Enteric parasites Cyclospora cayetanensis and Cryptosporidium hominis in domestic and wildlife animals in Ghana.

BACKGROUND: Enteric parasitic infections remain a major public health problem globally. Cryptosporidium spp., Cyclospora spp. and Giardia spp. are parasites that cause diarrhea in the general populations of both developed and developing countries. Information from molecular genetic studies on the speciation of these parasites and on the role of animals as vectors in disease transmission is lacking in Ghana. This study therefore investigated these diarrhea-causing parasites in humans, domestic rats and wildlife animals in Ghana using molecular tools. METHODS: Fecal samples were collected from asymptomatic school children aged 9-12 years living around the Shai Hills Resource Reserve (tourist site), from wildlife (zebras, kobs, baboons, ostriches, bush rats and bush bucks) at the same site, from warthogs at the Mole National Park (tourist site) and from rats at the Madina Market (a popular vegetable market in Accra, Ghana. The 18S rRNA gene (18S rRNA) and 60-kDa glycoprotein gene (gp60) for Cryptosporidium spp., the glutamate dehydrogenase gene (gdh) for Giardia spp. and the 18S rDNA for Cyclospora spp. were analyzed in all samples by PCR and Sanger sequencing as markers of speciation and genetic diversity. RESULTS: The parasite species identified in the fecal samples collected from humans and animals included the Cryptosporidium species C. hominis, C. muris, C. parvum, C. tyzzeri, C. meleagridis and C. andersoni; the Cyclopora species C. cayetanensis; and the Gardia species, G. lamblia and G. muris. For Cryptosporidium, the presence of the gp60 gene confirmed the finding of C. parvum (41%, 35/85 samples) and C. hominis (29%, 27/85 samples) in animal samples. Cyclospora cayetanensis was found in animal samples for the first time in Ghana. Only one human sample (5%, 1/20) but the majority of animal samples (58%, 51/88) had all three parasite species in the samples tested. CONCLUSIONS: Based on these results of fecal sample testing for parasites, we conclude that animals and human share species of the three genera (Cryptosporidium, Cyclospora, Giardia), with the parasitic species mostly found in animals also found in human samples, and vice-versa. The presence of enteric parasites as mixed infections in asymptomatic humans and animal species indicates that they are reservoirs of infections. This is the first study to report the presence of C. cayetanensis and C. hominis in animals from Ghana. Our findings highlight the need for a detailed description of these parasites using high-throughput genetic tools to further understand these parasites and the neglected tropical diseases they cause in Ghana where such information is scanty.

Aqueous Ozone Exposure Inhibits Sporulation in the Cyclospora cayetanensis Surrogate Eimeria acervulina.

Ozone is a potent disinfecting agent used to treat potable water and wastewater, effectively clearing protozoa such as Giardia and Cryptosporidium spp. It is unclear whether ozone treatment of water or fresh produce can reduce the spread of the emerging parasite Cyclospora cayetanensis, which causes cyclosporiasis in humans. Obtaining viable C. cayetanensis oocysts to evaluate inactivation methods is challenging because we lack the means to propagate them in vitro, because of delays in case reporting, and because health departments typically add inactivating fixatives to clinical specimens. Research in various surrogate organisms has sought to bolster understanding of the biology of C. cayetanensis. Among these surrogates is the poultry parasite Eimeria acervulina, a closely related and easily cultured parasite of economic significance. We used this surrogate to evaluate the consequences of ozone treatment, using the sporulation state as an indicator of infectious potential. Treating with ozonated water acidified with citric acid reduced sporulation ability in a dose-dependent manner; treatment with up to 4.93 mg/L initial concentration of ozone resulted in a 93% inactivation of sporulation by 7 days posttreatment. This developmental arrest was accompanied by transcriptional changes in genes involved in regulating the response to reactive oxygen species (ROS) in a time course that is consistent with the production of oxygen free radicals. This study shows that ozone is highly effective in preventing sporulation of E. acervulina, a model coccidian used as a surrogate for Cyclospora. Furthermore, ozone exposure induced molecular responses to general oxidative stress, documented with several well-characterized antioxidant enzymes.

