Incidental Detection of Cyclospora cayetanensis during General Health Screening: A Case Study from Singapore

Background: Cyclospora cayetanensis is a coccidian parasite recently recognized and characterized that cause enteritic infection worldwide. Endemic in tropical and subtropical countries where asymptomatic carriage is common, this parasite is also frequently associated with foodborne and waterborne outbreaks and reported from travellers in industrialized countries. Poorly characterized by routine laboratory procedures, the oocysts of C. cayetanensis are also discontinuously shed, altogether making this parasite difficult to detect. In Singapore, information about C. cayetanensis are scarce and the endemicity status is unknown, while the country seems vulnerable to outbreak occurrence. Case study: The present report describes the incidental laboratory finding of C. cayetanensis oocysts in the stool of an asymptomatic immunocompetent patient attending a general health screening. Initial suspicion about the diagnosis was confirmed by several morphological methods and concurrently by amplification and sequencing of the parasite DNA. Along the study, co-infections with Blastocytis sp. ST3, Cryptosporidium parvum/hominis and the nonpathogenic Entamoeba hartmanni were noticed and confirmed by molecular methods. Conclusions: This report could serve to raise awareness about Cyclospora cayetanensis and the asymsptomatic carriage of enteritic protozoa, and as a reminder about the risk of cyclosporiasis especially in the most susceptible populations such as: children, elderly and immunocompromised.


Introduction
Cyclospora cayetanensis is a Protozoan parasite of the human enteric tract only recently characterized and described [1,2] and now recognized as an important emerging cause of diarrhoea worldwide [3]. Probably first associated with human illness by [4] who reported an undescribed coccidian parasite related to Isospora, the identity of this pathogen remained controversial until 1993 when [1] managed to induce the sporulation of this organism and demonstrated that it has two sporocysts each containing two sporozoites and therefore it was an oocyst of a coccidian parasite related to the genus Cyclospora and proposed the name Cyclospora cayetanensis Ortega, Gilman & Sterling, 1994 [2]. Before, it might have been overlooked as Blastocystis [5], then subsequently considered as a coccidian parasite [6,7], a spore of fungus [6], an algae-like organism related to the blue-green algae [8,9] and was therefore also called cyanobacterium-like organism, abbreviated CLB [10][11][12][13]. Even after being identified as Cyclospora, it has remained reported as CLB for "coccidian/cyanobacterium-like body" [14,15] and sometime also for "cyclospora like body" [13]. Later, phylogenetic analysis has confirmed that C. cayetanensis truly is a coccidian parasite closely related to the avian Eimeria species [16].
C. cayetanensis is widely distributed worldwide but seems more common in tropical and subtropical regions [17]. In developing countries, it is regularly detected among local populations in both symptomatic and asymptomatic adults and children [17][18][19] and infections have been linked to different sources such as contaminated water, food and soil and also contact with animals, sanitation and low socioeconomic status [17]. In developed countries, it is usually detected in travellers returning from tropical and subtropical regions [20][21][22]; and it is also a recurrent cause of foodborne outbreaks related to consumption of imported raw fruits and vegetables in North America since the 1990s [21,23,24]. Frequently detected in water for human consumption in endemic countries [25,26], waterborne outbreaks have been reported in US [27] and in Nepal even after water chlorination [28].
As a major emerging cause of diarrhoea outbreaks, cyclosporiasis is a notifiable disease in US [21]. In Singapore, the Ministry of Health (MOH) doesn't collect information about this pathogen that is rarely reported (e.g. only 4 cases detected at National University Hospital in the last 4 years). To the authors knowledge, in the literature, Singapore was only listed as a visited country along with Indonesia in the first report of imported cyclosporiasis in Germany [29] and was also mentioned along with Malaysia as suspected port of embarkation of contaminated food potentially implicated in a cruise cyclosporiasis outbreak [30].
The present report describes the incidental laboratory finding of C. cayetanensis in the stool samples of a healthy, asymptomatic patient participating into a general health screening.

