Accepted Article This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/myc.12789 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has thein publication for approved and peer-reviewed has been that Article Accepted is an This Alireza Abdolrasouli title: Running complex in cystic fibrosis Airway persistence by the emerging multi-azole-resistant typeArticle :OriginalArticle (Orcid DR DARIUS ID :0000-0002-1014-7343)ARMSTRONG-JAMES 0000-0002-8934-2343) : (Orcid ID ABDOLRASOULIALIREZA DR B. Buil B. Nicholas J. NicholasSimmonds J. Stuart Elborn 7. Department of Microbiology, Royal Brompton and Harefield NHS Foundation trust, London, London, trust, Foundation NHS Harefield and Brompton Royal Microbiology, of Department 7. London, Trust, Foundation NHS Harefield and Brompton Royal Centre, Fibrosis Cystic Adult 6. The Utrecht, Institute, Biodiversity Fungal Westerdijk Mycology, Medical of Department 5. for Center Excellence ECMM and UMC/CWZ Radboud in Mycology Expertise of Centre 4. Hospital Canisius-Wilhelmina Diseases, Infectious and Microbiology Medical of Department 3. Health, Public of School College Imperial Epidemiology, Diseases Infectious of Department 2. FungalPathogens Laboratory, National Heart andLung Institute, Imperial College London, 1. 3,4 UK UK Netherlands Netherlands The Nijmegen, Mycology, Medical (CWZ), Nijmegen, The Netherlands UK London, UK London, , Alison, Y.Tang Y. 6 , Andrew Jones L.

Rasamsonia 1 , Amelia C.BercussonAmelia , 6 ,

Darius Armstrong-James 1 infection in cystic fibrosis in cystic fibrosis infection , LeonardBoer L.de 6 ,

Jacques F. Meis 1 , Johanna L. Rhodes Version of Record. Please Version as this cite of Record. article 1 , AnandShah, 1,7* 3,4 , Matthew, C.Fisher Rasamsonia argillacea 6 , Andrew J. Milburn 2 , Ferry Hagen 2 , Silke Schelenz , Silke 3,4,5 , Jochem 6 , J. 7 , Accepted Article This by is protectedarticle reserved. Allrights copyright. Cystic fibrosis (CF) Infections caused by Summary identified as with patients testing susceptibility Running title: title: Running Tel:Kingdom. +44207 5942746,[email protected] Fax:notavailable, E-mail: Building, and LungInstitute, National Heart SW7Imperial United CollegeLondon, 2AZ, *Corresponding author: Keywords: progression. disease on impact clinical obvious an have to appear not does it others In severity. disease of appearance the determine, to be difficult may function on lung patients, In certain Consistent with other fungi inthe CF, clinical impactof airway colonization by Rasamsonia. isolating of years three within died patients six of out Three response. clinical a exhibited one Antifungal cases. four of out three in isolation first first preceding year the in decline function to voriconazole and posaconazole and low MECs to echinocandins.

Rasamsonia argillacea Rasamsonia R. argillacea argillacea R. Rasamsonia Genotypingsuggests thatsimilar genotypes of Rasamsonia

Rasamsonia is one of the main underlying conditions. Anobservational cohort study ofCF Rasamsonia argillacea in respiratorysamples wasconducted. Eightisolates from six patients were complex and tested for antifungal susceptibility. All isolates had high MICs Darius Armstrong-James, Fungal Pathogens Laboratory, Flowers airway persistance may be able to drive clinical decline. In others, though a clear impact impact a clear though In others, decline. clinical drive to able be may species complex;cystic fibrosis; genotyping;outcome; antifungal Rasamsonia complex have been reported in various clinical settings. clinical invarious reported have been complex therapy was initiated in two patients, to which only isolation. This continued in the year following following year in the continued This isolation. Rasamsonia

Rasamsonia

Four patients experienced lung lung experienced patients Four can persist in CF airways. airways. in CF can persist acts as a marker of Rasamsonia is variable. is variable.

Accepted Article This by is protectedarticle reserved. Allrights copyright. case systematic a detailed, far thus However [17]. Kingdom United and Netherlands the Germany, France, Austria, including countries, European several across reported frequently more has been 2010, Since underestimated. prevalence true their and misidentified been often morphological similarities to to Due [14–16]. airways CF colonize to found been has pathogen fungal emerging this addition, [12]. Pulmonary and aorticvalve infectionin an immunocompetent patient has been described[13]. In disease graft-versus-host and [11] (CGD) disease granulomatous chronic with inpatients mycosis polyphyletic thermotolerant [6]. cultured are species bel species some and transmembrane conductance regulator ( Introduction diversity of fungal speciesin sputum samples from CF patients [8–10]. molecular techniques haverevealed higher a than previously recognized prevalenceand a greater and media culture fungal-selective using studies Recent [7]. susceptibility in genetic differences differences in the geographicalprevalence of species, local environmental factorsand population bedueto differences methods the in usedthein recoveryidentificationand of fungi,as well true as to prevalentclinically significant fungalspecies in CF patients [5]. Less frequently, [2–4]. microorganisms fungal and bacterial of variety a by infection and colonization airway chronic to prone is CF population The [1]. in CF mortality and morbidity of cause leading the is failure respiratory Progressive [1]. failure respiratory eventually and bronchiectasis of development the in results which isinitiated fibrosis obstruction and afailure to efficientlyclear pathogens. A vicious cycle ofinfection, inflammation and lungs. Low ASL volumeinhibits normal mucociliaryaction leading to mucus accumulation, airway transport across epithelial cell membranes resulting indepletion of airwaysurface liquid (ASL) in the Rasamsonia argillacea Rasamsonia Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutationsin the CF Aspergillus fumigatus, Exophiala dermatitidis dermatitidis Exophiala fumigatus, Aspergillus onging to thegenus

Prevalence rates vary considerably from onecentre to another, which may (previously (previously (family CFTR Alternaria spp. argillacea Trichocomacea ) ) gene. Mutations inthe

and and , Cladosporium and Scedosporium apiospermum apiospermum Scedosporium )

and is a causative agent of invasive invasive of agent is a causative and ) belongs to the emerging, spp., spp., , Penicillium CFTR Rasamsonia gene interfere with ion and and R. argillacea argillacea R. Paecilomyces variotii Paecilomyces Talaromyces species have have species are the most the are in CF CF in