The global prevalence of Cyclospora cayetanensis infection: A systematic review, meta-analysis, and meta-regression.

Cyclospora cayetanensis (C. cayetanensis) is a significant pathogen that causes diarrheal illness and causes large foodborne diarrhea outbreaks in the USA and Canada. However, there is currently a lack of published meta-analysis on the prevalence of C. cayetanensis infection in the global population. A real estimation of a disease prevalence should always be done on the basis of studies designed for that purpose. We conducted a comprehensive search of various databases for articles pertaining to the prevalence of C. cayetanensis infection in humans, spanning from the inception of these databases to March 10, 2023. Utilizing a random effects model, we estimated the prevalence of C. cayetanensis infection in humans. Our analysis included a total of 150 datasets sourced from 42 different countries, which were ultimately selected for the final quantitative assessment. The prevalence of C. cayetanensis infection in humans worldwide was estimated to be 3.4 % (5636/166,611). Notably, Africa exhibited the highest prevalence rate at 5.9 % (606/11,068). Further subgroup analysis revealed a significantly higher infection rate in humans residing in low-income countries (7.6 %, 83/921) compared to those in lower-middle-income countries (4.8 %, 3280/48,852), upper-middle-income countries (2.9 %, 2194/99,419), and high-income countries (0.4 %, 79/17,419). The results indicate that the global prevalence of C. cayetanensis infection in humans is relatively low, despite its extensive geographical distribution and children were found to be more susceptible to C. cayetanensis infection compared to those adults. Sensitivity analysis revealed that one study significantly affects the prevalence of C. cayetanensis, which was adjusted to 2.9 % (4017/160,049; 95 % CI: 2.7-3.1 %) by excluding this study. The findings highlight the relatively high prevalence of C. cayetanensis infection in low-income countries and among humans with diarrhea, particularly in Africa. Consequently, routine surveillance for intestinal protozoa is crucial in these regions.

The first Cyclospora cayetanensis lineage A genome from an isolate from Mexico.

BACKGROUND: Cyclospora cayetanensis is a protozoan parasite that causes intestinal illness in humans worldwide. Despite its global distribution, most genomic data for C. cayetanensis has been obtained from isolates collected in the United States, leaving genetic variability among globally distributed isolates underexplored. RESULTS: In the present study, the genome of an isolate of C. cayetanensis obtained from a child with diarrhea living in Mexico was sequenced and assembled. Evaluation of the assembly using a lineage typing system recently developed by the Centers for Disease Control and Prevention revealed that this isolate is lineage A. CONCLUSIONS: Given that the only other whole genome assembly available from Mexico was classified as lineage B, the data presented here represent an important step in expanding our knowledge of the diversity of C. cayetanensis isolates from Mexico at the genomic level.

Potent efficiency of the novel nitazoxanide-loaded nanostructured lipid carriers against experimental cyclosporiasis.

Cyclosporiasis is a ubiquitous infection caused by an obligate intracellular protozoan parasite known as Cyclospora cayetanensis (C. cayetanensis). The disease is characterized by severe diarrhea which may be regrettably fatal in immunosuppressed patients. The commercially available treatment options have either severe side effects or low efficiency. In the present study, the novel formula of nitazoxanide (NTZ)-loaded nanostructured lipid carriers (NLCs) was assessed for the first time for C. cayetanensis treatment in both immunocompetent and immunosuppressed mice in comparison to commercially available drugs (trimethoprim-sulfamethoxazole (TMP-SMX) and NTZ). Swiss Albino mice were orally infected by 104 sporulated oocysts. The experimental groups were treated with the gold standard TMP-SMX, NTZ, blank NLCs and NTZ-loaded NLCs. The results demonstrated that NTZ-loaded NLCs represented the highest significant parasite percent reduction of (>98% reduction) in both immunocompetent and immunosuppressed mice designating successful tissue penetration and avoiding recurrence of infection at the end of the study. Oocysts treated with NTZ-loaded NLCs demonstrated the most mutilated rapturing morphology via scanning electron microscope examination as well as representing the most profound improvement of the histopathological picture. In conclusion, NTZ-loaded NLCs exhibited the uppermost efficacy in the treatment of cyclosporiasis. The safe nature and the anti-parasitic effect of the novel formulation encourage its use as a powerful treatment for human cyclosporiasis.

