Analysis of Cryptosporidium spp. from clinical samples by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

AIM: To purify Cryptosporidium spp. oocysts from clinical stool samples and evaluate using an up-to-date mass spectrometry protocol producing high-quality reference spectra. METHODS AND RESULTS: A refined purification protocol was developed for oocysts from stools, involving salt flotation and potassium bromide density centrifugation. Purified oocysts were prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by formic acid extraction, and the extracts analysed using the Bruker MALDI Biotyper system. Individual spectral markers were identified by their specific mass peaks. Cryptosporidium parvum oocysts (Iowa strain) propagated in vivo, and C. parvum (n = 2) and Cryptosporidium hominis (n = 1) oocysts from clinical stool samples produced distinct spectra that were considered specific to Cryptosporidium spp. with no evidence of contamination. CONCLUSIONS: The production of distinct spectra demonstrated the utility of the purification method for oocysts from clinical stool samples and provided reference spectra. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of MALDI-TOF MS and other mass spectrometry techniques has been limited previously to C. parvum oocysts propagated in vivo. Appropriate purification of oocysts can achieve sufficient biomass, enabling analysis by MALDI-TOF MS and potentially other mass spectrometry platforms, facilitating peptide and protein discovery and identification of biomarkers from a much wider range of Cryptosporidium spp. from natural infections.

Health sequelae of human cryptosporidiosis-a 12-month prospective follow-up study.

To investigate long-term health sequelae of cryptosporidiosis, with especial reference to post-infectious irritable bowel syndrome (PI-IBS). A prospective cohort study was carried out. All patients with laboratory-confirmed, genotyped cryptosporidiosis in Wales, UK, aged between 6 months and 45 years of age, over a 2-year period were contacted. Five hundred and five patients agreed to participate and were asked to complete questionnaires (paper or online) at baseline, 3 and 12 months after diagnosis. The presence/absence of IBS was established using the Rome III criteria for different age groups. Two hundred and five of 505 cases completed questionnaires (40% response rate). At 12 months, over a third of cases reported persistent abdominal pain and diarrhoea, 28% reported joint pain and 26% reported fatigue. At both 3 and 12 months, the proportion reporting fatigue and abdominal pain after Cryptosporidium hominis infection was statistically significantly greater than after C. parvum. Overall, 10% of cases had sufficient symptoms to meet IBS diagnostic criteria. A further 27% met all criteria except 6 months' duration and another 23% had several features of IBS but did not fulfil strict Rome III criteria. There was no significant difference between C. parvum and C. hominis infection with regard to PI-IBS. Post-infectious gastrointestinal dysfunction and fatigue were commonly reported after cryptosporidiosis. Fatigue and abdominal pain were significantly more common after C. hominis compared to C. parvum infection. Around 10% of people had symptoms meriting a formal diagnosis of IBS following cryptosporidiosis. Using age-specific Rome III criteria, children as well as adults were shown to be affected.

First Cryptosporidium outbreak in Hungary, linked to a treated recreational water venue in 2015.

In June 2015, an outbreak of cryptosporidiosis with 35 cases (23 probable and 12 laboratory-confirmed) occurred among 191 attendees of a residential rehabilitation holiday for paediatric organ transplant patients (n = 49) and their families at a hotel in Somogy county, Hungary. The overall attack rate was 18%. Most of the cases were transplanted children who experienced severe acute disease and required adjustment to their tacrolimus immunosuppression. A retrospective case-control study suggested an association between recreational water exposures and illness: cases were seven times more likely than controls to have swum in the children's pool (odds ratio 7.17; 95% confidence interval 2.9-17.2; P < 0.0001) and five times more likely to have used the jetted whirlpool (odds ratio 5.25; 95% confidence interval 2.1-13.1; P < 0.0001). This was the first outbreak of cryptosporidiosis in Hungary and it is especially unfortunate that it affected vulnerable children who experienced severe symptoms. Cryptosporidium presents specific infection control difficulties in treated recreational water venues; the link to a whirlpool is unusual and highlights the importance of the age-appropriate use of these facilities and reminding users not to immerse their heads or swallow the water. Cryptosporidiosis is more commonly linked to children' pools where improved bather hygiene and promoting exclusion of diarrhoea cases could help to avoid similar outbreaks.

