No Change in the Mucosal Gut Microbiome is Associated With Celiac Disease-Specific Microbiome Alteration in Adult Patients

No Change in the Mucosal Gut Microbiome is Associated With Celiac Disease-Specific Microbiome Alteration in Adult Patients

theoretical hypothesis implicated Candida ( C .) albicans in CD onset, mainly because a hypthal wall protein 1 (HWP1) was found to share epitopes with gliadins ( 7 ). Very recently, a cross-reactivity between HWP1 and gliadin was reported in CD-and in C. albicans-aff ected patients ( 8 ). However, in our cohort, C. albicans infection did not seem to diff er between CD patients and controls. Th e lower fungal/bacterial equilibrium in CD patients than in controls or GFD patients ( P <0.01) ( Figure 2 ) may further support that bacterial dysbiosis has a role in active CD ( 1 ).
In conclusion, this is the fi rst study in which the mycobioma of the duodenal mucosa of CD patients has been studied using the high-throughput technology of NGS. Although our fi nding remains to be extended to other cohorts, it shows that changes in the gut mycobioma are not associated with CD-specifi c alterations in adults; however, the changes we found in bacterial dysbiosis of CD patients ( 1 ) appear to be indirectly confi rmed.

ACKNOWLEDGMENTS
We thank Jean Ann Gilder (Scientifi c Communication srl., Naples) for editing the text and Vittorio Lucignano (CEINGE-Biotecnologie Avanzate) for technical assistance related to graphics.

CONFLICT OF INTEREST
Guarantor of the article: Francesco Salvatore, MD, PhD. Specifi c author contributions: Valeria D' Argenio, Giorgio Casaburi, Francesco Salvatore, and Lucia Sacchetti conceived and designed the study; all the other authors contributed to diff erent aspects of the study (enrollment of patient and sample collection, molecular assays, microbiological evaluations, and bioinformatics studies). Valeria D' Argenio, Giorgio Casaburi, Francesco Salvatore, and Lucia Sacchetti analyzed the fi nal data and wrote the manuscript. All authors have read, revised, contributed to the writing, and approved the fi nal manuscript. Financial support: Th is work was supported by grant 007_FC_2014 from the "Fondazione Italiana Celiachia" (to Lucia Sacchetti), by DIAINTECH-Regione Campania (to Francesco Salvatore), by PRIN 2012 (n. 2012WJSX8K) and by POR Campania FSE 2007-2013, Project histological alterations. We obtained local Ethics Committee (Prot. N.36/13) approval for the study and informed consent from all enrolled subjects. We characterized the mycobioma from phylum to the species level by sequencing the highly variable internal transcribed spacer (ITS)1 DNA region. Duodenal biopsies were collected under sterile conditions, immediately cooled in dry ice, and stored at −80 °C until analysis. Total DNA was extracted and the ITS region amplifi ed with ITS1F ( CTTGGTCATTTAGAGGAAGTAA ) and ITS2 ( GCTGCGTTCTTCATCGATGC ) primers. Polymerase chain reaction and next-generation sequencing (NGS) conditions were as previously reported. Bioinformatics analysis was conducted in QIIME v.1.9.1 ( 5 ) using an open-reference operational taxonomic unit (OTU)-picking approach against the UNITE ITS database (January, 2016, ( 6 )). Diversity analyses were computed as alpha rarefaction curves considering the number of observed species and by principle coordinates analyses (PCoA) using Bray-Curtis (non-phylogenetic) distances. Using our previous bacterial data, we calculated the ITS1/16S species ratio using the Kruskal-Wallis test with Dunn's multiple comparison test to identify signifi cance in the three groups studied.
Th e genus Candida was present only in a few controls (2/10) and active CD patients (2/14) and not in comparable proportions (3.3%/30.7% and 85.7%/0.01%, respectively). A few years ago, an elegant To the Editor: Th e intestinal microbiota has a relevant role in human health, and its alteration has been implicated in several gastrointestinal disorders. We recently described variations, from phylum to the genus level, in duodenal microbiomas from adults with celiac disease (CD) and reported the presence of a peculiar Neisseria fl avescens species able to induce duodenal infl ammation ( 1 ). In addition to bacteria, fungi also contribute to the gut microbiota composition ( 2 ). Alterations in the fungal microbioma (mycobioma) have been recently described in stools and colonic mucosa of patients with infl ammatory bowel diseases ( 3,4 ), whereas data on the duodenal mycobioma of CD-patients are scarce. To probe the contribution of fungi to CD onset, we investigated the duodenal mycobioma in three groups of adults: 14 patients with active CD (CDantibodies/histology Marsh III, positivity), six patients on a gluten-free diet (GFD), and 10 non-CD controls; the latter two groups were negative for CD-antibodies/  , and patients on a gluten-free diet (GFD). The mycobioma composition did not differ signifi cantly between the three groups studied at phylum ( a ) or class ( b ), to species levels. Data are expressed as relative operational taxonomic unit (OTU) abundances in each subject. Diversity analysis computed as alpha-rarefaction curves ( c ) considering the number of observed species in the various groups showed no difference in fungal community richness between controls and gluten-free diet (GFD) patients, although the number of ITS OTUs was lower in the mycobiomas of celiac disease (CD) patients than in those of GFD patients and controls ( P =0.04 and P =0.02, respectively). ( d ) Principal coordinates analysis (PCoA) using Bray-Curtis distance matrices depicting the distribution between samples colored in red (controls), blue (active CD-patients), and green (GFD-patients). PCoA showed a weak correlation within fungal communities when samples were grouped by disease status ( R 2 =0.08, P =0.014). To the Editor: Th e pancreas is an uncommon site of metastasis for small cell lung cancer (SCLC). We report a case of extensive stage SCLC where acute pancreatitis was the initial manifestation of widely metastatic disease. A 79-year-old man with type 2 diabetes mellitus was referred for the evaluation of newly diagnosed chronic pancreatitis. He had presented with an episode of acute abdominal pain two months prior to referral. Blood tests at that time revealed a serum lipase elevated to greater than three times the upper limit of normal. An abdominal CT scan obtained during that episode demonstrated peripancreatic infl ammation and calcifi cation in the body of the pancreas with mild upstream dilation of the main pancreatic duct (MPD) suggestive of pre-existing chronic pancreatitis ( Figure 1a ). Th e patient had a longstanding history of diabetes mellitus with remote history of cigarette smoking and occasional alcohol use. He continued to experience intermittent abdominal pain and was referred for endoscopic interven-