Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1997 Mar;35(3):652–655. doi: 10.1128/jcm.35.3.652-655.1997

Specific PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal specimens from human immunodeficiency virus-infected patients.

C Ombrouck 1, L Ciceron 1, S Biligui 1, S Brown 1, P Marechal 1, T van Gool 1, A Datry 1, M Danis 1, I Desportes-Livage 1
PMCID: PMC229644  PMID: 9041406

Abstract

A routine assay based on the PCR was developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples. Two oligonucleotide primer pairs from a conserved region in the small-subunit rRNA genes of E. bieneusi (primer pair V1 and EB450) and E. intestinalis (primer pair V1 and SI500) were used to amplify microsporidian DNA. We achieved specific amplification of a 382-bp DNA fragment in E. intestinalis and a 353-bp DNA fragment in E. bieneusi. Boiling of the samples appeared to be most effective for DNA extraction. Fecal samples containing fewer than 10 microsporidia gave a positive result in the PCR assay. Fecal specimens from 30 human immunodeficiency virus-infected patients with microsporidiosis and fecal specimens from 42 patients suspected of having microsporidiosis were investigated by the PCR assay. The PCR assay was validated against standard staining methods (the Uvitex 2B and Chromotrope 2R staining methods) and immunofluorescence assay specific for E. intestinalis. This comparative study has shown that PCR improved species determination and can thus be considered a fast and reliable method for the detection and identification of each intestinal species.