Accepted Article Houbraken Houbraken in outlined procedures the following characterized morphologically were isolates The days. 7-10 for indark at 28-30°C incubated and (MEA) agar extract malt onto sub-cultured were isolates The interval. month 11 an had 2 case from analyzed isolates two while apart, months 4 obtained molecular sequencing to confirm their speciesidentification. For case 1, the selected isolates were recovered isolates were preserved, only eight available Fungal isolates appointments. clinic outpatient at records medical from collected was data function Lung review. note case through collected were patient each for outcomes characteristics. microscopic and features macroscopic using identified primarily was isolate fungal Any 4 weeks. to up for at 30°C incubated were cultures Fungal UK). Basingstoke, (Oxoid, chloramphenicol mg/L 100 with supplemented agar dextrose Sabouraud onto cultured then and agent monthly) and at times of clinical exacerbation. Respiratorysamples were first treated with a mucolytic Patients and sputum cultures Patients and Methods with hospital our attending patients clinical, mycological features and outcomes of airway colonization by Hospital Adult and PaediatricCF Centres in London, United Kingdom. Here we describedetailed This by is protectedarticle reserved. Allrights copyright. notperformed. been series-base analysisof clinical outcomes for cystic fibrosis-related all not As patients. CF six from obtained were cultures respiratory positive 32 In total, clinical and received treatments to relating data and characteristics clinical and Demographic 2–3 (i.e. appointment clinic follow-up every at collected routinely are samples Sputum Brompton Royal at the followed actively are patients paediatric 340 and adult 600 Nearly et al. et [17].

R. argillacea argillacea R. complex Rasamsonia

in their airways between 2013 and 2016. 2016. and 2013 between airways in their

Rasomsonia isolates were subjected to to subjected were isolates R. argillacea R. airwayinfection has complex in six in six complex CF Accepted Article v7.6.1 (Applied Maths NV, Sint-Martens-Latem, Belgium) and an AFLP banding patterns banding AFLP an and Belgium) Sint-Martens-Latem, NV, Maths (Applied v7.6.1 BioNumerics into imported was data Raw [21]. "TGAG" with replaced was HpyCH4IV-primer selective Zhang by described as analysis fingerprint (AFLP) ( Antifungal suscep clustering. with % similarity bar was generatedusing the ranked Pearson similarity coefficient and UPGMA This by is protectedarticle reserved. Allrights copyright. of the Part [19]. database barcoding DNA ITS reference sequences werecompared with the InternationalSociety of Human andAnimal Mycology (ISHAM) – ITS primers. PCR same the using MA) Waltham, Biosystems, (Applied Analyzer Sequence 3730xL ABI an on directions both in sequenced and kit Purification PCR QIAquick with purified were amplicons PCR [18]. ITS4 and ITS1 primers published using amplified was (rDNA) DNA ribosomal the of region (ITS) spacer transcribed internal The 3000. NanoDrop with quantified and moulds for instructions manufacturer’s the to according reagent Ultra Prepman with extracted DNA was analysis phylogenetic and identification Molecular amphotericin B, itraconazole, posaconazole, and voriconazole and to reduce growth by 80% for for by 80% growth reduce to and voriconazole and posaconazole, B, itraconazole, amphotericin for inspection onvisual of 100% growth to inhibit concentration lowest as the defined MIC was the Clinical and LaboratoryStandards Institute (CLSI)M38-A2 method for filamentous fungi [22]. The using using described elsewhere [20],visualised and FigTree in version 1.4.2.The final phylogenyrooted was phylogenetic analysisusing 500 replicates was carriedout using RAxML-HPC version8.2.4 as Expectation (MUSCLE), with the resulting alignment output in interleavedPhylip format.Bootstrapped Log- by Comparison Sequence Multiple using aligned were Netherlands) The Utrecht, Institute, Biodiversity Fungal and Westerdijk Netherlands; The Nijmegen, Hospital, (Canisius-Wilhelmina region. ITS the for above described as analyzed were Sequences ABI3500xL. onan sequenced bidirectionally and [14] T10 and Bt2B Bt2A, n =8), and The Netherland ( Netherland The and =8), R. emersonii Antifungal susceptibility testing was performed by meansof Isolates were grown onto MEA for 7–14 days at 28–30°C prior to DNA extraction. Genomic Genomic extraction. DNA to prior 28–30°C at days 7–14 MEA for onto grown were Isolates Concatenated ITS1 and and ITS1 Concatenated CBS 396.64 tibility and tibility n =16) was determined using amplified fragment length polymorphism polymorphism length fragment amplified using determined was =16) in vitro in T . Furthermore, the genetic diversity of of diversity genetic the Furthermore, . β -tubulin sequences from 8 UK isolates plus 55 additional isolates isolates additional 55 plus isolates 8UK from sequences -tubulin combination testing testing combination et al ., however, the 3'-side overhang "TGAC" of the of the "TGAC" overhang 3'-side the however, ., β -tubulin was amplified by using the primers primers the using by amplified was -tubulin

broth microdilution, according to to according microdilution, broth Rasamsonia isolates from UK

dendrogram dendrogram Accepted Article Nucleotide sequence accession numbers numbers accession sequence Nucleotide was >4. This by is protectedarticle reserved. Allrights copyright. a with cylindrical were phialides Rough penicilli. terverticillate or biverticillate, monoverticillate, bearing conidiophores with hyphae septate hyaline, revealed cultures the of examination Microscopic (Supplementary Figure 1). All isolates were thermophilic,displaying good growth at 37°C and 42°C. mm ~15–17 of growth arestricted demonstrated which Ras_RBH_7 of exception an 30°C with colour. All isolates produced colonies reaching a diameter of ~30–35 mmin 7–10day cultures at 28– Morphological characteristics inclusionin this study. where cultures fungal primary 32 From identified. were 2016 September and 2013 October between complex Results numbersKY649324 to KY649331. was FICI if the synergistic as hyphae for the echinocandins. Both the MIC and the MEC were determined at 48 hours. (MEC) was defined as the lowest concentrationseen to produce short,stubby, abnormally branched concentration effective minimal The well. control drug-free the to compared flucytosine and terbinafine lessthanactivities the ofthe individual agents, respectively.The summation ( dependingon whether the antifungal activityof thecombination was greater than, equivalent to, or vitro induplicate. tested was isolate each testing, combination of MECresults and MIC of reproducibility itraconazole-micafungin and terbinafine-micafungin as previously described [23]. Toascertain the interactions were calculated on the basis of the fractional inhibitory concentration (FIC) index, index, (FIC) concentration inhibitory fractional the of basis on the calculated were interactions The nucleotide sequences obtained in this work were submitted to GenBank under accession accession under to GenBank submitted were work this in obtained sequences nucleotide The Six CF patientsSix CF with for used was method microdilution A checkerboard Colonies on MEA were relatively slow growing, slightly floccose with cream-buff to pale brown brown pale to cream-buff with floccose slightly growing, slow relatively MEA were on Colonies Rasamsonia

