The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing | Enfermedades Infecciosas y Microbiología Clínica

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Para AMC se consideraron tanto los valores de resistencia como los de resistencia intermedia al antibiótico según CLSI<a class="elsevierStyleCrossRef" href="#bib0100">20</a>.AMC: amoxicilina/ácido clavulánico; AMK: amikacina; AMP: ampicilina; ATM: aztreonam; CAZ: ceftazidima; CF: cefalotina; CHL: cloranfenicol; CIP: ciprofloxacino; CTX: cefotaxima; FEP: cefepima; FOX: cefoxitina; GEN: gentamicina; KAN: kanamicina; NAL: ácido nalidíxico; STR: estreptomicina; SUL: sulfamidas; SXT: trimetoprim/sulfametoxazol; TET: tetraciclina, TOB: tobramicina; TRM: trimetoprim." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "María de Toro, Cristina Seral, Beatriz Rojo-Bezares, Carmen Torres, F. Javier Castillo, Yolanda Sáenz" "autores" => array:6 [ 0 => array:2 [ "nombre" => "María" "apellidos" => "de Toro" ] 1 => array:2 [ "nombre" => "Cristina" "apellidos" => "Seral" ] 2 => array:2 [ "nombre" => "Beatriz" "apellidos" => "Rojo-Bezares" ] 3 => array:2 [ "nombre" => "Carmen" "apellidos" => "Torres" ] 4 => array:2 [ "nombre" => "F. Javier" "apellidos" => "Castillo" ] 5 => array:2 [ "nombre" => "Yolanda" "apellidos" => "Sáenz" ] ] ] ] ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0213005X13000803?idApp=UINPBA00004N" "url" => "/0213005X/0000003200000001/v1_201401150043/S0213005X13000803/v1_201401150043/es/main.assets" ] "en" => array:19 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "11" "paginaFinal" => "17" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Leonel Pereira, Nicolina Dias, Cledir Santos, Nelson Lima" "autores" => array:4 [ 0 => array:4 [ "nombre" => "Leonel" "apellidos" => "Pereira" "email" => array:1 [ 0 => "leoneljpp@deb.uminho.pt" ] "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "¿" "identificador" => "cor0005" ] ] ] 1 => array:4 [ "nombre" => "Nicolina" "apellidos" => "Dias" "email" => array:1 [ 0 => "nidias@deb.uminho.pt" ] "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "¿" "identificador" => "cor0005" ] ] ] 2 => array:2 [ "nombre" => "Cledir" "apellidos" => "Santos" ] 3 => array:2 [ "nombre" => "Nelson" "apellidos" => "Lima" ] ] "afiliaciones" => array:1 [ 0 => array:1 [ "entidad" => "IBB/Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, Portugal" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "El uso de MALDI-TOF ICMS como una herramienta alternativa para la identificación y tipificación de Trichophyton rubrum" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2371 "Ancho" => 2167 "Tamanyo" => 287332 ] ] "descripcion" => array:1 [ "en" => "Dendrogram resulting from single-linkage cluster analysis mass spectra of T. rubrum strains and out-group strains T. interdigitale (MUM 10.136 and MUM 12.07) and reference strain T. mentagrophytes (ATCC MYA-4439) obtained by MALDI-TOF ICMS analysis. Distances were measured as percentage of mass similarity." ] ] ] "textoCompleto" => "IntroductionDermatophytes are the etiological agents of fungal infections of the skin, hair, and nails, globally known as tinea. Despite dermatophytes belong to a homogenous group of fungi closely related to each other phylogenetically, the identification of species, as well as strain level, remains an important issue for epidemiological and taxonomic purposes.<a class="elsevierStyleCrossRefs" href="#bib0005">1–4</a> Phenotypic characters and ecological features have been for a long time the basis for phylogenetic studies and taxonomic classification. Additionally, biochemical and physiological tests can be used as complementary tools.<a class="elsevierStyleCrossRef" href="#bib0025">5</a>Although morphology-based identification has been the main technique for the identification of dermatophytes, some limitations of the procedure could be pointed out. Firstly, it is based on the recognition of specific morphological features in macroscopic and microscopic observations; however, morphological similarity, intra-specific variability and polymorphism of dermatophytes are difficult for the identification procedure. Furthermore, confusion is caused by the atypical growth forms that occur frequently, and the requirement of a long incubation period (2–4 weeks) could be a significant drawback for rapid diagnosis and treatment. In addition, these approaches are time-and labour-consuming, expensive and requiring highly skilled-specific staff,<a class="elsevierStyleCrossRefs" href="#bib0015">3,6</a> which normally delays the diagnosis.PCR technology is a powerful method that enables the development of new techniques in molecular biology for accurate organism identification.<a class="elsevierStyleCrossRefs" href="#bib0035">7–9</a> Molecular biology-based techniques are useful to reveal intra-specific DNA polymorphism that enables defining clusters within the same species.<a class="elsevierStyleCrossRefs" href="#bib0010">2,9,10</a> Furthermore, they have the advantage, over traditional methods, of being fast, simple, sensitive, and reproducible.<a class="elsevierStyleCrossRef" href="#bib0015">3</a> Nevertheless, these methods remain time/labour consuming and relatively expensive.<a class="elsevierStyleCrossRef" href="#bib0055">11</a>Along the past decade matrix-assisted laser desorption/ionization time of flight intact cell mass spectrometry (MALDI-TOF ICMS) technique has been established as a valuable system for microorganisms identification to be introduced in laboratory routine.<a class="elsevierStyleCrossRefs" href="#bib0060">12,13</a> Potentialities of MALDI-TOF ICMS have been revised for filamentous fungi.<a class="elsevierStyleCrossRef" href="#bib0070">14</a> Some procedures for the identification of dermatophyte species, including Trichophyton rubrum, have been recently established. MALDI-TOF ICMS system was evaluated in comparison with sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions,<a class="elsevierStyleCrossRef" href="#bib0075">15</a> D2 28S rRNA,<a class="elsevierStyleCrossRef" href="#bib0080">16</a> and ITS region of the ribosomal DNA with V9D and LSU 266 primers.<a class="elsevierStyleCrossRef" href="#bib0085">17</a> High confidence level (99.9%) for identification was found. Authors found similar or even better specificity of MALDI-TOF ICMS than an ITS/PCR-based approach for species identification purposes. MALDI-TOF ICMS proved to be a rapid, low cost, accurate, and reliable alternative to morphological and PCR analysis for the identification of dermatophytes.<a class="elsevierStyleCrossRefs" href="#bib0075">15–19</a> Although MALDI-TOF ICMS technique for species identification is a widely investigated theme, only a few recent studies focalize on the potential capacity for intra-specific discrimination in fungi.<a class="elsevierStyleCrossRefs" href="#bib0065">13,20–23</a>The aim of this work was to examine the potential of MALDI-TOF ICMS for the identification of clinical isolates of T. rubrum at the species and strain level. Molecular typing using three different primers was compared with spectrometric analysis.Materials and methodsStrains and culture conditionsA set of 24 clinical isolates of T. rubrum from the fungal collection of Micoteca da Universidade do Minho (MUM), Portugal, were used for analysis. Two T. rubrum isolates were collected in Spain from human patients with tinea pedis (MUM 09.26) and onychomycosis (MUM 09.20). The remaining clinical isolates MUM 08.05, 08.09, 08.11, 08.12, 08.13, 08.15, 09.08, 09.09, 09.10, 09.12, 09.18, 09.29, 10.128, 10.133, 12.01, 12.02, 12.03, 12.04, 12.06, 12.08, 12. 09 and 12.10 were collected in Portugal from human patients with onychomycosis. Clinical isolates of Trichophyton interdigitale (MUM 10.136 and MUM 12.07) were also used as out-group. Two reference strains were obtained from the American Type Culture Collection (ATCC) and included in this study. Strain T. rubrum ATCC MYA-4438 was used as positive control and strain Trichophyton mentagrophytes ATCC MYA-4439 was used as out-group in MALDI-TOF analysis.Potato dextrose agar (PDA, OXOID, UK) was used to maintain the cultures and to grow the fungi for molecular and mass spectral analyses.Morphological identificationAll isolates were tested by conventional method, according to the standard procedures of morphological identification (macro-and micromorphology). Urease test was used as complementary biochemical test.Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry identificationSamples preparation: In a first attempt several experimental conditions were tested on four strains (ATCC-MYA 4438, MUM 09.08; MUM 08.11; MUM 10.133) and spectra were compared in order to select the best method. Two matrices were used: 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA). Different extraction protocols (including formic acid and sonication), as well as previous suspension of cells in water or solvent mixtures did not result in an improvement compared to the direct application of cells to the MALDI target. As no clear improvement in spectrum quality was observed in the preliminary study we followed the one described by Santos et al.<a class="elsevierStyleCrossRef" href="#bib0070">14</a> for the clinical isolates. A growing time of 5 days at 30°C was set for all cultures before MALDI-TOF analysis to avoid age-dependent variability. Each sample was spotted in quadruplicate to test reproducibility.MALDI-TOF analysis: The analysis was performed on an Axima LNR system (Kratos Analytical, Shimadzu, UK) equipped with a nitrogen laser (337nm), where the laser intensity was set just above the threshold for ion production. The mass range from 2000 to 20000Da was recorded using the linear mode with a delay of 104ns and an acceleration voltage of +20kV. Final spectra were generated by summing 20 laser shots accumulated per profile and 50 profiles produced per sample, leading to 1000 laser shots per summed spectrum.Spectral Identification and software analysis: Resulting peak lists were exported to the SARAMIS™ software package (Spectral Archiving and Microbial Identification System, AnagnosTec, Germany, <a class="elsevierStyleInterRef" href="http://www.anagnostec.eu/">www.anagnostec.eu</a>) where the final microbial identification was achieved. Peak lists of individual samples were compared on SARAMIS™ database generating a ranked list of matching spectra. This software uses a point system based on peak list with mass signals weighted according to their specificity. A dendrogram and matrix of spectral similarity between isolates were created using SARAMIS™ package. A minimum of 100 peaks, and at least 90% of mass similarity with species SuperSpectra was defined as the acceptance criteria to validate the result. The similarity between individual spectra was expressed as the relative or absolute number of matching mass signals after subjecting the data to a single link agglomerative clustering algorithm.Molecular identificationDNA isolation: Spores and mycelia from 5 days old culture grown on PDA medium were transferred into a sterile 1.5ml tube and 1ml of extraction buffer (1% SDS, 25mmol/l EDTA, 250mmol/l NaCl and 200mmol/l Tris–HCl, pH 8) was added. A sterile pestle was used to ground the sample. The content was then incubated for 1h at 65°C. After incubation samples were centrifuged at 14000rpm for 5min and the supernatant was aliquoted in 800μl volumes into 1.5ml tubes. Tubes were then cooled on ice, and protein extraction was performed with 800μl of phenol following with equal volumes of phenol–chloroform (1:1) and, finally, with 800μl chloroform. Purified nucleic acids were precipitated with 0.6 volumes of ice-cold 2-propanol. DNA was washed twice with 150μl of ice-cold 70% ethanol and air dried, suspended in 50μl of ultra-pure water, and stored at −20°C.ITS sequencing: The analysis was carried out using primer pair ITS1 5′-TCCGTAGGTGAACCTTGCGG-3′ and ITS4 5′-TCCTCCGCTTATTGATATGC-3′.