A review of Cryptosporidium spp. and their detection in water.

Cryptosporidium spp. are one of the most important waterborne pathogens worldwide and a leading cause of mortality from waterborne gastrointestinal diseases. Detection of Cryptosporidium spp. in water can be very challenging due to their low numbers and the complexity of the water matrix. This review describes the biology of Cryptosporidium spp. and current methods used in their detection with a focus on C. parvum and C. hominis. Among the methods discussed and compared are microscopy, immunology-based methods using monoclonal antibodies, molecular methods including PCR (polymerase chain reaction)-based assays, and emerging aptamer-based methods. These methods have different capabilities and limitations, but one common challenge is the need for better sensitivity and specificity, particularly in the presence of contaminants. The application of DNA aptamers in the detection of Cryptosporidium spp. oocysts shows promise in overcoming these challenges, and there will likely be significant developments in aptamer-based sensors in the near future.

Development and application of DNA-aptamer-coupled magnetic beads and aptasensors for the detection of Cryptosporidium parvum oocysts in drinking and recreational water resources.

Environmentally stable and disinfectant-resistant oocysts of Cryptosporidium spp. shed in the feces of infected humans and animals frequently contaminate water resources and are subsequently spread via potable and recreational waters. The current monoclonal-antibody-based methods for detecting them in water are slow, labor-intensive, and demand skills to interpret the results. We have developed DNA-aptamer-based aptasensors, coupled with magnetic beads, to detect and identify the oocysts of C. parvum for monitoring recreational and drinking water sources. A sensitive and specific electrochemical aptasensor (3'-biotinylated R4-6 aptamer) was used as a secondary ligand to bind the streptavidin-coated magnetic beads. This was incorporated into a probe using gold nanoparticle modified screen-printed carbon electrodes. Square wave voltammetry allowed for specific recognition of C. parvum oocysts. The aptamer-coated probes had an oocyst detection limit of 50. It did not bind to the cysts of Giardia duodenalis, another common waterborne pathogen, thus indicating its high specificity for the target pathogen. The system could successfully detect C. parvum oocysts in spiked samples of the raw lake and river waters. Therefore, the combined use of the aptasensor and magnetic beads has the potential to monitor water quality for C. parvum oocysts in field samples without relying on monoclonal antibodies and skill-demanding microscopy.

Fate of internalized Campylobacter jejuni and Mycobacterium avium from encysted and excysted Acanthamoeba polyphaga.

Association of the water- and foodborne pathogen Campylobacter jejuni with free-living Acanthamoeba spp. trophozoites enhances C. jejuni survival and resistance to biocides and starvation. When facing less than optimal environmental conditions, however, the Acanthamoeba spp. host can temporarily transform from trophozoite to cyst and back to trophozoite, calling the survival of the internalized symbiont and resulting public health risk into question. Studies investigating internalized C. jejuni survival after A. castellanii trophozoite transformation have neither been able to detect its presence inside the Acanthamoeba cyst after encystation nor to confirm its presence upon excystation of trophozoites through culture-based techniques. The purpose of this study was to detect C. jejuni and Mycobacterium avium recovered from A. polyphaga trophozoites after co-culture and induction of trophozoite encystation using three different encystation methods (Neff's medium, McMillen's medium and refrigeration), as well as after cyst excystation. Internalized M. avium was used as a positive control, since studies have consistently detected the organism after co-culture and after host excystation. Concentrations of C. jejuni in A. polyphaga trophozoites were 4.5 × 105 CFU/ml, but it was not detected by PCR or culture post-encystation. This supports the hypothesis that C. jejuni may be digested during encystation of the amoebae. M. avium was recovered at a mean concentration of 1.9 × 104 from co-cultured trophozoites and 4.4 × 101 CFU/ml after excystation. The results also suggest that M. avium recovery post-excystation was statistically significantly different based on which encystation method was used, ranging from 1.3 × 101 for Neff's medium to 5.4 × 101 CFU/ml for refrigeration. No M. avium was recovered from A. polyphaga cysts when trophozoites were encysted by McMillen's medium. Since C. jejuni internalized in cysts would be more likely to survive harsh environmental conditions and disinfection, a better understanding of potential symbioses between free-living amoebae and campylobacters in drinking water distribution systems and food processing environments is needed to protect public health. Future co-culture experiments examining survival of internalized C. jejuni should carefully consider the encystation media used, and include molecular detection tools to falsify the hypothesis that C. jejuni may be present in a viable but not culturable state.

