Swimming Pool-Related Outbreak of a Rare gp60 Subtype of Cryptosporidium hominis, England, October 2016

In October 2016, Public Health England was initially notified of four cases of cryptosporidiosis among users of two swimming pools. We investigated to identify further cases, the outbreak source, and ensure the implementation of appropriate control measures. Probable primary cases had diarrhoea and reported swimming in the pools 1–12 days prior to illness; confirmed cases were verified by the reference laboratory. Secondary cases had contact with primary cases 1–12 days prior to illness. We identified twenty-two cases: eleven were primary (eight confirmed) and eleven were secondary (five confirmed). Four cases were infected with C. parvum (different gp60 subtypes); all were primary and swam at two pools. Seven primary and secondary cases were infected with C. hominis gp60 subtype IdA16, and all were associated one pool. Failings in pool water treatment and management were identified that likely contributed to the load on the filters and their efficiency. Our investigation identified a complex outbreak, with secondary transmission, involving exposures to two swimming pools. C. hominis IdA16 is rare; it has been isolated from only three previous UK cases. We hypothesize that C. hominis cases arose from a common exposure, and the C. parvum cases were likely sporadic. This investigation highlights the value of integrating epidemiology and microbiology to investigate clusters of Cryptosporidium cases, defining the extent of the outbreak and the likely transmission pathways

Novel real-time PCR assays for the specific detection of human infective Cryptosporidium species

Cryptosporidium is a protozoan parasite causing gastrointestinal illness. Drinking waterborne outbreaks have been caused by C. hominis, C. parvum and C. cuniculus. Molecular detection techniques already exist for Cryptosporidium and usually target housekeeping genes. We set ourselves the task to identify species-specific genes. These genes are likely to be involved in host parasite interaction and virulence. Three subtelomeric species-specific putative virulence factor genes (Cops-2, Chos-1 and Chos-2) were identified in silico and used to develop novel real-time PCR assays. Our results show that Chos-2 is a suitable target for probe-based assays for the specific detection of C. hominis and C. cuniculus (two very closely related species) and that Cops-2 is a suitable target for specific detection of C. parvum

Multi-locus analysis of human infective Cryptosporidium species and subtypes using ten novel genetic loci

Background: Cryptosporidium is a protozoan parasite that causes diarrheal illness in a wide range of hosts including humans. Two species, C. parvum and C. hominis are of primary public health relevance. Genome sequences of these two species are available and show only 3-5% sequence divergence. We investigated this sequence variability, which could correspond either to sequence gaps in the published genome sequences or to the presence of species-specific genes. Comparative genomic tools were used to identify putative species-specific genes and a subset of these genes was tested by PCR in a collection of Cryptosporidium clinical isolates and reference strains. Results: The majority of the putative species-specific genes examined were in fact common to C. parvum and C. hominis. PCR product sequence analysis revealed interesting SNPs, the majority of which were species-specific. These genetic loci allowed us to construct a robust and multi-locus analysis. The Neighbour-Joining phylogenetic tree constructed clearly discriminated the previously described lineages of Cryptosporidium species and subtypes. Conclusions: Most of the genes identified as being species specific during bioinformatics in Cryptosporidium sp. are in fact present in multiple species and only appear species specific because of gaps in published genome sequences. Nevertheless SNPs may offer a promising approach to studying the taxonomy of closely related species of Cryptosporidia

Unusual Cryptosporidium Genotypes in Human Cases of Diarrhea

Several Cryptosporidium spp. are known to infect humans, but most cases of illness are caused by Cryptosporidium hominis or C. parvum. During a long-term genotyping in the United Kingdom, we identified 3 unusual Cryptosporidium genotypes (skunk, horse, and rabbit) in human patients with diarrhea

Development of a gp60-subtyping method for Cryptosporidium felis

Background: Feline cryptosporidiosis is an increasing problem, especially in catteries. In humans, close contact with cats could be a potential source of infection although the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low. Sequencing of the 60-kDa glycoprotein gene is a commonly used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium species. However, until now the sequence of gp60 from C. felis has not been available and genotyping has been limited to less discriminatory markers, such as 18S rRNA, COWP and HSP70. Methods: We have identified the gp60 orthologue within the genome sequence of C. felis, and used the sequence to design a nested PCR for subtyping purposes. A total of 128 clinical isolates of both feline and human origin, were used to evaluate the marker. Results: Sequence analysis revealed large variations between the different samples. The C. felis gp60 lack the characteristic serine-tract found in many other cryptosporidian orthologues, instead it has an insertion of variable length (361-742 nt). Also, two cases of suspected zoonotic transmission of C. felis between cats and humans were successfully confirmed. Conclusions: We have identified the gp60 gene in C. felis and show how this highly variable marker can be used in epidemiological investigations