<a class="elsevierStyleCrossRef" href="#bib0120">24</a> The PCR reactions were performed in a total volume of 50μl, containing the 1× PCR buffer (Promega, USA), 2.5mmol/l MgCl2 (Promega, USA), 0.2mmol/l of deoxynucleoside triphosphates (dNTPs) (Promega, USA), 0.5mmol/l of each primer, 1U of GoTaq® Hot Start Polymerase (Promega, USA) and 50ng of DNA. PCR was run in a MyCycler™ Thermal Cycler (Bio-Rad Laboratories, USA) using the following conditions: 2min, 95°C; 35 cycles of 1min, 95°C, 1min, 55°C, 1min, 72°C; and final extension, 5min, 72°C. Amplification products were separated by electrophoresis in 1.0% agarose gel, stained with SafeView DNA Stain (NBS Biologicals, UK), at 80V for 30min. The PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Germany) and then sequenced. The sequences were compared using the GenBank database (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/BLAST/">http://www.ncbi.nlm.nih.gov/BLAST/</a>). GenBank access numbers for the isolates MUM 08.11 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663974">JQ663974</a>), MUM 10.128 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663986">JQ663986</a>), MUM 08.05 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663971">JQ663971</a>), MUM 09.26 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663984">JQ663984</a>), MUM 09.20 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663983">JQ663983</a>), MUM 08.12 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663975">JQ663975</a>), MUM 09.09 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663979">JQ663979</a>), MUM 08.13 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663976">JQ663976</a>), MUM 08.15 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663977">JQ663977</a>) and MUM 09.29 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663985">JQ663985</a>) with the reference strain T. rubrum ATCC MYA-4438 (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=FJ746657">FJ746657</a>).Typing: For typing analysis oligonucleotides M13 5′-GAGGGTGGCGGTTCT-3′,<a class="elsevierStyleCrossRef" href="#bib0125">25</a> (GACA)4<a class="elsevierStyleCrossRef" href="#bib0130">26</a> and (AC)10<a class="elsevierStyleCrossRef" href="#bib0135">27</a> were used. PCR reactions were performed in a total volume of 50μl, containing the 1× PCR buffer, 2.5mmol/l MgCl2 0.2mmol/l of dNTPs, 0.5μmol/l of each primer, 2U of GoTaq® Hot Start Polymerase and 50ng of DNA. PCRs were run in a MyCycler™ Thermal Cycler using the following conditions: 2min, 95°C; 35 cycles of 1min, 95°C, 1min, 50°C, 2min, 72°C; and a final extension cycle, 5min, 72°C. Amplification products were separated by electrophoresis in 1.5% agarose gel, stained with SafeView DNA Stain, at 60V for 120min.ResultsTwenty six dermatophyte clinical isolates (24 T. rubrum and 2 T. interdigitale) and two ATCC reference strains were analyzed in quadruplicate, and the spectra were chosen according to the greatest number of peaks (data count) and the highest percentage of identity. Differences in the confidence level of identification between the replicates never exceeded 5%. A percentage identity over 99% was found in all strains, except MUM 08.11 (80.2%), MUM 09.10 (89.8%), MUM 10.128 (93.1%), MUM 12.10 (96.4%), MUM 08.05 (96.2%), and MUM 09.18 (97.1%). The identification of clinical isolates at the species level by MALDI-TOF ICMS was in agreement with the ID performed by the conventional method in 95.8% (23/24), of the T. rubrum isolates. Isolate MUM 12.01 was identified by conventional methods as Trichophyton spp., and as T. rubrum by MALDI-TOF ICMS. An agreement of 100% was found between conventional and MALDI-TOF ICMS results for T. interdigitale (2/2). Comparison of spectral analysis with molecular “gold standard” sequence of ITS region was performed, in ten T. rubrum strains, to confirm MALDI-TOF ICMS identification. Results of both methods agreed in 90% (9/10). Isolate MUM 08.11 was identified as Trichophyton megnini by sequence analysis of the ITS of the ribosomal DNA. Urease test was positive for this strain.A spectra-based dendrogram obtained by conventional identification algorithm of SARAMIS software had shown an evident separation between out-group cluster T. mentagrophytes ATCC MYA-4439 and T. interdigitale (MUM 10.136 and MUM 12.07) with T. rubrum cluster (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). A low percentage of mass similarity (40%) was obtained between T. rubrum cluster and out-group cluster. Within the T. rubrum cluster, strains were distributed into highly related sub-groups, with similarity values above 85%.<elsevierMultimedia ident="fig0005"></elsevierMultimedia>In order to understand the intra-specific differences inside T. rubrum cluster, the mass spectra profiles (MSP) were analyzed and compared. To perform this analysis, ten profiles of T. rubrum strains (MUM 09.29, MUM 08.09, MUM 08.13, MUM 09.09, MUM 08.12, MUM 09.20, MUM 09.26, MUM 08.05, MUM 10.128 and MUM 08.11) were selected randomly from different localizations of the dendrogram. Strain T. interdigitale MUM 10.136 was selected from out-group cluster. The spectra obtained for each selected strain were graphically reported highlighting specific and intra-specific variability (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>). Very similar mass patterns were generated, although slight variations were observed. The most relevant peaks were between 2000 and 11000 m/z, especially in the region encompassing 2500 to 9000 m/z, where the highest peaks were observed. A single peak with m/z 7380 stood out, due to its range of intensity in profile spectra along the strains. This peak was absent in out-group strain MUM 10.136 and was nearly present in T. rubrum strain MUM 08.11. Moreover, the percentage of identification of this strain reached 90% in only one spectrum.<elsevierMultimedia ident="fig0010"></elsevierMultimedia>All the spectra datasets were correlated in a similarity matrix, obtained from SARAMIS™ software (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>). The matrix displayed a relation between all the strains based on the percentage of similarity by a range colour scale from 0% (yellow) to 100% (blue). The out-group T. interdigitale (MUM 10.136 and MUM 12.07) obtained a maximum percentage of similarity of 32.7% and 33.9%, respectively, among all the strains analyzed. In T. rubrum cluster, it was possible to observe intra-specific variability between isolates. Comparing with the other isolates, the strain ATCC MYA-4438 presented a global low range of mass similarity percentage of 42.2%. Other strains such as MUM 08.05, MUM 08.11, and MUM 12.01 had also shown low mass similarity percentage. The remaining strains revealed an average mass similarity percentage ranging from 40 to 60% and a global average of 52.52%, excluding out-group strains.<elsevierMultimedia ident="fig0015"></elsevierMultimedia>ITS sequence analysis: Analysis of sequence data in GenBank database (<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/BLAST/">http://www.ncbi.nlm.nih.gov/BLAST/</a>) confirmed T. rubrum identification for the ten strains that profile spectra was analyzed and compared. The DNA sequence of ITS region was aligned and compared with T. rubrum reference strain ATCC MYA-4438. We found 100% nucleotide identity between clinical strains and reference strain, except for MUM 08.11 (99%), which presented 7 nucleotide differences (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>).<elsevierMultimedia ident="fig0020"></elsevierMultimedia>DNA strain typing: PCR amplification of single primer M13, and repetitive DNA sequences (GACA)4 and (AC)10 were performed in order to clarify if the results obtained by MALDI-TOF ICMS spectral analysis had a correspondence to molecular typing. Two profiles of DNA fragments were obtained for the primers M13 and (GACA)4. A single partner was obtained using the primer (AC)10 (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>). The (GACA)4 profiles varied between 0.6 and 2.3kb; the band-pattern of PCR using M13 varied between 0.5 and 3.0kb, whereas the banding pattern of the (AC)10 was from 0.3 to 3.5kb. Profiles of DNA fragments Ab and Ba were found for isolate MUM 08.11 while patterns Aa and Bb were observed for the other nine strains. A unique profile of 6 DNA fragments (lane C) was obtained for single repeat primer (AC)10 amplicons for all isolates.<elsevierMultimedia ident="fig0025"></elsevierMultimedia>DiscussionFungi species can be easily identified using MALDI-TOF ICMS by detecting a narrow number of specific biomarker peaks. Erhard et al.<a class="elsevierStyleCrossRef" href="#bib0075">15</a> stated that a minimum of 17 and 25 peaks matching the superspectrum of each species was enough to discriminate T. interdigitale and T. rubrum, respectively, with a confidence level of 99.9%. Different sample preparation methods were used to obtain reproducible and informative fungal spectra. Several methods have been reported to improve the protein extraction, and consequently the spectra obtained from the samples.