Species of Aspergillus section Aspergillus from clinical samples in the United States

Species of Aspergillus section Aspergillus from clinical samples in the United States Abstract The diversity of Aspergillus species in clinical samples is continuously increasing. Species under the former name Eurotium, currently accommodated in section Aspergillus of the genus Aspergillus, are xerophilic fungi widely found in the human environment and able to grow on substrates with low water activity. However, their prevalence in the clinical setting is poorly known. We have studied the presence of these species in a set of clinical samples from the United States using a multilocus sequence analysis based on the internal transcribed spacer (ITS) region of the rRNA, and fragments of the genes β-tubulin (BenA), calmodulin (CaM), and polymerase II second largest subunit (RPB2). A total of 25 isolates were studied and identified as follows: A. montevidensis (44%), A. chevalieri (36%), A. pseudoglaucus (8%), and A. costiformis (4%). A new species Aspergillus microperforatus is also proposed, which represented 8% of the isolates studied and is characterized by uniseriate conidial heads, subglobose to pyriform vesicles, rough conidia, globose to subglobose cleistothecia, and lenticular and smooth ascospores. The in vitro antifungal activity of eight clinically available antifungals was also determined against these isolates, with the echinocandins and posaconazole having the most potent activity. Aspergillus, phylogenetic analysis, antifungal susceptibility, taxonomy, Eurotium Introduction The number of species of Aspergillus involved in human infections is continuously increasing, and most of these species have nowadays been identified using modern molecular techniques. Until recently, the dual nomenclature system permitted different names for the sexual and asexual forms of Aspergillus. One such example is the genus Eurotium, the name for the sexual state for species in the former Aspergillus glaucus group.1 However, following the recent changes in fungal nomenclature2,3 and based on phylogenetic studies,4,5 all generic names for sexual states of Aspergillus are now included under the name Aspergillus, and former species of Eurotium now comprise the aspergilli in the section Aspergillus.4 A new approach to phylogenetic study supports the current broad concept of the genus Aspergillus,5 with A. glaucus (= E. herbariorum) being the type species of the genus. Species in the section Aspergillus are usually osmophilic, with optimum growth on substrates with high sugar or salt concentrations. Commonly the asexual morph has smooth conidiophores, with uniseriate, radiate to somewhat columnar conidial heads, and ellipsoidal to globose echinulate conidia.1,6 The sexual morph is usually characterized by globose to subglobose, thin-walled cleistothecia, eight-spored asci, and lenticular, smooth to rough-walled ascospores, generally showing an equatorial line or furrow.1,7 These species are found worldwide and often on organic materials, dust, and stored cereals and other food products.1,7 Although these aspergilli are of minimal clinical importance, some, such as A. glaucus, have been reported in orofacial8 and brain infections.9 In addition, A. montevidensis has been involved in cases of otitis, mycetoma, cerebral abscess, keratitis, and pulmonary infections,10 and A. glaucus and A. montevidensis can also cause hypersensitive pneumonitis11,12. Aspergillus chevalieri and A. pseudoglaucus have been linked to cutaneous aspergillosis13 and maxillary sinusitis,14 respectively. Hubka et al.15 recovered five species of section Aspergillus among isolates from probable cases of superficial infections (e.g., skin and nails), including A. montevidensis, A. costiformis, A. pseudoglaucus, A. proliferans, and A. ruber in Czech Republic. The antifungal susceptibility patterns of members of section Aspergillus are largely unknown,16 with little published data. Masih et al.17 demonstrated potent activity of posaconazole, anidulafungin, and micafungin against three strains of A. montevidensis (GM minimal inhibitory concentrations [MICs] of 0.04 μg/ml, 0.015 μg/ml, and 0.015 μg/ml, respectively) and one of A. chevalieri (MICs of 0.015 μg/ml, 0.03 μg/ml, and 0.015 μg/ml, respectively). García-Martos et al.18 also demonstrated low MIC values for amphotericin B, itraconazole, and voriconazole against three strains of A. glaucus (MICs of 0.125–0.5 μg/ml, 0.25–0.5 μg/ml, and 0.125–0.25 μg/ml, respectively) and against two strains of A. chevalieri (MICs of 0.125–0.5 μg/ml, 0.125–0.25 μg/ml, and 0.125–0.25 μg/ml, respectively). Wildfeuer et al.19 tested eight strains of A. glaucus against four drugs and observed that itraconazole exhibited the most potent activity (GM MIC of 0.39 μg/ml). In order to assess the diversity of species from Aspergillus section Aspergillus in the clinical setting and to observe their response to antifungal drugs, the aim of this study was to identify to the species level a set of clinical isolates from the United States using a multilocus phylogenetic study, and to determine the susceptibility pattern of eight clinically available antifungals against these species. Methods Fungal isolates A total of 25 isolates of section Aspergillus were investigated in this study. Most of them were from human clinical samples, primarily from the respiratory tract (BAL, sputum, and sinus), but also in fewer numbers from corneas, nails, stool, and lymph nodes. One of them was of environmental origin, and the origin of four were unknown (Table 1). These isolates were received at the Fungus Testing Laboratory of the University of Texas Health Science Center (San Antonio, TX, USA) from different institutions across the United States over a period of 11 years (2004–2015), for identification and/or antifungal susceptibility testing. Table 1. Origins, year of isolation, and GenBank/EMBL accession numbers of the Aspergillus strains included in this study. GenBank/EMBL accession number Species Isolate number Origin Year ITS BenA CaM RPB2 A. chevalieri (9) UTHSCSA DI15-18 BAL 2014 LT627247 LT627272 LT627297 LT627322 UTHSCSA DI16-375 Sputum 2004 LT627248 LT627273 LT627298 LT627323 UTHSCSA DI16-381 BAL 2006 LT627249 LT627274 LT627299 LT627324 UTHSCSA DI16-382 BAL 2008 LT627250 LT627275 LT627300 LT627325 UTHSCSA DI16-394 BAL 2007 LT627251 LT627276 LT627301 LT627326 UTHSCSA DI16-396 Corneal 2008 LT627252 LT627277 LT627302 LT627327 UTHSCSA DI16-397 Sinus 2008 LT627253 LT627278 LT627303 LT627328 UTHSCSA DI16-413 Unknown 2008 LT627254 LT627279 LT627304 LT627329 UTHSCSA DI16-414 Unknown 2008 LT627255 LT627280 LT627305 LT627330 A. costiformis (1) UTHSCSA DI15-16 Environmental 2014 LT627256 LT627281 LT627306 LT627331 A. microperforatus (2) UTHSCSA DI16-400 Toe nail 2009 LT627270 LT627295 LT627320 LT627345 UTHSCSA DI16-407 Lymph node 2011 LT627271 LT627296 LT627321 LT627346 A. montevidensis (11) UTHSCSA DI15-19 Ethmoid sinus 2014 LT627257 LT627282 LT627307 LT627332 UTHSCSA DI15-20 Sputum 2014 LT627258 LT627283 LT627308 LT627333 UTHSCSA DI15-21 BAL 2015 LT627259 LT627284 LT627309 LT627334 UTHSCSA DI15-22 Sputum 2015 LT627260 LT627285 LT627310 LT627335 UTHSCSA DI16-401 Lung tissue 2009 LT627261 LT627286 LT627311 LT627336 UTHSCSA DI16-403 Sputum 2009 LT627262 LT627287 LT627312 LT627337 UTHSCSA DI16-405 Sinus 2010 LT627263 LT627288 LT627313 LT627338 UTHSCSA DI16-406 Lung tissue 2010 LT627264 LT627289 LT627314 LT627339 UTHSCSA DI16-408 Paranasal 2013 LT627265 LT627290 LT627315 LT627340 UTHSCSA DI16-411 Unknown 2008 LT627266 LT627291 LT627316 LT627341 UTHSCSA DI16-412 Unknown 2008 LT627267 LT627292 LT627317 LT627342 A. pseudoglaucus (2) UTHSCSA DI15-17 Nasal 2011 LT627268 LT627293 LT627318 LT627343 UTHSCSA DI16-410 Stool 2014 LT627269 LT627294 LT627319 LT627344 GenBank/EMBL accession number Species Isolate number Origin Year ITS BenA CaM RPB2 A. chevalieri (9) UTHSCSA DI15-18 BAL 2014 LT627247 LT627272 LT627297 LT627322 UTHSCSA DI16-375 Sputum 2004 LT627248 LT627273 LT627298 LT627323 UTHSCSA DI16-381 BAL 2006 LT627249 LT627274 LT627299 LT627324 UTHSCSA DI16-382 BAL 2008 LT627250 LT627275 LT627300 LT627325 UTHSCSA DI16-394 BAL 2007 LT627251 LT627276 LT627301 LT627326 UTHSCSA DI16-396 Corneal 2008 LT627252 LT627277 LT627302 LT627327 UTHSCSA DI16-397 Sinus 2008 LT627253 LT627278 LT627303 LT627328 UTHSCSA DI16-413 Unknown 2008 LT627254 LT627279 LT627304 LT627329 UTHSCSA DI16-414 Unknown 2008 LT627255 LT627280 LT627305 LT627330 A. costiformis (1) UTHSCSA DI15-16 Environmental 2014 LT627256 LT627281 LT627306 LT627331 A. microperforatus (2) UTHSCSA DI16-400 Toe nail 2009 LT627270 LT627295 LT627320 LT627345 UTHSCSA DI16-407 Lymph node 2011 LT627271 LT627296 LT627321 LT627346 A. montevidensis (11) UTHSCSA DI15-19 Ethmoid sinus 2014 LT627257 LT627282 LT627307 LT627332 UTHSCSA DI15-20 Sputum 2014 LT627258 LT627283 LT627308 LT627333 UTHSCSA DI15-21 BAL 2015 LT627259 LT627284 LT627309 LT627334 UTHSCSA DI15-22 Sputum 2015 LT627260 LT627285 LT627310 LT627335 UTHSCSA DI16-401 Lung tissue 2009 LT627261 LT627286 LT627311 LT627336 UTHSCSA DI16-403 Sputum 2009 LT627262 LT627287 LT627312 LT627337 UTHSCSA DI16-405 Sinus 2010 LT627263 LT627288 LT627313 LT627338 UTHSCSA DI16-406 Lung tissue 2010 LT627264 LT627289 LT627314 LT627339 UTHSCSA DI16-408 Paranasal 2013 LT627265 LT627290 LT627315 LT627340 UTHSCSA DI16-411 Unknown 2008 LT627266 LT627291 LT627316 LT627341 UTHSCSA DI16-412 Unknown 2008 LT627267 LT627292 LT627317 LT627342 A. pseudoglaucus (2) UTHSCSA DI15-17 Nasal 2011 LT627268 LT627293 LT627318 LT627343 UTHSCSA DI16-410 Stool 2014 LT627269 LT627294 LT627319 LT627344 BenA, β-tubulin; CaM, calmodulin; ITS, internal transcribed spacer regions of the rDNA and 5.8S region; RPB2, partial RNA polymerase II second largest subunit; UTHSCSA, University of Texas Health Science Center (San Antonio, USA). View Large Table 1. Origins, year of isolation, and GenBank/EMBL accession numbers of the Aspergillus strains included in this study. GenBank/EMBL accession number Species Isolate number Origin Year ITS BenA CaM RPB2 A. chevalieri (9) UTHSCSA DI15-18 BAL 2014 LT627247 LT627272 LT627297 LT627322 UTHSCSA DI16-375 Sputum 2004 LT627248 LT627273 LT627298 LT627323 UTHSCSA DI16-381 BAL 2006 LT627249 LT627274 LT627299 LT627324 UTHSCSA DI16-382 BAL 2008 LT627250 LT627275 LT627300 LT627325 UTHSCSA DI16-394 BAL 2007 LT627251 LT627276 LT627301 LT627326 UTHSCSA DI16-396 Corneal 2008 LT627252 LT627277 LT627302 LT627327 UTHSCSA DI16-397 Sinus 2008 LT627253 LT627278 LT627303 LT627328 UTHSCSA DI16-413 Unknown 2008 LT627254 LT627279 LT627304 LT627329 UTHSCSA DI16-414 Unknown 2008 LT627255 LT627280 LT627305 LT627330 A. costiformis (1) UTHSCSA DI15-16 Environmental 2014 LT627256 LT627281 LT627306 LT627331 A. microperforatus (2) UTHSCSA DI16-400 Toe nail 2009 LT627270 LT627295 LT627320 LT627345 UTHSCSA DI16-407 Lymph node 2011 LT627271 LT627296 LT627321 LT627346 A. montevidensis (11) UTHSCSA DI15-19 Ethmoid sinus 2014 LT627257 LT627282 LT627307 LT627332 UTHSCSA DI15-20 Sputum 2014 LT627258 LT627283 LT627308 LT627333 UTHSCSA DI15-21 BAL 2015 LT627259 LT627284 LT627309 LT627334 UTHSCSA DI15-22 Sputum 2015 LT627260 LT627285 LT627310 LT627335 UTHSCSA DI16-401 Lung tissue 2009 LT627261 LT627286 LT627311 LT627336 UTHSCSA DI16-403 Sputum 2009 LT627262 LT627287 LT627312 LT627337 UTHSCSA DI16-405 Sinus 2010 LT627263 LT627288 LT627313 LT627338 UTHSCSA DI16-406 Lung tissue 2010 LT627264 LT627289 LT627314 LT627339 UTHSCSA DI16-408 Paranasal 2013 LT627265 LT627290 LT627315 LT627340 UTHSCSA DI16-411 Unknown 2008 LT627266 LT627291 LT627316 LT627341 UTHSCSA DI16-412 Unknown 2008 LT627267 LT627292 LT627317 LT627342 A. pseudoglaucus (2) UTHSCSA DI15-17 Nasal 2011 LT627268 LT627293 LT627318 LT627343 UTHSCSA DI16-410 Stool 2014 LT627269 LT627294 LT627319 