Quantifying Replication Slippage Error in Cryptosporidium Metabarcoding Studies.

Genetic variation in Cryptosporidium, a common protozoan gut parasite in humans, is often based on marker genes containing trinucleotide repeats, which differentiate subtypes and track outbreaks. However, repeat regions have high replication slippage rates, making it difficult to discern biological diversity from error. Here, we synthesized Cryptosporidium DNA in clonal plasmid vectors, amplified them in different mock community ratios, and sequenced them using next-generation sequencing to determine the rate of replication slippage with dada2. Our results indicate that slippage rates increase with the length of the repeat region and can contribute to error rates of up to 20%.

From modern-day parasitology to paleoparasitology: the elusive past record and evolution of Cryptosporidium.

Recent efforts have been made to review the state of the art on a variety of questions and targets in paleoparasitology, including protozoan taxa. Meanwhile, these efforts seemed to let aside Cryptosporidium, and we then intended to review its paleoparasitological record to assess its past distribution and favored detection methods, and eventually highlight needed research trajectories. This review shows that contrary to other parasites, most of the positive results came from South-American sites and coprolites rather than sediment samples, highlighting the need to test this kind of material, notably in Europe where many negative results were reported in the published literature from sediment samples. Moreover, aDNA-based detections are nearly absent from the paleoparasitological record of this parasite, though punctually shown successful. With their potential to address the evolutionary history of Cryptosporidium species, notably through their 18S rRNA tree, aDNA-based approaches should be encouraged in the future. In sum, and though the limits of currently used methods and materials remain unclear, this review highlights the potential role of coprolites and aDNA for the study of Cryptosporidium species in the past and how this history shaped their current diversity and distribution, notably among human populations but also farm animals.

Children develop Immunity to cryptosporidiosis in a high transmission intensity area.

Background: Cryptosporidium is one of the top causes of diarrhea in Bangladesh infants. Cryptosporidium infections lead to the production of antibody immune responses, which were associated with a decrease in parasite burden and decreased disease severity in subsequent infections. Methods: We conducted a longitudinal study of cryptosporidiosis from birth to five years of age in an urban slum of Dhaka Bangladesh. We then retrospectively tested the concentration of anti-Cryptosporidium Cp17 or Cp23 IgA in surveillance stool samples collected from 54 children during their first 3 years of life by enzyme-linked immunosorbent assay (ELISA). We also assessed the concentration of both IgA and IgG antibodies specific to Cryptosporidium Cp17 and Cp23 in the concentration of anti-Cryptosporidium Cp17 or Cp23 IgA and IgG antibodies in the children's plasma (1- 5 years). Results: The seroprevalence of both anti- Cp23 and Cp17 antibodies was high at ≤ one year of age and reflected the exposure of these children in this community to cryptosporidiosis. In Bangladesh, the prevalence of cryptosporidiosis is high during the rainy season (June to October) but decreases during the dry season. In younger infants' plasma anti-Cp17 and Cp23 IgG and anti-Cp17 IgA levels were markedly increased during the rainy season in line with the higher initial exposure to the parasite at this time. Both anti-Cp17, anti-Cp23 fecal IgA and the parasite burden declined during repeat infections. Conclusions: We found that anti-Cryptosporidium plasma and fecal antibody levels in children could contribute to the decrease in new infections in this study population.

Subtyping of Nonhuman Primate-Adapted Cryptosporidium hominis in Macaca Fascicularis and Macaca mulatta in Yunnan Province, Southwestern China.

Background: Cryptosporidium spp. are a type of protozoan parasite responsible for causing diarrheal illness worldwide. They infect a broad range of vertebrate hosts, including both non-human primates (NHPs) and humans. In fact, zoonotic transmission of cryptosporidiosis from NHPs to humans is frequently facilitated by direct contact between the two groups. However, there is a need to enhance the information available on the subtyping of Cryptosporidium spp. in NHPs in the Yunnan province of China. Materials and Methods: Thus, the study investigated the molecular prevalence and species of Cryptosporidium spp. from 392 stool samples of Macaca fascicularis (n = 335) and Macaca mulatta (n = 57) by using nested PCR targeting the large subunit of nuclear ribosomal RNA (LSU) gene. Of the 392 samples, 42 (10.71%) were tested Cryptosporidium-positive. Results: All the samples were identified as Cryptosporidium hominis. Further, the statistical analysis revealed that age is a risk factor for the infection of C. hominis. The probability of detecting C. hominis was found to be higher (odds ratio = 6.23, 95% confidence interval 1.73-22.38) in NHPs aged between 2 and 3 years, as compared with those younger than 2 years. Sequence analysis of the 60 kDa glycoprotein (gp60) identified six (IbA9 n = 4, IiA17 n = 5, InA23 n = 1, InA24 n = 2, InA25 n = 3, and InA26 n = 18) C. hominis subtypes with "TCA" repeats. Among these subtypes, it has been previously reported that the Ib family subtypes are also capable of infecting humans. Conclusion: The findings of this study highlight the genetic diversity of C. hominis infection among M. fascicularis and M. mulatta in Yunnan province. Further, the results confirm that both these NHPs are susceptible to C. hominis infection, posing a potential threat to humans.

