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PubMed1998

In vitro susceptibilities of the microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis to albendazole and its sulfoxide and sulfone metabolites.

In vitro comparisons demonstrated that the efficacy of albendazole, albendazole-sulfoxide, and albendazole-sulfone against pathogenic Encephalitozoon species was proportional to the degree of oxidation at a concentration of >10(-3) microgram/ml. Furthermore, at a concentration of <10(-2) microgram/ml, benzimidazoles were more effective against Encephalitozoon cuniculi and Encephalitozoon hellem than against Encephalitozoon intestinalis.

Albendazole↗

PubMedDate not supplied

Ribosomal DNA sequences of Encephalitozoon hellem and Encephalitozoon cuniculi: species identification and phylogenetic construction.

A segment of ribosomal DNA, about 1,350 base pairs long, was amplified from the microsporidian species Encephalitozoon hellem, isolated from AIDS patients, and Encephalitozoon cuniculi. The amplified DNA segment extends from position 530 in the small ribosomal RNA subunit to position 580 in the large ribosomal RNA subunit. A comparison of sequence data from this region for Encephalitozoon hellem and Encephalitozoon cuniculi shows relatively high sequence similarity, supporting the placement of these two organisms in the same genus. At the same time, sequence differences between the two organisms confirm that they are not the same species. Three separate isolates of E. hellem were sequenced in the highly variable intervening spacer region. The sequence was identical for all three isolates. Within the amplified DNA segment, regions were sequenced which yield highly variable, moderately variable and highly conserved sequence information, appropriate for comparison with other species in the phylum Microspora at all taxonomic levels. We suggest that sequence data from these regions be included in future species descriptions for the purposes of species identification and phylogenetic analysis. Restriction digests of the amplified region are presented and give a rapid method for distinguishing between the two Encephalitozoon species.

Animals↗

PubMed1996

Immunologic and molecular characteristics of Encephalitozoon-like microsporidia isolated from humans and rabbits indicate that Encephalitozoon cuniculi is a zoonotic parasite.

To assess the zoonotic potential of Encephalitozoon-like microsporidia, we isolated and cultivated spores from specimens of urine, respiratory secretions, and stool from six patients infected with human immunodeficiency virus and from nine rabbits. Because spores of Encephalitozoon-like species are indistinguishable by microscopy, we characterized the isolates by western blot analysis and by restriction enzyme analysis of the small subunit (SSU) rDNA after amplification by the polymerase chain reaction. We identified Septata intestinalis in one patient and Encephalitozoon hellem in two symptomatic patients. Encephalitozoon cuniculi was found in all rabbits and in three patients. One of these patients had clinical manifestations of infection with this parasite (severe interstitial pneumonitis). We observed abatement of symptoms and cessation of parasite excretion when these patients were treated with albendazole. Our findings suggest that E. cuniculi may be pathogenic in humans and that it is a zoonotic parasite.

AIDS-Related Opportunistic Infections↗

PubMed2002

[Encephalitozoon cuniculi and Encephalitozoon intestinalis--causes of opportunistic infections].

Microsporidia (phylum Microsporidia) are intracellular parasites that infect a wide range of protozoa, invertebrates and vertebrate hosts. Over a 1000 species have been classified into approximately 100 genera. Historically, microsporidial infections in silkworms, honey bees, and salmonid fish have been responsible for significant economic losses. More recently, microsporidiosis has been recognized as an important opportunistic infection in immunologically compromised patients. In this review there is information on the immunobiology of microsporidia Encephalitozoon cuniculi and Encephalitozoon intestinalis which were identified as the most common causative agents of microsporidiosis in mammals. Most of what is known about the immunology of microsporidiosis is based on experiments with the microsporidian Encephalitozoon cuniculi.

Animals↗

PubMed1993

Detection of microsporidian spores in clinical samples by indirect fluorescent-antibody assay using whole-cell antisera to Encephalitozoon cuniculi and Encephalitozoon hellem.

