Diagnostic Microbiology and Infectious Disease 84 (2016) 125–134

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Diagnostic Microbiology and Infectious Disease

j ourna l homepage: www.e lsev ie r .com/ locate /d iagmicrob io

Mycology

Genetic diversity of Aspergillus species isolated from onychomycosis and

Aspergillus hongkongensis sp. nov., with implications to antifungalsusceptibility testing

Chi-Ching Tsang a, Teresa W.S. Hui a,b, Kim-Chung Lee a, Jonathan H.K. Chen a, Antonio H.Y. Ngan a,Emily W.T. Tam a, Jasper F.W. Chan a,c,d,e, Andrea L. Wu a, Mei Cheung a,f, Brian P.H. Tse a,g, Alan K.L. Wu g,Christopher K.C. Lai f, Dominic N.C. Tsang f, Tak-Lun Que b, Ching-Wan Lam h, Kwok-Yung Yuen a,c,d,e,Susanna K.P. Lau a,c,d,e,⁎, Patrick C.Y. Woo a,c,d,e,⁎a Department of Microbiology, The University of Hong Kong, Hong Kongb Department of Clinical Pathology, Tuen Mun Hospital, Hong Kongc State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kongd Research Centre of Infection and Immunology, The University of Hong Kong, Hong Konge Carol Yu Centre for Infection, The University of Hong Kong, Hong Kongf Department of Pathology, Queen Elizabeth Hospital, Hong Kongg Department of Clinical Pathology, Pamela Youde Nethersole Eastern Hospital, Hong Kongh Department of Pathology, The University of Hong Kong, Hong Kong

a b s t r a c ta r t i c l e i n f o

⁎ Corresponding authors. Tel.: +852-2255-4892; fax: +E-mail addresses: skplau@hku.hk (S.K.P. Lau), pcywoo

http://dx.doi.org/10.1016/j.diagmicrobio.2015.10.0270732-8893/© 2016 Elsevier Inc. All rights reserved.

Article history:Received 23 May 2015Received in revised form 27 October 2015Accepted 30 October 2015Available online 3 November 2015

Keywords:AspergillusAspergillus hongkongensis sp. nov.OnychomycosisSequencingMetabolic fingerprintingMALDI-TOF MS

Thirteen Aspergillus isolates recovered from nails of 13 patients (fingernails, n = 2; toenails, n = 11) withonychomycosis were characterized. Twelve strains were identified by multilocus sequencing as Aspergillusspp. (Aspergillus sydowii [n = 4], Aspergillus welwitschiae [n = 3], Aspergillus terreus [n = 2], Aspergillus flavus[n = 1], Aspergillus tubingensis [n = 1], and Aspergillus unguis [n = 1]). Isolates of A. terreus, A. flavus, and A.unguis were also identifiable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The 13th isolate (HKU49T) possessed unique morphological characteristics different from other Aspergillusspp. Molecular characterization also unambiguously showed that HKU49T was distinct from other Aspergillusspp. We propose the novel species Aspergillus hongkongensis to describe this previously unknown fungus. Anti-fungal susceptibility testing showed most Aspergillus isolates had low MICs against itraconazole andvoriconazole, but all Aspergillus isolates had high MICs against fluconazole. A diverse spectrum of Aspergillusspecies is associatedwith onychomycosis. Itraconazole and voriconazole are probably better drug options for As-pergillus onychomycosis.

852-2855-1241.@hku.hk (P.C.Y. Woo).

© 2016 Elsevier Inc. All rights reserved.

1. Introduction

Aspergillus spp. are most well known as causes of invasive aspergillo-sis, which is 1 of themost important causes of morbidity andmortality inimmunocompromised patients (Bernardeschi et al., 2015; Chakrabartiet al., 2011; Jeurissen et al., 2013; Latgé, 1999; Lin et al., 2001; Yuenet al., 1997). In addition, they are also causes of aspergilloma, and a feware related with chronic pulmonary diseases, including chronic cavitary,fibrosing, and necrotizing pulmonary aspergillosis, in patients withpreexisting chronic lung diseases (Denning et al., 2003). Apart fromthese, Aspergillus spp. are occasional causes of other infectious diseasesyndromes, such as onychomycosis.

Among the known Aspergillus spp., Aspergillus fumigatus, Aspergillusflavus, Aspergillus niger, and Aspergillus terreus aremost commonly asso-ciated with Aspergillus infections, including Aspergillus onychomycosis(Bonifaz et al., 2007; Fernández et al., 2013; Lysková, 2007; Negroni,2010; Ng et al., 1999; Summerbell et al., 1989). Most of these caseswere diagnosed by isolation and morphological identification of thefungi involved. In recent years, as result of the use of gene sequencingfor molecular diagnosis in clinical microbiology laboratories, rare andnovel Aspergillus spp. as causes of Aspergillus infections have been in-creasingly reported (Brasch et al., 2009; Hubka et al., 2012, 2014;Sugui et al., 2010; Varga et al., 2008; Zotti et al., 2010, 2011). Recently,we observed that 3 out of 11 molds reported as A. flavus in our clinicalmicrobiology laboratorywere actually Aspergillus nomius andAspergillustamarii by internal transcribed spacer (ITS) as well as BenA and CaMgene sequencing, and metabolic fingerprinting (Tam et al., 2014). Asfor onychomycosis, Aspergillus melleus (Zotti et al., in press), Aspergillus

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sydowii (Gibas et al., 2002; Hubka et al., 2012; Lysková, 2007; Negroni,1942; Summerbell et al., 1989; Takahata et al., 2008; Wakeyama et al.,1998; Yamada et al., 2012), Aspergillus tubingensis (Nouripour-Sisakhtet al., 2015), Aspergillus unguis (Èmile-Weil and Gaudin, 1919; Hubkaet al., 2012; Lysková, 2007), and Aspergillus welwitschiae (synonym:A. awamori) (Hubka et al., 2012) have been reported as emerging causesof Aspergillus nail infections. In this study, using ITS and BenA, CaM, andRPB2 gene sequencing; metabolic fingerprinting; and matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we analyzed the spectrum of Aspergillus spp. associated withonychomycosis in a reference clinical microbiology laboratory. Antifun-gal susceptibility testing on the isolates was also performed, and the im-plications were discussed.

