Detection and genotyping of zoonotic microsporidia in the endangered Iberian lynx (Lynx pardinus).

Microsporidia is a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most frequently reported species in humans. Limited information is available about the presence and molecular diversity of microsporidian species in the endangered Iberian lynx (Lynx pardinus). Presence of Enterocytozoon bieneusi and Encephalitozoon spp. was investigated by molecular methods in wild and captive Iberian lynxes from Spain. Overall, E. bieneusi was detected in 3.2% (8/251) of the animals examined. None of the samples tested were positive for Encephalitozoon spp. Four known (D, EbfelA, PigEBITS7, and Type IV) and a novel (named as LynxSpEb1) E. bieneusi genotypes were identified. All the genotypes found belonged to the zoonotic Group 1 of E. bieneusi. This study provides the first genotyping data of E. bieneusi in Iberian lynx in Spain. Our result indicate that the Iberian lynx does not seem to play a relevant role in the epidemiology of Encephalitozoon spp., and that this endangered felid is likely acting as spillover host rather than a true reservoir of E. bieneusi. Additional studies should be conducted to assess the impact of this parasite in the health status of the endangered Iberian lynx.

Genetic diversity of Cryptosporidium spp., Encephalitozoon spp. and Enterocytozoon bieneusi in feral and captive pigeons in Central Europe.

Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon spp. are the most common protistan parasites of vertebrates. The results show that pigeon populations in Central Europe are parasitised by different species of Cryptosporidium and genotypes of microsporidia of the genera Enterocytozoon and Encephalitozoon. A total of 634 and 306 faecal samples of captive and feral pigeons (Columba livia f. domestica) from 44 locations in the Czech Republic, Slovakia and Poland were analysed for the presence of parasites by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (18S rDNA), 60 kDa glycoprotein (gp60) and internal transcribed spacer (ITS) of SSU rDNA. Phylogenetic analyses revealed the presence of C. meleagridis, C. baileyi, C. parvum, C. andersoni, C. muris, C. galli and C. ornithophilus, E. hellem genotype 1A and 2B, E. cuniculi genotype I and II and E. bieneusi genotype Peru 6, CHN-F1, D, Peru 8, Type IV, ZY37, E, CHN4, SCF2 and WR4. Captive pigeons were significantly more frequently parasitised with screened parasite than feral pigeons. Cryptosporidium meleagridis IIIa and a new subtype IIIl have been described, the oocysts of which are not infectious to immunodeficient mice, whereas chickens are susceptible. This investigation demonstrates that pigeons can be hosts to numerous species, genotypes and subtypes of the studied parasites. Consequently, they represent a potential source of infection for both livestock and humans.

First identification and coinfection detection of Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp. and Giardia duodenalis in diarrheic pigs in Southwest China.

BACKGROUND: Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp., and Giardia duodenalis (G. intestinalis) are enteric pathogens that cause diarrhea in pigs. This study aimed to determine the prevalence of these enteric parasites and their coinfection with E. bieneusi in diarrheic pigs in Southwest China (Chongqing and Sichuan) using nested polymerase chain reaction (nPCR) based methods. RESULTS: A total of 514 fecal samples were collected from diarrheic pigs from 14 pig farms in Chongqing (five farms) and Sichuan (nine farms) Provinces. The prevalence of Encephalitozoon spp., Cryptosporidium spp. and G. duodenalis was 16.14% (83/514), 0% (0/514), and 8.95% (46/514), respectively. Nested PCR revealed 305 mono-infections of E. bieneusi, six of E. cuniculi, two of E. hellem, and nine of G. duodenalis and 106 concurrent infections of E. bieneusi with the other enteric pathogens. No infections of E. intestinalis and Cryptosporidium species were detected. The highest coinfection was detected between E. bieneusi and E. cuniculi (10.5%, 54/514), followed by E. bieneusi and G. duodenalis (5.8%, 30/514) and E. bieneusi and E. hellem (2.9%, 15/514). E. bieneusi was the most frequently detected enteric pathogen, followed by E. cuniculi, G. duodenalis and E. hellem. There was a significant age-related difference in the prevalence of E. cuniculi in fattening pigs (χ2 = 15.266, df = 3, P = 0.002) and G. duodenalis in suckling pigs (χ2 = 11.92, df = 3, P = 0.008) compared with the other age groups. Sequence analysis of the ITS region of Encephalitozoon species showed two genotypes (II and III) for E. cuniculi and one (TURK1B) for E. hellem. Only G. duodenalis assemblage A was identified in all nested PCR-positive samples. E. bieneusi was found more often than other enteric pathogens. CONCLUSIONS: This study showed that E. bieneusi, Encephalitozoon spp. [E. cuniculi and E. hellem] and G. duodenalis were common enteric parasites in diarrheic pigs in Chongqing and Sichuan Provinces. In case of both mono-infection and coinfection, E. bieneusi was the most common enteric pathogen in diarrheic pigs. Thus, it may be a significant cause of diarrhea in pigs. Precautions should be taken to prevent the spread of these enteric parasites.

