Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted... Abstract An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media. Aspergillus, identification, cryptic species, MALDI-TOF MS, MALDI-TOF Bruker Introduction The genus Aspergillus includes multiple species widely distributed in the environment, which may be responsible for a wide spectrum of diseases such as allergic syndromes, chronic infection and acute invasive disease, particularly in people with compromised immune systems.1 Taxonomically, the genus Aspergillus includes four subgenera, which are subdivided into numerous sections. Each section corresponds to a specific “species complex,” with groups of related species2 that are almost indistinguishable by morphological methods and which have been designated as cryptic species. In clinical mycology laboratories, identification of Aspergillus species is routinely based on determination of macroscopic and microscopic morphological characteristics, such as colour, shape of conidia, spores and mycelial structures.3 These methods do not allow discrimination of close related species, usually from the same section. However, a correct identification at the species level could be clinically relevant because some of these cryptic species, such as A. calidoustus (Aspergillus section Usti) and A. lentulus (Aspergillus section Fumigati), show decreased susceptibility to multiple antifungal drugs.4,5 Current recommendations for identification at species level within the Aspergillus sections include the use of molecular methods based on comparative sequencing.6,7 Samson et al.8 recommended the use of the ribosomal internal transcribed spacer (ITS) of the nrDNA as the official DNA barcode for fungi and β-tubulin or calmodulin regions as secondary identification markers. However, sequencing is a slow and expensive process for routine Aspergillus species identification of isolates recovered from clinical samples. Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become in recent years a rapid, accurate and cost-effective technology for the identification of bacteria, yeast and mold species in the clinical microbiology laboratory.9–11 However, its use for the identification of molds is limited to date. This is due to several facts including that commercial databases have few fungal entries, the difficulty to obtain good quality mass spectra and the use of a non standardized and more cumbersome pretreatment of the samples to break the fungal cell wall, which is thicker and more robust than that of bacteria.12,13 The first commercial mould database for the Bruker MALDI Biotyper, the Filamentous Fungi Library 1.0 (Bruker Daltonik GmbH, Bremen, Germany), consists of 89 entries corresponding to 18 different species of the genus Aspergillus: 11 noncryptic species (A. candidus, A. clavatus, A. flavus, A. fumigatus, A. glaucus, A. nidulans, A. niger, A. ochraceus, A. terreus, A. ustus, and A. versicolor) and seven cryptic species (A. amstelodami, A. nomius, A. oryzae, A. parasiticus, A. sclerotiorum, A. tamarii, and A. unguis). This is a small representation of species considering than more of 300 have already been described.14 Some authors15–17 have attempted to use this commercial database for identification of Aspergillus spp.,but with unsatisfactory results due to the low number of introduced species and strains. The aim of this study was to supplement the Filamentous Fungi Library 1.0 with an in-house reference database and to validate it with the identification of a collection of clinical isolates of Aspergillus species grown on solid medium. Methods Reference strains and clinical isolates During a 2-year period, 379 strains of Aspergillus were isolated from clinical samples in the Clinical Microbiology Laboratory of the University Hospital Virgen del Rocío (HUVR)-Spain. All the strains were identified by morphological observation, and 179 strains were selected to be identified by gene sequencing (Table 1). Only strains that offered uncertain identification based on their phenotypic characteristics were sequenced. These strains were used to create the new database (n = 42) and for its later validation (n = 179, including the preceding 42). The remaining 200 strains were used in the prospective phase to demonstrate the usefulness of the in-house reference mould database. Table 1. Aspergillus isolates (n = 179) identified by gene sequencing. Number of Identification by Subgenus Section isolates sequencing Target GenBank accession no. Fumigati Fumigati 47 A. fumigatus β-tubulin KF921489, DQ438532, KF921476, KP784370, KU198177, KJ527255, KX165400, KU885422, KU737561 1 A. lentulus β-tubulin AB910085 1 Neosartorya fischeri β-tubulin EF669828 Circumdati Flavi 46 A. flavus β-tubulin KJ482657, KJ482657, KT354304, HQ400610, KT354304, KT275168, KU737557, KX306819 3 A. minisclerotigenes β-tubulin JX456195 1 A. nomius β-tubulin KJ767725 3 A. tamarii β-tubulin KP067209, KP641150, KJ767722 Nigri 15 A. niger β-tubulin KJ136073, KT805426, KM502177, HQ632737, LN482544, KT354315, HQ632737, KT965696, KT149875, KT965691 16 A. tubingensis β-tubulin LC000547, KT965707, KT354311, KT965718, KR064543, KJ938412 Circumdati 1 A. ochraceus β-tubulin KR737581 1 A. persii β-tubulin KT253228 1 A. pseudoelegans β-tubulin EU014095.1 Terrei Terrei 31 A. terreus β-tubulin GQ376137, LC000549, KJ777806, KP715156, LN835260, KR610363, KR051550, LC060787, KC190476, KR051541, KC473916, KU737559 Nidulantes Versicolores 1 A. creber β-tubulin KP329888 1 A. sydowii β-tubulin KT253232 1 A. tabacinus Calmodulin LT594402 Nidulantes 2 A. nidulans β-tubulin KP278196 5 A. quadrilineatus Calmodulin KU866794, KU866777, EF591681 1 A. rugulosus Calmodulin KU866801 Usti 1 A. calidoustus β-tubulin KJ777803 Number of Identification by Subgenus Section isolates sequencing Target GenBank accession no. Fumigati Fumigati 47 A. fumigatus β-tubulin KF921489, DQ438532, KF921476, KP784370, KU198177, KJ527255, KX165400, KU885422, KU737561 1 A. lentulus β-tubulin AB910085 1 Neosartorya fischeri β-tubulin EF669828 Circumdati Flavi 46 A. flavus β-tubulin KJ482657, KJ482657, KT354304, HQ400610, KT354304, KT275168, KU737557, KX306819 3 A. minisclerotigenes β-tubulin JX456195 1 A. nomius β-tubulin KJ767725 3 A. tamarii β-tubulin KP067209, KP641150, KJ767722 Nigri 15 A. niger β-tubulin KJ136073, KT805426, KM502177, HQ632737, LN482544, KT354315, HQ632737, KT965696, KT149875, KT965691 16 A. tubingensis β-tubulin LC000547, KT965707, KT354311, KT965718, KR064543, KJ938412 Circumdati 1 A. ochraceus β-tubulin KR737581 1 A. persii β-tubulin KT253228 1 A. pseudoelegans β-tubulin EU014095.1 Terrei Terrei 31 A. terreus β-tubulin GQ376137, LC000549, KJ777806, KP715156, LN835260, KR610363, KR051550, LC060787, KC190476, KR051541, KC473916, KU737559 Nidulantes Versicolores 1 A. creber β-tubulin KP329888 1 A. sydowii β-tubulin KT253232 1 A. tabacinus Calmodulin LT594402 Nidulantes 2 A. nidulans β-tubulin KP278196 5 A. quadrilineatus Calmodulin KU866794, KU866777, EF591681 1 A. rugulosus Calmodulin KU866801 Usti 1 A. calidoustus β-tubulin KJ777803 View Large Table 1. Aspergillus isolates (n = 179) identified by gene sequencing. Number of Identification by Subgenus Section isolates sequencing Target GenBank accession no. Fumigati Fumigati 47 A. fumigatus β-tubulin KF921489, DQ438532, KF921476, KP784370, KU198177, KJ527255, KX165400, KU885422, KU737561 1 A. lentulus β-tubulin AB910085 1 Neosartorya fischeri β-tubulin EF669828 Circumdati Flavi 46 A. flavus β-tubulin KJ482657, KJ482657, KT354304, HQ400610, KT354304, KT275168, KU737557, KX306819 3 A. minisclerotigenes β-tubulin JX456195 1 A. nomius β-tubulin KJ767725 3 A. tamarii β-tubulin KP067209, KP641150, KJ767722 Nigri 15 A. niger β-tubulin KJ136073, KT805426, KM502177, HQ632737, LN482544, KT354315, HQ632737, KT965696, KT149875, KT965691 16 A. tubingensis β-tubulin LC000547, KT965707, KT354311, KT965718, KR064543, KJ938412 Circumdati 1 A. ochraceus β-tubulin KR737581 1 A. persii β-tubulin KT253228 1 A. pseudoelegans β-tubulin EU014095.1 Terrei Terrei 31 A. terreus β-tubulin GQ376137, LC000549, KJ777806, KP715156, LN835260, KR610363, KR051550, LC060787, KC190476, KR051541, KC473916, KU737559 Nidulantes Versicolores 1 A. creber β-tubulin KP329888 1 A. sydowii β-tubulin KT253232 1 A. tabacinus Calmodulin LT594402 Nidulantes 2 A. nidulans β-tubulin KP278196 5 A. quadrilineatus Calmodulin KU866794, KU866777, EF591681 1 A. rugulosus Calmodulin KU866801 Usti 1 A. calidoustus β-tubulin KJ777803 Number of Identification by Subgenus Section isolates sequencing Target GenBank accession no. Fumigati Fumigati 47 A. fumigatus β-tubulin KF921489, DQ438532, KF921476, KP784370, KU198177, KJ527255, KX165400, KU885422, KU737561 1 A. lentulus β-tubulin AB910085 1 Neosartorya fischeri β-tubulin EF669828 Circumdati Flavi 46 A. flavus β-tubulin KJ482657, KJ482657, KT354304, HQ400610, KT354304, KT275168, KU737557, KX306819 3 A. minisclerotigenes β-tubulin JX456195 1 A. nomius β-tubulin KJ767725 3 A. tamarii β-tubulin KP067209, KP641150, KJ767722 Nigri 15 A. niger β-tubulin KJ136073, KT805426, KM502177, HQ632737, LN482544, KT354315, HQ632737, KT965696, KT149875, KT965691 16 A. tubingensis β-tubulin LC000547, KT965707, KT354311, KT965718, KR064543, KJ938412 Circumdati 1 A. ochraceus β-tubulin KR737581 1 A. persii β-tubulin KT253228 1 A. pseudoelegans β-tubulin EU014095.1 Terrei Terrei 31 A. terreus β-tubulin GQ376137, LC000549, KJ777806, KP715156, LN835260, KR610363, KR051550, LC060787, KC190476, KR051541, KC473916, KU737559 Nidulantes Versicolores 1 A. creber β-tubulin KP329888 1 A. sydowii β-tubulin KT253232 1 A. tabacinus Calmodulin LT594402 Nidulantes 2 A. nidulans β-tubulin KP278196 5 A. quadrilineatus Calmodulin KU866794, KU866777, EF591681 1 A. rugulosus Calmodulin KU866801 Usti 1 A. calidoustus β-tubulin KJ777803 View Large Protein spectra of 53 strains, 42 sequenced clinical isolates, and 11 reference strains (10 from the American Type Culture Collection [ATCC, Manassas, VA, USA] and one from the Colección Española de Cultivos Tipo [CECT, Valencia, Spain]) were used to construct an in-house reference database (HUVR) in the MALDI-TOF MS (Table 2). Table 2. Aspergillus isolates used to construct the HUVR database. Subgenus Section Species Total no. of isolates included (no. of reference strains)a Fumigati Fumigati A. fumigatus 5 (2) A. lentulus 1 