Green synthesis of zinc oxide/Allium sativum nano-composite and its efficacy against murine cryptosporidiosis.

Cryptosporidiosis is a global health problem threats life of immunocompromised patients. Allium sativum (A. sativum) is one of the therapeutic options for cryptosporidiosis. This study develops green synthesized ZnO-NPs based on A. sativum extract, and assesses its therapeutic application in treating experimental cryptosporidiosis in immunosuppressed mice. FTIR, scanning electron microscopy, and zeta analyzer were used for characterization of bio ZnO-NPs. The morphology of prepared materials appeared as sponge with many pores on the whole surface that allows the feasibility of bio ZnO-NPs for different biological activities. Its structural analysis was highly stabilized with negative charge surface which indicated for well distribution into the parasite matrix. Twenty-five immunosuppressed Cryptosporidium parvum infected mice, classified into 5 groups were sacrificed at 21th day after infection with evaluation of parasitological, histopathological, oxidative, and proinflammatory biomarkers. Treated mice groups with 50 and 100 mg/kg of AS/ZnO-NPs showed a highly significant decline (79.9% and 83.23%, respectively) in the total number of expelled oocysts. Both doses revealed actual amelioration of the intestinal, hepatic, and pulmonary histopathological lesions. They also significantly produced an increase in GSH values and improved the changes in NO and MDA levels, and showed high anti-inflammatory properties. This study is the first to report green synthesis of ZnO/A. sativum nano-composite as an effective therapy in treating cryptosporidiosis which gave better results than using A. sativum alone. It provides an economical and environment-friendly approach towards novel delivery synthesis for antiparasitic applications. RESEARCH HIGHLIGHTS: Green synthesis of ZnO-NPs was developed using A. sativum extract. The morphology of prepared ZnO-NPs appeared as sponge with many pores on SEM The study evaluates its therapeutic efficacy against murine cryptosporidiosis The green synthesized ZnO-NPs significantly reduced percent of oocyst shedding, improved the pathological changes, and showed high antioxidant and anti-inflammatory potentials.

Molecular prevalence and associated risk factors of Cryptosporidium spp. infection in dairy cattle in Khon Kaen, Thailand.

Background and Aim: Cryptosporidium spp. are important parasites in the small intestines of humans and animals, particularly cattle. The aim of this study was to estimate the molecular prevalence and associated risk factors of Cryptosporidium infection in dairy cattle in five districts of Khon Kaen province, Thailand, and to identify Cryptosporidium spp. Materials and Methods: From July 2020 to October 2021, 296 fecal samples were collected from three groups of dairy cattle: Calves aged <3 months, calves aged 3 months-1 year, and calves aged >1 year. Cryptosporidium spp. were detected by polymerase chain reaction (PCR) amplifying the 18s RNA gene. Both genus-specific and species-specific primers were used to identify Cryptosporidium confirmed by DNA sequencing. Age, house floor type, and water trough type were evaluated as risk factors. We analyzed all associated risk factor information using the logistic regression test in the Statistical Package for the Social Sciences. Results: PCR results showed that 40 (13.51%) out of 296 samples were positive for Cryptosporidium spp., including Cryptosporidium bovis (57.50%) and Cryptosporidium ryanae (2.50%). There was a significant association between Cryptosporidium incidence, cattle age, and house floor type (p < 0.05). National Center for Biotechnology Information Basic Local Alignment Search Tool displayed 99.48%-100% nucleotide similarity of each Cryptosporidium spp. isolate with references recorded on GenBank. Conclusion: C. bovis and C. ryanae are commonly found in dairy cattle, especially calves, in Khon Kaen, Thailand, and the incidence was associated with age and house floor type. A molecular technique may be influential for species identification. The results of the present study would provide useful information for veterinarians and animal owners to understand better Cryptosporidium spp. and how to manage farms properly.

Molecular characteristics of Cryptosporidium spp. in human cases in five Finnish hospital districts during 2021: first findings of Cryptosporidium mortiferum (Cryptosporidium chipmunk genotype I) in Finland.

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.

Increased number of cryptosporidiosis cases with travel history to Croatia might be related to swimming pools, Germany, 2023.

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.

Health impacts of poor water quality on an endangered shorebird breeding programme in Aotearoa New Zealand.

CASE HISTORY: Two clusters of mortality among endangered tuturuatu/tchuriwat'/shore plover (Thinornis novaeseelandiae) have occurred at captive breeding facilities around New Zealand in recent years. In the first, four chicks died at Pukaha 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.

