Multiplex PCR for bacterial, viral and protozoal pathogens in persistent diarrhoea or persistent abdominal pain in Côte d'Ivoire, Mali and Nepal.

In contrast to acute diarrhoea, the aetiology of persistent digestive disorders (≥ 14 days) is poorly understood in low-resource settings and conventional diagnostic approaches lack accuracy. In this multi-country study, we compared multiplex real-time PCR for enteric bacterial, parasitic and viral pathogens in stool samples from symptomatic patients and matched asymptomatic controls in Côte d'Ivoire, Mali and Nepal. Among 1826 stool samples, the prevalence of most pathogens was highest in Mali, being up to threefold higher than in Côte d'Ivoire and up to tenfold higher than in Nepal. In all settings, the most prevalent bacteria were EAEC (13.0-39.9%) and Campylobacter spp. (3.9-35.3%). Giardia intestinalis was the predominant intestinal protozoon (2.9-20.5%), and adenovirus 40/41 was the most frequently observed viral pathogen (6.3-25.1%). Significantly different prevalences between symptomatic and asymptomatic individuals were observed for Campylobacter, EIEC and ETEC in the two African sites, and for norovirus in Nepal. Multiple species pathogen infection was common in Côte d'Ivoire and Mali, but rarely found in Nepal. We observed that molecular testing detected multiple enteric pathogens and showed low discriminatory accuracy to distinguish between symptomatic and asymptomatic individuals. Yet, multiplex PCR allowed for direct comparison between different countries and revealed considerable setting-specificity.

Selection of ssDNA aptamers and construction of aptameric electrochemical biosensor for the detection of Giardia intestinalis trophozoite protein.

Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.

Microscopy detection and molecular characterisation of Giardia duodenalis infection in outpatients seeking medical care in Egypt.

Introduction: Giardiosis remains one of the most prevalent enteric parasitic infections globally. Earlier molecular-based studies conducted in Egypt have primarily focused on paediatric clinical populations and most were based on single genotyping markers. As a result, there is limited information on the frequency and genetic diversity of G. duodenalis infections in individuals of all age groups. Methods: Individual stool samples (n = 460) from outpatients seeking medical care were collected during January-December 2021 in Kafr El-Sheikh governorate, northern Egypt. Initial screening for the presence of G. duodenalis was conducted by coprological examination. Microscopy-positive samples were further confirmed by real-time PCR. A multilocus sequence typing approach targeted amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triose phosphate isomerase (tpi) genes was used for genotyping purposes. A standardised epidemiological questionnaire was used to gather basic sociodemographic and clinical features of the recruited patients. Results: Giardia duodenalis cysts were observed in 5.4% (25/460, 95% CI: 3.6-7.9) of the stool samples examined by conventional microscopy. The infection was more frequent in children under the age of 10 years and in individuals presenting with diarrhoea but without reaching statistical significance. Stool samples collected during the winter period were more likely to harbour G. duodenalis. All 25 microscopy-positive samples were confirmed by real-time PCR, but genotyping data was only available for 56.0% (14/25) of the isolates. Sequence analyses revealed the presence of assemblages A (78.6%, 11/14) and B (21.4%, 3/14). All assemblage A isolates were identified as sub-assemblage AII, whereas the three assemblage B sequences belonged to the sub-assemblage BIII. Patients with giardiosis presenting with diarrhoea were more frequently infected by the assemblage A of the parasite. Conclusion: This is one of the largest epidemiological studies evaluating G. duodenalis infection in individuals of all age groups in Egypt. Our molecular data suggest that G. duodenalis infections in the surveyed population are primarily of anthropic origin. However, because assemblages A and B are zoonotic, some of the infections identified can have an animal origin. Additional investigations targeting animal (domestic and free-living) and environmental (water) samples are warranted to better understand the epidemiology of giardiosis in Egypt.

Outcome of parasitological examinations in dogs in Germany: a retrospective survey.

