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The duodenal microbiota composition in children with active coeliac disease is influenced by the degree of enteropathy
La composición de la microbiota duodenal en niños con enfermedad celíaca activa está influenciada por el grado de enteropatía
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"apellidos" => "Sierra-Salinas" "email" => array:1 [ 0 => "carlos.sierra.sspa@juntadeandalucia.es" ] "referencia" => array:3 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">c</span>" "identificador" => "aff0015" ] 2 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:3 [ 0 => array:3 [ "entidad" => "Unidad de Gastroenterología y Nutrición Infantil, Hospital Materno Infantil, Málaga, Spain" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Departamento de Microbiología, Facultad de Ciencias, Málaga, Spain" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "IBIMA, Spain" "etiqueta" => "c" "identificador" => "aff0015" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "La composición de la microbiota duodenal en niños con enfermedad celíaca activa está influenciada por el grado de enteropatía" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1572 "Ancho" => 1500 "Tamanyo" => 122053 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Cluster analysis of DGGE patterns of bacterial group of the duodenal microbiota CD patients and HC.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Coeliac disease (CD) is a chronic inflammatory disorder of the small intestine characterized by a permanent intolerance to dietary gluten occurring in genetically predisposed individuals.<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">1</span></a> This disease can occur at any age and with a variety of clinical features, but typical cases often present in early childhood. Patients with CD may be asymptomatic, only with extraintestinal symptoms or silent forms. Currently, CD with a classical gastrointestinal symptoms is diagnosed by demonstrating mucosal villous atrophy, crypt hyperplasia and infiltration of intraepithelial lymphocytes.</p><p id="par0010" class="elsevierStylePara elsevierViewall">CD is a multifactorial disorder that involves interactions between genetic and environmental factors. Environmental factors include early life gluten exposure, short duration of breastfeeding, intestinal infections and changes in microbiota.</p><p id="par0015" class="elsevierStylePara elsevierViewall">Recently, scientific evidence showed changes in the intestinal microbiota composition in these patients.<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">2</span></a> So far, differences in the microbiota composition and related metabolites between CD patients and healthy controls (HC) have been reported mainly in feces.<a class="elsevierStyleCrossRefs" href="#bib0125"><span class="elsevierStyleSup">3,4</span></a> Increased bacterial diversity and changes in several bacterial groups in the duodenal microbiota of pediatric CD patients have also been reported.<a class="elsevierStyleCrossRefs" href="#bib0135"><span class="elsevierStyleSup">5–7</span></a> However, other studies have failed to show major microbiota differences between CD and HC.<a class="elsevierStyleCrossRefs" href="#bib0150"><span class="elsevierStyleSup">8–10</span></a> Cheng et al.<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">10</span></a> describe a similar microbiota composition in the duodenal mucosa between the CD and healthy children but a sub-population profile comprising eight genus-like bacterial groups was found to differ significantly between the study groups, probably with a specific role in the epithelial disruption in CD.</p><p id="par0020" class="elsevierStylePara elsevierViewall">Changes in the duodenal microbiota have been described in coeliac patients, adults and children, with active disease<a class="elsevierStyleCrossRefs" href="#bib0135"><span class="elsevierStyleSup">5,9,11</span></a> and when they were also under gluten free diet.<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">12</span></a> Wacklin et al.<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">13</span></a> found differences in the diversity and composition of the intestinal microbiota in adults with classic intestinal CD symptoms and extraintestinal symptoms; this indicated that the composition of mucosa-associated microbiota in the duodenum of the patients differed depending on the manifestations of CD.</p><p id="par0025" class="elsevierStylePara elsevierViewall">This study was designed to establish whether the duodenal mucosal microbiota of children with active CD and controls differ in composition and biodiversity in order to explain the differences in microbiota of pediatric CD patients and HC.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Material and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Subjects</span><p id="par0030" class="elsevierStylePara elsevierViewall">Duodenal biopsies were collected from children newly diagnosed with CD on a normal gluten-containing diet and from HC. The HC group consisted of children who were investigated for functional intestinal disorders of non-CD origin. All HC subjects had both negative celiac serology and showed normal small intestinal mucosa (Marsh 0).<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">14</span></a> CD patients had both positive celiac serology markers (anti-tissue transglutaminase antibodies IgA, deamidated gliadin antibodies IgG, and antiendomysium antibodies IgA) and histological lesions in duodenal biopsy.</p><p id="par0035" class="elsevierStylePara elsevierViewall">This study was conducted according to the guidelines established in the Helsinki Declaration according to EEC Good Clinical Practice Guidelines (document 111/3976/88, July 1990), and under the guidelines of current Spanish law which regulates clinical research in humans (Royal Decree 561/1993). The study protocol was approved by the Committee on Ethical Practice from CSIC and the Hospital.