Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation.

The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation.

Pyroptosis Tuning in Intestinal Cryptosporidiosis via the Natural Histone Deacetylase Inhibitor Romidepsin.

Cryptosporidium is an opportunistic protozoan, with many species of cross-human infectivity. It causes life-threatening diarrhoea in children and CD4-defective patients. Despite its limited efficacy, nitazoxanide remains the primary anti-cryptosporidial drug. Cryptosporidium infects the intestinal brush border (intracellular-extracytoplasmic) and down-regulates pyroptosis to prevent expulsion. Romidepsin is a natural histone deacetylase inhibitor that triggers pyroptosis. Romidepsin's effect on cryptosporidiosis was assessed in immunocompromised mice via gasdermin-D (GSDM-D) immunohistochemical expression, IFN-γ, IL-1ß and IL-18 blood levels by ELISA, and via parasite scanning by modified Ziehl-Neelsen staining and scanning electron microscopy (SEM). Oocyst deformity and local cytokines were also assessed in ex vivo ileal explants. Following intraperitoneal injection of romidepsin, oocyst shedding significantly reduced at the 9th, 12th and 15th d.p.i. compared with infected-control and drug-control (nitazoxanide-treated) mice. H&E staining of intestinal sections from romidepsin-treated mice showed significantly low intestinal scoring with marked reduction in epithelial hyperplasia, villous blunting and cellular infiltrate. SEM revealed marked oocyst blebbing and paucity (in vivo and ex vivo) after romidepsin compared with nitazoxanide. Regarding pyroptosis, romidepsin triggered significantly higher intestinal GSDM-D expression in vivo, and higher serum/culture IFN-γ, IL-1ß and IL-18 levels in romidepsin-treated mice than in the control groups. Collectively, in cryptosporidiosis, romidepsin succeeded in enhancing pyroptosis in the oocysts and infected epithelium, reducing infection and shifting the brush border towards normalisation.

Herbal-based compounds: A review on treatments of cryptosporidiosis.

Cryptosporidium, a monoxenous apicomplexan coccidia, is a prevalent diarrhetic and an opportunistic agent, mainly in immunocompromised individuals. As there are few chemotherapeutic compounds that have limited efficacy, we need to identify new compounds or specific parasite targets for designing more potent drugs to treat cryptosporidiosis. Herbal products with low toxicity, environmental compatibility, wide therapeutic potential, and abundant resources can be considered alternatives for treatment. The current review tried to summarize the studies on plants or herbal bioactive constituents with anti-cryptosporidial activities. Based on constituents, plants act via different mechanisms, and further investigations are needed to clarify the exact mechanisms by which they act on the developmental stages of the parasite or host-parasite relationships.

Azithromycin for Bacterial Watery Diarrhea: A Reanalysis of the AntiBiotics for Children With Severe Diarrhea (ABCD) Trial Incorporating Molecular Diagnostics.

BACKGROUND: Bacterial pathogens cause substantial diarrhea morbidity and mortality among children living in endemic settings, yet antimicrobial treatment is only recommended for dysentery or suspected cholera. METHODS: AntiBiotics for Children with severe Diarrhea was a 7-country, placebo-controlled, double-blind efficacy trial of azithromycin in children 2-23 months of age with watery diarrhea accompanied by dehydration or malnutrition. We tested fecal samples for enteric pathogens utilizing quantitative polymerase chain reaction to identify likely and possible bacterial etiologies and employed pathogen-specific cutoffs based on genomic target quantity in previous case-control diarrhea etiology studies to identify likely and possible bacterial etiologies. RESULTS: Among 6692 children, the leading likely etiologies were rotavirus (21.1%), enterotoxigenic Escherichia coli encoding heat-stable toxin (13.3%), Shigella (12.6%), and Cryptosporidium (9.6%). More than one-quarter (1894 [28.3%]) had a likely and 1153 (17.3%) a possible bacterial etiology. Day 3 diarrhea was less common in those randomized to azithromycin versus placebo among children with a likely bacterial etiology (risk difference [RD]likely, -11.6 [95% confidence interval {CI}, -15.6 to -7.6]) and possible bacterial etiology (RDpossible, -8.7 [95% CI, -13.0 to -4.4]) but not in other children (RDunlikely, -0.3% [95% CI, -2.9% to 2.3%]). A similar association was observed for 90-day hospitalization or death (RDlikely, -3.1 [95% CI, -5.3 to -1.0]; RDpossible, -2.3 [95% CI, -4.5 to -.01]; RDunlikely, -0.6 [95% CI, -1.9 to .6]). The magnitude of risk differences was similar among specific likely bacterial etiologies, including Shigella. CONCLUSIONS: Acute watery diarrhea confirmed or presumed to be of bacterial etiology may benefit from azithromycin treatment. CLINICAL TRIALS REGISTRATION: NCT03130114.

