Aspergillus tubingensis: a major filamentous fungus found in the airways of patients with lung disease

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1 Medical Mycology Advance Access published January 14, 2016 Medical Mycology, 2016, 0, 1 12 doi: /mmy/myv118 Advance Access Publication Date: Original Article Original Article Aspergillus tubingensis: a major filamentous fungus found in the airways of patients with lung disease Magali Gautier 1,, Anne-Cécile Normand 1, Coralie L Ollivier 1, Carole Cassagne 1, Martine Reynaud-Gaubert 2,3, Jean-Christophe Dubus 4, Fabienne Brégeon 3,5, Marijke Hendrickx 6, Carine Gomez 2,3, Stéphane Ranque 1,7 and Renaud Piarroux 1,7 1 Parasitology and Mycology, Assistance Publique-Hôpitaux de Marseille, CHU Timone-Adultes, Marseilles CEDEX 5, France, 2 Department of Respiratory diseases, CF Adult Centre and Lung Transplant Team; Assistance Publique-Hôpitaux de Marseille, CHU Nord, Marseilles, France, 3 URMITE CNRS IRD UMR 6236, IHU Méditerranée Infection, Aix-Marseille University, France, 4 Pediatric Pulmonology and CF Centre, Assistance Publique-Hôpitaux de Marseille, CHU Timone-Enfants, Marseilles CEDEX 5, France, 5 Service d Explorations Fonctionnelles Respiratoires, Assistance Publique-Hôpitaux de Marseille, CHU Nord, Marseilles, France, 6 BCCM/IHEM: Scientific Institute of Public Health, Mycology and Aerobiology Section, Brussels, Belgium and 7 Aix-Marseille University, UMR MD3 IP-TPT, Marseilles, France To whom correspondence should be addressed: Magali Gautier, Laboratoire de Parasitologie-Mycologie, CHU Timone. 264, rue Saint Pierre, Marseilles CEDEX 5, FRANCE. Tel: ;Fax: ; avellanmagali@hotmail.fr Received 30 August 2015; Revised 1 December 2015; Accepted 18 December 2015 Abstract The black Aspergillus group comprises A. niger and 18 other species, which are morphologically indistinguishable. Among this species subset, A. tubingensis, described in less than 30 human cases before 2014, is primarily isolated from ear, nose, and throat samples. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has emerged as a powerful technique to identify microbes in diagnostic settings. We applied this method to identify 1,720 filamentous fungi routinely isolated from clinical samples our laboratory over a two-year study period. Accordingly, we found 85 isolates of A. niger, 58ofA. tubingensis, and six other black Aspergillus (4 A. carbonarius and 2 A. japonicus). A. tubingensis was the fifth most frequent mold isolated in our mycology laboratory, primarily isolated from respiratory samples (40/58 isolates). In this study, we mainly aimed to describe the clinical pattern of Aspergillus tubingensis. We analyzed the clinical features of the patients in whom A. tubingensis had been isolated from 40 respiratory samples. Thirty patients suffered from cystic fibrosis, chronic obstructive pulmonary disease or other types of chronic respiratory failure. Strikingly, 20 patients were experiencing respiratory acute exacerbation at the time the sample was C The Author Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please journals.permissions@oup.com 1

2 2 Medical Mycology, 2016, Vol. 00, No. 00 collected. Antifungal susceptibility testing of 36 A. tubingensis isolates showed lower amphotericin B MICs (P < 10 4 ) and higher itraconazole and voriconazole MICs (P < 10 4 and P =.0331, respectively) compared with 36 A. niger isolates. Further studies are required to better establish the role that this fungus plays in human diseases, especially in the context of cystic fibrosis and chronic pulmonary diseases. Key words: colonization, aspergillosis, cystic fibrosis, chronic obstructive pulmonary disease, human. Introduction TheblackAspergillus group comprises 19 species. Together they constitute the most common etiological agent of fungal otomycosis and post-traumatic fungal endophthalmitis cases in humans. 1,2 A. niger, the most common black Aspergillus species isolated from humans, has been reported to cause allergic fungal rhinosinusitis 3 and invasive aspergillosis. 