Three polyclonal mouse antisera, to Encephalitozoon cuniculi, Nosema algerae, and Nosema corneum, and two polyclonal rabbit antisera, to E. cuniculi and Encephalitozoon hellem, were used in an indirect fluorescent-antibody assay (IFA) with Enterocytozoon bieneusi, E. cuniculi, and Encephalitgozoon. hellem spores (spores of the last two were taken from culture). Enterocytozoon bieneusi cannot be cultured. By IFA, antisera to E. cuniculi and E. hellem reacted strongly and equally with each other's spores. The mouse antisera reacted strongly with the homologous species, but for these there was segmental and particulate or "dot" staining of heterologous microsporidian spores, indicating cross-reactions with more selected antigens. In fecal samples, cross-reactions with both mouse and rabbit antisera were sometimes seen with different yeast species, with species of streptococci, and species of gram-negative rods. There were no cross-reactions to staphylococci. Enterocytozoon bieneusi was easily identified in duodenal and colonic biopsies, duodenal and colonic fluids, and feces of symptomatic AIDS patients by IFA. In a study of 12 AIDS patients with diarrhea, the new IFA identified microsporidia in all of 11 fecal samples, three colon fluids, six duodenal fluids, and three duodenal biopsy touch preparations. Although the fecal sample of 1 of the 12 was negative, the patient's duodenal fluid contained microsporidian spores by IFA.

Animals↗

PubMed1996

Effects of nifedipine, metronidazole, and nitric oxide donors on spore germination and cell culture infection of the microsporidia Encephalitozoon hellem and Encephalitozoon intestinalis.

Two species of microsporidia, Encephalitozoon hellem and Encephalitozoon intestinalis, were isolated from AIDS patients and cultured in green monkey kidney cells. A spore germination assay and a cultured-cell infection assay were used to test the efficacy of candidate antiparasitic agents. The calcium channel blocker nifedipine, metronidazole, and two nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, were tested in the two assays. Nifedipine (10(-8) M) significantly inhibited E. hellem spore germination in three of four germination media. Metronidazole (10(-5) M) inhibited germination weakly and significantly inhibited E. intestinalis germination in a single germination medium. The inhibitory effect of nifedipine and metronidazole used together was greater than the sum of the effects of the drugs used alone in all E. hellem germination assays. The NO donors also inhibited spore germination. The inhibitory effect of nifedipine and metronidazole could be reversed by washing the spores, while that of the NO donors was not reversible. In early cultured-cell infections, both nifedipine (10(-8) M) and metronidazole (10(-5) M) significantly reduced the number of cells being infected. As the infection spread, these agents were less effective. Some inhibition of the spread of the infection was also demonstrated with the NO donors at a concentration (10(-5) M) not obviously toxic to the cultured cells. These data suggest that combination drug therapy targeting spore germination and intracellular parasite development is promising.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi, Encephalitozoon hellem, and Vittaforma corneae.

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.

Aminopeptidases↗

PubMed2005

Phylogenetic relationships of methionine aminopeptidase 2 among Encephalitozoon species and genotypes of microsporidia.

This report describes the characterization and phylogenetic analysis of the deduced amino acid sequences of methionine aminopeptidase 2 (MetAP-2) enzymes from microsporidian species and genotypes of the genus Encephalitozoon. Fragments of DNA encoding 318 to 335 amino acid residues of the MetAP-2 genes were isolated from genomic DNA prepared from cultured spores of Encephalitozoon hellem, Encephalitozoon intestinalis, and Encephalitozoon cuniculi genotypes I-III. Sequence comparisons of the deduced amino acid residues indicated that the microsporidian sequences are MetAP-2-like rather than MetAP-1-like. Alignments demonstrated that the new Encephalitozoon sequences included sequences and structures conserved in eukaryotic MetAP-2s, including the five conserved, active site residues, Asp, Asp, His, Glu, and His, considered to be critical for catalysis and for coordinating the cation (e.g., cobalt) co-factor, and included residues known to interact with the antibiotic, fumagillin. The primary structure of the Encephalitozoon MetAP-2s, however, showed some dissimilarity with human and yeast MetAP-2s, including the absence of the NH2-terminal polylysine tract. Phylogenetic comparison of these Encephalitozoon MetAP-2s with orthologues from related species and from other informative taxa confirmed that the MetAP-2s of these Encephalitozoon species and strains are closely related to each other and cluster with MetAP-2s.

