwide. Cyclospora cayetanensis , which is distributed in the tropical and subtropical regions, and endemic in several developing countries, is the only species of Cyclospora resulting in cyclosporiasis in humans. The main objective of this review was to summarize the prevalence of Cyclospora in humans, animals, and environmental samples in Nepal. The overall prevalence of Cyclospora in humans has been reported to be 10.3% (3,785/36,570), including 3.6% (60/1,640) among HIV immunocompromised cases, 2.1% (210/9,733) among children, and 13.9% (3,515/25,197) in humans of all age categories, while the prevalence was calculated to be 2.9% (29/983) and 10.5% (9/85) in domestic and wild animals, respectively. The molecular characterization data on Cyclospora as related to Nepal is limited to a very few positive isolates from humans, dogs, chickens, and monkeys. Exposure to sewage water and contaminated fresh food items, and the presence of domestic animals appear to have been associated to an outbreak of Cyclospora in humans. Considering Cyclospora spp. as the etiologic agent for diarrhoea, a comprehensive, wider scale molecular epidemiologic study in humans, animals, and environmental samples is warranted to elucidate the exact distribution loci and probable outbreak of cyclosporiasis in Nepal.


INTRODUCTION
Cyclospora spp.are important medical and veterinary zoonotic enteric protozoan parasites in humans and a wide range of animals.C. cayetanensis, an emerging food-and water-borne enteric pathogen, has a one-host fecal-oral transmission resulting from the ingestion of sporulated oocysts through contaminated food and/ or water [1].C. cayetanensis is endemic in tropical and subtropical regions [2,3], and the only human pathogenic species of the genus Cyclospora [4].The other species within the genus, Cyclospora, result in infections in a wide array of animals from arthropods to non-human primates [5][6][7].Based on various animal samples, including myriapodes, vipers, moles, rodents, monkeys, and humans, 22 Cyclospora spp.have been documented [8,9].
The first publication on human cyclosporiasis dates back to 1979 [10] as a case report.C. cayetanensis has gained attention in the wake of first outbreak of Cyclospora-associated diarrhoea in the US in 1990 [11]. C. cayetanensis infection is characterized by prolonged diarrhoea, malaise, nausea, cramping, and anorexia in humans [12,13].The typical watery diarrhea in cyclosporiasis may last for several weeks and even months, and it may manifest more severe symptoms in young children and immunocompromised persons [14].Among the high-risk groups of people contracting Cyclospora infection are those living with severe immunocompromised state like HIV/AIDS [15].C. cayetanensis is considered to be an opportunistic pathogen in humans [16], and it has been documented from at least 54 countries [9].
The diagnostic stage of Cyclospora spp. is the sporulated or non-sporulated cyst.Microscopy with an emphasis on morphology or autof luorescence of oocysts is the common method to detect C. cayetanensis.However, these methods based on parasite morphology differ in diagnostic performance with respect to specificity and sensitivity, and may lead to false-positive or -negative results [9,17].To overcome this limitation, several molecular methods, including real-time qPCR assay targeting the 70 kDa heatshock protein (HSP70) gene [18], 18S rDNA [19], SSU rRNA sequence analysis [1,20,21], multilocus sequence typing (MLST), and population genetic analysis based on several mini-and micro-satellites [22], and different molecular markers, such as single locus, mt and MLST markers [23], have been used to link and track the sources of contamination.
Most Cyclospora-related infections have been recorded in travellers from various endemic regions [24], including Nepal, and the consequence is traveller's diarrhoea.Several instances of cyclosporiasis among travellers to Nepal [25][26][27][28][29][30] might be attributed to consumption of contaminated food and/or water while in Nepal.Nepal is one of the endemic regions for cyclosporiasis.Some studies involving Cyclospora infections in Nepal have been published in local journals, which are less accessible to the global scientific community.This review was intended to summarize the overall prevalence of Cyclospora in humans, animals, and environmental samples, and to identify the epidemiologic risk factors, research gaps, and further research prospects involving Cyclospora in Nepal.