Dog faecal samples examined from January 2019 to December 2019 were retrospectively analysed for frequency of endoparasites. The examinations were performed with several different methods: 29,219 samples were examined by flotation method and sodium acetate-acetic acid-formalin concentration (SAFC) technique, 1,330 samples by Baermann-Wetzel migration technique, 12,221 samples using a Giardia coproantigen enzyme-linked-immunosorbent assay (ELISA), 1,180 samples using a Cryptosporidium coproantigen ELISA, 1,671 samples by polymerase chain reaction (PCR) testing for Giardia duodenalis and 447 samples by PCR testing for Cryptosporidium spp.. A total of 7.1% of the samples were positive for parasites in the microscopical examination using the flotation method and SAFC technique. The parasites found included Cystoisospora spp. (2.8%), Giardia duodenalis (2.3%), Ancylostomatidae (1.8%), Toxocara canis (1.6%), Trichuris vulpis (0.7%), Toxascaris leonina (0.5%), Capillaria spp. (0.2%), Angiostrongylus vasorum (0.2%), Crenosoma vulpis (0.1%), Taeniidae (0.1%), Sarcocystis spp. (0.03%), Dipylidium caninum (0.01%), Diphyllobothrium latum (< 0.01%), Spirurida (< 0.01%) and Opisthorchiidae (< 0.01%). Using the Baermann-Wetzel migration technique, Angiostrongylus vasorum was found in 0.75% and Crenosoma vulpis in 0.3% of the samples. ELISAs for Giardia duodenalis and Cryptosporidium spp. revealed 13.9% and 1.0% positive faecal samples, and Giardia duodenalis and Cryptosporidium spp. PCRs 19.4% and 2.0%, respectively. Dogs in the first year of life were more frequently infected with parasites than older animals. In the microscopic examination using flotation method and SAFC technique, the significantly highest detection rates were found in dogs up to six months of age (p < 0.001).

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

PURPOSE: Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. METHODS: Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. RESULTS: Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. CONCLUSION: This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

Rabbits as reservoirs: An updated perspective of the zoonotic risk from Cryptosporidium and Giardia.

Rabbits are highly abundant in many countries and can serve as reservoirs of diseases for a diversity of pathogens including the enteric protozoan parasites, Cryptosporidium and Giardia. Both parasites shed environmentally robust environmental stages (oo/cysts) and have been responsible for numerous waterborne outbreaks of diseases. Cryptosporidium hominis and C. parvum are responsible for most infections in humans, while Giardia duodenalis assemblages A and B, cause most human cases of giardiasis. Cryptosporidium cuniculus, the dominant species infecting rabbits, is the only spceies other than C. hominis and C. parvum to have caused a waterborne outbreak of gastritis, which occurred in the United Kingdom in 2008. This review examines the prevalence of Cryptosporidium and Giardia species in rabbits to better understand the public health risks of contamination of water sources with Cryptosporidium and Giardia oo/cysts from rabbits. Despite the abundance of C. cuniculus in rabbits, reports in humans are relatively rare, with the exception of the United Kingdom and New Zealand, and reports of C. cuniculus in humans from the United Kingdom have declined substantially since the 2008 outbreak. Subtyping of C. cuniculus has supported the potential for zoonotic transmission. Relatively few studies have been conducted on Giardia, but assemblage B dominates. However, improved typing methods are required to better understand the transmission dynamics of Giardia assemblages in rabbits. Similarly, it is not well understood if pet rabbits or contaminated water are the main source of C. cuniculus infections in humans. Well-planned studies using high-resolution typing tools are required to understand the transmission dynamics better and quantify the public health risk of Cryptosporidium and Giardia from rabbits.

Detection of zoonotic enteropathogens in captive large felids in Italy.

AIMS: Within the One Health paradigm, infectious disease surveillance have been developed for domestic and wild animals, leaving the role of captive non-domestic populations, especially felids in zoos and circuses, less explored. This study addresses the proximity of these captive animals to urban areas, necessitating focused monitoring for potential zoonotic enteropathogens. The present work aimed to investigate the presence of such zoonotic enteropathogens in faecal samples from captive large felid populations. METHODS AND RESULTS: A total of 108 faecal samples were collected in three circuses, five zoos and one rescue centre across Italy. Salmonella spp. isolation, serotyping and antimicrobial susceptibility testing were conducted on all samples. Additionally, 60 samples were also examined for gastrointestinal parasites using standard coprological techniques. Giardia spp. detection employed direct immunofluorescent staining and specific PCR, while Toxoplasma gondii was detected using PCR targeting B1 gene. A total of 51 Salmonella enterica subsp. enterica were isolated, with predominant serovariants including Infantis (43.1%), Coeln (11.8%) and Newport (11.8%). The captive felids likely act as asymptomatic carriers of foodborne Salmonella, with notable resistance ampicillin and trimethoprim-sulfamethoxazole, no resistance to enrofloxacin was noted. Microscopic analysis revealed Toxascaris leonina eggs in 11 faecal samples (18.3%) and Giardia duodenalis cysts in one animal (1.7%). CONCLUSIONS: Captive animals in public settings may act as sources of Salmonella infection and enteroparasitosis for both occupational and general exposure. The study emphasizes the role of captive animals in antimicrobial resistance dynamics, highlighting the need for routine pathogen screening in the management practices of zoological structures.

