Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections.

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an understudied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.

A large cryptosporidiosis outbreak associated with an animal contact event in England: a retrospective cohort study, 2023.

Development of gastrointestinal illness after animal contact at petting farms is well described, as are factors such as handwashing and facility design that may modify transmission risk. However, further field evidence on other behaviours and interventions in the context of Cryptosporidium outbreaks linked to animal contact events is needed. Here, we describe a large outbreak of Cryptosporidium parvum (C. parvum) associated with a multi-day lamb petting event in the south-west of England in 2023 and present findings from a cohort study undertaken to investigate factors associated with illness. Detailed exposure questionnaires were distributed to email addresses of 647 single or multiple ticket bookings, and 157 complete responses were received. The outbreak investigation identified 23 laboratory-confirmed primary C. parvum cases. Separately, the cohort study identified 83 cases of cryptosporidiosis-like illness. Associations between illness and entering a lamb petting pen (compared to observing from outside the pen; odds ratio (OR) = 2.28, 95 per cent confidence interval (95% CI) 1.17 to 4.53) and self-reported awareness of diarrhoeal and vomiting disease transmission risk on farm sites at the time of visit (OR = 0.40, 95% CI 0.19 to 0.84) were observed. In a multivariable model adjusted for household clustering, awareness of disease transmission risk remained a significant protective factor (adjusted OR (aOR) = 0.07, 95% CI 0.01 to 0.78). The study demonstrates the likely under-ascertainment of cryptosporidiosis through laboratory surveillance and provides evidence of the impact that public health messaging could have.

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.

Cost-effective In Vivo and In Vitro Mouse Models for Evaluating Anticryptosporidial Drug Efficacy: Assessing Vorinostat, Docetaxel, and Baicalein.

BACKGROUND: Cryptosporidiosis is a significant diarrheal disease in humans and animals. Immunodeficient mice are the primary small animal models, but their high costs and specialized breeding/housing requirements limit in vivo drug testing. Numerous anticryptosporidial lead compounds identified in vitro remain untested in vivo. METHODS: Cryptosporidium tyzzeri, a natural mouse parasite closely related to Cryptosporidium parvum and Cryptosporidium hominis, was isolated to establish an infection model in immunocompetent mice. The model was validated using classic anticryptosporidial drugs (paromomycin and nitazoxanide) and then employed to assess the efficacy of 3 new leads (vorinostat, docetaxel, and baicalein). An in vitro culture of C. tyzzeri was also developed to complement the animal model. RESULTS: Chronic C. tyzzeri infection was established in chemically immunosuppressed wild-type mice. Paromomycin (1000 mg/kg/d) and nitazoxanide (100 mg/kg/d) demonstrated efficacy against C. tyzzeri. Vorinostat (30 mg/kg/d), docetaxel (25 mg/kg/d), and baicalein (50 mg/kg/d) were highly effective against C. tyzzeri infection. In vitro, nitazoxanide, vorinostat, docetaxel, and baicalein exhibited low to submicromolar efficacy against C. tyzzeri. CONCLUSIONS: Novel in vivo and in vitro models have been developed for cost-effective anticryptosporidial drug testing. Vorinostat, docetaxel, and baicalein show potential for repurposing and/or optimization for developing new anticryptosporidial drugs.

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.

Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis.

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

Cryptosporidium parvumcompetes with the intestinal epithelial cells for glucose and impairs systemic glucose supply in neonatal calves.

Cryptosporidiosis is one of the main causes of diarrhea in children and young livestock. The interaction of the parasite with the intestinal host cells has not been characterized thoroughly yet but may be affected by the nutritional demand of the parasite. Hence, we aimed to investigate the impact of C. parvum infection on glucose metabolism in neonatal calves. Therefore, N = 5 neonatal calves were infected with C. parvum on the first day of life, whereas a control group was not (N = 5). The calves were monitored clinically for one week, and glucose absorption, turnover and oxidation were assessed using stable isotope labelled glucose. The transepithelial transport of glucose was measured using the Ussing chamber technique. Glucose transporters were quantified on gene and protein expression level using RT-qPCR and Western blot in the jejunum epithelium and brush border membrane preparations. Plasma glucose concentration and oral glucose absorption were decreased despite an increased electrogenic phlorizin sensitive transepithelial transport of glucose in infected calves. No difference in the gene or protein abundance of glucose transporters, but an enrichment of glucose transporter 2 in the brush border was observed in the infected calves. Furthermore, the mRNA for enzymes of the glycolysis pathway was increased indicating enhanced glucose oxidation in the infected gut. In summary, C. parvum infection modulates intestinal epithelial glucose absorption and metabolism. We assume that the metabolic competition of the parasite for glucose causes the host cells to upregulate their uptake mechanisms and metabolic machinery to compensate for the energy losses.

