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Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia , Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards. DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction. For E. histolytica , this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium , this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.

The detection of Giardia duodenalis and Cryptosporidium spp. was performed, along with the identification of the ciliated protozoa biodiversity, to evaluate the correlation between these protozoa in freshwater quality monitoring. Water and sediment samples from two sites in the Atibaia River (Campinas, São Paulo, Brazil) were collected monthly for 2 years (n = 96). Pathogenic protozoa in water and sediment were detected by using immunomagnetic separation, followed by visualization by immunofluorescence assay (IFA). All positive aliquots in IFA were subjected to DNA extraction and subsequently nested PCR. Qualitative (in vivo observation and silver impregnation) and quantitative (in vivo enumeration) analyses were performed for the ciliated protozoa. Giardia cysts were detected in 62.5% of the surface water samples and Cryptosporidium spp. in 25.0%. In the sediment, cysts were detected in 35.4% samples and oocysts in 16.6%. A total of 57 samples positive for Giardia cysts were subjected to sequencing, 40 of which were harboring G. duodenalis (24 were characterized as sub-assemblage AII). For ciliated protozoa, 73 taxa belonging to 53 genera were identified over the period of the study. These results revealed a high degree of contamination by waterborne protozoa in the main water source which supplies drinking water for more than one million people in Campinas (São Paulo), highlighting the need for continuous monitoring of this catchment site. In addition, the present study provides important data regarding the sources of the water body degradation, i.e., fecal contamination of human origin, in addition to the survey of the ciliated protozoa., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Cryptosporidiosis is an infectious enteric disease caused by species (some of them zoonotic) of the genus Cryptosporidium that in many countries are under surveillance. Typing assays critical to the surveillance of cryptosporidiosis typically involve characterization of Cryptosporidium glycoprotein 60 genes (gp60). Here, we characterized the gp60 of Cryptosporidium suis from two samples-a human and a porcine faecal sample-based on which a preliminary typing scheme was developed. A conspicuous feature of the C. suis gp60 was a novel type of tandem repeats located in the 5' end of the gene and that took up 777/1635 bp (48%) of the gene. The C. suis gp60 lacked the classical poly-serine repeats (TCA/TCG/TCT), which is usually subject to major genetic variation, and the length of the tandem repeat made a typing assay incorporating this region based on Sanger sequencing practically unfeasible. We therefore designed a typing assay based on the post-repeat region only and applied it to C. suis-positive samples from suid hosts from Norway, Denmark, and Spain. We were able to distinguish three different subtypes; XXVa-1, XXVa-2, and XXVa-3. Subtype XXVa-1 had a wider geographic distribution than the other subtypes and was also observed in the human sample. We think that the present data will inform future strategies to develop a C. suis typing assay that could be even more informative by including a greater part of the gene, including the tandem repeat region, e.g., by the use of long-read next-generation sequencing., 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 © 2023. Published by Elsevier B.V.)

Pathogenic bacteria, viruses, and parasites can cause waterborne disease outbreaks. The study of coastal water quality contributes to identifying potential risks to human health and to improving water management practices. The Río de la Plata River, a wide estuary in South America, is used for recreational activities, as a water source for consumption and as a site for sewage discharges. In the present study, as the first step of a quantitative microbial risk assessment of the coastal water quality of this river, a descriptive study was performed to identify the microbial pathogens prevalent in its waters and in the sewage discharged into the river. Two sites, representing two different potential risk scenarios, were chosen: a heavily polluted beach and an apparently safe beach. Conductivity and fecal contamination indicators including enterococci, Escherichia coli, F + RNA bacteriophages, and human polyomaviruses showed high levels. Regarding enterococci, differences between sites were significant (p-values <0.001). 93.3% and 56.5% of the apparently safe beach exceeded the recreational water limits for E. coli and enterococci. Regarding pathogens, diarrheagenic E. coli, Salmonella, and noroviruses were detected with different frequencies between sites. The parasites Cryptosporidium spp. and Giardia duodenalis were frequently detected in both sites. The results regarding viral, bacterial, and parasitic pathogens, even without correlation with conventional indicators, showed the importance of monitoring a variety of microorganisms to determine water quality more reliably and accurately, and to facilitate further studies of health risk assessment. The taxonomic description of microbial pathogens in river waters allow identifying the microorganisms that infect the population living on its shores but also pathogens not previously reported by the clinical surveillance system., 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 © 2024 Elsevier Inc. All rights reserved.)

