Role of fungi in chronic rhinosinusitis through ITS sequencing

Abstract

Objective: Next-generation sequencing increases the sensitivity of fungal identification and may improve our understanding of the role that fungi play in sinus health and disease, which remains incompletely understood. We sequenced the internal transcribed spacer (ITS) amplicon to explore the role of the mycobiome in chronic rhinosinusitis (CRS).Methods: Swabs were collected intraoperatively from the middle meatus of 90 patients (63 with CRS; 27 controls). DNA was extracted, and ITS amplicon concentration was measured using fluorometry. Internal transcribed spacer amplicons were sequenced on the Illumina MiSeq (Illumina Inc San Diego CA). Sequencing data were analyzed using QIIME.Results: Using conventional detection techniques of culture and histology, fungi only were identified in nine of 63 (14.3%) CRS patients (fungus-identified group); the remaining 54 CRS patients and all controls did not have fungus identified using the traditional techniques. This fungus-identified group had a significantly higher average ITS concentration and a significantly lower Shannon's diversity index compared to the other two groups. The most abundant organism sequenced was Aspergillus (35.22% of all sequences). Multivariate analysis showed that positive fungal detection using traditional techniques and computed tomography (CT) double densities were the most important clinical predictors of a high fungal biomass, whereas Lund-Mackay score, polyps, eosinophilia, and eosinophilic mucus were not significant in comparison.Conclusion: Fungal biomass estimated through ITS amplicon concentration correlated with traditional fungal detection techniques and CT double densities. Our results suggest that fungal dysbiosis only occurs in the sinuses of a selected subset of patients, and therefore could not be a universal determinant of sinus disease pathogenesis in all CRS patients.

Characterizing the Human Mycobiota: A Comparison of Small Subunit rRNA, ITS1, ITS2, and Large Subunit rRNA Genomic Targets

“…Fungal species candidates were selected based on taxa previously identified in a broad range of published studies, including of the lung ( Agbetile et al, 2012 ; Charlson et al, 2012 ; Delhaes et al, 2012 ; Huffnagle and Noverr, 2013 ; Liu et al, 2013 ; van Woerden et al, 2013 ; Bittinger et al, 2014 ; Chotirmall and McElvaney, 2014 ; Pashley, 2014 ; Pourfathollah et al, 2014 ; Willger et al, 2014 ; Eickmeier et al, 2015 ; Nguyen et al, 2015 ; Sharpe et al, 2015 ), sinonasal cavity ( Aurora et al, 2013 ; Cleland et al, 2014 ; Jung et al, 2015 ; Comacle et al, 2016 ; Gelber et al, 2016 ; Zhao et al, 2017 ), oral cavity ( Ghannoum et al, 2010 ; Huffnagle and Noverr, 2013 ; Dupuy et al, 2014 ; Underhill and Iliev, 2014 ; Vesty et al, 2017 ), gastrointestinal tract ( Huffnagle and Noverr, 2013 ; Kim et al, 2014 ; Li et al, 2014 ; Underhill and Iliev, 2014 ; Wang Z.K. et al, 2014 ; Gerard et al, 2015 ; Tang et al, 2015 ; Hoarau et al, 2016 ; Liguori et al, 2016 ; Strati et al, 2016 ; Hallen-Adams and Suhr, 2017 ; Nash et al, 2017 ), vagina ( Underhill and Iliev, 2014 ), and skin ( Zhang et al, 2011 ; Park et al, 2012 ; Findley et al, 2013 ; Huffnagle and Noverr, 2013 ; Underhill and Iliev, 2014 ).…”

