Objectives: To do in vitro susceptibility testing and analyse the difference in the Antifungal susceptibility profile of Aspergillus isolates by Broth Microdilution method.
Methods: Aspergillus grown from various clinical samples (Ear swab, Bronchial wash, Endotracheal Aspiration, Paranasal sinus, BAL, Sputum) was subcultured on Sabouraud’s Dextrose Agar/Oat meal Agar. Tease mount/Slide culture was done to study the morphological features of the hyphae, size , shape and arrangement of the conidia. Antifungal susceptibility testing(AFST) was done according to CLSI guidelines M38-A2 2008 documented reference method for Broth Microdilution Antifungal susceptibility of Filamentous fungi. Antifungal stock solutions of AmphotericinB, Voriconazole, Posaconazole, Itraconazole and Caspofungin was prepared in di-methyl sulfoxide and stored at -700 C. RPMI medium was prepared , adjusted to pH 7 and filter sterilized. Sterility was checked and stored at 40 C. Dilution of the stock solution was done as per the guidelines. Inoculum was prepared by adjusting conidial suspension in sterile distilled water to 0.5 McFarland Turbidity standard; to 100 µl of drug solution, 100 µl of inoculum in RPMI 1640 medium was added. Test micro-titre plate was incubated for 48hrs at 350 C. Growth and sterility control wells were included in each test. AFSTREADING RESULT: For AmphotericinB, Voriconazole, Posaconazole, Itraconazole - MIC is the first well without visible growth. For Echinocandins, eg. Caspofungin- MEC is the point (lowest value) where a visible change in growth as compared to the positive growth is noted.
Results: A total of 30 Aspergillus isolates were collected-Aspergillus niger(14), Aspergillus flavus(9), Aspergillus fumigatus(6) & Aspergillus terreus(1). The result was compiled based on mean MIC values of 21 isolates are as follows. The MIC values of other isolates and Amphotericin B is yet to be completed.
Conclusion: The common Aspergillus species was Aspergillus niger which was obtained from Ear swab. The susceptibility profile showed high MIC for Itraconazole in case of Aspergillus niger and high MIC for Itraconazole, Voriconazole and Posaconazole in case of Aspergillus flavus. For Aspergillus fumigatus no such high MIC was recorded. In case of Aspergillus terreus, being resistant in nature the low MIC is recorded only with Caspofungin. Due to less susceptibility of Aspergillus to the common Antifungal agents, it is important to perform antifungal susceptibility testing to reduce the morbidity and mortality in patients especially with Invasive Aspergillosis.
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Full conference title:
- TIMM (2019)