Structure-Activity relationships of replacements for the triazolopyridazine of Anti-Cryptosporidium lead SLU-2633.

Cryptosporidiosis is a diarrheal disease particularly harmful to children and immunocompromised people. Infection is caused by the parasite Cryptosporidium and leads to dehydration, malnutrition, and death in severe cases. Nitazoxanide is the only FDA approved drug but is only modestly effective in children and ineffective in immunocompromised patients. To address this unmet medical need, we previously identified triazolopyridazine SLU-2633 as potent against Cryptosporidium parvum, with an EC50 of 0.17 µM. In the present study, we develop structure-activity relationships (SAR) for the replacement of the triazolopyridazine head group by exploring different heteroaryl groups with the aim of maintaining potency while reducing affinity for the hERG channel. 64 new analogs of SLU-2633 were synthesized and assayed for potency versus C. parvum. The most potent compound, 7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazine 17a, was found to have a Cp EC50 of 1.2 µM, 7-fold less potent than SLU-2633 but has an improved lipophilic efficiency (LipE) score. 17a was found to decrease inhibition in an hERG patch-clamp assay by about two-fold relative to SLU-2633 at 10 µM despite having similar inhibition in a [3H]-dofetilide competitive binding assay. While most other heterocycles were significantly less potent than the lead, some analogs such as azabenzothiazole 31b, have promising potency in the low micromolar range, similar to the drug nitazoxanide, and represent potential new leads for optimization. Overall, this work highlights the important role of the terminal heterocyclic head group and represents a significant extension of the understanding of the SAR for this class of anti-Cryptosporidium compounds.

A review and analysis of cryptosporidiosis outbreaks in New Zealand.

Cryptosporidium is a leading global cause of diarrhoea with many reported outbreaks related to water and zoonotic transmission. This study summarizes data from Public Health Surveillance reports since 2010 in New Zealand to describe exposures associated with human diarrhoea outbreaks caused by Cryptosporidium. We investigate the species and subtypes of cases involved in some of the outbreaks to elucidate transmission routes and the predominant aetiological agents of cryptosporidiosis. For the period 2010­2017, 318 cryptosporidiosis outbreaks were reported in New Zealand resulting in 1634 cases and 20 hospitalizations. The most important mode of transmission was person-to-person (primary infections and secondary or close contacts infections), relating to 260 outbreaks and 1320 cases, followed by 113 outbreaks associated with animals, resulting in 436 human cases. From 2018 to 2021, there were 37 cryptosporidiosis outbreaks associated with 324 cases. We identified the subtypes by using polymerase chain reaction targeting the gp60 gene and the likelihood of mixed subtype infections with the Tracking of Indels by DEcomposition (TIDE) algorithm. Subtype families Ib and Ig of Cryptosporidium hominis and IIa and IId of Cryptosporidium parvum were found among cases; however, C. hominis subtypes occurred in 8 of the 11 outbreaks reviewed where molecular data were available. Examination of the chromatograms showed no mixed subtype infections in the samples assessed. Subtyping data need to be routinely incorporated into national surveillance programmes to better understand the epidemiology, sources, transmission and extent of cryptosporidiosis outbreaks in New Zealand. Our study highlights the value of integrating epidemiological information and molecular typing to investigate and manage clusters of cryptosporidiosis cases.

Outbreak of severe diarrhea due to zoonotic Cryptosporidium parvum and C. xiaoi in goat kids in Chungcheongbuk-do, Korea.