Comparison of two novel one-tube nested real-time qPCR assays to detect human-infecting Cyclospora spp.

IMPORTANCE: Human-infecting Cyclospora spp. cause gastrointestinal distress among healthy individuals contributing to morbidity and putting stress on the economics of countries and companies in the form of produce recalls. Accessible and easy-to-use diagnostic tools available to a wide variety of laboratories would aid in the early detection of possible outbreaks of cyclosporiasis. This, in turn, will assist in the timely traceback investigation to the suspected source of an outbreak by informing the smallest possible recall and protecting consumers from contaminated produce. This manuscript describes two novel detection methods with improved performance for the causative agents of cyclosporiasis when compared to the currently used 18S assay.

Modeling Preharvest Cyclospora cayetanensis Sampling and Testing for Various Water and Produce Sampling Plans.

As of August 2023, the two U.S. Food and Drug Administration (FDA) official detection methods for C. cayetanensis are outlined in the FDA Bacteriological Analytical Manual (BAM) Chapters 19b (produce testing) and 19c (agricultural water testing). These newly developed detection methods have been shown to not always detect contamination when present at low levels. Yet, industry and regulators may choose to use these methods as part of their monitoring and verification activities while detection methods continue to be improved. This study uses simulation to better understand the performance of these methods for various produce and water sampling plans. To do so, we used published FDA test validation data to fit a logistic regression model that predicts the methods' detection rate given the number of oocysts present in a 10-L agricultural water or 25 g produce sample. By doing so, we were able to determine contamination thresholds at which different numbers of samples (n = 1, 2, 4, 8, 16, and 32) would be adequate for detecting contamination. Furthermore, to evaluate sampling plans in use cases, a simulation was developed to represent C. cayetanensis contamination in agricultural water and on cilantro throughout a 45-day growth cycle. The model included uncertainty around the contamination sources, including scenarios of unintentionally contaminated irrigation water or in-field contamination. The results demonstrate that in cases where irrigation water was the contamination source, frequent water testing proved to be more powerful than produce testing. In scenarios where contamination occurred in-field, conducting frequent produce testing or testing produce toward the end of the season more reliably detected contamination. This study models the power of C. cayetanensis detection methods to understand the sampling plan performance and how these methods can be better used to monitor this emerging food safety hazard.

Evaluation of nuclear and mitochondrial phylogenetics for the subtyping of Cyclospora cayetanensis.

Cyclospora cayetanensis is an enteric coccidian parasite responsible for gastrointestinal disease transmitted through contaminated food and water. It has been documented in several countries, mostly with low-socioeconomic levels, although major outbreaks have hit developed countries. Detection methods based on oocyst morphology, staining, and molecular testing have been developed. However, the current MLST panel offers an opportunity for enhancement, as amplification of all molecular markers remains unfeasible in the majority of samples. This study aims to address this challenge by evaluating two approaches for analyzing the genetic diversity of C. cayetanensis and identifying reliable markers for subtyping: core homologous genes and mitochondrial genome analysis. A pangenome was constructed using 36 complete genomes of C. cayetanensis, and a haplotype network and phylogenetic analysis were conducted using 33 mitochondrial genomes. Through the analysis of the pangenome, 47 potential markers were identified, emphasizing the need for more sequence data to achieve comprehensive characterization. Additionally, the analysis of mitochondrial genomes revealed 19 single-nucleotide variations that can serve as characteristic markers for subtyping this parasite. These findings not only contribute to the selection of molecular markers for C. cayetanensis subtyping, but they also drive the knowledge toward the potential development of a comprehensive genotyping method for this parasite.

