Comparative efficacy and safety of anti-cryptosporidial agents: an in vitro study on nitazoxanide, halofuginone lactate, KDU731, and paromomycin against Cryptosporidium parvum.

This study evaluated the in vitro effectiveness of anti-cryptosporidial agents nitazoxanide, halofuginone, the pyrazolopyridine analog KDU731, and paromomycin (PMC) in combating the significant zoonotic pathogen Cryptosporidium parvum. The study utilized HCT-8 host cells to culture C. parvum and fluorescent microscopy/quantitative PCR (qPCR) for detecting parasitic growth. The efficacy of the compounds was assessed by calculating their inhibitory concentrations (IC) against the total growth of C. parvum at 48 h post-infection. The study further investigated the impact of these compounds on early parasitophorous vacuole (PV) formation, merozoite egress, host cell viability, and cell growth cycle. KDU731 displayed the most promising profile, with low nanomolar (102 nM ± 2.28) activity and negligible host cell toxicity. This study offers new insights into the relative efficacy and safety of various anti-cryptosporidial compounds, highlighting their stage-specific effects on C. parvum and the consequential impacts on host cells. Identifying safe and effective anti-cryptosporidial agents contributes significantly to the One Health approach, which emphasizes the importance of integrated strategies in controlling zoonotic diseases.

Synergistic efficacy of ZnO quantum dots, Ag NPs, and nitazoxanide composite against multidrug-resistant human pathogens as new trend of revolutionizing antimicrobial treatment.

Antibiotic resistance is currently becoming a more serious threat to global health, especially in severe nosocomial infections treatment by multidrug-resistant bacteria. This research provides a new way of synergizing green-synthesis for zinc oxide quantum dots (ZnO-QDs with hexagonal crystals) that are 7 nm in diameter and zero-valent Ag cubic crystals that are 67 nm in size embedded with nitazoxanide substrate (NAZ). Instrumental characterization like SEM, TEM, EDAX, and FT-IR and comprehensive antimicrobial studies were conducted to study the incorporation behavior of composites based on Ag NPs/ZnO QDs/NAZ. This combination has not been hitherto addressed anywhere else in the published literature, as well as commercial viability. In this context, we have precisely tuned nanoparticle to nitazoxanide ratio for designing the formulation demonstrating potent activity against MDR infections. By employing nitazoxanide as a scaffold and careful decoration thereof antimicrobial potency has been unlocked overriding conventional therapies. In addition, Ag NPs/ZnO-QDs/nitazoxanide (G6) formula exhibited a therapeutic efficacy span of 96.15 ± 1.68% to 99.57 ± 0.20% against MDR human infections post 48 h incubation; a breakthrough in therapeutic efficacy levels has been achieved by our method. Accordingly, ZnO QDs/Ag NPs/NAZ composite offered potential multidrug resistant human pathogens as a new trend of revolutionizing antimicrobial treatment.

Development and characterization of nitazoxanide-loaded poly(ε-caprolactone) membrane for GTR/GBR applications.

OBJECTIVE: Guided tissue/guided bone regeneration (GTR/GBR) membranes are widely used for periodontal bone regeneration, but their success depends on a bacteria-free environment. Systemic antibiotic treatment often proves inadequate, moreover, the increasing prevalence of antibiotic resistance in oral infections exacerbates this challenge. This study aimed to fabricate antibacterial membranes using a new class of antibiotics for local drug delivery, to eradicate infections and promote tissue regeneration. METHODS: Membranes loaded with nitazoxanide (NTZ) were fabricated via electrospinning using poly(ε-caprolactone) (PCL) with varying concentrations of NTZ (0 %, 2.5 %, and 5 % w/w) relative to the polymer weight. Morphochemical of NTZ-loaded membranes were assessed using scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and Fourier Transform Infrared spectroscopy (FTIR). Mechanical properties were evaluated using universal testing machine and NTZ release profile from membranes was determined by spectrophotometer (λmax = 444) for 14 days. Antimicrobial efficacy against periodontal pathogens, cell compatibility and mineralization were evaluated using periodontal ligament stem cells (PDLSCs). RESULTS: Optimized spinning parameter maintained a uniform fiber diameter and successful loading of NTZ was confirmed by SEM-EDS and FTIR. NTZ incorporation did not significantly affect mechanical properties, whereas the drug release kinetics showed an initial burst, followed by sustained release over 14 days. NTZ-loaded membranes demonstrated antibacterial activity against Aggregatibacter actinomycetemcomitans (Aa) and Fusobacterium nucleatum (Fn). Importantly, the presence of NTZ showed minimal cell toxicity; however, it reduced the mineralization potential compared with that of the pure PCL membrane, which increased over time. SIGNIFICANCE: Taken together, these findings established that NTZ-loaded membranes could be promising barrier membrane to counteract microbial environment and aid periodontal bone regeneration.

