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A 44-year-old human immunodeficiency virus-infected (HIV+) female with severe immunodeficiency Category 3 (C3) diagnosed in 2010 was admitted to hospital with acute diarrhoea. She was non-adherent to antiretroviral therapy (ART) and had a previous suspicion of respiratory symptoms with a cough that had been persisting for 15 days. Clinical examination revealed severe immune deterioration (viral load: 109,655 copies/mL; CD4+ count: 14 cells/mm3), respiratory symptoms (negative sputum Gram stain and tuberculosis culture), and neurological deterioration (serological assays negative for Cryptococcus spp. and Toxoplasma gondii). A coproculture was negative for Campylobacter spp., Salmonella spp., and Shigella spp. Ziehl–Neelsen staining of faecal smears revealed the presence of Cryptosporidium spp. oocysts. PCR testing and sequencing confirmed a concomitant infection with C. meleagridis and Enterocytozoon bieneusi. The patient was treated with metronidazole (500 mg every 8 h for 5 days) and nitazoxanide (500 mg every 12 h for 14 days). After requesting voluntary discharge and abandoning ART and parasiticidal treatments, she experienced a dramatic deterioration of her state of health and contact with her was lost. Our results have demonstrated that molecular-based testing improves the detection of opportunistic pathogens that are difficult to detect by routine microscopy, allows for transmission dynamics investigations, and assists in choosing the best chemotherapeutical option.

Cryptosporidium spp., Giardia duodenalis and Entamoeba histolytica are species of protozoa- causing diarrhoea that are common worldwide, while Entamoeba dispar, Dientamoeba fragilis and Blastocystis sp. appear to be commensal parasites whose role in pathogenicity remains controversial. We conducted the clinical evaluation of five singleplex and one duplex CerTest VIASURE Real-Time PCR Assays against a large panel of positive DNA samples (n = 358), and specifically to Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25) E. dispar (n = 11), Blastocystis sp. (n = 42), D. fragilis (n = 37), and related parasitic phylum species such as Apicomplexa, Euglenozoa, Microsporidia and Nematoda. DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference centre. Estimated diagnostic sensitivity and specificity values were 0.94–1 for Cryptosporidium spp., 0.96–0.99 for G. duodenalis, 0.96–1 for E. histolytica, 1–1 for E. dispar, and 1–0.99 for D. fragilis in the evaluated singleplex assays. In the duplex assay for the simultaneous detection of Blastocystis sp. and D. fragilis these values were 1–0.98 and 1–0.99, respectively. Measures of diagnostic precision for repeatability and reproducibility were found to be under acceptable ranges. The assays identified six Cryptosporidium species (C. hominis, C. parvum, C. canis, C. felis, C. scrofarum, and C. ryanae), four G. duodenalis assemblages (A, B, C, and F), and six Blastocystis subtypes (ST1-ST5, and ST8). The evaluated singleplex and duplex VIASURE Real-Time PCR assays provide sensitive, practical, and cost-effective choices to the molecular diagnosis of the main diarrhoea-causing intestinal protists in clinical microbiology and research laboratories.

