Marie Desnos-Ollivier

| Corinne Maufrais

| Marc Pihet

Christine Aznar

Françoise Dromer

Epidemiological investigation for grouped cases of Trichosporon asahii using whole genome and IGS1 sequencing

Keywords: emerging yeast, epidemiology of trichosporonosis, IGS sequencing, Trichosporon asahii, whole genome sequencing DESNOS-OLLIVIER Et aL

Background:

Trichosporonosis is a rare invasive infection in humans mainly due to Trichosporon asahii, and especially recovered from patients having haematological malignancy. Since 2012, IGS1 region sequencing is used as a genotyping method to distinguish isolates, with high frequency of one haplotype worldwide and a geographic specificity for some haplotypes.

Objectives: We compared the IGS1 genotyping method and whole genome sequencing (WGS) to study the relationship between clinical isolates involved in two grouped cases in France.

Methods: IGS1 sequencing and antifungal susceptibility testing were performed for 54 clinical isolates. Clinical data for 28 isolates included in surveillance programs were analysed. Whole genome was sequenced for 32 clinical isolates and the type strain.

Results: All isolates were intrinsically resistant to flucytosine, while voriconazole had the most potent in vitro activity. The majority of the isolates was recovered from patients with haematological malignancies (42.86%), with a high proportion of children (<15 yrs-old, 32.14%) and a high mortality rate at three months (46.15%). Based on the WGS analysis, isolates exhibiting IGS1 haplotype 1, 3 and 7 belonged to different clades. Five isolates recovered during the first grouped cases had the same IGS1 haplotype and shared 99% of SNPs similarity. For the second grouped cases, four isolates had 98.7% of SNPs similarity while the isolate recovered 4 years earlier was totally unlinked.

Conclusions:

We confirmed the usefulness of IGS1 sequencing for grouped cases infection of T. asahii. We underlined its limitation for the study of population structure and the utility of WGS analysis for the study of epidemiologically unrelated isolates.

| INTRODUCTION

Trichosporon asahii is a basidiomycetous yeast-like species producing arthroconidia. It has a worldwide distribution in the environment, especially in the soil, pigeon droppings and houses (http://www. mycob ank.org). [START_REF] Sugita | Isolation of Trichosporon asahii from environmental materials[END_REF][START_REF] Sugita | The Yeasts, a Taxonomic Study[END_REF][START_REF] Medina | Pigeons and their droppings as reservoirs of Candida and other zoonotic yeasts[END_REF] This species is able to grow up to 40°C and to produce biofilms providing potential virulence factors for humans. [START_REF] Bonaventura | Biofilm formation by the emerging fungal pathogen Trichosporon asahii: development, architecture, and antifungal resistance[END_REF] Previous classification placed the species in the clade Ovoides Trichosporon asahii is considered to be part of the normal biota of the human skin [START_REF] Zhang | The opportunistic yeast pathogen Trichosporon asahii colonizes the skin of healthy individuals: analysis of 380 healthy individuals by age and gender using a nested polymerase chain reaction assay[END_REF] and gastrointestinal tract. [START_REF] Cho | Molecular evidence that the opportunistic fungal pathogen Trichosporon asahii is part of the normal fungal microbiota of the human gut based on rRNA genotyping[END_REF] This species is rarely involved in white piedra but is frequently recovered from patients with summer-type hypersensitivity pneumonitis (SHP), an allergy developed after repeated inhalation of arthroconidia. [START_REF] Sugita | Analysis of Trichosporon isolates obtained from the houses of patients with summer-type hypersensitivity pneumonitis[END_REF] It is also isolated from immunocompromised patients and is the major Trichosporon species responsible for life-threatening deep-seated infection called trichosporonosis, [START_REF] Garza | Disseminated Trichosporon asahii infection in a patient with acute myeloid leukemia[END_REF][START_REF] Galligan | Disseminated trichosporonosis with atypical histologic findings in a patient with acute lymphocytic leukemia[END_REF][START_REF] Gueho | Dupont B Trichosporon on humans: a practical account[END_REF][START_REF] Guo | Invasive Infections Due to Trichosporon: species distribution, genotyping, and antifungal susceptibilities from a multicenter study in China[END_REF][START_REF] De Almeida Junior | Invasive Trichosporon Infection: a systematic review on a re-emerging fungal pathogen[END_REF] mainly in patients with haematological malignancies. [START_REF] Bretagne | Predisposing factors and outcome of uncommon yeast species-related fungaemia based on an exhaustive surveillance programme (2002-14)[END_REF][START_REF] Colombo | Current knowledge of Trichosporon spp. and Trichosporonosis[END_REF][START_REF] Liao | Epidemiology and outcome of Trichosporon fungemia: a review of 185 reported cases from 1975 to 2014[END_REF] Fungaemia due to Trichosporon sp. are frequently associated with pre-exposure to caspofungin. [START_REF] Bretagne | Predisposing factors and outcome of uncommon yeast species-related fungaemia based on an exhaustive surveillance programme (2002-14)[END_REF][START_REF] Bayramoglu | Breakthrough Trichosporon asahii fungemia in neutropenic patient with acute leukemia while receiving caspofungin[END_REF] Trichosporon asahii is described in sporadic cases and also during local outbreaks of invasive infection. [START_REF] Bretagne | Predisposing factors and outcome of uncommon yeast species-related fungaemia based on an exhaustive surveillance programme (2002-14)[END_REF][START_REF] Vashishtha | A fatal outbreak of Trichosporon asahii sepsis in a neonatal intensive care Unit[END_REF] Infections can have exogenous (skin inoculation, inhalation) or endogenous (gut translocation) origins.

