JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1994, p. 684-690 Vol. 32, No. 30095-1 137/94/$04.00+0

Molecular Epidemiology of Nosocomial Invasive AspergillosisH. GIRARDIN,lt J. SARFATI,1 F. TRAORE,2 J. DUPOUY CAMET,3 F. DEROUIN,2 AND J. P. LATGEJ*

Unite de Mycologie, Institut Pasteur, 25, rue du Dr. Roux, 75015 Paris,1 Service de Parasitologie-Mycologie,H6pital St Louis, 75475 Paris Cedex 10,2 and Laboratoire Central de Parasitologie-Mycologie,

Groupe Hospitalier Cochin, 75679 Paris Cedex 14,3 France

Received 30 August 1993/Returned for modification 3 October 1993/Accepted 3 December 1993

Moderately repeated DNA sequences were used to fingerprint strains ofAspergillusfumigatus isolated frompatients with invasive aspergillosis and their hospital environment. Most strains sampled from the environ-ment displayed different Southern blot hybridization patterns. A temporal survey of air contaminants showedthat some strains can persist in the same environment for at least 6 months. Patients with invasiveaspergillosis were infected by a single strain. In two patients, a nosocomial origin of infection was suggested.

Invasive aspergillosis is a very severe nosocomial humandisease associated with heavy immunosuppressive treatments(6, 25). Aspergillus fumigatus is the species of Aspergillus mostfrequently responsible for this life-threatening disease. Highconcentrations of the conidia of A. fumigatus associated withcontaminated ventilation systems or hospital building workshave been correlated with aspergillosis outbreaks (2, 3, 14, 16,21). Until now, airborne transmission suggested by this circum-stancial evidence has not been demonstrated in the absence ofan efficient typing system for A. fumigatus (25).

Several fingerprinting systems based on phenotypic andgenomic differences have been reported for A. fumigatus.Biotyping methods based on the morphologies of sporulatingstructures (17), antigen patterns (8), and susceptibilities toyeast killer toxins (10) have the disadvantage of possiblyoveremphasizing the importance of the expression of pheno-typic characteristics, which may vary with the environmentalconditions. In contrast, genotyping methods that assess directlythe genetic relatedness among strains are the most accuratemethods for fingerprinting fungal isolates. Restriction frag-ment length polymorphism (RFLP) of total cellular DNAdigested by particular restriction enzymes (XbaI, XhoI, or Sall)discriminated strains of A. fumigatus to some extent (7, 9). Inaddition, two recently introduced systems have been proven tobe efficient in fingerprinting strains of this species. Thesemethods used randomly amplified polymorphic DNA markers(4, 19) and moderately repetitive nonribosomal DNA se-quences (11). This last method has proven to be very useful inassessing the absence of variability among strains sequentiallyisolated from individual patients with aspergilloma (12).

In the present study, moderately repetitive sequences(MRSs) have been used in different specific and unrelatedhospital situations in which cases of invasive aspergillosis weredocumented. The aim of the epidemiological study describedhere was mainly to investigate the putative diversity of isolatesfrom the same patient with invasive aspergillosis, the related-ness among isolates recovered from different patients and theirclose environment, and the persistence over time of a specificstrain in the ambient air of a hospital unit.

* Corresponding author.t Present address: Laboratoire de G6nie de l'Hygiene et des Pro-

cedes Alimentaires, Institut National de la Recherche Agronomique,91300 Massy, France.

MATERIALS AND METHODS

Patients. Five patients in hospital A were diagnosed withpulmonary or invasive aspergillosis. Three patients in unit Awere bone marrow transplant recipients; the underlying dis-ease was acute lymphoblastic leukemia in patients 1 and 2 andchronic lymphoblastic leukemia in patient 3. The two otherpatients from unit B had received chemotherapy for chronicmyelocytic leukemia (patient 4) or acute myeloblastic leukemia(patient 5).

In four patients, the diagnosis of pulmonary aspergillosis wassuspected clinically or radiologically, but A. fumigatus could beisolated antemortem from only three of them by culturingvarious pathological samples: blood (patient 1), open lungbiopsy specimens (patients 4 and 5), and bronchial aspirateand a cutaneous biopsy specimen (patient 5). In three patients,the diagnosis of aspergillosis was established or confirmedpostmortem on the isolation of A. fumigatus by culture ofbiopsy specimens of the lung (patients 1 to 3).