LT627344 GenBank/EMBL accession number Species Isolate number Origin Year ITS BenA CaM RPB2 A. chevalieri (9) UTHSCSA DI15-18 BAL 2014 LT627247 LT627272 LT627297 LT627322 UTHSCSA DI16-375 Sputum 2004 LT627248 LT627273 LT627298 LT627323 UTHSCSA DI16-381 BAL 2006 LT627249 LT627274 LT627299 LT627324 UTHSCSA DI16-382 BAL 2008 LT627250 LT627275 LT627300 LT627325 UTHSCSA DI16-394 BAL 2007 LT627251 LT627276 LT627301 LT627326 UTHSCSA DI16-396 Corneal 2008 LT627252 LT627277 LT627302 LT627327 UTHSCSA DI16-397 Sinus 2008 LT627253 LT627278 LT627303 LT627328 UTHSCSA DI16-413 Unknown 2008 LT627254 LT627279 LT627304 LT627329 UTHSCSA DI16-414 Unknown 2008 LT627255 LT627280 LT627305 LT627330 A. costiformis (1) UTHSCSA DI15-16 Environmental 2014 LT627256 LT627281 LT627306 LT627331 A. microperforatus (2) UTHSCSA DI16-400 Toe nail 2009 LT627270 LT627295 LT627320 LT627345 UTHSCSA DI16-407 Lymph node 2011 LT627271 LT627296 LT627321 LT627346 A. montevidensis (11) UTHSCSA DI15-19 Ethmoid sinus 2014 LT627257 LT627282 LT627307 LT627332 UTHSCSA DI15-20 Sputum 2014 LT627258 LT627283 LT627308 LT627333 UTHSCSA DI15-21 BAL 2015 LT627259 LT627284 LT627309 LT627334 UTHSCSA DI15-22 Sputum 2015 LT627260 LT627285 LT627310 LT627335 UTHSCSA DI16-401 Lung tissue 2009 LT627261 LT627286 LT627311 LT627336 UTHSCSA DI16-403 Sputum 2009 LT627262 LT627287 LT627312 LT627337 UTHSCSA DI16-405 Sinus 2010 LT627263 LT627288 LT627313 LT627338 UTHSCSA DI16-406 Lung tissue 2010 LT627264 LT627289 LT627314 LT627339 UTHSCSA DI16-408 Paranasal 2013 LT627265 LT627290 LT627315 LT627340 UTHSCSA DI16-411 Unknown 2008 LT627266 LT627291 LT627316 LT627341 UTHSCSA DI16-412 Unknown 2008 LT627267 LT627292 LT627317 LT627342 A. pseudoglaucus (2) UTHSCSA DI15-17 Nasal 2011 LT627268 LT627293 LT627318 LT627343 UTHSCSA DI16-410 Stool 2014 LT627269 LT627294 LT627319 LT627344 BenA, β-tubulin; CaM, calmodulin; ITS, internal transcribed spacer regions of the rDNA and 5.8S region; RPB2, partial RNA polymerase II second largest subunit; UTHSCSA, University of Texas Health Science Center (San Antonio, USA). View Large Morphological characterization The morphology of the fungi was characterized by the traditional criteria.4,20 Briefly, this is determined after 7 days of incubation on Czapek Yeast Autolysate agar (CYA, Becton, Dickinson and Company®, Sparks, MD, USA), CYA supplemented with 20% sucrose (CY20S), and Malt Extract agar (MEA, Pronadisa®, Madrid, Spain) at 25°C; and CY20S and Harrold's Agar containing 60% sucrose1 (M60Y) at 37°C. Colors match Kornerup and Wanscher.21 Microscopic features were examined and measured on MEA and CY20S cultures, after 10–14 days of incubation, on wet mounts with 60% lactic acid and a drop of ethanol 70% to wash out the excess conidia. Photomicrographs were taken with a DeltaPix Infinity X digital camera mounted on a Zeiss Axio Imager M1 light microscope (Zeiss, Oberkochen, Germany), using a Nomarski differential interference contrast and phase contrast optics. Scanning electron microscope (SEM) photographs were taken with a Jeol JSM- 6400 using techniques described previously.22 DNA extraction, amplification, and sequencing Total genomic DNA was extracted from MEA cultures after 7 days of incubation at 25°C, using the FastDNA® Kit and the FastPrep® Instrument (MP Biomedicals, Irvine CA, USA), according to the manufacturer's specifications. After extraction, four different genetic regions were amplified for each strain20,23; that is, the internal transcribed spacer (ITS) region of the rRNA, comprising ITS1, 5.8S gene, and ITS2 regions, using ITS5 and ITS4 primers24; a portion of β-tubulin gene (BenA), using Bt2a and Bt2b primers25; a portion of calmodulin gene (CaM), using Cmd5 and Cmd6 primers26; and a portion of RNA polymerase II second largest subunit gene (RPB2), using 5F and 7CR primers.27 Polymerase chain reaction (PCR) products were sequenced in both directions, using the same primers, at Macrogen Europe (Macrogen Inc., Amsterdam, the Netherlands). Sequences were assembled and edited using SeqMan v.7.0.0 (DNASTAR, Madison, WI, USA). Molecular identification and phylogenetic analysis The phylogeny was analyzed first individually for each partition and then in a concatenated study, once the topologies proved to be congruent. To give support to our analyses, sequences of the ex-type strains of all species of section Aspergillus obtained from GenBank were also included, and A. halophilicus (section Restricti) was used as the outgroup. To increase the robustness of the A. pseudoglaucus clade, sequences of 15 other strains of this species were additionally retrieved from GenBank and included in the analyses. A multiple sequence alignment was performed using ClustalW inside MEGA v.6 software.28 When necessary, the MUSCLE tool and manual adjustments were used to refine the alignment. Maximum Likelihood (ML) was conducted with MEGA v.6 software, as well as the estimation of the best nucleotide substitution method. Support values of the internal branches were assessed using the Bootstrap method with 1000 replications (values equal or higher than 70% were considered significant). Bayesian inference (BI) was performed using MrBayes v.3.1.2 software.29 The evolutionary models that best fit each partition were assessed by MrModel Test software.30 Markov chain Monte Carlo (MCMC) sampling was performed with two simultaneous runs for 1 million generations, with samples taken every 100 generations. The 50% majority rule consensus trees and posterior probability values (pp) were calculated after removing the first 25% of the resulting trees for burn-in. Values of 0.95 or higher were considered significant. Antifungal susceptibility testing The isolates were tested against eight antifungals, following the microdilution broth method.31 The antifungal agents tested were: amphotericin B (AMB) (Sigma Aldrich Quimica S.A., Madrid, Spain), itraconazole (ITC) (Jansen Pharmaceuticals, Beerse, Belgium), posaconazole (PSC) (Schering-Plough Res., Inst., Kenilworth, NJ, USA), voriconazole (VRC) (Pfizer S.A., Madrid, Spain), anidulafungin (AFG) (Pfizer S.A., Madrid, Spain), caspofungin (CFG) (Merk & Co., Inc., Rahway, NJ, USA), micafungin (MFG) (Astellas Pharma, Madrid, Spain), and terbinafine (TBF) (Sigma Aldrich Química S.A., Madrid, Spain). Readings were taken after 72 h to allow the strains to grow properly. Strains of A. pseudoglaucus (UTHSCSA DI15-17 and UTHSCSA DI16-410) and A. microperforatus (UTHSCSA DI16-400 and UTHSCSA DI16-407) were incubated at 30°C, while the others were incubated at 35°C to fit the growth requirements of the isolates under the CLSI protocol. Minimal inhibitory concentration (MIC) was defined as the lowest drug concentration that produced 100% inhibition of visible fungal growth for the AMB and azoles (ITC, PSC, and VRC) and 80% for TBF. For echinocandins (AFG, CFG, and MFG), the minimum effective concentrations (MEC) were determined microscopically as the lowest concentration of drug that allowed the growth of small, rounded, compact hyphal forms, as opposed to the long, unbranched hyphal clusters that are seen in the growth control. Candida krusei ATCC 6258 was used as the quality control strain in each test, and the MIC values were within the acceptable MIC range per the CLSI standard. All tests were carried out in duplicate, on different days, for reproducibility. Statistical analyses of the results were performed using the Prism software for Windows v.6.0 (GraphPad Software, San Diego, CA, USA). Nucleotide sequence accession numbers and alignments Newly generated sequences from this study have been deposited in GenBank/EMBL databases under the accession numbers listed on Table 1. The alignments were deposited in TreeBASE (submission number S20583). Results In the present study, as expected, the ITS region was the least informative marker, being unable to discriminate some of the species included in the analysis (Fig. S1 in supplemental material). Two main clades were defined using this genetic marker, one grouping the species A. chevalieri, A. intermedius, A. montevidensis, A. cristatus, and A. costiformis, and the second one grouping the species A. pseudoglaucus, A. glaucus, A. neocarnoyi, A. niveoglaucus, A. brunneus, A. proliferans, A. ruber, A. appendiculatus, A. cibarius, A. tonophilus, and A. sloanii. The other markers (BenA, CaM, and RPB2) were more informative, with better delineation in well-supported monophyletic groups (Figs. S2–S4 in supplemental material). The single phylogenetic analysis corresponding to the different genes showed very similar tree topologies, and a concatenated study was performed. The final concatenated sequence alignment consisted of 2,653 bases (ITS, 641; BenA, 433; CaM, 596; RPB2, 983), of which 794 were variable sites (ITS, 105; BenA, 176; CaM, 255; RPB2, 258) and 461 parsimony informative (ITS, 31; BenA, 104; CaM, 160; RPB2, 166). The ML tree (Fig. 1) shows significant support values for both phylogenetic methods (bootstrap/posterior probabilities). Figure 1. View largeDownload slide Maximum likelihood tree obtained from the combined ITS, BenA, CaM and RPB2 sequences of the isolates. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores over 70/0.95 are indicated on the nodes. The fully supported branches (100/1) and type strains are shown in bold. The new species is shown in the colored box. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Maximum likelihood tree obtained from the combined ITS, BenA, CaM and RPB2 sequences of the isolates. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores over 70/0.95 are indicated on the nodes. The fully supported branches (100/1) and type strains are shown in bold. The new species is shown in the colored box. This Figure is reproduced in color in the online version of Medical Mycology. The clinical isolates grouped together with the following species: A. montevidensis (11 isolates, 44%), A. chevalieri (9 isolates, 36%), and A. pseudoglaucus (2 isolates, 8%). The environmental isolate was identified as A. costiformis (4%). The isolates UTHSCSA DI16-400 (=CBS 142377) and UTHSCSA DI16-407 (=CBS 142376), from toenail and lymph node samples, respectively, and sequences of two environmental isolates (CCF 5387 and CCF 5388) retrieved from GenBank, formed a full-supported clade close to the A. pseudoglaucus clade, which represents an undescribed phylogenetic lineage for the section Aspergillus. Therefore, we propose the new species A. microperforatus. The morphology of the isolates shows the expected phenotypic characters that agree with the previous species descriptions.1,4,32–34Aspergillus montevidensis exhibits rough ascospores, with irregular crests; A. chevalieri shows smooth ascospores, with prominent crests; A. costiformis is the only species with smooth conidia; and A. pseudoglaucus and A. microperforatus demonstrated smooth ascospores, with no crests, and rough conidia. In fact, these last two species have a similar morphology, being differentiated by the slow growth and restricted sporulation of the novel species on CYA at 25°C and on M60Y at 37°C and the absence of soluble pigment in any of the culture media tested. Aspergillus pseudoglaucus isolates identified here grew and sporulated well on both media and temperatures, and produced a brownish soluble pigment on CYA at 25°C in 14 days of incubation. Table 2 shows the key phenotypic features of the species of genus Aspergillus already reported in clinic, including those recovered in this study. Table 2. Relevant features of the genus Aspergillus already reported in clinical setting. Growth rate (mm) at 7d Species 25°C 37°C Ascospores Conidia Long axis Equatorial Ornamen- Size Ornamen- CYA CY20S CY20S M60Y (μm) region tation (μm) tation References A. chevalieri 16–24 45–65 30–49 65–>70 4.5–6.5 crests prominent smooth 