Table 1

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Dec 2, 2015 - Christophe Lucet3,10, Francis Derouin1,8†. 1 AP-HP, Bichat-Claude Bernard Hospital, Infection Control Unit, F-75018, Paris, France, ...
RESEARCH ARTICLE

Relationship between Fungal Colonisation of the Respiratory Tract in Lung Transplant Recipients and Fungal Contamination of the Hospital Environment a11111

OPEN ACCESS Citation: Bonnal C, Leleu C, Brugière O, Chochillon C, Porcher R, Boelle P-Y, et al. (2015) Relationship between Fungal Colonisation of the Respiratory Tract in Lung Transplant Recipients and Fungal Contamination of the Hospital Environment. PLoS ONE 10(12): e0144044. doi:10.1371/journal. pone.0144044 Editor: Ritesh Agarwal, Postgraduate Institute of Medical Education and Research, INDIA

Christine Bonnal1☯*, Christopher Leleu2,3☯, Olivier Brugière4, Christian Chochillon5, Raphael Porcher6, Pierre-Yves Boelle2,7, Jean Menotti1,8, Sandrine Houze5,9, JeanChristophe Lucet3,10, Francis Derouin1,8† 1 AP-HP, Bichat-Claude Bernard Hospital, Infection Control Unit, F-75018, Paris, France, 2 University Paris Diderot, Sorbonne Paris Cité, Paris, France, 3 University Pierre et Marie Curie-Paris 6, Paris, France, 4 APHP, Service de Pneumologie B, Unité de Transplantation Pulmonaire, Centre Hospitalier Universitaire Bichat-Claude Bernard, Paris, France, 5 AP-HP, Laboratory of Parasitology and Mycology, Bichat-Claude Bernard University Hospital, Paris, France, 6 Centre de Recherche Epidémiologie et Statistique Sorbonne Paris Cité, UMR 1153, Inserm, Université Paris Descartes, Paris, France, 7 INSERM, Institut Pierre Louis d’Epidémiologie et de Santé Publique–U1136, Paris, France, 8 AP-HP, Laboratory of Parasitology and Mycology, Saint-Louis University Hospital, Paris, France, 9 UMR 216, Mère et enfants face aux infections tropicales, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France, 10 Paris Diderot University, IAME, UMR 1137, F-75018 Paris, France † Deceased. ☯ These authors contributed equally to this work. * [email protected]

Abstract

Received: August 8, 2015

Background

Accepted: November 12, 2015

Aspergillus colonisation is frequently reported after lung transplantation. The question of whether aspergillus colonisation is related to the hospital environment is crucial to prevention.

Published: December 2, 2015 Copyright: © 2015 Bonnal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funded by Programme de Recherche en Qualité Hospitalière, PREQHOS 2009 (04Preqhos09). (http://www.sante.gouv.fr/IMG/pdf/ projets_retenus_Preqhos_2009.pdf) and Doctoral grant from the Université Pierre et Marie Curie and the EHESP (Ecole des Hautes Etudes en Santé Publique, Rennes, France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Method To elucidate this question, a prospective study of aspergillus colonisation after lung transplantation, along with a mycological survey of the patient environment, was performed.

Results Forty-four consecutive patients were included from the day of lung transplantation and then examined weekly for aspergillus colonisation until hospital discharge. Environmental fungal contamination of each patient was followed weekly via air and surface sampling. Twelve patients (27%) had transient aspergillus colonisation, occurring 1–13 weeks after lung transplantation, without associated manifestation of aspergillosis. Responsible Aspergillus species were A. fumigatus (6), A. niger (3), A. sydowii (1), A. calidoustus (1) and Aspergillus sp. (1). In the environment, contamination by Penicillium and Aspergillus was predominant.

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Competing Interests: The authors have declared that no competing interests exist. Abbreviations: AC, aspergillus colonisation; LT, lung transplantation; SICU, surgical intensive care unit; LTU, lung transplant unit; GM, galactomannan; HSCT, Hematopoietic Stem Cell Transplantation.

Multivariate analysis showed a significant association between occurrence of aspergillus colonisation and fungal contamination of the patient’s room, either by Aspergillus spp. in the air or by A.fumigatus on the floor. Related clinical and environmental isolates were genotyped in 9 cases of aspergillus colonisation. For A. fumigatus (4 cases), two identical microsatellite profiles were found between clinical and environmental isolates collected on distant dates or locations. For other Aspergillus species, isolates were different in 2 cases; in 3 cases of aspergillus colonisation by A. sydowii, A. niger and A. calidoustus, similarity between clinical and environmental internal transcribed spacer and tubulin sequences was >99%.

Conclusion Taken together, these results support the hypothesis of environmental risk of hospital acquisition of aspergillus colonisation in lung transplant recipients.