An evaluation of health protection practices for the investigation and management of Cryptosporidium in England and Wales.

Background: Cryptosporidium is a major cause of gastroenteritis (cryptosporidiosis). Case and outbreak report rates vary geographically, which may in part reflect public health practice. Methods: To examine the public health management of cryptosporidiosis, an online questionnaire was administered to the 28 Health Protection Teams (HPTs) in England and Wales in 2014. Practices for investigation and management of cases and outbreaks were compared. Results: Practice varied among the 24 (86%) respondents in terms of who undertook actions (HPT or Local Authority) to investigate and manage cryptosporidiosis. HPTs without exceedance monitoring detected fewer outbreaks (1/5, 20%) than those with it (13/19, 68%) (P = 0.12), and those that always administered a risk-factor questionnaire detected more outbreaks (12/19, 63%) than those who did this only sometimes (2/5, 40%) (P = 0.62). Significantly more HPTs with a system to detect common exposures reported at least one outbreak (14/19, 74%) compared to HPTs with no system (0/5) (P = 0.01). Conclusions: Applying exceedance monitoring, using a standardized questionnaire taking into account the incubation period for Cryptosporidium, and having a structured system to detect common exposures increased outbreak detection. Information about all cases should be shared between local public health authorities, and current guidance used for the prevention of spread.

Cryptosporidiosis outbreak in visitors of a UK industry-compliant petting farm caused by a rare Cryptosporidium parvum subtype: a case-control study.

A case-control study was conducted to investigate an outbreak of 46 cases of cryptosporidiosis in visitors to a petting farm in England. Details of exposures on the farm were collected for 38 cases and 39 controls, recruited through snowball sampling. Multivariable logistic regression identified that cases were 5·5 times more likely than controls to have eaten without washing their hands [95% confidence interval (CI) 1·51-19·9, P = 0·01] and 10 times less likely to report being informed of risk of infection on arrival (odds ratio 0·10, 95% CI 0·01-0·71, P = 0·02). An uncommon Cryptosporidium parvum gp60 subtype (IIaA19G1R1) was identified in a lamb faecal sample and all subtyped cases (n = 22). We conclude that lack of verbal advice and non-compliance with hand washing are significantly associated with a risk of cryptosporidiosis on open farms. These findings highlight the public health importance of effectively communicating risk to petting farm visitors in order to prevent future outbreaks of zoonotic infections.

Development and Application of a gp60-Based Typing Assay for Cryptosporidium viatorum.

The apicomplexan intestinal parasites of the genus Cryptosporidium take a major toll on human and animal health and are frequent causes of waterborne outbreaks. Several species and genotypes can infect humans, including Cryptosporidium viatorum, which, to date, has only been found in humans. Molecular characterization of Cryptosporidium spp., critical to epidemiological analyses, is commonly based on gp60 gene analysis, which appears to require bespoke species- or group-specific PCR primers due to extensive genetic diversity across the genus. In this study, we amplified, sequenced, and characterized the gp60 gene of C. viatorum for the first time. Moreover, we developed and validated a gp60 typing assay for this species and applied it to 27 isolates originating from Asia, Africa, and Central America. A single subtype family, XVa, was identified containing multiple alleles.

Approaches to the detection of very small, common, and easily missed outbreaks that together contribute substantially to human Cryptosporidium infection.

