Concomitant presence of Aspergillus fumigatus and Stenotrophomonas maltophilia in the respiratory tract : a new risk for patients with liver disease ?

Correspondence Françoise Botterel francoise.botterel@aphp.fr Unité de Mycologie, DHU VIC, AP-HP, Hôpital Henri Mondor and Département de Microbiologie, Créteil, France Université Paris Est Créteil, LIC EA4393, Créteil, France Service de Santé publique, AP-HP, Hôpital Henri-Mondor, Créteil, France Unité de Bactériologie-Hygiène, DHU VIC, AP-HP, Hôpital Henri Mondor and Département de Microbiologie, Créteil, France Département d’anesthésie et de réanimation, DHU VIC, AP-HP, Hôpital Henri-Mondor, Créteil, France Unité de Pneumologie, Réanimation médicale Hôpital Henri Mondor, Centre Intercommunal de Créteil, Créteil, France


INTRODUCTION
Over the past decade, Aspergillus fumigatus has become the major air-borne fungal pathogen in developed countries, incurring an increasing incidence of aspergillosis (Lass-Flo ¨rl, 2009;Lortholary et al., 2011).This fungus is traditionally isolated in samples from patients with haematological diseases, stem cell or solid organ transplant, or on long-term corticotherapy, thus causing invasive infections (De Pauw et al., 2008).Recent studies have described A. fumigatus colonization in patients with chronic lung diseases, especially chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), probably due to lung structural changes and the heavy courses of antibiotics (Bafadhel et al., 2014;Sabino et al., 2014).Furthermore, co-morbidities such as alcoholism, diabetes, malnutrition and liver cirrhosis are often associated with this A. fumigatus colonization (Prodanovic et al., 2007).
One of the emerging micro-organisms concomitantly isolated with A. fumigatus from the respiratory tract of immunocompromised patients or those suffering from chronic respiratory diseases is Pseudomonas aeruginosa, a non-fermentative Gram-negative bacillus and one the most studied bacteria (Baxter et al., 2013).Other bacteria, such as Stenotrophomonas maltophilia, have recently emerged as important hospitalassociated pathogens colonizing the same type of patients.This intrinsically multidrug-resistant, saprophytic and ubiquitous micro-organism belongs to the 'Pseudomonas and parented' group of bacteria, and is increasingly encountered in human infectious diseases (Jacquier et al., 2011).Although not highly virulent, its environmental spread and resistance to antibiotic selective pressure promote its opportunistic pathogenicity in immunocompromised patients.Risk factors that are associated with S. maltophilia infections and/ or colonization are often shared with Aspergillus infections.These risk factors include immunosuppressive or corticosteroid therapy and history of long-term intake of broadspectrum antibiotics (Brooke, 2012).
Fungi and bacteria are often simultaneously cultivated from respiratory tract specimens, but the physiopathological, biological and clinical relevance of the microbial association remains controversial (Wargo & Hogan, 2006;Peleg et al., 2010;Frey-Klett et al., 2011).In CF, significant associations have been reported between the upper airway colonization by A. fumigatus and the presence of S. maltophilia (Marchac et al., 2004;Paugam et al., 2010), or the presence of P. aeruginosa or atypical mycobacteria (Amin et al., 2010;Paugam et al., 2010).Similarly, bronchial colonization by S. maltophilia is independently associated with the development of allergic bronchopulmonary aspergillosis (Ritz et al., 2005).If the association between A. fumigatus and S. maltophilia is well established in CF patients, it needs to be confirmed in other clinical settings and particularly in immunocompromised patients and other disease sufferers.
The aim of this work was to assess, beyond the CF context, the frequency of concomitant respiratory presence of A. fumigatus and S. maltophilia in hospitalized patients, and the factors associated with this co-presence.