Newly fabricated zinc oxide nanoparticles loaded materials for therapeutic nano delivery in experimental cryptosporidiosis.

Cryptosporidiosis is a global health problem that threatens the lives of immunocompromised patients. This study targets to fabricate and investigate the efficiency of zinc oxide nanoparticles (ZnO-NPs), nitazoxanide (NTZ)-loaded ZnO-NPs, and Allium sativum (A. sativum)-loaded ZnO-NPs in treating cryptosporidiosis. Further FTIR, SEM, XRD, and zeta analysis were used for the characterization of ZnO-NPs and loaded materials. The morphology of loaded materials for ZnO-NPs changed into wrapped layers and well-distributed homogenous particles, which had a direct effect on the oocyst wall. The charge surface of all particles had a negative sign, which indicated well distribution into the parasite matrix. For anti-cryptosporidiosis efficiency, thirty immunosuppressed Cryptosporidium parvum-infected mice, classified into six groups, were sacrificed on the 21st day after infection with an evaluation of parasitological, histopathological, and oxidative markers. It was detected that the highest reduction percent of Cryptosporidium oocyst shedding was (81.5%) in NTZ, followed by (71.1%) in A. sativum-loaded ZnO-NPs-treated groups. Also, treatment with A. sativum and NTZ-loaded ZnO-NPs revealed remarkable amelioration of the intestinal, hepatic, and pulmonary histopathological lesions. Furthermore, they significantly produced an increase in GSH values and improved the changes in NO and MDA levels. In conclusion, this study is the first to report ZnO-NPs as an effective therapy for treating cryptosporidiosis, especially when combined with other treatments that enhance their antioxidant activity. It provides an economical and environment-friendly approach to novel delivery synthesis for antiparasitic applications.

Identification of and Structural Insights into Hit Compounds Targeting N-Myristoyltransferase for Cryptosporidium Drug Development.

Each year, approximately 50,000 children under 5 die as a result of diarrhea caused by Cryptosporidium parvum, a protozoan parasite. There are currently no effective drugs or vaccines available to cure or prevent Cryptosporidium infection, and there are limited tools for identifying and validating targets for drug or vaccine development. We previously reported a high throughput screening (HTS) of a large compound library against Plasmodium N-myristoyltransferase (NMT), a validated drug target in multiple protozoan parasite species. To identify molecules that could be effective against Cryptosporidium, we counter-screened hits from the Plasmodium NMT HTS against Cryptosporidium NMT. We identified two potential hit compounds and validated them against CpNMT to determine if NMT might be an attractive drug target also for Cryptosporidium. We tested the compounds against Cryptosporidium using both cell-based and NMT enzymatic assays. We then determined the crystal structure of CpNMT bound to Myristoyl-Coenzyme A (MyrCoA) and structures of ternary complexes with MyrCoA and the hit compounds to identify the ligand binding modes. The binding site architectures display different conformational states in the presence of the two inhibitors and provide a basis for rational design of selective inhibitors.

