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Cryptosporidium poses significant public health risks as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized wastewater surveillance methods for Cryptosporidium spp. challenges implementation design and the comparability between studies. Thus, this study compared and contrasted Cryptosporidium wastewater surveillance methods for concentrating wastewater oocysts, extracting oocyst DNA, and detecting Cryptosporidium genetic markers. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome Particles, with and without additional immunomagnetic separation purification (except for the Nanotrap Microbiome Particles). Oocyst DNA extraction by either the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit were evaluated and the impact of bead beating and freeze-thaw pretreatments on DNA recoveries was assessed. Genetic detection via qPCR assays targeting either the Cryptosporidium 18S rRNA gene or the Cryptosporidium oocyst wall protein gene were tested. Oocyst recovery percentages were highest for centrifugation (39-77 %), followed by the Nanotrap Microbiome Particles (24 %), electronegative filtration with a PBST elution (22 %), and Envirocheck HV capsule filtration (13 %). Immunomagnetic separation purification was found to be unsuitable due to interference from the wastewater matrix. Bead-beating pretreatment enhanced DNA recoveries from both the DNeasy Powersoil Pro kit (314 gc/μL DNA) and the QIAamp DNA Mini kit (238 gc/μL DNA). In contrast, freeze-thaw pretreatment reduced DNA recoveries to under 92 gc/μL DNA, likely through DNA degradation. Finally, while both qPCR assays were specific to Cryptosporidium spp., the 18S rRNA assay had a 5-fold lower detection limit and could detect a wider range of Cryptosporidium spp. than the Cryptosporidium oocyst wall protein assay., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

A breeding colony of wild-origin eastern indigo snakes (EISs, Drymarchon couperi ) that is part of a reintroduction program has been impacted by gastric cryptosporidiosis. Gastric cryptosporidiosis is an insidious disease of squamates caused by an apicomplexan protozoan, Cryptosporidium serpentis . Viral coinfections have been implicated as possible immunosuppressant agents that allow for disease progression and both adenovirus and reovirus have been implicated in allowing for the progression of gastric cryptosporidiosis during coinfection in other snake species. Molecular (PCR) screening for adenoviruses and reoviruses was performed for both C. serpentis -positive and C. serpentis -negative EIS within the breeding colony. No reoviruses were detected in the collection. Adenoviruses were present in 11/68 (16.2%) EISs evaluated, and there was no significant difference between C. serpentis -positive and C. serpentis -negative EISs ( p = 0.196). There was no significant difference in adenovirus status between C. serpentis -positive EISs' lifespan ( p = 0.191) or survival rates ( p = 0.823). These findings suggest that the presence of the adenoviruses found in this study does not contribute to the formation or progression of gastric cryptosporidiosis in EISs.

PCR-based diagnostics has revealed the previously largely unknown Cryptosporidium transmission and infections in high-income countries. This study aimed to determine domestic and imported subtypes of Cryptosporidium species in Norway, evaluate their demographic distribution, and identify potential small outbreaks. Cryptosporidium -positive human faecal samples were obtained from six medical microbiology laboratories between February 2022 and January 2024, together with 22 Cryptosporidium -positive animal samples. Species and subtypes were identified by sequencing PCR products from gp60 and SSU rRNA genes. Most cryptosporidiosis cases occurred during late summer/early autumn, primarily in children and young adults. Of 550 human samples, 359 were successfully characterized molecularly (65%), revealing infection with 10 different Cryptosporidium species. C. parvum occurred in 245 (68%) human isolates with IIa and IId being major allele families, with distinct regional distribution patterns of common subtypes. A kindergarten outbreak with 5 cases was due to C. parvum IIaA14G1R1. C. mortiferum was identified in 33 (9.2%) human cases of which 24 were known to be of domestic origin, making it the second most common species in human autochthonous cases in Norway. All C. mortiferum isolates were of the same genotype; XIVaA20G2T1, including 13 cases from a suspected small outbreak in Trøndelag. C. hominis occurred in 68 typed cases (19%), but mostly in infections acquired abroad, with allele families Ib and If occurring most often. In conclusion, this study of recent Cryptosporidium spp. and subtypes in Norway, highlights the predominance of C. parvum and the emergence of C. mortiferum among autochthonous cases.

Background: Cryptosporidium parasites are an important cause of diarrhoea globally and sexual transmission may occur in men who have sex with men (MSM). We aimed to systematically review the literature to explore any factors associated with transmission of Cryptosporidium in MSM to provide insight for future guidelines and public health strategies., Methods: We searched MEDLINE, Embase, CINAHL and Web of Science for manuscripts published up to July 2023. A primary author conducted an initial screen of abstracts and full text eligibility, and risk of bias was assessed independently by two authors using the Joanna Briggs Institute critical appraisal tools. We used the nine-point synthesis without meta-analysis method to synthesise narrative data. The review was registered on PROSPERO (CRD42023374279)., Results: Six manuscripts were included in the final review from the USA ( n = 3), Europe ( n = 1) and Australia ( n = 2) and were case series ( n = 2), cross sectional ( n = 2) and case control studies ( n = 2) published between 1984 and 2021 and overall there were 1582 cases of Cryptosporidium in MSM. We identified demographic factors (living with HIV, younger age [<30 years old], geographical areas [Southern Europe v Northern Europe], previous Treponema pallidum , previous Entamoeba histolytica) and behavioural factors (recreational drug use, higher number of sexual partners and MSM attending sex on premises venues with spa/sauna) associated with Cryptosporidium in MSM., Conclusions: Despite a small number of manuscripts in this review, we identified demographic and behavioural factors associated with Cryptosporidium in MSM. These data will provide insight for public health interventions for future outbreaks of Cryptosporidium in MSM., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Intense rainfall and snowmelt events may affect the safety of drinking water, as large quantities of fecal material can be discharged from storm or sewage overflows or washed from the catchment into drinking water sources. This study used β-d-glucuronidase activity (GLUC) with microbial source tracking (MST) markers: human, bovine, porcine mitochondrial DNA markers (mtDNA) and human-associated Bacteroidales HF183 and chemical source tracking (CST) markers including caffeine, carbamazepine, theophylline and acetaminophen, pathogens (Giardia, Cryptosporidium, adenovirus, rotavirus and enterovirus), water quality indicators (Escherichia coli, turbidity) and hydrometeorological data (flowrate, precipitation) to assess the vulnerability of 3 drinking water intakes (DWIs) and identify sources of fecal contamination. Water samples were collected under baseline, snow and rain events conditions in urban and agricultural catchments (Québec, Canada). Dynamics of E. coli, HF183 and WWMPs were similar during contamination events, and concentrations generally varied over 1 order of magnitude during each event. Elevated human-associated marker levels during events demonstrated that urban DWIs were impacted by recent contamination from an upstream municipal water resource recovery facility (WRRF). In the agricultural catchment, mixed fecal pollution was observed with the occurrences and increases of enteric viruses, human bovine and porcine mtDNA during peak contaminating events. Bovine mtDNA qPCR concentrations were indicative of runoff of cattle-derived fecal pollutants to the DWI from diffuse sources following rain events. This study demonstrated that the suitability of a given MST or CST indicator depend on river and catchment characteristics. The sampling strategy using continuous online GLUC activity coupled with MST and CST markers analysis was a more reliable source indicator than turbidity to identify peak events at drinking water intakes., 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The IOWA strain of Cryptosporidium parvum is widely used in studies of the biology and detection of the waterborne pathogens Cryptosporidium spp. While several lines of the strain have been sequenced, IOWA-II, the only reference of the original subtype (IIaA15G2R1), exhibits significant assembly errors. Here we generated a fully assembled genome of IOWA-CDC of this subtype using PacBio and Illumina technologies. In comparative analyses of seven IOWA lines maintained in different laboratories (including two sequenced in this study) and 56 field isolates, IOWA lines (IIaA17G2R1) with less virulence had mixed genomes closely related to IOWA-CDC but with multiple sequence introgressions from IOWA-II and unknown lineages. In addition, the IOWA-IIaA17G2R1 lines showed unique nucleotide substitutions and loss of a gene associated with host infectivity, which were not observed in other isolates analyzed. These genomic differences among IOWA lines could be the genetic determinants of phenotypic traits in C. parvum. These data provide a new reference for comparative genomic analyses of Cryptosporidium spp. and rich targets for the development of advanced source tracking tools., 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.)