Search strategy
Relevant papers reporting the prevalence of Cyclospora infections in Nepal published in the English language until 31 July 2023 were searched using various databases (PubMed, Web of Science, ResearchGate, ScienceDirect, and Google Scholar) with the following search terms: "Cyclospora"; "cyclosporiasis"; " epidemiology"; " diagnosis"; "Nepal"; and combinations of the preceding terms.We extensively searched Nepal Journals Online (NepJOL) [https://www.nepjol.info] to identify articles published in local journals.In addition to the online databases, the reference lists of the published papers were manually searched for appropriate references.Information, including first author, year of publication, study location, study design, number of samples, human participant gender ratio, types of animals studied, environment samples, diagnostic methods, and prevalence of Cyclospora, were independently extracted by two authors.

Inclusion and exclusion criteria
Considering the lack of exclusive molecular prevalence studies involving Cyclospora in Nepal, full-length papers, short communications, abstracts based on cross-sectional studies, and clinical and cohort studies with explicit mention of the sampling location, sample size, detection methods, and positive results in humans, animals, or environment samples were included in the analysis (Fig 1).Review papers were not included in the analysis.

Study characteristics
Of the 39 papers included in this review, the highest number of studies on Cyclospora was conducted in humans (32 papers), especially in hospital-seeking patients and were case-control, seasonal prevalence, or cross-sectional studies.Thirteen studies were conducted exclusively among children < 15 years age, 12 studies involved humans of all ages, one study each was conducted exclusively on domestic (goats) and wild animals (Himalayan goral [Naemorhedus goral]), while the remaining studies were conducted in combination with human and environmental samples (soil, water, and fresh vegetables) and/or domestic or wild animals.Notably, no Cyclospora-related studies involving fresh fruit samples in Nepal have been reported.Moreover, an extensive study conducted exclusively with a large sample size on domestic or wild animals or environmental samples is not available.Nevertheless, it is clear that at least some attempts have been made to explore the general prevalence of Cyclospora among humans and their environments.Therefore, specific emphasis on a particular group of samples or a comprehensive study involving humans, domestic animals, wild animals, and the environmental interface need to be conducted to determine the prevalence, burden, and molecular epidemiology of Cyclospora spp. in Nepal.

Prevalence of Cyclospora infections in humans
Of the 36,570 human fecal samples analysed based on direct light microscopy of modified acid fast-stained preparations, phase contrast microscopy, differential interference contrast microscopy, UV-epif luorescence microscopy after concentration of oocysts by formalin ether or the Sheather's sucrose f loatation method, and enzyme immunoassay, 3,785 had Cyclospora oocysts (10.3%).The prevalence of C. cayetanensis in humans has been reported to be 3.6% (60/1,640) among HIV-immunocompromised patients, 2.1% (210/9,733) among children < 15 years of age, and 13.9% (3,515/25,197) among humans of all age groups (Table 1, Fig 2).C. cayetanensis is an opportunistic pathogen in humans [16,62] and is common in immunocompromised and immunocompetent individuals; however, C. cayetanensis is more severe in immunocompromised individuals, especially in HIV-infected patients [63].Cyclospora outbreaks in expatriates residing in developing countries, like Nepal, have been documented.Cyclosporiasis caused by C. cayetanensis, the so-called algalike organism, has been diagnosed among 55 British expatriates with prolonged diarrhoea in Nepal between June and November 1989 [64].With respect to the cause of diarrheal illnesses, coccidian-like organisms (CLOs) have been detected among travellers and foreign residents in Nepal [65].Scaglia et al. [25] reported co-infection with Cyclospora spp.and Cryptosporidium parvum in a 33-year-old AIDS patient with diarrhea who lived in Nepal between 1987 and 1989.Based on a cohort study conducted among expatriate adults with diarrhea who resided in Nepal for < 2 years, Cyclospora was identified as one of the causes [26].Cyclospora oocysts were isolated from British travellers who became ill in Nepal in 1989, and other travellers and foreign nationals who resided in Nepal in 1993 [27].C. cayetanensis was detected in a 39-year-old man who travelled from Tibet to Nepal with prolonged diarrhoea, abdominal discomfort, and significant weight loss [28].In a case-control study conducted in Nepal among visitors or residents from high socioeconomic countries, Cyclospora was detected in 8.1% (31/381) of the cases, especially during the monsoon season [29].Cyclospora was also detected in a female traveller who had been travelling around Nepal for approximately 4 months [66].In another study, 3 of 8 South Korean travellers (46-61 years of age) who returned from Nepal were diagnosed with C. cayetanensis infections based on nested PCR, followed by sequencing of an 18S rRNA gene [30].Such examples of Cyclospora infections among travellers associated with Nepal might be linked to the consumption of contaminated food and/or water, indicating that Nepal is one of the endemic regions for Cyclospora infection outbreaks.