Amino Acid Sequence↗

PubMed1997

Simple diagnosis of Encephalitozoon sp. microsporidial infections by using a panspecific antiexospore monoclonal antibody.

Microsporidia (phylum Microsproa) have recently become recognized as common opportunistic protozoans in the United States and worldwide, particularly affecting immunodeficient patients. Microsporidian organisms within the genus Encephalitozoon are the cause of nephrologic, ophthalmic, pneumologic, gastroenteric, and systemic infections. However, diagnosis of the small spores by light microscopy is difficult, even with newly developed and improved staining techniques. We have developed an anti-Encephalitozoon species monoclonal antibody-based immunoassay for easy diagnosis. A hybridoma was produced and selected following one main criterion: recognition by immunofluorescence of all known Encephalitozoon spores affecting humans. The selected monoclonal antibody-secreting hybridomas were characterized by enzyme-linked immunosorbent assay, immunofluorescence, Western blot, and immunoelectron microscopy using Encephalitozoon species from fresh and fixed samples from patients and from in vitro cultures. In the immunofluorescence assay, one monoclonal antibody, termed 3B6, strongly recognized Encephalitozoon cuniculi, E. hellem, and E. intestinalis. Monoclonal antibody 3B6 bound to other microsporidia (Nosema and Vairimorpha spp.) without cross-reacting with any other parasite, including Enterocytozoon bieneusi, fungus, or bacterium tested. In immunoelectron microscopy assays, monoclonal antibody 3B6 bound to the exospore of Encephalitozoon species, while in Western blot assays, it recognized three to seven antigens with molecular masses ranging from 34 to 117 kDa. We have developed a sensitive and specific monoclonal antibody-based immunoassay to diagnose common microsporidian infections, particularly with Encephalitozoon species. This is a new tool for identifying spores in bodily fluids and biopsy samples and is an efficient diagnostic test. Additionally, monoclonal antibody 3B6 can serve to assess the prevalence of microsporidial infections in immunodeficient and immunocompetent patients.

AIDS-Related Opportunistic Infections↗

PubMed1997

High seroprevalence of Encephalitozoon species in immunocompetent subjects.

Encephalitozoon species are important pathogens in human immunodeficiency virus-infected patients. However, in immunocompetent persons, little is known about Encephalitozoon infections, mainly because of the lack of reliable diagnostic tests. To improve diagnosis, three serologic techniques that use Encephalitozoon intestinalis as antigen were developed: an ELISA, an immunofluorescence technique (IFAT), and a counterimmunoelectrophoresis (CIE) method. The serologic response against E. intestinalis was studied in sera from 300 Dutch blood donors and 276 pregnant French women. For confirmation of specificity, sera from 150 subjects with various infectious and noninfectious diseases were examined. ELISA, IFAT, and CIE were specific for microsporidia infections, and IFAT and CIE were specific for Encephalitozoon infections. High antibody titers against Encephalitozoon organisms were found in 24 (8%) of 300 Dutch blood donors and in 13 (5%) of 276 pregnant French women. The high seroprevalence against Encephalitozoon species in Dutch blood donors and French women suggests that Encephalitozoon infection is common in immunocompetent subjects.

Adolescent↗

PubMed2004

Serodiagnostic studies in an immunocompetent individual infected with Encephalitozoon cuniculi.

Little is known about the prevalence and clinical significance of infection with Encephalitozoon species in immunocompetent individuals. In the present study, by using indirect immunofluorescence technique (IFAT), Western blot, and recombinant antigens of the spore wall (SWP1) and polar tube (PTP1, PTP2, and PTP3 ), we analyzed the IgG antibody response of a laboratory worker who was infected with Encephalitozoon cuniculi. Serum samples were analyzed 1, 20, 32, and 38 months after infection. After 1 month, by use of IFAT, only spore-wall antigens were recognized, an antibody reaction that changed toward both the spore wall and polar tube in the following months. By use of Western blot analysis, a characteristic pattern that recognized multiple bands was noticed. Reaction against SWP1 was present in all 4 serum samples. The IgG response against PTP1, PTP2, and PTP3 was not detectable 1 month after infection, but became evident in the follow-up serum samples. Serum samples showed cross-reactivity with the spore wall of Encephalitozoon hellem and Encephalitozoon intestinalis, but only little cross-reactivity with the polar tube of these parasites. This is the first study to our knowledge that provides full details about the antibody response against a specified Encephalitozoon species in an immunocompetent person. The results strongly encourage the development and use of reliable serodiagnostic methods, which will provide information about the prevalence and clinical significance of Encephalitozoon species infection in humans.

