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Human gut microbial communities are mainly composed of bacteria, but also include fungi, viruses, archaea, and protozoa, whose role in the gut ecosystem has only recently begun to be recognized. For example, humans colonized by Blastocystis (a gut protozoan with controversial pathogenicity) host a more diverse bacterial microbiota than individuals not carrying it, suggesting that its presence may be beneficial for the host. In parallel, the presence of non-pathogenic Entamoeba spp. has been associated with an increased diversity and compositional shifts in the bacterial microbiota of healthy rural individuals in Cameroon. However, Entamoeba and Blastocystis , the two most prevalent human gut protozoa, have never been studied in the same individuals, preventing the study of their interaction. As Blastocystis is one of the few gut protozoa commonly found in industrialized populations, which are otherwise mostly devoid of gut eukaryotes, we need to focus on rural “traditional” populations, who harbor a higher diversity of gut eukaryotes (whether pathogenic or commensal) in order to study protozoa interactions in the gut ecosystem. To this end, we profiled the gut bacterial microbiota of 134 healthy Cameroonian adults using 16S rRNA gene amplicon sequencing data. Entamoeba and Blastocystis presence and co-occurrence pattern in the same individuals were determined using metagenomic shotgun data. We found that, when taking into account both protozoa jointly, Blastocystis was associated with both a higher richness and a higher evenness of the gut bacterial microbiota, while Entamoeba was associated only with a higher richness. We demonstrated a cumulative influence of these protozoa on bacterial microbiome diversity. Furthermore, while the abundance of several common taxa (for example, Ruminococcaceae , Coprococcus and Butyrivibrio ) varied according to Blastocystis colonization, only a single Bacteroides amplicon sequence variant was found to be differentially abundant between Entamoeba -negative and Entamoeba -positive samples. Given the specific signature of each protozoan on the gut microbiota and the seemingly stronger association for Blastocystis , our results suggest that Blastocystis and Entamoeba interact with gut bacteria each in its own way, but experimental studies are needed to explore the precise mechanisms of these interactions.

Background A close connection between a protozoan parasite and the balance of the other gut microbes of the host has been demonstrated. The calves may be naturally co-infected with many parasites, and the co-effects of parasites on other intestinal microbes of calves remain unclear. This study aims to preliminarily reveal the relationship between intestinal parasites and other intestinal microbes in calves. Methods Fecal samples were collected from four calves with bloody diarrhea, four calves with watery diarrhea, and seven normal calves, and the microbial flora of the samples were analyzed by whole-genome sequencing. Protozoal parasites were detected in the metagenome sequences and identified using polymerase chain reaction (PCR). Results Cryptosporidium , Eimeria , Giardia , Blastocystis , and Entamoeba were detected by metagenomic analysis, and the identified species were Giardia duodenalis assemblage E, Cryptosporidium bovis , Cryptosporidium ryanae , Eimeria bovis , Eimeria subspherica , Entamoeba bovis , and Blastocystis ST2 and ST10. Metagenomic analysis showed that the intestinal microbes of calves with diarrhea were disordered, especially in calves with bloody diarrhea. Furthermore, different parasites show distinct relationships with the intestinal microecology. Cryptosporidium , Eimeria , and Giardia were negatively correlated with various intestinal bacteria but positively correlated with some fungi. However, Blastocystis and Entamoeba were positively associated with other gut microbes. Twenty-seven biomarkers not only were significantly enriched in bloody diarrhea, watery diarrhea, and normal calves but were also associated with Eimeria , Cryptosporidium , and Giardia . Only Eimeria showed a distinct relationship with seven genera of bacteria, which were significantly enriched in the healthy calves. All 18 genera of fungi were positively correlated with Cryptosporidium , Eimeria , and Giardia , which were also significantly enriched in calves with bloody diarrhea. Functional genes related to parasites and diseases were found mainly in fungi. Conclusions This study revealed the relationship between intestinal protozoan parasites and the other calf gut microbiome. Different intestinal protozoan parasites have diametrically opposite effects on other gut microecology, which not only affects bacteria in the gut, but also is significantly related to fungi and archaea. Graphical Abstract