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.)

The aims of the study were to characterise the distribution of Cryptosporidium spp. and subtypes causing infections in Finland during 2021. This was carried out with 60 clinical samples from the hospital districts of Helsinki and Uusimaa, Vaasa, Kymenlaakso, South Karelia, and Central Finland, as well as with Finnish Infectious Diseases Register (FIDR) data. Additionally, the study aimed to explore the potential exposures related to Cryptosporidium mortiferum (Cryptosporidium chipmunk genotype I) infections via interview. Species identification was carried out with quantitative real-time PCR (qPCR) and 18S sequencing. Further typing was performed with gp60 subtyping. Over 70% of the samples were identified as Cryptosporidium parvum and 20% as C. mortiferum, which had not been identified in Finland before. Two cases of Cryptosporidium hominis were identified from patients reported to have travelled outside Europe. The C. parvum subtype IIaA15G2R1 and the C. mortiferum subtype XIVaA20G2T1 were the most common subtypes identified. The interviewed C. mortiferum cases did not report shared exposures such as contact with wild rodents. In conclusion, C. parvum and C. mortiferum were the major causes of cryptosporidiosis in the five studied Finnish hospital districts., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Coccidiosis is an important disease in juvenile fish because of severe intestinal injury during infection. We first reported the mixed infection of intestinal coccidia and its association with health status and pathological findings in juvenile Asian seabass (Lates calcarifer) cultured in Thailand. Two groups of Asian seabass, 60-day fish and 90-day fish, were sampled to investigate prevalence and coccidian infection intensity using morphological characterization and PCR. Phylogenetic analysis of 18S rRNA gene amplified from the intestines revealed Eimeria sp. and Cryptosporidium sp. infection. The prevalence of Eimeria sp. and Cryptosporidium sp. in sampled fish was 100%. Clinical outcomes assessed, using health assessment index (HAI) scoring and semi-quantitative grading of intestinal lesions and inflammation, demonstrated that all fish developed variety of pathology and clinical illness; however, infection intensity in 60-day fish was significantly higher (p < .05) than 90-day fish. The HAI score of 60-day fish was poorer than 90-day fish, which correlated to a high infection intensity (r = .397), analysed by Pearson correlation coefficient. Overproduction of intestinal oxidants contributing to mucosal injury was examined by nitrotyrosine expression. The high production of reactive nitrogen species indicated severe inflammatory response, and intestinal injuries occurred mainly in the 60-day fish., (© 2024 John Wiley & Sons Ltd.)

Toxoplasma gondii is a widespread and specialized intracellular protozoan pathogen that affects one third of the world' s population, posing a great threat to public health. As the definitive host, cats excrete oocysts and play a crucial role in the transmission of toxoplasmosis. The current diagnostic tools usually require bulky equipment and expertize, which hinders the efficient diagnosis and intervention of Toxoplasma infection in cats. In this study, we combined (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to establish an easier method for the detection of T. gondii oocysts in cat fecal samples. The sensitivity, specificity, and practicability of the established RPA-CRISPR/Cas9 method were evaluated using a lateral flow strip, with the limitation of detection determined at 10 plasmid copies/μL (corresponding to about one oocyst), cross reactivity to none of Giardia lamblia, Cryptosporidium sp., Microsporidium biberi and Blastocystis hominis that also commonly found in cats, and comparable performance in detecting T. gondii in clinical samples to conventional PCR amplification. This RPA-CRISPR/Cas9 method provides an alternative to conventional molecular tools used in the clinical diagnosis of Toxoplasma infection in cats and other animals., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Permanent stains such as trichrome have better sensitivity but are time-consuming and the fixative includes toxic mercuric chloride. Thus, a newer modification was tested and found to be a superior, faster and safer staining technique for intestinal parasitic detection. Our study lasted 9 months and a single stool sample was collected from each enrolled patient. We evaluated classical trichrome (T1 - using Schaudinn fixative) with newer modifications, which involved different fixatives with mordant combinations (T2 - acetic acid + hydrated aluminium sulphate, T3 - citric acid + copper sulphate hydrate). Conventional PCR targeting Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. was taken as the reference. Out of 175 stool samples, 25.1% protozoa were identified by wet mount, 24% by each T1 and T2, 25.7% by T3. Statistically, T3 and T2 had higher sensitivity as compared to T1 and wet mount when PCR was used as reference., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Apicomplexan parasites comprise significant pathogens of humans, livestock and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. The study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting-edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure-expansion microscopy (U-ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here, we review the insights into apicomplexan cell biology that have been enabled through the use of U-ExM, with a specific focus on Plasmodium, Toxoplasma and Cryptosporidium. Further, we summarize emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Background: Ethno-veterinary practices could be used as a sustainable developmental tool by integrating traditional phytotherapy and husbandry. Phytotherapeutics are available and used worldwide. However, evidence of their antiparasitic efficacy is currently very limited. Parasitic diseases have a considerable effect on pig production, causing economic losses due to high morbidity and mortality. In this respect, especially smallholders and organic producers face severe challenges. Parasites, as disease causing agents, often outcompete other pathogens in such extensive production systems. A total of 720 faecal samples were collected in two farms from three age categories, i.e. weaners, fatteners, and sows. Flotation (Willis and McMaster method), modified Ziehl-Neelsen stained faecal smear, centrifugal sedimentation, modified Blagg technique, and faecal cultures were used to identify parasites and quantify the parasitic load., Results: The examination confirmed the presence of infections with Eimeria spp., Cryptosporidium spp., Balantioides coli (syn. Balantidium coli), Ascaris suum, Oesophagostomum spp., Strongyloides ransomi, and Trichuris suis, distributed based on age category. A dose of 180 mg/kg bw/day of Allium sativum L. and 90 mg/kg bw/day of Artemisia absinthium L. powders, administered for 10 consecutive days, revealed a strong, taxonomy-based antiprotozoal and anthelmintic activity., Conclusions: The results highlighted the therapeutic potential of both A. sativum and A. absinthium against gastrointestinal parasites in pigs. Their therapeutic effectiveness may be attributed to the content in polyphenols, tocopherols, flavonoids, sterols, sesquiterpene lactones, and sulfoxide. Further research is required to establish the minimal effective dose of both plants against digestive parasites in pigs., (© 2024. The Author(s).)

SUMMARYProtozoan parasite infection dramatically alters host metabolism, driven by immunological demand and parasite manipulation strategies. Immunometabolic checkpoints are often exploited by kinetoplastid and protozoan parasites to establish chronic infection, which can significantly impair host metabolic homeostasis. The recent growth of tools to analyze metabolism is expanding our understanding of these questions. Here, we review and contrast host metabolic alterations that occur in vivo during infection with Leishmania , trypanosomes, Toxoplasma , Plasmodium, and Cryptosporidium . Although genetically divergent, there are commonalities among these pathogens in terms of metabolic needs, induction of the type I immune responses required for clearance, and the potential for sustained host metabolic dysbiosis. Comparing these pathogens provides an opportunity to explore how transmission strategy, nutritional demand, and host cell and tissue tropism drive similarities and unique aspects in host response and infection outcome and to design new strategies to treat disease., Competing Interests: The authors declare no conflict of interest.

