Use of a case-control study and control bank to investigate an outbreak of locally acquired cyclosporiasis in Canada, 2016.
ABSTRACT: Background:Cyclospora is an intestinal parasite that is not endemic in Canada. However, national outbreaks of locally acquired cases have been reported since 2013. These outbreaks were suspected to be associated with consumption of produce imported from countries where Cyclospora is endemic. Identification of the source can be challenging because of reporting delays and limited traceability of produce. Objective:To report on a national outbreak of locally acquired cyclosporiasis, highlight the challenges of investigating these outbreaks and document the first time use of a control bank to recruit controls for a national outbreak case-control study in Canada. Methods:Cases of cyclosporiasis were identified through provincial laboratory testing and reported through provinces to the national level. Cases were interviewed about food exposures using a questionnaire and food exposures reported by cases were compared to Foodbook reference values. To narrow down the food items of interest, a matched case-control study was conducted. Controls for the study were recruited primarily from a control bank, that is, a list of individuals who had previously agreed to participate in public health-related surveys. Results:In total, 87 cases of locally acquired cyclosporiasis with onset or report dates between May 19, 2016 and August 10, 2016 were reported by four provinces. Comparing case exposures to Foodbook reference values identified several food items of interest, including blackberries, other berries, herbs and leafy greens. The case-control study identified only blackberries and mesclun greens as significantly more frequently consumed by cases than controls. Due to lack of product details for blackberries and mesclun greens, the source of the outbreak was not conclusively identified. Conclusion:Blackberries were the primary food item of interest, but could not be identified as the conclusive source due to lack of traceability. The control bank was found to be a useful tool for control recruitment.
Project description:Cyclosporiasis is an infection caused by Cyclospora cayetanensis, which is acquired by consumption of contaminated fresh food or water. In the United States, cases of cyclosporiasis are often associated with foodborne outbreaks linked to imported fresh produce or travel to disease-endemic countries. Epidemiologic investigation has been the primary method for linking outbreak cases. A molecular typing marker that can identify genetically related samples would be helpful in tracking outbreaks. We evaluated the mitochondrial junction region as a potential genotyping marker. We tested stool samples from 134 laboratory-confirmed cases in the United States by using PCR and Sanger sequencing. All but 2 samples were successfully typed and divided into 14 sequence types. Typing results were identical among samples within each epidemiologically defined case cluster for 7 of 10 clusters. These findings suggest that this marker can distinguish between distinct case clusters and might be helpful during cyclosporiasis outbreak investigations.
Project description:Outbreaks of cyclosporiasis, a food-borne illness caused by the coccidian parasite Cyclospora cayetanensis have increased in the USA in recent years, with approximately 2300 laboratory-confirmed cases reported in 2018. Genotyping tools are needed to inform epidemiological investigations, yet genotyping Cyclospora has proven challenging due to its sexual reproductive cycle which produces complex infections characterized by high genetic heterogeneity. We used targeted amplicon deep sequencing and a recently described ensemble-based distance statistic that accommodates heterogeneous (mixed) genotypes and specimens with partial genotyping data, to genotype and cluster 648 C. cayetanensis samples submitted to CDC in 2018. The performance of the ensemble was assessed by comparing ensemble-identified genetic clusters to analogous clusters identified independently based on common food exposures. Using these epidemiologic clusters as a gold standard, the ensemble facilitated genetic clustering with 93.8% sensitivity and 99.7% specificity. Hence, we anticipate that this procedure will greatly complement epidemiologic investigations of cyclosporiasis.