Cryptosporidium spp. and Giardia duodenalis are the main non-viral causes of diarrhoea in humans and domestic animals globally. Comparatively, much less information is currently available in free-ranging carnivore species in general and in the endangered Iberian lynx (Lynx pardinus) in particular. Cryptosporidium spp. and G. duodenalis were investigated with molecular (PCR and Sanger sequencing) methods in individual faecal DNA samples of free-ranging and captive Iberian lynxes from the main population nuclei in Spain. Overall, Cryptosporidium spp. and G. duodenalis were detected in 2.4% (6/251) and 27.9% (70/251) of the animals examined, respectively. Positive animals to at least one of them were detected in each of the analysed population nuclei. The analysis of partial ssu rRNA gene sequences revealed the presence of rodent-adapted C. alticolis (n = 1) and C. occultus (n = 1), leporid-adapted C. cuniculus (n = 2), and zoonotic C. parvum (n = 2) within Cryptosporidium, and zoonotic assemblages A (n = 5) and B (n = 3) within G. duodenalis. Subgenotyping analyses allowed for the identification of genotype VaA19 in C. cuniculus (gp60 locus) and sub-assemblages AI and BIII/BIV in G. duodenalis (gdh, bg, and tpi loci). This study represents the first molecular description of Cryptosporidium spp. and G. duodenalis in the Iberian lynx in Spain. The presence of rodent/leporid-adapted Cryptosporidium species in the surveyed animals suggests spurious infections associated to the Iberian lynx’s diet. The Iberian lynx seems a suitable host for zoonotic genetic variants of Cryptosporidium (C. parvum) and G. duodenalis (assemblages A and B), although the potential risk of human transmission is regarded as limited due to light parasite burdens and suspected low excretion of infective (oo)cysts to the environment by infected animals. More research should be conducted to ascertain the true impact of these protozoan parasites in the health status of the endangered Iberian lynx.

The wildcat (Felis silvestris) and the Iberian lynx (Lynx pardinus) are important species in Spain, considered as near-threatened and endangered, respectively. Both can be infected by Toxoplasma gondii, a parasite that can cause morbidity and mortality in transplacentally-infected or immunocompromised mammals. The data on the prevalence of this parasite in wild populations of these species in Spain are outdated. The objective of this study was to update information and evaluate the role of these felines in parasite epidemiology and the potential impact of the parasite on their conservation. Blood and fecal samples were collected from captured animals, as well as the tongue, diaphragm, and spleen, from animals killed in road accidents in central Spain. An indirect fluorescent antibody test (IFAT) was used to detect parasite antibodies in serum, microscopy and molecular analysis were used to detect oocysts in feces, and molecular analysis was used to determine the existence of tissue cysts. Seroprevalence was 85% in wildcats and 45% in lynx, and parasite DNA was detected in the feces of one wildcat and in tissue samples from 10 wildcats and 11 Iberian lynxes. These results highlight the epidemiological importance and high risk of T. gondii infection in animals and humans in the studied areas. Considering feline susceptibility to infection, monitoring programs are needed to assess the health status of wild felines.

Giardia duodenalis is a significant contributor to the burden of diarrheal disease in sub-Saharan Africa. This study assesses the occurrence and molecular diversity of G. duodenalis and other intestinal parasites in apparently healthy children (n = 311) in Ibadan, Nigeria. Microscopy was used as a screening method and PCR and Sanger sequencing as confirmatory and genotyping methods, respectively. Haplotype analyses were performed to examine associations between genetic variants and epidemiological variables. At microscopy examination, G. duodenalis was the most prevalent parasite found (29.3%, 91/311; 95% CI: 24.3–34.7), followed by Entamoeba spp. (18.7%, 58/311; 14.5–23.4), Ascaris lumbricoides (1.3%, 4/311; 0.4–3.3), and Taenia sp. (0.3%, 1/311; 0.01–1.8). qPCR confirmed the presence of G. duodenalis in 76.9% (70/91) of the microscopy-positive samples. Of them, 65.9% (60/91) were successfully genotyped. Assemblage B (68.3%, 41/60) was more prevalent than assemblage A (28.3%, 17/60). Mixed A + B infections were identified in two samples (3.3%, 2/60). These facts, together with the absence of animal-adapted assemblages, suggest that human transmission of giardiasis was primarily anthroponotic. Efforts to control G. duodenalis (and other fecal-orally transmitted pathogens) should focus on providing safe drinking water and improving sanitation and personal hygiene practices.

Three recent studies of Blastocystis epidemiology in mammalian hosts identified four novel sequences that appeared to share B. lapemi as the most similar sequence. However, full-length ssu rRNA gene sequences were not available to confirm the validity of these new subtypes. In the present study, Nanopore MinION sequencing was used to obtain full-length reference sequences for each of the new subtypes. Additionally, phylogenetic analyses and pairwise distance comparisons were performed to confirm the validity of each of these new subtypes. We propose that the novel sequences described in this study should be assigned the subtype designations ST35-ST38. The full-length reference sequences of ST35-ST38 will assist in accurate sequence descriptions in future studies of Blastocystis epidemiology and subtype diversity.

The enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis are—to various extents—contributors to the burden of gastrointestinal illness in high-income countries. Detection of these pathogens by microscopy examination is challenging because of the limited sensitivity and need for specific staining procedures. We developed and optimised a new multiplex real-time PCR assay for the simultaneous detection of Cryptosporidium spp., G. duodenalis and D. fragilis in clinical (stool) samples. The diagnostic performance of the assay was evaluated against a large panel of well-characterised DNA samples positive for Cryptosporidium spp. (n = 126), G. duodenalis (n = 132) and D. fragilis (n = 49). The specificity of the test was assessed against a DNA panel from other intestinal or phylogenetically related parasites (n = 105) and faecal DNA from individuals without clinical manifestations (n = 12). The assay exhibited a diagnostic sensitivity of 0.90–0.97 and a diagnostic specificity of 1. The limit of detection was estimated for Cryptosporidium (1 oocyst) and G. duodenalis (5 × 10−4 cysts). The method allowed the detection of four Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. cuniculus) and five G. duodenalis assemblages (A–E) without cross-reacting with other parasites belonging to the phyla Amoebozoa, Apicomplexa, Euglenozoa, Microsporidia, Nematoda and Platyhelminthes. This newly developed multiplex real-time PCR assay represents a novel alternative for the rapid and accurate detection of Cryptosporidium, G. duodenalis and D. fragilis in clinical settings.

Wild chimpanzee populations in West Africa (Pan troglodytes verus) have dramatically decreased as a direct consequence of anthropogenic activities and infectious diseases. Little information is currently available on the epidemiology, pathogenic significance, and zoonotic potential of protist species in wild chimpanzees. This study investigates the occurrence and genetic diversity of intestinal and blood protists as well as filariae in faecal samples (n = 234) from wild chimpanzees in the Dindefelo Community Nature Reserve, Senegal. PCR-based results revealed the presence of intestinal potential pathogens (Sarcocystis spp.: 11.5%; Giardia duodenalis: 2.1%; Cryptosporidium hominis: 0.9%), protist of uncertain pathogenicity (Blastocystis sp.: 5.6%), and commensal species (Entamoeba dispar: 18.4%; Troglodytella abrassarti: 5.6%). Entamoeba histolytica, Enterocytozoon bieneusi, and Balantioides coli were undetected. Blood protists including Plasmodium malariae (0.4%), Trypanosoma brucei (1.3%), and Mansonella perstans (9.8%) were also identified. Sanger sequencing analyses revealed host-adapted genetic variants within Blastocystis, but other parasitic pathogens (C. hominis, P. malariae, T. brucei, M. perstans) have zoonotic potential, suggesting that cross-species transmission between wild chimpanzees and humans is possible in areas where both species overlap. Additionally, we explored potential interactions between intestinal/blood protist species and seasonality and climate variables. Chimpanzees seem to play a more complex role on the epidemiology of pathogenic and commensal protist and nematode species than initially anticipated.

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi were observed in 32.1% (108/336, 95% CI: 27.2–37.4%) and 0.6% (2/336, 95% CI: 0.0–1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2–8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.