Introduction Solid-organ transplant recipients are at high risk of acquiring Aspergillus infection, with an average incidence of 6% in lung transplants (LT), higher than after other solid organ transplantations [1–3]. In LT recipients, aspergillosis possesses particularities linked to the type of transplanted organ [4]. The frequency of respiratory tract colonisation is high, between 22 and 85%, and most often occurs within the first 6-months post-transplant [5]. Whether or not it is associated with infection, colonisation results in increased mortality at 5 and 10 years [6]. The key to the prevention of aspergillosis lies in whether the infection is hospital- or community-acquired. If a relationship between fungal contamination of the hospital environment and the incidence of Aspergillus colonisation (AC) or other clinical manifestations of aspergillosis under our normal conditions of LT could be proven, specific measures should be taken to prevent aspergillosis contamination and colonisation during the hospitalization in post-transplantation period. In order to evaluate this potential relationship, we initiated a prospective study of fungal colonisation by Aspergillus spp. after LT together with patient environmental surveillance.

Patients, Materials and Methods Ethics Statement The Institutional Review Board (Comité d’Evaluation de l’Ethique des projets de Recherche Biomédicale, Hôpital Bichat Claude Bernard, 46, rue Henri Huchard, 75018 PARIS, France, 21 mai 2010) approved the study protocol and did not require written informed consent from the participants. However, patients were informed of the study's purpose.

Hospital Setting The study was carried out in the surgical intensive care unit (SICU) and the lung transplant unit (LTU) of Bichat-Claude Bernard Hospital (Paris, France). LT recipients were admitted to the SICU the day of transplantation. This unit is provided with HEPA-filtered air (99% efficiency) and maintained under positive pressure. Patients were admitted to the LTU after

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discharge from the SICU and maintained in this unit until discharge from the hospital. This unit is supplied with filtered air at 85% efficiency, similar to other conventional ward of the hospital. During the study period, no construction or renovation has been performed in the study wards or adjacent wards. Patient inclusion and follow-up. All consecutive LT patients were included prospectively, between April 2010 and September During the SICU stay, the policy of our LT center is to strictly restrict removal of LT patients from HEPA filtered system in place in each room of patient. In this way, only transfer for CT-scan in the radiology department are performed for respiratory events in this early postoperative period. When patients left their room for any purpose, they were required to wear a FFP2 protective mask to limit aspergillosis (or fungal) contamination. After LT, clinical, radiological and endoscopic signs, biological follow-up and antifungal treatments in relation to possible manifestation of aspergillosis were collected weekly until hospital discharge. Reports of endoscopy were recorded in patient’s medical file. A mycological examination was performed on all bronchial and alveolar sample (direct examination, culture on Sabouraud-chloramphenicol medium and identification). Detection of galactomannan (GM) antigen was performed on the serum using the Platelia Aspergillus technique (BioRad, Marnes-La Coquette, France). Patients with ischemic bronchitis and Candida spp. colonisation were all treated by fluconazole and when an invasive candidiasis was suspected or proven, an echinocandine was administered. If an aspergillosis infection was suspected or if the patient had a special risk of developing an aspergillosis infection (for example, administration of antilymphocyte serum), voriconazole was administered. Definition of colonisation and infection. Endoscopic signs of bronchial or anastomotic aspergillosis infection were assessed by senior pneumologists of LT department. Bronchial or anastomotic aspergillosis infections were defined as isolation of Aspergillus in culture with histopathologic evidence of tissue invasion or necrosis, ulceration or pseudomembranes on bronchoscopy, as previously reported in lung transplant recipients [7].AC was defined as identification of Aspergillus spp. via culture of a bronchopulmonary sample in patients with no radiologic or endoscopic signs of pulmonary or tracheobronchial invasive aspergillosis. Colonisation was considered certain if two consecutive cultures were positive in expectorations or aspirations, or if a single BAL was positive in culture [8]. Colonisation was considered probable when a culture was positive in a single expectoration or aspiration. Invasive aspergillosis was defined using EORTC/MSG 2008 criteria [9]. Possible, probable or proven aspergillosis is distinguished using these clinical, radiological and biological criteria. Monitoring of the environment. During the post-transplant hospitalization, air and surface samples were collected each week. All samples were performed by the same person using a standardized protocol over the study period. One air sample was collected in the patient's room, one in the corridor immediately adjacent to the room and one in the unit's care station. Each sample (500 L) was collected on an Air Test Omega impactor (LCB, LaSalle, France) loaded with a 90-mm diameter Petri dish containing maltose-agar chloramphenicol medium. Surface samples were taken from the patient's room via wet swabbing early in the morning prior to daily room cleaning. Five surfaces were sampled: the edge of the bed, the mobile table, the medical fluid supply and light ramp, the floor, television and windowsill. Air and surface samples were incubated for 3 to 5 days at 27°C. Colonies were counted and molds were identified by their macroscopic and microscopic appearance after lactophenol cotton blue staining. Results were expressed in cfu/m3 and % of positive surface samples (i.e. at least one cfu). Molecular identification and typing of Aspergillus isolates. This study was performed on Aspergillus isolates belonging to the same species or section based on mycological