Neosartorya fischeri 1 Circumdati Flavi A. flavus 5 (2) A. minisclerotigenes 3 A. nomius 1 A. oryzae 1 (1) A. parasiticus 1 (1) A. tamarii 4 (1) Nigri A. niger 4 (1) A. tubingensis 6 (1) Circumdati A. ochraceus 1 A. persii 1 A. pseudoelegans 1 Terrei Terrei A. terreus 5 (1) Nidulantes Versicolores A. creber 1 A. sydowii 1 A. tabacinus 1 A. versicolor 1 (1) Nidulantes A. nidulans 2 A. quadrilineatus 5 A. rugulosus 1 Usti A. calidoustus 1 Subgenus Section Species Total no. of isolates included (no. of reference strains)a Fumigati Fumigati A. fumigatus 5 (2) A. lentulus 1 Neosartorya fischeri 1 Circumdati Flavi A. flavus 5 (2) A. minisclerotigenes 3 A. nomius 1 A. oryzae 1 (1) A. parasiticus 1 (1) A. tamarii 4 (1) Nigri A. niger 4 (1) A. tubingensis 6 (1) Circumdati A. ochraceus 1 A. persii 1 A. pseudoelegans 1 Terrei Terrei A. terreus 5 (1) Nidulantes Versicolores A. creber 1 A. sydowii 1 A. tabacinus 1 A. versicolor 1 (1) Nidulantes A. nidulans 2 A. quadrilineatus 5 A. rugulosus 1 Usti A. calidoustus 1 aReference strains: Aspergillus flavus ATCC 76668 (CECT 2686), Aspergillus flavus ATCC 204304, Aspergillus fumigatus ATCC 204305, Aspergillus fumigatus var. fumigatus ATCC 16907 (CECT 20228), Aspergillus niger ATCC 9029 (CECT 2088), Aspergillus oryzae var. oryzae ATCC 1011 (CECT 2094), Aspergillus parasiticus ATCC 15517 (CECT 2680), Aspergillus tamarii CECT 20399, Aspergillus terreus var. terreus ATCC 10690 (CECT 2808), Aspergillus tubingensis ATCC 11394 (CECT 2089) and Aspergillus versicolor ATCC 42039 (CECT 1544). View Large Table 2. Aspergillus isolates used to construct the HUVR database. Subgenus Section Species Total no. of isolates included (no. of reference strains)a Fumigati Fumigati A. fumigatus 5 (2) A. lentulus 1 Neosartorya fischeri 1 Circumdati Flavi A. flavus 5 (2) A. minisclerotigenes 3 A. nomius 1 A. oryzae 1 (1) A. parasiticus 1 (1) A. tamarii 4 (1) Nigri A. niger 4 (1) A. tubingensis 6 (1) Circumdati A. ochraceus 1 A. persii 1 A. pseudoelegans 1 Terrei Terrei A. terreus 5 (1) Nidulantes Versicolores A. creber 1 A. sydowii 1 A. tabacinus 1 A. versicolor 1 (1) Nidulantes A. nidulans 2 A. quadrilineatus 5 A. rugulosus 1 Usti A. calidoustus 1 Subgenus Section Species Total no. of isolates included (no. of reference strains)a Fumigati Fumigati A. fumigatus 5 (2) A. lentulus 1 Neosartorya fischeri 1 Circumdati Flavi A. flavus 5 (2) A. minisclerotigenes 3 A. nomius 1 A. oryzae 1 (1) A. parasiticus 1 (1) A. tamarii 4 (1) Nigri A. niger 4 (1) A. tubingensis 6 (1) Circumdati A. ochraceus 1 A. persii 1 A. pseudoelegans 1 Terrei Terrei A. terreus 5 (1) Nidulantes Versicolores A. creber 1 A. sydowii 1 A. tabacinus 1 A. versicolor 1 (1) Nidulantes A. nidulans 2 A. quadrilineatus 5 A. rugulosus 1 Usti A. calidoustus 1 aReference strains: Aspergillus flavus ATCC 76668 (CECT 2686), Aspergillus flavus ATCC 204304, Aspergillus fumigatus ATCC 204305, Aspergillus fumigatus var. fumigatus ATCC 16907 (CECT 20228), Aspergillus niger ATCC 9029 (CECT 2088), Aspergillus oryzae var. oryzae ATCC 1011 (CECT 2094), Aspergillus parasiticus ATCC 15517 (CECT 2680), Aspergillus tamarii CECT 20399, Aspergillus terreus var. terreus ATCC 10690 (CECT 2808), Aspergillus tubingensis ATCC 11394 (CECT 2089) and Aspergillus versicolor ATCC 42039 (CECT 1544). View Large Strains collection The collection of Aspergillus strains was based on suspensions of conidia obtained from sporulated cultures grown on Sabouraud-chloramphenicol agar plates (SCA; Oxoid, Basingstoke, UK) after 5–7 d incubation, as described by Cassagne et al.13 The conidia were separated from the mycelium by passing a sterile cotton swab over its surface, which were then transferred to a screw-cap vial with 3 ml of sterile distilled water. The vials were stored at room temperature. Strains identification All 379 strains were characterized morphologically based on the macroscopic and microscopic features according to de Hoog et al.18 The identification by molecular methods of the 179 selected strains was performed by sequencing of beta-tubulin and calmodulin genes. For that, the fungal DNA was extracted with the QIAamp® DNA Mini Kit (Qiagen, Courtaboeuf, France) from the strains grown on SCA after 48 h of incubation at 30°C. Polymerase chain reaction (PCR) amplification and sequencing of the partial portions of the beta-tubulin and calmodulin genes were performed as previously described,19–21 with the primer pair of β-tub1 (5΄-AATTGGTGCCGCTTTCTGG-3΄) and β-tub4 (5΄-AGCGTCCATGGTACCGG-3΄) and the primer pair Cmd5 (5΄-GTCTCCGAGTACAAGGAGGC-3΄) and Cmd6 (5΄-TCGCCGATRGAGGTCATRACGTG-3΄), respectively. Sequencing of the partial calmodulin gene was only used for identification of cryptic species. A BLAST search analysis for species identification was carried out at the NCBI genomic database (http://blast.ncbi.nom.nih.gov/). Generation of the in-house reference mold database in MALDI-TOF MS Sample preparation The strains used to build the new database were cultured in liquid medium. To do this, we started with the suspension of conidia of each of the 53 selected isolates. A 300-μl aliquot of each suspended strain was inoculated to 3 ml of Sabouraud broth (BD, Franklin Lakes, NJ, USA) and was incubated at 30°C for 24–48 h. One ml of the liquid fungal culture was placed into a 1.5 ml Eppendorf tube and centrifuged at 15,000 g for 2 min. The supernatant was discarded and 500 μl of distilled water were added to the pellet. The mixture was aspirated several times with a pipette, trying to dissolve the pellet. Then, it was centrifuged at 15,000 g for 2 min. This washing process was carried out 3 times. Finally, the pellet was resuspended in a mixture of 300 μl distilled water and 900 μl ethanol. Each tube was then centrifuged at 15,000 g for 2 min, and the pellet was dried at 50°C and resuspended in 25 to 50 μl of 70% formic acid, depending on the fungal mass obtained. Not all Aspergillus strains grew at the same rate during incubation, so that the obtained fungus mass was variable and, therefore, also the added volume of formic acid. We always used the lowest volume of formic acid capable of covering the pellet. The objective was to dissolve the pellet and to dilute the released proteins as little as possible. After incubating for 5–10 min at room temperature, an equal volume of acetonitrile was added. Samples were incubated again at room temperature for 10 min and subsequently centrifuged at 15,000 g for 1 min. For the MALDI-TOF analysis the supernatants of the samples were transferred to a polished steel plate MSP 96 (Bruker Daltonik GmbH) that only allowed a maximum volume of 1 μl per sample spot. In our protocol, we used 2 μl per sample spot to increase the concentration of proteins and to improve the quality of the spectra. For this, 1 μl of the supernatant was spotted onto a sample splot and allowed to dry at room temperature, before repeating this step once more. Finally, after the samples had dried, 1 μl of the matrix solution (a saturated solution of α-cyano-4 hydroxy-cinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) was pipetted and dried at room temperature. MALDI-TOF MS Spectral analysis The acquisition and analysis of mass spectra was performed by a Microflex LT mass spectrometer (Bruker Daltonik GmbH) using the MALDI Biotyper software package (version 3.0). We worked with default parameter settings: positive linear mode; laser frequency, 60 Hz; ion source 1 voltage, 20 kV; ion source 2 voltage, 18.5 kV; lens voltage, 6.0 kV; mass range, 2000 to 20,000 Da. For each spectrum, 240 laser shots in 40-shot steps from different positions of the sample spot were accumulated and analyzed (automatic mode, default settings of MBT_AutoX method). The Bruker bacterial test standard (Bruker Daltonik GmbH) was used for calibration according to the instructions of the manufacturer. A reference spectrum was created from each of the 53 isolates, including 30 strains of cryptic species, which added to the in-house reference database. A total of 24 spectra and 1 mix spectrum (strain and BTS) were measured for each strain. The generated spectra were analyzed and calibrated using the FlexAnalysis software, uncertain spectra were excluded and a average of 18 to 24 spectra were used to generate a consensus reference spectrum using the MALDI Biotyper software. Finally, the main spectrum of each strain was included to the in-house reference database using the Biotyper software. Validation of the in-house reference database One hundred ninety sequenced strains were identified by the MALDI-TOF MS technology to validate the generated in-house reference database, including the 53 strains introduced into the database. An aliquot of distilled water suspension of conidia from each strain was inoculated on two SCA, making five marks per plate (Fig. 1). Thereby, we got maximum use of the surface of each plate, obtaining the largest amount of young mycelium. After 48 h of incubation at 30°C, the youngest mycelium of each colony (outermost area) was collected with a sterile cotton swab. This was passed through the periphery of the colonies to drag the mycelium, which was later transferred and emulsified in 1.0 ml distilled water in an Eppendorf tube, which was used for the MALDI-TOF MS identification, following the above described protocol. Figure 1. View largeDownload slide Aspect of an A. flavus strain after 48 hours of incubation on SCA (five marks plate). This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Aspect of an A. flavus strain after 48 hours of incubation on SCA (five marks plate). This Figure is reproduced in color in the online version of Medical Mycology. Prospective evaluation of the HUVR database Two hundred strains of the initial 379 clinical isolates, different from the 179 used for the creation and validation of the database, were used for the prospective evaluation. These strains grown on SCA were processed in the same manner as described above. MALDI-TOF MS data interpretation Each mass spectrum was analyzed against the reference mass spectra in the in-house reference database using the Biotyper software, which calculates an arbitrary unit score value (from 0 to 3) that reflects the similarity between sample and reference spectrum. For each isolate, the highest log (score) value of a match against an spectrum in the database was used for identification. Log (score) values ≥2.0 are rated as identification at the species level, whereas log (score) values between ≥1.7 and <2.0 are rated as identification at the genus level. Results with log (score) values of <1.7 are rated as not suitable for identification by the MALDI BioTyper database 3.0. All isolates were spotted and identified in triplicate by MALDI-TOF MS using the in-house reference database created, which included the reference spectra of 53 Aspergillus strains. The main spectrum comparison was based on the best log (score) values. Results Strains identification The 179 selected strains were morphologically identified as A. fumigatus (n = 41), A. flavus (n = 43), A. niger (n = 31), A. terreus (n = 26), A. nidulans (n = 3), A. versicolor (n = 1), and Aspergillus spp. (n = 34). Therefore, the conventional identification assigned 81% of the isolates to species level and the remaining 19% only to genus level. Gene sequencing identified 20 species (Table 1) belonging to eight sections, which included six noncryptic species and 14 cryptic species. Partial sequencing of the β-tubulin gene allowed the identification of all the species, except three (A. tabacinus, A. rugulosus and A. quadrilineatus), which were identified by partial calmodulin gene sequencing (Table 1). A 65.4% correlation between morphologic and molecular identification was found, mainly with A. fumigatus, A. flavus, and A. terreus. The remaining 200 strains, used in the prospective evaluation, were identified morphologically as A. fumigatus (n = 120), A. flavus (n = 24), A. niger (n = 28), A. terreus (n = 27), and A. nidulans (n = 1). Construction and validation of the in-house mould database The generated reference spectra with the MALDI Biotyper software, allowed us to construct an in-house reference database that included 53 entries (Table 2): 42 from clinical strains and 11 from reference strains, including 23 different species from eight sections: seven noncryptic species (with 23 entries), and 16 cryptic species (with 30 entries). To evaluate the HUVR database, 190 strains (179 clinical isolates previously identified by sequencing and 11 reference strains) (Table 3) were analyzed. In these 190 strains are included the 53 used for the construction of the database and with this bias a 100% correlation was obtained with both identification methods, sequencing and MALDI-TOF MS, and no discordant identification was obtained. Table 3. Identification of Aspergillus isolates using the HUVR database. Subgenus Section Species (identification by sequencing + MALDI-TOF MS)a No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 49 2.212 2.563 1.730 0.207 0 3 46 A. lentulus 1 2.067 2.120 1.980 0.076 0 0 1 N. fischeri 1 2.242 2.331 2.180 0.079 0 0 1 Circumdati Flavi A. flavus 48 2.224 2.674 1.717 0.181 0 1 47 A. minisclerotigenes 3 1.904 2.110 1.711 0.155 0 1 2 A. nomius 1 1.659 1.715 1.550 0.094 0 1 0 A. oryzae 1 2.020 2.040 2.010 0.017 0 0 1 A. parasiticus 1 1.703 1.750 1.670 0.042 0 1 0 A. tamarii 4 2.005 2.325 1.848 0.150 0 1 3 Nigri A. niger 16 2.407 2.695 1.445 0.202 0 0 16 A. tubingensis 17 2.219 2.632 1.815 0.227 0 3 14 Circumdati A. ochraceus 1 1.884 1.912 1.860 0.026 0 1 0 A. persii 1 1.933 1.950 1.910 0.021 0 1 0 A. pseudoelegans 1 1.973 2.010 1.950 0.032 0 0 1 Terrei Terrei A. terreus 32 2.056 2.364 1.804 0.171 0 7 25 Nidulantes Versicolores A. creber 1 2.618 2.621 2.615 0.003 0 0 1 A. sydowii 1 2.119 2.150 2.094 0.028 0 0 1 A. versicolor 1 2.023 2.110 1.970 0.076 0 0 1 A. tabacinus 1 2.010 2.050 1.970 0.057 0 0 1 Nidulantes A. nidulans 2 2.148 2.316 1.818 0.225 0 1 1 A. quadrilineatus 5 1.990 2.180 1.830 0.126 0 3 2 A. rugulosus 1 2.043 2.060 2.030 0.015 0 0 1 Usti A. calidoustus 1 1.940 1.990 1.890 0.071 0 1 0 Subgenus Section Specie (identification by MALDI-TOF MS)b No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 121 2.303 2.706 1.714 0.207 0 2 119 Circumdati Flavi A. flavus 24 2.275 2.573 1.803 0.201 0 0 24 Nigri A. niger 7 2.260 2.512 1.838 0.206 0 0 7 A. tubingensis 21 2.213 2.683 1.687 0.222 0 2 19 Terrei Terrei A. terreus 26 2.150 2.560 1.742 0.230 0 5 21 Nidulantes Nidulantes A. nidulans 1 2.161 2.273 2.056 0.108 0 0 1 Subgenus Section Species (identification by sequencing + MALDI-TOF MS)a No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 49 2.212 2.563 1.730 0.207 0 3 46 A. lentulus 1 2.067 2.120 1.980 0.076 0 0 1 N. fischeri 1 2.242 2.331 2.180 0.079 0 0 1 Circumdati Flavi A. flavus 48 2.224 2.674 1.717 0.181 0 1 47 A. minisclerotigenes 3 1.904 2.110 1.711 0.155 0 1 2 A. nomius 1 1.659 1.715 1.550 0.094 0 1 0 A. oryzae 1 2.020 2.040 2.010 0.017 0 0 1 A. parasiticus 1 1.703 1.750 1.670 0.042 0 1 0 A. tamarii 4 2.005 2.325 1.848 0.150 0 1 3 Nigri A. niger 16 2.407 2.695 1.445 0.202 0 0 16 A. tubingensis 17 2.219 2.632 1.815 0.227 0 3 14 Circumdati A. ochraceus 1 1.884 1.912 1.860 0.026 0 1 0 A. persii 1 1.933 1.950 1.910 0.021 0 1 0 A. pseudoelegans 1 1.973 2.010 1.950 0.032 0 0 1 Terrei Terrei A. terreus 32 2.056 2.364 1.804 0.171 0 7 25 Nidulantes Versicolores A. creber 1 2.618 2.621 2.615 0.003 0 0 1 A. sydowii 1 2.119 2.150 2.094 0.028 0 0 1 A. versicolor 1 2.023 2.110 1.970 0.076 0 0 1 A. tabacinus 1 2.010 2.050 1.970 0.057 0 0 1 Nidulantes A. nidulans 2 2.148 2.316 1.818 0.225 0 1 1 A. quadrilineatus 5 1.990 2.180 1.830 0.126 0 3 2 A. rugulosus 1 2.043 2.060 2.030 0.015 0 0 1 Usti A. calidoustus 1 1.940 1.990 1.890 0.071 0 1 0 Subgenus Section Specie (identification by MALDI-TOF MS)b No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 121 2.303 2.706 1.714 0.207 0 2 119 Circumdati Flavi A. flavus 24 2.275 2.573 1.803 0.201 0 0 24 Nigri A. niger 7 2.260 2.512 1.838 0.206 0 0 7 A. tubingensis 21 2.213 2.683 1.687 0.222 0 2 19 Terrei Terrei A. terreus 26 2.150 2.560 1.742 0.230 0 5 21 Nidulantes Nidulantes A. nidulans 1 2.161 2.273 2.056 0.108 0 0 1 aThis group include 190 strains (179 clinical strains + 11 reference strains) identified by sequencing and MALDI-TOF MS. In all cases, there was correlation between the two methods. bThis group include 200 strains identified by MALDI-TOF MS. View Large Table 3. Identification of Aspergillus isolates using the HUVR database. Subgenus Section Species (identification by sequencing + MALDI-TOF MS)a No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 49 2.212 2.563 1.730 0.207 0 3 46 A. lentulus 1 2.067 2.120 1.980 0.076 0 0 1 N. fischeri 1 2.242 2.331 2.180 0.079 0 0 1 Circumdati Flavi A. flavus 48 2.224 2.674 1.717 0.181 0 1 47 A. minisclerotigenes 3 1.904 2.110 1.711 0.155 0 1 2 A. nomius 1 1.659 1.715 1.550 0.094 0 1 0 A. oryzae 1 2.020 2.040 2.010 0.017 0 0 1 A. parasiticus 1 1.703 1.750 1.670 0.042 0 1 0 A. tamarii 4 2.005 2.325 1.848 0.150 0 1 3 Nigri A. niger 16 2.407 2.695 1.445 0.202 0 0 16 A. tubingensis 17 2.219 2.632 1.815 0.227 0 3 14 Circumdati A. ochraceus 1 1.884 1.912 1.860 0.026 0 1 0 A. persii 1 1.933 1.950 1.910 0.021 0 1 0 A. pseudoelegans 1 1.973 2.010 1.950 0.032 0 0 1 Terrei Terrei A. terreus 32 2.056 2.364 1.804 0.171 0 7 25 Nidulantes Versicolores A. creber 1 2.618 2.621 2.615 0.003 0 0 1 A. sydowii 1 2.119 2.150 2.094 0.028 0 0 1 A. versicolor 1 2.023 2.110 1.970 0.076 0 0 1 A. tabacinus 1 2.010 2.050 1.970 0.057 0 0 1 Nidulantes A. nidulans 2 2.148 2.316 1.818 0.225 0 1 1 A. quadrilineatus 5 1.990 2.180 1.830 0.126 0 3 2 A. rugulosus 1 2.043 2.060 2.030 0.015 0 0 1 Usti A. calidoustus 1 1.940 1.990 1.890 0.071 0 1 0 Subgenus Section Specie (identification by MALDI-TOF MS)b No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 121 2.303 2.706 1.714 0.207 0 2 119 Circumdati Flavi A. flavus 24 2.275 2.573 1.803 0.201 0 0 24 Nigri A. niger 7 2.260 2.512 1.838 0.206 0 0 7 A. tubingensis 21 2.213 2.683 1.687 0.222 0 2 19 Terrei Terrei A. terreus 26 2.150 2.560 1.742 0.230 0 5 21 Nidulantes Nidulantes A. nidulans 1 2.161 2.273 2.056 0.108 0 0 1 Subgenus Section Species (identification by sequencing + MALDI-TOF MS)a No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 49 2.212 2.563 1.730 0.207 0 3 46 A. lentulus 1 2.067 2.120 1.980 0.076 0 0 1 N. fischeri 1 2.242 2.331 2.180 0.079 0 0 1 Circumdati Flavi A. flavus 48 2.224 2.674 1.717 0.181 0 1 47 A. minisclerotigenes 3 1.904 2.110 1.711 0.155 0 1 2 A. nomius 1 1.659 1.715 1.550 0.094 0 1 0 A. oryzae 1 2.020 2.040 2.010 0.017 0 0 1 A. parasiticus 1 1.703 1.750 1.670 0.042 0 1 0 A. tamarii 4 2.005 2.325 1.848 0.150 0 1 3 Nigri A. niger 16 2.407 2.695 1.445 0.202 0 0 16 A. tubingensis 17 2.219 2.632 1.815 0.227 0 3 14 Circumdati A. ochraceus 1 1.884 1.912 1.860 0.026 0 1 0 A. persii 1 1.933 1.950 1.910 0.021 0 1 0 A. pseudoelegans 1 1.973 2.010 1.950 0.032 0 0 1 Terrei Terrei A. terreus 32 2.056 2.364 1.804 0.171 0 7 25 Nidulantes Versicolores A. creber 1 2.618 2.621 2.615 0.003 0 0 1 A. sydowii 1 2.119 2.150 2.094 0.028 0 0 1 A. versicolor 1 2.023 2.110 1.970 0.076 0 0 1 A. tabacinus 1 2.010 2.050 1.970 0.057 0 0 1 Nidulantes A. nidulans 2 2.148 2.316 1.818 0.225 0 1 1 A. quadrilineatus 5 1.990 2.180 1.830 0.126 0 3 2 A. rugulosus 1 2.043 2.060 2.030 0.015 0 0 1 Usti A. calidoustus 1 1.940 1.990 1.890 0.071 0 1 0 Subgenus Section Specie (identification by MALDI-TOF MS)b No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 121 2.303 2.706 1.714 0.207 0 2 119 Circumdati Flavi A. flavus 24 2.275 2.573 1.803 0.201 0 0 24 Nigri A. niger 7 2.260 2.512 1.838 0.206 0 0 7 A. tubingensis 21 2.213 2.683 1.687 0.222 0 2 19 Terrei Terrei A. terreus 26 2.150 2.560 1.742 0.230 0 5 21 Nidulantes Nidulantes A. nidulans 1 2.161 2.273 2.056 0.108 0 0 1 aThis group include 190 strains (179 clinical strains + 11 reference strains) identified by sequencing and MALDI-TOF MS. In all cases, there was correlation between the two methods. bThis group include 200 strains identified by MALDI-TOF MS. View Large The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.8%). No identification was made with a score lower than 1.7, and 25 isolates (13.2%) were identified to the genus level (score between 1.7 and <2.0). Among the 23 different species included in our database, 18 obtained scores ≥2.0, while in the remaining five species (A. calidoustus, A. nomius, A.ochraceus, A. parasiticus, and A. persii) the scores were between 1.7 and 2.0. Finally, a species identification (score of ≥2.0) was obtained in 136 (91.3%) of the noncryptic species and in 29 (70.7%) of the cryptic species. In