Domestic mould exposure and invasive aspergillosis air sampling of Aspergillus spp. spores in homes of hematological patients, a pilot study

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1 Medical Mycology, 2016, 54, doi: /mmy/myw007 Advance Access Publication Date: 3 March 2016 Original Article Original Article Domestic mould exposure and invasive aspergillosis air sampling of Aspergillus spp. spores in homes of hematological patients, a pilot study KE Schweer 1,2,BJakob 1,2,BLiss 1,2,HChrist 3, G Fischer 4, MJGT Vehreschild 1,2,5, OA Cornely 1,2,5,6,7 and JJ Vehreschild 1,2,5, 1 Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany, 2 Center for Integrated Oncology CIO KölnBonn, Medical Faculty, University of Cologne, Cologne, Germany, 3 Institute of Medical Statistics, Informatics and Epidemiology, University of Cologne, Cologne, Germany, 4 Landesgesundheitsamt Baden-Württemberg (local health authority Baden-Württemberg), Germany, 5 German Centre for Infection Research (DZIF), partner site Bonn-Cologne, Cologne, Germany, 6 Clinical Trials Centre Cologne, ZKS Köln, Medical Faculty, University of Cologne, Cologne, Germany and 7 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Medical Faculty, University of Cologne, Cologne, Germany To whom correspondence should be addressed. Jörg Janne Vehreschild, MD, Department I for Internal Medicine and Hematology and Oncology, University Hospital of Cologne, Herderstraße 52 54, Cologne, Germany. Tel: +49 (221) ;Fax:+49 (211) ; joerg-janne.vehreschild@uk-koeln.de Received 1 December 2015; Revised 12 January 2016; Accepted 13 January 2016 Abstract Aspergillus spp.-related morbidity and mortality remains a major challenge in the management of neutropenic patients. Little is known about the impact of domestic Aspergillus spp. exposure. In this controlled prospective study, fungal spores were collected from homes of neutropenic patients. Cases were defined as patients with probable or proven controls as patients with no invasive pulmonary aspergillosis, while patients with possible disease were evaluated as a third group. Forty patients were enrolled and returned questionnaires on high-risk activities and mould exposure. A. fumigatus was detected in concentrations of 0 to 76 cfu/m 3 in every home. A. terreus was detected in nine (18%) homes. Mean Aspergillus spp. cfu/m 3 according to EORTC criteria were: proven/probable IA (15 patients) 36; possible IA (12 patients) 42; no IA (13 patients) 42. Of the seven patients with self-reported moulded walls at home, four had probable and three had possible aspergillosis; the risk ratio of developing IA was 1.65 (95% CI: ). In conclusion self-reported domestic mould exposure was associated with a high incidence of IA and may be a feasible tool for identifying high-risk patients. There was no correlation between domestic ambient-air spore counts and IA. 576 C The Author Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please journals.permissions@oup.com

2 Schweer et al. 577 Key words: domestic exposure, spores, pathogenesis, risk factors, hygiene. Introduction As treatment options for patients with hematological, oncological, or immunological diseases multiply, more patients under severe immunosuppression have to be managed in the hospital as well as in the outpatient setting. A significant number of these patients experience lifethreatening opportunistic infections. Among them, invasive aspergillosis (IA) is an important clinical entity, 1 especially for patients with hematological malignancies receiving anticancer chemotherapy. Aspergillus spp. are ubiquitous opportunistic pathogens. The clinically most relevant species comprise A. fumigatus, A. flavus, A. niger, and A. terreus. Conidia, the sporal form of Aspergillus spp. with high environmental resistance, can be detected in and outside of buildings year round. 2 4 Air concentrations of conidia are expressed as colony-forming units (cfu) per volume and are reportedly influenced by numerous environmental factors such as temperature, humidity, and precipitation. 5 7 They are also often present during periods of construction work. 8 The infection mechanism of aspergillosis has been subject to numerous studies; airborne infection by inhalation of Aspergillus conidia is a common hypothesis. Never the less both the role of quantity and duration of exposure to Aspergillus conidia remain unclear. 9 Some observational studies reported that infection control measures focussing on air purification might reduce airborne Aspergillus spore counts and the number of hospitalacquired IA among neutropenic patients. 8,10 14 It is an ongoing debate whether all IA are hospital-acquired, as some authors suggest Another hypothesis would be that patients arriving in the hospital after domestic mould exposure are later infected by dormant conidia or bronchial mould colonization. Aspergillus air-sampling has been used for over two decades to gain estimates on air contamination Analysis of Aspergillus spp. air samples from patient homes and surroundings has rarely and only in the recent past been correlated with development of IA, even though exposition to moulds at home might be an important risk factor to consider. 