Severe diarrhea was reported in goat kids in Chungcheongbuk-do, Korea, from 2021 to 2023, and Cryptosporidium infection was suspected. To confirm the cause of this outbreak, fecal samples were collected from goat farms where diarrhea had been reported and analyzed for Cryptosporidium infection using a molecular assay. A total of 65 fecal samples, including 37 from goats with diarrhea and 28 from goats without diarrhea, were collected from six goat farms. Forty-eight of the goats were kids (<2 months) and 17 were adults (>1 year). Cryptosporidium was identified in 53.8% (35/65) of total samples. Overall, 86.5% (32/37) of the diarrheic fecal samples tested positive; however, Cryptosporidium was not detected in any fecal sample from non-diarrheic adult goats. Therefore, cryptosporidiosis was significantly associated with diarrhea in goat kids, and adult goats were not responsible for transmission of Cryptosporidium to them. Phylogenetic analysis and molecular characterization revealed two Cryptosporidium species, namely, C. parvum (n = 28) and C. xiaoi (n = 7). In the C. parvum-positive samples, gp60 gene analysis revealed three zoonotic subtypes-IIaA18G3R1, IIdA15G1, and IIdA16G1. To the best of our knowledge, this study is the first to identify C. parvum IIaA18G3R1 and IIdA16G1 in goats, as well as the first to identify C. xiaoi in goats in Korea. These results suggest that goat kids play an important role as reservoir hosts for different Cryptosporidium species and that continuous monitoring with biosecurity measures is necessary to control cryptosporidiosis outbreaks.

Detection of unusual Cryptosporidium parvum subtype in patients with gastrointestinal cancer in Egypt.

While the importance of cryptosporidiosis in immunocompromised persons is well known, the prevalence of Cryptosporidium spp. in cancer patients is not clear. The current study was designed to assess the occurrence and genetic characteristics of Cryptosporidium spp. in patients with gastrointestinal (GI) cancer in Egypt. Stool samples were collected from 100 patients with GI malignancies and 20 healthy individuals without any GI manifestations (control group). They were screened by microscopy and the immunochromatographic RIDA®QUICK Cryptosporidium kit. Subtyping of Cryptosporidium spp. was conducted by sequence analysis of the glycoprotein 60 (gp60) locus. Sociodemographic, environmental data and information on GI symptoms, cancer types, and clinical treatment were obtained via a questionnaire. By microscopy and RIDA®QUICK, only 7% (7/100) of GI cancer patients were positive for Cryptosporidium, compared with 40% (40/100) by gp60 nPCR. No positives were obtained from the control group. Male sex (P = 0.02) and younger age (P = 0.004) were major Cryptosporidium risk factors for infection. The occurrence of Cryptosporidium was also significantly more frequent (P = 0.003) in watery stool samples. Sequence analysis of the gp60 amplicons (~ 400 bp) identified a novel C. parvum subtype with nine TCA repeats and eleven ACATCA repeats. A formal subtype designation could not be made due to the short sequence length. More studies should be conducted to verify the common occurrence of this unusual C. parvum subtype and establish its genetic identity.

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.

RNA-Based Therapy for Cryptosporidium parvum Infection: Proof-of-Concept Studies.

Cryptosporidium is a leading cause of moderate-to-severe diarrhea in children, which is one of the major causes of death in children under 5 years old. Nitazoxanide is the only FDA-approved treatment for cryptosporidiosis. However, it has limited efficacy in immunosuppressed patients and malnourished children. Therefore, it is urgent to develop novel therapies against this parasite. RNA interference-mediated therapies are emerging as novel approaches for the treatment of infectious diseases. We have developed a novel method to silence essential genes in Cryptosporidium using single-stranded RNA (ssRNA)/Argonaute (Ago) complexes. In this work we conducted proof-of-concept studies to test the anticryptosporidial activity of these complexes by silencing Cryptosporidium parvum nucleoside diphosphate kinase (NDK) using in vitro and in vivo models. We demonstrated that a 3-day treatment with anti-sense NDK ssRNA/Ago decreased parasite burden by ~98% on infected cells. In vivo studies showed that ssRNA/Ago complexes encapsulated in lipid nanoparticles can be delivered onto intestinal epithelial cells of mice treated orally. In addition a cryptosporidiosis-mouse model showed that treatment with NDK ssRNA/Ago complexes reduced oocyst shedding in 4/5 SCID/beige mice during the acute phase of the infection. Our findings highlight the potential use of antisense RNA-based therapy as an alternative approach to cryptosporidiosis treatment.