Pathogens are ubiquitously detected in various natural and engineered water systems, posing potential threats to public health. However, it remains unclear which human-accessible waters are hotspots for pathogens, how pathogens transmit to these waters, and what level of health risk associated with pathogens in these environments. This review collaboratively focuses and summarizes the contamination levels of pathogens on the 5 water systems accessible to humans (natural water, drinking water, recreational water, wastewater, and reclaimed water). Then, we showcase the pathways, influencing factors and simulation models of pathogens transmission and survival. Further, we compare the health risk levels of various pathogens through Quantitative Microbial Risk Assessment (QMRA), and assess the limitations of water-associated QMRA application. Pathogen levels in wastewater are consistently higher than in other water systems, with no significant variation for Cryptosporidium spp. among five water systems. Hydraulic conditions primarily govern the transmission of pathogens into human-accessible waters, while environmental factors such as temperature impact pathogens survival. The median and mean values of computed public health risk levels posed by pathogens consistently surpass safety thresholds, particularly in the context of recreational waters. Despite the highest pathogens levels found in wastewater, the calculated health risk is significantly lower than in other water systems. Except pathogens concentration, variables like the exposure mode, extent, and frequency are also crucial factors influencing the public health risk in water systems. This review shares valuable insights to the more accurate assessment and comprehensive management of public health risk in human-accessible water environments., 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 © 2024 Elsevier Ltd. All rights reserved.)

The association between the parasitic illnesses and the consumption of contaminated water has been largely reported. However, there is still a lack of studies investigating the extent of parasitic contamination in water in Morocco. This is the first study in Morocco that aimed at assessing the presence of protozoan parasites, namely Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii, in drinking water consumed in the region of Marrakech. Samples processing was performed by membrane filtration and qPCR detection. A total of 104 drinking water samples (tap water, well, and spring waters) was collected between 2016 and 2020. The analysis revealed an overall protozoa contamination rate of 67.3% (70/104), of which 35 samples were positive for Giardia duodenalis, 18 for Toxoplasma gondii, and 17 for both parasites, whereas no sample was positive for Cryptosporidium spp. This first study showed that drinking water in the region of Marrakech contained parasites which could represent a risk for consumers. For a better understanding and estimation of the risk encountered by local inhabitants, further studies concerned with (oo)cyst viability, infectivity, and genotype identification need to be performed., 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 © 2023. Published by Elsevier Inc.)

It is important to know the characteristics of the immunological response in newborn calf diarrhea, which is often caused by bacterial, viral and protozoal pathogens. Cytokinesare proteins that serve as chemical messengers to regulate theinnate and adaptive arms of theimmune response. Changes in circulatory cytokine levels provide valuable information for understanding the pathophysiological process and monitoring disease progression and inflammation. Vitamin D has important immunomodulatory effects, which include enhancing the innate immune system and inhibiting adaptative immune responses. The objective of this study was to evaluate the relationship between serum cytokine profile and vitamin D level in neonatal calves with diarrhea. The study population was comprised of 40 neonatal calves, 32 of which had diarrhea and 8 of which were healthy calves. The calves with diarrhea were allocated to four groups according to bacterial (Escherichia coli), viral (Rotavirus, Coronavirus) and protozoal (Cryptosporidium parvum) etiologies. Circulatory vitamin D metabolites (25-hydroxyvitamin D, 1,25-dihydroxyvitamin D) and cytokines (TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and IL-17) in the calves were determined. There was no statistically significant difference among the groups in 25-hydroxyvitamin D levels. 1,25-dihydroxyvitamin D levels were higher in Coronavirus and E. coli groups compared to the controls. Serum levels of all cytokines except for IL-13, were higher in E. coli group than those of the control group. As a result, differences in serum cytokines and vitamin D levels according to etiological factors in calf diarrhea indicate that vitamin D may play a role in the immune response in the disease., 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 © 2023 Elsevier Ltd. All rights reserved.)

