Thanks to Dr Fan and coworkers for their letter and interests in our article.
The conclusion drawn by us was that confocal microscopy was a rapid and sensitive diagnostic tool for both early diagnosis and non-invasive follow -up of fungal keratitis, not that it was a superior to culture and corneal biopsy staining techniques in the early stage of fungal keratitis. It is rapid compared to culture and biopsy staining techniques, since we were able to detect fungal hyphae in all rabbit eyes 2 days after fungal inoculation, but at least 2-3 days had to be elapsed to determine any fungal growth on Sabouraud's agar. Moreover, O'Day et al reported that about one fourth of fungal cultures became positive only after 2 weeks. Confocal microscopy is also rapid compared to biopsy staining, since to perform calcofluor staining some time had to be elapsed. As stated in our article  'Although in our model Sabouraud's agar and corneal biopsy techniques showed similar sensitivity (100%) in the early stage, confocal microscopy appears to have a definitive advantage in the later stages of infection, since not all cases of fungal keratitis could be cultured.' In the abstract section we wrote that 'on days 14 and 22 confocal microscopy was more sensitive than culture technique in both treated and untreated animals, since not all cases of fungal keratitis could be cultured.' I think the conclusion drawn is valid in lights of the data provided in the study. In the second experiment, 6 rabbits were treated with topical fluconazole, 7 rabbits were treated with oral fluconazole and 7 rabbits were left untreated. On day 14, we observed hyphal fragments (broken in treated corneas and full size in untreated ones) in each 20 corneas by confocal microscopy. However, only eight of 20 scrapings grew Aspergillus fumigatus on Sabouraud's agar culture. The difference between groups was statistically significant as appeared in the text by utilizing chi square test. Similarly, on day 22 confocal microscopy revelaed hyphal fragments totally 14 corneas out of 20 (three in the topically treated, four in the orally treated, and 7 in the untreated groups). At this stage only 5 corneal scrapings grew fungus on culture. The difference was statistically significant again as appeared in the article by utilizing chi square test. Thus, superiority of confocal microscopy over culture technique on days 14 and 22 in treated and untreated rabbits was supported well by the data presented in the article.
In the result section, we were attempting to determine the efficacies of topical and oral fluconazole treatment by culture. However, p values were not correct as a result of typewriting error. The typewriting errors must be escaped both our and reviewer's attentions. However, this part of result section does not contain any information that could affect any conclusion drawn as a result of study data. Actually, this part was not directly linked to the main aim of the study. The authors wish to thank to Dr Fan and coworkers for their careful attentions. The correct p values were given below.
On day 14 (p=0,383 and p=0,296)
On day 22 (p=0342 and p=0,279).
(1) Dorothy SP Fan, David TL Liu, Wai-Man Chan, Dennis SC Lam. Comments on Confocal Microscopy of Aspergillus Fumigatus Keratitis [electronic response to Avunduk et al Confocal microscopy of Aspergillus fumigatus keratitis] bjophthalmol.com 2003http://bjo.bmjjournals.com/cgi/eletters/87/4/409#212
(2) O'Day DM, Akrabawi PL, Head WS, et al. Laboratory isolation techniques in human and experimental fungal infections. Am J Ophthalmol. 1979;87:688-93.
(3) Avunduk AM, Beuerman RW, Varnell ED, Kaufman HE. Confocal microscopy of aspergillus fumigatus keratitis. Br J Ophthalmol 2003; 87: 409-10.
Comments on Confocal Microscopy of Aspergillus Fumigatus Keratitis
We read with great interest the article of Avunduk and coworkers, who conducted a study in using confocal microscopy to evaluate Aspergillus fumigatus keratitis in treated and untreated rabbits eyes. They concluded that "confocal microscopy is a rapid and sensitive diagnostic tool for both the early diagnosis and non-invasive follow-up of fungal keratitis". In order to justify the statement, two issues of concern on the early diagnosis have to be addressed.
The first is about the sensitivity in having positive diagnosis in the untreated eyes of first experiment. On day 2, all 14 samples were smear and culture positive for the Asperigilus fumigatus, therefore confocal microscopy could not demonstrate any superiority in early diagnosis in term of sensitivity. On days 14 and 22, their conclusion that "confocal microscopy was more sensitive than culture technique" also could not be drawn unless the authors could enlighten us with supplementary data on the percentages of positive culture in those periods together with their p values.
Another issue is about computation of statistical values in the second experiment. The authors implied that topical and orally treated eyes had significantly lower positive culture growth than the control group receiving no treatment on days 14 and 22 by listing p values of 0.002 and 0.003. However, in performing the Chi-Square analysis again with the data provided, we can only achieve p= 0.391 and p= 0.280 on Day 14 and p= 0.308 and p= 0.237 on Day 22. We would suggest that statistical differences cannot be demonstrated in these parts of study, at least, with such a sample size.
(1) Avunduk AM, Beuerman RW, Varnell ED, Kaufman HE. Confocal microscopy of Aspergillus fumigatus keratitis. Br J Ophthalmol. 2003;87:409-10.
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