Genetic Diversity among Clinical and Environmental Isolates of ...

INFECTION AND IMMUNITY, Aug. 1997, p. 3080–3085 0019-9567/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 65, No. 8

Genetic Diversity among Clinical and Environmental Isolates of Aspergillus fumigatus ´ RIE CHAZALET, JEAN-PAUL DEBEAUPUIS, JACQUELINE SARFATI, VALE ´* AND JEAN-PAUL LATGE Laboratoire des Aspergillus, Institut Pasteur, 75724 Paris Cedex 15, France Received 21 January 1997/Returned for modification 6 April 1997/Accepted 26 May 1997

To determine if cases of invasive aspergillosis (IA) were caused by strains of Aspergillus fumigatus with unique characteristics, strains from immunosuppressed patients with IA were compared to strains obtained from sputa of patients with cystic fibrosis and to strains from the environment. An extremely high genomic diversity was observed among the 879 strains typed by Southern blotting with a retrotransposon-like element from A. fumigatus (C. Neuve´glise, J. Sarfati, J. P. Latge´, and S. Paris, Nucleic Acids Res. 24:1428–1434, 1996). Analysis of Southern blot hybridization patterns showed the absence of clustering between environmental isolates and clinical isolates from patients with IA or cystic fibrosis. In addition, strains could not be clustered depending on their geographical location. This study implies that practically any strain of A. fumigatus is potentially pathogenic and can provoke a case of IA when it encounters a favorable environment in an immunosuppressed host. the study was to investigate the relatedness between clinical isolates and the known capacity to cause disease in order to test for the presence of a secondary adaptation of the clinical isolates to a parasitic condition.

Aspergillus fumigatus is a saprobic fungus playing an essential role in recycling carbon and nitrogen. This fungal species is a soil inhabitant which colonizes organic debris and decaying vegetation and releases into the atmosphere a high number of conidia. Besides its saprobic life, A. fumigatus has also become one of the most important human pathogens in industrialized countries. It causes different diseases like allergic bronchopulmonary aspergillosis, aspergilloma, and invasive aspergillosis depending on the underlying disease as well as the immunological status of the host (5, 32). A. fumigatus is today the most threatening aerial fungal pathogen because nosocomially acquired invasive aspergillosis typically occurs in the treatment setting for hematological malignancy. The pathogenic behavior of this opportunistic fungus results from (i) specific biological features of the fungus, such as the release into the air of a high concentration of conidia of small size which are easily inhaled and able to germinate and grow at temperatures higher than 37°C without any specific nutritional requirement, and (ii) the increase in the use of immunosuppressive therapies which result in the lack of efficiency of the normal phagocytic host response (1, 15). Recent epidemiological studies using molecular techniques have shown that, at least in some cases, infection occurs as a consequence of exogenous acquisition of the fungus (26). Two typing methods have been used until now. The method most currently used is the random amplification of polymorphic DNAs (4, 6, 16, 18, 30), although such amplified patterns are difficult to repeat or interpret (3, 20). In contrast, a restriction fragment length polymorphism method which uses a speciesspecific inactive retrotransposon-like repeated sequence as a marker has shown high reproducibility and reliability, allowing computer-aided analysis of a large population of A. fumigatus strains (11–13, 23). Previous epidemiological studies have investigated the nosocomial origin of aspergillosis cases in limited geographical areas and included fewer than 30 strains (6, 13, 16, 18). The present study includes over 800 isolates with different hosts and geographical origins. The main purpose of

