Wastewater-based intestinal protozoa monitoring in Shanghai, China.

Intestinal protozoa Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi have been implicated in serious waterborne outbreaks worldwide. Wastewater-based epidemiology (WBE) is a promising approach for evaluating the disease prevalence in a catchment population in that it monitors the contamination level of the intestinal pathogens in wastewater. We collected 48 urban wastewater samples (24 from influents and 24 from effluents) from the Yangpu Wastewater Treatment Plant (YPWTP) in Shanghai, China. We identified Cryptosporidium spp., G. duodenalis, and E. bieneusi by nested polymerase chain reaction (PCR) amplification. Cryptosporidium hominis and subtype IdA14 were identified in two samples by analyzing the sequences of small subunit ribosomal RNA (SSU rRNA) and 60-kDa glycoprotein (gp60) genes, respectively. The G. duodenalis sub-assemblage AII (n = 8) and assemblage C (n = 4) in 12 samples were determined by analyzing triosephosphate isomerase (tpi) gene sequences. The E. bieneusi genotype A was identified in one sample by analyzing the sequence of the internal transcribed spacer (ITS) region of the rRNA gene. These findings suggest that improving wastewater treatment and monitoring the virility of pathogens in effluents is critical. We observed similar prevalence and genotypes/subtypes of the three intestinal protozoa in our wastewater samples as those reported in previous studies, providing evidence that WBE can be used as an effective epidemic management tool.IMPORTANCECryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common intestinal protozoa causing diarrhea. The infective oocysts, cysts, and spores released in feces can survive in different environments, including multiple types of water bodies. Humans can acquire these intestinal protozoan infections via the fecal-oral route as in waterborne transmission. Wastewater-based epidemiology can rapidly and reliably detect and monitor the emergence and spread of waterborne diseases. We detected Cryptosporidium spp., G. duodenalis, and E. bieneusi in a wastewater treatment plant in Shanghai, China, reflecting the occurrence and genetic characterizations of the three intestinal pathogens from community members served by the wastewater treatment plant.

Molecular detection and identification of Enteromonas species in human and animal hosts using polymerase chain reaction and DNA sequencing.

Enteromonas hominis, a human intestinal protozoan parasite of the diplomonad group, has been overlooked because of its commensal features; therefore, molecular studies on this parasite are limited. To address this gap, we designed a molecular screening protocol using polymerase chain reaction (PCR) and DNA sequencing targeting the 18S small subunit ribosomal RNA gene and applied this screening method to the molecular epidemiological analysis of Enteromonas spp. in humans and various livestock. We validated our methodology using stool samples collected from 215 humans and 270 animal hosts (buffaloes, pigs, dogs, goats, horses, rodents, chickens, and ducks) during an annual epidemiological investigation conducted from 2013 to 2016 on Sumba Island, Indonesia. The overall prevalences of Enteromonas spp. were 33.9 % (n = 73/215) in humans and 25.2 % (n = 68/270) in mammals and avians. The positive predictive value of this PCR method for Enteromonas spp., as evaluated through sequencing, was 90.1 % in human samples and 58.1 % in non-human samples (particularly low, 11.4 % in rodents). Although the specificity of the PCR approach may not be perfect, in combination with DNA sequencing, it was effective in detecting and identifying a partial sequence (1458 bp) of the target gene region in Enteromonas species.

Large-Scale Molecular Epidemiological Survey of Blastocystis sp. among Herbivores in Egypt and Assessment of Potential Zoonotic Risk.