METHODS
Patients, settings and specimens.The study had a cross-sectional, retrospective design and was conducted from February 2007 to December 2011 in Henri Mondor University Hospital.This 900-adult bed, tertiary care institution, which is located in the suburb of Paris (France), provides care to a large panel of immunocompromised patients [solid organ transplantation, haematopoietic stem cell transplantation, immunosuppressive treatment and intensive care unit (ICU)].
The case definition was a patient who had at least one respiratory sample (sputum, tracheal aspirate, plugged telescoping catheter, protected sample brush, bronchoalveolar lavage or sinusal sample) yielding an A. fumigatuspositive culture during the study period.For each case patient, the presence of bacteria in the same respiratory sample or in another sample drawn during a 1-month period from the culture of A. fumigatus was collected.The presence, in the same period, of these two micro-organisms in the airway reflects either colonization or infection.Invasive aspergillosis was classified as proven, probable, possible or excluded according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria (De Pauw et al., 2008).The microbiological data were extracted from the database of the microbiology laboratory.The medical charts were retrospectively reviewed and the following clinical data were collected: demographic features, underlying disease, history of invasive procedure or 1 month of antimicrobial therapy preceding the A. fumigatus and/or S. maltophilia colonization, time period between hospitalization and A. fumigatus and/or S. maltophilia identification, duration of hospital stay, and clinical outcome.
Processing of the respiratory samples in the microbiology laboratory.The respiratory samples were cultured for both filamentous fungi and bacteria following the European Society of Clinical Microbiology and Infectious Diseases Guidelines (Freymuth et al., 2012).As such, Sabouraud chloramphenicol gentamicin agar (bioMe ´rieux) was inoculated in order to identify filamentous fungi.Considering the bacteria, the specimens were cultured as described previously (Nebbad-Lechani et al., 2013) with Trypticase soy, Drigalski, colistin nalidixic acid sheep blood and chocolate agar supplemented with PolyViteX.
Statistical analysis.The clinical and microbiological characteristics of the micro-organism population were presented as n (%), mean+SD or median (25th-75th percentiles), as appropriate.Frequencies of colonization by A. fumigatus/S.maltophilia and other bacteria were expressed as percentage and 95% confidence interval (95% CI) based on binomial distribution.
The clinical and microbiological characteristics of patients with and without concomitant A. fumigatus/S.maltophilia colonization were compared using the Pearson x 2 -test or Fisher test for qualitative variables and the Student t-test or Wilcoxon-Mann-Whitney test for quantitative variables, as appropriate.
First, all variables with Pv0.15 in the univariate analysis were selected for the multivariate analysis.Second, a bivariate analysis was performed to identify potential confounders.In the final multivariate logistic regression model, only variables which remained significantly and independently associated with colonization by A. fumigatus/ S. maltophilia at Pv0.05 were included.The same methodology was used to assess factors associated with in-hospital death.
Interactions were checked.Adjusted odd ratios (ORs) with their 95 % CIs were estimated.All tests were two-tailed.The threshold of significance was fixed at Pv0.05.No imputation of missing data was made.The data were analysed using R software (www.r-project.org).