Examination of ELISA against PCR for assessing treatment efficacy against Cryptosporidium in a clinical trial context.

BACKGROUND: Cryptosporidium is a gastrointestinal pathogen that presents a serious opportunistic infection in immunocompromised individuals including those living with human immunodeficiency syndrome. The CRYPTOFAZ trial, previously published, was conducted in Malawi to evaluate the efficacy of clofazimine in response to an unmet need for drugs to treat cryptosporidiosis in HIV populations. A combination of rapid diagnostic tests, ELISA, qPCR, and conventional sequencing were employed to detect Cryptosporidium in 586 individuals during pre-screening and monitor oocyst shedding and identify enteric co-pathogens in 22 enrolled/randomized participants during the in-patient period and follow-up visits. METHODOLOGY: Oocyst shedding as measured by qPCR was used to determine primary trial outcomes, however pathogen was detected even at trial days 41-55 in individuals randomized to either clofazimine or placebo arms of the study. Therefore, in this work we re-examine the trial outcomes and conclusions in light of data from the other diagnostics, particularly ELISA. ELISA data was normalized between experiments prior to comparison to qPCR. The amount of all identified enteric pathogens was examined to determine if co-pathogens other than Cryptosporidium were major causative agents to a participant's diarrhea. CONCLUSION: ELISA had higher sample-to-sample variability and proved to be equally or less sensitive than qPCR in detecting Cryptosporidium positive samples. Compared to qPCR, ELISA had equal or greater specificity in detecting Cryptosporidium negative samples. Sequencing identified several Cryptosporidium species including viatorum which has never been identified in Malawi and Southern Africa. In addition to Cryptosporidium, enterotoxigenic E. coli was also identified as a pathogen in diarrheagenic amounts in 4 out of 22 participants.

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.

Study on the Therapeutic Effect of Coconut Oil Extracts as An Alternative Medicinal Plant in Cryptosporidium Infected Mice.

OBJECTIVE: Cryptosporidiosis caused by Cryptosporidium sp. is a globally spreading disease. Nowadays, new researches are moving towards an effective treatment without side effects, especially for young and immune-compromised patients. The current study was designed to evaluate the therapeutic effect of the coconut oil extracts as an alternative medicinal plant in Cryptosporidium infected immunocompromised mice. METHODS: Sixty white albino mice were classified into six groups; Group I: Infected with Cryptosporidium oocysts treated with Nitazoxanide, Group II: Infected with Cryptosporidium oocysts and treated with coconut water extract, Group III: Infected with Cryptosporidium oocysts and treated with coconut Hexan extract, Group IV: Infected with Cryptosporidium oocysts and treated with coconut ethanol extract, Group V: Positive control, Group VI: Negative control. Stool samples were collected and examined; histopathological and immune-histochemical assessment using anti caspase-3 and anti CDX2 monoclonal antibodies were performed. RESULTS: Coconut oil extracts results revealed a significant decrease of oocyst count, correlated with an amelioration of histopathological and confirmed by immunohistochemical changes in ileal tissue. CONCLUSION: The present study has opened fresh avenues for development of natural therapy like coconut oil extracts, which have a potential therapeutic efficacy against Cryptosporidiosis. That was confirmed by different methodologies, parasitological examination, histopathological examination, and immunohistochemical assays. It paves the way for being a promising anti-parasitic agent for infection eradication. However, further studies are still required to gain more knowledge about different coconut extracts in order to reach the best treatment efficacy.

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.

The controverted therapeutic efficacy of Allium sativum and Artemisia herba-alba extracts on Cryptosporidium-infected mice.