Water supply-associated cryptosporidiosis outbreaks have decreased in England since the application of risk reduction measures to public water supplies. We hypothesized that smaller outbreaks were occurring which could be better detected by enhanced surveillance. Rolling analysis of detailed questionnaire data was applied prospectively in a population of 2·2 million in the south of England in 2009 and 2010. Detection of spatiotemporal clusters using SaTScan was later undertaken retrospectively. Together these approaches identified eight outbreaks, compared to an expectation of less than one based on national surveillance data. These outbreaks were small and associated with swimming pool use or, less commonly, direct (e.g. petting-farm) contact with animals. These findings suggest that frequent small-scale transmission in swimming pools is an important contributor to disease burden. Identification of swimming pool-level risk factors may inform preventative measures. These findings and the approaches described may be applicable to many other populations and to some other diseases.

An outbreak of cryptosporidiosis at a swimming club--can rapid field epidemiology limit the spread of illness?

In September 2010, an outbreak of cryptosporidiosis affected members of a swimming club. A cohort study was undertaken to identify the number affected and risk factors for infection. Of 101 respondents, 48 met the case definition for probable cryptosporidiosis. Multivariate analysis demonstrated a strong and highly significant association between illness and attendance at a training session on 13 September 2010 (adjusted odds ratio 28, P < 0.0001). No faecal incidents were reported and pool monitoring parameters were satisfactory. The competitive nature of club swimming requires frequent training and participation in galas, potentially facilitating contamination into other pools and amplification of outbreaks among wider groups of swimmers. There was a lack of awareness of the 2-week exclusion rule among swimmers and coaches, and a high level of underreporting of illness. The study demonstrates the benefits of rapid field epidemiology in identifying the true burden of illness, the source of infection and limiting spread.

Seizure secondary to cardiac arrythmias.

BACKGROUND: The relationship between seizure activity and arrhythmias is not well established. Sinus tachycardia is commonly seen post seizure. Bradyarrythmias leading to seizure activity is rarely reported in the literature thus far. CASE PRESENTATION: A 62-year-old lady presented with nocturnal seizures and was subsequently started on oral anti-convulsant with no relief of her symptoms. Further investigations led to outpatient ambulatory ECGs which showed marked sinus bradycardia prior to seizure activity and post seizure atrial fibrillation with fast ventricular rate. The patient was commenced on oral anticoagulant and beta blocker for ventricular rate control in view of atrial fibrillation. A backup dual chamber permanent pacemaker was implanted to treat the bradycardia. The patient became seizure free since the implantation of permanent pacemaker. CONCLUSION: This case stresses the importance of investigating and treating cardiac arrhythmias underlying seizure activity.

Glycine receptor antibody mediated Progressive Encephalomyelitis with Rigidity and Myoclonus (PERM): a rare but treatable neurological syndrome.

A 40-year-old man presented with respiratory compromise and was intubated. After tracheostomy, he was found to have ophthalmoplegia, severe limb rigidity, stimulus-sensitive myoclonus and autonomic dysfunction. For 1 week before admission, there had been a prodromal illness with low mood, hallucinations and limb myoclonus. Serum glycine receptor antibodies were strongly positive: we diagnosed progressive encephalomyelitis with rigidity and myoclonus. Despite a relapse, he has done well following immunotherapies. The clinical syndrome of encephalomyelitis with rigidity, described in 1976, often has a severe progressive course. A minority of patients have glutamic acid decarboxylase antibodies. The association with glycine receptor antibody was first reported in 2008, and we briefly review subsequent case reports to illustrate the range of clinical features. The antibody is likely to be disease mediating, although this remains unproven. The spectrum of diagnosable and treatable antibody mediated neurological syndromes is expanding. It is vital to recognise these conditions early to reduce morbidity and mortality.

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Phylogenomic reconstruction of Cryptosporidium spp. captured directly from clinical samples reveals extensive genetic diversity.