Since 2002, sequencing of the IGS1 region (intergenic spacer localised in the rDNA gene) is used as a genotyping method to differentiate T. asahii isolates. [START_REF] Rodriguez-Tudela | Genotype distribution of clinical isolates of Trichosporon asahii based on sequencing of intergenic spacer 1[END_REF][START_REF] Sugita | Sequence analysis of the ribosomal DNA intergenic spacer 1 regions of Trichosporon species[END_REF] Up to now, 12 IGS1 haplotypes have been identified and a correlation between haplotype and geographical origin of the isolates has been described. Indeed, haplotype 1 is ubiquitous but predominant in Japan, China, South America, Turkey and Thailand, while haplotype 3 is more frequent in USA, Thailand and also in houses of SHP patients in Japan. [START_REF] Guo | Invasive Infections Due to Trichosporon: species distribution, genotyping, and antifungal susceptibilities from a multicenter study in China[END_REF][START_REF] Sugita | Sequence analysis of the ribosomal DNA intergenic spacer 1 regions of Trichosporon species[END_REF] Haplotype 5 is also frequent in USA and haplotype 7 in Thailand. [START_REF] Sugita | Sequence analysis of the ribosomal DNA intergenic spacer 1 regions of Trichosporon species[END_REF][START_REF] Mekha | Genotyping and antifungal drug susceptibility of the pathogenic yeast Trichosporon asahii isolated from Thai patients[END_REF] Other haplotypes are described in Spain, Japan and Brazil. [START_REF] Sugita | Sequence analysis of the ribosomal DNA intergenic spacer 1 regions of Trichosporon species[END_REF][START_REF] Mekha | Genotyping and antifungal drug susceptibility of the pathogenic yeast Trichosporon asahii isolated from Thai patients[END_REF] In 2012, Yang et al sequenced the whole genome of two strains of T. asahii (an environmental isolate, CBS8904, recovered from maize cobs, in 1998, Illinois, USA [START_REF] Yang | Genome sequence of the Trichosporon asahii environmental strain CBS 8904[END_REF] and a clinical isolate, CBS2479, the type strain, recovered in Japan in 1929 from a psoriasis lesion). [START_REF] Yang | Draft genome sequence of CBS 2479, the standard type strain of Trichosporon asahii[END_REF] In 2014, two French hospitals notified to the NRCMA, grouped cases of colonisation and/or invasive infections due to T. asahii over a short period of time. At that time, the clinicians wondered if a unique strain was involved in each of these grouped cases. Given that the majority of the isolates recovered worldwide is of IGS1-haplotype 1, we considered that IGS1 sequencing was not sufficient to confirm link between isolates. We decided to sequence the whole genome of these isolates and others from our collection to study their genetic diversity and the relationship between isolates involved in these grouped cases.

