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Protozoa have long been considered undesirable residents of the human gut, but recent findings suggest that some of them may positively affect the gut ecosystem. To better understand the role and ecological dynamics of these commensal and potentially beneficial protozoan symbionts, we need efficient methods to detect them, as well as accurate estimates of their prevalence across human populations. Metagenomics provides such an opportunity, allowing simultaneous detection of multiple symbionts in a single analytical procedure. In this study, we collected fecal samples of 68 individuals from three Cameroonian populations with different subsistence modes and compared metagenomics-based and targeted methods of detection for two common protozoan genera: Blastocystis and Entamoeba. In addition, we analyzed our data along with publicly available fecal metagenomes from various worldwide populations to explore the prevalence and association patterns of ten protozoan genera. Regarding the detection method, microscopy was much less sensitive than metagenomics for Entamoeba, whereas qPCR was at least as sensitive as metagenomics for Blastocystis sp. However, metagenomics was more likely to detect co-colonizations by multiple subtypes. Out of the ten examined genera in 127 individuals from Cameroon, Tanzania, Peru, Italy or USA, only three (Blastocystis, Entamoeba and Enteromonas) had an overall prevalence exceeding 10%. All three genera were more common in less industrialized populations and their prevalence differed between continents and subsistence modes, albeit not in a straightforward manner. The majority (72.5%) of colonized individuals carried at least two protozoan species, indicating that mixed-species colonizations are common. In addition, we detected only positive and no negative association patterns between different protozoa. Despite the pitfalls of the metagenomic approach, ranging from the availability of good-quality sequencing data to the lack of standard analytical procedures, we demonstrated its utility in simultaneous detection of multiple protozoan genera, and especially its ability to efficiently detect mixed-species colonizations. Our study corroborates and expands prevalence results previously obtained for Blastocystis sp. and provides novel data for Entamoeba spp. and several other protozoan genera. Furthermore, it indicates that multiple protozoa are common residents of the healthy human gut worldwide.

Purpose Epidemiological studies on amoebic infections are complicated due to morphologically identical and clinically important Entamoeba species. Therefore, newer, simpler, and more economical diagnostic techniques are required for differentiating clinically important Entamoeba species. Methods We developed a single-round multiplex PCR assay to identify E. histolytica , E. moshkovskii , E. dispar , E. bangladeshi , and E. coli . Primers were designed based on variations in 18 S rRNA sequences. Sensitivity and specificity were assessed using known positive and negative samples. Furthermore, we screened 472 diarrheal samples using this technique alongside the reference PCR method to evaluate its suitability for epidemiological studies and clinical diagnosis. DNA sequencing and phylogenetic analysis of the isolates were conducted. All statistical analyses of the data were performed using GraphPad Prism. Results The designed primers successfully yielded species-specific PCR products of different sizes as expected. We did not observe any non-specific amplifications of the primer set. The diagnostic performance was also convincing. After screening clinical samples using the method, we observed that 2.33% ( n  = 11) tested positive for E. moshkovskii , 1.06% ( n  = 5) tested positive for E. histolytica , and 0.85% ( n  = 4) tested positive for E. bangladeshi in the studied area. DNA sequencing further confirmed the identified species. The constructed phylogenetic tree also demonstrated clear separation of the detected species lineages. Conclusion The study suggests the multiplex PCR assay could be a reliable diagnostic tool for amoebic infections. This study is particularly significant as it marks the first reported occurrence of E. bangladeshi since its documentation in South Africa and its native Bangladesh.

