Widespread distribution of human-infective Enterocytozoon bieneusi genotypes in small rodents in northeast China and phylogeny and zoonotic implications revisited.

Enterocytozoon bieneusi features high genetic diversity among host species and environmental sources and over 500 genotypes in 11 phylogenetic groups have been defined. Here we investigated 291 small rodents in Heilongjiang province, northeast China, for the presence of E. bieneusi by PCR of the ribosomal internal transcribed spacer (ITS). Nine of 60 (15.0 %) gray squirrels from a park in Harbin, 120 of 201 (59.7 %) guinea pigs from a pet shop in Harbin, and two of 30 (6.7 %) peridomestic rats from a pasture in Qiqihar were positive for the parasite. Six known genotypes (EbpB, SCC-1, SCC-2, D, S7 and HLJ-CP1) and two novel genotypes (NESQ1 and NEGP1) were identified by sequence analysis of the ITS, with EbpB, SCC-1, SCC-2 and NESQ1 found in squirrels, D, S7 and NEGP1 in guinea pigs, and EbpB and HLJ-CP1 in rats. Widespread distribution of human-infective Group 10 genotype S7 and Group 1 genotype D in guinea pigs raised our concerns about the importance of pet animals as zoonotic reservoirs of microsporidiosis. Co-occurrence of Group 1 genotypes D and HLJ-CP1 in cancer patients and rodents in Heilongjiang indicated a possibility of zoonotic transmission. The host range of Group 1 genotype EbpB previously considered pig-adapted was extended. A potential variant of genotype S7, namely NESQ1, went into the existing Group 10 in phylogenetic analysis. The other new genotype, NEGP1, was clustered in an undefined clade we proposed as Group 15. With the emerging epidemiologic evidence, the host specificity of existing E. bieneusi genotypes is now being challenged.

Enterocytozoon bieneusi Infection after Hematopoietic Stem Cell Transplant in Child, Argentina.

We report a case of Enterocytozoon bieneusi infection in a pediatric hematopoietic stem cell transplant recipient in Argentina. Spores were visualized in feces using Calcofluor White and modified trichrome stainings. PCR and sequencing identified E. bieneusi genotype D in fecal samples and liver samples, confirming extraintestinal dissemination of the parasite.

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.

Detection of a novel microsporidium with intranuclear localization in farmed Penaeus vannamei from Latin America.

Microsporidia are emerging intracellular parasites of most known animal phyla in all ecological niches. In shrimp aquaculture, the microsporidium Enterocytozoon hepatopenaei (EHP) is a major cause of concern inflicting tremendous losses to shrimp producers in southeast Asia. During a histopathological examination of Penaeus vannamei samples originating in a country from Latin America presenting slow growth, we observed abnormal nuclei in the epithelial cells of the hepatopancreas. A PCR screening of the samples using DNA isolated from paraffin embedded tissues for the SSU rRNA gene of EHP provided a 149 bp amplicon. In situ hybridization using the SSU rRNA gene probe provided a positive signal in the nuclei instead of the cytoplasm. Sequence analysis of the SSU rRNA gene product revealed a 91.3 %, 89.2 % and 85.4 % sequence identity to Enterocytozoon bieneusi, E. hepatopenaei and Enterospora canceri respectively. Furthermore, phylogenetic analysis revealed the newly discovered microsporidium clustered with E. bieneusi. Considering the intranuclear location of the novel microsporidium and the differences in the sequence of the SSU rRNA, we tentatively consider this parasite a new member of the genus Enterospora sp. The pathogenicity and distribution of the shrimp Enterospora sp. are currently unknown. Our future efforts are focused on the characterization and development of diagnostic tools for this parasite to understand if it acts as an emergent pathogen that might require surveillance to prevent its spread.

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.

An isothermal enzymatic recombinase amplification (ERA) assay for rapid and accurate detection of Enterocytozoon hepatopenaei infection in shrimp.

