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Molecular characterization of microsporidia indicates that wild mammals Harbor host-adapted Enterocytozoon spp. as well as human-pathogenic Enterocytozoon bieneusi.

Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of Enterocytozoon spp., with the use of primers complementary to the conserved regions of published nucleotide sequences. Fifty-nine PCR-positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of Enterocytozoon spp. (WL1 to WL17); of these, 15 have not been reported before. Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described Enterocytozoon bieneusi genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic E. bieneusi. However, there are also host-adapted Enterocytozoon genotypes in wildlife, which may represent species different from E. bieneusi and have no apparent public health significance. This is the first report of E. bieneusi in wildlife.

Animals↗

Comparative development of two microsporidian species: Enterocytozoon bieneusi and Enterocytozoon salmonis, reported in AIDS patients and salmonid fish, respectively.

Enterocytozoon bieneusi and Enterocytozoon salmonis are reported in HIV-infected patients and in salmonid fish, respectively. Both species share the early development of the extrusion apparatus of the spores, which is completed prior to fission of the sporogonic syncytium into sporoblasts, and the early synthesis of polar tube constituents, but they differ in other developmental and sporogenetic processes. Enterocytozoon bieneusi develops in direct contact with the cytoplasm of epithelial cells whereas E. salmonis occurs only in the nucleus of leucocytes and epithelioid cells. Sporogonic nuclei, which are scattered throughout the sporont in E. bieneusi, are located in the periphery in E. salmonis. The multilamellar structures associated with the nuclear envelopes and the endoplasmic reticulum cisternae are specific for E. bieneusi. Additionally, the evolution of the polar tube precursors proceeds differently in the two parasites. In E. bieneusi, they transform into electron-dense bodies associated with a reticulum and polar tubes derive from these structures according to a process similar to that reported in other microsporidia. In E. salmonis, polar tube precursors fuse directly at their ends and the polar tubes appear to be formed by the assemblage of these fused precursors with a material previously synthesized in the vicinity of nuclei. In conclusion, both species appear to be less closely related than was supposed in earlier descriptions.

Acquired Immunodeficiency Syndrome↗

Detection of new Enterocytozoon genotypes in faecal samples of farm dogs and a cat.

The microsporidian species Enterocytozoon bieneusi had emerged as opportunistic pathogen in AIDS patients causing chronic diarrhoea and was found with high prevalences in faeces of asymptomatic pigs. Analysis of the ribosomal RNA gene internal transcribed spacer (rDNA ITS) had revealed that nine distinct but closely related genotypes occur in humans and in swine. Using primers that were designed to be specific for E. bieneusi, we obtained amplicons from the faecal samples of one from twelve cats and from three out of 36 farm dogs. Sequence analysis of the rDNA ITS, which is part of the diagnostic PCR product, revealed that the isolate from the cat is very closely related to the E. bieneusi genotypes of human or swine origin. The corresponding sequence of all three dog-derived isolates were identical among each other and had a sequence similarity to known sequences of only 47.6-48.2%. In addition, part of the small subunit rRNA gene was amplified and sequenced from one dog-derived isolate revealing a similarity to known sequences of human-derived E. bieneusi of 96-98%. Enterocytozoon-like spores could be detected by light microscopy in one canine sample. Together with recent reports of detection of Enterocytozoon in environmental samples, our findings suggest that microsporidia of the genus. Enterocytozoon seem to be ubiquitous and consist of many genotypes in various naturally infected animal species.

AIDS-Related Opportunistic Infections↗

The presence of Enterocytozoon bieneusi spores in the lamina propria of small bowel biopsies with no evidence of disseminated microsporidiosis. Enteric Opportunistic Infections Working Group.

Enterocytozoon bieneusi is the most frequently reported microsporidial infection of humans. In patients with the acquired immunodeficiency syndrome, Enterocytozoon infects the lining epithelial cells of the small intestine, hepatobiliary tract, and gallbladder. Because Enterocytozoon has been thought to be limited to infecting lining epithelial cells, the mechanism of spread of E bieneusi within the intestine, to the biliary tract, and, in two case reports, to distant organs remains unknown. This report describes a patient with acquired immunodeficiency syndrome and intestinal microsporidiosis due to E bieneusi. Histopathologic examination of well-oriented biopsies from the duodenum and jejunum revealed both intra- and extracellular spores of Enterocytozoon extending deeply into the lamina propria, where they were located adjacent to capillaries. The patient has not developed disseminated disease 20 months after the initial diagnosis. In this patient, the demonstration of E bieneusi spores in extraepithelial tissues does not appear to be associated with development of subsequent systemic infection.