spp. was isolated, eight isolates from sixpatients were available for ≥ ≤ 1 sputum culture-positive (mean 5 isolates per case) for for case) per 5 isolates (mean culture-positive 1 sputum 0.5, no interactionif the FICI was >0.5-4, and antagonistic if the FICI in vitro in

combination testing with with testing combination Σ FIC) was interpreted interpreted was FIC) R. argillacea R.

In Accepted Article amphotericin B was 0.25–2 mg/L. High MIC ranges were observed for triazole agents: triazole for observed were ranges MIC High mg/L. 0.25–2 B was amphotericin Antifungal su 2). (Fig. similar highly 2were case from apart months 11 isolated Ras_RBH_6 and and Ras_RBH_2 1 case from apart months 4 isolated and Ras_RBH_3 Of note, AFLP fingerprint analysis clearlyconfirmed that two sets of sequentialisolates Ras_RBH_1 Ras_RBH_8. and Ras_RBH_5 Ras_RBH_4, of identification AFLP the corroborate not does analysis Phylogenetic Ras_RBH_7). and Ras_RBH_6 Ras_RBH_3, Ras_RBH_2, (Ras_RBH_1, isolates confirming the ITS1 and This by is protectedarticle reserved. Allrights copyright. aegroticola as identified were isolates identified as were Ras_RBH_6, and Ras_RBH_2 isolates Two reproduction. asexual and clonality of degree analysis phylogenetic and identification Molecular as identified were isolates eight all features microscopic and characteristics colonial on Based conidia. ovoid or cylindrical smooth-walled producing tip tapering triazoles. combinationsWhen of micafungin with terbinafine oritraconazole were s Rasamsonia our general, In respectively. mg/L 0.031–0.125 and mg/L of <0.015–0.062 ranges MEC had caspofungin and Anidulafungin mg/L. <0.015 of MECs micafungin low exhibited isolates all contrast, ranges of flucytosine and terbinafine were <0.062–32 mg/Land 0.25–8 mg/L, respectively. In MIC drugs. triazole more or two to MICs raised had Ras_RBH_6) and Ras_RBH_4, (Ras_RBH_2, for itraconazole, 4 tomg/L >16 forvoriconazole, and 0.5 to 4 mg/L for posaconazole.Three isolates R. eburnea R. the within species distinguish andpartial ITS1 that demonstrates analysis Phylogenetic investigations. molecular for included were strains) type including Netherlands The from isolates The results of the antifungal susceptibility testing are summarized in Table 1. MIC ranges of Concatenated ITS1 and and ITS1 Concatenated ( and n R. argillacea sensu stricto sensu argillacea R. spp. =14), sceptibility and combination testing andcombination sceptibility R. piperina

isolates demonstrated lowest MECswith echinocandinsand highest MICs to R. argillacea β -tubulin combined identifications, and the phylogenetic placement of 5 R. aegroticola R. species are highly related to each other, which is suggestive of a high ahigh of suggestive is which other, each to related highly are species Rasamsonia ( β n -tubulin sequences (8 isolates from this study plus 55 additional =5) and , Ras_RBH_7 wasidentified as . UPGMA analysis grouped the isolates into three clusters; clusters; three into isolates the grouped analysis UPGMA . (Fig. 1). The analysis also shows that isolates in the the in isolates that shows also analysis The 1). (Fig. genus R. piperina R.

( n =5) (Fig. 1). AFLP fingerprint analysis analysis fingerprint AFLP 1). (Fig. =5) β -tubulin gene sequences can adequately adequately can sequences gene -tubulin R. argilacea sensu lato sensu argilacea R.

R. piperina R. , and five remaining eparately tested for for tested eparately .

1 to1 >16 mg/L R. R. Accepted Article wasof 48% predicted. Four outof patients 6 chronically were infected with This by is protectedarticle reserved. Allrights copyright. episodes infection and characteristics Clinical combinations. drug both for interaction no demonstrated isolates 8 all 1), Table (Supplementary synergy a history of azole use prior to first isolation of of isolation first to prior use azole of a history for culture negative despite presentation clinical and serology additionalpatient diagnosed was with allergic bronchopulmonaryaspergillosis (ABPA) based on for other fungal organisms and 2 6 out of with complex. The most common CF-related mutation was was mutation CF-related common The most complex. diverse collection of 55 of 55 collection diverse and where complex reported. been also have [13] hosts immunocompetent and [12] recipients [11,13] to airway colonization in patients with CF [15,16, 25-28]. Infectionsin bone marrow transplant Discussion 3. Figure in depicted is time over change function lung and given therapy anti-infective growth, fungal of summary A transplantation. post-lung sepsis from one and failure respiratory from two period, study the within died 6 patients of out Three azithromycin. long-term taking 6were of out 4 and antibiotics inhaled 3 were homozygous (Table 2). This is similar to the prevalence of of prevalence the to similar is This 2). (Table homozygous 3 were first isolation was 21 kg/m 21 was isolation first mass i body Mean replacement. enzyme for need the by defined insufficient, pancreatic were (83%) 6 patients of out Five [24]. whole a as population CF UK the within homogeneity

R. piperina R. The patients had an age range of 11–53 years (median 30.5) at first isolation of of isolation first at 30.5) (median years 11–53 of range age an had patients The Eightisolates frompatients 6 in our CFcohort were identified as members of of presence The . We compared concatenated ITS1 and and ITS1 concatenated compared We . R. aegroticola Staphylococcus aureus. Rasamsonia R. argillacea 2

. Mean. forced expiratoryvolumesecond 1 in (FEV in the year prior to first isolation of was the most common species, followed by by followed species, common most the was isolates from the Netherlands. The same pattern of species species of pattern same The Netherlands. the from isolates complex has been reported in invasive infections in CGD in CGD infections ininvasive has reported been complex Three out of six patients had positive respiratory cultures cultures respiratory positive had patients six of out Three R. argillacea. R.