4 Black Aspergillus species are difficult, and sometimes impossible, to differentiate from each other using conventional laboratory methods based on macroscopic and microscopic morphological criteria. 5 Therefore, in the clinical routine laboratory practice, black aspergilli are generally identified as Aspergillus section Nigri. 6 Multilocus DNA sequencing has recently been used to reidentify strains stored in national or international collections, thereby demonstrating that in addition to A. niger sensu stricto, other so-called cryptic species such as A. awamori, A. aculeatus, A. acidus, A. foetidus, and A. tubingensis have been isolated from humans. 7,8 However, very limited clinical information on these culture collection strains was available. Furthermore, these studies did not provide estimates of each species repartition based on the patient clinical presentation. Consequently, data on the implication of each Aspergillus section Nigri cryptic species in human pathology and the spectrum of diseases they might be involved in are lacking. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful technique for microbial identification in diagnostic settings and has revolutionized bacterium and yeast identification. 9 Several studies have demonstrated that this technique may be applicable in routine laboratory practice to identify filamentous fungi At Marseilles University Hospital, we have consistently performed MALDI-TOF-based identification on each clinical fungus isolated since Such a diagnostic approach enables the differentiation of many Aspergillus species that were previously indistinguishable, including black Aspergillus species. 5 In this report, we described the results of the MALDI-TOF-based identification of 149 Aspergillus section Nigri identified at the Parasitology and Mycology laboratory (Marseilles University Hospital) in 2012 and We found that some species, which were previously considered rare, proved to be much more common than suspected. Strikingly, Aspergillus tubingensis was found in 58 samples, including 40 respiratory samples from 37 patients. Nevertheless, very few cases have been described in the literature, especially in pulmonology. Therefore, we aimed to investigate whether our finding was associated or not with an epidemic and to describe the clinical and microbiologic features of the patients in whom A. tubingensis had been isolated from respiratory samples. Materials and methods Fungal isolates The study was carried out at Marseilles University Hospital. We characterized all filamentous fungi routinely isolated at the Parasitology and Mycology laboratory during 2012 and 2013, regardless of the type or origin of samples (n = 1,659). A few (n = 91) strains that were sent for identification by laboratories of other health care facility in Marseilles were also included in the study. In summary, from an exhaustive panel of 1,750 filamentous fungi isolated from patients hospitalized in Marseilles health care facilities in 2012 and 2013, we studied 149 Aspergillus section Nigri, 90% of which were isolated from respiratory samples. Culture and identification Clinical samples were incubated at 30 C for two to ten days onto Sabouraud Chloramphenicol Gentamicin plates (OX- OID, France). As soon as the culture grew, mould colonies were subjected to a formic acid and acetonitrile pretreatment. The extracts were deposited in four replicas onto the MALDI-TOF MS target and covered with 1 μl of the manufacturer s matrix. MALDI-TOF MS spectra were acquired with a Microflex LT R instrument (Bruker Daltonics, Germany). Identification was performed by comparing the four spectra obtained from each isolate to an extensive in-house filamentous fungi reference spectrum library including Aspergillus Nigri strains 13 via the MALDI Biotyper R v3.0 software (Bruker Daltonics, Germany), as described in Cassagne et al. 14 A subset of Aspergillus tubingensis isolates was selected for molecular confirmation. Thus, MALDI-TOF MS

