www.elsevier.com/locate/jcfJournal of Cystic Fibrosis xx (2014) xxx–xxx

JCF-01120; No of Pages 8

Original Article

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

Raquel Sabino a,b,c,⁎, Jose A.G. Ferreira b,c,d, Richard B. Moss e, Joana Valente a,Cristina Veríssimo a, Elisabete Carolino f, Karl V. Clemons b,c, Cassie Everson e, Niaz Banaei g,

John Penner c, David A. Stevens b,c

a National Institute of Health Dr. Ricardo Jorge–Infectious Diseases Department, Lisbon, Portugalb Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, CA, United States

c California Institute for Medical Research, San Jose, CA, United Statesd School of Medicine, Faculdade da Saúde e Ecologia Humana–FASEH, Vespasiano, Brazil

e Department of Pediatrics, Division of Pulmonology, Stanford University, Stanford, CA, United Statesf Scientific Area of Mathematics, Lisbon School of Health Technology, Polytechnic Institute of Lisbon, Lisbon, Portugal

g Clinical Microbiology Laboratory, Stanford University, Stanford, CA, United States

Received 5 July 2014; revised 16 October 2014; accepted 19 October 2014Available online xxxx

Abstract

Background: Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary functionand allergic disease.Methods: Fifty-three Aspergillus isolates recovered fromCF patients were identified to species by Internal Transcribed Spacer Region (ITS),β-tubulin,and calmodulin sequencing.Results: Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensustricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found.Conclusions: To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A.fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likelydisplays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeuticapproach geared to this pathogen.© 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Keywords: Cystic fibrosis; Aspergillus; Cryptic species

1. Introduction

Cystic fibrosis (CF) is the result of a genetic mutation thatresults in chronic illness and a shortened life span, with currentmedian survival still less than half the general population [1]. Themutation is in the CF transmembrane conductance regulator(CFTR) affecting anion transport and mucociliary clearance. The

⁎ Corresponding author at: National Institute of Health Dr. Ricardo Jorge –Mycology Laboratory, Infectious Diseases Department Av. Padre Cruz1649-016, Lisbon, Portugal. Tel.: +351 217519247; fax: +351 217516400.

E-mail address: raquelsabino@hotmail.com (R. Sabino).

http://dx.doi.org/10.1016/j.jcf.2014.10.0051569-1993/© 2014 European Cystic Fibrosis Society. Published by Elsevier B

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disease particularly affects the lungs, resulting in airwayobstruction, chronic infection and inflammation, and recurrentexacerbations that progressively impair pulmonary function andultimately lead to respiratory failure and death [2]. Aspergillusspecies are the dominant group of fungi [3–5], particularly late inthe course of these patients [6]. Data indicate Aspergillus mayreside in the airways as a biofilm [7,8], within mucus plugs. Othermicrobes, such as Pseudomonas aeruginosa, may also be presentin the airways. Epidemiologic data suggest that CF patientsinfected chronically with Aspergillus have significantly accelerat-ed loss of lung function compared to non-infected CF patients [9].Aspergillus fumigatus can also cause allergic bronchopulmonary

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aspergillosis (ABPA) in up to 15% of CF patients [10], acomplication that causes repeated acute exacerbations, institutionof immunosuppressive therapy, and accelerated decline in lungfunction.

Historically, mycologists have relied on macroscopic colonialmorphology and microscopic morphology to identify the genusAspergillus and its species. Because of overlap in thesecharacteristics, molecular methods have become useful in theidentification of Aspergillus. Using these methods, specificallythe DNA sequence of the internal transcribed spacer (ITS) region,Aspergillus can be identified to the species complex level (e.g.,Fumigati, Flavi, Terrei, etc.). Within each species complex lieseparate species, based on molecular genetic characteristics ofgene sequences, which cannot be identified using morphologicalmethods. Thus, the species within a species complex are termed“cryptic species” [11]. The importance of the cryptic species liesin differences in drug susceptibility and the potential of refractorydisease caused by a species that is resistant to the treatment beingused.

Aspergillus species identification based on molecular methodscan be cost-effective, and rapid, with a good discriminatoryapproach for delineating species [12]. Genes encoding ribosomalRNA and spacers occur in tandem repeats and multiple copies arepresent in each fungal genome. Sequence comparison of theinternal transcribed spacer (ITS) region is widely used intaxonomy and molecular phylogeny because it is easy to amplifyfrom small quantities of DNA (due to the high copy number ofrDNA genes). The ITSDNA sequence is highly conserved withina species but has a high degree of variation between even closelyrelated species [13,14]. Thus, identification based on comparativesequencing of the ITS region can identify the organism inquestion to the Aspergillus species complex level. However, ITSsequence is not sufficiently discriminatory to allow for identifi-cation to the species level. Identification to the species level hasbeen accomplished by DNA sequencing of protein encoding loci.These loci include a β-tubulin region, and calmodulin, amongothers. For identification of species within the species complexesof Aspergillus, DNA sequencing of regions of the β-tubulin andcalmodulin genes provides sufficient variation to identify thespecies within a species complex [11,15]. Thus, ITS sequencefollowed by sequence of β-tubulin and calmodulin has beenrecommended for Aspergillus species identification [11].

Recently, it has been recognized via the use of molecularidentification methods that there are cryptic species closely relatedto A. fumigatus, which had hitherto been misidentified on amorphological basis as A. fumigatus, or otherwise grouped withA. fumigatus as an “umbrella” complex or family [15]. These novelspecies include A. lentulus, A. novofumigatus, A. viridinutans, andNeosartorya udagawae, among others [16]. Discrimination amongthese species is important because marked differences within thisfamily in antifungal susceptibility and growth temperatures havebeen described [15–17]. In addition, there are probable differencesin toxin production, biofilm formation, ability to compete withother microbes, etc., which could be important for a fungal speciescausing a chronic infection and playing a role in pathogenesis.

