Giardia duodenalis infection is a common cause of diarrhoea in returning travellers from low-income regions. The increasing prevalence of resistance to nitroimidazoles, as first-line therapy, are of global concern. The interaction between Giardia and the host’s gut microbiota plays a crucial role in the pathophysiology of giardiasis. Yet, longitudinal data assessing the course of the infection are scarce, and non-existent in patients refractory to treatment. Here, we provide the first longitudinal bacterial community study, carried out in two Giardia-infected Spanish adult patients refractory to treatment after travelling to India, a Giardia-hyperendemic country, which offers insights into the gut microbiota's response to infection and therapy over time. A significant reduction in alpha diversity was evidenced during and post-infection, accompanied by changes in bacterial taxa contribution, with an important individual effect. We evidenced microbial shifts that may be associated with infection, treatment effectiveness or microbial restoration after infection, which can potentially be used to monitor disease evolution and adjust the therapeutic strategy.

Schistosomiasis is one of the most prevalent neglected tropical diseases in Sub-Saharan Africa, causing substantial morbidity and millions of disability-adjusted life years (DALYs). Although the WHO aims to eliminate schistosomiasis as a public health problem in several countries by 2030, limited data on infection prevalence in Zambia has hindered effective Mass Drug Administration (MDA) coverage, contributing to the persistence and resurgence of the disease. We assessed the prevalence of urogenital schistosomiasis in two peri-urban districts of Lusaka (Chongwe and Kafue). A total of 208 participants were enrolled, and infection status was determined using microscopy, serological assays, and molecular diagnostics. Prevalence was significantly higher in Chongwe than in Kafue, as detected by both microscopy and qPCR, demonstrating a strong association between infection and district location. No association was found between sex and infection in either district. However, a significant association between age and Schistosoma haematobium infection was observed across both sites. Molecular characterization of individual eggs revealed that S. haematobium × S. curassoni hybrids were the most prevalent species circulating in the study population. Our findings reveal a high prevalence of urogenital schistosomiasis in at least two peri-urban areas of Lusaka, Zambia, indicating that transmission remains highly active and may be underestimated in national surveillance. Furthermore, the presence of hybrid species infecting humans highlights the need to consider livestock reservoirs when designing elimination strategies. These results provide updated information on the epidemiological situation of urogenital schistosomiasis in Zambia and will support planning and implementation within the WHO NTD agenda. Urogenital schistosomiasis remains a major public health concern in many parts of Sub-Saharan Africa, yet recent data from Zambia are limited, particularly in communities surrounding Lusaka. In this study, we assessed infection levels in two peri-urban districts, Chongwe and Kafue, using a combination of microscopy, serology, and molecular techniques to ensure accurate detection. We found substantial differences in prevalence between the two locations, with significantly higher infection rates in Chongwe, as well as a clear association between age and risk of infection. Molecular characterisation of parasite eggs revealed that hybrid Schistosoma haematobium × S. curassoni forms were the dominant circulating genotypes, indicating potential involvement of animal reservoirs in transmission. These findings demonstrate that transmission remains active and likely underestimated, and they highlight the importance of incorporating updated epidemiological data and potential zoonotic sources into future control and elimination strategies in Zambia.

Giardia duodenalis and Cryptosporidium spp. are environmentally resistant protozoan parasites transmitted via the faecal-oral route, often through contaminated water or food. Although companion animals may contribute to environmental dissemination of infective stages, their role in zoonotic transmission remains debated. In this study, 75 Giardia-positive (65 dogs, 10 cats) and 20 Cryptosporidium-positive (11 dogs, 9 cats) faecal samples from central Spain were molecularly characterized at the gdh and bg loci for G. duodenalis and at the ssu rRNA and gp60 loci for Cryptosporidium spp. Host-adapted Giardia assemblages predominated, with assemblages C (33.9%) and D (47.7%) in dogs and assemblage F (50.0%) in cats, while zoonotic assemblages A and B occurred at moderate frequencies (10.8%-40.0%). Cryptosporidium canis and Cryptosporidium felis were the predominant species in dogs and cats, respectively, whereas C. parvum and C. hominis were sporadically detected. These findings expand molecular data on enteric protozoa in companion animals in Spain and highlight their potential relevance within a One Health context.