MATERIALS AND METHODS Isolates. The origins of the strains of A. fumigatus used in this study are given in Table 1. All strains from Denmark were isolated from cows with aspergillosis. Clinical isolates of A. fumigatus were from immunosuppressed patients suffering mainly from pulmonary or invasive aspergillosis with the exception of the strains from the Necker and Trousseau hospitals, which were from cystic fibrosis (CF) patients. All isolates were kept on 2% malt agar and were eventually stored as a conidial suspension on dry silica gel (Prolabo) at room temperature (28). DNA isolation and characterization. Protocols for DNA extraction and EcoRI digestion were as previously described (11). Digested DNAs were loaded onto 20-cm long 0.8% agarose gels (SeaKem LE; FMC) at a ratio of 500 ng of DNA per well (4 by 4 mm). Electrophoresis and transfer were done on the same automated multiblotting device (Mark II; Bertin, France) at 14°C in 0.53 Trisborate-EDTA buffer. After 20 min of premigration at 180 V, electrophoresis was performed for 15 h at 80 V. Transfer of DNA to Pall Biodyne B membranes lasted 40 min at 250 V. Eight to twelve gels of 15 slots each (12 restriction digests and 3 size markers) were run simultaneously. Membranes were treated with 0.4 M NaOH, neutralized in 0.5 M Tris-HCl (pH 7.5)–1.5 M NaCl, washed with water, and fixed at 80°C for 30 min. Southern blot hybridization was performed with the l3.9 probe containing the recently characterized (23) repeated sequence Afut1 isolated from A. fumigatus. Hybridization was performed as previously described (11) with 25 ng of probe DNA labelled with 2.5 ml of [32P]dCTP and two exposure times of 12 and 24 h with X-Omat film (Kodak) in a cassette without an intensifying screen. Analysis of fingerprints. Autoradiographs were scanned with an HP Scanjet IIcx/T (Hewlett-Packard) driven by a 466DX2/Sp personal computer (IBM). The resulting digital images (Tagged Image Format File) were converted and normalized with the GelCompar Software (Applied Maths BVBA, Kortrijk, Belgium). Each track corresponding to one slot was converted to a densitometric curve (700 points with values between 0 and 255, corresponding to the 256 gray levels). The conversion process included a background subtraction step and alignment on reference positions determined by size markers (corresponding to phage l digested by XhoI, BstEII, and BssHII). In addition, an undigested phage l giving a band of 45 kb was added to each A. fumigatus DNA sample, allowing for the best alignment at the highest molecular weight bands. An example of a blot and its computer treatment are shown in Fig. 1. Densitometric profiles were clustered by the software with a matrix of similarity based on the Pearson product-moment correlation coefficient (2). As this method compares curves as a whole, it is independent of band definition and is not troubled by peakshoulders mismatches. The clustering method used was the unweighted pair group method with arithmetic averages. Groups of genotypic patterns were compared pairwise by principal component analysis (PCA). Definition of the groups was made a priori and was based on the geographical location (ranging from a localized area, e.g., one Parisian hospital

* Corresponding author. Mailing address: Laboratoire des Aspergillus, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: 33 140 61 35 18. Fax: 33 140 61 34 19. E-mail: jplatge @pasteur.fr. 3080

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TABLE 1. Origin of A. fumigatus isolates used for this study Country

Denmark France France France France France France France France Germany Netherlands United Statesb Total a b

City

Angers Paris Paris Paris Paris Paris Paris Paris Frankfurt Nijmegen

Hospital

Cochin Ho ˆtel-Dieu Necker St. Antoine St. Louis Tenon Trousseau

No. of environmental isolates

22 85 65 21 38 9 4 36 280

No. of isolates from human or animal sources (no. of patients)

26 (14a) 23 (5) 10 (7) 15 (8) 37 (18) 14 (6) 53 (35) 12 (4) 338 (6) 48 (32) 23 (6) 599 (141)

Provider

H. Jensen J. P. Bouchara J. Dupouy-Camet B. Me´chin, J. Lortholary C. Hennequin J. L. Poirot F. Traore´ P. Roux H. Vu Thien P. Shah P. Verweij B. Lasker

Samples from cows infected by A. fumigatus. Origin not further defined.

to a more general origin, e.g., Europe) and host origin (human versus nonhuman and clinical versus environmental) of the isolates. PCA is a nonhierarchical method which allows densitometric data to be used directly without the application of any similarity coefficient (29, 31). The arrays of normalized densitometric values were used directly as input data for PCA. The three principal axes were used to produce a three-dimensional representation in which each single genotype occupied a separate place, shown as a dot. The degree of discrimination between the groups defined as indicated above was statistically evaluated with the software. Between each pair of groups, two values were calculated: the mean percentage relatedness (MPR) of the groups and the ratio of divergence (RD) (2). MPR is the sum of all Pearson product-moment correlation values (rm) of the entries of the first group (A) with the entries of the second group (B) divided by the total number of correlation values calculated: rm,AB 5 SriA jB/nAnB, where riA jB is the product-moment correlation between the ith and jth entries of groups A and B, respectively, and nA and nB are the number of entries in groups A and B, respectively. Within a group (A), this value (rm,A) is reduced to a measure of internal homogeneity: rm,A 5 Srij/n2. The ratio of divergence (RD) is calculated as the distance 12rm,AB between two groups divided by the weighted internal distances 12rm,A and 12rm,B:

RD 5

~nA 1 nB!~1 2 rm, A B! nA~1 2 rm, A! 1 nB~1 2 rm, B!

The MPR values provide information about the internal homogeneity of groups and the overall relatedness between groups. Values of MPR higher than 90 indicate very compact groups. In the case of an intragroup analysis, such a value would indicate that a specific hybridization profile can be associated with all of the isolates of the group. Values between 70 and 80 indicate an even scattering of the patterns analyzed inside a group or between groups. MPR data do not give information about the degree of discrimination between groups. The relative distance between groups, as revealed by the RD, gives an estimate of the degree of discrimination between two groups, taking into account their internal homogeneity. RD values of 1 to 1.5 indicate that the two clusters of points from the two groups are superimposed. RD values of greater than 2 (and up to 10 depending on how compact the groups being compared are) indicate that the cloud of points belongs to two statistically different groups. Preliminary experiments on a random sample totalling 5% of the isolates showed that the same pattern was obtained from 10 single-spore strains isolated

FIG. 1. (A) Southern blot hybridization patterns in a blot with 12 A. fumigatus isolates hybridized with the l3.9 probe. The first, eighth, and fifteenth lanes contain molecular size markers. Lanes A and B contain identical isolates (99% homology), the isolate in lane C has 90% homology with the isolates in lanes A and B, and the isolate in lane D has 56% homology with those in lanes A and B. (B and C) Densitometric curves obtained from digitalized images of the blot shown in panel A. Lanes are as defined above.

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FIG. 2. Dendrogram (unweighted pair group method with arithmetic averages) of the 424 strains analyzed. Note that clinical isolates of A. fumigatus (marked by vertical lines underneath dendrogram) are widespread throughout the dendrogram.

from the original slant, justifying our decision to consider every slant received as a single strain. In addition, on .20% of the strains, repeated extraction and hybridization of DNA samples from the same strain have shown the reproducibility of the method, with a coefficient of similarity of .95% between two replicates. Similarly, analysis of 75 identical strains isolated over a 3-year period from the same CF patient gave a 94.3% similarity coefficient. As a result of this analysis, genotypic profiles presenting a similarity coefficient of $92% were always compared individually by superimposition of densitometric curves and by direct visual examination of the autoradiograms to validate the software analysis and to confirm the identicalness of strains. Different exposure times were used to match the intensity levels of the bands and the background and to make sure that missing bands were not due to too little target DNA. For better accuracy in pattern comparison, profiles with fuzzy bands were always discarded and DNA extraction and hybridization were repeated. (Data are not presented in their entirety due to the considerable number of hybridization patterns analyzed but are available upon request.)

RESULTS From the 879 isolates included in this study, clustering analysis of the Southern blot patterns obtained with the l3.9 probe gave distinct and unique genotypes for 424 strains (Fig. 2). The presence of identical genotypes among isolates was due to our nonrandomized sampling method and corresponded to particular epidemiological situations: (i) isolates from the same patients (see below), (ii) environmental isolates from the same location, and (iii) environmental and clinical isolates obtained during nosocomial invasive aspergillosis outbreaks (8). For PCA analysis, only one representative of the genotype of each series of identical isolates was kept. Among the 424 strains studied, only 14 identities (14 couples) were found between strains of different origins (for example, a sample from a patient in Frankfurt, Germany, and a sample from a U.S. environment). In some cases, the possibility of laboratory contamination could not be ruled out; in other cases, contamination could be eliminated when, for example, DNA samples were prepared separately in the United States and in France. Examination of the dendrogram in Fig. 2 shows that fingerprints are not grouped in very distinct groups but are in a number of small groups, indicating a gradual variability between all strains. Comparison of clinical and environmental strains. Several (2 to 15) isolates were obtained per patient for 56 of the 140 patients from which Aspergillus was isolated (Table 2). The interval between the first and the last sampling dates varied from 1 day to 6 months for patients with aspergillosis and from 12 to 32 months for patients with CF. The number of strains isolated from CF patients was much higher than that from aspergillosis patients. In the CF patient group, no one patient was colonized by a single strain. Colonization of every CF patient was due to several genotypes which were repeatedly isolated from the same patient (data not shown). In contrast, in