RESULTS
Between February 2007 and December 2011, 257 patients with at least one positive A. fumigatus culture were enrolled in the study (Group A patients).The origin of the specimen was sputum (n5129), tracheal aspirate (n553), plugged telescoping catheter (n519), protected sample brush (n51), bronchoalveolar lavage (n5156) or a sinusal sample (n514).The clinical and microbiological characteristics of this population are summarized in Table 1.The mean+SD age was 59.4+14.6 years and 176 patients (68 %) were male.
Most of the patients were immunocompromised (71 %) due to different diseases.They received immunosuppressive therapy for haematological malignancy (n5118), including 38 haematopoietic stem cell receivers, solid organ transplantation (n539), cancer (n545) or other causes (Table 1).Of the 39 patients with solid organ transplantation, 16 received liver, 16 kidney, one liver/kidney, one pancreas/ kidney, two heart/kidney, two heart and one pancreas transplantation.Of the non-immunocompromised patients, 69 (32 %) had chronic respiratory disease, such as bronchiectasis, COPD and asthma, but none had CF.At inclusion, the most commonly performed invasive procedures on the study patients were mechanical ventilation (n538), central In Group AS, both micro-organisms were isolated in the same respiratory sample in 11 out of the 20 cases (55 %), during the same week in five cases (25 %) or the same month in four cases (20 %).However, 16 patients (80 %) were colonized by at least one other different species.
Factors associated with the co-culture of A. fumigatus and S. maltophilia No significant difference was found for age, sex, immunosuppressive or anti-neoplasic therapy, and corticosteroid regimen between Group A and Group AS (Table 1).
In the univariate analysis, significant differences between Group A and Group AS were found for liver disease (cirrhosis, liver transplantation, hepatitis, etc.) ( 2).
In Group AS, all of the seven patients with liver disease had EORTC/MSG criteria for probable invasive aspergillosis (De Pauw et al., 2008).These patients were all immunocompromised and hospitalized in the ICU when A. fumigatus and S. maltophilia were isolated.Amongst them, four patients had undergone liver transplantation (three for cirrhosis and one for fulminant hepatitis), and two patients had alcoholic cirrhosis and one hepatic acute graft versus host disease after allogenic stem cell transplantation for acute myeloid leukaemia.None had chronic respiratory diseases.S. maltophilia was isolated before A. fumigatus in four cases, A. fumigatus was isolated before S. maltophilia in two cases and both were found in the same sample in one case.All patients were treated for A. fumigatus and S. maltophilia infections with appropriate medicines.Five patients (71 %) died during hospitalization.
In the multivariate analysis, factors which independently associated with in-hospital death were P. aeruginosa, liver disease and orotracheal intubation (Table 3).Age w61 years was borderline significant.However, A. fumigatus/ S. maltophilia was no longer associated with in-hospital death after adjustment for the three main factors, i.e.P. aeruginosa, liver disease and orotracheal intubation.

