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Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction


In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for speciesspecific detection ofEncephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, andEnterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected withEnterocytozoon bieneusi (n=9),Encephalitozoon spp. (n=2), andEncephalitozoon intestinalis (n=1) as well as stool spiked with spores ofEncephalitozoon cuniculi andEncephalitozoon hellem and tissue cultures ofEncephalitozoon cuniculi andEncephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection ofEnterocytozoon bieneusi andEncephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected withEncephalitozoon cuniculi andEncephalitozoon hellem. Moreover, identification ofEncephalitozoon spp. could be specified asEncephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates speciesspecific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.

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  1. 1.

    Weber R, Bryan RT, Schwartz DA, Owen RL: Human microsporidial infections. Clinical Microbiology Reviews 1994, 7: 426–461.

  2. 2.

    Nägeli KW: Über die neue Krankheit der Seidenraupe und verwandte Organismen. Botanische Zeitung 1857, 15: 760–761.

  3. 3.

    Matsubayashi H, Koike T, Mikata T, Hagiwara S: A case ofEncephalitozoon-like body infection in man. Archives of Pathology 1959, 67: 181–187.

  4. 4.

    Schwartz DA, Sobottka I, Leitch GJ, Cali A, Visvesvara GS: Pathology of microsporidiosis. Archives of Pathology and Laboratory Medicine 1996, 120: 173–188.

  5. 5.

    Sobottka I, Albrecht H, Schottelius J, Schmetz C, Bentfeld M, Laufs R, Schwartz DA: Self-limited traveller's diarrhea due to a dual infection withEnterocytozoon bieneusi andCryptosporidium parvum in an immunocompetent HIV-negative child. European Journal of Clinical Microbiology & Infectious Diseases 1995, 14: 919–920.

  6. 6.

    Margileth AM, Strano AJ, Chandra R, Neafie R, Blum M, McCully RM: Disseminated nosematosis in an immunologically compromised infant. Archives of Pathology 1973, 95: 145–150.

  7. 7.

    Ledford DK, Overman MD, Gonzalo A, Cali A, Mester W, Lockey RF: Microsporidiosis myositis in a patient with acquired immunodeficiency syndrome. Annals of Internal Medicine 1985, 102: 628–630.

  8. 8.

    Hollister WS, Canning EU, Weidner E, Field AS, Kench J, Marriott DJ: Development and ultrastructure ofTrachipleistophora hominis n.g., n.sp. after in vitro isolation from an AIDS patient and inoculation into athymic mice. Parasitology 1996, 112: 143–154.

  9. 9.

    Silveira H, Canning EU:Vittaforma corneae n.comb. for the human microsporidiumNosema corneum Shadduck, Meccoli, Davis & Font, 1990, based on its ultrastructure in the liver of experimentally infected athymic mice. Journal of Eukaryotic Microbiology 1995, 42: 158–165.

  10. 10.

    Desportes I, Le Charpentier Y, Galian A, Bernard F, Cochand-Priollet B, Lavergne A, Ravisse P, Modigliani R: Occurrence of a new microsporidian:Enterocytozoon bieneusi n.g., n.sp., in the enterocytes of a human patient with AIDS. Journal of Protozoology 1985, 32: 250–254.

  11. 11.

    Levaditi C, Nicolau S, Schoen R: L'agent étiologique de l'encephalite épizootique du lapin (Encephalitozoon cuniculi). Comptes Rendus des Séances de la Société de Biologie et de ses Filiales 1923, 89: 984–986.

  12. 12.

    Didier ES, Didier PJ, Friedberg DN, Stenson SM, Orenstein JM, Yee RW, Tio FO, Davis RM, Vossbrinck CR, Millichamp N, Shadduck JA: Isolation and characterization of a new human microsporidian,Encephalitozoon hellem (n.sp.), from three AIDS patients with keratoconjunctivitis. Journal of Infectious Diseases 1991, 163: 617–621.

  13. 13.

    Cali A, Kotler DR, Orenstein JM:Septata intestinalis n.g., n.sp., an intestinal microsporidian associated with chronic diarrhea and dissemination in AIDS patients. Journal of Eukaryotic Microbiology 1993, 40: 101–112.