Background: Consuming contaminated raw vegetables and fruits is one of the primary means of parasite transmission to humans. Periodic monitoring of parasitic contamination in these food items is a crucial step in preventing the spread of parasitic disease in the community. This study was aimed at detecting intestinal parasitic contamination caused by consuming raw vegetables and fruits sold in three open-air markets and its associated factors in peri-urban areas of Jimma City, Oromia, Ethiopia., Methods: A cross-sectional study was conducted on fruits and vegetables collected from three peri-urban open-aired markets (namely; Hora Gibe, Bore and Jiren markets) in peri-urban areas of Jimma City between July and September 2021. A total of 187 fresh vegetable samples and 188 fruits were collected and examined for intestinal parasite contamination. About 200g of fruit and vegetable samples were processed and examined microscopically for parasite contamination, utilizing direct wet mount and modified Zeihl-Neelson staining methods in accordance with standard protocols. A structured questionnaire was used to collect data on the socio-demographic characteristics of vendors and risk factors for fruit and vegetable contamination. All data were analyzed using SPSS version 20.0., Result: Of the 187 fresh vegetable samples and 188 fresh vegetable samples, 105 (56.1%) and 68/188 (36.2%) of vegetables and fruit samples, respectively, were found contaminated with one or more intestinal parasites. Remarkably, high level of contamination in fresh vegetable samples was recorded both in Carrot (Daucus carota) 63.8% (30/46) and Lettuce (Lactuca sativa) 63.1% (29/46) while Green pepper (Capsicum spp.) is the least contaminated. In fruit samples, Avokado (Persea americana) 42.6% (20/47) and Banana (Musa acuminata) 14.9% (7/47) were the most and the least commonly contaminated items respectively. The identified helminthes and protozoans were Ascaris lumbricoides, Strongyloides stercoralis, Hymenolepis nana, Entamoeba histolytica/dispar, Giardia lamblia, Cryptosporidium spp., Toxocara spp. And Fasciola spp. The most predominant parasite encountered was A. lumbricoides 46(12.3%) whereas both Toxocara spp. 12(6.9) and Fasciola spp. 2(0.5) were the least detected parasites. It is worth-mentioned that the rate of contamination in Bore market (38.15%) was higher compared with Jiren market (34.7%) and Hora Gibe market (27%). However, the rate of contamination in vegetables and fruit obtained from the three district was non-significant (p = 0.19). Contamination was more common in vegetables than fruits (AOR = 5.78, p<0.001). It was also observed that decreased parasitic contamination was significantly associated with washing the products before displaying it for selling (p < 0.001)., Conclusion: The study has identified a high rate of raw vegetables and fruits contaminated with intestinal helminthes and protozoan. Contaminated fresh vegetables and fruits in open-aired peri-urban markets of Jimma city, Ethiopia may play a significant role in transmission of intestinal parasitic infections to humans, particularly A. lumbricoides infection. Therefore, it is urgently needed for health authorities to educate the public on the proper handling of vegetables and fruits prior to consumption., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zeynudin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dog faecal samples examined from January 2019 to December 2019 were retrospectively analysed for frequency of endoparasites. The examinations were performed with several different methods: 29,219 samples were examined by flotation method and sodium acetate-acetic acid-formalin concentration (SAFC) technique, 1,330 samples by Baermann-Wetzel migration technique, 12,221 samples using a Giardia coproantigen enzyme-linked-immunosorbent assay (ELISA), 1,180 samples using a Cryptosporidium coproantigen ELISA, 1,671 samples by polymerase chain reaction (PCR) testing for Giardia duodenalis and 447 samples by PCR testing for Cryptosporidium spp.. A total of 7.1% of the samples were positive for parasites in the microscopical examination using the flotation method and SAFC technique. The parasites found included Cystoisospora spp. (2.8%), Giardia duodenalis (2.3%), Ancylostomatidae (1.8%), Toxocara canis (1.6%), Trichuris vulpis (0.7%), Toxascaris leonina (0.5%), Capillaria spp. (0.2%), Angiostrongylus vasorum (0.2%), Crenosoma vulpis (0.1%), Taeniidae (0.1%), Sarcocystis spp. (0.03%), Dipylidium caninum (0.01%), Diphyllobothrium latum (< 0.01%), Spirurida (< 0.01%) and Opisthorchiidae (< 0.01%). Using the Baermann-Wetzel migration technique, Angiostrongylus vasorum was found in 0.75% and Crenosoma vulpis in 0.3% of the samples. ELISAs for Giardia duodenalis and Cryptosporidium spp. revealed 13.9% and 1.0% positive faecal samples, and Giardia duodenalis and Cryptosporidium spp. PCRs 19.4% and 2.0%, respectively. Dogs in the first year of life were more frequently infected with parasites than older animals. In the microscopic examination using flotation method and SAFC technique, the significantly highest detection rates were found in dogs up to six months of age (p < 0.001)., (© 2024. The Author(s).)

Background: Raw vegetables have been considered vehicles of enteroparasites. South American countries are among the most important exporters of fresh vegetables, including Ecuador, which has a tropical climate and soils rich in organic matter that allow it to harvest throughout the year for sale to different countries. The aim of the study was to assess the occurrence of the parasitic contamination of fruits, vegetables and leafy greens grown in an agricultural area of the Ecuadorian Andes., Methods: A cross-sectional field study was conducted with snowball sampling on 1,416 samples (516 fruits, 488 vegetables, and 412 leafy greens). Each sample were washed with water, and the resulting solution after removing the vegetables, was subjected to 24-hour sedimentation. The concentrated sediment underwent microscopic analysis., Results: The overall positivity for parasitic contamination was 63.4%, with leafy greens having the highest contamination rate (76.9%) (P<0.0001), surpassing vegetables (67.8%) and fruits (48.4%). Cabbage (100%), onions (84%), and strawberries (60.2%) emerged as the most contaminated within their respective groups. Protozoa were more prevalent (49.6%) than helminths (15.5%) (P<0.0001). Blastocystis sp. (33.5%) ranked highest, followed by Eimeria spp. (26.3%), Entamoeba spp. (10.3%), Giardia spp. (8.3%), Balantidium spp. (6.9%), Cryptosporidium spp. (6.6%), Cyclospora spp. (4.4%), Cystoisospora spp. (0.5%), Strongylida (15.5%), and Ascaris spp. (0.4%)., Conclusions: The study reveals that vegetables and fruits for human consumption from this area of the Ecuadorian Andes are highly contaminated with various parasites, constituting a possible source of infection for humans and animals in this area, or in non-endemic areas where these products are marketed. The finding emphasizes the need for strict hygienic measures in agricultural crops, which will be properly achieved through the treatment of soil, manure and water used for cultivation., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 González-Ramírez LC et al.)

The dependence of Cryptosporidium parasites on host cell metabolites suggests that the development of nutritional interventions to limit parasite proliferation should be feasible. Based on this concept, we are testing dietary interventions to affect the enterocytes' metabolism in a manner that limits intracellular multiplication of the parasite. We hypothesize that changes in the metabolic pathways encoded by the gastro-intestinal tract microbiota may restrict parasite proliferation. To identify taxonomic and metabolic features of the microbiota associated with severity of cryptosporidiosis, as determined by estimating oocyst output, we characterized the fecal microbiota from mice experimentally infected with Cryptosporidium parvum. To eliminate the confounding effect of the interaction between co-housed mice, as well as facilitate the identification of microbiota markers associated with severity of cryptosporidiosis, fecal microbiota from individually caged mice were analyzed. Variation partitioning analysis applied to 16S sequence data from 25 mice belonging to four experiments shows that experiment was by far the biggest source of microbiota variation. Severity of cryptosporidiosis explained a smaller, though significant, fraction of microbiota variation. Notably, this effect was significant in the pre-patent phase of the infection, before mice excreted oocysts. These results are consistent with the pre-patent intestinal microbiota having a modest, but measurable, effect on cryptosporidiosis., (© 2024. The Author(s).)