We determined the prevalence of Entamoeba (E.) histolytica, E. dispar and E. moshkovskii in patients with chronic diarrhoea associated with abdominal pain or discomfort mimicking irritable bowel syndrome. Stool samples were collected from 161 patients with chronic diarrhoea and from 157 healthy controls. Stool microscopy with modified trichrome stain, culture and polymerase chain reaction (PCR) for Entamoeba spp. differentiation was performed. Microscopy demonstrated Entamoeba cysts in 44% (57/129) of patients with diarrhoea compared to 29% (44/151) of controls (P=0·009). In patients with diarrhoea, PCR for E. histolytica was positive in 9% (11/129) (P=0·008), E. dispar in 19% (24/129) (P=0·117) and E. moshkovskii in 19% (24/129) (P<0·001). E. histolytica and E. moshkovskii were significantly associated with diarrhoea while E. dispar was found equally in both groups.

Three species of Entamoeba were common in South Africa: E. dispar, E. histolytica, and the recently described E. bangladeshi. In E. histolytica–positive samples, changes in both parasite and P. copri levels were associated with alterations in gastrointestinal status. Abstract Background Diarrhea is frequent in communities without clean water, which include low-income South African populations in Giyani and Pretoria. In these populations, the amount of diarrhea caused by Entamoeba histolytica, inclusive of all ages, sexes, and human immunodeficiency virus status, is uncertain. Infection with E. histolytica can modulate the host microbiota, and a key species indicative of this is the Prevotella copri pathobiont. Methods A cross-sectional study of patients attending gastroenterology clinics was conducted to determine the frequency and burden of 4 Entamoeba species and P. copri. Results Entamoeba species were present in 27% of patients (129/484), with E. histolytica detected in 8.5% (41), E. dispar in 8% (38), E. bangladeshi in 4.75% (23), and E. moshkovskii in 0%. This is the first description of E. bangladeshi outside Bangladesh. In E. histolytica–positive samples, the levels of both the parasite and P. copri were lower in nondiarrheal samples, validating the results of a study in Bangladesh (P = .0034). By contrast, in E. histolytica–negative samples positive for either of the nonpathogenic species E. dispar or E. bangladeshi, neither P. copri nor Entamoeba levels were linked to gastrointestinal status. Conclusions Nonmorphologic identification of this parasite is essential. In South Africa, 3 morphologically identical Entamoeba were common, but only E. histolytica was linked to both disease and changes in the microbiota.

The families Naryaviridae (order Rivendellvirales), Nenyaviridae (order Rohanvirales), and Vilyaviridae (order Cirlivirales), all within the class Arfiviricetes of the phylum Cressdnaviricota, include single-stranded DNA viruses associated with protozoan parasites of the genera Entamoeba and Giardia as well as viruses found in various environmental samples, also likely infecting protozoans. Here, we provide a taxonomic update for these three families, which were recently expanded with multiple new members. In particular, we established seven new genera and nine new species in the family Naryaviridae, one new genus with one new species in the family Nenyaviridae, and three new genera and nine new species in the family Vilyaviridae. We also summarize the genomic properties and protein characteristics, including conserved motifs of the rolling-circle replication initiation proteins, of the viruses in the three families. Notably, the high GC content of vilyavirids (51-61%) and considerably lower GC content of naryavirids and nenyavirids (33-44%) appear to represent an adaptation to their hosts, Giardia and Entamoeba species, respectively.

Parasitic infections, especially intestinal protozoan parasites (IPPs) remain a significant public health issue in Africa, where many conditions favour the transmission and children are the primary victims. This systematic review and meta-analysis was carried out with the objective of assessing the prevalence of IPPs among school children in Africa. Relevant studies published between January 2000 and December 2020 were identified by systematic online search on PubMed, Web of Science, Embase and Scopus databases without language restriction. Pooled prevalence was estimated using a random-effects model. Heterogeneity of studies were assessed using Cochrane Q test and I2 test, while publication bias was evaluated using Egger's test. Of the 1,645 articles identified through our searches, 46 cross-sectional studies matched our inclusion criteria, reported data from 29,968 school children of Africa. The pooled prevalence of intestinal protozoan parasites amongst African school children was 25.8% (95% CI: 21.2%-30.3%) with E. histolytica/ dispar (13.3%; 95% CI: 10.9%-15.9%) and Giardia spp. (12%; 95% CI: 9.8%-14.3%) were the most predominant pathogenic parasites amongst the study participants. While E. coli was the most common non-pathogenic protozoa (17.1%; 95% CI: 10.9%-23.2%). This study revealed a relatively high prevalence of IPPs in school children, especially in northern and western Africa. Thus, poverty reduction, improvement of sanitation and hygiene and attention to preventive control measures will be the key to reducing protozoan parasite transmission.