  14. 14.

    Hartskeerl RA, van Gool T, Schuitema ARJ, Didier ES, Terpstra WJ: Genetic and immunological characterization of the microsporidianSeptata intestinalis Cali, Kotler and Orenstein, 1993: reclassification toEncephalitozoon intestinalis. Parasitology 1995, 110: 277–285.

  15. 15.

    Bryan RT: Microsporidiosis as an AIDS-related opportunistic infection. Clinical Infectious Diseases 1995, 21, Supplement 1: 62–65.

  16. 16.

    Schottelius J, Lo Y, Schmetz C:Septata intestinalis andEncephalitozoon cuniculi: cross-reactivity between two microsporidian species. Folia Parasitologica 1995, 42: 169–172.

  17. 17.

    Sobottka I, Albrecht H, Schäfer H, Schottelius J, Visvesvara GS, Laufs R, Schwartz DA: DisseminatedEncephalitozoon (Septata) intestinalis infection in a patient with AIDS: novel diagnostic approaches and autopsy-confirmed parasitological cure following treatment with albendazole. Journal of Clinical Microbiology 1995, 33: 2948–2952.

  18. 18.

    Asmuth DM, de Girolami PC, Federman M, Ezratty CR, Pleskow DK, Desai G, Wanke CA: Clinical features of microsporidiosis in patients with AIDS. Clinical Infectious Diseases 1994, 18: 819–825.

  19. 19.

    Kotler DP: Gastrointestinal manifestations of human immunodeficiency virus infection. Advances in Internal Medicine 1995, 40: 197–242.

  20. 20.

    Albrecht H, Stellbrink HJ, Sobottka I: Failure of itraconazole to preventEnterocytozoon bieneusi infection. Genitourinary Medicine 1995, 71: 325–326.

  21. 21.

    Didier PJ, Didier ES, Orenstein JM, Shadduck JA: Fine structure of a new human microsporidian,Encephalitozoon hellem, in culture. Journal of Protozoology 1991, 38: 502–507.

  22. 22.

    Fedorko DP, Nelson NA, Cartwright CP: Identification of microsporidia in stool specimens by using PCR and restriction endonucleases. Journal of Clinical Microbiology 1995, 33: 1739–1741.

  23. 23.

    Franzen C, Müller A, Hegener P, Salzberger B, Hartmann P, Fätkenheuer G, Diehl V, Schrappe M: Detection of microsporidia (Enterocytozoon bieneusi) in intestinal biopsy specimens from human immunodeficiency virus-infected patients by PCR. Journal of Clinical Microbiology 1995, 33: 2294–2296.

  24. 24.

    Franzen C, Küppers R, Müller A, Salzberger B, Fätkenheuer G, Vetten B, Diehl V, Schrappe M: Genetic evidence for latentSeptata intestinalis infection in human immunodeficiency virus-infected patients with intestinal microsporidiosis. Journal of Infectious Diseases 1996, 173: 1038–1040.

  25. 25.

    Katzwinkel-Wladarsch S, Lieb M, Heise W, Löscher T, Rinder H: Direct amplification and species determination of microsporidian DNA from stool specimens. Tropical Medicine and International Health 1996, 1: 373–378.

  26. 26.

    Visvesvara GS, Leitch GL, Da Silva AJ, Croppo GP, Moura H, Wallace S, Slemenda SB, Schwartz DA, Moss D, Bryan RT, Pieniazek NJ: Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian,Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. Journal of Clinical Microbiology 1994, 32: 2760–2768.

  27. 27.

    Weiss LM, Zhu X, Cali A, Tanowitz HB, Wittner M: Utility of microsporidian rRNA in diagnosis and phylogeny: a review. Folia Parasitologica 1994, 41: 81–90.

  28. 28.

    Zhu X, Wittner M, Tanowitz HB, Kotler D, Cali A, Weiss LM: Small subunit rRNA sequence ofEnterocytozoon bieneusi and its potential diagnostic role with use of the polymerase chain reaction. Journal of Infectious Diseases 1993, 168: 1570–1575.