Case Study
Patient: A 35 years old immunocompetent Indonesian male without any symptoms presented at the National University Hospital (NUH) for a general health screening. The physical examination was unremarkable and there were no complaints of gastrointestinal disturbance noted. As part of the health screening a stool specimen was collected for screening for ova, cysts and parasites.
NUH laboratory: Initially, a direct examination by saline wet mounts was made by mixing a small volume of stool sample with a drop of physiological saline on a glass microscope slide according to standard procedure [31]. With light microscope at low (100X) then medium (400X) magnification some unidentified cysts like bodies were noted.
Thin faecal smears were made and fixed in methanol prior permanent staining by Kinyoun acid-fast and modified trichrome stains respectively following standard procedures [31]. The smears were observed with light microscope at medium (400X) then high (1000X) magnification.
They revealed cysts of 8-10 µm, totally unstained in trichrome ( Figures 1A-1C) and only partly acid-fast with some cysts stained in light to dark pink while the wide majority remained unstained ( Figures  1D-1F). This result was suggestive of C. cayetanensis. The original smears and two aliquots of the faecal material in sodium acetate-acetic acid-formalin (SAF) and absolute ethanol respectively were passed to the National Public Health Laboratory (NPHL) for confirmation.
NPHL microscopy: Morphological observation was performed with an Olympus CX31 microscope and microphotographs were taken with a Nikon Eclipse 80i microscope equipped with a Nikon DS Ri1 camera and the Elements-D Imaging Software at medium (400X) and high (1000X) magnification. The original smears were confirmed positive for C. cayetanensis ( Figures 1A-1F).
Additional thin faecal smears were prepared from the SAF fixed sample for UV fluorescence microscopy and safranin staining tests. The safranin stained smears were prepared according to [32] procedure with microwave heating and showed a uniform staining of all the oocysts (Figures 1G-1I).
Under UV light, the oocysts were autofluorescent even after fixation, and displayed a typical blue colour with UV excitation set at 330-365 nm (Figures 2A-2D) and green colour with UV excitation set at 450-490 nm ( Figures 2B-2E). The safranin being a fluorophore itself, the oocysts stained with this dye were also visible under UV light and displayed a red fluorescence with excitation set at 528-552 nm ( Figures  2C-2F).
Taken all together these results confirmed the nature of the organism as oocyst of C. cayetanensis. Additional findings were made on the trichrome stained smear with the detection of numerous Entamoeba cysts morphologically compatible with Entamoeba hartmanni ( Figure 1J), some Blastocystis sp. cysts ( Figure 1K) and some rare oocysts suggestive of Cryptosporidium spp. ( Figure 1L). DNA extraction was performed with the QIAmp® DNA stool mini kit (Qiagen®) after 10 freeze/thaw cycles (1 min -196°C and 1 min 100°C). DNA was eluted in AE buffer and stored at -30°C. DNA amplifications for each organism were performed by nested-PCR protocols targeting the 18S ribosomal RNA (18S rRNA) gene.
The first reaction was common to all organisms and used a pair of 18S rRNA universal oligonucleotide primers as described by [33].
For the second amplifications four separate specific reactions were performed according to the protocols proposed by [34] (with the primers pair: ExCycF/ExCycR), [35] (with the primers pair: Eg-SS-F1/Eg-SS-CR1), [36] (with the primers set B); and [37] (with primers pair: SSU-F2/SSU-R2); for Cyclospora, Entamoeba spp., Blastocystis sp. and Cryptosporidium spp. respectively. All the PCRs reactions were run on Veriti® Thermal Cycler (Applied Biosystems®), in a total volume of 20µL containing 2µL of original DNA template for the first reaction and 1µL of the PCR product of the first reaction as template for each second reaction.
All PCR products were visualised by electrophoresis on QIAxcel® Advanced instrument (Qiagen®) prior purification by the QIAquick® PCR Purification Kit (Qiagen®); eluted in 30µL of EB buffer; frozen at -30°C; then prepared for sequencing in both directions using the BigDye® Terminator v3.1 cycle sequencing Kit (Applied Biosystems®) and the respective oligonucleotide primers. The BigDye® reaction's products were purified using the BigDye® XTerminator™ Purification Kit (Applied Biosystems®) before being sequenced on a 3500xl Genetic Analyzer (Applied Biosystems®).