</p><p id="par0040" class="elsevierStylePara elsevierViewall">Written informed consent was obtained from the parents of the children included in the study. No case included in the study received antibiotic treatment within 2 months before the completion of the endoscopy. Four duodenal mucosa biopsies were taken from each patient. Two samples from each case were sent for histological examination using the Marsh score and two other samples were immediately frozen at −20<span class="elsevierStyleHsp" style=""></span>°C and kept until processing for bacteriological study. These were collected with 1<span class="elsevierStyleHsp" style=""></span>ml of PBS and centrifuged at 1000<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 5<span class="elsevierStyleHsp" style=""></span>min.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">DNA extraction</span><p id="par0045" class="elsevierStylePara elsevierViewall">Total DNA was extracted from each sample as described by Martínez et al.,<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">15</span></a> with some modifications, as reported by Tapia-Paniagua et al.<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">16</span></a> Twenty microlitres of DNA were treated with 2<span class="elsevierStyleHsp" style=""></span>μl of sodium acetate 3<span class="elsevierStyleHsp" style=""></span>M and 46<span class="elsevierStyleHsp" style=""></span>μl of isopropanol. Samples were centrifuged 12,000<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 6<span class="elsevierStyleHsp" style=""></span>min. Pellet was cleaned with ethanol 70% and centrifuged 12,000<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 6<span class="elsevierStyleHsp" style=""></span>min. Pellet was dried and resuspended in water.</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">PCR-denaturing gradient gel electrophoresis (DGGE)</span><p id="par0050" class="elsevierStylePara elsevierViewall">In order to compare DGGE patterns of the duodenal microbiota, DNA was amplified using the 16S rDNA bacterial domain-specific primers 968-GC-F and 1401-R. These primers were used to amplify V6-V8 regions of 16S rDNA. PCR mixtures and conditions to perform PCR were those previously described.<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">12</span></a> Specific amplicons were also used for the genus <span class="elsevierStyleItalic">Lactobacillus</span> (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). PCR mixtures (50<span class="elsevierStyleHsp" style=""></span>μl) contained 1.25<span class="elsevierStyleHsp" style=""></span>U Taq polymerase (Life Technologies Gaithersburg, MD, USA), 20<span class="elsevierStyleHsp" style=""></span>mM Tris–HCl (pH 8.5), 50<span class="elsevierStyleHsp" style=""></span>mM KCl, 3<span class="elsevierStyleHsp" style=""></span>mM MgCl<span class="elsevierStyleInf">2</span>, 200<span class="elsevierStyleHsp" style=""></span>μM of each deoxynucleoside triphosphate, 5<span class="elsevierStyleHsp" style=""></span>pmol of the primers, 1<span class="elsevierStyleHsp" style=""></span>μl of DNA template, and UV-sterilized water. The PCR was performed in a T1 thermocycler (Whatman Biometra, Göttingen, Germany) using 1 cycle of 94<span class="elsevierStyleHsp" style=""></span>°C for 2<span class="elsevierStyleHsp" style=""></span>min, 35 cycles of 95<span class="elsevierStyleHsp" style=""></span>°C for 30<span class="elsevierStyleHsp" style=""></span>s, 56<span class="elsevierStyleHsp" style=""></span>°C for 40<span class="elsevierStyleHsp" style=""></span>s, and 72<span class="elsevierStyleHsp" style=""></span>°C for 1<span class="elsevierStyleHsp" style=""></span>min, followed by 1 cycle of 72<span class="elsevierStyleHsp" style=""></span>°C for 5<span class="elsevierStyleHsp" style=""></span>min. Aliquots (5<span class="elsevierStyleHsp" style=""></span>μl) were analyzed by electrophoresis on 1.5% (w/v) agarose gels containing ethidium bromide to check for product size and quantity.</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0055" class="elsevierStylePara elsevierViewall">The amplicons obtained were separated by DGGE according to the specifications of Muyzer et al.<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">17</span></a> using a Dcode TM system (Bio-Rad Laboratories, Hercules, CA). Electrophoresis was performed in 8% polyacrylamide gels (37.5:1 acrylamide–bisacrylamide; dimensions, 200<span class="elsevierStyleHsp" style=""></span>mm<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>200<span class="elsevierStyleHsp" style=""></span>mm<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>1<span class="elsevierStyleHsp" style=""></span>mm) using a 30–55% denaturing gradient for separation of PCR products. The gels contained a 30–55% gradient of urea and formamide increasing in the direction of the electrophoresis. A 100% denaturing solution contained 7<span class="elsevierStyleHsp" style=""></span>M urea and 40% (vol/vol) deionized formamide. PCR samples were applied to gels in aliquots of 13<span class="elsevierStyleHsp" style=""></span>μl per lane. The gels were electrophoresed for 16<span class="elsevierStyleHsp" style=""></span>h at 85<span class="elsevierStyleHsp" style=""></span>V in 0.5× TAE (20<span class="elsevierStyleHsp" style=""></span>mM Tris acetate [pH 7.4], 10<span class="elsevierStyleHsp" style=""></span>mM sodium acetate, 0.5<span class="elsevierStyleHsp" style=""></span>mM Na<span class="elsevierStyleInf">2</span>-EDTA) buffer at a constant temperature of 60<span class="elsevierStyleHsp" style=""></span>°C and subsequently stained with AgNO<span class="elsevierStyleInf">3</span>.