  29. 29.

    Ombrouck C, Ciceron L, Biligui S, Brown S, Marechal P, van Gool T, Datry A, Danis M, Desportes-Livage I: Specific PCR assay for direct detection of intestinal microsporidiaEnterocytozoon bieneusi andEncephalitozoon intestinalis in fecal specimens from human immunodeficiency virus-infected patients. Journal of Clinical Microbiology 1997, 35: 652–655.

  30. 30.

    Velásquez JN, Carnevale S, Guarnera EA, Labbé JH, Chertcoff A, Cabrera MG, Rodriguez MI: Detection of the microsporidian parasiteEnterocytozoon bieneusi in specimens from patients with AIDS by PCR. Journal of Clinical Microbiology 1996, 34: 3230–3232.

  31. 31.

    Deplazes P, Mathis A, Baumgartner R, Tanner I, Weber R: Immunologic and molecular characteristics ofEncephalitozoon-like microsporidia isolated from humans and rabbits indicate thatEncephalitozoon cuniculi is a zoonotic parasite. Clinical Infectious Diseases 1996, 22: 557–559.

  32. 32.

    Van Gool T, Canning EU, Dankert J: An improved practical and sensitive technique for the detection of microsporidian spores in stool samples. Transactions of the Royal Society of Tropical Medicine and Hygiene 1994, 88: 189–190.

  33. 33.

    Vavra J, Dahbiova R, Hollister WS, Canning EU: Staining of microsporidian spores by optical brighteners with remarks on the use of brighteners for the diagnosis of AIDS-associated human microsporidioses. Folia Parasitologica 1993, 40: 267–272.

  34. 34.

    Weber R, Bryan RT, Owen RL, Wilcox CM, Gorelkin L, Visvesvara GS: Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. New England Journal of Medicine 1992, 326: 161–166.

  35. 35.

    Visvesvara GS, Da Silva AJ, Croppo GR, Pieniazek NJ, Leitch GJ, Ferguson D, de Moura H, Wallace S, Slemenda SB, Tyrrell I, Moore DF, Meador J: In vitro culture and serologic and molecular identification ofSeptata intestinalis isolated from urine of a patient with AIDS. Journal of Clinical Microbiology 1995, 33: 930–936.

  36. 36.

    Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-Van Dillen PME, van der Noordaa J: Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 1990, 28: 495–503.

  37. 37.

    Vossbrinck CR, Baker MD, Didier ES, Debrunner-Vossbrinck BA, Shadduck JA: Ribosomal DNA sequences ofEncephalitozoon hellem andEncephalitozoon cuniculi: species identification and phylogenetic construction. Journal of Eukaryotic Microbiology 1993, 40: 354–362.

  38. 38.

    Baker MD, Vossbrinck CR, Didier ES, Maddox JV, Shadduck JA: Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms. Journal of Eukaryotic Microbiology 1995, 42: 564–570.

  39. 39.

    Weitz JC, Bothelo R, Bryan RT: Microsporidiosis en pacientes con diarrea cronica y SIDA, HIV asintomaticos y pacientes con diarrea aguda. Revista Medica de Chile 1995, 123: 849–856.

  40. 40.

    Blanshard C: Hollister WS, Peacock CS, Tovey DG, Ellis DS, Canning EU, Gazzard BG: Simultaneous infection with two types of intestinal microsporidia in a patient with AIDS. Gut 1992, 33: 418–420.

  41. 41.

    Van Gool T, Canning EU, Gilis H, van den Bergh-Weerman MA, Eeftinck-Schattenkerk JK, Dankert J:Septata intestinalis frequently isolated from stool of AIDS patients with a new cultivation method. Parasitology 1994, 109: 281–289.

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Correspondence to N. P. Kock.

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Kock, N.P., Petersen, H., Fenner, T. et al. Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction. Eur. J. Clin. Microbiol. Infect. Dis. 16, 369–376 (1997).

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  • Polymerase Chain Reaction
  • Polymerase Chain Reaction Assay
  • Stool Sample
  • Nest Polymerase Chain Reaction
  • Stool Specimen