Cryptosporidium sp. and Giardia sp. are parasites that cause diseases in the population. Most of parasite diseases regarding the consumption of drinking water polluted with sewage are caused by Cryptosporidium sp. or Giardia sp. it is because of the incomplete disinfection of the wastewater treatment. Therefore, in this work the removal or inactivation efficiency of different treatment technologies presented by around 40 scientific studies was evaluated, with a view to water circularity. For Cryptosporidium sp., we conclude that the most efficient secondary technologies are aerobic technologies, which remove between 0.00 and 2.17 log units (Ulog), with activated sludge presenting the greatest efficiency, and that the tertiary technologies with the greatest removal are those that use ultrasound, which reach removal values of 3.17 Ulog. In the case of Giardia sp., the secondary technologies with the greatest removal are anaerobic technologies, with values between 0.00 and 3.80 Ulog, and the tertiary technologies with the greatest removal are those that combine filtration with UV or a chemical disinfection agent. Despite the removal values obtained, the greatest concern remains detecting and quantifying the infectious forms of both parasites in effluents; therefore, although the technologies perform adequately, discharge effluents must be monitored with more sensitive techniques, above all aiming for circularity of the treated water in a context of the water scarcity that affects some parts of the world., 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 Ltd. All rights reserved.)

Background: Integrated surveillance for multiple diseases can be an efficient use of resources and advantageous for national public health programs. Detection of IgG antibodies typically indicates previous exposure to a pathogen but can potentially also serve to assess active infection status. Serological multiplex bead assays have recently been developed to simultaneously evaluate exposure to multiple antigenic targets. Haiti is an island nation in the Caribbean region with multiple endemic infectious diseases, many of which have a paucity of data for population-level prevalence or exposure., Methods: A nationwide serosurvey occurred in Haiti from December 2014 to February 2015. Filter paper blood samples ( n = 4,438) were collected from participants in 117 locations and assayed for IgG antibodies on a multiplex bead assay containing 15 different antigens from 11 pathogens: Plasmodium falciparum, Toxoplasma gondii , lymphatic filariasis roundworms, Strongyloides stercoralis , chikungunya virus, dengue virus, Chlamydia trachomatis, Treponema pallidum , enterotoxigenic Escherichia coli, Entamoeba histolytica , and Cryptosporidium parvum ., Results: Different proportions of the Haiti study population were IgG seropositive to the different targets, with antigens from T. gondii, C. parvum , dengue virus, chikungunya virus, and C. trachomatis showing the highest rates of seroprevalence. Antibody responses to T. pallidum and lymphatic filariasis were the lowest, with <5% of all samples IgG seropositive to antigens from these pathogens. Clear trends of increasing seropositivity and IgG levels with age were seen for all antigens except those from chikungunya virus and E. histolytica . Parametric models were able to estimate the rate of seroconversion and IgG acquisition per year for residents of Haiti., Conclusions: Multiplex serological assays can provide a wealth of information about population exposure to different infectious diseases. This current Haitian study included IgG targets for arboviral, parasitic, and bacterial infectious diseases representing multiple different modes of host transmission. Some of these infectious diseases had a paucity or complete absence of published serological studies in Haiti. Clear trends of disease burden with respect to age and location in Haiti can be used by national programs and partners for follow-up studies, resource allocation, and intervention planning., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chan, Martin, Mace, Jean, Stresman, Drakeley, Chang, Lemoine, Udhayakumar, Lammie, Priest and Rogier.)