INTRODUCTION: Cryptosporidiosis has become an issue of great interest being life-threatening among immunocompromised hosts worldwide. This study explored the curative effect of Allium sativum (garlic) and Artemisia herba-alba ethanolic extract versus Nitazoxanide drug on both immunocompetent and immunosuppressed-Cryptosporidium experimentally-infected mice. METHODOLOGY: One hundred male Swiss albino mice were divided into the following groups: (GI) non-infected non-treated group, (GII) infected non-treated group, (GIII) garlic treated group, (GIV) A. herba-alba treated group, (GV) Nitazoxanide treated group, each group subdivided into two subgroups (a) Immunocompetent, (b) immunosuppressed. The assessment was performed by parasitological counting of fecal oocysts, histological examination of intestinal tissue, immunological detection of interferon-gamma levels in mice sera, and ultrastructural study by transmission electron microscopy. RESULTS: Garlic and A. herbal-alba extracts showed a decrease in the mean oocyst counts through all days of follow-up. This was associated with significant up-regulation of interferon-gamma cytokine levels in serum and histological improvement in intestinal tissues of mice compared to control groups and the results were confirmed by transmission electron microscopy. The highest efficacy was obtained by garlic, then by A. herbal-alba extracts followed by Nitazoxanide treated group; where the immunocompetent groups showed better improvement than immunosuppressed ones. CONCLUSIONS: Garlic has a perfect effect as a promising therapeutic agent against Cryptosporidiosis and therefore validates their traditional use in parasitic infections. Accordingly, it may offer a good option for cryptosporidium treatment in immunocompromised patients. They could be used as a natural safe product for the preparation of a new therapeutic agent.

Treatment of Urticaria caused by severe cryptosporidiosis in a 17-month-old child - a case report.

BACKGROUND: Cryptosporidium is an intracellular protozoan that causes gastrointestinal symptoms in humans and animals. In immunocompromised patients and children under 5 years of age, the infection is severe and can be life-threatening due to severe diarrhea. CASE PRESENTATION: We report a case of urticaria associated with Cryptosporidium in a 17-month-old female Iranian child. The patient had moderate diarrhea (> 3 loose, watery stools but not more than 10 diarrhea stools in a day), weight loss, and acute urticarial (rash clears completely within 6 weeks). Since the child's father worked in livestock farming, the parasite may have been transferred from the cow or calve to the house and the child. Several Cryptosporidium oocysts were detected in the modified acid-fast staining of the child's stool sample. The patient was successfully treated with nitazoxanide (100 mg twice daily) and became negative for parasites three days after treatment and one week after discharge from the hospital. The child was observed to produce < 3 loose stools in the previous 24 h after 1-week post-treatment and after 6 months of follow-up. CONCLUSION: A number of parasites are associated with urticaria, but to our knowledge, there is no information on Cryptosporidium-induced urticaria. Therefore, our result may be evidence for the role of this parasite in the development of urticaria if other causes such as food allergies, autoimmune diseases and etc. don't role in urticaria.

Dual mechanism (sunlight/dark) of the self-assembly nitazoxanide drug on cellulose nanocrystal surface for destroying the Cryptosporidium parvum oocysts.

Destruction of the cryptosporidium parvum (C. parvum) Oocysts is the main target of the work via the improvement effect of the nitazoxanide (NTZ) drug by increasing the drug adsorption process without changing the cell viability. The synthesis of a self-assembly nanocomposite (NCP) of cellulose nanocrystals (CNC) and NTZ drug was performed successfully via the chemical precipitation methods without utilizing the temperature. Also, the characterization of the fabricated NCP was achieved by different techniques to confirm the natural formation of the NCP. The efficient loading of the NTZ drug on the CMC surface and the release process of NCP was calculated by a UV-Visible spectroscopy device, and the loading efficiency is 37 %. The release efficiency is displayed at 66.3 % after 6 h, and 97 % after 48 h at pH 7.4 with NTZ pure, while the release efficiency of CNC@NTZ at the same pH is 61 % after 6 h, and 86 % after 48 h at pH 7.4. The cytotoxicity of different concentrations of NCP was conducted on normal mouse liver cells (BNL) via the quick screening cytotoxicity method (SRB). The effect of NCP on C. parvum was detected with an in-vivo study in the dark and under sunlight conditions. Compared to the NTZ and CNC, the fabricated NCP was able to destroy 89.3 % of the oocyst wall after 96 h. Moreover, a sporulation inhibition percentage of 53.97 % ± 0.63 % was achieved by a maximum concentration of 7 mg/mL after 9.5 h. The results are very encouraging to use the modified NCP as an alternative NTZ drug, although further research is required in terms of clinical trials.

