Search

Researchers describe a rapid method for genotyping Cryptosporidium parvum from clinical and environmental samples and used it to investigate an outbreak of cryptosporidiosis in schoolchildren who had visited a farm. The method is based on a nested PCR-RFLP technique that identifies polymorphisms of the bacterial gene for thrombospondin-related adhesive protein.

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. [ABSTRACT FROM AUTHOR]

Objectives To determine at what glycated haemoglobin (HbA1c) level physicians from eight European countries would initiate insulin in type 2 diabetes, which physician or practice related factors influenced this level and whether physicians would differentiate between a younger uncomplicated patient and an older patient with comorbidities.Design Cross-sectional study with data from the Guideline Adherence to Enhance Care study.Setting and participants 410 physicians from both primary and secondary care from Belgium, France, Germany, Italy, Ireland, Sweden, the Netherlands and the UK.Outcome measures Physicians were asked at which HbA1c level they would initiate insulin for a young, uncomplicated patient (vignette 1) and for an older, complicated patient (vignette 2). We evaluated differences in HbA1c levels between physicians from different countries using analysis of variance. To identify physician and practice related factors associated with HbA1c level at initiation of insulin, we performed multivariable linear regression. Multiple imputation was used to deal with missing data.Results In Germany, Ireland, Sweden, the Netherlands and the UK, the HbA1c levels for initiating insulin in vignette 2 (range: 60.0 to 66.0 mmol/mol; 7.6% to 8.2%) were higher than for vignette 1 (range: 57.2 to 64.2 mmol/mol; 7.4% to 8.0%). In multivariable analysis, the HbA1c level at which insulin was initiated only differed between countries (vignette 1): Dutch physicians initiated insulin at a lower HbA1c level compared with Belgium, France and the UK. No physician or practice factors were independently associated with HbA1c level at insulin initiation.Conclusions When deciding on individualised HbA1c targets for insulin initiation, physicians from five countries took patient’s age and comorbidity into account. The HbA1c level at which physicians would initiate insulin therapy differed between countries.

UK Cryptosporidium hominis isolates have previously shown slight PCR fragment length polymorphism at multiple loci. To further investigate transmission, we conducted a case-control study and sequenced the GP60 locus from 115 isolates. Nine subtypes were identified; IbA10G2 predominated. Having a non-IbA10G2 subtype was significantly linked to recent travel outside Europe. [ABSTRACT FROM AUTHOR]

The article focuses on the proposition that in the military architecture of the early seventeenth century it is not only mathematics that plays an important part in the planning and layout of the fortifications. The site, the existing infrastructure and cost-reducing measures also play important and even decisive roles. This proposition is defended on the basis of the analysis of the layout of the new fortifications around the town of Groningen as they were planned and built in the first quarter of the seventeenth century. The first part describes the planning process, the discussions between the Frisian stadtholder Willem-Lodewijk, prince Maurits, the States General and the town councilors of Groningen. In addition, a number of alternative plans are brought up: Johan van Valckenburg, Hindrick van der Merwede and Garwer Peters. In the second part a reconstruction follows of the variables that the planners had to take into account. This chiefly concerns differences in height occurring on the site. This assumption is supported by the discovery of three cartographic documents from various collections. In the fourth part the form plays an important part. How large should the town become, and what was feasible within the scope of the theory and practice of fortress building? The distance between the bastions is relatively fixed, but this is also the case with the exit roads from the existing town. Choices had to be made, but which? The eventual, reconstructed form of Groningen is surprising: the ellipse. The next phase in the total planning process was the financing. The town would become larger, and not everyone is pleased with this. Nevertheless, also through investments in large-scale projects in the peat districts, but especially through the support of the States General, the town managed to arrange the financing. The article shows the complex planning process, including a number of decisions that were not taken on the basis of the theory of fortress building, but on the basis of the practical experience of the ‘man in the field’. Was it a well-considered concept or a question of ad hoc adjustments made later on?