Toxoplasma gondii infection in stranded St. Lawrence Estuary beluga Delphinapterus leucas in Quebec, Canada.

The St. Lawrence Estuary (SLE) beluga Delphinapterus leucas in Quebec, Canada, is endangered due to intensive hunting in the 19th and 20th centuries and subsequent anthropogenic contamination and human activities in the region. Infectious disease is a primary cause of death in this population. The protozoan parasite Toxoplasma gondii is reported in numerous marine mammal species, including beluga. In the present study, 55 tissue samples (heart and brain) collected from 34 stranded SLE beluga were analysed by PCR followed by DNA sequencing and restriction fragment length polymorphism analysis (RFLP) to determine the PCR prevalence and genotypes of T. gondii in these beluga. Of 34 beluga tested, 44% were positive for T. gondii by PCR, with males having a higher prevalence of infection than females and with more infected neonates and juveniles than adults. Molecular analyses indicated that all T. gondii infecting stranded SLE beluga grouped into genotype II, which predominates in humans. While our results indicate that a high prevalence of stranded beluga are PCR-positive for T. gondii infection, very few deaths are attributed to toxoplasmosis based on published necropsy results. Toxoplasma gondii can cause a range of diseases, including neurological deficits, and more data are needed to investigate this parasite's effect on population recovery.

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation.

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-µm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.

Toxoplasma gondii DNA in Tissues of Anadromous Arctic Charr, Salvelinus alpinus, Collected From Nunavik, Québec, Canada.

BACKGROUND: Toxoplasma gondii is a very common zoonotic parasite in humans and animals worldwide. Human seroprevalence is high in some regions of Canada's North and is thought to be associated with the consumption of traditionally prepared country foods, such as caribou, walrus, ringed seal and beluga. While numerous studies have reported on the prevalence of T. gondii in these animals, in the general absence of felid definitive hosts in the North there has been considerable debate regarding the source of infection, particularly in marine mammals. It has been proposed that fish could be involved in this transmission. AIMS: The objectives of the present study were to perform a targeted survey to determine the prevalence of T. gondii DNA in various tissues of anadromous Arctic charr sampled in Nunavik, Québec, and to investigate the possible role of this commonly consumed fish in the transmission of infection to humans and marine mammals in Canada's North. METHODS AND RESULTS: A total of 126 individual Arctic charr were sampled from several sites in Nunavik, and various tissues were tested for the presence of T. gondii DNA using PCR. Overall, 12 out of 126 (9.5%) Arctic charr tested in the present study were PCR-positive, as confirmed by DNA sequencing. Brain tissue was most commonly found to be positive, followed by heart tissue, while none of the dorsal muscle samples tested were positive. CONCLUSIONS: Although the presence of T. gondii DNA in brain and heart tissues of Arctic charr is very intriguing, infection in these fish, and their possible role in the transmission of this parasite to humans and marine mammals, will need to be confirmed using mouse bioassays. Arctic charr are likely exposed to T. gondii through the ingestion of oocysts transported by surface water and ocean currents from more southerly regions where the definitive felid hosts are more abundant. If infection in Arctic charr can be confirmed, it is possible that these fish could play an important role in the transmission of toxoplasmosis to Inuit, either directly through the consumption of raw fish or indirectly through the infection of fish-eating marine mammals harvested as country foods.

Foodborne protozoan parasites in fresh mussels and oysters purchased at retail in Canada.

Studies worldwide have reported the presence of protozoan parasites in a variety of commercial bivalve shellfish. The uptake of these parasites by shellfish occurs during filter feeding in faecally-contaminated waters. The objective of the present study was to determine the prevalence of Giardia, Cryptosporidium and Toxoplasma in fresh, live shellfish purchased in three Canadian provinces as part of the retail surveillance activities led by FoodNet Canada (Public Health Agency of Canada). Packages containing mussels (n = 253) or oysters (n = 130) were purchased at grocery stores in FoodNet Canada sentinel sites on a biweekly basis throughout 2018 and 2019, and shipped in coolers to Health Canada for testing. A small number of packages were not tested due to insufficient quantity or poor quality. Following DNA extraction from homogenized, pooled tissues, nested PCR and DNA sequencing were used to detect parasite-specific sequences. Epifluorescence microscopy was used to confirm the presence of intact cysts and oocysts in sequence-confirmed PCR-positive samples. Giardia duodenalis DNA was present in 2.4 % of 247 packages of mussels and 4.0 % of 125 packages of oysters, while Cryptosporidium parvum DNA was present in 5.3 % of 247 packages of mussels and 7.2 % of 125 packages of oysters. Toxoplasma gondii DNA was only found in mussels in 2018 (1.6 % of 249 packages). Parasite DNA was detected in shellfish purchased in all three Canadian provinces sampled, and there was no apparent seasonal variation in prevalence. While the present study did not test for viability, parasites are known to survive for long periods in the marine environment, and these findings suggest that there is a risk of infection, especially when shellfish are consumed raw.