“…Primers were selected based on representative primer pairs for each of the four target genomic regions that have been commonly used in published mammalian mycobiota studies: Euk1a – Euk516r (SSU) ( Sogin and Gunderson, 1987 ; Amann et al, 1990 ; van Woerden et al, 2013 ); ITS1F – ITS2 (ITS1) ( White et al, 1990 ; Gardes and Bruns, 1993 ; Ghannoum et al, 2010 ; Charlson et al, 2012 ; Bittinger et al, 2014 ; Dupuy et al, 2014 ; Wang X. et al, 2014 ; Hoarau et al, 2016 ; Botschuijver et al, 2017 ; Hamad et al, 2017 ; Huseyin et al, 2017 ; Motooka et al, 2017 ; Schei et al, 2017 ; Zhao et al, 2017 ); ITS3 – ITS4 (ITS2) ( White et al, 1990 ; Wang X. et al, 2014 ; Strati et al, 2016 ; Gao et al, 2017 ; Hamad et al, 2017 ); NL1 – NL4 (LSU) ( O’Donnell, 1992 ; Zhang et al, 2011 ; Park et al, 2012 ; Table 1 ). Target regions were amplified and sequenced for each of the 21 mock community taxa individually, and as a mixed community which was constructed by pooling equal concentrations of DNA to generate an even 21 species mock community.…”

“…This was further supported in a later study by Tedersoo et al (2015) , highlighting ITS2 in particular as the strongest candidate. Nonetheless, an array of marker targets have been used in human microbiota studies, including both ITS regions ( Ghannoum et al, 2010 ; Charlson et al, 2012 ; Delhaes et al, 2012 ; Findley et al, 2013 ; Bittinger et al, 2014 ; Dupuy et al, 2014 ; Wang X. et al, 2014 ; Willger et al, 2014 ; Hoarau et al, 2016 ; Liguori et al, 2016 ; Strati et al, 2016 ; Botschuijver et al, 2017 ; Gao et al, 2017 ; Hamad et al, 2017 ; Huseyin et al, 2017 ; Motooka et al, 2017 ; Schei et al, 2017 ; Zhao et al, 2017 ) as well as SSU ( Aurora et al, 2013 ; van Woerden et al, 2013 ; Cleland et al, 2014 ; Li et al, 2014 ) and LSU ( Zhang et al, 2011 ; Park et al, 2012 ). The use of different methodological approaches makes meaningful comparisons between studies difficult, and it is unclear whether observed differences are genuine (e.g., due to geographical influences affecting the fungal exposure of different populations) or simply biases associated with different methods.…”

Rinosinusitis crónica: Una revisión de su etiopatogenia

“…A pesar de lo anterior, la hipótesis fúngica ha perdido fuerza debido a que se ha objetivado que las PBMC producen IL-5 ante la exposición a Alternaria, tanto en pacientes como en controles, sin existir alguna correlación con la presencia RSC 16 , y, además, la producción de IL-13, principal quimiotáctico de eosinófilos, no difiere entre RSC y controles al ser expuestos a Alternaria 17 . Otro argumento en contra de esta hipótesis se muestra en un estudio reciente en que se obtuvieron muestras de meato medio de pacientes con RSC, detectándose en el 14,3% de las RSC alguna especie de hongo, por lo que solo podrían tener algún rol en la patogenia de un pequeño grupo de pacientes 18 . Sumado a lo anterior, un metaanálisis que incluyó cinco ensayos clínicos randomizados, doble ciego, que evaluaban la efectividad de antifúngicos tópicos, y uno de antifúngico sistémico en RSC, evidenció que no existían beneficios significativos de la terapia antifúngica tópica o sistémica en relación al placebo, en ninguno de los resultados evaluados 19 .…”

Fungal Microbiota in Chronic Airway Inflammatory Disease and Emerging Relationships with the Host Immune Response

“…Fungi found in culture-based studies of CRS patients include members of Aspergillus, Alternaria, Candida, Cladosporium , and Penicillium (Ponikau et al, 1999 ). Molecular studies have expanded this list to include fastidious fungi Malassezia, Curvularia, Schizophyllum , and Neocosmospora (Cleland et al, 2014 ; Gelber et al, 2016 ; Zhao et al, 2017 ). While there are few studies on host-fungal interactions in the upper airways, recent research suggests that defects in epithelial genes might compromise immune barrier function and lead to dysfunctional host immune responses to bacterial or fungal colonization (Tieu et al, 2009 ).…”