Gastrointestinal protist (GP) and soil-transmitted helminth (STH) infections cause significant morbidity among children in poor-resource settings of tropical and sub-tropical countries including Colombia. Few prospective transversal studies investigating how GP and STH infections affect growth development and nutritional status during childhood have been conducted in this country, none of them in the Antioquia Department. This microscopy-based study estimated the prevalence of GP and helminth (including STH) infections in faecal samples from schoolchildren (n = 384) collected during April-May 2019 in three municipalities of the Antioquia Department. Demographic, epidemiological, and household data were elicited through face-to-face interviews. Parasite detection was carried out by direct microscopic examination of both fresh smears and concentrated faecal material. Children (aged 6-15 years) also had their haemoglobin (Hb) levels, height and weight data collected, and BMI estimated. Data were analysed using bivariate and multivariate logistic regression analysis. Overall, 60.7 % (233/384) of schoolchildren were infected by at least one intestinal parasitic species. Among GPs, Blastocystis sp. was the most common species found (47.7 %, 95 % CI: 42.6-52.8), followed by G. duodenalis (15.9 %, 95 % CI: 12.4-19.9). Cryptosporidium spp. and Cyclospora cayetanensis were sporadically identified (0.3 %, 95 % CI: 0.1-1.4 each). Among helminths, the most prevalent species found were Trichuris trichiura (6.0 %, 95 % CI: 3.8-8.9) and Enterobius vermicularis (1.0 %, 95 % CI: 0.3-2.6). Hookworms, Ascaris lumbricoides, and Strongyloides stercoralis were found at prevalence rates <1 %. Underweight, overweigh, or obese schoolchildren had 1.2 times greater chance of being infected with intestinal parasites than their counterparts with a healthy weight (P-value: 0.015). Variables significantly associated with an increased likelihood of being infected by intestinal parasites include living in a household with unfinished flouring, not wearing shoes, being in close proximity to rodents, and having improper waste disposal. Relatively simple interventional measures directed towards the improvement of household conditions, access to sanitary toilets, and promoting shoe wearing can significantly reduce childhood infections by GP and helminths in the Antioquia Department., 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. Published by Elsevier B.V.)

Rodents represent the single largest group within mammals and host a diverse array of zoonotic pathogens. Urbanisation impacts wild mammals, including rodents, leading to habitat loss but also providing new resources. Urban-adapted (synanthropic) rodents, such as the brown rat (R. norvegicus), black rat (R. rattus), and house mouse (Mus musculus), have long successfully adapted to living close to humans and are known carriers of zoonotic pathogens. Two important enteric, zoonotic protozoan parasites, carried by rodents, include Cryptosporidium and Giardia. Their environmental stages (oocysts/cysts), released in faeces, can contaminate surface and wastewaters, are resistant to common drinking water disinfectants and can cause water-borne related gastritis outbreaks. At least 48 species of Cryptosporidium have been described, with C. hominis and C. parvum responsible for the majority of human infections, while Giardia duodenalis assemblages A and B are the main human-infectious assemblages. Molecular characterisation is crucial to assess the public health risk linked to rodent-related water contamination due to morphological overlap between species. This review explores the global molecular diversity of these parasites in rodents, with a focus on evaluating the zoonotic risk from contamination of water and wasterwater with Cryptosporidium and Giardia oocysts/cysts from synanthropic rodents. Analysis indicates that while zoonotic Cryptosporidium and Giardia are prevalent in farmed and pet rodents, host-specific Cryptosporidium and Giardia species dominate in urban adapted rodents, and therefore the risks posed by these rodents in the transmission of zoonotic Cryptosporidium and Giardia are relatively low. Many knowledge gaps remain however, and therefore understanding the intricate dynamics of these parasites in rodent populations is essential for managing their impact on human health and water quality. This knowledge can inform strategies to reduce disease transmission and ensure safe drinking water in urban and peri‑urban areas., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Una Ryan reports financial support was provided by Water Research Australia. If there are other authors, they 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Despite zoonotic potential, data are lacking on enteric infection diversity in wild apes. We employed a novel molecular diagnostic platform to detect enteric infections in wild chimpanzees and gorillas. Prevalent Cryptosporidium parvum, adenovirus, and diarrheagenic Escherichia coli across divergent sites and species demonstrates potential widespread circulation among apes in Africa.

Case History: Two clusters of mortality among endangered tūturuatu/tchūriwat'/shore plover ( Thinornis novaeseelandiae ) have occurred at captive breeding facilities around New Zealand in recent years. In the first, four chicks died at Pūkaha National Wildlife Centre (Mount Bruce, NZ) in February 2016, and in the second five adult birds at the Cape Sanctuary (Cape Kidnappers, NZ) died in 2022., Clinical Findings: In 2016, four chicks were noted to become weak, have increased vocalisations and closed eyes prior to death. The remaining chicks were treated for 5 days with amoxycillin/clavulanate orally twice daily. Water containers and brooders were cleaned and disinfected with chlorhexidine. No further mortality was seen.In the 2022 cluster, three adult breeding birds died acutely and five others showed inappetence, weight loss and diarrhoea approximately 10 days after heavy rains flooded the local river. The five birds were treated with amoxycillin/clavulanate orally twice daily and oral fluids for 5 days. Two birds died and three survived. No breeding occurred in the aviaries in the following season., Pathological Findings: In 2016, the chicks showed pulmonary changes ranging from congestion and oedema to heterophilic inflammation consistent with septicaemia.In 2022, the adult birds showed proliferation of bacteria in the distal small intestine associated with mucosal ulceration and heterophilic infiltration. Acid-fast staining of the caecal contents in one bird showed organisms consistent with Cryptosporidium spp., Laboratory Findings: Aerobic bacterial cultures of the lung and liver of two affected chicks carried out in 2016 showed heavy growth of Plesiomonas shigelloides. The same organism was cultured from water trays and holding tanks containing water boatmen ( Sigara arguta ) on which the chicks were fed.In 2022, cultures from the livers of three dead birds each showed a mixed bacterial growth with differing dominant organisms ( Aeromonas sobria , Hafnia alvei , Citrobacter freundii and an Enterococcus sp.). PCR and sequencing confirmed Cryptosporidium parvum in the caecum of one bird . Fresh faeces from 24 breeding birds from the captive breeding facilities were negative by PCR for Cryptosporidium spp.The captive breeding facilities obtain water for the aviaries and aquatic invertebrates to feed to the chicks from local freshwater sources. Water quality testing at the Cape Sanctuary revealed concentrations of faecal indicator bacteria in excess of safe drinking water guidelines, with peaks following heavy rainfall., Clinical Relevance: Fluctuations in water quality associated with mammalian faecal bacteria can adversely affect bird health and impact on captive rearing of endangered wildlife.