Analysis of the Cryptosporidium spp. and gp60 subtypes linked to human outbreaks of cryptosporidiosis in England and Wales, 2009 to 2017

Abstract Background Cryptosporidium spp. are important causes of gastroenteritis that can be transmitted from humans and animals. We elucidated the distribution of species and gp60 subtypes in human outbreaks classified by transmission vehicle. Methods We used a combined database of national outbreak surveillance and reference unit data to analyse outbreaks by setting, vehicle, season, and linkage with suspected sources. Results A total of 178 outbreaks involving 4031 laboratory confirmed cases were identified; 82 (46%) outbreaks involved recreational waters, 74 (42%) animal contact, 4 (2%) environmental contact, 4 (2%) person-to-person spread, 3 (2%) food, 2 (1%) drinking water supplies, and 9 (5%) were of unknown source. The infecting Cryptosporidium sp. was identified in 131 (74%) outbreaks; 69 were C. parvum, 60 C. hominis, and in two outbreaks cases were infected with either species. Animal contact, environmental contact, and food-borne outbreaks were exclusively C. parvum and were mainly in first half of the year. Recreational water outbreaks were predominantly C. hominis and were mainly in the second half of the year. Outbreaks attributed to person-to-person spread were exclusively C. hominis and all occurred in October. Both C. parvum and C. hominis caused drinking waterborne outbreaks. Gp60 subtypes were identified from patients in 48 C. parvum and 38 C. hominis outbreaks, revealing more subtypes among C. parvum (n = 14) than C. hominis (n = 7) outbreaks. Cryptosporidium hominis IbA10G2 predominated (30 outbreaks). Of C. parvum subtypes, IIaA15G2R1 predominated (17 outbreaks), followed by IIaA17G1R1 (12 outbreaks), IIaA19G1R1 (four outbreaks), and other subtypes caused three or fewer outbreaks each. Linkage between cases and suspected sources by gp60 subtype was established in nine animal contact, three swimming pool, and one drinking water outbreak. Conclusions The public health benefit of identifying infecting species and subtypes was twofold: (i) identifying and strengthening epidemiologic links between cases; and (ii) indicating possible exposures and sources to inform outbreak management. Gp60 subtype refined the epidemiological investigations, but a multilocus genotyping scheme would provide further benefit. Characterisation of Cryptosporidium spp. and subtypes needs to shift from predominantly supporting outbreak investigations to becoming nationally systematic

Sporadic Human Cryptosporidiosis Caused by Cryptosporidium cuniculus, United Kingdom, 2007–2008

To investigate sporadic human cryptosporidiosis trends in the United Kingdom, we tested 3,030 Cryptosporidium spp.–positive fecal samples, submitted for routine typing in 2007–2008, for C. cuniculus. C. cuniculus prevalence was 1.2%; cases were mostly indigenous and occurred across all age groups. Most occurred during August–October and may be linked to exposure opportunities

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

BACKGROUND: Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. RESULTS: The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. CONCLUSION: This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples

Meeting Report: Application of Genotyping Methods to Assess Risks from Cryptosporidium in Watersheds

A workshop titled “Application of Genotyping Methods to Assess Pathogen Risks from Cryptosporidium in Drinking Water Catchments” was held at the International Water Association biennial conference, Marrakech, Morocco, 23 September 2004. The workshop presented and discussed the findings of an interlaboratory trial that compared methods for genotyping Cryptosporidium oocysts isolated from feces. The primary goal of the trial and workshop was to assess the utility of current Cryptosporidium genotyping methods for determining the public health significance of oocysts isolated from feces in potable-water–supply watersheds. An expert panel of 16 watershed managers, public health practitioners, and molecular parasitologists was assembled for the workshop. A subordinate goal of the workshop was to educate watershed management and public health practitioners. An open invitation was extended to all conference delegates to attend the workshop, which drew approximately 50 interested delegates. In this report we summarize the peer consensus emerging from the workshop. Recommendations on the use of current methods by watershed managers and public health practitioners were proposed. Importantly, all the methods that were reported in the trial were mutually supporting and found to be valuable and worthy of further utility and development. Where there were choices as to which method to apply, the small-subunit ribosomal RNA gene was considered to be the optimum genetic locus to target. The single-strand conformational polymorphism method was considered potentially the most valuable for discriminating to the subtype level and where a large number of samples were to be analyzed. A research agenda for protozoan geneticists was proposed to improve the utility of methods into the future. Standardization of methods and nomenclature was promoted