3 Gautier et al. 3 identification of the 25 A. tubingensis isolates from respiratory samples collected in 2012 were further subjected to a complementary molecular identification involving sequence analysis of the ITS2 (5 -GCATCGATGAAGAACGCAGC- 3 ; 5 -TCCTCCGCTTATTGATATGC-3 ; Hybridation temperature (Ht): 58 C), 28S D1-D2 variable region (5 -AACTTAAGCATATCAATAAGCGGAGGA- 3 ; 5 - GGTCCGTGTTTCAAGACGG-3 ; Ht : 53 C) as well as the beta-tubulin (5 - GGTAACCAAATCGGTGCTGCTTTC-3 ; 5 - ACCCTCAGTGTAGTGACCCTTGGC-3 ; Ht: 58 C) and calmodulin (5 -CCGAGTACAAGGAGGCCTTC- 3 ; 5 -CCGATAGAGGTCATAACGTGG-3 ; Ht: 58 C) regions Sequences were compared to reference sequences in the NCBI database via the nucleotide BLAST TM algorithm to obtain a definitive identification. DNA extraction was performed using a QIAamp DNA kit (QIAGEN, France), and the nucleotide sequences were obtained using a 3130 Genetic Analyzer (Applied Biosystems, Inc., Courtaboeuf, France). Each locus sequences were aligned using the ClustalW program in the BioEdit version software. A dendogram was generated from the concatenated DNA sequences of the four loci using MEGA version 5 (Tamura, Peterson Stecher, Nei, and Kumar 2011). A neighbor-joining tree was constructed based on the number of differences using the substitution model. Gaps were treated as relevant to calculate branch length. The support for each clade was determined via 1,000 bootstrap replicates. Antifungal susceptibility testing The minimum inhibitory concentrations (MICs) of amphotericin B, itraconazole, voriconazole, and posaconazole were determined using the Etest TM assay (biomérieux, France) as recommended by the manufacturer. MIC 50 and MIC 90 refer to the MICs that inhibited the growth of 50% and 90% of the isolates, respectively. The in vitro antifungal susceptibility of 36 of the 40 A. tubingensis isolates and 36 A. niger isolates collected from respiratory samples during the two-year study period were compared via the Conover score test. Among the four A. tubingensis isolates without antifungal susceptibility tests, three came from a patient with a fourth tested isolate. Two-sided exact P value <.05 was considered statistically significant. The analyses were done with the SAS 9.2 (SAS Institute Inc., Cary, NC, USA) statistical software. Results During the two-year study period, a total of 1,750 filamentous fungi isolates were subjected to MALDI-TOF MS identification. Among those, 1,311 isolates were classified in the Aspergillus genus, including 149 Aspergillus section Nigri (85 A. niger, 58 A. tubingensis, 4 A. carbonarius and 2 A. japonicus isolates). A. tubingensis was the fifth most common Aspergillus species identified after A. fumigatus (771 isolates), A. flavus (202 isolates), A. niger (85 isolates), and A. terreus (74 isolates). Interestingly, the majority of A. tubingensis isolates had been collected between August and November of each year(fig. 1). Indeed 62% (2012: 66% and 2013: 57%) of A. section Nigri were isolated in this period. Indeed, during this period A. section Nigri incidence was statistically significantly higher than the incidence of other filamentous fungi species (38%, P<10 3 ) and, particularly, A. fumigatus (35%, P<10 3 ). Forty A. section Nigri isolates (71%) were collected from respiratory samples (25 isolates during 2012 and 15 in 2013), 15 isolates were collected from ear samples, two isolates were collected from mouth samples and one isolate was derived from a cutaneous sample. The 25 A. tubingensis isolates collected from respiratory samples during the year 2012, which were identified via MALDI-TOF MS, were subjected to a complementary molecular identification involving sequence analysis of the partial beta-tubulin and calmodulin genes. In 22 cases, molecular analysis yielded indisputable identification of A. tubingensis ( 99% similarity obtained with A. tubingensis deposited nucleotide sequences). In the three remaining cases, although the isolate sequences matched with A. tubingensis ( 99% similarity), they also closely matched with A. phoenicis, A. foetidus, A. piperis, and A. acidus sequences available on the NCBI database. Therefore, it was impossible to obtain an unambiguous