<a class="elsevierStyleCrossRefs" href="#bib0140">28–30</a> In the present study, some of these sample preparation methods were evaluated on a small scale (data not shown), but no clear improvement in spectrum quality was observed.Analysis of mass spectra-based dendrogram obtained by MALDI-TOF ICMS revealed an intra-specific variability within T. rubrum cluster, where clinical strains MUM 09.12, MUM 08.11, MUM 10.133 and reference strain ATCC MYA-4438 were more distant from the remaining strains. Nevertheless, high percentage of similarity (60%) was observed revealing a very high phylogenetic proximity. Molecular strain typing profiles confirmed the lack of genetic variability and DNA polymorphism of T. rubrum although the distribution of strains on clusters was different from MALDI-TOF ICMS, showing less discriminative power than spectral analysis. Among the ten selected strains for this study, MUM 08.11 had shown a different fingerprinting pattern using primers M13 and (GACA)4. Mass spectral data suggest the existence of intra-specific variability between different strains of T. rubrum. These results agreed with clonal mode of reproduction of T. rubrum stated by other authors.<a class="elsevierStyleCrossRefs" href="#bib0030">6,10,31,32</a> In fact, it is referred that recent adaptation to a highly specialized ecological niche, the skin of the human host, combined with exclusively asexual reproduction of T. rubrum could explain genetic uniformity within the species.Spectra profiles of ten T. rubrum strains were obtained showing a similar pattern of mass peaks, and at least one peak with m/z 7380 was consistently present in all T. rubrum being absent in T. interdigitale. Strain MUM 08.11 lack mass signal at m/z 7380, and was identified with a percentage of identity of 80.2%. The result suggested that the presence/absence of only one mass signal is critical for the accurate identification of the strain. Interestingly, this peak has been previously identified as a marker of T. rubrum species.<a class="elsevierStyleCrossRef" href="#bib0075">15</a>The mutual relation of 27 strains analyzed by SARAMIS™ software was represented by the colour similarity matrix. Values corresponded to the percentage of similar peaks between strains. A maximum value of 100% was found as the highest intrinsic mass relation (each strain with itself) and 19.3% was the lowest intrinsic mass relation. It suggested that a maximum of 20 peaks was enough to identify T. rubrum specie, and at least a part of the remaining peaks might be associated with intra-specific variability. A global average of similarity of 52.52% suggested that about 50% of the peaks was associated with strain variability.Molecular identification using sequence data from the ITS region is shown to be accurate but unable to clearly discriminate species that exhibited slight genetic variation such as T. rubrum.<a class="elsevierStyleCrossRef" href="#bib0050">10</a> It is worthy to refer that molecular biology-based methods such as sequencing and typing are time-consuming and expensive.MALDI-TOF ICMS has herein proved to be sensitive and accurate for the discrimination between species of dermatophytes.<a class="elsevierStyleCrossRefs" href="#bib0075">15–19</a> Thus, it can be regarded as an extra step to the polyphasic scheme of identification<a class="elsevierStyleCrossRef" href="#bib0165">33</a> being an objective and fast analytical methodology. Furthermore, relating to labour and consumables, it is cost-effective when compared to other biological techniques.This study provided new insights into mass spectra analysis and the potentialities of strain typing for MALDI-TOF ICMS. Analysis of the mass spectral profiles and comparison with molecular typing profiles provide new insights in the proteomic approach, namely the determination of phenotypic similarity and variability of strains for T. rubrum. Moreover, results suggested that MALDI-TOF ICMS is an alternative discriminating tool for intraspecific variability determination of T. rubrum at the strain level than strain DNA typing. MALDI-TOF ICMS allows a fast, reliable and low-cost identification over molecular biology approach.FundingL. Pereira was granted by the EMbaRC project. The research leading to these results has received partial funding from the European Community's Seventh Framework Programme (FP7, 2007–2013), Research Infrastructures action, under the grant agreement No. FP7-228310 (EMbaRC project).Conflicts of interestThe authors report no conflicts of interest." "textoCompletoSecciones" => array:1 [ "secciones" => array:11 [ 0 => array:2 [ "identificador" => "xres303527" "titulo" => array:5 [ 0 => "Abstract" 1 => "Aims" 2 => "Methods" 3 => "Results" 4 => "Conclusions" ] ] 1 => array:2 [ "identificador" => "xpalclavsec286668" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "xres303526" "titulo" => array:5 [ 0 => "Resumen" 1 => "Objetivo" 2 => "Métodos" 3 => "Resultados" 4 => "Conclusiones" ] ] 3 => array:2 [ "identificador" => "xpalclavsec286669" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Strains and culture conditions" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Morphological identification" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry identification" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Molecular identification" ] ] ] 6 => array:2 [ "identificador" => "sec0035" "titulo" => "Results" ] 7 => array:2 [ "identificador" => "sec0040" "titulo" => "Discussion" ] 8 => array:2 [ "identificador" => "sec0045" "titulo" => "Funding" ] 9 => array:2 [ "identificador" => "sec0050" "titulo" => "Conflicts of interest" ] 10 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2012-10-10" "fechaAceptado" => "2013-01-07" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec286668" "palabras" => array:5 [ 0 => "Identification" 1 => "MALDI-TOF ICMS" 2 => "Molecular biology" 3 => "Proteomics" 4 => "Trichophyton rubrum and typing" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec286669" "palabras" => array:5 [ 0 => "Identificación" 1 => "MALDI-TOF ICMS" 2 => "Biología molecular" 3 => "Proteómica" 4 => "Trichophyton rubrum y tipificación" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "AimsIn this study, the potential of matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) was investigated for the identification of clinical isolates. The isolates were analyzed at the species and strain level. MethodsSpectral identification by MALDI-TOF ICMS was performed for all strains, and compared with the results of sequencing of the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA region. PCR fingerprinting analysis using primers M13, (GACA)4, and (AC)10 was performed in order to assess the intra-specific variability of Trichophyton rubrum strains. ResultsThe identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis. Sequence data confirmed spectral mass identification at species level. Intra-specific variability was assessed. Within the T. rubrum cluster, strains were distributed into smaller highly related sub-groups with a similarity values above 85%. ConclusionsMALDI-TOF ICMS was shown to be a rapid, low-cost and accurate alternative tool for the identification and strain typing of T. rubrum." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "ObjetivoEn este estudio se investigó el potencial de matrix-assisted laser desorption/ionization time-of-flight intact cell mass (MALDI-TOF ICMS) para la identificación de aislados clínicos. Los aislados fueron analizados al nivel de especie y de cepa. MétodosSe realizó una identificación espectral mediante MALDI-TOF ICMS de todas las cepas que se comparó con los resultados de secuenciación de la región espaciadores transcritos internos (ITS1 e ITS2) y el ADNr 5,8S. Los análisis PCR fingerprinting usando los primers M13, (GACA)4 y (AC)10 se realizaron con la intención de evaluar la variabilidad intraespecífica de las cepas de Trichophyton rubrum. ResultadosLa identificación de las cepas al nivel de especie mediante MALDI-TOF ICMS concordó con la ID realizada retrospectivamente en el análisis morfológico y bioquímico. La secuencia de datos confirmó la identificación por espectro de masas a nivel de especie. Se evaluó la variabilidad intraespecífica. Dentro del clúster de T. rubrum, las cepas se distribuyeron en subgrupos menores y muy relacionados con valores de similaridad superiores a 85%. ConclusionesSe demuestra que MALDI-TOF ICMS es una herramienta alternativa rápida, de bajo coste y precisa para la identificación y tipificación de cepas de T. rubrum." ] ] "multimedia" => array:5 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2371 "Ancho" => 2167 "Tamanyo" => 287332 ] ] "descripcion" => array:1 [ "en" => "Dendrogram resulting from single-linkage cluster analysis mass spectra of T. rubrum strains and out-group strains T. interdigitale (MUM 10.136 and MUM 12.07) and reference strain T. mentagrophytes (ATCC MYA-4439) obtained by MALDI-TOF ICMS analysis. Distances were measured as percentage of mass similarity." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1672 "Ancho" => 3334 "Tamanyo" => 257896 ] ] "descripcion" => array:1 [ "en" => "MALDI-TOF ICMS mass spectra of T. rubrum strains (MUM 09.29, MUM 08.09, MUM 08.13, MUM 09.09, MUM 08.12, MUM 09.20, MUM 09.26, MUM 08.05, MUM 10.128 and MUM 08.11) and the out-group MUM 10.136 T. interdigitale strain." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Fig. 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1696 "Ancho" => 3333 "Tamanyo" => 776371 ] ] "descripcion" => array:1 [ "en" => "The colour similarity matrix representing the mutual relation of the different strains analyzed in MALDI-TOF ICMS. The colour bar indicates the colours associated with the different percentage of mass similarity." ] ] 3 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Fig. 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 625 "Ancho" => 1658 "Tamanyo" => 76520 ] ] "descripcion" => array:1 [ "en" => "DNA sequence alignment of internal transcribed spacers (ITS1 and ITS4) and the 5.8S rRNA region of strain MUM 08.11. View conservation by plotting identities to a standard as a dot and showing the differences between the strains by the respective representative nucleotide symbols." ] ] 4 => array:7 [ "identificador" => "fig0025" "etiqueta" => "Fig. 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 1364 "Ancho" => 2000 "Tamanyo" => 137348 ] ] "descripcion" => array:1 [ "en" => "PCR pattern for primers M13, (GACA)4, and (AC)10. Lane M, 0.1–10kb ladder; lanes Aa and Bb as DNA profiles for reference strain, MUM 09.09, MUM 08.05, MUM 08.12, MUM 08.13, MUM 08.15, MUM 10.128, MUM 09.20, MUM 09.26 and MUM 09.29. Lanes Ab and Ba as DNA profile for isolates MUM 08.11. 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The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing

El uso de MALDI-TOF ICMS como una herramienta alternativa para la identificación y tipificación de Trichophyton rubrum

Leonel Pereira

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leoneljpp@deb.uminho.pt

Corresponding author.

, Nicolina Dias

Corresponding author

nidias@deb.uminho.pt

Corresponding author.

, Cledir Santos, Nelson Lima

IBB/Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, Portugal

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    "titulo" => "The use of MALDI-TOF ICMS as an alternative tool for <span class="elsevierStyleItalic">Trichophyton rubrum</span> identification and typing"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Dermatophytes are the etiological agents of fungal infections of the skin&#44; hair&#44; and nails&#44; globally known as tinea&#46; Despite dermatophytes belong to a homogenous group of fungi closely related to each other phylogenetically&#44; the identification of species&#44; as well as strain level&#44; remains an important issue for epidemiological and taxonomic purposes&#46;<a class="elsevierStyleCrossRefs" href="#bib0005"><span class="elsevierStyleSup">1&#8211;4</span></a> Phenotypic characters and ecological features have been for a long time the basis for phylogenetic studies and taxonomic classification&#46; Additionally&#44; biochemical and physiological tests can be used as complementary tools&#46;<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">Although morphology-based identification has been the main technique for the identification of dermatophytes&#44; some limitations of the procedure could be pointed out&#46; Firstly&#44; it is based on the recognition of specific morphological features in macroscopic and microscopic observations&#59; however&#44; morphological similarity&#44; intra-specific variability and polymorphism of dermatophytes are difficult for the identification procedure&#46; Furthermore&#44; confusion is caused by the atypical growth forms that occur frequently&#44; and the requirement of a long incubation period &#40;2&#8211;4 weeks&#41; could be a significant drawback for rapid diagnosis and treatment&#46; In addition&#44; these approaches are time-and labour-consuming&#44; expensive and requiring highly skilled-specific staff&#44;<a class="elsevierStyleCrossRefs" href="#bib0015"><span class="elsevierStyleSup">3&#44;6</span></a> which normally delays the diagnosis&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">PCR technology is a powerful method that enables the development of new techniques in molecular biology for accurate organism identification&#46;<a class="elsevierStyleCrossRefs" href="#bib0035"><span class="elsevierStyleSup">7&#8211;9</span></a> Molecular biology-based techniques are useful to reveal intra-specific DNA polymorphism that enables defining clusters within the same species&#46;<a class="elsevierStyleCrossRefs" href="#bib0010"><span class="elsevierStyleSup">2&#44;9&#44;10</span></a> Furthermore&#44; they have the advantage&#44; over traditional methods&#44; of being fast&#44; simple&#44; sensitive&#44; and reproducible&#46;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> Nevertheless&#44; these methods remain time&#47;labour consuming and relatively expensive&#46;<a class="elsevierStyleCrossRef" href="#bib0055"><span class="elsevierStyleSup">11</span></a></p><p id="par0020" class="elsevierStylePara elsevierViewall">Along the past decade matrix-assisted laser desorption&#47;ionization time of flight intact cell mass spectrometry &#40;MALDI-TOF ICMS&#41; technique has been established as a valuable system for microorganisms identification to be introduced in laboratory routine&#46;<a class="elsevierStyleCrossRefs" href="#bib0060"><span class="elsevierStyleSup">12&#44;13</span></a> Potentialities of MALDI-TOF ICMS have been revised for filamentous fungi&#46;<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">14</span></a> Some procedures for the identification of dermatophyte species&#44; including <span class="elsevierStyleItalic">Trichophyton rubrum</span>&#44; have been recently established&#46; MALDI-TOF ICMS system was evaluated in comparison with sequencing the internal transcribed spacer 1 &#40;ITS1&#41;&#44; ITS2 and the 5&#46;8S rDNA regions&#44;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">15</span></a> D2 28S rRNA&#44;<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">16</span></a> and ITS region of the ribosomal DNA with V9D and LSU 266 primers&#46;<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">17</span></a> High confidence level &#40;99&#46;9&#37;&#41; for identification was found&#46; Authors found similar or even better specificity of MALDI-TOF ICMS than an ITS&#47;PCR-based approach for species identification purposes&#46; MALDI-TOF ICMS proved to be a rapid&#44; low cost&#44; accurate&#44; and reliable alternative to morphological and PCR analysis for the identification of dermatophytes&#46;<a class="elsevierStyleCrossRefs" href="#bib0075"><span class="elsevierStyleSup">15&#8211;19</span></a> Although MALDI-TOF ICMS technique for species identification is a widely investigated theme&#44; only a few recent studies focalize on the potential capacity for intra-specific discrimination in fungi&#46;<a class="elsevierStyleCrossRefs" href="#bib0065"><span class="elsevierStyleSup">13&#44;20&#8211;23</span></a></p><p id="par0025" class="elsevierStylePara elsevierViewall">The aim of this work was to examine the potential of MALDI-TOF ICMS for the identification of clinical isolates of <span class="elsevierStyleItalic">T&#46; rubrum</span> at the species and strain level&#46; Molecular typing using three different primers was compared with spectrometric analysis&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Strains and culture conditions</span><p id="par0030" class="elsevierStylePara elsevierViewall">A set of 24 clinical isolates of <span class="elsevierStyleItalic">T&#46; rubrum</span> from the fungal collection of Micoteca da Universidade do Minho &#40;MUM&#41;&#44; Portugal&#44; were used for analysis&#46; Two <span class="elsevierStyleItalic">T&#46; rubrum</span> isolates were collected in Spain from human patients with tinea pedis &#40;MUM 09&#46;26&#41; and onychomycosis &#40;MUM 09&#46;20&#41;&#46; The remaining clinical isolates MUM 08&#46;05&#44; 08&#46;09&#44; 08&#46;11&#44; 08&#46;12&#44; 08&#46;13&#44; 08&#46;15&#44; 09&#46;08&#44; 09&#46;09&#44; 09&#46;10&#44; 09&#46;12&#44; 09&#46;18&#44; 09&#46;29&#44; 10&#46;128&#44; 10&#46;133&#44; 12&#46;01&#44; 12&#46;02&#44; 12&#46;03&#44; 12&#46;04&#44; 12&#46;06&#44; 12&#46;08&#44; 12&#46; 09 and 12&#46;10 were collected in Portugal from human patients with onychomycosis&#46; Clinical isolates of <span class="elsevierStyleItalic">Trichophyton interdigitale</span> &#40;MUM 10&#46;136 and MUM 12&#46;07&#41; were also used as out-group&#46; Two reference strains were obtained from the American Type Culture Collection &#40;ATCC&#41; and included in this study&#46; Strain <span class="elsevierStyleItalic">T&#46; rubrum</span> ATCC MYA-4438 was used as positive control and strain <span class="elsevierStyleItalic">Trichophyton mentagrophytes</span> ATCC MYA-4439 was used as out-group in MALDI-TOF analysis&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">Potato dextrose agar &#40;PDA&#44; OXOID&#44; UK&#41; was used to maintain the cultures and to grow the fungi for molecular and mass spectral analyses&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Morphological identification</span><p id="par0040" class="elsevierStylePara elsevierViewall">All isolates were tested by conventional method&#44; according to the standard procedures of morphological identification &#40;macro-and micromorphology&#41;&#46; Urease test was used as complementary biochemical test&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Matrix-assisted laser desorption&#47;ionization time-of-flight intact cell mass spectrometry identification</span><p id="par0045" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Samples preparation&#58;</span> In a first attempt several experimental conditions were tested on four strains &#40;ATCC-MYA 4438&#44; MUM 09&#46;08&#59; MUM 08&#46;11&#59; MUM 10&#46;133&#41; and spectra were compared in order to select the best method&#46; Two matrices were used&#58; 2&#44;5-dihydroxybenzoic acid &#40;DHB&#41; and &#945;-cyano-4-hydroxycinnamic acid &#40;CHCA&#41;&#46; Different extraction protocols &#40;including formic acid and sonication&#41;&#44; as well as previous suspension of cells in water or solvent mixtures did not result in an improvement compared to the direct application of cells to the MALDI target&#46; As no clear improvement in spectrum quality was observed in the preliminary study we followed the one described by Santos et al&#46;<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">14</span></a> for the clinical isolates&#46; A growing time of 5 days at 30<span class="elsevierStyleHsp" style=""></span>&#176;C was set for all cultures before MALDI-TOF analysis to avoid age-dependent variability&#46; Each sample was spotted in quadruplicate to test reproducibility&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">MALDI-TOF analysis&#58;</span> The analysis was performed on an Axima LNR system &#40;Kratos Analytical&#44; Shimadzu&#44; UK&#41; equipped with a nitrogen laser &#40;337<span class="elsevierStyleHsp" style=""></span>nm&#41;&#44; where the laser intensity was set just above the threshold for ion production&#46; The mass range from 2000 to 20000<span class="elsevierStyleHsp" style=""></span>Da was recorded using the linear mode with a delay of 104<span class="elsevierStyleHsp" style=""></span>ns and an acceleration voltage of &#43;20<span class="elsevierStyleHsp" style=""></span>kV&#46; Final spectra were generated by summing 20 laser shots accumulated per profile and 50 profiles produced per sample&#44; leading to 1000 laser shots per summed spectrum&#46;</p><p id="par0055" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Spectral Identification and software analysis&#58;</span> Resulting peak lists were exported to the SARAMIS&#8482; software package &#40;Spectral Archiving and Microbial Identification System&#44; AnagnosTec&#44; Germany&#44; <a class="elsevierStyleInterRef" href="http://www.anagnostec.eu/">www&#46;anagnostec&#46;eu</a>&#41; where the final microbial identification was achieved&#46; Peak lists of individual samples were compared on SARAMIS&#8482; database generating a ranked list of matching spectra&#46; This software uses a point system based on peak list with mass signals weighted according to their specificity&#46; A dendrogram and matrix of spectral similarity between isolates were created using SARAMIS&#8482; package&#46; A minimum of 100 peaks&#44; and at least 90&#37; of mass similarity with species SuperSpectra was defined as the acceptance criteria to validate the result&#46; The similarity between individual spectra was expressed as the relative or absolute number of matching mass signals after subjecting the data to a single link agglomerative clustering algorithm&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Molecular identification</span><p id="par0060" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">DNA isolation&#58;</span> Spores and mycelia from 5 days old culture grown on PDA medium were transferred into a sterile 1&#46;5<span