Patient 6 was a 30-year-old patient hospitalized in hospitalB, unit A, for acute respiratory distress syndrome complicatinga refractory asthma. Although patient 6 was not severelyimmunosuppressed, the patient died of disseminated aspergil-losis on 16 November 1992, 3 weeks after admission to thehospital. Invasive aspergillosis was proven by the presence ofhyphae in a bronchoalveolar lavage and in a pulmonar biopsyspecimen, which were found 72 and 24 h, respectively, beforedeath. Hyphae were also seen on postmortem histologic sec-tions of heart and lung tissues from patient 6. A. fumigatus wasisolated from all these specimens by culture.

Strain isolation. Isolates from bronchial aspirate or lavagesand antemortem or postmortem biopsy specimens from eachpatient were plated onto Sabouraud agar (2% glucose, 1%peptone, 0.05% chloramphenicol, 2% agar). The number ofstrains, the body location, and the day of isolation of eachstrain from each patient are presented in Table 1.Most environmental isolates were obtained from the air by

using an automated air sampling device (most often anAndersen apparatus that samples 56 liters of air). In addition,surface contaminants were isolated by brushing the walls of thepatients' rooms or ventilation fences (inside or outside theventilation unit at the air entrance or exit). The locations anddates of isolation of the environmental isolates are also givenin Table 1.

All strains were identified as A. fumigatus by their culturalcharacteristics and the morphologies of their conidiophoresand conidia.

684

EPIDEMIOLOGY OF NOSOCOMIAL INVASIVE ASPERGILLOSIS 685

TABLE 1. Origins of strains collected from patients with invasive aspergillosis and from the environment of their respective hospital units

Date of culture Strain Abbreviation inHospital Unit Origin Location" (mo.day.yr) collection no. Fig. I to 4

A A Patient 2Patient 3Patient 1

Environment

Lung biopsy (pm)Lung biopsy (pm)Lung biopsy (pm)

AirAirAirAirAir

09.02.91 520112.17.91 814012.19.91 8212

3.16.92 46-1633.23.92 30-2338.24.92 4-2488.24.92 SAS-2489.07.92 SAS-079

A B Patient 4 Bronchial washing (trachea)Right lung (inferior lobe)CavernomaLingulaThoracic drainSputumThoracic drain

A B Patient 5 Bronchial aspirationLung (median lobe)Cutaneous biopsyLungTrachea

1.08.92 3861.08.92 3901.08.92 3931.09.92 4341.12.92 601

EnvironmentRoom of patient 5 Air

AirAirAirAirAirAirAirAirAir

Outside room of patient 5 Unit entrance, airVentilation system, surface brushingVentilation system, surface brushingVentilation system, surface brushing

1.09.92 145-136.22.92 W 38-26611.09.92 38-911 N11.09.92 38-911 BC11.16.92 W 38-161111.24.92 38-241111.24.92 W 38-5-241112.15.92 38-151212.15.92 W 38-1512 C12.15.92 W 38-1512 F

6.29.921.28.921.28.921.28.92

ES-2963636-23640-203644-8

B A Patient 6 Bronchial washingLung am biopsyLung pm biopsyHeart pm biopsy

11.13.92 469111.15.92 470711.16.92 471511.16.92 4714

EnvironmentSame unit Air

Surface of disinfection apparatusVentilation unit grillVentilation unit grillVentilation unit grill

Outside the unit

11.16.92 Gas211.16.92 Gas 111.16.92 Gas 311.17.92 Gas 411.17.92 Gas 5

Surface of ventilation system prefilter 11.30.92 PF1Surface of ventilation system prefilter 11.30.92 PF2Surface of ventilation system prefilter 11.30.92 PF3Decaying leaves on grill of air entrance 11.30.92 FlDecaying leaves on grill of air entrance 11.30.92 F2Wall next to grill of air entrance 11.30.92 RI

(" am, antemortem; pm, postmortem.