Project description:Cyclosporiasis is caused by the coccidian parasite Cyclospora cayetanensis and is associated with large and complex food-borne outbreaks worldwide. Associated symptoms include severe watery diarrhea, particularly in infants, and immune dysfunction. With the globalization of human food supply, the occurrence of cyclosporiasis has been increasing in both food growing and importing countries. As well as being a burden on the health of individual humans, cyclosporiasis is a global public health concern. Currently, no vaccine is available but early detection and treatment could result in a favorable clinical outcome. Clinical diagnosis is based on cardinal clinical symptoms and conventional laboratory methods, which usually involve microscopic examination of wet smears, staining tests, fluorescence microscopy, serological testing, or DNA testing for oocysts in the stool. Detection in the vehicle of infection, which can be fresh produce, water, or soil is helpful for case-linkage and source-tracking during cyclosporiasis outbreaks. Treatment with trimethoprim-sulfamethoxazole (TMP-SMX) can evidently cure C. cayetanensis infection. However, TMP-SMX is not suitable for patients having sulfonamide intolerance. In such case ciprofloxacin, although less effective than TMP-SMX, is a good option. Another drug of choice is nitazoxanide that can be used in the cases of sulfonamide intolerance and ciprofloxacin resistance. More epidemiological research investigating cyclosporiasis in humans should be conducted worldwide, to achieve a better understanding of its characteristics in this regard. It is also necessary to establish in vitro and/or in vivo protocols for cultivating C. cayetanensis, to facilitate the development of rapid, convenient, precise, and economical detection methods for diagnosis, as well as more effective tracing methods. This review focuses on the advances in clinical features, diagnosis, and therapeutic intervention of cyclosporiasis.
Project description:Shiga toxin-producing Escherichia coli (STEC) cause substantial and costly illnesses. Leafy greens are the second most common source of foodborne STEC O157 outbreaks. We examined STEC outbreaks linked to leafy greens during 2009-2018 in the United States and Canada. We identified 40 outbreaks, 1,212 illnesses, 77 cases of hemolytic uremic syndrome, and 8 deaths. More outbreaks were linked to romaine lettuce (54%) than to any other type of leafy green. More outbreaks occurred in the fall (45%) and spring (28%) than in other seasons. Barriers in epidemiologic and traceback investigations complicated identification of the ultimate outbreak source. Research on the seasonality of leafy green outbreaks and vulnerability to STEC contamination and bacterial survival dynamics by leafy green type are warranted. Improvements in traceability of leafy greens are also needed. Federal and state health partners, researchers, the leafy green industry, and retailers can work together on interventions to reduce STEC contamination.
Project description:Cyclospora spp. in nonhuman primates are most closely related to Cyclospora cayetanensis, an emerging human pathogen causing outbreaks of cyclosporiasis in North America. Studies thus far indicate the possible existence of host specificity in Cyclospora spp. In this study, 411 fecal specimens from free-range rhesus monkeys (Macaca mulatta) were collected and examined for Cyclospora by sequence analysis of the small subunit rRNA gene. A novel Cyclospora species was identified in 28 (6.8%) specimens and named Cyclospora macacae based on morphologic and molecular characterizations. The oocyst of C. macacae is spherical and measures 8.49 ± 0.55 × 8.49 ± 0.49 μm in diameter. Phylogenetic analysis grouped this species together with the other four Cyclospora species infecting primates, including C. cayetanensis in humans, forming a monophyletic group closely related to avian Eimeria species. In addition, C. cayetanensis was detected in one specimen, although whether rhesus monkeys can serve as a natural reservoir host of C. cayetanensis needs further investigation.
Project description:In recent years, human cyclosporiasis has emerged as an important infection, with large outbreaks in the United States and Canada. Understanding the biology and epidemiology of Cyclospora has been difficult and slow and has been complicated by not knowing the pathogen s origins, animal reservoirs (if any), and relationship to other coccidian parasites. This report provides morphologic and molecular characterization of three parasites isolated from primates and names each isolate: Cyclospora cercopitheci sp.n. for a species recovered from green monkeys, C. colobi sp.n. for a parasite from colobus monkeys, and C. papionis sp.n. for a species infecting baboons. These species, plus C. cayetanensis, which infects humans, increase to four the recognized species of Cyclospora infecting primates. These four species group homogeneously as a single branch intermediate between avian and mammalian Eimeria. Results of our analysis contribute toward clarification of the taxonomic position of Cyclospora and its relationship to other coccidian parasites.
Project description:BACKGROUND:Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990's. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing. METHODS:We developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline. RESULTS:This approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study. CONCLUSIONS:Our results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.