The article focuses on cryptosporidiosis, an enteric disease in human beings and animal species caused by cryptosporidium parvum. It features a research by G.C. Pritchard and his colleagues on orphan lambs in Great Britain. Faecal samples were subjected to diagnostic postmortem examination. Multivariable analysis and other facts about the epidemiological survey of flocks are also presented.

Abstract 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.

Background: We report clinical, epidemiological, and laboratory features of a large diarrhea outbreak caused by a novel Cryptosporidium hominis subtype during British military training in Kenya between February and April 2022., Methods: Data were collated from diarrhea cases, and fecal samples were analyzed on site using the multiplex polymerase chain reaction (PCR) BioFire FilmArray. Water was tested using Colilert kits (IDEXX, UK). DNA was extracted from feces for molecular characterization of Cryptosporidium A135 , Lib13 , ssu rRNA , and gp60 genes., Results: One hundred seventy-two of 1200 (14.3%) personnel at risk developed diarrhea over 69 days. One hundred six primary fecal samples were tested, and 63/106 (59.4%; 95% CI, 0.49%-0.69%) were positive for Cryptosporidium spp. Thirty-eight had Cryptosporidium spp. alone, and 25 had Cryptosporidium spp. with ≥1 other pathogen. A further 27/106 (25.5%; 95% CI, 0.18%-0.35%) had non- Cryptosporidium pathogens only, and 16/106 (15.1%; 95% CI, 0.09%-0.23%) were negative. C. hominis was detected in 58/63 (92.1%) Cryptosporidium spp.-positive primary samples, but the others were not genotypable. Twenty-seven C. hominis specimens were subtypable; 1 was gp60 subtype IeA11G3T3, and 26 were an unusual subtype, ImA13G1 (GenBank accession OP699729), supporting epidemiological evidence suggesting a point source outbreak from contaminated swimming water. Diarrhea persisted for a mean (SD) of 7.6 (4.6) days in Cryptosporidium spp. cases compared with 2.3 (0.9) days in non- Cryptosporidium cases ( P = .001)., Conclusions: Real-time multiplex PCR fecal testing was vital in managing this large cryptosporidiosis outbreak. The etiology of a rare C. hominis gp60 subtype emphasizes the need for more genotypic surveillance to identify widening host and geographic ranges of novel C. hominis subtypes., Competing Interests: Potential conflicts of interest. All authors: no reported conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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.

Introduction. Cryptosporidium presents one of the main waterborne public health threats due to its resistance to chlorine disinfection and ability to cause large-scale outbreaks. The standard method used in the UK water industry for detection and enumeration of Cryptosporidium is based on fluorescence microscopy and is laborious and expensive. Molecular methods such as quantitative polymerase chain reaction (qPCR) can be more amenable to streamlining through automation, improving workflows and standardizing procedures. Hypothesis. The null hypothesis was that there was no difference in the detection or enumeration between the standard method and a qPCR. Aim. We aimed to develop and evaluate a qPCR for the detection and enumeration of Cryptosporidium in drinking water, and to compare the assay with the standard method used in the UK. Methodology. We first developed and evaluated a qPCR method by incorporating an internal amplification control and calibration curve into a real-time PCR currently used for Cryptosporidium genotyping. Then we compared the qPCR assay with the standard method of immunofluorescent microscopy for the detection and enumeration of 10 and 100 Cryptosporidium oocysts in 10 l of artificially contaminated drinking water. Results. The results demonstrated that detection of Cryptosporidium by this qPCR was reliable at low numbers of oocysts; however, enumeration was less reliable and more variable than immunofluorescence microscopy. Conclusions. Despite these results, qPCR offers practical advantages over microscopy. There is potential for the use of PCR-based methods for Cryptosporidium analysis if parts of the upstream sample preparation are revised, and alternative technologies for enumeration (such as digital PCR) are also explored to improve analytical sensitivity.