This work aims to: (1) elucidate the immune response exhibited by CD4 + and CD8 + T lymphocyte cells in response to various infectious agents in calves suffering with neonatal diarrhea; and (2) determine and investigate the association between serum selenium levels and T lymphocyte subtypes in neonatal calves afflicted with neonatal diarrhea and infected with various infectious agents. The study encompassed a cohort of 50 calves, encompassing both sexes and various breeds, within the neonatal age range (1-28 days old). Subdivided into distinct groups, the calves were categorized based on the causative agents of neonatal diarrhea, including Rotavirus (n = 10), Cryptosporidium parvum (C.parvum) (n = 10), Coronavirus (n = 5), Rotavirus+C.parvum (n = 5), and a Control group (n = 20). Blood samples were meticulously obtained from the vena jugularis of all animals utilizing specific techniques-8 ml in tubes devoid of anticoagulant and 3 ml in blood collection tubes containing EDTA. Serum selenium levels were analyzed by ICP-MS. Flow Cytometry device was used to determine CD4 + and CD8 +T lymphocyte levels. In this study, although there was no statistically significant difference in serum selenium levels between all study groups, it was found that the selenium level in the control group was not sufficient. CD4 T lymphocyte levels, the rotavirus+C.parvum group exhibited a statistically significant elevation compared to the coronavirus group. Regarding CD8 + T lymphocyte levels, the coronavirus group demonstrated a statistically significant increase when compared to the control group. In intragroup analyses of CD8 + T lymphocyte levels, the coronavirus group exhibited a significant elevation compared to the rotavirus group, C.parvum group, and the C.parvum + Rotavirus group. A significant negative correlation was detected between selenium levels and CD4 + T lymphocytes, while no correlation was found between CD8 + T lymphocytes. Fibrinogen concentration exhibited statistical significance, being higher in the Rotavirus group (p < 0.008) compared to the control group, in the C.parvum group (p < 0.004) compared to the control group, and in the Coronavirus group (p < 0.001) compared to the control group. The leukocyte count demonstrated statistical significance, being higher in the Rotavirus group compared to the control group (p < 0.001), in the Rotavirus+C.parvum group compared to the control group (p < 0.002), and in the Coronavirus group compared to the control group (p < 0.011). In conclusion, the data derived from this study illuminate discernible disparities in CD4 + and CD8 + T lymphocyte immune responses, contingent upon the specific etiological agent associated with neonatal diarrhea. Furthermore, the study underscores the importance of considering selenium deficiency as a relevant factor in calves affected by neonatal diarrhea., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Oral drug absorption kinetics are usually established in populations with a properly functioning gastrointestinal tract. However, many diseases and therapeutics can alter gastrointestinal physiology and cause diarrhea. The extent of diarrhea-associated impact on drug pharmacokinetics has not been quantitatively described. To address this knowledge gap, we used a population pharmacokinetic modeling approach with data collected in a phase IIa study of matched human immunodeficiency virus (HIV)-infected adults with/without cryptosporidiosis and diarrhea to examine diarrhea-associated impact on oral clofazimine pharmacokinetics. A population pharmacokinetic model was developed with 428 plasma samples from 23 HIV-infected adults with/without Cryptosporidium infection using nonlinear mixed-effects modeling. Covariates describing cryptosporidiosis-associated diarrhea severity (e.g., number of diarrhea episodes, diarrhea grade) or HIV infection (e.g., viral load, CD4+ T cell count) were evaluated. A two-compartment model with lag time and first-order absorption and elimination best fit the data. Maximum diarrhea grade over the study duration was found to be associated with a more than sixfold reduction in clofazimine bioavailability. Apparent clofazimine clearance, intercompartmental clearance, central volume of distribution, and peripheral volume of distribution were 3.71 L/h, 18.2 L/h (interindividual variability [IIV] 45.0%), 473 L (IIV 3.46%), and 3434 L, respectively. The absorption rate constant was 0.625 h -1 (IIV 149%) and absorption lag time was 1.83 h. In conclusion, the maximum diarrhea grade observed for the duration of oral clofazimine administration was associated with a significant reduction in clofazimine bioavailability. Our results highlight the importance of studying disease impacts on oral therapeutic pharmacokinetics to inform dose optimization and maximize the chance of treatment success., (© 2023 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

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.)

Despite tremendous advances in the prevention and treatment of infectious diseases, only few antiparasitic drugs have been developed to date. Protozoan infections such as malaria, leishmaniasis, and trypanosomiasis continue to exact an enormous toll on public health worldwide, underscoring the need to discover novel antiprotozoan drugs. Recently, there has been an explosion of research into the antiprotozoan properties of quercetin, one of the most abundant flavonoids in the human diet. In this review, we tried to consolidate the current knowledge on the antiprotozoal effects of quercetin and to provide the most fruitful avenues for future research. Quercetin exerts potent antiprotozoan activity against a broad spectrum of pathogens such as Leishmania spp., Trypanosoma spp., Plasmodium spp., Cryptosporidium spp., Trichomonas spp., and Toxoplasma gondii . In addition to its immunomodulatory roles, quercetin disrupts mitochondrial function, induces apoptotic/necrotic cell death, impairs iron uptake, inhibits multiple enzymes involved in fatty acid synthesis and the glycolytic pathways, suppresses the activity of DNA topoisomerases, and downregulates the expression of various heat shock proteins in these pathogens. In vivo studies also show that quercetin is effective in reducing parasitic loads, histopathological damage, and mortality in animals. Future research should focus on designing effective drug delivery systems to increase the oral bioavailability of quercetin. Incorporating quercetin into various nanocarrier systems would be a promising approach to manage localized cutaneous infections. Nevertheless, clinical trials are needed to validate the efficacy of quercetin in treating various protozoan infections., Competing Interests: The authors have no relevant financial or nonfinancial interests to disclose. The authors declare no conflicts of interest., (Copyright © 2024 Hamed Memariani et al.)

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.

While in much of the Western world snakes are feared, in the small, rural, mountainous town of Cocullo, in the middle of central Italy, snakes are annually collected and celebrated in a sacro-profane ritual. Every 1st of May, Serpari (snake catchers) capture and showcase dozens of non-venomous snakes to celebrate the ritual of San Domenico. In order to detect potential zoonotic pathogens within this unique epidemiological context, parasites and microorganisms of snakes harvested for the "festa dei serpari" ritual were investigated. Snakes (n = 112) were examined and ectoparasites collected, as well as blood and feces sampled. Ectoparasites were identified morpho-molecularly, and coprological examination conducted through direct smear and flotation. Molecular screenings were performed to identify parasites and microorganisms in collected samples (i.e., Mesostigmata mites, Anaplasma/Ehrlichia spp., Rickettsia spp., Borrelia burgdorferi sensu lato, Coxiella burnetii, Babesia/Theileria spp., Cryptosporidium spp., Giardia spp., Leishmania spp. and helminths). Overall, 28.5% (32/112) of snakes were molecularly positive for at least one parasite and/or microorganism. Endosymbiont Wolbachia bacteria were identified from Macronyssidae mites and zoonotic vector-borne pathogens (e.g., Rickettsia, Leishmania), as well as orally transmitted pathogens (i.e., Cryptosporidium, Giardia, Proteus vulgaris, Pseudomonas), were detected from blood and feces. Thus, given the central role of the snakes in the tradition of Cocullo, surveys of their parasitic fauna and associated zoonotic pathogens may aid to generate conservation policies to benefit the human-snake interactions, whilst preserving the cultural patrimony of this event., Competing Interests: The authors report there are no competing interests to declare., (Copyright: © 2024 Mendoza-Roldan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Despite extensive research, little is known about the composition of eukaryotic protists in environmental samples. This is due to low parasite concentrations, the complexity of parasite diversity, and a lack of suitable reference databases and standardized protocols. To bridge this knowledge gap, this study used 18S rRNA short amplicon and shotgun metagenomic sequencing approaches to profile protozoan microbial communities as well as their functional pathways in treated and untreated wastewater samples collected from different regions of South Africa. Results demonstrated that protozoan diversity (Shannon index P-value = 0.03) and taxonomic composition (PERMANOVA, P-value = 0.02) was mainly driven by the type of wastewater samples (treated & untreated) and geographic location. However, these WWTPs were also found to contain a core community of protozoan parasites. The untreated wastewater samples revealed a predominant presence of free-living, parasitic, and potentially pathogenic protists typically found in humans and animals, ranging from Alveolata (27 %) phylum (Apicomplexa and Ciliophora) to Excavata (3.88 %) (Discoba and Parasalia) and Amoebozoa (2.84 %) (Entamoeba and Acanthamoeba). Shotgun metagenomics analyses in a subset of the untreated wastewater samples confirmed the presence of public health-importance protozoa, including Cryptosporidium species (3.48 %), Entamoeba hystolitica (6.58 %), Blastocystis hominis (2.91 %), Naegleria gruberi (2.37 %), Toxoplasma gondii (1.98 %), Cyclospora cayetanensis (1.30 %), and Giardia intestinalis (0.31 %). Virulent gene families linked to pathogenic protozoa, such as serine/threonine protein phosphatase and mucin-desulfating sulfatase were identified. Additionally, enriched pathways included thiamine diphosphate biosynthesis III, heme biosynthesis, Methylerythritol 4-Phosphate Pathway, methyl erythritol phosphate (MEP), and pentose phosphate pathways. These findings suggest that protozoan pathogens may possess metabolic and growth potential within WWTPs, posing a severe risk of transmission to humans and animals if inadequately disinfected before release. This study provides a baseline for the future investigation of diverse protozoal communities in wastewater, which are of public health importance., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Cryptosporidium spp. are common protozoa causing diarrhea in humans and animals. There are currently only one FDA-approved drug and no vaccines for cryptosporidiosis, largely due to the limited knowledge of the molecular mechanisms involved in the invasion of the pathogens. Previous studies have shown that GP60, which is cleaved into GP40 and GP15 after expression, is an immunodominant mucin protein involved in the invasion of Cryptosporidium. The protein is highly O-glycosylated, and recombinant proteins expressed in prokaryotic systems are non-functional. Therefore, few studies have investigated the function of GP40 and GP15., Methods: To obtain recombinant GP40 with correct post-translational modifications, we used CRISPR/Cas9 technology to insert GP40 and GP15 into the UPRT locus of Toxoplasma gondii, allowing heterologous expression of Cryptosporidium proteins. In addition, the Twin-Strep tag was inserted after GP40 for efficient purification of GP40., Results: Western blotting and immunofluorescent microscopic analyses both indicated that GP40 and GP15 were stably expressed in T. gondii mutants. GP40 localized not only in the cytoplasm of tachyzoites but also in the parasitophorous vacuoles, while GP15 without the GPI anchor was expressed only in the cytoplasm. In addition, a large amount of recTgGP40 was purified using Strep-TactinXT supported by a visible band of ~ 50 kDa in SDS-PAGE., Conclusions: The establishment of a robust and efficient heterologous expression system of GP40 in T. gondii represents a novel approach and concept for investigating Cryptosporidium mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs., (© 2024. The Author(s).)

