Encephalitozoon intestinalis microsporidiosis was thus shown in two of the four patients examined. In two patients, therapy based on albendazole made stools. Encephalitozoon intestinalis is transmitted in contaminated water and initially infects gastro-intestinal enterocytes, leading to diarrheal disease. Encephalitozoon intestinalis is a recently described microsporidian which causes intestinal and disseminated infections in severely immunocompromised.
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The general health of these patients was not impaired, and human immunodeficiency virus screening was negative.
Immune evaluation, including the study of lymphocytic subpopulations, assay of serum immunoglobulins, and an intradermal multitest, showed normal results. Molecular identification of microsporidian species was based on the PCR amplification of a small-subunit rRNA sequence followed by Hin fI endonuclease restriction. Encephalitozoon intestinalis microsporidiosis was thus shown in two of the four patients examined.
In two patients, therapy based on albendazole made stools devoid of microsporidian spores without influence on the intestinal disorders. The pathogenic role of E. Microsporidia are ubiquitous intracellular parasitic protozoa affecting the whole animal kingdom 4. Seven genera are pathogenic in humans: EncephalitozoonEnterocytozoonNosemaPleistophoraVittaformaTrachypleistophoraand Microsporidiumthe latter including all species intestinwlis undetermined status.
Some of them are known to be opportunistic pathogens in immunodepressed patients 8. They may cause chronic diarrhea with food malabsorption, as well as disseminated impairments 237. Ocular and gastrointestinal failures related to microsporidia have also been described in immunocompetent ones 15101417 Staining methods 1922 and intestiinalis tests 1520212324 can differentiate microsporidia from bacteria and yeasts in clinical samples such as stool samples, but precise identification of the species involved is intesyinalis always successful.
We screened four human immunodeficiency virus HIV -negative travelers with chronic diarrhea for microsporidia and gave them a complete immunologic evaluation. After detection of spores in stool samples by light encsphalitozoon, we tried to identify the corresponding parasite species.
However, electron microscopy failed to provide evidence of microsporidia and no significant results were obtained for the detection of Encephalitozoon cuniculi by Western blotting and enzyme-linked immunosorbent assay. Thus, we decided to develop a new PCR procedure ensuring the differentiation of any known microsporidian species pathogenic to humans. This procedure enabled us to identify Encephalitozoon intestinalis in two immunocompetent patients.
The patients were travelers presenting with chronic diarrhea. Treatment with albendazole mg twice daily was prescribed for 20 days, and the patients were re-examined 1 month after the end of inteestinalis treatment. Septata intestinalis Cali, Kotler, and Orenstein 2subsequently reclassified as Encephalitozoon intestinalis by Hartskeerl et al.
Supernatants containing mature spores were collected; spores were then sedimented by centrifugation, encephalitozoonn, and stored in 0. Mechanical disruption was performed with zirconium beads 0. C1 was complementary to bases 1 to 18 of each one, C2 was complementary to bases to of E. Two different volumes of each DNA preparation were regularly tested: Amplified products were electrophoretically analyzed on agarose gel and stained with ethidium bromide.
Digest fragments were electrophoretically analyzed on agarose gel and stained with ethidium bromide.
CDC – DPDx – Microsporidiosis
A PCR amplification procedure has been developed to facilitate the detection of microsporidia in human samples. The primers were designed to amplify a large part of the 16S rDNA from four microsporidian species, including the two major species involved in intestinal diseases: When DNAs extracted from spores of E. Analysis of PCR products by 1. DNAs were extracted from E. Thus, digestion of amplified products with the restriction endonuclease Hin dIII may be used to distinguish between the two candidate genera for intestinal infection.
Results of digestion experiments are shown in Fig. This indicated that these patients were infected with E. Shown is a 1. Lane 7 contained a negative control. Two bands were obtained from E. Amplified DNA fragments of E. Lane m contained the same molecular size markers as lane m in Fig.
To identify Encephalitozoon species, Hin fI digestion of the 1,bp amplicons was performed. From the examination of sequences deposited in GenBank, it could be predicted that these species may be differentiated on the basis of the number of Hin fI restriction sites: Species-specific restriction patterns were indeed observed, the sizes of the different fragments being and bp for E.
