Respiratory infection Original research Thorax: first published as 10.1136/thoraxjnl-2020-216179 on 25 June 2021. Downloaded from Lung microbiota predict invasive pulmonary aspergillosis and its outcome in immunocompromised patients Anaïs Hérivaux,1,2 Jesse R Willis,3,4 Toine Mercier,5,6 Katrien Lagrou,6,7 Samuel M Gonçalves,1,2 Relber A Gonçales,1,2 Johan Maertens,5,6 Agostinho Carvalho,1,2 Toni Gabaldón,3,4,8 Cristina Cunha 1,2 ► Additional supplemental ABStract material is published online Rationale Recent studies have revealed that the lung Key messages only. To view, please visit the journal online (http:// dx. doi. microbiota of critically ill patients is altered and predicts org/ 10. 1136/ thoraxjnl- 2020- clinical outcomes. The incidence of invasive fungal What is the key question? 216179). infections, namely, invasive pulmonary aspergillosis ► Does the lung microbiota modulate (IPA), in immunocompromised patients is increasing, but susceptibility to invasive pulmonary For numbered affiliations see the clinical significance of variations in lung bacterial aspergillosis (IPA) and predict its clinical end of article. communities is unknown. outcome? Objectives To define the contribution of the lung Correspondence to What is the bottom line? microbiota to the development and course of IPA. Dr Cristina Cunha, Life and ► We show that variation in the lung microbiota Health Sciences Research Methods and measurements We performed an is associated with the development of IPA in Institute (ICVS), University observational cohort study to characterise the lung critically ill patients. Patients with IPA display of Minho, Braga 4710-057, microbiota in 104 immunocompromised patients using Portugal; a microbiota signature in the lower airways bacterial 16S ribosomal RNA gene sequencing on cristinacunha@ med. uminho. characterised by a decreased bacterial diversity bronchoalveolar lavage samples sampled on clinical pt and Dr Toni Gabaldón, that predicts clinical outcomes after infection. Comparative Genomics Group, suspicion of infection. Associations between lung Moreover, we describe associations between Barcelona Supercomputing dysbiosis in IPA and pulmonary immunity were evaluated Centre (BSC-CNS), 08034 lung dysbiosis in IPA and pulmonary antifungal by quantifying alveolar cytokines and chemokines and Barcelona, Spain; immunity, including alveolar cytokines and immune cells. The contribution of microbial signatures toni. gabaldon. bcn@ gmail. com chemokines and neutrophil counts. to patient outcome was assessed by estimating overall http://thorax.bmj.com/ AH and JRW contributed survival. Why read on? equally. Main results Patients diagnosed with IPA displayed ► Recent studies have unveiled alterations in the a decreased alpha diversity, driven by a markedly lung microbiota of critically ill patients, which TG and CC are joint senior increased abundance of the Staphylococcus, Escherichia, were associated with alveolar and systemic authors. Paraclostridium and Finegoldia genera and a decreased inflammation to predict disease outcomes. IPA Received 9 September 2020 proportion of the Prevotella and Veillonella genera. is frequently observed in critically ill patients. Accepted 26 May 2021 The overall composition of the lung microbiome was However, no study to date has determined influenced by the neutrophil counts and associated with whether the lung microbiota is altered in on September 27, 2021 by guest. Protected copyright. differential levels of alveolar cytokines. Importantly, the patients with IPA or predict clinical outcomes degree of bacterial diversity at the onset of IPA predicted after infection. the survival of infected patients. Conclusions Our results reveal the lung microbiota as an understudied source of clinical variation in patients at predictive of disease outcomes across numerous risk of IPA and highlight its potential as a diagnostic and chronic respiratory diseases. For example, the diver- therapeutic target in the context of respiratory fungal sity of sputum microbiota predicts exacerbations in diseases. bronchiectasis,6 disease progression and lung func- tion decline in cystic fibrosis (CF)7 and mortality after acute exacerbations of chronic obstructive © Author(s) (or their pulmonary disease (COPD).8 employer(s)) 2021. Re- use INTRODUCTION Although lung dysbiosis is established as a major permitted under CC BY- NC. No In the past decade, advances in molecular methods trigger of exacerbations in inflammatory airway commercial re- use. See rights and permissions. Published for the quantification and sequencing of bacterial diseases, only a few studies have addressed the by BMJ. DNA have revealed that the lungs, previously consid- mechanisms whereby specific bacterial commu- ered sterile, instead harbour complex and dynamic nities contribute to disease severity. Nonetheless, To cite: Hérivaux A, Willis JR, communities of bacteria.1 The lung microbiome is mounting evidence supports a key contribution of Mercier T, et al. Thorax Epub 2 ahead of print: [please known to diverge significantly across healthy and the lung microbiota to the activation