| MATERIAL AND METHODS

| Isolates

| Species identification

Purity was first checked using chromogenic medium (BBL™ CHROMagar™ Candida; BD Company, USA). After DNA extraction using NucleoMag plant kit extraction (Macherey-Nagel, Germany) in a KingFisher TM Flex system (Thermo Fisher Scientific), D1D2, ITS1/ ITS2 and IGS1 regions were amplified and sequenced using panfungal primers (NL1/NL4, 24 V9D/LS266, [START_REF] De Hoog | Molecular diagnostics of clinical strains of filamentous Basidiomycetes[END_REF][START_REF] Masclaux | Phylogenetic relationships of human-pathogenic Cladosporium ( Xylohypha) species inferred from partial LS rRNA sequences[END_REF] 26SF/5SR, 20 respectively). Sequences were compared with sequences of type strains of Trichosporon species to confirm species identification. Haplotype of IGS1 was determined after comparison with the 12 haplotypes of T. asahii available in the literature. [START_REF] Rodriguez-Tudela | Genotype distribution of clinical isolates of Trichosporon asahii based on sequencing of intergenic spacer 1[END_REF] Sequences of IGS1 region were aligned with MUSCLE using Mega v. 6, [START_REF] Tamura | MEGA6: molecular evolutionary genetics analysis version 6.0[END_REF] and maximum-likelihood tree was constructed with matrix alignment.

| Antifungal susceptibility testing

Minimal inhibitory concentrations (MICs) were determined for 5 antifungal agents (flucytosine, fluconazole, posaconazole, voriconazole and amphotericin B; all purchased from Alsachim, a Shimadzu Group Company) according to the EUCAST standardised microdilution broth method [START_REF] Arendrup | EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST)[END_REF] except for amphotericin B testing since it was diluted in AM3 medium. ATCC22019 and ATCC6258 reference strains were used as quality control strains. MIC50 and MIC90, corresponding to the MIC value inhibiting at least 50% and 90% of the isolates, were calculated for each antifungal agent.

| Whole Genome Sequencing (WGS)

Epidemiologically related (n = 11 involved in the two grouped cases) or unrelated (n = 20) clinical isolates were selected in the NRCMA collection for WGS based on anatomical, geographical and temporal origins as well as IGS1 haplotype. The selected isolates were recovered from 29 patients diagnosed in 18 different hospitals between 2003 and 2016 and

Strain

Site of isolation

Date of isolation

Haplotype IGS1

Patient

H#

Continent of diagnosis NGS

ETHICS STATEMENT

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as the research in this article related to micro-organisms. MIC50 and MIC90 were high for flucytosine (32 mg/L and ≥64 mg/L, respectively), fluconazole (4 mg/L and 16 mg/L) and amphotericin B (2 mg/L and ≥8 mg/L) and lower for posaconazole (0.25 mg/L and 0.5 mg/L) and voriconazole (0.06 mg/L and 0.25 mg/L) (Table 1).

| RESULTS

| Characteristics of the isolates

| IGS1 sequencing

Six different IGS1 haplotypes were identified among the 54 isolates studied (Figure 1

| Whole genome sequencing data

To better assess genetic relatedness, WGS was performed on selected isolates including all the grouped cases isolates. The percentage of mapping for isolates with reference genome of CBS 2479 varied between 93% and 99%, confirming the good species identification for all (Table S2). The percentage of GC varied between 55% and 59% for the whole genome. The number of indels was 1,156 for the genome of CBS 2479 re-sequenced in our laboratory and varied between 57,451 and 67,919 for the 32 clinical isolates. The number of SNPs positions was 2,039 in the 're-sequenced' genome of CBS 2479 and varied between 148,294 and 175,236 for the clinical isolates. The Ts/Tv ratio was approximately equal to 2.