<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">18</span></a></p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Analysis of DGGE patterns</span><p id="par0060" class="elsevierStylePara elsevierViewall">A DGGE analysis was performed and the banding patterns were analyzed using FPQuest Software version 4.0 (Applied Maths BVBA, Sint-Martens-Latem, Belgium). A matrix of similarities for the densitometric curves of the band patterns was calculated using the band-based Pearson coefficient, and clustering of DGGE patterns was achieved by construction of dendrograms using the Unweighted Pair Groups Method using Arithmetic Averages (UPGMA). The values of Pearson coefficients obtained for each treatment were compared using multiple range tests for similarity by diet. In order to determine the structural diversity of the microbial community corresponding to the DGGE banding patterns several parameters were calculated: (1) the specific richness (<span class="elsevierStyleItalic">r</span>) was calculated based on the total number of bands; (2) the Shannon index (<span class="elsevierStyleItalic">H</span>′) was calculated following the function: <span class="elsevierStyleItalic">H</span>′=−ΣPi<span class="elsevierStyleHsp" style=""></span>log<span class="elsevierStyleHsp" style=""></span>Pi, where Pi is defined as (ni/<span class="elsevierStyleItalic">N</span>), ni is the peak surface of each band, and <span class="elsevierStyleItalic">N</span> is the sum of all the peak surfaces of all bands; (3) range-weighted richness (Rr) calculated as the total number of bands multiplied by the percentage of denaturing gradient needed to describe the total diversity of the sample analyzed, according to the following formula: Rr<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>(<span class="elsevierStyleItalic">N</span><span class="elsevierStyleSup">2</span><span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">D</span><span class="elsevierStyleInf">g</span>), where <span class="elsevierStyleItalic">N</span> represents the total number of bands in the pattern, and <span class="elsevierStyleItalic">D</span><span class="elsevierStyleInf">g</span> the denaturing gradient comprised between the first and the last band of the pattern.<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">19</span></a></p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Sequencing of DGGE bands</span><p id="par0065" class="elsevierStylePara elsevierViewall">In order to determine the principal bacteria, predominant bands in the DGGE gels were retrieved for sequencing with sterile pipette tips, placed in 100<span class="elsevierStyleHsp" style=""></span>μl of double distilled water (ddH<span class="elsevierStyleInf">2</span>O) and incubated at 4<span class="elsevierStyleHsp" style=""></span>°C overnight. Volumes of 5<span class="elsevierStyleHsp" style=""></span>μl were used as template in PCR amplification reaction performed as described above. The product was re-run on DGGE to confirm its position and further subjected to cycle sequencing with primers 968-without the GC clamp (5′-AACGCGAAGAACCTTAC-3′) and 1401-R and (5′-ACG′GCTACCTTGTTACGACTT-3′). The PCR was performed in a T1 thermocycler (Whatman Biometra, Göttingen, Germany) using one cycle of 94<span class="elsevierStyleHsp" style=""></span>°C for 2<span class="elsevierStyleHsp" style=""></span>min, 28 cycles of 95<span class="elsevierStyleHsp" style=""></span>°C for 30<span class="elsevierStyleHsp" style=""></span>s, 56<span class="elsevierStyleHsp" style=""></span>°C for 40<span class="elsevierStyleHsp" style=""></span>s and 72<span class="elsevierStyleHsp" style=""></span>°C for 1<span class="elsevierStyleHsp" style=""></span>min, followed by one cycle of 72<span class="elsevierStyleHsp" style=""></span>°C for 5<span class="elsevierStyleHsp" style=""></span>min. Products were purified using the High Pure Spin Kit PCR purification kit (Roche). The sequencing of the amplicons was performed on the ABI PRISM 377 sequencer (Perkin-Elmer). The sequence was read from both directions with primers RV-M and M13-47, respectively. The resulting sequences (∼500<span class="elsevierStyleHsp" style=""></span>bp) were compared with the sequences from the National Center for Biotechnology Information (NCBI) or Greengenes DNA sequence database using the BLAST<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">21</span></a> sequence algorithm. Database sequences showing the highest identity were used to infer identity. Although the high resolution of DGGE does not exclude the possibility that two different 16S rDNA sequences might migrate to exactly the same position, all sequences that migrated to the same position were sequenced. Chimeric sequences were identified by using the CHECK CHIMERA program of the Ribosomal Database Project.<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">22</span></a></p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Principal components analysis (PCA)</span><p id="par0070" class="elsevierStylePara elsevierViewall">Principal component analysis (PCA) is a mathematical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables. It has been used to summarize RFLP profiles.<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">23</span></a> In the present study it has been applied to correlate different such as ecological parameters (<span class="elsevierStyleItalic">H</span>′, <span class="elsevierStyleItalic">R</span> and Rr), histological parameters and the proportion of the DNA of each microbial species detected in the intestinal microbiota assayed with the intestinal microbiota of these patients. PCA has been performed using the LXSTAT 2012 software (Addinsoft, Spain) calculated with the help of the MS excel software XLSTAT 2012.1.01; Addinsoft The influence of the variables in the PCA was determined by the distance of each variable from the center of the axis d ≥√2/<span class="elsevierStyleItalic">n</span>, where <span class="elsevierStyleItalic">n</span> is the number of variables. For determining the quantity of DNA, the intensities obtained from each of the DGGE bands were used.