The main goal of this study was to perform a molecularcharacterization of Aspergillus species collected from cystic

Please cite this article as: Sabino R, et al, Molecular epidemiology of Aspergillus collej.jcf.2014.10.005

fibrosis patients, to understand better whether different speciesare involved in infection in the CF patients. This informationmay consequently allow a better prophylactic or therapeuticapproach.

2. Materials and methods

2.1. Sample collection and laboratory processing

Sputum and bronchoalveolar lavage (BAL) samples werecollected from 46 patients with cystic fibrosis. The patientsenrolled in this study were from Stanford University Hospitalsand Clinics in 2012–2013. The samples analyzed in this studywere obtained by standard bronchoscopy and bronchoalveolarlavage procedure protocols in use at Stanford hospitals. Briefly,appropriately sized fiberoptic bronchoscopes were utilized afterthe patient was sedated (by the attending anesthesiologist)with propofol and fentanyl. The airway was anesthetized with2–4 cc of 1% lidocaine at the vocal cords, midtrachea, andcarina. Bronchoalveolar lavage was performed in three aliquotsof 1 ml/kg normal saline with lavage return of the second andthird aliquots pooled for cultures and other microbiologicstudies, as ordered by the attending physician. The sampleswere immediately sent to the microbiology laboratory andprocessed for culture by standard laboratory methods.

All patients had respiratory samples processed with selectivemedia for CF respiratory bacterial, fungal, and mycobacterialcultures. The samples were plated undiluted, approximately 1 to2 h after collection. For fungal culture, a cotton swab applicatorwas used to transfer an aliquot of sputum onto Stanford PotatoAgar (potato dextrose agar supplemented with 500 mg/lchloramphenicol) [18]. The swab was rolled onto the agar andstreaked out with a disposable loop. For the BAL specimens, adrop of the sediment was placed onto the agar and then streaked.The plates were then sealed with 3 M™ Blenderm™ MedicalTape (3 M™, St. Paul, MN, USA). Plates were incubated atroom temperature for up to 8 h and then moved to a 30 °Cincubator. Cultures were checked daily for growth during the first7 days and then weekly for up to 21 days. Moulds wereidentified by macroscopic and microscopic morphology. AllAspergillus spp. isolates were subcultured on Sabouraud agarslants (Becton Dickinson & Co., Cockeysville, MD, USA) andsent to the California Institute for Medical Research. The isolateswere then subcultured on potato dextrose agar plates (BectonDickinson & Co., Cockeysville, MD, USA) and incubated for7 days at 37 °C. Conidia were harvested by flooding the surfaceof the agar plates with 5 ml phosphate-buffered saline (Lonza,Walkersville, MD, USA) containing 0.05% (v/v) Tween 80 androcking gently. Conidial suspensions were stored at −80 °C inMicrobankTM tubes (Pro-Lab Diagnostics, Texas, USA) untilprocessed.

2.2. Molecular analysis

Aspergillus species were identified using a polyphasicapproach, already employed by our research group in a previousstudy in non-CF-patients [19]. DNA extraction was performed

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using the High Pure PCRTemplate kit (Roche Diagnostics Corp.,Indianapolis, Indiana) according to the manufacturer's instruc-tions. The universal fungal primers, ITS1 and ITS4, were used toamplify DNA from all isolates of Aspergillus as describedpreviously [13]. Amplifications were performed in a 25 μlvolume containing PCR buffer (Applied Biosystems Inc., FosterCity, California), 1.5 mMMgCl2 (Applied Biosystems), 0.1 mMeach of dATP, dGTP, dCTP, and dTTP (Applied Biosystems),0.3 μM of each primer, 1.5 U of Taq DNA polymerase (AppliedBiosystems), and 20 to 50 ng of Aspergillus genomic DNA. PCRamplification was done in a thermal cycler (My Cycler ThermalCycler, Bio-Rad Laboratories Inc., Berkeley, CA, USA) usingone initial step at 95 °C for 4.5 min, and then 40 cycles at 95 °Cfor 30 s, 50 °C for 30 s, and 72 °C for 1 min. A final extensionstep at 72 °C for 3 min was added.

Amplifications of the partial β-tubulin gene were performedusing primers Bt2a and Bt2b, as described previously [20], withslight modifications, described in the following: PCR was carriedout in a 25 μl volume containing PCR buffer; 2 mM MgCl2;0.2 mM each of dATP, dGTP, dCTP, and dTTP; 0.4 μM of eachprimer Bt2a and Bt2b; 1 U of Taq DNA polymerase; and 20 to50 ng of Aspergillus genomic DNA. PCR conditions were asfollows: an initial denaturation at 94 °C for 5 min, followed by30 cycles of 94 °C for 30 s, 55 °C for 45 s, and a final extensionstep of 72 °C for 2 min.

Calmodulin gene amplification was performed using the set ofprimers Cmd5 and Cmd6 [15]. Amplifications were performed ina 25 μl volume reaction of Illustra PuReTaq Read-to-Go PCRbeads (GE Healthcare, Buckinghamshire, UK), containing0.6 μM of each primer and 20 to 50 ng of Aspergillus genomicDNA. Amplifications were carried out with an initial denatur-ation at 95 °C for 10 min, followed by 38 cycles of 95 °C for30 s, 55 °C for 30 s, and 72 °C for 1 min, and a last finalextension step of 72 °C for 7 min.