Repurposing Nitazoxanide for Potential Treatment of Rare Disease Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare genetic lung disease. Unfortunately, treatment with the mTORC1 inhibitor Rapamycin only slows disease progression, and incomplete responses are common. Thus, there remains an urgent need to identify new targets for the development of curative LAM treatments. Nitazoxanide (NTZ) is an orally bioavailable antiprotozoal small molecule drug approved for the treatment of diarrhea caused by Giardia lamblia or Cryptosporidium parvum in children and adults, with a demonstrated mTORC1 inhibitory effect in several human cell lines. NTZ's excellent safety profile characterized by its more than 20 years of clinical use makes it a promising candidate for repurposing. Our rationale for this study was to further investigate NTZ's effect using in vitro and in vivo LAM models and to elucidate the underlying molecular mechanism beyond mTORC1 inhibition. For this purpose, we investigated cell proliferation, cell viability, and changes in protein phosphorylation and expression in primary human cell cultures derived from LAM lung samples before translating our results into a syngeneic mouse model utilizing Tsc2-null cells. NTZ reduced cell growth for all tested cell lines at a dose of about 30 µM. Lower doses than that had no effect on cell viability, but doses above 45 µM lowered the viability by about 10 to 15% compared to control. Interestingly, our western blot revealed no inhibition of mTORC1 and only a mild effect on active ß-Catenin. Instead, NTZ had a pronounced effect on reducing pAkt. In the mouse model, prophylactic NTZ treatment via the intraperitoneal and oral routes had some effects on reducing lung lesions and improving body weight retention, but the results remain inconclusive.

Antiviral Activity of Nitazoxanide and Miltefosine Against FeHV-1 In Vitro.

Feline herpesvirus type 1 (FeHV-1) is a primary pathogen in cats responsible for respiratory and ocular signs. There are presently no antiviral drugs that are officially licensed for veterinary use in several countries. Consequently, veterinarians must depend on off-label antivirals designed for human use. Recent advances in virus-host cell interaction have resulted in new insights into FeHV-1 replication, establishing the importance of the PI3K/Akt axis. The aim of this study was to employ this new information to assess the efficacy of two compounds whose activities involve this pathway. The antiviral properties of miltefosine and nitazoxanide were examined using seven different concentrations, evaluating cell viability and viral titers after 24 h of infection. Furthermore, selected concentrations were supplied at different time points to investigate the influence of the timing of the addition. The best results were obtained when the drugs were added both before and after viral adsorption (in particular for nitazoxanide). Each compound was further investigated by real-time PCR, western blot, and immunofluorescence. Nitazoxanide was the most effective treatment, reducing the expression of viral glycoproteins as measured by western blot and immunofluorescence, as well as reducing the release of virions in the supernatant (measured by real-time PCR). Moreover, treatment with nitazoxanide and miltefosine was associated with a decrease in Akt phosphorylation. This work emphasized the significance of comprehending the pathways necessary for viral replication and their use in the assessment of novel and effective antivirals.

Joint Screening and Identification of Potential Targets of Nitazoxanide by Affinity Chromatography and Label-Free Techniques.