Molecular insights into anti-Protozoal action of natural compounds against Cryptosporidium parvum: a molecular simulation study.

The current study used the major target protein lactate dehydrogenase Cryptosporidium parvum to identify potential binders. Our approach was a comprehensive three-step screening of 2,569 natural compounds. First, we used molecular docking techniques, followed by an advanced DeepPurpose ML model for virtual screening. The final step involved meticulous re-docking and detailed interaction analysis. The known inhibitor FX11 was considered as a control that was used for comparative analysis. Our screening process led to the identification of three promising compounds: 5353794, 18475114, and 25229652. These compounds were chosen due to their exceptional ability to form hydrogen bonds and their high binding scores with the protein. Here, all three hits showed H-bonds with the functional residues (Asn122 and Thr231) of protein, while 25229652 also showed H-bond with the catalytic site residue (His177). RMSD behaviour reflected stable and consistent complex formation for all the compounds in their last 30 ns trajectories. Principal component analysis (PCA) and free energy landscape (FEL) showed a high frequency of favourable low free energy states. Using the MM/GBSA calculation, compounds 5353794 (ΔGTOTAL = -34.92 kcal/mol) and 18475114 (ΔGTOTAL = -34.66 kcal/mol) had the highest binding affinity with the protein however, 25229652 (ΔGTOTAL = -22.62 kcal/mol) had ΔGTOTAL comparable to the control FX11. These natural compounds not only show the potential for hindering C. parvum lactate dehydrogenase but also open new avenues in its drug development. Their strong binding properties and stable interactions mark them as the prime candidates for further research and experimental validation as anti-cryptosporidiosis agents.Communicated by Ramaswamy H. Sarma.

Evidence of the Zoonotic Transmission of Cryptosporidium among Children and Pets.

We investigated the zoonotic transmission of Cryptosporidium among the children (n = 188), dogs (n = 133), and cats (n = 55) living in 188 households. Fecal samples were examined using ELISA and confirmed via nested PCR. Coproantigens oocysts were detected in 3.7% of children, 8.3% of dogs, and 5.5% of cats. We found strong evidence of two cases of the zoonotic transmission of Cryptosporidium canis between children and dogs. Furthermore, four children and their respective pets (one dog and three cats) were infected with Cryptosporidium parvum, but we cannot exclude the hypotheses that the oocysts were transmitted from children to animals or that both hosts were infected by a shared source, such as contaminated water or food. The presence of an infected animal elevated the risk of zoonotic transmission by 129.7-fold (95% CI: 13.92-1209.68). Furthermore, sharing a bed with pets was identified as a risk factor for infection in children (OR: 9.9, 95% CI: 1.37-71.2). In conclusion, the zoonotic transmission of Cryptosporidium among children and pets cohabiting in the same household may be quite common, especially when infected animals lie or sleep on children's beds. These findings unequivocally highlight the public health concern surrounding C. canis.

Case Report: The impact of severe cryptosporidiosis on the gut microbiota of a pediatric patient with CD40L immunodeficiency.

Cryptosporidium parvum is a protozoan parasite and one of the leading causes of gastroenteritis in the world, primarily affecting very young children and immunocompromised patients. While infection is usually self-limiting, it can become chronic and even lethal in these vulnerable populations, in whom Cryptosporidium treatments are generally ineffective, due to their acting in concert with a functioning immune system. Here, we describe a case of chronic cryptosporidiosis in a European child with severe CD40L immunodeficiency infected with Cryptosporidium parvum of the IIa20G1 subgenotype, a lineage which has thus far only ever been described in the Middle East. After years of on-off treatment with conventional and non-conventional anti-parasitic drugs failed to clear parasitosis, we performed targeted metagenomics to observe the bacterial composition of the patient's gut microbiota (GM), and to evaluate fecal microbiota transplantation (FMT) as a potential treatment option. We found that C. parvum infection led to significant shifts in GM bacterial composition in our patient, with consequent shifts in predicted intestinal functional signatures consistent with a state of persistent inflammation. This, combined with the patient's poor prognosis and increasing parasitic burden despite many rounds of anti-parasitic drug treatments, made the patient a potential candidate for an experimental FMT procedure. Unfortunately, given the many comorbidities that were precipitated by the patient's immunodeficiency and chronic C. parvum infection, FMT was postponed in favor of more urgently necessary liver and bone marrow transplants. Tragically, after the first liver transplant failed, the patient lost his life before undergoing FMT and a second liver transplant. With this case report, we present the first description of how cryptosporidiosis can shape the gut microbiota of a pediatric patient with severe immunodeficiency. Finally, we discuss how both our results and the current scientific literature suggest that GM modulations, either by probiotics or FMT, can become novel treatment options for chronic Cryptosporidium infection and its consequent complications, especially in those patients who do not respond to the currently available anti-parasitic therapies.