Given the proven zoonotic potential of the intestinal protozoan Blastocystis sp., a fast-growing number of surveys are being conducted to identify potential animal reservoirs for transmission of the parasite. Nevertheless, few epidemiological studies have been conducted on farmed animals in Egypt. Therefore, a total of 1089 fecal samples were collected from herbivores (sheep, goats, camels, horses, and rabbits) in six Egyptian governorates (Dakahlia, Gharbia, Kafr El Sheikh, Giza, Aswan, and Sharqia). Samples were screened for the presence of Blastocystis sp. by real-time PCR followed by sequencing of positive PCR products and phylogenetic analysis for subtyping of the isolates. Overall, Blastocystis sp. was identified in 37.6% of the samples, with significant differences in frequency between animal groups (sheep, 65.5%; camels, 62.2%; goats, 36.0%; rabbits, 10.1%; horses, 3.3%). Mixed infections were reported in 35.7% of the Blastocystis sp.-positive samples. A wide range of subtypes (STs) with varying frequency were identified from single infections in ruminants including sheep (ST1-ST3, ST5, ST10, ST14, ST21, ST24, ST26, and ST40), goats (ST1, ST3, ST5, ST10, ST26, ST40, ST43, and ST44), and camels (ST3, ST10, ST21, ST24-ST26, ST30, and ST44). Most of them overlapped across these animal groups, highlighting their adaptation to ruminant hosts. In other herbivores, only three and two STs were evidenced in rabbits (ST1-ST3) and horses (ST3 and ST44), respectively. The greater occurrence and wider genetic diversity of parasite isolates among ruminants, in contrast to other herbivores, strongly suggested that dietary habits likely played a significant role in influencing both the colonization rates of Blastocystis sp. and ST preference. Of all the isolates subtyped herein, 66.3% were reported as potentially zoonotic, emphasizing the significant role these animal groups may play in transmitting the parasite to humans. These findings also expand our knowledge on the prevalence, genetic diversity, host specificity, and zoonotic potential of Blastocystis sp. in herbivores.

Zoonotic assemblages A and B of Giardia duodenalis in Chiroptera from Brazilian Amazon biome.

Bats are important reservoirs and spreaders of pathogens. Giardia duodenalis is a globally important protozoan that infects humans and other mammals with considerable public health burden, particularly on the child development. Based on genetic variation and host specificity, G. duodenalis is categorized into eight genotypes/assemblages A-H. Assemblages A and B are widespread globally and are associated with human and animal disease. There is evidence of Giardia in the bat feces from diverse geographic regions, but the G. duodenalis assemblages are unknown, which is a key point for the One Health view. Here, we successfully amplified the BG/GDH/DIS3/HCMP2/HCMP3 targets of G. duodenalis from five bat species captured in the Brazilian Amazon biome revealing the presence of zoonotic G. duodenalis assemblages A and B in the feces of these flying mammals. Our study reveals that bats may play a role in transmission of zoonotic G. duodenalis, at least in this biome.

The epidemiology of intestinal protozoa in the Israeli population based on molecular stool test: a nationwide study.

Stool examination using microscopy was the traditional method for the diagnosis of intestinal parasites. Recently, the use of molecular tests to identify stool protozoa has become the main tool used in most clinical laboratories in Israel. This study aimed to evaluate the prevalence of intestinal parasites in Israel and to compare this prevalence in laboratories that use molecular tests vs a laboratory that uses microscopy. Samples collected from January to October 2021 at seven laboratories were analyzed by real-time PCR (RT-PCR) or by microscopy. The multiplex panel included the following pathogens: Giardia lamblia, Entamoeba histolytica, Cryptosporidium spp., Cyclospora, Dientamoeba fragilis, and Blastocystis spp. Overall, 138,415 stool samples were tested by RT-PCR and 6,444 by microscopy. At least one protozoa species was identified in 28.4% of the PCR-tested samples compared to 4.6% of the microscopy-tested samples. D. fragilis was the most common PCR-identified species (29%). D. fragilis, G. lamblia, and Cryptosporidium spp. were mainly found in pediatric population, while Blastocystis spp. was most prevalent among adults (P < 0.001). In a sub-cohort of 21,480 samples, co-infection was found in 4,113 (19.15%) samples, with Blastocystis spp. and D. fragilis being the most common (14.9%) pair. Molecular stool testing proved more sensitive compared to microscopy. D. fragilis was the most commonly detected pathogen. The above profile was identified during the COVID pandemic when traveling was highly restricted and most likely represents the locally circulating protozoa. IMPORTANCE: This study sheds light on the prevalence of stool parasites in Israel. Additionally, this study indicates that the shift from microscope analysis to molecular tests improved protozoa diagnosis.

Blastocystis spp. and Other Intestinal Parasites in Polish Soldiers Deployed to Lebanon and Iraq.