Giardia lamblia Diagnosed During Upper Gastrointestinal Endoscopy: Clinical Manifestation, Histopathologic Findings and the Association With Celiac Disease.

BACKGROUND: Giardia lamblia may be found incidentally during upper gastrointestinal (GI) endoscopy, including when biopsies are taken for celiac disease (CeD) diagnosis. We aimed to study the clinical presentation and histopathology of G. lamblia and determine its association with CeD. METHODS: A retrospective case series of pediatric patients diagnosed with G. lamblia based on intestinal biopsies between January 1999 and January 2023. Baseline data; demographics, symptoms, celiac serology, stool testing, macroscopic and histopathologic findings. Follow-up data; treatment and repeated celiac serology. RESULTS: Of 38 patients with G. lamblia , 15 (39.5%) were female, mean age of 6.7 (±4.8 SD) years. Clinical symptoms; GI 19/38 (50%), growth retardation and/or iron deficiency anemia 8/38 (21.1%) or a combination 11/38 (28.9%). Celiac serology was positive in 13/38 (34.2%). Duodenal endoscopic findings; normal (n = 23, 60.5%), nodularity (n = 12, 32.4%), erosions in 2 (5.4%) and scalloping in 1 (2.7%). Histopathology; normal villi 24/38 (63.2%), villous shortening with increased intraepithelial lymphocytes (IEL) 5/38 (13.2%), isolated IEL 3/38 (7.9%) and duodenitis in 6/38 (15.8%). Children with positive CeD serology were younger (4 vs. 8.1 years, P = 0.019), had fewer GI symptoms (23.1% vs. 64%, P = 0.017) and a higher rate of villous shortening with increased IEL (38.5% vs. 0, P < 0.001) versus children with negative serology. On follow-up, metronidazole treatment was recommended to all but was documented to be given in 22/38 (57.9%). Among the 13 children with positive CeD serology, serology normalized in 10 (77%). CONCLUSIONS: G. lamblia is a rare histopathologic finding in children. It may be an incidental finding in CeD or may cause false positive celiac serology.

The risk of wild birds contaminating source water with zoonotic Cryptosporidium and Giardia is probably overestimated.

Cryptosporidium and Giardia are important waterborne protozoan parasites that are resistant to disinfectants commonly used for drinking water. Wild birds, especially wild migratory birds, are often implicated in the contamination of source and wastewater with zoonotic diseases, due to their abundance near water and in urban areas and their ability to spread enteric pathogens over long distances. This review summarises the diversity of Cryptosporidium and Giardia in birds, with a focus on zoonotic species, particularly in wild and migratory birds, which is critical for understanding zoonotic risks. The analysis revealed that both avian-adapted and zoonotic Cryptosporidium species have been identified in birds but that avian-adapted Cryptosporidium species dominate in wild migratory birds. Few studies have examined Giardia species and assemblages in birds, but the non-zoonotic Giardia psittaci and Giardia ardeae are the most commonly reported species. The identification of zoonotic Cryptosporidium and Giardia in birds, particularly C. parvum and G. duodenalis assemblages A and B in wild migratory birds, is likely due to mechanical carriage or spillback from birds co-grazing pastures contaminated with C. parvum from livestock. Therefore, the role of wild migratory birds in the transmission of zoonotic Cryptosporidium and Giardia to source water is likely overestimated. To address knowledge gaps, it is important to conduct more extensive studies on the prevalence of Cryptosporidium and Giardia in a broader range of migratory wild birds. There is also a need to investigate the extent to which zoonotic infections with C. hominis/C. parvum and G. duodenalis assemblages A and B are mechanical and/or transient, and to assess the load and viability of zoonotic oo/cysts shed in avian faeces. Understanding the contribution of birds to zoonoses is essential for effective disease surveillance, prevention, and control.

The detection and phylogenetic characterization of Cryptosporidium, Cystoisospora, and Giardia duodenalis of cats in South Korea.