3.5–5.5 rough this study A. costiformis 18–21 33–38 36–39 >70 6–8 crests irregular rough 4–8 (12) smooth this study A. glaucus 3–20 30–45 0 0 6–7.5 crests absent smooth 4.5–8.5 rough [1, 4] A. microperforatus 8–15 40–46 0 28–32 4–5.5 crests absent smooth 6–9.5(11) rough this study A. montevidensis 17–21 36–48 39–55 68–>70 3.5–5.5 crests irregular rough 4.5–5.5 rough this study A. proliferans NA 15–22 0 0 4.5–6 crests absent smooth 5–9 rough [1, 4] A. pseudoglaucus 22–24 38–44 0 41–46 3.5–5.5 crests absent smooth 7–8.5 rough this study A. ruber NA >30 0 NA 5–6 crests absent smooth 5–7.5 rough [1, 4] Growth rate (mm) at 7d Species 25°C 37°C Ascospores Conidia Long axis Equatorial Ornamen- Size Ornamen- CYA CY20S CY20S M60Y (μm) region tation (μm) tation References A. chevalieri 16–24 45–65 30–49 65–>70 4.5–6.5 crests prominent smooth 3.5–5.5 rough this study A. costiformis 18–21 33–38 36–39 >70 6–8 crests irregular rough 4–8 (12) smooth this study A. glaucus 3–20 30–45 0 0 6–7.5 crests absent smooth 4.5–8.5 rough [1, 4] A. microperforatus 8–15 40–46 0 28–32 4–5.5 crests absent smooth 6–9.5(11) rough this study A. montevidensis 17–21 36–48 39–55 68–>70 3.5–5.5 crests irregular rough 4.5–5.5 rough this study A. proliferans NA 15–22 0 0 4.5–6 crests absent smooth 5–9 rough [1, 4] A. pseudoglaucus 22–24 38–44 0 41–46 3.5–5.5 crests absent smooth 7–8.5 rough this study A. ruber NA >30 0 NA 5–6 crests absent smooth 5–7.5 rough [1, 4] CYA, Czapek yeast autolysate agar; CY20S, CYA supplemented with 20% sucrose; MEA, malt extract agar; M60Y, Harrold's agar; NA, not available. View Large Table 2. Relevant features of the genus Aspergillus already reported in clinical setting. Growth rate (mm) at 7d Species 25°C 37°C Ascospores Conidia Long axis Equatorial Ornamen- Size Ornamen- CYA CY20S CY20S M60Y (μm) region tation (μm) tation References A. chevalieri 16–24 45–65 30–49 65–>70 4.5–6.5 crests prominent smooth 3.5–5.5 rough this study A. costiformis 18–21 33–38 36–39 >70 6–8 crests irregular rough 4–8 (12) smooth this study A. glaucus 3–20 30–45 0 0 6–7.5 crests absent smooth 4.5–8.5 rough [1, 4] A. microperforatus 8–15 40–46 0 28–32 4–5.5 crests absent smooth 6–9.5(11) rough this study A. montevidensis 17–21 36–48 39–55 68–>70 3.5–5.5 crests irregular rough 4.5–5.5 rough this study A. proliferans NA 15–22 0 0 4.5–6 crests absent smooth 5–9 rough [1, 4] A. pseudoglaucus 22–24 38–44 0 41–46 3.5–5.5 crests absent smooth 7–8.5 rough this study A. ruber NA >30 0 NA 5–6 crests absent smooth 5–7.5 rough [1, 4] Growth rate (mm) at 7d Species 25°C 37°C Ascospores Conidia Long axis Equatorial Ornamen- Size Ornamen- CYA CY20S CY20S M60Y (μm) region tation (μm) tation References A. chevalieri 16–24 45–65 30–49 65–>70 4.5–6.5 crests prominent smooth 3.5–5.5 rough this study A. costiformis 18–21 33–38 36–39 >70 6–8 crests irregular rough 4–8 (12) smooth this study A. glaucus 3–20 30–45 0 0 6–7.5 crests absent smooth 4.5–8.5 rough [1, 4] A. microperforatus 8–15 40–46 0 28–32 4–5.5 crests absent smooth 6–9.5(11) rough this study A. montevidensis 17–21 36–48 39–55 68–>70 3.5–5.5 crests irregular rough 4.5–5.5 rough this study A. proliferans NA 15–22 0 0 4.5–6 crests absent smooth 5–9 rough [1, 4] A. pseudoglaucus 22–24 38–44 0 41–46 3.5–5.5 crests absent smooth 7–8.5 rough this study A. ruber NA >30 0 NA 5–6 crests absent smooth 5–7.5 rough [1, 4] CYA, Czapek yeast autolysate agar; CY20S, CYA supplemented with 20% sucrose; MEA, malt extract agar; M60Y, Harrold's agar; NA, not available. View Large In general, all isolates were inhibited by each of the antifungal drugs tested, with overall geometric mean (GM) values lower than 1.0 μg/ml. The most potent activity was observed with the echinocandins (GM of 0.03 μg/ml), while VRC had the highest MIC values (GM of 1.0 μg/ml for A. pseudoglaucus, and 0.77 μg/ml for A. montevidensis, with individual values up to 2.0 μg/ml). The results of the in vitro susceptibility test are summarized in Table 3. Table 3. Results of in vitro antifungal susceptibility test for 25 isolates of Aspergillus section Aspergillus. Species (no. of isolates) MIC or MEC (μg/ml) for: AMB AFG CFG MFG ITC PSC VRC TBF A. chevalieri (9) GM 0.14 0.03 0.03 0.03 0.24 0.03 0.37 0.09 MIC range 0.06–0.5 0.03 0.03 0.03 0.12–1.0 0.03 0.12–0.5 0.03–0.12 Mode 0.12 0.03 0.03 0.03 0.5 0.03 0.5 0.12 A. costiformis (1) Values 0.25 0.03 0.03 0.03 0.25 0.06 0.5 0.12 A. microperforatus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 A. montevidensis (11) GM 0.25 0.03 0.03 0.03 0.19 0.03 0.77 0.13 MIC range 0.12–0.5 0.03–0.06 0.03 0.03 0.12–0.5 0.03–0.06 0.5–2.0 0.06–0.25 Mode 0.25 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.25 0.03 1.0 0.25 A. pseudoglaucus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 Total (25) GM 0.14 0.03 0.03 0.03 0.2 0.03 0.57 0.1 MIC range 0.03–0.5 0.03–0.06 0.03 0.03 0.12–1.0 0.03–0.06 0.12–2.0 0.03–0.25 Mode 0.12 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.5 0.03 1.0 0.12 Species (no. of isolates) MIC or MEC (μg/ml) for: AMB AFG CFG MFG ITC PSC VRC TBF A. chevalieri (9) GM 0.14 0.03 0.03 0.03 0.24 0.03 0.37 0.09 MIC range 0.06–0.5 0.03 0.03 0.03 0.12–1.0 0.03 0.12–0.5 0.03–0.12 Mode 0.12 0.03 0.03 0.03 0.5 0.03 0.5 0.12 A. costiformis (1) Values 0.25 0.03 0.03 0.03 0.25 0.06 0.5 0.12 A. microperforatus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 A. montevidensis (11) GM 0.25 0.03 0.03 0.03 0.19 0.03 0.77 0.13 MIC range 0.12–0.5 0.03–0.06 0.03 0.03 0.12–0.5 0.03–0.06 0.5–2.0 0.06–0.25 Mode 0.25 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.25 0.03 1.0 0.25 A. pseudoglaucus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 Total (25) GM 0.14 0.03 0.03 0.03 0.2 0.03 0.57 0.1 MIC range 0.03–0.5 0.03–0.06 0.03 0.03 0.12–1.0 0.03–0.06 0.12–2.0 0.03–0.25 Mode 0.12 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.5 0.03 1.0 0.12 AMB, amphotericin B; AFG, anidulafungin; CFG, caspofungin; MFG, micafungin; ITC, itraconazole; PSC, posaconazole; VRC, voriconazole; TBF, terbinafine; MIC, minimum inhibitory concentration; MEC, minimum effective concentration, for AFG, CFG, and MFG; GM, geometric mean. View Large Table 3. Results of in vitro antifungal susceptibility test for 25 isolates of Aspergillus section Aspergillus. Species (no. of isolates) MIC or MEC (μg/ml) for: AMB AFG CFG MFG ITC PSC VRC TBF A. chevalieri (9) GM 0.14 0.03 0.03 0.03 0.24 0.03 0.37 0.09 MIC range 0.06–0.5 0.03 0.03 0.03 0.12–1.0 0.03 0.12–0.5 0.03–0.12 Mode 0.12 0.03 0.03 0.03 0.5 0.03 0.5 0.12 A. costiformis (1) Values 0.25 0.03 0.03 0.03 0.25 0.06 0.5 0.12 A. microperforatus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 A. montevidensis (11) GM 0.25 0.03 0.03 0.03 0.19 0.03 0.77 0.13 MIC range 0.12–0.5 0.03–0.06 0.03 0.03 0.12–0.5 0.03–0.06 0.5–2.0 0.06–0.25 Mode 0.25 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.25 0.03 1.0 0.25 A. pseudoglaucus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 Total (25) GM 0.14 0.03 0.03 0.03 0.2 0.03 0.57 0.1 MIC range 0.03–0.5 0.03–0.06 0.03 0.03 0.12–1.0 0.03–0.06 0.12–2.0 0.03–0.25 Mode 0.12 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.5 0.03 1.0 0.12 Species (no. of isolates) MIC or MEC (μg/ml) for: AMB AFG CFG MFG ITC PSC VRC TBF A. chevalieri (9) GM 0.14 0.03 0.03 0.03 0.24 0.03 0.37 0.09 MIC range 0.06–0.5 0.03 0.03 0.03 0.12–1.0 0.03 0.12–0.5 0.03–0.12 Mode 0.12 0.03 0.03 0.03 0.5 0.03 0.5 0.12 A. costiformis (1) Values 0.25 0.03 0.03 0.03 0.25 0.06 0.5 0.12 A. microperforatus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 A. montevidensis (11) GM 0.25 0.03 0.03 0.03 0.19 0.03 0.77 0.13 MIC range 0.12–0.5 0.03–0.06 0.03 0.03 0.12–0.5 0.03–0.06 0.5–2.0 0.06–0.25 Mode 0.25 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.25 0.03 1.0 0.25 A. pseudoglaucus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 Total (25) GM 0.14 0.03 0.03 0.03 0.2 0.03 0.57 0.1 MIC range 0.03–0.5 0.03–0.06 0.03 0.03 0.12–1.0 0.03–0.06 0.12–2.0 0.03–0.25 Mode 0.12 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.5 0.03 1.0 0.12 AMB, amphotericin B; AFG, anidulafungin; CFG, caspofungin; MFG, micafungin; ITC, itraconazole; PSC, posaconazole; VRC, voriconazole; TBF, terbinafine; MIC, minimum inhibitory concentration; MEC, minimum effective concentration, for AFG, CFG, and MFG; GM, geometric mean. View Large Taxonomy Aspergillus microperforatus J.P.Z. Siqueira, Deanna A. Sutton & Gené, sp. nov. (MycoBank MB 820080, Fig. 2). Figure 2. View largeDownload slide Morphological features of Aspergillus microperforatus sp. nov. (UTHSCSA DI16-407). Panels: a, b, c, f, g, h, front and reverse of colonies on CY20S, DG18, and YES, respectively, after 7 days at 25ºC; d, e, front of colonies on CYA, and MEA, respectively, after 7 days at 25ºC; i, front of colonies on M60Y after 7 days at 37ºC; j, front of colonies on CYA after 14 days at 25º; k, l, ascoma; m, n, asci; o, p, ascospores; q, r, s, conidial heads; t, u, conidia. Scale bars: k, 100 μm, l–u, 10 μm. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Morphological features of Aspergillus microperforatus sp. nov. (UTHSCSA DI16-407). Panels: a, b, c, f, g, h, front and reverse of colonies on CY20S, DG18, and YES, respectively, after 7 days at 25ºC; d, e, front of colonies on CYA, and MEA, respectively, after 7 days at 25ºC; i, front of colonies on M60Y after 7 days at 37ºC; j, front of colonies on CYA after 14 days at 25º; k, l, ascoma; m, n, asci; o, p, ascospores; q, r, s, conidial heads; t, u, conidia. Scale bars: k, 100 μm, l–u, 10 μm. This Figure is reproduced in color in the online version of Medical Mycology. Colonies on CYA 8–15 mm diameter in 7 days at 25°C, floccose, yellowish white (3A2) at the center, white toward the periphery, sporulation scarce, margin entire; reverse pale (2A2) to olive (3A3); exudate and soluble pigment absent. On CY20S, colonies 30–45 mm diameter in 7 days at 25°C, granulose due to the presence of ascomata, sporulation abundant, conidial masse greyish green (25E5); reverse brownish orange (7C5) to olive (3A6) at the center, pale yellow (2A3) to yellow (2A6) toward the periphery; exudate and soluble pigment absent. On MEA, colonies 11 mm diameter in 7 days at 25°C; sporulation absent in UTHSCSA DI16–407, abundant in UTHSCSA DI16–400, with conidial masse brown (5A5), margin entire; reverse pale (2A2) to brownish orange (5C3); exudate and soluble pigment absent. On YES, colonies 33–40 mm in 7 days at 25°C, velutinous to downy, slightly granulose at the center due to the presence of ascomata, sporulation abundant, with conidial masse dark green (27F5), margin entire; reverse pale yellow (4A3) to light yellow (4A4). On DG18, colonies 30–45 mm in 7 days at 25°C, fuzzy, white to light orange (5A5), sporulation abundant, conidial mass honey yellow (4D6); reverse light yellow (1A4) to yellow (3A7). On CREA, colonies up to 5 mm in 7 days at 25°C, acid production absent. No growth on OA at 25°C or on CY20S at 37°C. Conidiophores up to 550 μm long, with uniseriate and radiating conidial heads; stipes occasionally septate, 260–500 × 6.5–9.5 μm, hyaline to subhyaline, smooth to finely roughened; vesicles subglobose to pyriform, 24–36 μm diameter; phialides variable in shape and size, ampulliform to cylindrical, 7–18 (30) × 2–5 μm; conidia globose to elongate, sometimes pyriform, 6–9.5(–11) × 4.5–9 μm, in shades of brown, rough. Cleistothecia globose to subglobose, 90–130 μm diameter, light yellow (2A5) to deep yellow (4A8); asci globose, 10–14 μm in diameter; ascospores lenticular, 4–5.5 × 2.5–4.5 μm, hyaline, with a slight furrow in the equatorial region, convex surface smooth with very small pits only visible under SEM. Etymology Referring to the presence of small pits in the ascospore wall under SEM. Type usa, Texas, isolated from human lymph node, D.A. Sutton, 2011 (CBS H-22998 