</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Statistical analysis</span><p id="par0075" class="elsevierStylePara elsevierViewall">The data expressed as mean and standard deviation were analyzed with SPSS (Statistical Package for Social Science for Windows, version 15.0, SPSS Inc., Chicago, IL, USA). The ecological indices were analyzed with the Kruskal–Wallis test using the Statgraphics Plus 5.0 software (Statgraphics Corporation, Rockville, MD, USA). Differences were considered statistically significant when <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>≤<span class="elsevierStyleHsp" style=""></span>0.05.</p></span></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Results</span><p id="par0080" class="elsevierStylePara elsevierViewall">Duodenal biopsy specimens were collected from 11 children with newly diagnosed CD on a normal gluten-containing diet, and 6 HC (<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>).</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Microbiota diversity and composition by DGGE</span><p id="par0085" class="elsevierStylePara elsevierViewall">The values of richness, diversity, and range-weighted richness did not show significant differences among HC subjects and CD patients (<a class="elsevierStyleCrossRef" href="#tbl0015">Table 3</a>), whereas the three values were significantly lower for CD compared to HC when the <span class="elsevierStyleItalic">Lactobacillus</span> genus was analyzed (<a class="elsevierStyleCrossRef" href="#tbl0015">Table 3</a>).</p><elsevierMultimedia ident="tbl0015"></elsevierMultimedia><p id="par0090" class="elsevierStylePara elsevierViewall">Furthermore, a clustering analysis was applied to the duodenal microbiota DGGE patterns. This resulted in high intragroup similarity percentages (98%) in patients that presented histology associated with Marsh 3c type. The other patients showed a similarity from 80% until 30% and the group was not based on other characteristics (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>).</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0095" class="elsevierStylePara elsevierViewall">A second dendrogram was done only with the amplicons of <span class="elsevierStyleItalic">Lactobacillus</span> genus showing two separate clusters with a 40% of similarity, approximately but the groups were not based on any analyzed characteristic. (Data not shown.)</p><p id="par0100" class="elsevierStylePara elsevierViewall">Bands showing higher intensities in DGGE gels of samples of biopsies were sequenced and compared with BLAST references based on the phylogenetic relationship of the ∼500<span class="elsevierStyleHsp" style=""></span>bp partial 16S rDNA sequence (<a class="elsevierStyleCrossRef" href="#tbl0020">Table 4</a>). The bands were mainly related to species such as <span class="elsevierStyleItalic">Streptococcus</span>, <span class="elsevierStyleItalic">Bacteroides</span> and <span class="elsevierStyleItalic">Escherichia coli</span> in CD patients, while in HC the predominant bands <span class="elsevierStyleItalic">were Bifidobacterium</span>, <span class="elsevierStyleItalic">Acinetobacter</span>, and <span class="elsevierStyleItalic">Lactobacillus</span>. Other groups like <span class="elsevierStyleItalic">Streptococcus</span> (17–13%) or Bacteroides (16–11%) were minor.</p><elsevierMultimedia ident="tbl0020"></elsevierMultimedia><p id="par0105" class="elsevierStylePara elsevierViewall">The PCA shows that two factors (F1 and F2) are able to explain 83.44% of the variability found between the microbiota and the other variables studied in patients and controls. It is noteworthy that the variables related to the richness, diversity and habitability of the genus <span class="elsevierStyleItalic">Lactobacillus</span> change from reverse to the other variables used by the F1 addressed. Also the F1 separates both axes, HC from CD patients, so that F1 is a factor related to the presence or absence of disease (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>).</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Discussion</span><p id="par0110" class="elsevierStylePara elsevierViewall">The structural diversity of the microbial community corresponding to the DGGE banding patterns did not show significant differences between HC and CD. DGGE analysis with universal primers did not show a higher bacterial diversity associated with the small intestinal microbiota of CD but the ecological parameters, (<span class="elsevierStyleItalic">H</span>′, <span class="elsevierStyleItalic">R</span> and Rr), were significantly lower for CD compared to HC when the <span class="elsevierStyleItalic">Lactobacillus</span> genus was analyzed.</p><p id="par0115" class="elsevierStylePara elsevierViewall">Nadal et al.,<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">5</span></a> conducted bacteriological analyses of duodenal biopsy specimens based on pediatric patients with CD. Their results showed that patients with active CD had significantly higher numbers of total bacteria, especially Gram-negative bacteria, compared with asymptomatic patients and healthy subjects and the ratio of <span class="elsevierStyleItalic">Lactobacillus-Bifidobacterium</span> to <span class="elsevierStyleItalic">Bacteroides-E. coli</span> was lower in patients with CD. Nistal et al.<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">9</span></a> analyzed the bacterial 16S rRNA gene sequencing of DNA extracted from duodenal biopsies and showed that the diversity of duodenal microbiota was significantly different between treated and untreated adults with CD.</p><p id="par0120" class="elsevierStylePara elsevierViewall">In our study the patients with Marsh 3c lesion were clustered separately in PCA of the DGGE profiles. The other patients with Marsh 3a and Marsh 3b and one case of Marsh 2, shared different microbiota as compared with more closely clustered patients with Marsh 3c. This indicated that the composition of duodenal microbiota differed depending on the grade of intestinal damage.