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Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis
JCF-01016; No of Pages 7
Journal of Cystic Fibrosis xx (2014) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis☆,☆☆ Perrine Parize a,b,⁎, Sandrine Billaud c , Anne L. Bienvenu d,e , Stéphanie Bourdy f , Marie A. le Pogam f , Philippe Reix b,g , Stéphane Picot d,e , Raymond Robert c , Olivier Lortholary h , Jean-Philippe Bouchara c,i , Isabelle Durieu a a
h
Hospices Civils de Lyon, Centre Hospitalier Lyon Sud, Adult Cystic Fibrosis Reference Center, 69495 Pierre-Bénite, France b Université de Lyon, Lyon, France c L'UNAM Université, Université d'Angers, Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, Institut de Biologie en Santé-IRIS, 49933 Angers, France d Hospices Civils de Lyon, Service de Parasitologie et de Mycologie Médicale, 69317 Lyon, France e University Lyon 1, Malaria Research Unit, ICBMS, CNRS UMR, 69373 Lyon, France f Hospices Civils de Lyon, Pôle IMER, Université de Lyon, 69424 Lyon, France g Hospices Civils de Lyon, Hôpital Femme-Mère-Enfant, Pediatric Cystic Fibrosis Reference Center, 69500 Bron, France Université Paris-Descartes, Hôpital Necker-Enfants malades, Service des Maladies Infectieuses et Tropicales, Centre d'Infectiologie Necker-Pasteur, IHU Imagine, 75015 Paris, France i Laboratoire de Parasitologie-Mycologie, Institut de Biologie en Santé-PBH, Centre Hospitalier Universitaire, 49933 Angers, France Received 6 December 2013; received in revised form 20 January 2014; accepted 27 January 2014
Abstract Background: Species of the Scedosporium apiospermum complex (S. a complex) are emerging fungi responsible for chronic airway colonization in cystic fibrosis (CF) patients. Recent studies performed on Aspergillus fumigatus suggest that the colonization of the airways by filamentous fungi may contribute to the progressive deterioration of lung function. Methods: We studied S. a complex seroprevalence, as a marker of close contact between patient and the fungi, in a large monocentric cohort of CF patients attended in a reference centre in Lyon, France. Results: Serum samples from 373 CF patients were analysed. Antibodies against S. a complex were detected in 35 patients (9.4%). In multivariate analysis, S. a complex seropositivity was only associated with seropositivity to A. fumigatus. Conclusions: This study does not suggest an association between sensitization against S. a complex and poorer lung function in CF. Prospective studies are needed to evaluate the impact of both seropositivity and S. a complex colonization on the course of CF. © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Scedosporium apiospermum; Cystic fibrosis/microbiology; Respiratory function tests; Serologic tests
☆ Abstract presentation: European Cystic Fibrosis Society conference, June 2010, Valencia, Spain. ☆☆ S. Billaud, R. Robert and J.P. Bouchara are members of the ECMM/ ISHAM working group on fungal respiratory infections in Cystic Fibrosis (FriCF). ⁎ Corresponding author at: Université Paris-Descartes, Hôpital Necker-Enfants malades, Service des Maladies Infectieuses et Tropicales, Centre d'Infectiologie Necker-Pasteur, 149 rue de Sèvres, 75015 Paris, France. Tel.: +33 1 42 19 26 63; fax: +33 1 44 49 54 40. E-mail address: [email protected] (P. Parize).
1. Introduction Scedosporium apiospermum is a ubiquitous filamentous fungus initially considered the anamorph state of Pseudallescheria boydii. However, recent taxonomic studies indicate that S. apiospermum is a member of a species complex, called S. apiospermum complex (S. a complex), which comprises at least five closely related but distinct species, i.e. S. apiospermum sensu stricto (anamorph state of Pseudallescheria apiosperma), Scedosporium boydii (anamorph
1569-1993/$ -see front matter © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcf.2014.01.011 Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
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state of P. boydii), Scedosporium aurantiacum, Scedosporium dehoogii and Pseudallescheria minutispora [1,2]. These fungi are now considered as emerging opportunist pathogens due to the recent increase in reported infections [3–5]. The spectrum of S. a complex infections is broad, ranging from localized diseases to multi-organ involvement and is closely related to the immunological status of patients [6]. Along with Aspergillus fumigatus, species of the S. a complex are currently identified as the main fungal agents responsible for chronic airway colonization in CF patients, with a prevalence ranging from 3.4% to 15.9% in limited monocentric studies [7–10]. Several cases of lethal invasive S. a. complex infections have been reported in CF patients having undergone lung transplantation [11–14]. These patients usually developed disseminated infections, affecting mostly the lungs, heart, eyes and central nervous system in the first year post-transplantation and died few weeks after diagnosis. Most of these patients were colonized before transplantation which is thought to be a risk factor of infection once the patients become immunocompromised. Indeed, because mortality of Scedosporium infections is high, S. a. complex colonization is debated as a contraindication to transplantation by some centres. Diagnosis of Scedosporium infections is difficult and often delayed because the clinical symptoms are aspecific and because a positive culture of a pulmonary specimen (i.e. sputum or bronchial aspirate) can't allow the distinction between colonization and infection. The diagnosis is confirmed once the fungus has been identified in culture from a specimen obtained by a sterile procedure or when histopathological examination reveals the presence of tissue damages in association with hyphae. Management of S. a complex infections is also complex due to the poor intrinsic activity of echinocandins and triazoles against these species. Treatment may thus require a combination of optimized antifungal therapy and surgery to reduce the fungal burden. Additionally a reduction of immunosuppression is discussed when feasible. In contrast to the severity of Scedosporium infections in immunocompromised patients, colonization is mostly asymptomatic prior to lung transplantation, although allergic bronchopulmonary disease [8] and rare Scedosporium invasive infections have been described in patients with diabetes mellitus [15,16]. However, the impact of long-term airway colonization by S. a complex on respiratory function in CF patients is still unclear. It is suggested that prolonged colonization of the airways by A. fumigatus in CF patients may accelerate lung function deterioration, thus increasing the risk of hospitalizations for pulmonary exacerbations and radiological abnormalities [17–19]. Therefore one may suspect that airway colonization by species of the S. a complex also contributes to the progressive deterioration of lung function by enhancing the local inflammatory response [20]. A study evaluating the impact of airway colonization on CF lung function is actually hardly achievable due to the low apparent incidence of colonization by S. a complex among the CF population. The rate of respiratory colonization by S. a complex is difficult to evaluate in non-specialised centres because the routine use of non-selective culture media for mycological examination of sputum samples leads to a significant underestimation of S. a complex detection [9,10].