Preservation and conservation of biological specimens, including faecal samples, is a challenge in remote areas or poor-resource settings where the cold chain cannot be maintained. This study aims at evaluating the suitability of filter cards for long-term storage of faecal samples of animal and human origin positive to the diarrhoea-causing protozoan parasites, Giardia duodenalis and Cryptosporidium hominis. Three commercially available Whatman® Filter Cards were comparatively assessed: the FTA® Classic Card, the FTA® Elute Micro Card, and the 903 Protein Saver Card. Human faecal samples positive to G. duodenalis (n = 5) and C. hominis (n = 5) were used to impregnate the selected cards at given storage (1 month, 3 months, and 6 months) periods and temperature (−20 °C, 4 °C, and room temperature) conditions. Parasite DNA was detected by PCR-based methods. Sensitivity assays and quality control procedures to assess suitability for genotyping purposes were conducted. Overall, all three Whatman® cards were proven useful for the detection and molecular characterisation of G. duodenalis and C. hominis under the evaluated conditions. Whatman® cards represent a simple, safe, and cost-effective option for the transportation, preservation, and storage of faecal samples without the need of the cold chain.

Background: The aim of the present study is to describe the occurrence of Blastocystis sp. detection among asymptomatic subjects and patients with irritable bowel syndrome in order to evaluate the potential association between irritable bowel syndrome and the parasitic infection. Methods: Cross-sectional study where adult patients with irritable bowel syndrome diagnosed according to Rome IV criteria were included. A control group was formed by asymptomatic subjects older than 18 years. Exclusion criteria were: immunosuppressive condition or having received any drug with demonstrated activity against Blastocystis sp. within the last 6 months before study inclusion. Epidemiological and clinical information was collected from all included participants. Two stool samples were obtained from all participants: one sample for microscopic examination and one sample for Blastocystis sp. PCR detection. Blastocystis sp. infection was defined by the positivity of any of the diagnostic techniques. Results: Seventy-two participants were included (36 asymptomatic subjects and 36 patients with irritable bowel syndrome). Thirty-five (48.6%) were men, and median age of participants was 34 (IQR 29–49) years. The overall rate of Blastocystis sp. carriage was 27.8% (20/72). The prevalence assessed through microscopic examination was 22.2% (16/72), while the prevalence measured by PCR was 15.3% (11/72). When comparing the presence of Blastocystis sp. between asymptomatic subjects and IBS patients, we did not find any statistically significant difference (36.1% vs. 19.4% respectively, p = 0.114). Conclusions: regarding the occurrence of Blastocystis sp., no differences were found between asymptomatic participants and patients with irritable bowel disease irrespective of the diagnostic technique performed.

Little information is currently available on the epidemiology of parasitic and commensal protist species in captive non-human primates (NHP) and their zoonotic potential. This study investigates the occurrence, molecular diversity, and potential transmission dynamics of parasitic and commensal protist species in a zoological garden in southern Spain. The prevalence and genotypes of the main enteric protist species were investigated in faecal samples from NHP (n = 51), zookeepers (n = 19) and free-living rats (n = 64) by molecular (PCR and sequencing) methods between 2018 and 2019. The presence of Leishmania spp. was also investigated in tissues from sympatric rats using PCR. Blastocystis sp. (45.1%), Entamoeba dispar (27.5%), Giardia duodenalis (21.6%), Balantioides coli (3.9%), and Enterocytozoon bieneusi (2.0%) (but not Troglodytella spp.) were detected in NHP. Giardia duodenalis (10.5%) and Blastocystis sp. (10.5%) were identified in zookeepers, while Cryptosporidium spp. (45.3%), G. duodenalis (14.1%), and Blastocystis sp. (6.25%) (but not Leishmania spp.) were detected in rats. Blastocystis ST1, ST3, and ST8 and G. duodenalis sub-assemblage AII were identified in NHP, and Blastocystis ST1 in zookeepers. Giardia duodenalis isolates failed to be genotyped in human samples. In rats, four Cryptosporidium (C. muris, C. ratti, and rat genotypes IV and V), one G. duodenalis (assemblage G), and three Blastocystis (ST4) genetic variants were detected. Our results indicate high exposure of NHP to zoonotic protist species. Zoonotic transmission of Blastocysts ST1 was highly suspected between captive NHP and zookeepers.