P3P1P2

E3ElE2E4E5

1.07.931.12.931.12.931.12.931.16.931.20.931.22.93

23154454517987911111199

P4aP4bP4cP4dP4eP4fP4g

P5aP5bP5cP5dP5e

E6E7E8E9EIOEllE12E13E14E15

E16E17E18E19

P6aP6bP6cP6d

E27E26E28E29E30

E23E24E25E20E21E22

VOL. 32, 1994

686 GIRARDIN ET AL.

P4

a b c d e f g

P5

a b c d e

23,1 0-

9,4~6,6 -

4,3 m-

2,3 1-

2,0

Patient 4 Patient 5

Patient 6

FIG. 1. Southern blot patterns of EcoRI-digested DNAs of strains of A. fumigatus isolated from patients 4, 5, and 6. Molecular size markers(in kilobases) are presented to the left of each panel. Each lane corresponds to a different isolate (see Table 1 for a description of strains).

The original slant was kept at room temperature, andmonospore isolates were obtained by the limiting dilutiontechnique. All strains were grown on 2% malt agar and werekept at room temperature until further analysis.DNA fingerprinting. Conidia from each monospore isolate

were inoculated into 50 ml of Sabouraud liquid medium in150-ml Erlenmeyer flasks and were shaken at 37°C at 200 rpmovernight. The mycelium was harvested, and DNA was ex-tracted basically by following the protocol described previously(11). The only modification concerned the amount of myce-lium and the volumes of solvent used; the mycelial mat (300 mg[fresh weight] after drying the cells under filter paper) wasground in liquid nitrogen and was suspended in 0.8 ml of lysisbuffer, and all further solvent extraction steps were performedin 2-ml microcentrifuge tubes. The DNA pellet was resus-pended in 100 pLI of TE buffer and was quantified by afluorimetric technique (5). Under these extraction conditions,the DNA concentrations of the different samples varied from200 to 1,000 ,ug/ml.

After complete digestion by the EcoRI enzyme, DNA was

loaded onto a 20-cm-long agarose gel in TBE buffer (0.7% in89 mM Tris, 89 mM borate, 2 mM EDTA buffer) at a

concentration of 1.2 ,ug of DNA per 7-by-1.5-mm well and was

separated for 24 h at 1.12 V/cm of gel. The DNA was thentransferred to a nylon membrane and hybridized with themoderately repetitive sequence XAF3.9 (described as 3.9 inreference 11). XAF3.9 was radiolabeled with [ax-32P]dCTP by a

random priming method (specific activity, 2 x 109 to 4 x 109cpm/,ug of DNA). After washing, hybridization membraneswere exposed for 3 to 5 h against an X-OMAT film in a cassettecontaining a Cronex Lightning Plus intensifying screen. TheSouthern blot patterns of the different isolates were comparedon the basis of the presence or absence of bands. Band

positions were identified by comparison with bacteriophageHindlIl molecular size markers and DNA fragments from theA. fumigatus AF1 reference strain which was included in allgels. The stabilities of the fingerprints during strain subcultur-ing were assessed previously (11). Two strains were consideredto be identical when the Southern blot patterns were totallysuperimposable. Although it was shown in a previous study(11) that comparison of different Southern blot patterns isamenable to computerized analysis, quantification of strainrelatedness was not undertaken in the present study since wewere looking only for the identities between the Southern blotpatterns of strains of different origins.

RESULTS

For each patient or environmental A. fumigatus isolate, 5 to18 monospore clones were fingerprinted by Southern blothybridization. All clones of the same isolate gave identicalpatterns (data not shown). Only isolates 38-911 andW 38-1512produced two distinct Southern blot patterns from the mono-

spore strains from each isolate, suggesting that the originalculture began with a mixture of two spores. For these isolates,the two strains that gave different patterns were included in thepanel of strains used for comparison (strains E8 and E9 andstrains E14 and E15 in Table 1). The Southern blot patterns ofthe single-spore strain representative of each isolate are pre-sented in Fig. 1 to 4.

Fingerprinting of patient isolates. Multiple isolates were

obtained from three patients with invasive aspergillosis ondifferent dates or from different body locations (Table 1). TheSouthern blots of isolates from patients 4 to 6 are presented inFig. 1. The seven isolates from patient 4 collected antemortemover a period of 15 days produced identical patterns. The four

P6

a b c d

2 3, 1

9,4 _ i

6,6 --

4,3

... .. .........

2,33

2,0 ~

2z3,

-~~~~~~~~~~~~~~~~~~~~. ...