Intestinal diseases caused by protistan parasites of the genera Giardia (giardiasis), Entamoeba (amoebiasis), Cryptosporidium (cryptosporidiosis) and Blastocystis (blastocystosis) represent a major burden in human and animal populations worldwide due to the severity of diarrhea and/or inflammation in susceptible hosts. These pathogens interact with epithelial cells, promoting increased paracellular permeability and enterocyte cell death (mainly apoptosis), which precede physiological and immunological disorders. Some cell-surface-anchored and molecules secreted from these parasites function as virulence markers, of which peptide hydrolases, particularly cysteine proteases (CPs), are abundant and have versatile lytic activities. Upon secretion, CPs can affect host tissues and immune responses beyond the site of parasite colonization, thereby increasing the pathogens’ virulence. The four intestinal protists considered here are known to secrete predominantly clan A (C1- and C2-type) CPs, some of which have been characterized. CPs of Giardia duodenalis (e.g., Giardipain-1) and Entamoeba histolytica (EhCPs 1-6 and EhCP112) degrade mucin and villin, cause damage to intercellular junction proteins, induce apoptosis in epithelial cells and degrade immunoglobulins, cytokines and defensins. In Cryptosporidium, five Cryptopains are encoded in its genome, but only Cryptopains 4 and 5 are likely secreted. In Blastocystis sp., a legumain-activated CP, called Blastopain-1, and legumain itself have been detected in the extracellular medium, and the former has similar adverse effects on epithelial integrity and enterocyte survival. Due to their different functions, these enzymes could represent novel drug targets. Indeed, some promising results with CP inhibitors, such as vinyl sulfones (K11777 and WRR605), the garlic derivative, allicin, and purified amoebic CPs have been obtained in experimental models, suggesting that these enzymes might be useful drug targets.

Background Urinary schistosomiasis and intestinal helminth infections remain a public health problem in sub-Saharan Africa. In Burkina Faso, prevalence varies by region. This study aimed to determine the prevalence of urinary schistosomiasis and intestinal helminth infections and the associated risk factors for transmission. Methods A cross-sectional survey was conducted from October to December 2023 among 807 individuals, aged 6 to 92 years, from five villages around the Soum Dam in the Nanoro area. Transmission-related associated risk factors were precise using a structured questionnaire. Stool samples were analyzed to detect intestinal parasites using the Kato-Katz technique and Formol-Ether/Ethyl Acetate Concentration Technique (FECT). Urine samples were examined for Schistosoma haematobium through urine filtration and microhematuria detection using Haemastix reagent strips. Results Out of the 807 individuals enrolled in the study, 84 (10.4%) were found to be infected with at least one parasitic species (95% Confidence Interval (CI): 8.5%–12.7%). Nematode infections were the most prevalent, affecting 47 (5.8%) of participants (95% CI: 4.4%–7.7%), and included Ascaris lumbricoides , Trichuris trichiura , Ancylostoma spp., and Enterobius vermicularis . Intestinal protozoa ( Entamoeba histolytica/dispar and Entamoeba coli) were detected in 15 (1.9%) of individuals (95% CI: 1.0%–3.3%). Platyhelminth infections were observed in participants, including 19 (2.4%) (95% CI: 1.4%–4.0%) due to cestodes ( Hymenolepis nana ) and 4 (0.05%) (95% CI: 0.1%–1.4%) due to trematodes ( S. haematobium ). Overall, people aged 15 years and older were the most infected population. The greatest proportion of infections was due to nematodes, which are transmitted by food and soil (soil-transmitted helminths). The bivariate analysis revealed several important trends, particularly with regard to geographical location, living in Séguédin (Prevalence Ratio (PR) = 0.4; 95% CI: 0.2%–0.9%) and Soala (PR = 0.3; 95% CI: 0.1%–0.9%) was associated with a lower prevalence of parasitic infections. Conclusion This study showed that the prevalence of urinary schistosomiasis and intestinal parasitosis are low among populations living around Soum dam agropole. However, soil-transmitted helminths were the most frequent parasites highlighting the need to improve water, sanitation, and hygiene practices in this area. Clinical trial number Not applicable.

The human gastrointestinal microbiota contains a diverse consortium of microbes, including bacteria, protozoa, viruses, and fungi. Through millennia of co-evolution, the host–microbiota interactions have shaped the immune system to both tolerate and maintain the symbiotic relationship with commensal microbiota, while exerting protective responses against invading pathogens. Microbiome research is dominated by studies describing the impact of prokaryotic bacteria on gut immunity with a limited understanding of their relationship with other integral microbiota constituents. However, converging evidence shows that eukaryotic organisms, such as commensal protozoa, can play an important role in modulating intestinal immune responses as well as influencing the overall health of the host. The presence of several protozoa species has recently been shown to be a common occurrence in healthy populations worldwide, suggesting that many of these are commensals rather than invading pathogens. This review aims to discuss the most recent, conflicting findings regarding the role of intestinal protozoa in gut homeostasis, interactions between intestinal protozoa and the bacterial microbiota, as well as potential immunological consequences of protozoa colonization.