F508del β -tubulin sequences from 8 UK isolates plus a plus 8 UK isolates from sequences -tubulin All6 patients were takingcontinuous R. argillacea A. fumigatus

, present, inpatients 6 5out of ofwhom ∆ F508 heterogeneity and and heterogeneity F508 complex and one one and complex . Four out of 6 patients had patients 6 of out Four . R. argillacea sensu stricto sensu R. argillacea Pseudomonas aeruginosa 1 ) at time of acquisition acquisition of time at ) R. argillacea ndex (BMI)at R. argillacea R.

Accepted Article R. argillacea R. other the to compared isolate an atypical is this that indicates which profile fingerprint AFLP a distinct (Ras_RBH_4) that wasidentified by AFLPas by ITS/ referred from another centre for declining lung function in the context of recurrent growth of of growth recurrent of context the in function lung declining for centre another from referred Rasamsonia This by is protectedarticle reserved. Allrights copyright. similarto thatisolatean of thatbysequencing was foundtobe be to found was it while cluster resolution but that does not explainthat isolate Ras_RBH_8 fell by AFLP analysis in the ITS/ to compared used when is discrimination [17]. However, by applying AFLP fingerprinting a larger part of thegenomic information ITS/ as former ITS/ major three of set a onto before applied argillacea to belonging isolates 116 of study multi-centre large a in described was recently prevalence will elucidate how this mould adapts to the host microenvironment during prolo during microenvironment host the to adapts mould this how elucidate will with colonized chronically patients CF individual from isolates serial of sequencing ability of of ability isolates is inline with the results of a recentcomprehensive genotyping analysis which identified the sequential our of sets two among observed similarities of level high However, testing. for available longer no were isolates fungal these as patients our from isolates sequential all among diversity genotypic the examine to able not were we Unfortunately, airways. CF from isolated consistently be that suggests 2 respectively, and 1 cases in apart, months 11 and 4 obtained isolates. our among β -tubulin sequencing identified as a different species which may question the suitability of the the of suitability the question may which species different a as identified sequencing -tubulin β Of the six cases described here, there was one whose history clearly suggests that that suggests clearly history whose one was there here, described cases six the Of AFLP analysis has been widely used as a genotyping and identification tool, but it was not not was it but tool, identification and genotyping a as used widely been has analysis AFLP Genotypic similarities observed by AFLP analysis among two sets of sequential isolates isolates sequential of sets two among analysis AFLP by observed similarities Genotypic -tubulin sequencing. -tubulin R. aegroticola complex collected from 26 CF patients [14]. Similarly, we did not find any any not find did we Similarly, [14]. patients 26 CF from collected complex Rasamsonia Rasamsonia spp. contributed directly to the patient’s clinical deterioration. In case 2, the patient was was patient 2, the case In deterioration. clinical patient’s the to directly contributed spp.

isolates. It remains enigmatic that all three discrepancies were found to be be to found were discrepancies three all that enigmatic remains It isolates. β -tubulin sequencing has been observed beto areliable tool for and and clusters were observed, two clusterscontain isolates that were by combined R. argillacea Rasamsonia

Using single-nucleotidepolymorphism-based typing with whole-genome β R. aegroticola R. -tubulin-based identification, hence AFLP analysis gives more to chronically infectthe CF airway[14]. spp. isolates. With this rapid molecular genotyping method method genotyping molecular rapid this With isolates. spp. by sequencing as the AFLP fingerprint profile was R. argillacea and by sequencing as as sequencing by and

R. piperina R. . One ofthe two isolates Rasamsonia Rasamsonia nged colonization. Rasamsonia R. aegroticola R. Rasamsonia R. eburnea R. R. aegroticola R. R. piperina R. spp. can can spp. species species P. variotii R.

spp. spp. , had had ,

- .

Accepted Article This by is protectedarticle reserved. Allrights copyright. of culture positive function has continued to decline.case In 6,a man with late-diagnosiscystic fibrosis, singlea Rasamsonia Although benefit. clinical clear any confer not did this but voriconazole of a trial given was 5, case four exacerbations in the on-goingpresence of change when long-termitraconazole andco-amoxiclav were stopped. Over the followingyear he had administered to treat was isolated recurrently over the next 4 years while the patient was on itraconazole, which had been [30]. reported has been disseminated fatal a Notably, transplantation. subsequently received a lung transplant but died of aGram-negativebacterial sepsispost- two the This isconsistent with the low echinocandin MECs highand itraconazole andvoriconazole MICs of atime. at year toa up for culture-negative became he which after caspofungin, with treated was spp. the when achieved best was stability Clinical function. lung of his decline in the reduction with increasedof risk exacerbation. note, Of hadhe received treatment for Stenotrophomonas maltophilia 2was case of samples respiratory from cultured pathogen other only The [29]. documented as only centre our in but accuracy, its confirm to unable were we institution different a in performed was identification species As the Aspergillus fumigatus Aspergillus as such fungi, opportunistic other for observations with consistent entirely is This disease. lung of severity underlying the upon dependent is that manner a in fibrosis, cystic in decline respiratory Taken together, these observationssuggest that airway persistence of fumigatus 1, case accelerated decline inlung function, that was variably responsive to Paecilomyces Rasamsonia argillacea R. Rasamsonia In cases 1, 3, 4 and 5 the persistence of of persistence 5 the and 3, 4 1, In cases and spp. has not been isolated from her respiratory cultures for two years now, her lung lung her now, years two for cultures respiratory her from isolated been not has spp. P. variotii P. and/or and/or Rasomsonia spp. was isolated for the first time during a period of declining lung function. She She function. lung declining of period a during time first the for isolated was spp. and further deteriorated He samples. his respiratory from cultured isolates spp. was isolated in the presence of multiple other fungal pathogens including including pathogens fungal other multiple of presence the in isolated was spp. A. fumigatus , where a spectrum of disease from simple colonisation to ABPA and invasive invasive and to ABPA colonisation simple from disease of spectrum a , where as well as intermittent intermittent as well as Talaromyces , for which there is only limited evidence that infection is associated associated is infection that evidence limited is only there which for , was not associated with clinical deterioration andhe remained well. Rasamsonia . He remained stable for the first three years up until a medication a medication until up years three first the for stable remained . He due to their morphological similarities has been well spp. was detected. Misidentification of of Misidentification detected. was spp. Rasamsonia P. aeruginosa P. Rasamsonia Rasamsonia infection in CF post lung transplantation transplantation lung post inCF infection spp.and absence of and and in the airway was associated with