DISCUSSION
In the present study, we have shown that 7.8 % of the hospitalized patients who were carriers of Aspergillus spp. in their lung also harboured S. maltophilia.This concurrent colonization was associated with excess mortality.In this retrospective study, it was sometimes difficult to separate colonization and infection for these two micro-organisms.We identified three risk factors for this microbial association: liver disease, orotracheal intubation and the culture of other pathogenic bacteria in the respiratory specimens, particularly P. aeruginosa.
To the best of our knowledge, this work is the first study interested in the co-culture of A. fumigatus and S. maltophilia in respiratory samples from patients other than CF sufferers.Two previous studies have found an association between the presence of A. fumigatus and subsequent S. maltophilia infection in patients with CF (Marchac et al., 2004;Paugam et al., 2010).In these two studies, the factors associated with contracting S. maltophilia were: more than two consecutive courses of intravenous antibiotics, identification of A. fumigatus in the sputum and oral steroid treatment.With the exclusion of CF, the factors associated with S. maltophilia identification in patients are well known: underlying malignancy, presence of indwelling devices (e.g.catheters), chronic respiratory disease, immunocompromised host, prior use of antibiotics and long-term hospitalization or ICU stay (Brooke, 2012).We found that P. aeruginosa was significantly associated with the co-presence of A. fumigatus and S. maltophilia in the multivariate analysis.Polymicrobial infections by S. maltophilia and other organisms such as P. aeruginosa, Burkholderia spp., Staphylococcus aureus or E. coli in the CF patient's lung were reported previously (Brooke, 2012).P. aeruginosa, associated with S. maltophilia, has the ability to form biofilms on the lung cells in vitro (Pompilio et al., 2010).P. aeruginosa may provide a more hospitable environment for the adherence, invasion and persistence of S. maltophilia in the CF patient's lung (De Vidipo ´et al., 2001).This may help explain their tendency to cause persistent infections and the vast damage of the epithelial mucosa induced by the exoproducts they release (Looney et al., 2009;Brooke, 2012).Furthermore, S. maltophilia or P. aeruginosa colonization may increase the probability of respiratory tract colonization by A. fumigatus, thus degrading the mucosa and leading to subsequent worsening of respiratory function.These interactions could be compared to the effect of influenza H1N1 infection that provokes invasive pulmonary aspergillosis by impairing the airway epithelium and releasing innate cytokine/chemokine (Martin-Loeches & Valles, 2012).
Interaction of S. maltophilia with other micro-organisms is currently under study.A. fumigatus and S. maltophilia could communicate through secreted factors (quorum sensing molecules, secondary metabolites, carbohydrates and proteins) that would influence the production of biofilm (Pompilio et al., 2010).
S. maltophilia commonly colonizes the lungs of patients with pulmonary disease, particularly CF patients.In our group of 69 patients who had chronic respiratory diseases, the co-presence of both A. fumigatus and S. maltophilia was not significantly higher than in patients without pulmonary diseases.No independent association was found between S. maltophilia and death during hospitalization.
In the multivariate analysis, liver disease was a factor independently associated with such co-presence.All of the seven patients with liver disease suffered from invasive aspergillosis, which reflected their immunocompromised status and the presence of additional risk factors for S. maltophilia colonization, such as antibiotic therapy.As such, those seven patients are at risk of infections precipitated by the significant impairment of the neutrophil defence mechanism, frequent use of corticosteroids and invasive procedures, and malnutrition (Panasiuk et al., 2005;Cheruvattath & Balan, 2007;Falcone et al., 2011;Fishman, 2011;Barchiesi et al., 2015).However, to the best of our knowledge, our study is the first description of an association between co-infection by A. fumigatus and S. maltophilia and patients with liver disease.This study has several limitations.First, this work was a single-centre retrospective study.Nevertheless, its first results encourage us to propose a larger-scale study on A. fumigatus and S. maltophilia co-culture in patients without respiratory diseases.Second, we were not able to distinguish between S. maltophilia infection and colonization, which made it difficult to reach conclusions on the specific role of the bacterium in the pathological process.However, A. fumigatus infections were detected in a specific group of patients, i.e. the liver transplant patients, who presented a propensity to be co-colonized by S. maltophilia.These results should be confirmed in a largerscale study.

CONCLUSION
In this study, we identified the prevalence of concomitant colonization by A. fumigatus and S. maltophilia, and the factors independently associated with it.These results support the hypothesis of a particular susceptibility of patients with end-stage liver disease to this co-colonization.Additional complementary epidemiological and physiopathological investigations are needed.
Abbreviations: CF, cystic fibrosis; CI, confidence interval; COPD, chronic obstructive pulmonary disease; EORTC/MSG, European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group; ICU, intensive care unit; OR, odds ratio.

Table 1 .
Clinical and bacteriological characteristics of the 257 enrolled in-patients *Data are presented as n (%) for qualitative variables and as mean¡SD or median (25th-75th percentile) for quantitative variables as appropriate.*Pearson x 2 -test/Fisher test or Student t-test/Wilcoxon-Mann-Whitney test as appropriate.DLiver disease: cirrhosis (n515) or others (n521).dIn samples taken within the 1-month period from A. fumigatus culture.
OR53.92, P50.014) and mechanical ventilation (OR53.42,95 % CI 1.17-9.96,P50.024) were independently associated with the co-culture of S. maltophilia and A. fumigatus within the same month (Table and different bacteria in the respiratory specimen, and (2) between invasive procedures and different bacteria in the respiratory specimen.No significant interactions were found.The multivariate logistic regression model, adjusted for Pv0.15 in the univariate analysis, showed that the simultaneous presence of P. aeruginosa in the respiratory tract (OR53.19,95 % CI 1.11-9.14,P50.031), liver disease (

Table 3 .
Factors associated with in-hospital fatality risk in 257 in-patients with already A. fumigatus-positive culture