holotype; cultures ex-type: UTHSCSA DI16-407, CBS 142376, FMR 14071). Discussion Although the diversity of genus Aspergillus species is well known in osmophilic substrates, house dust, indoor air, or stored products, in the clinical setting it is poorly documented. As previously noted, the taxonomy and nomenclature of the species of section Aspergillus has recently changed. In addition to that, recent advances in molecular tools have allowed for the description of new cryptic species that are almost impossible to differentiate using classical morphological tools.35 Clinically, identification of Aspergillus isolates at the species level may be important given that susceptibilities to antifungal drugs vary for different species and that species identity can influence the choice of appropriate antifungal therapy.36 In the present study, a total of 25 isolates of genus Aspergillus were used, most of them being recovered from human respiratory specimens, although further studies are needed to elucidate the role of these fungi as pathogens. Five different species of the section have been identified here, including a novel one (i.e., A. chevalieri, A. costiformis, A. microperforatus, A. montevidensis and A. pseudoglaucus). To facilitate comparison, their key morphological features are presented in Table 2. Aspergillus montevidensis was the most prevalent (44%). Clinically, this species may be the most relevant pathogen of this group because it has been isolated from different bodies sites, from superficial to deep tissue infections.10,15,37 It was first reported and described from a case of human otomycosis.38 This species currently includes strains that were formerly accepted as different but now considered conspecific. Aspergillus hollandicus (incorrectly associated to the sexual state Eurotium amstelodami), A. heterocaryoticus, and A. vitis are all synonyms of A. montevidensis, and these names should no longer be used.4 The nine isolates of A. montevidensis in the present study, with the exception of two of unknown origins, were from respiratory specimens (i.e., sputum, sinuses, and lung biopsies). The second most prevalent species in the present study is A. chevalieri (36%), known until recently by its teleomorph name, Eurotium chevalieri. This species has been reported from a case of cutaneous aspergillosis13 and more recently was the cause of fatal cerebral aspergillosis acquired by traumatic inoculation.17 It is worth noting that A. chevalieri and A. montevidensis represent 80% of the isolates included in this study, in fact they are also some of the most commonly species found in indoor environments.4 These two species, and the others reported here, were able to grow well at 37°C in vitro (Table 2), the basic pathogenic feature that enables them to invade deep tissue.39 The isolate of A. costiformis identified in this study is the third known strain of this species since its description. It was originally recovered from a mouldy paper-box in China.34 The second strain was isolated from a human nail in the Czech Republic,15 and the present study has recovered a third strain from hospital environment. The difficulty in the phenotypic characterization of this species might explain why it is rarely reported. The key characteristic that identifies A. costiformis is the presence of smooth conidia (Table 2) and the asexual morph is not usually formed in standard culture conditions. To overcome this problem, the production of conidial heads can be induced on M60Y at 37°C, as previously reported.4 The remaining isolates included in this study were genetically and morphologically similar but they could be distinguished as two different species (each representing 8% of the isolates). First, isolates UTHSCSA DI15-17 and UTHSCSA DI16-410 were identified as A. pseudoglaucus, a species described by Blochwitz in 1929.33 This species was more recently delineated phylogenetically by Hubka et al.4 in whose study other species were shown to be conspecific with A. pseudoglaucus, that is, A. glaucoaffinis/E. pseudoglaucum,40A. glaber,40A. fimicola,41 and A. reptans/E. repens.40 The phylogenetic tree constructed in the present study includes sequences of the ex-type strains of synonymous species and of numerous reference strains, giving more support to the A. pseudoglaucus clade. Our analysis shows that the genetic similarity among all those strains is 99.9% or higher in the concatenated alignment, in agreement with the proposal mentioned above. Aspergillus pseudoglaucus is commonly found in stored products7 and produces metabolites that are potentially toxic.42 There are few clinical reports involving A. pseudoglacus; it has been reported from a mixed infection in a case of maxillary sinusitis14 and occasionally recovered from human skin and nails,15 although its pathogenicity has not been confirmed. The two isolates of A. pseudoglaucus identified in the present study were from nasal and stool samples. Second, the isolates UTHSCSA DI16-400 and UTHSCSA DI16-407, recovered from toenail and lymph node, respectively, and sequences of two isolates retrieved from GenBank group in a clade close to A. pseudoglaucus. Although these latter isolates were labelled as A. pseudoglaucus, both phylogenetic methods (ML and BI) and the three most informative markers (BenA, CaM, and RPB2) all show that they represent together with our isolates investigated a distinct lineage in the genus.4 Thus, they have been proposed as the novel species A. microperforatus. The possible role of this species in the clinical disease is yet unknown. Although genus Aspergillus includes some species that are closely related, some characteristics can be useful for discriminating A. microperforatus (Table 2). For example, the novel species shows restricted growth on CYA at 25°C (up to 15 mm), in contrast to A. pseudoglaucus (up to 24 mm), which also differs by growing better on M60Y at 37°C (41 to 46 mm, vs. 28 to 32 mm diameter in A. microperforatus) and, according to our results, on CYA at 25°C it produces a diffusible brown pigment that is absent in A. microperforatus. The novel species can be differentiated from A. glaucus and A. proliferans by its ability to grow on M60Y at 37°C and A. glaucus has larger ascospores (6.0 to 7.5 μm, vs. 4.0 to 5.5 μm in A. microperforatus). Based on the descriptions and other reports, it might be difficult to differentiate the morphologies of A. microperforatus and A. ruber although the ascospores of A. ruber have an evident furrow and the conidia are usually ellipsoidal.1,4 Although A. glaucus is a known opportunistic pathogen, being reported from many types of infections,8,9,11 it was not recovered in the present study, as was the case in a study of clinical aspergilli in the Czech Republic.15 Therefore, it is possible that the clinical prevalence of this species has been overestimated, probably due to the limitations of diagnostic tools for Aspergillus identification and for filamentous fungi in general. The CLSI have established epidemiological cut-off values for triazoles (ITC, VRC, and POS) and AMB for only six Aspergillus species, that is, A. fumigatus, A. flavus, A. niger, A. terreus, A. nidulans, and A. versicolor.43,44 However, the limited number of isolates of other species available in different clinical laboratories precludes the determination of epidemiologic cutoff values, and members of genus Aspergillus have only been rarely tested for antifungal susceptibility.16 With few exceptions, the eight antifungals used in this study showed good activity against the aspergilli tested, with MIC values equal to or less than 1.0 μg/ml (Table 3). Recently, Masih et al.17 provided the in vitro antifungal susceptibility profiles of rare Aspergillus species in clinical samples from India. Although they did not test TBF, the MIC values for the other seven antifungals against three isolates of A. montevidensis and one strain of A. chevalieri were similar to the values observed in the current study. Most available in vitro data are with A. glaucus. Wildfeuer et al.21 and García-Martos et al.22 include eight and three clinical isolates of A. glaucus, respectively, and both reported good activity for ITC (MIC range of 0.25–0.5 μg/ml), VRC (0.125–0.78 μg/ml), and AMB (0.125–1.56 μg/ml). Although we did not study any A. glaucus strains, these values are similar to the overall values observed for the strains tested here. However, our MIC values were slightly lower for AMB and higher for VRC. Furthermore, García-Martos et al.21 also included two isolates of A. chevalieri and report the same MIC range (0.125–0.25 μg/ml) for ITC, VRC, and AMB. These results are very similar to those found in our A. chevalieri isolates, with the exception of one isolate that had an ITC MIC of 1.0 μg/ml. In summary, this study has assessed the species diversity of genus Aspergillus from a set of clinical isolates from the United Statesand demonstrated that A. montevidensis and A. chevalieri were the most frequently identified species. We also describe A. microperforatus as a new species. The antifungals tested showed potent activity against these isolates, especially the echinocandins and PSC. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgements This study was supported by the Spanish Ministerio de Economía y Competitividad, grant CGL2013-43789-P and by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil), grant BEX 0623/14-8. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. 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Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics . In: Innis MA , Gelfand DH , Sninsky JJ , White TJ , eds. PCR Protocols: A Guide to Methods and Applications. New York : Academic Press Inc .; 1990 : 315 – 322 . CrossRef Search ADS 25. Glass NL , Donaldson GC . Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes . Appl Environ Microbiol . 1995 ; 61 ( 4 ): 1323 – 1330 . Google Scholar PubMed 26. Hong SB , Go SJ , Shin HD , Frisvad JC , Samson RA . Polyphasic taxonomy of Aspergillus fumigatus and related species . Mycologia . 2005 ; 97 ( 6 ): 1316 – 1329 . Google Scholar CrossRef Search ADS PubMed 27. Liu YJ , Whelen S , Hall BD . Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit . Mol Biol Evol . 1999 ; 16 ( 12 ): 1799 – 1808 . Google Scholar CrossRef Search ADS PubMed 28. Tamura K , Stecher G , Peterson D , Filipski A , Kumar S . MEGA6: molecular evolutionary genetics analysis version 6.0 . Mol Biol Evol . 2013 ; 30 ( 12 ): 2725 – 2729 . Google Scholar CrossRef Search ADS PubMed 29. Ronquist F , Huelsenbeck JP . MRBAYES 3: Bayesian phylogenetic inference under mixed models . Bioinformatics . 2003 ; 19 : 1572 – 1574 . Google Scholar CrossRef Search ADS PubMed 30. Nylander JAA. MrModeltest v2. Program distributed by the author . Evolutionary Biology Centre , Uppsala University . 2004 . 31. Clinical and Laboratory Standards Institute . Reference method for broth dilution antifungals susceptibility testing of conidium-forming filamentous fungi: approved standard , 2nd ed . M38-A2 . Wayne, PA : CLSI ; 2008 . 32. Mangin L. Quest-ce que L’Aspergillus glaucus? Etude critique et experimentale des formes groupers sous ce nom . Ann Sci Nat Bot . 1909 ; 9 : 303 – 371 . 33. Gattung Blochwitz VA. Die Aspergillus. Neue Species. Diagnosen. Synonyme . Ann Mycol . 1929 ; 27 ( 3–4 ): 205 – 240 . 34. Kong H , Qi Z . Two new species of Eurotium Link. Acta Mycol Sin . 1995 ; 14 ( 1 ): 10 – 16 . 35. Alastruey-Izquierdo A , Mellado E , Cuenca-Estrella M . Current section and species complex concepts in Aspergillus: recommendations for routine daily practice . Ann N Y Acad Sci . 2012 ; 1273 : 18 – 24 . Google Scholar CrossRef Search ADS PubMed 36. Balajee SA , Houbraken J , Verweij PE et al. Aspergillus species identification in the clinical setting . Stud Mycol . 