3D reconstructions of parasite development and the intracellular niche of the microsporidian pathogen Encephalitozoon intestinalis.

Microsporidia are an early-diverging group of fungal pathogens with a wide host range. Several microsporidian species cause opportunistic infections in humans that can be fatal. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on host metabolites for successful replication and development. Our knowledge of microsporidian intracellular development remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has relied on 2D TEM images and light microscopy. Here, we use serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis, within host cells. We track E. intestinalis development through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in developing spores. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Our data provide insights into parasite development, polar tube assembly, and microsporidia-induced host mitochondria remodeling.

Case series of intestinal microsporidiosis in non-HIV patients caused by Encephalitozoon hellem.

Intestinal microsporidiosis is most often caused by Enterocytozoon bieneusi, and to a lesser extent by species of the genus Encephalitozoon. Until now, Encephalitozoon hellem was not clearly known to induce disease restricted to the intestine, or rarely in HIV subjects or in tropical countries. We report here 11 cases of delineated intestinal microsporidioses due to E. hellem diagnosed in France in non-HIV patients. Briefly, all patients were immunocompromised. They all suffered from diarrhoea, associated in nearly 50% of cases with weight loss. Concerning treatment, 5/11 patients had a discontinuation or a decrease of their immunosuppressive therapy, and 4/11 received albendazole. All patients recovered. Five different genotypes were identified based on the rRNA ITS sequence.

Microscopic and Molecular Investigation of Intestinal Microsporidia in HIV + /AIDS and Cancer Patients Undergoing Chemotherapy in Mazandaran Province, North of Iran.

BACKGROUND: The aim of this study was to identify Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples of HIV + /AIDS and cancer patients undergoing chemotherapy, and comparing the results to healthy individuals in Mazandaran province, north of Iran. METHODS: Stool samples were collected from 50 HIV + /AIDS patients, 50 cancer patients, and 50 healthy samples referred to medical centers in north of Iran. Stool samples were kept in 2.5% potassium dichromate at 4 °C, and stained by modified trichrome for light microscopy examination. The multiplex/nested-PCR targeted the small subunit ribosomal RNA (SSU rRNA) gene. To characterize genotypes, the nested PCR products sequenced by Bioneer Company and was subjected to phylogenetic analyses. RESULTS: Ten of 50 samples (20%) of HIV + /AIDS patients, 5 of 50 samples (10%) of cancer patients, and 1 of healthy individuals (2%) were microscopically positive. From 50 HIV + / AIDS patients, E. bieneusi and Encephalitozoon spp. were detected in 10 (20%) and 6 (12%) cases, respectively. Furthermore, among cancer patients, 7 (14%) and 2 (4%) cases were E. bieneusi and Encephalitozoon spp., respectively. Out of 50 samples of healthy individuals, only 3 (6%) cases of E. bieneusi were observed. The genotypes D and M were detected among positive samples of E. bieneusi. CONCLUSIONS: E. bieneusi and then Encephalitozoon spp. are common intestinal microsporidia in HIV + /AIDS patients and cancer patients undergoing chemotherapy in Mazandaran province. E. bieneusi genotype D seems to be the predominant genotype in Mazandaran province. Due to the considerable prevalence of intestinal microsporidia, physicians are advised to pay more attention to this opportunistic infection in high-risk groups.

Molecular Detection of Toxoplasma gondii, Neospora caninum and Encephalitozoon spp. in Vespertilionid Bats from Central Europe.