Study of the economic impact of cryptosporidiosis in calves after implementing good practices to manage the disease on dairy farms in Belgium, France, and the Netherlands.

Cryptosporidium spp. are widespread parasitic protozoans causing enteric infections in humans and animals. The parasites cause neonatal diarrhoea in calves, leading to a high mortality rate in the first three weeks. Losses are significant for farmers, but the cost of cryptosporidiosis remains poorly documented. In the absence of a vaccine, only preventive measures are available to farmers to combat the infection. This study, conducted between 2018 and 2021, aimed to evaluate the economic impact of Cryptosporidium spp. on European dairy farms and monitor changes in costs after implementing disease management measures. First, a field survey was carried out and questionnaires administered to 57 farmers in Belgium, France, and the Netherlands. The aim of the survey was to assess the losses associated with the occurrence of diarrhoea in calves aged between 3 days and 3 weeks. The economic impact of diarrhoea was calculated based on mortality losses, health expenditures, and additional labour costs. To refine the cost estimation specifically for Cryptosporidium spp., stool samples were collected from 10 calves per farm. The prevalence of Cryptosporidium spp. was determined, and the economic impact of diarrhoea was adjusted accordingly. The assumption was made that a certain percentage of costs was attributed to cryptosporidiosis based on the prevalence. These protocols were repeated at the end of the study to observe changes in costs. In the three years, the cost of diarrhoea for the 28 farms that stayed in the panel all along the study improved from €140 in 2018 to €106 on average per diarrhoeic calf in 2021. With a stable prevalence at 40%, the cost of cryptosporidiosis per infected calf decreased from €60.62 to €45.91 in Belgium, from €43.83 to €32.14 in France, and from €58.24 to €39.48 in the Netherlands. This represented an average of €15 saved per infected calf. The methodology employed in this study did not allow us to conclude that the improvement is strictly due to the implementation of preventive measures. However, with 11 million calves raised in the Interreg 2 Seas area covered by the study, it provided valuable insights into the economic burden of Cryptosporidium spp.

Is Personal Protective Equipment Worth the Hassle? Annual Risk of Cryptosporidiosis to Dairy Farmers and How Personal Protective Equipment and Handwashing Can Mitigate It.

Cows are known carriers of Cryptosporidium parvum (C. parvum), a protozoa that can cause the gastrointestinal illness cryptosporidiosis in humans. Despite this potential exposure, dairy farmers tend to wear personal protective equipment (PPE) to protect the milk from contamination, rather than to protect themselves from zoonotic diseases, such as cryptosporidiosis. In this study, cow feces were collected from individual cattle on dairy farms and analyzed for C. parvum using qPCR. Quantitative microbial risk assessment (QMRA) was used to determine the risk of cryptosporidiosis to the dairy farmer with and without the use of handwashing and PPE (gloves and masks). The annualized risk of cryptosporidiosis to dairy farmers was 29.08% but was reduced significantly in each of the three interventions. Among the individual interventions, glove use provided the greatest reduction in risk, bringing the annual risk of cryptosporidiosis to 4.82%. Implementing regular handwashing, the use of gloves and a mask brought the annual risk of cryptosporidiosis to 1.29%. This study provides evidence that handwashing and PPE use can significantly reduce the risk of cryptosporidiosis to farmers and is worth implementing despite potential barriers such as discomfort and cost.

Cryptosporidiosis, an Uncommon Complication after Allogeneic Stem-Cell Transplantation.