Apicomplexa encompasses a large number of intracellular parasites infecting a wide range of animals. Cyclic nucleotide signaling is crucial for a variety of apicomplexan life stages and cellular processes. The cyclases and kinases that synthesize and respond to cyclic nucleotides (i.e., 3',5'-cyclic guanosine monophosphate and 3',5'-cyclic adenosine monophosphate) are highly conserved and essential throughout the parasite phylum. Growing evidence indicates that phosphodiesterases (PDEs) are also critical for regulating cyclic nucleotide signaling via cyclic nucleotide hydrolysis. Here, we discuss recent advances in apicomplexan PDE biology and opportunities for therapeutic interventions, with special emphasis on the major human apicomplexan parasite genera Plasmodium , Toxoplasma , Cryptosporidium , and Babesia . In particular, we show a highly flexible repertoire of apicomplexan PDEs associated with a wide range of cellular requirements across parasites and lifecycle stages. Despite this phylogenetic diversity, cellular requirements of apicomplexan PDEs for motility, host cell egress, or invasion are conserved. However, the molecular wiring of associated PDEs is extremely malleable suggesting that PDE diversity and redundancy are key for the optimization of cyclic nucleotide turnover to respond to the various environments encountered by each parasite and life stage. Understanding how apicomplexan PDEs are regulated and integrating multiple signaling systems into a unified response represent an untapped avenue for future exploration., Competing Interests: The authors declare no conflict of interest.

L-tryptophan (l-Trp), a vital amino acid for the survival of various organisms, is synthesized by the enzyme tryptophan synthase (TS) in organisms such as eubacteria, archaebacteria, protista, fungi, and plantae. TS, a pyridoxal 5'-phosphate (PLP)-dependent enzyme, comprises α and β subunits that typically form an α 2 β 2 tetramer. The enzyme's activity is regulated by the conformational switching of its α and β subunits between the open (T state) and closed (R state) conformations. Many microorganisms rely on TS for growth and replication, making the enzyme and the l-Trp biosynthetic pathway potential drug targets. For instance, Mycobacterium tuberculosis , Chlamydiae bacteria, Streptococcus pneumoniae , Francisella tularensis , Salmonella bacteria, and Cryptosporidium parasitic protozoa depend on l-Trp synthesis. Antibiotic-resistant salmonella strains have emerged, underscoring the need for novel drugs targeting the l-Trp biosynthetic pathway, especially for salmonella-related infections. A single amino acid mutation can significantly impact enzyme function, affecting stability, conformational dynamics, and active or allosteric sites. These changes influence interactions, catalytic activity, and protein-ligand/protein-protein interactions. This study focuses on the impact of mutating the βGln114 residue on the catalytic and allosteric sites of TS. Extensive molecular dynamics simulations were conducted on E(PLP), E(AEX 1 ), E(A-A), and E(C 3 ) forms of TS using the WT, βQ114A, and βQ114N versions. The results show that both the βQ114A and βQ114N mutations increase protein backbone root mean square deviation fluctuations, destabilizing all TS forms. Conformational and hydrogen bond analyses suggest the significance of βGln114 drifting away from cofactor/intermediates and forming hydrogen bonds with water molecules necessary for l-Trp biosynthesis. The βQ114A mutation creates a gap between βAla114 and cofactor/intermediates, hindering hydrogen bond formation due to short side chains and disrupting β-sites. Conversely, the βQ114N mutation positions βAsn114 closer to cofactor/intermediates, forming hydrogen bonds with O3 of cofactors/intermediates and nearby water molecules, potentially disrupting the l-Trp biosynthetic mechanism.

Phosphatidylinositol 4-kinases (PI4Ks) could phosphorylate phosphatidylinositol (PI) to produce phosphatidylinositol 4-phosphate (PI4P) and maintain its metabolic balance and location. PI4P, the most abundant monophosphate inositol in eukaryotic cells, is a precursor of higher phosphoinositols and an essential substrate for the PLC/PKC and PI3K/Akt signaling pathways. PI4Ks regulate vesicle transport, signal transduction, cytokinesis, and cell unity, and are involved in various physiological and pathological processes, including infection and growth of parasites such as Plasmodium and Cryptosporidium, replication and survival of RNA viruses, and the development of tumors and nervous system diseases. The development of novel drugs targeting PI4Ks and PI4P has been the focus of the research and clinical application of drugs, especially in recent years. In particular, PI4K inhibitors have made great progress in the treatment of malaria and cryptosporidiosis. We describe the biological characteristics of PI4Ks; summarize the physiological functions and effector proteins of PI4P; and analyze the structural basis of selective PI4K inhibitors for the treatment of human diseases in this review. Herein, this review mainly summarizes the developments in the structure and enzyme activity of PI4K inhibitors., 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 Inc. All rights reserved.)

The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l-methionine S-adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S-adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species-selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog-selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum., (© 2023 The Authors. Drug Development Research published by Wiley Periodicals LLC.)

Information on the causative agent in an enteric disease outbreak can be used to generate hypotheses about the route of transmission and possible vehicles, to guide environmental assessments, and to target outbreak control measures. However, only about 40% of outbreaks reported in the United States include a confirmed etiology. The goal of this project was to identify clinical and demographic characteristics that can be used to predict the causative agent in an enteric disease outbreak and to use these data to develop an online tool for investigators to use during an outbreak when hypothesizing about the causative agent. Using data on enteric disease outbreaks from all transmission routes (animal contact, environmental contamination, foodborne, person-to-person, waterborne, unknown) reported to the U.S. Centers for Disease Control and Prevention, we developed random forest models to predict the etiology of an outbreak based on aggregated clinical and demographic characteristics at both the etiology category (i.e., bacteria, parasites, toxins, viruses) and individual etiology ( Clostridium perfringens , Campylobacter , Cryptosporidium , norovirus, Salmonella , Shiga toxin-producing Escherichia coli , and Shigella ) levels. The etiology category model had a kappa of 0.85 and an accuracy of 0.92, whereas the etiology-specific model had a kappa of 0.75 and an accuracy of 0.86. The highest sensitivities in the etiology category model were for bacteria and viruses; all categories had high specificities (>0.90). For the etiology-specific model, norovirus and Salmonella had the highest sensitivity and all etiologies had high specificities. When laboratory confirmation is unavailable, information on the clinical signs and symptoms reported by people associated with the outbreak, with other characteristics including case demographics and illness severity, can be used to predict the etiology or etiology category. An online publicly available tool was developed to assist investigators in their enteric disease outbreak investigations.