Bats may carry various viruses and bacteria which can be harmful to humans, but little is known about their role as a parasitic source with zoonotic potential. The aim of this study was to test wild bats for the presence of selected parasites: Toxoplasma gondii, Neospora caninum and microsporidia Encephalitozoon spp. In total, brain and small intestine tissues of 100 bats (52 Myotis myotis, 43 Nyctalus noctula and 5 Vespertilio murinus) were used for the DNA isolation and PCR detection of the abovementioned agents. Toxoplasma gondii DNA was detected by real-time PCR in 1% of bats (in one male of M. myotis), while all bats were negative for N. caninum DNA. Encephalitozoon spp. DNA was detected by nested PCR in 25% of bats, including three species (twenty-two M. myotis, two N. noctula and one V. murinus). Positive samples were sequenced and showed homology with the genotypes Encephalitozoon cuniculi II and Encephalitozoon hellem 2C. This is the first study on wild vespertilionid bats from Central Europe and worldwide, with a relatively high positivity of Encephalitozoon spp. detected in bats.

Telomere-to-Telomere genome assemblies of human-infecting Encephalitozoon species.

BACKGROUND: Microsporidia are diverse spore forming, fungal-related obligate intracellular pathogens infecting a wide range of hosts. This diversity is reflected at the genome level with sizes varying by an order of magnitude, ranging from less than 3 Mb in Encephalitozoon species (the smallest known in eukaryotes) to more than 50 Mb in Edhazardia spp. As a paradigm of genome reduction in eukaryotes, the small Encephalitozoon genomes have attracted much attention with investigations revealing gene dense, repeat- and intron-poor genomes characterized by a thorough pruning of molecular functions no longer relevant to their obligate intracellular lifestyle. However, because no Encephalitozoon genome has been sequenced from telomere-to-telomere and since no methylation data is available for these species, our understanding of their overall genetic and epigenetic architectures is incomplete. METHODS: In this study, we sequenced the complete genomes from telomere-to-telomere of three human-infecting Encephalitozoon spp. -E. intestinalis ATCC 50506, E. hellem ATCC 50604 and E. cuniculi ATCC 50602- using short and long read platforms and leveraged the data generated as part of the sequencing process to investigate the presence of epigenetic markers in these genomes. We also used a mixture of sequence- and structure-based computational approaches, including protein structure prediction, to help identify which Encephalitozoon proteins are involved in telomere maintenance, epigenetic regulation, and heterochromatin formation. RESULTS: The Encephalitozoon chromosomes were found capped by TTAGG 5-mer telomeric repeats followed by telomere associated repeat elements (TAREs) flanking hypermethylated ribosomal RNA (rRNA) gene loci featuring 5-methylcytosines (5mC) and 5-hemimethylcytosines (5hmC), themselves followed by lesser methylated subtelomeres and hypomethylated chromosome cores. Strong nucleotide biases were identified between the telomeres/subtelomeres and chromosome cores with significant changes in GC/AT, GT/AC and GA/CT contents. The presence of several genes coding for proteins essential to telomere maintenance, epigenetic regulation, and heterochromatin formation was further confirmed in the Encephalitozoon genomes. CONCLUSION: Altogether, our results strongly support the subtelomeres as sites of heterochromatin formation in Encephalitozoon genomes and further suggest that these species might shutdown their energy-consuming ribosomal machinery while dormant as spores by silencing of the rRNA genes using both 5mC/5hmC methylation and facultative heterochromatin formation at these loci.

Enterocytozoon bieneusi and Encephalitozoon intestinalis (microsporidia) in HIV-positive patients in central Spain.

Microsporidia are fungi-related eukaryotic intracellular parasites that opportunistically infect immunocompromised individuals such as those infected by the human immunodeficiency virus (HIV). Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most clinically relevant species. We investigated the occurrence and genetic diversity of microsporidial and protist infections in mostly immunocompetent HIV-positive patients in Madrid, Spain. A structured questionnaire was used to retrieve data on factors potentially associated with an increased risk of infection, including sexual attitudes and sex-risk behaviour. Faecal samples (n = 96) from 81 HIV-positive patients were collected and analysed by molecular (PCR and Sanger sequencing) methods. Two microsporidial pathogens were detected: Ent. bieneusi (2.5%, 95% CI: 0.3-8.6) and Enc.intestinalis (4.9%, 95% CI: 1.4-12.2). The two Ent. bieneusi isolates were identified as zoonotic genotype A. Among protists, Entamoeba dispar was the species most prevalently found (33.3%, 95% CI: 23.2-44.7), followed by Blastocystis spp. (19.8%, 95% CI: 11.7-30.1), Giardia duodenalis (13.6%, 95% CI: 7.0-23.0), and Cryptosporidium spp. and Entamoeba histolytica (2.5%, 95% CI: 0.3-8.6 each). Cyclospora cayetanensis and Cystoisospora belli were not detected. Subtypes ST1 (70.6%, 12/17) and ST3 (29.4%, 5/17) were identified within Blastocystis sp., sub-assemblages AII and BIII (50%, 1/2 each) within G. duodenalis, and Cry. parvum and canine-adapted Cry. canis (50%, 1/2 each) within Cryptosporidium spp. Microsporidial and protist parasites were frequent in well-controlled, mostly immunocompetent HIV-positive patients and should be included in diagnostic algorithms when diarrhoea is present.