the aspergillosis group, the number of genotypes isolated per patient was close to 1. The recovery of a single genotype per patient among 18 of the 34 patients studied indicated that in more than 50% of the patients, infection was due to a single strain (Table 2). PCA analysis of clinical and environmental isolates included 328 genotypes from among three groups: (i) 136 strains isolated from 115 neutropenic patients with evidence of Aspergillus infection, (ii) 97 strains obtained from 26 patients with CF and which apparently were not implicated in an infectious process, and (iii) 155 randomly chosen environmental strains, mainly isolated in hospitals. PCA comparison showed no differences among the three groups of strains. Figure 3 shows a two-dimensional representation of the three-dimensional analysis. The absence of clustering was seen at all rotating angles. Pairwise comparisons gave values of MPR and RD which were not statistically different (Fig. 3, inset). In conclusion, although the clinical isolates were indeed the infective strains, no difference was seen between the saprobic and clinical isolates. This result means that every strain present in the environment can become pathogenic, if it encounters the appropriate host. Comparison of strains based on their geographical origin. The 12 groups of strains (a total of 424 strains) gathered on the basis of their common geographical origin were compared by the different clustering methods available with the software (dendrograms not shown) and by PCA. The PCA results showed that the genetic variability encountered in the species A. fumigatus was extremely high and that no discrimination between strains was possible on the basis of their geographical origin. Figure 4 gives an example of a comparison of strains from (i) a Parisian hospital, (ii) the Paris area, (iii) Europe, and (iv) the United States. Similar MPRs and RDs were found for comparisons between 56 strains from one Parisian hospital and either a random selection of 244 strains from the rest of

TABLE 2. A. fumigatus isolates obtained from patients by repeated sampling Patient status

No. of patients

No. of isolates per patient

No. of genotypes

Strain/ patient ratio

P1/PT ratioa

Aspergillus infection CF

34 22

2.20 17.13

46 85

1.35 4.58

18/34 0/26

a P1, number of patients from whom only one genotype of A. fumigatus was isolated; PT, total number of patients.


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FIG. 3. Comparison by PCA of A. fumigatus strains isolated from patients with Aspergillus infections (PA) (■) or CF (p) or from the environment (EN) (h). The MPR and RD values between the groups are shown in the inset.

the world (MPR, 74.9; RD, 1.3) or 140 strains from France and 160 strains from outside of France (MPR, 73.4; RD, 1.1). DISCUSSION This is the first large-scale study of the genetic variability occurring in the opportunistic fungal pathogen A. fumigatus.

Although this species has been shown to display some phenotypic variations (17, 19, 25), A. fumigatus looks taxonomically homogeneous on the basis of its morphology (27). In contrast, the intraspecies variability seen at the genomic level looks very high. As a result, comparison of different genotypes with either a clinical or an environmental origin showed no difference between the strains from either origin, suggesting that any

FIG. 4. PCA of A. fumigatus strains from different geographical locations. (A) A total of 62 strains from Ho ˆtel-Dieu, Paris, France (■), versus 200 randomly chosen strains from other Parisian hospitals (h) (MPR, 76.1; RD, 1.3). (B) A total of 150 randomly chosen strains from Paris (■) versus 124 strains from other origins in Europe (h) (MPR, 78.9; RD, 1.0). (C) A total of 36 isolates from the United States (■) versus 200 randomly chosen strains from Europe (h) (MPR, 76.4; RD, 1.0).