identification. Nevertheless, none of these 25 Aspergillus isolates closely aligned with sequences of A. niger sensu stricto. To complete the identification process and detect potentially epidemiologically related clones, we constructed a neighbor-joining tree using the nucleotide sequence (1,668 bases) of the four concatenated loci (Fig. 2). This tree highlighted the genetic heterogeneity of the 25 A. tubingensis isolates. The patient characteristics are detailed in Table 1. Thirteen patients suffered from cystic fibrosis, including four children of one, two, 11, and 15 years of age. Nine of the cystic fibrosis patients presented with respiratory acute exacerbation (including aggravation of dyspnea, obstructive syndrome, decrease in respiratory function, or onset of purulent expectorations) at the time the sample was collected. One patient suffered from pulmonary fibrosis associated with rheumatoid polyarthritis, three patients had noncystic fibrosis bronchiectasis and four patients suffered from chronic respiratory failure (due to graft-versus-hostdisease, pulmonary adenocarcinoma lobotomized, histiocytosis, and light chain deposition disease). Six of these eight

4 4 Medical Mycology, 2016, Vol. 00, No. 00 Figure 1. Monthly distribution of Aspergillus section Nigri isolates collected over the two-year study period. patients also experienced acute exacerbation at the time the sample was collected. Nine patients suffered from chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis, six of which had acute exacerbation. Three patients originated from hematology and oncology wards. The four remaining samples corresponded to one kidney transplant patient with concomitant legionellosis, one patient with community-acquired pneumonia, and two comatose patients, one with a basilar artery stroke and the other with septic shock complicating a Klebsiella pneumoniae endocarditis. Twelve patients were concomitantly colonized with Pseudomonas aeruginosa, and seven individuals had Staphylococcus aureus infection, including four patients who were colonized with both Pseudomonas aeruginosa and Staphylococcus aureus. Pulmonary function testing was performed at the time of A. tubingensis isolation for 14 patients. Seven of these cases displayed a significant decrease in forced expiratory volume in one second. One patient had recently undergone transplantation, thereby rendering it inappropriate to compare the function test results. Three patients had already tested positive for Aspergillus section Nigri, which had been isolated in 2011 and stored in the Marseilles hospital laboratory. Reidentification of these three isolates showed that one was A. niger and two were A. tubingensis. For one patient, DNA sequence analysis of two A. tubingensis isolates collected 19 months apart yielded a 100% identical match, whereas two isolates differed at nine base pairs in the other patient. The antifungal susceptibility results of 36 clinical isolates were compared with he antifungal susceptibility results of

5 Gautier et al. 5 Figure 2. Neighbor-joining tree based on the DNA sequence data (1,668 bases) from the concatenated loci of the ITS2, 28S D1-D2 variable region as well as the beta tubulin and calmodulin regions. The included samples were 25 A. tubingensis isolates collected from respiratory samples in 2012 and isolates 1bis and 9bis, which were collected before the study period (in 2011). 36 A. niger sensu stricto isolates collected from other patients in Marseilles. Antifungal susceptibility results were unavailable for four A. tubingensis isolates, including three from the same patient, who has a fourth isolate included in the study. When compared to A. niger, A. tubingensis isolates displayed significantly lower amphotericin B MICs (P < 10 4 ) and higher itraconazole and voriconazole MICs (P < 10 4 and P =.0331, respectively), although the MIC values largely overlapped (Table 2). Posaconazole MICs did not statistically significantly differed between species. Discussion This study presents the largest A. tubingensis case series ever described. Indeed, a review of the medical literature revealed only eight clinical studies or case reports of A. tubingensis infection in humans, describing a total of 26 patients. The majority of these patients presented with invasive aspergillosis (11 cases), cutaneous infection (seven cases) 19,23 or otomycosis (five cases). 