We decided to perform a large monocentric retrospective study of the impact of S. a complex seroprevalence on CF patients' outcome and to determine clinical factors associated with seropositivity. 2. Methods 2.1. Subject population Eligible subjects for this study were patients diagnosed with CF who had at least one serum sample collected during a routine annual visit to the pediatric or adult CF reference centre in Lyon, France in 2008. A total of 394 patients were eligible for this study. Patients who had undergone lung transplantation were excluded (n = 21). Thus, 373 patients were included in this study which was approved by the hospital Ethical Committee (2009, Hospices Civils de Lyon). 2.2. Study design and data collection Data were extracted from the electronic medical records of Lyon Centre Cystic Fibrosis Database. During each visit, a sputum is systematically collected for bacterial and fungal examination and a serum sample is collected and stored at −20 °C in the sera bank of our laboratory at least once a year. For each patient we selected in the bank a serum sample collected in 2008 to assess the S. a complex serology. Patients' clinical characteristics collected during the same visit as the selected serum sample were extracted from the Database. Studied parameters were age, weight, size, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutations, history of previous bacterial or fungal airway colonization, history of diabetes, ABPA (allergic bronchopulmonary aspergillosis), medications, serological status towards A. fumigatus, results from sputum fungal culture, and outcomes of pulmonary function testing. 2.3. Serology Serum antibodies to the S. a complex were determined in July 2009 by ELISA using a cytosolic antigenic extract obtained from strain IHEM 15.155 (Institute of Hygiene and Epidemiology– Mycology section; Institute of Public Health, Brussels, Belgium). This strain originally isolated from a bronchoalveolar fluid in the laboratory of mycology of Angers University hospital and already used in previous studies [8,21,22] was identified by beta-tubulin gene sequencing as P. boydii, which is the most common species of the S. a complex in CF in France [23]. Antigenic extract was prepared from cultures grown under stationary phase in yeast extract–peptone–dextrose broth (containing in g per litre: yeast extract, 10; peptone, 10 and glucose, 20). After incubation for two weeks at 37 °C without agitation, the mycelium was harvested by centrifugation, washed twice with sterile water, and mechanically disrupted for 2 min with glass beads (mix of 0.25-mm and 1-mm diameter; Braun Melsungen, Melsungen, Germany) [8]. Debris were removed by centrifugation at 12,000 g for 30 min at 4 °C, and the supernatant which constitutes the antigenic extract was stored at − 80 °C as aliquots until used.
Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
P. Parize et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
ELISA was performed in 96-well flat-bottom microtiter plates coated with the antigenic extract diluted in 50 mM carbonate buffer pH 9.6 (25 μg proteins per ml, 100 μl per well). After a 1hour incubation at 37 °C, followed by three washes in 0.15 M phosphate buffered saline (PBS) pH 7.2, 200 μl of a 3% bovine serum albumin solution in PBS was added to each well. The plates were incubated overnight at 4 °C, washed three times in PBS, and incubated for 1 h at 37 °C with sera from CF patients diluted 1:100 in PBS (100 μl per well). After washes in PBS containing 0.05% (vol/vol) Tween 20 (PBST), 100 μl of horseradish peroxidase-conjugated goat anti-human immunoglobulin γ chain antibodies (Rockland Immunochemicals Inc., Gilbertsville) or anti-human immunoglobulin μ chain antibodies (Biosys Technology, Paris, France) diluted 1:2000 in PBS was added to each well for determination of IgG and IgM, respectively. The plates were incubated for 1 h at 37 °C and finally washed three times in PBST. OPD (o-phenylenediamine dihydrochloride) in a buffered hydrogen peroxide solution was used for colour development. The absorbance was measured spectrophotometrically at 492 nm. A 0.9 absorbance unit was chosen as positivity threshold, as a result of prior unpublished analysis of sera from 49 non-CF healthy patients with a median age of 17 years [15–18 years] consulting for voluntary interruption of pregnancy or for sexually transmitted diseases screening and from CF- or non CF-patients with proven scedosporiosis (n = 5) or proven aspergillosis (n = 15), defined by the isolation of the fungus by culture and the presence of precipitin lines by counter-immunoelectrophoresis (CIE) using the corresponding antigenic extract (from P. boydii or A. fumigatus). All tests were performed in triplicate. Controls included the use of uncoated wells and addition of PBS instead of CF sera in coated wells. 2.4. Mycological examination of sputum samples The results of mycological examination of sputum samples were extracted from the mycology laboratory Database. Sputa were inoculated onto Sabouraud dextrose agar containing chloramphenicol and gentamicin. Plates were incubated at 37 °C and examined twice weekly for 7 days. Fungi were identified on the basis of their cultural features and microscopic morphology according to standard descriptions [24]. 2.5. Statistical analysis The initial analysis used descriptive statistics to characterize the study subjects. A univariate analysis was performed to compare the clinical characteristics of S. a complex seropositive and seronegative patients. Normality of all data was tested by Shapiro–Wilk test. Comparison of means was done using Student's t test when data were Gaussian and using Mann and Whitney test in the case of non-Gaussian data. Categorical variables were analysed using the Fisher's exact test or the χ2 test. Continuous data were compared using Student's t test. p values b 0.05 were considered to be statistically significant. A multivariable regression model was constructed using Hosmer–Lemeshow test to evaluate the effect of S. a complex seropositivity on covariates of interest defined by p b 0.2 in
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univariate: detail what is entered in the model. A cut-off of p b 0.2 was chosen to limit confusion factors which are frequent in CF. 3. Results 3.1. Cohort characteristics The demographic and clinical features of the 373 patients included are summarized in Table 1. Patients' median age was 15.4 years (range, 0.8–53.3). They were 199 males and 174 females. Bacterial airway colonization was reported for 272/373 patients (72.9%), mostly plurimicrobial with 203 patients colonized by Staphylococcus aureus, 156 by Pseudomonas aeruginosa and 31 by Stenotrophomonas maltophilia. During the study period, 119/ 373 patients (31.9%) had at least one sputum culture growing with one or more Aspergillus species including 111 patients colonized with A. fumigatus, 25 with Aspergillus flavus, 15 with Aspergillus terreus and 10 with Aspergillus niger. Among these patients 36 were colonized with several species of Aspergillus. Eight patients had S. a complex identified in sputum culture and 4 of them had concomitant A. fumigatus colonization. Of note, two patients had positive sputum samples for Scedosporium prolificans. Patients' medical history included diabetes mellitus for 35 patients and ABPA for 28 (among these patients, 21 have had at Table 1 Demographic and clinical characteristics of the 373 CF patients evaluated in 2008. Characteristics
Patients (n = 373)
Age, median (min–max), yr Sex ratio (M/F) CFTR mutations: F508del homozygosity a Bronchial colonization by S. aureus P. aeruginosa S. maltophilia Aspergillus fumigatus Scedosporium apiospermum complex Scedosporium prolificans FEV1% for patients b 18 (n = 222) b, mean (SD) FEV1% for patients ≥ 18 (n = 151), mean (SD) Prior ABPA Prior diabetes mellitus Treatment by Azithromycin Systemic steroids Itraconazole Voriconazole Inhaled treatment Inhaled steroids Inhaled RhDNAse Inhaled antibiotics Inhaled bronchodilator
15.4 (0,8;53,3) (0–53) 1.14 146 (39.1%) 203 (54.4%) 156 (41.8%) 31 (8.3%) 111 (29.8%) 8 (2.1%) 2 (0.5%) 87 (20.4) 60 (23.6) 28 (7.5%) 35 (9.4%) 127 (34.0%) 18 (4.8%) 16 (4.3%) 5 (1.3%) 287 (76.9%) 183 (49.0%) 175 (46.9%) 157 (42.1%) 202 (54.2%)
CFTR — Cystic Fibrosis Transmembrane Conductance Regulator. FEV1 — Forced Expiratory Volume in one second. ABPA — Allergic bronchopulmonary aspergillosis. a Missing data for 14 patients. b Missing data for 41 patients since spirometric measures were not available for patients younger than 6 years.
Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
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least one sputum culture growing with an Aspergillus species in Lyon). 21 patients received antifungal therapy, 16 had itraconazole and 5 voriconazole. Other therapeutics included long-term azithromycin therapy for 127 patients and systemic steroids for 18 patients. Most patients (287/373) had one or several inhaled treatments: 202 received inhaled bronchodilator, 183 inhaled steroids, 175 inhaled recombinant human deoxyribonuclease and 157 received at least one course of inhaled antibiotics during 2008. 3.2. Serology Prior analysis of sera from non-CF healthy patients and from CF- or non-CF patients with proven scedosporiosis or proven aspergillosis permitted to define a cut-off value of 0.9. None of the 49 patients in the control group as well as patients with proven aspergillosis were assessed positive for S. a complex serology by both CIE which could be considered as a “gold standard” serological test for fungi of the complex S. apiospermum and ELISA, whereas all tested sera from patients with confirmed scedosporiosis were found positive by CIE and ELISA with absorbance values always exceeding the threshold of 0.9. Using this threshold, antibodies against S. a complex were detected in 35 patients (9.4%) including 3 out of 8 patients with documented S. a complex airway colonization. The median age of seropositive patients was 20.2 years [1.7–47.2 years] compared to16.3 years [0.8–53.4] in seronegative patients. Distribution of seroprevalence in the different age classes is shown in Fig. 1. Seropositivity among patients increased during teenage years, then was stable during adulthood (χ2 test for trend p nonsignificant). The univariate analysis indicated that detection of serum antibodies against S. a complex was associated with older age, more frequent and prolonged hospitalizations, P. aeruginosa and S. a complex airway colonization, positive A. fumigatus serology, medical history of diabetes mellitus and ABPA, and at last with decreased pulmonary functions defined by a Forced Expiratory Volume in one second (FEV1) below 70% of the predicted value (Table 2). A regression model was generated to independently evaluate the effect of S. a complex seropositivity on those parameters.
Results of multivariate analysis revealed that seropositivity to S. a complex was significantly associated with seropositivity to A. fumigatus only. 4. Conclusions Here we demonstrate that immune response against S. a complex is relatively frequent in a large cohort of CF patients. Nearly 10% of the patients in our sample were found seropositive. This result is consistent with reported prevalence rates of 14.3% to 15.9% for the airway colonization by these fungi based on sputum cultures performed on highly selective culture media [9,10]. Currently, very few centres are using a selective culture medium to detect species of the S. a complex in CF patients. The heterogeneity of the procedures used for mycological examination of sputum samples (including the use of agar slants or agar plates, the number and nature of the culture media, the temperature and duration of incubation) leads to great variations in colonization prevalence estimates between centres and more generally to underestimating the burden of these fungi in CF patients [25]. The distribution of seroprevalence shows that the first exposure to the fungus is more likely to occur during teenage years as sensitization increases after the age of 10. This finding is consistent with the mean age of first detection of S. apiospermum colonization in CF patients, observed at 14.5 years (range, 7.8–21) by Cimon et al. [8]. In addition, first isolation of A. fumigatus in sputum of CF patients was also observed during teenage years, between 12.3 and 14.1 years [8]. After the age of 10 years, the S. a complex seroprevalence seems stable over time suggesting repetitive close contacts between the fungus and the patient during adulthood or that sensitization persists following the first exposure. Exposure to soil organisms could be the major source of contamination of the patients since species of the S. a complex are mainly recovered from ground (soil from the outdoor environment or from potted plants). Nevertheless, presence of potted plants at home of our patients was not recorded in the Database, and environmental studies were not performed. So it is not possible at present to establish a link between the S. a complex seropositivity and a higher exposure to these moulds. In addition, it is likely that genetic factors also determine the establishment of
Fig. 1. Seroprevalence of S. apiospermum complex by age class in 373 CF patients Chi2 test for trend: p not significant. Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
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Table 2 Univariate analysis and multivariate logistic regression analysis of factors associated with seropositivity to S. a complex. Characteristics
S. a complex seronegative patients (n = 338)
S. a complex seropositive patients (n = 35)
Age, median (min–max), yr Hospital admissions in 2008, mean number (SD) Number of hospitalisation days in 2008, mean (SD) Bronchial colonization S. aureus P. aeruginosa S. maltophilia S. a complex colonization Aspergillus fumigatus colonization Aspergillus fumigatus positive serology Medical history of ABPA Diabetes mellitus FEV1% predicted b 70 Treatment Itraconazole Voriconazole Azithromycin Systemic steroids Aerosol therapy
16.3 (0.8;53.4) 0.4 (1.0) 2.9 (10.8)
20.2 (1.7;47.2) 1.0 (1.6) 9.8 (21.8)
Simple logistic regression
Multiple logistic regression
p
p
OR(95%CI)
188 (55.6%) 136 (40.2%) 30 (8.9%) 5 (1.5%) 99 (29.3%) 39 (11.5%) 22 (6.5%) 27 (8%) 107 (31.6%)
15 20 1 3 12 11 6 8 20
(42.9%) (57.1%) (2.9%) (8.6%) (34.3%) (31.4%) (17.1%) (22.9%) (57.1%)
0.15 0.05 NS 0.03 NS 0.003 0.04 0.009 0.002
16 (4.7%) 5 (1.5%) 111 (33.4%) 14 (4.2%) 257 (77.4%)
0 0 16 4 30
(0%) (0%) (47.1%) (11.8%) (88.2%)
NS NS 0.11 0.07 0.14
OR(95%CI)
0.42 0.36 0.16
0.04 0.003 0.004
1.9 [1.0–4.0]
0.11 0.83
6.2 [1.4–27.3] 3.5 [1.6–7.7] 2.9 [1.1–7.9] 3.2 [1.5–6.9]
0.003 0.32 0.65 0.24
4.2 [1.6–10.8]
0.78 0.95 0.76
FEV1 — Forced Expiratory Volume in one second. ABPA — Allergic bronchopulmonary aspergillosis.
these fungi in the respiratory tract, such as the CFTR genotype and some polymorphism in the IL-10 gene as suggested for the airway colonization by A. fumigatus [26]. The association between S. a complex seropositivity and decrease in lung function or hospital admissions found in univariate analysis was no longer significant after adjustment for covariates. The association appeared to be explained mainly by age and confounders known to be related to lung function decline in CF patients. However, the study population may be too small to reveal correlations between S. a complex seropositivity and these particular outcomes. Interestingly, the analysis revealed no association between sensitization to S. a complex and inhaled antibiotics while the use of prophylactic inhaled antibiotics has been suggested to predispose CF patients to A. fumigatus colonization [27,28]. Finally, the results of multivariate analysis showed that seropositivity to S. a complex was significantly associated with seropositivity to A. fumigatus. This could be explained by the common occurrence of mixed colonization by S. a complex and A. fumigatus. As A. fumigatus is commonly found in soil and in the air, patients exposed to S. a complex probably have high risks of having been exposed to A. fumigatus as well. In the study conducted by Cimon et al. [8], almost all the patients colonized by species of the S. a complex were also colonized by A. fumigatus (10 out of 11 patients culture-positive for the S. a complex were colonized by both fungal species). Nevertheless, immune crossreactions between the S. a complex and other fungi such as A. fumigatus could also not be excluded. Although some of the cell wall polysaccharides differ from one fungus to another (peptidorhamnomannans in the S. a complex and galactomannans in A. fumigatus), they certainly share common epitopes in their
mannan moieties. Moreover, some of the fungal cell wall polysaccharides (chitin and ß-glucans) are common to almost all fungi, and some cell wall, membrane, of intracellular proteins needed for essential physiological processes in the fungal cell like respiration, glucose catabolism and energy production or synthesis of membrane ergosterol or cell wall polysaccharides, may also exhibit common epitopes. The role of fungal colonization on the evolution of the CF lung function is currently debated [29]. Recently, Chotirmall et al. [30] reported that repeated isolation of Candida albicans from sputum samples predicts both FEV1 decline and hospital-treated exacerbations in CF. Importantly, Candida airway colonization may be linked to an increased risk of P. aeruginosa ventilator-associated pneumonia in non-CF patients who received mechanical ventilation for more than 2 days [31]. Likewise, a longitudinal study has shown that CF patients persistently colonized by A. fumigatus had a higher rate of hospital admissions for pulmonary exacerbations and a decreased FEV1% predicted than controls [17]. A relationship between lung condition and fungal colonization was suggested by the authors but not further evidenced. A later retrospective cohort study evaluated the longitudinal effect of A. fumigatus colonization on lung function of CF patients over a 5-year period [28]. Differences in FEV1 between patients colonized by A. fumigatus and non-colonized subjects were explained by confounding factors known to be associated with lung disease severity. The present study is the first to evaluate the potential impact of S. a complex seropositivity on the course of CF lung disease. It made use of a large Database of children and adult patients representative of the French CF registry [32].
Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
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We acknowledge the retrospective nature of our study as a potential source of bias. Nevertheless, our data were extracted from a pre-existing Database filled prospectively at every patient's routine visit which ensures the quality of the data, and the number of patients included is appropriate for precise conclusions. In addition, the use of a non selective culture medium for mycological culture of sputum samples may have lead to an underestimation of the airway colonization by species of the S. a complex in our cohort, due to their common association with the more rapidly and more extensively growing fungus A. fumigatus. Finally, the specificity of the serological method used in the study should also be questioned as seropositivity to S. a complex was significantly associated with seropositivity to A. fumigatus. The pathogenicity of the S. a complex in CF patients remains controversial before lung transplantation. To our knowledge, this investigation represents the first large scale study attempting to assess a relationship between exposure to fungi of the S. a complex and CF lung disease. This study shows that a sensitization against S. a complex is frequent in CF patients with rate in agreement with the incidence of the airway colonization estimated by culture on highly selective media, but not independently associated with lung function deterioration or hospitalizations. Further researches are needed to evaluate the impact of both sensitization to these fungi and a chronic colonization of the airways on the course of the CF disease. Conflict of interest statement Olivier Lortholary reports being part of speakers' bureau of Pfizer, Merck, Astellas and Gilead Sciences and being consultant for Gilead Sciences. None of the other authors report having conflicts of interest to disclose. Funding Grant from the non-profit patients' organization «Vaincre la Mucoviscidose» (RCB0902). The study sponsor had no involvement in the study design, data analysis or decision to submit the manuscript for publication. Acknowledgements Authors thank Dr Florence Persat for collecting the sera samples and for the Aspergillosis serology. References [1] Gilgado F, Cano J, Gené J, Sutton DA, Guarro J. Molecular and phenotypic data supporting distinct species statuses for Scedosporium apiospermum and Pseudallescheria boydii and the proposed new species Scedosporium dehoogii. J Clin Microbiol 2008;46:766–71. [2] Gilgado F, Gene J, Cano J, Guarro J. Heterothallism in Scedosporium apiospermum and description of its teleomorph Pseudallescheria apiosperma sp. nov. Med Mycol 2010;48:122–8. [3] Cortez KJ, Roilides E, Quiroz-Telles F, Meletiadis J, Antachopoulos C, Knudsen T, et al. Infections caused by Scedosporium spp. Clin Microbiol Rev 2008;21:157–97.
[4] Guarro J, Kantarcioglu AS, Horré R, Rodriguez-Tudela JL, Cuenca Estrella M, Berenguer J, et al. Scedosporium apiospermum: changing clinical spectrum of a therapy-refractory opportunist. Med Mycol 2006;44: 295–327. [5] Bouchara JP, Horré R, de Hoog S. Pseudallescheria and Scedosporium: emerging opportunists. Med Mycol 2009;47:341–2. [6] Husain S, Muñoz P, Forrest G, Alexander BD, Somani J, Brennan K, et al. Infections due to Scedosporium apiospermum and Scedosporium prolificans in transplant recipients: clinical characteristics and impact of antifungal agent therapy on outcome. Clin Infect Dis 2005;40:89–99. [7] Paugam A, Baixench MT, Demazes-Dufeu N, Burgel PR, Sauter E, Kanaan R, et al. Characteristics and consequences of airway colonization by filamentous fungi in 201 adult patients with cystic fibrosis in France. Med Mycol 2010;48:32S–6S. [8] Cimon B, Carrère J, Vinatier JF, Chazalette JP, Chabasse D, Bouchara JP. Clinical significance of Scedosporium apiospermum in patients with cystic fibrosis. Eur J Clin Microbiol Infect Dis 2000;19:53–6. [9] Horré R, Marklein G, Siekmeier R, Nidermajer S, Reiffert SM. Selective isolation of Pseudallescheria and Scedosporium species from respiratory tract specimens of cystic fibrosis patients. Respiration 2009;77:320–4. [10] Blyth CC, Harun A, Middleton PG, Sleiman S, Lee O, Sorrell TC, et al. Detection of occult Scedosporium species in respiratory tract specimens from patients with cystic fibrosis by use of selective media. J Clin Microbiol 2010;48:314–6. [11] Castiglioni B, Sutton DA, Rinaldi MG, Fung J, Kusne S. Pseudallescheria boydii (Anamorph Scedosporium apiospermum). Infection in solid organ transplant recipients in a tertiary medical center and review of the literature. Medicine (Baltimore) 2002;81:333–48. [12] Symoens F, Knoop C, Schrooyen M, Denis O, Estenne M, Nolard N, et al. Disseminated Scedosporium apiospermum infection in a cystic fibrosis patient after double-lung transplantation. J Heart Lung Transplant 2006;25:603–7. [13] Sahi H, Avery RK, Minai OA, Hall G, Mehta AC, Raina P, et al. Scedosporium apiospermum (Pseudoallescheria boydii) infection in lung transplant recipients. J Heart Lung Transplant 2007;26:350–6. [14] Morio F, Horeau-Langlard D, Gay-Andrieu F, Talarmin JP, Haloun A, Treilhaud M, et al. Disseminated Scedosporium/Pseudallescheria infection after double-lung transplantation in patients with cystic fibrosis. J Clin Microbiol 2010;48:1978–82. [15] Borghi E, Iatta R, Manca A, Montagna MT, Morace G. Chronic airway colonization by Scedosporium apiospermum with a fatal outcome in a patient with cystic fibrosis. Med Mycol 2010;48:108S–13S. [16] Guignard S, Hubert D, Dupont B, Anract P, Alioua D, Guerini H, et al. Multifocal Scedosporium apiospermum spondylitis in a cystic fibrosis patient. J Cyst Fibros 2008;7:89–91. [17] Amin R, Dupuis A, Aaron SD, Ratjen F. The effect of chronic infection with Aspergillus fumigatus on lung function and hospitalization in patients with cystic fibrosis. Chest 2010;137:171–6. [18] Fillaux J, Brémont F, Murris M, Cassaing S, Rittié JL, Tétu L, et al. Assessment of Aspergillus sensitization or persistent carriage as a factor in lung function impairment in cystic fibrosis patients. Scand J Infect Dis 2012;44:842–7. [19] McMahon MA, Chotirmall SH, McCullagh B, Branagan P, McElvaney NG, Logan PM. Radiological abnormalities associated with Aspergillus colonization in a cystic fibrosis population. Eur J Radiol 2012;81:e197–202. [20] Pihet M, Carrere J, Cimon B, Chabasse D, Delhaes L, Symoens F, et al. Occurrence and relevance of filamentous fungi in respiratory secretions of patients with cystic fibrosis—a review. Med Mycol 2009;47:387–97. [21] Larcher G, Cimon B, Symoens F, Tronchin G, Chabasse D, Bouchara JP. A 33 kDa serine proteinase from Scedosporium apiospermum. Biochem J 1996;315:119–26. [22] Lima OC, Larcher G, Vandeputte P, Lebouil A, Chabasse D, Simoneau P, et al. Molecular cloning and biochemical characterization of a Cu, Znsuperoxide dismutase from Scedosporium apiospermum. Microbes Infect 2007;9:558–65. [23] Zouhair R, Rougeron A, Razafimandimby B, Kobi A, Bouchara JP, Giraud S. Distribution of the different species of the Pseudallescheria boydii/Scedosporium apiospermum complex in French patients with cystic fibrosis. Med Mycol 2013;51:603–13.