Enterocytozoon hepatopenaei (EHP) is a kind of microsporidian parasite belonging to fungi, and poses a serious threat to prawn farmers. Due to the lack of effective treatments for EHP, the establishment of a rapid and sensitive detection method would be beneficial to the control and prevention of this prawn parasitic disease. In this study, an isothermal enzymatic recombinase amplification (EHP-ERA) assay that could diagnose EHP within 20 min at 42 °C was developed and evaluated. The determined final concentrations of primers and probe in the reaction system were 400 nM and 120 nM, respectively. EHP-ERA was carried out within 13 min (24.31 ± 0.37 Ct) with a detection limit of 10 copies/µL. The results of specificity test showed that EHP-ERA had no cross-reactivity with white spot syndrome virus (WSSV), Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (VpAHPND), and infectious hypodermal and hematopoietic necrosis virus (IHHNV) and specific pathogen free (SPF) shrimp. Using 32 clinical samples, the practical diagnostic results of EHP-ERA was consistent with nested PCR and real-time PCR (qPCR) under the premise of less time-consuming and simpler operation. In summary, we established a simple, rapid, and effective ERA assay for the detection of EHP, which had great potential to be widely used in both lab and practical usage.

Molecular identification and genetic characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in human immunodeficiency virus/acquired immunodeficiency syndrome patients in Shanghai, China.

BACKGROUND: Opportunistic infections are a ubiquitous complication in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients. Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans. In China, despite the number of HIV/AIDS patients being extremely large, only a few studies have investigated opportunistic infections caused by intestinal pathogens in this patient population. The aims of this study were to elucidate the occurrence and genetic characteristics of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients. METHODS: We collected fecal specimens from 155 HIV/AIDS patients (one from each patient). All of the specimens were examined for the presence of the pathogens by genotyping using polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene for Cryptosporidium spp.; the triosephosphate isomerase, ß-giardin and glutamate dehydrogenase genes for G. duodenalis; and the internal transcribed spacer region of the rRNA gene for E. bieneusi. The Cryptosporidium-positive specimens were further subtyped by polymerase chain reacion and sequencing of the 60-kDa glycoprotein gene. RESULTS: Six (3.9%), three (1.9%), and eight (5.2%) HIV/AIDS patients were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. No statistical differences were observed in occurrence rate between the groups by gender, clinical symptom (diarrhea), and CD4+ cell count. Four Cryptosporidium species were identified: Cryptosporidium hominis (n = 2), Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). Furthermore, two C. hominis subtypes (IeA12G3T3 and IaA28R4) were detected. Three G. duodenalis-positive specimens were successfully amplified and sequenced at the triosephosphate isomerase and ß-giardin loci, which led to the identification of assemblages C and B, respectively. Seven genotypes (D, Type IV, EbpC, Peru11, EbpD, A, and I) were identified in E. bieneusi-positive specimens. CONCLUSIONS: Our findings should increase awareness of AIDS-related opportunistic intestinal pathogens, and indicate the need for routine examination in clinical practice for the detection of Cryptosporidium spp., G. duodenalis, and E. bieneusi. Homology analyses of the three intestinal pathogens at the nucleotide and/or amino acid levels indicated their zoonotic potential.

Concurrent infection of a novel genotype of hepatopancreatic parvovirus and Enterocytozoon hepatopenaei in Penaeus vannamei in Taiwan.

Hepatopancreatic parvovirus (HPV) and Enterocytozoon hepatopenaei (EHP) are emerging and reemerging pathogens in shrimps. In the present study, a novel genotype of HPV concurrently infected with EHP in Penaeus vannamei in Taiwan leading to severe atrophy and damage of hepatopancreas were confirmed by histopathology, in situ hybridization, and PCR. The novel genotype of HPV exhibited 66%-69.5% sequence identities with all known HPVs and carried unique amino acid deletions and insertions in the VP gene. According to phylogenetic analysis, the Taiwan HPV isolates were classified as the genotype IV. The present study not only provided the histopathological and molecular proof of HPV and EHP co-infection in Taiwan, but also revealed the importance of investigating the geographical expansion of novel HPV genotypes.

Establishment and application of a TaqMan probe-based qPCR for the detection of Enterocytozoon hepatopenaei in shrimp Litopenaeus vannamei.