Project description:BACKGROUND:Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. For effective outbreak identification and investigation, it is essential to rapidly assess the genetic heterogeneity of C. cayetanensis specimens from cluster cases and identify the likely occurrence of outbreaks. METHODS:In this study, we developed a quantitative PCR (qPCR) targeting the polymorphic link region between copies of the mitochondrial genome of C. cayetanensis, and evaluated the genetic heterogeneity among 36 specimens from six countries using melt curve, gel electrophoresis, and sequence analyses of the qPCR products. RESULTS:All specimens were amplified successfully in the qPCR and produced melt peaks with different Tm values in the melt curve analysis. In gel electrophoresis of the qPCR products, the specimens yielded bands of variable sizes. Nine genotypes were identified by DNA sequencing of the qPCR products. Geographical segregation of genotypes was observed among specimens analyzed, which could be useful in geographical source-tracking. CONCLUSIONS:The length and nucleotide sequence variations in the mitochondrial genome marker allow rapid assessment of the genetic heterogeneity among C. cayetanensis specimens by melt curve, gel electrophoresis, or DNA sequence analysis of qPCR products. The sequence data generated could be helpful in the initial source-tracking of the pathogen.
Project description:Cyclospora cayetanensis is an important cause for diarrhea in children in developing countries and foodborne outbreaks of cyclosporiasis in industrialized nations. To improve understanding of the basic biology of Cyclospora spp. and development of molecular diagnostic tools and therapeutics, we sequenced the complete apicoplast and mitochondrial genomes of C. cayetanensis.The genome of one Chinese C. cayetanensis isolate was sequenced using Roche 454 and Illumina technologies. The assembled genomes of the apicoplast and mitochondrion were retrieved, annotated, and compared with reference genomes for other apicomplexans to infer genome organizations and phylogenetic relationships. Sequence variations in the mitochondrial genome were identified by comparison of two C. cayetanensis nucleotide sequences from this study and a recent publication.The apicoplast and mitochondrial genomes of C. cayetanensis are 34,155 and 6,229 bp in size and code for 65 and 5 genes, respectively. Comparative genomic analysis showed high similarities between C. cayetanensis and Eimeria tenella in both genomes; they have 85.6% and 90.4% nucleotide sequence similarities, respectively, and complete synteny in gene organization. Phylogenetic analysis of the genomic sequences confirmed the genetic similarities between cecum-infecting avian Eimeria spp. and C. cayetanensis. Like in other coccidia, both genomes of C. cayetanensis are transcribed bi-directionally. The apicoplast genome is circular, codes for the complete machinery for protein biosynthesis, and contains two inverted repeats that differ slightly in LSU rRNA gene sequences. In contrast, the mitochondrial genome has a linear concatemer or circular mapping topology. Eight single-nucleotide and one 7-bp multiple-nucleotide variants were detected between the mitochondrial genomes of C. cayetanensis from this and recent studies.The apicoplast and mitochondrial genomes of C. cayetanensis are highly similar to those of cecum-infecting avian Eimeria spp. in both genome organization and sequences. The availability of sequence data beyond rRNA and heat shock protein genes could facilitate studies of C. cayetanensis biology and development of genotyping tools for investigations of cyclosporiasis outbreaks.
Project description:Due to recent outbreaks of cyclosporiasis associated with consumption of fresh berries, producers are demanding modern microbiological tools for the rapid and accurate identification of the human pathogen Cyclospora cayetanensis in berries and environmental samples. The aim of the present work was to develop a molecular tool based on a PCR approach for the rapid and accurate detection of C. cayetanensis. A nested PCR assay was validated for the amplification of a 294 bp size region of the 18S rRNA gene from C. cayetanensis. The limit of detection for the nested PCR assay was validated using 48 berry samples spiked with ~0, 10, 100, and 1000 oocyst per gram of sample. With this assay, it was possible to detect as few as 1 oocyst per gram of berry, in a 50 g sample. Sanger DNA sequencing and phylogenetic analysis were carried out to confirm the presence of C. cayetanensis in berry (n = 17) and soil (n = 5) samples. The phylogenetic analysis revealed that the C. cayetanensis sequences obtained from Mexico clustered within a group recovered from China, Peru, Guatemala-Haiti, and Japan. The PCR protocol designed in the present study could be an important tool for the rapid and accurate detection of this human pathogen in environmental and food samples.