and regula- 3 4 include Day Month Year]. diseased states and is associated with variations tion of lung immunity. For instance, the dysbiosis in doi:10.1136/ in both alveolar and systemic immunity.5 The struc- the lower respiratory tract of patients with COPD thoraxjnl-2020-216179 ture and composition of the lung microbiome are revealed an expansion of streptococcal genera that, Hérivaux A, et al. Thorax 2021;0:1–9. doi:10.1136/thoraxjnl-2020-216179 1 Respiratory infection by suppressing the antimicrobial peptide cathelicidin, impaired patients were randomly selected from the patients indicated for Thorax: first published as 10.1136/thoraxjnl-2020-216179 on 25 June 2021. Downloaded from the pulmonary clearance of bacteria.4 Moreover, the disruption bronchoscopy at around the same time as the cases of IPA to of the lung microbiota in patients with idiopathic pulmonary obtain a 1:1 case–control ratio. Patients with ‘possible’ disease fibrosis was described to promote increased levels of alveolar were excluded from the study, and no mold-active antifungals profibrotic cytokines underlying local inflammation and aber- were administered by the treating physician(s) before sample rant repair to drive disease progression.9 collection. This study was approved by the Ethics Committee for The lung microbiome of critically ill patients also differs Research in Life and Health Sciences of the University of Minho, profoundly from that of healthy individuals10–12 and is associ- Portugal, and the Ethics Committee of the University Hospitals ated with alveolar inflammation and injury.10 11 Such features of Leuven, Belgium (IRB number 126/014). Written informed of lung dysbiosis were found to predict the clinical outcome consent was obtained from the patient or a representative prior of patients with sepsis and acute respiratory distress syndrome to collection of airway samples. Additional details, including (ARDS).13 14 A history of smoking was related to the composi- tion of the lung microbiome in intensive care patients, and devel- opment of ARDS was associated with a respiratory microbial Table 1 Demographics and clinical characteristics of the study cohort community structure characterised by a relative enrichment of taxa more abundant in smokers at baseline.11 Collectively, these Variables IPA Controls P value observations highlight the composition of the lung microbiome (n=54) (n=50) as a key factor involved in the development of complications in Age, mean±SD 63.6±12.6 60.9±13.1 0.26 the intensive care setting. Man, no (%) 34 (63) 35 (70) 0.53 Owing to advances in critical care medicine and the intro- Underlying disease, no (%)* duction of broad- spectrum antibiotics, the incidence of inva- sive fungal infections in critical care is on the rise, namely, Acute leukaemia 16 (29.6) 14 (28) 0.08 among haematological patients under chemotherapy or patients Allogeneic HSCT 10 (18.5) 16 (32) undergoing solid organ or allogeneic haematopoietic stem- Chronic lymphoproliferative 8 (14.8) 10 (20) cell transplantation (HSCT).15 In particular, invasive pulmo- diseases nary aspergillosis (IPA) remains a major cause of mortality in SOT 5 (9.3) 4 (8) these clinical settings with rates estimated between 20% and Influenza A (H1N1) 8 (14.8) 0 (0) 30%16 17 and more than 80% when involving cases of infection with azole- resistant strains.18 The relevance of these numbers Solid tumours 2 (3.7) 4 (8) is further highlighted by the lack of any licensed vaccine and Liver cirrhosis 3 (5.6) 2 (4) limitations in currently available tests for the diagnosis of IPA. COPD 2 (3.7) 0 (0) An improved understanding of the pathogenesis of IPA is there- ICU admission 28 (51.9) 12 (24) 0.009 fore required for the development of more effective diagnostic or control measures for this infection. Mechanical ventilation 18 (33.3) 5 (10) 0.005 Although the importance of the lung microbiome has already Severe neutropenia, no (%)† 20 (37) 11 (22) 0.13 http://thorax.bmj.com/ 19 20 been put forward in the context of IPA, no study to date has BAL cell counts, mean (range)‡ determined whether the lung microbiota is altered in patients Neutrophils 4.9 (0–30.3) 4.0 (0–24.6) 0.28 with IPA and whether it predicts clinical outcomes after infec- Lymphocytes 4.0 (0–175.7) 1.3 (0.1–17.4) 0.004 tion. In light of these observations, we hypothesised that varia- tion in the lung microbiota is a relevant factor contributing to the CMV IgG serostatus development of IPA and its outcome in immunocompromised Positive 18 (33.3) 20 (40) 0.80 patients. We report that the dysbiosis of the lung microbiota Negative 14 (25.9) 12 (24) in immunocompromised patients is associated with the devel- on September 27, 2021 by guest. Protected copyright. Unknown 22 (40.7) 18 (36) opment of IPA and that a decreased bacterial diversity in IPA predicts poorer outcomes. Moreover, we describe associations Immunosuppression, no (%) between lung dysbiosis and pulmonary immunity, as revealed by Steroids 25 (46.3) 14 (28) 0.06 the differential effects on the levels of alveolar cytokines and the Other immunosuppressive 4 (7.4) 10 (20) number of neutrophils.