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. 1996 Nov;34(11):2670–2673. doi: 10.1128/jcm.34.11.2670-2673.1996

Modified technique to recover microsporidian spores in sodium acetate-acetic acid-formalin-fixed fecal samples by light microscopy and correlation with transmission electron microscopy.

P L Carter 1, D W MacPherson 1, R A McKenzie 1
PMCID: PMC229383  PMID: 8897162

Abstract

Microsporidia are an emerging cause of significant disease, particularly in the immunocompromised host. Until recently, the diagnosis of enteric infections has required invasive sampling, the use of expensive technology, and considerable technological expertise. The purpose of the present study was to examine three modifications to the processing of fecal specimens for light microscopy (LM) examination for microsporidian spores: the use of pretreatment with potassium hydroxide, modified centrifugation conditions, and a modified staining technique. A sodium acetate-acetic acid-formalin-fixed fecal sample containing numerous microsporidian spores confirmed to be positive by transmission electron microscopy (TEM) was used in all studies performed. A simulation of a heavy to lightly infected individual was used. The results of LM were correlated with those of TEM. Duplicate smears were stained with Weber's modified trichrome and Giemsa (GS) stains. The stained slides were randomized and examined blindly by LM at x 625 and x 1,250 magnifications. A portion of the dilutions after centrifugation were fixed for TEM. The Weber modified trichrome stain performance rating was higher than the Giemsa stain rating because of ease of interpretation, and material stained with Weber modified trichrome stain required less examination time at a lower magnification. The number of positive smears and the quantity of spores detected were significantly higher following pretreatment of the sample with KOH. TEM was positive only when numerous spores were present, but the quality of the photomicrographs was superior after pretreatment with KOH. Pretreatment of sodium acetate-acetic acid-formalin-fixed fecal samples with 10% KOH and then a 5-min centrifugation time and staining with Weber modified trichrome stain provide for the excellent recovery of microsporidia in the routine diagnostic parasitology laboratory.