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P. Parize et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx [24] De Hoog GS, Guarro J, Gené J, Figueras MJ. Atlas of clinical fungi. 2nd ed. Utrecht, The Netherlands: Centraal bureau voor Schimmelcultures; 2000. p. 1124 [Rovira i Virgili University, Reus, Spain]. [25] Borman AM, Palmer MD, Delhaes L, Carrère J, Favennec L, Ranque S, et al. Lack of standardization in the procedures for mycological examination of sputum samples from CF patients: a possible cause for variations in the prevalence of filamentous fungi. Med Mycol 2010;48:88S–97S. [26] Brouard J, Knauer N, Boelle PY, Corvol H, Henrion-Caude A, Flamant C, et al. Influence of interleukin-10 on Aspergillus fumigatus infection in patients with cystic fibrosis. J Infect Dis 2005;191:1988–91. [27] Bargon J, Dauletbaev N, Köhler B, Wolf M, Posselt HG, Wagner TO. Prophylactic antibiotic therapy is associated with an increased prevalence of Aspergillus colonization in adult cystic fibrosis patients. Respir Med 1999;93:835–8.
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[28] de Vrankrijker AM, van der Ent CK, van Berkhout FT, et al. Aspergillus fumigatus colonization in cystic fibrosis: implications for lung function? Clin Microbiol Infect 2011;17:1381–6. [29] Speirs JJ, van der Ent CK, Beekman JM. Effects of Aspergillus fumigatus colonization on lung function in cystic fibrosis. Curr Opin Pulm Med 2012;18:632–8. [30] Chotirmall SH, O'Donoghue E, Bennett K, Gunaratnam C, O'Neill SJ, McElvaney NG. Sputum Candida albicans presages FEV1 decline and hospital-treated exacerbations in cystic fibrosis. Chest 2010;138:1186–95. [31] Azoulay E, Timsit JF, Tafflet M, de Lassence A, Darmon M, Zahar JR, et al. Candida colonization of the respiratory tract and subsequent pseudomonas ventilator-associated pneumonia. Chest 2006;129:110–7. [32] Registre Français de la Mucoviscidose. Bilan des données 2008. Paris: Vaincre la mucoviscidose et Ined; 2011.
Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
Journal of Cystic Fibrosis xx (2014) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis☆,☆☆ Perrine Parize a,b,⁎, Sandrine Billaud c , Anne L. Bienvenu d,e , Stéphanie Bourdy f , Marie A. le Pogam f , Philippe Reix b,g , Stéphane Picot d,e , Raymond Robert c , Olivier Lortholary h , Jean-Philippe Bouchara c,i , Isabelle Durieu a a
h
Hospices Civils de Lyon, Centre Hospitalier Lyon Sud, Adult Cystic Fibrosis Reference Center, 69495 Pierre-Bénite, France b Université de Lyon, Lyon, France c L'UNAM Université, Université d'Angers, Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, Institut de Biologie en Santé-IRIS, 49933 Angers, France d Hospices Civils de Lyon, Service de Parasitologie et de Mycologie Médicale, 69317 Lyon, France e University Lyon 1, Malaria Research Unit, ICBMS, CNRS UMR, 69373 Lyon, France f Hospices Civils de Lyon, Pôle IMER, Université de Lyon, 69424 Lyon, France g Hospices Civils de Lyon, Hôpital Femme-Mère-Enfant, Pediatric Cystic Fibrosis Reference Center, 69500 Bron, France Université Paris-Descartes, Hôpital Necker-Enfants malades, Service des Maladies Infectieuses et Tropicales, Centre d'Infectiologie Necker-Pasteur, IHU Imagine, 75015 Paris, France i Laboratoire de Parasitologie-Mycologie, Institut de Biologie en Santé-PBH, Centre Hospitalier Universitaire, 49933 Angers, France Received 6 December 2013; received in revised form 20 January 2014; accepted 27 January 2014
Abstract Background: Species of the Scedosporium apiospermum complex (S. a complex) are emerging fungi responsible for chronic airway colonization in cystic fibrosis (CF) patients. Recent studies performed on Aspergillus fumigatus suggest that the colonization of the airways by filamentous fungi may contribute to the progressive deterioration of lung function. Methods: We studied S. a complex seroprevalence, as a marker of close contact between patient and the fungi, in a large monocentric cohort of CF patients attended in a reference centre in Lyon, France. Results: Serum samples from 373 CF patients were analysed. Antibodies against S. a complex were detected in 35 patients (9.4%). In multivariate analysis, S. a complex seropositivity was only associated with seropositivity to A. fumigatus. Conclusions: This study does not suggest an association between sensitization against S. a complex and poorer lung function in CF. Prospective studies are needed to evaluate the impact of both seropositivity and S. a complex colonization on the course of CF. © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Scedosporium apiospermum; Cystic fibrosis/microbiology; Respiratory function tests; Serologic tests
☆ Abstract presentation: European Cystic Fibrosis Society conference, June 2010, Valencia, Spain. ☆☆ S. Billaud, R. Robert and J.P. Bouchara are members of the ECMM/ ISHAM working group on fungal respiratory infections in Cystic Fibrosis (FriCF). ⁎ Corresponding author at: Université Paris-Descartes, Hôpital Necker-Enfants malades, Service des Maladies Infectieuses et Tropicales, Centre d'Infectiologie Necker-Pasteur, 149 rue de Sèvres, 75015 Paris, France. Tel.: +33 1 42 19 26 63; fax: +33 1 44 49 54 40. E-mail address: [email protected] (P. Parize).
