Comparative genomics of Cryptosporidium parvum reveals the emergence of an outbreak-associated population in Europe and its spread to the United States.

The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g., IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water- and food-borne outbreaks. In this study, we generated new whole-genome sequence (WGS) data from 123 human- and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China, and the United States (n = 72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analyzed a data set from 141 isolates from the zoonotic groups IIa (n = 119) and IId (n = 22). Based on 28,047 high-quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: Isolates from China (IId) and Egypt (IIa and IId) formed population 1; a minority of European isolates (IIa and IId) formed population 2; and the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the United States (IIa) clustered in population 3. Based on analyses of the population structure, population genetics, and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the United Kingdom, reach the United States, where it also expanded. The reason(s) for the successful spread of population 3 remain elusive, although genes under selective pressure uniquely in this population were identified.

Global Population Genomics of Two Subspecies of Cryptosporidium hominis during 500 Years of Evolution.

Cryptosporidiosis is a major global health problem and a primary cause of diarrhea, particularly in young children in low- and middle-income countries (LMICs). The zoonotic Cryptosporidium parvum and anthroponotic Cryptosporidium hominis cause most human infections. Here, we present a comprehensive whole-genome study of C. hominis, comprising 114 isolates from 16 countries within five continents. We detect two lineages with distinct biology and demography, which diverged circa 500 years ago. We consider these lineages two subspecies and propose the names C. hominis hominis and C. hominis aquapotentis (gp60 subtype IbA10G2). In our study, C. h. hominis is almost exclusively represented by isolates from LMICs in Africa and Asia and appears to have undergone recent population contraction. In contrast, C. h. aquapotentis was found in high-income countries, mainly in Europe, North America, and Oceania, and appears to be expanding. Notably, C. h. aquapotentis is associated with high rates of direct human-to-human transmission, which may explain its success in countries with well-developed environmental sanitation infrastructure. Intriguingly, we detected genomic regions of introgression following secondary contact between the subspecies. This resulted in high diversity and divergence in genomic islands of putative virulence genes, including muc5 (CHUDEA2_430) and a hypothetical protein (CHUDEA6_5270). This diversity is maintained by balancing selection, suggesting a co-evolutionary arms race with the host. Finally, we find that recent gene flow from C. h. aquapotentis to C. h. hominis, likely associated with increased human migration, maybe driving the evolution of more virulent C. hominis variants.

Preliminary investigation of a significant national Cryptosporidium exceedance in the United Kingdom, August 2023 and ongoing.

Routine laboratory surveillance has identified an unprecedented and ongoing exceedance of Cryptosporidium spp. across the United Kingdom, notably driven by C. hominis transmission, since 14 August 2023. Information from 477 reported cases in England and Wales, followed up with a standardised exposure questionnaire as of 25 September 2023, identified foreign travel in 250 (54%) of 463 respondents and swimming in 234 (66%) of 353 cases. A significant, common exposure has not yet been identified in first analyses.

TIDE Analysis of Cryptosporidium Infections by gp60 Typing Reveals Obscured Mixed Infections.

BACKGROUND: Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. METHODS: Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. RESULTS: For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. CONCLUSIONS: We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

Cross-sectional household transmission study of Cryptosporidium shows that C. hominis infections are a key risk factor for spread.

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.

An outbreak of Cryptosporidium parvum linked to pasteurised milk from a vending machine in England: a descriptive study, March 2021.

We describe the investigations and management of a Cryptosporidium parvum outbreak of linked to consumption of pasteurised milk from a vending machine. Multiple locus variable number of tandem repeats analysis was newly used, confirming that C. parvum detected in human cases was indistinguishable from that in a calf on the farm. This strengthened the evidence for milk from an on-farm vending machine as the source of the outbreak because of post-pasteurisation contamination. Bacteriological indicators of post-pasteurisation contamination persisted after the initial hygiene improvement notice. We propose that on-farm milk vending machines may represent an emerging public health risk.

Development and evaluation of a real-time PCR for genotyping of Cryptosporidium spp. from water monitoring slides.