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environmental strain of A. fumigatus is a putative infectious strain. This conclusion has some practical implications in that it indicates that prevention measures should be applied to any environmental Aspergillus conidia. The absence of strains of A. fumigatus with different pathogenicity potentials is in agreement with the results of previous biochemical, molecular, and immunological studies which were unable to identify a key factor responsible for the pathogenicity of A. fumigatus (15). For example, until now, no avirulent or hypervirulent mutant has been found or constructed by genetic manipulation. However, the failure of this molecular approach may just reflect the very few proteins purified from A. fumigatus and the low number of disrupted genes. The ability of any strain of A. fumigatus to become pathogenic in contact with an appropriate host can also be linked to the absence of host specificity. No marked difference was seen between strains isolated from cows or humans. MPR and RD values between the two groups of strains were 78.3 and 1.2, respectively, whereas the MPR values for the intragroup variability of the 98 strains isolated from humans and the 14 strains isolated from cow strains were 81.7 and 78.2, respectively. This is in contrast with true phytopathogenic fungi where pathotypes are genetically different and host-associated pathotypes can be separated through diverse typing methods on the basis of their host or geographical origin (21, 22). All these studies suggest that virulence in A. fumigatus is multifactorial and that in vivo development requires the activation of multiple genes. Questions regarding the identity of the genes expressed and their differential levels of expression in vivo and in vitro have never been asked seriously until now. The potential pathogenicity of strains with very different genetic backgrounds is in agreement with the absence of any selective pressure for this fungus resulting from the accidental and rare development of A. fumigatus in a human being. This fungus does not need a human host to complete its biological cycle, and encountering an immunocompetent human host is indeed a deadly cul-de-sac for it. A. fumigatus is a true saprobic fungus which becomes pathogenic only when the human defense reactions are very weakened (for example, in the terminal stage of an AIDS infection or as a result of heavy immunosuppressive therapies). Virulence would seem to represent more a lack of resistance of the human host than the expression of specific disease-related proteins. Experimental intranasal infection of mice never succeeds even at the maximal possible dose (108 conidia per mouse) unless a mouse is immunosuppressed (unpublished data). The question of the origin of the variability encountered in this species remains open. No teleomorph has been found, and the possibility that Neosartorya fischeri represents the sexual stage of A. fumigatus has been definitely discarded (14, 24). Three hypotheses can be put forward. (i) Continuous genetic exchange can occur through the parasexual cycle. A. fumigatus remains today a continuously evolving species. Vegetative compatible groups have been found in A. fumigatus (9), indicating the possibility of a parasexual cycle in this species. However, it has never been shown that nonmeiotic parasexual recombination can account for recombination in a natural population of any fungus (7). (ii) The variability could have been fixed a long time ago through meiotic exchange at a time when A. fumigatus had a sexual stage. Continuous exchange of conidia through air currents all around the world, in addition to the absence of any adaptation to a parasitic condition, would explain the lack of recovery of identical multilocus genotypes from geographically and temporally nonassociated hosts (6). (iii) The third hypothesis concerns the presence of a sexual stage in A. fumigatus which has gone undetected. Recent pop-

INFECT. IMMUN.