Cyclospora spp. in domestic animals
Molecular tools have detected Cyclospora oocysts in the faeces of animals, such as dairy cattle [70], dogs, chickens, and rhesus monkeys [71].Studies have confirmed that living in the proximity of domestic animals or sharing a similar habitat may pose a risk of sporadic cyclosporiasis in endemic regions [69,72], as evidenced by the contiguity with chickens [31,55,59,72], other birds, guinea pigs, rabbits [73], and livestock [46].A human-animal nexus in the transmission of Cyclospora oocysts has been reported in Nepal (Fig 3).Because Cyclospora has been detected in some animals, the Cyclospora spp.identified in many domestic animals remains controversial owing to the lack of a sophisticated molecular approach for identification.This review has summarized the overall prevalence of Cyclospora infections in 2.9% (29/983) different domestic animals investigated in Nepal (Table 2).Among the domestic animals, chickens    [4].These animals and environmental agents, as mentioned in published papers incorporated in this review, and represented in this picture were investigated for Cyclospora oocysts.The presence of Cyclospora oocysts in the animal faeces does not indicate infection and cannot be considered the reservoir of C. cayetanensis; however, Cyclospora oocysts might act as paratenic hosts [14] to disseminate this pathogen to the surroundings where humans can be infected.A biopsy is the only means by which whether the animals are actually infected can be ascertained.
of Cyclospora are capable of infecting different animals.A microscopic examination is considered crucial for the diagnosis of most pathogenic enteric protozoa, including Cyclospora; however, microscopic examinations are labor-intensive and require trained and skilled human resources [75].

Cyclospora spp. in wild animals
Cyclospora oocysts have been identified in monkeys [54,59] and Himalayan goral [76] (Table 3) in Nepal.According to one review [14], some attempts have been successful in infecting oysters, Asian fresh water clams, Swiss albino mice, and guinea pigs through artificial inoculation of C. cayetanensis oocysts.Based on the appearance of oocysts in the faeces, it cannot be concluded that the animals are infected by Cyclospora.This finding simply implies that the parasite must have colonized the gut [77] and is released outside the animal without resulting in an infection.However, it should not be overlooked that animals can potentially contribute as paratenic hosts for dissemination of C. cayetanensis oocysts by mechanical spread, which results in contamination of water resources and food, leading to possible infection of humans [14].

Seasonality of cyclosporiasis
Cyclosporiasis caused by C. cayetanensis has a marked seasonality [20,78,79]; however, the seasonality effect is deferred in various regions, owing to anthropogenic habits, chances of environmental contamination, and optimal circumstances required for sporulation [80].The prevalence of Cyclospora in Nepal also has a seasonal pattern.A marked higher prevalence of Cyclospora infections has been observed in summer months (rainy season, 12.6%) with a sharp decline in the spring (dry season, 1.8%) and closely related to the amount of precipitation [57].Cyclospora contamination has been recorded in June and July in water samples of ponds and irrigation canals [59].In a study conducted among Nepali children (1 month to  15 years of age) to determine the seasonality of Cyclospora prevalence, oocysts were only detected during the monsoon season ( July and August) with a prevalence of 19.0% (37/195); however the prevalence was zero in the pre-monsoon season (April and May) [39].In another study, of 2,138 human faecal samples, a higher prevalence of Cyclospora (26.3%) was recorded in June and < 1.0% in winter months (December, January, and February) [54].
The monthly prevalence of Cyclospora was noted to be higher from May (3.6%) to July (12.09%) with a decline in October (3.8%) [55].Similarly, Ghimire et al. [56] noticed a rise in Cyclospora prevalence from May to July, followed by gradual decline until November 2002.The highest prevalence of Cyclospora was recorded in June 2003 and 2004.The summer/rainy season ( June and July) had the highest infection rate [59], which had an increasing trend from May to July and a gradual fall [56].The prevalence of Cyclospora infections peaked in June and July [53], June to August [57], and August [49].