Enterocytozoon hepatopenaei (EHP) is a common parasite that invades the epithelial cytoplasm in the hepatopancreas of shrimp Litopenaeus vannamei and results in slow growth of the host shrimps to cause significant economic loss in shrimp aquaculture. In this study, a TaqMan probe-based qPCR for quantitative detection of EHP was established. A pair of specific primers and a TaqMan probe were designed based on the sequence of cysteine desulfurase gene (NFS1) of EHP. The standard curve between cycle threshold (Ct) and the logarithmic starting quantity (SQ) of the template was determined as Ct = - 3.231 lg (SQ) + 40.638, with a correlation coefficient (R2) of 0.998 and an amplification efficiency of 103.9%. The lower limit of quantification was 1.67 × 101 copies/µL for this TaqMan probe-based qPCR and 1.67 × 103 copies/µL for the conventional PCR. The TaqMan probe-based qPCR established in the research was 100 times more sensitive than the conventional PCR method. In addition, the results of clinical sample detection indicated that the present technique was efficient in detecting EHP in the hepatopancreas, feces, water, and pond bottom mud samples. Therefore, the established TaqMan probe-based qPCR is a suitable technique for detecting EHP in both shrimp and aquatic environment samples.

Identification of two potential aetiological agents of chronic diarrhoea in an immunocompromised patient in Cuba using conventional and molecular diagnostic techniques.

The aetiology of diarrhoea in a patient in Cuba with HIV was investigated. Although molecular diagnostics are still not used in many under-resourced settings, here traditional methods were supported by use of PCR. This approach enabled detection of a dual infection (Cystoisospora belli and Enterocytozoon bieneusi), the latter of which was not identified by microscopy with Didier's trichromic staining.

The successful treatment of Enterocytozoon bieneusi Microsporidiosis with nitazoxanide in a patient with B-ALL: A Case Report.

Introduction: Enterocytozoon bieneusi (E. bieneusi) Microsporidia can cause opportunistic infections in immunocompromised patients and is also an emerging disease in these individuals. Its clinical manifestations are chronic diarrhea and severe wasting syndrome, these can be extremely debilitating and carry a significant risk of death for immunocompromised patients. Often, microsporidia cannot be confirmed immediately by routine examination and culture. Effective and available treatment options are limited for infections caused by E. bieneusi in humans. Such cases are very rare in Chinese Mainland. Case presentation: A 47-year-old male had recurrent, profuse watery diarrhea and abdominal discomfort for more than 7 months, with a fever for 5 days. Two years earlier, he received treatment with a modified BFM-90 protocol for acute B cell lymphoblastic leukemia and is currently in the final stages of maintenance therapy with oral methotrexate and mercaptopurine. The leukemia was assessed as still in remission two months ago. PET/CT showed massive peritoneal fluid accumulation and a high uptake area in the diffused peritoneum (SUVmax 12.57), suggesting tumor invasion or microbial infections. However, broad-spectrum antibacterial therapies were ineffective. Metagenomic sequencing of plasma and peritoneal fluid showed no suggestion of the existence of a tumor but instead showed a high sequence number of DNA and RNA of the Microsporidia. His albendazole treatment failed and subsequent treatment with nitazoxanide successfully resolved the infection. Conclusion: This case shows that we should consider the possibility of atypical pathogen infection in patients with hematologic malignancy who repeatedly develop unexplained diarrhea with wasting. mNGS can help rule out malignant neoplasms and diagnose infections. Our results suggest that nitazoxanide effectively treats E. bieneusi microsporidia infections.

Microscopio and PCR-based detection of microsporidia spores in human stool samples

Abstract Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

Resumen Los microsporidios son hongos intracelulares obligados con una notable capacidad para infectar una amplia gama de hospedadores invertebrados y vertebrados. Enterocytozoon bieneusi es el microsporidio más frecuentemente reportado en todo el mundo, principalmente tricrómicaasociado con diarrea crónica y síndrome debilitante en pacientes con sida. Las técnicas dedetección basadas en microscopía y PCR son útiles para el diagnóstico y la identificación deespecies y genotipos, pero estos métodos deben estar estandarizados en cada laboratorio.En este estudio evaluamos técnicas de microscopía y PCR anidada, con secuenciación de losproductos, para detectar E. bieneusi en muestras de heces humanas. Estas técnicas, usadas con-juntamente, podrían ser útiles para su aplicación en el diagnóstico de microsporidiosis intestinaly para realizar estudios epidemiológicos de esta afección en Argentina.