Structure-Activity Relationship Studies of the Aryl Acetamide Triazolopyridazines against Cryptosporidium Reveals Remarkable Role of Fluorine.

Our previous work identified compound 1 (SLU-2633) as a potent lead compound toward the identification of a novel treatment for cryptosporidiosis, caused by the parasite Cryptosporidium (EC50 = 0.17 µM). While this compound is potent and orally efficacious, the mechanism of action and biological target(s) of this series are currently unknown. In this study, we synthesized 70 compounds to develop phenotypic structure-activity relationships around the aryl "tail" group. In this process, we found that 2-substituted compounds are inactive, confirmed that electron withdrawing groups are preferred over electron donating groups, and that fluorine plays a remarkable role in the potency of these compounds. The most potent compound resulting from this work is SLU-10482 (52, EC50 = 0.07 µΜ), which was found to be orally efficacious with an ED90 < 5 mg/kg BID in a Cryptosporidium-infection mouse model, superior to SLU-2633.

Chlorogenic acid derived from Moringa oleifera leaf as a potential antiinflammatory agent against cryptosporidiosis in mice.

Cryptosporidiosis is a serious illness in immunodeficient patients, and there is still no drug that can completely remove the parasite from the host. The present study represents the first report investigating the impact of the active molecule chlorogenic acid (CGA), naturally isolated from Moringa oleifera leaf extract (EMOLE), on immunosuppressed, Cryptosporidium parvum-infected BALB/c mice. Mice were divided into five groups: normal mice, infected immunosuppressed mice, and infected immunosuppressed mice treated with EMOLE, CGA, and nitazoxanide (NTZ) drugs. Parasitological, immunological, and histopathological investigations were recorded besides differences in the mice' body weight. Infected control mice showed elevated levels of oocyst shedding throughout the study. The EMOLE- and CGA-treated groups showed 84.2% and 91.0% reductions in oocyst shedding, respectively, with no significant difference compared to the drug control. The inflammatory markers IFN-γ, IL-6, IL-1ß, and TNF-α were significantly higher in the infected control group. Treatment with 300 mg/kg/day of EMOLE or 30 mg/kg/day of CGA significantly downregulated pro-inflammatory cytokine levels compared to the infected group, although they did not change significantly compared to the NTZ-treated group. Histopathology of intestinal sections showed inflammatory and pathological changes in the infected control group. Low-grade tissue changes and an obvious improvement in villi structure were seen in mice treated with CGA. This study highlighted the role of CGA, isolated and purified from EMOLE, as an effective anti-inflammatory agent in eradicating C. parvum infection.

Cryptosporidiosis in India and the World: A Review.

Cryptosporidiosis is caused by infection with a coccidian parasite belonging to the genus Cryptosporidium. Initially, human cryptosporidiosis was believed to be caused only by one species, but since the advent of molecular studies, 15 more species have been discovered to cause this infection. Among them, Cryptosporidium hominis and Cryptosporidium parvum are the most common species involved. This mainly affects children and causes diarrhea in most cases. It is mainly diagnosed by microscopy, especially in low-middle-income countries. This review covers the epidemiology, life cycle, risk factors, clinical manifestations, different diagnostic methods and treatment of this disease.

A case of septic shock due to delayed diagnosis of Cryptosporidium infection after liver transplantation.