S. aureus Rasamsonia Rasomsonia S. maltophilia S. growth. growth. -directed therapy. In In therapy. -directed A. fumigatus Rasamsonia may contribute to contribute may Rasamsonia without any any without Rasamsonia . In . In spp. spp. spp. spp. A. A.

Accepted Article This by is protectedarticle reserved. Allrights copyright. against MEC lowest the had it as echinocandins of the representative class a as micafungin chose We combinations. terbinafine-micafungin and micafungin our Furthermore, in [25]. therapy micafungin prolonged and high-dose with only obtained was eradication patient, the French reportedcase, althoughcaspofungin treatmentresulted in improvementthe in clinicalstatus of echinocandins may have potential as treatment for eight all for caspofungin thermophilic in emerging also is resistance Azole . emerging this for options treatment viable are isavuconazole fungus. closely-related currently available, but may help further determine if similar sensitization syndromesoccur for this Unfortunately, fibrosis. cystic in observed is disease single and combination therapyis needed to determine its potential of eradication given. In a French case report from 2012, a CF patientwas treated with echinocandins resultingin the that show not did [16] Barton series, another In [15]. patient the to benefit clinical any brought this whether of details without though caspofungin, with eradication of case one and alone posaconazole with patients argillacea R. unknown. largely remains agent echinocandin an and flucytosine using therapy combination of value clinical Similarly, infection. this for warranted treat to used been previously has it as terbinafine and CF in infection fungal for agent line first used commonly most the in isolates for isolates of andreference clinical 47 of collection a tested study recent A species. specific any to association complexstudied here. The MICs ofitraconazole and posaconazole were variable with noobvious Voriconazoleno had There is little evidence in the literature as to the Rasamsonia species. Giraud Rasamsonia R. argillacea R. Rasamsonia in vitro belongingseven to speciesand found high isavuconazole MICs of >32 mg/L Rasamsonia Penicillium Penicillium Rasamsonia combination testing we did not observe any synergy for itraconazole- for synergy any observe not did we testing combination and improvement in lung function [25]. Further research into antifungal antifungal into research Further [25]. function lung in improvement and in vitro in et al. complex [31]. These findings suggest that neither voriconazole nor nor voriconazole neither that suggest findings These [31]. complex infection. These findings suggest combination therapyis not reported variable success of success variable reported activity (MIC >16 mg/L) against the eight isolates of of isolates eight the against mg/L) >16 (MIC activity species [32]. MECs of micafungin, anidulafungin and isolates tested in this study were low, indicating that that indicating low, were study this in tested isolates was having any clinical impact and antifungal therapy was not not was therapy antifungal and impact any clinical having was Rasamsonia Rasomsonia Rasamsonia in vivo

efficacy of antifungal agents against against agents antifungal of efficacy R. argillacea argillacea R. spp. infections. Notably, in the –specific IgE or IgG testing is not not is testing IgG or IgE –specific spp., itraconazole as it is the in vivo efficacy. eradication from CF CF from eradication R. argillacea R. et al.

Accepted Article cases and3 4.still In others, as incase 6, it appears to have no obvious clinical impact.The in as driver, sole the being clearly without function lung declining of rapidly context inthe appears case1, driving the inflammatoryprocess that leads to lung function decline; in other patientsit Rasamsonia of feature remarkable most isthe which presentation, clinical of heterogeneity the is it however, colonizer in CF patients.Similar to other fungal pathogens that have been identified in CF airways, of this manuscript have no conflicts of interest to disclose. disclose. to interest of conflicts no have manuscript of this authors The Award. Seed a Wellcome by supported is A-J. D. TEVA. and Medical United Sciences, Gilead from fees speaker and Scynexis from fees consultancy received J.F.M. Mycology. Medical for Award Strategic Trust a by Welcome A. supported B. is C. Gilead. from fees and consultancy lecture received has J. S. N. Gilead. from support travel received and talks for paid been A. has A. Conflict of interest study. this for received has been funding No specific Funding Centre. Sciences Health Academic Healthcare College Fibrosis Stichting (Dutch Cystic Fibrosis Foundation; NCFS). This work was supportedby the Imperial Trust forassistance in fungal isolate collection. J.F.M was supportedby theNederlandse Cystic thank the microbiology staff at the Royal Brompton and Harefield National Health Service Foundation we Additionally, Director). Centre CF (Paediatric Balfour-Lynn Ian Dr thank to like would authors The Acknowledgements strategies. treatment effective develop to and infection fungal of impact clinical the emerging fungal pathogen.Larger scale multi-centre prospective studies are needed to investigate of this management for effective is essential colonisation to this in response active infections difficult. more strategies treatment determining of significance the of uncertainty This by is protectedarticle reserved. Allrights copyright. Our data demonstrates that that demonstrates data Our Understanding what predisposes certain CF patients to fungal colonisation and to develop develop to and colonisation fungal to patients CF certain predisposes what Understanding colonization. While in some patientscan it act aggressively,ascase2inpossibly and

Rasamsonia Rasamsonia spp. isolation from respiratory samples makes makes samples respiratory from isolation spp. spp.

can become established as a chronic airway airway achronic as established become can