Environmental risk factors
Surveillance studies in endemic areas have reported C. cayetanensis contamination in fresh produce [80].
Both food-producing and -importing countries have experienced an increased occurrence of cyclosporiasis due to the globalized human food supply [69,81].A review indicated that different types of human pathogenic enteric protozoan parasites (amoebae, microsporidia, and coccidia), including C. cayetanensis, have been isolated from various types of raw vegetables and fruits worldwide [82].
Cyclospora oocysts have been identified in sewage water and some vegetable items, such as cabbage, lettuce, and mustard leaves collected from various areas in Kathmandu valley [53].In several other parts involving Nepal, raddishes, caulif lower, cabbage, lettuce, spinach, mustard, and basil leaves were shown to contain Cyclospora oocysts [52,56,59] (Table 4).Cyclospora spp., along with C. cayetanensis, have been detected in different types of wastewater treatment plants [83], as well as inf luent and eff luent wastewater samples [84] in some developed countries.Therefore, drinking or irrigation water is potentially contaminated by the oocysts carried along with wastewater.Cyclosporiasis can even be attributed to exposure to recreational water contaminated with C. cayetanensis oocysts [85].In Nepal, water samples from irrigation canals and ponds have also been shown to contain Cyclospora oocysts, mainly in June and July [59].This finding indicates a health threat of contracting cyclosporiasis in the community by contaminated foods.
A study was conducted involving the Cyclospora prevalence among HIV patients in relation to the probable environmental risk factors in and around their residences [31].Ghimire et al. [31] identified Cyclospora oocysts from sewage, river water, soil samples, and human faecal samples collected from the streets and some domestic animals, like chicken and dogs.In a study conducted in rural settings in various parts of Nepal, the highest risk groups for Cyclospora infection were children and students [59].
Similarly, the higher prevalence in children 3-5 years of age (10.15% [13/128]) with a peak in August have been attributed to the presence of livestock and consuming fresh produce without proper washing [46].Exposure to the soil is another important means of transmission of C. cayetanensis, and thereby contributes to food contamination [86][87][88] because disposal of faeces is considered to be evidence of soil contamination.Therefore, sewage drainage, presence of domestic animals (mainly chickens) in the household, and contact with the soil, irrigation water, and some wild animals, like monkeys, could potentially be potential risk factors for transmission of cyclosporiasis (Fig 2).Indeed, studies tracking the sources and possible routes of transmission of Cyclospora to humans and animals need to be conducted in various endemic locations within the country.

Molecular characterization of Cyclospora spp.
A molecular analysis of Cyclosporia-positive faecal specimens of humans, dogs, chickens, and monkeys obtained from Nepal has been carried out in international laboratories (Table 5).Even though C. cayetanensis was confirmed in two dogs, and one each in chicken and monkey faecal samples, these animals cannot be designated as the natural reservoir hosts without evidence of biopsy analysis [71].In a sequence analysis of the HSP70  ▲ Specific location within Nepal not specified; ◊ A follow-up study for Sulaiman et al. [89] in which the 18S RNA locus of C. cayetanensis was characterized; # Analysis of stool samples supplied by the Microbiology and Public Health Research Laboratory, Tribhuvan University Teaching Hospital, Kathmandu, Nepal; ◙ Multilocus sequencing typing (MLST) data obtained in the previous study [22] were analysed.
two of which had complete multi-locus sequence typing (MLST) and were found to be genetically more aligned to the specimens under investigation from Peru and the United States [91].The number of human and animal faecal specimens and the diversity of animals included in the molecular characterization of Cyclospora spp.are very low in context to Nepal.It is worthwhile to get a broader consensus between the responsible authorities to elucidate the geographic distribution and molecular epidemiologic investigation of C. cayetanensis in humans and Cyclospora spp. in a wide array of domestic and wild animals.