class="elsevierStyleHsp" style=""></span>ml tube and 1<span class="elsevierStyleHsp" style=""></span>ml of extraction buffer &#40;1&#37; 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and&#44; finally&#44; with 800<span class="elsevierStyleHsp" style=""></span>&#956;l chloroform&#46; Purified nucleic acids were precipitated with 0&#46;6 volumes of ice-cold 2-propanol&#46; DNA was washed twice with 150<span class="elsevierStyleHsp" style=""></span>&#956;l of ice-cold 70&#37; ethanol and air dried&#44; suspended in 50<span class="elsevierStyleHsp" style=""></span>&#956;l of ultra-pure water&#44; and stored at &#8722;20<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">ITS sequencing&#58;</span> The analysis was carried out using primer pair ITS1 5&#8242;-TCCGTAGGTGAACCTTGCGG-3&#8242; and ITS4 5&#8242;-TCCTCCGCTTATTGATATGC-3&#8242;&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">24</span></a> The PCR reactions were performed in a total volume of 50<span class="elsevierStyleHsp" style=""></span>&#956;l&#44; containing the 1&#215; PCR buffer &#40;Promega&#44; USA&#41;&#44; 2&#46;5<span class="elsevierStyleHsp" style=""></span>mmol&#47;l MgCl<span class="elsevierStyleInf">2</span> &#40;Promega&#44; USA&#41;&#44; 0&#46;2<span class="elsevierStyleHsp" style=""></span>mmol&#47;l of deoxynucleoside triphosphates &#40;dNTPs&#41; &#40;Promega&#44; USA&#41;&#44; 0&#46;5<span class="elsevierStyleHsp" style=""></span>mmol&#47;l of each primer&#44; 1<span class="elsevierStyleHsp" style=""></span>U of GoTaq<span class="elsevierStyleSup">&#174;</span> Hot Start Polymerase &#40;Promega&#44; USA&#41; and 50<span class="elsevierStyleHsp" style=""></span>ng of DNA&#46; PCR was run in a MyCycler&#8482; Thermal Cycler &#40;Bio-Rad Laboratories&#44; USA&#41; using the following conditions&#58; 2<span class="elsevierStyleHsp" style=""></span>min&#44; 95<span class="elsevierStyleHsp" style=""></span>&#176;C&#59; 35 cycles of 1<span class="elsevierStyleHsp" style=""></span>min&#44; 95<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 1<span class="elsevierStyleHsp" style=""></span>min&#44; 55<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 1<span class="elsevierStyleHsp" style=""></span>min&#44; 72<span class="elsevierStyleHsp" style=""></span>&#176;C&#59; and final extension&#44; 5<span class="elsevierStyleHsp" style=""></span>min&#44; 72<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Amplification products were separated by electrophoresis in 1&#46;0&#37; agarose gel&#44; stained with SafeView DNA Stain &#40;NBS Biologicals&#44; UK&#41;&#44; at 80<span class="elsevierStyleHsp" style=""></span>V for 30<span class="elsevierStyleHsp" style=""></span>min&#46; The PCR products were purified using QIAquick PCR Purification Kit &#40;Qiagen&#44; Germany&#41; and then sequenced&#46; The sequences were compared using the GenBank database &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/BLAST/">http&#58;&#47;&#47;www&#46;ncbi&#46;nlm&#46;nih&#46;gov&#47;BLAST&#47;</a>&#41;&#46; GenBank access numbers for the isolates MUM 08&#46;11 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663974">JQ663974</a>&#41;&#44; MUM 10&#46;128 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663986">JQ663986</a>&#41;&#44; MUM 08&#46;05 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663971">JQ663971</a>&#41;&#44; MUM 09&#46;26 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663984">JQ663984</a>&#41;&#44; MUM 09&#46;20 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663983">JQ663983</a>&#41;&#44; MUM 08&#46;12 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663975">JQ663975</a>&#41;&#44; MUM 09&#46;09 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663979">JQ663979</a>&#41;&#44; MUM 08&#46;13 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663976">JQ663976</a>&#41;&#44; MUM 08&#46;15 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663977">JQ663977</a>&#41; and MUM 09&#46;29 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=JQ663985">JQ663985</a>&#41; with the reference strain <span class="elsevierStyleItalic">T&#46; rubrum</span> ATCC MYA-4438 &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=FJ746657">FJ746657</a>&#41;&#46;</p><p id="par0070" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Typing&#58;</span> For typing analysis oligonucleotides M13 5&#8242;-GAGGGTGGCGGTTCT-3&#8242;&#44;<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">25</span></a> &#40;GACA&#41;<span class="elsevierStyleInf">4</span><span class="elsevierStyleHsp" style=""></span><a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">26</span></a> and &#40;AC&#41;<span class="elsevierStyleInf">10</span><span class="elsevierStyleHsp" style=""></span><a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">27</span></a> were used&#46; PCR reactions were performed in a total volume of 50<span class="elsevierStyleHsp" style=""></span>&#956;l&#44; containing the 1&#215; PCR buffer&#44; 2&#46;5<span class="elsevierStyleHsp" style=""></span>mmol&#47;l MgCl<span class="elsevierStyleInf">2</span> 0&#46;2<span class="elsevierStyleHsp" style=""></span>mmol&#47;l of dNTPs&#44; 0&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;mol&#47;l of each primer&#44; 2<span class="elsevierStyleHsp" style=""></span>U of GoTaq<span class="elsevierStyleSup">&#174;</span> Hot Start Polymerase and 50<span class="elsevierStyleHsp" style=""></span>ng of DNA&#46; PCRs were run in a MyCycler&#8482; Thermal Cycler using the following conditions&#58; 2<span class="elsevierStyleHsp" style=""></span>min&#44; 95<span class="elsevierStyleHsp" style=""></span>&#176;C&#59; 35 cycles of 1<span class="elsevierStyleHsp" style=""></span>min&#44; 95<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 1<span class="elsevierStyleHsp" style=""></span>min&#44; 50<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 2<span class="elsevierStyleHsp" style=""></span>min&#44; 72<span class="elsevierStyleHsp" style=""></span>&#176;C&#59; and a final extension cycle&#44; 5<span class="elsevierStyleHsp" style=""></span>min&#44; 72<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Amplification products were separated by electrophoresis in 1&#46;5&#37; agarose gel&#44; stained with SafeView DNA Stain&#44; at 60<span class="elsevierStyleHsp" style=""></span>V for 120<span class="elsevierStyleHsp" style=""></span>min&#46;</p></span></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Results</span><p id="par0075" class="elsevierStylePara elsevierViewall">Twenty six dermatophyte clinical isolates &#40;24 <span class="elsevierStyleItalic">T&#46; rubrum</span> and 2 <span class="elsevierStyleItalic">T&#46; interdigitale</span>&#41; and two ATCC reference strains were analyzed in quadruplicate&#44; and the spectra were chosen according to the greatest number of peaks &#40;data count&#41; and the highest percentage of identity&#46; Differences in the confidence level of identification between the replicates never exceeded 5&#37;&#46; A percentage identity over 99&#37; was found in all strains&#44; except MUM 08&#46;11 &#40;80&#46;2&#37;&#41;&#44; MUM 09&#46;10 &#40;89&#46;8&#37;&#41;&#44; MUM 10&#46;128 &#40;93&#46;1&#37;&#41;&#44; MUM 12&#46;10 &#40;96&#46;4&#37;&#41;&#44; MUM 08&#46;05 &#40;96&#46;2&#37;&#41;&#44; and MUM 09&#46;18 &#40;97&#46;1&#37;&#41;&#46; The identification of clinical isolates at the species level by MALDI-TOF ICMS was in agreement with the ID performed by the conventional method in 95&#46;8&#37; &#40;23&#47;24&#41;&#44; of the <span class="elsevierStyleItalic">T&#46; rubrum</span> isolates&#46; Isolate MUM 12&#46;01 was identified by conventional methods as <span class="elsevierStyleItalic">Trichophyton</span> spp&#46;&#44; and as <span class="elsevierStyleItalic">T&#46; rubrum</span> by MALDI-TOF ICMS&#46; An agreement of 100&#37; was found between conventional and MALDI-TOF ICMS results for <span class="elsevierStyleItalic">T&#46; interdigitale</span> &#40;2&#47;2&#41;&#46; Comparison of spectral analysis with molecular &#8220;gold standard&#8221; sequence of ITS region was performed&#44; in ten <span class="elsevierStyleItalic">T&#46; rubrum</span> strains&#44; to confirm MALDI-TOF ICMS identification&#46; Results of both methods agreed in 90&#37; &#40;9&#47;10&#41;&#46; Isolate MUM 08&#46;11 was identified as <span class="elsevierStyleItalic">Trichophyton megnini</span> by sequence analysis of the ITS of the ribosomal DNA&#46; Urease test was positive for this strain&#46;</p><p id="par0080" class="elsevierStylePara elsevierViewall">A spectra-based dendrogram obtained by conventional identification algorithm of SARAMIS software had shown an evident separation between out-group cluster <span class="elsevierStyleItalic">T&#46; mentagrophytes</span> ATCC MYA-4439 and <span class="elsevierStyleItalic">T&#46; interdigitale</span> &#40;MUM 10&#46;136 and MUM 12&#46;07&#41; with <span class="elsevierStyleItalic">T&#46; rubrum</span> cluster &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46; A low percentage of mass similarity &#40;40&#37;&#41; was obtained between <span class="elsevierStyleItalic">T&#46; rubrum</span> cluster and out-group cluster&#46; Within the <span class="elsevierStyleItalic">T&#46; rubrum</span> cluster&#44; strains were distributed into highly related sub-groups&#44; with similarity values above 85&#37;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0085" class="elsevierStylePara elsevierViewall">In order to understand the intra-specific differences inside <span class="elsevierStyleItalic">T&#46; rubrum</span> cluster&#44; the mass spectra profiles &#40;MSP&#41; were analyzed and compared&#46; To perform this analysis&#44; ten profiles of <span class="elsevierStyleItalic">T&#46; rubrum</span> strains &#40;MUM 09&#46;29&#44; MUM 08&#46;09&#44; MUM 08&#46;13&#44; MUM 09&#46;09&#44; MUM 08&#46;12&#44; MUM 09&#46;20&#44; MUM 09&#46;26&#44; MUM 08&#46;05&#44; MUM 10&#46;128 and MUM 08&#46;11&#41; were selected randomly from different localizations of the dendrogram&#46; Strain <span class="elsevierStyleItalic">T&#46; interdigitale</span> MUM 10&#46;136 was selected from out-group cluster&#46; The spectra obtained for each selected strain were graphically reported highlighting specific and intra-specific variability &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; Very similar mass patterns were generated&#44; although slight variations were observed&#46; The most relevant peaks were between 2000 and 11000 <span class="elsevierStyleItalic">m</span>&#47;<span class="elsevierStyleItalic">z</span>&#44; especially in the region encompassing 2500 to 9000 <span class="elsevierStyleItalic">m</span>&#47;<span class="elsevierStyleItalic">z</span>&#44; where the highest peaks were observed&#46; A single peak with <span class="elsevierStyleItalic">m</span>&#47;<span class="elsevierStyleItalic">z</span> 7380 stood out&#44; due to its range of intensity in profile spectra along the strains&#46; This peak was absent in out-group strain MUM 10&#46;136 and was nearly present in <span class="elsevierStyleItalic">T&#46; rubrum</span> strain MUM 08&#46;11&#46; Moreover&#44; the percentage of identification of this strain reached 90&#37; in only one spectrum&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0090" class="elsevierStylePara elsevierViewall">All the spectra datasets were correlated in a similarity matrix&#44; obtained from SARAMIS&#8482; software &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46; The matrix displayed a relation between all the strains based on the percentage of similarity by a range colour scale from 0&#37; &#40;yellow&#41; to 100&#37; &#40;blue&#41;&#46; The out-group <span class="elsevierStyleItalic">T&#46; interdigitale</span> &#40;MUM 10&#46;136 and MUM 12&#46;07&#41; obtained a maximum percentage of similarity of 32&#46;7&#37; and 33&#46;9&#37;&#44; respectively&#44; among all the strains analyzed&#46; In <span class="elsevierStyleItalic">T&#46; rubrum</span> cluster&#44; it was possible to observe intra-specific variability between isolates&#46; Comparing with the other isolates&#44; the strain ATCC MYA-4438 presented a global low range of mass similarity percentage of 42&#46;2&#37;&#46; Other strains such as MUM 08&#46;05&#44; MUM 08&#46;11&#44; and MUM 12&#46;01 had also shown low mass similarity percentage&#46; The remaining strains revealed an average mass similarity percentage ranging from 40 to 60&#37; and a global average of 52&#46;52&#37;&#44; excluding out-group strains&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0095" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">ITS sequence analysis&#58;</span> Analysis of sequence data in GenBank database &#40;<a class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/BLAST/">http&#58;&#47;&#47;www&#46;ncbi&#46;nlm&#46;nih&#46;gov&#47;BLAST&#47;</a>&#41; confirmed <span class="elsevierStyleItalic">T&#46; rubrum</span> identification for the ten strains that profile spectra was analyzed and compared&#46; The DNA sequence of ITS region was aligned and compared with <span class="elsevierStyleItalic">T&#46; rubrum</span> reference strain ATCC MYA-4438&#46; We found 100&#37; nucleotide identity between clinical strains and reference strain&#44; except for MUM 08&#46;11 &#40;99&#37;&#41;&#44; which presented 7 nucleotide differences &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia><p id="par0100" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">DNA strain typing&#58;</span> PCR amplification of single primer M13&#44; and repetitive DNA sequences &#40;GACA&#41;<span class="elsevierStyleInf">4</span> and &#40;AC&#41;<span class="elsevierStyleInf">10</span> were performed in order to clarify if the results obtained by MALDI-TOF ICMS spectral analysis had a correspondence to molecular typing&#46; Two profiles of DNA fragments were obtained for the primers M13 and &#40;GACA&#41;<span class="elsevierStyleInf">4</span>&#46; A single partner was obtained using the primer &#40;AC&#41;<span class="elsevierStyleInf">10</span> &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>&#41;&#46; The &#40;GACA&#41;<span class="elsevierStyleInf">4</span> profiles varied between 0&#46;6 and 2&#46;3<span class="elsevierStyleHsp" style=""></span>kb&#59; the band-pattern of PCR using M13 varied between 0&#46;5 and 3&#46;0<span class="elsevierStyleHsp" style=""></span>kb&#44; whereas the banding pattern of the &#40;AC&#41;<span class="elsevierStyleInf">10</span> was from 0&#46;3 to 3&#46;5<span class="elsevierStyleHsp" style=""></span>kb&#46; Profiles of DNA fragments Ab and Ba were found for isolate MUM 08&#46;11 while patterns Aa and Bb were observed for the other nine strains&#46; A unique profile of 6 DNA fragments &#40;lane C&#41; was obtained for single repeat primer &#40;AC&#41;<span class="elsevierStyleInf">10</span> amplicons for all isolates&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Discussion</span><p id="par0105" class="elsevierStylePara elsevierViewall">Fungi species can be easily identified using MALDI-TOF ICMS by detecting a narrow number of specific biomarker peaks&#46; Erhard et al&#46;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">15</span></a> stated that a minimum of 17 and 25 peaks matching the superspectrum of each species was enough to discriminate <span class="elsevierStyleItalic">T&#46; interdigitale</span> and <span class="elsevierStyleItalic">T&#46; rubrum</span>&#44; respectively&#44; with a confidence level of 99&#46;9&#37;&#46; Different sample preparation methods were used to obtain reproducible and informative fungal spectra&#46; Several methods have been reported to improve the protein extraction&#44; and consequently the spectra obtained from the samples&#46;<a class="elsevierStyleCrossRefs" href="#bib0140"><span class="elsevierStyleSup">28&#8211;30</span></a> In the present study&#44; some of these sample preparation methods were evaluated on a small scale &#40;data not shown&#41;&#44; but no clear improvement in spectrum quality was observed&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">Analysis of mass spectra-based dendrogram obtained by MALDI-TOF ICMS revealed an intra-specific variability within <span class="elsevierStyleItalic">T&#46; rubrum</span> cluster&#44; where clinical strains MUM 09&#46;12&#44; MUM 08&#46;11&#44; MUM 10&#46;133 and reference strain ATCC MYA-4438 were more distant from the remaining strains&#46; Nevertheless&#44; high percentage of similarity &#40;60&#37;&#41; was observed revealing a very high phylogenetic proximity&#46; Molecular strain typing profiles confirmed the lack of genetic variability and DNA polymorphism of <span class="elsevierStyleItalic">T&#46; rubrum</span> although the distribution of strains on clusters was different from MALDI-TOF ICMS&#44; showing less discriminative power than spectral analysis&#46; Among the ten selected strains for this study&#44; MUM 08&#46;11 had shown a different fingerprinting pattern using primers M13 and &#40;GACA&#41;<span class="elsevierStyleInf">4</span>&#46; Mass spectral data suggest the existence of intra-specific variability between different strains of <span class="elsevierStyleItalic">T&#46; rubrum&#46;</span> These results agreed with clonal mode of reproduction of <span class="elsevierStyleItalic">T&#46; rubrum</span> stated by other authors&#46;<a class="elsevierStyleCrossRefs" href="#bib0030"><span class="elsevierStyleSup">6&#44;10&#44;31&#44;32</span></a> In fact&#44; it is referred that recent adaptation to a highly specialized ecological niche&#44; the skin of the human host&#44; combined with exclusively asexual reproduction of <span class="elsevierStyleItalic">T&#46; rubrum</span> could explain genetic uniformity within the species&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">Spectra profiles of ten <span class="elsevierStyleItalic">T&#46; rubrum</span> strains were obtained showing a similar pattern of mass peaks&#44; and at least one peak with <span class="elsevierStyleItalic">m</span>&#47;<span class="elsevierStyleItalic">z</span> 7380 was consistently present in all <span class="elsevierStyleItalic">T&#46; rubrum</span> being absent in <span class="elsevierStyleItalic">T&#46; interdigitale</span>&#46; Strain MUM 08&#46;11 lack mass signal at <span class="elsevierStyleItalic">m</span>&#47;<span class="elsevierStyleItalic">z</span> 7380&#44; and was identified with a percentage of identity of 80&#46;2&#37;&#46; The result suggested that the presence&#47;absence of only one mass signal is critical for the accurate identification of the strain&#46; Interestingly&#44; this peak has been previously identified as a marker of <span class="elsevierStyleItalic">T&#46; rubrum</span> species&#46;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">15</span></a></p><p id="par0120" class="elsevierStylePara elsevierViewall">The mutual relation of 27 strains analyzed by SARAMIS&#8482; software was represented by the colour similarity matrix&#46; Values corresponded to the percentage of similar peaks between strains&#46; A maximum value of 100&#37; was found as the highest intrinsic mass relation &#40;each strain with itself&#41; and 19&#46;3&#37; was the lowest intrinsic mass relation&#46; It suggested that a maximum of 20 peaks was enough to identify <span class="elsevierStyleItalic">T&#46; rubrum</span> specie&#44; and at least a part of the remaining peaks might be associated with intra-specific variability&#46; A global average of similarity of 52&#46;52&#37; suggested that about 50&#37; of the peaks was associated with strain variability&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">Molecular identification using sequence data from the ITS region is shown to be accurate but unable to clearly discriminate species that exhibited slight genetic variation such as <span class="elsevierStyleItalic">T&#46; rubrum</span>&#46;<a class="elsevierStyleCrossRef" href="#bib0050"><span class="elsevierStyleSup">10</span></a> It is worthy to refer that molecular biology-based methods such as sequencing and typing are time-consuming and expensive&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">MALDI-TOF ICMS has herein proved to be sensitive and accurate for the discrimination between species of dermatophytes&#46;<a class="elsevierStyleCrossRefs" href="#bib0075"><span class="elsevierStyleSup">15&#8211;19</span></a> Thus&#44; it can be regarded as an extra step to the polyphasic scheme of identification<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">33</span></a> being an objective and fast analytical methodology&#46; Furthermore&#44; relating to labour and consumables&#44; it is cost-effective when compared to other biological techniques&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">This study provided new insights into mass spectra analysis and the potentialities of strain typing for MALDI-TOF ICMS&#46; Analysis of the mass spectral profiles and comparison with molecular typing profiles provide new insights in the proteomic approach&#44; namely the determination of phenotypic similarity and variability of strains for <span class="elsevierStyleItalic">T&#46; rubrum</span>&#46; Moreover&#44; results suggested that MALDI-TOF ICMS is an alternative discriminating tool for intraspecific variability determination of <span class="elsevierStyleItalic">T&#46; rubrum</span> at the strain level than strain DNA typing&#46; MALDI-TOF ICMS allows a fast&#44; reliable and low-cost identification over molecular biology approach&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Funding</span><p id="par0140" class="elsevierStylePara elsevierViewall">L&#46; Pereira was granted by the EMbaRC project&#46; The research leading to these results has received partial funding from the <span class="elsevierStyleGrantSponsor">European Community&#39;s Seventh Framework Programme</span> &#40;FP7&#44; 2007&#8211;2013&#41;&#44; <span class="elsevierStyleGrantSponsor" id="gs0005">Research Infrastructures action</span>&#44; under the grant agreement No&#46; <span class="elsevierStyleGrantNumber" refid="gs0005">FP7-228310</span> &#40;EMbaRC project&#41;&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Conflicts of interest</span><p id="par0145" class="elsevierStylePara elsevierViewall">The authors report no conflicts of interest&#46;</p></span></span>"
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        "titulo" => "Abstract"