Enterocytozoon bieneusi is the most frequently detected microsporidian in humans and infects a wide range of animals. Data integrating human, animal, and environmental compartments under a One Health framework remains scarce. We conducted a molecular survey in a rural area of Adana Province, south-central Türkiye (October–November 2023). A total of 394 samples were screened, including stool from apparently healthy individuals (n = 124) from 64 households and livestock such as cattle (n = 75), sheep (n = 95), goats (n = 60) and environmental samples (lake water, n = 24; mud, n = 16). Detection and genotyping targeted the Internal Transcribed Spacer (ITS) region by nested PCR and Sanger sequencing. No human or environmental samples were positive, this result may be influenced by sampling limitations, seasonality, and methodological sensitivity rather than reflecting true absence. Overall E. bieneusi prevalence in livestock was 5.2

Blastocystis is a common anaerobic protist of uncertain pathogenicity that inhabits the human intestine. Rare cases reported in the literature suggest possible extraintestinal dissemination by unclear mechanisms. We followed a 43-year-old Peruvian woman presenting with low-grade fever and gastrointestinal symptoms who was diagnosed with a liver abscess by ultrasound and CT imaging. Identification of the causative agents was achieved through a combination of mass spectrometry, molecular methods (PCR and Sanger sequencing) and culture-based methods in blood, tissue and faecal samples. We detected gut-associated aerobic and anaerobic bacteria, including Escherichia coli, Eikenella corrodens and Bacteroides fragilis. Active forms of Blastocystis subtype ST1 were identified in the liver abscess fluid and successfully cultured in vitro. These findings suggest translocation of gut microbiota into the portal circulation as the underlying route of infection. Blastocystis can act as an opportunistic invasive microorganism in locations other than the intestinal tract.

Gastrointestinal protist (GIP) infections, mainly caused by Giardia duodenalis and Cryptosporidium spp., are major contributors to childhood morbidity in resource-limited settings, yet their epidemiology in Madagascar remains poorly documented. We conducted a microscopy-based prospective cross-sectional study in 3 deprived areas of southern Madagascar to investigate pathogenic and commensal protists among children and adolescents (n = 318), young women (n = 57) and their children (n = 61). Epidemiological questionnaires were collected, and selected species were characterized using PCR and Sanger sequencing. Risk factors were analysed using binary logistic regression. A total of 436 stool samples and 221 questionnaires were analysed. G. duodenalis was the most prevalent pathogenic species (81.9%), followed by Cryptosporidium spp. (1.2%). Balantioides coli was not detected. Potentially pathogenic species included Blastocystis sp. (70.4%) and Dientamoeba fragilis (0.2%). The most frequent commensals were Entamoeba coli (37.4%), Endolimax nana (15.4%) and Iodamoeba b & uuml;tschlii (12.6%). Assemblages A, B and A + B were identified for G. duodenalis, and C. hominis, C. felis and C. parvum for Cryptosporidium spp.; Blastocystis subtypes ST1-ST3 were detected. G. duodenalis infection was associated with abdominal pain and loss of appetite, but not diarrhoea. Geographical origin predicted G. duodenalis infection and Blastocystis sp. and E. coli colonization. GIP infections by G. duodenalis and, to a lesser extent, by Cryptosporidium spp., are a public health concern in resource-limited settings in Madagascar. Our results are the most important contribution made in the country in terms of molecular epidemiology of intestinal protist.

Purpose Enterocytozoon bieneusi is an obligate intracellular eukaryotic parasite of the phylum Microsporidia that infects a wide range of hosts, including humans. In Algeria, epidemiological information on microsporidian infections caused by E. bieneusi is scarce, with only three molecular studies conducted in equines, cattle, and dromedary camels. Here, we investigated the prevalence and genotypic diversity of E. bieneusi in cattle (n = 288), including cows (n = 182) and their calves (n = 106), from 54 farms in central Algeria. Materials and methods The initial screening of cattle faecal samples was conducted using a nested PCR targeting a partial sequence of the internal transcribed spacer (ITS) region. Results Enterocytozoon bieneusi was identified in 16.0

Mediterranean mountain ecosystems are characterized by strong seasonal climatic variation and environmental constraints that may substantially influence parasite transmission dynamics in free-ranging herbivores. However, little information is available regarding gastrointestinal parasite circulation in unmanaged equids inhabiting alpine grazing systems. This study investigated the seasonal dynamics of gastrointestinal helminths and the occurrence of Strongylus vulgaris in a feral horse population inhabiting the Sierra Nevada Natural Space (southern Spain), the southernmost alpine ecosystem in Europe. Faecal samples were collected across four seasons and analysed using quantitative coprological and molecular methods. Strongylid eggs were detected in almost all examined horses throughout the study period, indicating sustained parasite circulation under free-ranging conditions and in the absence of anthelmintic intervention. Although generalized linear mixed models did not detect statistically significant seasonal differences in egg shedding intensity, the highest egg counts were recorded during summer, coinciding with the period of maximum use of alpine grazing areas. Molecular analyses revealed a consistently high prevalence of S. vulgaris across seasons, whereas Parascaris spp . and Anoplocephala spp . were only sporadically detected. The predominance and persistence of strongylids likely reflect the ecological advantages conferred by direct transmission cycles in Mediterranean mountain grazing systems. These findings provide novel insights into parasite ecology in unmanaged equids inhabiting high-altitude ecosystems and highlight the value of feral horse populations as natural models for investigating host–parasite interactions under changing mountain environmental conditions.