</p><p id="par0125" class="elsevierStylePara elsevierViewall">Probably the conditions of the intestinal environment in cases of severe villous atrophy cause a profound change in the microbial community. Some changes in the duodenal microbial composition may be due to the destructive inflammatory consequences pattern, most evident in greater duodenal mucosa villous atrophy. In these cases the intestinal inflammatory effect exerted by the gluten may further activate the intestinal inflammation, a consequence of the destructive pattern of atrophic lesion related to Marsh 3c type. We speculate that the most intense atrophy of the duodenal mucosa in untreated CD, as Marsh 3c, promotes a colonization of mucosal similar between these patients.</p><p id="par0130" class="elsevierStylePara elsevierViewall">More studies are necessary to understand the microbiota–host interaction and unravel the significance of specific microbiota to determine if it is just a consequence of the EC or are involved in the variability and the different degrees of enteropathy of the disease. In this respect it has recently been associated with alterations in intestinal microbiota as the reduction of microbial richness, with persistent symptoms in treated CD.<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">24</span></a></p><p id="par0135" class="elsevierStylePara elsevierViewall">We expect further research to find bacteriological markers in the duodenal microbiota of CD for diagnostic and therapeutic reasons.</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Conflict of interest</span><p id="par0140" class="elsevierStylePara elsevierViewall">The authors declare no conflict of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:10 [ 0 => array:3 [ "identificador" => "xres623597" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Objectives" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Material and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusions" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec637227" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres623598" "titulo" => "Resumen" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0025" "titulo" => "Objetivo" ] 1 => array:2 [ "identificador" => "abst0030" "titulo" => "Material y Métodos" ] 2 => array:2 [ "identificador" => "abst0035" "titulo" => "Resultados" ] 3 => array:2 [ "identificador" => "abst0040" "titulo" => "Conclusiones" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec637226" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Material and methods" "secciones" => array:7 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Subjects" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "DNA extraction" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "PCR-denaturing gradient gel electrophoresis (DGGE)" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Analysis of DGGE patterns" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Sequencing of DGGE bands" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Principal components analysis (PCA)" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Statistical analysis" ] ] ] 6 => array:3 [ "identificador" => "sec0050" "titulo" => "Results" "secciones" => array:1 [ 0 => array:2 [ "identificador" => "sec0055" "titulo" => "Microbiota diversity and composition by DGGE" ] ] ] 7 => array:2 [ "identificador" => "sec0060" "titulo" => "Discussion" ] 8 => array:2 [ "identificador" => "sec0065" "titulo" => "Conflict of interest" ] 9 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2015-05-04" "fechaAceptado" => "2015-06-04" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec637227" "palabras" => array:4 [ 0 => "Coeliac disease" 1 => "Microbiota" 2 => "MARSH" 3 => "<span class="elsevierStyleItalic">Lactobacillus</span>" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec637226" "palabras" => array:4 [ 0 => "Enfermedad celíaca" 1 => "Microbiota" 2 => "MARSH" 3 => "<span class="elsevierStyleItalic">Lactobacillus</span>" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Objectives</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">To establish whether the duodenal mucosa microbiota of children with active coelic disease (CD) and healthy controls (HC) differ in composition and biodiversity.</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Material and methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Samples of duodenal biopsies in 11 CD patients were obtained at diagnosis, and in 6 HC who were investigated for functional intestinal disorders of non-CD origin. Total duodenal microbiota and that belonging to the genus <span class="elsevierStyleItalic">Lactobacillus</span> using PCR-denaturing gradient gel electrophoresis (DGGE) were analyzed. The banding patterns obtained in the resulting gels were analyzed to determine the differences between the microbiota of CD patients and HC (FPQuest 4.5) while environmental indexes (richness, diversity and habitability) were calculated with the past version 2.17 program.</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">The intestinal microbiota of patients with Marsh 3c lesion showed similarity of 98% and differs from other CD patients with other type of histologic lesion as Marsh 3a, Marsh 3b and Marsh 2. The main differences were obtained in ecological indexes belonging to the genus <span class="elsevierStyleItalic">Lactobacillus</span>, with significant richness, diversity and habitability reduction in CD patients. In CD bands were categorized primarily with <span class="elsevierStyleItalic">Streptococcus</span>, <span class="elsevierStyleItalic">Bacteroides</span> and <span class="elsevierStyleItalic">Escherichia coli</span> species. In HC the predominant bands were <span class="elsevierStyleItalic">Bifidobacterium</span>, <span class="elsevierStyleItalic">Lactobacillus</span> and <span class="elsevierStyleItalic">Acinetobacter</span>, though the <span class="elsevierStyleItalic">Streptococcus</span> and <span class="elsevierStyleItalic">Bacteroides</span> were lower.</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">The celiac patients with major histological affectation presented a similar microbiota duodenal. The ecological indexes applied to the genus <span class="elsevierStyleItalic">Lactobacillus</span> were significantly reduced in CD.