PCR products were analyzed by electrophoresis through 2%agarose gels and the resultant PCR amplicons were purified usingthe ExoSAP-IT enzyme system (USB Corporation, Cleveland,OH, USA), according to the manufacturer's instructions.

Sequencing of both strands was performed with the BigDyeterminator v 1.1 Cycle sequencing kit (Applied Biosystems) inthe thermal cycler using the same primers as those used in thePCR amplification for ITS (ITS1 and ITS4) or calmodulin (cmd5and cmd6) amplification. For both regions, the conditions were asfollows: an initial denaturation at 96 °C for 5 s, followed by30 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min,followed by one cycle of 72 °C for 5 min.

For β-tubulin sequencing, another set of primers (Btub1 andBtub4) was used [16], and the amplification was done under thefollowing conditions: an initial denaturation at 94 °C for 3 min,followed by 25 cycles of 96 °C for 10 s, 50 °C for 5 s, and52 °C for 4 min, followed by one cycle of 60 °C for 5 min.

Nucleotide sequences were edited using the programChromas2 and aligned using the program CLUSTAL X2 [21].The sequences obtained were compared with sequences depos-ited in the GenBank and CBS-KNAW Fungal BiodiversityCentre databases. The ITS sequences were used to identifyisolates to the species complex level, and the partialβ-tubulin and

Please cite this article as: Sabino R, et al, Molecular epidemiology of Aspergillus collej.jcf.2014.10.005

calmodulin sequences were used to identify the isolate to thespecies level.

2.3. Statistical analysis

The chi-square test by Monte Carlo simulation was used tocompare proportions and analyze differences in speciesdistribution. Two-sided p-values from tests were used tosummarize the comparability and a 5% significance level wasset. SPSS v21.0 program for Windows was used to perform thestatistical analysis

3. Results

The clinical characteristics of the sample cohort for thisstudy are shown in Table 1. Fifty-three respiratory culturesamples were obtained from 46 CF patients attending theStanford CF Center. All but four of the samples were obtainedfrom spontaneous or induced sputum, while four patients hadsamples obtained for culture during clinically indicatedbronchoscopy with bronchoalveolar lavage, processed formicrobiology immediately post-procedure. Clinical correlativedata were available for 45 of the 46 patients. The sample cohortwas widely distributed in age (mean 25.5, range 7–53 years)with 35 adults (≥18 years old at time of sample acquisition)and 10 pediatric patients; there was a female preponderance (17males, 28 females). Twenty-nine patients were homozygous forF508del, 6 were heterozygous for deltaF508, and 10 patientshad two non-deltaF508 CFTR mutations. There was also a widerange of pulmonary disease severity with mean FEV1 of 62.6%predicted (range, 15–117). Most of the samples were obtainedwhen patients were in clinically stable status (n = 30), while 13patients had samples obtained during an exacerbation, and 2patients were at end of life in respiratory failure. The mostcommon co-colonizing microbes were P. aeruginosa (found in25 samples), Candida spp. (22), and Staphylococcus aureus(15). Two patients were co-infected by non-tuberculousmycobacteria, one with Burkholderia, and 3 had other moulds.Eight patients had a diagnosis of ABPA. In summary, thissample cohort represents a broad spectrum of CF patients withrespect to age, clinical status, genotype, and microbiology,suggesting generalizability of the Aspergillus molecularepidemiological findings.

Fifty-three isolates of Aspergillus were obtained from CFpatients. Identifications were done on the basis of microscopicmorphology and through the use of molecular tools (i.e., ITS,β-tubulin, and calmodulin sequencing) (Table 1). Fifty-twoisolates were identified by morphology as belonging toA. fumigatus complex and one to A. niger complex. ITSsequencing showed that three species complexes were presentamong these 53 isolates: Terrei (strain ID 13-54), Nigri (strainID 13-67), and Fumigati (all other strains). Molecular identifi-cation by β-tubulin and calmodulin sequencing identified 51(96%) of the isolates as A. fumigatus sensu stricto and detectedone misidentification (2%) at the species complex level (strainID 13-54); this isolate was morphologically identified asA. fumigatus, whereas molecular methods indicated it was

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able 1haracterization of the cystic fibrosis patients enrolled in this study and morphological and molecular identification of the Aspergillus isolates collected from those patients.

train ID Sample Age at timeof sample

Gender CFTR genotype ABPADiagnosis +/−

FEV1 (L and% predicted)

Concomitant colonization Clinical status Morphologicalidentification(complex)

ITS sequencing(complex)

Species(β-tubulinand calmodulinsequencing)

3-54 Sputum 37 Female deltaF508/deltaF508 ABPA − 0.91 L 28% Candida albicans, Pseudomonasaeruginosa mucoid

Stable A. fumigatus Terrei A. terreus

3-55 (a) Sputum 53 Male deltaF508/deltaF508 ABPA − 2.36 L 66% P. aeruginosa mucoid, P. aeruginosanon-mucoid, C. albicans,Candida parapsilois

Stable A. fumigatus Fumigati A. fumigatus

3-87 (a) Sputum 53 Male deltaF508/deltaF508 ABPA − 2.35 L 66% P. aeruginosa non-mucoid Stable A. fumigatus Fumigati A. fumigatus3-56 BAL 41 Female deltaF508/D1152H ABPA − 0.95 L 35% P. aeruginosa non-mucoid Exacerbation A. fumigatus Fumigati A. fumigatus3-57 Sputum 23 Female deltaF508/deltaF508 ABPA + 0.85 L 30% P. aeruginosa mucoid End stage A. fumigatus Fumigati A. fumigatus3-58 BAL n/a n/a n/a n/a n/a n/a n/a A. fumigatus Fumigati A. fumigatus3-59 Sputum 28 Male deltaF508/G542X ABPA − 3.62 L 84% Staphylococcus aureus,