Acquired Immunodeficiency Syndrome↗

Cryptosporidium, enterocytozoon, and cyclospora infections in pediatric and adult patients with diarrhea in Tanzania.

Cryptosporidiosis, microsporidiosis, and cyclosporiasis were studied in four groups of Tanzanian inpatients: adults with AIDS-associated diarrhea, children with chronic diarrhea (of whom 23 of 59 were positive [+] for human immunodeficiency virus [HIV]), children with acute diarrhea (of whom 15 of 55 were HIV+), and HIV control children without diarrhea. Cryptosporidium was identified in specimens from 6/86 adults, 5/59 children with chronic diarrhea (3/5, HIV+), 7/55 children with acute diarrhea (0/7, HIV+), and 0/20 control children. Among children with acute diarrhea, 7/7 with cryptosporidiosis were malnourished, compared with 10/48 without cryptosporidiosis (P < .01). Enterocytozoon was identified in specimens from 3/86 adults, 2/59 children with chronic diarrhea (1 HIV+), 0/55 children with acute diarrhea, and 4/20 control children. All four controls were underweight (P < .01). Cyclospora was identified in specimens from one adult and one child with acute diarrhea (HIV-). Thus, Cryptosporidium was the most frequent and Cyclospora the least frequent pathogen identified. Cryptosporidium and Enterocytozoon were associated with malnutrition. Asymptomatic fecal shedding of Enterocytozoon in otherwise healthy, HIV children has not been described previously.

Acute Disease↗

Identification of an Enterocytozoon bieneusi-like microsporidian parasite in simian-immunodeficiency-virus-inoculated macaques with hepatobiliary disease.

Enterocytozoon bieneusi is a common opportunistic pathogen of human patients with acquired immune deficiency syndrome (AIDS) causing significant morbidity and mortality. In a retrospective analysis utilizing conventional histochemical techniques, in situ hybridization, polymerase chain reaction, and ultrastructural examination, we identified 18 simian-immunodeficiency-virus-infected macaques (16 Macaca mulatta, 1 M. nemestrina, and 1 M. cyclopis) with Enterocytozoon infection of the hepatobiliary system and small intestine. The organisms were readily identified in the bile ducts and gall bladder by special stains and by in situ hybridization using a probe directed against the small subunit ribosomal RNA of human origin E. bieneusi. Infection of the biliary system was associated with a nonsuppurative and proliferative cholecystitis and choledochitis. Hepatic involvement was characterized by bridging portal fibrosis and nodular hepatocellular regeneration accompanied by marked bile ductular and septal duct hyperplasia. Ultrastructurally, all developmental stages of the organism were found in direct contact with the host cell cytoplasm; spores and sporoblasts contained a double layer of polar tubes. Sequencing of a 607-bp segment of the small subunit ribosomal RNA revealed 97 and 100% identity to two clones of small subunit ribosomal RNA derived from E. bieneusi of human origin. Extensive morphological and genetic similarities between the simian and human enterocytozoons suggest that experimentally infected macaques may serve as a useful model of microsporidial infection in AIDS.

Animals↗

Detection of Cryptosporidium, Giardia and Enterocytozoon bieneusi in surface water, including recreational areas: a one-year prospective study.

Accidental ingestion of natural waters while bathing carries a risk of infection by waterborne protozoa such as Cryptosporidium, Giardia and, possibly, microsporidia. In order to evaluate this risk, we conducted a one-year prospective study of two recreational lakes and three river sites located near Paris, where bathing and boating are frequent. Twenty-litre water samples were collected monthly from each site. Concentrated samples were submitted to immunomagnetic separation followed by immunofluorescence (IMS-IF) for Cryptosporidium and Giardia detection. PCR and PCR restriction fragment length polymorphism (PCR-RFLP) were used for the genetic characterization of Cryptosporidium species on IMS-IF-positive samples. PCR were systematically performed to detect Enterocytozoon bieneusi. Bacteria counts were also determined. IMS-IF revealed low counts of Giardia cysts and Cryptosporidium oocysts in the recreational lakes, with occasional peaks (max. 165 cysts/10 L and 9 oocysts/10 L). By contrast, the river sites were consistently and sometimes heavily contaminated throughout the year. Enterocytozoon bieneusi was found in only two river samples. PCR-RFLP genotyping showed the presence of C. hominis and C. parvum. No correlation was found between the presence or counts of parasites and bacteria, except between the presence of Giardia and high counts of Escherichia coli and enterococci. Based on a previously developed model for quantitative risk assessment of waterborne parasitic infections, we estimated that the mean risk of infection by Cryptosporidium and Giardia associated with swimming was <10(-4) in the recreational lakes, and frequently higher at the river sites.