Cryptosporidium, an intestinal protozoan pathogen, is one of the leading causes of death in children and diarrhea in healthy adults. Detection of Cryptosporidium has become a high priority to prevent potential outbreaks. In this paper, a simple, easy to fabricate, and cost-effective on-chip-based electrochemical biosensor has been developed for the sensitive and label-free detection of Cryptosporidium oocysts in water samples. The sensor was fabricated using standard lithography using a mask with a 3-electrode design and modified by self-assembling a hybrid of a thiolated protein/G and the specific anti-Cryptosporidium monoclonal antibodies (IgG3). The electrochemical impedance spectroscopy (EIS) was employed to quantitate C. parvum in the range of 0 to 300 oocysts, with a detection limit of approximately 20 oocysts/5 µL. The high sensitivity and specificity of the developed label-free electrochemical biosensor suggest that this novel platform is a significant step towards the development of fast, real-time, inexpensive and label-free sensing tool for early warning and immediate on-site detection of C. parvum oocysts in water samples, as compared to the traditional methods (such as PCR and microscopy). Furthermore, under optimized conditions, this label-free biosensor can be extended to detect other analytes and biomarkers for environmental and biomedical analyses., (© 2022. The Author(s).)

Cryptosporidium is an intracellular protozoan parasite, globally distributed and capable of infecting various vertebrate species, including humans as well as domestic and wild animals. Cryptosporidium is increasingly gaining attention as a human and an animal pathogen mainly due to its dominant involvement in worldwide waterborne outbreaks. The present paper reviews the current knowledge and understanding of Cryptosporidium spp. in terrestrial and water animals in Azerbaijan.

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water.

Background: Cryptosporidium is a waterborne intestinal parasite that causes diarrhoea in low and middle-income countries worldwide. Reports from Mediterranean countries have documented the prevalence of cryptosporidiosis in children at various ages, also among cancer patients, and in cases of chronic kidney disease, haemodialysis, and organ transplant. Untill now, modified-acid staining preceded by stool concentration preparation remains the leading screening diagnostic test for the infection. In Jordan, few studies for cryptosporidiosis have been performed during the last 3 decades., Aims: This paper reviewed the status of cryptosporidiosis in Jordan and tracked recent updates for this emerging protozoal infection among different population groups., Methods: In this study, an online search was conducted on Google Scholar and PubMed databases using the keywords: Jordan, cryptosporidiosis and Cryptosporidium to inspect studies done on this parasite in Jordan., Results: Only 9 articles were identified from 1994 to 2019. These were analysed in terms of population group, demography, clinical history and the diagnostic tools used., Conclusion: Cryptosporidiosis is still neglected in Jordan as indicated by the low number of studies over the last 3 decades and the prevalence is diverse depending on the diagnostic test used and socioeconomic status., (Copyright © World Health Organization (WHO) 2020. Open Access. Some rights reserved. This work is available under the CC BY-NC-SA 3.0 IGO license (https://creativecommons.org/licenses/by-nc-sa/3.0/igo).)

The Roma are Europe's largest minority. They are also one of its most disadvantaged, with low levels of education and health and high levels of poverty. Research on Roma health often reveals higher burdens of disease in the communities studied. This paper aims to review the literature on communicable diseases among Roma across Eastern and Central Europe. A PubMed search was carried out for communicable diseases among Roma in these parts of Europe, specifically in Romania, Bulgaria, Hungary, Serbia, Slovakia, the Czech Republic and North Macedonia. The papers were then screened for relevance and utility. Nineteen papers were selected for review; most of them from Slovakia. Roma continue to have a higher prevalence of communicable diseases and are at higher risk of infection than the majority populations of the countries they live in. Roma children in particular have a particularly high prevalence of parasitic disease. However, these differences in disease prevalence are not present across all diseases and all populations. For example, when Roma are compared to non-Roma living in close proximity to them, these differences are often no longer significant.