Burkholderia gladioliStable A. fumigatus Fumigati A. fumigatus

3-60 BAL 7 Female 2055del9 N A; 3199del6 ABPA − 0.74 L 66% Yeast (non-Candida), S. aureus,non-fermenting rod(non-P. aeruginosa )

Stable A. fumigatus Fumigati A. fumigatus

3-61 (b) Sputum 26 Male deltaF508/deltaF508 ABPA − 2.95 L 64% S. aureus, P. aeruginosamucoid, C. albicans

Stable A. fumigatus Fumigati A. fumigatus

3-121 (b) Sputum 26 Male deltaF508/deltaF508 ABPA − 3.07 L 68% S. aureus, P. aeruginosamucoid, Mycobacterium abcessus

Stable A. fumigatus Fumigati A. fumigatus

3-62 Sputum 30 Female deltaF508/deltaF508 ABPA + 1.12 L 35% Candida glabrata, P. aeruginosamucoid

Exacerbation A. fumigatus Fumigati A. fumigatus

3-63 Sputum 13 Female deltaF508/deltaF508 ABPA − 0.64 L 31% C. albicans, P. aeruginosa mucoid Exacerbation A. fumigatus Fumigati A. fumigatus3-64 (c) Sputum 22 Female deltaF508/deltaF508 ABPA − 0.93 L 33% S. aureus, P. aeruginosa mucoid Exacerbation A. fumigatus Fumigati A. fumigatus4-53 (c) Sputum 22 Female deltaF508/deltaF508 ABPA − 1.04 L 37% Methicillin-resistant S. aureus,

P. aeruginosa mucoid,Stenotrophomonas maltophilia

Stable A. fumigatus Fumigati A. fumigatus

3-77 (c) Sputum 22 Female deltaF508/deltaF508 ABPA − 0.93 L 33% Methicillin-resistant S. aureus,P. aeruginosa mucoid,C. albicans

Stable A. fumigatus Fumigati A. fumigatus

3-65 Sputum 45 Male deltaF508/deltaF508 ABPA − 3.26 L 86% Normal flora Stable A. fumigatus Fumigati A. fumigatus3-66 BAL 42 Female deltaF508/deltaF508 ABPA − 1.78 L 66% P. aeruginosa mucoid Exacerbation A. fumigatus Fumigati A. fumigatus3-67 Sputum 41 Female deltaF508/R347H ABPA − 2.68 L 92% P. aeruginosa mucoid Stable A. niger Nigri A. niger3-68 Sputum 53 Female deltaF508/R347P ABPA − 2.44 L 90% C. albicans, Mycobacterium avium Stable A. fumigatus Fumigati A. fumigatus3-69 Sputum 23 Female deltaF508/deltaF508 ABPA − 4.12 L 117% S. aureus, P. aeruginosa mucoid,

S. maltophilia,Cryseobacterium indologenes

Stable A. fumigatus Fumigati A. fumigatus

3-70 Sputum 11 Male M1303K; 621 + 1G N T ABPA − 1.80 L 60% Scedosporium/Pseudoallescheria,C. albicans, S. aureus

Exacerbation A. fumigatus Fumigati A. fumigatus

3-71 Sputum 30 Female deltaF508/deltaF508 ABPA − 2.09 L 72% C. albicans, Methicillin-resistantS. aureus

Stable A. fumigatus Fumigati A. fumigatus

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Sample Age attimeofsample

Gender CFTR genotype ABPADiagnosis +/−

FEV1 (L and% predicted)

Concomitant colonization Clinical status Morphologicalidentification(complex)

ITS sequencing(complex)

Species(β-tubulinand calmodulinsequencing)

13-72 Sputum 53 Male deltaF508/deltaF508 ABPA − 1.41 L 44% Normal flora Stable A. fumigatus Fumigati A. fumigatus13-73 (d) Sputum 22 Male deltaF508/1717-1G N A ABPA − 4.34 L 99% C. albicans, P. aeruginosa non-mucoid Stable A. fumigatus Fumigati A. fumigatus13-132 (d) Sputum 22 Male deltaF508; 1717-1G N A ABPA − 4.08 L 94% C. albicans Stable A. fumigatus Fumigati A. fumigatus13-74 Sputum 13 Female G542X; 1717-G N A ABPA − 1.53 L 54% C. albicans, P. aeruginosa mucoid Stable A. fumigatus Fumigati A. fumigatus13-75 Sputum 22 Female deltaF508/deltaF508 ABPA − 2.56 L 88% C. albicans, C. indologenes, S. aureus,

P. aeruginosa non-mucoid (2 strains)Stable A. fumigatus Fumigati A. fumigatus

13-76 Sputum 18 Female deltaF508/deltaF508 ABPA + 3.20 L 104% C. albicans Stable A. fumigatus Fumigati A. fumigatus13-78 Sputum 22 Female deltaF508/deltaF508 ABPA − 2.61 L 87% P. aeruginosa mucoid, P. aeruginosa

non-mucoid, C. albicans,Basidiomycete fungi

Stable A. fumigatus Fumigati A. fumigatus

13-79 Sputum 42 Male (TG)12-5T/(TG)13-5T ABPA + 2.18 L 52% P. aeruginosa non-mucoid Stable A. fumigatus Fumigati A. fumigatus13-80 Sputum 41 Male deltaF508/deltaF508 ABPA − 2.42 62% Proteus mirabilis (2 strains), C. albicans Exacerbation A. fumigatus Fumigati A. fumigatus13-81 Sputum 11 Male W1282X; I1234V ABPA + 2.31 L 101% S. aureus Stable A. fumigatus Fumigati A. fumigatus13-82 Sputum 20 Female 365_366insT and G542X ABPA − 0.40 L 15% C. albicans, Trichospororon sp.,