both groups, an identification at the genus level (score between 1.7 and <2.0) was found in 13 (8.7%) strains and in 12 (29.3%) strains, respectively. Even for those identifications with scores between 1.7 and <2.0, MALDI-TOF MS results always agreed with partial sequencing data. Prospective evaluation of the HUVR database A prospective evaluation of the routine MALDI-TOF MS analysis was performed with 200 Aspergillus clinical isolates grown on solid medium and with the new database. A species identification (score of ≥2.0) was obtained in 191 strains (95.5%) and only nine strains (4.5%) were not identified at species level, using the manufacturer recommended cut-off values (Table 3). We identified species belonging to five sections (Flavi, Fumigati, Nidulantes, Nigri, and Terrei). The strains identified at species level, with scores ranging from 2.273 to 2.706, were A. fumigatus (n = 119), A. flavus (n = 24), A. niger (n = 7), A. tubingensis (n = 19), A. terreus (n = 21), and A. nidulans (n = 1); while scores between 1.7 and <2.0 were seen with A. fumigatus (n = 2), A. tubingensis (n = 2), and A. terreus (n = 5). Among the 200 clinical isolates A. tubingensis was the only cryptic species identified. Additionally, MALDI-TOF MS identified 28 aspergilli of the section Nigri to species level: 19 (67.9%) A. tubingensis and seven (25%) A. niger, while the remaining two were identified to the genus level (score between 1.7 and <2.0). Discussion In this study, we constructed and validated an in-house reference database in the MALDI-TOF MS, which significantly expanded the original commercially-available database and allowed the accurate identification of a large number of Aspergillus species isolated in the clinical setting, including both noncryptic and cryptic species. The original library with 89 entries was supplemented with 53 additional entries of 23 species, which allowed us to include 12 new cryptic species and to increase the number of entries of 11 species already included. Separately, the HUVR database provided 16 cryptic species while the Filamentous Fungi Library 1.0 only seven cryptic species, representing 69.6% and 38.9% of the total species included in each one of the libraries. Finally, the supplemented library had 142 entries that allowed the identification of 30 different species of Aspergillus from 11 sections: 11 noncryptic species and 19 cryptic species. The prevalence of the cryptic species present in our strain collection (15.7%), with A. tubingensis as the most frequent species, was similar to the ones published in previous studies, ranging from 10 to 15%.22,23 Several authors21,24–26 pointed out the need to expand the Filamentous Fungi Library 1.0 library with new species and a greater number of entries to improve the sensitivity of this technology, and for this reason we have validated our database by identifying 390 strains but without comparing the identification results with those obtained only using the original database. The prospective study showed that the Aspergillus identification grown on solid medium and the use of the HUVR database in the MALDI-TOF MS Biotyper were highly reliable. An identification in 95.5% of the isolates at the species level was obtained, with the remaining 4.5% of the isolates identified at the genus level. These results are similar to those previously described.27–29 Schulthess et al.15 significantly enhanced identification from 54.2% to 71.1% at species level using a score ≥1.7 instead of ≥2.0, without increasing misidentifications. If we applied this score in our results, 100% of identifications at the species level would have been obtained. To create the new reference spectra, we used liquid cultivation to obtain a more homogeneous mycelium,15 easier to be emulsified, and rendering better quality spectra. The validation of the database and the prospective study were done using colonies grown on solid medium, where culture of the clinical samples is performed routinely, so that no extra time is needed to get results by an additional subculture in liquid medium. Although several authors13,15,30 recommended the application of a lower cut-off value (≥1.7) to increase identification rates, our procedure with strains grown on solid medium, identified most of them with scores of ≥2.0. In the case of clinical isolates with scores between 1.7 and 2.0, the presumptive identification can be confirmed performing the procedure from a subculture in liquid medium. Aspergillus calidoustus, A. nomius, A. ochraceus, A. parasiticus, and A. persii are the species in which the scores between 1.7 and <2.0 were found, and it can be due to the particular characteristics of their mycelium and to the presence of a single entry of each of them in the library. When the identifications were made from liquid medium, all these species were correctly identified with scores ranging from 2.43 to 2.63 (data not shown). Therefore, the new library shows its feasibility, regardless of the medium type (i.e., solid or liquid). Nonetheless, better identification scores were obtained if the molds were grown in liquid medium, because those were the conditions for which the library was generated. We demonstrated the feasibility and usefulness of a new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species from different sections, grown either on solid or liquid media. This procedure could be incorporated as a routine mold identification procedure in the mycology laboratory to improve the accuracy of the identification of clinically relevant species of Aspergillus and significantly shortening the response time. Howewer, future studies carried out with our database are necessary to confirm our results, avoiding the bias committed by including strains used in the construction of the database for its validation. The creation of databases in the MALDI-TOF MS is a slow and expensive process, but necessary if we want to benefit from a better identification capacity. As we have shown, this research has improved the identification of Aspergillus species in our laboratory. For this reason, we have made it available to the manufacturer for the benefit of the medical community, in case it is considered appropriate its inclusion in future software updates. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. This work was funded by the Autonomous Government of Andalusia. References 1. Fortún J , Meije Y , Fresco G , Moreno S . Aspergillosis: clinical forms and treatment . Enfermedades Infecc Microbiol Clínica . 2012 ; 30 : 201 – 208 [in Spanish] . Google Scholar Crossref Search ADS 2. Peterson SW , Varga J , Frisvad JV , Samson RA . Phylogeny and subgeneric taxonomy of Aspergillus . In: Varga J , Samson RA , eds. Aspergillus in the Genomic Era . Wageningen : Wageningen Academic Publishers , 2008 : 33 – 53 . 3. Samson RA , Hong SB , Frisvad JC . Old and new concepts of species differentiation in Aspergillus . Med Mycol . 2006 ; 44 : 133 – 148 . Google Scholar Crossref Search ADS PubMed 4. Balajee SA , Gribskov JL , Hanley E , Nickle D , Marr KA . Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus . Eukaryot Cell . 2005 ; 4 : 625 – 632 . Google Scholar Crossref Search ADS PubMed 5. Varga J , Houbraken J , Van Der Lee HA , Verweij PE , Samson RA . Aspergillus calidoustus sp. nov., causative agent of human infections previously assigned to Aspergillus ustus . Eukaryot Cell . 2008 ; 7 : 630 – 638 . Google Scholar Crossref Search ADS PubMed 6. Balajee SA , Borman AM , Brandt ME et al. Sequence-based identification of Aspergillus, Fusarium, and Mucorales species in the clinical mycology laboratory: where are we and where should we go from here? J Clin Microbiol . 2009 ; 47 : 877 – 884 . Google Scholar Crossref Search ADS PubMed 7. Balajee SA , Houbraken J , Verweij PE et al. Aspergillus species identification in the clinical setting . Stud Mycol . 2007 ; 59 : 39 – 46 . Google Scholar Crossref Search ADS PubMed 8. Samson RA , Visagie CM , Houbraken J et al. Phylogeny, identification and nomenclature of the genus Aspergillus . Stud Mycol . 2014 ; 78 : 141 – 173 . Google Scholar Crossref Search ADS PubMed 9. Bille E , Dauphin B , Leto J et al. MALDI-TOF MS Andromas strategy for the routine identification of bacteria, mycobacteria, yeasts, Aspergillus spp. and positive blood cultures . Clin Microbiol Infect . 2012 ; 18 : 1117 – 1125 . Google Scholar Crossref Search ADS PubMed 10. Patel R . MALDI-TOF MS for the diagnosis of infectious diseases . Clin Chem . 2015 ; 61 : 100 – 111 . Google Scholar Crossref Search ADS PubMed 11. Wieser A , Schneider L , Jung J , Schubert S . MALDI-TOF MS in microbiological diagnostics-identification of microorganisms and beyond (mini review). Appl Microbiol Biotechnol . 2012 ; 93 : 965 – 974 . Google Scholar Crossref Search ADS PubMed 12. Bader O . MALDI-TOF-MS-based species identification and typing approaches in medical mycology . Proteomics . 2013 ; 13 : 788 – 799 . Google Scholar Crossref Search ADS PubMed 13. Cassagne C , Ranque S , Normand AC et al. Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry . PLoS One . 2011 ; 6 : e28425 . Google Scholar Crossref Search ADS PubMed 14. Gautier M , Normand A-C , Ranque S . Previously unknown species of Aspergillus . Clin Microbiol Infect . 2016 ; 22 : 662 – 669 . Google Scholar Crossref Search ADS PubMed 15. Schulthess B , Ledermann R , Mouttet F et al. Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory . J Clin Microbiol . 2014 ; 52 : 2797 – 2803 . Google Scholar Crossref Search ADS PubMed 16. Riat A , Hinrikson H , Barras V , Fernandez J , Schrenzel J . Confident identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without subculture-based sample preparation . Int J Infect Dis . 2015 ; 35 : 43 – 45 . Google Scholar Crossref Search ADS PubMed 17. Sleiman S , Halliday CL , Chapman B et al. Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of Aspergillus, Scedosporium, and Fusarium spp. in the Australian clinical setting . J Clin Microbiol . 2016 ; 54 : 2182 – 2186 . Google Scholar Crossref Search ADS PubMed 18. de Hoog GS , Guarro J , Tan CS , Wintermans RGF , Gené J . Hyphomycetes . In: de Hoog GS , Guarro J , Gené J , Figueras MJ , eds. Atlas of Clinical Fungi . Baarn, The Netherlands : Centraalbureau voor Schimmel-cultures , 2000 : 380 – 1007 . 