Background: Cryptosporidiosis is an emerging infectious disease of public health significance worldwide. The burden of disease caused by Cryptosporidium varies between and within countries/areas. To have a comprehensive understanding of epidemiological status and characteristics of human Cryptosporidium infection in China since the first report in 1987, a retrospective epidemiological analysis was conducted by presenting differences in the prevalence of Cryptosporidium by province, year, population, living environment and season and possible transmission routes and risk factors as well as genetic characteristics of Cryptosporidium in humans., Methodology/principal Findings: A systematic search was conducted to obtain epidemiological papers of human Cryptosporidium infection/cryptosporidiosis from PubMed and Chinese databases. Finally, 164 papers were included in our analysis. At least 200,054 people from 27 provinces were involved in investigational studies of Cryptosporidium, with an average prevalence of 2.97%. The prevalence changed slightly over time. Variable prevalences were observed: 0.65-11.15% by province, 1.89-47.79% by population, 1.77-12.87% and 0-3.70% in rural and urban areas, respectively. The prevalence peak occurred in summer or autumn. Indirect person-to-person transmission was documented in one outbreak of cryptosporidiosis in a pediatric hospital. 263 Cryptosporidium isolates were obtained, and seven Cryptosporidium species were identified: C. hominis (48.3%), C. andersoni (22.43%), C. parvum (16.7%), C. meleagridis (8.36%), C. felis (3.04%), C. canis (0.76%) and C. suis (0.38%)., Conclusions/significances: This systematic review reflects current epidemiological status and characteristics of Cryptosporidium in humans in China. These data will be helpful to develop efficient control strategies to intervene with and prevent occurrence of human Cryptosporidium infection/cryptosporidiosis in China as well as have a reference effect to other countries. Further studies should focus on addressing a high frequency of C. andersoni in humans and a new challenge with respect to cryptosporidiosis with an increasing population of elderly people and patients with immunosuppressive diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: Cryptosporidium spp. are causative agents of gastrointestinal diseases in a wide variety of vertebrate hosts. Mortality resulting from the disease is low in livestock, although severe cryptosporidiosis has been associated with fatality in young animals., Methods: The goal of this systematic review and meta-analysis was to review the prevalence and molecular data on Cryptosporidium infections in selected terrestrial domestic and wild ungulates of the families Bovidae (bison, buffalo, cattle, goat, impala, mouflon sheep, sheep, yak), Cervidae (red deer, roe deer, white-tailed deer), Camelidae (alpaca, camel), Suidae (boar, pig), Giraffidae (giraffes) and Equidae (horses). Data collection was carried out using PubMed, Scopus, Science Direct and Cochran databases, with 429 papers being included in this systematic analysis., Results: The results show that overall 18.9% of ungulates from the investigated species were infected with Cryptosporidium spp. Considering livestock species (cattle, sheep, goats, pigs, horses and buffaloes), analysis revealed higher Cryptosporidium infection prevalence in ungulates of the Cetartiodactyla than in those of the Perissodactyla, with cattle (29%) being the most commonly infected farm animal., Conclusions: Overall, the investigated domestic ungulates are considered potential sources of Cryptosporidium contamination in the environment. Control measures should be developed to reduce the occurrence of Cryptosporidium infection in these animals. Furthermore, literature on wild populations of the named ungulate species revealed a widespread presence and potential reservoir function of wildlife.

Cryptosporidium , an intestinal protozoan pathogen, is one of the leading causes of diarrhea in healthy adults and death in children. Detection of Cryptosporidium oocysts has become a high priority to prevent potential outbreaks. In this paper, a label-free interdigitated-based capacitive biosensor has been introduced for the detection of Cryptosporidium oocysts in water samples. Specific anti- Cryptosporidium monoclonal antibodies (IgG3) were covalently immobilized onto interdigitated gold electrodes as the capture probes, and bovine serum albumin was used to avoid non-specific adsorption. The immobilization of the antibodies was confirmed by measuring the change in the contact angle. The detection was achieved by measuring the relative change in the capacitive/dielectric properties due to the formation of Cryptosporidium -antibody complex. The biosensor has been tested for different concentrations of Cryptosporidium . The results show that the biosensor developed can accurately distinguish different numbers of captured cells and densities on the surface of the biosensor. The number of Cryptosporidium oocysts captured on the electrode surface was confirmed using a fluorescein isothiocyanate (FITC) immunofluorescence assay. The response from the developed biosensor has been mainly dependent on the concentration of Cryptosporidium under optimized conditions. The biosensor showed a linear detection range between 15 and 153 cells/mm² and a detection limit of 40 cells/mm². The label-free capacitive biosensor developed has a great potential for detecting Cryptosporidium in environmental water samples. Furthermore, under optimized conditions, this label-free biosensor can be extended for detection of other biomarkers for biomedical and environmental analyses.