Animals↗

PubMed1996

Diagnosis of disseminated microsporidian Encephalitozoon hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin.

A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identified to the E. hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates were identical to each other and to E. hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly, with no recurrence of ophthalmologic signs. The results of this study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. hellem, and E. intestinalis in clinical specimens.

AIDS-Related Opportunistic Infections↗

PubMed2005

Direct agglutination test for Encephalitozoon cuniculi.

Encephalitozoon cuniculi is a small protozoan parasite in the phylum Microspora. It has been shown to naturally infect several host species, including humans. Infection with microsporidia is usually asymptomatic, except in young or immunocompromised hosts. Currently, serological diagnosis of infection is made using the indirect immunofluorescent antibody assay (IFA) or enzyme-linked immunosorbent assay (ELISA). Although these methods are sensitive and reliable, there are several drawbacks to the IFA and ELISA tests. Cross-reactivity between other Encephalitozoon species is common, and specialized equipment is required to conduct these tests. This paper reports the development of a direct agglutination test for detecting IgG antibodies to E. cuniculi. The utility of the agglutination test was examined in CD-1 and C3H/He mice infected with E. cuniculi or one of 2 other Encephalitozoon species. Test sera were incubated overnight with eosin-stained microsporidia spores in round-bottom microtiter plates. In positive samples, agglutination of spores with antibodies in test sera resulted in an opaque mat spread across the well. The results indicate that the agglutination test is 86% sensitive and 98% specific for E. cuniculi, with limited cross-reactivity to Encephalitozoon intestinalis. No cross-reactivity to Encephalitozoon hellem was observed. The test is fast and easy to conduct, and species-specific antibodies are not required.

Agglutination Tests↗

PubMed1995

Genetic and immunological characterization of the microsporidian Septata intestinalis Cali, Kotler and Orenstein, 1993: reclassification to Encephalitozoon intestinalis.

The relationships between the Encephalitozoon-like Septata intestinalis and other microsporidia that occur in humans; notably Encephalitozoon cuniculi and Encephalitozoon hellem, is insufficiently documented using morphological descriptions alone. To assess mutual relationships, we have examined other phenotypic as well as genetic aspects of S. intestinalis, obtained both from tissue culture and clinical specimens, in comparison with a number of other microsporidia. Phenotypic characterization was performed by analysis of the protein composition and antigenic structure of various microsporidian spores by SDS-PAGE and Western blotting. The genetic characterization consisted of the determination of the sequence of the S. intestinalis rrs gene encoding the small subunit ribosomal RNA (srRNA), restriction fragment length polymorphism (RFLP) analysis of amplified rrs genes and establishment of the degree of sequence identity between rrs genes of various microsporidian species. The unique sequence of rrs of S. intestinalis as well as the distinct RFLP and SDS-PAGE profiles indicate that S. intestinalis is clearly different from other human microsporidian species. However, its rrs gene shared about 90% sequence identity with rrs of both Encephalitozoon spp., E. cuniculi and E. hellem. This is remarkably higher than the about 70% identity observed between rrs of microsporidian species which belong to different genera and thus suggests that S. intestinalis should be regarded as a species of the genus Encephalitozoon.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Inter- and intra-species karyotype variations among microsporidia of the genus Encephalitozoon as determined by pulsed-field gel electrophoresis.

Disseminated infections due to microsporidia of the genus Encephalitozoon are detected increasingly, especially in patients with AIDS. Identification of microsporidia can be achieved by a variety of immunological and molecular methods. This study evaluates the feasibility of pulsed-field gel electrophoresis (PFGE) for the analysis of karyotypes of the 3 known species of this genus (Encephalitozoon cuniculi, Encephalitozoon hellem and Encephalitozoon intestinalis) and of 2 of the 3 known E. cuniculi strains (strains I and III). Eleven chromosomal DNA bands were resolved for E. cuniculi and 10 chromosomal DNA bands for E. hellem and E. intestinalis, with molecular sizes ranging from 231 to 320 kb, from 197 to 288 kb and from 195 to 285 kb, respectively, resulting in estimated genome sizes of about 3.0 Mb, 2.5 Mb and 2.4 Mb. Different PFGE chromosomal banding patterns indicate that not only E. cuniculi, as previously described, but also E. hellem, represent a heterogeneous entity. PFGE is a valuable method of evaluating inter- and intra-species variations among Encephalitozoon species that may enable the identification of environmental sources of infection and modes of transmission.