2007 ; 59 : 39 – 46 . Google Scholar CrossRef Search ADS PubMed 37. Marr KA , Patterson T , Denning D . Aspergillosis: pathogenesis, clinical manifestations, and therapy . Infect Dis Clin North Am . 2002 ; 16 ( 4 ): 875 – 894 . Google Scholar CrossRef Search ADS PubMed 38. Talice R V. , MaKinnon JE. Aspergillus (Eurotium) montevidensis, n. sp. isolé d’un cas d’otomycose chez l’homme . Compt Rend Soc Biol . 1931 ; 108 : 1007 – 1009 . 39. Kobayashi GS. Disease of mechanisms of fungi . In: Baron S , ed. Medical Microbiology . 4th ed . Galveston : University of Texas Medical Branch at Galveston ; 1996 . 40. Samson RA , Gams W . Typification of the species of Aspergillus and associated teleomorphs . In: Samson RA , Pitt JI , eds. Advances in Penicillium and Aspergillus Systematics . New York : Plenum Press ; 1985 : 31 – 54 . Google Scholar CrossRef Search ADS 41. Kong H , Qi Z . Two new species of Eurotium isolated from Xizang (Tibet), China . Acta Microbiol Pol . 1995 ; 14 ( 2 ): 86 – 91 . 42. Podojil M , Sedmera P , Vokoun J et al. Eurotium (Aspergillus) repens metabolites and their biological activity . Folia Microbiol (Praha) . 1978 ; 23 ( 6 ): 438 – 443 . Google Scholar CrossRef Search ADS PubMed 43. Espinel-Ingroff A , Diekema DJ , Fothergill A et al. Wild-type MIC distributions and epidemiological cutoff values for the triazoles and six Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document) . J Clin Microbiol . 2010 ; 48 ( 9 ): 3251 – 3257 . Google Scholar CrossRef Search ADS PubMed 44. Espinel-Ingroff A , Cuenca-Estrella M , Fothergill A et al. Wild-type MIC distributions and epidemiological cutoff values for amphotericin b and Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document) . Antimicrob Agents Chemother . 2011 ; 55 ( 11 ): 5150 – 5154 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Species of Aspergillus section Aspergillus from clinical samples in the United States

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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1369-3786
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1460-2709
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10.1093/mmy/myx085
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Abstract

Abstract The diversity of Aspergillus species in clinical samples is continuously increasing. Species under the former name Eurotium, currently accommodated in section Aspergillus of the genus Aspergillus, are xerophilic fungi widely found in the human environment and able to grow on substrates with low water activity. However, their prevalence in the clinical setting is poorly known. We have studied the presence of these species in a set of clinical samples from the United States using a multilocus sequence analysis based on the internal transcribed spacer (ITS) region of the rRNA, and fragments of the genes β-tubulin (BenA), calmodulin (CaM), and polymerase II second largest subunit (RPB2). A total of 25 isolates were studied and identified as follows: A. montevidensis (44%), A. chevalieri (36%), A. pseudoglaucus (8%), and A. costiformis (4%). A new species Aspergillus microperforatus is also proposed, which represented 8% of the isolates studied and is characterized by uniseriate conidial heads, subglobose to pyriform vesicles, rough conidia, globose to subglobose cleistothecia, and lenticular and smooth ascospores. The in vitro antifungal activity of eight clinically available antifungals was also determined against these isolates, with the echinocandins and posaconazole having the most potent activity. Aspergillus, phylogenetic analysis, antifungal susceptibility, taxonomy, Eurotium Introduction The number of species of Aspergillus involved in human infections is continuously increasing, and most of these species have nowadays been identified using modern molecular techniques. Until recently, the dual nomenclature system permitted different names for the sexual and asexual forms of Aspergillus. One such example is the genus Eurotium, the name for the sexual state for species in the former Aspergillus glaucus group.1 However, following the recent changes in fungal nomenclature2,3 and based on phylogenetic studies,4,5 all generic names for sexual states of Aspergillus are now included under the name Aspergillus, and former species of Eurotium now comprise the aspergilli in the section Aspergillus.4 A new approach to phylogenetic study supports the current broad concept of the genus Aspergillus,5 with A. glaucus (= E. herbariorum) being the type species of the genus. Species in the section Aspergillus are usually osmophilic, with optimum growth on substrates with high sugar or salt concentrations. Commonly the asexual morph has smooth conidiophores, with uniseriate, radiate to somewhat columnar conidial heads, and ellipsoidal to globose echinulate conidia.1,6 The sexual morph is usually characterized by globose to subglobose, thin-walled cleistothecia, eight-spored asci, and lenticular, smooth to rough-walled ascospores, generally showing an equatorial line or furrow.1,7 These species are found worldwide and often on organic materials, dust, and stored cereals and other food products.1,7 Although these aspergilli are of minimal clinical importance, some, such as A. glaucus, have been reported in orofacial8 and brain infections.9 In addition, A. montevidensis has been involved in cases of otitis, mycetoma, cerebral abscess, keratitis, and pulmonary infections,10 and A. glaucus and A. montevidensis can also cause hypersensitive pneumonitis11,12. Aspergillus chevalieri and A. pseudoglaucus have been linked to cutaneous aspergillosis13 and maxillary sinusitis,14 respectively. Hubka et al.15 recovered five species of section Aspergillus among isolates from probable cases of superficial infections (e.g., skin and nails), including A. montevidensis, A. costiformis, A. pseudoglaucus, A. proliferans, and A. ruber in Czech Republic. The antifungal susceptibility patterns of members of section Aspergillus are largely unknown,16 with little published data. Masih et al.17 demonstrated potent activity of posaconazole, anidulafungin, and micafungin against three strains of A. montevidensis (GM minimal inhibitory concentrations [MICs] of 0.04 μg/ml, 0.015 μg/ml, and 0.015 μg/ml, respectively) and one of A. chevalieri (MICs of 0.015 μg/ml, 0.03 μg/ml, and 0.015 μg/ml, respectively). García-Martos et al.18 also demonstrated low MIC values for amphotericin B, itraconazole, and voriconazole against three strains of A. glaucus (MICs of 0.125–0.5 μg/ml, 0.25–0.5 μg/ml, and 0.125–0.25 μg/ml, respectively) and against two strains of A. chevalieri (MICs of 0.125–0.5 μg/ml, 0.125–0.25 μg/ml, and 0.125–0.25 μg/ml, respectively). Wildfeuer et al.19 tested eight strains of A. glaucus against four drugs and observed that itraconazole exhibited the most potent activity (GM MIC of 0.39 μg/ml). In order to assess the diversity of species from Aspergillus section Aspergillus in the clinical setting and to observe their response to antifungal drugs, the aim of this study was to identify to the species level a set of clinical isolates from the United States using a multilocus phylogenetic study, and to determine the susceptibility pattern of eight clinically available antifungals against these species. Methods Fungal isolates A total of 25 isolates of section Aspergillus were investigated in this study. Most of them were from human clinical samples, primarily from the respiratory tract (BAL, sputum, and sinus), but also in fewer numbers from corneas, nails, stool, and lymph nodes. One of them was of environmental origin, and the origin of four were unknown (Table 1). These isolates were received at the Fungus Testing Laboratory of the University of Texas Health Science Center (San Antonio, TX, USA) from different institutions across the United States over a period of 11 years (2004–2015), for identification and/or antifungal susceptibility testing. Table 1. Origins, year of isolation, and GenBank/EMBL accession numbers of the Aspergillus strains included in this study. GenBank/EMBL accession number Species Isolate number Origin Year ITS BenA CaM RPB2 A. chevalieri (9) UTHSCSA DI15-18 BAL 2014 LT627247 LT627272 LT627297 LT627322 UTHSCSA DI16-375 Sputum 2004 LT627248 LT627273 LT627298 LT627323 UTHSCSA DI16-381 BAL 2006 LT627249 LT627274 LT627299 LT627324 UTHSCSA DI16-382 BAL 2008 LT627250 LT627275 LT627300 LT627325 UTHSCSA DI16-394 BAL 2007 LT627251 LT627276 LT627301 LT627326 UTHSCSA DI16-396 Corneal 2008 LT627252 LT627277 LT627302 LT627327 UTHSCSA DI16-397 Sinus 2008 LT627253 LT627278 LT627303 LT627328 UTHSCSA DI16-413 Unknown 2008 LT627254 LT627279 LT627304 LT627329 UTHSCSA DI16-414 Unknown 2008 LT627255 LT627280 LT627305 LT627330 A. costiformis (1) UTHSCSA DI15-16 Environmental 2014 LT627256 LT627281 LT627306 LT627331 A. microperforatus (2) UTHSCSA DI16-400 Toe nail 2009 LT627270 LT627295 LT627320 LT627345 UTHSCSA DI16-407 Lymph node 2011 LT627271 LT627296 LT627321 LT627346 A. montevidensis (11) UTHSCSA DI15-19 Ethmoid sinus 2014 LT627257 LT627282 LT627307 LT627332 UTHSCSA DI15-20 Sputum 2014 LT627258 LT627283 LT627308 LT627333 UTHSCSA DI15-21 BAL 2015 LT627259 LT627284 LT627309 LT627334 UTHSCSA DI15-22 Sputum 2015 LT627260 LT627285 LT627310 LT627335 UTHSCSA DI16-401 Lung tissue 2009 LT627261 LT627286 LT627311 LT627336 UTHSCSA DI16-403 Sputum 2009 LT627262 LT627287 LT627312 LT627337 UTHSCSA DI16-405 Sinus 2010 LT627263 LT627288 LT627313 LT627338 UTHSCSA DI16-406 Lung tissue 2010 LT627264 LT627289 LT627314 LT627339 UTHSCSA DI16-408 Paranasal 2013 LT627265 LT627290 LT627315 LT627340 UTHSCSA DI16-411 Unknown 2008 LT627266 LT627291 LT627316 LT627341 UTHSCSA DI16-412 Unknown 2008 LT627267 LT627292 LT627317 LT627342 A. pseudoglaucus (2) UTHSCSA DI15-17 Nasal 2011 LT627268 LT627293 LT627318 LT627343 UTHSCSA DI16-410 Stool 2014 LT627269 LT627294 LT627319 LT627344 GenBank/EMBL accession number Species Isolate number Origin Year ITS BenA CaM RPB2 A. chevalieri (9) UTHSCSA DI15-18 BAL 2014 LT627247 LT627272 LT627297 LT627322 UTHSCSA DI16-375 Sputum 2004 LT627248 LT627273 LT627298 LT627323 UTHSCSA DI16-381 BAL 2006 LT627249 LT627274 LT627299 LT627324 UTHSCSA DI16-382 BAL 2008 LT627250 LT627275 LT627300 LT627325 UTHSCSA DI16-394 BAL 2007 LT627251 LT627276 LT627301 LT627326 UTHSCSA DI16-396 Corneal 2008 LT627252 LT627277 LT627302 LT627327 UTHSCSA DI16-397 Sinus 2008 LT627253 LT627278 LT627303 LT627328 UTHSCSA DI16-413 Unknown 2008 LT627254 LT627279 LT627304 LT627329 UTHSCSA DI16-414 Unknown 2008 LT627255 LT627280 LT627305 LT627330 A. costiformis (1) UTHSCSA DI15-16 Environmental 2014 LT627256 LT627281 LT627306 LT627331 A. microperforatus (2) UTHSCSA DI16-400 Toe nail 2009 LT627270 LT627295 LT627320 LT627345 UTHSCSA DI16-407 Lymph node 2011 LT627271 LT627296 LT627321 LT627346 A. montevidensis (11) UTHSCSA DI15-19 Ethmoid sinus 2014 LT627257 LT627282 LT627307 LT627332 UTHSCSA DI15-20 Sputum 2014 LT627258 LT627283 LT627308 LT627333 UTHSCSA DI15-21 BAL 2015 LT627259 LT627284 LT627309 LT627334 UTHSCSA DI15-22 Sputum 2015 LT627260 LT627285 LT627310 LT627335 UTHSCSA DI16-401 Lung tissue 2009 LT627261 LT627286 LT627311 LT627336 UTHSCSA DI16-403 Sputum 2009 LT627262 LT627287 LT627312 LT627337 UTHSCSA DI16-405 Sinus 2010 LT627263 LT627288 LT627313 LT627338 UTHSCSA DI16-406 Lung tissue 2010 LT627264 LT627289 LT627314 LT627339 UTHSCSA DI16-408 Paranasal 2013 LT627265 LT627290 LT627315 LT627340 UTHSCSA DI16-411 Unknown 2008 LT627266 LT627291 LT627316 LT627341 UTHSCSA DI16-412 Unknown 2008 LT627267 LT627292 LT627317 LT627342 A. pseudoglaucus (2) UTHSCSA DI15-17 Nasal 2011 LT627268 LT627293 LT627318 LT627343 UTHSCSA DI16-410 Stool 2014 LT627269 LT627294 LT627319 LT627344 BenA, β-tubulin; CaM, calmodulin; ITS, internal transcribed spacer regions of the rDNA and 