A total of 154,051 SNP positions were concatenated, and a multiple alignment was constructed for the 32 clinical isolates using the genome of CBS 2479 as reference. Then, a maximum-likelihood tree with 1,000 bootstraps was constructed using this SNP alignment (Figure 2). The tree included three major clades We then analysed the haplotype 1 isolates involved in the grouped cases. For hospital #1, the five isolates recovered from two patients were grouped in a subclade with more than 99% of SNPs similarity. For hospital#12, four isolates were grouped in another subclade with 98.7% of SNPs similarity, while the two other isolates were unlinked with less than 37% of SNPs similarity. The two subclades shared 60% of SNPs similarity.

A maximum-likelihood tree was constructed by using indels information, and similar topology was obtained compared to the maximum-likelihood tree constructed based on SNPs matrix alignment (data not shown).

| DISCUSSION

We took advantage of our assignments as National Reference Center for Invasive Mycoses & Antifungals to assess the usefulness of IGS1 genotyping for the study of the genetic relatedness between isolates of T. asahii recovered during two grouped cases, using our collection of epidemiologically unrelated isolates.

Among the 54 clinical isolates of T. asahii analysed here, 28 were received in the context of active epidemiological surveillance programs (YEASTS and RESSIF programs). In a previous study, [START_REF] Bretagne | Predisposing factors and outcome of uncommon yeast species-related fungaemia based on an exhaustive surveillance programme (2002-14)[END_REF] 19 isolates of Trichosporon species (T. asahii, T. inkin, T. dermatis, T. japonicum, T. loubieri, T. mucoides), grouped in the 'Trichosporon sp.' group, represented 5.9% of the bloodstream infections between October 2002 and December 2014 in the Paris area. These rare species were significantly associated with haematological malignancies and pre-exposure to caspofungin, and the mortality rate was similar to that of patients infected with C albicans. In the present study concerning only T. asahii, similar results were obtained with a high percentage (42.86%, 12/28) of patients with haematological malignancies and especially with acute leukaemia (50%, 6/12), followed by patients with recent surgery (21.43%, 6/28). We also found a high proportion of patient under 15 years (32.14%, 9/28) and a high mortality rate at three months (46.15%, 12/26). As reported in the literature, [START_REF] Guo | Invasive Infections Due to Trichosporon: species distribution, genotyping, and antifungal susceptibilities from a multicenter study in China[END_REF][START_REF] Bretagne | Predisposing factors and outcome of uncommon yeast species-related fungaemia based on an exhaustive surveillance programme (2002-14)[END_REF][START_REF] Pfaller | Wild-type MIC distribution and epidemiological cutoff values for Aspergillus fumigatus and three triazoles as determined by the Clinical and Laboratory Standards Institute broth microdilution methods[END_REF][START_REF] Rodriguez-Tudela | Susceptibility patterns and molecular identification of Trichosporon species[END_REF][START_REF] Arendrup | ESCMID and ECMM joint clinical guidelines for the diagnosis and management of rare invasive yeast infections[END_REF] FIGURE 2 Maximum-likelihood tree constructed using matrix alignment, with IQTree, for the whole genome sequences of 32 clinical isolates and the type strain CBS2479 of Trichosporon asahii. The isolates previously identified as belonging to a given IGS1 haplotype are shown in red (haplotype 1), blue (haplotype 3), green (haplotype 4), orange (haplotype 7), pink (haplotype 5) and black (new haplotype) H1 H12 H12 H12 all T. asahii isolates were intrinsically resistant to flucytosine and less susceptible to fluconazole and amphotericin B while voriconazole had the most potent in vitro activity with MIC50 and MIC90 of 0.06 and 0.25 mg/L, respectively. Voriconazole is the treatment usually recommended for deep-seated trichosporonosis. [START_REF] Arendrup | ESCMID and ECMM joint clinical guidelines for the diagnosis and management of rare invasive yeast infections[END_REF] All isolates were genotyped using IGS1 sequencing. The majority (51.9%, 28/54) belonged to IGS1 haplotype 1, which is also the most frequent haplotype found worldwide in clinical isolates. Five other haplotypes were found (haplotypes 3, 4, 5, 7 and one unknown).

