Investigation of azole resistance in Aspergillus species isolated from clinical specimens by azole agar screening method

<b>Introduction.</b> Aspergillosis represents a significant global health threat, with increasing concerns about azole resistance.<b>Hypothesis/Gap Statement.</b> There is limited evidence on the prevalence and distribution of azole resistance among clinical <i>Aspergillus</i> isolates.<b>Aim.</b> This study investigated the prevalence of azole resistance in clinical <i>Aspergillus</i> isolates and evaluated different susceptibility testing methods.<b>Methodology.</b> A total of 125 <i>Aspergillus</i> spp. isolates were collected from clinical samples (abscess, corneal abscess, biopsy, tissue and respiratory samples). Species identification was performed using conventional morphological methods and Matrix assisted laser desorption Ionization time of flight massspectrometry (MALDI-TOF) MS. Azole susceptibility testing was conducted using the gradient test (E-test), the agar plate screening method and broth microdilution for voriconazole (VOR), itraconazole (ITR) and posaconazole (POS). Molecular analysis was performed to detect <i>cyp51A</i> gene mutations associated with resistance.<b>Results.</b> Among 125 isolates, species distribution was 44% <i>Aspergillus fumigatus</i>, 33.6% <i>Aspergillus flavus</i>, 5% <i>Aspergillus terreus</i>, 3% <i>Aspergillus niger</i> and 14% <i>Aspergillus</i> spp. Using gradient testing, <i>A. fumigatus</i> showed 1.8% resistance to VOR, 5.45% to ITR and 1.8% to POS, with one isolate resistant to all azoles. <i>A. terreus</i> showed 16.7% resistance to VOR, <i>A. niger</i> 25% resistance to ITR and <i>Aspergillus</i> spp. showed various resistance patterns. The agar plate method detected resistance with 100% susceptibility/specificity for <i>non-fumigatus</i> species but 33.3% susceptibility for <i>A. fumigatus</i> ITR resistance. CypA-L98H mutations were detected in six isolates and CypA-M220 mutations in seven isolates.<b>Conclusion.</b> This study confirms the presence of azole resistance in clinical <i>Aspergillus</i> isolates with species-specific variations. The agar plate screening method shows promise for <i>non-fumigatus</i> species but requires optimization for <i>A. fumigatus</i>.