Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 26, Num. 4, 2008, pp. 407-407

Indian Journal of Medical Microbiology, Vol. 26, No. 4, October-December, 2008, pp. 407

Correspondence

Comparison of weber green and ryan blue modified trichrome staining for the diagnosis of microsporidial spores from stool samples of HIV-positive patients with diarrhoea

Patil K, De A, Mathur M

Department of Microbiology, LTM Medical College, Sion, Mumbai - 400 022, Maharashtra
Correspondence Address:Department of Microbiology, LTM Medical College, Sion, Mumbai - 400 022, Maharashtra
pati_kg_79@rediffmail.com

Date of Submission: 21-Nov-2007
Date of Acceptance: 20-Mar-2008

Code Number: mb08131

Dear Editor,

Microsporidia is increasingly being reported in the recent years in HIV-positive patients with diarrhoea. ^[1],[2] Electron microscopy, though a gold standard for detection of microsporidial spores, ^[2] can only be used in sophisticated laboratories. Intestinal biopsy, though better, is not advocated in HIV-infected patients with diarrhoea because of its invasive nature. ^[3] therefore, trichrome staining still remains the most commonly used method for detection of microsporidial spores in routine laboratories. Therefore, on 30 stool samples of HIV-positive patients with diarrhoea, modified trichrome staining, i.e., Chromotrope 2R staining, was performed in duplicate - one method using fast green (Weber Green method) ^[4] and the other method using aniline blue (Ryan Blue method). ^[5] The procedure of staining was similar for both. Smears were examined under oil immersion lens for pink-coloured spores 1 to 2 µm in diameter. Background appeared green in case of fast green and blue when aniline blue was used. Photographs of microsporidial spores by two staining methods are shown (Fig.) .

Weber Green method could detect microsporidia in 10% of the samples, whereas Ryan Blue method detected the same in 6.67% of the samples. By validity method, sensitivity and specificity of Ryan Blue method were 60% and 50.9% respectively taking Weber Green method as the gold standard. The positive predictive value was 10%, and negative predictive value was 93.3%.

Ryan et al. ^[5] reported a good contrast between microsporidial spores by using aniline blue as an alternative to fast green in Chromotrope 2R stain. They observed that fast green is quite pale and fades rapidly as compared to aniline blue, and use of aniline blue has the advantage of yeast cells and pseudohyphae staining greyish blue. In contrast, results of this study showed better results with fast green staining. However, the sample size was very small and further study is required in this regard. Other modifications of trichrome stain, like Gram Chromotrope staining, need to be tried.

References

1.	Florez AC, Garcia DA, Moncada L, Beltran M. Prevalence of icrosporidia and other intestinal parasites in patients with HIV infection. Biomedica 2003;23:274-82.  Back to cited text no. 1
2.	Satheeshkumar S, Ananthan S. Electron microscopy identification of microsporidia ( Enterocytozoon bieneusi ) and Cyclospora cayatenensis from stool samples of HIV infected patients. Indian J Med Microbiol 2004;22:119-22.  Back to cited text no. 2  [PUBMED]
3.	Conteas CN, Sowerby T, Berlin G, Dahlan F, Nguyen A, Porschen R, et al . Fluorescence techniques for diagnosing intestinal microsporidiosis in stool, enteric fluid and biopsy specimens from acquired immunodeficiency syndrome patients with chronic diarrhoea. Arch Pathol Lab Med 1996;120:847-53.  Back to cited text no. 3
4.	Weber R, Bryan RT, Schwartz DA, Owen RL. Human microsporidial infections. Clin Microbiol Rev 1994;7:426-61.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.	Ryan NJ, Sutherland G, Coughlan K, Globan M, Doultree J, Marshall J, 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.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]

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