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.

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.

Giardia duodenalis typing from stools: a comparison of three approaches to extracting DNA, and validation of a probe-based real-time PCR typing assay.

A weighted, multi-attribute approach was used to compare three methods for direct extraction of Giardia duodenalis DNA from 15 microscopy-positive stools: (1) a QIAamp spin-column method for stools including a 10 min incubation at 95 °C, (2) method 1 preceded by five freeze-thaw cycles and (3) bead beating with guanidine thiocyanate using a FastPrep-28 machine followed by liquid-phase silica purification of DNA. The attributes compared included DNA yield measured using a new triose phosphate isomerase (tpi) gene probe-based real-time PCR, also described here. All three methods shared 100 % PCR positivity, while the bead-beating method provided the highest G. duodenalis DNA yield (P<0.01). However, when other weighted attributes, including biocontainment, resources and technical requirements, were also considered, spin-column extraction with prior freeze-thaw treatment (method 2) was deemed the most desirable and was selected for use. The tpi real-time PCR typing assay was designed to discriminate between the main human infectious assemblages of G. duodenalis (A and B) and was evaluated initially using standard isolates. Validation using microscopy-positive stools from 78 clinical giardiasis cases revealed 100 % typability; 20 (26 %) samples contained assemblage A, 56 (72 %) assemblage B and two (3 %) assemblages A and B. While the epidemiological significance of assemblage distribution will be revealed as more isolates are typed and analysed with patient demographic and exposure data, the utility of this assay and its ready application in our laboratory workflow and result turnaround margins is already evident.

A comparison of two approaches to extracting Cryptosporidium DNA from human stools as measured by a real-time PCR assay.

Direct extraction of Cryptosporidium DNA from 46 stools by bead-beating, guanidine thiocyanate and silica purification provided slightly lower PCR positivity (93.5% vs. 100%) and higher threshold cycle values (mean 34.93 vs. 28.03; P=0.00) than spin-column extraction from boiled, semi-purified oocyst suspensions. However, direct extraction is cheaper, and amenable to automation.

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).

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.

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.

Investigation of farms linked to human patients with cryptosporidiosis in England and Wales.

The study investigates farms suspected of being sources of zoonotic human cryptosporidiosis. A variety of implicated farm animal species were sampled and tested to detect Cryptosporidium oocysts and investigate genetic linkage with human patients. Risk factor information was collected from each farm and analysed by multivariable logistic regression to detect significant associations between factors and Cryptosporidium in animals. The results showed that average sample prevalence of Cryptosporidium infection was highest in cattle, sheep and pigs ( approximately 40-50%), in the mid-range in goats and horses (20-25%) and lowest in rabbits/guinea pigs, chickens and other birds ( approximately 4-7%). A single sample from a farm dog was also positive. Cryptosporidium parvum, which has zoonotic potential, was the commonest species and was most likely to be present in cattle and, to a lesser extent, in sheep. In particular, young calves and lambs shed C. parvum and this finding was corroborated in a statistical model which demonstrated that samples from groups of preweaned animals were 11 times, and immature animal groups six times, more likely to be positive than groups of adult animals, and that samples from a farm with a cattle enterprise were twice as likely to be positive than farms without a cattle enterprise. On seven out of eight farms, at least one C. parvum isolate from an animal sample was indistinguishable at the gp60 locus from those found in the human patients, indicating that farm animals are a likely source of infection for humans.

Long-term Cryptosporidium typing reveals the aetiology and species-specific epidemiology of human cryptosporidiosis in England and Wales, 2000 to 2003.

To improve understanding of the aetiology and epidemiology of human cryptosporidiosis, over 8,000 Cryptosporidium isolates were submitted for typing to the species level over a four year period. The majority were either Cryptosporidium parvum (45.9%) or Cryptosporidium hominis (49.2%). Dual infection occurred in 40 (0.5%) cases and six other known Cryptosporidium species or genotypes were found in 67 (0.9%) cases. These were Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis, and the Cryptosporidium cervine, horse and skunk genotypes. The remaining 3.5% were not typable. Epidemiology differed between infecting species. C. parvum cases were younger, although C. hominis was more prevalent in infants under one year and in females aged 15 to 44 years. Spring peaks in cases reported to national surveillance were due to C. parvum, while C. hominis was more prevalent during the late summer and early autumn as well as in patients reporting recent foreign travel. Temporal and geographical differences were observed and a decline in C. parvum cases persisted from 2001. Typing of isolates allowed outbreaks to be more clearly delineated, and demonstrated anthroponotic spread of C. parvum as well as C. hominis. Our findings suggest that national surveillance for Cryptosporidium should be conducted at the species level.