The limit of detection of the BioFire® FilmArray® gastrointestinal panel for the foodborne parasite Cyclospora cayetanensis.

Cyclosporiasis is a foodborne diarrheal illness caused by the parasite Cyclospora cayetanensis. The BioFire® FilmArray® gastrointestinal (FilmArray GI) panel is a common method for diagnosing cyclosporiasis from clinical stool samples. The currently published limit of detection (LOD) of this panel is in genome equivalents; however, it is unclear how this relates to the number of C. cayetanensis oocysts in a clinical sample. In this study, we developed a technique to determine the LOD in terms of oocysts, using a cell sorter to sort 1 to 50 C. cayetanensis oocyst(s) previously purified from three human stool sources. We found the FilmArray GI panel detected samples with ≥20 C. cayetanensis oocysts in 100% of replicates, with varying detection among samples with 1, 5, or 10 C. cayetanensis oocysts. This method provides a parasitologically relevant LOD that should enable comparison among C. cayetanensis detection techniques, including the FilmArray GI panel.

First case report of Cyclosporiasis from eastern India: Incidence of Cyclospora cayetanensis in a patient with unusual diarrheal symptoms.

Cyclospora cayetanensis, a recently described coccidian parasite causes severe gastroenteric disease worldwide. Limited studies are found on the incidence of C. cayetanensis infection from India; hence remains largely unknown. To date, no case of cyclosporiasis from eastern India has been reported. In this study, we described an incidental case of C. cayetanensis in a 30 years old Bengali female patient with no travel history from eastern India. In June 2022, the patient presented with a history of diarrhoea persisting for more than two months with continuous passage foul smelling stools for which she took multiple antibiotics that were ineffective. There were no Salmonella, Shigella, or Vibrio-like organisms in the patient's faecal sample, and Toxin A/B of Clostridium difficile was also not detected by ELISA. The patient was HIV-negative. Finally, UV autofluorescence and DNA-based diagnosis confirmed the presence of C. cayetanensis, and the treatment with a combination of appropriate antibiotics was successful. This case report could raise awareness about C. cayetanensis associated diarrhoeal cases in India.

An Unlikely Guest With an Overstayed Welcome: Cyclospora-Induced Postinfectious Irritable Bowel Syndrome.

INTRODUCTION: Postinfectious irritable bowel syndrome is a phenomenon that can occur following bouts of acute gastroenteritis. While bacterial pathogens are typically implicated in the development of postinfectious irritable bowel syndrome, viral and parasitic infections should also be considered as inciting pathogens. CASE PRESENTATION: An immunocompetent, 65-year-old woman presented with several weeks of watery diarrhea, which polymerase chain reaction testing confirmed to be a Cyclospora infection. Resolution of diarrhea was achieved with antibiotic treatment, however, months later she presented to the gastroenterology service with persistence of loose stools and abdominal cramping consistent with a diagnosis of postinfectious irritable bowel syndrome. DISCUSSION: Postinfectious irritable bowel syndrome has a similar presentation to sporadic irritable bowel syndrome, with diagnosis aided by the identification of an inciting pathogen. To our knowledge, this is the first documented case of Cyclospora-induced postinfectious irritable bowel syndrome. While parasitic infections typically aren't implicated in cases of postinfectious irritable bowel syndrome, this case highlights the value of considering this condition as a cause of protracted diarrhea in patients previously diagnosed with Cyclospora.

Retrospective evaluation of an integrated molecular-epidemiological approach to cyclosporiasis outbreak investigations - United States, 2021.