The occurrence and zoonotic potential of Cryptosporidium species in freshwater biota.

BACKGROUND: Protozoan pathogens from the genus Cryptosporidium cause the diarrhoeal disease cryptosporidiosis in humans and animals globally. Freshwater biota could act as potential reservoirs or zoonotic sources of Cryptosporidium infections for livestock and people, but Cryptosporidium occurrence in aquatic biota is largely unexplored. The aim of this study was to investigate the occurrence of Cryptosporidium in a range of freshwater organisms in upland rivers across England and Wales. METHODS: Fish were sampled by electrofishing, invertebrate larvae by kick sampling and the otter Lutra lutra and mink Mustela vison through faecal samples collected opportunistically as part of a nation-wide study. PCR targeting the small subunit ribosomal RNA gene was used to detect Cryptosporidium species. RESULTS: Cryptosporidium occurred in just 0.8% of all the samples and in none of 73 samples from nine invertebrate genera. Cryptosporidium was detected in two of 2/74 fish samples (2.7%), both salmonids, and in 2/92 otter faecal samples (2.17%), but there were no positive samples in mink (0/24) or the bullhead Cottus gobio (0/16). CONCLUSIONS: Low detection rate of human-infective Cryptosporidium species in aquatic fauna indicates they may present a low risk of contamination of some upland freshwaters.

Mammal-related Cryptosporidium infections in endemic reptiles of New Zealand.

New Zealand's endemic reptile fauna is highly threatened and pathogens causing infectious diseases may be a significant risk to already endangered species. Here, we investigate Cryptosporidium infection in captive endemic New Zealand reptiles. We found two mammal-related Cryptosporidium species (C. hominis and C. parvum) and six subtypes from three gp60 families (Ib, Ig and IIa) in 12 individuals of captive endemic Tuatara, Otago and Grand skinks, and Jewelled and Rough geckos. Cryptosporidium serpentis was identified in two Jewelled geckos using 18S. In New Zealand, C. hominis and C. parvum are associated with infections in humans and introduced domestic animals but have also been recently found in wildlife. Our finding of Cryptosporidium infection in endemic reptiles can help inform strategies to monitor the conservation of species and manage potential introductions of pathogens to in-situ and ex-situ populations.

An immunoinformatics analysis: design of a multi-epitope vaccine against Cryptosporidium hominis by employing heat shock protein triggers the innate and adaptive immune responses.

Cryptosporidium hominis, an anthropologically transferred species in the Cryptosporidium genus, represents many clinical studies in several countries. Its growth in the recent decade is primarily owing to epidemiologic studies. This parasite has complicated life cycles that require differentiation through a variety of phases of development and passage across two or more hosts throughout their lifetimes. As they move from host to host and environment to environment, pathogenic organisms are continually exposed to unexpected changes in the circumstances under which they develop. Heat shock proteins (HSPs) are targets of the host immune response; they are involved in the progression of diseases and play a significant part in this process. It has been discovered that the immunodominant immunogenic antigens in parasite infections HSPs. In this study, we have generated a multi-epitope vaccine against Cryptosporidium hominis (C. hominis) by using heat shock proteins. The epitopes that were selected had a substantial binding affinity for the B- and T-cell reference set of alleles, a high antigenicity score, a nature that was not allergic, a high solubility, non-toxicity and good binders. The epitopes were incorporated into a chimeric vaccine by using appropriate linkers. In order to increase the immunogenicity of the connected epitopes and effectively activate both innate and adaptive immunity, an adjuvant was attached to the epitopes. We have also analyzed the physiochemical characteristics of the vaccine which were satisfactory and then lead to the development of a 3D model. In addition, the binding confirmation of the vaccine to the TLR-4 innate immune receptor was also determined using molecular docking and molecular dynamics (MD) simulation. The results of this simulation show that the vaccine has a strong binding affinity for TLR4, which indicates that the vaccine is highly effective. In general, the vaccine that has been described here has a good potential for inducing protective and targeted immunogenicity, however, this hypothesis is contingent upon more experimental testing.Communicated by Ramaswamy H. Sarma.

