Epidemiology and Antifungal Susceptibility Profile of Aspergillus Species: Comparison between Environmental and Clinical Isolates from Patients with Hematologic Malignancies

Abstract

Global data on the epidemiology and susceptibility of Aspergillus are crucial in the management of invasive aspergillosis. Here, we aimed to determine the characteristics of clinical and environmental Aspergillus isolates, focusing mainly on hematologic malignancy patients. We prospectively collected all consecutive cases and clinical isolates of culture-positive proven/probable invasive aspergillosis patients from January 2016 to April 2018 and sampled the air inside and outside the hospital. Cryptic species-level identification of Aspergillus, antifungal susceptibilities, and cyp51 gene sequencing were performed, and clinical data were analyzed. This study was conducted as part of the Catholic Hematology Hospital Fungi Epidemiology (CAFÉ) study. A total of 207 proven/probable invasive aspergillosis and 102 clinical and 129 environmental Aspergillus isolates were included in this analysis. The incidence of proven/probable invasive aspergillosis was 1.3 cases/1,000 patient-days during the study period. Cryptic Aspergillus species accounted for 33.8%, with no differences in proportions between the clinical and environmental isolates. Section Nigri presented a high proportion (70.5%) of cryptic species, mainly from A. tubingensis and A. awamori: the former being dominant in environmental samples, and the latter being more common in clinical isolates (P < 0.001). Of 91 A. fumigatus isolates, azole-resistant A. fumigatus was found in 5.3% of all A. fumigatus isolates. Three isolates presented the TR34/L98H mutation of the cyp51A gene. Patients with invasive aspergillosis caused by azole-resistant A. fumigatus showed 100% all-cause mortality at 100 days. This study demonstrates the significant portion of cryptic Aspergillus species and clinical implications of azole resistance and underscores the comparison between clinical and environmental isolates.

Development of multiplex real-time PCR for rapid identification and quantitative analysis of Aspergillus species

“…Wild-type isolates and mutant isolates with TR34 showed different melting temperatures. In particular, A. tubingensis revealed intrinsically higher azole minimum inhibitory concentrations according to previous studies [2,3,5]. The A. tubingensis-specific primers and probe designed in this study showed that only A. tubingensis was amplified without affecting other Aspergillus in section Nigri.…”

“…The fungal isolates used in this study were representative strains of clinical and environmental isolates stored anonymously, and standard strains including A. fumigatus (ATCC 16424; American Type Culture Collection, Manassas, VA, USA), A. terreus (ATCC 10690), A. flavus (ATCC 16883), and A. niger (ATCC 16888) [2,15]. The representative Aspergillus isolates were selected for each sequence type according to benA and cyp51A sequencing results.…”

“…The representative Aspergillus isolates were selected for each sequence type according to benA and cyp51A sequencing results. The Aspergillus isolates used in this study included 20 strains of A. fumigatus, 2 strains of A. lentulus, 1 strain of A. turcosus, 1 strain of A. udagawae, 2 strains of A. luchuensis, 11 strains of A. awamori, 11 strains of A. niger, 12 strains of A. tubingensis, 4 strains of A. flavus, 1 strain of A. nidulans, 2 strains of A. sydowii, 3 strains of A. terreus, and 1 strain of A. subramanianii, which were registered to GenBank in our previous study [2]. In addition, 38 non-Aspergillus filamentous ascomycetes (No.…”

“…Invasive aspergillosis (IA) is a fatal disease caused by Aspergillus species that occurs mainly in immunocompromised patients [1]. Above all, rapid and accurate fungal diagnosis is important, as the morbidity and mortality of IA remain high, and diagnosis and treatment impart a significant economic burden [2][3][4][5]. The method of Aspergillus species identification includes morphologic identification of fungi through culture and molecular identification via the polymerase chain reaction (PCR) [5,6].…”

“…The method of Aspergillus species identification includes morphologic identification of fungi through culture and molecular identification via the polymerase chain reaction (PCR) [5,6]. The former method is currently the gold standard; however, its use can depend largely on clinical specimen quality and the proficiency of the microbiology test personnel [6,7] The latter method, molecular identification of filamentous ascomycetes, is mainly conducted through sequence analysis of the internal transcribed spacer (ITS) region, and β-tubulin (benA) and calmodulin (CaM) genes are also used for cryptic species-level identification [2,8,9]. However sequencing-based identification is time-consuming, as it takes 5-17 days to reach the final diagnosis; specifically, it takes 3-14 days to produce conidia following fungal culture from the clinical sample, followed by an additional 2-3 days to extract DNA from the conidia and obtain sequencing analysis results [10,11].…”