2. Materials and methods

2.1. Patients and fungal isolates

This study has been approved by the Institutional Review Board ofthe University of Hong Kong/Hospital Authority. Thirteen Aspergillusisolates, recovered from nail specimens collected during 2012–2013,were sent from 3 different hospitals in Hong Kong. All specimens werehandled according to the guidelines by the CLSI (2012), and all work in-volving the processing and inoculation of the specimenswas performedin a class II biosafety cabinet in order to avoid possible environmentalcontamination. All the Aspergillus isolates were present at the primaryinocula after direct inoculation of the nail samples on Sabouraud dex-trose agar (SDA) (Oxoid, Basingstoke, UK) supplementedwith chloram-phenicol (50 μg/mL) (Sigma-Aldrich, St Louis, MO, USA). All clinical dataof the patients were collected by retrieving and analyzing the patients'hospital records. The reference strains Aspergillus austroafricanus NRRL233T, A. flavus NRRL 1957T, A. niger NRRL 326T, Aspergillus protuberusNRRL 3505T, A. sydowii NRRL 250T, A. terreus NRRL 255T, A. tubingensisNRRL 4875T, A. unguis NRRL 216, and A. welwitschiae NRRL 4948 wereobtained from the Agricultural Research Service (ARS) Culture Collec-tion (NRRL), Department of Agriculture, USA,while the reference strainsIssatchenkia orientalis (synonym: Candida krusei) ATCC 6258T and Can-dida parapsilosis ATCC 22019T were obtained from the American TypeCulture Collection (ATCC), USA.

2.2. Molecular characterization

DNA extraction, PCR, and sequencing of the ITS and partial BenA,CaM, and RPB2 genes for all the case isolates; as well as the partialMcm7 and Tsr1 genes for HKU49T, and the partial Tsr1 gene for the ref-erence strain A. protuberus NRRL 3505T were performed according toour previous publication (Woo et al., 2013), where the primer pairsITS1/ITS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995),CAL-228/CAL-737R (Carbone and Kohn, 1999) or CF1M/CF4 (Peterson,2008), fRPB2-5F/fRPB2-7cR (Liu et al., 1999), Mcm7-709for/Mcm7-1447rev (Schmitt et al., 2009), and Tsr1-1453for/Tsr1-2308rev(Schmitt et al., 2009) were used for the ITS, partial BenA, CaM, RPB2,Mcm7, and Tsr1 genes, respectively. The sequences of the PCR productswere compared with sequences of closely related species from theDDBJ/ENA/GenBank databases by multiple sequence alignment usingMUSCLE 3.8 (Edgar, 2004) and were then end-trimmed. The end-trimmed sequences of the PCR products were comparatively analyzedby pairwise alignment, with the optimal GLOBAL alignment parameters,using BioEdit 7.2.0 (Hall, 1999). For phylogenetic analyses, poorlyaligned or divergent regions of the multiply aligned, end-trimmedDNA sequences were removed using Gblocks 0.91b (Castresana, 2000;Talavera and Castresana, 2007) with relaxed parameters. Tests for sub-stitution models and phylogenetic trees construction, by the maximumlikelihood method, were performed using MEGA 6.06 (Tamura et al.,2013).

2.3. Metabolic fingerprinting by UHPLC-ESI-Q-TOF-MS analysis

Metabolic fingerprinting was performed according to our previouspublication (Tamet al., 2014)withmodifications. Briefly, for eachAsper-gillus strain, 2 × 106 conidia were cultured in 10mL of RPMI 1640medi-um buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(Gibco, Grand Island, NY, USA) supplemented with 2% glucose (w/v)(Sigma-Aldrich) at 25 °C with shaking at 250 rpm for 48–72 h. The cul-ture supernatant was filtered using a mixed cellulose esters membranewith a pore size of 0.22 μm (MerckMillipore, Darmstadt, Germany) andquenched immediately in liquid nitrogen for 10min. The samples werethen lyophilized and stored at−80 °C until use. For each strain, sampleswere collected in duplicate for subsequent analysis.

In order to prepare for analysis by ultra-high-performance liquidchromatography (UHPLC) and electrospray ionization quadrupole–time-of-flight mass spectrometry (ESI-Q-TOF-MS), lyophilized sampleswere reconstituted in 600 μL of solvent mixture containing 1 volume ofliquid chromatography mass spectrometry (LC-MS) grade water (J. T.Baker, Center Valley, PA, USA), 2 volumes of LC-MS grade acetonitrile(J. T. Baker), and 2 volumes of LC-MS grade methanol (J. T. Baker). Thesamples were then vortexed for 1 min followed by sonication for10 min at room temperature. After centrifugation at 16,100 rcf for10min at 4 °C, the supernatantswere collected and stored at−80 °C im-mediately until analysis, which was performed no later than 1 weekafter the preparation. The thawed samples were analyzed within 48 h.To prevent batch effect, all sampleswere analyzed in a randommanner.