Background: Diversity in clinical signs and symptoms are associated with soil transmitted diseases (STD), which are spread to humans by intestinal worms and transmitted in a variety of ways. There is a need for the present study, which aimed to investigate the prevalence of intestinal parasites and to compare between the common detection techniques for soil-transmitted helminths (STHs) among newly arrived expatriate labors in Jeddah, Saudi Arabia., Methods: A total of 188 stool samples were analyzed by macroscopic examination, and microscopic examination using direct iodine smear and the formal ether sedimentation technique. Trichrome and modified Kinyoun's stains were used to confirm the morphology of any detected protozoa stages and oocyst of Cryptosporidium , respectively. A chromatographic immunoassay kit was used for Entamoeba histolytica , Giardia lamblia and Cryptosporidium . In addition, real-time PCR was employed only to identify various STHs., Results: Out of 188, several types of parasites were detected in 35 samples (18.62%), of which some with multiple infections. Nine samples (4.79%) were positive for Entamoeba coli , seven samples (3.72%) for Trichuris trichiura , six samples (3.19%) for Necator americanus , four samples (2.13%) for Strongyloides stercoralis , four samples (2.13%) for Ascaris lumbricoides , four samples (2.13%) for E. histolytica , three samples (1.60%) for Blastocystis hominis and two samples (1.06%) for Ancylostoma duodenale . In comparison between laboratory techniques for STHs, real-time PCR was able to detect the DNA of 19 samples (10.1%) followed by Ritchie sedimentation technique (18, 9.6%), and direct smear (7, 3.7%) ( p > 0.05)., Conclusion: The high rate of newly arrived foreign workers infected with intestinal parasites could lead to a risk to society. Continuous and regular surveys are needed to deal with the occurrence of intestinal parasitic infections including STHs. To improve the identification of these infections, we recommend a supporting infrastructure for the application of concentration methods and molecular assays., Competing Interests: The authors declare that they have no competing interests., (© 2024 Al-Refai and Wakid.)

One of the leading causes of infectious diarrhea in newborn calves is the apicomplexan protozoan Cryptosporidium parvum ( C. parvum ). However, little is known about its immunopathogenesis. Using next generation sequencing, this study investigated the immune transcriptional response to C. parvum infection in neonatal calves. Neonatal male Holstein-Friesian calves were either orally infected (N = 5) or not (CTRL group, N = 5) with C. parvum oocysts (gp60 subtype IIaA15G2R1) at day 1 of life and slaughtered on day 7 after infection. Total RNA was extracted from the jejunal mucosa for short read. Differentially expressed genes (DEGs) between infected and CTRL groups were assessed using DESeq2 at a false discovery rate < 0.05. Infection did not affect plasma immunohematological parameters, including neutrophil, lymphocyte, monocyte, leucocyte, thrombocyte, and erythrocyte counts as well as hematocrit and hemoglobin concentration on day 7 post infection. The immune-related DEGs were selected according to the UniProt immune system process database and were used for gene ontology (GO) and pathway enrichment analysis using Cytoscape (v3.9.1). Based on GO analysis, DEGs annotated to mucosal immunity, recognizing and presenting antigens, chemotaxis of neutrophils, eosinophils, natural killer cells, B and T cells mediated by signaling pathways including toll like receptors, interleukins, tumor necrosis factor, T cell receptor, and NF-KB were upregulated, while markers of macrophages chemotaxis and cytosolic pattern recognition were downregulated. This study provides a holistic snapshot of immune-related pathways induced by C. parvum in calves, including novel and detailed feedback and feedforward regulatory mechanisms establishing the crosstalk between innate and adaptive immune response in neonate calves, which could be utilized further to develop new therapeutic strategies., 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 © 2024 Veshkini, Dengler, Bachmann, Liermann, Helm, Ulrich, Delling, Kühn and Hammon.)

Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the tricarboxylic acid cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves., (© 2024. The Author(s).)

Patients with diabetes are at an increased risk of intestinal parasitic infections (IPIs). We evaluated the pooled prevalence and OR of IPIs in patients with diabetes through a systematic review and meta-analysis. A systematic search was performed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol for studies reporting IPIs in patients with diabetes through 1 August 2022. The collected data were analyzed using comprehensive meta-analysis software version 2. Thirteen case-control studies and nine cross-sectional studies were included in this study. The overall prevalence of IPIs in patients with diabetes was calculated to be 24.4% (95% CI 18.8 to 31%). Considering the case-control design, the prevalence of IPIs in case (25.7%; 95% CI 18.4 to 34.5%) was higher than controls (15.5%; 95% CI 8.4 to 26.9%) and a significant correlation was observed (OR, 1.80; 95% CI 1.08 to 2.97%). Moreover, a significant correlation was seen in the prevalence of Cryptosporidium spp. (OR, 3.30%; 95% CI 1.86 to 5.86%), Blastocystis sp. (OR, 1.57%; 95% CI 1.11 to 2.22%) and hookworm (OR, 6.09%; 95% CI 1.11 to 33.41%) in the cases group. The present results revealed a higher prevalence of IPIs in patients with diabetes than in controls. Therefore, the results of this study suggest a proper health education program to preventing measures for the acquisition of IPIs in patients with diabetes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Background: Global studies show intestinal parasitic infections (IPIs) have been introduced and spread with refugee inflows from low to high socio-economic countries. However, there is relatively limited information on the prevalence of infections among the community., Methods: A 2-year cross-sectional study was conducted to determine the prevalence and associated risk factors for infections among urban refugees in the Klang Valley, Malaysia. A total of 418 faecal samples were collected and examined by microscopy., Results: Faecal screening revealed moderate levels (32.3%) of infections in the community. Three nematode (Ascaris lumbricoides, Trichuris trichiura and hookworm) and three protozoan species (Entamoeba, Giardia and Cryptosporidium) were recorded, with the highest prevalence being A. lumbricoides (20.6%) followed by T. trichiura (10.3%), while other infections were <5%. Statistical analysis found that young males with less education were more likely to be infected with helminths. Additionally, living near waste disposal sites, the presence of stray animals, eating with bare hands, bare footedness, poor handwashing practices and no anthelmintic treatment constituted significant risk factors for helminth infections. Protozoan infections were linked to drinking tap water or from water dispensers and poor handwashing practices., Conclusions: These findings emphasize the importance of health education in addition to introduction of biannual anthelmintic treatment to promote community health and well-being., (© The Author(s) 2023. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

The aim of this study was to determine the occurrence of zoonotic enteroparasites in the feces of dogs from public shelters, squares, parks, and public roads in the city of Guarapuava, Paraná, Brazil, and to evaluate the perception of dog guardians regarding zoonoses and their risk to public health. Fecal samples were collected, coproparasitological examinations were performed to detect zoonotic enteroparasites, and polymerase chain reaction (PCR) was used to identify Giardia spp. and Cryptosporidium spp. Questionnaires were given to guardians who walked their dogs in parks, squares, and public roads, as to assess their perception of zoonoses. A total of 333 samples were collected, of these 75, 123, and 135 of them were from public shelters, squares and parks, and public roads, respectively. One or more parasites were identified in 166 (50 %) samples, of which 58/75 (77 %) were from public shelters, 50/123 (41 %) from squares and parks, and 58/135 (43 %) from public roads. The parasites detected included Ancylostoma spp., Giardia spp., Trichuris spp., Toxocara spp., and Cystoisospora spp., with Ancylostoma spp. having the highest occurrence. PCR was performed on 161 samples for convenience due to financial limitations, because only a portion of the study was funded by the municipal government, of which 15.6 % were positive for Giardia spp., and all were negative for Cryptosporidium spp. In total, 246 guardians were interviewed, of which 36 % said they did not collect their animals' feces during walks, 20 % did not use anti-helminthics on their dogs, and 23 % did not know which diseases could be transmitted by feces. Therefore, we conclude that there is a high infection rate of parasites with zoonotic potential in public places, showing the need to raise awareness among guardians about the diseases transmitted by dog feces, correct vermifugation and the importance of collecting feces in public places., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cryptosporidium, Shigella, toxin-producing Escherichia coli, and rotavirus were reported to be the most responsible for severe and fatal diarrhea among infants. This study aimed to investigate the presence of these pathogens in infants' drinking water samples and analyzing using water quality determinants in eastern Ethiopia. A molecular (LAMP)-based cross-sectional study design was employed. A total of 410 and 37 water samples were tested from infant point-of-use at household and corresponding water source, respectively, from June 2020 to May, 2021. Cryptosporidium, Shigella, toxin-producing E. coli, and rotavirus were detected in 28.5, 30.0, 26.3, and 32.2%, of water samples tested from infant point-of-use, respectively. About 13.2% of the water samples were positive for all (four) pathogens together. Cryptosporidium, Shigella, toxin-producing E. coli, and rotavirus were detected in 27.0, 32.4, 29.7, and 37.8%, of water samples tested from water sources, respectively. Positive significant correlation was observed between infant point-of-consumption and water sources from which it is drawn toward the presence of each targeted pathogen. Unimproved water source showed a strong significant association with the presence of Cryptosporidium, Shigella and toxin-producing E. coli. Therefore, efforts should be made in development of improved water sources, source protection safety and health education to caretakers of infants.