Interestingly, the 3 isolates recovered in 2 hospitals localised in the Caribbean islands were haplotype 3 which is also the most frequent haplotype in the USA. There was no other evidence of an association between geographic origin and haplotype among the isolates studied here.

The IGS1 sequencing was initially used to investigate grouped cases. In hospital#1, the multiple isolates recovered from two patients belonged to haplotype 1 suggesting a potential common source of infection that was not investigated. In hospital#12, a total of 8 patients were infected with T. asahii over 5 years. Two patients (#13 and 38) sampled the same day were infected by isolates exhibiting two different haplotypes (haplotypes 4 and 7), as did the last patient diagnosed 4 years later with haplotype 3 infection. This suggested different sources of infection that could include endogenous ones. The four remaining patients diagnosed over 4 months were infected with haplotype 1 with the hypothesis that a common source of infection could be responsible for these grouped cases. However, given that the majority of the isolates shared haplotype 1 and that it is also the most frequent haplotype worldwide, we were not confident to assert a potential link between isolates. We thus decided to sequence whole genome and selected 32 isolates representative of our collection in terms of the diversity of anatomical, temporal and geographic origins, as well as IGS1 diversity. The genome of the type strain CBS2479 was used as reference and was also re-sequenced.

The GC content can be of interest for the study of the orphan proteins, [START_REF] Basile | High GC content causes orphan proteins to be intrinsically disordered[END_REF] mutation rate, spectrum of mutations. [START_REF] Long | Similar mutation rates but highly diverse mutation spectra in Ascomycete and Basidiomycete yeasts[END_REF] The GC content for the reference strain CBS 2479 was 59%, as previously reported and varied between 55% and 59% for the clinical isolates. These values confirm data of the literature showing that Basidiomycetous yeast have usually a GC content higher than Ascomycetous yeast: 48.2% for Cryptococcus neoformans var. grubii, 37 62% for Rhodotorula toruloides [START_REF] Long | Similar mutation rates but highly diverse mutation spectra in Ascomycete and Basidiomycete yeasts[END_REF] while Candida glabrata and Saccharomyces cerevisiae are AT-rich with GC content of 38.8% and 38.3%, respectively. [START_REF] Dujon | Genome evolution in yeasts[END_REF] The number of SNPs positions for the clinical isolates varied between 148,294 and 174,873. Given the 24 MB size of the genome, the SNPs positions corresponded to 0.62% of the genome. The number of transitions and transversions was calculated with a Ts/Tv ratio of ~2 confirming data obtained by Long et al demonstrating a predominance of transitions relative to transversions for Basidiomycetes. [START_REF] Long | Similar mutation rates but highly diverse mutation spectra in Ascomycete and Basidiomycete yeasts[END_REF] The concatenation of the 154,050 SNP positions was used for construction of matrix alignment and of a maximum-likelihood tree. This tree confirmed the results obtained with IGS1 sequencing for the isolates involved in the two grouped cases. Indeed, the five isolates recovered in hospital#1 shared 99% of SNPs similarity, suggesting a common source of infection. No specific investigation was carried out, but the hypothesis of a transmission through a healthcare worker seemed more likely than inter-patient transmission (since the patients did no share time in the same ward). For the second grouped cases, the four isolates recovered over 4 months had 98.7% of SNPs similarity suggesting also a common source of infection while the isolate recovered 4 years earlier was totally unlinked. In addition, nine isolates were grouped by 2 or 3, with more than 97% of SNPs identical. In one instance, it concerned two haplotype 1 isolates recovered 16 months apart from the same patient (Pt#21) grouped with an isolate recovered from a patient hospitalised 13 months earlier 700 kms away. For the other six isolates, the two pairs of IGS1 haplotype 4 isolates and one pair of haplotype 1 isolates were also uncovered with no temporal or spatial link. Environmental investigations were missing here to identify exogenous source of infection during the grouped cases. In conclusion, our study suggests that IGS1 sequencing of T. asahii is a good tool to investigate grouped cases but it is not sufficient to study the genetic diversity of the species and potential link between isolates. Although the presence of a rare species in several patients in the same hospital over a short period of time suggests a common source of infection, our results show that this is not always the case especially for a species that is part of the normal biota of the skin and/or gastrointestinal tract.