Introduction. In England and Wales, cryptosporidiosis cases peak in spring and autumn, associated with zoonotic/environmental exposures ( Cryptosporidium parvum, spring/autumn) and overseas travel/water-based activities ( Cryptosporidium hominis, autumn). Coronavirus disease 2019 (COVID-19) restrictions prevented social mixing, overseas travel and access to venues (swimming pools/restaurants) for many months, potentially increasing environmental exposures as people sought alternative countryside activities. Hypothesis. COVID-19 restrictions reduced incidence of C. hominis cases and potentially increased incidence of C. parvum cases. Aim. To inform/strengthen surveillance programmes, we investigated the impact of COVID-19 restrictions on the epidemiology of C. hominis and C. parvum cases. Methodology. Cases were extracted from the Cryptosporidium Reference Unit (CRU) database (1 January 2015 to 31 December 2021). We defined two periods for pre- and post-COVID-19 restrictions implementation, corresponding to before and after the first UK-wide lockdown on 23 March 2020. We conducted a time series analysis, assessing differences in C. parvum and C. hominis incidence, trends and periodicity between these periods. Results. There were 21 304 cases ( C. parvum =12 246; C. hominis =9058). Post-restrictions implementation incidence of C. hominis dropped by 97.5 % (95 % CI: 95.4-98.6 %; P <0.001). The decreasing incidence trend pre-restrictions was not observed post-restrictions implementation due to lack of cases. No periodicity change was observed post-restrictions implementation. There was a strong social gradient; there was a higher proportion of cases in deprived areas. For C. parvum , post-restrictions implementation incidence fell by 49.0 % (95 % CI: 38.4-58.3 %; P <0.001). There was no pre-restrictions incidence trend but an increasing incidence trend post-restrictions implementation. A periodicity change was observed post-restriction implementation, peaking 1 week earlier in spring and 2 weeks later in autumn. The social gradient was the inverse of that for C. hominis . Where recorded, 22 % of C. hominis and 8 % of C. parvum cases had travelled abroad. Conclusion. C. hominis cases almost entirely ceased post-restrictions implementation, reinforcing that foreign travel seeds infections. C. parvum incidence fell sharply but recovered post-restrictions implementation, consistent with relaxation of restrictions. Future exceedance reporting for C. hominis should exclude the post-restriction implementation period but retain it for C. parvum (except the first 6 weeks post-restrictions implementation). Infection prevention and control advice should be improved for people with gastrointestinal illness (GI) symptoms to ensure hand hygiene and swimming pool avoidance.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

Cryptosporidium is a leading cause of gastroenteritis (cryptosporidiosis), with significant morbidity and mortality worldwide. Irish cryptosporidiosis incidence rates are consistently the highest reported in Europe. A retrospective, longitudinal study of clinical Cryptosporidium isolates was conducted from 2015 to 2018 in Cork, southern Ireland. Overall, 86.5% of cases were attributed to C. parvum , while the remaining 13.5% were caused by C. hominis Despite the widespread implications of this protozoan parasite in sporadic and outbreak-related illness in Ireland, the current dearth of species-level epidemiological surveillance and clinical studies needs to be addressed in order to elucidate the national impact of this enteric pathogen., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Foodborne parasites with zoonotic potential are of particular concern for human health, being responsible for serious and potentially life threatening diseases. In the last decades, the development of molecular biology techniques have been successfully implemented for clinical diagnosis of FBPs in animal or human samples providing cheaper, less labor intensive, reliable and more sensitive tests. It is apparent from recent publications that unsubstantiated molecular methods for parasite detection that have undergone scant evaluation for sensitivity and specificity are becoming increasingly common. The aim of the organized Training Schools was to transfer knowledge on application, optimization and troubleshooting for methods used to extract, amplify, and sequence nucleic acids from contaminated matrices and isolated FBPs. The organized Training Schools fulfilled the trainees' expectations, whom acquired useful knowledge for their research activities., (Copyright © 2020 Elsevier Inc. All rights reserved.)