The rapid diagnosis of infectious diarrhea is lifesaving for intensive care unit (ICU) patients. This study evaluated a commercially available multiplex polymerase chain reaction (PCR) (BioFire FilmArray) for the diagnosis of parasitic and bacterial infections in ICU patients with secretory diarrhea in comparison to other traditional methods. This cross-sectional study included 50 subjects with infectious diarrhea. Their stool samples were subjected to macroscopic and microscopic examinations, concentration techniques, permanent staining techniques, stool culture, identification of bacterial infection by the Vitek 2 Compact 15 System, and molecular diagnosis of bacterial or parasitic infections by BioFire FilmArray multiplex PCR. Parasitological examination showed that the sensitivity and specificity of BioFire FilmArray multiplex PCR in the diagnosis of Cryptosporidium oocysts were 83.33% and 100.0%, respectively compared with 100% and 92.5% in diagnosis of G. lamblia cysts. Bacteriological examination showed that the sensitivity and specificity of BioFire FilmArray multiplex PCR in the diagnosis of E. coli and salmonella were 100% and 100.0%, respectively. The BioFire FilmArray multiplex PCR gastrointestinal (GI) panel assay was more sensitive and specific in the diagnosis of bacterial infections than parasitic infections. The BioFire FilmArray multiplex PCR GI panel assay was less sensitive in the detection of Cryptosporidium oocysts than traditional methods. In conclusion, the BioFire FilmArray multiplex PCR may be useful for rapid diagnosis of ICU patients with infectious diarrhea., (Copyright© by the Egyptian Association of Immunologists.)

Emerging organoid research is paving way for studies in infectious diseases. Described here is a technique for the generation of stem-cell derived organoids for human small intestine and lung together with methods to infect such organoids with a mock pathogen (Cryptosporidium parvum). Such systems are amenable to imaging and processing for molecular biological analyses. It is the intent of this chapter to provide a simple, routine organoid procedure so that in vitro studies with Borrelia such as cell invasion and dissemination can be conducted., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In August and September 2023, an unusually high number of cryptosporidiosis cases identified by routine German surveillance had travelled to Croatia (n = 23). Nine cases had stayed in the same camping resort and seven further cases had stayed at other camping sites within 15 km. Based on our standardised questionnaires, the most likely source of infection was swimming pools (93%). Further environmental investigations on site might reveal potential common sources of contamination that could be targeted by control measures.

Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. Cryptosporidium infection induced a strong interferon response from enterocytes, possibly driven, in part, by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

There is a paucity of information regarding the health status of free-ranging eastern indigo snakes (EIS; Drymarchon couperi) in heavily modified and developing landscapes. As a component of regional Florida Everglades restoration efforts, several areas occupied by EIS are being converted from agricultural lands to reservoirs. From 2020 to 2022, 28 EIS were opportunistically captured at two of these sites and brought into captivity to join a captive breeding colony; however, 11 snakes died within 5 mo of capture. Health assessments were performed on 28 individuals and included hematology and plasma biochemistry analysis, as well as screening for pesticide contaminant levels, parasites, and other pathogens. Overall, the presence of pathogens was relatively high, suggesting immunosuppression secondary to stress: 25/28 (89.4%) Kalicephalus sp.; 12/28 (42.9%) Raillietiella orientalis; 11/28 (39.2%) Ochetosoma validum; 7/28 (25.0%) Cryptosporidium serpentis; 3/28 (10.7%) snake adenovirus 1; and 1/28 (3.6%) Ferlavirus genotype C. Stress may have been caused by physical displacement, habitat modification, and noise pollution. These potential stressors (including the presence of remnant harmful chemicals from previous land use and the impacts on this federally threatened species) should be considered further when making restoration or construction decisions., (© Wildlife Disease Association 2024.)

Asthma is characterized by chronic airway inflammation. In addition to allergens, microorganisms can affect the clinical course of asthma. It has been shown that some fungi play an important role in the progression of asthma. However, the effects of Pneumocystis jirovecii and Cryptosporidium spp., on the disease are little known. We investigated P. jirovecii and Cryptosporidium spp. in the sputum and stool sample of patients with asthma (n = 40) by microscopy and PCR compared to the healthy group (n = 40). P. jirovecii (12.5 %), and Cryptosporidium spp. (12.5 %) were detected in the sputum samples of only asthmatic patients (p = 0.029 and 0.029 respectively). However, Crpytosporidium spp. was detected equally in stool samples of both groups (p = 0.682). Our results indicate that P. jirovecii and Cryptosporidium spp. should be considered in patients with asthma and molecular screening of these neglected eukaryotes in respiratory tract samples may be beneficial in the clinical management of the disease., Competing Interests: Declaration of Competing Interest The authors have declared no conflict of financial and non-financial interests, (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: The aim of this study is to determine the prevalence of intestinal parasites in patients admitted to University of Health Sciences Türkiye (UHS) Van Training and Research Hospital., Methods: A total of 300 patients between the ages of 18-90 who applied to UHS Van Training and Research Hospital with gastrointestinal complaints and were referred to the parasitology laboratory between September 2021 and December 2021, and 100 patients without any chronic disease and gastrointestinal complaints in the control group were included in the study. Stool samples taken from patients included in the study and individuals in the control group were analyzed by native-lugol and modified acid-fast staining methods., Results: In the study, intestinal parasites were detected in 41 (13.3%) of 300 patients in the patient group and in seven (7%) of 100 individuals in the control group. The highest rate of Blastocystis species ( Blastocystis spp.) (5.7%) was found in the patient group. Entamoeba coli 3%, G. intestinalis 2.7% and Cryptosporidium species ( Cryptosporidium spp). 2.3% were found among the other species detected. In addition, a statistically significant correlation was found between the incidence of parasites and abdominal pain (p=0.022) and nausea (p=0.029)., Conclusion: As a result; it was concluded that intestinal parasites are still an important health problem in patients with gastrointestinal complaints and intestinal parasites should definitely be considered in this patient group.

Purpose: Inherited deficiencies of CD40 and CD40 ligand (CD40L) reflect the crucial immunological functions of CD40-CD40L interaction/signaling. Although numerous studies have provided a detailed description of CD40L deficiency, reports of CD40 deficiency are scarce. Herein, we describe the characteristics of all reported patients with CD40 deficiency., Methods: The PubMed, Embase and Web of Science databases were searched for relevant literature published till 7th August 2023. Study deduplication and identification of relevant reports was performed using the online PICO Portal. The data were extracted using a pre-designed data extraction form and the SPSS software was used for analysis., Results: Systematic literature review revealed 40 unique patients with CD40 deficiency. Respiratory tract and gastrointestinal infections were the predominant clinical manifestations (observed in 93% and 57% patients, respectively). Sclerosing cholangitis has been reported in nearly one-third of patients. Cryptosporidium sp. (29%) and Pneumocystis jirovecii (21%) were the most common microbes identified. Very low to undetectable IgG levels and severely reduced/absent switch memory B cells were observed in all patients tested/reported. Elevated IgM levels were observed in 69% patients. Overall, splice-site and missense variants were the most common (36% and 32%, respectively) molecular defects identified. All patients were managed with immunoglobulin replacement therapy and antimicrobial prophylaxis was utilized in a subset. Hematopoietic stem cell transplantation (HSCT) has been performed in 45% patients (curative outcome observed in 73% of these patients). Overall, a fatal outcome was reported in 21% patients., Conclusions: We provide a comprehensive description of all important aspects of CD40 deficiency. HSCT is a promising curative treatment option for CD40 deficiency., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)