24,25 The three remaining case reports described two cases of keratitis 26 and one case of osteomyelitis of the maxillary bone. 27 In addition to these clinical case reports, additional information was provided by reidentifying isolates stored in collection or epidemiological series. 28,29 However, clinical information in such studies is scarce and the organ involved varied greatly. For instance, all A. tubingensis strains re-identified from the BCCM collection corresponded to ear, nose, and throat (ENT) disorders (nine otomycosis cases, eight sinusitis cases and one nose obstruction case) and one eye disorder case, while A. tubingensis strains re-identified from a Spanish collection primarily corresponded to respiratory samples (10 of 17 samples, with no additional data provided). In the present study, A. tubingensis represented the fifth most common Aspergillus species isolated from patients (primarily isolated from respiratory samples), which accounted for 3.3% of the clinical samples identified (58 isolates over the course of two years). The distribution over the two-year period suggests that A. tubingensis infection is seasonal, with maximum infection rates between August

6 6 Medical Mycology, 2016, Vol. 00, No. 00 Table 1. Clinical description of the 37 patients harboring A. tubingensis identified in respiratory samples collected during the year Date of isolation Age (year/month/day) Sex (years) Sample Clinical context Symptoms Concomitant microorganism /10/01 M 42 Exp Cystic fibrosis Respiratory function: obstructive syndrome increased, FEV1: -7.5%. Pseudomonas aeruginosa /10/05 M 1 Exp Cystic fibrosis Respiratory function: stable. Candida parapsilosis /10/03 M 18 Exp Cystic fibrosis Exacerbation,FEV1: -20%, PEFR: -20%. Stenotrophomonas maltophilia, Aspergillus fumigatus /09/24 M 2 Exp Cystic fibrosis Respiratory function: stable. Streptococcus pneumonia, C. parapsilosis /08/03 F 21 Exp Cystic fibrosis Exacerbation, obstructive respiratory failure, oxygen required /10/12 F 39 Exp Cystic fibrosis, lung and liver transplantation (2002) /10/22 M 63 Exp Cystic fibrosis, tuberculosis Re-transplant assessment,fev1: 11%, PEFR: -7%. Exacerbation bronchial congestion, purulent expectoration, sinusitis /09/07 M 15 Exp Cystic fibrosis Exacerbation (reason for referral) /09/24 M 11 Exp Cystic fibrosis Exacerbation (reason for referral) /01/02 F 47 Exp Cystic fibrosis, lung transplantation (2007) /11/19 F 31 Exp Cystic fibrosis, lung transplantation (2007) /05/07 M 70 Exp Pulmonary fibrosis followed rheumatoid polyarthritis /09/17 F 20 Exp Non-cystic fibrosis bronchiectasis Staphylococcus aureus, Achromobacter xylosoxidans, Candida albicans P. aeruginosa, Candida dubliniensis, C. lusitaniae C. albicans S. aureus, P. aeruginosa, Trichoderma koningii S. aureus, Acinetobacter Chronic sinusitis exacerbation. S. aureus, P. aeruginosa, Enterobacter kobei, C. albicans Exacerbation dyspnea and congestion, purulent expectoration, chest pain, sibilants, FEV1: 27% and +4% two months later, sinusitis. Exacerbation, respiratory distress. Exacerbation treated by voriconazole, sinusitis history. P. aeruginosa, Aspergillus sydowii Serratia marcescens (blood culture), A. fumigatus A. fumigatus, A. flavus