Animals↗

Detection of Enterocytozoon bieneusi (Microsporidia) by polymerase chain reaction (PCR) using species-specific primer in stool samples of HIV patients.

BACKGROUND & OBJECTIVE: Microsporidia are obligate intracellular parasites causing infections predominantly in immunocompromised patients. Enterocytozoon bieneusi is the most important microsporidian causing chronic diarrhoea in AIDS patients. The current method used for diagnosing the microsporidia spores is based on light microscopy using stained smears, which do not differentiate spores at species level. The present study was undertaken to detect microsporidia and confirm at species level (E. bieneusi) by PCR from stool samples of HIV positive patients. METHODS: During September 2002 to April 2003, stool samples from 153 HIV-positive patients (with chronic diarrhoea n = 105; without diarrhoea n=48) were collected and examined microscopically for microsporidia spores using modified Weber's chromotrope stain. Stool samples were subjected to PCR assay using species-specific primer EBIEFI/EBIER1, which amplifies small subunit ribosomal RNA (SSU rRNA) of this microsporidian RESULTS: A total of 10 HIV positive patients with chronic diarrhoea were positive for microsporidia by microscopic analysis and confirmed as Enterocytozoon bieneusi by PCR. No false positive results were observed. A diagnostic DNA fragment of 607 bp of the unique SSU rRNA was amplified from all samples infected with E. bieneusi. INTERPRETATION & CONCLUSION: The study revealed that polymerase chain reaction is a useful tool for accurate species identification of microsporidia in stool samples, which serves the benefit of treatment to the patients.

Base Sequence↗

Identification of the microsporidian parasite, Enterocytozoon bieneusi in faecal samples and intestinal biopsies from an AIDS patient.

The microsporidian parasite, Enterocytozoon bieneusi, is currently recognized as a potentially important cause of chronic diarrhoea in patients infected with the human immunodeficiency virus (HIV). Faecal concentrates from a 38-year-old, HIV-seropositive patient examined by light and electron microscopy revealed the presence of numerous microsporidian spores. The structural characteristics of the spores were consistent with those previously described for Enterocytozoon bieneusi. Each spore contained a single nucleus, a posterior vacuole and a polar filament with 6-7 overlapping coils which appeared in cross-section as a series of 3 doublets. Mature spores were surrounded by an inner unit membrane, an electron-lucent endospore and a thin, electron-dense exospore. The identity of the parasite was confirmed by the detection of unique endogenous developmental stages in duodenal biopsies. Both proliferative and sporogonial plasmodia (meronts and sporonts) were observed and all stages were monokaryotic (single nucleus) and apansporoblastic (sporophorous vesicle absent). Proliferative and sporogonial plasmodia divided by plasmotomy and spore organelles (polar filament, attachment disc and polaroplast) were well developed prior to fission of the sporogonial plasmodium.

AIDS-Related Opportunistic Infections↗

Enterocytozoon salmonis n. sp.: an intranuclear microsporidium from salmonid fish.

The developmental stages of a recently described microsporidian from the nucleus of hematopoietic cells of salmonid fish were found to be unique among the Microsporida. All observed stages, including meronts, sporonts, and spores were in direct contact with the host cell nucleus (principally hematopoietic cells) of chinook salmon (Oncorhynchus tshawytscha). There is no parasitophorous vacuole and sporogony does not involve formation of a pansporoblastic membrane as with other members of the suborder Apansporoblastina. The extrusion apparatus differentiates prior to division of sporogonial plasmodia. The spores are ovoid (1 x 2 microns) and uninucleate, and possess a coiled polar tube with 8-12 turns. Developmental stages of the salmonid microsporidian are similar to those described for Enterocytozoon bieneusi as found in the intestinal mucosa of human AIDS patients. However, the intranuclear development, different cell types, and host infected clearly separate the salmonid and human parasites. Accordingly, the intranuclear parasite of salmonids is given the name Enterocytozoon salmonis n. sp. within the suborder Apansporoblastina.

