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.

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.

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.

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.

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.

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.

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.

Is there any change in the prevalence of intestinal or cardiopulmonary parasite infections in companion animals (dogs and cats) in Germany between 2004-2006 and 2015-2017? An assessment of the impact of the first ESCCAP guidelines.

Main objective of the present nationwide study was to assess the impact of the ESCCAP guideline for the control of worm infections in dogs and cats 8-10 years after its first publication in Germany. A secondary aim was to determine the prevalence of canine and feline cardiopulmonary nematodes and intestinal protozoa. Faecal samples of 53,693 dogs and 26,491 cats in 2004-2006 as well as of 129,578 dogs and 45,709 cats in 2015-2017 routinely submitted by veterinarians to a private veterinary laboratory were examined using appropriate parasitological methods. In dogs, the prevalence of Toxocara and taeniid egg shedding was significantly lower in 2015-2017 (3.8 % and 0.16 %, respectively) than in 2004-2006 (4.6 % and 0.27 %, respectively). The prevalence of hookworm and Capillaria eggs was higher in the second study period (2.3 % and 0.77 %, respectively) than in the first (1.3 % and 0.6 %, respectively). For Toxascaris leonina (0.55-0.6 %) and Trichuris (0.8-0.9 %), the difference was not significant between the study periods. Dogs shed more often Angiostrongylus vasorum larvae in the second study (3.1 %) than in the first (1.0 %), whereas the prevalence of Crenosoma vulpis did not change significantly (2.2-2.6 %). Cystoisospora canis and C. ohioensis-like infections were less detected in the second study period (1.0 % and 2.1 %, respectively) than in the first (1.8 % and 2.7 %, respectively). Neospora-like oocysts and Sarcocystis sporocysts were more prevalent in the second study period (0.19 % and 0.13 %, respectively) than in the first (0.13 % and 0.06 %, respectively). The percentage of Giardia or Cryptosporidium coproantigen-positive samples was lower in the second study period (18.9 % and 6.7 %, respectively) than in the first (22.8 % and 10.0 %, respectively). In cats, the prevalence of egg shedding of T. cati, Capillaria and taeniids was significantly lower in 2015-2017 (3.5 %, 0.25 % and 0.1 %, respectively) than in 2004-2006 (4.8 %, 0.54 % and 0.22 %, respectively). No difference was recorded for hookworms (0.12-0.13 %) and Ts. leonina (0.04-0.05 %). Aelurostrongylus-like larvae were detected more often in the second study period (6.5 %) than in the first (2.6 %). Infections with Cystoisospora felis, C. rivolta, Toxoplasma-like coccids and Sarcocystis were less prevalent in the second study period (1.9 %, 0.7 %, 0.24 % and 0.02 %, respectively) than in the first (2.7 %, 1.1 %, 0.36 % and 0.1 %, respectively). The percentage of Giardia or Cryptosporidium coproantigen-positive samples was significantly lower in the second study period (10.6 % and 4.8 %, respectively) than in the first (15.4 % and 8.3 %, respectively). Although these results indicate a decline of the occurrence of most canine and feline intestinal parasites in Germany over the years, a transmission risk of zoonotic parasites remains. Therefore, the control of helminth infections in domestic dogs and cats continues to be a challenge for veterinarians and pet owners.

Prevalence, Subtype Distribution and Zoonotic Significance of Blastocystis sp. Isolates from Poultry, Cattle and Pets in Northern Egypt.

Blastocystis sp. is a widespread enteric protozoan that frequently infects human and animal groups. Despite its burden and zoonotic potential worldwide, epidemiological investigations remain limited in animal groups that come in contact with humans. Therefore, the largest survey ever conducted in North Africa was performed in Egypt with the aim to investigate the prevalence and subtype (ST) distribution of Blastocystis sp. in animals. For this purpose, a total of 889 fecal specimens were collected from chickens (217), cattle (373), dogs (144) and cats (155) from six governorates of northern Egypt. These specimens were then screened for the presence of Blastocystis sp. using a quantitative real-time PCR, followed by subtyping the isolates. The overall prevalence of Blastocystis sp. reached 9.2% (82/889), with the highest infection rates reported in chickens (17.0%) and domestic cattle (11.0%), highlighting an active circulation of the parasite in both animal groups. In contrast, the low prevalence in cats (2.6%) and the absence of the parasite in dogs suggested that pets are not natural hosts of Blastocystis sp. ST10 and ST14 were largely predominant in cattle, confirming that both STs represented cattle-adapted STs. The report of one ST3 and one ST4 isolate in this animal group could be explained by an accidental zoonosis from humans to animals. All but one of the subtyped isolates in poultry belonged to ST7, which was considered as an avian ST. The presence of a remaining isolate of ST14 likely reflected a transient infection from contact between birds and cattle feces. The same environmental contamination was also likely the source of the ST14 infection in three of the four positive cats, with the remaining animals infected by ST3 as the result of human-to-animal transmission. These occurrences and subtyping data, combined with those previously collected in the Egyptian population, implies that poultry could play a significant role as reservoir for zoonotic transmission, which would not be the case for cattle and pets.