7 Gautier et al. 7 Table 1. continued. Date of isolation Age (year/month/day) Sex (years) Sample Clinical context Symptoms Concomitant microorganism /09/21 F 60 Exp Pulmonary adenocarcinoma lobectomized, chronic respiratory failure Bronchitis and pleural infection, undocumented spondylodiscitis /10/12 M 34 Exp Hodgkin s disease Autologous bone marrow transplantation (Day 0) /11/26 M 75 BrA Pulmonary neoplasia Deterioration of general condition and febrile cough resisting ATB /09/28 M 64 Exp Chronic myeloid leukemia /08/02 F 17 Exp Chronic respiratory failure due to graft-versus-host-disease (acute myeloid leukemia, 2008) /09/06 M 59 BAL COPD, pulmonary neoplasia A. flavus, bacteriology not available Not available Negative culture, antigenemia-negative Aspergillus Interstitial pneumonia. Not available Exacerbation, acute respiratory distress with hypercapnic decompensation. Chest pain led to discovery of pulmonary neoplasia /09/19 M 58 Exp COPD Exacerbation. Respiratory decompensation, FEV1: -34% and +43% after three months, PEFR: -22% and +50% after three months. Lung transplantation two years later /09/03 M 50 Exp Terminal COPD Pre-transplant assessment (cough, expectorations and wheezing) /01/07 M 76 Exp Terminal COPD Exacerbation despite antibiotics (dyspnea and expectorations increased), death /01/06 M 70 BAL Kidney transplantation Respiratory decompensation, legionellosis, aspergillosis. C. albicans, direct exam: mycelium S. aureus, P. aeruginosa, T. koningii Bacteriology: negative culture C. albicans P. aeruginosa, Enterococcus faecalis, C. albicans Legionella pneumophila /08/10 M 58 BrA Basilar artery stroke Mechanical ventilation, death. Bacteriology: negative culture

8 8 Medical Mycology, 2016, Vol. 00, No. 00 Table 1. continued. Date of isolation Age (year/month/day) Sex (years) Sample Clinical context Symptoms Concomitant microorganism /11/02 M 66 BrA Endocarditis,septic shock Coma, mechanical ventilation. Klebsiella pneumonia (blood culture) /01/23 F 24 Exp Cystic fibrosis Exacerbation bronchial congestion, purulent expectoration, sinusitis.lung transplantation after eight months /07/16 M 62 Exp Idiopathic pulmonary fibrosis /07/18 M 56 Exp Idiopathic pulmonary fibrosis, bronchiolitis obliterans syndrome after lung transplantation (2008) /09/06 M 61 Exp COPD, lung transplantation (2011) /09/05 F 49 Exp Histiocytosis with obstructive respiratory failure Exacerbation: Respiratory function decreased (PEFR -16%), expectorations increased. Lung transplantation one month later. Death two months later. Respiratory function: stable. Death eight months later. Respiratory dysfunction due to mechanical fall. Death five months later /09/11 M 19 Exp Cystic fibrosis Exacerbation: Respiratory function decreased (PEFR -14%), purulent expectorations, cough and dyspnea increased. Improvement with anti-pseudomonal antibiotics. Lung transplantation 11 months later. P. aeruginosa, C. albicans P. aeruginosa, Candida inconspicua, Kluyveromyces marxianus P. aeruginosa, C. albicans, Aspergillus flavus Providencia, Candida tropicalis, A. fumigates, A. flavus Respiratory function: stable. C. albicans P. aeruginosa, S aureus,proteus mirabilis, C. tropicalis, A. fumigatus

9 Gautier et al. 9 Table 1. continued. Date of isolation Age (year/month/day) Sex (years) Sample Clinical context Symptoms Concomitant microorganism /10/25 F 40 BrA Light chain deposition disease, lung transplantation (2012: June and December) Exacerbation: acute decrease of respiratory function (FEV1: 27%) without acute rejection and after gradually increasing dyspnea for one month. Death five months later /08/27 M 71 BAL COPD Exacerbation: fever, dyspnea then respiratory failure.aspergillosis (positive serology and compatible CT scan features). Death despite antifungal treatment /01/17 M 68 Exp COPD Exacerbation: acute respiratory failure /04/23 F 71 BAL Bronchiectasis, Mycobacterium avium eight years earlier /10/ /11/12 F 52 Abr and three Exp Bronchiectasis after treated tuberculosis Respiratory function: stable. Exacerbation: hemoptysis, acute respiratory distress, hemodynamic choc.aspergillosis (compatible CT scan features) /09/28 M 23 Exp Not available Community-acquired pneumonia, persistent despite antibiotics. Bacteriology: negative culture S. aureus, C. albicans Serratia marcescens, P. aeruginosa C. glabrata Bacteriology: negative culture Note: Exp: expectoration, BrA: bronchial aspiration, BAL: bronchoalveolar lavage, M: male, F: female, FEV1: forced expiratory volume in one second, PEFR: peak expiratory flow rate, COPD: chronic obstructive pulmonary disease.