For UHPLC, the separationwas performed using the Agilent 1290 In-finity LC System (Agilent Technologies, Santa Clara, CA, USA) with theACQUITY HSS T3 column (3.0mm× 100mm, 1.8 μm) (Waters, Milford,MA, USA) and the ACQUITY UPLCHSS T3 VanGuard precolumn (2.1mm× 5 mm, 1.8 μm) (Waters). The injection volume was 5.0 μL. The tem-peratures of the column and autosampler were maintained at 45 °Cand 15 °C, respectively. The separation was performed at a flow rate of0.4 mL/min under a gradient program in which mobile phase A was0.1% (v/v) formic acid (Sigma-Aldrich) in water and mobile phase Bwas methanol. The gradient program was applied as follows: time(t) = 0 min, 1% phase B; t = 2.0 min, 1% phase B; t = 12 min, 38%phase B; t = 30 min, 99.5% phase B; t = 35 min, 99.5% phase B; t =35.1 min, 1% phase B; and t = 38 min, 1% phase B. Detection was per-formed with the Agilent 6540 Accurate-Mass Q-TOFmass spectrometer(Agilent Technologies) operating in the positive ion mode using theAgilent Jet Stream ESI source (Agilent Technologies). The capillary volt-age was set at +3800 V with a nozzle voltage of 0 V. Other source con-ditions were kept constant in all the experiments, with the gastemperature maintained at 320 °C, the drying gas (nitrogen) set at arate of 8 L/min, the pressure of the nebulizer gas (nitrogen) set at40 psi, and the flow rate and temperature of the sheath gas maintainedat 10 L/min and 380 °C, respectively. The voltages of the Fragmentor,Skimmer 1, and OctopoleRFPeak were 135 V, 60 V, and 500 V, respec-tively. The scan range was adjusted to m/z 80–1700 at an acquisitionrate of 2 spectra/s.

The raw data from UHPLC-ESI-Q-TOF-MS were analyzed usingMassHunter Qualitative Analysis B.05.00 (Agilent Technologies). Usingmolecular feature extraction algorithm for automated baseline correc-tion, noise calculation, peak detection and chromatogramdeconvolution,molecular features (MFs) characterized by retention times (RTs), chro-matographic peak intensities, and accurate masses were obtained. Datawere converted into the compound exchange file format (.cef) and an-alyzed using Mass Profiler Professional B.02.02 (Agilent Technologies)for data filtering, peak alignment, and statistical analysis. For data filter-ing, MFs which either had an abundance lower than 8000 counts persecond (cps) or which had less than 2 isobaric mass peaks were re-moved. Alignment of RTs and m/z values was performed across thesample set with a tolerancewindow of±0.2min and±10mDa, respec-tively. Data were normalized, and baseline transformation was per-formed according to the median expression level of all data and was

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then examined by multivariate analysis. After stepwise reduction ofdata dimensionality, principle component analysis (PCA) was per-formed using the unsupervised pattern recognition technique, and un-supervised hierarchical cluster analysis (HCA)was performed using theEuclidean correlation metric and complete linkage algorithm.

2.4. MALDI-TOF MS

MALDI-TOFMSwas performed by the formic acid extractionmethodaccording to the manufacturer's instruction and our previous publica-tion (Lau et al., 2014) with slight modifications, and all the chemical re-agents usedwere of LC-MS grade. Briefly, each Aspergillus sample isolatewas cultured in 10 mL of Sabouraud dextrose broth (Sigma-Aldrich).Cells were then harvested by centrifugation at 16,100 rcf for 2 minand washed with 1 mL of water (Sigma-Aldrich). The pellet was resus-pended in 300 μL of water and 900 μL of absolute ethanol (MerckMillipore). The mixture was vortexed and centrifuged at 16,100 rcf for2 min. The supernatant was removed, and the pellet was air dried for30 min. The pellet was then resuspended in 30 μL of 70% formic acid(Merck Millipore) and an equal volume of acetonitrile (Fluka, Buchs,Switzerland), followed by centrifugation at 16,100 rcf for 2 min. Usingthe IVD Bacterial Test Standard (Bruker Daltonics, Bremen, Germany)as a control, 1 μL of the supernatant was transferred to an individualspot on the MSP 96 polished steel BC targets plate (Bruker Daltonics);and each sample isolate was analyzed on four different spots. Eachspot was further overlaid with the α-cyano-4-hydroxycinnamic acidmatrix (Sigma-Aldrich) andwas air dried. The target plate was then an-alyzed by the microflex LT system (Bruker Daltonics), where each spotwas read 6 times, 40 shots each. For each isolate, 24 mass spectra withm/z values of 2000–20,000 were generated. Fungal identification wasachieved using FlexControl 3.4 (Bruker Daltonics) with the database Fil-amentous Fungi Library v1.0 (Bruker Daltonics). Moreover, for each iso-late, themass spectra generatedwere integrated to give a sumspectrumusingMALDI Biotyper 3.1 (Bruker Daltonics), and the sum spectra of allthe isolates obtained were processed and analyzed for generation ofdendrogram by HCA using MALDI Biotyper 3.1.