Animals↗

Short-term in vitro culture and molecular analysis of the microsporidian, Enterocytozoon bieneusi.

The microsporidium, Enterocytozoon bieneusi, causes a severe, debilitating, chronic diarrhea in patients with the acquired immunodeficiency syndrome. Specific diagnosis of intestinal microsporidiosis, especially due to Enterocytozoon, is difficult and there is no known therapy that can completely eradicate this parasite. Preliminary studies indicate that a short term (about 6 months) in vitro culture of this parasite yielding low numbers of spores, may be established by inoculating human lung fibroblasts and/or monkey kidney cell cultures with duodenal aspirates and or biopsy from infected patients. The cultures may subsequently be used for the isolation and molecular analysis of parasite DNA.

AIDS-Related Opportunistic Infections↗

Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water.

Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of Encephalitozoon intestinalis was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of Enterocytozoon bieneusi was confirmed in surface water; and the presence of Vittaforma corneae was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.

Animals↗

Localization of persistent Enterocytozoon bieneusi infection in normal rhesus macaques (Macaca mulatta) to the hepatobiliary tree.

Enterocytozoon bieneusi is the most common microsporidian parasite recognized in human patients with AIDS. Recently, we identified a virtually identical organism causing a spontaneous infection associated with hepatobiliary and intestinal disease in simian immunodeficiency virus (SIV)-infected macaques. To examine the natural history of the infection, we examined captive rhesus macaques for E. bieneusi by PCR, in situ hybridization, and cytochemical techniques. PCR performed on fecal DNA detected enterocytozoon infection in 22 (16.7%) of 131 normal rhesus macaques (Macaca mulatta), compared to 18 (33.8%) of 53 rhesus macaques experimentally inoculated with SIV. In normal rhesus macaques, persistence of infection was demonstrated for up to 262 days and was usually not associated with clinical signs. In six of seven normal rhesus animals, E. bieneusi was detected by PCR in bile obtained through percutaneous cholecystocentesis but not by in situ hybridization performed on endoscopic biopsies of duodenum and proximal jejunum.

Animals↗

[Identification of Enterocytozoon bieneusi in a patient with sclerosing cholangitis and AIDS].

Enterocytozoon bieneusi is the most common microsporidian parasite found in patients with AIDS. We report the clinical features of a patient with chronic diarrhea, pancreatitis, and AIDS-related sclerosing cholangitis. Ultrasonography and endoscopic retrograde cholangiopancreatography disclosed intrahepatic and extrahepatic bile duct changes identical to those seen in sclerosing cholangitis. Enterocytozoon bieneusi was found in duodenum and peripapillary duodenum by means of light microscopy, and confirmed by PCR amplification of paraffin-embedded tissues with species-specific primers. Microsporidian infection should be suspected in patients with advanced immunodeficiency and AIDS-related sclerosing cholangitis in our country.

AIDS-Related Opportunistic Infections↗

Use of furazolidone for the treatment of microsporidiosis due to Enterocytozoon bieneusi in patients with AIDS.

The efficacy of furazolidone for treatment of intestinal microsporidiosis due to Enterocytozoon bieneusi was studied in three patients with AIDS. All patients had chronic diarrhoea and weight loss. Mean CD4 cell count was 34.6/mm3. A course of furazolidone (100 mg orally four times a day) was given for 20 days. The drug was well tolerated and neither side effects nor alterations in the laboratory parameters were noted. Diarrhoea ceased within a mean of 12 days of starting treatment and clearance of microsporidian shedding in stool was observed. In one of the patients, however, symptomatic microsporidiosis recurred. Therefore furazolidone seems to have a transient but significant effect on intestinal infection due to Enterocytozoon bieneusi.

Acquired Immunodeficiency Syndrome↗

[Persistent diarrhea in HIV infected patients: role of Enterocytozoon bieneusi].