Background Preventive chemotherapy with donated anthelminthic drugs is the cornerstone for the control of helminthiases. However, reinfection can occur rapidly in the absence of clean water and sanitation coupled with unhygienic behaviour. The purpose of this study was to assess the effect of an integrated package of interventions, consisting of preventive chemotherapy, community-led total sanitation (CLTS) and health education, on the prevalence of helminth and intestinal protozoa infections and on participants’ knowledge, attitude, practice and beliefs (KAPB) towards these diseases including water, sanitation and hygiene (WASH). Methods A cross-sectional survey was carried out in nine communities of south-central Côte d’Ivoire to assess people’s infection with helminths and intestinal protozoa and KAPB. Subsequently, interventions were targeted to five communities, while the remaining communities served as control. The intervention encouraged latrine construction and an evaluation was done 6–7 months later to determine open defecation status of the respective communities. Anthelminthic treatment was provided to all community members. A follow-up cross-sectional survey was conducted approximately one year later, using the same procedures. Results Overall, 810 people had complete baseline and follow-up data and were given anthelminthic treatment. The baseline prevalence of hookworm, Schistosoma haematobium , Trichuris trichiura , Schistosoma mansoni and Ascaris lumbricoides was 31.1%, 7.0%, 2.0%, 1.0% and 0.3%, respectively. Four of the five intervention communities were classified open-defecation free. For hookworm infection, we observed higher negative changes in terms of proportion of decrease (-0.10; 95% confidence interval (CI): - 0.16, -0.04) and higher egg reduction rate (64.9 vs 15.2%) when comparing intervention with control communities. For intestinal protozoa, prevalence reduction was higher in intervention compared to control communities (8.2 vs 2.6%) and WASH indicators and intervention outcomes associated with lower odds for infection at follow-up. The intervention significantly impacted on reported latrine use (before: 15.5%, after: 94.6%), open defecation in the community surroundings (before: 75.0%, after: 16.7%) and awareness for environmental contamination through open defecation (before: 20.4%, after: 52.2%). Conclusions An integrated package of interventions consisting of preventive chemotherapy, health education and CLTS reduces the prevalence of helminth and intestinal protozoa infection. Additional studies in other social-ecological settings are warranted to confirm our findings.

The aim of this study is to determine the frequency of intestinal protozoa in disabled patients attending a rehabilitation center, thereby highlighting the significance of intestinal protozoa in individuals with disabilities. The study included a total of 300 individuals, comprising 200 disabled patients and 100 non-disabled individuals. Stool samples were collected from all participants and examined using the native-Lugol and modified acid-fast methods. In the study, intestinal protozoa were detected in 41% of disabled individuals and in 9% of individuals in the control group (p=0.001). was detected in 18% of patients, spp. in 15%, in 9%, in 5%, and in 4%. Among the subgroups, spina bifida patients had a protozoan infection rate of 83.3%, while the rates in other groups were lower. A significant relationship was detected between the presence of parasites and diarrhea, constipation, and loss of appetite (p<0.05). Disabled individuals, especially those with spina bifida, are at risk for intestinal protozoa, and regular screening for opportunistic protozoa is essential for these patients.