2.5. Antifungal susceptibility test

The in vitro susceptibilities against fluconazole, itraconazole,voriconazole, and terbinafine (test range: 0.0156–16mg/L) were deter-mined by the microbroth dilution method according to the guidelinesby the European Committee on Antimicrobial Susceptibility Testing(Arendrup et al., 2014). Briefly, fluconazole (Sigma-Aldrich) was dis-solved in sterile water, while itraconazole, voriconazole, and terbinafine(all Sigma-Aldrich)were dissolved in sterile dimethyl sulfoxide (Sigma-Aldrich) for the preparation of stock solutions (3.2 g/L), which werestored in polypropylene vials (Axygen Scientific, Union City, CA, USA)at−80 °C until use. For the preparation of microdilution plates, the an-tifungal agent stock solutions were diluted using double-strength RPMI1640medium (Gibco) bufferedwith 3-(N-morpholino)propanesulfonicacid (Gibco) supplementedwith 2% glucose (w/v), and for each antifun-gal agent, a dilution series at 2 times the final concentrations was pro-duced and dispensed into the flat-bottomed wells of polypropylene96-well microdilution plates (Eppendorf, Hamburg, Germany). Themicrodilution plates were sealed and stored at −80 °C until use. Forthe preparation of inoculum, conidia were harvested from fungal cul-tures on SDA incubated at 35 °C (25 °C for HKU49T because of poorgrowth at 35 °C) for 2–5 days and were resuspended in 0.1% Tween20 (Sigma-Aldrich). The conidial suspensions were then filteredusing cell strainers with a pore size of 10 μm (pluriSelect, Leipzig,Germany) to remove large hyphal fragments. The turbidity of each ofthe conidial suspension was then adjusted to 0.5 McFarland standard(OD625 nm=0.08–0.13), and the conidial suspensionswere then diluted10 times with sterile distilled water before being inoculated into thewells of the microdilution plates. The inoculated plates were incubated

at 35 °C, and the MIC results were read at 48 h. I. orientalis ATCC 6258T

and C. parapsilosis ATCC 22019T were used as quality controls.

2.6. Phenotypic characterization of HKU49T

Colonymorphology and growth rateswere characterized by culturingHKU49T on Czapek's agar (CZA), Czapek yeast autolysate agar (CYA), CYAwith 20% sucrose (CY20S), malt extract agar (MEA), MEA with 20% su-crose (M20S), oatmeal agar (OA), and yeast extract sucrose agar (YES).CZA, CYA, CY20S, and YES were prepared according to Samson et al.(2014), while the MEA base and OA were obtained from Oxoid and BDDiagnostic Systems (Sparks,MD, USA), respectively. Slides formicroscop-ic examination of HKU49T were prepared by the adhesive tape prepara-tion method using Fungi-Tape (Scientific Device Laboratory, DesPlaines, IL, USA) with 70% lactic acid (BDH Chemicals, Poole, UK) or 5%potassium hydroxide (Merck Millipore) with calcofluor white stain(Sigma-Aldrich) as the mounting media. Scanning electron microscopy(SEM) of HKU49T was performed according to our previous publication(Woo et al., 2010) with modifications. Briefly, HKU49T was cultured ona round filter membrane (10 mm in diameter with a pore size of 1 μm).The membrane was fixed in 2.5% glutaraldehyde (w/v) for 1 h andwashed once in 0.1 mol/L sodium cacodylate buffer. Fixed material wasdehydrated every 15 min using an increasing concentration of ethanolfrom 30% to 90% (20% increment each time), followed by 2 subsequentdehydration steps, of 15min each, in absolute ethanol. Dehydratedmate-rial in absolute ethanol was critical point dried in the CPD 030 CriticalPoint Drier (Bal-Tec, Balzers, Liechtenstein) using carbon dioxide as thedrying agent. Critical point–dried material was mounted onto an alumi-num stub and coated with palladium using the SCD 005 Cool SputterCoater (Bal-Tec). Coatedmaterialwas examined in theS-4800field emissionscanning electron microscope (Hitachi High-Technologies, Tokyo, Japan).

2.7. Nucleotide sequence accession numbers

The ITS andpartialBenA,CaM, andRPB2 gene sequences of the case iso-lates; as well as the partial Mcm7 and Tsr1 gene sequences of HKU49T andthe partial Tsr1 gene sequence of A. protuberus NRRL 3505T have been de-posited to the DDBJ/ENA/GenBank databases with the accession numbersAB987899-AB987911, LC000544-LC000584, and LC004923.

3. Results

3.1. Clinical spectrum of Aspergillus onychomycosis

The clinical characteristics of the 13 patients with Aspergillus spp.isolated from nails are shown in Table 1. The male to female ratio of pa-tients with Aspergillus nail infections was 4:9, with a median age of 56years (range, 27–79 years). Seven of the patients had underlying dis-eases that may predispose them to the infection. Fingernails and toe-nails were affected in 2 and 11 patients, respectively. None of thepatients had previously received any antifungal therapy.

3.2. Molecular characterization

PCR of the ITS and partial BenA, CaM, RPB2,Mcm7, and Tsr1 genes ofthe Aspergillus isolates yielded DNA fragments of about 530–570 bp,370–500 bp, 350–730 bp, 1050 bp, 750 bp, and 850 bp, respectively.Phylogenetic analysis of the partial BenA gene showed that the 13 caseisolates were scattered in 4 different Aspergillus sections, namely, sec-tions Flavi, Nidulantes, Nigri, and Terrei (Fig. 1 and SupplementaryFig. 1). Among these 4 sections, comparative sequence analyses andphylogenetic analyses of the ITS and partial BenA, CaM, and RPB2genes showed that 12 of the 13 isolates were known Aspergillus spp.(Supplementary Table 1 and Supplementary Fig. 2). As for the 13th iso-late (HKU49T), pairwise alignments of the ITS and partial BenA, CaM,

Table 1Cases of onychomycosis caused by Aspergillus spp. reported in this study.