Cryptosporidium is a leading cause of severe diarrhea and mortality in young children and infants in Africa and southern Asia. More than twenty Cryptosporidium species infect humans, of which C. parvum and C. hominis are the major agents causing moderate to severe diarrhea. Relatively few genetic markers are typically applied to genotype and/or diagnose Cryptosporidium. Most infections produce limited oocysts making it difficult to perform whole genome sequencing (WGS) directly from stool samples. Hence, there is an immediate need to apply WGS strategies to 1) develop high-resolution genetic markers to genotype these parasites more precisely, 2) to investigate endemic regions and detect the prevalence of different genotypes, and the role of mixed infections in generating genetic diversity, and 3) to investigate zoonotic transmission and evolution. To understand Cryptosporidium global population genetic structure, we applied Capture Enrichment Sequencing (CES-Seq) using 74,973 RNA-based 120 nucleotide baits that cover ~92% of the genome of C. parvum. CES-Seq is sensitive and successfully sequenced Cryptosporidium genomic DNA diluted up to 0.005% in human stool DNA. It also resolved mixed strain infections and captured new species of Cryptosporidium directly from clinical/field samples to promote genome-wide phylogenomic analyses and prospective GWAS studies.

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Subclinical infection and asymptomatic carriage of gastrointestinal zoonoses: occupational exposure, environmental pathways, and the anonymous spread of disease.

Asymptomatic carriage of gastrointestinal zoonoses is more common in people whose profession involves them working directly with domesticated animals. Subclinical infections (defined as an infection in which symptoms are either asymptomatic or sufficiently mild to escape diagnosis) are important within a community as unknowing (asymptomatic) carriers of pathogens do not change their behaviour to prevent the spread of disease; therefore the public health significance of asymptomatic human excretion of zoonoses should not be underestimated. However, optimal strategies for managing diseases where asymptomatic carriage instigates further infection remain unresolved, and the impact on disease management is unclear. In this review we consider the environmental pathways associated with prolonged antigenic exposure and critically assess the significance of asymptomatic carriage in disease outbreaks. Although screening high-risk groups for occupationally acquired diseases would be logistically problematical, there may be an economic case for identifying and treating asymptomatic carriage if the costs of screening and treatment are less than the costs of identifying and treating those individuals infected by asymptomatic hosts.

Simultaneous increase of Cryptosporidium infections in the Netherlands, the United Kingdom and Germany in late summer season, 2012.

Starting August 2012, an increase in Cryptosporidium infections was reported in the Netherlands, the United Kingdom and Germany. It represented a 1.8 to 4.9-fold increase compared to previous years. Most samples were C. hominis IbA10G2. A case­control study was performed in the Netherlands but did not identify an endemic source. A case­case study in the north of England found travel abroad to be the most common risk factor.

A conserved sorting-associated protein is mutant in chorea-acanthocytosis.

Chorea-acanthocytosis (CHAC, MIM 200150) is an autosomal recessive neurodegenerative disorder characterized by the gradual onset of hyperkinetic movements and abnormal erythrocyte morphology (acanthocytosis). Neurological findings closely resemble those observed in Huntington disease. We identified a gene in the CHAC critical region and found 16 different mutations in individuals with chorea-acanthocytosis. CHAC encodes an evolutionarily conserved protein that is probably involved in protein sorting.

The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000-2008.

Routine typing of 14 469 isolates from human cryptosporidiosis cases between 2000 and 2008 revealed that 7439 (51·4%) were Cryptosporidium (C.) hominis, 6372 (44·0%) C. parvum, 51 (0·4%) both C. hominis and C. parvum, 443 (3·1%) were not typable and 164 (1·1%) were other Cryptosporidium species or genotypes. Of the latter, 109 were C. meleagridis, 38 C. felis, 11 C. ubiquitum, one C. canis, two horse, two novel and one skunk genotype. C. hominis monkey genotype and C. cuniculus were identified in a separate study. Patients with unusual infections were older than those with C. hominis (P<0·01) or C. parvum (P<0·01) and were more likely to be immunocompromised (Fisher's exact P<0·01). Forty-one percent of unusual cases had travelled abroad, mainly to the Indian subcontinent. Significant risk factors in those with unusual species were travel abroad (C. meleagridis, P<0·01), being immunocompromised (C. felis, Fisher's exact P=0·02), and contact with cats (C. felis, Fisher's exact P=0·02).