5.8S region; RPB2, partial RNA polymerase II second largest subunit; UTHSCSA, University of Texas Health Science Center (San Antonio, USA). View Large Table 1. Origins, year of isolation, and GenBank/EMBL accession numbers of the Aspergillus strains included in this study. GenBank/EMBL accession number Species Isolate number Origin Year ITS BenA CaM RPB2 A. chevalieri (9) UTHSCSA DI15-18 BAL 2014 LT627247 LT627272 LT627297 LT627322 UTHSCSA DI16-375 Sputum 2004 LT627248 LT627273 LT627298 LT627323 UTHSCSA DI16-381 BAL 2006 LT627249 LT627274 LT627299 LT627324 UTHSCSA DI16-382 BAL 2008 LT627250 LT627275 LT627300 LT627325 UTHSCSA DI16-394 BAL 2007 LT627251 LT627276 LT627301 LT627326 UTHSCSA DI16-396 Corneal 2008 LT627252 LT627277 LT627302 LT627327 UTHSCSA DI16-397 Sinus 2008 LT627253 LT627278 LT627303 LT627328 UTHSCSA DI16-413 Unknown 2008 LT627254 LT627279 LT627304 LT627329 UTHSCSA DI16-414 Unknown 2008 LT627255 LT627280 LT627305 LT627330 A. costiformis (1) UTHSCSA DI15-16 Environmental 2014 LT627256 LT627281 LT627306 LT627331 A. microperforatus (2) UTHSCSA DI16-400 Toe nail 2009 LT627270 LT627295 LT627320 LT627345 UTHSCSA DI16-407 Lymph node 2011 LT627271 LT627296 LT627321 LT627346 A. montevidensis (11) UTHSCSA DI15-19 Ethmoid sinus 2014 LT627257 LT627282 LT627307 LT627332 UTHSCSA DI15-20 Sputum 2014 LT627258 LT627283 LT627308 LT627333 UTHSCSA DI15-21 BAL 2015 LT627259 LT627284 LT627309 LT627334 UTHSCSA DI15-22 Sputum 2015 LT627260 LT627285 LT627310 LT627335 UTHSCSA DI16-401 Lung tissue 2009 LT627261 LT627286 LT627311 LT627336 UTHSCSA DI16-403 Sputum 2009 LT627262 LT627287 LT627312 LT627337 UTHSCSA DI16-405 Sinus 2010 LT627263 LT627288 LT627313 LT627338 UTHSCSA DI16-406 Lung tissue 2010 LT627264 LT627289 LT627314 LT627339 UTHSCSA DI16-408 Paranasal 2013 LT627265 LT627290 LT627315 LT627340 UTHSCSA DI16-411 Unknown 2008 LT627266 LT627291 LT627316 LT627341 UTHSCSA DI16-412 Unknown 2008 LT627267 LT627292 LT627317 LT627342 A. pseudoglaucus (2) UTHSCSA DI15-17 Nasal 2011 LT627268 LT627293 LT627318 LT627343 UTHSCSA DI16-410 Stool 2014 LT627269 LT627294 LT627319 LT627344 GenBank/EMBL accession number Species Isolate number Origin Year ITS BenA CaM RPB2 A. chevalieri (9) UTHSCSA DI15-18 BAL 2014 LT627247 LT627272 LT627297 LT627322 UTHSCSA DI16-375 Sputum 2004 LT627248 LT627273 LT627298 LT627323 UTHSCSA DI16-381 BAL 2006 LT627249 LT627274 LT627299 LT627324 UTHSCSA DI16-382 BAL 2008 LT627250 LT627275 LT627300 LT627325 UTHSCSA DI16-394 BAL 2007 LT627251 LT627276 LT627301 LT627326 UTHSCSA DI16-396 Corneal 2008 LT627252 LT627277 LT627302 LT627327 UTHSCSA DI16-397 Sinus 2008 LT627253 LT627278 LT627303 LT627328 UTHSCSA DI16-413 Unknown 2008 LT627254 LT627279 LT627304 LT627329 UTHSCSA DI16-414 Unknown 2008 LT627255 LT627280 LT627305 LT627330 A. costiformis (1) UTHSCSA DI15-16 Environmental 2014 LT627256 LT627281 LT627306 LT627331 A. microperforatus (2) UTHSCSA DI16-400 Toe nail 2009 LT627270 LT627295 LT627320 LT627345 UTHSCSA DI16-407 Lymph node 2011 LT627271 LT627296 LT627321 LT627346 A. montevidensis (11) UTHSCSA DI15-19 Ethmoid sinus 2014 LT627257 LT627282 LT627307 LT627332 UTHSCSA DI15-20 Sputum 2014 LT627258 LT627283 LT627308 LT627333 UTHSCSA DI15-21 BAL 2015 LT627259 LT627284 LT627309 LT627334 UTHSCSA DI15-22 Sputum 2015 LT627260 LT627285 LT627310 LT627335 UTHSCSA DI16-401 Lung tissue 2009 LT627261 LT627286 LT627311 LT627336 UTHSCSA DI16-403 Sputum 2009 LT627262 LT627287 LT627312 LT627337 UTHSCSA DI16-405 Sinus 2010 LT627263 LT627288 LT627313 LT627338 UTHSCSA DI16-406 Lung tissue 2010 LT627264 LT627289 LT627314 LT627339 UTHSCSA DI16-408 Paranasal 2013 LT627265 LT627290 LT627315 LT627340 UTHSCSA DI16-411 Unknown 2008 LT627266 LT627291 LT627316 LT627341 UTHSCSA DI16-412 Unknown 2008 LT627267 LT627292 LT627317 LT627342 A. pseudoglaucus (2) UTHSCSA DI15-17 Nasal 2011 LT627268 LT627293 LT627318 LT627343 UTHSCSA DI16-410 Stool 2014 LT627269 LT627294 LT627319 LT627344 BenA, β-tubulin; CaM, calmodulin; ITS, internal transcribed spacer regions of the rDNA and 5.8S region; RPB2, partial RNA polymerase II second largest subunit; UTHSCSA, University of Texas Health Science Center (San Antonio, USA). View Large Morphological characterization The morphology of the fungi was characterized by the traditional criteria.4,20 Briefly, this is determined after 7 days of incubation on Czapek Yeast Autolysate agar (CYA, Becton, Dickinson and Company®, Sparks, MD, USA), CYA supplemented with 20% sucrose (CY20S), and Malt Extract agar (MEA, Pronadisa®, Madrid, Spain) at 25°C; and CY20S and Harrold's Agar containing 60% sucrose1 (M60Y) at 37°C. Colors match Kornerup and Wanscher.21 Microscopic features were examined and measured on MEA and CY20S cultures, after 10–14 days of incubation, on wet mounts with 60% lactic acid and a drop of ethanol 70% to wash out the excess conidia. Photomicrographs were taken with a DeltaPix Infinity X digital camera mounted on a Zeiss Axio Imager M1 light microscope (Zeiss, Oberkochen, Germany), using a Nomarski differential interference contrast and phase contrast optics. Scanning electron microscope (SEM) photographs were taken with a Jeol JSM- 6400 using techniques described previously.22 DNA extraction, amplification, and sequencing Total genomic DNA was extracted from MEA cultures after 7 days of incubation at 25°C, using the FastDNA® Kit and the FastPrep® Instrument (MP Biomedicals, Irvine CA, USA), according to the manufacturer's specifications. After extraction, four different genetic regions were amplified for each strain20,23; that is, the internal transcribed spacer (ITS) region of the rRNA, comprising ITS1, 5.8S gene, and ITS2 regions, using ITS5 and ITS4 primers24; a portion of β-tubulin gene (BenA), using Bt2a and Bt2b primers25; a portion of calmodulin gene (CaM), using Cmd5 and Cmd6 primers26; and a portion of RNA polymerase II second largest subunit gene (RPB2), using 5F and 7CR primers.27 Polymerase chain reaction (PCR) products were sequenced in both directions, using the same primers, at Macrogen Europe (Macrogen Inc., Amsterdam, the Netherlands). Sequences were assembled and edited using SeqMan v.7.0.0 (DNASTAR, Madison, WI, USA). Molecular identification and phylogenetic analysis The phylogeny was analyzed first individually for each partition and then in a concatenated study, once the topologies proved to be congruent. To give support to our analyses, sequences of the ex-type strains of all species of section Aspergillus obtained from GenBank were also included, and A. halophilicus (section Restricti) was used as the outgroup. To increase the robustness of the A. pseudoglaucus clade, sequences of 15 other strains of this species were additionally retrieved from GenBank and included in the analyses. A multiple sequence alignment was performed using ClustalW inside MEGA v.6 software.28 When necessary, the MUSCLE tool and manual adjustments were used to refine the alignment. Maximum Likelihood (ML) was conducted with MEGA v.6 software, as well as the estimation of the best nucleotide substitution method. Support values of the internal branches were assessed using the Bootstrap method with 1000 replications (values equal or higher than 70% were considered significant). Bayesian inference (BI) was performed using MrBayes v.3.1.2 software.29 The evolutionary models that best fit each partition were assessed by MrModel Test software.30 Markov chain Monte Carlo (MCMC) sampling was performed with two simultaneous runs for 1 million generations, with samples taken every 100 generations. The 50% majority rule consensus trees and posterior probability values (pp) were calculated after removing the first 25% of the resulting trees for burn-in. Values of 0.95 or higher were considered significant. Antifungal susceptibility testing The isolates were tested against eight antifungals, following the microdilution broth method.31 The antifungal agents tested were: amphotericin B (AMB) (Sigma Aldrich Quimica S.A., Madrid, Spain), itraconazole (ITC) (Jansen Pharmaceuticals, Beerse, Belgium), posaconazole (PSC) (Schering-Plough Res., Inst., Kenilworth, NJ, USA), voriconazole (VRC) (Pfizer S.A., Madrid, Spain), anidulafungin (AFG) (Pfizer S.A., Madrid, Spain), caspofungin (CFG) (Merk & Co., Inc., Rahway, NJ, USA), micafungin (MFG) (Astellas Pharma, Madrid, Spain), and terbinafine (TBF) (Sigma Aldrich Química S.A., Madrid, Spain). Readings were taken after 72 h to allow the strains to grow properly. Strains of A. pseudoglaucus (UTHSCSA DI15-17 and UTHSCSA DI16-410) and A. microperforatus (UTHSCSA DI16-400 and UTHSCSA DI16-407) were incubated at 30°C, while the others were incubated at 35°C to fit the growth requirements of the isolates under the CLSI protocol. Minimal inhibitory concentration (MIC) was defined as the lowest drug concentration that produced 100% inhibition of visible fungal growth for the AMB and azoles (ITC, PSC, and VRC) and 80% for TBF. For echinocandins (AFG, CFG, and MFG), the minimum effective concentrations (MEC) were determined microscopically as the lowest concentration of drug that allowed the growth of small, rounded, compact hyphal forms, as opposed to the long, unbranched hyphal clusters that are seen in the growth control. Candida krusei ATCC 6258 was used as the quality control strain in each test, and the MIC values were within the acceptable MIC range per the CLSI standard. All tests were carried out in duplicate, on different days, for reproducibility. Statistical analyses of the results were performed using the Prism software for Windows v.6.0 (GraphPad Software, San Diego, CA, USA). Nucleotide sequence accession numbers and alignments Newly generated sequences from this study have been deposited in GenBank/EMBL databases under the accession numbers listed on Table 1. The alignments were deposited in TreeBASE (submission number S20583). Results In the present study, as expected, the ITS region was the least informative marker, being unable to discriminate some of the species included in the analysis (Fig. S1 in supplemental material). Two main clades were defined using this genetic marker, one grouping the species A. chevalieri, A. intermedius, A. montevidensis, A. cristatus, and A. costiformis, and the second one grouping the species A. pseudoglaucus, A. glaucus, A. neocarnoyi, A. niveoglaucus, A. brunneus, A. proliferans, A. ruber, A. appendiculatus, A. cibarius, A. tonophilus, and A. sloanii. The other markers (BenA, CaM, and RPB2) were more informative, with better delineation in well-supported monophyletic groups (Figs. S2–S4 in supplemental material). The single phylogenetic analysis corresponding to the different genes showed very similar tree topologies, and a concatenated study was performed. The final concatenated sequence alignment consisted of 2,653 bases (ITS, 641; BenA, 433; CaM, 596; RPB2, 983), of which 794 were variable sites (ITS, 105; BenA, 176; CaM, 255; RPB2, 258) and 461 parsimony informative (ITS, 31; BenA, 104; CaM, 160; RPB2, 166). The ML tree (Fig. 1) shows significant support values for both phylogenetic methods (bootstrap/posterior probabilities). Figure 1. View largeDownload slide Maximum likelihood tree obtained from the combined ITS, BenA, CaM and RPB2 sequences of the isolates. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores over 70/0.95 are indicated on the nodes. The fully supported branches (100/1) and type strains are shown in bold. The new species is shown in the colored box. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Maximum likelihood tree obtained from the combined ITS, BenA, CaM and RPB2 sequences of the isolates. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores over 70/0.95 are indicated on the nodes. The fully supported branches (100/1) and type strains are shown in bold. The new species is shown in the colored box. This Figure is reproduced in color in the online version of Medical Mycology. The clinical isolates grouped together with the following species: A. montevidensis (11 isolates, 44%), A. chevalieri (9 isolates, 36%), and A. pseudoglaucus (2 isolates, 8%). The environmental isolate was identified as A. costiformis (4%). The isolates UTHSCSA DI16-400 (=CBS 142377) and UTHSCSA DI16-407 (=CBS 142376), from toenail and lymph node samples, respectively, and sequences of two environmental isolates (CCF 5387 and CCF 5388) retrieved from GenBank, formed a full-supported clade close to the A. pseudoglaucus clade, which represents an undescribed phylogenetic lineage for the section Aspergillus. Therefore, we propose the new species A. microperforatus. The morphology of the isolates shows the expected phenotypic characters that agree with the previous species descriptions.1,4,32–34Aspergillus montevidensis exhibits rough ascospores, with irregular crests; A. chevalieri shows smooth ascospores, with prominent crests; A. costiformis is the only species with smooth conidia; and A. pseudoglaucus and A. microperforatus demonstrated smooth ascospores, with no crests, and rough conidia. In fact, these last two species have a similar morphology, being differentiated by the slow growth and restricted sporulation of the novel species on CYA at 25°C and on M60Y at 37°C and the absence of soluble pigment in any of the culture media tested. Aspergillus pseudoglaucus isolates identified here grew and sporulated well on both media and temperatures, and produced a brownish soluble pigment on CYA at 25°C in 14 days of incubation. Table 2 shows the key phenotypic features of the species of genus Aspergillus already reported in clinic, including those recovered in this study. Table 2. Relevant features of the genus Aspergillus already reported in clinical setting. Growth rate (mm) at 7d Species 25°C 37°C Ascospores Conidia Long axis Equatorial Ornamen- Size Ornamen- CYA CY20S CY20S M60Y (μm) region tation (μm) tation References A. chevalieri 16–24 45–65 30–49 65–>70 4.5–6.5 crests prominent smooth 3.5–5.5 rough this study A. costiformis 18–21 33–38 36–39 >70 6–8 crests irregular rough 4–8 (12) smooth this study A. glaucus 3–20 30–45 0 0 6–7.5 crests absent smooth 4.5–8.5 rough [1, 4] A. microperforatus 8–15 40–46 0 28–32 4–5.5 crests absent smooth 6–9.5(11) rough this study A. montevidensis 17–21 36–48 39–55 68–>70 3.5–5.5 crests irregular rough 4.5–5.5 rough this study A. proliferans NA 15–22 0 0 4.5–6 crests absent smooth 5–9 rough [1, 4] A. pseudoglaucus 22–24 38–44 0 41–46 3.5–5.5 crests absent smooth 7–8.5 rough this study A. ruber NA >30 0 NA 5–6 crests absent smooth 5–7.5 rough [1, 4] Growth rate (mm) at 7d Species 25°C 37°C Ascospores Conidia Long axis Equatorial Ornamen- Size Ornamen- CYA CY20S CY20S M60Y (μm) region tation (μm) tation References A. chevalieri 16–24 45–65 30–49 65–>70 4.5–6.5 crests prominent smooth 3.5–5.5 rough this study A. costiformis 18–21 33–38 36–39 >70 6–8 crests irregular rough 4–8 (12) smooth this study A. glaucus 3–20 30–45 0 0 6–7.5 crests absent smooth 4.5–8.5 rough [1, 4] A. microperforatus 8–15 40–46 0 28–32 4–5.5 crests absent smooth 6–9.5(11) rough this study A. montevidensis 17–21 36–48 39–55 68–>70 3.5–5.5 crests irregular rough 4.5–5.5 rough this study A. proliferans NA 15–22 0 0 4.5–6 crests absent smooth 5–9 rough [1, 4] A. pseudoglaucus 22–24 38–44 0 41–46 3.5–5.5 crests absent smooth 7–8.5 rough this study A. ruber NA >30 0 NA 5–6 crests absent smooth 5–7.5 rough [1, 4] CYA, Czapek yeast autolysate agar; CY20S, CYA supplemented with 20% sucrose; MEA, malt extract agar; M60Y, Harrold's agar; NA, not available. View Large Table 2. Relevant features of the genus Aspergillus already reported in clinical setting. Growth rate (mm) at 7d Species 25°C 37°C Ascospores Conidia Long axis Equatorial Ornamen- Size Ornamen- CYA CY20S CY20S M60Y (μm) region tation (μm) tation References A. chevalieri 16–24 45–65 30–49 65–>70 4.5–6.5 crests prominent smooth 3.5–5.5 rough this study A. costiformis 18–21 33–38 36–39 >70 6–8 crests irregular rough 4–8 (12) smooth this study A. glaucus 3–20 30–45 0 0 6–7.5 crests absent smooth 4.5–8.5 rough [1, 4] A. microperforatus 8–15 40–46 0 28–32 4–5.5 crests absent smooth 6–9.5(11) rough this study A. montevidensis 17–21 36–48 39–55 68–>70 3.5–5.5 crests irregular rough 4.5–5.5 rough this study A. proliferans NA 15–22 0 0 4.5–6 crests absent smooth 5–9 rough [1, 4] A. pseudoglaucus 22–24 38–44 0 41–46 3.5–5.5 crests absent smooth 7–8.5 rough this study A. ruber NA >30 0 NA 5–6 crests absent smooth 5–7.5 rough [1, 4] Growth rate (mm) at 7d Species 25°C 37°C Ascospores Conidia Long axis Equatorial Ornamen- Size Ornamen- CYA CY20S CY20S M60Y (μm) region tation (μm) tation References A. chevalieri 16–24 45–65 30–49 65–>70 4.5–6.5 crests prominent smooth 3.5–5.5 rough this study A. costiformis 18–21 33–38 36–39 >70 6–8 crests irregular rough 4–8 (12) smooth this study A. glaucus 3–20 30–45 0 0 6–7.5 crests absent smooth 4.5–8.5 rough [1, 4] A. microperforatus 8–15 40–46 0 28–32 4–5.5 crests absent smooth 6–9.5(11) rough this study A. montevidensis 17–21 36–48 39–55 68–>70 3.5–5.5 crests irregular rough 4.5–5.5 rough this study A. proliferans NA 15–22 0 0 4.5–6 crests absent smooth 5–9 rough [1, 4] A. pseudoglaucus 22–24 38–44 0 41–46 3.5–5.5 crests absent smooth 7–8.5 rough this study A. ruber NA >30 0 NA 5–6 crests absent smooth 5–7.5 rough [1, 4] CYA, Czapek yeast autolysate agar; CY20S, CYA supplemented with 20% sucrose; MEA, malt extract agar; M60Y, Harrold's agar; NA, not available. View Large In general, all isolates were inhibited by each of the antifungal drugs tested, with overall geometric mean (GM) values lower than 1.0 μg/ml. The most potent activity was observed with the echinocandins (GM of 0.03 μg/ml), while VRC had the highest MIC values (GM of 1.0 μg/ml for A. pseudoglaucus, and 0.77 μg/ml for A. montevidensis, with individual values up to 2.0 μg/ml). The results of the in vitro susceptibility test are summarized in Table 3. Table 3. Results of in vitro antifungal susceptibility test for 25 isolates of Aspergillus section Aspergillus. Species (no. of isolates) MIC or MEC (μg/ml) for: AMB AFG CFG MFG ITC PSC VRC TBF A. chevalieri (9) GM 0.14 0.03 0.03 0.03 0.24 0.03 0.37 0.09 MIC range 0.06–0.5 0.03 0.03 0.03 0.12–1.0 0.03 0.12–0.5 0.03–0.12 Mode 0.12 0.03 0.03 0.03 0.5 0.03 0.5 0.12 A. costiformis (1) Values 0.25 0.03 0.03 0.03 0.25 0.06 0.5 0.12 A. microperforatus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 A. montevidensis (11) GM 0.25 0.03 0.03 0.03 0.19 0.03 0.77 0.13 MIC range 0.12–0.5 0.03–0.06 0.03 0.03 0.12–0.5 0.03–0.06 0.5–2.0 0.06–0.25 Mode 0.25 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.25 0.03 1.0 0.25 A. pseudoglaucus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 Total (25) GM 0.14 0.03 0.03 0.03 0.2 0.03 0.57 0.1 MIC range 0.03–0.5 0.03–0.06 0.03 0.03 0.12–1.0 0.03–0.06 0.12–2.0 0.03–0.25 Mode 0.12 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.5 0.03 1.0 0.12 Species (no. of isolates) MIC or MEC (μg/ml) for: AMB AFG CFG MFG ITC PSC VRC TBF A. chevalieri (9) GM 0.14 0.03 0.03 0.03 0.24 0.03 0.37 0.09 MIC range 0.06–0.5 0.03 0.03 0.03 0.12–1.0 0.03 0.12–0.5 0.03–0.12 Mode 0.12 0.03 0.03 0.03 0.5 0.03 0.5 0.12 A. costiformis (1) Values 0.25 0.03 0.03 0.03 0.25 0.06 0.5 0.12 A. microperforatus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 A. montevidensis (11) GM 0.25 0.03 0.03 0.03 0.19 0.03 0.77 0.13 MIC range 0.12–0.5 0.03–0.06 0.03 0.03 0.12–0.5 0.03–0.06 0.5–2.0 0.06–0.25 Mode 0.25 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.25 0.03 1.0 0.25 A. pseudoglaucus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 Total (25) GM 0.14 0.03 0.03 0.03 0.2 0.03 0.57 0.1 MIC range 0.03–0.5 0.03–0.06 0.03 0.03 0.12–1.0 0.03–0.06 0.12–2.0 0.03–0.25 Mode 0.12 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.5 0.03 1.0 0.12 AMB, amphotericin B; AFG, anidulafungin; CFG, caspofungin; MFG, micafungin; ITC, itraconazole; PSC, posaconazole; VRC, voriconazole; TBF, terbinafine; MIC, minimum inhibitory concentration; MEC, minimum effective concentration, for AFG, CFG, and MFG; GM, geometric mean. View Large Table 3. Results of in vitro antifungal susceptibility test for 25 isolates of Aspergillus section Aspergillus. Species (no. of isolates) MIC or MEC (μg/ml) for: AMB AFG CFG MFG ITC PSC VRC TBF A. chevalieri (9) GM 0.14 0.03 0.03 0.03 0.24 0.03 0.37 0.09 MIC range 0.06–0.5 0.03 0.03 0.03 0.12–1.0 0.03 0.12–0.5 0.03–0.12 Mode 0.12 0.03 0.03 0.03 0.5 0.03 0.5 0.12 A. costiformis (1) Values 0.25 0.03 0.03 0.03 0.25 0.06 0.5 0.12 A. microperforatus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 A. montevidensis (11) GM 0.25 0.03 0.03 0.03 0.19 0.03 0.77 0.13 MIC range 0.12–0.5 0.03–0.06 0.03 0.03 0.12–0.5 0.03–0.06 0.5–2.0 0.06–0.25 Mode 0.25 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.25 0.03 1.0 0.25 A. pseudoglaucus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 Total (25) GM 0.14 0.03 0.03 0.03 0.2 0.03 0.57 0.1 MIC range 0.03–0.5 0.03–0.06 0.03 0.03 0.12–1.0 0.03–0.06 0.12–2.0 0.03–0.25 Mode 0.12 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.5 0.03 1.0 0.12 Species (no. of isolates) MIC or MEC (μg/ml) for: AMB AFG CFG MFG ITC PSC VRC TBF A. chevalieri (9) GM 0.14 0.03 0.03 0.03 0.24 0.03 0.37 0.09 MIC range 0.06–0.5 0.03 0.03 0.03 0.12–1.0 0.03 0.12–0.5 0.03–0.12 Mode 0.12 0.03 0.03 0.03 0.5 0.03 0.5 0.12 A. costiformis (1) Values 0.25 0.03 0.03 0.03 0.25 0.06 0.5 0.12 A. microperforatus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.06 A. montevidensis (11) GM 0.25 0.03 0.03 0.03 0.19 0.03 0.77 0.13 MIC range 0.12–0.5 0.03–0.06 0.03 0.03 0.12–0.5 0.03–0.06 0.5–2.0 0.06–0.25 Mode 0.25 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.25 0.03 1.0 0.25 A. pseudoglaucus (2) GM 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 MIC range 0.03 0.03 0.03 0.03 0.12 0.03 1.0 0.12 Total (25) GM 0.14 0.03 0.03 0.03 0.2 0.03 0.57 0.1 MIC range 0.03–0.5 0.03–0.06 0.03 0.03 0.12–1.0 0.03–0.06 0.12–2.0 0.03–0.25 Mode 0.12 0.03 0.03 0.03 0.12 0.03 0.5 0.12 MIC90 0.5 0.03 0.03 0.03 0.5 0.03 1.0 0.12 AMB, amphotericin B; AFG, anidulafungin; CFG, caspofungin; MFG, micafungin; ITC, itraconazole; PSC, posaconazole; VRC, voriconazole; TBF, terbinafine; MIC, minimum inhibitory concentration; MEC, minimum effective concentration, for AFG, CFG, and MFG; GM, geometric mean. View Large Taxonomy Aspergillus microperforatus J.P.Z. Siqueira, Deanna A. Sutton & Gené, sp. nov. (MycoBank MB 820080, Fig. 2). Figure 2. View largeDownload slide Morphological features of Aspergillus microperforatus sp. nov. (UTHSCSA DI16-407). Panels: a, b, c, f, g, h, front and reverse of colonies on CY20S, DG18, and YES, respectively, after 7 days at 25ºC; d, e, front of colonies on CYA, and MEA, respectively, after 7 days at 25ºC; i, front of colonies on M60Y after 7 days at 37ºC; j, front of colonies on CYA after 14 days at 25º; k, l, ascoma; m, n, asci; o, p, ascospores; q, r, s, conidial heads; t, u, conidia. Scale bars: k, 100 μm, l–u, 10 μm. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Morphological features of Aspergillus microperforatus sp. nov. (UTHSCSA DI16-407). Panels: a, b, c, f, g, h, front and reverse of colonies on CY20S, DG18, and YES, respectively, after 7 days at 25ºC; d, e, front of colonies on CYA, and MEA, respectively, after 7 days at 25ºC; i, front of colonies on M60Y after 7 days at 37ºC; j, front of colonies on CYA after 14 days at 25º; k, l, ascoma; m, n, asci; o, p, ascospores; q, r, s, conidial heads; t, u, conidia. Scale bars: k, 100 μm, l–u, 10 μm. This Figure is reproduced in color in the online version of Medical Mycology. Colonies on CYA 8–15 mm diameter in 7 days at 25°C, floccose, yellowish white (3A2) at the center, white toward the periphery, sporulation scarce, margin entire; reverse pale (2A2) to olive (3A3); exudate and soluble pigment absent. On CY20S, colonies 30–45 mm diameter in 7 days at 25°C, granulose due to the presence of ascomata, sporulation abundant, conidial masse greyish green (25E5); reverse brownish orange (7C5) to olive (3A6) at the center, pale yellow (2A3) to yellow (2A6) toward the periphery; exudate and soluble pigment absent. On MEA, colonies 11 mm diameter in 7 days at 25°C; sporulation absent in UTHSCSA DI16–407, abundant in UTHSCSA DI16–400, with conidial masse brown (5A5), margin entire; reverse pale (2A2) to brownish orange (5C3); exudate and soluble pigment absent. On YES, colonies 33–40 mm in 7 days at 25°C, velutinous to downy, slightly granulose at the center due to the presence of ascomata, sporulation abundant, with conidial masse dark green (27F5), margin entire; reverse pale yellow (4A3) to light yellow (4A4). On DG18, colonies 30–45 mm in 7 days at 25°C, fuzzy, white to light orange (5A5), sporulation abundant, conidial mass honey yellow (4D6); reverse light yellow (1A4) to yellow (3A7). On CREA, colonies up to 5 mm in 7 days at 25°C, acid production absent. No growth on OA at 25°C or on CY20S at 37°C. Conidiophores up to 550 μm long, with uniseriate and radiating conidial heads; stipes occasionally septate, 260–500 × 6.5–9.5 μm, hyaline to subhyaline, smooth to finely roughened; vesicles subglobose to pyriform, 24–36 μm diameter; phialides variable in shape and size, ampulliform to cylindrical, 7–18 (30) × 2–5 μm; conidia globose to elongate, sometimes pyriform, 6–9.5(–11) × 4.5–9 μm, in shades of brown, rough. Cleistothecia globose to subglobose, 90–130 μm diameter, light yellow (2A5) to deep yellow (4A8); asci globose, 10–14 μm in diameter; ascospores lenticular, 4–5.5 × 2.5–4.5 μm, hyaline, with a slight furrow in the equatorial region, convex surface smooth with very small pits only visible under SEM. Etymology Referring to the presence of small pits in the ascospore wall under SEM. Type usa, Texas, isolated from human lymph node, D.A. Sutton, 2011 (CBS H-22998 holotype; cultures ex-type: UTHSCSA DI16-407, CBS 142376, FMR 14071). Discussion Although the diversity of genus Aspergillus species is well known in osmophilic substrates, house dust, indoor air, or stored products, in the clinical setting it is poorly documented. As previously noted, the taxonomy and nomenclature of the species of section Aspergillus has recently changed. In addition to that, recent advances in molecular tools have allowed for the description of new cryptic species that are almost impossible to differentiate using classical morphological tools.35 Clinically, identification of Aspergillus isolates at the species level may be important given that susceptibilities to antifungal drugs vary for different species and that species identity can influence the choice of appropriate antifungal therapy.36 In the present study, a total of 25 isolates of genus Aspergillus were used, most of them being recovered from human respiratory specimens, although further studies are needed to elucidate the role of these fungi as pathogens. Five different species of the section have been identified here, including a novel one (i.e., A. chevalieri, A. costiformis, A. microperforatus, A. montevidensis and A. pseudoglaucus). To facilitate comparison, their key morphological features are presented in Table 2. Aspergillus montevidensis was the most prevalent (44%). Clinically, this species may be the most relevant pathogen of this group because it has been isolated from different bodies sites, from superficial to deep tissue infections.10,15,37 It was first reported and described from a case of human otomycosis.38 This species currently includes strains that were formerly accepted as different but now considered conspecific. Aspergillus hollandicus (incorrectly associated to the sexual state Eurotium amstelodami), A. heterocaryoticus, and A. vitis are all synonyms of A. montevidensis, and these names should no longer be used.4 The nine isolates of A. montevidensis in the present study, with the exception of two of unknown origins, were from respiratory specimens (i.e., sputum, sinuses, and lung biopsies). The second most prevalent species in the present study is A. chevalieri (36%), known until recently by its teleomorph name, Eurotium chevalieri. This species has been reported from a case of cutaneous aspergillosis13 and more recently was the cause of fatal cerebral aspergillosis acquired by traumatic inoculation.17 It is worth noting that A. chevalieri and A. montevidensis represent 80% of the isolates included in this study, in fact they are also some of the most commonly species found in indoor environments.4 These two species, and the others reported here, were able to grow well at 37°C in vitro (Table 2), the basic pathogenic feature that enables them to invade deep tissue.39 The isolate of A. costiformis identified in this study is the third known strain of this species since its description. It was originally recovered from a mouldy paper-box in China.34 The second strain was isolated from a human nail in the Czech Republic,15 and the present study has recovered a third strain from hospital environment. The difficulty in the phenotypic characterization of this species might explain why it is rarely reported. The key characteristic that identifies A. costiformis is the presence of smooth conidia (Table 2) and the asexual morph is not usually formed in standard culture conditions. To overcome this problem, the production of conidial heads can be induced on M60Y at 37°C, as previously reported.4 The remaining isolates included in this study were genetically and morphologically similar but they could be distinguished as two different species (each representing 8% of the isolates). First, isolates UTHSCSA DI15-17 and UTHSCSA DI16-410 were identified as A. pseudoglaucus, a species described by Blochwitz in 1929.33 This species was more recently delineated phylogenetically by Hubka et al.4 in whose study other species were shown to be conspecific with A. pseudoglaucus, that is, A. glaucoaffinis/E. pseudoglaucum,40A. glaber,40A. fimicola,41 and A. reptans/E. repens.40 The phylogenetic tree constructed in the present study includes sequences of the ex-type strains of synonymous species and of numerous reference strains, giving more support to the A. pseudoglaucus clade. Our analysis shows that the genetic similarity among all those strains is 99.9% or higher in the concatenated alignment, in agreement with the proposal mentioned above. Aspergillus pseudoglaucus is commonly found in stored products7 and produces metabolites that are potentially toxic.42 There are few clinical reports involving A. pseudoglacus; it has been reported from a mixed infection in a case of maxillary sinusitis14 and occasionally recovered from human skin and nails,15 although its pathogenicity has not been confirmed. The two isolates of A. pseudoglaucus identified in the present study were from nasal and stool samples. Second, the isolates UTHSCSA DI16-400 and UTHSCSA DI16-407, recovered from toenail and lymph node, respectively, and sequences of two isolates retrieved from GenBank group in a clade close to A. pseudoglaucus. Although these latter isolates were labelled as A. pseudoglaucus, both phylogenetic methods (ML and BI) and the three most informative markers (BenA, CaM, and RPB2) all show that they represent together with our isolates investigated a distinct lineage in the genus.4 Thus, they have been proposed as the novel species A. microperforatus. The possible role of this species in the clinical disease is yet unknown. Although genus Aspergillus includes some species that are closely related, some characteristics can be useful for discriminating A. microperforatus (Table 2). For example, the novel species shows restricted growth on CYA at 25°C (up to 15 mm), in contrast to A. pseudoglaucus (up to 24 mm), which also differs by growing better on M60Y at 37°C (41 to 46 mm, vs. 28 to 32 mm diameter in A. microperforatus) and, according to our results, on CYA at 25°C it produces a diffusible brown pigment that is absent in A. microperforatus. The novel species can be differentiated from A. glaucus and A. proliferans by its ability to grow on M60Y at 37°C and A. glaucus has larger ascospores (6.0 to 7.5 μm, vs. 4.0 to 5.5 μm in A. microperforatus). Based on the descriptions and other reports, it might be difficult to differentiate the morphologies of A. microperforatus and A. ruber although the ascospores of A. ruber have an evident furrow and the conidia are usually ellipsoidal.1,4 Although A. glaucus is a known opportunistic pathogen, being reported from many types of infections,8,9,11 it was not recovered in the present study, as was the case in a study of clinical aspergilli in the Czech Republic.15 Therefore, it is possible that the clinical prevalence of this species has been overestimated, probably due to the limitations of diagnostic tools for Aspergillus identification and for filamentous fungi in general. The CLSI have established epidemiological cut-off values for triazoles (ITC, VRC, and POS) and AMB for only six Aspergillus species, that is, A. fumigatus, A. flavus, A. niger, A. terreus, A. nidulans, and A. versicolor.43,44 However, the limited number of isolates of other species available in different clinical laboratories precludes the determination of epidemiologic cutoff values, and members of genus Aspergillus have only been rarely tested for antifungal susceptibility.16 With few exceptions, the eight antifungals used in this study showed good activity against the aspergilli tested, with MIC values equal to or less than 1.0 μg/ml (Table 3). Recently, Masih et al.17 provided the in vitro antifungal susceptibility profiles of rare Aspergillus species in clinical samples from India. Although they did not test TBF, the MIC values for the other seven antifungals against three isolates of A. montevidensis and one strain of A. chevalieri were similar to the values observed in the current study. Most available in vitro data are with A. glaucus. Wildfeuer et al.21 and García-Martos et al.22 include eight and three clinical isolates of A. glaucus, respectively, and both reported good activity for ITC (MIC range of 0.25–0.5 μg/ml), VRC (0.125–0.78 μg/ml), and AMB (0.125–1.56 μg/ml). Although we did not study any A. glaucus strains, these values are similar to the overall values observed for the strains tested here. However, our MIC values were slightly lower for AMB and higher for VRC. Furthermore, García-Martos et al.21 also included two isolates of A. chevalieri and report the same MIC range (0.125–0.25 μg/ml) for ITC, VRC, and AMB. These results are very similar to those found in our A. chevalieri isolates, with the exception of one isolate that had an ITC MIC of 1.0 μg/ml. In summary, this study has assessed the species diversity of genus Aspergillus from a set of clinical isolates from the United Statesand demonstrated that A. montevidensis and A. chevalieri were the most frequently identified species. We also describe A. microperforatus as a new species. The antifungals tested showed potent activity against these isolates, especially the echinocandins and PSC. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgements This study was supported by the Spanish Ministerio de Economía y Competitividad, grant CGL2013-43789-P and by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil), grant BEX 0623/14-8. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. 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Medical MycologyOxford University Press

Published: Oct 9, 2017

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