Gastrointestinal helminth infections are widespread in free-ranging horses, but information on their seasonal occurrence in Mediterranean mountain ecosystems remains limited. We investigated gastrointestinal helminth occurrence in a feral horse population inhabiting the Sierra Nevada Natural Space, southern Spain, using faecal egg counts and molecular detection of Strongylus vulgaris. A total of 166 faecal samples were collected from 61 individually identified horses during four seasonal campaigns between spring 2024 and winter 2025. Strongylid infections were highly prevalent throughout the study, with sample-level prevalence ranging from 95.2 to 100%, while S. vulgaris was detected by PCR in 86.4–100% of faecal samples across seasons. In contrast, S. vulgaris PCR status was not significantly associated with faecal egg counts after accounting for season and repeated sampling. These findings provide a seasonal baseline for gastrointestinal helminth infections in an unmanaged feral horse population and highlight marked seasonal variation in strongylid egg shedding in a Mediterranean mountain ecosystem.

Dientamoeba fragilis is a cosmopolitan intestinal protist with an insufficiently understood life cycle and pathogenicity. Diagnosis by light microscopy is challenging, leading to significant underdiagnosis. Two genotypes (1 and 2) have been described, with genotype 1 being the most frequently detected worldwide. As no molecularly characterized isolates from Spain have been reported, we performed a molecular characterization and evaluated sociodemographic factors of D. fragilis in patients coinfected with Blastocystis sp. and/or Giardia duodenalis. Using a multiplex qPCR assay (Allplex™ GI-Parasite Assay, Seegene), we analyzed 354 stool samples previously identified as positive for Blastocystis sp. (n = 276), G. duodenalis (n = 63), or both (n = 15) by light microscopy. Eligible positive samples were selected for conventional PCR targeting the SSU rRNA gene and subsequent Sanger sequencing. D. fragilis was detected in 34.5

Sarcocystis spp. (Apicomplexa: Sarcocystidae) are globally distributed protozoan parasites with an obligatory two-host prey-predator life cycle involving intermediate (IHs) and definitive hosts (DHs). Canids, including the red fox (Vulpes vulpes), often serve as DHs for species infecting various ungulates. This study identified Sarcocystis species in red foxes from Croatia, Lithuania, and Portugal. Between 2021 and 2024, 164 faecal samples (80 from Croatia, 50 from Portugal, and 34 from Lithuania) were analysed using nested PCR targeting the cox1 gene. Twelve Sarcocystis species were detected: S. arieticanis, S. capracanis, S. capreolicanis, S. cruzi, S. gracilis, S. hjorti, S. iberica, S. linearis, S. miescheriana, S. morae, S. rossii, and S. tenella. The overall prevalence was highest in Croatia (78%) and Lithuania (62%) and lowest in Portugal (30%). Phylogenetic and haplotype analyses revealed high homogeneity and absence of geographic structuring. These results confirm the red fox as a key DH for multiple Sarcocystis species infecting European ungulates and underscore its epidemiological importance in parasite transmission across diverse ecosystems.

Gastroenteritis is a major cause of morbidity in young children worldwide. A prospective multicentre study was conducted from September 2020 to September 2021 in three tertiary hospitals in Spain to assess the burden of the gastrointestinal pathogens causing paediatric gastroenteritis in different Spanish regions, to understand the impact of COVID-19 pandemic in their incidence, and to assess potential risk factors for infection. Stool samples from children aged 0–59 months with acute or chronic diarrhoea were analysed by PCR to detect and characterise gastrointestinal bacteria, parasites, and viruses. Whole-genome sequencing was carried out on enteroaggregative Escherichia coli isolates. Epidemiological and clinical data were analysed using univariable and multivariable logistic regression models. A total of 1114 children were included (median age, 16 months; 57.3