Multiple introductions and recombination events underlie the emergence of a hyper-transmissible Cryptosporidium hominis subtype in the USA.

The parasite Cryptosporidium hominis is a leading cause of the diarrheal disease cryptosporidiosis, whose incidence in the United States has increased since 2005. Here, we show that the newly emerged and hyper-transmissible subtype IfA12G1R5 is now dominant in the United States. In a comparative analysis of 127 newly sequenced and 95 published C. hominis genomes, IfA12G1R5 isolates from the United States place into three of the 14 clusters (Pop6, Pop13, and Pop14), indicating that this subtype has multiple ancestral origins. Pop6 (IfA12G1R5a) has an East Africa origin and has recombined with autochthonous subtypes after its arrival. Pop13 (IfA12G1R5b) is imported from Europe, where it has recombined with the prevalent local subtype, whereas Pop14 (IfA12G1R5c) is a progeny of secondary recombination between Pop6 and Pop13. Selective sweeps in invasion-associated genes have accompanied the emergence of the dominant Pop14. These observations offer insights into the emergence and evolution of hyper-transmissible pathogens.

Exploratory Algorithm of a Multi-epitope-based Subunit Vaccine Candidate Against Cryptosporidium hominis: Reverse Vaccinology-Based Immunoinformatic Approach.

Cryptosporidiosis is the leading protozoan-induced cause of diarrheal illness in children, and it has been linked to childhood mortality, malnutrition, cognitive development, with retardation of growth. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in numerous nations, as well as its emergence in the last decade is largely due to detections obtained through noteworthy epidemiologic research. Nevertheless, there is no vaccine available, and the only licensed medication, nitazoxanide, has been demonstrated to have efficacy limitations in a number of patient groups recognized to be at high risk of complications. Therefore, current study delineates the computational vaccine design for Cryptosporidium hominis , the notable pathogen for enteric diarrhea. Firstly, a comprehensive literature search was conducted to identify six proteins based on their toxigenicity, allergenicity, antigenicity, and prediction of transmembrane helices to make up a multi-epitope-based subunit vaccine. Following that, antigenic non-toxic HTL epitope, CTL epitope with B cell epitope were predicted from the selected proteins and construct a vaccine candidate with adding an adjuvant and some linkers with immunologically superior epitopes. Afterwards, the constructed vaccine candidates and TLR2 receptor were put into the ClusPro server for molecular dynamic simulation to know the binding stability of the vaccine-TLR2 complex. Following that, Escherichia coli strain K12 was used as a cloning host for the chosen vaccine construct via the JCat server. As a result of the findings, it was resolute that the proposed chimeric peptide vaccine could improve the immune response to Cryptosporidium hominis.

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.

Cryptosporidium hominis Phylogenomic Analysis Reveals Separate Lineages With Continental Segregation.

Cryptosporidium is a leading cause of waterborne outbreaks globally, and Cryptosporidium hominis and C. parvum are the principal cause of human cryptosporidiosis on the planet. Thanks to the advances in Next-Generation Sequencing (NGS) sequencing and bioinformatic software development, more than 100 genomes have been generated in the last decade using a metagenomic-like strategy. This procedure involves the parasite oocyst enrichment from stool samples of infected individuals, NGS sequencing, metagenomic assembly, parasite genome computational filtering, and comparative genomic analysis. Following this approach, genomes of infected individuals of all continents have been generated, although with striking different quality results. In this study, we performed a thorough comparison, in terms of assembly quality and purity, of 100+ de novo assembled genomes of C. hominis. Remarkably, after quality genome filtering, a comprehensive phylogenomic analysis allowed us to discover that C. hominis encompasses two lineages with continental segregation. These lineages were named based on the observed continental distribution bias as C. hominis Euro-American (EA) and the C. hominis Afro-Asian (AA) lineages.