Molecular epidemiology of Enterocytozoon bieneusi and Encephalitozoon sp., among immunocompromised and immunocompetent subjects in Iran.

Intestinal microsporidiosis is known as an opportunistic infection in immunocompromised patients. The current study aimed to investigate intestinal microsporidia infection in human subjects with/without immunodeficiency. Totally, 600 stool samples were collected from immunocompromised (254) and immunocompetent (346) subjects. DNA extraction was performed and the SSU rRNA and the ITS genes were amplified to detect and characterize microsporidia and the relevant genotypes. Phylogenetic trees were drawn using MEGA7 software to illustrate the correlation between isolates. From 600 enrolled subjects, 283 and 317 were male and female, respectively. The average age ± SD of all tested subjects was 28.85 ± 26.92. The results of PCR demonstrated the presence of E. bieneusi and Encephalitozoon sp., among 10/600 (1.67%) and 26/600 (4.33%) of samples, respectively. Accordingly, E. bieneusi was seen among 4/346 (1.15%), 1/53 (1.88%), 3/124 (2.42%), and 2/63 (3.17%), and Encephalitozoon sp., was detected from 17/346 (4.91%), 3/53 (5.36%), 4/124 (3.22%) and 2/63 (3.17%) of healthy subjects, RA patients, cancer patients, and transplantation recipients, respectively. Statistical significant correlation was not seen between the presence of microsporidia and age, gender, stool appearance, and geographical region. Molecular analysis showed that all E. bieneusi were the genotype D. Phylogenetic tree demonstrated no classification according to the presence/absence of immunodeficiency, geographical locations and presence of diarrhea. The high prevalence of Encephalitozoon sp., in comparison to E. bieneusi in this study suggested the importance of this genus alongside with E. bieneusi in Iran. In addition, predominance of the genotype D highlighted the wide distribution of this genotype in Iran.

Genotyping Approach for Potential Common Source of Enterocytozoon bieneusi Infection in Hematology Unit.

Microsporidiosis is a fungal infection that generally causes digestive disorders, especially in immunocompromised hosts. Over a 4-day period in January 2018, 3 patients with hematologic malignancies who were admitted to the hematology unit of a hospital in France received diagnoses of Enterocytozoon bieneusi microsporidiosis. This unusually high incidence was investigated by sequence analysis at the internal transcribed spacer rDNA locus and then by 3 microsatellites and 1 minisatellite for multilocus genotyping. The 3 isolates had many sequence similarities and belonged to a new genotype closely related to genotype C. In addition, multilocus genotyping showed high genetic distances with all the other strains collected from epidemiologically unrelated persons; none of these strains belonged to the new genotype. These data confirm the epidemiologic link among the 3 patients and support a common source of infection.

Detection of Enterocytozoon hepatopenaei using an invasive but non-lethal sampling method in shrimp (Penaeus vannamei).

The detection of enteric pathogens that cause diseases in shrimp involves the sacrifice of the host to obtain tissue samples for diagnosis. In this study, we describe an invasive but non-lethal sampling methodology using a syringe to collect biopsy samples from the hepatopancreas (HP) of Penaeus vannamei to detect the microsporidian pathogen, Enterocytozoon hepatopenaei (EHP), by qPCR and transmission electron microscopy. EHP was detected in all the infected shrimp by qPCR. The shrimp infected by the microsporidian showed 65% survival at 7 days post-sampling. Transmission electron microscopic examination of the biopsy samples revealed numerous spores of the pathogen. The presence of EHP was further confirmed by histology and in situ hybridization from HP tissue samples. The data shows that a hepatopancreas biopsy could be a viable means of detecting enteric pathogens in shrimp, and the method could be valuable in sampling broodstock and natural populations without the need to sacrifice the animals.