BACKGROUND: Nitazoxanide not only exhibits a broad spectrum of activities against various pathogens infecting animals and humans but also induces cellular autophagy. Currently, the pattern of action and subcellular targets of nitazoxanide-induced cellular autophagy are still unclear. METHODS: To identify potential targets of nitazoxanide in mammalian cells, we developed an af-finity chromatography system using tizoxanide, a deacetyl derivative of nitazoxanide, as a ligand. Affinity chromatography was performed using VERO cell extracts on tizoxanide-biotin, and the isolated binding proteins were identified by mass spectrometry. Candidate target proteins ob-tained using affinity chromatography were co-analysed with the drug affinity response target sta-bility method. Fluorescent probes obtained by coupling rhodamine B to nitazoxanide were used for intracellular localisation of the binding targets. Solvent-induced protein precipitation profiling and thermal proteome profiling were used to further validate the binding proteins. RESULTS: The joint analysis of the drug affinity response target stability method and affinity chro-matography resulted in the screening of six possible candidate target proteins. Fluorescent probes localised the nitazoxanide-binding protein around the nuclear membrane. Molecular docking re-vealed that the binding proteins mainly formed hydrogen bonds with the nitro group of nitazoxa-nide. Solvent-induced protein precipitation profiling and thermal proteome profiling further vali-dated SEC61A, PSMD12, and PRKAG1 as potential target proteins of nitazoxanide. CONCLUSION: The data supports the idea that nitazoxanide is a multifunctional compound with multiple targets.

The clinical antiprotozoal drug nitazoxanide and its metabolite tizoxanide extend Caenorhabditis elegans lifespan and healthspan.

The drugs extending healthspan in clinic have always been searched. Nitazoxanide is an FDA-approved clinical antiprotozoal drug. Nitazoxanide is rapidly metabolized to tizoxanide after absorption in vivo. Our previous studies find that nitazoxanide and its metabolite tizoxanide induce mild mitochondrial uncoupling and activate cellular AMPK, oral nitazoxanide protects against experimental hyperlipidemia, hepatic steatosis, and atherosclerosis. Here, we demonstrate that both nitazoxanide and tizoxanide extend the lifespan and healthspan of Caenorhabditis elegans through Akt/AMPK/sir 2.1/daf16 pathway. Additionally, both nitazoxanide and tizoxanide improve high glucose-induced shortening of C. elegans lifespan. Nitazoxanide has been a clinical drug with a good safety profile, we suggest that it is a novel anti-aging drug.

Anti-cancer effects of nitazoxanide in epithelial ovarian cancer in-vitro and in-vivo.

Epithelial ovarian cancer is one of the most lethal gynecologic malignancies and poses a considerable threat to women's health. Although the progression-free survival of patients has been prolonged with the application of anti-angiogenesis drugs and Poly (ADP-ribose) polymerases (PARP) inhibitors, overall survival has not substantially improved. Thus, new therapeutic strategies are essential for the treatment of ovarian cancer. Nitazoxanide (NTZ), an FDA-approved anti-parasitic drug, has garnered attention for its potential anti-cancer activity. However, the anti-tumor effects and possible underlying mechanisms of NTZ on ovarian cancer remain unclear. In this study, we investigated the anti-tumor effects and the mechanism of NTZ on ovarian cancer in vitro and in vivo. We found that NTZ inhibited the proliferation of A2780 and SKOV3 epithelial ovarian cancer cells in a time- and concentration-dependent manner; Furthermore, NTZ suppressed the metastasis and invasion of A2780 and SKOV3 cells in vitro, correlating with the inhibition of epithelial-mesenchymal transition; Additionally, NTZ suppressed the Hippo/YAP/TAZ signaling pathway both in vitro and in vivo and demonstrated a good binding activity with core genes of Hippo pathway, including Hippo, YAP, TAZ, LATS1, and LATS2. Oral administration of NTZ inhibited tumor growth in xenograft ovarian cancer mice models without causing considerable damage to major organs. Overall, these data suggest that NTZ has therapeutic potential for treating epithelial ovarian cancer.

Glucuronidation of tizoxanide, an active metabolite of nitazoxanide, in liver and small intestine: Species differences in humans, monkeys, dogs, rats, and mice and responsible UDP-glucuronosyltransferase isoforms in humans.

