Comparison of Three Staining Methods for the Detection of Intestinal Microspora Spp.


 Background: This study aimed to compare three staining methods including: Calcofluor white, Chromotrope and Quick Hot Gram chromotrope used in diagnosis of intestinal microsporidial spores.

Methods: One hundred and seventy five stool specimens were collected from patients referred to Laboratory of Intestinal Protozoology at the School of Public Health, Tehran university of Medical Sciences during 2012-2013. All of specimens were evaluated by nested PCR. The formalin–fixed stool samples were prepared from each specimen and dried at room temperature for 10 min, followed by 10 min methanol fixation. All the collected stoolsamples were evaluated blindly by calcofluor white, Chromotrope and Quick Hot Gram chromotrope staining methods separately.

Results : Microsporidial spores were recognized using Chromotrope, Quick Hot Gram chromotrope and Calcofluor white, in16 of 18 (88.8%), 17 of 18 (94.4%) and 18 of18(100%) samples that were positive by nested PCR respectively. Regarding 14 stool samples that were negative by nested PCR, 14 cases were negative bychromotrope and Quick hot Gram chromotrope and 13 samples were negative by Calcofluor white. One discordant sample interpreted as false positive.

Conclusion: Calcofluor white staining had the best performance for the detection of intestinal Microsprora spores and can be used as initial screen test for the detection of intestinal Microspora spp


Weber R, Bryan RT, Owen RL, Wilcox CM, etal. Improved light-microcopical detection of microsporidia spores in stool and duodenal aspirates.N Engl J Med. 1992; 326: 161-6.

Garcia LS. Laboratory Identification of the Microsporidia. J Clin Microbiol. 2002; 40(6):1892-1901.

Muller A, Stellermann K, Hartmann P,Schrappe M, et al. A powerful DNA extraction method and PCR for detection of microsporidiain clinical stool specimens. Clin Diag Lab Immunol. 1999; 6:243-6.

Subrungruang I, Mungthin M, Chavalitshewinkoon-Petmitr P, Rangsin R, et al. Evaluation of DNA Extraction and PCR Methods forDetection of Enterocytozoon bienuesi in StoolSpecimens. J Clin Microbiol. 2004; 42(8): 3490-4.

Saigal K, Khurana S, Shama A, Sehgal R, Malla N. Comparison of staining techniques and multiplex nested PCR for diagnosis of intestinalmicrosporidiosis. Diagn Microbiol Infect Dis. 2013; 77(3): 248-9.

Ryan NJ, Sutherland G, Coughlan K, Globan M, et al. A new trichrome-blue stain for detection of microsporidial species in urine, stool,and nasopharyngeal specimens. J Clin Microbiol.1993; 31: 3264-9.

Kokoskin E, Gyorkos T, Camus A, Celidotte L, Purtill T, Ward B. Modified technique for efficient detection of microsporidia. J Clin Microbiol.1994; 32:1074-5.

Vavra J, Dahbiova R. Hollister WS, Canning EU. Staining of microporidian spores by optical brighteners with remarks on the use ofbrighteners for the diagnosis of AIDS associated human microsporidiosis. Folia Parasitol.1993; 40:267-72.

Vavra J, Chalupsky J. Fluorescence staining of microporidian spores with the brightener calcofluor white M2R. J Protozool. 1982;29(Suppl.):503.

Vicki AL, Stewart BK, Bergeron DL, et al. Use of the fluorochrome calcofluor white in the screening of stool specimens for spores of microsporidia.Am J Clin Pathol. 1995; 103:656-9.

Moura H, Da Silva JL, Sodre FC, Brasil P, et al. Gram-Chromotrope: a New Technique that Enhances Detection of Microsporidial Sporesin Clinical Samples. J Euk Microbiol. 1996;43(5):94S-95S.

Moura H, Schwartz DA, Bornay-Llinares F,Sodré FC, et al. A new and improved "quickhot Gram-Chromotrope" technique that differentiallystains microporidian spores in clinicalsamples, including paraffin-embedded tissue sections. Arch Pathol Lab Med. 1997; 121(8):888-93.

Mirjalali H, Mohebali M, Mirhendi H, Gholami R, et al. Emerging intestinal microsporidial infection in HIV+/AIDS patients in Iran: microscopicand molecular detection. Iran J Parasitol.2014; 9(2):149-54.

Orenstein JM. Microsporidiosis in the acquired immunodeficiency Syndrome. J Parasitol. 1991;77: 843–64.

15. Wuhib T, Silva TM, Newman RD, et al.Cryptosporidial and microsporidial infections in human immunodeficiency virus-infected patientsin northeastern Brazil. J Infect Dis. 1994;170:494-7.

Shadduck JA. Human microsporidiosis in AIDS. Rev Infect Dis.1989; 11:203-7.

Kotler DP, Orenstein JM. Prevalence of intestinal microsporidiosis in HIV-infected individ referred for gastrointestinal evaluation. Am J Gastroenterol. 1994; 89:1998-2002.

Molina JM, Sarfati C, Beauvais B, Lemann M,et al. Intestinal microsporidiosis in human immunodeficiency virus infected patients withchronic unexplained diarrhea: prevalence and clinical and biologic features. J Infect Dis.1993;167:217- 21.

Isenberg HD. Clinical microbiology procedures handbook. American Society for MicrobiologyPress: Washington, D.C; 1992.

Isenberg HD. Essential procedures for clinical microbiology. American Society for Microbiology Press: Washington, D.C; 1995.

Didier SD, Orenstein JM, Aldras A, et al.Comparison of three staining methods for detecting microsporidia in fluids. J Clin Microbiol.1995; 33: 3138-45.

Harrington BJ, Hageage GJ. Calcofluor white:A Review of its Uses and Applications in Clinical Mycology and Parasitology. Lab Med. 2003;5(34):361-7.

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Calcofluor Chromotrope Gram stain Microspora Staining

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Khanaliha K, Mirjalali H, Mohebali M, Tarighi F, Rezaeian M. Comparison of Three Staining Methods for the Detection of Intestinal Microspora Spp. Iran J Parasitol. 1;9(4):445-451.