1. Introduction Scedosporium apiospermum is a ubiquitous filamentous fungus initially considered the anamorph state of Pseudallescheria boydii. However, recent taxonomic studies indicate that S. apiospermum is a member of a species complex, called S. apiospermum complex (S. a complex), which comprises at least five closely related but distinct species, i.e. S. apiospermum sensu stricto (anamorph state of Pseudallescheria apiosperma), Scedosporium boydii (anamorph
1569-1993/$ -see front matter © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcf.2014.01.011 Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
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state of P. boydii), Scedosporium aurantiacum, Scedosporium dehoogii and Pseudallescheria minutispora [1,2]. These fungi are now considered as emerging opportunist pathogens due to the recent increase in reported infections [3–5]. The spectrum of S. a complex infections is broad, ranging from localized diseases to multi-organ involvement and is closely related to the immunological status of patients [6]. Along with Aspergillus fumigatus, species of the S. a complex are currently identified as the main fungal agents responsible for chronic airway colonization in CF patients, with a prevalence ranging from 3.4% to 15.9% in limited monocentric studies [7–10]. Several cases of lethal invasive S. a. complex infections have been reported in CF patients having undergone lung transplantation [11–14]. These patients usually developed disseminated infections, affecting mostly the lungs, heart, eyes and central nervous system in the first year post-transplantation and died few weeks after diagnosis. Most of these patients were colonized before transplantation which is thought to be a risk factor of infection once the patients become immunocompromised. Indeed, because mortality of Scedosporium infections is high, S. a. complex colonization is debated as a contraindication to transplantation by some centres. Diagnosis of Scedosporium infections is difficult and often delayed because the clinical symptoms are aspecific and because a positive culture of a pulmonary specimen (i.e. sputum or bronchial aspirate) can't allow the distinction between colonization and infection. The diagnosis is confirmed once the fungus has been identified in culture from a specimen obtained by a sterile procedure or when histopathological examination reveals the presence of tissue damages in association with hyphae. Management of S. a complex infections is also complex due to the poor intrinsic activity of echinocandins and triazoles against these species. Treatment may thus require a combination of optimized antifungal therapy and surgery to reduce the fungal burden. Additionally a reduction of immunosuppression is discussed when feasible. In contrast to the severity of Scedosporium infections in immunocompromised patients, colonization is mostly asymptomatic prior to lung transplantation, although allergic bronchopulmonary disease [8] and rare Scedosporium invasive infections have been described in patients with diabetes mellitus [15,16]. However, the impact of long-term airway colonization by S. a complex on respiratory function in CF patients is still unclear. It is suggested that prolonged colonization of the airways by A. fumigatus in CF patients may accelerate lung function deterioration, thus increasing the risk of hospitalizations for pulmonary exacerbations and radiological abnormalities [17–19]. Therefore one may suspect that airway colonization by species of the S. a complex also contributes to the progressive deterioration of lung function by enhancing the local inflammatory response [20]. A study evaluating the impact of airway colonization on CF lung function is actually hardly achievable due to the low apparent incidence of colonization by S. a complex among the CF population. The rate of respiratory colonization by S. a complex is difficult to evaluate in non-specialised centres because the routine use of non-selective culture media for mycological examination of sputum samples leads to a significant underestimation of S. a complex detection [9,10].
We decided to perform a large monocentric retrospective study of the impact of S. a complex seroprevalence on CF patients' outcome and to determine clinical factors associated with seropositivity. 2. Methods 2.1. Subject population Eligible subjects for this study were patients diagnosed with CF who had at least one serum sample collected during a routine annual visit to the pediatric or adult CF reference centre in Lyon, France in 2008. A total of 394 patients were eligible for this study. Patients who had undergone lung transplantation were excluded (n = 21). Thus, 373 patients were included in this study which was approved by the hospital Ethical Committee (2009, Hospices Civils de Lyon). 2.2. Study design and data collection Data were extracted from the electronic medical records of Lyon Centre Cystic Fibrosis Database. During each visit, a sputum is systematically collected for bacterial and fungal examination and a serum sample is collected and stored at −20 °C in the sera bank of our laboratory at least once a year. For each patient we selected in the bank a serum sample collected in 2008 to assess the S. a complex serology. Patients' clinical characteristics collected during the same visit as the selected serum sample were extracted from the Database. Studied parameters were age, weight, size, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutations, history of previous bacterial or fungal airway colonization, history of diabetes, ABPA (allergic bronchopulmonary aspergillosis), medications, serological status towards A. fumigatus, results from sputum fungal culture, and outcomes of pulmonary function testing. 2.3. Serology Serum antibodies to the S. a complex were determined in July 2009 by ELISA using a cytosolic antigenic extract obtained from strain IHEM 15.155 (Institute of Hygiene and Epidemiology– Mycology section; Institute of Public Health, Brussels, Belgium). This strain originally isolated from a bronchoalveolar fluid in the laboratory of mycology of Angers University hospital and already used in previous studies [8,21,22] was identified by beta-tubulin gene sequencing as P. boydii, which is the most common species of the S. a complex in CF in France [23]. Antigenic extract was prepared from cultures grown under stationary phase in yeast extract–peptone–dextrose broth (containing in g per litre: yeast extract, 10; peptone, 10 and glucose, 20). After incubation for two weeks at 37 °C without agitation, the mycelium was harvested by centrifugation, washed twice with sterile water, and mechanically disrupted for 2 min with glass beads (mix of 0.25-mm and 1-mm diameter; Braun Melsungen, Melsungen, Germany) [8]. Debris were removed by centrifugation at 12,000 g for 30 min at 4 °C, and the supernatant which constitutes the antigenic extract was stored at − 80 °C as aliquots until used.
Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
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ELISA was performed in 96-well flat-bottom microtiter plates coated with the antigenic extract diluted in 50 mM carbonate buffer pH 9.6 (25 μg proteins per ml, 100 μl per well). After a 1hour incubation at 37 °C, followed by three washes in 0.15 M phosphate buffered saline (PBS) pH 7.2, 200 μl of a 3% bovine serum albumin solution in PBS was added to each well. The plates were incubated overnight at 4 °C, washed three times in PBS, and incubated for 1 h at 37 °C with sera from CF patients diluted 1:100 in PBS (100 μl per well). After washes in PBS containing 0.05% (vol/vol) Tween 20 (PBST), 100 μl of horseradish peroxidase-conjugated goat anti-human immunoglobulin γ chain antibodies (Rockland Immunochemicals Inc., Gilbertsville) or anti-human immunoglobulin μ chain antibodies (Biosys Technology, Paris, France) diluted 1:2000 in PBS was added to each well for determination of IgG and IgM, respectively. The plates were incubated for 1 h at 37 °C and finally washed three times in PBST. OPD (o-phenylenediamine dihydrochloride) in a buffered hydrogen peroxide solution was used for colour development. The absorbance was measured spectrophotometrically at 492 nm. A 0.9 absorbance unit was chosen as positivity threshold, as a result of prior unpublished analysis of sera from 49 non-CF healthy patients with a median age of 17 years [15–18 years] consulting for voluntary interruption of pregnancy or for sexually transmitted diseases screening and from CF- or non CF-patients with proven scedosporiosis (n = 5) or proven aspergillosis (n = 15), defined by the isolation of the fungus by culture and the presence of precipitin lines by counter-immunoelectrophoresis (CIE) using the corresponding antigenic extract (from P. boydii or A. fumigatus). All tests were performed in triplicate. Controls included the use of uncoated wells and addition of PBS instead of CF sera in coated wells. 2.4. Mycological examination of sputum samples The results of mycological examination of sputum samples were extracted from the mycology laboratory Database. Sputa were inoculated onto Sabouraud dextrose agar containing chloramphenicol and gentamicin. Plates were incubated at 37 °C and examined twice weekly for 7 days. Fungi were identified on the basis of their cultural features and microscopic morphology according to standard descriptions [24]. 2.5. Statistical analysis The initial analysis used descriptive statistics to characterize the study subjects. A univariate analysis was performed to compare the clinical characteristics of S. a complex seropositive and seronegative patients. Normality of all data was tested by Shapiro–Wilk test. Comparison of means was done using Student's t test when data were Gaussian and using Mann and Whitney test in the case of non-Gaussian data. Categorical variables were analysed using the Fisher's exact test or the χ2 test. Continuous data were compared using Student's t test. p values b 0.05 were considered to be statistically significant. A multivariable regression model was constructed using Hosmer–Lemeshow test to evaluate the effect of S. a complex seropositivity on covariates of interest defined by p b 0.2 in