Accepted Article 6. Pihet M, Carrere J, Cimon B, Chabasse D, Delhaes L, Symoens F, F, Symoens L, Delhaes D, B, Chabasse Cimon J, M, Carrere Pihet 6. in cystic therapy antifungal and fungi pathogenic of Characteristics M. Seidler FM, Müller 5. of study Cohort MG. Höfle WR, Abraham CA, T, Guzman A, Welte R, Sauer-Heilborn Kramer 4. polymicrobial A MG. Surette JC, Norgaard K, Duan HR, Rabin MD, Parkins CD, Sibley 3. Gibson BurnsRL, JL, Ramsey BW. Pathophysiology and managementof pulmonary infections 2. fibrosis. JS. Cystic Elborn 1. References of kin. next contact to able not was team clinical the manuscript, this of publication to prior deceased unfortunately that patients two For obtained. were consents informed cases, In four Consent forpublication . BormanAM, Palmer MD,Delhaes L, CarrèreJ, Favennec L, RanqueS, 7. This by is protectedarticle reserved. Allrights copyright. FigureS1,Table S1and six case histories are available as Supplementary dataat Supplementary data the drafting and revisionofthis manuscript. contributed All authors AYYT experiments. performed JB, FH, JLR, AA, research. of the design and conception the to contributed DAJ and NS ACB, AA, Author contributions standardization in the procedures for mycological examination of sputum samples from CF review. fibrosis--a cystic with patients of secretions in respiratory fungi filamentous of relevance fibrosis. 53:2900–2907. fungibacteria and reveal predominanceoftransient fungal elements. between structure community in Differences patients: fibrosis cystic in adult mycobiome airway Proc NatlSc Acad patients. fibrosis in cystic pathogen enigmatic an exposes infections of pulmonary perspective in cystic fibrosis. Med Mycol Med Expert Rev Anti Infect Ther Ther Infect Anti Rev Expert Am J Respir Crit Care Med Care Crit J Respir Am USA 2008; 105:1570–1575. 2009; 47:387–397. Lancet 2016; 388:2519-2531. 388:2519-2531. 2016; 2010; 8:957 2010; 2003; 168:918–951. 168:918–951. 2003; to the analysis and interpretation of data and data and of interpretation and analysis to the –964.

et al et J Clin Microbiol J Clin . Occurrence and and Occurrence . et al et JAC . Lack of . Lack Online. Online. 2015; Accepted Article 17. Houbraken J, Giraud S, Meijer M, Bertout S, Frisvad JC, JC, Meis JF, S, Frisvad M,Bertout S, J, Meijer Giraud Houbraken 17. 16. Barton RC, Borman AM, Johnson EM, Houbraken J, Hobson RP, Denton M, M, Denton RP, Hobson J, Houbraken EM, Johnson AM, Borman RC, Barton 16. 15. Giraud S, Pihet M, Razafimandimby B, Carrère J, Degand N, Mely L, L, Mely N, Degand J, B, Carrère M, Razafimandimby Pihet S, Giraud 15. This by is protectedarticle reserved. Allrights copyright. 8. Middleton PG, Chen SC, Meyer W. Fungal infections and treatment in cystic fibrosis. fibrosis. cystic in treatment and infections Fungal W. Meyer SC, Chen PG, Middleton 8. 1 De RavinSS, Challipalli M, Anderson V, Shea YR, MarcianoB, Hilligoss D, 11. fibrosis cystic in diversity and epidemiology Fungal L. Sedlacek S, Suerbaum S, Ziesing 10. fibrosis: in cystic infections respiratory Fungal J-P. Bouchara L, Delhaes F, Symoens R, Horré 9. 13. Doyon JB, Sutton DA, Theodore P, Dhillon G, Jones KD, Thompson EH, EH, Thompson KD, Jones G, Dhillon P, Theodore DA, Sutton JB, Doyon 13. D, Haas C, Koidl A, Rohn M, Pichler P, Neumeister T, Valentin 12. 14. Mouhajir A, Matray O, Giraud S, Mély L, Marguet C, Sermet-Gaudelus I, I, C, Sermet-Gaudelus Marguet L, Mély S, Giraud O, Matray A, Mouhajir 14. antifungal susceptibility of clinically important important clinically of susceptibility antifungal 48:2615–2617. 2010; the fungus 48:2381–2386. argillacea patients. patients. 1:S88–97. Suppl 48 2010; fungi. filamentous of prevalence in the variations for cause apossible patients: patients over a 5-year period in a national reference center. center. reference national a in period a 5-year over patients a growing problem. Opin Pulm Med argillacea Transplant argillacea Geosmithia Dis Infect Clin argillacea Rasamsonia argillacea Rasamsonia Microbiol 2013; 51:719–722. 51:719–722. 2013; J Clin Microbiol J Clin : An emerging pathogen in patients with cystic fibrosis. fibrosis. cystic with patients in pathogen Anemerging : pulmonary and aortic graft infection in an immune-competent patient. patient. immune-competent inan infection graft aortic and pulmonary disease. granulomatous chronic human in mycosis invasive of cause Anemerging : 2012; 47:734–736. Geosmithia argillacea 2011; 52:e136–143. 2013; 19:670–675. 19:670–675. 2013; Med Mycol infection in a patient with gastrointestinal GvHD. complex species colonization revealed by rep-PCR in cystic fibrosis 2016;

2010; 48 Suppl 1:S1–3. 54:2804-2812. 54:2804-2812. insputum of people with cystic fibrosis. Rasamsonia

species. Med Mycol et al J Clin Microbiol J Clin et al J Clin Microbiol J Clin . Disseminated et al et . and and Taxonomy . 2016; 54:781–786. 54:781–786. 2016; Bone Marrow et al et et al J ClinMicrobiol . Geosmithia et al et et al et . Long-term Long-term . . Rasamsonia Med Mycol J Clin J Clin . 2010; 2010; . Isolation of of Isolation . Geosmithia 2013; 2013; Curr