Parasitic infections by Cryptosporidium species are rare but can be life-threatening disease after allogeneic stem-cell transplantation (allo-SCT). Here, we reported a case of cryptosporidiosis occurring after a reduced-intensity conditioning and allo-SCT in a 64-year-old farmer with diffuse large B-cell lymphoma. Around day 70 after allo-SCT, he presented with diarrhea attributed to graft-versus-host disease (GvHD) and was treated with immunosuppressive therapy. Due to the patient's worsening clinical condition, a biopsy review was performed, revealing evidence of cryptosporidiosis. Therefore, immunosuppressive therapy was progressively decreased, and antimicrobial therapy including paromomycin and azithromycin was initiated. Following an increase in diarrhea, a second-line treatment with nitazoxanide was administered, resulting in gradual improvement of symptoms. However, recurrence of cryptosporidiosis occurred despite treatment with paromomycin 6 months after transplant and after an episode of GvHD recurrence and colic cytomegalovirus reactivation. Antiparasitic treatment was stopped and azithromycin and rifaximine were started. Immunosuppressive therapy was also reduced. The good clinical evolution allowed for the cessation of all medications. In conclusion, Cryptosporidium infection can complicate allo-SCT and be mistaken for GvHD at the clinical and histologic levels. Early and accurate diagnosis is all the more important as the therapeutic approach for the two conditions is opposite: reduction versus intensification of immunosuppressive therapy. Nitazoxanide, paromomycin, and azithromycin are the first therapeutic options.

Magnetic Resonance Cholangiopancreatography (MRCP) Findings in a Patient With AIDS Cholangiopathy and Cryptosporidiosis.

AIDS cholangiopathy is a rare condition characterized by intra- and extra-hepatic ductal strictures causing biliary obstruction primarily in individuals with advanced HIV infection and low clusters of differentiation 4 (CD4) count. This case report presents a male patient with a history of HIV, poor adherence to antiretroviral therapy (ART), and chronic cryptosporidiosis infection, who exhibited clinical and radiological findings consistent with advanced immunocompromise and AIDS cholangiopathy. The patient presented with respiratory symptoms, weight loss, renal dysfunction, and elevated liver enzymes. Imaging studies, including ultrasound and magnetic resonance cholangiopancreatography (MRCP), revealed diffuse biliary dilatation and stricturing, indicative of cholangiopathy. Subsequent endoscopic retrograde cholangiopancreatography (ERCP) with stent placement was performed to manage the biliary obstruction. This case highlights the importance of considering AIDS cholangiopathy in HIV patients with poor ART compliance who present with biliary obstruction symptoms or cholestatic liver enzyme abnormalities. Prompt diagnostic evaluation using MRCP or ERCP can aid in confirming the diagnosis and guiding appropriate therapeutic interventions, including endoscopic management and initiation of ART.

Preliminary investigation of a significant national Cryptosporidium exceedance in the United Kingdom, August 2023 and ongoing.

Routine laboratory surveillance has identified an unprecedented and ongoing exceedance of Cryptosporidium spp. across the United Kingdom, notably driven by C. hominis transmission, since 14 August 2023. Information from 477 reported cases in England and Wales, followed up with a standardised exposure questionnaire as of 25 September 2023, identified foreign travel in 250 (54%) of 463 respondents and swimming in 234 (66%) of 353 cases. A significant, common exposure has not yet been identified in first analyses.

Specific pathway abundances in the neonatal calf faecal microbiome are associated with susceptibility to Cryptosporidium parvum infection: a metagenomic analysis.

BACKGROUND: Cryptosporidium parvum is the main cause of calf scour worldwide. With limited therapeutic options and research compared to other Apicomplexa, it is important to understand the parasites' biology and interactions with the host and microbiome in order to develop novel strategies against this infection. The age-dependent nature of symptomatic cryptosporidiosis suggests a link to the undeveloped immune response, the immature intestinal epithelium, and its associated microbiota. This led us to hypothesise that specific features of the early life microbiome could predict calf susceptibility to C. parvum infection. RESULTS: In this study, a single faecal swab sample was collected from each calf within the first week of life in a cohort of 346 animals. All 346 calves were subsequently monitored for clinical signs of cryptosporidiosis, and calves that developed diarrhoea were tested for Rotavirus, Coronavirus, E. coli F5 (K99) and C. parvum by lateral flow test (LFT). A retrospective case-control approach was taken whereby a subset of healthy calves (Control group; n = 33) and calves that went on to develop clinical signs of infectious diarrhoea and test positive for C. parvum infection via LFT (Cryptosporidium-positive group; n = 32) were selected from this cohort, five of which were excluded due to low DNA quality. A metagenomic analysis was conducted on the faecal microbiomes of the control group (n = 30) and the Cryptosporidium-positive group (n = 30) prior to infection, to determine features predictive of cryptosporidiosis. Taxonomic analysis showed no significant differences in alpha diversity, beta diversity, and taxa relative abundance between controls and Cryptosporidium-positive groups. Analysis of functional potential showed pathways related to isoprenoid precursor, haem and purine biosynthesis were significantly higher in abundance in calves that later tested positive for C. parvum (q ≤ 0.25). These pathways are either absent or streamlined in the C. parvum parasites. Though the de novo production of isoprenoid precursors, haem and purines are absent, C. parvum has been shown to encode enzymes that catalyse the downstream reactions of these pathway metabolites, indicating that C. parvum may scavenge those products from an external source. CONCLUSIONS: The host has previously been put forward as the source of essential metabolites, but our study suggests that C. parvum may also be able to harness specific metabolic pathways of the microbiota in order to survive and replicate. This finding is important as components of these microbial pathways could be exploited as potential therapeutic targets for the prevention or mitigation of cryptosporidiosis in bovine neonates.