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Objectives" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Material and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusions" ] ] ] "es" => array:3 [ "titulo" => "Resumen" "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Objetivo</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Comprobar diferencias en la microbiota duodenal al diagnóstico de la EC en relación con grupo control.</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Material y Métodos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Se obtuvieron muestras de biopsias duodenales en 11 pacientes con EC al diagnostico y en 6 controles. Se analizó la microbiota duodenal total así como la perteneciente al género <span class="elsevierStyleItalic">Lactobacillus</span> mediante la técnica molecular PCR-Electroforesis en gel con gradiente desnaturalizante (DGGE). Los patrones de bandas obtenidos en los geles resultantes fueron analizados para determinar las diferencias presentes entre la microbiota de pacientes con EC y controles (FPQuest 4.5) mientras que los índices ecológicos (riqueza, diversidad y habitabilidad) fueron calculados con el programa Past versión 2.17.</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">La microbiota intestinal de los individuos con histología Marsh 3c presentó similitud del 98% y fue diferente del resto de pacientes celiacos. Las principales diferencias se obtuvieron en los índices ecológicos pertenecientes al género <span class="elsevierStyleItalic">Lactobacillus</span>, con importante reducción de especies en los celiacos respecto al grupo control (riqueza, diversidad y habitabilidad). En los pacientes con EC las bandas principalmente fueron catalogadas con las especies <span class="elsevierStyleItalic">Streptococcus</span>, <span class="elsevierStyleItalic">Bacteroides</span> y <span class="elsevierStyleItalic">E. coli.</span> En los controles las bandas predominantes fueron <span class="elsevierStyleItalic">Bifidobacterium</span>, <span class="elsevierStyleItalic">Acinetobacter</span> y <span class="elsevierStyleItalic">Lactobacillus</span>, sin embargo los <span class="elsevierStyleItalic">Streptococcus</span> y Bacteroides fueron más bajos.</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclusiones</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Los índices ecológicos aplicados al género <span class="elsevierStyleItalic">Lactobacillus</span> fueron significativamente reducidos en los pacientes celíacos. Los casos con mayor afectación histológica presentaron una microbiota duodenal similar.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0025" "titulo" => "Objetivo" ] 1 => array:2 [ "identificador" => "abst0030" "titulo" => "Material y Métodos" ] 2 => array:2 [ "identificador" => "abst0035" "titulo" => "Resultados" ] 3 => array:2 [ "identificador" => "abst0040" "titulo" => "Conclusiones" ] ] ] ] "NotaPie" => array:1 [ 0 => array:2 [ "etiqueta" => "☆" "nota" => "<p class="elsevierStyleNotepara" id="npar0010">Please cite this article as: Girón Fernández-Crehuet F, Tapia-Paniagua S, Morinigo-Gutiérrez MA, Navas-López VM, Serrano MJ, Blasco-Alonso J, et al. La composición de la microbiota duodenal en niños con enfermedad celíaca activa está influenciada por el grado de enteropatía. An Pediatr (Barc). 2016;84:224–230.</p>" ] ] "multimedia" => array:6 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1572 "Ancho" => 1500 "Tamanyo" => 122053 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Cluster analysis of DGGE patterns of bacterial group of the duodenal microbiota CD patients and HC.</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1418 "Ancho" => 3167 "Tamanyo" => 231845 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">PCA applied to microbiota composition of biopsies and parameters analyzed in CD patients and in healthy controls (HC). Correlation of assayed variables and principal components explaining 83.44% of variability. Distribution of subjects of CD and HC along axis F1 and F2.</p>" ] ] 2 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Primers \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Sequence (5′–3′) \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Size product (bp) \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Ref. \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry " colspan="4" align="left" valign="top"><span class="elsevierStyleItalic">Universal primers for Bacteria</span></td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>968-GC-F \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′GGGAACGCGAAGAACCTTAC-3 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " rowspan="2" align="char" valign="middle">470</td><td class="td" title="table-entry " rowspan="2" align="left" valign="middle"><a class="elsevierStyleCrossRef" href="#bib0210">[20]</a></td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>1401-R \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′CGGTGTGTACAAGACCC-3′ \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " colspan="4" align="left" valign="top"><span class="elsevierStyleVsp" style="height:0.5px"></span></td></tr><tr title="table-row"><td class="td" title="table-entry " colspan="4" align="left" valign="top"><span class="elsevierStyleItalic">Primers for Lactobacillus genus</span></td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>Lac1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">AGCAGTAGGGAATCTTCCA \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " rowspan="2" align="char" valign="middle">380</td><td class="td" title="table-entry " rowspan="2" align="left" valign="middle"><a class="elsevierStyleCrossRef" href="#bib0130">[4]</a></td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>Lac2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">GCclamp-ATTYCACCGCT ACACATG \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1023040.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Primers used for analysis of intestinal microbiota by DGGE.