Tizoxanide (TZX) is an active metabolite of nitazoxanide (NTZ) originally developed as an antiparasitic agent, and is predominantly metabolized into TZX glucuronide. In the present study, TZX glucuronidation by the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice, and recombinant human UDP-glucuronosyltransferase (UGT) were examined. The kinetics of TZX glucuronidation by the liver and intestinal microsomes followed the Michaelis-Menten or biphasic model, with species-specific variations in the intrinsic clearance (CLint). Rats and mice exhibited the highest CLint values for liver microsomes, while mice and rats were the highest for intestinal microsomes. Among human UGTs, UGT1A1 and UGT1A8 demonstrated significant glucuronidation activity. Estradiol and emodin inhibited TZX glucuronidation activities in the human liver and intestinal microsomes in a dose-dependent manner, with emodin showing stronger inhibition in the intestinal microsomes. These results suggest that the roles of UGT enzymes in TZX glucuronidation in the liver and small intestine differ extensively across species and that UGT1A1 and/or UGT1A8 mainly contribute to the metabolism and elimination of TZX in humans. This study presents the relevant and novel-appreciative report on TZX metabolism catalyzed by UGT enzymes, which may aid in the assessment of the antiparasitic, antibacterial, and antiviral activities of NTZ for the treatment of various infections.

Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy.

The rapid expansion of antibiotic-resistant bacterial diseases is a global burden on public health. It makes sense to repurpose and reposition already-approved medications for use as supplementary agents in synergistic combinations with existing antibiotics. Here, we demonstrate that the anthelmintic drug nitazoxanide (NTZ) synergistically enhances the effectiveness of the lipopeptide antibiotic polymyxin B in inhibiting gram-negative bacteria, including those resistant to polymyxin B. Mechanistic investigations revealed that nitazoxanide inhibited calcium influx and cell membrane depolarization, enhanced the affinity between polymyxin B and the extracellular membrane, and promoted intracellular ATP depletion and an increase in reactive oxygen species (ROS), thus enhancing the penetration and disruption of the Escherichia coli cell membrane by polymyxin B. The transcriptomic analysis revealed that the combination resulted in energy depletion by inhibiting both aerobic and anaerobic respiration patterns in bacterial cells. The increased bactericidal effect of polymyxin B on the E. coli ∆nuoC strain further indicates that NuoC could be a promising target for nitazoxanide. Furthermore, the combination of nitazoxanide and polymyxin B showed promising therapeutic effects in a mouse infection model infected with E. coli. Taken together, these results demonstrate the potential of nitazoxanide as a novel adjuvant to polymyxin B, to overcome antibiotic resistance and improve therapeutic outcomes in refractory infections.IMPORTANCEThe rapid spread of antibiotic-resistant bacteria poses a serious threat to public health. The search for potential compounds that can increase the antibacterial activity of existing antibiotics is a promising strategy for addressing this issue. Here, the synergistic activity of the FDA-approved agent nitazoxanide (NTZ) combined with polymyxin B was investigated in vitro using checkerboard assays and time-kill curves. The synergistic mechanisms of the combination of nitazoxanide and polymyxin B were explored by fluorescent dye, transmission electron microscopy (TEM), and transcriptomic analysis. The synergistic efficacy was evaluated in vivo by the Escherichia coli and mouse sepsis models. These results suggested that nitazoxanide, as a promising antibiotic adjuvant, can effectively enhance polymyxin B activity, providing a potential strategy for treating multidrug-resistant bacteria.

Asparagus officinalis Herb-Derived Carbon Quantum Dots: Luminescent Probe for Medical Diagnostics.

The utilization of natural materials for the synthesis of highly fluorescent carbon quantum dots (CQDs) presents a sustainable approach to overcome the challenges associated with traditional chemical precursors. Here, we report the synthesis of novel S,N-self-doped CQDs (S,N@CQDs) derived from asparagus officinalis herb. These S,N@CQDs exhibit 16.7 % fluorescence quantum yield, demonstrating their potential in medical diagnostics. We demonstrate the efficacy of S,N@CQDs as luminescent probes for the detection of anti-pathogenic medications metronidazole (MTZ) and nitazoxanide (NTZ) over concentration ranges of 0.0-180.0 µM (with a limit of detection (LOD) of 0.064 µM) and 0.25-40.0 µM (LOD of 0.05 µM), respectively. The probes were successfully applied to determine MTZ and NTZ in medicinal samples, real samples, and spiked human plasma, with excellent recovery rates ranging from 99.82 % to 103.03 %. Additionally, S,N@CQDs demonstrate exceptional efficacy as diagnostic luminescent probes for hemoglobin (Hb) detection over a concentration range of 0-900 nM, with a minimal detectability of 9.24 nM, comparable to commercially available medical laboratory diagnostic tests. The eco-friendly synthesis and precise detection limits of S,N@CQDs meet necessary analytical requirements and hold promise for advancing diagnostic capabilities in clinical settings. This research signifies a significant step towards sustainable and efficient fluorescence-based medical diagnostics.