Strain Sex/age (y) Underlying diseases Nail involved Identity by molecular studies

HKU49T M/54 Big toenail A. hongkongensisPW3037 F/56 Gouty arthritis Left big toenail A. sydowiiPW3048 F/49 Toenail A. sydowiiPW3050 F/73 Cirrhosis, hypothyroidism, hypertension, diabetes mellitus, hyperlipidemia Left ring fingernail A. welwitschiaePW3161 F/57 Toenail A. tubingensisPW3162 F/79 Adenocarcinoma of lung, hypertension, osteoarthritis of knees Toenail A. welwitschiaePW3163 M/27 Right thumbnail A. terreusPW3164 M/35 Diabetic nephropathy, hypertension, gout, chronic hepatitis B carrier Toenail A. sydowiiPW3165 F/51 Bilateral lower limb varicose veins Right big toenail A. terreusPW3168 F/28 Toenail A. sydowiiPW3169 F/56 Hypertension Toenail A. unguisPW3170 M/58 Depression, poliomyelitis, obstructive sleep apnea Toenail A. flavusPW3171 F/56 Toenail A. welwitschiae

F = female; M = male.

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RPB2, Mcm7, and Tsr1 gene sequences showed HKU49T possessed thehighest sequence identities to the ex-type strains of Aspergillus amoenus(ITS, 99.4%), A. austroafricanus (CaM, 99.7%; RPB2, 99.2%; Tsr1, 99.3%),

0.02

Fig. 1. Phylogenetic tree showing the relationship of the 13 case isolates to other members ofpartial BenA gene sequence data by the maximum likelihood method with the substitution momated proportion of invariable sites (I) and rooted using Talaromyces flavus CBS 310.38T, and 23scale bar indicates the estimated numbers of substitutions per base. Numbers at nodes, expressestrap values lower than 70 are not displayed. Only the names and sequence accession numbersand each Aspergillus section is color coded. The tree is presented in full details in Supplementa

Aspergillus fructus (ITS, 99.4%; Mcm7, 99.6%), A. protuberus (ITS, 99.4%;BenA, 98.0%), Aspergillus tabacinus (ITS, 99.4%), and Aspergillus versicolor(ITS, 99.4%) (Supplementary Table 1). Moreover, phylogenetic analyses

Section AeniSection AspergillusSection BisporiSection CandidiSection CerviniSection CircumdatiSection ClavatiSection CremeiSection FlaviSection FlavipedesSection FumigatiSection NidulantesSection NigriSection RestrictiSection OchraceoroseiSection SilvatiSection SparsiSection TerreiSection Usti

the genus Aspergillus as accepted by Samson et al. (2014). The tree was inferred from thedel Kimura 2-parameter model (K2) with gamma-distributed rate variation (G) and esti-4 nucleotide positions of the trimmed aligned sequenceswere included in the analysis. Thed in percentage, indicate levels of bootstrap support calculated from 1000 trees, and boot-as cited in the DDBJ/ENA/GenBank databases (in blankets) of the case isolates are shown,ry Fig. 1.

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of the 6 gene loci showed that although HKU49T was the most closelyrelated to these Aspergillus spp., it occupied a unique phylogenetic posi-tion in the ITS and BenA, RPB2,Mcm7, and Tsr1 genes trees, suggesting itis a novel Aspergillus spp. (Supplementary Fig. 2).

b

a

A. austroafricanus NRRL 233 T

A. sydowii NRRL 250 T

A. terreus PW3165

A. unguis NRRL 216

A. unguis PW3169

A. flavus NRRL 1957 T

A. flavus PW3170

A. welwitschiae NRRL 4948

A. welwitschiae PW3162

A. welwitschiae PW3171

A. welwitschiae PW3050

A. niger NRRL 326 T

A. tubingensis NRRL 4875 T

A. tubingensis PW3161

A. sydowii PW3168

A. sydowii PW3037

A. terreus NRRL 255 T

A. terreus PW3163

A. protuberus NRRL 3505 T

A. hongkongensis HKU49 T

A. sydowii PW3048

A. sydowii PW3164

PC-1

PC-2

Fig. 2. (a) PCAofmetabolicfingerprints of the case isolates and the reference strains. A total of 14principle component (PC) 1 versus component PC-2 versus component PC-3 is presented. Thepe26.11%, 16.02%, and 14.04%, respectively. (b) Heat map visualizing the differences in concentratgenerated from HCA of the metabolic fingerprints. Red and blue colors of the heat map indicat

3.3. Metabolic fingerprinting by UHPLC-ESI-Q-TOF-MS analysis

The 3-dimensional (3D) PCA score plot (Fig. 2a) revealed thatA. austroafricanus, A. flavus, A. niger, A. tubingensis, and A. welwitschiae

A. hongkongensis

A. austroafricanus

PC-3

A. flavus

A. welwitschiae

A. niger

A. sydowii

A. terreus

A. tubingensis

A. unguis

A. protuberus

08MFs defined by retention times andmass pairswere included. The 3DPCA score plot forrcentages of variance in thedata set reflected byPC-1, PC-2, and PC-3 for each samplewereions of different metabolites of the case isolates and the reference strains and dendrograme up-regulated and down-regulated metabolites, respectively.