Little information is available on the occurrence and genetic variability of the diarrhoea-causing enteric protozoan parasite Giardia duodenalis in indigenous communities in Brazil. This cross-sectional epidemiological survey describes the frequency, genotypes, and risk associations for this pathogen in Tapirapé people (Brazilian Amazon) at four sampling campaigns during 2008–2009. Microscopy was used as a screening test, and molecular (PCR and Sanger sequencing) assays targeting the small subunit ribosomal RNA, the glutamate dehydrogenase, the beta-giardin, and the triosephosphate isomerase genes as confirmatory/genotyping methods. Associations between G. duodenalis and sociodemographic and clinical variables were investigated using Chi-squared test and univariable/multivariable logistic regression models. Overall, 574 individuals belonging to six tribes participated in the study, with G. duodenalis prevalence rates varying from 13.5–21.7%. The infection was positively linked to younger age and tribe. Infected children G. duodenalis infections and showed a high genetic diversity. No association between assemblage and age or occurrence of diarrhoea was demonstrated. These data indicate that the most likely source of infection was anthropic and that different pathways (e.g., drinking water) may be involved in the transmission of the parasite.

Infections by the protist enteroparasites Giardia duodenalis, Cryptosporidium spp., and, to a much lesser extent, Blastocystis sp. are common causes of childhood diarrhoea in low-income countries. This molecular epidemiological study assesses the frequency and molecular diversity of these pathogens in faecal samples from asymptomatic schoolchildren (n = 807) and symptomatic children seeking medical attention (n = 286) in Zambézia province, Mozambique. Detection and molecular characterisation of pathogens was conducted by polymerase chain reaction (PCR)-based methods coupled with Sanger sequencing. Giardia duodenalis was the most prevalent enteric parasite found [41.7%, 95% confidence interval (CI): 38.8–44.7%], followed by Blastocystis sp. (14.1%, 95% CI: 12.1–16.3%), and Cryptosporidium spp. (1.6%, 95% CI: 0.9–2.5%). Sequence analyses revealed the presence of assemblages A (7.0%, 3/43) and B (88.4%, 38/43) within G. duodenalis-positive children. Four Cryptosporidium species were detected, including C. hominis (30.8%; 4/13), C. parvum (30.8%, 4/13), C. felis (30.8%, 4/13), and C. viatorum (7.6%, 1/13). Four Blastocystis subtypes were also identified including ST1 (22.7%; 35/154), ST2 (22.7%; 35/154), ST3 (45.5%; 70/154), and ST4 (9.1%; 14/154). Most of the genotyped samples were from asymptomatic children. This is the first report of C. viatorum and Blastocystis ST4 in Mozambique. Molecular data indicate that anthropic and zoonotic transmission (the latter at an unknown rate) are important spread pathways of diarrhoea-causing pathogens in Mozambique.

Enteric parasites including Giardia duodenalis, Cryptosporidium spp., and to a lesser extent, Blastocystis sp. and Enterocytozoon bieneusi, are major worldwide contributors to diarrhoeal disease. Assessing their molecular frequency and diversity is important to ascertain the sources of infection, transmission dynamics, and zoonotic potential. Little molecular information is available on the genotypes of these pathogens circulating in apparently healthy children. Here, we show that asymptomatic carriage of G. duodenalis (17.4%, 95% CI: 15.5‒19.4%), Blastocystis sp. (13.0%, 95% CI: 11.4‒14.8%), and Cryptosporidium spp. (0.9%, 95% CI: 0.5‒1.5%) is common in children (1‒16 years; n = 1512) from Madrid, Spain. Our genotyping data indicate that; (i) the observed frequency and diversity of parasite genetic variants are very similar to those previously identified in Spanish clinical samples, so that the genotype alone does not predict the clinical outcome of the infection, (ii) anthroponotic transmission accounts for a large proportion of the detected cases, highlighting that good personal hygiene practices are important to minimizing the risk of infection, (iii) Blastocystis ST4 may represent a subtype of the parasite with higher pathogenic potential, and (iv) Enterocytozoon bieneusi does not represent a public health concern in healthy children.