Escherichia coliEnd stage A. fumigatus Fumigati A. fumigatus

13-83 (e) Sputum 26 Female deltaF508/deltaF508 ABPA + 1.33 L 44% P. aeruginosa mucoid, P. aeruginosanon-mucoid, S. maltophilia,yeast (non-Candida)

Stable A. fumigatus Fumigati A. fumigatus

13-120 (e) Sputum 26 Female deltaF508/deltaF508 ABPA + 1.17 L 40% Yeast (non-Candida), P. aeruginosamucoid, P. aeruginosa non-mucoid

Exacerbation A. fumigatus Fumigati A. fumigatus

13-84 Sputum 37 Female deltaF508/deltaF508 ABPA − 0.73 22% P. aeruginosa mucoid Exacerbation A. fumigatus Fumigati A. fumigatus13-85 Sputum 24 Male deltaF508/deltaF508 ABPA − 1.90 L 47% C. albicans, Candida sp., S. aureus,

P. aeruginosa mucoidStable A. fumigatus Fumigati A. fumigatus

13-86 Sputum 47 Male deltaF508/G551D ABPA − 2.85 L 64% C. albicans, Candida parapsilosis Stable A. fumigatus Fumigati A. fumigatus13-106 Sputum 34 Male P.G542X (C.1624G N T);

P.G500D (c.1799G N A)ABPA − 2.34 L 63% S. aureus (2 strains) Stable A. fumigatus Fumigati A. fumigatus

13-107 Sputum 19 Female deltaF508/deltaF508 ABPA − 1.33 L 45% P. aeruginosa non-mucoid (2 strains) Stable A. fumigatus Fumigati A. fumigatus13-108 Sputum 44 Female deltaF508; D1152H ABPA − 1.53 L 50% C. albicans, P. aeruginosa mucoid Exacerbation A. fumigatus Fumigati A. fumigatus13-109 Sputum 21 Male deltaF508/deltaF508 ABPA− 3.19 L 69% Methicillin-resistant S. aureus,

C. albicans, C. glabrataStable A. fumigatus Fumigati A. fumigatus

13-110 Sputum 15 Female deltaF508; E1371 ABPA − 3.53 L 102% S. aureus (2 strains) Stable A. fumigatus Fumigati A. fumigatus13-111 Sputum 17 Female deltaF508/deltaF508 ABPA - 2.61 L 88% S. aureus, P. aeruginosa mucoid Stable A. fumigatus Fumigati A. fumigatus13-112 Sputum 7 Female 2055 del9 N A; 3199del6 ABPA − 1.17 L 50% P. aeruginosa mucoid Exacerbation A. fumigatus Fumigati A. fumigatus13-113 (f) Sputum 12 Male W1282X; I1234V ABPA + 2.89 L 74% C. albicans, S. aureus Stable A. fumigatus Fumigati A. fumigatus13-131(f) Sputum 13 Male W1282X; I1234V ABPA + 2.97 L 82% C. albicans, S. aureus Stable A. fumigatus Fumigati A. fumigatus13-114 Sputum 29 Female deltaF508/deltaF508 ABPA − 1.39 L 46% Methicillin-resistant S. aureus,

P. aeruginosa mucoidExacerbation A. fumigatus Fumigati A. fumigatus

13-115 Sputum 20 Female deltaF508/deltaF508 ABPA − 2.48 L 93% C. albicans, S. aureus (2 strains) Stable A. fumigatus Fumigati A. fumigatus13-116 Sputum 23 Female deltaF508/deltaF508 ABPA − 1.60 L 50% S. aureus, P. aeruginosa mucoid Exacerbation A. fumigatus Fumigati A. fumigatus13-117 Sputum 28 Male deltaF508/deltaF508 ABPA − 3.26 L 69% C. albicans, yeast, P. aeruginosa

mucoid, P. aeruginosa non-mucoidStable A. fumigatus Fumigati A. fumigatus

13-118 Sputum 11 Female deltaF508/deltaF508 ABPA + 0.65 L 37% C. albicans, S. aureus (2 strains) Exacerbation A. fumigatus Fumigati A. fumigatus13-119 Sputum 41 Male deltaF508/deltaF508 ABPA − 2.26 L 57% Normal flora Stable A. fumigatus Fumigati A. fumigatus(a), (b), (c), (d), (e), (f) Strains with the same letter were collected from the same patient.n/a—not available.

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A. terreus sensu stricto. The isolate identified to the A. nigercomplex was identified to species as A. niger sensu stricto. Thus,among all of the isolates analyzed, only three different specieswere identified (from three different species complexes identified

Table 2Morphological and molecular identification of the Aspergillus isolates collected from