Introduction: Cryptosporidium, Cystoisospora, and Giardia duodenalis are gastrointestinal protozoa parasites that cause diarrhea in various animals. However, information regarding the detection and phylogenetic characterization of gastrointestinal protozoa parasites in cats is limited throughout South Korea. Therefore, this study aimed to determine the detection and identify subspecies of gastrointestinal protozoa parasites in cats from South Korea. Methods: A total of 290 fecal samples were collected from stray, companion, and shelter cats in six provinces. Cryptosporidium, Cystoisospora, and G. duodenalis were identified by PCR. All positive samples were subtyped by PCR and sequencing of gp60, ITS-1, tpi, bg, and gdh. Results: The overall detection of gastrointestinal protozoan parasitic infection was 17.93%. G. duodenalis was the most prevalent, with 7.93%, followed by Cystoisospora spp. (7.24%) and Cryptosporidium spp. (4.48%). In addition, C. felis (n=10), C. parvum (n=2), C. ryanae (n=1), Cystoisospora felis (n=14), Cystoisospora suis (n=5), Cystoisospora ohioensis (n=1), Cystoisospora spp. were identified in subspecies analysis of positive samples. C. felis showed a significant association with diarrhea (7.81%) and living condition (6.04%), and Cystoisospora felis in diarreha (9.38%) according to detection. Through phylogenetic analysis of the tpi, bg, and gdh genes from 23 G. duodenalispositive samples, it was confirmed that the samples of present study belonged to assemblage A, B, C, and D. Discussion: South Korean cats have a high rate of gastrointestinal protozoan parasites infection with cat-specific Cryptosporidium and Cystoisospora, which are associated with living conditions and diarrhea symptoms. Moreover, zoonotic and other animal-specific subtype of protozoan parasites have been detected in cat feces.

Comparison of cysteine content in whole proteomes across the three domains of life.

An empirical observation suggests that Giardia lamblia proteins have larger cysteine content than their counterparts in other organisms. As this parasite lacks conventional antioxidant stress systems, it is generally accepted that high cysteine content helps G. lamblia cope with oxygen toxicity, a strategy apparently shared by other organisms. Here, we question whether the high cysteine content in some organisms is genuine or just a simple assumption based on singular observations. To this end, we analyzed the cysteine content in 78 proteomes of organisms spanning the three domains of life. The results indicate that the cysteine content in eukaryota is approximately double that in archaea and bacteria, with G. lamblia among the highest. Atypical cysteine contents were found in a few organisms correlating with specific environmental conditions, supporting the evolutionary amino acid-level selection of amino acid composition.

Respiratory allergy symptoms and cytokine profiles in the presence of anti-Ascaris antibody in Giardia lamblia-infected children.

Anti-Ascaris lumbricoides (Asc) IgE and IgG can immunomodulate the allergy; however, the influence of these isotypes has not been investigated in the giardiasis and allergy. Therefore, the frequency of respiratory allergy (RA) symptoms in Giardia lamblia-infected children, with or without anti-Asc IgE, IgG1, or IgG4 and Th1, Th2/Treg, and Th17 cytokine production, was evaluated. We performed a case-control study with children aged 2-10 years old selected by questionnaire and stool exams to form the groups: infected or uninfected with RA (G-RA, n = 55; nG-RA, n = 43); infected and uninfected without RA (G-nRA, n = 59; nG-nRA, n = 54). We performed blood leukocyte counts and in vitro culture. Cytokine levels in the supernatants (CBA), serum total IgE and anti-Asc IgE (ImmunoCAP), IgG1, IgG4, and total IgA (ELISA) were measured. Infection was not associated with allergy. Infected children showed increased levels of anti-Asc IgG1, IL-2, IFN-γ, IL-4, and IL-10. There was a lower frequency of allergy-related symptoms in anti-Asc IgG1-positive children than IgG1-negative (OR = 0.38; CI = 0.17-0.90, p = 0.027) and few eosinophils in G-RA than in G-nRA and more in G-nRA than in nG-nRA, whereas TNF-α levels were higher in the G-RA than in the nG-nRA group. For infected and positive anti-Asc IgG1, there was higher TNF-α and IL-10 production than G/-IgG1. IL-10 levels were lower in nG/ + IgG1 than in infected or non-infected, and both were negative for anti-Asc IgG1. Th1/Th2/IL-10 profiles were stimulated in the infected patients, and in those with circulating anti-Asc IgG1, the TNF-α production was strengthened with a lower risk for respiratory allergy symptoms.