Modeling nuclear energy's future role in decarbonized energy systems.

Increased attention has been focused on the potential role of nuclear energy in future electricity markets and energy systems as stakeholders target rapid and deep decarbonization and reductions in fossil fuel use. This paper examines models of electric sector planning and broader energy systems optimization to understand the prospective roles of nuclear energy and other technologies. In this perspective, we survey modeling challenges in this environment, illustrate opportunities to propagate best practices, and highlight insights from the deep decarbonization literature on the range of visions for nuclear energy's role. Nuclear energy deployment is highest with combinations of stringent emissions policies, nuclear cost reductions, and constraints on the deployment of other technologies, which underscores model dimensions related to these areas. New modeling capabilities are needed to adequately address emerging issues, including representing characteristics and applications of nuclear energy in systems models, and to ensure the relevance of models for policy and planning as deeper decarbonization is explored.

Aptamer-Based Electrochemical Microfluidic Biosensor for the Detection of Cryptosporidium parvum.

Cryptosporidium parvum is a high-risk and opportunistic waterborne parasitic pathogen with highly infectious oocysts that can survive harsh environmental conditions for long periods. Current state-of-the-art methods are limited to lengthy imaging and antibody-based detection techniques that are slow, labor-intensive, and demand trained personnel. Therefore, the development of new sensing platforms for rapid and accurate identification at the point-of-care (POC) is essential to improve public health. Herein, we propose a novel electrochemical microfluidic aptasensor based on hierarchical 3D gold nano-/microislands (NMIs), functionalized with aptamers specific to C. parvum. We used aptamers as robust synthetic biorecognition elements with a remarkable ability to bind and discriminate among molecules to develop a highly selective biosensor. Also, the 3D gold NMIs feature a large active surface area that provides high sensitivity and a low limit of detection (LOD), especially when they are combined with aptamers,. The performance of the NMI aptasensor was assessed by testing the biosensor's ability to detect different concentrations of C. parvum oocysts spiked in different sample matrices, i.e., buffer, tap water, and stool, within 40 min detection time. The electrochemical measurements showed an acceptable LOD of 5 oocysts mL-1 in buffer medium, as well as 10 oocysts mL-1 in stool and tap water media, over a wide linear range of 10-100,000 oocysts mL-1. Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibiting no significant cross-reactivity to other related coccidian parasites. The specific feasibility of the aptasensor was further demonstrated by the detection of the target C. parvum in patient stool samples. Our assay showed coherent results with microscopy and real-time quantitative polymerase chain reaction, achieving high sensitivity and specificity with a significant signal difference (p < 0.001). Therefore, the proposed microfluidic electrochemical biosensor platform could be a stepping stone for the development of rapid and accurate detection of parasites at the POC.

Draft Hybrid Genome Assembly of a Canadian Cyclospora cayetanensis Isolate.

The apicomplexan parasite Cyclospora cayetanensis causes foodborne gastrointestinal disease in humans. Here, we report the first hybrid assembly for C. cayetanensis, which uses both Illumina MiSeq and Oxford Nanopore Technologies MinION platforms to generate genomic sequence data. The final genome assembly consists of 44,586,677 bases represented in 313 contigs.

Genotyping Canadian Cyclospora cayetanensis Isolates to Supplement Cyclosporiasis Outbreak Investigations.

Cyclospora cayetanensis is an emerging foodborne parasite that causes cyclosporiasis, an enteric disease of humans. Domestically acquired outbreaks have been reported in Canada every spring or summer since 2013. To date, investigations into the potential sources of infection have relied solely on epidemiological data. To supplement the epidemiological data with genetic information, we genotyped 169 Canadian cyclosporiasis cases from stool specimens collected from 2010 to 2021 using an existing eight-marker targeted amplicon deep (TADS) scheme specific to C. cayetanensis as previously described by the US Centers for Disease Control and Prevention (CDC). This is the first study to genotype Canadian Cyclospora cayetanensis isolates, and it focuses on evaluating the genotyping performance and genetic clustering. Genotyping information was successfully collected with at least part of one of the markers in the TADS assay for 97.9% of specimens, and 81.1% of cyclosporiasis cases met the minimum requirements to genetically cluster into 20 groups. The performance of the scheme suggests that examining cyclosporiasis cases genetically will be a valuable tool for supplementing epidemiological outbreak investigations and to minimize further infections. Further research is required to expand the number of discriminatory markers to improve genetic clustering.