Cryptosporidium is an important cause of gastroenteritis globally and the main agent of waterborne outbreaks caused by protozoan parasites. Water monitoring for Cryptosporidium oocysts is by detection and enumeration using stained slide microscopy. Species identification (known as genotyping) may be undertaken post hoc and remains a specialist test, only undertaken in some laboratories. The benchmark method is nested PCR-sequencing of part of the SSU rRNA gene, but not all slides are typable and the workflow is cumbersome. We report the development, in-house validation and application of a real-time PCR-sequencing assay based on that gene, using a hydrolysis probe, for the detection and genotyping of all Cryptosporidium spp. The assay was investigated in two formats; a high volume DNA template for analysing all the DNA extracted from Cryptosporidium-positive water monitoring slides with <5 oocysts seen, and a lower volume DNA template permitting several technical replicates from slides with ≥5 oocysts seen where multiple species are more likely to be present. Each format conformed to the MIQE guidelines for amplification dynamics and was specific for Cryptosporidium spp. With high sensitivity, being capable of detecting and genotyping single oocysts by sequencing of a 435 bp amplicon. When 65 water monitoring slides with <5 oocysts seen were tested, slide typeability varied by sending laboratory (n = 9), and ranged from 22 to 60%. Typeability was 75% for slides with ≥5 oocysts seen that were submitted by a single laboratory. The laboratory workflow was improved by using real-time PCR, and decreased the time to result compared with nested PCR-sequencing. In practical application, there was no loss of typeability when the ≥5 oocysts assay was applied to all slides, irrespective of the number of oocysts present.

Cryptosporidium.

The protozoan Cryptosporidium is notorious for its resistance to chlorine disinfection, a mainstay of water treatment. Human infections, mainly of the small intestine, arise from consumption of faecally contaminated food or water, environmental exposure, and person-to-person or animal-to-person spread. Acute gastrointestinal symptoms can be prolonged but are usually self-limiting. Problems arise with immune-deficient, including malnourished, people including chronic diarrhoea, hepato-biliary tree and extra-gastrointestinal site infection, and few options for treatment or prevention exist. Although genomics has enabled refined classification, identification of chemotherapeutic targets and vaccine candidates, and putative factors for host adaption and pathogenesis, their confirmation has been hampered by a lack of biological tools.

Role of filtration in managing the risk from Cryptosporidium in commercial swimming pools - a review.

Most commercial swimming pools use pressurised filters, typically containing sand media, to remove suspended solids as part of the water treatment process designed to keep water attractive, clean and safe. The accidental release of faecal material by bathers presents a poorly quantified risk to the safety of swimmers using the pool. The water treatment process usually includes a combination of maintaining a residual concentration of an appropriate biocide in the pool together with filtration to physically remove particles, including microbial pathogens, from the water. However, there is uncertainty about the effectiveness of treatment processes in removing all pathogens, and there has been growing concern about the number of reported outbreaks of the gastrointestinal disease cryptosporidiosis, caused by the chlorine-resistant protozoan parasite Cryptosporidium. A number of interacting issues influence the effectiveness of filtration for the removal of Cryptosporidium oocysts from swimming pools. This review explains the mechanisms by which filters remove particles of different sizes (including oocyst-sized particles, typically 4-6 µm), factors that affect the efficiency of particle removal (such as filtration velocity), current recommended management practices, and identifies further work to support the development of a risk-based management approach for the management of waterborne disease outbreaks from swimming pools.

Genetic Diversity of Cryptosporidium hominis in a Bangladeshi Community as Revealed by Whole-Genome Sequencing.

We studied the genetic diversity of Cryptosporidium hominis infections in slum-dwelling infants from Dhaka over a 2-year period. Cryptosporidium hominis infections were common during the monsoon, and were genetically diverse as measured by gp60 genotyping and whole-genome resequencing. Recombination in the parasite was evidenced by the decay of linkage disequilibrium in the genome over <300 bp. Regions of the genome with high levels of polymorphism were also identified. Yet to be determined is if genomic diversity is responsible in part for the high rate of reinfection, seasonality, and varied clinical presentations of cryptosporidiosis in this population.

Cryptosporidium genotyping in Europe: The current status and processes for a harmonised multi-locus genotyping scheme.