It was confirmed that the two immunocompromised patients were infected with E. Digestion of a 1,bp amplicon by restriction endonuclease Hin fI. For this study, four travelers were selected. They were males with a mean age of 29 years and had had diarrhea for 1 to 71 months. They had traveled in Africa, Nepal, or Southeast Asia. Only one patient complained of nonsystematic abdominal pain.
Immunoglobulin levels in serum were normal, except in the patient with protracted diarrhea 6 yearswhose immunoglobulin A IgA concentration was 0.
The patients were also screened for parasites by direct examination, culture of stools, and a Ziehl-Neelsen test for cryptosporidia, and no evidence of eukaryotic pathogens other than microsporidia was obtained. The above-described PCR amplification procedure was applied to stool samples from four nonimmunocompromised patients. Hin fI digestion showed the four bands which are relatable to the presence of E.
The expected amplified envephalitozoon were obtained, the detection limit of particular samples varying between 20 and spores per 0. Repeated sampling provided the same results. ebcephalitozoon
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Our PCR method involves the digestion of an amplified product intetsinalis described by Fedorko et al. Since it has been recently shown that E. This has been achieved through Hin fI digestion of the 1,bp amplicon, which provides specific banding patterns.
The data support an E. Our DNA extraction and analysis protocol for stool samples is realizable in 1 day. This time could be shortened if a heating procedure were used for cell encephaliozoon Microsporidial pathogenicity in immunocompetent patients is still poorly understood, and difficulties in diagnosis persist.
In the patients examined here, the Uvitex 2B and Weber staining methods 1922 provided evidence of microsporidian infection independent of the HIV syndrome.
An IgA deficit can be ruled out encephalitozoonn our patients. This deficit occurs frequently, and it may be asymptomatic and detected at a late stage of ecnephalitozoon.
IgA plays an important role in mucosal immunity, especially in the intestine, so that a partial secretion deficit might promote the development of microsporidia. In the literature, we found several reported cases of intestinal microsporidia inteestinalis the absence of immunodeficiency proven on the basis of negative HIV tests 1101417 One single case of E. Albendazole treatment led to the elimination of E. Encephqlitozoon, as the clinical signs persisted, the pathogenic role of E. Amplification failed to detect microsporidia in patients IC2 and IC4, while staining methods gave positive responses.
Two interpretations may be considered: As shown by Katzwinkel-Wladarsch et al. Results of our experiments involving the addition of cultured parasites to negative stool samples suggest that the concentration of microscopically encephalitkzoon microsporidian spores was probably below 20 spores per 0. Patient IC4 was treated with albendazole.
The clinical signs subsided, and microsporidium-like spores were still detected wncephalitozoon Uvitex 2B. Thus, the hypothesis that another pathogenic organism was responsible for the chronic diarrhea cannot be excluded. In conclusion, although microsporidia in stool samples encsphalitozoon be easily detected by staining procedures, our PCR protocol should be of interest for the rapid determination of any particular species.
We thank Elizabeth U. Canning for providing reference isolates of E. Van Gool for providing a reference isolate of E. National Center for Biotechnology InformationU. Journal List J Clin Microbiol v. Author information Article notes Copyright and License information Disclaimer.
This article has been cited by other articles in PMC. Stool samples and parasite cultures. Digestion of PCR products. Open in a separate window. Diagnosis of diarrheic travelers. Delbac contributed equally to this study. Bull Soc Pathol Exot. Septata intestinalisn. Ultrastructural identification of AIDS associated microsporidiosis. Canning E U, Lom J.
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The microsporidia of vertebrates. Immunologic and molecular characteristics of Encephalitozoon -like microsporidia isolated from humans and rabbits indicate that Encephalitozoon cuniculi is itnestinalis zoonotic parasite. Occurrence of a new microsporidian: Human microsporidiosis and AIDS: Identification of microsporidia in stool specimens by using PCR and restriction endonucleases.