Cryptosporidium spp. diagnosis and research in the 21st century.

The protozoan parasite Cryptosporidium has emerged as a leading cause of diarrhoeal illness worldwide, posing a significant threat to young children and immunocompromised patients. While endemic in the vast majority of developing countries, Cryptosporidium also has the potential to cause waterborne epidemics and large scale outbreaks in both developing and developed nations. Anthroponontic and zoonotic transmission routes are well defined, with the ingestion of faecally contaminated food and water supplies a common source of infection. Microscopy, the current diagnostic mainstay, is considered by many to be suboptimal. This has prompted a shift towards alternative diagnostic techniques in the advent of the molecular era. Molecular methods, particularly PCR, are gaining traction in a diagnostic capacity over microscopy in the diagnosis of cryptosporidiosis, given the laborious and often tedious nature of the latter. Until now, developments in the field of Cryptosporidium detection and research have been somewhat hampered by the intractable nature of this parasite. However, recent advances in the field have taken the tentative first steps towards bringing Cryptosporidium research into the 21st century. Herein, we provide a review of these advances.

Long-Term Preservation and Storage of Faecal Samples in Whatman® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis.

Preservation and conservation of biological specimens, including faecal samples, is a challenge in remote areas or poor-resource settings where the cold chain cannot be maintained. This study aims at evaluating the suitability of filter cards for long-term storage of faecal samples of animal and human origin positive to the diarrhoea-causing protozoan parasites, Giardia duodenalis and Cryptosporidium hominis. Three commercially available Whatman® Filter Cards were comparatively assessed: the FTA® Classic Card, the FTA® Elute Micro Card, and the 903 Protein Saver Card. Human faecal samples positive to G. duodenalis (n = 5) and C. hominis (n = 5) were used to impregnate the selected cards at given storage (1 month, 3 months, and 6 months) periods and temperature (-20 °C, 4 °C, and room temperature) conditions. Parasite DNA was detected by PCR-based methods. Sensitivity assays and quality control procedures to assess suitability for genotyping purposes were conducted. Overall, all three Whatman® cards were proven useful for the detection and molecular characterisation of G. duodenalis and C. hominis under the evaluated conditions. Whatman® cards represent a simple, safe, and cost-effective option for the transportation, preservation, and storage of faecal samples without the need of the cold chain.

A novel genotyping method for Cryptosporidium hominis.

Cryptosporidiosis remains the leading protozoan induced cause of diarrhoea-associated mortality worldwide. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in many countries. This study applied high resolution melting analysis, a post-real-time PCR application, to the differentiation of six globally prevalent C. hominisgp60-subtypes. This novel method targeted three microsatellite, tandem repeat containing genetic markers, gp60, the genetic marker upon which current Cryptosporidium subtype nomenclature is based, MSB, and MSE, by which to differentiate between C. hominis isolates. This multi-locus approach successfully differentiated between all six C. hominisgp60-subtypes studied, some of which, such as IbA10G2, are known to exhibit global ubiquity. Thus, this method has the potential to be universally employed as a sensitive, cost effective and highly reproducible means to rapidly differentiate between C. hominisgp60-subtypes. Such a method would be of particular utility in epidemiological studies and outbreak scenarios, providing cost effective, clinically accessible alternative to DNA sequencing. The success of this preliminary study also supports further analysis of an expanded C. hominisgp60-subtype range and the potential expansion of the multi-locus panel in order to improve the discriminatory power of this approach.

Molecular epidemiology of Cryptosporidium spp. in an agricultural area of northern Vietnam: A community survey.

The purpose of this study was to investigate the occurrence of Cryptosporidium infection and the potential for transmission of Cryptosporidium spp. between animals and humans in northern Vietnam. A total of 2715 samples (2120 human diarrheal samples, 471 human non-diarrheal samples, and 124 animal stool samples) were collected through our community survey in an agricultural area. All samples were tested for Cryptosporidium spp. by direct immunofluorescence assay (DFA) using a fluorescent microscope. DNA extraction, PCR amplification of three genes (COWP, SSU-rRNA, and GP60), and sequencing analysis were performed to identify Cryptosporidium spp. Of 2715 samples, 15 samples (10 diarrheal samples, 2 non-diarrheal samples, and 3 animal stool samples) tested positive by PCR for the COWP gene. Three species of Cryptosporidium spp. were identified as C. canis (from six human diarrheal samples, two human non-diarrheal samples, and one dog sample), C. hominis (from four human diarrheal samples), and C. suis (from two pig samples) by sequencing the amplified COWP and/or SSU-rRNA genes. In terms of C. hominis, the GP60 subtype IeA12G3T3 was detected in all four human diarrheal samples. Although the number of positive samples was very small, our epidemiological data showed that the emerging pattern of each of the three species (C. canis, C. hominis, and C. suis) was different at this study site. While C. hominis and C. suis were only detected in human and pig samples, respectively, C. canis was detected in samples from both dogs and humans. We suspect that C. canis infections in humans at this study site may be due to environmental contamination with animal and human feces.