130 C.-C. Tsang et al. / Diagnostic Microbiology and Infectious Disease 84 (2016) 125–134

could be distinguished clearly from each other and from other Aspergil-lus spp. while strains within each of these species were closely related.The 3D PCA score plot (Fig. 2a) also revealed that although HKU49T,A. protuberus, A. sydowii, A. terreus, and A. unguis could be distinguishedfrom other Aspergillus spp. clearly, these Aspergillus strains were closelyclustered. Similar to PCA, HCA indicated that A. austroafricanus, A. flavus,A. niger, A. tubingensis, and A.welwitschiae formedunambiguous clustersdistinguishable from each other and from other Aspergillus spp. whereA. niger, A. tubingensis, and A. welwitschiae possessed relatively similarextracellular metabolite profiles, while HKU49T, A. protuberus,A. sydowii,A. terreus, andA. unguiswere ambiguously clustered together,having A. austroafricanus as the outgroup (Fig. 2b).

3.4. MALDI-TOF MS

Among the 13 case isolates and the 9 reference strains, 9 isolates(PW3050, PW3161, PW3162, PW3163, PW3165, PW3168, PW3169,PW3170, and PW3171) and 6 reference strains (A. flavus NRRL 1957T,A. niger NRRL 326T, A. terreus NRRL 255T, A. tubingensis NRRL 4875T,A. unguis NRRL 216, and A. welwitschiae NRRL 4948) showed interpret-able protein profiles by MALDI-TOF MS, whereas the other 4 case iso-lates (HKU49T, PW3037, PW3048, and PW3164) as well as the other 3reference strains (A. austroafricanus NRRL 233T, A. protuberus NRRL3505T, and A. sydowii NRRL 250T) did not show any interpretable pro-tein profile. For the 9 case isolates and 6 reference strains with inter-pretable protein profiles, HCA of their MALDI-TOF mass spectrashowed that the 15 strains were separated into 4 distinct clusters(Fig. 3), where PW3170 was clustered with A. flavus NRRL 1957T;PW3163 and PW3165 were clustered with A. terreus NRRL 255T;PW3168 and PW3169 were clustered with A. unguis NRRL 216; andPW3050, PW3161, PW3162, and PW3171 were clustered with A. nigerNRRL 326T, A. tubingensis NRRL 4875T, and A. welwitschiae NRRL 4948.As for species identification using MALDI-TOF MS, 4 case isolates(PW3163, PW3165, PW3169, and PW3170) and 4 reference strains(NRRL 216, NRRL 1957T, NRRL 326T, and NRRL 3505T) with interpret-able protein profiles could be correctly identified down to the specieslevel with topmatch scores of ≥1.850 (Supplementary Table 2). Howev-er, for the other 5 case isolates with interpretable protein profiles,PW3168 was identified as A. versicolor, while PW3050, PW3161,PW3162, and PW3171 were identified as A. niger (SupplementaryTable 2). In addition, the reference strains A. tubingensis NRRL 4875T

0040001 900 800 700 600 500

Distance level (Arbitrary unit)

Fig. 3. Dendrogram generated from HCA of MALDI-TOF mas

and A. welwitschiae NRRL 4948 were also identified as A. niger byMALDI-TOF MS (Supplementary Table 2).

3.5. Antifungal susceptibility test

The MICs of the Aspergillus isolates for fluconazole, itraconazole,voriconazole, and terbinafine are presented in Table 2. All Aspergillusisolates had high MICs of N16 mg/L for fluconazole. Most isolates hadlow MICs of ≤0.5 mg/L for itraconazole, except for A. tubingensisPW3161 and A. unguis PW3169 whose MICs for itraconazole wereN16 mg/L, while all isolates had MICs between 0.5 and 2 mg/L forvoriconazole. As for terbinafine, A. unguis PW3169 as well asA. welwitschiae PW3050, PW3162, and PW3171 had MICs of ≤1 mg/L,while all other isolates had MICs of ≥2 mg/L.

4. Taxonomy

4.1. Aspergillus hongkongensis

Tsang, Hui, Lee, Chen, Ngan, Tam, Chan, Wu, Cheung, Tse, Wu,Lai, Tsang, Que, Lam, Yuen, Lau & Woo, sp. nov. MycoBank accessionnumber: MB 810279. Known distribution: Hong Kong. Etymology:named after Hong Kong, where the holotype was isolated. Specimenexamined: HKU49T; isolated from the big toenail of a man withonychomycosis in Hong Kong in 2013. Holotype: dried culture speci-men in the Herbarium of Biological Resource Center, National Instituteof Technology and Evaluation (NBRC), Japan,with the accession numberNBRC H-13268. Ex-type cultures: HKU49T (= NBRC 110693T = NCPF7870T = BCRC FU30360T).

Colonies on CYA attained a diameter of about 26 mm in 7 d at 25 °Candwere white-to-creamy, with a pale bluish green, raised center, withradial grooves, and without diffusing pigment (Fig. 4a and b). Growthwas similar at 30 °C (diameter 25 mm in 7 d) on CYA, but growth waspoor at 35 °C (diameter 3mm in 7 d) on CYA and colonies became gray-ish brown in 14 d. No growth was observed at 37 °C. Colonies on MEAattained a diameter of about 22 mm in 7 d at 25 °C, and were velvetyand sulfate-green with patches of red color and a thick white rim(Fig. 4c). Colonies on CZA attained a diameter of about 18 mm in 7 dat 25 °C and were white with no observable diffusing pigment and abrown reverse (Fig. 4d and e). Growth on YES was similar to that onCYA butwith a faster growth rate (diameter 31mm in 7 d) (Fig. 4f). Col-ony morphology on OA was similar to that on MEA, but with a darker

A. tubingensis PW3161

A. tubingensis NRRL 4875 T

A. welwitschiae PW3162

A. welwitschiae NRRL 4948

A. welwitschiae PW3050

A. welwitschiae PW3171

A. niger NRRL 326 T

A. sydowii PW3168

A. unguis PW3169

A. unguis NRRL 216

A. terreus PW3163

A. terreus PW3165

A. terreus NRRL 255 T

A. flavus PW3170

A. flavus NRRL 1957 T

002300 100 0

s spectra of the case isolates and the reference strains.

Table 2MIC of the Aspergillus isolates for fluconazole, itraconazole, voriconazole, and terbinafine.

MIC (mg/L)

Species Isolate Fluconazole Itraconazole Voriconazole Terbinafine

A. hongkongensis HKU49T N16 0.25 0.5 4A. flavus PW3170 N16 0.25 1 4A. sydowii PW3037 N16 0.5 1 4

PW3048 N16 0.5 1 2PW3164 N16 0.5 1 4PW3168 N16 0.5 1 2

A. terreus PW3163 N16 0.25 1 4PW3165 N16 0.125 1 8

A. tubingensis PW3161 N16 N16 2 8A. unguis PW3169 N16 N16 1 0.5A. welwitschiae PW3050 N16 0.25 1 1

PW3162 N16 0.25 1 0.125PW3171 N16 0.5 1 1

131C.-C. Tsang et al. / Diagnostic Microbiology and Infectious Disease 84 (2016) 125–134

green color and a slower growth rate (diameter 18mm in 7 d) (Fig. 4g).Diffusing red pigment could also be observed after 14 d of incubation onOA. Growth on CY20S and M20S was similar to that on CYA and MEA,respectively, but with faster growth rates (diameters 28 mm and34 mm in 7 d, respectively) (Fig. 4h and i). Yellow-to-brown exudatescould be observed on the colonies incubated on CYA, CZA, MEA, OA,and YES but not on CY20S or M20S.

Microscopically, Aspergillus heads were biseriate, with medium-to-long conidiophores (usually more than 100 μm in length) (Fig. 4j–m)and small, oval vesicles with a length of about 15 μm. Conidia werespherical, with a diameter of about 2 μm, and with a very rough surfaceas observed in SEM (Fig. 4n).

Genotypically A. hongkongensis is themost closely related to, but dis-tinct from, A. austroafricanus, A. amoenus, A. fructus, A. protuberus,A. tabacinus, and A. versicolor, all belonging to Aspergillus sectionNidulantes (Supplementary Fig. 2). Phenotypically, A. hongkongensis dif-fers from A. austroafricanus by the lack of diffusing magenta pigmentwhen cultured on CYA, CY20S, or CZA. The presence of red patches onthe colonies on MEA and OA also made A. hongkongensis differentfrom A. austroafricanus. In addition, A. hongkongensis differs fromA. protuberus by its ability to grow at 35 °C, from A. amoenus andA. versicolor by its inability to grow at 37 °C (Jurjevic et al., 2012), andfrom A. fructus and A. tabacinus by its smaller conidia (A. fructus,2.5–3.5 μm; A. tabacinus, 3–4 μm) (Jurjevic et al., 2012).

5. Discussion

We documented a broad spectrum of Aspergillus spp. associated withonychomycosis. Nail infection is most commonly caused by dermato-phytes, including Trichophyton spp.,Microsporum spp., and Epidermophytonfloccosum. Occasionally, it is associated with yeasts, such as Candida spp.,andnondermatophyticmolds, includingmainly Scopulariopsis brevicaulis,Hendersonula toruloidea, Aspergillus spp., Acremonium spp., and Fusariumspp., which account for about 2–22% of all onychomycosis cases(Clayton, 1992; Hwang et al., 2012; Kaur et al., 2008; Ng et al., 1999;Ramani et al., 1993). As a result of the recent use of molecular technolo-gies for identification of fungi, fungal species that have never been report-ed to be isolated from nails, including novel fungal species, are nowrecognized to be causes of onychomycosis. For example, in our recent re-port on the spectrumof Exophiala infections in our hospital, we describedthe first reported cases of onychomycosis caused by Exophiala bergeri,Exophiala oligosperma, and a novel Exophiala sp., E. hongkongensis(Woo et al., 2013). In this study, using a polyphasic approach of classifica-tion and identification, we observed that A. sydowii (n = 4),A. welwitschiae (n = 3), A. terreus (n = 2), A. flavus (n = 1),A. tubingensis (n = 1), and A. unguis (n = 1) were associated withonychomycosis. In addition, we found that more Aspergillus isolateswere recovered from toenails than from fingernails (Table 1). This

observation is in line with previous reports that nondermatophyticmolds aremore often associatedwith toenail infection than fingernail in-fection (Clayton, 1992; Walshe and English, 1966). This broad spectrumof Aspergillus spp. associated with onychomycosis was also observed in2 recent studies on a variety of Aspergillus infections, which the isolateswere also characterized using gene sequencing (Hubka et al., 2012,2014). Interestingly, in 1 of the studies, the authors specifically chose tostudyAspergillus spp. of the section Candidi (Hubka et al., 2014), and3 dif-ferent Aspergillus spp. were found to cause onychomycosis. All theseraised the concern on the accuracy of identification of the Aspergillusspp. in previous studies which only relied on phenotypic tests for identi-fying their isolates. For example, many strains of A. tubingensis andA. welwitschiaemight have been reported as A. niger in many clinical mi-crobiology laboratories. One limitation of the current study is the relative-ly small number of isolates involved. If more isolates could be obtainedfor molecular identification and characterization, more emerging orpreviously unrecognized Aspergillus spp. may be revealed as causes ofnondermatophytic onychomycosis.