22 Methods Study design In this prospective case-control pilot study, we aimed to assess if differences in Aspergillus spp. exposure in the homes and domestic surroundings of neutropenic haematological patients exist between patients with or without later diagnosis of IA. Patients with probable or proven IA according to the revised EORTC/MSG criteria 23 were compared to patients without signs for IA. Patient selection Key inclusion criterion was a galactomannan (GM) test performed in bronchoalveolar lavage (BAL) fluid or serum of a patient with clinical suspicion of IA. Patients with GM 0.5 optical density (OD) index were matched to patients with a GMresult of 0.5 OD according to underlying disease, duration of neutropenia, and age. We then classified patients according to the EORTC criteria for possible, probable, proven or no aspergillosis. Intervention All eligible patients were informed about the study and written informed consent was obtained before participation. If the patient agreed, we performed air sampling with the MAS-100 eco R (Merck Millipore, Merck KGaA, Darmstadt, Germany) air sampler in and around patients homes on two occasions separated by at least one week. The used device previously had shown comparable results with new generation impactors For each sampling inside and outside, 500 l of air were impacted on one culture plate (malt extract agar with chloramphenicol or Sabouraud agar with gentamycin and chloramphenicol) in the MAS-100 Eco. Exact places for measurement of the Aspergillus-spore air concentration were determined together with the patients on the spot. We performed measurements in rooms or home surroundings where patients spent most of their time (such as bedroom, living room, balcony, garden next to the compost, etc.) throughout the year. If patients were not able to specify their preferred room, we used the room with the highest expected mould concentration. The investigator recorded places of measurement and climatic conditions (season, clouds, wind, rain, temperature). Some examples can be found in Table 2. Patients completed a questionnaire as presented in the appendix, providing information on housekeeping habits, housing interior, and outside conditions, including possible surrounding Aspergillus spp. sources, (urban vs. rural neighbourhood, close-by park, forest, farming, waste disposal site, factory, or waste water treatment plant, presence of a bio-waste container,

3 578 Medical Mycology, 2016, Vol. 54, No. 6 compost, damp or mouldy walls, mouldy basement, type of flooring like PCV, parquet, tiles, carpet or laminate, potted plants, pets, frequency and method of cleaning, age of mattress, vacuum cleaner and building, duration of residence). Potential Aspergillus spp. sources in the surrounding area were confirmed by analysing satellite images with metadata of a three kilometre radius surrounding the patient s home using GOOGLE Earth TM (Google Inc., Mountain View, United States). Analysis We expressed Aspergillus spp. findings as cfu/plate (colonyforming units per agar plate), which corresponds to analysis of 500 l of air impacted on one plate. Fungi were cultured on the impacted agar plates at 37 C for 2 5 days (depending on colony growth speed), thus only the few species relevant for infections were able to grow. For quality control plates were photographed and sent electronically to the coauthor Dr. G. Fischer at the LGA in Stuttgart, where identification by macro-morphology is routinely performed. In a later stage, identification was validated only in doubtful cases. Statistics We used IBM R SPSS R Statistics, version 22 (IBM, Armonk, New York, US) for data procession and analysis. Fisher s exact test, two sided was used for contingencytable data. We expected considerable curtosis for cfu data, so the Mann Whitney-U was used to test for differences in cfu/plate by EORTC group, for possible influential factors on cfu/plate and for differences in the EORTC groups. We used two-sided tests and allowed a type I error likelihood of 0.05 in this pilot study. Given the small sample size, all analyses were exploratory in nature. For the same reason, we decided to refrain from performing multivariate statistics. Ethical considerations This study was approved by the ethics committee of the University Hospital of Cologne. All patients included received detailed oral and written information and signed a written informed consent. Results Demographics Patients at risk for IA were consented during inpatient periods and followed up during discharge periods. From a total of 40 patients enrolled, 15 had probable or proven IA (IA positives) and 13 had no IA (IA negatives). Because of the intrinsic lack of diagnostic accuracy, the 12 patients with possible IA were excluded from analysis of risk factors for IA. Patient age ranged from 19 to 75 years (median 49). GM levels were >0.5 optical density index (OD) for 14 patients and <0.5 for 26 patients, levels varied between 0.1 and 5.2 OD for BAL testing and between 0.1 and 144 OD in serological testing. Aspergillus spp. were detected by histology from lobectomy and BAL PCR in one patient each. Mean duration of neutropenia was longer for IA positive patients compared to IA negative patients (25 days vs. 16 days, onesided P-value.16). Distribution of sex, age, and underlying disease was roughly even between cases and controls (IA negatives vs. IA positives). Table 1 gives an overview of the patient demographics. Aspergillus air sampling Of 158 incubated impact plates, two could not be analysed. One patient died before the second measurement. We performed analysis for the five most common species (A. fumigatus, A. flavus, A. terreus, A. niger, A. nidulans). Air sampling yielded between 0 and 38 (median 6) Aspergillus spp. cfu per plate and measurement. Aspergillus fumigatus was the most frequently detected species; it was found in the homes of all 40 patients. The A. fumigatus cfu counts ranged from 0 to 38 (median 4). Other Aspergillus spp. were found in lower quantities and less frequently, for example, A. terreus in nine (23%), and A. flavus in six (15%) homes. We measured lower Aspergillus spore counts inside than outside patients homes. Median cfu/plate inside vs. outside were two vs. three (first measurement) and one vs. five (second measurement), two-sided P- values were.104 and.003, respectively. Median cfu/plate were not higher during autumn than during other seasons. Peak values, however, were recorded during autumn season (Table 2, a detailed overview of the results per season is provided in the appendix 4). Of note, these patients did not report mouldy walls. There was no significant correlation between results from the first and second measurement or between outside and inside spore counts from one measurement. Univariate analysis also did not show a correlation between Aspergillus spore counts and any weather condition, housekeeping habits or any interior design. Mean Aspergillus spp. cfu counts inside and outside of the homes of patients with IA were not elevated compared to spore counts from the homes of patients without IA (Table 3).

4 Schweer et al. 579 Table 1. Patient demographics according to EORTC criteria. Absolute patient numbers IA negative IA possible IA positive Total Mean GM in BAL 0.29 (SD 0.44) (SD 0.05) 1.35 (SD 1.34) Mean GM in serum (SD 0.05) (SD 0,03) (SD 37) Female sex Mean age Mean duration of neutropenia Autologous stem cell transplantation Allogeneic stem cell transplantation AML/MDS ALL Lymphoma Other hematologic malignancy Positive culture Positive histology Admission to ICU Death after 90 days of follow up Abbreviations: ALL = acute lymphoblastic leukemia; AML = acute myeloid leukemia; BAL = bronchoalveolar lavage; EORTC = European Organisation for Research and Treatment of Cancer; GM = galactomannan (Aspergillus antigen); IA = invasive Aspergillosis; IA positive = probable and proven IA according to EORTC criteria; ICU = intensive care unit; MDS = myelodysplastic syndrome; SD = standard deviation. Note: N = absolute number of patients, except for GM (as optical density index) age (in years), neutropenia (in days), in total: 40 patients enrolled. mean age of total study population: 49 years. mean duration of neutropenia in total study population: 24 days. Table 2. Peak spore counts, their site of measurement and weather conditions. Type of sampling Peak Site of measurement + weather conditions INDOOR Aspergillus fumigatus 24 Living room, October, 22 C, cloudy, dry (patient 26) Aspergillus niger 15 Bedroom, September, 23 C, cloudy, dry Aspergillus terreus 4 Dairy Counter in patient s cheese parlor, January, 8 C, cloudy, dry Aspergillus flavus 11 Living room, April, 10 C, cloudy, dry Aspergillus nidulans 3 Living room, July, 13 C, cloudy, rainy OUTDOOR Aspergillus fumigatus 38 Balcony, October, 22 C, cloudy, dry (patient 26)Garden, November, 5 C, cloudy, dry (patient 28) Aspergillus niger 14 Compost in driveway, September, 21 C, cloudy, dry Aspergillus terreus 3 Front door, January, 0 C, cloudy, dry Aspergillus flavus 2 Dustbin at front door, August, 25 C, cloudy, rainy Aspergillus nidulans 2 Site not specified, July, 13 C, cloudy, rainy Note: Peak Aspergillus spore counts expressed as cfu/plate (500 l of ambient air impacted on each plate) for Aspergillus air samplings indoors and outdoors (exemplary single patient data from a pool of 40 domestic measurements on two separate occasions). Questionnaire data All patients filled an extensive questionnaire as presented in the supplementary files. Data from questionnaires were analysed for statistical differences between patients with and without IA. Of the seven patients who reported mouldy walls in their home, four were diagnosed with probable/proven IA (total cfu/plate from both measurements 7, 13, 21, 48) and the other three with possible IA (total cfu/plate from both measurements 24, 38, 48). From 33 patients without visible mould growth at home, 13 did not have IA, 9 had possible IA, and 11 had probable or proven IA. The risk ratio of developing possible, probable, or proven IA after self-reported domestic mould exposure was 1.65 (95% CI: ). Mean indoor spore counts in mouldy homes were 6 cfu/plate (standard deviation (SD) = 7) compared to 3 cfu/plate (SD = 5) in homes without mould growths (Figure 1, a detailed overview of the results is provided in the appendix 5). This difference did not reach statistical significance (two-sided P-value.3).