Molecular characterization of Cryptosporidium isolates from humans in Ontario, Canada.

BACKGROUND: Cryptosporidiosis is a gastrointestinal disease with global distribution. It has been a reportable disease in Canada since 2000; however, routine molecular surveillance is not conducted. Therefore, sources of contamination are unknown. The aim of this project was to identify species and subtypes of Cryptosporidium in clinical cases from Ontario, the largest province in Canada, representing one third of the Canadian population, in order to understand transmission patterns. METHODS: A total of 169 frozen, banked, unpreserved stool specimens that were microscopy positive for Cryptosporidium over the period 2008-2017 were characterized using molecular tools. A subset of the 169 specimens were replicate samples from individual cases. DNA was extracted directly from the stool and nested PCR followed by Sanger sequencing was conducted targeting the small subunit ribosomal RNA (SSU) and glycoprotein 60 (gp60) genes. RESULTS: Molecular typing data and limited demographic data were obtained for 129 cases of cryptosporidiosis. Of these cases, 91 (70.5 %) were due to Cryptosporidium parvum and 24 (18.6%) were due to Cryptosporidium hominis. Mixed infections of C. parvum and C. hominis occurred in four (3.1%) cases. Five other species observed were Cryptosporidium ubiquitum (n = 5), Cryptosporidium felis (n = 2), Cryptosporidium meleagridis (n = 1), Cryptosporidium cuniculus (n = 1) and Cryptosporidium muris (n = 1). Subtyping the gp60 gene revealed 5 allelic families and 17 subtypes of C. hominis and 3 allelic families and 17 subtypes of C. parvum. The most frequent subtype of C. hominis was IbA10G2 (22.3%) and of C. parvum was IIaA15G2R1 (62.4%). CONCLUSIONS: The majority of isolates in this study were C. parvum, supporting the notion that zoonotic transmission is the main route of cryptosporidiosis transmission in Ontario. Nonetheless, the observation of C. hominis in about a quarter of cases suggests that anthroponotic transmission is also an important contributor to cryptosporidiosis pathogenesis in Ontario.

The first UK report of a rare Cryptosporidium hominis genetic variant isolated during a complex Scottish swimming pool outbreak.

Cryptosporidium species are responsible for causing the majority of parasite-related gastrointestinal infections in the UK. This report describes an outbreak of 12 laboratory-confirmed cryptosporidiosis cases identified as part of a Scottish swimming pool investigation, with 9 primary and 3 secondary cases occurring over an 8-week period. Molecular speciation was successful for 11/12 cases, which revealed 10 Cryptosporidium hominis cases and 1 Cryptosporidium parvum case. Of the 10 C. hominis cases, further typing identified 7 as being an unusual sub-type, IbA6G3, which is the first description in the UK of this rare variant. The remaining three C. hominis cases were identified as the common IbA10G2 subtype. Following implementation of control measures on two occasions, no further cases were reported. This report highlights the importance of molecular typing to identify and characterize outbreaks, and emphasizes the need to adhere to swimming pool guidance. It also raises awareness of the potential for outbreaks to involve multiple species/sub-types, and emphasizes the importance of strong public health leadership to ensure effective multi-agency investigations and management of outbreaks.

Comparative genomics revealed adaptive admixture in Cryptosporidium hominis in Africa.