Intestinal parasitic infections are one of the most common infectious diseases worldwide, particularly in developing countries. A distinct group at increased risk of infection is military personnel deployed overseas for extended periods, typically six months at a time. The aim of this study was to determine the prevalence of Blastocystis spp. and other intestinal parasites in Polish military personnel returning from deployments to Lebanon (n = 206) and Iraq (n = 220). In this group of subjects, we found Blastocystis spp. (13.6%), Dientamoeba fragilis (3.3%), Entamoeba coli (0.9%), and Endolimax nana (0.5%). Entamoeba histolytica sensu lato and Chilomastix mesnili infections were detected only in one soldier returning from Lebanon and Iraq, respectively. Blastocystis subtype (ST) 3 was predominant in soldiers returning from Lebanon, followed by ST2 and ST1. ST1 infection was predominant in soldiers returning from Iraq, followed by ST3 and ST2. Our study affirms that, deployment abroad is of no influence of the prevalence of parasitic protozoa. However, it would be worth to monitor parasite infection in military personnel returning from tropical zone even if they have no actual symptoms. In addition, it is very important to determine the subtypes of Blastocystis-this may help to clearly define their pathogenicity, especially considering the scarcity of studies on Blastocystis genotypes in Iraqi and Lebanese residents.

Beyond schistosomiasis: unraveling co-infections and altered immunity.

Schistosomiasis is a neglected tropical disease caused by the helminth Schistosoma spp. and has the second highest global impact of all parasites. Schistosoma are transmitted through contact with contaminated fresh water predominantly in Africa, Asia, the Middle East, and South America. Due to the widespread prevalence of Schistosoma, co-infection with other infectious agents is common but often poorly described. Herein, we review recent literature describing the impact of Schistosoma co-infection between species and Schistosoma co-infection with blood-borne protozoa, soil-transmitted helminths, various intestinal protozoa, Mycobacterium, Salmonella, various urinary tract infection-causing agents, and viral pathogens. In each case, disease severity and, of particular interest, the immune landscape, are altered as a consequence of co-infection. Understanding the impact of schistosomiasis co-infections will be important when considering treatment strategies and vaccine development moving forward.

Modified trichrome stain for faster and improved detection of intestinal protozoan parasites.

Permanent stains such as trichrome have better sensitivity but are time-consuming and the fixative includes toxic mercuric chloride. Thus, a newer modification was tested and found to be a superior, faster and safer staining technique for intestinal parasitic detection. Our study lasted 9 months and a single stool sample was collected from each enrolled patient. We evaluated classical trichrome (T1 - using Schaudinn fixative) with newer modifications, which involved different fixatives with mordant combinations (T2 - acetic acid + hydrated aluminium sulphate, T3 - citric acid + copper sulphate hydrate). Conventional PCR targeting Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. was taken as the reference. Out of 175 stool samples, 25.1% protozoa were identified by wet mount, 24% by each T1 and T2, 25.7% by T3. Statistically, T3 and T2 had higher sensitivity as compared to T1 and wet mount when PCR was used as reference.

Evaluation of a rapid immunochromatographic test for the diagnosis of intestinal protozoan infections among patients attending a rural outreach outpatient department in Northern India.

Despite great efforts, intestinal protozoan infections remain a significant healthcare concern worldwide. Although many point-of-care (POC) tests are increasingly being used, microscopic examination of stool specimens remains the mainstay for their diagnosis, especially in resource-limited settings. We assessed the utility of rapid POC tests based on immunochromatography among patients from rural Northern India. A total of 78 patients were enrolled in the study. Out of nine specimens that tested positive for Giardia duodenalis on microscopy, an immunochromatographic test (ICT) could detect only five (55.55%). Entamoeba histolytica/dispar was demonstrated in two specimens on microscopy, both of which were missed by ICT. Its overall sensitivity, specificity, and positive and negative predictive value were 50%, 98.5%, 83.3%, and 93%, respectively. Its performance was considered unsatisfactory. Although ICT-based tests provide a relatively rapid and less labor-intensive alternative, they should be used to supplement and not replace stool microscopy.

Corrigendum: Exploring genetic variability of Giardia duodenalis and Enterocytozoon bieneusi in raw vegetables and fruits: implications for food safety and public health in Mozambique.

[This corrects the article DOI: 10.3389/fmicb.2023.1223151.].