Development of Two Mouse Models for Vaccine Evaluation against Cryptosporidiosis.

Cryptosporidiosis was shown a decade ago to be a major contributor to morbidity and mortality of diarrheal disease in children in low-income countries. A serious obstacle to develop and evaluate immunogens and vaccines to control this disease is the lack of well-characterized immunocompetent rodent models. Here, we optimized and compared two mouse models for the evaluation of vaccines: the Cryptosporidium tyzzeri model, which is convenient for screening large numbers of potential mixtures of immunogens, and the Cryptosporidium parvum-infected mouse pretreated with interferon gamma-neutralizing monoclonal antibody.

Horse-Specific Cryptosporidium Genotype in Human with Crohn's Disease and Arthritis.

We identified an unusual subtype of a Cryptosporidium sp. horse genotype as the cause of cryptosporidiosis in a 13-year-old girl in Poland who was undergoing immunosuppressive treatment for juvenile rheumatoid arthritis and Crohn's disease. The same subtype was identified in a horse the girl had ridden.

Pharmacokinetics and Pharmacodynamics of Clofazimine for Treatment of Cryptosporidiosis.

Infection with Cryptosporidium spp. can cause severe diarrhea, leading to long-term adverse impacts and even death in malnourished children and immunocompromised patients. The only FDA-approved drug for treating cryptosporidiosis, nitazoxanide, has limited efficacy in the populations impacted the most by the diarrheal disease, and safe, effective treatment options are urgently needed. Initially identified by a large-scale phenotypic screening campaign, the antimycobacterial therapeutic clofazimine demonstrated great promise in both in vitro and in vivo preclinical models of Cryptosporidium infection. Unfortunately, a phase 2a clinical trial in HIV-infected adults with cryptosporidiosis did not identify any clofazimine treatment effect on Cryptosporidium infection burden or clinical outcomes. To explore whether clofazimine's lack of efficacy in the phase 2a trial may have been due to subtherapeutic clofazimine concentrations, a pharmacokinetic/pharmacodynamic modeling approach was undertaken to determine the relationship between clofazimine in vivo concentrations and treatment effects in multiple preclinical infection models. Exposure-response relationships were characterized using Emax and logistic models, which allowed predictions of efficacious clofazimine concentrations for the control and reduction of disease burden. After establishing exposure-response relationships for clofazimine treatment of Cryptosporidium infection in our preclinical model studies, it was unmistakable that the clofazimine levels observed in the phase 2a study participants were well below concentrations associated with anti-Cryptosporidium efficacy. Thus, despite a dosing regimen above the highest doses recommended for mycobacterial therapy, it is very likely the lack of treatment effect in the phase 2a trial was at least partially due to clofazimine concentrations below those required for efficacy against cryptosporidiosis. It is unlikely that clofazimine will provide a remedy for the large number of cryptosporidiosis patients currently without a viable treatment option unless alternative, safe clofazimine formulations with improved oral absorption are developed. (This study has been registered in ClinicalTrials.gov under identifier NCT03341767.).

Transition to PCR diagnosis of cryptosporidiosis and giardiasis in the Norwegian healthcare system: could the increase in reported cases be due to higher sensitivity or a change in the testing algorithm?

Cryptosporidiosis has been a notifiable infection in Norway since 2012 and giardiasis since 1977. For both infections, there has been an increase in notified cases. We used a questionnaire to explore whether this may be associated with implementation of molecular diagnostic methods. We received responses from 14 of 16 laboratories, most of which had implemented molecular diagnostic methods for these parasites. Algorithms for testing had also been modified, and several laboratories now test more faecal samples than previously for both parasites. The increase in reported cases may reflect not only higher sensitivity of diagnostic methods, but also more sample testing.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.