Investigation of the role of companion animals in the zoonotic transmission of cryptosporidiosis.

Companion animals owned by human patients with cryptosporidiosis (cases) and those animals owned by the wider human population (controls), were studied to determine whether Cryptosporidium was more likely to be excreted by case animals than controls. A total of 280 recently voided faecal samples (114 case animals and 166 control animals) were collected and tested by immunomagnetic separation and immunofluorescent microscopy. A multivariable model was also created to identify pet characteristics, contacts and management factors associated with Cryptosporidium infection in animals, using information collected by a standardized questionnaire. The model was designed to take into account the clustering of samples at the owner level and whether the sampled animal was a case or control.

Cryptosporidiosis in two alpaca (Lama pacos) holdings in the South-West of England.

Cryptosporidiosis was investigated on two alpaca (Lama pacos) holdings in the South-West of England. Diagnosis was initially confirmed in a cria with diarrhoea from each holding. Cohort faeces samples were subsequently collected and examined for presence of Cryptosporidium oocysts by immunofluorescence microscopy. On the first holding, 30 samples (24 adults, 6 crias) were tested, and oocysts were detected in three of the cria samples but in none of the adults. On the second holding, 14 floor faeces samples representing apparently healthy crias and one faeces sample from a cria with diarrhoea were collected. Oocysts were detected in four of the "healthy" faeces samples and the sample of diarrhoeic faeces. All isolates were confirmed as Cryptosporidium parvum using polymerase chain reaction restriction fragment length polymorphism of the cryptosporidium oocyst wall protein (COWP) and ssu rRNA genes. Sequence analysis of a 741bp region of ssu rDNA was carried out on nine of these and revealed high sequence homology with previously reported C. parvum isolates. This investigation highlights the possibility of alpaca crias subclinically shedding oocysts, which has implications for epidemiology and transmission in animals as well as raising zoonotic concerns for human contacts. Gene sequencing of UK isolates from South American camelids is also described for the first time.

Serological responses to Cryptosporidium in human populations living in areas reporting high and low incidences of symptomatic cryptosporidiosis.

One approach to investigating differences in the reported incidence of disease is to measure the extent of exposure to the organism in question by testing for a specific antibody response. IgG responses to Cryptosporidium sporozoite antigens of low molecular size in adults have been shown to be consistent and of sufficient intensity to act as reliable markers of exposure. This study used a western blot procedure to investigate the relative intensity of IgG antibody responses to the 15/17-kDa Cryptosporidium sporozoite antigen complex and the 27-kDa antigen in sera from two cities in north-west England: Liverpool (low numbers of clinical cases reported) and Preston (high numbers reported). The intensity of antibody response to the 15/17-kDa antigen complex was significantly greater in the Liverpool sera, but there was no significant difference in intensity of response to the 27-kDa antigen. The relationship between diagnosed and reported cryptosporidiosis infections and infections identified by serological testing is complex, but could indicate a protective effect resulting from either exposure to non-pathogenic strains or from repeated low-level exposure to pathogenic strains.

Sample prevalence and molecular characterisation of Cryptosporidium andersoni within a dairy herd in the United Kingdom.

Bovine cryptosporidiosis is usually an acute diarrhoeal disease of young calves caused by Cryptosporidium parvum. However, chronic infection with Cryptosporidium andersoni has been associated with gastritis, reduced milk yield and poor weight gain in adult cattle. Here we describe the first genetic confirmation and characterisation of C. andersoni from cattle in the United Kingdom and its sample prevalence within a dairy herd. Oocysts measured 7.5+/-0.4 microm x 5.5+/-0.4 microm (7.0-8.5 microm x 4.5-6.5 microm) with a length-to-width ratio of 1.37 (1.08-1.60). The within-herd sample prevalence was 16% (95% confidence intervals=10.4-21.6%). Nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and sequence analysis of the small subunit rDNA was used to confirm the species and characterise the isolates. Due to the lack of overt, acute, clinical symptoms, the incidence, prevalence and importance of this parasite is probably currently underestimated in cattle in the UK. The potential for zoonotic transmission is unknown.