5 580 Medical Mycology, 2016, Vol. 54, No. 6 Table 3. Mean spore counts in and around IA-positive and IA-negative patients homes. Total Aspergillus spp. in cfu/plate A. Fumigatus in cfu/plate IA negatives IA positives IA negatives IA positives 1 st inside measurement 5 (1 9) 3 (0 6) 2 (1 4) 3 (0 5) 2 nd inside measurement 3 (0 5) 3 (2 4) 2 (0 3) 2 (1 3) 1 st outside measurement 5 (2 8) 5 (2 8) 5 (2 7) 4 (1 7) 2 nd outside measurement 8 (1 14) 7 (3 10) 7 (1 13) 6 (3 9) Abbreviations: EORTC = European Organisation for Research and Treatment of Cancer; IA = invasive aspergillosis. Note: Mean spore counts expressed as cfu/plate, with 500 l of ambient air impacted on each plate, 95% CI given in brackets, IA positive = probable and proven invasive aspergillosis according to EORTC criteria; IA negative = no suspicion of invasive aspergillosis. Figure 1. Aspergillus spore counts given in absolute numbers of cfu/plate (500 l of ambient air impacted on culture plate), data from 38 participants. No statistically significant difference between the spore counts from homes with mouldy walls and those without was found. Patients who had ongoing construction work in their homes during their oncologic treatment were not diagnosed with IA more often than those who did not. Patients with pets did not develop IA more frequently than those without. Likewise, no difference was observed for any reported housekeeping routine or different types of flooring or bedding (appendix 1, 2). Between the proven/probable IA patients and those without IA, we found no differences in home surroundings (appendix 3). Discussion To our knowledge, this is one of the first studies exploring potential influences of home mould exposure on development of IA. We were able to confirm that Aspergillus fumigatus, the predominant pathogen causing IA, was isolated most frequently and in highest quantities in and around the homes of our patients. In addition, we found Aspergillus terreus in 23% and Aspergillus flavus in 15% of the homes investigated. Mean cfu/m 3 with MAS 100 eco were comparable to values from air samplings around the globe by different samplers (Table 4). By air-sampling in this pilot study, we found no correlation between airborne Aspergillus spores in patients homes and development of probable or proven IA. Attempts to show a correlation between cfu/air volume and IA incidence have failed repetitively in different study set-ups, 2,24 while other evidence supports a direct connection. 8,10 14 Correlation of spore counts and IA in the domestic setting is generally more difficult to interpret since the precise time spent in the surroundings by a patient is significantly more variable than in the hospital. Further inference is limited by the difficulty of establishing diagnosis of IA, 23 and the fact that the infectious inoculum and exposure time-frame

6 Schweer et al. 581 Table 4. Mean indoor cfu/ m 3 in different countries measured by different air samplers. Sampler Impact plate Country Year cfu/m 3 indoors This study MAS 100 eco Germany Boff et al. 2 Six-Stage Andersen Brazil Hospenthal et al. 24 Single-Stage Andersen USA Cornet et al. 8 Bio-Impactor France Note: Exemplary comparison of mean spore counts acquired with different air samplers in different countries during different years. (NB: Results given in cfu/m 3, values from the literature were acquired in hospitals, we measured in patient homes.) for development of IA are not known. 9 In our study, for all but one patient, the causative Aspergillus spp. remained unknown and we were not able to perform correlation analysis on species level. Of note, patients reporting walls with visible mouldinfestation in their home had a higher chance of developing IA. These results are far from statistical relevance and unfortunately wall smears and detailed information on wall covering (wall paper, emulsion, etc.) were not obtained. Still IA may be home-borne, as is also suggested by the findings of Rocchi et al. 22 Explanations for the difficulty of proving a clinically established risk factor (exposure to mouldy walls or ongoing construction) by air sampling of Aspergillus spp. are manifold: spore counts may be highly variable even during one day and in one place and are easily influenced by external factors as simple as type and frequency of ventilation. 25 In this pilot study, we found no correlation of spore counts between the two measurements, suggesting that we recorded some transient high counts and missed others. Dust analysis is complementary to air sampling as it reflects the presence of fungi over an accumulated period of time and might in fact be more accurate than air sampling because it is less influenced by wind and movement. 18 Gravity plates on the other hand might be an alternative to capture peak spore levels during a set time period. 