2, 0 W' _

J. CLIN. MICROBIOL.

EPIDEMIOLOGY OF NOSOCOMIAL INVASIVE ASPERGILLOSIS 687

P E

1 2 3 1 2 3 4 5

E P

8 9 10 12 11 14 15 13 6 17 19 7 4b

23,1 w-

9,4 i-

6,6 0-

4,3-

2,3-2,0-

*-. gFs : - w =* g * Iw; Z" " u uEES __ EFXw E: Ww WF_ X s w-<-__ .._._

WF w w.t S4. ..qS.q; F

_wl .,W, 3t.__ *

FIG. 2. Southern blot patterns of EcoRI-digested DNAs of strainsof A. fumigatus collected in unit A of hospital A from patients withinvasive aspergillosis (lanes P1 to P3) and from the environment (lanesEl to E5) (see Table 1 for a description of the strains). Molecular sizemarkers (in kilobases) are presented to the left of each panel.

isolates from patient 6 sampled from the lung and the heartante- or postmortem also gave identical patterns. In contrast,among the five isolates from patient 5, the three isolatescollected on 8 January 1993 (isolates PSa, P5b, and P5c) frombody locations as distant as the lung or a skin lesion were

identical, whereas two isolates sampled 2 and 4 days later(isolates P5d and PSe, respectively) exhibited different South-ern blot hybridization patterns.Comparison between environmental and clinical isolates. In

unit A of hospital A, two patients (infected with isolates P1 andP2) died of invasive aspergillosis during the same week at a

2-day interval. The Southern blot hybridization patterns dis-played by the isolates from these two patients, as well as by anisolate from another patient (infected with isolate P3) whodied of invasive aspergillosis 3 months earlier, differed mark-edly (Fig. 2). No strain was isolated from the environmentduring the period of infection. Environmental surveys under-taken 3 months after the last patient died in unit A resulted inthe isolation of five strains of A. fumigatus from the atmo-sphere over a period of 6 months. Three different patternswere identified among the environmental isolates that werecollected (Fig. 2). The two isolates (isolates E2 and E3) fromtwo different rooms collected over a 5-month interval betweenthe sampling times were indistinguishable. The two isolatessampled in the entrance of the unit after 2 weeks of delay alsodisplayed the same patterns (isolates E4 and E5). Mostinterestingly, the isolate (isolate El) from the environment ofa room of unit A where patient 2 never stayed displayed thesame hybridization pattern as the isolate collected from patient2 (lane P3 in Fig. 2) more than 6 months earlier.

Eight colonies were obtained from the air of the same room(the room where patient 5 stayed) in unit B of hospital A overa period of 6 months. Preliminary fingerprinting of monosporeisolates obtained from each colony showed that two of the

FIG. 3. Southern blot patterns of EcoRI-digested DNA of strainsof A. fumigatus collected in the environment of unit B of hospital A(lanes E6 to E19). Strains from patient 4 (P4), who stayed in unit B,were included to show the similarity between isolates P4 and E7. Allstrains are presented in Table 1. Molecular size markers (in kilobases)are presented to the left of each panel.

isolates contained two different strains. The analysis of theenvironmental strains was therefore performed on 10 strains(E6 to E15). Among the strains sampled from the same room,strains E9 and E14, which were isolated in November andDecember 1992, respectively, were indistinguishable on thebasis of their hybridization patterns (Fig. 3). Two other strains(strains E8 and E15) collected at a 1-month interval were alsoidentical.The Southern hybridization patterns of the 14 environmen-

tal strains collected from the same room (strains E6 to E15)and the close outer environment of the room (strains E16 toE19) were different from the patterns displayed by the isolatessampled from patient 5 (isolates PSa to P5e), who stayed in theroom. In contrast, one of the isolates from the environment ofunit B collected at the end of June 1992 (isolate E7) wasidentical to the strain recovered in January 1993 from patient4 (strain P4), who stayed in this unit.Four of the five isolates from the inner environment of unit

A of hospital B displayed different Southern blot hybridizationpatterns (Fig. 4, lanes E26 to E30). Among all of these isolates,isolate E30, which was collected by brushing the ventilationsystem filter inside the unit, gave a pattern identical to that ofstrain P6 from the patient hospitalized in this unit.