Background: Cryptosporidium is a protozoan parasite of humans and other animals worldwide and is one of the greatest contributors to human diarrhoeal illness. Transmission can occur indirectly via contaminated food or water, or directly via contact with animals or other infected people. Risk exposures are often identified from outbreak investigations, but a subset of cases remains unexplained, and sources for sporadic disease and pathways to infection are still unclear. Given the few systematic syntheses of reported evidence in industrialised populations, the aim of this review is to consolidate the literature to describe exposures associated with human cryptosporidiosis in industrialised countries, specifically including the UK, and describe any differences between outbreak-associated and sporadic disease., Methods/design: Where relevant, methods will follow the recommendations made in the Cochrane Handbook for Systematic Reviews of Interventions. Three steps will be used to identify the literature including electronic database searching using PubMed, Scopus, Embase and Web of Science; reference list trawling; and an exploration of the grey literature. Screening of results will be undertaken by two reviewers using pre-defined criteria. Studies conducted in industrialised countries and reporting on human subjects will be included. All observational studies will be included where they report exposures and relevant quantitative results. Data will be extracted using a standardised form. Study quality will be assessed using the ROBINS-I tool. Data will be summarised presenting the papers' main findings including population under study, outcomes, and exposures, and whether these were considered outbreak or sporadic cases. A narrative summary will also be included. Where populations are appropriate, available data will be pooled in a meta-analysis combining the significant exposures across studies., Discussion: This review aims to consolidate the evidence for transmission routes and exposures for Cryptosporidium in industrialised countries, with particular reference to how these may apply to the UK. In addition, the review will seek to describe differences between outbreak and sporadic cases. This will help to identify those most vulnerable, highlighting pathways where interventions and public health response may be appropriate., Systematic Review Registration: PROSPERO number CRD42017056589 .

IntroductionThis paper reviews the current knowledge and understanding of Cryptosporidium spp . and Giardia spp. in humans, animals and the environment in 10 countries in the eastern part of Europe: Bosnia and Herzegovina, Croatia, Czech Republic, Estonia, Hungary, Latvia, Poland, Romania, Serbia and Slovenia. Methods: Published scientific papers and conference proceedings from the international and local literature, official national health service reports, national databases and doctoral theses in local languages were reviewed to provide an extensive overview on the epidemiology, diagnostics and research on these pathogens, as well as analyse knowledge gaps and areas for further research. Results: Cryptosporidium spp. and Giardia spp. were found to be common in eastern Europe, but the results from different countries are difficult to compare because of variations in reporting practices and detection methodologies used. Conclusion: Upgrading and making the diagnosis/detection procedures more uniform is recommended throughout the region. Public health authorities should actively work towards increasing reporting and standardising reporting practices as these prerequisites for the reported data to be valid and therefore necessary for appropriate control plans.

Background: In a long-term program to monitor pathogens in water catchments serving the City of Melbourne in the State of Victoria in Australia, we detected and genetically characterised Cryptosporidium and Giardia in faecal samples from various animals in nine water reservoir areas over a period of 4 years (July 2011 to November 2015)., Methods: This work was conducted using PCR-based single-strand conformation polymorphism (SSCP) and phylogenetic analyses of portions of the small subunit of ribosomal RNA (SSU) and 60 kDa glycoprotein (gp60) genes for Cryptosporidium, and triose-phosphate isomerase (tpi) gene for Giardia., Results: The prevalence of Cryptosporidium was 1.62 % (69 of 4,256 samples); 25 distinct sequence types were defined for pSSU, and six for gp60 which represented C. hominis (genotype Ib - subgenotype IbA10G2), C. cuniculus (genotype Vb - subgenotypes VbA26, and VbA25), and C. canis, C. fayeri, C. macropodum, C. parvum, C. ryanae, Cryptosporidium sp. "duck" genotype, C. suis and C. ubiquitum as well as 12 novel SSU sequence types. The prevalence of Giardia was 0.31 % (13 of 4,256 samples); all three distinct tpi sequence types defined represented assemblage A of G. duodenalis., Conclusions: Of the 34 sequence types (genotypes) characterized here, five and one have been recorded previously for Cryptosporidium and Giardia, respectively, from humans. Novel genotypes of Cryptosporidium and Giardia were recorded for SSU (n = 12), gp60 (n = 4) and tpi (n = 1); the zoonotic potential of these novel genotypes is presently unknown. Future work will continue to monitor the prevalence of Cryptosporidium and Giardia genotypes in animals in these catchments, and expand investigations to humans. Nucleotide sequences reported in this paper are available in the GenBank database under accession nos. KU531647-KU531718.