Accepted Article 20. Abdolrasouli A, Rhodes J, Beale M, Hagen F, Rogers TR, Chowdhary A, Chowdhary TR, Rogers F, Hagen M, Beale J, Rhodes A, Abdolrasouli 20. This by is protectedarticle reserved. Allrights copyright. 19. Irinyi L, Serena C, Garcia-Hermoso D, Arabatzis M, Desnos-Ollivier M, Vu D, D, Vu M, Desnos-Ollivier M, Arabatzis D, C, Garcia-Hermoso Serena L, Irinyi 19. and significance Clinical A. Chowdhary JF, Meis SN, K, Gaur Agarwal S, Kathuria PK, Singh 18. 21. Zhang Y, Hagen F, Stielow B, Rodrigues AM, Samerpitak K, Zhou X, X, K,Zhou Samerpitak AM, B, Rodrigues Stielow F, Hagen Y, Zhang 21. 3 MeletiadisJ, PournarasS,Roilides E, Walsh TJ. Defining fractional inhibitoryconcentration 23. Antifungal Dilution Broth for Method Reference Institute. Standards Laboratory and Clinical 22. 25. Marguet C, Favennec L, Matray O, Bertout S, Giraud S, Couderc L, L, S,Couderc Giraud S, Bertout O, Matray L, C, Favennec Marguet 25. 2016;(August). 2015. report data Annual Registry Fibrosis Cystic UK Trust. Fibrosis Cystic 24. sequencing. context of azole-resistance mutations in pathogenic fungi. fungi. pathogenic animal and of human identification routine for tool standard controlled quality -The database barcoding DNA reference (ISHAM)-ITS Mycology Animal and of Human Society International 51:3331–3337. 2013; bronchopulmonarymycosis patients with special referencetobasidiomycetes. of tracts respiratory the from isolated molds of nonsporulating characterization molecular 51:22–30. reports. reports. in patterns and evolutionary simulation analysis, and analysis, simulation index cutoffs for additive interactions based on self-drug additivecombinations, Monte Carlo M38-A2. Pennsylvania;2008. Edition—Document Standard—Second Approved fungi; Filamentous of Testing Susceptibility resources. resources. Available from: https://www.cysticfibrosis.org.uk/the-work-we-do/uk-cf-registry/reporting-and- against patient. patient. microbiological efficacy ofmicafunginon Aspergillus fumigatus Med Mycol Case Rep Case Mycol Med Persoonia MBio Med Mycol 2015;6: 2015; 35:1–20. in vitro-in vivo vitro-in in

e00536. e00536. Sporothrix 2015; 53:313–337. 53:313–337. 2015; . 2012; 1:79–81. 1:79–81. 2012; Antimicrob Agents Chemother correlationantifungal for combinations drug data spanning more than 14 000 human and animalcase Aspergillus fumigatus Aspergillus Geosmithia argillacea Geosmithia

2010; 54:602–609. 54:602–609. 2010; using whole-genome in acystic infection fibrosis et al et et al . Clinical and Clinical . et al et . Phylogeography . Phylogeography et al . Genomic Genomic . J Clin Microbiol J Clin .

Accepted Article 31. Steinmann J, Dittmer S, Houbraken J, Buer J, Rath P. P. Rath J, Buer J, S, Houbraken Dittmer J, Steinmann 31. This by is protectedarticle reserved. Allrights copyright. Voriconazole- JF. Meis S, Jain PK, Singh N, Sachdeva K, Agarwal S, Kathuria A, Chowdhary 32. disseminated Fatal al. et N, Lechtzin M, White G, Hong 30. JC. H, Frisvad J, Spierenburg Houbraken 29. E,Feierl Eber A, Badura L, Masoud-Landgraf 26. 28. Steinmann J, Giraud S, Schmidt D, Sedlacek L, Hamprecht A, Houbraken J, Houbraken A, L,Hamprecht Sedlacek D, S, Schmidt Giraud J, Steinmann 28. of recovery M. First Pirš U, M,Krivec Praprotnik T, Cerar T, Matos 27. against against transplantation. post-lung Leeuwenhoek Forum Infect Dis Infect Forum resistant thermotolerant and thermophilic thermophilic and thermotolerant patients. fibrosis cystic from secretions a novel real-time PCR for detecting of review literature. and report Slovenia--case in fibrosis cystic with patient in adolescent isolated complex species 186. patients. fibrosis cystic in species fungal of diversity high a detects Rasamsonia Penicillium oxalicum: Penicillium 2012; 101:403–421. 101:403–421. 2012; . 2014;1(2):ofu029. 2014;1(2):ofu029. . Mycoses species. species. J Cystic Fibrosis J Cystic 2015; 58:506–510. 58:506–510. 2015; Antimicrob Agents Chemother Agents Antimicrob An emerging pathogen in immunocompromised hosts. hosts. immunocompromised in pathogen emerging An Talaromyces Rasamsonia argillacea Rasamsonia New MicrobesNew Infect Rasamsonia 2017; 16:e3-e7. and and method culture Modified W. Buzina E, Marth G, Geosmithia , a new genus comprising comprising genus new a , In Vitro In

Rasamsonia species complex in respiratory in respiratory complex species 2016; 60:6890–6891. Activity of isavuconazole species. 2014; 2:72–78. 2:72–78. 2014; Rasamsonia argillacea Rasamsonia Med Mycol infection in cystic fibrosis cystic in fibrosis infection Antonie van van Antonie et al et 2014; 52:179– . Validation of of Validation . Open

Accepted ArticleRas_RBH_8 Ras_RBH_7 Ras_RBH_6 Ras_RBH_5 Ras_RBH_4 Ras_RBH_3 Ras_RBH_2 Ras_RBH_1 Isolate no This by is protectedarticle reserved. Allrights copyright. Caspofungin CFG: Anidulafungin, AFG: Micafungin, MFG: Terbinafine, TRB: Fluorocytosine, 5- 5FC: Posaconazole, PSC: Voriconazole, VRC: Itraconazole, B, ITC: Amphotericin AMP: 1. Table

included in the study Results of Results 6 5 2 4 3 1 2 1 Case

R. aegroticolaR. piperinaR. R. argillacea aegroticolaR. aegroticolaR. aegroticolaR. R. argillacea aegroticolaR. in vitro Species antifungal susceptibility testing for 8 clinical isolates of of isolates 8clinical for testing susceptibility antifungal

03/02/2015 28/11/2014 20/10/2014 22/07/2014 14/03/2014 20/01/2014 29/11/2013 30/09/2013 Isolation date

0.25 4 2 2 2 2 2 1 AMB 1 >16 2 16 2 >16 2 1 ITC >16 >16 >16 >16 >16 >16 >16 4.0 VRC

1 4 2 2 2 2 4 0.5 I rMC(gm)for: (µg/ml) MEC or MIC PSC 32.0 0.25 0.06 <0.06 <0.06 <0.06 <0.06 <0.06 5FC 2 8 4 0.25 2 8 1 8 TRB Rasamsonia <0.015 <0.015 <0.015 <0.015 <0.015 <0.015 <0.015 <0.015

MFG

<0.015 <0.015 0.06 0.06 0.03 <0.015 0.06 0.06 AFG

spp. spp.