19. Alastruey-Izquierdo A , Mellado E , Pelaez T et al. Population-based survey of filamentous fungi and antifungal resistance in Spain (FILPOP Study). Antimicrob Agents Chemother . 2013 ; 57 : 3380 – 3387 . Google Scholar Crossref Search ADS PubMed 20. Hong SB , Go SJ , Shin HD , Frisvad JC , Samson RA . Polyphasic taxonomy of Aspergillus fumigatus and related species . Mycologia . 2005 ; 97 : 1316 – 1329 . Google Scholar Crossref Search ADS PubMed 21. Park JH , Shin JH , Choi MJ et al. Evaluation of matrix-assisted laser desorption/ionization time-of-fight mass spectrometry for identification of 345 clinical isolates of Aspergillus species from 11 Korean hospitals: comparison with molecular identification . Diagn Microbiol Infect Dis . 2017 ; 87 : 28 – 31 . Google Scholar Crossref Search ADS PubMed 22. Alastruey-Izquierdo A , Mellado E , Cuenca-Estrella M . Current section and species complex concepts in Aspergillus: recommendations for routine daily practice . Ann NY Acad Sci . 2012 ; 1273 : 18 – 24 . Google Scholar Crossref Search ADS PubMed 23. Balajee SA , Kano R , Baddley JW et al. Molecular identification of Aspergillus species collected for the Transplant-Associated Infection Surveillance Network . J Clin Microbiol . 2009 ; 47 : 3138 – 3141 . Google Scholar Crossref Search ADS PubMed 24. Sanguinetti M , Posteraro B . MALDI-TOF mass spectrometry: any use for Aspergilli? Mycopathologia . 2014 ; 178 : 417 – 426 . Google Scholar Crossref Search ADS PubMed 25. Cassagne C , Normand A-C , L’Ollivier C , Ranque S , Piarroux R . Performance of MALDI-TOF MS platforms for fungal identification . Mycoses . 2016 ; 59 : 678 – 690 . Google Scholar Crossref Search ADS PubMed 26. Masih A , Singh PK , Kathuria S , Agarwal K , Meis JF , Chowdhary A . Identification by molecular methods and matrix-assisted laser-desorption ionization-time of flight and antifungal susceptibility profiles of clinically significant rare Aspergillus species in a referral chest hospital in Delhi, India. J Clin Microbiol . 2016 ; 54 : 2354 – 2364 . Google Scholar Crossref Search ADS PubMed 27. Ranque S , Normand AC , Cassagne C et al. MALDI-TOF mass spectrometry identification of filamentous fungi in the clinical laboratory . Mycoses . 2014 ; 57 : 135 – 140 . Google Scholar Crossref Search ADS PubMed 28. Lau AF , Drake SK , Calhoun LB , Henderson CM , Zelazny AM . Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by matrix-assisted laser desorption-ionization time of flight mass spectrometry . J Clin Microbiol . 2013 ; 51 : 828 – 834 . Google Scholar Crossref Search ADS PubMed 29. De CE , Posteraro B , Lass-Florl C et al. Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry . Clin Microbiol Infect . 2012 ; 18 : 475 – 484 . Google Scholar Crossref Search ADS PubMed 30. Normand A-C , Cassagne C , Ranque S et al. Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi . BMC Microbiol . 2013 ; 13 : 76 . Google Scholar Crossref Search ADS PubMed © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

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Oxford University Press
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© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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1369-3786
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Abstract

Abstract An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media. Aspergillus, identification, cryptic species, MALDI-TOF MS, MALDI-TOF Bruker Introduction The genus Aspergillus includes multiple species widely distributed in the environment, which may be responsible for a wide spectrum of diseases such as allergic syndromes, chronic infection and acute invasive disease, particularly in people with compromised immune systems.1 Taxonomically, the genus Aspergillus includes four subgenera, which are subdivided into numerous sections. Each section corresponds to a specific “species complex,” with groups of related species2 that are almost indistinguishable by morphological methods and which have been designated as cryptic species. In clinical mycology laboratories, identification of Aspergillus species is routinely based on determination of macroscopic and microscopic morphological characteristics, such as colour, shape of conidia, spores and mycelial structures.3 These methods do not allow discrimination of close related species, usually from the same section. However, a correct identification at the species level could be clinically relevant because some of these cryptic species, such as A. calidoustus (Aspergillus section Usti) and A. lentulus (Aspergillus section Fumigati), show decreased susceptibility to multiple antifungal drugs.4,5 Current recommendations for identification at species level within the Aspergillus sections include the use of molecular methods based on comparative sequencing.6,7 Samson et al.8 recommended the use of the ribosomal internal transcribed spacer (ITS) of the nrDNA as the official DNA barcode for fungi and β-tubulin or calmodulin regions as secondary identification markers. However, sequencing is a slow and expensive process for routine Aspergillus species identification of isolates recovered from clinical samples. Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become in recent years a rapid, accurate and cost-effective technology for the identification of bacteria, yeast and mold species in the clinical microbiology laboratory.9–11 However, its use for the identification of molds is limited to date. This is due to several facts including that commercial databases have few fungal entries, the difficulty to obtain good quality mass spectra and the use of a non standardized and more cumbersome pretreatment of the samples to break the fungal cell wall, which is thicker and more robust than that of bacteria.12,13 The first commercial mould database for the Bruker MALDI Biotyper, the Filamentous Fungi Library 1.0 (Bruker Daltonik GmbH, Bremen, Germany), consists of 89 entries corresponding to 18 different species of the genus Aspergillus: 11 noncryptic species (A. candidus, A. clavatus, A. flavus, A. fumigatus, A. glaucus, A. nidulans, A. niger, A. ochraceus, A. terreus, A. ustus, and A. versicolor) and seven cryptic species (A. amstelodami, A. nomius, A. oryzae, A. parasiticus, A. sclerotiorum, A. tamarii, and A. unguis). This is a small representation of species considering than more of 300 have already been described.14 Some authors15–17 have attempted to use this commercial database for identification of Aspergillus spp.,but with unsatisfactory results due to the low number of introduced species and strains. The aim of this study was to supplement the Filamentous Fungi Library 1.0 with an in-house reference database and to validate it with the identification of a collection of clinical isolates of Aspergillus species grown on solid medium. Methods Reference strains and clinical isolates During a 2-year period, 379 strains of Aspergillus were isolated from clinical samples in the Clinical Microbiology Laboratory of the University Hospital Virgen del Rocío (HUVR)-Spain. All the strains were identified by morphological observation, and 179 strains were selected to be identified by gene sequencing (Table 1). Only strains that offered uncertain identification based on their phenotypic characteristics were sequenced. These strains were used to create the new database (n = 42) and for its later validation (n = 179, including the preceding 42). The remaining 200 strains were used in the prospective phase to demonstrate the usefulness of the in-house reference mould database. Table 1. Aspergillus isolates (n = 179) identified by gene sequencing. Number of Identification by Subgenus Section isolates sequencing Target GenBank accession no. Fumigati Fumigati 47 A. fumigatus β-tubulin KF921489, DQ438532, KF921476, KP784370, KU198177, KJ527255, KX165400, KU885422, KU737561 1 A. lentulus β-tubulin AB910085 1 Neosartorya fischeri β-tubulin EF669828 Circumdati Flavi 46 A. flavus β-tubulin KJ482657, KJ482657, KT354304, HQ400610, KT354304, KT275168, KU737557, KX306819 3 A. minisclerotigenes β-tubulin JX456195 1 A. nomius β-tubulin KJ767725 3 A. tamarii β-tubulin KP067209, KP641150, KJ767722 Nigri 15 A. niger β-tubulin KJ136073, KT805426, KM502177, HQ632737, LN482544, KT354315, HQ632737, KT965696, KT149875, KT965691 16 A. tubingensis β-tubulin LC000547, KT965707, KT354311, KT965718, KR064543, KJ938412 Circumdati 1 A. ochraceus β-tubulin KR737581 1 A. persii β-tubulin KT253228 1 A. pseudoelegans β-tubulin EU014095.1 Terrei Terrei 31 A. terreus β-tubulin GQ376137, LC000549, KJ777806, KP715156, LN835260, KR610363, KR051550, LC060787, KC190476, KR051541, KC473916, KU737559 Nidulantes Versicolores 1 A. creber β-tubulin KP329888 1 A. sydowii β-tubulin KT253232 1 A. tabacinus Calmodulin LT594402 Nidulantes 2 A. nidulans β-tubulin KP278196 5 A. quadrilineatus Calmodulin KU866794, KU866777, EF591681 1 A. rugulosus Calmodulin KU866801 Usti 1 A. calidoustus β-tubulin KJ777803 Number of Identification by Subgenus Section isolates sequencing Target GenBank accession no. Fumigati Fumigati 47 A. fumigatus β-tubulin KF921489, DQ438532, KF921476, KP784370, KU198177, KJ527255, KX165400, KU885422, KU737561 1 A. lentulus β-tubulin AB910085 1 Neosartorya fischeri β-tubulin EF669828 Circumdati Flavi 46 A. flavus β-tubulin KJ482657, KJ482657, KT354304, HQ400610, KT354304, KT275168, KU737557, KX306819 3 A. minisclerotigenes β-tubulin JX456195 1 A. nomius β-tubulin KJ767725 3 A. tamarii β-tubulin KP067209, KP641150, KJ767722 Nigri 15 A. niger β-tubulin KJ136073, KT805426, KM502177, HQ632737, LN482544, KT354315, HQ632737, KT965696, KT149875, KT965691 16 A. tubingensis β-tubulin LC000547, KT965707, KT354311, KT965718, KR064543, KJ938412 Circumdati 1 A. ochraceus β-tubulin KR737581 1 A. persii β-tubulin KT253228 1 A. pseudoelegans β-tubulin EU014095.1 Terrei Terrei 31 A. terreus β-tubulin GQ376137, LC000549, KJ777806, KP715156, LN835260, KR610363, KR051550, LC060787, KC190476, KR051541, KC473916, KU737559 Nidulantes Versicolores 1 A. creber β-tubulin KP329888 1 A. sydowii β-tubulin KT253232 1 A. tabacinus Calmodulin LT594402 Nidulantes 2 A. nidulans β-tubulin KP278196 5 A. quadrilineatus Calmodulin KU866794, KU866777, EF591681 1 A. rugulosus Calmodulin KU866801 Usti 1 A. calidoustus β-tubulin KJ777803 View Large Table 1. Aspergillus isolates (n = 179) identified by gene