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examination, and that were found in both clinical and environmental samples taken at the same period. DNA was extracted for isolates to be genotyped and A. fumigatus strains were typed with a set of 4 microsatellites as previously described [10]. Signals were read with an automatic sequencer (ABI Prism 310, Applied Biosystems) and data were stored and analyzed with Genescan software (version 2.1, Applied Biosystems). Multilocus sequence typing was used for Aspergillus isolates for which microsatellite typing was not available. Internal transcribed spacer (ITS) 1 and 2 loci, the β-tubulin gene and the actin gene were amplified and sequenced via the fluorescent dideoxynucleoside triphosphate method [11–13]. Sequences were aligned using Clustalw software (http://www.ebi.ac.uk/Tools/ msa/clustalw2/), and detection of homologous sequences in GenBank's nucleotide database was carried out using Nucleotide Basic Local Alignment Search Tool software (BLASTn) (http://blast.ncbi.nlm.nih.gov/).

Statistical Analysis Statistical analysis of environmental data included a comparison of frequencies of positive samples (i.e. presence of at least one colony) in air and surface samples collected in the SICU and LTU. Descriptive and comparative analyses of patients’ characteristics according to their colonisation status were carried out using Fisher's exact test for categorical variables and the MannWhitney U test for continuous variables. The Pearson test was used to analyze the correlation between average weekly contamination of air (cfu/m3) and % positive cultures on surfaces in the SICU and LTU. A discrete-time Cox’s proportional hazards model was used to determine the role of individual characteristics and environmental data in risk of AC. In this analysis, we focused on time to first certain or probable AC sample in patients after transplant, ignoring repeat episodes in the same patient. Patients who remained negative were censored at their date of discharge or death. Environmental variables were entered in the model as time dependent, changing at each date of sampling. All variables were first tested in a univariable model. Covariates significant at the P0,25

9 (75)

24 (75)

33 (75)

>0,25

Pulmonary emphysema

7 (58)

10 (31)

17 (39)

>0,25

Idiop. pulm. fibrosis

3 (25)

1 (3)

4 (9)

0,19

COPD

4 (34)

16 (50)

20 (45)

>0,25

Age (y), mean ± SD (range) Male gender Underlying disease

0

2 (6,5)

2 (5)

>0,25

Smoking

11 (92)

24 (75)

35 (80)

>0,25

Diabetes

3 (25)

4 (13)

7 (16)

>0,25

Positive CMV status

8 (67)

20 (63)

28 (64)

>0,25

Other

Before transplantation status 0

2 (6)

2 (5)

>0,25

Aspergillus colonisation

1 (8)

4 (13)

5 (11)

>0,25

Antifungal treatment

2 (17)

5 (16)

7 (16)

>0,25

Anti- Aspergillus Ab

Type of transplantation Single-lung

7 (58)

19 (59)

26 (59)

>0,25

Double-lung

5 (42)

13 (41)

18 (41)

>0,25

Mortality

1 (8)

13 (41)

14 (32)

0.06

0

2 (6)

2 (4.5)

>0.25

Two-year follow-up Invasive aspergillosis All values are n (%), unless indicated Abbreviations: SD, standard deviation; Idiop. pulm. Fibrosis, idiopathic pulmonary fibrosis; COPD, chronic obstructive pulmonary disease, CMV, cytomegalovirus; Ab, antibody Eight patients (18%) died during their post-operative hospital stay, but none from fungal infection. Non-colonised patients had a higher mortality than colonised patients (p = 0,06). No patient developed invasive aspergillosis during the study period. Twelve patients (27%) had transient AC (certain: 2, probable: 10), without associated manifestation of aspergillosis. All colonised patients had single species colonisation. Their clinical and biological characteristics are presented in Table 2. AC occurred 1 to 13 weeks after transplant (median, 5 weeks); it lasted from 1 to 2 weeks. AC occurred in the SICU and LTU in 4 and 8 cases, respectively. doi:10.1371/journal.pone.0144044.t001

All patients were followed for 2 years after transplantation and 14 died during the surveillance period; 1 had AC during hospitalization, while 13 did not (p = 0.06). Among survivors, one developed sinusal aspergilloma 13 months after LT and one pulmonary aspergillosis 17 months after LT; these two patients were not colonised during initial hospitalization. Environmental contamination. Mycological surveillance of the environment included 198 series of samples in the SICU (1,592 samples) and 228 (1,848 samples) in the LTU. Average fungal air contamination was significantly higher in the LTU (15.8 cfu/m3 ± 24.2) than in the SICU (4.6 cfu/m3 ± 6.8) (p