Opportunistic microsporidial and protist intestinal infections were relatively common in well-controlled HIV-positive patients in Madrid, Spain. These agents should be suspected and appropriately diagnosed in HIV-positive patients presenting with diarrhoea regardless of their immunological status.

Occurrence and molecular characterization of Enterocytozoon bieneusi in water buffaloes (Bubalus bubalis) in Turkey.

Microsporidia are obligate intracellular fungus-like parasites that infect humans and animals worldwide. However, there is limited epidemiological data on the occurrence and molecular diversity of microsporidia in buffaloes worldwide. In the present study, fecal samples of 300 water buffaloes (Bubalus bubalis) in Kayseri, Sivas, and Samsun provinces of Turkey were investigated using two nested PCR assays targeting the rRNA of E. bieneusi and Encephalitozoon spp. All the fecal samples from water buffalo were found to be negative for Encephalitozoon spp. PCR positive isolates of E. bieneusi were bidirectionally sequenced for genotyping and phylogenetic analyses. Enterocytozoon bieneusi was the only microsporidian species identified in 8 water buffaloes with an overall molecular prevalence of 2.7%. Two known genotypes, YNDCEB-90 (n = 5) and J (n = 3) were identified by ITS sequence analysis. The YNDCEB-90 and J genotypes fall into zoonotic Group 1 and 2 of E. bieneusi in the phylogenetic tree, respectively. These findings suggested that water buffalo in Turkey are harbouring zoonotic genotypes of E. bieneusi and may have a significant risk for zoonotic transmission to humans. This is the first report of detecting E. bieneusi genotypes J and YNDCEB-90 in water buffaloes. Further insight into the epidemiology of E. bieneusi in water buffaloes in different geographical areas in Turkey will be highly important to have determined the public health significance of this pathogen.

Molecular diagnosis of intestinal microsporidia infection in HIV/AIDS-patients in Zahedan city, Southeast of Iran.

Microsporidia belong to the intracellular spore-like pathogen, that can cause infection in invertebrates and vertebrates, including humans. Encephalitozoon spp. and Enterocytozoon bieneusi, are important causes of chronic diarrhea, especially in patients with HIV/AIDS. Therefore, in this study, modified trichrome staining (MTS) and nested polymerase chain reaction (nested PCR) methods were used for the diagnosis of common intestinal microsporidia in faecal samples of patients with HIV/AIDS in Zahedan, southeastern Iran, for the first time. Stool samples were collected from 50 HIV/AIDS-infected patients with gastrointestinal symptoms whose infections were confirmed by serology test. Prepared smears from each stool sample were stained using the MTS method. Nested PCR was used to amplify 440 bp and 629 bp fragments of 16S rRNA genes in E. bieneusi and Encephalitozoon spp., respectively. Based on the MTS method and the nested PCR, 8 (16%) and 12 (24%) stool samples were positive, respectively. According to the results of nested PCR, eight, three, and one case were infected with E. bieneusi, Encephalitozoon spp., and both of them, respectively. Findings indicated microsporidiosis in HIV/AIDS-infected patients in Zahedan is an important health problem. Therefore, this opportunistic microorganism in HIV/AIDS-infected patients should be diagnosed using sensitive and accurate methods.

Molecular Identification of Encephalitozoon hellem from Companion Birds Kept in Pet Shops, Japan.

To evaluate the role of companion birds as a reservoir of Encephalitozoon hellem infection in humans, the present study determined the prevalence and genotypes of E. hellem from 269 birds in 4 pet shops using polymerase chain reaction (PCR) assay. E. hellem was identified in 4.8% (13/269) of the birds and was detected in all pet shops. Every positive sample corresponded to zoonotic genotype 1A. Considering the low prevalence of E. hellem infection, it is likely that the risk of zoonotic transmission from companion birds kept in pet shops to humans is low in Japan.