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis.

BACKGROUND: The unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported. METHODS: To examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1ß secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. RESULTS: VSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1ß secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated. CONCLUSIONS: Overall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity.

Tubulin as a potential molecular target for resveratrol in Giardia lamblia trophozoites, in vitro and in silico approaches.

Giardia lamblia is a globally distributed protozoan parasite that causes intestinal disease. Recently, there is an increase in refractory cases of giardiasis to chemotherapeutic agents, and drugs available cause side effects that may limit its use or cause therapeutic non-compliance. Therefore, search for alternative and less harmful drugs to treat giardiasis is an important task. In this sense, resveratrol (RSV) is a polyphenol with a wide range of pharmacological effects such as antimicrobial, anticarcinogenic and antioxidant. The aim of this study was to evaluate the effects of RSV on Giardia lamblia trophozoites in vitro and in silico, focusing on tubulin affectation, a major protein of the Giardia cytoskeleton which participates in relevant processes for cell survival. In vitro determinations showed that RSV inhibits parasite growth and adherence, causes morphological changes, and induces apoptosis-like cell death through tubulin alterations demonstrated by immunolocalization and Western blot assays. Bioinformatic analysis by molecular docking suggested that RSV binds to Giardia tubulin interface heterodimer, sharing binding residues to those reported with depolymerization inhibitors. These findings suggest that RSV affects microtubular dynamics and make it an interesting compound to study for its safety and antigiardiasic potential.

Prevalence and public health relevance of enteric parasites in domestic dogs and cats in the region of Madrid (Spain) with an emphasis on Giardia duodenalis and Cryptosporidium sp.

BACKGROUND: Pet dogs and cats exert an unquestionable beneficial effect in the well-being of their owners, but can also act as a source of zoonotic infections if improperly cared. OBJECTIVES: We investigated the occurrence, risk factors, genetic variability and zoonotic potential of intestinal parasites in dogs and cats attended in a clinical veterinary setting in Spain. METHODS: Canine (n = 252) and feline (n = 35) faecal samples were collected during 2017-2019 and analysed by coproparasitological methods. A rapid lateral immunochromatographic test (ICT) was used for detecting Giardia duodenalis and Cryptosporidium sp. Samples positive at microscopy examination and/or ICT were reassessed by molecular methods. RESULTS: Overall, 48.8% (123/252) of dogs and 48.6% (17/35) of cats were infected by enteric parasites. In dogs, G. duodenalis was the most prevalent species (40.9%), followed by Cystoisospora sp. (7.1%), and Toxocara canis (5.2%). In cats, Joyeuxiella sp. and Toxocara cati were the dominant species (20.0% each), followed by G. duodenalis (14.3%), D. caninum (5.7%) and Cystoisospora felis and Toxascaris leonina (2.9% each). Pups and kittens were more likely to harbour intestinal parasites and develop clinical signs. Sequence analyses of dog isolates revealed the presence of assemblages A (n = 1), C (n = 4), D (n = 4) and C+D (n = 1) within G. duodenalis; C. parvum (n = 1) and C. canis (n = 4) within Cryptosporidium and PtEb IX (n = 1) in Enterocytozoon bieneusi. A novel C. canis subtype family, named XXi, is reported. CONCLUSIONS: Our results highlight that (i) well-cared dogs carry zoonotic enteric protozoan parasites of public health relevance, (ii) proper hygiene practices and routine veterinary treatment are essential to prevent zoonotic infections, (iii) vulnerable populations should avoid contact with pups/kittens with diarrhoea and (iv) infected dogs might be major contributors to the environmental contamination with soil-transmitted helminths (STHs) eggs.

Nitazoxanide Inhibits the Bifunctional Enzyme GlG6PD::6PGL of Giardia lamblia: Biochemical and In Silico Characterization of a New Druggable Target.