Due to the occurrence of genetic recombination, a reliable and discriminatory method to genotype Cryptosporidium isolates at the intra-species level requires the analysis of multiple loci, but a standardised scheme is not currently available. A workshop was held at the Robert Koch Institute, Berlin in 2016 that gathered 23 scientists with appropriate expertise (in either Cryptosporidium genotyping and/or surveillance, epidemiology or outbreaks) to discuss the processes for the development of a robust, standardised, multi-locus genotyping (MLG) scheme and propose an approach. The background evidence and main conclusions were outlined in a previously published report; the objectives of this further report are to describe 1) the current use of Cryptosporidium genotyping, 2) the elicitation and synthesis of the participants' opinions, and 3) the agreed processes and criteria for the development, evaluation and validation of a standardised MLG scheme for Cryptosporidium surveillance and outbreak investigations. Cryptosporidium was characterised to the species level in 7/12 (58%) participating European countries, mostly for human outbreak investigations. Further genotyping was mostly by sequencing the gp60 gene. A ranking exercise of performance and convenience criteria found that portability, biological robustness, typeability, and discriminatory power were considered by participants as the most important attributes in developing a multilocus scheme. The major barrier to implementation was lack of funding. A structured process for marker identification, evaluation, validation, implementation, and maintenance was proposed and outlined for application to Cryptosporidium, with prioritisation of Cryptosporidium parvum to support investigation of transmission in Europe.

Suitability of loci for multiple-locus variable-number of tandem-repeats analysis of Cryptosporidium parvum for inter-laboratory surveillance and outbreak investigations.

Cryptosporidium parvum is the major cause of livestock and zoonotically-acquired human cryptosporidiosis. The ability to track sources of contamination and routes of transmission by further differentiation of isolates would assist risk assessment and outbreak investigations. Multiple-locus variable-number of tandem-repeats (VNTR) analysis provides a means for rapid characterization by fragment sizing and estimation of copy numbers, but structured, harmonized development has been lacking for Cryptosporidium spp. To investigate potential for application in C. parvum surveillance and outbreak investigations, we studied nine commonly used VNTR loci (MSA, MSD, MSF, MM5, MM18, MM19, MS9-Mallon, GP60 and TP14) for chromosome distribution, repeat unit length and heterogeneity, and flanking region proximity and conservation. To investigate performance in vitro, we compared these loci in 14 C. parvum samples by capillary electrophoresis in three laboratories. We found that many loci did not contain simple repeat units but were more complex, hindering calculations of repeat unit copy number for standardized reporting nomenclature. However, sequenced reference DNA enabled reproducible fragment sizing and inter-laboratory allele assignation based on size normalized to that of the sequenced fragments by both single round and nested polymerase chain reactions. Additional Cryptosporidium loci need to be identified and validated for robust inter-laboratory surveillance and outbreak investigations.

Cyclosporiasis in travellers returning to the United Kingdom from Mexico in 2017: lessons from the recent past to inform the future.

During the summers of 2015 and 2016, the United Kingdom experienced large outbreaks of cyclosporiasis in travellers returning from Mexico. As the source of the outbreaks was not identified, there is the potential for a similar outbreak to occur in 2017; indeed 78 cases had already been reported as at 27 July 2017. Early communication and international collaboration is essential to provide a better understanding of the source and extent of this recurring situation.

Discovery of new variable number tandem repeat loci in multiple Cryptosporidium parvum genomes for the surveillance and investigation of outbreaks of cryptosporidiosis.

Cryptosporidium parvum is a protozoan parasite causing gastro-intestinal disease (cryptosporidiosis) in humans and animals. The ability to investigate sources of contamination and routes of transmission by characterization and comparison of isolates in a cost- and time-efficient manner will help surveillance and epidemiological investigations, but as yet there is no standardised multi-locus typing scheme. To systematically identify variable number tandem repeat (VNTR) loci, which have been shown to provide differentiation in moderately conserved species, we interrogated the reference C. parvum Iowa II genome and seven other C. parvum genomes using a tandem repeat finder software. We identified 28 loci that met criteria defined previously for robust typing schemes for inter-laboratory surveillance, that had potential for generating PCR amplicons analysable on most fragment sizing platforms: repeats ≥6 bp, occurring in tandem in a single repeat region, and providing a total amplicon size of <300 bp including 50 bp for the location of the forward and reverse primers. The qualifying loci will be further investigated in vitro for consideration as preferred loci in the development of a robust VNTR scheme.