For each step, kits and equipment were used according to the manufacturer's recommendations.
Despite multiples attempts the detection of Cryptosporidium spp. remained unsuccessful on the 18S rRNA gene.
However Cryptosporidium parvum/hominis DNA was detected in all replicates of a real-time PCR (rt-PCR) assay targeting a gene encoding a DNA-J like protein with high Ct value (x̅ 36.45). The rt-PCR was performed according to the protocol proposed by [38].
Additional rt-PCR assays for Dientamoeba fragilis and Enterocytozoon bieneusi remained negative. All rt-PCRs were performed using the QuantiFast® Probe RT-PCR Kit (Qiagen®) and run on the StepOnePlus™ Real-Time PCR System (Applied Biosystems®).
Alignment and cross-checking of the sequences were performed with CLC Main Workbench v7.6 (CLC Bio-Qiagen®) and consensus sequences of 1793 bp, 1910 bp and 1769 bp representing the nearly complete sequence of the 18S rRNA gene were obtained for Cyclospora, Entamoeba spp. and Blastocystis sp. respectively. There was no sequence obtained for Cryptosporidium parvum/hominis as this organism was only detected by rt-PCR protocol.
Basic Local Alignment Search Tool (BLAST) [39] confirmed the identity of these organisms as Cyclospora cayetanensis, Entamoeba hartmanni and Blastocystis sp. subtype 3 (ST3). The respective sequences were deposited in GenBank under the accession numbers: KX618190 to KX618192.

Discussion
The present study reports the incidental detection of C. cayetanensis in Singapore during the voluntary health screening of a healthy patient. Being asymptomatic the patient didn't take any treatment and was found negative in NUH laboratory during a follow-up appointment two weeks later. This situation highlights the difficulty to detect C. cayetanensis oocysts from stool samples because of their intermittent shedding and the low sensitivity of the routine procedures [17,20], while it also highlight the usefulness of special staining procedure, fluorescence microscopy as well as molecular methods.
Similarly to some other industrialized country, C. cayetanensis remains rarely reported and largely understudied in Singapore. It might due to a lack of awareness and/or the difficulty to detect this pathogen [41]. This situation seem surprising as Singapore, is geographically located in a favourable region for the environmental maturation of C. cayetanensis oocysts, is surrendered by endemic countries that supply the majority of its food and water; and is visited by hundreds of migrants such as workers, tourists and travellers coming or returning from these endemic countries. For all these reasons Singapore seems to be a vulnerable country for the occurrence of cyclosporiasis outbreaks.
As the prevalence of C. cayetanensis in diarrheic patients as well as in in susceptible populations such as the children, elderly, travellers and immunocompromised in Singapore, is unknown the endemicity of the infection for the nation is unknown and could only be partly reflected through the report and study of sporadic cases among local residents and tourists visiting the country. In the present case study, it was not possible to confidently identify the origin of the infection as the patient originated from Indonesia which is an endemic country [15,20,29,[41][42][43] and there was no information about a past onset of symptoms.
Given the particularities in the biological cycles of the different parasitic protozoa detected, it is likely that the patient had multiple exposures to faecally contaminated water and/or food. The present report about an asymptomatic carrier of multiple enteritic protozoa should serve as a reminder that introduction and dissemination of these organisms could possibly lead to contamination of soil, water sources and perhaps even food especially for C. cayetanensis as its oocysts are resistant to basic food and water treatments [20,27,28].
This report should also raise awareness locally to all clinicians, medical practitioners, laboratory staffs and public health officers about C. cayetanensis. Until now, cyclosporiasis outbreaks in Singapore could have been overlooked or really avoided by the strict control of food and water quality. In the future, in the perspective of an increase of both the population and its inherent needs of importation of fresh products, C. cayetanensis could potentially become a public health problem in Singapore. To assess the risk and monitor the situation, it would be desirable to evaluate the endemicity status in both symptomatic and asymptomatic individuals.