BACKGROUND: Cryptosporidium is recognized as a significant pathogen of diarrhea disease in immunocompromised hosts, and studies have shown that Cryptosporidium infection is high in solid organ transplantation (SOT) patients and often has serious consequences. Because of the lack of specificity of diarrheasymptoms cased by Cryptosporidium infection, it is rarely reported in patients undergoing liver transplantation (LT). It frequently delays diagnosis, coming with severe consequences. In clinical work, diagnosing Cryptosporidium infection in LT patients is also complex but single, and the corresponding anti-infective treatment regimen has not yet been standardized. A rare case of septic shock due to a delayed diagnosis of Cryptosporidium infection after LT and relevant literature are discussed in the passage. CASE PRESENTATION: A patient who had received LT for two years was admitted to the hospital with diarrhea more than 20 days after eating an unclean diet. After failing treatment at a local hospital, he was admitted to Intensive Care Unit after going into septic shock. The patient presented hypovolemia due to diarrhea, which progressed to septic shock. The patient's sepsis shock was controlled after receiving multiple antibiotic combinations and fluid resuscitation. However, the persistent diarrhea, as the culprit of the patient's electrolyte disturbance, hypovolemia, and malnutrition, was unsolved. The causative agent of diarrhea, Cryptosporidium infection, was identified by colonoscopy, faecal antacid staining, and blood high-throughput sequencing (NGS). The patient was treated by reducing immunosuppression and Nitazoxanide (NTZ), which proved effective in this case. CONCLUSION: When LT patients present with diarrhea, clinicians should consider the possibility of Cryptosporidium infection, in addition to screening for conventional pathogens. Tests such as colonoscopy, stool antacid staining and blood NGS sequencing can help diagnose and treat of Cryptosporidium infection early and avoid serious consequences of delayed diagnosis. In treating Cryptosporidium infection in LT patients, the focus should be on the patient's immunosuppressive therapy, striking a balance between anti-immunorejection and anti-infection should be sought. Based on practical experience, NTZ therapy in combination with controlled CD4 + T cells at 100-300/mm3 was highly effective against Cryptosporidium without inducing immunorejection.

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.

Successful haploidentical hematopoietic stem cell transplantation for activated phosphoinositide 3-kinase δ syndrome: Case report and literature review.

RATIONALE: Activated phosphoinositide 3-kinase δ syndrome (APDS), a recently described primary immunodeficiency,is caused by autosomal dominant mutation in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta(PIK3CD) gene encoding the p110δ catalytic subunit of PI3Kδ (APDS1) or the PIK3R1 gene that encodes the p85α regulatory subunit of PI3Kδ (APDS2). Gain-of-function mutation of PIK3CD in APDS1 leads to p110δ hyperactivity, with the result of the hyperphosphorylation of downstream mediators of Akt and mammalian target of rapamycin that cause a series of clinical symptoms. Few cases with APDS were reported in Asia. PATIENT CONCERNS: We report a 6-year-old patient with a recurrent respiratory infection, cryptosporidium enteritis, lymphoproliferation, high serum immunoglobulin-M level, anemia, and inverted CD4+/CD8+ ratio. The whole exome sequencing confirmed a heterozygous missense mutation c.3061G>A（p.E1021K）in patient and her mother. Her mutant gene is inherited from her mother, but her mother has not any clinical symptoms. DIAGNOSES: Activated phosphoinositide 3-kinase δ syndrome. INTERVENTIONS: The patient was received immunoglobulin (Ig) replacement therapy, antibiotics, and rapamycin treatment. Through effectively controlling infection and optimal timing of transplantation by adjusting the conditioning regimen, haploidentical Hematopoietic Stem Cell Transplantation(haplo-HSCT) from her brother was successfully performed. OUTCOMES: The patient is in good condiion with a good quality of life after 20 months of follow-up. LESSONS: We reported a rare APDS1 case with PIK3CD E1021K gene mutation, Successfully treated with haplo-HSCT. This case provided a reference for treating APDS with haplo-HSCT.