Age-specific seroprevalence to an immunodominant Cryptosporidium sporozoite antigen in a Brazilian population.

The seroepidemiology of Cryptosporidium infection was investigated in a representative sample of a normal population in the State of Sao Paulo, Brazil using a recombinant form of the immunodominant 27-kDa sporozoite antigen. IgG seropositivity was low in infants following loss of maternal antibody but quickly increased to approximately 60% by 5 years, then 80% by the age of 10 years, after which prevalence remained constant. The broad range of antibody concentrations is consistent with previous reports that the IgG response to C. parvum is short-lived. There is also evidence that average antibody concentrations increase with age. Results suggest that the recombinant antigen may be a more sensitive method of measuring seroprevalence than the native antigen in Western blot. Although cross-sectional studies can provide an insight into the epidemiology of C. parvum in normal populations, further studies investigating the dynamics of the humoral immune responses to Cryptosporidium and the use of serology in epidemiological studies are required.

Blinded evaluation of DNA extraction and genotyping of stained Cryptosporidium on glass slides.

AIMS: A blinded trial was performed on Cryptosporidium genotyping using polymerase chain reaction (PCR)/restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein (COWP) gene between DNA extracted from oocyst suspensions as compared with DNA from fixed and stained faecal smears on glass microscope slides. METHODS AND RESULTS: Sixty-five faecal smears on slides were stained by one of three different methods comprising 50 positives and 15 negatives as determined by the observation of Cryptosporidium oocysts by microscopy. The expected result in terms of detection and the COWP genotype detected was achieved using DNA extracted from 94% of the slides tested. CONCLUSIONS: This study shows that DNA, which can be amplified by PCR, is present in stained smears on glass microscope slides. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be useful for molecular epidemiological studies on a range of gastrointestinal pathogens where samples are collected from locations remote from the testing laboratory.

Cryptosporidium in farmed animals: the detection of a novel isolate in sheep.

We describe the discovery of polymorphisms in the Cryptosporidium oocyst wall protein (COWP) gene conferring a novel restriction fragment length polymorphism (RFLP) pattern in 26/60 (43%) isolates from a flock of sheep sampled following a waterborne outbreak of human cryptosporidiosis. The sheep isolates showed identical PCR-RFLP patterns to each other by COWP genotyping but different from those of most currently recognised genotypes, including the major Cryptosporidium parvum genotypes 1 and 2. Sequence analysis of the 550bp amplicon from the COWP gene was compared with a DNA coding region employed in previous studies and showed the novel isolate to differ from other Cryptosporidium species and C. parvum isolates by 7-21%. The sheep-derived isolates were compared at this and further three Cryptosporidium gene loci with isolates from other farmed animals. The loci employed were one in the thrombospondin related adhesive protein (TRAP-C2) gene and two in the 70kDa heat shock protein (HSP70) gene (CPHSP1 and 2). Other animal samples tested in our laboratory were from clinically ill animals and all contained C. parvum genotype 2. The sheep in which the novel isolate was identified were healthy and showed no symptoms of cryptosporidiosis, and the novel sheep isolate could represent a non-pathogenic strain. Our studies suggest that a previously undetected Cryptosporidium sub-type may exist in sheep populations, reflecting the increasingly recognised diversity within the parasite genus.

Modification of a rapid method for the identification of gene-specific polymorphisms in Cryptosporidium parvum and its application to clinical and epidemiological investigations.

The application of genotyping to clinical isolates of Cryptosporidium has increased significantly our knowledge and understanding of the distribution and epidemiology of this parasite. However, some methods can be laborious and demand specialist technical expertise. PCR-restriction fragment length polymorphism (RFLP) techniques represent a more rapid and simple method of genotyping to support epidemiological and clinical investigations than conventional DNA analytical techniques. We describe a nested PCR-RFLP technique that identifies polymorphisms in the C. parvum thrombospondin-related adhesive protein gene locus; this method offers a sensitive and specific tool for the confirmation and investigation of disease associated with C. parvum. The potential of this enhanced method is demonstrated by its application to the confirmation and epidemiological investigation of an outbreak of cryptosporidiosis associated with a school visit to an open farm.