sequencing. Number of Identification by Subgenus Section isolates sequencing Target GenBank accession no. Fumigati Fumigati 47 A. fumigatus β-tubulin KF921489, DQ438532, KF921476, KP784370, KU198177, KJ527255, KX165400, KU885422, KU737561 1 A. lentulus β-tubulin AB910085 1 Neosartorya fischeri β-tubulin EF669828 Circumdati Flavi 46 A. flavus β-tubulin KJ482657, KJ482657, KT354304, HQ400610, KT354304, KT275168, KU737557, KX306819 3 A. minisclerotigenes β-tubulin JX456195 1 A. nomius β-tubulin KJ767725 3 A. tamarii β-tubulin KP067209, KP641150, KJ767722 Nigri 15 A. niger β-tubulin KJ136073, KT805426, KM502177, HQ632737, LN482544, KT354315, HQ632737, KT965696, KT149875, KT965691 16 A. tubingensis β-tubulin LC000547, KT965707, KT354311, KT965718, KR064543, KJ938412 Circumdati 1 A. ochraceus β-tubulin KR737581 1 A. persii β-tubulin KT253228 1 A. pseudoelegans β-tubulin EU014095.1 Terrei Terrei 31 A. terreus β-tubulin GQ376137, LC000549, KJ777806, KP715156, LN835260, KR610363, KR051550, LC060787, KC190476, KR051541, KC473916, KU737559 Nidulantes Versicolores 1 A. creber β-tubulin KP329888 1 A. sydowii β-tubulin KT253232 1 A. tabacinus Calmodulin LT594402 Nidulantes 2 A. nidulans β-tubulin KP278196 5 A. quadrilineatus Calmodulin KU866794, KU866777, EF591681 1 A. rugulosus Calmodulin KU866801 Usti 1 A. calidoustus β-tubulin KJ777803 Number of Identification by Subgenus Section isolates sequencing Target GenBank accession no. Fumigati Fumigati 47 A. fumigatus β-tubulin KF921489, DQ438532, KF921476, KP784370, KU198177, KJ527255, KX165400, KU885422, KU737561 1 A. lentulus β-tubulin AB910085 1 Neosartorya fischeri β-tubulin EF669828 Circumdati Flavi 46 A. flavus β-tubulin KJ482657, KJ482657, KT354304, HQ400610, KT354304, KT275168, KU737557, KX306819 3 A. minisclerotigenes β-tubulin JX456195 1 A. nomius β-tubulin KJ767725 3 A. tamarii β-tubulin KP067209, KP641150, KJ767722 Nigri 15 A. niger β-tubulin KJ136073, KT805426, KM502177, HQ632737, LN482544, KT354315, HQ632737, KT965696, KT149875, KT965691 16 A. tubingensis β-tubulin LC000547, KT965707, KT354311, KT965718, KR064543, KJ938412 Circumdati 1 A. ochraceus β-tubulin KR737581 1 A. persii β-tubulin KT253228 1 A. pseudoelegans β-tubulin EU014095.1 Terrei Terrei 31 A. terreus β-tubulin GQ376137, LC000549, KJ777806, KP715156, LN835260, KR610363, KR051550, LC060787, KC190476, KR051541, KC473916, KU737559 Nidulantes Versicolores 1 A. creber β-tubulin KP329888 1 A. sydowii β-tubulin KT253232 1 A. tabacinus Calmodulin LT594402 Nidulantes 2 A. nidulans β-tubulin KP278196 5 A. quadrilineatus Calmodulin KU866794, KU866777, EF591681 1 A. rugulosus Calmodulin KU866801 Usti 1 A. calidoustus β-tubulin KJ777803 View Large Protein spectra of 53 strains, 42 sequenced clinical isolates, and 11 reference strains (10 from the American Type Culture Collection [ATCC, Manassas, VA, USA] and one from the Colección Española de Cultivos Tipo [CECT, Valencia, Spain]) were used to construct an in-house reference database (HUVR) in the MALDI-TOF MS (Table 2). Table 2. Aspergillus isolates used to construct the HUVR database. Subgenus Section Species Total no. of isolates included (no. of reference strains)a Fumigati Fumigati A. fumigatus 5 (2) A. lentulus 1 Neosartorya fischeri 1 Circumdati Flavi A. flavus 5 (2) A. minisclerotigenes 3 A. nomius 1 A. oryzae 1 (1) A. parasiticus 1 (1) A. tamarii 4 (1) Nigri A. niger 4 (1) A. tubingensis 6 (1) Circumdati A. ochraceus 1 A. persii 1 A. pseudoelegans 1 Terrei Terrei A. terreus 5 (1) Nidulantes Versicolores A. creber 1 A. sydowii 1 A. tabacinus 1 A. versicolor 1 (1) Nidulantes A. nidulans 2 A. quadrilineatus 5 A. rugulosus 1 Usti A. calidoustus 1 Subgenus Section Species Total no. of isolates included (no. of reference strains)a Fumigati Fumigati A. fumigatus 5 (2) A. lentulus 1 Neosartorya fischeri 1 Circumdati Flavi A. flavus 5 (2) A. minisclerotigenes 3 A. nomius 1 A. oryzae 1 (1) A. parasiticus 1 (1) A. tamarii 4 (1) Nigri A. niger 4 (1) A. tubingensis 6 (1) Circumdati A. ochraceus 1 A. persii 1 A. pseudoelegans 1 Terrei Terrei A. terreus 5 (1) Nidulantes Versicolores A. creber 1 A. sydowii 1 A. tabacinus 1 A. versicolor 1 (1) Nidulantes A. nidulans 2 A. quadrilineatus 5 A. rugulosus 1 Usti A. calidoustus 1 aReference strains: Aspergillus flavus ATCC 76668 (CECT 2686), Aspergillus flavus ATCC 204304, Aspergillus fumigatus ATCC 204305, Aspergillus fumigatus var. fumigatus ATCC 16907 (CECT 20228), Aspergillus niger ATCC 9029 (CECT 2088), Aspergillus oryzae var. oryzae ATCC 1011 (CECT 2094), Aspergillus parasiticus ATCC 15517 (CECT 2680), Aspergillus tamarii CECT 20399, Aspergillus terreus var. terreus ATCC 10690 (CECT 2808), Aspergillus tubingensis ATCC 11394 (CECT 2089) and Aspergillus versicolor ATCC 42039 (CECT 1544). View Large Table 2. Aspergillus isolates used to construct the HUVR database. Subgenus Section Species Total no. of isolates included (no. of reference strains)a Fumigati Fumigati A. fumigatus 5 (2) A. lentulus 1 Neosartorya fischeri 1 Circumdati Flavi A. flavus 5 (2) A. minisclerotigenes 3 A. nomius 1 A. oryzae 1 (1) A. parasiticus 1 (1) A. tamarii 4 (1) Nigri A. niger 4 (1) A. tubingensis 6 (1) Circumdati A. ochraceus 1 A. persii 1 A. pseudoelegans 1 Terrei Terrei A. terreus 5 (1) Nidulantes Versicolores A. creber 1 A. sydowii 1 A. tabacinus 1 A. versicolor 1 (1) Nidulantes A. nidulans 2 A. quadrilineatus 5 A. rugulosus 1 Usti A. calidoustus 1 Subgenus Section Species Total no. of isolates included (no. of reference strains)a Fumigati Fumigati A. fumigatus 5 (2) A. lentulus 1 Neosartorya fischeri 1 Circumdati Flavi A. flavus 5 (2) A. minisclerotigenes 3 A. nomius 1 A. oryzae 1 (1) A. parasiticus 1 (1) A. tamarii 4 (1) Nigri A. niger 4 (1) A. tubingensis 6 (1) Circumdati A. ochraceus 1 A. persii 1 A. pseudoelegans 1 Terrei Terrei A. terreus 5 (1) Nidulantes Versicolores A. creber 1 A. sydowii 1 A. tabacinus 1 A. versicolor 1 (1) Nidulantes A. nidulans 2 A. quadrilineatus 5 A. rugulosus 1 Usti A. calidoustus 1 aReference strains: Aspergillus flavus ATCC 76668 (CECT 2686), Aspergillus flavus ATCC 204304, Aspergillus fumigatus ATCC 204305, Aspergillus fumigatus var. fumigatus ATCC 16907 (CECT 20228), Aspergillus niger ATCC 9029 (CECT 2088), Aspergillus oryzae var. oryzae ATCC 1011 (CECT 2094), Aspergillus parasiticus ATCC 15517 (CECT 2680), Aspergillus tamarii CECT 20399, Aspergillus terreus var. terreus ATCC 10690 (CECT 2808), Aspergillus tubingensis ATCC 11394 (CECT 2089) and Aspergillus versicolor ATCC 42039 (CECT 1544). View Large Strains collection The collection of Aspergillus strains was based on suspensions of conidia obtained from sporulated cultures grown on Sabouraud-chloramphenicol agar plates (SCA; Oxoid, Basingstoke, UK) after 5–7 d incubation, as described by Cassagne et al.13 The conidia were separated from the mycelium by passing a sterile cotton swab over its surface, which were then transferred to a screw-cap vial with 3 ml of sterile distilled water. The vials were stored at room temperature. Strains identification All 379 strains were characterized morphologically based on the macroscopic and microscopic features according to de Hoog et al.18 The identification by molecular methods of the 179 selected strains was performed by sequencing of beta-tubulin and calmodulin genes. For that, the fungal DNA was extracted with the QIAamp® DNA Mini Kit (Qiagen, Courtaboeuf, France) from the strains grown on SCA after 48 h of incubation at 30°C. Polymerase chain reaction (PCR) amplification and sequencing of the partial portions of the beta-tubulin and calmodulin genes were performed as previously described,19–21 with the primer pair of β-tub1 (5΄-AATTGGTGCCGCTTTCTGG-3΄) and β-tub4 (5΄-AGCGTCCATGGTACCGG-3΄) and the primer pair Cmd5 (5΄-GTCTCCGAGTACAAGGAGGC-3΄) and Cmd6 (5΄-TCGCCGATRGAGGTCATRACGTG-3΄), respectively. Sequencing of the partial calmodulin gene was only used for identification of cryptic species. A BLAST search analysis for species identification was carried out at the NCBI genomic database (http://blast.ncbi.nom.nih.gov/). Generation of the in-house reference mold database in MALDI-TOF MS Sample preparation The strains used to build the new database were cultured in liquid medium. To do this, we started with the suspension of conidia of each of the 53 selected isolates. A 300-μl aliquot of each suspended strain was inoculated to 3 ml of Sabouraud broth (BD, Franklin Lakes, NJ, USA) and was incubated at 30°C for 24–48 h. One ml of the liquid fungal culture was placed into a 1.5 ml Eppendorf tube and centrifuged at 15,000 g for 2 min. The supernatant was discarded and 500 μl of distilled water were added to the pellet. The mixture was aspirated several times with a pipette, trying to dissolve the pellet. Then, it was centrifuged at 15,000 g for 2 min. This washing process was carried out 3 times. Finally, the pellet was resuspended in a mixture of 300 μl distilled water and 900 μl ethanol. Each tube was then centrifuged at 15,000 g for 2 min, and the pellet was dried at 50°C and resuspended in 25 to 50 μl of 70% formic acid, depending on the fungal mass obtained. Not all Aspergillus strains grew at the same rate during incubation, so that the obtained fungus mass was variable and, therefore, also the added volume of formic acid. We always used the lowest volume of formic acid capable of covering the pellet. The objective was to dissolve the pellet and to dilute the released proteins as little as possible. After incubating for 5–10 min at room temperature, an equal volume of acetonitrile was added. Samples were incubated again at room temperature for 10 min and subsequently centrifuged at 15,000 g for 1 min. For the MALDI-TOF analysis the supernatants of the samples were transferred to a polished steel plate MSP 96 (Bruker Daltonik GmbH) that only allowed a maximum volume of 1 μl per sample spot. In our protocol, we used 2 μl per sample spot to increase the concentration of proteins and to improve the quality of the spectra. For this, 1 μl of the supernatant was spotted onto a sample splot and allowed to dry at room temperature, before repeating this step once more. Finally, after the samples had dried, 1 μl of the matrix solution (a saturated solution of α-cyano-4 hydroxy-cinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) was pipetted and dried at room temperature. MALDI-TOF MS Spectral