DISCUSSION

The results of the present study confirmed the efficiency andsensitivity of MRSs for fingerprinting strains of A. fumigatus.Strain differentiation obtained by this method can be made onthe basis of very slight and unique differences such as theswitching or disappearance of a very few bands (11, 12). Theresults presented here demonstrate the requirement of a verysensitive fingerprinting system for A. fumigatus not only toregister differences among isolates but, most importantly, to

23,1 a-

9,4 _6,6 m-_m

4,3 -

2,3~2,0~

VOL. 32, 1994

j,,s"

0Imq.*i: I., 4. W;-

Adlwmh6..qw4mw

688 GIRARDIN ET AL.

E

20 21 22 23 24 25 26 27

p

28 29 30 6b

23,1 -

9,4-

6,6-

4,3 00-

2,3

2,00 .,

. .

FIG. 4. Southern blot patterns of EcoRI-digested DNAs of strainsof A. fumigatus collected from the environment of unit A of hospital B(lanes E20 to E30). The strain from patient 6 (lane P6) was added tothe panel to show the similarity of the patterns obtained with thepatient strain and environmental strain E30. See Table 1 for descrip-tions of the strains. Molecular size markers (in kilobases) are pre-sented to the left of each panel.

assess the identities between two isolates of different origins,for example, from the environment and from a patient.

Other genomic fingerprinting systems such as RFLPs (7, 9),patterns of chromosomes, or Southern blots probed withribosomal (11, 23) or mitochondrial DNA or even randomlyamplified polymorphic DNA (4, 19) are less precise thanMRSs and are efficient only for assessing marked differencesamong strains. For example, strains of A. fumigatus indistin-guishable on the basis of RFLP or randomly amplified poly-morphic DNA patterns have proven to be different by using anMRS as a probe (6a, 11). The use of MRSs allowed us toidentify infecting strains, to study the persistence of A. fumiga-tus strains in a hospital environment, and to assess theirputative roles in the development of nosocomial invasiveaspergillosis infections.

Isolates from the same patient. The results presented herestrongly suggest that each of the patients with invasive aspergil-losis was infected with a single different strain. This was shownfor at least two of the three patients studied (patients 4 and 6),in which only one pattern was found for all the isolatessampled from each patient. In the third patient (patient 5),three isolates from very different body locations such as thelung or the skin were similar. However, in this patient, twosupplementary isolates characterized by different Southernblot hybridization patterns were also isolated. Since the conidiaofA. fumigatus are always present in all hospital environments(6, 22, 25), it could be possible that these two isolates werecontaminating strains isolated during plating or samplingprocedures (26). The presence of such contaminants has also

been evoked in a study of patients with aspergilloma fromwhich sequential isolates of A. fumigatus were obtained andfingerprinted (12). Although it was shown that each patientwith aspergilloma was infected by a single and unique strain, 4of 43 strains sampled from the lung and fingerprinted hadpatterns different from those of the infecting strains and weresuggested to be contaminating strains.The putative presence of contaminating strains isolated

during sample collection from a single patient stresses the needto fingerprint a large number of isolates from each patient toidentify the infecting strain reliably. This may represent aserious drawback in the study of invasive aspergillosis, becauseit is technically and ethically very difficult to collect numerousclinical samples from patients.

Nevertheless, the possibility of the presence of two or moreinfecting strains in a single patient cannot be ruled out. In theonly previous study of patients with invasive aspergillosis,Denning et al. (9) have shown that two isolates from each ofthe two patients whom they studied displayed different RFLPpatterns and were considered different strains. Moreover, weshowed that one severely immunocompromised patient withaspergilloma can harbor one isolate in the lung and a differentone on a skin lesion (12). More patients should be surveyed inorder to confirm the uniqueness of the infective strains inpatients with aspergillosis.

In the case of A. fumigatus, MRSs did not identify a patternwhich looked to be specific for strains of human origin. This isin agreement with experimental infections in a murine modelin which all strains tested were infective to mice, whatever theorigin of the strain (unpublished data). These results wouldconfirm the opportunistic nature of the pathological behaviorof A. fumigatus. This conclusion is in opposition to those ofstudies with truly pathogenic phytopathogenic fungi in whichthe MRS was able to resolve pathotypic diversity (13, 18, 20).

