Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.

Cryptosporidium infects gastrointestinal epithelium and is a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. There are no vaccines and no fully effective therapy available for the infection. Type II and III interferon (IFN) responses are important determinants of susceptibility to infection but the role for type I IFN response remains obscure. Cryptosporidium parvum virus 1 (CSpV1) is a double-stranded RNA (dsRNA) virus harbored by Cryptosporidium spp. Here we show that intestinal epithelial conditional Ifnar1-/- mice (deficient in type I IFN receptor) are resistant to C. parvum infection. CSpV1-dsRNAs are delivered into host cells and trigger type I IFN response in infected cells. Whereas C. parvum infection attenuates epithelial response to IFN-γ, loss of type I IFN signaling or inhibition of CSpV1-dsRNA delivery can restore IFN-γ-mediated protective response. Our findings demonstrate that type I IFN signaling in intestinal epithelial cells is detrimental to intestinal anti-C. parvum defense and Cryptosporidium uses CSpV1 to activate type I IFN signaling to evade epithelial antiparasitic response.

Cryptosporidium rhoptry effector protein ROP1 injected during invasion targets the host cytoskeletal modulator LMO7.

The parasite Cryptosporidium invades and replicates in intestinal epithelial cells and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms that underlie infection and pathogenesis are largely unknown. Here, we delineate the events of host cell invasion and uncover a mechanism unique to Cryptosporidium. We developed a screen to identify parasite effectors, finding the injection of multiple parasite proteins into the host from the rhoptry organelle. These factors are targeted to diverse locations within the host cell and its interface with the parasite. One identified effector, rhoptry protein 1 (ROP1), accumulates in the terminal web of enterocytes through direct interaction with the host protein LIM domain only 7 (LMO7) an organizer of epithelial cell polarity and cell-cell adhesion. Genetic ablation of LMO7 or ROP1 in mice or parasites, respectively, impacts parasite burden in vivo in opposite ways. Taken together, these data provide molecular insight into how Cryptosporidium manipulates its intestinal host niche.

Circular RNA ciRS-7 affects the propagation of Cryptosporidium parvum in HCT-8 cells by sponging miR-1270 to activate the NF-κB signaling pathway.

BACKGROUND: Cryptosporidium is an important zoonotic pathogen responsible for severe enteric diseases in humans and animals. However, the molecular mechanisms underlying host and Cryptosporidium interactions are still not clear. METHODS: To study the roles of circRNAs in host cells during Cryptosporidium infection, the expression profiles of circRNAs in HCT-8 cells infected with C. parvum were investigated using a microarray assay, and the regulatory role of a significantly upregulated circRNA, ciRS-7, was investigated during C. parvum infection. RESULTS: C. parvum infection caused notable alterations in the expression profiles of circRNAs in HCT-8 cells, and a total of 178 (including 128 up- and 50 downregulated) circRNAs were significantly differentially expressed following C. parvum infection. Among them, ciRS-7 was significantly upregulated and regulated the NF-κB signaling pathway by sponging miR-1270 during C. parvum infection. Furthermore, the ciRS-7/miR-1270/relA axis markedly affected the propagation of C. parvum in HCT-8 cells. CONCLUSIONS: Our results revealed that ciRS-7 would promote C. parvum propagation by regulating the miR-1270/relA axis and affecting the NF-κB pathway. To the best of our knowledge, this is the first study to investigate the role of circRNA during Cryptosporidium infection, and the findings provide a novel view for implementing control strategies against Cryptosporidium infection.

Cryptosporidium parvum infection induces autophagy in intestinal epithelial cells.

Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP-induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin-4 and E-cadherin. Therefore, we utilised confluent Caco-2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco-2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 106 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co-treated with CP + CQ as compared to CP or CQ alone, suggesting that CP-induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin-4 and E-cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP-induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis.

Homeostatic mini-intestines through scaffold-guided organoid morphogenesis.

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.

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

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

Demonstrating the Benefits of Predictive Bayesian Dose-Response Relationships Using Six Exposure Studies of Cryptosporidium parvum.