Molecular epidemiology and genotype/subtype distribution of Blastocystis sp., Enterocytozoon bieneusi, and Encephalitozoon spp. in livestock: concern for emerging zoonotic infections.

Intestinal parasitic infections have high prevalence rate in many regions especially in developing countries. The aim of this study was to determine the presence and genotype/subtype of some intestinal protozoa in livestock in Iran. Stool samples were collected from cattle, sheep, chickens, and horses. The presence of targeted parasites was evaluated using real-time PCR. Genotyping/subtyping of positive samples was characterized using sequencing of the ITS and barcoding region, respectively. Blastocystis sp., 27.7% (48/173) and Enterocytozoon bieneusi 26.0% (45/173) were the most frequent protozoa followed by Encephalitozoon spp., 0.57% (1/173). Cryptosporidium spp. were not detected among samples. Encephalitozoon spp., was detected only in chickens 2.2% (1/45). A statistically correlation was seen between animals and the prevalence of targeted protozoa. E. bieneusi genotypes I (9/38; 23.68%), BEB6 (22/38; 57.89%), D (6/38; 15.79%), and horse1 (1/38; 2.63%) were detected among samples. A statistically significant correlation was seen between the genotypes and animals (P ≤ 0.05). Blastocystis sp., ST1 (1/45; 2.22%), ST5 3/45; 6.66%), ST7 (1/45; 2.22%), ST10 (24/45; 53.33%), and ST14 (16/45; 35.55%) were characterized among samples. There was no significant correlation between certain subtypes and animals (P = 0.173). The presence of zoonotic potential genotypes of E. bieneusi in animals and zoonotic potential subtypes ST1 and ST7 among our samples provide a clue about the transmission dynamic of E. bieneusi and Blastocystis sp. between animals-animals and humans-animals.

Von Willebrand Factor Facilitates Intravascular Dissemination of Microsporidia Encephalitozoon hellem.

Microsporidia are a group of spore-forming, fungus-related pathogens that can infect both invertebrates and vertebrates including humans. The primary infection site is usually digestive tract, but systemic infections occur as well and cause damages to organs such as lung, brain, and liver. The systemic spread of microsporidia may be intravascular, requiring attachment and colonization in the presence of shear stress. Von Willebrand Factor (VWF) is a large multimeric intravascular protein and the key attachment sites for platelets and coagulation factors. Here in this study, we investigated the interactions between VWF and microsporidia Encephalitozoon hellem (E. hellem), and the modulating effects on E. hellem after VWF binding. Microfluidic assays showed that E. hellem binds to ultra-large VWF strings under shear stress. In vitro germination assay and infection assay proved that E. hellem significantly increased the rates of germination and infection, and these effects would be reversed by VWF blocking antibody. Mass spectrometry analysis further revealed that VWF-incubation altered various aspects of E. hellem including metabolic activity, levels of structural molecules, and protein maturation. Our findings demonstrated that VWF can bind microsporidia in circulation, and modulate its pathogenicity, including promoting germination and infection rate. VWF facilitates microsporidia intravascular spreading and systemic infection.

Advancement in our understanding of immune response against Encephalitozoon infection.

Microsporidia are a group of obligate, intracellular, spore-forming eukaryotic pathogens, which predominantly infects immunocompromised individuals worldwide. Encephalitozoon spp. is one of the most prevalent microsporidia known to infect humans. Host immune system plays a major role in combating pathogens including Encephalitozoon spp. infecting humans. Both innate and adaptive arms of host immune system work together in combating Encephalitozoon infection. Researchers are conducting studies to elucidate the role of both arms of immune system against Encephalitozoon infection. In addition to cell-mediated adaptive immunity, role of innate immunity is also being highlighted in clearance of Encephalitozoon spp. from host body. Therefore, the current review will give a clear and consolidated update on the role of innate as well as adaptive immunity in protection against Encephalitozoon spp.

The use of flow cytometry for fungal nuclear DNA quantification.

