Esma KAYA, Hatice ÖZBİLGE

Determination of the effect of fluconazole against Candida albicans and Candida glabrata by using microbroth kinetic assay

Amaç: Bu çalışmada, Candida albicans (ATCC 90028) ve Candida glabrata (RSKK 04019) suşlarının model antifungal olarak seçilen flukonazole karşı antifungal aktivitesi, fungal üremedeki optik dansite değişikliklerini sürekli olarak izlemeye olanak veren kinetik bir sistemle belirlenmiştir. Antifungal duyarlılık testleri son yıllarda gerek fungal enfeksiyonlardaki artış gerekse artan antifungal ilaç direnci nedeniyle giderek daha önemli hale gelmiştir. Yöntem ve gereç: C. albicans ve C. glabratanın flukonazolün artan konsantrasyonları varlığında, multi-detection microplate okuyucu kullanılarak 405 nmde 35 °Cde 24 saat süreyle her 15 dakikada bir optik dansiteleri ölçüldü. Bu kinetik sistemle suşların turbidimetrik üreme eğrileri elde edildi ve fl ukonazolün minimal inhibitör konsantrasyon (MİK) değerleri belirlendi. Ayrıca CLSI referans yöntem M27-A2ye göre konvansiyonel sıvı mikrodilüsyon yöntemi uygulandı. Bulgular: Flukonazol MİK90 değerleri C. albicans için 0,31 μg/mL; C. glabrata için 16,32 μg/mL olarak belirlendi. Sonuç: Kinetik ölçüm yapan yöntemler kullanılarak antifungal ilaçların mayalar üzerindeki etkileri inkübasyon süresince istenilen herhangi bir zamanda gözlenebilir.

Candida albicans ve Candida glabrata ya karşı flukonazolün etkisinin belirlenmesinde mikrobroth kinetik yöntemin kullanılması

Aim: To evaluate the antifungal activity of a model antifungal (fluconazole) against Candida albicans (ATCC 90028) and Candida glabrata (RSKK 04019) strains using a microbroth kinetic assay based on continuous monitoring of changes in the optical density of fungal growth. Recently, antifungal susceptibility testing has become more important because of the increasing incidence of both fungal infections and antifungal drug resistance. Materials and methods: The optical growth densities of C. albicans and C. glabrata in the presence of increasing concentrations of fluconazole were measured every 15 min for 24 h at 35 °C, using a multidetection microplate reader at 405 nm. The turbidimetric growth curves of these strains were obtained, and the minimal inhibitory concentration (MIC) values for fluconazole were determined with this kinetic assay. Furthermore, the conventional broth microdilution method was performed according to Clinical and Laboratory Standards Institute reference method M27-A2. Results: MIC90 values for fluconazole were determined as 0.31 μg/mL for C. albicans and 16.32 μg/mL for C. glabrata. Conclusion: Using kinetic procedures, the effects of antifungal drugs on target yeasts can be determined at any desired time-point of the incubation period.

PDF

___

1. Hadley S, Martinez JA, McDermott L, Rapino B, Syndman DR. Real-time antifungal susceptibility screening aids management of invasive yeast infections in immunocompromised patients. J Antimicrob Chemother 2002; 49: 415-9.
2. Pfaller MA, Diekema DJ, Sheehan DJ. Interpretive breakpoints for fluconazole and Candida revisited: a blueprint for the future of antifungal susceptibility testing. Clin Microbiol Rev 2006; 19: 435-47.
3. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard M27-A2. Wayne (PA): CLSI; 2002.
4. Espinel-Ingroff A, Barchiesi F, Cuenca-Estrella M, Fothergill A, Pfaller MA, Rinaldi M et al. Comparison of visual 24-hour and spectrophotometric 48-hour MICs to CLSI reference microdilution MICs of fluconazole, itraconazole, posaconazole, and voriconazole for Candida spp.: a collaborative study. J Clin Microbiol 2005; 43: 4535-40.
5. Arthington-Skaggs BA, Lee-Yang W, Ciblak MA, Frade JP, Brandt ME, Hajjeh RA et al. Comparison of visual and spectrophotometric methods of broth microdilution MIC endpoint determination and evaluation of sterol quantitation method for in vitro susceptibility testing of fluconazole and itraconazole against trailing and nontrailing Candida isolates. Antimicrob Agents Chemother 2002; 46: 2477-81.
6. Chou HH, Lo HJ, Chen KW, Liao MH, Li SY. Multilocus sequence typing of Candida tropicalis shows clonal cluster enriched in isolates with resistance or trailing growth of fluconazole. Diagn Microbiol Infect Dis 2007; 58: 427-33.
7. Meletiadis J, Meis JF, Mouton JW, Verweij PE. Analysis of growth characteristics of filamentous fungi in different nutrient media. J Clin Microbiol 2001; 39: 478-84.
8. Meletiadis J, Dorsthorst D, Verweij PE. Use of turbidimetric growth curves for early determination of antifungal drug resistance of filamentous fungi. J Clin Microbiol 2003; 41: 4718-25.
9. Lehtinen J, Jarvinen S, Virta M, Lilius EM. Real-time monitoring of antimicrobial activity with the multiparameter microplate assay. J Microbiol Methods 2006; 66: 381-9.
10. Hosphental DR, Murray CK, Rinaldi MG. The role of antifungal susceptibility testing in the therapy of candidiasis. Diagn Microbiol Infect Dis 2004; 48: 153-60.
11. Ostrosky-Zeichner L, Rex JH, Pfaller MA, Diekema DJ, Alexander BD, Andes D et al. Rationale for reading fluconazole MICs at 24 hours rather than 48 hours when testing Candida spp. by the CLSI M27-A2 standard method. Antimicrob Agents Chemother 2008; 52: 4175-7.
12. Balajee SA, Imhof A, Gribskov JL, Marr KA. Determination of antifungal drug susceptibilities of Aspergillus species by a fl uorescence-based microplate assay. J Antimicrob Chemother 2005; 55: 102-5.