Corrigendum: In silico and in vivo evaluation of the anti-cryptosporidial activity of eugenol.

[This corrects the article DOI: 10.3389/fvets.2024.1374116.].

Oxfendazole Nitazoxanide combination in experimental neurocysticercosis - Anti-inflammatory and cysticidal effects.

Neurocysticercosis (NCC) is a parasitic infection caused by the larval stage of the pork tapeworm, Taenia solium. The complications of NCC include seizures, headaches, cognitive impairment, and focal neurological deficits. In addition to antiparasitic drugs and surgery, the management of NCC includes the use of corticosteroids to reduce inflammation and control symptoms. The traditional treatment with albendazole and praziquantel has not been altered over 30 years and present several side effects. There are other anti-helminthic drugs such as oxfendazole and nitazoxanide that may show efficacy in NCC treatment. The aim of this study was to determine the histopathologic aspects of experimental NCC after in vivo treatment with the combination of oxfendazole and nitazoxanide. Balb/c mice were infected with T. crassiceps cysticerci and divided into groups of 10 animals each that received a single dose through gavage as follows: group treated with NaCl 0.9% (control group); group treated by monotherapy of the anti-helminthic drugs, 30 mg/kg in single dose of oxfendazole (OXF) or nitazoxanide (NTZ); and groups treated with the combination of the drugs (OXF/NTZ group). Macroscopic and microscopic analysis were performed. There was greater presence of final stage cysticerci after treatment. The microscopic analysis of the general pathological processes showed that the monotherapy with all treatment groups induced higher perivasculitis than what was observed in the control group. In contrast, the combination treatment showed a lower observation of PMN and MN inflammatory infiltration in comparison to the other treatments and to the control one. These results show that indeed the association of benzimidazole derivatives which present both anti-helminthic and anti-inflammatory properties with other cysticidal drugs are beneficial for the NCC treatment in which the aim is to destroy parasite without inducing inflammatory damage in the brain tissue.

Nitazoxanide protects against experimental ulcerative colitis through improving intestinal barrier and inhibiting inflammation.

Ulcerative colitis is a chronic disease with colonic mucosa injury. Nitazoxanide is an antiprotozoal drug in clinic. Nitazoxanide and its metabolite tizoxanide have been demonstrated to activate AMPK and inhibit inflammation, therefore, the aim of the present study is to investigate the effect of nitazoxanide on dextran sulfate sodium (DSS)-induced colitis and the underlying mechanism. Oral administration of nitazoxanide ameliorated the symptoms of mice with DSS-induced colitis, as evidenced by improving the increased disease activity index (DAI), the decreased body weight, and the shortened colon length. Oral administration of nitazoxanide ameliorated DSS-induced intestinal barrier dysfunction and reduced IL-6 and IL-17 expression in colon tissues. Mechanistically, nitazoxanide and its metabolite tizoxanide treatment activated AMPK and inhibited JAK2/STAT3 signals. Nitazoxanide and tizoxanide treatment increased caudal type homeobox 2 (CDX2) expression, increased alkaline phosphatase (ALP) activity and promoted tight junctions in Caco-2 cells. Nitazoxanide and tizoxanide treatment restored the decreased zonula occludens-1(ZO-1) and occludin protein levels induced by LPS or IL-6 in Caco-2 cells. On the other hand, nitazoxanide and tizoxanide regulated macrophage bias toward M2 polarization, as evidenced by the increased arginase-1expression in bone marrow-derived macrophages (BMDM). Nitazoxanide and tizoxanide reduced the increased IL-6, iNOS and CCL2 pro-inflammatory gene expressions and inhibited JAK2/STAT3 activation in BMDM induced by LPS. In conclusion, nitazoxanide protects against DSS-induced ulcerative colitis in mice through improving intestinal barrier and inhibiting inflammation and the underlying mechanism involves AMPK activation and JAK2/STAT3 inhibition.

Pharmacokinetics of Nitazoxanide Dry Suspensions After Single Oral Doses in Healthy Subjects: Food Effects Evaluation and Bioequivalence Study.

