Identification and Susceptibility of Aspergillus Section Nigri in China: Prevalence of Species and Paradoxical Growth in Response to Echinocandins

Abstract

Molecular identification and in vitro antifungal susceptibility tests of 43Aspergillus section Nigri isolates from China were performed. Aspergillus niger and Aspergillus tubingensis were present in almost equal numbers. All of the isolates had low MIC/ MECs (minimum effective concentrations) for the 7 common antifungals, and a paradoxical effect was observed for the first time in response to caspofungin and micafungin.A spergillus section Nigri, which includes 26 species of black Aspergillus, is important in medical mycology, food science, and biotechnology (1). More importantly, in the clinical setting, Aspergillus niger, which is the most commonly described species of Aspergillus section Nigri, is the third most common pathogen that causes invasive pulmonary aspergillosis and the most frequent etiological agent of otomycosis (2, 3). However, Aspergillus section Nigri is one of the most difficult groups to classify; species that belong to this group are typically identified as A. niger based on morphological observations in the clinical laboratory (4). In recent years, sequence-based molecular methods have been used successfully for species identification in this group, particularly methods using the calmodulin gene, which can distinguish all species within section Nigri (4-6). Molecular studies indicated that several species in addition to A. niger were able to cause human infections; different regions had different species of pathogenic black Aspergillus, and A. niger and Aspergillus tubingensis were the most frequently identified pathogens in previous studies (2,(7)(8)(9)(10)(11)(12)(13)(14). Additionally, it is worth noting in a clinical context that azoles exhibited different activity against the two species and that isolates from different geographical regions exhibited remarkable differences in susceptibility to azoles and amphotericin B (1-13, 15). In light of this situation, accurate identification and susceptibility testing are necessary in the clinic.To date, no reports are available concerning the identification and antifungal susceptibility testing of black Aspergillus in China. The aim of this study was to reidentify black Aspergillus isolates from Chinese clinical patients and environments based on sequence analysis of the calmodulin gene. Furthermore, in vitro drug susceptibility was tested to evaluate the same isolates.A total of 43 isolates that belong to Aspergillus section Nigri and had been preidentified as A. niger based on morphology were collected nationally by the Research Center for Medical Mycology at Peking University between 1997 and 2014. These isolates included 27 from clinical samples and 16 from the environment. For details, see Table S1 in the supplemental material. Isolates were cultured on 2% malt extract agar (MEA) at 28°C for 5 days and were subsequently investigated using molecular analysis and antifungal susceptibility tests.Genomic DNA was extracted and purified according to the instructions provided with the DNeasy plant minikit (Qiagen, Hilden, Germany), with modifica...

Drug Sensitivity and Resistance Mechanism in Aspergillus Section Nigri Strains from Japan

“…Our finding suggests that A. tubingensis isolates are intrinsically resistant to azoles, but this does not depend on exposure to azoles. Several reports from other countries, so far, have supported the idea of less susceptibility in A. tubingensis (6,7,9,21). Considering the lowered azole susceptibilities in A. tubingensis, it must be further stressed that identification of the detailed species causing mycoses is important for appropriate drug therapy.…”

“…Importantly, several reports on the antifungal drug susceptibility of strains of Aspergillus section Nigri have revealed that strains showing higher MICs of itraconazole were often isolated from A. niger and A. tubingensis in a clinical setting as well as from the environment (6)(7)(8)(9). This is suggestive of an intrinsic resistance to itraconazole by these species.…”

Aspergillus Species in Lower Respiratory Tract of Hospitalized Patients from Shanghai, China: Species Diversity and Emerging Azole Resistance

“…and cryptic species within the genus Aspergillus, have been reported as causative agents of aspergillosis worldwide, including China. [16][17][18] The recommended first-line drugs to treat aspergillosis are triazoles. 19 However, triazole-resistant isolates of Aspergillus spp.…”

Species Distribution and In Vitro Azole Susceptibility of Aspergillus Section Nigri Isolates from Clinical and Environmental Settings

“…The management and prophylaxis of aspergillosis is mainly performed using triazole drugs, although longterm therapy and the indiscriminate use of azoles in agriculture have raised concerns about resistance to these compounds (8)(9)(10). In spite of the large number of reports of triazole resistance phenomena worldwide, mainly in Aspergillus fumigatus isolates from environmental and clinical settings (9,10), data on the in vitro antifungal susceptibilities of members of Aspergillus section Nigri are scant and those available show that clinical isolates from different geographical regions exhibited remarkably different azole susceptibilities (5,7,11,12). Azole resistance mechanisms have been described mainly in A. tubingensis (5), the most common being attributed to point mutations in the cyp51A gene encoding the azole target protein (14␣-sterol demethylase [CYP51A]) (9,10).…”

“…Species belonging to Aspergillus section Nigri have been difficult to classify due to their phenotypic similarities (4), whereas molecular tools such as the sequencing of calmodulin and ␤-tubulin genes have been successfully employed for species identification and discrimination within section Nigri. Molecular data have shown that Aspergillus tubingensis is the species most frequently distributed in various environments (5)(6)(7). The management and prophylaxis of aspergillosis is mainly performed using triazole drugs, although longterm therapy and the indiscriminate use of azoles in agriculture have raised concerns about resistance to these compounds (8)(9)(10).…”