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Selected References

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  1. 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]
  2. Blanshard C., Hollister W. S., Peacock C. S., Tovey D. G., Ellis D. S., Canning E. U., Gazzard B. G. Simultaneous infection with two types of intestinal microsporidia in a patient with AIDS. Gut. 1992 Mar;33(3):418–420. doi: 10.1136/gut.33.3.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cali A., Kotler D. P., Orenstein J. M. Septata intestinalis N. G., N. Sp., an intestinal microsporidian associated with chronic diarrhea and dissemination in AIDS patients. J Eukaryot Microbiol. 1993 Jan-Feb;40(1):101–112. doi: 10.1111/j.1550-7408.1993.tb04889.x. [DOI] [PubMed] [Google Scholar]
  4. Cali A., Owen R. L. Intracellular development of Enterocytozoon, a unique microsporidian found in the intestine of AIDS patients. J Protozool. 1990 Mar-Apr;37(2):145–155. doi: 10.1111/j.1550-7408.1990.tb05885.x. [DOI] [PubMed] [Google Scholar]
  5. Curry A., Canning E. U. Human microsporidiosis. J Infect. 1993 Nov;27(3):229–236. doi: 10.1016/0163-4453(93)91923-d. [DOI] [PubMed] [Google Scholar]
  6. DeGirolami P. C., Ezratty C. R., Desai G., McCullough A., Asmuth D., Wanke C., Federman M. Diagnosis of intestinal microsporidiosis by examination of stool and duodenal aspirate with Weber's modified trichrome and Uvitex 2B strains. J Clin Microbiol. 1995 Apr;33(4):805–810. doi: 10.1128/jcm.33.4.805-810.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Dore G. J., Marriott D. J., Hing M. C., Harkness J. L., Field A. S. Disseminated microsporidiosis due to Septata intestinalis in nine patients infected with the human immunodeficiency virus: response to therapy with albendazole. Clin Infect Dis. 1995 Jul;21(1):70–76. doi: 10.1093/clinids/21.1.70. [DOI] [PubMed] [Google Scholar]
  9. Doultree J. C., Maerz A. L., Ryan N. J., Baird R. W., Wright E., Crowe S. M., Marshall J. A. In vitro growth of the microsporidian Septata intestinalis from an AIDS patient with disseminated illness. J Clin Microbiol. 1995 Feb;33(2):463–470. doi: 10.1128/jcm.33.2.463-470.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Garcia L. S., Shimizu R. Y., Bruckner D. A. Detection of microsporidial spores in fecal specimens from patients diagnosed with cryptosporidiosis. J Clin Microbiol. 1994 Jul;32(7):1739–1741. doi: 10.1128/jcm.32.7.1739-1741.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Molina J. M., Oksenhendler E., Beauvais B., Sarfati C., Jaccard A., Derouin F., Modaï J. Disseminated microsporidiosis due to Septata intestinalis in patients with AIDS: clinical features and response to albendazole therapy. J Infect Dis. 1995 Jan;171(1):245–249. doi: 10.1093/infdis/171.1.245. [DOI] [PubMed] [Google Scholar]
  15. Ombrouck C., Romestand B., da Costa J. M., Desportes-Livage I., Datry A., Coste F., Bouix G., Gentilini M. Use of cross-reactive antigens of the microsporidian Glugea atherinae for the possible detection of Enterocytozoon bieneusi by western blot. Am J Trop Med Hyg. 1995 Jan;52(1):89–93. doi: 10.4269/ajtmh.1995.52.89. [DOI] [PubMed] [Google Scholar]
  16. Orenstein J. M. Microsporidiosis in the acquired immunodeficiency syndrome. J Parasitol. 1991 Dec;77(6):843–864. [PubMed] [Google Scholar]
  17. Orenstein J. M., Zierdt W., Zierdt C., Kotler D. P. Identification of spores of Enterocytozoon bieneusi in stool and duodenal fluid from AIDS patients. Lancet. 1990 Nov 3;336(8723):1127–1128. doi: 10.1016/0140-6736(90)92600-m. [DOI] [PubMed] [Google Scholar]
  18. Pol S., Romana C. A., Richard S., Amouyal P., Desportes-Livage I., Carnot F., Pays J. F., Berthelot P. Microsporidia infection in patients with the human immunodeficiency virus and unexplained cholangitis. N Engl J Med. 1993 Jan 14;328(2):95–99. doi: 10.1056/NEJM199301143280204. [DOI] [PubMed] [Google Scholar]
  19. Rijpstra A. C., Canning E. U., Van Ketel R. J., Eeftinck Schattenkerk J. K., Laarman J. J. Use of light microscopy to diagnose small-intestinal microsporidiosis in patients with AIDS. J Infect Dis. 1988 Apr;157(4):827–831. doi: 10.1093/infdis/157.4.827. [DOI] [PubMed] [Google Scholar]
  20. Schwartz D. A., Bryan R. T., Hewan-Lowe K. O., Visvesvara G. S., Weber R., Cali A., Angritt P. Disseminated microsporidiosis (Encephalitozoon hellem) and acquired immunodeficiency syndrome. Autopsy evidence for respiratory acquisition. Arch Pathol Lab Med. 1992 Jun;116(6):660–668. [PubMed] [Google Scholar]
  21. Shadduck J. A., Greeley E. Microsporidia and human infections. Clin Microbiol Rev. 1989 Apr;2(2):158–165. doi: 10.1128/cmr.2.2.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Visvesvara G. S., Leitch G. J., da Silva A. J., Croppo G. P., Moura H., Wallace S., Slemenda S. B., Schwartz D. A., Moss D., Bryan R. T. Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. J Clin Microbiol. 1994 Nov;32(11):2760–2768. doi: 10.1128/jcm.32.11.2760-2768.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Wang J. T., Lin J. T., Sheu J. C., Wang T. H., Chen D. S. Hepatitis E virus and posttransfusion hepatitis. J Infect Dis. 1994 Jan;169(1):229–230. doi: 10.1093/infdis/169.1.229. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Weber R., Kuster H., Keller R., Bächi T., Spycher M. A., Briner J., Russi E., Lüthy R. Pulmonary and intestinal microsporidiosis in a patient with the acquired immunodeficiency syndrome. Am Rev Respir Dis. 1992 Dec;146(6):1603–1605. doi: 10.1164/ajrccm/146.6.1603. [DOI] [PubMed] [Google Scholar]
  27. Yang J., Scholten T. A fixative for intestinal parasites permitting the use of concentration and permanent staining procedures. Am J Clin Pathol. 1977 Mar;67(3):300–304. doi: 10.1093/ajcp/67.3.300. [DOI] [PubMed] [Google Scholar]
  28. van Gool T., Hollister W. S., Schattenkerk W. E., Van den Bergh Weerman M. A., Terpstra W. J., van Ketel R. J., Reiss P., Canning E. U. Diagnosis of Enterocytozoon bieneusi microsporidiosis in AIDS patients by recovery of spores from faeces. Lancet. 1990 Sep 15;336(8716):697–698. doi: 10.1016/0140-6736(90)92198-q. [DOI] [PubMed] [Google Scholar]
  29. 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]

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