Strain ID Source Morphological identification

12–26 Sputum A. fumigatus complex12–27 Sputum A. fumigatus complex12–28 Bronchial secretions A. fumigatus complex12–29 Bronchial secretions A. fumigatus complex12–30 Sputum A. fumigatus complex12–31 Bronchoalveolar lavage A. fumigatus complex12–32 Bronchoalveolar lavage A. flavus complex12–33 Bronchoalveolar lavage A. flavus complex12–34 Sputum A. flavus complex12–35 Sputum A. niger complex12–36 Unknown A. flavus complex12–37 Bronchial secretions A. flavus complex12–38 Stool A. niger complex12–39 Bronchoalveolar lavage A. fumigatus complex12–40 Auricular exudate A. fumigatus complex12–41 Sputum A. niger complex12–42 Auricular pus A. fumigatus complex12–43 Bronchial secretions A. fumigatus complex12–44 Sputum A. fumigatus complex12–45 Sputum A. fumigatus complex12–46 Palate biopsy A. fumigatus complex12–47 Bronchoalveolar lavage Aspergillus sp.12–48 Sputum A. fumigatus complex12–49 Bronchial secretions A. fumigatus complex12–50 Sputum A. fumigatus complex12–51 Bronchoalveolar lavage A. fumigatus complex12–52 Sputum A. fumigatus complex12–53 Ascitic fluid A. fumigatus complex12–54 Auricular exudate A. flavus complex12–55 Skull biopsy A. flavus complex12–56 Bronchial secretions A. flavus complex12–57 Unknown A. flavus complex12–59 Bronchoalveolar lavage A. candidus complex12–60 Auricular exudate A. candidus complex12–62 Bronchial secretions A. niger complex12–63 Nasal sinus aspirate A. flavus complex12–64 Bronchoalveolar lavage A. fumigatus complex12–65 Auricular exudate A. niger complex12–66 Auricular exudate A. niger complex12–67 Auricular exudate A. niger complex12–68 Bronchial secretions A. fumigatus complex12–69 Sputum A. fumigatus complex12–70 Sputum A. terreus complexINSA1 Bronchoalveolar lavage A. flavus complexINSA2 Bronchoalveolar lavage A. fumigatus complexINSA3 Unknown A. fumigatus complexINSA4 Bronchoalveolar lavage A. fumigatus complexINSA5 Unknown A. nidulans complexINSA6 Unknown A. fumigatus complexINSA7 Bronchoalveolar lavage Aspergillus sp.INSA8 Unknown A. fumigatus complexINSA9 Unknown A. nidulans complexINSA10 Sputum A. niger complexINSA11 Unknown A. nidulans complexINSA12 Sputum A. niger complexINSA13 Mammary gland biopsy A. fumigatus complexINSA14 Bronchial secretions A. terreus complex

Please cite this article as: Sabino R, et al, Molecular epidemiology of Aspergillus collej.jcf.2014.10.005

by ITS sequencing) (Table 1). Of the 51 isolates identifiedcorrectly as part of the A. fumigatus complex, no cryptic specieswere detected, and all 51 isolates were identified as beingA. fumigatus sensu stricto. Recently, our group [19] analyzed 57

non-cystic fibrosis patients of different sources.

Species (β-tubulin and calmodulin sequencing) Hospital wards

A. fumigatus SurgeryA. fumigatus PulmonaryA. fumigatus SurgeryA. fumigatus Internal medicineA. fumigatus PulmonaryA. fumigatus PulmonaryNot identified to species level PulmonaryA. flavus Internal medicineNot identified to species level Outpatient clinicA. niger Outpatient clinicA. flavus Data not availableA. flavus Data not availableA. tubigensis Data not availableA. fumigatus PulmonaryA. fumigatus OtorhinolaryngologyA. awamorii PulmonaryA. fumigatus Data not availableA. fumigatus Data not availableA. fumigatus Data not availableA. fumigatus Internal medicineA. fumigatus OncologyA. sydowii Data not availableA. fumigatus Infectious diseasesA. fumigatus Infectious diseasesA. fumigatus Infectious diseasesA. fumigatus Infectious diseasesA. fumigatus Infectious diseasesA. fumigatus Infectious diseasesA. flavus Infectious diseasesA. flavus Infectious diseasesA. flavus Infectious diseasesA. flavus Infectious diseasesA. terreus Infectious diseasesA. flavus Infectious diseasesA. tubigensis Infectious diseasesA. flavus Infectious diseasesA. fumigatus Data not availableA. awamorii OtorhinolaryngologyA. niger OtorhinolaryngologyA. brasiliensis OtorhinolaryngologyA. fumigatus PulmonaryNot identified to species level Outpatient clinicA. terreus Outpatient clinicA. flavus OncologyA. fumigatus PulmonaryA. fumigatus PulmonaryA. fumigatus Data not availableEmmericella echinulata Internal MedicineA. fumigatus Intensive care unitA. fructus PulmonaryA. fumigatus PulmonaryA. lentulus PulmonaryA. awamorii Internal medicineEmericella nidulans Data not availableA. tubigensis Data not availableA. fumigatus OncologyA. terreus Data not available

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7R. Sabino et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx

isolates of Aspergillus from samples collected from non-CFpatients with different infectious processes from 10 health careinstitutions, using the same molecular methods (Table 2).Significant differences (p b 0.001) were found between CFpatients and non-CF patients with respect to the number ofAspergillus species complexes and also the number of speciesfound in each group) (Table 3). When only species isolated fromrespiratory samples from non-CF patients were considered,significant differences (p b 0.001) were also found between CFpatients and non-CF patients regarding both parameters.

4. Discussion

Chronic lung infections in CF patients predominantlyinfluence morbidity and mortality. In addition to bacteria, thelungs of CF patients are often infected by Aspergillus. Severalstudies have been performed to evaluate the importance ofAspergillus colonization of the respiratory airways in this groupof patients [9,22], as well as the epidemiology of the speciesinvolved in the colonization or infection. As summarized byMortensen et al. [23], A. fumigatus is the predominant species,with reported prevalence rates from 6% to nearly 60%; A. terreushas been reported as the second most common Aspergillusspecies.

To our knowledge, there has only been one priorinvestigation specifically searching for Aspergillus crypticspecies in CF airways [24]. That study was, in contrast to ourpresent study, not a prospective study. Four CF patients withcryptic species in the Aspergillus complex were described, butno denominator was provided, to be able to assess frequency.Moreover, the isolates reported were obtained from 1999 to2009, whereas changing antifungal drug use and increasingdrug resistance could have produced a mix of aspergillidifferent than that in the present day.