Combination of inhibitors for two glycolytic enzymes portrays high synergistic efficacy against Cryptosporidium parvum.

Cryptosporidium is an intracellular protozoan parasite that causes serious enteric disease in humans and in a wide range of animals worldwide. Despite its high prevalence, no effective therapeutic drugs are available against life-threatening cryptosporidiosis in at-risk populations including malnourished children, immunocompromised patients, and neonatal calves. Thus, new efficacious drugs are urgently needed to treat all susceptible populations with cryptosporidiosis. Unlike other apicomplexans, Cryptosporidium parvum lacks the tricarboxylic acid cycle and the oxidative phosphorylation steps, making it solely dependent on glycolysis for metabolic energy production. We have previously reported that individual inhibitors of two unique glycolytic enzymes, the plant-like pyruvate kinase (CpPyK) and the bacterial-type lactate dehydrogenase (CpLDH), are effective against C. parvum, both in vitro and in vivo. Herein, we have derived combinations of CpPyK and CpLDH inhibitors with strong synergistic effects against the growth and survival of C. parvum, both in vitro and in an infection mouse model. In infected immunocompromised mice, compound combinations of NSC303244 + NSC158011 and NSC252172 + NSC158011 depicted enhanced efficacy against C. parvum reproduction and ameliorated intestinal lesions of cryptosporidiosis at doses fourfold lower than the total effective doses of individual compounds. Importantly, unlike individual compounds, NSC303244 + NSC158011 combination was effective in clearing the infection completely without relapse in immunocompromised mice. Collectively, our study has unveiled compound combinations that simultaneously block two essential catalytic steps for metabolic energy production in C. parvum to achieve improved efficacy against the parasite. These combinations are, therefore, lead compounds for the development of a new generation of efficacious anti-cryptosporidial drugs.

Case Report: Severe Diarrhea Caused by Cryptosporidium Diagnosed by Metagenome Next-Generation Sequencing in Blood.

Background: Cryptosporidium is one of the major pathogens causing diarrhea worldwide. At present, cryptosporidiosis is difficult to prevent and control, especially in immunocompromised hosts. It may cause life-threatening diarrhea and malabsorption among children and immunocompromised patients. Therefore, it is very important to explore rapid diagnostic tools and treatment methods for Cryptosporidium infection. Case Presentation: We reported a case of severe diarrhea caused by cryptosporidiosis in a liver transplant recipient, whose condition was finally confirmed by metagenomic next-generation sequencing (mNGS) and fecal microscopy. His illness was resolved with immunosuppression regulation, nitazoxanide administration, and infection control. Conclusion: So far, nitazoxanide is still the first choice for the treatment of cryptosporidiosis. Our institutional experience suggested that nitazoxanide alone may be effective on the basis of adjusting immunosuppressant. In addition, even though diagnosis of Cryptosporidium infection is a challenge, mNGS can serve as a rapid screening tool in low-prevalence setting.

Changing Molecular Profiles of Human Cryptosporidiosis Cases in Scotland as a Result of the Coronavirus Disease, COVID-19 Pandemic.