CONFLICT OF INTEREST STATEMENT

Authors have no conflict of interest.

ACKNOWLEDGMENTS

The following investigators are members of the French Mycoses Study Group: N. Brieu (Aix-en-Provence), T. Chouaki (Amiens), M.

2

 2 with other pathogenic species such as Trichosporon inkin and Trichosporon ovoides. In 2015, a new classification of the Tremellomycetes class divided Trichosporon species into five genera in the new family Trichosporonaceae: Trichosporon, Apiotrichum, Cutaneotrichosporon, Effuseotrichosporon, Haglerozyma. Trichosporon asahii and other species affiliated to the clade Ovoides were maintained in the Trichosporon genus. 5

  Since 2003, 54 clinical isolates were sent to the National Reference Center for Invasive Mycoses & Antifungals (NRCMA, Institut Pasteur, Paris, France) in the context of the active surveillance of yeast fungaemia in the Paris area (YEASTS program, n = 17) or that of the nationwide Surveillance Network of Invasive Fungal Infections (RESSIF, n = 11). Other isolates (n = 26) were sent only for expertise (confirmation of species identification and/or determination of antifungal susceptibility). Clinical data (age, gender, underlying disease, prior exposure to antifungal agents, antifungal treatment and outcome at 90 days) when available were collected. The type strain of T. asahii CBS2479 was included in the analysis.

  used to indicate isolates from a same hospital. Abbreviations: H, hospital; Fluco, fluconazole; Vori, voriconazole; Posa, posaconazole; AmB, amphotericinB; Flucy, Flucytosin; CSF, cerebrospinal fluid.

  morphism (SNPs) and insertion/deletion (indels) positions were called using Genome Analysis Toolkit version3.1-1 30 according to the GATK Best Practices. VariantFiltration, QD <2.0, LowQD, ReadPosRankSum<-8.0, LowRankSum, FS >60.0, HightFS, MQRankSum<-12.5, MQRankSum, MQ <40.0, LowMQ, HaplotypeScore >13.0, HaploScore parameters were used to filter SNPs and indels. SNPs that varied between isolates were combined into single multiple alignments. Phylogenetic analyses were performed with IQTree version 1.6.12 31 with the TVM+F+R2 substitution model, the perturbation strength 0.5 and 1000 bootstraps replicates. Number of transitions [Ts, interchanges of purines (A ↔ G) or of pyrimidines (C ↔ T)] and transversions (Tv, interchanges of purine for pyrimidine bases) were determined, and ratio of Ts to Tv was calculated for SNPs positions, using vcftools v. 0.1.13.

  . The isolates belonging to IGS1 haplotype 4 (n = 6) were found grouped in one clade, while the majority (15/18) of isolates belonging to haplotype 1 composed a second clade. A third clade contained isolates belonging to haplotype 3 (n = 5), haplotype 1 (n = 2), haplotype 7 (n = 2)and the new haplotype. When looking at haplotype 1 isolates, three (CBS2479, CNRMA10.64 and CNRMA12.448) were unlinked while inside the clade containing the majority of them, subclades were observed with isolates sharing more than 98% of SNPs similarity.

FIGURE

  FIGURE 1 Maximum-likelihood tree constructed using matrix alignment (MUSCLE) with MEGA v. 6 for the IGS1 sequence of the 54 clinical isolates, and the 12 haplotypes of IGS1 deposited in public database. Bootstrap values are indicated. Black dots correspond to isolates recovered in South America, Black squares to Caribbean Islands and black triangles to Africa

  IGS1 sequencing was thus helpful to rule out a common source of infection in several cases like in hospital #8 with 3 different haplotypes for 3 different patients including two (pt#14 and 15) diagnosed a few months apart. Analysis of the SNPs showed that sharing the same IGS1 haplotype does not always mean genetic relatedness (hospital #12) and that isolates can be genetically linked without obvious spatial or temporal relatedness complicating the investigation of grouped cases based only on IGS1 sequencing.