Animals↗

PubMed1997

Use of a fluorescent probe to assess the activities of candidate agents against intracellular forms of Encephalitozoon microsporidia.

Microsporidia are obligate intracellular protozoan parasites. Three species of the genus Encephalitozoon are among the microsporidia that infect immunodeficient humans. These species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis, all develop in a parasitophorous vacuole within a host cell. The present study describes a method that uses the fluorescent probe calcein and confocal microscopy to detect drug-induced effects in Encephalitozoon-infected green monkey kidney cells. The effects were as follows: (i) changes in parasite organization within the parasitophorous vacuole; (ii) swelling and gross morphological changes of parasite developing stages in situ; (iii) killing of developing parasite stages in situ, detected by their uptake of the fluorescent probe; and (iv) reduction in the viability of the host cell population, assessed by the loss of the probe. Verapamil and itraconazole were used to increase the vital dye loading by both uninfected and infected cells. Agents with known antimicrosporidial activity, albendazole and fumagillin, caused all three types of parasite changes at concentrations that had no detectable effect on host cell viability. The effective doses of albendazole and fumagillin that caused swelling and disorganization of parasite developing stages were 5 x 10(-7) and 10(-6) M respectively. Killing of developing stages was detected at 10-fold-higher concentrations for these agents and at 10(-5) M for metronidazole. This method can be used to screen candidate antimicrosporidial agents in infected cultured cells.

Albendazole↗

PubMed2005

Application of specific primers in the diagnosis of Encephalitozoon spp.

In our experiment, 3 species-specific primer pairs cultivated in cell lines were used: Encephalitozoon cuniculi -specific primer pairs (ECUNF and ECUNR), Encephalitozoon hellem -specific primer pairs (EHELF and EHELR), and Encephalitozoon intestinalis -specific primer pairs (SINTF and SINTR). The PCR products were estimated to be 550 bp in E. cuniculi , 547 bp in E. hellem and 545 bp in E. intestinalis respectively, which can prove the precision and reliability of this method in the species identification of the genus Encephalitozoon. All 3 primer pairs were species-specific and none of them amplified gene sequences from other Encephalitozoon spp.

AIDS-Related Opportunistic Infections↗

PubMed1992

Disseminated microsporidiosis (Encephalitozoon hellem) and acquired immunodeficiency syndrome. Autopsy evidence for respiratory acquisition.

Microsporidia are obligate intracellular protozoal parasites that infect a variety of cell types in a broad range of invertebrates and vertebrates. They have recently come to medical attention due to the increased frequency with which members of two microsporidian genera, Enterocytozoon and Encephalitozoon, are being diagnosed in patients with the acquired immunodeficiency syndrome (AIDS). The majority of published reports of human microsporidiosis describe Enterocytozoon infection of small intestinal enterocytes. In addition, a growing number of AIDS patients have been identified with infection due to the two species of Encephalitozoon-Encephalitozoon cuniculi and Encephalitozoon hellem, observed in conjunctival, corneal, and, recently, sinonasal tissues. However, there are scant data regarding the systemic pathology and epidemiology of these infections. This article describes a patient with AIDS who died with systemic Encephalitozoon infection. The etiologic microsporidian was found to be E hellem by using antemortem biochemical and antigenic analyses. A complete autopsy, the first to be reported in a patient with this infection, revealed organisms in the eyes, urinary tract, and respiratory tract. A surprising observation was the occurrence of numerous organisms within the lining epithelium of almost the entire length of the tracheobronchial tree, suggestive of respiratory acquisition. Detailed light and electron microscopic findings and the biological and diagnostic features of microsporidiosis are discussed.

Acquired Immunodeficiency Syndrome↗