Apicomplexan parasites cause infectious diseases that are either a severe public health problem or an economic burden. In this paper we will shed light on how oxidative stress can influence the host-pathogen relationship by focusing on three major diseases: babesiosis, coccidiosis, and toxoplasmosis.

Cryptosporidium and Giardia pose a threat to human health in rural environments where water supplies are commonly untreated and susceptible to contamination from agricultural animal waste/manure, animal wastewater, septic tank effluents and septage. Our goals for this paper are to: (1) explore the prevalence of these protozoan parasites, where they are found, in what quantities, and which genotypes are present; (2) examine relationships between disease and land use comparing human health risks between rural and urban environments; and (3) synthesize available information to gain a better understanding of risk and risk management for rural water supplies. Our results indicate that Cryptosporidium and Giardia were more prevalent in rural versus urban environments based on the number of positive samples. Genotyping showed that both the human and animal types of the parasites are found in rural and urban environments. Rural areas had a higher incidence of disease compared to urban areas based on the total number of disease cases. Cryptosporidiosis and giardiasis were both positively correlated (p < 0.001) with urban area, population size, and population density. Finally, a comprehensive strategy that creates knowledge pathways for data sharing among multiple levels of management may improve decision-making for protecting rural water supplies.

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

Water-borne pathogens such as Cryptosporidium pose a significant human health risk and catchments provide the first critical pollution 'barrier' in mitigating risk in drinking water supply. In this paper we apply an adaptive management framework to mitigating Cryptosporidium risk in source water using a case study of the Myponga catchment in South Australia. Firstly, we evaluated the effectiveness of past water quality management programs in relation to the adoption of practices by landholders using a socio-economic survey of land use and management in the catchment. The impact of past management on the mitigation of Cryptosporidium risk in source water was also evaluated based on analysis of water quality monitoring data. Quantitative risk assessment was used in planning the next round of management in the adaptive cycle. Specifically, a pathogen budget model was used to identify the major remaining sources of Cryptosporidium in the catchment and estimate the mitigation impact of 30 alternative catchment management scenarios. Survey results show that earlier programs have resulted in the comprehensive adoption of best management practices by dairy farmers including exclusion of stock from watercourses and effluent management from 2000 to 2007. Whilst median Cryptosporidium concentrations in source water have decreased since 2004 they remain above target levels and put pressure on other barriers to mitigate risk, particularly the treatment plant. Non-dairy calves were identified as the major remaining source of Cryptosporidium in the Myponga catchment. The restriction of watercourse access of non-dairy calves could achieve a further reduction in Cryptosporidium export to the Myponga reservoir of around 90% from current levels. The adaptive management framework applied in this study was useful in guiding learning from past management, and in analysing, planning and refocusing the next round of catchment management strategies to achieve water quality targets.