analysis The acquisition and analysis of mass spectra was performed by a Microflex LT mass spectrometer (Bruker Daltonik GmbH) using the MALDI Biotyper software package (version 3.0). We worked with default parameter settings: positive linear mode; laser frequency, 60 Hz; ion source 1 voltage, 20 kV; ion source 2 voltage, 18.5 kV; lens voltage, 6.0 kV; mass range, 2000 to 20,000 Da. For each spectrum, 240 laser shots in 40-shot steps from different positions of the sample spot were accumulated and analyzed (automatic mode, default settings of MBT_AutoX method). The Bruker bacterial test standard (Bruker Daltonik GmbH) was used for calibration according to the instructions of the manufacturer. A reference spectrum was created from each of the 53 isolates, including 30 strains of cryptic species, which added to the in-house reference database. A total of 24 spectra and 1 mix spectrum (strain and BTS) were measured for each strain. The generated spectra were analyzed and calibrated using the FlexAnalysis software, uncertain spectra were excluded and a average of 18 to 24 spectra were used to generate a consensus reference spectrum using the MALDI Biotyper software. Finally, the main spectrum of each strain was included to the in-house reference database using the Biotyper software. Validation of the in-house reference database One hundred ninety sequenced strains were identified by the MALDI-TOF MS technology to validate the generated in-house reference database, including the 53 strains introduced into the database. An aliquot of distilled water suspension of conidia from each strain was inoculated on two SCA, making five marks per plate (Fig. 1). Thereby, we got maximum use of the surface of each plate, obtaining the largest amount of young mycelium. After 48 h of incubation at 30°C, the youngest mycelium of each colony (outermost area) was collected with a sterile cotton swab. This was passed through the periphery of the colonies to drag the mycelium, which was later transferred and emulsified in 1.0 ml distilled water in an Eppendorf tube, which was used for the MALDI-TOF MS identification, following the above described protocol. Figure 1. View largeDownload slide Aspect of an A. flavus strain after 48 hours of incubation on SCA (five marks plate). This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Aspect of an A. flavus strain after 48 hours of incubation on SCA (five marks plate). This Figure is reproduced in color in the online version of Medical Mycology. Prospective evaluation of the HUVR database Two hundred strains of the initial 379 clinical isolates, different from the 179 used for the creation and validation of the database, were used for the prospective evaluation. These strains grown on SCA were processed in the same manner as described above. MALDI-TOF MS data interpretation Each mass spectrum was analyzed against the reference mass spectra in the in-house reference database using the Biotyper software, which calculates an arbitrary unit score value (from 0 to 3) that reflects the similarity between sample and reference spectrum. For each isolate, the highest log (score) value of a match against an spectrum in the database was used for identification. Log (score) values ≥2.0 are rated as identification at the species level, whereas log (score) values between ≥1.7 and <2.0 are rated as identification at the genus level. Results with log (score) values of <1.7 are rated as not suitable for identification by the MALDI BioTyper database 3.0. All isolates were spotted and identified in triplicate by MALDI-TOF MS using the in-house reference database created, which included the reference spectra of 53 Aspergillus strains. The main spectrum comparison was based on the best log (score) values. Results Strains identification The 179 selected strains were morphologically identified as A. fumigatus (n = 41), A. flavus (n = 43), A. niger (n = 31), A. terreus (n = 26), A. nidulans (n = 3), A. versicolor (n = 1), and Aspergillus spp. (n = 34). Therefore, the conventional identification assigned 81% of the isolates to species level and the remaining 19% only to genus level. Gene sequencing identified 20 species (Table 1) belonging to eight sections, which included six noncryptic species and 14 cryptic species. Partial sequencing of the β-tubulin gene allowed the identification of all the species, except three (A. tabacinus, A. rugulosus and A. quadrilineatus), which were identified by partial calmodulin gene sequencing (Table 1). A 65.4% correlation between morphologic and molecular identification was found, mainly with A. fumigatus, A. flavus, and A. terreus. The remaining 200 strains, used in the prospective evaluation, were identified morphologically as A. fumigatus (n = 120), A. flavus (n = 24), A. niger (n = 28), A. terreus (n = 27), and A. nidulans (n = 1). Construction and validation of the in-house mould database The generated reference spectra with the MALDI Biotyper software, allowed us to construct an in-house reference database that included 53 entries (Table 2): 42 from clinical strains and 11 from reference strains, including 23 different species from eight sections: seven noncryptic species (with 23 entries), and 16 cryptic species (with 30 entries). To evaluate the HUVR database, 190 strains (179 clinical isolates previously identified by sequencing and 11 reference strains) (Table 3) were analyzed. In these 190 strains are included the 53 used for the construction of the database and with this bias a 100% correlation was obtained with both identification methods, sequencing and MALDI-TOF MS, and no discordant identification was obtained. Table 3. Identification of Aspergillus isolates using the HUVR database. Subgenus Section Species (identification by sequencing + MALDI-TOF MS)a No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 49 2.212 2.563 1.730 0.207 0 3 46 A. lentulus 1 2.067 2.120 1.980 0.076 0 0 1 N. fischeri 1 2.242 2.331 2.180 0.079 0 0 1 Circumdati Flavi A. flavus 48 2.224 2.674 1.717 0.181 0 1 47 A. minisclerotigenes 3 1.904 2.110 1.711 0.155 0 1 2 A. nomius 1 1.659 1.715 1.550 0.094 0 1 0 A. oryzae 1 2.020 2.040 2.010 0.017 0 0 1 A. parasiticus 1 1.703 1.750 1.670 0.042 0 1 0 A. tamarii 4 2.005 2.325 1.848 0.150 0 1 3 Nigri A. niger 16 2.407 2.695 1.445 0.202 0 0 16 A. tubingensis 17 2.219 2.632 1.815 0.227 0 3 14 Circumdati A. ochraceus 1 1.884 1.912 1.860 0.026 0 1 0 A. persii 1 1.933 1.950 1.910 0.021 0 1 0 A. pseudoelegans 1 1.973 2.010 1.950 0.032 0 0 1 Terrei Terrei A. terreus 32 2.056 2.364 1.804 0.171 0 7 25 Nidulantes Versicolores A. creber 1 2.618 2.621 2.615 0.003 0 0 1 A. sydowii 1 2.119 2.150 2.094 0.028 0 0 1 A. versicolor 1 2.023 2.110 1.970 0.076 0 0 1 A. tabacinus 1 2.010 2.050 1.970 0.057 0 0 1 Nidulantes A. nidulans 2 2.148 2.316 1.818 0.225 0 1 1 A. quadrilineatus 5 1.990 2.180 1.830 0.126 0 3 2 A. rugulosus 1 2.043 2.060 2.030 0.015 0 0 1 Usti A. calidoustus 1 1.940 1.990 1.890 0.071 0 1 0 Subgenus Section Specie (identification by MALDI-TOF MS)b No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 121 2.303 2.706 1.714 0.207 0 2 119 Circumdati Flavi A. flavus 24 2.275 2.573 1.803 0.201 0 0 24 Nigri A. niger 7 2.260 2.512 1.838 0.206 0 0 7 A. tubingensis 21 2.213 2.683 1.687 0.222 0 2 19 Terrei Terrei A. terreus 26 2.150 2.560 1.742 0.230 0 5 21 Nidulantes Nidulantes A. nidulans 1 2.161 2.273 2.056 0.108 0 0 1 Subgenus Section Species (identification by sequencing + MALDI-TOF MS)a No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 49 2.212 2.563 1.730 0.207 0 3 46 A. lentulus 1 2.067 2.120 1.980 0.076 0 0 1 N. fischeri 1 2.242 2.331 2.180 0.079 0 0 1 Circumdati Flavi A. flavus 48 2.224 2.674 1.717 0.181 0 1 47 A. minisclerotigenes 3 1.904 2.110 1.711 0.155 0 1 2 A. nomius 1 1.659 1.715 1.550 0.094 0 1 0 A. oryzae 1 2.020 2.040 2.010 0.017 0 0 1 A. parasiticus 1 1.703 1.750 1.670 0.042 0 1 0 A. tamarii 4 2.005 2.325 1.848 0.150 0 1 3 Nigri A. niger 16 2.407 2.695 1.445 0.202 0 0 16 A. tubingensis 17 2.219 2.632 1.815 0.227 0 3 14 Circumdati A. ochraceus 1 1.884 1.912 1.860 0.026 0 1 0 A. persii 1 1.933 1.950 1.910 0.021 0 1 0 A. pseudoelegans 1 1.973 2.010 1.950 0.032 0 0 1 Terrei Terrei A. terreus 32 2.056 2.364 1.804 0.171 0 7 25 Nidulantes Versicolores A. creber 1 2.618 2.621 2.615 0.003 0 0 1 A. sydowii 1 2.119 2.150 2.094 0.028 0 0 1 A. versicolor 1 2.023 2.110 1.970 0.076 0 0 1 A. tabacinus 1 2.010 2.050 1.970 0.057 0 0 1 Nidulantes A. nidulans 2 2.148 2.316 1.818 0.225 0 1 1 A. quadrilineatus 5 1.990 2.180 1.830 0.126 0 3 2 A. rugulosus 1 2.043 2.060 2.030 0.015 0 0 1 Usti A. calidoustus 1 1.940 1.990 1.890 0.071 0 1 0 Subgenus Section Specie (identification by MALDI-TOF MS)b No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 121 2.303 2.706 1.714 0.207 0 2 119 Circumdati Flavi A. flavus 24 2.275 2.573 1.803 0.201 0 0 24 Nigri A. niger 7 2.260 2.512 1.838 0.206 0 0 7 A. tubingensis 21 2.213 2.683 1.687 0.222 0 2 19 Terrei Terrei A. terreus 26 2.150 2.560 1.742 0.230 0 5 21 Nidulantes Nidulantes A. nidulans 1 2.161 2.273 2.056 0.108 0 0 1 aThis group include 190 strains (179 clinical strains + 11 reference strains) identified by sequencing and MALDI-TOF MS. In all cases, there was correlation between the two methods. bThis group include 200 strains identified by MALDI-TOF MS. View Large Table 3. Identification of Aspergillus isolates using the HUVR database. Subgenus Section Species (identification by sequencing + MALDI-TOF MS)a No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 49 2.212 2.563 1.730 0.207 0 3 46 A. lentulus 1 2.067 2.120 1.980 0.076 0 0 1 N. fischeri 1 2.242 2.331 2.180 0.079 0 0 1 Circumdati Flavi A. flavus 48 2.224 2.674 1.717 0.181 0 1 47 A. minisclerotigenes 3 1.904 2.110 1.711 0.155 0 1 2 A. nomius 1 1.659 1.715 1.550 0.094 0 1 0 A. oryzae 1 2.020 2.040 2.010 0.017 0 0 1 A. parasiticus 1 1.703 1.750 1.670 0.042 0 1 0 A. tamarii 4 2.005 2.325 1.848 0.150 0 1 3 Nigri A. niger 16 2.407 2.695 1.445 0.202 0 0 16 A. tubingensis 17 2.219 2.632 1.815 0.227 0 3 14 Circumdati A. ochraceus 1 1.884 1.912 1.860 0.026 0 1 0 A. persii 1 1.933 1.950 1.910 0.021 0 1 0 A. pseudoelegans 1 1.973 2.010 1.950 0.032 0 0 1 Terrei Terrei A. terreus 32 2.056 2.364 1.804 0.171 0 7 25 Nidulantes Versicolores A. creber 1 2.618 2.621 2.615 0.003 0 0 1 A. sydowii 1 2.119 2.150 2.094 0.028 0 0 1 A. versicolor 1 