        "resumen" => "<span class="elsevierStyleSectionTitle">Aims</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">In this study&#44; the potential of matrix-assisted laser desorption&#47;ionization time-of-flight intact cell mass spectrometry &#40;MALDI-TOF ICMS&#41; was investigated for the identification of clinical isolates&#46; The isolates were analyzed at the species and strain level&#46;</p> <span class="elsevierStyleSectionTitle">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Spectral identification by MALDI-TOF ICMS was performed for all strains&#44; and compared with the results of sequencing of the internal transcribed spacers &#40;ITS1 and ITS2&#41;&#44; and the 5&#46;8S rDNA region&#46; PCR fingerprinting analysis using primers M13&#44; &#40;GACA&#41;<span class="elsevierStyleInf">4</span>&#44; and &#40;AC&#41;<span class="elsevierStyleInf">10</span> was performed in order to assess the intra-specific variability of <span class="elsevierStyleItalic">Trichophyton rubrum</span> strains&#46;</p> <span class="elsevierStyleSectionTitle">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">The identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis&#46; Sequence data confirmed spectral mass identification at species level&#46; Intra-specific variability was assessed&#46; Within the <span class="elsevierStyleItalic">T&#46; rubrum</span> cluster&#44; strains were distributed into smaller highly related sub-groups with a similarity values above 85&#37;&#46;</p> <span class="elsevierStyleSectionTitle">Conclusions</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">MALDI-TOF ICMS was shown to be a rapid&#44; low-cost and accurate alternative tool for the identification and strain typing of <span class="elsevierStyleItalic">T&#46; rubrum</span>&#46;</p>"
      ]
      "es" => array:2 [
        "titulo" => "Resumen"
        "resumen" => "<span class="elsevierStyleSectionTitle">Objetivo</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">En este estudio se investig&#243; el potencial de <span class="elsevierStyleItalic">matrix-assisted laser desorption&#47;ionization time-of-flight intact cell mass</span> &#40;MALDI-TOF ICMS&#41; para la identificaci&#243;n de aislados cl&#237;nicos&#46; Los aislados fueron analizados al nivel de especie y de cepa&#46;</p> <span class="elsevierStyleSectionTitle">M&#233;todos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Se realiz&#243; una identificaci&#243;n espectral mediante MALDI-TOF ICMS de todas las cepas que se compar&#243; con los resultados de secuenciaci&#243;n de la regi&#243;n espaciadores transcritos internos &#40;ITS1 e ITS2&#41; y el ADNr 5&#44;8S&#46; Los an&#225;lisis PCR <span class="elsevierStyleItalic">fingerprinting</span> usando los <span class="elsevierStyleItalic">primers</span> M13&#44; &#40;GACA&#41;<span class="elsevierStyleInf">4</span> y &#40;AC&#41;<span class="elsevierStyleInf">10</span> se realizaron con la intenci&#243;n de evaluar la variabilidad intraespec&#237;fica de las cepas de <span class="elsevierStyleItalic">Trichophyton rubrum&#46;</span></p> <span class="elsevierStyleSectionTitle">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">La identificaci&#243;n de las cepas al nivel de especie mediante MALDI-TOF ICMS concord&#243; con la ID realizada retrospectivamente en el an&#225;lisis morfol&#243;gico y bioqu&#237;mico&#46; La secuencia de datos confirm&#243; la identificaci&#243;n por espectro de masas a nivel de especie&#46; Se evalu&#243; la variabilidad intraespec&#237;fica&#46; Dentro del cl&#250;ster de <span class="elsevierStyleItalic">T&#46; rubrum</span>&#44; las cepas se distribuyeron en subgrupos menores y muy relacionados con valores de similaridad superiores a 85&#37;&#46;</p> <span class="elsevierStyleSectionTitle">Conclusiones</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Se demuestra que MALDI-TOF ICMS es una herramienta alternativa r&#225;pida&#44; de bajo coste y precisa para la identificaci&#243;n y tipificaci&#243;n de cepas de <span class="elsevierStyleItalic">T&#46; rubrum</span>&#46;</p>"
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Dendrogram resulting from single-linkage cluster analysis mass spectra of <span class="elsevierStyleItalic">T&#46; rubrum</span> strains and out-group strains <span class="elsevierStyleItalic">T&#46; interdigitale</span> &#40;MUM 10&#46;136 and MUM 12&#46;07&#41; and reference strain <span class="elsevierStyleItalic">T&#46; mentagrophytes</span> &#40;ATCC MYA-4439&#41; obtained by MALDI-TOF ICMS analysis&#46; Distances were measured as percentage of mass similarity&#46;</p>"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">MALDI-TOF ICMS mass spectra of <span class="elsevierStyleItalic">T&#46; rubrum</span> strains &#40;MUM 09&#46;29&#44; MUM 08&#46;09&#44; MUM 08&#46;13&#44; MUM 09&#46;09&#44; MUM 08&#46;12&#44; MUM 09&#46;20&#44; MUM 09&#46;26&#44; MUM 08&#46;05&#44; MUM 10&#46;128 and MUM 08&#46;11&#41; and the out-group MUM 10&#46;136 <span class="elsevierStyleItalic">T&#46; interdigitale</span> strain&#46;</p>"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">The colour similarity matrix representing the mutual relation of the different strains analyzed in MALDI-TOF ICMS&#46; The colour bar indicates the colours associated with the different percentage of mass similarity&#46;</p>"
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          "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">DNA sequence alignment of internal transcribed spacers &#40;ITS1 and ITS4&#41; and the 5&#46;8S rRNA region of strain MUM 08&#46;11&#46; View conservation by plotting identities to a standard as a dot and showing the differences between the strains by the respective representative nucleotide symbols&#46;</p>"
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          "en" => "<p id="spar0065" class="elsevierStyleSimplePara elsevierViewall">PCR pattern for primers M13&#44; &#40;GACA&#41;<span class="elsevierStyleInf">4</span>&#44; and &#40;AC&#41;<span class="elsevierStyleInf">10</span>&#46; Lane M&#44; 0&#46;1&#8211;10<span class="elsevierStyleHsp" style=""></span>kb ladder&#59; lanes Aa and Bb as DNA profiles for reference strain&#44; MUM 09&#46;09&#44; MUM 08&#46;05&#44; MUM 08&#46;12&#44; MUM 08&#46;13&#44; MUM 08&#46;15&#44; MUM 10&#46;128&#44; MUM 09&#46;20&#44; MUM 09&#46;26 and MUM 09&#46;29&#46; Lanes Ab and Ba as DNA profile for isolates MUM 08&#46;11&#46; Lane C&#44; DNA profile for all isolate&#46;</p>"
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    "bibliografia" => array:2 [
      "titulo" => "References"
      "seccion" => array:1 [
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                      "titulo" => "DNA and the classical way&#58; identification of medically important molds in the 21st century"
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                        0 => array:2 [
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                            0 => "S&#46;A&#46; Balajee"
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                          "etal" => false
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                          ]
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                  ]
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                      "doi" => "10.1007/s11046-008-9099-y"
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                        "tituloSerie" => "Mycopathologia"
                        "fecha" => "2008"
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                    0 => array:2 [
                      "titulo" => "Identification of common dermatophytes &#40;<span class="elsevierStyleItalic">Trichophyton</span>&#44; <span class="elsevierStyleItalic">Microsporum</span>&#44; Epidermophyton&#41; using polymerase chain reactions"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:5 [
                            0 => "Y&#46; Gr&#228;ser"
                            1 => "M&#46; el Fari"
                            2 => "W&#46; Presber"
                            3 => "W&#46; Sterry"
                            4 => "H&#46;J&#46; Tietz"
                          ]
                        ]
                      ]
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              ]
            ]
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              "etiqueta" => "8"
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                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:5 [
                            0 => "F&#46; Seyfarth"
                            1 => "M&#46; Ziemer"
                            2 => "Y&#46; Gr&#228;ser"
                            3 => "P&#46; Elsner"
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                          ]
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                      ]
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                  ]
                  "host" => array:1 [
                    0 => array:2 [
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                        "fecha" => "2007"
                        "volumen" => "50"
                        "numero" => "Suppl&#46; 2"
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                          ]
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                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:4 [
                            0 => "C&#46;J&#46; Jackson"
                            1 => "R&#46;C&#46; Barton"
                            2 => "S&#46;L&#46; Kelly"
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                          ]
                        ]
                      ]
                    ]
                  ]
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                          ]
                        ]
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                ]
              ]
            ]
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              "referencia" => array:1 [
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                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:4 [
                            0 => "Y&#46; Graser"
                            1 => "A&#46;F&#46; Kuijpers"
                            2 => "W&#46; Presber"
                            3 => "G&#46;S&#46; de Hoog"
                          ]
                        ]
                      ]
                    ]
                  ]
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                    0 => array:1 [
                      "Revista" => array:6 [
                        "tituloSerie" => "J Clin Microbiol"
                        "fecha" => "2000"
                        "volumen" => "38"
                        "paginaInicial" => "3329"
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                          ]
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                    ]
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              ]
            ]
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              "etiqueta" => "11"
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                  "contribucion" => array:1 [
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                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "N&#46;D&#46; Padliya"
                            1 => "B&#46; Cooper"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1002/pmic.200600146"
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                        "tituloSerie" => "Proteomics"
                        "fecha" => "2006"
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                        "paginaInicial" => "4069"
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Article information

