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. 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. 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. 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.

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. 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. This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.

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.

Background: Infection with the Cryptosporidium parasite causes over 4000 cases of diagnosed illness (cryptosporidiosis) in England and Wales each year. The incidence of sporadic disease has not been sufficiently established, and how frequently this arises from contact with other infected people is not well documented. This project aimed to explore potential transmission in the home and attempt to identify asymptomatic infections, which might play a role in transmission. Risk factors and characteristics associated with spread of infection in the home were described including any differences between Cryptosporidium species. Methods: The study identified cryptosporidiosis cases from North West England and Wales over a year and invited them and their household to take part. Each household was sent a study pack containing study information and a questionnaire, and stool sample kits to provide samples from consenting household members. Cryptosporidium-positive stool samples, identified by immunofluorescence microscopy, were characterised using molecular methods to help describe any patterns of transmission. Characteristics of households with and without additional cases were described, and compared using odds ratios (OR) and a multivariable logistic regression identified independent risk factors for household transmission. Data collection ran for one year, beginning in September 2018 with an initial pilot phase. Results: We enrolled 128 index cases and their households. Additional illness occurred in over a quarter of homes, each reporting an average of two additional cases. The majority of these were undiagnosed and unreported to surveillance. This burden was even greater in households where the index case was infected with C. hominis versus C. parvum, or the index case was under five years old, with mums and siblings most at risk of secondary infection. Only having an index case of C. hominis was independently associated with transmission in the multivariable model (OR 4.46; p = 0.01). Conclusions: Cryptosporidium was a considerable burden in the home. At-risk homes were those where the index was less than five years old and/or infected with C. hominis. Of particular risk were female caregivers and siblings. Hygiene advice should be specifically directed here. This work provides evidence for humans as sources of C. hominis infection and that person-person is a key pathway. We recommend that all stools submitted for the investigation of gastrointestinal pathogens are tested for Cryptosporidium to better capture cases, inclusion of speciation data in routine surveillance, and the consideration of specific clinical advice on prevention for high-risk homes.

Cryptosporidium has a wide range of hosts and routes of transmission, so public health investigations require Cryptosporidium species identification and discriminatory typing to enhance the microbiological evidence provided by routine diagnostic tests. A pioneering seven-locus genotyping scheme, based on multilocus variable number of tandem repeats analysis, has been validated and implemented for Cryptosporidium parvum by the national Cryptosporidium Reference Unit for England and Wales. In this paper, the journey to implementation as a service to support disease surveillance, epidemiology and outbreak investigations, and the resultant benefits to public health activities are described.

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.