</p>" ] ] 3 => array:7 [ "identificador" => "tbl0010" "etiqueta" => "Table 2" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "leyenda" => "<p id="spar0065" class="elsevierStyleSimplePara elsevierViewall">Modified Marsh classification of CD: Marsh 0, normal mucosa; Marsh 1, infiltrative lesion: increased numbers of intraepithelial lymphocytes in the villous epithelium in an otherwise normal mucosa with normal villous to crypt ratio; Marsh 2, hyperplasic type: crypt hyperplasia with normal villi showing increased numbers of intraepithelial lymphocytes; Marsh 3, atrophic lesion with crypt hyperplasia and increased numbers of intraepithelial lymphocytes (Marsh 3a: mild villous atrophy; Marsh 3b: marked villous atrophy; Marsh 3c: flat mucosa).<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">14</span></a></p>" "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Characteristic \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">CD \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">HC \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Number</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">11 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">6 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Mean (SD) age (yr)</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5.0 (3.4) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">8.8 (3.1) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Sex (male/female)</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">4/7 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">3/3 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Positive anti-tissue transglutaminase antibodies IgA</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">11 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Positive deamidated gliadin antibodies IgG</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">11 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Positive antiendomysium antibodies IgA</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">11 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Duodenal biopsy</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>Marsh classification (number of cases) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Marsh 2 (1)<br>Marsh 3a (2)<br>Marsh 3b (2)<br>Marsh 3c (6) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Marsh 0 (6) \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1023037.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">Clinical characteristics of study subjects: untreated coeliac patients and healthy controls.</p>" ] ] 4 => array:7 [ "identificador" => "tbl0015" "etiqueta" => "Table 3" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Richness \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Diversity \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Range-weighted richness \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">CD \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">22.1<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>5.04 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2.91<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>0.2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">100.36<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>47.44 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">HC \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">19.87<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>5.1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2.50<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>0.29 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">74.38<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>37.38 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " colspan="4" align="left" valign="top"><span class="elsevierStyleItalic">Lactobacillus genus from biopsies</span></td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>CD \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">6<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>2<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">*</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.5<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>0.2<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">*</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">9<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>1<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">*</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>HC \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">15<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>3 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2.2<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>0.2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">56.25<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>2.25 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1023039.png" ] ] ] "notaPie" => array:1 [ 0 => array:3 [ "identificador" => "tblfn0005" "etiqueta" => "*" "nota" => "<p class="elsevierStyleNotepara" id="npar0005"><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.01.</p>" ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0070" class="elsevierStyleSimplePara elsevierViewall">Richness, diversity and range-weighted values obtained from microbiota of biopsies in CD patients and in healthy controls.