A conventional dose-response function can be refitted as additional data become available. A predictive dose-response function in contrast does not require a curve-fitting step, only additional data and presents the unconditional probabilities of illness, reflecting the level of information it contains. In contrast, the predictive Bayesian dose-response function becomes progressively less conservative as more information is included. This investigation evaluated the potential for using predictive Bayesian methods to develop a dose-response for human infection that improves on existing models, to show how predictive Bayesian statistical methods can utilize additional data, and expand the Bayesian methods for a broad audience including those concerned about an oversimplification of dose-response curve use in quantitative microbial risk assessment (QMRA). This study used a dose-response relationship incorporating six separate data sets for Cryptosporidium parvum. A Pareto II distribution with known priors was applied to one of the six data sets to calibrate the model, while the others were used for subsequent updating. While epidemiological principles indicate that local variations, host susceptibility, and organism strain virulence may vary, the six data sets all appear to be well characterized using the Bayesian approach. The adaptable model was applied to an existing data set for Campylobacter jejuni for model validation purposes, which yielded results that demonstrate the ability to analyze a dose-response function with limited data using and update those relationships with new data. An analysis of the goodness of fit compared to the beta-Poisson methods also demonstrated correlation between the predictive Bayesian model and the data.

Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.

Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.

Long-term production effects of clinical cryptosporidiosis in neonatal calves.

Cryptosporidiosis can have a devastating effect in neonatal calves, resulting in diarrhoea, dehydration and, in severe cases, death of the animal. The disease is caused by Cryptosporidium spp. and is one of the most common causes of calf enteritis in the UK. The parasite is very difficult to remove from the farm, as the oocysts have a tough outer wall which enables the parasite to survive for several months in moist temperate environmental conditions and it is difficult to kill oocysts with common disinfectants used on a farm. If appropriate management practises are applied, the disease is usually self-limiting and most calves will recover. It has been shown, in studies with children and in lambs, that severe clinical cryptosporidiosis can result in long-term growth and cognitive impairment compared with individuals with no obvious signs of the disease. This study measured the long-term growth rate of beef calves on farm by comparing groups of animals that had suffered differing degrees of clinical severity of cryptosporidiosis as neonates. A group of 27 beef calves were enrolled in the study and monitored from birth to 6 months of age. The calves were scored for severity of cryptosporidiosis and weighed at regular intervals. The average difference in weight gain, at 6 months, between a group of calves that had severe cryptosporidiosis as neonates and a group of calves with no clinical signs of infection was 34 kg. Those calves that had experienced severe cryptosporidiosis as neonates showed a significantly reduced live weight gain compared with those calves showing no clinical signs of infection (P = 0.034). Therefore, the impact of severe cryptosporidiosis in neonatal calves has longer term effects on weight gain and production efficiency, resulting in the parasite having a greater impact on cattle production than previously thought.

Calcium-dependent Protein Kinases in Malaria Parasite Development and Infection.

Apicomplexan parasites have challenged researchers for nearly a century. A major challenge to developing efficient treatments and vaccines is the parasite's ability to change its cellular and molecular makeup to develop intracellular and extracellular niches in its hosts. Ca2+ signaling is an important messenger for the egress of the malaria parasite from the infected erythrocyte, gametogenesis, ookinete motility in the mosquito, and sporozoite invasion of mammalian hepatocytes. Calcium-dependent protein kinases (CDPKs) have crucial functions in calcium signaling at various stages of the parasite's life cycle; this therefore makes them attractive drug targets against malaria. Here, we summarize the functions of the various CDPK isoforms in relation to the malaria life cycle by emphasizing the molecular mechanism of developmental progression within host tissues. We also discuss the current development of anti-malarial drugs, such as how specific bumped kinase inhibitors (BKIs) for parasite CDPKs have been shown to reduce infection in Toxoplasma gondii, Cryptosporidium parvum, and Plasmodium falciparum. Our suggested combinations of BKIs, artemisinin derivatives with peroxide bridge, and inhibitors on the Ca(2+)-ATPase PfATP6 as a potential target should be inspected further as a treatment against malaria.

In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers.

Cryptosporidium parvum has a complex life cycle consisting of asexual and sexual phases that culminate in oocyst formation in vivo. The most widely used cell culture platforms to study C. parvum only support a few days of growth and do not allow the parasite to proceed past the sexual stages to complete oocyst formation. Additionally, these cell culture platforms are mostly adenocarcinoma cell lines, which do not adequately model the parasite's natural environment in the small intestine. We describe here a method to create primary intestinal epithelial cell monolayers that support long-term C. parvum growth. Monolayers were derived from mouse intestinal stem cells grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an "air-liquid interface" that enhanced host cell differentiation and supported long-term C. parvum growth. The use of primary intestinal cells to grow C. parvum in vitro will be a valuable tool for studying host-parasite interactions using a convenient in vitro model that more closely resembles the natural niche in the intestine.

Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection.

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

Bovine Cryptosporidium parvum field isolates differ in cytopathogenicity in HCT-8 monolayers.