Phenotypic and genotypic analyses revealed that HKU49T is a novelspecies in the genus Aspergillus, which we propose its name to beAspergillus hongkongensis. Using 6 independent DNA regions widelyused for phylogenetic analysis, including ITS and 5 house-keepinggenes (BenA, CaM, RPB2,Mcm7, and Tsr1 genes), it was shown unambig-uously that A. hongkongensis is closely related to, but distinct from, otherAspergillus spp. of the sectionNidulantes (Supplementary Fig. 2). Amongthe Aspergillus spp. of the section Nidulantes, A. hongkongensis ismost closely related to A. amoenus, A. austroafricanus, A. fructus,A. protuberus, A. tabacinus, and A. versicolor, as shown in the BenA,CaM, RPB2,Mcm1, and Tsr1 treeswith high bootstrap supports. Compar-ison of phenotypic characteristics between A. hongkongensis and thoseof other closely related Aspergillus spp. shown by phylogenetic analysesalso revealed unique phenotypic characteristics of A. hongkongensis,which distinguished this novel fungus from other closely related Asper-gillus spp. Notably, in contrast to the commonly known Aspergillus spp.associated with invasive aspergillosis or aspergilloma, such asA. fumigatus, A. flavus, A. niger, and A. terreus, A. hongkongensis, as wellas Aspergillus pragensis (Hubka et al., 2014), do not grow at 37 °C. Thisproperty may explain why these Aspergillus spp. were only isolatedfrom infected nails, but not deep-seated Aspergillus infections.

Multilocus sequencing is by far the most reliable method for theidentification of Aspergillus spp. In this study, sequencing multiplegene loci is able to unambiguously differentiate all the Aspergillus spp.Although metabolic fingerprinting can confidently differentiateA. austroafricanus, A. flavus, A. niger, A. tubingensis, and A. welwitschiae,A. hongkongensis and 4 other Aspergillus spp. (A. protuberus, A. sydowii,A terreus, andA. unguis)were grouped into a single cluster (Fig. 2). Inter-estingly, although molecular studies have grouped A. austroafricanus,A. hongkongensis, A. protuberus, A. sydowii, and A. unguis into the sectionNidulantes, while A. terreus has been placed in the section Terrei, these 6species belonging to 2 different sections were clustered together in theHCA plot generated from metabolic fingerprinting (Fig. 2b), indicatingthat species of these 2 sections may possess relatively similar metabolicprofiles despite being phylogenetically distinct. As for MALDI-TOF MS,althoughA. flavus, A. terreus, and A. unguis could potentially be identifiedusing this technology, A. tubingensis and A. welwitschiae weremisidentified as A. niger, suggesting that species of the A. niger cladecould not be differentiated from each other usingMALDI-TOFMS. In ad-dition, A. sydowii PW3168 was misidentified as A. versicolor, and this isprobably due to the inadequate number of reference spectra in theMALDI-TOF MS database. Furthermore, A. hongkongensis HKU49T, 3 clin-ical strains of A. sydowii, and the reference strains A. austroafricanusNRRL233T, A. protuberus NRRL 3505T, and A. sydowii NRRL 250T, which belongto the sameAspergillus section, did not generate any interpretable proteinprofile by MALDI-TOF MS. This is likely due to problems during proteinextraction as a result of the difficulty in breaking down the cell clumpsduring the process of formic acid digestion.

Fig. 4.Macroscopic andmicroscopic morphology of A. hongkongensisHKU49T after 7 d of incubation at 25 °C on different culture media. (a) Colony surface and (b) reverse of colony of onCYA. Colony surfaces on (c)MEA and (d) CZA and (e) reverse of colony on CZA. Colony surfaces on (f) YES, (g) OA, (h) CY20S, and (i)M20S. (j–m)Biseriate Aspergillusheadswithmedium-to-long conidiophores and small, oval vesicles (lightmicrograph, originalmagnification [400×], 70% lactic acid [j and k] or 5% KOHwith calcofluorwhite stain [l andm]). (n) Round conidiawith a very rough surface (SEM, scale bar = 5 μm).

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Accurate identification of the Aspergillus isolates to the species levelis important because it will affect the choice of antifungal agent. Themost widely used antifungal agents for the treatment of onychomycosisdue to nondermatophyticmolds are terbinafine and itraconazole. In thisstudy, we showed that fluconazole had very low activities against mostAspergillus spp. Isolates of 2 Aspergillus spp. (A. tubingensis andA. unguis)had high MICs against itraconazole, but only A. tubingensis had a rela-tively higher MIC against voriconazole (Table 2). As for terbinafine,most Aspergillus spp. had MICs between 2 and 8 mg/L, and only A.welwitschiae and A. unguis had low MICs against this drug. Based onthese susceptibility results, we envisaged that itraconazole andvoriconazolemaybebetter choices of antifungal agents for onychomycosiscaused by Aspergillus spp.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.diagmicrobio.2015.10.027.

Conflict of interest

None.

Acknowledgments

Thiswork is partly supported by theResearch Fund for the Control ofInfectious Diseases (commissioned project), Food and Health Bureau,the Government of the Hong Kong Special Administrative Region,Hong Kong; the Strategic Research Theme Fund, Small Project Fund,Mary Sun Medical Scholarship, Wong Ching Yee Medical PostgraduateScholarship, and University Postgraduate Scholarship, The Universityof Hong Kong, Hong Kong; and the Croucher Senior Medical ResearchFellowship, Croucher Foundation, Hong Kong.

We also thank the curators of NRRL for providing the referencestrains for free.

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