0.125 0.125 0.125 0.125 0.125 0.06 0.03 0.03 CFG Accepted Article This by is protectedarticle reserved. Allrights copyright. BMI: body mass index, FEV colonizationin8 CFpatients 2. Table Date of first isolate No of positive cultures acquisition History of azole prior use to Fungal co-isolation co-infection NTM Other bacterial co-infection aeruginosaP. FEV CFdiabetes related Pancreatic insufficiency (kg/m acquisition of time at BMI Genotype acquisition/Sex Age (yrs) at time of Characteristics Clinical outcome Co-therapy azithromycin antibiotics Co-therapy inhaled culture positive Ongoing acquisition 1 % predicted at time of 2 )

Summary of characteristic features of reported 6 cases of of cases 6 reported of features characteristic of Summary

co-infection

No sodium Colistimethate 30/09/2013 10 Itraconazole aureus us Staphylococc Intermittent Intermittent No Yes percentile) 16.1, (24th 80 Stable 2016 November growth Yes, last fumi Aspergills No ∆ 10/male 1

F508/

1

: forcedexpiratory volume one in second

g

atu

∆

s F508

maltophilia onas Stenotrophom No No Yes Yes Tobramycin 08/10/13 7 Itraconazole 22.1 38 varioti Paecilomyces ∆ ∆ 20/male 2016 September died transplant, Lung No 2 2016 February growth Yes, last

F508 F508/

i

No Yes No No No 14/03/2014 22.6 16 ∆ lysine aztreonam Tobramycin + 4 Itraconazole No 3 46/male 2015 Died January fumi Aspergillus 2014 September growth last No,

F508/?

g

atu

s

Case 07/07/2014 aureus Staphylococcus Yes Yes Yes Yes 22.5 25 G542X/Q493X sodium Colistimethate 4 Itraconazole abscessu M. 4 32/male 2014 December Died No November 2014 LastNo, growth Rasamsonia Rasamsonia

s

species chronic chronic species 31/01/2014 aureus Staphylococcus Yes No Yes Yes 19 39 sodium colistimethate Tobramycin + ∆ 5 Nil a Scedosporium 5 30/female lung functionlung Slow decline in November 2014 No, last growth No

p

F508/Q493X

ios

p ermu

m

03/02/2015 No Yes Yes Yes Yes 23.8 91 e sodium Colistimethat ∆ ∆ 2 Nil No 6 53/male Stable 2016 growth June No, last No

F508 F508/

Accepted Article shown. are 70% This by is protectedarticle reserved. Allrights copyright. rooted phylogeny, a maximum-likelihood generate to data Netherlands). Bootstrap (500 replicates) analysis was performed on ITS1 and Hospital (Nijmegen,The Netherlands) and Westerdijk Fungal Biodiversity Institute (Utrecht,The highlightedin red) and 55 isolates (including the type strains)from collectionsin Canisius-Wilhelmina Figure 1 . Phylogenetic. tree ofsequences obtainedfrom 8 R. emersonii. R. Rasamsonia

clinical isolates (UK, isolates clinical Bootstrap valuesgreater than β -tubulin sequence

Accepted Article This by is protectedarticle reserved. Allrights copyright. of isolates of pattern

( 2. Figure n

=8), compared to those from Nijmegen, The Netherlands ( Netherlands The Nijmegen, from those to compared =8), Similarity scale [%] AFLP Rasamsonia species

Strain number Species Localitly 30 40 50 60 70 80 90 100 Ras_RBH_8 R. piperina London, United Kingdom

Amplified fragment length polymorphism (AFLP) profiles of of profiles (AFLP) polymorphism length fragment Amplified 61-7613 R. piperina Slovenia CF4219 R. piperina The Netherlands Ras_RBH_7 R. piperina London, United Kingdom CF2634 R. piperina The Netherlands Ras_RBH_6 R. argillacea London, United Kingdom Ras_RBH_2 R. argillacea London, United Kingdom CF3086 R. argillacea The Netherlands Rasamsonia Ras_RBH_5 R. argillacea London, United Kingdom Ras_RBH_4 R. argillacea London, United Kingdom CF1145 R. aegroticola The Netherlands CF0090 R. aegroticola The Netherlands CF2577 R. aegroticola The Netherlands CF1810 R. aegroticola The Netherlands CF0013 R. aegroticola The Netherlands

was done by UPGMA. Scale bar indicates the percentage similarity. percentage the indicates bar Scale UPGMA. by done was CF0636 R. aegroticola The Netherlands CF1015 R. aegroticola The Netherlands CF1766 R. aegroticola The Netherlands CF2266 R. aegroticola The Netherlands CF1099 R. aegroticola The Netherlands CF0969 R. aegroticola The Netherlands CF0964 R. aegroticola The Netherlands Ras_RBH_3 R. aegroticola London, United Kingdom Ras_RBH_1 R. aegroticola London, United Kingdom

n =16). Clustering of AFLP banding banding AFLP of Clustering =16).

R .argillacea complex fromUK

Accepted Article samples. respiratory from p each for time over function Lung Right, allergic bronchopulmonaryaspergillosis; itraconazole; CFG,caspofungin; PSC, p Figure 3. Left, clinical course of six case of course Left, clinical o o s a NTM, non-tuberculose mycobacteria; VRC, voriconaz VRC, mycobacteria; non-tuberculose NTM, saconazole; TRB, terbinafine; FLC, fluconazole; ABP fluconazole; FLC, terbinafine; TRB, saconazole; of chronic chronic of tient, red arrows show the first isolation of of isolation first the show arrows red tient, R. argillacea R. complex colonization in CF. in I colonization complex Rasamso n n I o A TC, i a le. le.

, spp. spp.