The wildlife-livestock-environment interface is a complex system with implications for biodiversity and diseases. Environmental nucleic acid detection (ENAD) is a non-invasive method for monitoring DNA/RNA from microorganisms, meiofauna, and hosts. However, how environmental variables influence ENAD in heterogeneous geographic contexts remains poorly explored. In this study, 18 sites were evaluated in Iberian Peninsula, collecting 10 surface sponge samples per site and a total of 146 environmental fecal samples to investigate how environmental, mammal community and wildlife health variables may influence ENAD, using different pathogen-related markers as the study model. Differences in ENAD were assessed among sponge sampling methods and between sponge and fecal samples. The results show that environmental characteristics influence ENAD, at least for pathogen-related markers and at larger geographic scales. Greater pathogen marker diversity and richness was observed at higher latitudes. Most markers found in feces were also detectable in surface sponges. Combining different sponge sampling methodologies provided the best overall coverage of detectable environmental DNA. A predictive map linking ENAD in sponges to environmental factors was developed.

Blastocystis is a globally prevalent gut protist colonising over a billion people worldwide, yet its epidemiology, transmission dynamics, and clinical significance remain underexplored. This protocol represents the first step of a large-scale effort to map Blastocystis epidemiology and diagnostic practices across Europe through the COST Action CA21105: Blastocystis under One Health. By assessing diagnostic methodologies across clinical, veterinary, and environmental sectors, this work sets the foundation for future research and standardisation. Here, we highlight key findings, challenges, and a roadmap for improving Blastocystis detection, ultimately influencing global health policies and microbial ecology studies.

Cryptosporidiosis is a leading cause of morbidity and mortality due to diarrhoea during early childhood in low-income countries, including Ethiopia. Human Cryptosporidium infections can be anthroponotically or zoonotically transmitted. This study assesses the diversity and frequency of Cryptosporidium species and genotypes circulating in children and young livestock in eastern Ethiopia, with the aim of tracing the sources of infection. Cryptosporidium isolates from young children (n = 152), calves (n = 8), lambs (n = 4), goat kids (n = 4), and camels (n = 11) were subjected to PCR targeting the SSU rRNA and gp60 loci. Species and subtypes were determined by Sanger sequencing. Two Cryptosporidium species were found in children: C. hominis (75.3%, 67/89) and C. parvum (24.7%, 22/89). Three gp60 subtype families, Ia (30.0%, 9/30), Ib (10.0%, 3/30), and Id (60.0%, 18/30), were identified within C. hominis whereas gp60 subtype families IIe (55.6%, 10/18) and IIc (44.4%, 8/18) were identified within C. parvum. The predominant subtypes were C. hominis IdA15 and C. parvum IIeA10G1. Among livestock, C . ubiquitum was found in two camels and a goat kid, C. xiaoi in a lamb and goat kid, and C. ryanae in a calf. We did not identify any C. parvum among the animal samples. The goat isolate of C. xiaoi was characterised as subtype XXIIIg. Human cryptosporidiosis cases in eastern Ethiopia are primarily caused by anthroponotically transmitted Cryptosporidium species and subtypes, whereas zoonotic transmission events seem infrequent.

Blastocystis, a common protist in the human gastrointestinal tract, exhibits substantial genetic diversity and has been linked to varying clinical outcomes. However, its role in human health remains debated, with studies suggesting both commensal and pathogenic interactions. This scoping review aims to systematically map the existing evidence on the association between Blastocystis presence and human clinical outcomes. Herein, we present our proposed protocol, where, using systematic search methods, studies will be identified from multiple databases, focusing on diagnostic procedures, clinical outcomes, and treatment options. Findings will provide a comprehensive evidence map, highlighting knowledge gaps and guiding future research. The resulting data is intended to inform clinical and public health perspectives on Blastocystis and its potential implications for human health.

Background/Objectives: Many Tropical diseases such as Malaria, Chagas, Human African Trypanosomiasis, and Lymphatic filariasis coexist in endemic countries, affecting more than 1 billion people worldwide, and are recognized as major global vector-borne diseases. Tackle this disease lies in a correct diagnosis, sensitive, specific, and fast. This study aims to describe and validate a new highly sensitive and specific multiple-analysis system that can effectively detect numerous etiological agents in a single test. Methods: A total of 230 human blood samples were evaluated retrospectively for parasite characterization and a total of 58 stool samples from Non-Human Primates (NHP). Results: The analytical specificity of the presented method was 100%, with no unspecific amplifications or cross-reactions with other blood parasitic diseases. The detection limit obtained was between 0.6 to 3.01 parasites/µl for Plasmodium species, 1,8 parasite/ml for Trypanosomatidae, and 2 microfilariae/ml in the case of Filariae. The sensitivity, specificity, predictive values, and kappa coefficient reached almost 100%, except for the sensitivity for Filariae, which dropped to 93,9%, and its NPV to 89.5%. The operational features described a turnaround and a hands-on time notably shorter than the compared methods with a lower cost per essay. Conclusions: This work presents a cost-effective and highly sensitive multiplexed tool (RT-PCR-bp) capable of performing a simultaneous detection for blood parasitic diseases using specific fluorescence probes, enabling the diagnosis of low parasite loads and co-infections.