Nosocomial origin of the aspergillosis infection. The noso-comial origin of invasive aspergillosis was strongly suggested inthe case of patient 6. A strain with a pattern similar to that ofthe only strain found in patient 6 was isolated in the unit wherepatient 6 died. The strain was isolated the day following thedeath of the patient. Although it cannot be ruled out that thisisolate originated from the patient (sputum or contaminationof the clinical devices), such a possibility seems rather improb-able. The isolate was collected in the grid of the ventilationsystem, which suggested contamination of the ambient air bythis strain. Moreover, in contrast to patients with aspergilloma,in patients with invasive aspergillosis, the Aspergillus straingrows as a mycelium in the colonized tissues and does notsporulate in the infected lung. It is therefore unlikely thatconidia originating from such patients would contaminate theambient air.

In the case of patient 4, a nosocomial origin of the infectioncould be suspected since the patient was infected with a strainthat was also isolated from the environment of the samehospital unit. However, the considerable time lapse betweenthe two samplings (more than 6 months) weakens this putativeconclusion of a nosocomial origin of infection.

In the case of patient 5, no isolate resembling any of thestrains from the patient was found in the environment of theroom or the unit where the patient stayed. This result was notunexpected, since this patient was ambulatory and had spenttime in other hospitals or different departments of hospital A,where he could have been infected with Aspergillus spores.

In the case of unit A of hospital A, sampling of theenvironment was undertaken 3 months after the last case ofinvasive aspergillosis was diagnosed. This delay and the veryfew environmental isolates gathered from this unit are respon-

J. CLIN. MICROBIOL.

EPIDEMIOLOGY OF NOSOCOMIAL INVASIVE ASPERGILLOSIS 689

sible for the inconclusive results from the fingerprinting of theenvironmental isolates. The two isolates from the two patients(Fig. 2, lanes P1 and P2, respectively) who had invasiveaspergillosis at the same time in unit A were different. If thesepatients were infected in the hospital, the infection was due totwo different strains that contaminated the same hospitalenvironment. Another alternative would be that these patientscame to the unit with conidia resident in particular niches oftheir bodies, for example, their sinuses or lymph nodes. Theseconidia would not grow in the presence of efficient hostimmune defenses. The administration of potent immunosup-pressive drugs would result in the activation and growth of thefungus in host tissues. Under these conditions, each patientwould be infected by his or her own specific strain, which wouldlogically be different for each patient since they came fromdifferent geographical locations. Although the search forconidia in different body locations has not been seriouslyundertaken with humans (1), in other animals the presence ofAspergillus conidia has been demonstrated on several occasionsin organs other than the lung (15, 24).The results of our study show that the nosocomial origin of

an infection can be irrefutably demonstrated if environmentalstrains identical to the strains from patients are isolated in aprospective survey a few days before the patient's aspergillosisis diagnosed. Such study would require the isolation andfingerprinting of large numbers of strains, since all hospitalenvironments screened in the present study were contaminatedwith numerous different strains with no common Southernblotting patterns. In addition, the absence of dominant strainsin the environment surrounding the patients raises the ques-tion of the origin of a nosocomial infection and the capacityof an environmental strain to promote an aspergillosis infec-tion.

Persistence of a strain in a hospital environment. Until now,the 70 unrelated strains of A. fumigatus fingerprinted with themoderately repetitive sequence XAF3.9 have displayed distinctand unique Southern hybridization patterns (unpublisheddata). Therefore, the recovery of the same strain duringsuccessive environmental samplings strongly suggests that aparticular strain can persist for some time in the hospitalenvironment. The first example was seen in unit A of hospitalA, where two different strains were recovered within a 2-weekinterval at the entrance of the unit or a 5-month interval in aroom of the unit, respectively. In unit B of hospital A, a strainwas isolated from the environment (strain E7) and from apatient (strain P4) within a 6-month interval. A systematicsearch is now necessary to identify the source of the inoculumand trace the routes of contamination in the hospital environ-ment by the strains which can be pathogenic to humans.

ACKNOWLEDGMENTS

This study could not have been conducted without the help andinterest of E. Gluckman (Unite de Greffe de Moelle, H6pital StLouis), G. Schaison (Hematologie Pediatrique, H6pital St Louis), andJ. F. Dhainaut (Reanimation Medicale, H6pital Cochin), who allowedus to collect isolates of A. fumigatus in their respective units.

Research was funded by CNAMTS-INSERM grant 3AM05 1 toJ.P.L.

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