OBJECTIVES: To determine the epidemiologic, clinical and aetiologic features of chronic diarrhoea in patients with human immunodeficiency virus (HIV) infection. METHODS: Between January 1992 and April 1993, all HIV positive patients with chronic diarrhoea were enrolled in a prospective study. There were 46 patients in the study group including 33 (22 homosexuals) in stage C according to the 1993 Centers for Disease Control classification. RESULTS: Thirty-four pathogenic agents were isolated in 26 of the patients (57%). Enterocytozoon bieneusi was the most frequently found pathogen (11 patients, 24%) followed by Cryptosporidium sp (8 patients). Enterocytozoon bieneusi was found in association with other pathogens in 7 patients. All patients with microsporidiosis were in stage C (p = 0.04) and had a longer duration of diarrhoea (19.6 vs 9.8 weeks, p = 0.03), greater weight loss (9.6 vs 2.1 kg, p = 0.0003) and a lower Karnofsky index (48% vs 67%, p = 0.01). Prophylaxy with dapsone or pyrimethamine during the 3 months prior to inclusion had been more frequent (p = 0.04 and p = 0.05 respectively) in patients without microsporidiosis. CONCLUSION: Microsporidiosis should be suspected as a probable cause of chronic diarrhoea in HIV positive patients with a CD4 count < 100/mm3. Multiple infections may be a factor leading to treatment failure. Microsporidiosis seems to be linked to homosexual intercourse.

AIDS-Related Opportunistic Infections↗

Coinfection with Giardia lamblia and Enterocytozoon bieneusi in a patient with acquired immunodeficiency syndrome and chronic diarrhea.

Diarrhea is an important clinical problem in immunosuppressed patients with acquired immunodeficiency syndrome (AIDS). There are numerous classical as well as emerging enteric pathogens that can produce diarrhea; however, these agents can be missed when only one method, such as microbiological examination of stool, is used for diagnosis. The endoscopic biopsy is a sensitive method for diagnosis of many viral, fungal, and parasitic infections of the gastrointestinal tract. Although only one agent is often identified in mucosal biopsies from these immunosuppressed patients, coinfection with multiple microbial agents is being increasingly recognized. Giardia infection is not as prevalent as other pathogens in patients with AIDS, but it remains an important diarrheal agent that is potentially curable. However, there have been only rare reports of coinfections with giardiasis and other infectious agents. This report describes a patient with AIDS and chronic diarrhea who had repeated negative stool examinations for ova and parasites. Light and electron microscopic examination of subsequent endoscopically obtained small intestinal biopsies revealed coinfection with two parasites, Enterocytozoon bieneusi and Giardia lamblia. Following treatment with metronidazole for giardiasis, the diarrhea persisted, but was less severe. This report also describes the diagnostic features of Giardia and Enterocytozoon infections in biopsy tissues and emphasizes the importance of identifying enteric coinfections in patients with AIDS to ensure timely and specific modes of therapy.

AIDS-Related Opportunistic Infections↗

Diagnosis of Enterocytozoon bieneusi (microsporidia) infections by polymerase chain reaction in stool samples using primers based on the region coding for small-subunit ribosomal RNA.

OBJECTIVE: Enterocytozoon bieneusi is the most prevalent microsporidian causing chronic diarrhea in patients with acquired immunodeficiency syndrome. The current methods used for routine diagnosis of infections caused by microsporidia are based on microscopic detection of the microorganism spores in stained smears. We evaluated the usefulness of the polymerase chain reaction (PCR) technique as a tool to diagnose Enterocytozoon bieneusi infections, using the species-specific diagnostic primer pair EBIEF1/EBIER1 on stool samples that were also analyzed by optical microscopy. DESIGN: To perform PCR in such samples, we developed a novel protocol to obtain DNA free of PCR inhibitors. This protocol was based on disruption of spores using glass beads and overnight digestion with proteinase K; final purification was accomplished with the RapidPrep Micro Genomic DNA isolation Kit for Cells and Tissues (Pharmacia Biotech Inc, Piscataway, NJ). We also evaluated this approach on aliquots of a sample fixed in formalin from 1 to 10 days. PATIENTS AND SAMPLES: We evaluated the PCR technique on 64 stool samples obtained from patients with acquired immunodeficiency syndrome who had persistent chronic diarrhea. Patients were from Spain, Brazil, Germany, and the United States. RESULTS: Using this approach, we could confirm the presence of E bieneusi in all 17 positive samples; no false-positive results were observed. We could also amplify E bieneusi DNA in 10 aliquots of one sample fixed up to 10 days in 10% formalin. CONCLUSION: We conclude that PCR technology is very suitable for species identification of microsporidia in stool samples and may have a potential application in prospective studies in formalin-fixed samples.

Animals↗