Nitazoxanide (NTZ) is an effective antiparasitic drug with potent antiviral and antimicrobial activity. This randomized, open-label, 2-sequence, 2-period crossover trial was designed to evaluate the bioequivalence (BE) of the NTZ dry suspension in healthy subjects and investigated the effect of food intake on the pharmacokinetic (PK) properties of tizoxanide (an active metabolite of NTZ, TIZ). Sixty healthy Chinese subjects were enrolled and received a single dose of 500 mg/25 mL of preparations on days 1 and 4 under overnight fasting or fed conditions, respectively. The plasma concentration of TIZ was determined using high-performance liquid chromatography/tandem mass spectrometry. PK parameters were calculated using WinNonlin 8.2 and BE was evaluated using SAS 9.4. The 90% confidence intervals for the geometric mean ratio (test/reference) of maximum concentration (Cmax), the area under the curve from time 0 to the time of the last quantifiable concentration (AUC0-t), and the area under the curve from time 0 to extrapolation to infinity (AUC0-∞) were all within the equivalent interval of 80%-125%, compliant with BE requirements. In comparison with fasting, on taking the reference and test preparations of the NTZ dry suspension after a meal, the AUC0-t increased by 48.9% and 47.3%, respectively, the AUC0-∞ increased by 48.4% and 48.3%, respectively, and the post-meal Tmax was prolonged by 1.8-2 hours. Our results demonstrate that the test and reference preparations were bioequivalent. High-fat meals significantly improve the degree of drug absorption and delay the rate of drug absorption.

The antiprotozoal drug nitazoxanide improves experimental liver fibrosis in mice.

Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous studies find that nitazoxanide and its metabolite tizoxanide affect AMPK, STAT3, and Smad2/3 signals which are involved in the pathogenesis of liver fibrosis, therefore, in the present study, we examined the effect of nitazoxanide on experimental liver fibrosis and elucidated the potential mechanisms. The in vivo experiment results showed that oral nitazoxanide (75, 100 mg·kg-1) significantly improved CCl4- and bile duct ligation-induced liver fibrosis in mice. Oral nitazoxanide activated the inhibited AMPK and inhibited the activated STAT3 in liver tissues from liver fibrosis mice. The in vitro experiment results showed that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 signals in LX-2 cells (human hepatic stellate cells). Nitazoxanide and tizoxanide inhibited cell proliferation and collagen I expression and secretion of LX-2 cells. Nitazoxanide and tizoxanide inhibited transforming growth factor-ß1 (TGF-ß1)- and IL-6-induced increases of cell proliferation, collagen I expression and secretion, inhibited TGF-ß1- and IL-6-induced STAT3 and Smad2/3 activation in LX-2 cells. In mouse primary hepatic stellate cells, nitazoxanide and tizoxanide also activated AMPK, inhibited STAT3 and Smad2/3 activation, inhibited cell proliferation, collagen I expression and secretion. In conclusion, nitazoxanide inhibits liver fibrosis and the underlying mechanisms involve AMPK activation, and STAT3 and Smad2/3 inhibition.

In silico and in vivo evaluation of the anti-cryptosporidial activity of eugenol.

Background: Cryptosporidiosis is an opportunistic parasitic disease widely distributed worldwide. Although Cryptosporidium sp. causes asymptomatic infection in healthy people, it may lead to severe illness in immunocompromised individuals. Limited effective therapeutic alternatives are available against cryptosporidiosis in this category of patients. So, there is an urgent need for therapeutic alternatives for cryptosporidiosis. Recently, the potential uses of Eugenol (EUG) have been considered a promising novel treatment for bacterial and parasitic infections. Consequently, it is suggested to investigate the effect of EUG as an option for the treatment of cryptosporidiosis. Materials and methods: The in silico bioinformatics analysis was used to predict and determine the binding affinities and intermolecular interactions of EUG and Nitazoxanide (NTZ) toward several Cryptosporidium parvum (C. parvum) lowa II target proteins. For animal study, five groups of immunosuppressed Swiss albino mice (10 mice each) were used. Group I was left uninfected (control), and four groups were infected with 1,000 oocysts of Cryptosporidium sp. The first infected group was left untreated. The remaining three infected groups received NTZ, EUG, and EUG + NTZ, respectively, on the 6th day post-infection (dpi). All mice were sacrificed 30 dpi. The efficacy of the used formulas was assessed by counting the number of C. parvum oocysts excreted in stool of infected mice, histopathological examination of the ileum and liver tissues and determination of the expression of iNOS in the ileum of mice in different animal groups. Results: treatment with EUG resulted in a significant reduction in the number of oocysts secreted in stool when compared to infected untreated mice. In addition, oocyst excretion was significantly reduced in mice received a combination therapy of EUG and NTZ when compared with those received NTZ alone. EUG succeeded in reverting the histopathological alterations induced by Cryptosporidium infection either alone or in combination with NTZ. Moreover, mice received EUG showed marked reduction of the expression of iNOS in ileal tissues. Conclusion: Based on the results, the present study signified a basis for utilizing EUG as an affordable, safe, and alternative therapy combined with NTZ in the management of cryptosporidiosis.

