In Vitro Activities of Five Antifungal Drugs Against Opportunistic Agents of Aspergillus Nigri Complex

Species of Aspergillus belonging to the section Nigri known as black aspergilli, i.e., A. niger, A. tubingensis, A. brasilliensis, A. carbonarius, A. foetidus, A. ibericus, A. heteromorphus, A. acidus, A. aculeatinus, A. heteromorphus, A. homomorphus, A. ibericus, A. uvarum, A. japonicus, A. lacticoffeatus, A. costaricaensis, A. ellipticus, A. Japonicas and A. aculeatu are widely distributed in nature [1, 2, 3]. They are widely known for their ability to produce different sorts of enzymes, organic compounds, mycotoxins, and being able to cause food spoilage [1]. The two former species are mostly common etiological agents of otomycosis followed by being involved in onychomycosis, pulmonary aspergillosis and aspergilloma [4, 5, 6, 7, 8]. The clinical implications of cryptic species such as A. awamori, A. uvarum and A. foetidus have been rarely reported [9, 10, 11]. Triazole antifungals either in mono or combination therapy are the mainstay in the management and prophylaxis of aspergillosis, but recently triazole-resistant isolates of Aspergillus species have emerged, which can be due to the long-term azole therapy or through the exposure of the fungus in the environment with the azole fungicides used in agriculture [12, 13, 14]. Black aspergilli have been reported to be the most common etiological agent of aspergillosis, but systematic study on its antifungal susceptibility profile is lacking [9, 11, 15]. The fungus has not been identified to the species level in the majority of cases reported so far. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent and they are generally misidentified. However, with development of molecular methods, numerous novel species have been described within the genus Aspergillus; thus, the term of species complex has been applied [16]. Small series of in vitro antifungal susceptibility studies have shown high MIC value in Nigri isolates, although informative data are limited to small numbers of isolates tested in sporadic case reports. Therefore, in the present investigation we compiled the first comprehensive study on antifungal susceptibility data of a large collection of environmental and clinically significant Aspergillus complex Nigri (black-sporing aspergilli) to five marketed antifungal drugs, viz amphotericin B, itraconazole, voriconazole, posaconazole, and caspofungin.

Remarkably, in vitro antifungal profiles for the black Aspergillus are relatively scarce and based on low numbers of test strains. The in vivo efficacy of antifungal therapy against the black aspergilli and cryptic species is as yet undetermined, and also in vitro data from different sources are limited. Strains analyzed were pooled from divergent clinical and environmental sources and originated from different samples in Iran [10, 18]. Recently high prevalence of itraconazole resistance A. fumigatus strains has been reported from different continent (Europe, USA and Asia) [12, 13, 14, 20, 21]. In contrast, itraconazole resistance in black aspergilla (MIC > 16 µg/ml) was reported in more than 50 % of cases to be without any known resistant mechanisms [9]. Overall, up to 40 % of our isolates had itraconazole MICs >1 µg/ml, there was a particularly high frequency of itraconazole resistance of black aspergilli; its cause remains to be unknown. In contrast, our results showed that high itraconazole susceptibilities were more prevalence in cryptic species, i.e., A. acidus, A. uvarum and A. sydowii, suggesting that itraconazole resistance may be more obvious in common species of black aspergilli such as A. niger and A. tubingensis. In similarity our investigation showed that A. tubingensis and other isolates were more susceptible for itraconazole rather than A. niger isolates. A. niger and A. tubingensis related to mild to systemic aspergillosis are mostly susceptible to amphotericin B in vitro conditions, but treatment is a big challenge because frequent relapses and failures are observed despite the application of antifungal therapy. Amphotericin B MICs ranged between 0.25 and 4 µg/ml for most black aspergilla, but the correlation between MICs and the outcome of treatment with amphotericin B is not very well known. Although in the present study low MICs of posaconazole and caspofungin with potent activity were observed, the highest MICs in increasing order were consistently found with amphotericin B, voriconazole, and itraconazole. Low MICs against A. niger complex lead to the poor outcome of most patients treated with these drugs. Itraconazole resistance was numerous in certain Nigri species, indicating that species identification may help the selection of antifungal therapy [8, 17]. Based on CLSI guidelines, most of the filamentous fungi are not susceptible to fluconazole and most MICs were >32 µg/ml, but black aspergilli are resistant to itraconazole as shown in Table 1, posaconazole was the most active compound in vitro, and itraconazole was the least active in this data set. Isolates with itraconazole MICs >8 µg/ml showed reduced susceptibility to the other azoles. Verweij et al. [22] have compared the itraconazole resistant groups of black aspergilli to the itraconazole-susceptible group and showed that the voriconazole geometric mean MICs was 2.08 and 0.91 µg/ml, respectively. They also reported that 12 and 6 % of isolates were resistant to voriconazole and posaconazole, respectively, and 59 and 6 % fell into the intermediate range, respectively [22]. Our results showed obvious differences among itraconazole-susceptible and resistant black aspergilla, although for better understanding more informative data are required. In this study numerous azole cross-resistance were observed in A. niger and A. tubingensis with high itraconazole MICs (Table 1), in concordant with other studies [11] and concordant with other reports [9, 22]. Fortunately, the antifungal armamentarium has been extended with high efficacy, e.g., posaconazole and ravuconazole. In conclusion we found that not only posaconazole but also caspofungin as an echinocandin agents showed potent activity in vitro. Thus, the relevance of these in vitro findings for clinical efficacy has not been established and remains to be determined and further study on resistance mechanisms and for an optimal susceptibility testing methodology is highly essential in this group of fungi. These reports emphasize a number of key issues for clinical management.