ulation genetic studies have suggested that such a cryptic sexual stage exists in the case of another human pathogen, Coccidioides imitis, which has been found to be almost completely recombining (7). The presence of such a sexual stage would be correlated with variations occurring through meiotic recombination. In a study with different heterokaryon compatibility groups of Aspergillus nidulans (thus incapable of mitotic recombination), genetic variations were found indicating that heterocaryon groups represent recently diverged lineages that arose via meiotic recombination (10). ACKNOWLEDGMENTS Although they are not coauthors of this report, this study would not have been possible without the help of the investigators who provided us with strains from various origins. A special thanks is due to the main providers: J. P. Bouchara (Laboratoire de Parasitologie-Mycologie, Angers, France); H. Jensen (The Royal Veterinary and Agricultural University, Department of Pharmacology and Pathobiology, Copenhagen, Denmark); J. Dupouy-Camet (Laboratoire de Parasitologie-Mycologie, Ho ˆpital Cochin, Paris, France); B. Me´chin (Laboratoire de Mycologie-Parasitologie, Ho ˆtel-Dieu, Paris, France); C. Hennequin (Laboratoire de Microbiologie, Ho ˆ pital Necker, Paris, France); F. Traore´ (Laboratoire de Mycologie, Ho ˆ pital Saint-Louis, Paris, France); G. Bru ¨cker, G. Rykner, and J. Lortholary (SEHP, Assistance Publique, Ho ˆpitaux de Paris, Paris, France); J. L. Poirot (Laboratoire de Parasitologie, Ho ˆ pital Saint-Antoine, Paris, France); P. Roux (Laboratoire de Parasitologie-Mycologie, Ho ˆ pital Tenon, Paris, France); H. Vu Thien (Laboratoire de Parasitologie-Mycologie, Ho ˆ pital Trousseau, Paris, France); P. Shah (Zim-Infektiologie Klinikum der J. W. Goethe, Universita¨t Theodore-Stern-Kai, Frankfurt, Germany); P. Verweij (Department of Medical Microbiology, University Hospital, Nijmegen, The Netherlands); and B. Lasker (Centers for Disease Control and Prevention, Division of Bacterial and Mycotic Diseases, Atlanta, Georgia). We thank J. Taylor for helpful comments on the manuscript and L. Vauterin (Applied Maths, Kortrijk, Belgium) for providing data and explanations on the GelCompar software. Research was partly funded by CNAMTS-INSERM grant 3AM051 to J.-P.L. REFERENCES 1. Anaissie, E. J., G. P. Bodey, H. Kantarjian, J. Ro, S. E. Vartivarian, R. Hopfer, J. Hoy, and K. Rolston. 1989. New spectrum of fungal infections in patients with cancer. Rev. Infect. Dis. 11:369–378. 2. Applied Maths BVBA. 1996. GelCompar manual (version 4.0). Applied Maths BVBA, Kortrijk, Belgium. 3. Arbeit, R. D., J. N. Maslow, and M. E. Mulligan. 1994. Polymerase chain reaction-mediated genotyping in microbial epidemiology. Clin. Infect. Dis. 18:1018–1019. 4. Aufauvre-Brown, A., J. Cohen, and D. Holden. 1992. Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus. J. Clin. Microbiol. 30:2991–2993. 5. Bodey, G. P., and S. Vartivarian. 1989. Aspergillosis. Eur. J. Clin. Microbiol. Infect. Dis. 8:413–437. 6. Buffington, J., R. Reporter, B. A. Lasker, M. M. McNeil, J. M. Lanson, L. A. Ross, L. Mascola, and W. R. Jarvis. 1994. Investigation of an epidemic of invasive aspergillosis: utility of molecular typing with the use of random amplified polymorphic DNA probes. Pediatr. Infect. Dis. J. 13:386–393. 7. Burt, A., D. A. Carter, G. L. Koenig, T. J. White, and J. W. Taylor. 1996. Molecular markers and sex in the human pathogen Coccidioides immitis (Ascomycota). Proc. Natl. Acad. Sci. USA 93:770–773. 8. Chazalet, V., J. P. Debeaupuis, J. Sarfati, J. Lortholary, P. Ribaud, P. Shah, F. Gluckman, G. Bru ¨cker, and J. P. Latge´. Unpublished data. 9. Chazalet, V., and J. P. Latge´. Unpublished data. 10. Geiser, D. M., M. L. Arnold, and W. E. Timberlake. 1994. Sexual origins of British Aspergillus isolates. Proc. Natl. Acad. Sci. USA 91:1–4. 11. Girardin, H., J. P. Latge´, T. Srikantha, B. Morrow, and D. R. Soll. 1993. Development of DNA probes for fingerprinting Aspergillus fumigatus. J. Clin. Microbiol. 31:1547–1554. 12. Girardin, H., J. Sarfati, H. Kobayashi, J. P. Bouchara, and J. P. Latge´. 1994. Use of DNA moderately repetitive sequence to type Aspergillus fumigatus isolates from aspergilloma patients. J. Infect. Dis. 169:683–685. 13. Girardin, H., J. Sarfati, F. Traore´, J. Dupouy-Camet, F. Derouin, and J. P. Latge´. 1994. Molecular epidemiology of nosocomial invasive aspergillosis. J. Clin. Microbiol. 32:684–690. 14. Girardin, H., M. Monod, and J. P. Latge´. 1995. Molecular characterization

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