Giardiasis, which is caused by Giardia lamblia infection, is a relevant cause of morbidity and mortality worldwide. Because no vaccines are currently available to treat giardiasis, chemotherapeutic drugs are the main options for controlling infection. Evidence has shown that the nitro drug nitazoxanide (NTZ) is a commonly prescribed treatment for giardiasis; however, the mechanisms underlying NTZ's antigiardial activity are not well-understood. Herein, we identified the glucose-6-phosphate::6-phosphogluconate dehydrogenase (GlG6PD::6PGL) fused enzyme as a nitazoxanide target, as NTZ behaves as a GlG6PD::6PGL catalytic inhibitor. Furthermore, fluorescence assays suggest alterations in the stability of GlG6PD::6PGL protein, whereas the results indicate a loss of catalytic activity due to conformational and folding changes. Molecular docking and dynamic simulation studies suggest a model of NTZ binding on the active site of the G6PD domain and near the structural NADP+ binding site. The findings of this study provide a novel mechanistic basis and strategy for the antigiardial activity of the NTZ drug.

Giardiasis in an Infant With Fibrosarcoma: A Case Report.

INTRODUCTION: Giardia lamblia is a neglected parasitic infection that typically affects the developing nations of the world. It is a microscopic intestinal parasite that is known to cause stomach cramps, bloating, nausea and bouts of diarrhoea. CASE PRESENTATION: Here, we are presenting the case of a 1.5 years-old-baby with an immunocompromised condition who got infected by Giardia lamblia. The baby with fibrosarcoma was receiving treatment in our tertiary care centre, and later developed abdominal and minor systemic complaints. Stool samples were collected, which showed trophozoites and cysts of Giardia. DISCUSSION: To the best of our knowledge, this is the first case of Giardia lamblia infection in a paediatric patient with fibrosarcoma. The patient improved after taking metronidazole for ten days. CONCLUSION: It is critical to keep a watch out for this neglected parasite, and suggested samples, particularly stool samples, must be sent for investigation in order to diagnose and manage these cases properly.

Investigation of the Presence of Toxoplasma gondii, Giardia duodenalis, and Cryptosporidium spp. in Drinking Waters in the Region of Marrakech, Morocco.

The association between the parasitic illnesses and the consumption of contaminated water has been largely reported. However, there is still a lack of studies investigating the extent of parasitic contamination in water in Morocco. This is the first study in Morocco that aimed at assessing the presence of protozoan parasites, namely Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii, in drinking water consumed in the region of Marrakech. Samples processing was performed by membrane filtration and qPCR detection. A total of 104 drinking water samples (tap water, well, and spring waters) was collected between 2016 and 2020. The analysis revealed an overall protozoa contamination rate of 67.3% (70/104), of which 35 samples were positive for Giardia duodenalis, 18 for Toxoplasma gondii, and 17 for both parasites, whereas no sample was positive for Cryptosporidium spp. This first study showed that drinking water in the region of Marrakech contained parasites which could represent a risk for consumers. For a better understanding and estimation of the risk encountered by local inhabitants, further studies concerned with (oo)cyst viability, infectivity, and genotype identification need to be performed.

Occurrence and limited zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Balantioides coli infections in free-ranging and farmed wild ungulates in Spain.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1-3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2-6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3-1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulate-adapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate species would not presumably play a significant role as source of human infections by these pathogens. Wild ruminants do not seem to be susceptible hosts for B. coli.

Trophozoite fitness dictates the intestinal epithelial cell response to Giardia intestinalis infection.

Giardia intestinalis is a non-invasive, protozoan parasite infecting the upper small intestine of most mammals. Symptomatic infections cause the diarrhoeal disease giardiasis in humans and animals, but at least half of the infections are asymptomatic. However, the molecular underpinnings of these different outcomes of the infection are still poorly defined. Here, we studied the early transcriptional response to G. intestinalis trophozoites, the disease-causing life-cycle stage, in human enteroid-derived, 2-dimensional intestinal epithelial cell (IEC) monolayers. Trophozoites preconditioned in media that maximise parasite fitness triggered only neglectable inflammatory transcription in the IECs during the first hours of co-incubation. By sharp contrast, "non-fit" or lysed trophozoites induced a vigorous IEC transcriptional response, including high up-regulation of many inflammatory cytokines and chemokines. Furthermore, "fit" trophozoites could even suppress the stimulatory effect of lysed trophozoites in mixed infections, suggesting active G. intestinalis suppression of the IEC response. By dual-species RNA-sequencing, we defined the IEC and G. intestinalis gene expression programs associated with these differential outcomes of the infection. Taken together, our results inform on how G. intestinalis infection can lead to such highly variable effects on the host, and pinpoints trophozoite fitness as a key determinant of the IEC response to this common parasite.