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univariate: detail what is entered in the model. A cut-off of p b 0.2 was chosen to limit confusion factors which are frequent in CF. 3. Results 3.1. Cohort characteristics The demographic and clinical features of the 373 patients included are summarized in Table 1. Patients' median age was 15.4 years (range, 0.8–53.3). They were 199 males and 174 females. Bacterial airway colonization was reported for 272/373 patients (72.9%), mostly plurimicrobial with 203 patients colonized by Staphylococcus aureus, 156 by Pseudomonas aeruginosa and 31 by Stenotrophomonas maltophilia. During the study period, 119/ 373 patients (31.9%) had at least one sputum culture growing with one or more Aspergillus species including 111 patients colonized with A. fumigatus, 25 with Aspergillus flavus, 15 with Aspergillus terreus and 10 with Aspergillus niger. Among these patients 36 were colonized with several species of Aspergillus. Eight patients had S. a complex identified in sputum culture and 4 of them had concomitant A. fumigatus colonization. Of note, two patients had positive sputum samples for Scedosporium prolificans. Patients' medical history included diabetes mellitus for 35 patients and ABPA for 28 (among these patients, 21 have had at Table 1 Demographic and clinical characteristics of the 373 CF patients evaluated in 2008. Characteristics
Patients (n = 373)
Age, median (min–max), yr Sex ratio (M/F) CFTR mutations: F508del homozygosity a Bronchial colonization by S. aureus P. aeruginosa S. maltophilia Aspergillus fumigatus Scedosporium apiospermum complex Scedosporium prolificans FEV1% for patients b 18 (n = 222) b, mean (SD) FEV1% for patients ≥ 18 (n = 151), mean (SD) Prior ABPA Prior diabetes mellitus Treatment by Azithromycin Systemic steroids Itraconazole Voriconazole Inhaled treatment Inhaled steroids Inhaled RhDNAse Inhaled antibiotics Inhaled bronchodilator
15.4 (0,8;53,3) (0–53) 1.14 146 (39.1%) 203 (54.4%) 156 (41.8%) 31 (8.3%) 111 (29.8%) 8 (2.1%) 2 (0.5%) 87 (20.4) 60 (23.6) 28 (7.5%) 35 (9.4%) 127 (34.0%) 18 (4.8%) 16 (4.3%) 5 (1.3%) 287 (76.9%) 183 (49.0%) 175 (46.9%) 157 (42.1%) 202 (54.2%)
CFTR — Cystic Fibrosis Transmembrane Conductance Regulator. FEV1 — Forced Expiratory Volume in one second. ABPA — Allergic bronchopulmonary aspergillosis. a Missing data for 14 patients. b Missing data for 41 patients since spirometric measures were not available for patients younger than 6 years.
Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
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P. Parize et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
least one sputum culture growing with an Aspergillus species in Lyon). 21 patients received antifungal therapy, 16 had itraconazole and 5 voriconazole. Other therapeutics included long-term azithromycin therapy for 127 patients and systemic steroids for 18 patients. Most patients (287/373) had one or several inhaled treatments: 202 received inhaled bronchodilator, 183 inhaled steroids, 175 inhaled recombinant human deoxyribonuclease and 157 received at least one course of inhaled antibiotics during 2008. 3.2. Serology Prior analysis of sera from non-CF healthy patients and from CF- or non-CF patients with proven scedosporiosis or proven aspergillosis permitted to define a cut-off value of 0.9. None of the 49 patients in the control group as well as patients with proven aspergillosis were assessed positive for S. a complex serology by both CIE which could be considered as a “gold standard” serological test for fungi of the complex S. apiospermum and ELISA, whereas all tested sera from patients with confirmed scedosporiosis were found positive by CIE and ELISA with absorbance values always exceeding the threshold of 0.9. Using this threshold, antibodies against S. a complex were detected in 35 patients (9.4%) including 3 out of 8 patients with documented S. a complex airway colonization. The median age of seropositive patients was 20.2 years [1.7–47.2 years] compared to16.3 years [0.8–53.4] in seronegative patients. Distribution of seroprevalence in the different age classes is shown in Fig. 1. Seropositivity among patients increased during teenage years, then was stable during adulthood (χ2 test for trend p nonsignificant). The univariate analysis indicated that detection of serum antibodies against S. a complex was associated with older age, more frequent and prolonged hospitalizations, P. aeruginosa and S. a complex airway colonization, positive A. fumigatus serology, medical history of diabetes mellitus and ABPA, and at last with decreased pulmonary functions defined by a Forced Expiratory Volume in one second (FEV1) below 70% of the predicted value (Table 2). A regression model was generated to independently evaluate the effect of S. a complex seropositivity on those parameters.
Results of multivariate analysis revealed that seropositivity to S. a complex was significantly associated with seropositivity to A. fumigatus only. 4. Conclusions Here we demonstrate that immune response against S. a complex is relatively frequent in a large cohort of CF patients. Nearly 10% of the patients in our sample were found seropositive. This result is consistent with reported prevalence rates of 14.3% to 15.9% for the airway colonization by these fungi based on sputum cultures performed on highly selective culture media [9,10]. Currently, very few centres are using a selective culture medium to detect species of the S. a complex in CF patients. The heterogeneity of the procedures used for mycological examination of sputum samples (including the use of agar slants or agar plates, the number and nature of the culture media, the temperature and duration of incubation) leads to great variations in colonization prevalence estimates between centres and more generally to underestimating the burden of these fungi in CF patients [25]. The distribution of seroprevalence shows that the first exposure to the fungus is more likely to occur during teenage years as sensitization increases after the age of 10. This finding is consistent with the mean age of first detection of S. apiospermum colonization in CF patients, observed at 14.5 years (range, 7.8–21) by Cimon et al. [8]. In addition, first isolation of A. fumigatus in sputum of CF patients was also observed during teenage years, between 12.3 and 14.1 years [8]. After the age of 10 years, the S. a complex seroprevalence seems stable over time suggesting repetitive close contacts between the fungus and the patient during adulthood or that sensitization persists following the first exposure. Exposure to soil organisms could be the major source of contamination of the patients since species of the S. a complex are mainly recovered from ground (soil from the outdoor environment or from potted plants). Nevertheless, presence of potted plants at home of our patients was not recorded in the Database, and environmental studies were not performed. So it is not possible at present to establish a link between the S. a complex seropositivity and a higher exposure to these moulds. In addition, it is likely that genetic factors also determine the establishment of
Fig. 1. Seroprevalence of S. apiospermum complex by age class in 373 CF patients Chi2 test for trend: p not significant. Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
P. Parize et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
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Table 2 Univariate analysis and multivariate logistic regression analysis of factors associated with seropositivity to S. a complex. Characteristics
S. a complex seronegative patients (n = 338)
S. a complex seropositive patients (n = 35)
Age, median (min–max), yr Hospital admissions in 2008, mean number (SD) Number of hospitalisation days in 2008, mean (SD) Bronchial colonization S. aureus P. aeruginosa S. maltophilia S. a complex colonization Aspergillus fumigatus colonization Aspergillus fumigatus positive serology Medical history of ABPA Diabetes mellitus FEV1% predicted b 70 Treatment Itraconazole Voriconazole Azithromycin Systemic steroids Aerosol therapy
16.3 (0.8;53.4) 0.4 (1.0) 2.9 (10.8)
20.2 (1.7;47.2) 1.0 (1.6) 9.8 (21.8)
Simple logistic regression
Multiple logistic regression
p
p
OR(95%CI)
188 (55.6%) 136 (40.2%) 30 (8.9%) 5 (1.5%) 99 (29.3%) 39 (11.5%) 22 (6.5%) 27 (8%) 107 (31.6%)
15 20 1 3 12 11 6 8 20
(42.9%) (57.1%) (2.9%) (8.6%) (34.3%) (31.4%) (17.1%) (22.9%) (57.1%)
0.15 0.05 NS 0.03 NS 0.003 0.04 0.009 0.002
16 (4.7%) 5 (1.5%) 111 (33.4%) 14 (4.2%) 257 (77.4%)
0 0 16 4 30
(0%) (0%) (47.1%) (11.8%) (88.2%)
NS NS 0.11 0.07 0.14
OR(95%CI)
0.42 0.36 0.16
0.04 0.003 0.004
1.9 [1.0–4.0]
0.11 0.83
6.2 [1.4–27.3] 3.5 [1.6–7.7] 2.9 [1.1–7.9] 3.2 [1.5–6.9]
0.003 0.32 0.65 0.24
4.2 [1.6–10.8]
0.78 0.95 0.76
FEV1 — Forced Expiratory Volume in one second. ABPA — Allergic bronchopulmonary aspergillosis.