2.023 2.110 1.970 0.076 0 0 1 A. tabacinus 1 2.010 2.050 1.970 0.057 0 0 1 Nidulantes A. nidulans 2 2.148 2.316 1.818 0.225 0 1 1 A. quadrilineatus 5 1.990 2.180 1.830 0.126 0 3 2 A. rugulosus 1 2.043 2.060 2.030 0.015 0 0 1 Usti A. calidoustus 1 1.940 1.990 1.890 0.071 0 1 0 Subgenus Section Specie (identification by MALDI-TOF MS)b No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 121 2.303 2.706 1.714 0.207 0 2 119 Circumdati Flavi A. flavus 24 2.275 2.573 1.803 0.201 0 0 24 Nigri A. niger 7 2.260 2.512 1.838 0.206 0 0 7 A. tubingensis 21 2.213 2.683 1.687 0.222 0 2 19 Terrei Terrei A. terreus 26 2.150 2.560 1.742 0.230 0 5 21 Nidulantes Nidulantes A. nidulans 1 2.161 2.273 2.056 0.108 0 0 1 Subgenus Section Species (identification by sequencing + MALDI-TOF MS)a No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 49 2.212 2.563 1.730 0.207 0 3 46 A. lentulus 1 2.067 2.120 1.980 0.076 0 0 1 N. fischeri 1 2.242 2.331 2.180 0.079 0 0 1 Circumdati Flavi A. flavus 48 2.224 2.674 1.717 0.181 0 1 47 A. minisclerotigenes 3 1.904 2.110 1.711 0.155 0 1 2 A. nomius 1 1.659 1.715 1.550 0.094 0 1 0 A. oryzae 1 2.020 2.040 2.010 0.017 0 0 1 A. parasiticus 1 1.703 1.750 1.670 0.042 0 1 0 A. tamarii 4 2.005 2.325 1.848 0.150 0 1 3 Nigri A. niger 16 2.407 2.695 1.445 0.202 0 0 16 A. tubingensis 17 2.219 2.632 1.815 0.227 0 3 14 Circumdati A. ochraceus 1 1.884 1.912 1.860 0.026 0 1 0 A. persii 1 1.933 1.950 1.910 0.021 0 1 0 A. pseudoelegans 1 1.973 2.010 1.950 0.032 0 0 1 Terrei Terrei A. terreus 32 2.056 2.364 1.804 0.171 0 7 25 Nidulantes Versicolores A. creber 1 2.618 2.621 2.615 0.003 0 0 1 A. sydowii 1 2.119 2.150 2.094 0.028 0 0 1 A. versicolor 1 2.023 2.110 1.970 0.076 0 0 1 A. tabacinus 1 2.010 2.050 1.970 0.057 0 0 1 Nidulantes A. nidulans 2 2.148 2.316 1.818 0.225 0 1 1 A. quadrilineatus 5 1.990 2.180 1.830 0.126 0 3 2 A. rugulosus 1 2.043 2.060 2.030 0.015 0 0 1 Usti A. calidoustus 1 1.940 1.990 1.890 0.071 0 1 0 Subgenus Section Specie (identification by MALDI-TOF MS)b No. of isolates Score No. of isolates with: Standard Score between Mean Maximum Minimum deviation Score <1.7 1.7 and <2.0 Score ≥2.0 Fumigati Fumigati A. fumigatus 121 2.303 2.706 1.714 0.207 0 2 119 Circumdati Flavi A. flavus 24 2.275 2.573 1.803 0.201 0 0 24 Nigri A. niger 7 2.260 2.512 1.838 0.206 0 0 7 A. tubingensis 21 2.213 2.683 1.687 0.222 0 2 19 Terrei Terrei A. terreus 26 2.150 2.560 1.742 0.230 0 5 21 Nidulantes Nidulantes A. nidulans 1 2.161 2.273 2.056 0.108 0 0 1 aThis group include 190 strains (179 clinical strains + 11 reference strains) identified by sequencing and MALDI-TOF MS. In all cases, there was correlation between the two methods. bThis group include 200 strains identified by MALDI-TOF MS. View Large The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.8%). No identification was made with a score lower than 1.7, and 25 isolates (13.2%) were identified to the genus level (score between 1.7 and <2.0). Among the 23 different species included in our database, 18 obtained scores ≥2.0, while in the remaining five species (A. calidoustus, A. nomius, A.ochraceus, A. parasiticus, and A. persii) the scores were between 1.7 and 2.0. Finally, a species identification (score of ≥2.0) was obtained in 136 (91.3%) of the noncryptic species and in 29 (70.7%) of the cryptic species. In both groups, an identification at the genus level (score between 1.7 and <2.0) was found in 13 (8.7%) strains and in 12 (29.3%) strains, respectively. Even for those identifications with scores between 1.7 and <2.0, MALDI-TOF MS results always agreed with partial sequencing data. Prospective evaluation of the HUVR database A prospective evaluation of the routine MALDI-TOF MS analysis was performed with 200 Aspergillus clinical isolates grown on solid medium and with the new database. A species identification (score of ≥2.0) was obtained in 191 strains (95.5%) and only nine strains (4.5%) were not identified at species level, using the manufacturer recommended cut-off values (Table 3). We identified species belonging to five sections (Flavi, Fumigati, Nidulantes, Nigri, and Terrei). The strains identified at species level, with scores ranging from 2.273 to 2.706, were A. fumigatus (n = 119), A. flavus (n = 24), A. niger (n = 7), A. tubingensis (n = 19), A. terreus (n = 21), and A. nidulans (n = 1); while scores between 1.7 and <2.0 were seen with A. fumigatus (n = 2), A. tubingensis (n = 2), and A. terreus (n = 5). Among the 200 clinical isolates A. tubingensis was the only cryptic species identified. Additionally, MALDI-TOF MS identified 28 aspergilli of the section Nigri to species level: 19 (67.9%) A. tubingensis and seven (25%) A. niger, while the remaining two were identified to the genus level (score between 1.7 and <2.0). Discussion In this study, we constructed and validated an in-house reference database in the MALDI-TOF MS, which significantly expanded the original commercially-available database and allowed the accurate identification of a large number of Aspergillus species isolated in the clinical setting, including both noncryptic and cryptic species. The original library with 89 entries was supplemented with 53 additional entries of 23 species, which allowed us to include 12 new cryptic species and to increase the number of entries of 11 species already included. Separately, the HUVR database provided 16 cryptic species while the Filamentous Fungi Library 1.0 only seven cryptic species, representing 69.6% and 38.9% of the total species included in each one of the libraries. Finally, the supplemented library had 142 entries that allowed the identification of 30 different species of Aspergillus from 11 sections: 11 noncryptic species and 19 cryptic species. The prevalence of the cryptic species present in our strain collection (15.7%), with A. tubingensis as the most frequent species, was similar to the ones published in previous studies, ranging from 10 to 15%.22,23 Several authors21,24–26 pointed out the need to expand the Filamentous Fungi Library 1.0 library with new species and a greater number of entries to improve the sensitivity of this technology, and for this reason we have validated our database by identifying 390 strains but without comparing the identification results with those obtained only using the original database. The prospective study showed that the Aspergillus identification grown on solid medium and the use of the HUVR database in the MALDI-TOF MS Biotyper were highly reliable. An identification in 95.5% of the isolates at the species level was obtained, with the remaining 4.5% of the isolates identified at the genus level. These results are similar to those previously described.27–29 Schulthess et al.15 significantly enhanced identification from 54.2% to 71.1% at species level using a score ≥1.7 instead of ≥2.0, without increasing misidentifications. If we applied this score in our results, 100% of identifications at the species level would have been obtained. To create the new reference spectra, we used liquid cultivation to obtain a more homogeneous mycelium,15 easier to be emulsified, and rendering better quality spectra. The validation of the database and the prospective study were done using colonies grown on solid medium, where culture of the clinical samples is performed routinely, so that no extra time is needed to get results by an additional subculture in liquid medium. Although several authors13,15,30 recommended the application of a lower cut-off value (≥1.7) to increase identification rates, our procedure with strains grown on solid medium, identified most of them with scores of ≥2.0. In the case of clinical isolates with scores between 1.7 and 2.0, the presumptive identification can be confirmed performing the procedure from a subculture in liquid medium. Aspergillus calidoustus, A. nomius, A. ochraceus, A. parasiticus, and A. persii are the species in which the scores between 1.7 and <2.0 were found, and it can be due to the particular characteristics of their mycelium and to the presence of a single entry of each of them in the library. When the identifications were made from liquid medium, all these species were correctly identified with scores ranging from 2.43 to 2.63 (data not shown). Therefore, the new library shows its feasibility, regardless of the medium type (i.e., solid or liquid). Nonetheless, better identification scores were obtained if the molds were grown in liquid medium, because those were the conditions for which the library was generated. We demonstrated the feasibility and usefulness of a new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species from different sections, grown either on solid or liquid media. This procedure could be incorporated as a routine mold identification procedure in the mycology laboratory to improve the accuracy of the identification of clinically relevant species of Aspergillus and significantly shortening the response time. Howewer, future studies carried out with our database are necessary to confirm our results, avoiding the bias committed by including strains used in the construction of the database for its validation. The creation of databases in the MALDI-TOF MS is a slow and expensive process, but necessary if we want to benefit from a better identification capacity. As we have shown, this research has improved the identification of Aspergillus species in our laboratory. For this reason, we have made it available to the manufacturer for the benefit of the medical community, in case it is considered appropriate its inclusion in future software updates. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. This work was funded by the Autonomous Government of Andalusia. References 1. Fortún J , Meije Y , Fresco G , Moreno S . Aspergillosis: clinical forms and treatment . 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Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry . Clin Microbiol Infect . 2012 ; 18 : 475 – 484 . Google Scholar Crossref Search ADS PubMed 30. Normand A-C , Cassagne C , Ranque S et al. Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi . BMC Microbiol . 2013 ; 13 : 76 . Google Scholar Crossref Search ADS PubMed © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Medical MycologyOxford University Press

Published: Oct 1, 2018

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