Surface-water treatment plants provide water to approximately 67 percent of the residents of Pennsylvania. Industrial activities and agricultural practices significantly contribute to the chemical and microbiological load of surface-water systems. For the study reported here, surface water from three sites along Two Lick Creek in Indiana County in Pennsylvania were sampled and analyzed for nitrate, iron, total and fecal coliforms, and Cryptosporidium oocysts. Mean nitrate concentrations were 2.88, 3.15, and 3.83 mg/L for Site 1, Site 2, and Site 3, respectively, while mean total-coliform counts ranged from 6.89 x 10(2) to 22.40 x 10(2) CFUs per 100 mL, and fecal coliform counts ranged from 0.30 x 10(2) to 11.1 x 10(2) CFUs per 100 mL. Cryptosporidium oocysts ranged from a low of <46 to a high of 91 per 100 L depending on sample turbidity. The recovery rates of oocysts from spiked samples ranged from 22 percent (low-turbidity samples) to a low of 0.76 percent (high-turbidity samples). This paper discusses the impact of environmental factors on nitrate concentration, coliforms, and Cryptosporidium, as well as the health significance of these water quality indicators.

This paper reports the investigation carried out on the occurrence of Cryptosporidium and Giardia (oo)cysts in water samples of two municipal treatment plants, and in surface water and ground water wells of the province of Palermo. The wastewater samples taken before and after treatment process were assayed over the course of one year Giardia cysts were detected in all samples throught the year at higher concentration levels than Cryptosporidium oocysts, with a peak observed in spring. The overall removal efficiency of (oo)cysts in the treatment plants was about of 90%. Their presence were also searched in surface waters (three artificial lakes and one river); (oo)cysts were detected in one lake at very low concentration; on the contrary, both parasites were found at high concentration levels in all the samples collected throught one year from the water of the river. The pattern of occurrence of both parasites appears temporally related to the level of rainfall trend. Cryptosporidium and Giardia were also found in ground water wells; their presence occurred only in waters taken from wells at a depth lower than 31 meters with concomitant presence of faecal bacteria. These results may provide further insight into the possible source of Cryptosporidium and Giardia in natural environmental and stress the potential risk associated with the use of waters for recreational and agricultural purposes.

Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.

Before 1982, only eight case reports of human cryptosporidiosis and fewer than 30 papers on Cryptosporidium spp. appeared in the biomedical literature. At that time, cryptosporidiosis was thought to be an infrequent infection in animals and rarely an opportunistic infection in humans. The concept of Cryptosporidium spp. as pathogens has changed dramatically within the past 8 years because of improved diagnostic techniques, increased awareness within the biomedical community, and the development of basic research programs in numerous laboratories. Presently, greater than 1,000 publications including over 400 case reports in the biomedical literature address Cryptosporidium spp. and cryptosporidiosis. Cryptosporidium parvum is now thought to be one of the three most common enteropathogens causing diarrheal illness in humans worldwide, especially in developing countries. It is likely that cryptosporidiosis was previously included in the 25 to 35% of diarrheal illness with unknown etiology. Because of the severity and length of diarrheal illness and because no effective therapy has been identified, cryptosporidiosis is one of the most ominous infections associated with AIDS. The role of C. parvum as an enteropathogen is well established; documentation of its role as a cause of hepatobiliary and respiratory diseases is now appearing in the literature. Our present understanding of the natural history, epidemiology, biology, and immunology of Cryptosporidium spp. as well as the clinical features, pathogenicity, and treatment of cryptosporidiosis are reviewed here.

The antigenic constituents of sporulated Cryptosporidium parvum oocyst antigens were characterized with antisera from mice immunized against C. parvum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining defined the major proteins. Six of seven lectins used recognized as many as 15 bands. The lectins concanavalin A, Dolichos biflorus, and wheat germ agglutinin showed strong activity against the same eight bands with molecular weights ranging from 72,000 to greater than 100,000. An enzyme-linked immunosorbent assay was used to detect antibody to C. parvum. Antibody binding was significantly decreased by heat and enzymatic treatment with trypsin, protease, and mixed glycosidases. C. parvum antigens were further defined by the reactivity of immune sera with a C. parvum sonicate preparation separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. Antisera from orally infected mice consistently recognized four antigens with molecular weights ranging from 72,000 to greater than 100,000. These antigens also bound concanavalin A. Treatment of the antigen preparation with mixed glycosidases reduced the reactivity of antisera with most antigens with molecular weights greater than 60,000. The data suggest that the antigenic composition of C. parvum is complex and that carbohydrates alone or in association with lipids or proteins may be important in the immune response to C. parvum.