In the CF samples reported herein, three Aspergillus speciescomplexes were detected, as opposed to the six speciescomplexes (Fumigati, Flavi, Nigri, Terrei, Nidulantes, andVersicolores) isolated from the non-CF group [19]. In thatstudy, only 51% of the non-CF patient isolates were A. fumigatusspecies complex. If only respiratory samples were compared,there were 37 in non-CF patients of which only 54% wereA. fumigatus complex. Regarding the number of speciesidentified, the non-CF group showed a number of speciesfour-times higher than in CF group (12 versus 3). It should bealso noted that no cryptic species of A. fumigatus were found in

Table 3Comparison of Aspergillus identification between cystic fibrosis patients andnon-cystic fibrosis patients groups.

Cystic fibrosispatients

Non-cystic fibrosispatients

p-value

No. patients 52 53No. isolates analyzed 53 57No. Aspergillus species

complexes3 6 p b 0.001

No. Aspergillus Species 3 12 p b 0.001

Please cite this article as: Sabino R, et al, Molecular epidemiology of Aspergillus collej.jcf.2014.10.005

the CF group and one (4%), an A. lentulus, was found in thenon-CF group.

We also studied, using the same molecular methods, 109non-hospital environmental Aspergillus isolates. Of these, 10species complexes were found and only 20% of the isolates wereof the A. fumigatus complex. Without complete speciation of thegroup, there were four cryptic species among the A. fumigatuscomplex isolates, including A. lentulus and Neosartorya fischeri.We also studied an additional 80 environmental isolates fromhospitals. These included 10 species complexes, of which only9% of the isolates were A. fumigatus complex.

Two recent studies [25,26] of Aspergillus species in non-CFpatients reported 9 and 8 species complexes found among 278and 218 isolates studied by comparable molecular methods,respectively. In these two studies, the percentage of isolates thatwere A. fumigatus complex were 58% and 67%, respectively,or overall, 62%. These authors found 6 and 8 cryptic species ofA. fumigatus complex isolates, respectively, for an overallincidence of 5% of the A. fumigatus complex, consistent withthe 4% we found in non-CF patients, and a comparablefrequency in our environmental isolates.

The paucity of cryptic species of Aspergillus, andnon-A. fumigatus species of Aspergillus, among our CF patientisolates, compared to those previous studies [25,26], ournon-CF study, and those isolates found in the environment[19], has potential implications about the selectivity of A.fumigatus sensu stricto for CF airways. It would suggest thereare species characteristics of A. fumigatus sensu stricto thatmake it suitable for residence in the unique environment that isthe CF airway (e.g., ionic milieu, biofilm, mucus plugs,bacterial colonization, aerosol or systemic use of antibacterials,etc.).

In conclusion, the isolates of Aspergillus collected fromrespiratory products of CF patients were almost all identified asA. fumigatus sensu stricto, and no cryptic species were foundamong them. These data, taken together, can help strengthenthe clinician's therapeutic approach to treating fungal infectionsin CF patients. It would be important to understand why A.fumigatus sensu stricto is uniquely dominant in CF airwayscompared to the environmental reservoirs of Aspergillus and toother patient groups. Understanding the factors involved wouldimprove our understanding of the pathobiology of A. fumigatussensu stricto in CF and possibly lead to prevention oreradication procedures.

Acknowledgments

Raquel Sabino was financially supported by a fellowshipfrom Fundação para a Ciência e Tecnologia (FCT) Portugal(contract SFRH/BPD/72775/2010).

This study was partially supported by a grant from the ChildHealth Research Institute, Stanford University, and a gift fromMr. John Flatley.

José A.G. Ferreira was partially supported by a grant fromthe Brazilian National Council for Scientific and TechnologicalDevelopment (CNPq).

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8 R. Sabino et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx

References

[1] Cystic Fibrosis Foundation Patient Registry. 2012 Annual Data Report;2013 [Bethesda, Maryland].

[2] Boucher RC. An overview of the pathogenesis of cystic fibrosis lungdisease. Adv Drug Deliv Rev 2002;54(11):1359–71. http://dx.doi.org/10.1016/S0169-409X(02)00144-8.

[3] Pihet M, Carrere J, Cimon B, Chabasse D, Delhaes L, Symoens F, et al.Occurrence and relevance of filamentous fungi in respiratory secretions ofpatients with cystic fibrosis—a review. Med Mycol 2009;47(4):387–97.http://dx.doi.org/10.1080/13693780802609604.

[4] Horré R, Symoens F, Delhaes L, Bouchara J-P. Fungal respiratoryinfections in cystic fibrosis: a growing problem. Med Mycol 2010;48(1):S1–3. http://dx.doi.org/10.3109/13693786.2010.529304.

[5] Masoud-Landgraf L, Badura A, Eber E, Feierl G, Marth E, Buzina W.Modified culture method detects a high diversity of fungal species incystic fibrosis patients. Med Mycol 2014;52(2):179–86. http://dx.doi.org/10.3109/13693786.2013.792438.

[6] Middleton PG, Chen SC, Meyer W. Fungal infections and treatment incystic fibrosis. Curr Opin Pulm Med 2013;19(6):670–5. http://dx.doi.org/10.1097/MCP.0b013e328365ab74.

[7] Mowat E, Rajendran R, Williams C, McCulloch E, Jones B, Lang S, et al.Pseudomonas aeruginosa and their small diffusible extracellular moleculesinhibit Aspergillus fumigatus biofilm formation. FEMSMicrobiol Lett 2010;313(2):96–102. http://dx.doi.org/10.1111/j.1574-6968.2010.02130.x.