Anthelmintic efficacy of nitazoxanide in dogs naturally infected with Toxocara canis.

Toxocara canis (T. canis) is a gastrointestinal nematode in dogs, and its larvae also infect humans, causing severe larval migratory disease. Anthelmintic drugs have become the primary means to combat T. canis. In this study, the efficacy of nitazoxanide (NTZ) was tested against all the internal stages of T. canis, including L3 larval stage in vitro experiments and gastrointestinal worm in vivo experiments. In the in vitro experiment, after treatment with NTZ at 7.81 and 62.5 µg/mL for 12 h, the larval mortality efficacy reached 90.0 and 100.0%, respectively. In the in vivo experiments, 100 mg/kg NTZ possessed good anthelmintic efficacy against T. canis, with an egg per gram (EPG) reduction of 99.19%, and 90.00% of dogs cleared with residual worms. These results were comparable to those of the positive control drug. The highest anthelmintic efficacy was observed in the group treated with 150 mg/kg NTZ. Based on faecal egg counts, the number of T. canis eggs decreased by 100.00%, and the percentage of dogs cleared with residual worms achieved 90.00% after 7 days of treatment in the 150-mg/kg NTZ treatment group. In general, NTZ showed great potential to be applied as an anthelmintic against T. canis.

Nitazoxanide refractory cryptosporidiosis complicating Burkitt lymphoma in a child.

Cryptosporidium species cause watery diarrhea in several vertebrate hosts, including humans. Most apparently, immunocompetent-infected individuals remain asymptomatic, whereas immunocompromised may develop severe or chronic cryptosporidiosis. We report here the case of a 6-year-old girl undergoing chemotherapy for Burkitt lymphoma who experienced multiple episodes of watery diarrhea during her hospital stay. Microscopic examination of her stool sample revealed oocysts of Cryptosporidium species. The rapid immunochromatographic test was also positive for Cryptosporidium species. She was treated with nitazoxanide for 3 weeks, which failed to provide both clinical improvement and parasitological clearance. This case highlights the importance of treatment failure in human cryptosporidiosis.

Transcriptional Differential Analysis of Nitazoxanide-Mediated Anticanine Parvovirus Effect in F81 Cells.

Canine parvovirus (CPV) is a single-stranded DNA virus that can cause typical hemorrhagic enteritis, and it is one of the common canine lethal viruses. In previous studies, we screened the Food and Drug Administration (FDA)'s drug library and identified nitazoxanide (NTZ), which has anti-CPV capabilities. To investigate the potential antiviral mechanisms, we first reconfirmed the inhibitory effect of NTZ on the CPV by inoculating with different doses and treating for different lengths of time. Then, the differences in the transcription levels between the 0.1%-DMSO-treated virus group and the NTZ-treated virus group were detected using RNA-seq, and a total of 758 differential expression genes (DEGs) were finally identified. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs revealed that these genes are involved in a variety of biological processes and/or signaling pathways, such as cell cycle, mitosis and cell proliferation and differentiation. A protein-protein interaction (PPI) analysis further identified hub genes associated with cell cycle and division among the DEGs. In addition, the expression levels of some of the enriched genes were detected, which were consistent with the high-throughput sequencing results. Moreover, when the cell cycle was regulated with cell cycle checkpoint kinase 1 (Chk1) inhibitor MK-8776 or Prexasertib HCl, both inhibitors inhibited the CPV. In summary, the transcriptome differential analysis results presented in this paper lay the foundation for further research on the molecular mechanism and potential targets of NTZ anti-CPV.