Dynamics of protozoal excretion in the faeces of calves during the first 28 days after arrival at the fattening farm indicate infection before regrouping and show poor temporal correlation with diarrhoea.

BACKGROUND: Calves in dairy cattle production in Switzerland are transported to a fattening farm at the age of 3-5 weeks, and frequently suffer from diarrhoea within the first 14 days after arrival. To characterise the role of intestinal protozoa in this, we investigated the excretion dynamics of Eimeria, Cryptosporidium and Giardia during the first 28 days after the arrival and regrouping of calves at fattening farms. METHODS: A total of 610 faecal samples from 122 calves (mean age 37.3 days; mean body weight 79.8 kg) were collected on seven different fattening farms during the first 28 days after the arrival and regrouping of the animals. The farms were visited between January and April (cold season; n = 4) and between June and August (warm season; n = 3). The samples were collected rectally on days 1, 4, 7, 14 and 28, assessed for consistency, and analysed using the McMaster method for quantitative determination of the number of Eimeria oocysts per gram of faeces (OPG), flotation for morphological differentiation of the unsporulated Eimeria oocysts, a concentration method for the semi-quantitative determination of Giardia cysts, and modified Ziehl-Neelsen staining for semi-quantitative determination of Cryptosporidium oocysts. RESULTS: Overall, 50.8% (62/122) of the animals had diarrhoea during the study period. However, the faecal excretion of protozoal pathogens was neither associated with diarrhoea nor with body weight gain of the animals. Altogether, 90.2% (110/122) of the calves were Eimeria positive. Eimeria zuernii was excreted by 51 (41.8%) and Eimeria bovis by 68 (55.7%) animals. In the warm season more animals tested positive for Eimeria and OPGs were higher than in the cold season. There was no correlation between the age of the calves and the OPG values. Overall, 64.8% (79/122) of the calves excreted Eimeria oocysts within the first 7 days, indicating that they had been infected with the parasite on the dairy farm of origin. Eighty-nine calves (73.0%) excreted Giardia cysts, with more positive animals in the cold (80.3%) compared with the warm season (64.3%). Only Giardia duodenalis assemblage E was identified. Cryptosporidium oocysts were microscopically detected in 14 animals (11.5%) on five farms. Cryptosporidium spp. were present in a total of 12 animals, i.e. Cryptosporidium parvum in nine, Cryptosporidium ryanae in two, and Cryptosporidium bovis in one animal. CONCLUSIONS: A better understanding of the temporal dynamics of protozoal infections in calves is helpful for the implementation of appropriate measures to protect the health of these animals at a critical phase in their lives. Our results indicate that factors other than those examined in the present study contributed to the onset of diarrhoea in the calves.

Extracellular Cysteine Proteases of Key Intestinal Protozoan Pathogens-Factors Linked to Virulence and Pathogenicity.

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.

Exploring genetic variability of Giardia duodenalis and Enterocytozoon bieneusi in raw vegetables and fruits: implications for food safety and public health in Mozambique.

Giardia duodenalis and Enterocytozoon bieneusi are etiological agents of enteric diseases characterized by diarrhea that can progress to chronicity in humans, especially in children and in immunocompromised patients. This study aims to assess the genetic pattern of G. duodenalis and E. bieneusi detected in vegetables and fruits commercialized in Maputo markets, Mozambique and determine their public health importance. Eight study points were sampled: a farmer zone, a wholesale, four retail markets, and two supermarkets in Maputo city, where eight types of horticultural products were purchased. Using nested-PCR methods, 2.8% (9/321) and 1.3% (4/321) of samples monitored were positive for G. duodenalis and E. bieneusi, respectively. Based on the analysis of the ß-giardin and ITS rRNA sequences of G. duodenalis and E. bieneusi detected, respectively, four different sequences of G. duodenalis (three novel sequences: BgMZ1, BgMZ2, and BgMZ3, and one known sequence) all from assemblage B and three genotypes of E. bieneusi (two novel sequences: EbMZ4 and EbMZ5, and one known sequence: KIN-1) from group 1. These microorganisms were found and characterized for the first time in horticultural products in Maputo markets. All identified G. duodenalis and E. bieneusi display high genetic similarity within their ß-giardin and ITS rRNA sequences, respectively, having been clustered into assemblages and genotypes with high zoonotic transmission potential. Our study may represent a relevant step in the understanding of these intestinal pathogens in association with fresh vegetables and fruits for human consumption, for a better and broader "One Health" approach.