ISSN: 0213005X
Original language: English

DOI:10.1016/j.eimc.2013.01.009

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 October	15	4	19
2024 September	26	7	33
2024 August	21	7	28
2024 July	18	7	25
2024 June	35	14	49
2024 May	38	8	46
2024 April	44	23	67
2024 March	39	17	56
2024 February	23	10	33
2024 January	11	5	16
2023 December	16	10	26
2023 November	32	18	50
2023 October	26	51	77
2023 September	15	25	40
2023 August	22	15	37
2023 July	25	44	69
2023 June	36	7	43
2023 May	45	4	49
2023 April	63	7	70
2023 March	43	15	58
2023 February	22	6	28
2023 January	20	8	28
2022 December	22	21	43
2022 November	20	27	47
2022 October	16	16	32
2022 September	28	57	85
2022 August	29	32	61
2022 July	19	12	31
2022 June	12	14	26
2022 May	20	19	39
2022 April	18	21	39
2022 March	25	15	40
2022 February	23	11	34
2022 January	48	11	59
2021 December	39	19	58
2021 November	40	12	52
2021 October	26	22	48
2021 September	16	10	26
2021 August	18	5	23
2021 July	30	13	43
2021 June	23	5	28
2021 May	24	17	41
2021 April	37	14	51
2021 March	68	13	81
2021 February	26	14	40
2021 January	26	17	43
2020 December	21	10	31
2020 November	16	16	32
2020 October	11	9	20
2020 September	22	15	37
2020 August	17	13	30
2020 July	23	18	41
2020 June	18	12	30
2020 May	29	16	45
2020 April	17	8	25
2020 March	30	12	42
2020 February	32	10	42
2020 January	17	20	37
2019 December	53	27	80
2019 November	24	23	47
2019 October	32	7	39
2019 September	34	17	51
2019 August	34	3	37
2019 July	22	25	47
2019 June	42	29	71
2019 May	137	125	262
2019 April	59	27	86
2019 March	19	9	28
2019 February	23	20	43
2019 January	21	13	34
2018 December	17	23	40
2018 November	32	13	45
2018 October	37	19	56
2018 September	23	3	26
2018 August	15	0	15
2018 July	19	3	22
2018 June	12	2	14
2018 May	18	3	21
2018 April	7	0	7
2018 March	12	2	14
2018 February	11	1	12
2018 January	17	3	20
2017 December	13	1	14
2017 November	19	4	23
2017 October	19	5	24
2017 September	22	10	32
2017 August	23	2	25
2017 July	26	3	29
2017 June	25	1	26
2017 May	40	12	52
2017 April	22	17	39
2017 March	26	13	39
2017 February	40	3	43
2017 January	29	2	31
2016 December	57	7	64
2016 November	35	1	36
2016 October	66	5	71
2016 September	52	1	53
2016 August	33	1	34
2016 July	35	4	39
2016 June	39	4	43
2016 May	26	7	33
2016 April	21	5	26
2016 March	32	6	38
2016 February	22	9	31
2016 January	32	8	40
2015 December	38	11	49
2015 November	26	5	31
2015 October	46	6	52
2015 September	28	7	35
2015 August	50	8	58
2015 July	41	4	45
2015 June	23	5	28
2015 May	35	4	39
2015 April	41	9	50
2015 March	36	2	38
2015 February	35	5	40
2015 January	21	1	22
2014 December	24	5	29
2014 November	16	3	19
2014 October	23	7	30
2014 September	15	7	22
2014 August	14	2	16
2014 July	17	6	23
2014 June	7	6	13
2014 May	13	6	19
2014 April	16	9	25
2014 March	64	21	85
2014 February	73	32	105
2014 January	45	52	97

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