[8] Kaur S, Singh S. Biofilm formation by Aspergillus fumigatus. Med Mycol2014;52(1):2–9. http://dx.doi.org/10.3109/13693786.2013.819592.

[9] Amin R, Dupuis A, Aaron SD, Ratjen F. The effect of chronic infection withAspergillus fumigatus on lung function and hospitalization in patients withcystic fibrosis. Chest 2010;137(1):171–6. http://dx.doi.org/10.1378/chest.09-1103.

[10] Stevens DA, Moss RB, Kurup VP, Knutsen AP, Greenberger P, JudsonMA, et al. Allergic bronchopulmonary aspergillosis in cystic fibrosis—state of the art: Cystic Fibrosis Foundation Consensus Conference. ClinInfect Dis 2003;37(Suppl3):S225–64. http://dx.doi.org/10.1086/376525.

[11] Balajee SA, Houbraken J, Verweij PE, Hong S-B, Yaghuchi T, Varga J,et al. Aspergillus species identification in the clinical setting. Stud Mycol2007;59:39–46.

[12] Deak E, Balajee SA. Molecular methods for identification of Aspergillusspecies. In: Pasqualotto AC, editor. Aspergillosis: From Diagnosis toPrevention, 1. London: Springer; 2010. p. 75–85.

[13] White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct sequencingof fungal ribosomal RNA genes for phylogenetics. In: Innis MA, GelfandDH, Sninsky JJ, White TJ, editors. PCR Protocols: A Guide to Methods andApplications. New York: Academic Press, Inc.; 1990. p. 315–22.

[14] Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA,et al. Nuclear ribosomal internal transcribed spacer (ITS) region as auniversal DNA barcode marker for fungi. Proc Natl Acad Sci U S A 2012;109(16):6241–6.

Please cite this article as: Sabino R, et al, Molecular epidemiology of Aspergillus collej.jcf.2014.10.005

[15] Hong SB, Go SJ, Shin HD, Frisvad JC, Samson RA. Polyphasic taxonomyof Aspergillus fumigatus and related species. Mycologia 2005;97(6):1316–29. http://dx.doi.org/10.3852/mycologia.97.6.1316.

[16] Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Cuenca-Estrella M,Rodriguez-Tudela JL. Aspergillus section Fumigati: antifungal susceptibilitypatterns and sequence-based identification. Antimicrob Agents Chemother2008;52(4):1244–51. http://dx.doi.org/10.1128/AAC. 00942-07.

[17] Balajee SA, Nickle D, Varga J, Marr KA. Molecular studies reveal frequentmisidentification of Aspergillus fumigatus by morphotyping. Eukaryot Cell2006;5(10):1705–12. http://dx.doi.org/10.1128/EC.00162-06.

[18] Burtelow MA, Merker JD, Baron EJ. Growth of Histoplasma capsulatumisolates is better on potato dextrose agar with chloramphenical than onbrain heart infusion agar. J Mycol Med 2009;19(3):197–9. http://dx.doi.org/10.1016/j.mycmed.2009.04.002.

[19] Sabino R, Veríssimo C, Parada H, Brandão J, Viegas C, Carolino E, et al.Molecular screening of 246 Portuguese Aspergillus isolates amongdifferent clinical and environmental sources. Med Mycol 2014;52(5):519–29. http://dx.doi.org/10.1093/mmy/myu006.

[20] Glass NL, Donaldson GC. Development of primer sets designed for usewith the PCR to amplify conserved genes from filamentous ascomycetes.Appl Environ Microbiol 1995;61(4):1323–30.

[21] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving thesensitivity of progressive multiple sequence alignment through sequenceweighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res 1994;22(22):4673–80. http://dx.doi.org/10.1093/nar/22.22.4673.

[22] de Vrankrijker AM, van der Ent CK, van Berkhout FT, Stellato RK,Willems RJ, Bonten MJ, et al. Aspergillus fumigatus colonization in cysticfibrosis: implications for lung function? Clin Microbiol Infect 2011;17(9):1381–6. http://dx.doi.org/10.1111/j.1469-0691.2010.03429.x.

[23] Mortensen KL, Jensen RH, Johansen HK, Skov M, Pressler T, Howard SJ,et al. Aspergillus species and other molds in respiratory samples frompatients with cystic fibrosis: a laboratory-based study with focus onAspergillus fumigatus azole resistance. J Clin Microbiol 2011;49(6):2243–51. http://dx.doi.org/10.1128/JCM. 00213-11.

[24] Symoens F, Haase G, Pihet M, Carrere J, Beguin H, Degand N, et al. UnusualAspergillus species in patients with cystic fibrosis. Med Mycol 2010;48(Suppl. 1):S10–6. http://dx.doi.org/10.3109/13693786.2010.501345.

[25] Balajee SA, Kano R, Baddley JW, Moser SA, Marr KA, Alexander BD,et al. Molecular identification of Aspergillus species collected for thetransplant-associated infection surveillance network. J Clin Microbiol2009;47(10):3138–41. http://dx.doi.org/10.1128/JCM. 01070-09.

[26] Alastruey-Izquierdo A, Mellado E, Peláez T, Pemán J, Zapico S, AlvarezM, et al. Population-based survey of filamentous fungi and antifungalresistance in Spain (FILPOP Study). Antimicrob Agents Chemother 2013;57(7):3380–7. http://dx.doi.org/10.1128/AAC.00383-13.

cted from cystic fibrosis patients, J Cyst Fibros (2014), http://dx.doi.org/10.1016/