OBJECTIVES:Traditionally, the detection of intestinal parasites, including Cryptosporidium, has relied on microscopic examination of bulk stool samples. However, obtaining bulk stool samples requires waiting time, collection can be messy, and children may not readily produce a sample, particularly if severely dehydrated. This study aimed to evaluate the diagnostic performance of rectal swab samples in comparison with bulk stool samples for the point-of-care diagnosis of cryptosporidiosis using light-emitting diode auramine-phenol (LED-AP) fluorescence microscopy. METHODS:A cross-sectional study was conducted among diarrhoeic children aged 1-59 months attending six health centres in Dire Dawa and Shinile, Eastern Ethiopia. Both rectal swabs and bulk stool samples were collected, stained with auramine-phenol and examined for Cryptosporidium spp. oocysts using LED-AP fluorescence microscopy, blinded to the results obtained from the corresponding sample. The diagnostic performance of rectal swabs was evaluated against bulk stool results by estimating sensitivity, specificity, positive and negative predictive values, and the kappa coefficient. McNemar's test was used to assess whether discordant results between rectal swabs and bulk stools were statistically significant. RESULTS:Stool and rectal swab samples were obtained from 485 children with median age of 16 months (interquartile range: 12-28). Most children (71.5%, 347/485) were below 2 years of age. Cryptosporidium was detected in 19.4% (94/485) of stool and 17.1% (83/485) of rectal swab samples by LED-AP fluorescence microscopy, with 96.9% (95% CI 95.0-98.3%) overall concordance. Compared with bulk stools, rectal swabs showed 86.2% (95% CI 83.1-89.2%) sensitivity, 99.5% (95% CI 98.5-100.0%) specificity, and excellent agreement (κ = 0.90, 95% CI 0.81-0.99). Oocyst numbers were consistently higher in bulk stool samples than in rectal swabs. CONCLUSIONS:Rectal swabs demonstrated high diagnostic accuracy and excellent agreement with bulk stools, supporting their use as a reliable and practical alternative when rapid results are needed or stool collection is challenging.

Background/Objectives: Many tropical diseases such as malaria, Chagas, human African Trypanosomiasis, and Lymphatic filariasis coexist in endemic countries, affecting more than 1 billion people worldwide, and are recognised as major global vector-borne diseases. Tackling this disease requires an accurate diagnosis that is sensitive, specific, and rapid. This study aimed to describe and validate a new highly sensitive and specific multiple-analysis system that can effectively detect numerous etiological agents in a single test. Methods: A total of 230 human blood samples were assessed retrospectively for parasite characterisation, as well as 58 stool samples from non-human primates. Primers and probes were designed in the small subunit ribosomal RNA gene, except for Plasmodium spp., for which the novel target was Cytochrome Oxidase Subunit 1. Results: The analytical specificity of the presented method was 100%, with no unspecific amplifications or cross-reactions with other blood parasitic diseases. The detection limit obtained was between 0.6 and 3.01 parasites/µL for Plasmodium species, 1.8 parasites/mL for Trypanosomatidae, and 2 microfilariae/mL in the case of Filariae. The sensitivity, specificity, predictive values, and kappa coefficient reached almost 100%, except for Filariae, whose sensitivity dropped to 93.9% and whose negative predicted value dropped to 89.5%. The operational features described a turnaround and a hands-on time shorter than the compared methods with a lower cost per essay. Conclusions: This work presents a cost-effective and highly sensitive multiplexed tool (RT-PCR-bp) capable of performing simultaneous detection for blood parasitic diseases using specific fluorescence probes, enabling the diagnosis of low parasite loads and coinfections.

Although superficially persuasive, claims suggesting a causal link between Blastocystis and colorectal cancer (CRC) lack robust scientific support. As Blastocystis is the most common gut protist found in human populations globally, its detection in CRC patients is unsurprising and does not imply pathogenicity. Current claims championing a causal role for Blastocystis in CRC are based on speculative correlations, a single poorly controlled animal study, and inconsistent subtype associations. We argue that linking Blastocystis to CRC is premature, misleading, and may give rise to unnecessary concern in patients that are colonised by or test positive for Blastocystis. We emphasise the need for rigorously designed investigations to establish causal roles for any microorganism in disease and the importance of conclusions being based on solid evidence, particularly in matters of public health.