Occurrence of zoonotic Enterocytozoon bieneusi in cats in Brazil / Ocorrência de Enterocytozoon bieneusi zoonótico em gatos no Brasil

Abstract Enterocytozoon bieneusi is an opportunistic intestinal pathogen that infects humans and a wide variety of animals worldwide. Our aim in this study was to investigate the occurrence of E. bieneusi in a domestic cat population in Campo Grande, Mato Grosso do Sul, Brazil. Sixty fecal samples from diarrheic cats were subjected to polymerase chain reaction (PCR) and the amplicons were sequenced for identification. E. bieneusi was detected in two samples (3.3%), both identified as genotype D. This genotype has already been reported in animals and humans and is considered a zoonotic genotype. Our findings represent the first report of E. bieneusi in domestic cats in Brazil, reinforcing the importance of identifying this agent as a source of infection in animals and humans.

Resumo Enterocytozoon bieneusi é um patógeno intestinal oportunista que infecta humanos e uma variedade de animais em todo o mundo. O objetivo no presente estudo foi investigar a ocorrência de E. bieneusi em uma população de gatos domésticos em Campo Grande, Mato Grosso do Sul, Brasil. Sessenta amostras fecais de gatos diarréicos foram submetidas a reação em cadeia da polimerase (PCR) e os produtos de amplificação foram sequenciados para identificação molecular. E. bieneusi foi detectado em duas amostras (3,3%), ambos identificados como genótipo D. Esse genótipo tem sido relatado em animais e humanos e é considerado um genótipo zoonótico. Nossos resultados representam a primeira descrição de E. bieneusi em gatos domésticos no Brasil, reforçando a importância desse agente como fonte de infecção para animais e humanos.

Experimental horizontal transmission of Enterocytozoon hepatopenaei in post-larvae of whiteleg shrimp, Litopenaeus vannamei.

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that causes hepatopancreatic microsporidiosis (HPM) in penaeid shrimp. HPM was observed in several countries, including Thailand and India; it has become a prominent pathogen in shrimp culture. Based on observations on EHP infection in the wild, the route of transmission has been hypothesized. Identification of artificial EHP infection procedures can facilitate our understanding of EHP transmission. Experimental transmission of EHP was attempted using the immersion and oral infections of infection. In the immersion mode, post-larvae (PL) were exposed to an EHP tissue homogenate (0.2%) by immersion for 48 hr. Experimental samples were collected at various time points, and infection was confirmed using polymerase chain reaction, haematoxylin and eosin staining, transmission electron microscopy and modified trichrome staining. All test results revealed successful EHP transmission. Similar results were obtained through oral infection (oral infection). Innate immune gene expression patterns during infection were analysed; prophenoloxidase, crustin and superoxide dismutase were upregulated at 6, 6 and 48 hr post-challenge, respectively. Experimental infection procedures facilitate the development of diagnostic and prevention strategies. This is the first study demonstrating the experimental transmission of EHP in shrimp PL.

Molecular Diagnosis of Microsporidia among Immunocompromised Patients in Kuala Lumpur, Malaysia.

Microsporidia are obligate intracellular parasitic fungi causing chronic diarrhea, particularly among immunocompromised patients. The current method used for diagnosis is based on conventional microscopy, which does not differentiate parasites at the species level. The present study was carried out to identify microsporidian species in immunocompromised patients. From March 2016 to March 2017, a total of 289 archived stool samples were examined microscopically for microsporidian spores using Gram-chromotrope Kinyoun (GCK) stain. Positive stool samples by microscopy were subjected to polymerase chain reaction and sequencing for species identification. Based on microscopy examination, the overall prevalence of microsporidian infections was 32.2% (93/289; 95% CI = 27.1-37.8). Of these stool samples, 45 samples were successfully amplified and confirmed as Enterocytozoon bieneusi. No Encephalitozoon intestinalis was detected. Accurate identification of species might help clinicians to decide appropriate management strategies as dissemination risks and treatment response vary for different species, hence improving the management of microsporidian infections.

Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP).

BACKGROUND: The microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp. RESULTS: We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein. CONCLUSIONS: Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.

Enterocytozoon bieneusi detected by molecular methods in raw sewage and treated effluent from a combined system in Brazil

BACKGROUND Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.