  Pihet (Angers), S. Bland (Annecy), V. Blanc (Antibes), A. P. Bellanger, F. Grenouillet, L. Millon (Besançon), S. Brun (Bobigny), I. Poilane (Bondy), F. Gabriel (Bordeaux), A. L. Roux (Boulogne-Billancourt), D. Quinio, E. Moalic (Brest), J. Bonhomme (Caen), P. Poirier, C. Nourrisson (Clermont-Ferrand), F. Botterel (hôpital Henri Mondor, Créteil), N. Fauchet (Centre Intercommunal, Créteil), E. Forget (Clichy), F. Dalle (Dijon), P. Cahen (Foch), C. Lawrence (Garches), O. Faure, D. Maubon, M. Cornet (Grenoble), M. Nicolas (Guadeloupe), M. Demar, C. Nabet (Guyane), A. Angoulvant (Kremlin-Bicêtre), S. Picot, N. Traversier (La Réunion), O. Eloy (Le Chesnay), B. Sendid (Lille), B. Bouteille (Limoges), F. Persat, M. Wallon (Lyon), S. Ranque, H. Piarroux (Marseille), N. Desbois (Fort de France, Martinique), L. Collet (Mayotte), N. Bourgeois, L. Lachaux (Montpellier), F. Morio

TABLE 1 Characteristics of the 54 clinical isolates and the type strain of Trichosporon asahii Strain Site of isolation Date of isolation Haplotype IGS1 Patient H# Continent of diagnosis NGS MIC (mg/L) Fluco Vori Posa AmB Flucy

 1

	8	16	≥64	16	≥64	32	8	≥64	16	≥64	32	≥64	≥64
	2	≥8	1	1	2	≥8	0.5	0.5	1	2	1	2	≥8
	0.06	0.25	0.125	0.06	0.03	0.03	0.06	0.5	0.5	0.125	0.25	0.5	0.125
	0.125	0.125	≤0.015	0.03	0.03	≤0.015	0.06	0.25	0.06	0.25	0.06	0.5	0.06
	2	8	2	2	2	0.25	0.5	8	1	32	2	16	4
	Yes	Yes	Yes	Yes	Yes		Yes	Yes	Yes	Yes		Yes
	Europe	Europe	Europe	Europe	Europe	Europe	Europe	Europe	Europe	Europe	Europe	Europe	Europe
	1	1	1	1	1	1	2	3	4	5	6	7	8
	10	10	7	7	7	28	45	11	6	9	24	16	31
	1	1	1	1	1	1	3	1	4	1	1	7	1
	05/07/2013	26/06/2013	23/12/2013	20/01/2014	27/01/2014	11/01/2016	16/12/2012	01/05/2014	14/04/2015	29/11/20 09	27/0 4/2018	18/09/20 08	20/07/2008
	Lung	Blood	Urinary tract	Digestive tract	Blood	Blood	Blood	Skin	Blood	Blood	Blood	Blood	Blood
	CNRMA14.90	CNRMA14.89	CNRMA14.717	CNRMA14.718	CNRMA14.716	CNRMA17.64	CNRMA12.1406	CNRMA14.394	CNRMA15.287	CNRMA9.1206	CNRMA18.365	CNRMA8.1157	CNRMA8.907	CNRMA8.972

TABLE 1 (

 1 Continued)involved in deep and superficial infections (53% blood culture, 16% urine, 9% lung, 6% skin). They belong to the 5/12 known IGS1 haplotypes found in the NRCMA collection, and one was of a yet undescribed haplotype. Thus, the whole genome of 32 strains including CBS 2479, the type strain of T. asahii, was sequenced at the Institut Pasteur P2M platform, using an Illumina NextSeq 500 sequencer. Libraries were Nextera® DNA Library

	Preparation Kit and sequencing using a 2 × 150 nucleotide paired-end
	strategy. Library adaptators of paired-ends read were trimmed with
	Cutadapt version 1.18 (https://doi.org/10.14806 /ej.17.1.200) and then
	mapped to the 78 scaffolds of the reference genome of CBS2479 23
	(GenBank accession number ALBS00000000) using the Burrows-
	Wheeler Alignment tool, BWA version 0.7.13 29 with the BWA-mem algo-
	rithm. SAMtools version 1.9 and Picard tools version 1.94 (http://broad
	insti tute.github.io/picard) were then used to filter, convert SAM files
	and assign read groups and mark duplicate reads. Single-nucleotide poly-