Cryptosporidiosis is a major cause of diarrhoeal illness among African children, and is associated with childhood mortality, malnutrition, cognitive development and growth retardation. Cryptosporidium hominis is the dominant pathogen in Africa, and genotyping at the glycoprotein 60 (gp60) gene has revealed a complex distribution of different subtypes across this continent. However, a comprehensive exploration of the metapopulation structure and evolution based on whole-genome data has yet to be performed. Here, we sequenced and analysed the genomes of 26 C. hominis isolates, representing different gp60 subtypes, collected at rural sites in Gabon, Ghana, Madagascar and Tanzania. Phylogenetic and cluster analyses based on single-nucleotide polymorphisms showed that isolates predominantly clustered by their country of origin, irrespective of their gp60 subtype. We found a significant isolation-by-distance signature that shows the importance of local transmission, but we also detected evidence of hybridization between isolates of different geographical regions. We identified 37 outlier genes with exceptionally high nucleotide diversity, and this group is significantly enriched for genes encoding extracellular proteins and signal peptides. Furthermore, these genes are found more often than expected in recombinant regions, and they show a distinct signature of positive or balancing selection. We conclude that: (1) the metapopulation structure of C. hominis can only be accurately captured by whole-genome analyses; (2) local anthroponotic transmission underpins the spread of this pathogen in Africa; (3) hybridization occurs between distinct geographical lineages; and (4) genetic introgression provides novel substrate for positive or balancing selection in genes involved in host-parasite coevolution.

Health sequelae of human cryptosporidiosis in industrialised countries: a systematic review.

BACKGROUND: Cryptosporidium is a protozoan parasite which is a common cause of gastroenteritis worldwide. In developing countries, it is one of the most important causes of moderate to severe diarrhoea in young children; in industrialised countries it is a cause of outbreaks of gastroenteritis associated with drinking water, swimming pools and other environmental sources and a particular concern in certain immunocompromised patient groups, where it can cause severe disease. However, over recent years, longer-term sequelae of infection have been recognised and a number of studies have been published on this topic. The purpose of this systematic review was to examine the literature in order to better understand the medium- to long-term impact of cryptosporidiosis. METHODS: This was a systematic review of studies in PubMed, ProQuest and Web of Science databases, with no limitations on publication year or language. Studies from any country were included in qualitative synthesis, but only those in industrialised countries were included in quantitative analysis. RESULTS: Fifteen studies were identified for qualitative analysis which included 3670 Cryptosporidium cases; eight studies conducted in Europe between 2004-2019 were suitable for quantitative analysis, including five case-control studies. The most common reported long-term sequelae were diarrhoea (25%), abdominal pain (25%), nausea (24%), fatigue (24%) and headache (21%). Overall, long-term sequelae were more prevalent following infection with Cryptosporidium hominis, with only weight loss and blood in stool being more prevalent following infection with Cryptosporidium parvum. Analysis of the case-control studies found that individuals were 6 times more likely to report chronic diarrhoea and weight loss up to 28 months after a Cryptosporidium infection than were controls. Long-term abdominal pain, loss of appetite, fatigue, vomiting, joint pain, headache and eye pain were also between 2-3 times more likely following a Cryptosporidium infection. CONCLUSIONS: This is the first systematic review of the long-term sequelae of cryptosporidiosis. A better understanding of long-term outcomes of cryptosporidiosis is valuable to inform the expectations of clinicians and their patients, and public health policy-makers regarding the control and prevention of this infection. Systematic review registration PROSPERO Registration number CRD42019141311.

Targeting the TS dimer interface in bifunctional Cryptosporidium hominis TS-DHFR from parasitic protozoa: Virtual screening identifies novel TS allosteric inhibitors.

Effective therapies are lacking to treat gastrointestinal infections caused by the genus Cryptosporidium, which can be fatal in the immunocompromised. One target of interest is Cryptosporidium hominis (C. hominis) thymidylate synthase-dihydrofolate reductase (ChTS-DHFR), a bifunctional enzyme necessary for DNA biosynthesis. Targeting the TS-TS dimer interface is a novel strategy previously used to identify inhibitors against the related bifunctional enzyme in Toxoplasma gondii. In the present study, we target the ChTS dimer interface through homology modeling and high-throughput virtual screening to identifying allosteric, ChTS-specific inhibitors. Our work led to the discovery of methylenedioxyphenyl-aminophenoxypropanol analogues which inhibit ChTS activity in a manner that is both dose-dependent and influenced by the conformation of the enzyme. Preliminary results presented here include an analysis of structure activity relationships and a ChTS-apo crystal structure of ChTS-DHFR supporting the continued development of inhibitors that stabilize a novel pocket formed in the open conformation of ChTS-TS.