Detection and characterization of Giardia duodenalis and Cryptosporidium spp. on swine farms in Ontario, Canada.

As part of the C-EnterNet surveillance program of the Public Health Agency of Canada, 122 pooled swine manure samples from 10 farms in Ontario, Canada were collected and tested for Giardia and Cryptosporidium. Giardia duodenalis cysts and Cryptosporidium spp. oocysts were detected using immunofluorescence microscopy. Nested-polymerase chain reaction protocols were performed to amplify the small subunit rRNA gene and the ß-giardin gene for G. duodenalis, and the small subunit rRNA gene and the heat shock protein-70 gene for Cryptosporidium spp. The DNA amplicons were sequenced to determine genotypes and species. A mixed multivariable method was used to compare the presence of Giardia and Cryptosporidium in different stages of production. Both Giardia cysts and Cryptosporidium oocysts were present on all tested farms, with 50.8% of the samples positive for G. duodenalis and 44.3% positive for Cryptosporidium spp. by microscopy, and 66.4% and 55.7%, respectively, positive by polymerase chain reaction (PCR). No significant agreement was observed between microscopy and PCR method to detect Giardia and Cryptosporidium (p<0.05). The prevalence of Giardia in manure pits and finisher pigs did not differ (p>0.05), however, it was less frequent (odds ratio, OR=0.21 [0.07, 0.63]) among sows. Cryptosporidium was more likely (OR=3.6 [1.3, 9.9]) to be detected in manure pits and weaners (OR=3.3 [1.1, 10.0]) compared to finisher pigs, and it was less frequent (OR=0.06 [0.007, 0.55]) in sows than in finishers (p<0.05). DNA sequencing demonstrated that 92.1% of the Giardia isolates were Assemblage B and 7.9% were Assemblage E. The most prevalent Cryptosporidium were Cryptosporidium parvum (55.4%), and Cryptosporidium sp. pig genotype II (37.5%). These findings indicate that the occurrence of zoonotic isolates of G. duodenalis and Cryptosporidium is very high on swine farms in southern Ontario, and that there is a potential for transmission between swine and humans by means of cyst and oocyst contaminated water or foods.

Prevalence and genotypes of Giardia duodenalis in dairy and beef cattle in farms around Charlottetown, Prince Edward Island, Canada.

Prevalence of Giardia duodenalis in dairy and beef cattle on farms around Charlottetown, Prince Edward Island (Canada) was determined by analyzing feces using direct immunofluorescence antibody microscopy. Genotypes were determined by 16S-rRNA sequencing. Fecal samples (n = 892) were collected from adult cattle in dairy tie-stall, dairy free-stall, and beef herds (10 herds each), and from calves (n = 183) from 11 dairy farms. Prevalence rates were 38% and 51% in cows and calves, respectively. Giardia duodenalis was present in all dairy herds, in 9/10 beef herds and in calves from 10/11 herds examined. Prevalence rates were 40% and 41% for cows in tie- and free-stall herds, respectively, and 27% for beef cows. Zoonotic Assemblage A was found in 12.2% of calves concomitantly infected with Assemblage E. All successfully sequenced samples (114/128) from cows corresponded to Assemblage E. Giardia duodenalis is highly prevalent in cattle herds in Prince Edward Island and Assemblage A in calves is a potential public health concern.

Giardia duodenalis in humans and animals - Transmission and disease.

Giardia duodenalis is a protozoan parasite infecting the upper intestinal tract of humans, as well as domestic and wild animals worldwide. Transmission of giardiasis occurs through the faecal-oral route, and may be either direct (i.e., person-to-person, animal-to-animal or zoonotic) or indirect (i.e., waterborne or foodborne). While asymptomatic infections are common in both humans and animals, a wide range of enteric symptoms have been reported, along with extra-intestinal and post-infectious complications. A definitive diagnosis of giardiasis is generally made by detection of cysts in stool specimens through microscopical examination of wet mounts, or through the use of permanent or fluorescent antibody stains. More recently, molecular methods have become popular for diagnosis and for testing environmental samples. Symptomatic giardiasis is often treated to reduce the duration of symptoms, to prevent complications, and to minimize transmission of the parasite to other hosts. Direct faecal-oral transmission of giardiasis can be largely controlled thorough improved hygiene and sanitation. In the case of waterborne transmission, a multiple barrier approach, including limiting access of people and animals to watersheds and reservoirs, and treatment using flocculation, filtration and disinfection, is necessary to minimize the risk. Since foodborne transmission is often associated with the consumption of fresh produce, a number of control measures can be taken during pre- and post-harvest, as well as at the food handler/consumer level to minimize the risk of contamination, or for removing or inactivating parasites. Good husbandry and farm management practices are important in controlling the spread of giardiasis in livestock and companion animals.