Cryptosporidium, the most frequently reported parasite in Scotland, causes gastrointestinal illness resulting in diarrhoea, nausea and cramps. Two species are responsible for most cases: Cryptosporidium hominis (C. hominis) and Cryptosporidium parvum (C. parvum). Transmission occurs faecal-orally, through ingestion of contaminated food and water, or direct contact with faeces. In 2020, the COVID-19 pandemic led to global restrictions, including national lockdowns to limit viral transmission. Such interventions led to decreased social mixing, and reduced/no local and international travel, which are factors associated with transmission of multiple communicable diseases, including cryptosporidiosis. This report assessed the impact of the pandemic on Scottish cryptosporidiosis cases, and identified changes in circulating molecular variants of Cryptosporidium species. Molecular data generated using real time PCR and GP60 nested-PCR assays on laboratory-confirmed cryptosporidiosis cases reported during 2018-22 were analysed. The Scottish Microbiology Reference Laboratories (SMiRL), Glasgow, received 774 Cryptosporidium-positive faeces during 2018-22, of which 486 samples were successfully subtyped. During this time period, C. hominis (n = 155; 21%) and C. parvum (n = 572; 77%) were the most commonly detected species. The total number of cases during 2020, which was greatly affected by the pandemic, was markedly lower in comparison to case numbers in the 2 years before and after 2020. The most predominant C. hominis family detected prior to 2020 was the Ib family which shifted to the Ie family during 2022. The most common C. parvum variant during 2018-22 was the IIa family, however a rise in the IId family was observed (n = 6 in 2018 to n = 25 in 2022). The dominant C. hominis subtype IbA10G2, which accounted for 71% of C. hominis subtypes in 2018-19 was superseded by three rare subtypes: IeA11G3T3 (n = 15), IdA16 (n = 8) and IbA9G3 (n = 3) by 2022. Frequently reported C. parvum subtypes in 2018-19 were IIaA15G2R1 and IIaA17G1R1, accounting for 59% of total C. parvum subtypes. By 2022, IIaA15G2R1 remained the most common (n = 28), however three unusual subtypes in Scotland emerged: IIdA24G1 (n = 7), IIaA16G3R1 (n = 7) and IIaA15G1R2 (n = 7). Continuous monitoring of Cryptosporidium variants following the pandemic will be essential to explore further changes and emergence of strains with altered virulence.

The Gut-Wrenching Effects of Cryptosporidiosis and Giardiasis in Children.

Cryptosporidium species and Giardia duodenalis are infectious intestinal protozoan pathogens that cause alarming rates of morbidity and mortality worldwide. Children are more likely to have clinical symptoms due to their less developed immune systems and factors such as undernutrition, especially in low- and middle-income countries. The severity of the symptoms and clinical manifestations in children may vary from asymptomatic to life-threatening depending on the Cryptosporidium species/G. duodenalis strains and the resulting complex stepwise interactions between the parasite, the host nutritional and immunologic status, and the gut microbiome profile. Structural damages inflicted by both parasites to epithelial cells in the large and small intestines could severely impair children's gut health, including the ability to absorb nutrients, resulting in stunted growth, diminished neurocognitive development, and other long-term effects. Clinically approved cryptosporidiosis and giardiasis drugs have broad antimicrobial effects that have incomprehensible impacts on growing children's gut health.

Respiratory cryptosporidiosis detected by commercial multiplex-PCR in immunosuppressed pediatric patients.

We report the off-label use of a commercial gastrointestinal protozoa multiplex-PCR panel for bronchoalveolar lavage samples, detecting respiratory cryptosporidiosis in 2 immunocompromised pediatric patients. We suggest that implying this readily available assay in cases in which systemic cryptosporidiosis is suspected, may widen our understanding regarding this rarely reported syndrome.

Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium.

Introduction: Cryptosporidiosis is a leading cause of diarrheal-associated morbidity and mortality, predominantly affecting children under 5 years old in low-and-middle-income countries. There is no effective treatment and no vaccine. New therapeutics are emerging from drug discovery efforts. It is critical that mode of action studies are performed alongside drug discovery to ensure the best clinical outcomes. Unfortunately, technology to identify and validate drug targets for Cryptosporidium is severely lacking. Methods: We used C. parvum lysyl-tRNA synthetase (CpKRS) and DDD01510706 as a target-compound pair to develop both chemical and genetic tools for mode of action studies for Cryptosporidium. We adapted thermal proteome profiling (TPP) for Cryptosporidium, an unbiased approach for target identification. Results: Using TPP we identified the molecular target of DDD01510706 and confirm that it is CpKRS. Genetic tools confirm that CpKRS is expressed throughout the life cycle and that this target is essential for parasite survival. Parasites genetically modified to over-express CpKRS or parasites with a mutation at the compound-binding site are resistant to treatment with DDD01510706. We leveraged these mutations to generate a second drug selection marker for genetic modification of Cryptosporidium, KRSR. This second selection marker is interchangeable with the original selection marker, NeoR, and expands the range of reverse genetic approaches available to study parasite biology. Due to the sexual nature of the Cryptosporidium life cycle, parental strains containing different drug selection markers can be crossed in vivo. Discussion: Selection with both drug markers produces highly efficient genetic crosses (>99% hybrid progeny), paving the way for forward genetics approaches in Cryptosporidium.