Distribution of Blastocystis subtypes isolated from various animal hosts in Thailand.

Blastocystis is a parasitic protist of a variety of hosts, including humans. Mapping the distribution of Blastocystis and its genetic variants across different host species can help us understand the epidemiology of this organism and its role in health and disease. This study aimed to identify subtypes of Blastocystis detected in different animal hosts in Thailand. A total of 825 fecal samples belonging to 18 vertebrate orders, 36 families, 68 genera, and 80 species were collected. Of these, 111 specimens were Blastocystis-positive by culture. Seventy-nine samples were subjected to small subunit (SSU) ribosomal DNA amplification by PCR, and reliable subtype data were obtained for 61 specimens. At least 14 subtypes (ST), namely ST1 to ST10, ST14/ST24/ST25 complex, ST23, ST26, and ST29 were detected. In addition, Blastocystis was found in tortoises. ST1 (3.2%) and ST5 (11.5%) were found in pigs, ST2 (1.6%) and ST3 (3.2%) in non-human primates, ST4 (14.7%) in rodents and ruminants, ST6 (4.9%), ST7 (30%), ST9 (1.6%), and ST29 (1.6%) in birds, ST8 (6.6%) in Green peafowl and East Asian Porcupine, and ST10 (4.9%), ST14/ST24/ST25 (9.8%), ST23 (1.6%) and ST26 (1.6%) in ruminants. The sequence recovered from the elongated tortoises (Indotestudo elongata) (3.2%) was phylogenetically placed within the reptilian cluster of Blastocystis, for which no subtype system is available yet. Of note, we did not obtain Blastocystis sequences from any of the many canids and felids sampled in the study, and our data are in support of host specificity of Blastocystis, according to both colonization and subtype distribution.

Metagenomic analysis reveals the relationship between intestinal protozoan parasites and the intestinal microecological balance in calves.

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.

Clues on the dynamics of DNA replication in Giardia lamblia.

Genomic replication is a critical, regulated process that ensures accurate genetic information duplication. In eukaryotic cells, strategies have evolved to prevent conflicts between replication and transcription. Giardia lamblia, a binucleated protozoan, alternates between tetraploid and octaploid genomes during its cell cycle. Using single-molecule techniques like DNA combing and nanopore-based sequencing, we investigated the spatio-temporal organization of DNA replication, replication fork progression and potential head-on replication-transcription collisions in Giardia trophozoites. Our findings indicate that Giardia chromosomes are replicated from only a few active origins, which are widely spaced and exhibit faster replication rates compared to those in other protozoan parasites. Immunofluorescence assays revealed that ∼20% of trophozoites show asynchronous replication between nuclei. Forksense and gene ontology analyses disclosed that genes in regions with potential head-on collisions are linked to chromatin dynamics, cell cycle regulation and DNA replication/repair pathways, possibly explaining the observed asynchronous replication in part of the population. This study offers the first comprehensive view of replication dynamics in Giardia, which is the pathogen that causes giardiasis, a diarrheal disease impacting millions worldwide.

First Epidemiological Survey on the Prevalence and Subtypes Distribution of the Enteric Parasite Blastocystis sp. in Vietnam.