PCR-based screenings on the presence of diarrhoea-causing intestinal protist species are limited in Zambia, resulting in inaccurate current prevalence and epidemiological data. Sensitive PCR-based methods are particularly well suited for detecting subclinical infections in apparently healthy carriers. In this prospective cross-sectional study, we investigated the occurrence of the most common intestinal protists in an apparently healthy paediatric population (5-18 years) in Lusaka Province, Zambia. We collected single stool samples (n = 256) and epidemiological questionnaires on demographics, behavioural habits, drinking water and toilet access from participating children. We used PCR for the initial screening of samples for the presence of intestinal protist species and Sanger and next-generation sequencing for genotyping. We conducted statistical analyses to assess the association of the gathered variables with an increased likelihood of the investigated pathogens. Blastocystis sp. was the most prevalent intestinal protist found (37.9%, 97/256; 95% CI: 31.9-44.1), followed by Giardia duodenalis (30.9%, 79/256; 95% CI: 25.3-36.90), Entamoeba dispar (13.3%, 34/256; 95% CI: 9.4-18.1), and Cryptosporidium spp. (4.3%, 11/256, 95% CI: 2.2-7.6). Entamoeba histolytica was not detected. Based on Sanger sequencing results, subtypes ST2 (44.3%, 43/97), ST1 (35.1%, 34/97), and ST3 (20.6%, 20/97) were identified within Blastocystis sp. and assemblages B (71.0%), A+B (16.1%), and A (12.9%) within G. duodenalis. Cryptosporidium parvum (81.8%) and C. hominis (18.2%) were the only two Cryptosporidium species found. Living in the Kafue District was positively associated with higher infection rates by G. duodenalis and Blastocystis sp. Schoolchildren living in Chongwe District were more likely to be infected by Cryptosporidium spp. Intestinal protist infection/colonization is a common finding in apparently healthy children in Lusaka Province, Zambia. Asymptomatic carriers may play an underestimated role as spreaders of gastrointestinal parasitic infections. This study improves our current understanding of the epidemiology of diarrhoea-causing protists in Zambia and sub-Saharan Africa and indicates that the role of asymptomatic carriers of gastrointestinal parasites in transmission should be further explored.

To address the molecular diversity and occurrence of pathogenic species of the genus Entamoeba spp. in wild non-human primates (NHP) we conducted molecular-phylogenetic analyses on Entamoeba from wild chimpanzees living in the Issa Valley, Tanzania. We compared the sensitivity of molecular [using a genus-specific polymerase chain reaction (PCR)] and coproscopic detection (merthiolate-iodine-formaldehyde concentration) of Entamoeba spp. We identified Entamoeba spp. in 72 chimpanzee fecal samples (79%) subjected to species-specific PCRs for six Entamoeba species/groups (Entamoeba histolytica, Entamoeba nuttalli, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli and Entamoeba polecki ST2). We recorded three Entamoeba species: E. coli (47%), E. dispar (16%), Entamoeba hartmanni (51%). Coproscopically, we could only distinguish the cysts of complex E. histolytica/dispar/moshkovskii/nuttalli and E. coli. Molecular prevalence of entamoebas was higher than the prevalence based on the coproscopic examination. Our molecular phylogenies showed that sequences of E. dispar and E. coli from Issa chimpanzees are closely related to sequences from humans and other NHP from GenBank. The results showed that wild chimpanzees harbour Entamoeba species similar to those occurring in humans; however, no pathogenic species were detected. Molecular-phylogenetic methods are critical to improve diagnostics of entamoebas in wild NHP and for determining an accurate prevalence of Entamoeba species.