10 10 Medical Mycology, 2016, Vol. 00, No. 00 Table 2. Comparison of minimum inhibitory concentrations (MICs) of amphotericin B, itraconazole, voriconazole and posaconazole for 36 of the 40 A. tubingensis isolates and 36 A. niger isolates collected from respiratory samples during the period. The MICs were determined using the Etest TM assay. Antifungal MIC (mg/l) A. niger A. tubingensis Conover score test s exact P-value Amphotericin B Itraconazole Voriconazole Posaconazole Range <10 4 Mean (SD) (1.351) (0.440) Mode MIC MIC Range <10 4 Mean (SD) (4.771) (9.866) Mode MIC MIC Range Mean (SD) (0.194) (0.237) Mode MIC MIC Range Mean (SD) (0.196) (0.161) Mode MIC MIC and November, despite no increase in the number of samples assessed. Additional data concerning previous years is required to confirm this tendency. Nevertheless, seasonal mold exposure has previously been suggested in studies concerning children with asthma or allergies, transplant patients with invasive aspergillosis or HIV-positive patients with penicilliosis As many cases were pulmonology ward patients diagnosed over the course of a limited period of time in 2012, we initially suspected an epidemic. However, the DNA sequence analysis highlighted the genetic heterogeneity of the isolates and allowed to rule out both outbreak and laboratory contamination hypotheses. Indeed, in these two cases, a single clone would have been identified. In contrast to the medical literature, most of the patients in the current study were not complaining of ENT disorders but were rather affected by chronic respiratory diseases, including cystic fibrosis (13 cases) and other chronic respiratory disease (16 cases). Interestingly, this study revealed the presence of A. tubingensis in four children suffering from cystic fibrosis. This species has never been previously reported in pediatrics wards. While a wide variety of filamentous fungi may be encountered in cystic fibrosis patients and other patients with chronic pulmonary diseases, the clinical relevance of filamentous fungi isolation in such patients merits further clinical and experimental investigations. It is widely admitted that the abnormal airway condition in cystic fibrosis patients facilitates the entrapment of inhaled fungi and provides a suitable environment for the growth of microorganisms. 33 Most observations regarded Aspergillus fumigatus with a 57% incidence, followed by Scedosporium apiospermum. However, other Aspergillus species have been transiently isolated from cystic fibrosis respiratory secretions. 34 We demonstrated a patient harboring the same A. tubingensis strain during a 19-month period. Mold colonization is associated with various adverse effects in patients diagnosed with chronic pulmonary diseases. Horré etal. have identified a correlation between fungal colonization and increased number of hospital admissions among cystic fibrosis patients. 35 Filamentous fungi are responsible for an increasing rate of allergic bronchopulmonary aspergillosis (ABPA), a condition that has been mainly described in cystic fibrosis patients with A. fumigatus colonization. According to the European Register of Cystic Fibrosis, the prevalence of ABPA is 7.8% in cystic

11 Gautier et al. 11 fibrosis patients in Europe and 2% in cystic fibrosis patients in North America. 35 The Cystic Fibrosis Foundation Consensus Conference has redefined the diagnostic criteria of ABPA for the particular case of cystic fibrosis. 36 Filamentous fungi may also be responsible for direct damage of the respiratory mucosa, similar to that caused by bacteria in asthma, bronchitis or aspergilloma patients. As half of the pulmonary patients with positive A. tubingensis cultures were experiencing respiratory acute exacerbations, our findings suggest that colonization by this fungus may either contribute to or be an indicator of respiratory function deterioration. As most of these patients were also colonized with other germs, the individual effect of each microorganism is particularly difficult to assess. Concerning antifungal susceptibility, azoles activity was lower on A. tubingensis compared with A. niger isolates, as reported in Van Der Linden et al. 37 Elevated MICs of itraconazole and voriconazole would impact the treatment of aspergillosis patients. In contrast, amphotericin B MICs were lower for A. tubingensis compared with A. niger, as reported in Alastruey-Izquierdo et al. 29 Overall, this study shows that A. tubingensis is a major mold associated with bronchial colonization in patients with pulmonary conditions. The impact of such bronchial colonization on lung function is difficult to ascertain in these patients with underlying lung diseases, which themselves could worsen their respiratory function. Nevertheless, this report demonstrates that precise identification of fungi isolated from colonized lung disease patients is essential to efficiently diagnose potentially pathogenic molds and subsequently optimize patient care. As shown in recent studies, the progress of MALDI-TOF MS-based identification will soon facilitate the accurate identification of pathological fungal species. It is likely that, as already observed in the field of bacteriology, the introduction of mass spectrometry in mycology laboratories will lead to a revolution in mycoses diagnosis. 9 Acknowledgements We thank Sandra Moore for proofreading the article. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Vennewald I, Klemm E. Otomycosis: Diagnosis and treatment. Clin Dermatol 2010; 28: Chhablani J. Fungal endophthalmitis. Expert Rev Anti Infect Ther 2011; 9: Montone KT, Livolsi VA, Feldman MD et al. Fungal rhinosinusitis: a retrospective microbiologic and pathologic review of 400 patients at a single university medical center. Int J Otolaryngol 2012; 2012: Person AK, Chudgar SM, Norton BL et al. 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