Suckling calves are prone to Cryptosporidium infection. The variable degree of clinical disease is influenced by keeping conditions and immune status of the host, but diversity of isolate virulence may also contribute. The aim of the current study was to evaluate the cytopathogenic effects of 26 C. parvum field isolates by using a MTT assay in HCT-8 cell monolayers. Cell viability of monolayers inoculated with oocysts of the field isolates varied considerably with values of 17.7% (± 5.1%) to 99.5% (± 7.1%). A standard deviation of 18.6% was detected for cell viability of the in house reference strain, which were tested alongside in every assay. Field isolates were grouped in three categories of cytopathogenicity. Probably the length of storage has an effect on the level of the cell destruction category detected post infection in vitro. The applied tool may help to better understand the variable course of cryptosporidiosis in the field.

Novel lactate dehydrogenase inhibitors with in vivo efficacy against Cryptosporidium parvum.

Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic protozoan parasite that causes a diarrheal syndrome in children and neonatal livestock, culminating in growth retardation and mortalities. Despite the high prevalence of C. parvum, there are no fully effective and safe drugs for treating infections, and there is no vaccine. We have previously reported that the bacterial-like C. parvum lactate dehydrogenase (CpLDH) enzyme is essential for survival, virulence and growth of C. parvum in vitro and in vivo. In the present study, we screened compound libraries and identified inhibitors against the enzymatic activity of recombinant CpLDH protein in vitro. We tested the inhibitors for anti-Cryptosporidium effect using in vitro infection assays of HCT-8 cells monolayers and identified compounds NSC158011 and NSC10447 that inhibited the proliferation of intracellular C. parvum in vitro, with IC50 values of 14.88 and 72.65 µM, respectively. At doses tolerable in mice, we found that both NSC158011 and NSC10447 consistently significantly reduced the shedding of C. parvum oocysts in infected immunocompromised mice's feces, and prevented intestinal villous atrophy as well as mucosal erosion due to C. parvum. Together, our findings have unveiled promising anti-Cryptosporidium drug candidates that can be explored further for the development of the much needed novel therapeutic agents against C. parvum infections.

Amixicile Reduces Severity of Cryptosporidiosis but Does Not Have In Vitro Activity against Cryptosporidium.

Cryptosporidium species cause significant morbidity in malnourished children. Nitazoxanide (NTZ) is the only approved treatment for cryptosporidiosis, but NTZ has diminished effectiveness during malnutrition. Here, we show that amixicile, a highly selective water-soluble derivative of NTZ diminishes Cryptosporidium infection severity in a malnourished mouse model despite a lack of direct anticryptosporidial activity. We suggest that amixicile, by tamping down anaerobes associated with intestinal inflammation, reverses weight loss and indirectly mitigates infection-associated pathology.

Revisiting the infectivity and pathogenicity of Cryptosporidium avium provides new information on parasitic sites within the host.

BACKGROUND: Cryptosporidium spp. are protozoans that cause diarrheal illness in humans and animals, including birds, worldwide. The present study was aimed to revisit the infectivity and pathogenicity of C. avium, recently considered to be a valid avian-infecting species of Cryptosporidium, and foster further understanding of its biological characteristics. RESULTS: Results showed that no Cryptosporidium oocysts were detected in the feces of experimentally inoculated BALB/c mice, Mongolian gerbils, quail or budgerigars within 30 days post-infection (dpi). Oocysts were first detected in feces of 3-day-old and 40-day-old hens at 8 and 9 dpi, respectively. In ducks infected with C. avium, oocysts were first detected at 9 dpi. Oocysts of infected animals were studied using a nested-polymerase chain reaction (PCR) technique for the SSU rRNA gene, actin gene, HSP70 gene and Cryptosporidium oocyst wall protein gene (COWP) detection. Restriction fragment length polymorphism (RFLP), using SspI and VspI restriction enzymes, was carried out to genotype the species and obtained amplification products were sequenced. Cryptosporidium developmental stages were found in the longitudinal plica of the bursa fabricii (BF) of hens, with high levels observed in histological sections and scanning electron microscopy. No pathological changes were observed. CONCLUSIONS: These findings indicate that the bursa fabricii may be the primary site of C. avium infection. More biological data are needed to support the establishment of new species and contribute to the taxonomy of Cryptosporidium.

Localization of Fe-S Biosynthesis Machinery in Cryptosporidium parvum Mitosome.