Although Blastocystis sp. is the most common enteric protozoan in human stools worldwide, various geographical areas remain to be investigated regarding the frequency and circulation of this parasite. Such is the case of some developing countries in Southeast Asia that exhibit a higher risk for parasitic infections due to unsanitary conditions. While several epidemiological surveys have been conducted, for instance, in Thailand, little or no data are available from neighboring countries, such as Vietnam. Therefore, in order to determine the prevalence and subtype (ST) distribution of Blastocystis sp. and to clarify the transmission of the parasite, the first molecular epidemiological survey ever conducted in this country was performed. For this purpose, a total of 310 stool specimens were collected from patients enrolled at the Family Hospital of Da Nang and then tested for the presence of Blastocystis sp. by real-time Polymerase Chain Reaction (qPCR), followed by subtyping of the isolates. The overall prevalence of the parasite reached 34.5% in this Vietnamese cohort. No significant association was found between parasite infection and gender, age, symptomatic status, contact with animals or source of drinking water. Out of the 107 positive patients, nearly half presented mixed infections. Therefore, some of the corresponding samples were reanalyzed by end-point PCR, followed by PCR products cloning and sequencing. Of the 88 total subtyped isolates, ST3 was predominant, followed by ST10, ST14, ST7, ST1, ST4, ST6 and ST8. Our study was, thus, the first to report ST8, ST10 and ST14 in the Southeast Asian population. The predominance of ST3 within this Vietnamese cohort, coupled with its low intra-ST genetic variability, reflected a large inter-human transmission, while ST1 transmission was suggested to be not only anthroponotic, but also likely correlated to animal or environmental sources. Strikingly, isolates considered of animal origin (ST6-ST8, ST10 and ST14) accounted for more than 50% of the subtyped isolates. These findings improved our knowledge of the epidemiology and circulation of Blastocystis sp. in Southeast Asia, and in particular, in Vietnam, and highlighted both a major burden of the parasite in this country and a high risk of zoonotic transmission, mainly from poultry and livestock.

Development of a new multiplex PCR to detect fecal coccidian parasite.

BACKGROUND: Cryptosporidium spp., Cystoisospora belli and Cyclospora cayetanensis are common intestinal coccidian parasites causing gastroenteritis. The clinical presentation caused by each parasite is indistinguishable from each other. Uniplex polymerase chain reaction (PCR) for these three groups of intestinal coccidian parasites was developed by us in our laboratory. Thereafter, we planned to develop a single-run multiplex polymerase chain reaction (mPCR) assay to detect Cryptosporidium spp., C. belli and C. cayetanensis simultaneously from a stool sample and described it here as coccidian mPCR. METHODS: New primers for C. belli and C. cayetanensis were designed and uniplex PCRs were standardized. The coccidian mPCR was standardized with known positive DNA control isolates. It was validated with 58 known positive and 58 known negative stool samples, which were previously identified by uniplex PCR. RESULTS: The coccidian mPCR was standardized with earlier primers designed by us for Cryptosporidium spp. and C. cayetanensis, and a newly designed primer for the internal transcribed spacer-1 (ITS-1) gene for C. belli. The coccidian mPCR was 92.1% sensitive for Cryptosporidium spp., and 100% sensitive for C. belli and C. cayetanensis each, when tested on 116 known samples. It was 100% specific for all intestinal coccidian parasites. Two representative PCR products of the newly designed ITS-1 primer for C. belli were sequenced and submitted to the GenBank, which best match with the sequences of C. belli. CONCLUSION: A highly sensitive, specific, cost-effective, indigenous, single-run coccidian mPCR has been developed, which can simultaneously detect Cryptosporidium spp., C. belli and C. cayetanensis.

Clues on the dynamics of DNA replication in Giardia lamblia Icon for the Forest of Biologists

Genomic replication is a critical, regulated process that ensures accurate genetic information duplication. In eukaryotic cells, strategies have evolved to prevent conflicts between replication and transcription. Giardia lamblia, a binucleated protozoan, alternates between tetraploid and octoploid genomes during its cell cycle. Using single-molecule techniques like DNA combing and nanopore-based sequencing, we investigated the spatio-temporal organization of DNA replication, replication fork progression, and potential head-on replication-transcription collisions in Giardia trophozoites. Our findings indicate that Giardia chromosomes are replicated from few active origins, which are widely spaced and exhibit faster replication rates compared to other protozoan parasites. Immunofluorescence assays revealed that around 20% of trophozoites show asynchronous replication between nuclei. Forksense and gene ontology analyses disclosed that genes in regions with potential head-on collisions are linked to chromatin dynamics, cell cycle regulation, and DNA replication/repair pathways, possibly explaining the observed asynchronous replication in part of the population. This study offers the first comprehensive view of replication dynamics in Giardia, the cause of giardiasis, a diarrheal disease impacting millions worldwide.

Commensal Intestinal Protozoa-Underestimated Members of the Gut Microbial Community.

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.