26 In addition, especially in the supposedly less rigorously supervised and cleaned domestic surroundings, it might be worth considering water-borne IA While some publications report peaks of conidial airconcentrations in autumn season measured by air sampling, 2 4,35 37 others 24 could not reproduce this finding. From our data we cannot deduce that spore counts are generally elevated in autumn season; however, peak spore levels were more often measured during autumn season than during the rest of the year. Panackal et al. came to the conclusion that geoclimatic influences such as temperature and precipitation have different effects on spore counts and IA in geographically distinct areas with differences in baseline spore counts. 5 While we investigated the impact of weather conditions on air mould concentrations, we cannot provide data on humidity, an important external factor for fungal growth. 6,7 Our analysis was not powered to test the impact of interior design aspects such as type of ventilation (natural like open windows, circulatory like heat-exchanger, etc.), flooring, wall covering, bedding and pillows on the incidence of IA. Still, this might be an important factor, should more evidence emerge that a significant part of IA is home-borne. 38 Future studies assessing development of IA following environmental fungal exposure should combine multiple methods to further determine the actual mould exposure in patients homes. We propose the complementary use of gravity plates, 24 hour sampling, dust, and wall samples as well as water sampling coupled with on the spot visits and questionnaires as presented in this pilot study. To evaluate a possible correlation of domestic mould burden and clinical infection, ideally DNA of the domestic and clinical patient isolates should be quantified and sequenced. Data on humidity should be obtained, as well as objective information on interior design and dimension (size and type) of possible home mould infestation. Even though we could not proof a correlation between outside and inside spore counts the dominant effects of outdoor air on indoor mould incidence seems obvious. 38 Habits and ways of home ventilation should be recorded. Roussel et al. even propose to exclude measurement during summer months and to close all windows 1 h before air sampling for spores. 25 Supplementary home counselling of patients at risk, as proposed by Rocchi et al. 22 may be useful. Acknowledgments This study was supported by an unrestricted grant from Pfizer. Declaration of Interests OAC is supported by the German Federal Ministry of Research and Education and the European Commission, and has received research grants from, is an advisor to, or received lecture honoraria from 3 M, Actelion, Anacor, Astellas, AstraZeneca, Basilea, Bayer, Celgene, Cidara, Cubist/Optimer, Da Volterra, Daiichi Sankyo, Duke University (NIH M1AI104681), F2G, Genentech, Genzyme, Gilead, GSK, Leeds University, Matinas, Medpace, Merck Serono, MSD, Miltenyi, NanoMR, Novartis, Parexel, Pfizer, Quintiles, Roche, Sanofi Pasteur, Scynexis, Seres, Summit, Vical, Vifor, Viropharma.

7 582 Medical Mycology, 2016, Vol. 54, No. 6 JJV is supported by the German Centre for Infection Research and has received research grants from Astellas, Gilead Sciences, Infectopharm, Merck Sharp Dohme/Merck, Pfizer, and Essex/Schering-Plough; has consulted Merck Sharp Dohme/Merck; and served on the speakers bureau of Merck Sharp Dohme/Merck, Gilead Sciences, Pfizer, and Essex/Schering-Plough. MJGTV has served at the speakers bureau of Pfizer, Merck/MSD, Gilead Sciences, and Astellas Pharma, received research funding from 3 M, DaVolterra, Gilead Sciences and Astellas Pharma and been a consultant to Astellas Pharma, Berlin Chemie, Merck/MSD and DaVolterra. KS has served on the speakers bureau of Astellas. GF, HC, BJ, and BL have declared to have no conflicts of interest. Supplementary Material Supplementary material is available at Medical Mycology online ( Author s Contributions KES analysed data and wrote the manuscript. BJ consented patients, conducted measurements, identified fungi, collected, processed and analysed data from patient records, and revised the manuscript. BL consented patients, conducted measurements, and revised the manuscript. GF identified fungi and revised the manuscript. HC analysed data and revised the manuscript. MJGTV selected patients, analysed data and revised the manuscript OAC designed study, analysed data, and revised the manuscript. JJV selected patients, analysed data, and prepared the final manuscript. References 1. Segal BH. Aspergillosis. NEnglJMed2009; 360: Boff C, Zoppas BCDA, Aquino VR et al. The indoor air as a potential determinant of the frequency of invasive aspergillosis in the intensive care. Mycoses 2013; 56: Vonberg RP, Gastmeier P. Nosocomial aspergillosis in outbreak settings. J Hosp Infect 2006; 63: Brenier-Pinchart MP, Lebeau B, Quesada JL et al. Influence of internal and outdoor factors on filamentous fungal flora in hematology wards. 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