Background Pathogenic intestinal protozoa exhibit a global distribution and are significant causes of diarrhea, estimated to affect approximately 3.5 billion individuals annually. These intestinal infections continue to pose formidable diagnostic challenges. Microscopy remains the reference diagnostic method for intestinal protozoa, but is limited in terms of sensitivity, specificity and the ability to differentiate closely related species. Additionally, microscopy requires an experienced microbiologist. Emerging diagnostic methods, such as immunochromatography and enzyme-linked immunosorbent assay (ELISA), are regarded as suitable techniques for rapid screening. Molecular diagnostic technologies, particularly real-time PCR (RT-PCR), are gaining traction in non-endemic areas characterised by low parasitic prevalence owing to their enhanced sensitivity and specificity, although these techniques still face various technical challenges. Methods In this multicentre study involving 18 Italian laboratories, we compared the performance of a commercial RT-PCR test (AusDiagnostics) and an in-house RT-PCR assay against traditional microscopy for identifying infections with Giardia duodenalis, Cryptosporidium spp., Entamoeba histolytica and Dientamoeba fragilis . Results The study analysed 355 stool samples, of which 230 samples were freshly collected and 125 had been stored in preservation media. The data from our analyses show complete agreement between the AusDiagnostics and in-house PCR methods for the detection of G. duodenalis , with both methods demonstrating high sensitivity and specificity, similar to those of conventional microscopy. For Cryptosporidium spp. and D. fragilis detection, both methods showed high specificity but limited sensitivity, likely due to inadequate DNA extraction from the parasite. Molecular assays seem to be critical for the accurate diagnosis of E. histolytica . Overall, PCR results from preserved stool samples were better than those from fresh samples, likely due to better DNA preservation in the former. Conclusions Molecular methods show promise for the diagnosis of intestinal protozoan infections. The molecular assays tested in this investigation performed well for G. duodenalis and Cryptosporidium spp. in fixed faecal specimens, while D. fragilis detection was inconsistent. These results suggest that although PCR techniques are promising in terms of reliable and cost-effective parasite identification, further standardisation of sample collection, storage and DNA extraction procedures is necessary for consistent results. Graphical Abstract

'Blastocystis' sp. is known to be the most prevalent parasite in fecal samples of humans worldwide. In the present report, a case-control study (1:9.89 (≈10)) was performed, by analyzing data from 3682 patients who attended a public hospital in the northern area of Spain showing gastrointestinal symptoms. Diagnosis was performed in human fecal samples by means of optical microscopy. The prevalence of 'Blastocystis' sp. in patients with gastrointestinal symptoms was 9.18% (338/3682). Most of the 'Blastocystis' sp.-infected patients tested negative for protozoa and helminths, and were underweight and foreign-born (26.4%), mainly from Africa and Central/South America. Gastrointestinal symptoms, such as abdominal pain, anorexia, halitosis, plus relative eosinophilia, as well as co-infections with pathogenic bacteria were associated with 'Blastocystis' sp. infection. Both type 2 diabetes and treatment with immunosuppressive medicines at the time of 'Blastocystis' sp. detection were associated with a higher proportion of infected patients. This is the first case-control study of 'Blastocystis' sp. in humans in northern Spain and may contribute to surveillance and intervention strategies by public health authorities.

Background In Mozambique, infection by intestinal parasites is reported all over the country. However, infection in children with diarrhoea is mostly focused in the southern region of Mozambique. This work aims to determine the frequency and potential risk factors for infection by Cryptosporidium spp., Giardia lamblia, and Entamoeba histolytica in children under-five years hospitalized with diarrhoea in Hospital Central de Nampula , northern Mozambique. Methods A cross-sectional hospital-based surveillance was conducted between March 2015 and January 2018 in children admitted with diarrhoea in Hospital Central de Nampula . Sociodemographic information was obtained through semi-structured interviews applied to the children’s caregivers. A single stool sample was collected from each child to detect antigens from Cryptosporidium spp., G. lamblia, and E. histolytica using an immune-enzymatic technique. Crude and adjusted odds ratios (with 95% Confidence Intervals) were obtained by logistic regression models to identify factors associated with infection by Cryptosporidium spp. and G. lamblia . Results The median age and interquartile intervals of our sample population was 12 months (8–20). Intestinal protozoa were detected in 21.4% (59/276). Cryptosporidium spp. was the most common protozoa (13.9% - 38/274), followed by G. lamblia (9.1% - 25/274) and E. histolytica (0.4% - 1/275). Children with illiterate caregiver’s ( p -value = 0.042) and undernourished ( p -value = 0.011) were more likely to be infected by Cryptosporidium spp. G. lamblia was more common in children living in households with more than four members (p-value = 0.039). E. histolytica was detected in an eleven month’s child, co-infected with Cryptosporidium spp. and undernourished. Conclusion Cryptosporidium spp. and Giardia lamblia were the most common pathogenic intestinal protozoa detected in children with diarrhoea hospitalized in the Hospital Central de Nampula . Our findings obtained highlight the importance of exploring the caregiver’s education level, children’s nutritional status for infections with Cryptosporidium spp., and living conditions, namely crowded households for infections with G. lamblia in children younger than five years.