Pitfalls in sampling and analyzing low-biomass human nasal microbiome samples

Abstract

We read with great interest the original article by Liang et al on alterations of the microbiome in eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP).1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar Unlike the authors of previous reports who did not observe any changes or reduced α-diversity in patients with CRS versus in healthy controls, Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar report a higher α-diversity in patients with eCRSwNP and suggest using the nasal microbiome as a novel diagnostic classifier for eCRSwNP. Despite these interesting findings, we would like to raise some concerns. First, the group of patients with eCRSwNP may have been biased toward patients with severe type 2 disease. The cutoff value of an absolute tissue count of 55 eosinophils per hpf used by Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar has been evaluated for predicting the recurrence of polyps in eCRSwNP2Lou H. Meng Y. Piao Y. Wang C. Zhang L. Bachert C. Predictive significance of tissue eosinophilia for nasal polyp recurrence in the Chinese population.Am J Rhinol Allergy. 2015; 29: 350-356Crossref PubMed Scopus (136) Google Scholar but is much higher than the European Position Paper on Rhinosinusitis and Nasal Polyps–based cutoff of 10 eosinophils per hpf defining eosinophilic chronic rhinosinusitis.3Fokkens W.J. Lund V.J. Hopkins C. Hellings P.W. Kern R. Reitsma S. et al.European position paper on rhinosinusitis and nasal polyps 2020.Rhinology. 2020; 58: 1-464Google Scholar Additionally, the population of individuals without CRSwNP used by Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar also shows signs of type 2 inflammation (eg, ≤500 eosinophils/μL blood).3Fokkens W.J. Lund V.J. Hopkins C. Hellings P.W. Kern R. Reitsma S. et al.European position paper on rhinosinusitis and nasal polyps 2020.Rhinology. 2020; 58: 1-464Google Scholar Furthermore, we would like to voice our concerns with regard to the quality of the presented microbiome data. Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar do not clarify whether any controls were performed during sampling, DNA extraction, and 16S rRNA gene amplicon preparation and sequencing, which is a critical step during microbiome profiling of low-biomass samples.4Kennedy K.M. de Goffau M.C. Perez-Munoz M.E. Arrieta M.C. Backhed F. Bork P. et al.Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.Nature. 2023; 613: 639-649Crossref PubMed Scopus (9) Google Scholar Even if such controls were performed, they were neither deposited in the Sequence Read Archive alongside the sample data (PRJNA785109) nor appropriately documented in the article's Methods section. Although the aforementioned information would be crucial to comprehensively evaluate the data set, we would like to point out 2 further considerations. First, a number of the taxa discussed in the context of differential abundance and correlation with clinical parameters are most probably not native to the nasal mucosa. On the basis of the ecology of these microorganisms, they are most likely cross-contaminations from distinct sample types (ie, anaerobes abundant in human gut samples [Akkermansia, Blautia, Desulfovibrio, and Sutterella]).4Kennedy K.M. de Goffau M.C. Perez-Munoz M.E. Arrieta M.C. Backhed F. Bork P. et al.Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.Nature. 2023; 613: 639-649Crossref PubMed Scopus (9) Google Scholar Such contaminations may have inflated α-diversity measures (see Fig 1, A in Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar), which here are atypically high compared with those of other nasal microbiome data sets.5Wilson M.T. Hamilos D.L. The nasal and sinus microbiome in health and disease.Curr Allergy Asthma Rep. 2014; 14: 485Crossref PubMed Scopus (55) Google Scholar Second, the β-diversity presented in Fig 1, B in Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar displays a clustering in 3 groups not consistent with the patient groups, which is a common feature observed with sequencing batch effects. To evaluate this further, it would be necessary to disclose in the Methods section whether samples were sequenced in a single run or as multiple batches. Third, although the relative abundance for significantly differentially abundant taxa (see Fig 2 in Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar) or taxa displaying correlations with clinical factors (see Fig 3 in Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar) is not explicitly discussed, on the basis of the coarse taxonomic profile, it has to be assumed that they are predominantly relatively low-abundant and sporadically occurring (see Fig 1, C in Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar). This is problematic if these low-abundant sporadic taxa occur because of contamination and sample cross talk, as may partly be the case in the study by Liang et al.1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar In conclusion, the article by Liang et al1Liang Y. Xie R. Xiong X. Hu Z. Mao X. Wang X. et al.Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.J Allergy Clin Immunol. 2023; 151: 1286-1295Abstract Full Text Full Text PDF Scopus (0) Google Scholar clearly raises the urgent need for stringent and uniform guidelines for sampling and processing low-biomass human microbiome samples to achieve comparable data and explore the diagnostic potential of the human microbiome further. Alterations of nasal microbiome in eosinophilic chronic rhinosinusitisJournal of Allergy and Clinical ImmunologyVol. 151Issue 5PreviewExposure to microbes may be important in the development of chronic rhinosinusitis (CRS). Dysbiosis of the nasal microbiome is considered to be related to CRS with nasal polyps (CRSwNP). The link between the nasal microbiota and eosinophilic CRSwNP (eCRSwNP) has rarely been studied. Full-Text PDF Open AccessReplyJournal of Allergy and Clinical ImmunologyPreviewWe thank Pjevac et al1 for their interest in our article “Alterations of nasal microbiome in eosinophilic chronic rhinosinusitis.”2 From the clinician's perspective, the eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP) endotype is frequently associated with poorer treatment effectiveness and higher polyp recurrence rates. Identifying specific risk factors for polyp recurrence may provide valuable guidance for treatment selection and appropriate management of nasal polyps. Therefore, the cutoff value of mucosal eosinophils, which can accurately identify polyp recurrence, is of more interest to clinicians. Full-Text PDF