Genome size information is sparse across fungi, with information being available for less than 2000 species. So far, most records have been obtained using static, microscope-based cytometry methods or derived from genome sequencing projects. Flow cytometry is now considered the state-of-the-art method for obtaining genome size measurements, and appropriate methods and DNA standards are available, enabling the analysis of most genome size ranges in a rapid, robust and inexpensive way. The average fungal genome size is 60 Mbp, but sizes vary across phylogeny, ranging from 2.2 (Encephalitozoon romaleae) to 3706 Mbp (Jafnea semitosta). In several fungal clades, genome size expansion seems to accompany evolution either to plant mutualism or to plant parasitism (particularly biotrophy), and fungi that interact with plants seem to have larger genomes than saprobes and those that interact with animals. Whereas flow cytometry for nuclear DNA quantification is routinely employed in plant sciences for genome size and ploidy studies, its use in fungal biology is still infrequent. Appropriate standards, methods and best practices are described here, with the aim of stimulating a more generalized and widespread use of flow cytometry for fungal genome size measurement.

Human microsporidian pathogen Encephalitozoon intestinalis impinges on enterocyte membrane trafficking and signaling.

Microsporidia are a large phylum of obligate intracellular parasites. Approximately a dozen species of microsporidia infect humans, where they are responsible for a variety of diseases and occasionally death, especially in immunocompromised individuals. To better understand the impact of microsporidia on human cells, we infected human colonic Caco2 cells with Encephalitozoon intestinalis, and showed that these enterocyte cultures can be used to recapitulate the life cycle of the parasite, including the spread of infection with infective spores. Using transmission electron microscopy, we describe this lifecycle and demonstrate nuclear, mitochondrial and microvillar alterations by this pathogen. We also analyzed the transcriptome of infected cells to reveal host cell signaling alterations upon infection. These high-resolution imaging and transcriptional profiling analysis shed light on the impact of the microsporidial infection on its primary human target cell type.This article has an associated First Person interview with the first authors of the paper.

Assessment of a Multiplex PCR for the Simultaneous Diagnosis of Intestinal Cryptosporidiosis and Microsporidiosis: Epidemiologic Report from a French Prospective Study.

Microsporidiosis and cryptosporidiosis are associated with chronic diarrhea in immunocompromised patients. The objectives of this study were to: i) assess a multiplex quantitative PCR assay targeting Cryptosporidium spp and the microsporidian Enterocytozoon bieneusi and Encephalitozoon spp, and ii) provide an update on the epidemiology of these pathogens. A prospective study was conducted from January 2017 to January 2019. Performance of the assay was assessed, and all cryptosporidia and microsporidia isolates were genotyped. The sensitivity of the multiplex PCR method reached 1 copy/µL for each targeted pathogen. The sensitivity of co-proantigen testing in the diagnosis of cryptosporidiosis was 73%. The sensitivity of microscopy in the diagnosis of cryptosporidiosis was 64%, and microsporidiosis, 50%. Among the 456 patients included, 14 were positive for Cryptosporidium spp (4 different species); 5, for E. bieneusi; and 2, for Encephalitozoon intestinalis. The overall prevalence of cryptosporidia was 3.1%, and of microsporidia, 1.5%; in kidney transplant recipients (n = 82), corresponding values were 7.3% and 2.4% (6 and 2 patients), respectively. Two cases of E. intestinalis infection were diagnosed in children who had traveled to the tropics. This study is the first to assess a multiplex quantitative PCR method for the simultaneous diagnosis of intestinal microsporidiosis and cryptosporidiosis. The highest prevalences of both pathogens were observed in kidney transplant recipients.

Encephalitozoon intestinalis Infection Impacts the Expression of Apoptosis-Related Genes in U937 Macrophage Cells.

PURPOSE: Encephalitozoon intestinalis affects many physiological processes of host cells to survive, proliferate, and spread to different regions within the body. In this study, the effects of the parasite on host cell apoptosis and proliferation were investigated. METHODS: To determine the impact of the parasite on the host cell apoptosis, changes in the expression profile of genes were investigated with the qPCR array using the Human Apoptosis Panel in infected and non-infected macrophage cells. Also, the rate of apoptosis in the cells was determined by Giemsa staining method. Cell proliferation was determined by measuring the DNA concentration in infected and non-infected cells. RESULTS: The thirty-six of apoptosis-related genes were down-regulated, while 20 of apoptosis-related genes were up-regulated in infected cells compared to uninfected cells. However, there were no significant changes detected in 32 analyzed genes between infected and control groups. E. intestinalis was determined to decrease cell proliferation in U937 macrophage cells. Unexpectedly, Giemsa staining showed an increase in the rate of apoptosis in infected cells. CONCLUSION: Regulated genes after infection are involved in many different biological pathways and various components of the cell. This suggests that the parasite uses highly sophisticated ways to maintain the viability of the cell.