Epidemiology of anthroponotic and zoonotic human cryptosporidiosis in England and Wales, 2004-2006.

In order to monitor epidemiological trends, Cryptosporidium-positive samples (n=4509) from diarrhoeic patients were typed. Compared to the previous 4 years, the proportion of Cryptosporidium hominis cases in 2004-2006 increased to 57·3%, while 38·5% were C. parvum. The remaining 4·2% cases included mixed C. parvum and C. hominis infections, C. meleagridis, C. felis, C. ubiquitum and a novel genotype. When the typing results were combined with enhanced surveillance data to monitor risk exposures, C. hominis was linked to urban dwelling, previous diarrhoea in the household, any travel especially abroad, and using a swimming or paddling pool. C. parvum was linked to having a private water supply, contact with surface water, visiting or living on a farm, and contact with farm animal faeces. The proportion of laboratory-confirmed indigenous cases acquired from direct contact with farm animals was estimated to be 25% for C. parvum and 10% of all reported Cryptosporidium cases.

Longitudinal and spatial distribution of GP60 subtypes in human cryptosporidiosis cases in Ireland.

Within Europe, Ireland has one of the highest reported infection rates with the diarrhoeal protozoan pathogen Cryptosporidium. In this study 249 Cryptosporidium parvum isolates collected from Irish patients between 2000 and 2009 were subtyped by sequence analysis of the GP60 locus. A subsample of 127 isolates was also typed at the MS1 and ML1 loci. GP60 subtype IIaA18G3R1 was the predominant subtype in every year and every season throughout the country. Over the 10-year period there was no evidence that host immunity to the predominant subtype caused a shift in its prevalence. Length frequency distributions of the GP60 TCA/TCG repeats compiled from published data, showed distinct patterns for countries with predominantly zoonotic or anthroponotic transmission cycles, respectively. Although considered to be mostly affected by zoonotic cryptosporidiosis, the GP60 fragment length of Irish C. parvum isolates mirrored that of countries with predominantly human-to-human transmission, indicating more complex routes of infection between livestock and humans. Due to their homogeneity, ML1 and MS1 were not considered useful loci for subtyping C. parvum strains in Ireland.

A whole water catchment approach to investigating the origin and distribution of Cryptosporidium species.

AIMS: Investigating the distribution and origin of Cryptosporidium species in a water catchment affected by destocking and restocking of livestock as a result of a foot and mouth disease epidemic. METHODS AND RESULTS: Surface water, livestock and wildlife samples were screened for Cryptosporidium and oocysts characterised by sequencing SSU rRNA and COWP loci, and fragment analysis of ML1, ML2 and GP60 microsatellite loci. Oocyst concentrations in water samples (0-20.29 per 10 l) were related to rainfall events, amount of rainfall and topography. There was no detectable impact from catchment restocking. Cryptosporidium spp. found in water were indicative of livestock (Cryptosporidium andersoni and Cryptosporidium parvum) and wildlife (novel genotypes) sources. However, C. andersoni was not found in any animals sampled. Calf infections were age related; C. parvum was significantly more common in younger animals (<4 weeks old). Older calves shared Cryptosporidium bovis, Cryptosporidium ryanae and C. parvum. Wildlife shed C. parvum, Cryptosporidium ubiquitum, muskrat genotype II and deer genotype. CONCLUSIONS: Several factors affect the occurrence of Cryptosporidium within a catchment. In addition to farmed and wild animal hosts, topography and rainfall patterns are particularly important. SIGNIFICANCE AND IMPACT OF THE STUDY: These factors must be considered when undertaking risk-based water safety plans.