</p>" ] ] 5 => array:7 [ "identificador" => "tbl0020" "etiqueta" => "Table 4" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">HC (%) \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">CD (%) \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Lactobacillus</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">7 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Streptococcus</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">17 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Bacteroides \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">11 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">16 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Acinetobacter</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">10 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">E. coli</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">10 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">16 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Bifidobacterium</span> (<span class="elsevierStyleItalic">B. brevis</span>, <span class="elsevierStyleItalic">B. bifidum</span>, <span class="elsevierStyleItalic">B. lactis</span>) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">10 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">5 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Clostridium</span> (<span class="elsevierStyleItalic">C. coccoides</span>) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">9 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">8 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Eubacterium</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">9 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">7 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Not identified \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">9 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">16 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Not cultivated \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">6 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">8 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1023038.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0075" class="elsevierStyleSimplePara elsevierViewall">Relative abundance of different microbial groups sequenced from the duodenal microbiota in HC and in CD.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:24 [ 0 => array:3 [ "identificador" => "bib0115" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Celiac disease" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:2 [ 0 => "P.H. 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Lähdeaho" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1038/ajg.2014.355" "Revista" => array:6 [ "tituloSerie" => "Am J Gastroenterol" "fecha" => "2014" "volumen" => "109" "paginaInicial" => "1933" "paginaFinal" => "1941" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/25403367" "web" => "Medline" ] ] ] ] ] ] ] ] ] ] ] ] ] "idiomaDefecto" => "en" "url" => "/23412879/0000008400000004/v1_201603300938/S2341287915001520/v1_201603300938/en/main.assets" "Apartado" => array:4 [ "identificador" => "26005" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Original Articles" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/23412879/0000008400000004/v1_201603300938/S2341287915001520/v1_201603300938/en/main.pdf?idApp=UINPBA00005H&text.app=https://analesdepediatria.org/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S2341287915001520?idApp=UINPBA00005H" ]
| Year/Month | Html | Total | |
|---|---|---|---|
| 2024 November | 7 | 10 | 17 |
| 2024 October | 35 | 45 | 80 |
| 2024 September | 52 | 23 | 75 |
| 2024 August | 71 | 64 | 135 |
| 2024 July | 47 | 25 | 72 |
| 2024 June | 51 | 17 | 68 |
| 2024 May | 50 | 30 | 80 |
| 2024 April | 54 | 36 | 90 |
| 2024 March | 42 | 18 | 60 |
| 2024 February | 44 | 19 | 63 |
| 2024 January | 38 | 24 | 62 |
| 2023 December | 17 | 21 | 38 |
| 2023 November | 39 | 16 | 55 |
| 2023 October | 30 | 23 | 53 |
| 2023 September | 22 | 22 | 44 |
| 2023 August | 31 | 18 | 49 |
| 2023 July | 30 | 20 | 50 |
| 2023 June | 27 | 30 | 57 |
| 2023 May | 38 | 22 | 60 |
| 2023 April | 20 | 17 | 37 |
| 2023 March | 22 | 21 | 43 |
| 2023 February | 22 | 10 | 32 |
| 2023 January | 16 | 20 | 36 |
| 2022 December | 33 | 38 | 71 |
| 2022 November | 35 | 36 | 71 |
| 2022 October | 42 | 43 | 85 |
| 2022 September | 45 | 41 | 86 |
| 2022 August | 40 | 38 | 78 |
| 2022 July | 30 | 37 | 67 |
| 2022 June | 33 | 34 | 67 |
| 2022 May | 39 | 39 | 78 |
| 2022 April | 38 | 37 | 75 |
| 2022 March | 46 | 57 | 103 |
| 2022 February | 33 | 26 | 59 |
| 2022 January | 34 | 42 | 76 |
| 2021 December | 43 | 34 | 77 |
| 2021 November | 45 | 50 | 95 |
| 2021 October | 40 | 50 | 90 |
| 2021 September | 26 | 40 | 66 |
| 2021 August | 30 | 34 | 64 |
| 2021 July | 18 | 35 | 53 |
| 2021 June | 31 | 31 | 62 |
| 2021 May | 39 | 36 | 75 |
| 2021 April | 83 | 73 | 156 |
| 2021 March | 37 | 34 | 71 |
| 2021 February | 31 | 19 | 50 |
| 2021 January | 47 | 27 | 74 |
| 2020 December | 35 | 22 | 57 |
| 2020 November | 23 | 19 | 42 |
| 2020 October | 35 | 13 | 48 |
| 2020 September | 30 | 19 | 49 |
| 2020 August | 35 | 15 | 50 |
| 2020 July | 32 | 14 | 46 |
| 2020 June | 30 | 13 | 43 |
| 2020 May | 34 | 23 | 57 |
| 2020 April | 35 | 11 | 46 |
| 2020 March | 50 | 16 | 66 |
| 2020 February | 41 | 26 | 67 |
| 2020 January | 20 | 11 | 31 |
| 2019 December | 55 | 24 | 79 |
| 2019 November | 19 | 18 | 37 |
| 2019 October | 37 | 14 | 51 |
| 2019 September | 25 | 13 | 38 |
| 2019 August | 53 | 19 | 72 |
| 2019 July | 22 | 17 | 39 |
| 2019 June | 23 | 25 | 48 |
| 2019 May | 31 | 15 | 46 |
| 2019 April | 30 | 27 | 57 |
| 2019 March | 27 | 19 | 46 |
| 2019 February | 36 | 14 | 50 |
| 2019 January | 19 | 22 | 41 |
| 2018 December | 36 | 22 | 58 |
| 2018 November | 78 | 30 | 108 |
| 2018 October | 75 | 19 | 94 |
| 2018 September | 25 | 10 | 35 |
| 2018 August | 4 | 0 | 4 |
| 2018 July | 1 | 0 | 1 |
| 2018 June | 5 | 0 | 5 |
| 2018 May | 5 | 0 | 5 |
| 2018 April | 13 | 0 | 13 |
| 2018 March | 16 | 0 | 16 |
| 2018 February | 11 | 0 | 11 |
| 2018 January | 14 | 0 | 14 |
| 2017 December | 12 | 0 | 12 |
| 2017 November | 22 | 0 | 22 |
| 2017 October | 11 | 0 | 11 |
| 2017 September | 13 | 0 | 13 |
| 2017 August | 12 | 0 | 12 |
| 2017 July | 16 | 0 | 16 |
| 2017 June | 19 | 6 | 25 |
| 2017 May | 18 | 4 | 22 |
| 2017 April | 16 | 3 | 19 |
| 2017 March | 28 | 4 | 32 |
| 2017 February | 24 | 3 | 27 |
| 2017 January | 12 | 3 | 15 |
| 2016 December | 28 | 17 | 45 |
| 2016 November | 25 | 14 | 39 |
| 2016 October | 22 | 9 | 31 |
| 2016 September | 36 | 6 | 42 |
| 2016 August | 41 | 9 | 50 |
| 2016 July | 17 | 4 | 21 |
| 2016 June | 1 | 0 | 1 |
| 2016 April | 2 | 0 | 2 |
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