these fungi in the respiratory tract, such as the CFTR genotype and some polymorphism in the IL-10 gene as suggested for the airway colonization by A. fumigatus [26]. The association between S. a complex seropositivity and decrease in lung function or hospital admissions found in univariate analysis was no longer significant after adjustment for covariates. The association appeared to be explained mainly by age and confounders known to be related to lung function decline in CF patients. However, the study population may be too small to reveal correlations between S. a complex seropositivity and these particular outcomes. Interestingly, the analysis revealed no association between sensitization to S. a complex and inhaled antibiotics while the use of prophylactic inhaled antibiotics has been suggested to predispose CF patients to A. fumigatus colonization [27,28]. Finally, the results of multivariate analysis showed that seropositivity to S. a complex was significantly associated with seropositivity to A. fumigatus. This could be explained by the common occurrence of mixed colonization by S. a complex and A. fumigatus. As A. fumigatus is commonly found in soil and in the air, patients exposed to S. a complex probably have high risks of having been exposed to A. fumigatus as well. In the study conducted by Cimon et al. [8], almost all the patients colonized by species of the S. a complex were also colonized by A. fumigatus (10 out of 11 patients culture-positive for the S. a complex were colonized by both fungal species). Nevertheless, immune crossreactions between the S. a complex and other fungi such as A. fumigatus could also not be excluded. Although some of the cell wall polysaccharides differ from one fungus to another (peptidorhamnomannans in the S. a complex and galactomannans in A. fumigatus), they certainly share common epitopes in their
mannan moieties. Moreover, some of the fungal cell wall polysaccharides (chitin and ß-glucans) are common to almost all fungi, and some cell wall, membrane, of intracellular proteins needed for essential physiological processes in the fungal cell like respiration, glucose catabolism and energy production or synthesis of membrane ergosterol or cell wall polysaccharides, may also exhibit common epitopes. The role of fungal colonization on the evolution of the CF lung function is currently debated [29]. Recently, Chotirmall et al. [30] reported that repeated isolation of Candida albicans from sputum samples predicts both FEV1 decline and hospital-treated exacerbations in CF. Importantly, Candida airway colonization may be linked to an increased risk of P. aeruginosa ventilator-associated pneumonia in non-CF patients who received mechanical ventilation for more than 2 days [31]. Likewise, a longitudinal study has shown that CF patients persistently colonized by A. fumigatus had a higher rate of hospital admissions for pulmonary exacerbations and a decreased FEV1% predicted than controls [17]. A relationship between lung condition and fungal colonization was suggested by the authors but not further evidenced. A later retrospective cohort study evaluated the longitudinal effect of A. fumigatus colonization on lung function of CF patients over a 5-year period [28]. Differences in FEV1 between patients colonized by A. fumigatus and non-colonized subjects were explained by confounding factors known to be associated with lung disease severity. The present study is the first to evaluate the potential impact of S. a complex seropositivity on the course of CF lung disease. It made use of a large Database of children and adult patients representative of the French CF registry [32].
Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011
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P. Parize et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
We acknowledge the retrospective nature of our study as a potential source of bias. Nevertheless, our data were extracted from a pre-existing Database filled prospectively at every patient's routine visit which ensures the quality of the data, and the number of patients included is appropriate for precise conclusions. In addition, the use of a non selective culture medium for mycological culture of sputum samples may have lead to an underestimation of the airway colonization by species of the S. a complex in our cohort, due to their common association with the more rapidly and more extensively growing fungus A. fumigatus. Finally, the specificity of the serological method used in the study should also be questioned as seropositivity to S. a complex was significantly associated with seropositivity to A. fumigatus. The pathogenicity of the S. a complex in CF patients remains controversial before lung transplantation. To our knowledge, this investigation represents the first large scale study attempting to assess a relationship between exposure to fungi of the S. a complex and CF lung disease. This study shows that a sensitization against S. a complex is frequent in CF patients with rate in agreement with the incidence of the airway colonization estimated by culture on highly selective media, but not independently associated with lung function deterioration or hospitalizations. Further researches are needed to evaluate the impact of both sensitization to these fungi and a chronic colonization of the airways on the course of the CF disease. Conflict of interest statement Olivier Lortholary reports being part of speakers' bureau of Pfizer, Merck, Astellas and Gilead Sciences and being consultant for Gilead Sciences. None of the other authors report having conflicts of interest to disclose. Funding Grant from the non-profit patients' organization «Vaincre la Mucoviscidose» (RCB0902). The study sponsor had no involvement in the study design, data analysis or decision to submit the manuscript for publication. Acknowledgements Authors thank Dr Florence Persat for collecting the sera samples and for the Aspergillosis serology. References [1] Gilgado F, Cano J, Gené J, Sutton DA, Guarro J. Molecular and phenotypic data supporting distinct species statuses for Scedosporium apiospermum and Pseudallescheria boydii and the proposed new species Scedosporium dehoogii. J Clin Microbiol 2008;46:766–71. [2] Gilgado F, Gene J, Cano J, Guarro J. Heterothallism in Scedosporium apiospermum and description of its teleomorph Pseudallescheria apiosperma sp. nov. Med Mycol 2010;48:122–8. [3] Cortez KJ, Roilides E, Quiroz-Telles F, Meletiadis J, Antachopoulos C, Knudsen T, et al. Infections caused by Scedosporium spp. Clin Microbiol Rev 2008;21:157–97.
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Please cite this article as: Parize P, et al, Impact of Scedosporium apiospermum complex seroprevalence in patients with cystic fibrosis, J Cyst Fibros (2014), http:// dx.doi.org/10.1016/j.jcf.2014.01.011