TABLE 2 Clinical

 2 characteristics of the 28 patients identified in the YEASTS and RESSIF surveys between 2003 and 2018

	Characteristic of the patients	No./N a	%
	Male gender		64.29
	Mean age in years ± SD	35 ± 26.47
	Age
	<15 years	9	32.14
	[15-44 years]	7	25
	[45-64 years]	9	32.14
	[65-79 years]	3	10.71
	>80 years	0	0
	Underlying disease
	Haematological malignancies	12	42.86
	Lymphoma	4/12	33.33
	Acute leukaemia	6/12	50
	Others	2/12	16.67
	Solid tumours	1	3.57
	Other disease	7	25
	Surgery within 30 days	6	21.43
	Solid organ transplantation	1	3.57
	Pre-exposure to antifungal	11	39.29
	drugs
	Pre-exposure to echinocandins	7	25
	Initial treatment (first 48 h)
	None	2	7.14
	Voriconazole alone	14	50
	Caspofungin alone	4	14.29
	Lip-AmB alone	4	14.29
	Combination	1	3.57
	Lip-AmB + Caspo
	Other azoles	3	10.71
	Death rate at 3 months	12/26	46.15
	Abbreviation: Lip-AmB, liposomal Amphotericin B.

a N = 28 except when value is indicated. haplotype 1. Of note, an isolate recovered more than a year earlier (pt#32) was also haplotype 1, whereas another isolate responsible for fungaemia (pt#1) 4 years later was haplotype 3.

  1 Maximum-likelihood tree constructed using matrix alignment (MUSCLE) with MEGA v. 6 for the IGS1 sequence of the 54 clinical isolates, and the 12 haplotypes of IGS1 deposited in public database. Bootstrap values are indicated. Black dots correspond to isolates recovered in South America, Black squares to Caribbean Islands and black triangles to Africa

		CNRMA14.826
		IGS1 haplotype 10
		CNRMA10.64
		CNRMA4.1415
		CNRMA15.424
		CNRMA11.1137
		CNRMA11.755
		CNRMA6.842
		CNRMA15.564
		CNRMA17.259
		CNRMA9.1206
		CNRMA14.401
		CNRMA14.393
		CNRMA14.391
		CNRMA14.165
		CNRMA14.164
		CNRMA12.448
		CNRMA8.907
		CNRMA8.972
		CNRMA14.394
		CNRMA17.531
		CNRMA17.64
		CNRMA16.701
	70	CNRMA14.718
		CNRMA14.717
		CNRMA14.716
		CNRMA14.90
		CNRMA14.89
		CNRMA18.365
		IGS1 haplotype 1
		CNRMA16.422
		IGS1 haplotype 2
			IGS1 haplotype 12
		CNRMA14.672
				CNRMA7.368
				CNRMA14.72
				CNRMA12.1055
				CNRMA13.273
				CNRMA8.358
				99	CNRMA14.483
				CNRMA12.1406
				CNRMA18.15
				CNRMA17.857
				CNRMA16.827
				CNRMA16.785
				IGS1 haplotype 3
				CNRMA14.166
		77	66	CNRMA8.1157
				IGS1 haplotype 7
				IGS1 haplotype 6
				91	IGS1 haplotype 5
				CNRMA3.276
				IGS1 haplotype 4
				93	CNRMA18.102
				CNRMA17.767
				CNRMA17.910
				CNRMA15.287
				72	CNRMA14.841
				CNRMA14.167
				CNRMA14.475
				CNRMA8.212
				CNRMA14.203
	IGS1 haplotype 9
				IGS1 haplotype 8
	0.005

DATA AVAIL ABILIT Y STATEMENT

The sequence of isolate CNRMA14.672 with an unknown IGS1 haplotype is deposited to GenBank (Accession no. MN936175).