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.

Population structure of natural and propagated isolates of Cryptosporidium parvum, C. hominis and C. meleagridis.

The three protozoan species Cryptosporidium parvum, C. meleagridis and C. hominis (phylum Apicomplexa) are enteric pathogens of humans. The former two species are zoonotic and the latter is thought to infect only humans. To better characterize the structure and transmission of natural and laboratory-propagated isolates, we analyzed a collection of archived human and animal isolates of these three species by deep-sequencing polymerase chain reaction products amplified from a polymorphic sequence on chromosome 1. Thousands of screened 200-nucleotide sequences were analyzed to compare the diversity among samples, to assess the impact of laboratory propagation on population complexity and to identify taxonomically mixed isolates. Contrary to our expectation, repeated propagation in animals did not reduce intra-isolate diversity nor was diversity associated with host species. Significantly, in most samples, sequences characteristic of a different species were identified. The presence of C. hominis alleles in C. parvum and C. meleagridis isolates confirms earlier reports of mixed isolates and raises the possibility that the host range of C. hominis is broader than typically assumed. In a genetically divergent isolate of C. parvum, a majority of sequences was found to be recombinant, suggesting that this genotype originated from a C. parvum × C. hominis recombination event.

Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water.

The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples.

Outbreak of Cryptosporidium hominis following river flooding in the city of Halle (Saale), Germany, August 2013.

BACKGROUND: During weeks 32-33, 2013, 24 cases of cryptosporidiosis were notified in the city of Halle (annual mean 2008-2012: 9 cases). We investigated the outbreak to identify the source and recommend control measures, considering that between weeks 23-25 the river Saale which flows through the city centre overflowed the floodplain, parts of the city centre and damaged sewage systems. METHODS: We defined a case as a resident of Halle with gastroenteritis, Cryptosporidium-positive stool and disease onset weeks 27 through 47. In a case-control study among kindergarten children, we compared cases and controls regarding environmental exposure, use of swimming pools, zoo visits and tap water consumption 14 days pre-onset or a corresponding 14-days-period (controls) and adjusted for residence. Stool specimens were tested by microscopy and PCR, and Cryptosporidium DNA was sequenced. Samples from public water system, swimming pools and river Saale were examined for Cryptosporidium oocysts (microscopy and PCR). RESULTS: Overall, 167 cases were detected, 40/167 (24%) were classified as secondary cases. First disease onsets occurred during week 29, numbers peaked in week 34 and started to decrease in week 36. Median age was 8 years (range: 0-77). Compared to controls (n = 61), cases (n = 20) were more likely to report visits to previously flooded areas (OR: 4.9; 95%-CI: 1.4-18) and the zoo (OR: 2.6; 95%-CI: 0.9-7.6). In multivariable analysis visits to the floodplain remained the sole risk factor (OR: 5.5; 95%-CI: 1.4-22). Only C.hominis of a single genotype (IbA9G2) was detected in stools. Oocysts were detected in samples from the river, two local lakes and three public swimming pools by microscopy, but not in the public water supply. CONCLUSIONS: Evidence suggests that activities in the dried out floodplain led to infection among children. Secondary transmissions may be involved. Consequently, authorities recommended to avoid playing, swimming and having picnics in the flood-affected area. Health authorities should consider the potential health risks of long-term surviving parasites persisting on flooded grounds and in open waters even several weeks after the flooding and of bathing places close to sewage spill-overs. Preventive measures comprise water sampling (involving parasites), information of the public and prolonged closures of potentially contaminated sites.

Cryptosporidium in small water systems in Puerto Rico: a pilot study.

A pilot study was undertaken to investigate the occurrence of Cryptosporidium in four very small drinking water systems supplying communities in rural Puerto Rico. Water samples (40 L) were collected and oocysts were concentrated by calcium carbonate flocculation, recovered by immunomagnetic separation and detected by immunofluorescence microscopy. Cryptosporidium oocysts were identified in all four systems. This is the first report of evidence of the potential public health risk from this chlorine-resistant pathogen in Puerto Rican small water systems. Further work is warranted to fully assess the health risks that Cryptosporidium and other protozoa pose to populations served by community-managed small drinking water systems.