Background The intestinal protozoa Entamoeba spp. can infect humans and various animals, including donkeys, causing diarrhea and malabsorption and presenting significant risks to animal husbandry and public health. Most Entamoeba species are not pathogenic except for Entamoeba histolytica . China has among the highest rates of donkey farming worldwide. Donkey ( Equus asinus ) farming is increasingly important in China because of their draft and medicinal value; however, epidemiological data on Entamoeba spp. in donkeys remains limited globally. This study aimed to investigate the prevalence of Entamoeba in donkeys in Shanxi Province, North China, and assess associated risk factors using a molecular approach. Methods Fecal samples of 815 donkeys from three representative geographical locations in Shanxi Province were collected to investigate the presence of Entamoeba spp. A portion of the small-subunit rRNA gene (SSU rRNA) was amplified and sequenced to determine the prevalence and species/genotypes of Entamoeba spp. Statistical analysis of possible risk factors was performed using Statistical Product and Service Solutions (SPSS) 26.0 software. The phylogenetic relationship of Entamoeba spp. was reconstructed using the neighbor-joining (NJ) method in Molecular Evolutionary Genetics Analysis (Mega) 7.0 software. Results The overall prevalence of Entamoeba spp. in donkeys in Shanxi Province was 7.12% (58/815). Two species ( Entamoeba sp. RL9 and Entamoeba equi ) were identified by sequence analysis; of these, Entamoeba sp. RL9 was the most prevalent species in donkeys in this study. Statistical analysis revealed that the donkeys' sex, region, age, and altitude are the risk factors associated with Entamoeba spp. prevalence ( P  < 0.05). Phylogenetic analysis indicated that the sequences of Entamoeba sp. RL9 and E. equi isolated from donkeys in this study were clustered with previously reported animal-derived Entamoeba sp. RL9 and E. equi sequences, respectively. Conclusions This study reports the occurrence and prevalence of Entamoeba spp. in donkeys worldwide for the first time to our knowledge. This not only expands the geographical distribution but also broadens the host range of Entamoeba spp., addressing the knowledge gap regarding the prevalence of Entamoeba spp. in donkeys, providing baseline data for carrying out prevention and control of Entamoeba spp. in donkeys in China. Graphical Abstract

This study aimed to carry out a molecular screening for the presence of Giardia , Cryptosporidium , and/or Entamoeba in the feces of pet and stray/feral cats in Jordan. G. duodenalis was found in 27.9% (95% CI, 23.2–32.9) of the 348 sampled cats overall; E. histolytica was found in only 0.6% (95% CI, 0.1–2.1) of the cats, while none of the sampled cats had Cryptosporidium infections. The infection rate of G. duodenalis among indoor cats (32.3%) did not differ significantly from that among outdoor cats (24.1%). There were significantly more infections ( p  = 0.0004) geographically in the cold semiarid areas (67%) than in the cold desert areas (24%). Multilocus sequence typing analysis of amplicons based on the bg , tpi , and gdh genes revealed that the majority of G. duodenalis infections were zoonotic assemblage B (65.9%; 64 of 97 positive samples); followed by feline-specific assemblage F (18.5%, 18/97); cattle-specific assemblage E (5.2%, 5/97); and then assemblage C that was shared with canids (1.0%; 1/97). Within Giardia isolates, a substitution mutation (A/G) was found at position 297 of the complete protein coding sequence (cds) of tpi -assemblage B, which may represent a new spreading mutation within this gene among the cat population in Jordan. The results of the present study suggest that close human-cat interactions could play a role in zoonotic transmission of Giardia , but further research is needed to determine the possible contribution of cats to the transmission of other protozoa to humans.