Comparison of Different Copromicroscopic Techniques in the Diagnosis of Intestinal and Respiratory Parasites of Naturally Infected Dogs and Cats.

Several copromicroscopic techniques, including tools belonging to the FLOTAC group, are available for the qualitative and/or quantitative diagnosis of canine and feline parasitoses. The present study was carried out to compare the diagnostic performance of different copromicroscopic methods for detecting common intestinal and extra-intestinal parasites of dogs and cats. Fecal samples of 100 dogs and 105 cats were randomly selected from different regions of Italy. All samples were subjected to conventional flotation, McMaster, Mini-FLOTAC, and Baermann. Fifty-six dogs and twenty-five cats were found positive to at least one technique, and, among them, flotation (55% and 20.9% of the dogs and cats, respectively) and Mini-FLOTAC (52% and 20.9% of the dogs and cats, respectively) detected the highest number of positive samples. Larvae of the feline metastrongyloids Aelurostrongylus abstrusus and Troglostrongylus brevior were identified only using the Baermann test in two (1.9%) and one (0.9%) cat respectively. No larvae were found with the Baermann examination of dog feces or any of the other methods. The present results show that the Mini-FLOTAC represents a possible alternative to conventional flotation in clinical settings for the detection of intestinal and respiratory parasites e.g., Toxocara spp., Toxascaris leonina, Ancylostomatidae, Cystoisospora spp., Trichuris vulpis and Capillaria spp., although Baermann's test remains the most recommended technique for the diagnosis of infections caused by metastrongyloid lungworms.

Prevalence and Associated Factors of Blastocystis sp. Infection in Patients with Gastrointestinal Symptoms in Spain: A Case-Control Study.

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

Standardization of molecular techniques for the detection and characterization of intestinal protozoa and other pathogens in humans.

Background: The intrinsic sensitivity limitations of basic parasitological methods, along with the particular biological characteristics of parasites, make these methods ineffective to differentiate morphologically indistinguishable species. Molecular detection and characterization techniques could be used to overcome these problems. The purpose of this work was to standardize molecular polymerase chain reaction (PCR) techniques, described in the literature, for the detection and molecular characterization of intestinal protozoa and other pathogens in humans. Methods: DNA was extracted from human or animal feces, previously washed or cultured in Boeck Drbohlav's Modified Medium. DNA extraction was performed with Machery-Nagel extraction kits. The standardization of the PCR, nested-PCR or RFLP techniques was carried out according to the literature. For each molecular technique performed, the sensitivity of the test was determined based on the minimun quantity required of DNA (sensitivity A) and the minimum quantity of life forms that the test detected (sensitivity B). Results: Sensitivity A was 10 fg for G. duodenalis, 12.5 pg for Entamoeba histolytica or Entamoeba dispar, 50 fg for Cryptosporidium spp., 225 pg for Cyclospora spp. and 800 fg or 8 fg for Blastocystis spp. after performing a 1780 bp PCR or 310 bp nested PCR, respectively. The sensitivity B was 100 cysts for G. duodenalis, 500 cysts for E. histolytica or E. dispar, 1000 oocysts for Cyclospora spp. and 3600 or four vegetatives forms for PCR or nested PCR of Blastocystis spp., respectively. Conclusions: The molecular detection of protozoa and chromist was achieved and the molecular characterization allowed the genotyping of some of the parasites such as Giardia duodenalis, Cryptosporidium spp., and Blastocystis spp. This study summarizes the molecular techniques for epidemiological studies in humans and animals, and helps in the investigation of their transmission sources in countries where intestinal parasites are a public health problem.