Aspergillus welwitschiae; an otomycosis predominant agent, new epidemiological and antifungal susceptibility data from Iran

Background: Black aspergilli (section Nigri) are predominate etiologic agents of otomycosis, however, there is controversy in the exact differentiation of species. For several decades Aspergillus niger is considered the main otomycosis etiologic agent. Recently calmodulin gene has been accepted as a more suitable gene for the accurate assignment of Aspergillus species. Therefore, it is found that A. welwitschiae and A. tubingensis are the main otomycosis agents based on calmodulin gene sequencing. Aims: The objective of the study was to isolate and identify black aspergilli from otomycosis using the calmodulin gene and their susceptibility was evaluated against several antifungals. Methods: 134 clinically confirmed patients with otomycosis were sampled and specimens were cultured on Sabouraud dextrose agar (SDA) at ambient temperature. Black aspergilli were screened based on colony morphology on SDA and microscopy features and then subjected to sequencing using calmodulin primers. Moreover, antifungal susceptibility for isolates was applied based on CLSI M38 3rd edition. Results: 132 (98.5%) of patients had positive cultures for different species of molds or yeasts. Most of the patients (30.3%) ranged from 31 to 40 years, and 56.1% of them were female. Aspergillus section Nigri was the most prevalent fungal pathogen and of 86 isolates, 60.5% isolates were identified as A. welwitschiae, A. tubingensis, 31 (36.0%), A. niger (sensu stricto), 2 (2.3%), and A. neoniger 1 (1.2%). According to the maximum likelihood method, all isolates of A. tubingensis and one isolate of A. neoniger were included in the A. tubingensis clade. On the other hand, the clade of A. niger/A. welwitschiae contains, all isolates of A. welwitschiae, two A. niger (sensu stricto) isolates, and 36 isolates from other countries. Aspergillus welwitschiae was more sensitive to luliconazole, voriconazole, and amphotericin B compared to A. tubingensis. 78.8% of A. welwitschiae strains were classified as nonwild type to nystatin compared to 35.5% of A. tubingensis. Moreover, 3.2% of A. tubingensis strains were non-wild type against amphotericin B. The isolates of A. tubingensis were more sensitive to itraconazole than A. welwitschiae. Conclusions: It is concluded that in contrast, to the previous study A. welwitschiae from section Nigri is the most causative agent of otomycosis followed by A. tubingensis. In addition, the isolates of A. welwitschiae were more sensitive to luliconazole, voriconazole, and amphotericin B compared to A. tubingensis. Whereas, the isolates of A. tubingensis were more sensitive to itraconazole than A. welwitschiae. On the other hand, 78.8% and 35.5% of A. welwitschiae and A. tubingensis strains were classified as a non-wild type against nystatin. Also, 3.2% of A. tubingensis strains were non-wild type against amphotericin B. All A. welwitschiae were included in the A. niger/ A. welwitschiae clade, associated with different clinical and environmental species from different countries.