Cryptosporidium is a protozoan, apicomplexan, parasite that poses significant risk to humans and animals, as a common cause of potentially fatal diarrhea in immunodeficient hosts. The parasites have evolved a number of unique biological features that allow them to thrive in a highly specialized parasitic lifestyle. For example, the genome of Cryptosporidium parvum is highly reduced, encoding only 3,805 proteins, which is also reflected in its reduced cellular and organellar content and functions. As such, its remnant mitochondrion, dubbed a mitosome, is one of the smallest mitochondria yet found. While numerous studies have attempted to discover the function(s) of the C. parvum mitosome, most of them have been focused on in silico predictions. Here, we have localized components of a biochemical pathway in the C. parvum mitosome, in our investigations into the functions of this peculiar mitochondrial organelle. We have shown that three proteins involved in the mitochondrial iron-sulfur cluster biosynthetic pathway are localized in the organelle, and one of them can functionally replace its yeast homolog. Thus, it seems that the C. parvum mitosome is involved in iron-sulfur cluster biosynthesis, supporting the organellar and cytosolic apoproteins. These results spearhead further research on elucidating the functions of the mitosome and broaden our understanding in the minimalistic adaptations of these organelles.

First detection of Cryptosporidium parvum in falcons (Falconiformes): Diagnosis, molecular sequencing, therapeutic trial and epidemiological assessment of a possible emerging disease in captive falcons.

Cryptosporidiosis in raptors and falcons is well-known to be caused by Cryptosporidium baileyi and associated mainly with respiratory pathology. This report presents the diagnosis of an atypical cryptosporidiosis event caused by Cryptosporidium parvum, that to the authors' knowledge, is a case observed for the first time in falcons. Two falcons (Gyrfalcon x Peregrine hybrids) were presented for annual check without any clinical signs. Hematology, biochemistry, fecal and crop parasitology, radiographic and endoscopic examinations were performed. Endoscopy revealed microcystic formation of the caudal lung field in the two falcons, adhesions and air sac alterations. Sampling and subsequent cytology revealed fungal spores and acid fast stain organisms (identified as Cryptosporidium spp.). Feces and affected lung tissue was further send for Cryptosporidium spp.-DNA detection. Fecal samples and lung tissue tested positive for Cryptosporidium spp. gp60 gene by PCR. By sequence analysis of the gp60 gene locus, diagnosis of C. parvum was confirmed with 100% homology. Despite the fact that falcons didn't recover after 1 month of therapy, eight months after the initial examination they were clinically healthy and had satisfactory flying performance. No other falcons were observed with C. parvum infections in the facility so far. The possible source, infection route and implications are discussed.

Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples.

OBJECTIVE: Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1® (Qiagen) and the manual QIAamp® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi. RESULTS: Whereas EZ1® (Qiagen) and QIAamp® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1® on the five remaining pathogens. DNA extraction using the semi-automated EZ1® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

A Novel Piperazine-Based Drug Lead for Cryptosporidiosis from the Medicines for Malaria Venture Open-Access Malaria Box.

Cryptosporidiosis causes life-threatening diarrhea in children under the age of 5 years and prolonged diarrhea in immunodeficient people, especially AIDS patients. The standard of care, nitazoxanide, is modestly effective in children and ineffective in immunocompromised individuals. In addition to the need for new drugs, better knowledge of drug properties that drive in vivo efficacy is needed to facilitate drug development. We report the identification of a piperazine-based lead compound for Cryptosporidium drug development, MMV665917, and a new pharmacodynamic method used for its characterization. The identification of MMV665917 from the Medicines for Malaria Venture Malaria Box was followed by dose-response studies, in vitro toxicity studies, and structure-activity relationship studies using commercial analogues. The potency of this compound against Cryptosporidium parvum Iowa and field isolates was comparable to that against Cryptosporidium hominis Furthermore, unlike nitazoxanide, clofazimine, and paromomycin, MMV665917 appeared to be curative in a NOD SCID gamma mouse model of chronic cryptosporidiosis. MMV665917 was also efficacious in a gamma interferon knockout mouse model of acute cryptosporidiosis. To determine if efficacy in this mouse model of chronic infection might relate to whether compounds are parasiticidal or parasitistatic for C. parvum, we developed a novel in vitro parasite persistence assay. This assay suggested that MMV665917 was parasiticidal, unlike nitazoxanide, clofazimine, and paromomycin. The assay also enabled determination of the concentration of the compound required to maximize the rate of parasite elimination. This time-kill assay can be used to prioritize early-stage Cryptosporidium drug leads and may aid in planning in vivo efficacy experiments. Collectively, these results identify MMV665917 as a promising lead and establish a new method for characterizing potential anticryptosporidial agents.