A cloth-based hybridization array system for rapid detection of the food- and waterborne protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii.

Protozoan parasites in food or water samples are generally detected using microscopy or PCR followed by Sanger sequencing. However, microscopy is subjective, requires a high degree of expertise and has limited sensitivity, while DNA sequencing requires expensive and specialized equipment and facilities. This study describes a cloth-based hybridization array system (CHAS) that is an alternative to Sanger sequencing to confirm PCR-positive samples. CHAS is an inexpensive, rapid and reliable method for the simultaneous detection of multiple protozoan parasite species based on the colorimetric detection of PCR amplicons on a polyester cloth. PCR primers and CHAS hybridization probes were developed to detect the protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii. In addition, CHAS probes were designed for the differentiation of G. duodenalis Assemblages A and B. In artificially contaminated fresh produce (lettuce, parsley) and water samples (river water, wastewater), this CHAS assay allowed for the successful detection of G. duodenalis, Cryptosporidium spp., and T. gondii. The present study demonstrates that the CHAS detection method is a simple and inexpensive alternative to DNA sequencing for the confirmation of PCR-positive results in laboratories testing for parasites in food or water samples. This assay may also be beneficial in developing countries, where DNA sequencing facilities may not be readily available.

Highly sensitive magnetic-microparticle-based aptasensor for Cryptosporidium parvum oocyst detection in river water and wastewater: Effect of truncation on aptamer affinity.

Many methods have been reported to detect Cryptosporidium parvum (C. parvum) oocysts in the water environment using monoclonal antibodies. Herein, we report the use of DNA aptamers as an alternative ligand. We present the highly sensitive detection of C. parvum oocysts in wastewater samples based on aptamer-conjugated magnetic beads. A previously selected DNA aptamer (R4-6) that binds to C. parvum oocysts with high affinity and selectivity was rationally truncated into two minimer aptamers (Min_Crypto1 and Min_Crypto2), and conjugated to micro-magnetic beads. In flow cytometry tests with phosphate buffer, river water, and wastewater samples, both the minimers showed improved affinity and specificity toward C. parvum oocysts than the parent R4-6. Moreover, Min_Crypto2 showed higher affinity to its target than the parent aptamer when testing in wastewater, indicating superior binding properties in a complex matrix. Using a fluorescence microplate-based assay, and when incubated with different numbers of oocysts, Min_Crypto2 showed a limit of detection as low as 5 C. parvum oocysts in 300 µL of wastewater. Results described here indicate that Min_Crypto2 has superior specificity and sensitivity for the detection of C. parvum oocysts, and has a strong potential to be used successfully in a sensor.

Microbiological quality of blue mussels (Mytilus edulis) in Nunavik, Quebec: a pilot study.

This pilot study was aimed at documenting the presence of fecal indicators and enteric pathogens in blue mussels (Mytilus edulis) from 6 communities in Nunavik, Quebec. One to four 2 kg samples of mussels were collected at low tide in each community. Samples were investigated by enumeration methods for the fecal indicators enterococci, Escherichia coli, F-specific coliphages, Clostridium perfringens, and by molecular identification for the pathogens norovirus, Salmonella spp., Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, verocytotoxin-producing E. coli (particularly serovar O157:H7), Shigella spp., and Yersinia enterocolitica. In 5 communities, the presence of Giardia duodenalis and Cryptosporidium spp. was also tested by microscopy and molecular methods and that of Toxoplasma gondii was tested by molecular methods. Apart from small quantities of Clostridium perfringens in 2 samples, no bacterial or viral pathogens were detected in the mussels. Toxoplasma gondii was also not detected. However, G. duodenalis and Cryptosporidium spp. were present in 18% and 73% of the samples investigated for these pathogens, respectively. When considering the indicators and the viral and bacterial pathogens investigated, the mussels examined were of good microbiological quality, but considering the presence of potentially zoonotic protozoa, it should be recommended that consumers cook the molluscs well before eating them.