Cyclosporiasis results from an infection of the small intestine by Cyclospora parasites after ingestion of contaminated food or water, often leading to gastrointestinal distress. Recent developments in temporally linking genetically related Cyclospora isolates demonstrated effectiveness in supporting epidemiological investigations. We used 'temporal-genetic clusters' (TGCs) to investigate reported cyclosporiasis cases in the United States during the 2021 peak-period (1 May - 31 August 2021). Our approach split 655 genotyped isolates into 55 genetic clusters and 31 TGCs. We linked two large multi-state epidemiological clusters (Epidemiologic Cluster 1 [n = 136 cases, 54 genotyped] and Epidemiologic Cluster 2 [n = 42 cases, 15 genotyped]) to consumption of lettuce varieties; however, product traceback did not identify a specific product for either cluster due to the lack of detailed product information. To evaluate the utility of TGCs, we performed a retrospective case study comparing investigation outcomes of outbreaks first detected using epidemiological methods with those of the same outbreaks had TGCs been used to first detect them. Our study results indicate that adjustments to routine epidemiological approaches could link additional cases to epidemiological clusters of cyclosporiasis. Overall, we show that CDC's integrated genotyping and epidemiological investigations provide valuable insights into cyclosporiasis outbreaks in the United States.

Investigation of Giardia spp., Cryptosporidium spp. and Cyclospora cayetanensis in Samples Collected from Different Spring Waters Igdir, Türkiye

OBJECTIVE: In this study, it was aimed to investigate the physical and chemical properties of different spring waters and parasitic factors with different methods. METHODS: This study was carried out on 69 water samples collected from different spring waters in and around Igdir Province in April and June 2021. The samples were analyzed by native-Lugol, modified acid-fast staining, and nested polymerase chain reaction (nPCR). In addition, altitude (meter) and pressure (mmHg) measurements were made at the point where the water samples were taken. RESULTS: One or more parasites were detected in 27.5% of the 69 water samples examined. Only C. cayetanensis was found in 13% of the samples, only Cryptosporidium spp. in 10.1%, only Giardia spp. in 1.4%, only C. cayetanensis and Giardia spp. in 1.4%, only C. cayetanensis, Cryptosporidium spp., and Giardia spp. in 1.4%. Only Giardia spp. cyst (4.3%) was detected by the direct examination method. While C. cayetanensis and Cryptosporidium spp. oocysts were detected in 8.7% and 7.2% of the samples by the modified acid-fast staining method, C. cayetanensis was detected in 15.9% and Cryptosporidium spp. was detected in 11.6% of the samples by nPCR. When the C. cayetanensis and Cryptosporidium spp. positivity rates were compared according to the characteristics of the water, there was no statistical difference between the altitude, salinity, pH, mmHg, and temperature (kelvin) values, but a significant correlation was found between the amount of dissolved oxygen and Cryptosporidium spp. positivity (p=0.047). CONCLUSION: Cryptosporidium spp., C. cayetanensis, and G. intestinalis are important waterborne pathogens that can cause epidemics. It is our belief that in order to reduce the risk of contamination of these parasitic factors with spring waters, public awareness should be raised, infrastructures should be improved, and new water treatment techniques, such as ultraviolet, ozonation and monitoring systems, should be used.

Monitoring the Trends in Intestinal Parasite Frequencies; 2018 and 2022 Data

Objective: Monitoring intestinal parasite frequencies is effective on diagnosis, treatment, and prevention strategies to be developed against these parasites. In this study, it was aimed to reveal the parasite species and frequency data of stool samples in parasitology direct diagnosis laboratory. Methods: Stool parasitological examination results were obtained retrospectively from our laboratory internal quality control data tables. Data belonging to the year 2018 and 2022 were compared retrospectively. Results: Annual parasites detected in stool samples were 388 of 4.518, and 710 of 3.537, in 2018 and 2022, respectively. Frequency of parasite detection in stool samples was found to be significantly higher in 2022 (p<0.0001). Number of stools with more than one parasite was 12 and 30 in 2018 and 2022, respectively. Incidence of infection with more than one parasite was significantly higher in 2022 (p=0.0003). Five most common parasite species were Blastocystis spp., Enterobius vermicularis, Cryptosporidium spp., Giardia intestinalis and Entamoeba histolytica in 2018, respectively; and Cryptosporidium spp., Blastocystis spp., Cyclospora spp., Entamoeba dispar and Giardia intestinalis, in 2022, respectively. Cryptosporidium spp., Cyclospora spp. and Entamoeba dispar increased significantly, while Blastocystis spp. and Enterobius vermicularis decreased significantly, in 2022. Conclusion: According to the data obtained, causative agents for intestinal parasitic infections were protozoans, especially Cryptosporidium spp. It has been concluded that tightening the measures for protection of water with one health approach and improving the education and habits of society on personal hygiene and food safety can be effective in reducing the frequency of intestinal parasite infections in our region.

Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce.

Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.

Development of a new multiplex PCR to detect fecal coccidian parasite.

BACKGROUND: Cryptosporidium spp., Cystoisospora belli and Cyclospora cayetanensis are common intestinal coccidian parasites causing gastroenteritis. The clinical presentation caused by each parasite is indistinguishable from each other. Uniplex polymerase chain reaction (PCR) for these three groups of intestinal coccidian parasites was developed by us in our laboratory. Thereafter, we planned to develop a single-run multiplex polymerase chain reaction (mPCR) assay to detect Cryptosporidium spp., C. belli and C. cayetanensis simultaneously from a stool sample and described it here as coccidian mPCR. METHODS: New primers for C. belli and C. cayetanensis were designed and uniplex PCRs were standardized. The coccidian mPCR was standardized with known positive DNA control isolates. It was validated with 58 known positive and 58 known negative stool samples, which were previously identified by uniplex PCR. RESULTS: The coccidian mPCR was standardized with earlier primers designed by us for Cryptosporidium spp. and C. cayetanensis, and a newly designed primer for the internal transcribed spacer-1 (ITS-1) gene for C. belli. The coccidian mPCR was 92.1% sensitive for Cryptosporidium spp., and 100% sensitive for C. belli and C. cayetanensis each, when tested on 116 known samples. It was 100% specific for all intestinal coccidian parasites. Two representative PCR products of the newly designed ITS-1 primer for C. belli were sequenced and submitted to the GenBank, which best match with the sequences of C. belli. CONCLUSION: A highly sensitive, specific, cost-effective, indigenous, single-run coccidian mPCR has been developed, which can simultaneously detect Cryptosporidium spp., C. belli and C. cayetanensis.

Immunogenic multi-epitope-based vaccine development to combat cyclosporiasis of immunocompromised patients applying computational biology method.

Cyclospora cayetanensis infections, also known as cyclosporiasis, persist to be the prevalent emerging protozoan parasite and an opportunist that causes digestive illness in immunocompromised individuals. In contrast, this causal agent can affect people of all ages, with children and foreigners being the most susceptible populations. For most immunocompetent patients, the disease is self-limiting; in extreme circumstances, this illness can manifest as severe or persistent diarrhea as well as colonize on secondary digestive organs leading to death. According to recent reports, worldwide 3.55% of people are infected by this pathogen, with Asia and Africa being more prevalent. For the treatment, trimethoprim-sulfamethoxazole is the only licensed drug and does not appear to work as well in some patient populations. Therefore, the much more effective strategy to avoid this illness is immunization through the vaccine. This present study uses immunoinformatics for identifying a computational multi-epitope-based peptide vaccine candidate for Cyclospora cayetanensis. Following the review of the literature, a highly efficient, secure, and vaccine complex based on multi-epitopes was designed by utilizing the identified proteins. These selected proteins were then used to predict non-toxic and antigenic HTL-epitopes, B-cell-epitopes, and CTL-epitopes. Ultimately, both a few linkers and an adjuvant were combined to create a vaccine candidate with superior immunological epitopes. Then, to establish the vaccine-TLR complex binding constancy, the TLR receptor and vaccine candidates were placed into the FireDock, PatchDock, and ClusPro servers for molecular docking and iMODS server for molecular-dynamic simulation. Finally, this selected vaccine construct was cloned into Escherichia coli strain-K12; thus, the constructed vaccines against Cyclospora cayetanensiscould improve the host immune response and can be produced experimentally.