PERSPECTIVES AND CONCLUSIONS
Nepal is facing a dearth of authentic data on the epidemiology and transmission dynamics of Cyclospora spp.owing to the lack of sufficient molecular characterization studies among humans, animals, and environmental samples.Extensive studies on clinical, epidemiologic, and molecular aspects of Cyclospora need to be carried out at the national level to configure the exact status on the basis of which effective prophylactic measures can be implemented against this ubiquitous enteric parasite.This review has synthesized and provided a glimpse of Cyclospora and its prevalence in humans, domestic animals, wild animals, and environmental samples in Nepal.The prevalence of Cyclospora infections could be much higher if specific and sensitive molecular techniques were used to carry out the diagnostic procedure.Contaminated sewage water, fresh produce (especially vegetables), exposure to contaminated soil, and the presence of domestic animals could be attributed to be the causes of cyclosporiasis outbreaks in humans.Consideration of Cyclospora as the cause of childhood diarrhea and implementation of routine diagnosis of this parasite for diarrheal stool samples in the hospitals and further molecular characterization could determine the epidemic loci of outbreaks and probable risk management.Comprehensive studies using molecular techniques under the support of public health authorities are necessary to provide significant information regarding the probable circulation of Cyclospora in humans, animals, and environmental samples.

LIMITATIONS
Because the molecular prevalence reports on Cyclospora in Nepal are limited, the data incorporated in this review were mainly obtained from studies carried out using microscopic methods.

FIGURE 1 |
FIGURE 1 | Flowchart of the publication screening.

FIGURE 2 |
FIGURE 2 | An epidemiologic map showing the Cyclospora-prevalent districts in humans in Nepal.The original sample map was obtained from the Survey Department of the Government of Nepal and Ministry of Land Management (https://dos.gov.np).

FIGURE 3 |
FIGURE 3 | Life cycle of Cyclospora and associated risk factors in transmission.The green cycle represents the transmission of C. cayetanensis in humans and the blue cycle indicates the circulation of Cyclospora spp. in animals.It is widely accepted that C. cayetanensis only infects humans[4].These animals and environmental agents, as mentioned in published papers incorporated in this review, and represented in this picture were investigated for Cyclospora oocysts.The presence of Cyclospora oocysts in the animal faeces does not indicate infection and cannot be considered the reservoir of C. cayetanensis; however, Cyclospora oocysts might act as paratenic hosts[14] to disseminate this pathogen to the surroundings where humans can be infected.A biopsy is the only means by which whether the animals are actually infected can be ascertained.

TABLE 1 |
Cyclospora detected in different categories of humans (HIV immunocompromised cases, children, and common humans).

TABLE 2 |
Cyclospora detection in domestic animals in Nepal.
‡The specific number of samples from each district not mentioned.

TABLE 3 |
Cyclospora detection in wild animals of Nepal.
‡The specific number of samples from each district not mentioned.

TABLE 4 |
Evidence of Cyclospora contamination in various environmental samples in Nepal.
NA: Sample number and prevalence not provided; only presence of Cyclospora was mentioned.
[90] to characterize six human C. cayetanensis isolates supplied from Nepal, genetic polymorphisms were not demonstrated among the isolates at the target locus[89].Considering the 18S rRNA locus in the follow-up study of Sulaiman et al.[89], evidence supported the lack of geographic separation and existence of genetically homogenous population of C. cayetanensis[18].Cinar et al.[90]performed an apicoplast genome sequence analysis for the C. cayetanensis-positive specimens provided by the Tribhuvan University Teaching Hospital in Kathmandu.Only three C. cayetanensis-positive specimens obtained from Nepal were included in the population genetic characterization,

TABLE 5 |
Molecular studies on Cyclospora positive samples/isolates.