Temporal changes in the prevalence and shedding patterns of Giardia duodenalis cysts and Cryptosporidium spp. oocysts in a herd of dairy calves in Ontario.

Giardia duodenalis and Cryptosporidium spp. infections, and the patterns of cyst and oocyst shedding, were observed in a herd of dairy calves in Ontario over a period of 3 mo. Cysts and oocysts were detected and enumerated in fecal samples using immunofluorescence microscopy; Giardia and Cryptosporidium DNA was detected using the polymerase chain reaction. The prevalence of G. duodenalis increased during the course of the study, reaching a peak of 93.1% when calves were 43 to 54 d old, and then decreased. Conversely, Cryptosporidium spp. prevalence was highest (75.9%) when calves were 11 to 22 d old, and subsequently decreased. The numbers of cysts and oocysts shed per gram of feces were positively correlated over time with the respective prevalence rates. Along with genotyping data, temporal changes in prevalence and shedding patterns should be considered when testing dairy calves for the presence and concentrations of cysts and oocysts, and when considering the potential for zoonotic transmission.

Cryptosporidium spp.: Human incidence, molecular characterization and associated exposures in Québec, Canada (2016-2017).

The aim of this study was to describe the epidemiology of human cryptosporidiosis in Québec from 2016 to 2017 and to identify possible exposures associated with the disease, and the dominant Cryptosporidium species in circulation. A descriptive analysis was performed on data collected from the provincial notifiable infectious diseases registry and the epidemiological investigation. Fecal sample were sent to the Laboratoire de santé publique du Québec for molecular characterization. In Québec, from January 1, 2016 to December 31, 2017, a total of 201 confirmed cases of cryptosporidiosis were notified. A peak in the number of reported cases was observed at the end of the summer. The regional public health department with the highest adjusted incidence rate for sex and age group for both years was that of Nunavik, in the north of Québec. A higher average annual incidence rate was observed for females between the ages of 20 to 34 years compared to males. Overall, for both males and females the distribution appeared to be bimodal with a first peak in children younger than five years old and a second peak in adults from 20 to 30 years of age. Molecular characterization showed that 23% (11/47) of cases were infected with C. hominis while 74% (35/47) were infected with C. parvum. Meanwhile, subtyping results identified by gp60 sequencing, show that all C. parvum subtypes belonged to the IIa family, whereas the subtypes for C. hominis belonged to the Ia, Ib, and Id families. Finally, the epidemiological investigation showed that diarrhea was the most common reported symptom with 99% (72/73) of investigated cases having experienced it. This first brief epidemiological portrait of cryptosporidiosis in Québec has allowed for the description, both at the provincial and regional level, of the populations that could be particularly vulnerable to the disease.

Benchmarking hybrid assemblies of Giardia and prediction of widespread intra-isolate structural variation.

BACKGROUND: Currently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genomes. Subsequent addition of highly accurate short reads to long-read assemblies further improves assembly quality. Using this hybrid approach, we assembled genomes for three Giardia isolates, two with published assemblies and one novel, to evaluate the improvement in genome quality gained from long reads. We then used the long reads to predict structural variants to examine this previously unexplored source of genetic variation in Giardia. METHODS: With MinION reads for each isolate, we assembled genomes using several assemblers specializing in long reads. Assembly metrics, gene finding, and whole genome alignments to the reference genomes enabled direct comparison to evaluate the performance of the nanopore reads. Further improvements from adding Illumina reads to the long-read assemblies were evaluated using gene finding. Structural variants were predicted from alignments of the long reads to the best hybrid genome for each isolate and enrichment of key genes was analyzed using random genome sampling and calculation of percentiles to find thresholds of significance. RESULTS: Our hybrid assembly method generated reference quality genomes for each isolate. Consistent with previous findings based on SNPs, examination of heterozygosity using the structural variants found that Giardia BGS was considerably more heterozygous than the other isolates that are from Assemblage A. Further, each isolate was shown to contain structural variant regions enriched for variant-specific surface proteins, a key class of virulence factor in Giardia. CONCLUSIONS: The ability to generate reference quality genomes from a single MinION run and a multiplexed MiSeq run enables future large-scale comparative genomic studies within the genus Giardia. Further, prediction of structural variants from long reads allows for more in-depth analyses of major sources of genetic variation within and between Giardia isolates that could have effects on both pathogenicity and host range.