Full Text

The Full Text of this article is available as a PDF (543.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aldras A. M., Orenstein J. M., Kotler D. P., Shadduck J. A., Didier E. S. Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies. J Clin Microbiol. 1994 Mar;32(3):608–612. doi: 10.1128/jcm.32.3.608-612.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asmuth D. M., DeGirolami P. C., Federman M., Ezratty C. R., Pleskow D. K., Desai G., Wanke C. A. Clinical features of microsporidiosis in patients with AIDS. Clin Infect Dis. 1994 May;18(5):819–825. doi: 10.1093/clinids/18.5.819. [DOI] [PubMed] [Google Scholar]
  3. Challier S., Brown S., Ombrouck C., Desportes-Livage I., De Nay D., Gentilini M. Flow cytometry as a possible method of isolation of spores of the microsporidian Enterocytozoon bieneusi. J Eukaryot Microbiol. 1994 Sep-Oct;41(5):27S–27S. [PubMed] [Google Scholar]
  4. Desportes I., Le Charpentier Y., Galian A., Bernard F., Cochand-Priollet B., Lavergne A., Ravisse P., Modigliani R. Occurrence of a new microsporidan: Enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with AIDS. J Protozool. 1985 May;32(2):250–254. doi: 10.1111/j.1550-7408.1985.tb03046.x. [DOI] [PubMed] [Google Scholar]
  5. Didier E. S., Orenstein J. M., Aldras A., Bertucci D., Rogers L. B., Janney F. A. Comparison of three staining methods for detecting microsporidia in fluids. J Clin Microbiol. 1995 Dec;33(12):3138–3145. doi: 10.1128/jcm.33.12.3138-3145.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fedorko D. P., Nelson N. A., Cartwright C. P. Identification of microsporidia in stool specimens by using PCR and restriction endonucleases. J Clin Microbiol. 1995 Jul;33(7):1739–1741. doi: 10.1128/jcm.33.7.1739-1741.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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. J Clin Microbiol. 1995 Sep;33(9):2294–2296. doi: 10.1128/jcm.33.9.2294-2296.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hartskeerl R. A., Schuitema A. R., van Gool T., Terpstra W. J. Genetic evidence for the occurrence of extra-intestinal Enterocytozoon bieneusi infections. Nucleic Acids Res. 1993 Aug 25;21(17):4150–4150. doi: 10.1093/nar/21.17.4150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hartskeerl R. A., Van Gool T., Schuitema A. R., Didier E. S., Terpstra W. J. Genetic and immunological characterization of the microsporidian Septata intestinalis Cali, Kotler and Orenstein, 1993: reclassification to Encephalitozoon intestinalis. Parasitology. 1995 Apr;110(Pt 3):277–285. doi: 10.1017/s0031182000080860. [DOI] [PubMed] [Google Scholar]
  10. Kokoskin E., Gyorkos T. W., Camus A., Cedilotte L., Purtill T., Ward B. Modified technique for efficient detection of microsporidia. J Clin Microbiol. 1994 Apr;32(4):1074–1075. doi: 10.1128/jcm.32.4.1074-1075.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kotler D. P. Gastrointestinal manifestations of human immunodeficiency virus infection. Adv Intern Med. 1995;40:197–242. [PubMed] [Google Scholar]
  12. Ombrouck C., Ciceron L., Desportes-Livage I. Specific and rapid detection of Microsporidia in stool specimens from AIDS patients by PCR. Parasite. 1996 Mar;3(1):85–86. doi: 10.1051/parasite/1996031085. [DOI] [PubMed] [Google Scholar]
  13. Ombrouck C., Desportes-Livage I., Achbarou A., Gentilini M. Specific detection of the microsporidia Encephalitozoon intestinalis in AIDS patients. C R Acad Sci III. 1996 Jan;319(1):39–43. [PubMed] [Google Scholar]
  14. Orenstein J. M. Microsporidiosis in the acquired immunodeficiency syndrome. J Parasitol. 1991 Dec;77(6):843–864. [PubMed] [Google Scholar]
  15. Van Eys G. J., Gravekamp C., Gerritsen M. J., Quint W., Cornelissen M. T., Schegget J. T., Terpstra W. J. Detection of leptospires in urine by polymerase chain reaction. J Clin Microbiol. 1989 Oct;27(10):2258–2262. doi: 10.1128/jcm.27.10.2258-2262.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Visvesvara G. S., da Silva A. J., Croppo G. P., Pieniazek N. J., Leitch G. J., Ferguson D., de Moura H., Wallace S., Slemenda S. B., Tyrrell I. In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS. J Clin Microbiol. 1995 Apr;33(4):930–936. doi: 10.1128/jcm.33.4.930-936.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Vávra J., Dahbiová R., Hollister W. S., Canning E. U. Staining of microsporidian spores by optical brighteners with remarks on the use of brighteners for the diagnosis of AIDS associated human microsporidioses. Folia Parasitol (Praha) 1993;40(4):267–272. [PubMed] [Google Scholar]
  18. Weber R., Bryan R. T., Owen R. L., Wilcox C. M., Gorelkin L., Visvesvara G. S. Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. The Enteric Opportunistic Infections Working Group. N Engl J Med. 1992 Jan 16;326(3):161–166. doi: 10.1056/NEJM199201163260304. [DOI] [PubMed] [Google Scholar]
  19. Weiss L. M., Zhu X., Cali A., Tanowitz H. B., Wittner M. Utility of microsporidian rRNA in diagnosis and phylogeny: a review. Folia Parasitol (Praha) 1994;41(2):81–90. [PubMed] [Google Scholar]
  20. Widjojoatmodjo M. N., Fluit A. C., Torensma R., Verdonk G. P., Verhoef J. The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples. J Clin Microbiol. 1992 Dec;30(12):3195–3199. doi: 10.1128/jcm.30.12.3195-3199.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zhu X., Wittner M., Tanowitz H. B., Kotler D., Cali A., Weiss L. M. Small subunit rRNA sequence of Enterocytozoon bieneusi and its potential diagnostic role with use of the polymerase chain reaction. J Infect Dis. 1993 Dec;168(6):1570–1575. doi: 10.1093/infdis/168.6.1570. [DOI] [PubMed] [Google Scholar]
  22. Zierdt C. H., Gill V. J., Zierdt W. S. Detection of microsporidian spores in clinical samples by indirect fluorescent-antibody assay using whole-cell antisera to Encephalitozoon cuniculi and Encephalitozoon hellem. J Clin Microbiol. 1993 Nov;31(11):3071–3074. doi: 10.1128/jcm.31.11.3071-3074.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. da Silva A. J., Schwartz D. A., Visvesvara G. S., de Moura H., Slemenda S. B., Pieniazek N. J. Sensitive PCR diagnosis of Infections by Enterocytozoon bieneusi (microsporidia) using primers based on the region coding for small-subunit rRNA. J Clin Microbiol. 1996 Apr;34(4):986–987. doi: 10.1128/jcm.34.4.986-987.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. van Gool T., Canning E. U., Gilis H., van den Bergh Weerman M. A., Eeftinck Schattenkerk J. K., Dankert J. Septata intestinalis frequently isolated from stool of AIDS patients with a new cultivation method. Parasitology. 1994 Sep;109(Pt 3):281–289. doi: 10.1017/s0031182000078318. [DOI] [PubMed] [Google Scholar]
  25. van Gool T., Snijders F., Reiss P., Eeftinck Schattenkerk J. K., van den Bergh Weerman M. A., Bartelsman J. F., Bruins J. J., Canning E. U., Dankert J. Diagnosis of intestinal and disseminated microsporidial infections in patients with HIV by a new rapid fluorescence technique. J Clin Pathol. 1993 Aug;46(8):694–699. doi: 10.1136/jcp.46.8.694. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES