# Multicenter Outbreak of Infections by Saprochaete clavata, an Unrecognized Opportunistic Fungal Pathogen

Sophie Vaux, Alexis Criscuolo, Marie Desnos-Ollivier, Laure Diancourt, Chloé Tarnaud, Matthias Vandenbogaert, Sylvain Brisse, Bruno Coignard, Françoise Dromer

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Sophie Vaux, Alexis Criscuolo, Marie Desnos-Ollivier, Laure Diancourt, Chloé Tarnaud, et al. Mul- ticenter Outbreak of Infections by Saprochaete clavata, an Unrecognized Opportunistic Fungal Pathogen. mBio, 2014, 5 (6), pp.e02309-14. 10.1128/mBio.02309-14. hal-01484251

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# Multicenter Outbreak of Infections by *Saprochaete clavata*, an Unrecognized Opportunistic Fungal Pathogen

Sophie Vaux,ª Alexis Criscuolo,ᵇ Marie Desnos-Ollivier,ᶜ,>d Laure Diancourt,ᵇ Chloé Tarnaud,ª Matthias Vandenbogaert,ᵇ Sylvain Brisse,ᵇ,⁰,f Bruno Coignard,ª Françoise Dromer,ᶜ,>d The Geotrichum Investigation Group

Institut de veille sanitaire, département des Maladies Infectieuses, Saint-Maurice, Franceª; Institut Pasteur, Genotyping of Pathogens and Public Health, Paris, Franceᵇ; Institut Pasteur, Molecular Mycology Unit, Centre National de Référence Mycoses Invasives et Antifongiques, Paris, Franceᶜ; CNRS URA3012, Paris, Franceᵈ; Institut Pasteur, Microbial Evolutionary Genomics, Paris, Franceᵉ; CNRS UMR 3525, Paris, Francef

S.V., A.C., and M.D.-O. are co-first authors.

S.B., B.C., and F.D. are co-senior authors.

**ABSTRACT** Rapidly fatal cases of invasive fungal infections due to a fungus later identified as *Saprochaete clavata* were reported in France in May 2012. The objectives of this study were to determine the clonal relatedness of the isolates and to investigate possible sources of contamination. A nationwide alert was launched to collect cases. Molecular identification methods, whole-genome sequencing (WGS), and clone-specific genotyping were used to analyze recent and historical isolates, and a case-case study was performed. Isolates from thirty cases (26 fungemias, 22 associated deaths at day 30) were collected between September 2011 and October 2012. Eighteen cases occurred within 8 weeks (outbreak) in 10 health care facilities, suggesting a common source of contamination, with potential secondary cases. Phylogenetic analysis identified one clade (clade A), which accounted for 16/18 outbreak cases. Results of microbiological investigations of environmental, drug, or food sources were negative. Analysis of exposures pointed to a medical device used for storage and infusion of blood products, but no fungal contamination was detected in the unused devices. Molecular identification of isolates from previous studies demonstrated that *S. clavata* can be found in dairy products and has already been involved in monocentric outbreaks in hematology wards. The possibility that *S. clavata* may transmit through contaminated medical devices or can be associated with dairy products as seen in previous European outbreaks is highly relevant for the management of future outbreaks due to this newly recognized pathogen. This report also underlines further the potential of WGS for investigation of outbreaks due to uncommon fungal pathogens.

**IMPORTANCE** Several cases of rapidly fatal infections due to the fungus *Saprochaete clavata* were reported in France within a short period of time in three health care facilities, suggesting a common source of contamination. A nationwide alert collected 30 cases over 1 year, including an outbreak of 18 cases over 8 weeks. Whole-genome sequencing (WGS) was used to analyze recent and historical isolates and to design a clade-specific genotyping method that uncovered a clone associated with the outbreak, thus allowing a case-case study to analyze the risk factors associated with infection by the clone. The possibility that *S. clavata* may transmit through contaminated medical devices or can be associated with dairy products as seen in previous European outbreaks is highly relevant for the management of future outbreaks due to this newly recognized pathogen.

**Received** 13 November 2014 **Accepted** 14 November 2014 **Published** 16 December 2014

**Citation** Vaux S, Criscuolo A, Desnos-Ollivier M, Diancourt L, Tarnaud C, Vandenbogaert M, Brisse S, Coignard B, Dromer F, The Geotrichum Investigation Group. 2014. Multicenter outbreak of infections by *Saprochaete clavata*, an unrecognized opportunistic fungal pathogen. mBio 5(6):e02309-14. doi:10.1128/mBio.02309-14.

**Editor** Joseph Heitman, Duke University

**Copyright** © 2014 Vaux et al. This is an open-access article distributed under the terms of the [Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license](http://creativecommons.org/licenses/by-nc-nd/3.0/), which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Address correspondence to Françoise Dromer, dromer@pasteur.fr.

This article is a direct contribution from a Fellow of the American Academy of Microbiology.

Invasive fungal infections are well described in immunocompromised patients and represent a still-underestimated public health burden (1, 2). They can also occur in the setting of natural disasters (3) or as sporadic cases or epidemic health care-associated infections, possibly traced to a common source (4). Until now, detection of fungal outbreaks has been hampered by the diversity of fungal species involved, the small number of genomes annotated, and the lack of reliable typing tools for every pathogenic species. These reasons often preclude detection of a clonal dissemination and its source, at least for rare species. Genomic comparisons using whole-genome sequencing (WGS)

have been successfully used to investigate outbreaks of bacterial pathogens (5) but have so far rarely been applied to fungal outbreaks (6, 7). As noted by Litvintseva and colleagues (8), who investigated by WGS the recent outbreak of fungal meningitis due to *Exserohilum rostratum*, rapid laboratory methods to establish the relatedness of different strains may be the critical factor in determining whether a cluster of rare fungal infections represents a true outbreak or just an unusual coincidence. We here report a case where WGS was also a powerful tool for the investigation of a multicenter outbreak of infections due to a rare fungal pathogen, *Saprochaeta clavata*.
<figure><img src="image_4.png" /><figcaption>FIG 1 Cases of *Saprochaeta clavata* infections, France, September 2011 to October 2012. (A) Epidemiological curve of *S. clavata* infections in France. Centers are designated by numbers (see Table 1 for details), with boxes corresponding to the date of the first positive culture for cases infected by clade A, clade B, and other lineages. Index cases are outlined with thick black lines. (B) Administrative districts where the cases of *S. clavata* infections were diagnosed.</figcaption></figure>

*Saprochaeta clavata*, an ascomycetous fungus, formerly called *Geotrichum clavatum*, is phylogenetically and phenotypically closely related to *Magnusiomyces capitatus*, formerly called *Geotrichum capitatum*. Both species are often misidentified (9). Both are intrinsically resistant to echinocandins, which are recommended in hematology wards in cases of *Candida* fungemia or febrile neutropenia of unknown origin (10). Sporadic cases and outbreaks of fungemia due to *M. capitatus* have been reported, mostly in Europe, in patients with acute hematological malignancies, and these outbreaks have sometimes been connected to contamination of dairy products (11–13) by the use of nonstringent typing methods (14). In contrast, *S. clavata* has rarely been reported to be responsible for infection (15).

## RESULTS

**Initial investigation.** Of the 39 cases collected between September 2011 and October 2012, 30 were due to *S. clavata* and 9 to *M. capitatus*. A peak of 18 cases due to *S. clavata* was observed over 2 months in 2012 (week 15 [W15] to W22; Fig. 1A) in 10 health care facilities (HCF) located in 10 different regions (Fig. 1B).

Half of the 30 cases were male patients, with a median age of 63 years (Table 1). The majority (70%) of the patients were hospitalized for acute myeloid leukemia. *Saprochaete clavata* was recovered from blood (26/30, 87%) and bronchoalveolar fluid or tracheal aspirates (12/30, 40%). Overall, 18/30 (60%) patients had multiple body sites infected. Diarrhea before or associated with fungemia was reported for 16/26 (61.5%) cases. Stool cultures yielding a fungus exhibiting arthrospores were reported in 11/19 patients (57.9%). The case fatality rate at day 60 was 80%, with death occurring at a median of 7 days after diagnosis.

**Microbiological investigation.** Overall, 73 isolates of *S. clavata*, collected during this study and through the surveillance programs at the National Reference Centre for Invasive Mycoses & Antifungals (NRCMA) and including the European isolates and the type strain, were studied (see Table S1 in the supplemental material). All *S. clavata* isolates exhibited similar antifungal susceptibility profiles, with no significant difference according to period of isolation (Table 2). WGS was performed on a panel of 17 French *S. clavata* clinical isolates collected before (n = 6) and during (n = 11) the investigation period as well as on the type strain (see Table S1). In the 18 isolates, the gene sequences coding for a set of 175 fungal proteins as well as the 18S rRNA sequences (~324,000 aligned nucleotide characters) were totally identical (<0.00034% polymorphic sites), showing that *S. clavata* is a genetically highly monomorphic species. We therefore searched for single nucleotide polymorphisms (SNPs) over the entire genome sequence using a mapping approach, which led to the discovery of 312 high-quality SNPs. The resulting 103 phylogenetically informative characters were compatible with each other (homoplasy index = 0 [16]), therefore allowing a robust phylogenetic tree to be inferred (Fig. 2). Two particular clades, denoted A and B, were identified as homogeneous phylogenetic sublineages containing 7 and 5 of the 18 isolates, respectively. Clade A isolates differed by at most four SNPs among each other (Fig. 2), indicating that this clade is a young clone. Clades A and B were characterized by 15 and 9 unique characters, respectively. The clade-specific polymorphisms (see Table S2) were confirmed by Sanger sequencing, and eight of the SNPs were used to classify all isolates as belonging to clade A or B or to other lineages.

Among the 15 clade A-specific nucleotide changes, four were
<table><caption>TABLE 1 Characteristics of the *S. clavata* cases and major exposures prior to infection, France, September 2011 to October 2012</caption><thead><tr><th>Characteristic(s) of the case</th><th>All cases (n = 30)</th><th>A cases (n = 19)</th><th>Non-A cases (n = 11)</th><th>P value</th><th>A index cases (n = 10)</th><th>Non-A index cases (n = 10)</th><th>P value</th></tr></thead><tbody><tr><td>No. (%) of occurrences during the outbreak period (weeks 15–22, 2012)</td><td>18 (60.0)</td><td>16 (84.2)</td><td>2 (18.2)</td><td>0.001</td><td>9 (90.0)</td><td>2 (20.0)</td><td>0.005</td></tr><tr><td>Median age in yrs (95% CI)<sup>a</sup></td><td>63 (55–65)</td><td>63 (49–69)</td><td>63 (36–69)</td><td>0.846</td><td>62 (48–70)</td><td>63.5 (34–73)</td><td>0.821</td></tr><tr><td>Male gender, n<sup>b</sup> (%)</td><td>15 (50.0)</td><td>7 (36.8)</td><td>8 (72.7)</td><td>0.128</td><td>4 (40.0)</td><td>7 (70.0)</td><td>0.37</td></tr><tr><td>Underlying disease, n (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Acute myeloid leukemia</td><td>21 (70.0)</td><td>13 (68.4)</td><td>8 (72.7)</td><td>0.919</td><td>8 (80.0)</td><td>7 (70.0)</td><td>0.582</td></tr><tr><td>Acute lymphoid leukemia</td><td>6 (20.0)</td><td>3 (15.8)</td><td>3 (27.3)</td><td></td><td>1 (10.0)</td><td>3 (30.0)</td><td></td></tr><tr><td>Chronic lymphoid leukemia</td><td>1 (3.3)</td><td>1 (5.3)</td><td></td><td></td><td>1 (10.0)</td><td></td><td></td></tr><tr><td>Others</td><td>2 (6.7)</td><td>2 (10.5)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Immune status</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Severe neutropenia (&lt;500 neutrophils/mm³), n (%)</td><td>27 (90.0)</td><td>17 (89.5)</td><td>10 (90.9)</td><td>1</td><td>10 (100.0)</td><td>10 (100.0)</td><td>1</td></tr><tr><td>Median duration (days) of neutropenia at the time of positive culture (95% CI)</td><td>14 (13–20)<br>(n = 25)</td><td>14 (11–20)<br>(n = 15)</td><td>15.5 (10–32)<br>(n = 10)</td><td>0.579</td><td>13 (8–17)</td><td>15.5 (12–32)</td><td>1</td></tr><tr><td>Prolonged corticosteroid treatment without aplasia, n (%)</td><td>2 (6.7)</td><td>2 (10.5)</td><td></td><td></td><td>1 (9.1)</td><td></td><td></td></tr><tr><td>Clinical signs at the time of positive culture, n (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Diarrhea</td><td>16/26 (61.5)</td><td>8/15 (53.3)</td><td>8/11 (72.7)</td><td>0.428</td><td>4/9 (44.4)</td><td>7/10 (70.0)</td><td>0.37</td></tr><tr><td>Pulmonary symptoms</td><td>16 (53.3)</td><td>10 (66.7)</td><td>6/9 (66.7)</td><td>1</td><td>4/8 (50.0)</td><td>5/8 (62.5)</td><td>1</td></tr><tr><td>Positive culture (S. clavata), n (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Blood</td><td>26 (86.7)</td><td>16 (84.2)</td><td>10 (90.9)</td><td>1</td><td>10 (100.0)</td><td>9 (90.0)</td><td>1</td></tr><tr><td>Respiratory tract (bronchoalveolar lavage, tracheal aspirate)</td><td>12 (40.0)</td><td>9 (47.4)</td><td>3 (27.3)</td><td>0.442</td><td>4 (40.0)</td><td>3 (30)</td><td>1</td></tr><tr><td>Stools<sup>c</sup></td><td>11/19 (57.9)</td><td>7/10 (70.0)</td><td>4/9 (44.4)</td><td>0.370</td><td>2/5 (40.0)</td><td>4/8 (50.0)</td><td>1</td></tr><tr><td>Multiple noncontiguous sites</td><td>18 (60.0)</td><td>11 (57.9)</td><td>7 (63.6)</td><td>1</td><td>6 (60.0)</td><td>6 (60.0)</td><td>1</td></tr><tr><td>Median interval (days) between admission and first positive culture (95% CI)</td><td>21 (18–27)</td><td>22 (18–29)</td><td>20 (15–29)</td><td>0.667</td><td>24.5 (16–35)</td><td>22 (15–32)</td><td>0.647</td></tr><tr><td>Mortality</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Crude mortality at day 30, n (%)</td><td>22 (73.3)</td><td>15 (79.0)</td><td>7 (63.6)</td><td>0.417</td><td>9 (90.0)</td><td>6 (60.0)</td><td>0.303</td></tr><tr><td>Crude mortality at day 60, n (%)</td><td>24 (80.0)</td><td>16 (84.2)</td><td>8 (72.7)</td><td>0.641</td><td>9 (90.0)</td><td>7 (70.0)</td><td>0.582</td></tr><tr><td>Median interval (days) between first culture and death (95% CI)</td><td>7 (5–16)<br>(n = 24)</td><td>8 (3.5–21)<br>(n = 16)</td><td>6.5 (2–31)<br>(n = 8)</td><td>0.668</td><td>7 (2–19)<br>(n = 9)</td><td>7 (1–41)<br>(n = 7)</td><td>0.958</td></tr><tr><td>Major exposure prior to infection, n (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Echinocandins</td><td>16 (63.3)</td><td>10 (52.6)</td><td>6 (54.6)</td><td>1</td><td>6 (60.0)</td><td>5 (50.0)</td><td>1</td></tr><tr><td>Cytarabine<sup>d</sup></td><td>18/23 (78.3)</td><td>13/17 (76.5)</td><td>5/6 (83)</td><td>1</td><td>8/9 (89)</td><td>4/6 (83)</td><td>0.5</td></tr><tr><td>Apheresis platelet concentrates (APC)</td><td>21/25 (84)</td><td>15/17 (88)</td><td>6/8 (75.0)</td><td>0.57</td><td>10 (100)</td><td>5 (50.0)</td><td>0.03</td></tr><tr><td>Prepared with medical device from manufacturer X</td><td>10/25 (40)</td><td>6/17 (35)</td><td>4/8 (50)</td><td>0.7</td><td>3/9 (33)</td><td>3/7 (43)</td><td>1</td></tr><tr><td>Prepared with medical device from manufacturer Y</td><td>19 (63.3)</td><td>14 (73.7)</td><td>5 (45.4)</td><td>0.238</td><td>10 (100.0)</td><td>5 (50.0)</td><td>0.03</td></tr></tbody></table>

located in sequences homologous to those of protein-coding genes from *Yarrowia lipolytica*, the most closely related species with an annotated genome (17). All these changes impacted the deduced amino acid sequence, suggesting positive selection. SNP9 (see Table S2 in the supplemental material) caused a premature stop codon in a sequence similar to that of mycelial growth factor-1 from *Y. lipolytica*. The carbon assimilation pattern and

antifungal susceptibility profile and mycelium growth did not reveal clade-specific characteristics (data not shown).

Of the 30 cases recorded during the investigation period, 19 belonged to clade A, 7 to clade B, and 4 to other sublineages (Fig. 1). Of note, 18 of 19 cases belonging to clade A occurred in 2012 between weeks 15 and 28, and in that time frame, clade A was identified in 18/20 (90%) cases reported (including the 9 original

<footnote>a 95% CI = 95% confidence interval.</footnote>

<footnote>b n where the denominator represents the number of patients for whom the information was available (not stated when it was available for all patients).</footnote>

<footnote>c Stools were not cultured for all patients.</footnote>

<footnote>d Analysis taking into account brands or producers gave only low exposure rates.</footnote>
<table><caption>TABLE 2 MICs of the major antifungal drugs for all *Saprochaete clavata* isolates tested at the French National Reference Center for Invasive Mycoses & Antifungals</caption><thead><tr><th rowspan="2">Isolate group</th><th rowspan="2">No. of isolates</th><th colspan="5">MIC (mg/liter) (median [range])</th></tr><tr><th>Flucytosine</th><th>Voriconazole</th><th>Posaconazole</th><th>Amphotericin B</th><th>Caspofungin</th></tr></thead><tbody><tr><td>All isolates</td><td>45</td><td>0.25 [&lt;0.125-1]</td><td>1 [0.06-2]</td><td>0.5 [0.125-1]</td><td>0.5 [0.125-1]</td><td>8 [1-8]</td></tr><tr><td>Isolates recovered before September 2011</td><td>15</td><td>0.25 [&lt;0.125-1]</td><td>1 [0.06-2]</td><td>0.5 [0.125-1]</td><td>0.5 [0.125-1]</td><td>8 [1-8]</td></tr><tr><td>Isolates recovered between September 2011 and October 2012</td><td>30</td><td>0.5 [&lt;0.125-1]</td><td>1 [0.06-2]</td><td>1 [0.125-1]</td><td>0.5 [0.25-1]</td><td>8 [1-8]</td></tr><tr><td>Clade A isolates</td><td>19</td><td>0.5 [&lt;0.125-0.5]</td><td>1 [0.5-2]</td><td>1 [0.25-1]</td><td>0.5 [0.25-1]</td><td>8 [1-8]</td></tr><tr><td>Non-clade A isolates</td><td>26</td><td>0.25 [&lt;0.125-1]</td><td>1 [0.06-2]</td><td>0.5 [0.125-1]</td><td>0.5 [0.125-1]</td><td>8 [1-8]</td></tr></tbody></table>

cases that alerted us to the issue; see Table S1 in the supplemental material). This allowed defining an outbreak period (W15 to W28). Isolates belonging to clade B or other sublineages have been responsible for sporadic cases since 2005 and occurred with no increase during the outbreak period.

Of the 31 European isolates sent as *M. capitatus*, reidentification at the NRCMA showed that *S. clavata* accounted for 12/17 clinical and 13/14 environmental sources, none belonging to clade A or B (see Table S1 in the supplemental material). The remaining isolates were confirmed to be *M. capitatus*. No notification of an outbreak of *S. clavata* infection was sent to the Centers for Diseases Control in the United States (CDC) or Stockholm (eCDC).

**Assessment of risk factors for infection.** The case-case study compared 19 clade A cases to 11 non-clade A cases (Table 1). Characteristics of the patients did not differ significantly between the two groups except for a trend toward a lower proportion of men in the clade A group. Clade A was associated with more clusters (defined as 2 or more cases in a single HCF within 8 weeks) than other sublineages (5 clusters of 2 to 4 cases versus 1 cluster of 2 cases).

Analysis of exposures prior to positive culture did not show a significant difference between the two groups or identify a common source of contamination (see Table S3 in the supplemental material). All cases received blood products. Clade A cases (14/19, 74%) were more frequently exposed than non-clade A cases (5/11, 45%) to apheresis platelet concentrates (APCs) prepared by the use of manufacturer Y's medical device (not significant). This trend was significant for the group consisting solely of index cases (10/10 [100%] versus 5/10 [50%], respectively, *P* = 0.033). Of note, 10/13 (1 missing information) patients with clade A infection cases exposed to the suspected medical device were also exposed to cytarabine. The mean interval between the last infusion of APC prepared with this medical device and positive culture results was 3.0 days for clade A cases (2.0 days for index cases).

The market share in France of this specific medical device is high (50% to 60% of APC in France are prepared with it), which is consistent with the exposure rates recorded among non-clade A cases. Overall, 11 lots of this medical device—produced abroad on the same production line and distributed only in France—were linked to the 30 cases, and more than 12,300 APCs were prepared with the 11 suspected lots. Other lots were produced on the same line and distributed to other countries.

Applying precautionary principles, imposition of a quarantine was decided by health authorities in September 2012 for unused

blood products prepared with the suspected lots of the medical device, but very few were still stored. Two unused medical devices (see Fig. S1 in the supplemental material) as well as 3 APCs and 2 frozen plasma samples prepared with the suspected lots were analyzed and showed no evidence of fungal contamination. Results of local investigations of fungal contamination of food and environmental samples were also negative.

**Experimental infections.** Knowing the epidemiology of fungal infections helps in understanding their pathophysiology and the risk of person-to-person transmission. Here we had indirect evidence that index cases could have been contaminated through APC and we knew that *S. clavata* was recovered from stool specimens in humans, representing therefore a source for transmission to secondary cases. Translocation from gut to blood after local proliferation is well known and has been demonstrated, for example, for *Candida albicans* (18) in experimental models and in humans. The reverse process (translocation from blood to gut) is usually not a concern and has not been systematically studied for other fungal pathogens. To assess whether it was possible, intravenous inoculation of *S. clavata* clade A was performed in nonimmunocompromised mice and fungal growth recorded in blood and feces samples from infected animals (Fig. 3A and B). We first checked that no fungal colony was observed in samples from uninfected control mice. From infected mice, all stool specimens on days 2, 5, and 7, as well as 4/5 blood samples on days 2 and 5 and 3/5 blood samples on day 7, grew *S. clavata*.

We also compared the levels of virulence of clades A and B, which differed in mice after intravenous inoculation, and found a significant difference (*P* = <0.001, log-rank test) (Fig. 3C). All mice infected with clade B isolate died before day 25 after inoculation; no differences between males and females were seen. All female mice infected with clade A survived on day 30, whereas two male mice infected with clade A died (1 on day 10 and 1 on day 24).

## DISCUSSION

While the French national surveillance of invasive fungal infections recorded only sporadic cases of *S. clavata* until 2012, the events observed in the spring of 2012 (epidemic curve and geographic distribution) suggested a common-source outbreak during which patients were exposed to a nationally distributed source over a relative brief period. This multicenter outbreak affected patients with severe hematological malignancies and was associated with a high short-term case fatality rate. As the potential
<figure><img src="image_5.png" /><figcaption>FIG 2 Phylogenetic tree inferred for 18 *Saprochaete clavata* isolates based on genome-wide SNPs. An unrooted BioNJ (40) tree constructed from pairwise nucleotide p-distances is shown. The number of nucleotide changes which were unique to the corresponding branch is indicated below each branch. The scale bar represents the SNP rate based on the estimated genome length (~17.5 Mb). Thick branches are 100% supported by bootstrap analysis results. Clades A and B are indicated with dashed boxes. Isolates recovered during the outbreak are indicated with a black star.</figcaption></figure>

source of *S. clavata* was unknown, an investigation of food, drugs, and blood products was started based on the assumption that dairy products might be involved, as seen with *M. capitatus*, and that the incubation period should be short in cases of oral infection with digestive translocation.

Multilocus sequence typing (MLST) can now be replaced by

whole-genome SNP typing (whole-genome sequence typing [WGST]) for inferring the relatedness of both bacteria and fungi. Whole-genome sequencing is indeed a powerful approach to characterize microbial strains for outbreak investigation and molecular epidemiology purposes. Indeed, genomic sequencing provides maximal discriminatory power while uncovering at the same
<figure><img src="image_6.png" /><figcaption>FIG 3 Experimental infections with *Saprochaete clavata*. (A) Macroscopic image of *S. clavata* on SDA petri dish after 48 h of incubation at 30°C. (B) *Saprochaete clavata* from a culture grown on 2% malt extract agar examined with Nomarski interphase contrast (×100), after 48 h of incubation at 30°C. (C) Survival curves for OF1 mice (dashed lines for males, solid lines for females) infected intravenously by strains of *Saprochaete clavata* belonging to clade B (green lines) or clade A (red lines).</figcaption></figure>

time genomic variations that can be linked to increased patho-
genic potential. Here, in the absence of validated methods for
genotyping S. clavata isolates, the first complete genome sequence
of this fungal species was determined and used to design a novel
genotyping method. Comparison of outbreak and sporadic iso-
lates clearly demonstrated that the outbreak involved one single
strain (clade A), which represents a recently emerged clone, while
other lineages were responsible for sporadic cases in France and
for monocentric outbreaks in Europe (12, 14, 19), outbreaks pre-
viously attributed to M. capitatus. Given the small amount of poly-
morphism uncovered, any classical typing approach was deemed
likely to fail to recognize the outbreak clone, underlining the im-
portance of WGS as a novel tool for outbreak investigations of
fungal pathogens.

Four clade A-specific nucleotide changes were located in se-
quences homologous to those of protein-coding genes from Y. li-
polytica and impacted the deduced amino acid sequence, suggest-
ing positive selection. SNP9 caused a premature stop codon in a
sequence similar to that of mycelial growth factor-1 from Y. lipo-
lytica. The issue of whether these changes conferred a selective
advantage to clade A isolates or increased their pathogenic poten-
tial was intriguing. Carbon assimilation pattern and mycelium
growth analyses did not reveal clade-specific characteristics. We
ruled out major changes in antifungal susceptibility profiles that
could explain the emergence of clade A. A nonsignificant increase
in the voriconazole MIC was observed for clade A isolates (1 dilu-
tion) that may have impacted its fitness. However, this was not
confirmed by *in vivo* experiments, where clade A was less virulent
in mice than clade B. It must be kept in mind, though, that we used
intravenous inoculation of immunocompetent mice and not neu-

tropenic mice or mice with damaged intestinal tract or disturbed
microflora (18).

The discovery that a single clade of S. clavata was associated
with the outbreak and was distinct from sporadic isolates pro-
vides, however, additional if not definite evidence for a common
source of contamination. Clade A was associated with a higher
number of clusters than other sublineages, indicating high expo-
sure to a common source or a higher capability of person-to-
person transmission. The majority (almost 80%) of cases were
also exposed to cytarabine, which is known to alter digestive mu-
cosa. Diarrhea associated with fungemia was reported in more
than half of the patients. Furthermore, gut colonization was re-
corded in approximately 60% of patients for whom we had the
relevant information. Gut colonization may have been underesti-
mated, given the knowledge that *Galactomyces candidus* (synonym
of *Geotrichum candidum*), which has the same morphology in cul-
ture as S. clavata, is considered a contaminant and is not always
reported. We found here that, after experimental intravenous in-
oculation of S. clavata into immunocompetent mice, S. clavata
can translocate from the blood to the gut even when the gut mu-
cosa is intact. Even if the proportion of patients recorded with
diarrhea and positive stools was relatively low, our observations
suggested that index patients with fungemia could have experi-
enced subsequent gut translocation due to cytarabine or diarrhea.
These findings are compatible with cross-contamination within
HCF.

We considered the possibility of a source of contamination that
was ingested orally, given the high frequency of diarrhea and pos-
itive stool specimen results. Furthermore, we discovered that
S. clavata can be recovered from dairy products (12, 19) and dish-
washers (20) as determined on the basis of reidentification of iso-
lates previously labeled as M. capitatus. Finally, the low percentage
of men infected with clade A (37%), which is different from the
usual male predominance recorded during periods of yeast fun-
gemia (60.7% [2,317/3,818] for all yeast species and 77.8% [7/9]
for non-clade A S. clavata fungemia in our YEASTS surveillance
program [2] [NRCMA, unpublished data]), could suggest the in-
volvement of a product preferentially consumed by women, as in
the recent Shiga-toxin-producing Escherichia coli O104:H4 out-
break in Germany (21). However, epidemiological and microbio-
logical investigations did not lead to a documented source of in-
fection.

Case-case comparisons have been instrumental in deciphering,
for example, outbreaks of listeriosis (22, 23). Our genotyping
technique allowed differentiation of the cases into two meaningful
groups (clade A versus other sublineages), thus allowing a case-
case study. All patients came from the same source population and
were susceptible to S. clavata infection. A classical case-control
approach selecting controls among patients in the same wards
might have introduced bias due to similar probabilities of expo-
sure to contaminating sources. A significantly higher exposure
frequency among clade A index cases than among non-clade A
index cases led to the hypothesis of a role for APC as an infection
source and of possible transmission to secondary cases as de-
scribed above. The end of the nationwide outbreak coincided with
the exhaustion of the APC stock before recall of the suspected lots,
consistent with our hypothesis. Exposure to the medical device
used for APC collection could explain 75% of the cases. However,
the APC contamination hypothesis is challenged by the fact that
the same production line was used to produce medical devices that
were exported to various countries, which makes the possibility
that contamination occurred only in lots distributed to France less
likely. Neither the European CDC nor the U.S. CDC recorded
outbreaks due to S. clavata, and no fungal contamination was
evidenced in the medical devices of the same lots and in the blood
products tested.

The proposed association between APCs and *S. clavata* cases remains hypothetical, as no cases occurred in HCF outside the study area that also used products from the same lots; only a few cases were identified despite widespread use of the device, and no fungal contamination of blood products or associated with the storage or processing of medical devices was uncovered. Our study included only a small number of cases, which lowered the power of the analysis and limited the choice of the controls. The retrospective nature of the investigation precluded collection of all data and microbiological testing of all potential sources of contamination (especially food). Overall, the source of the contamination remains unidentified.

Further studies are needed to provide insight into the process
of how a microorganism probably ingested or inoculated with a
low inoculum can cause severe invasive disease once critical com-
ponents of the host immune system are compromised. However,
the possibility that *S. clavata* may be transmitted through contam-
inated medical devices or can be associated with dairy products as
in previous European outbreaks is highly relevant for the manage-
ment of future outbreaks due to this newly recognized pathogen.

MATERIALS AND METHODS

**Epidemiological investigation.** The French National Institute for Public Health Surveillance (Institut de veille sanitaire [InVS]) coordinates na-

tional surveillance for health care-associated infections (24). During the
spring of 2012, three clusters of three cases each of invasive infections due
to *M. capitatus* were reported to InVS from 3 HCF located in three regions
(25). The isolates were subsequently identified as *S. clavata* at the National
Reference Centre for Invasive Mycoses & Antifungals (NRCMA).

The occurrence of nine cases in three remote centers over a limited period suggested a multicentric outbreak, possibly associated with a common source. Indeed, only sporadic infections due to these species (15 cases between January 2005 and September 2011) had been recorded through the nationwide ongoing surveillance programs on invasive fungal infections implemented by the NRCMA. These programs include active surveillance of fungemia (including those due to *M. capitatus* and *S. clavata*) in the Paris area (YEASTS program [26]) and passive nationwide surveillance of rare invasive fungal infections, collecting both clinical information and isolates (https://epidemio.pasteur.fr/resomyc_portail/).

Thus, because *S. clavata* epidemiology and ecology were largely un-
known and to confirm the outbreak and identify its onset, a national alert
was launched in all hematology wards and HCF laboratories in metropol-
itan France, with the objective to identify all cases due to one of the two
species (investigation period, September 2011 to October 2012). A stan-
dardized questionnaire was used to collect information from treating phy-
sicians. The cases were defined as patients with a positive culture for a
fungus identified as *M. capitatus* or *S. clavata* or *Geotrichum* sp. from a
sterile site or from the respiratory tract when it was associated with clinical
and/or radiological symptoms in a patient with underlying predisposing
conditions (27). All isolates were to be sent to NRCMA for further anal-
ysis.

The objectives were to describe case characteristics, medical histories, and outcomes and to identify risk factors, including a potential common source. Data corresponding to food intake, including but not limited to dairy products, and drug administration, including mouthwashes, medical fluids, and drugs, within 1 week (1 month for antifungals) before positive culture were recorded. Exposure within 1 month to blood products was specifically investigated with the cooperation of the French national agencies for blood products (Etablissement Français du Sang) and for drug safety (Agence de Sécurité du Médicament et Produits de Santé). The single-use medical devices used to collect and store blood products (see Fig. S1 for an example) and the specific lots used for the infused blood products were also recorded. In parallel, the Centers for Diseases Control in the United States (CDC) and in Stockholm (eCDC) were informed and asked if similar events had occurred recently in other countries.

This investigation was considered to be a public health response and deemed to be nonresearch; thus, requirements for approval by an institutional review board or for written informed consent from patients were waived. Epidemiological data were recorded anonymously except when tracking blood products lots (approval 341194v42 of the French Commission for Data Protection [CNIL]).

**Laboratory investigation.** Food samples (samples from similar dishes prepared at the same time as those offered to the patients were blended and plated on Sabouraud dextrose agar [SDA] plates) and environmental samples (swabs on hard surfaces, air samples plated on SDA) were analyzed in a few HCF. Because of the high fatality rate, these investigations were limited to a few cases. At the NRCMA, washes of unused medical devices and blood products from the incriminated lots were tested for fungal contamination by culture (pellets of centrifugation incubated for 30 days on Sabouraud agar at 30°C) and amplification of internal transcribed spacer (ITS) regions with panfungal primers after DNA extraction was performed (28).

**Strains studied.** European colleagues who previously identified *M. capitatus* from clinical sources (blood) or environmental sources (dishwashers, milk, or containers) (12, 19, 20) were contacted and asked to share their isolates for comparisons. All these “European” isolates together with all the French isolates collected during and before the investigation period through the surveillance programs were reidentified at the NRCMA using nucleotide sequencing of the D1D2 and ITS1-5.8S-ITS2
regions (9, 29–31). Antifungal susceptibility testing was performed according to the EUCAST method (32). Type strains of *S. clavata* (CBS425.71) and *M. capitatus* (CBS162.80) (see Table S1 in the supplemental material) were studied.

**Illumina sequencing and data preprocessing.** Since genotyping methods for *S. clavata* were lacking, WGS was performed to compare isolates and allowed the subsequent design of a genotyping method by PCR and Sanger sequencing.

A first batch of 11 clinical isolates, including 5 outbreak isolates (see Table S1 in the supplemental material), was sequenced in July 2012. Libraries were constructed using a Nextera DNA sample preparation kit (Illumina) and sequenced on an Illumina HiSeq 2000 sequencer with a 100-nucleotide single-end protocol. Two lanes from these tagged libraries resulted in ~26 million reads per strain on average. Genomic libraries for a second batch of 6 clinical isolates plus the *S. clavata* type strain were prepared in August 2012 following the same protocol, except that the libraries, with an insertion size of ~250 bp, were sequenced with a paired-end protocol that generated 2× ~7 million reads of 100 bases per strain on average. Finally, to improve genome assembly, one isolate (CNRMA12.647; see Table S1) was resequenced using an Illumina TruSeq library with a 2× 100-nucleotide paired-end module (insertion size of ~400 bp), leading to 2× ~190 million reads. All reads were preprocessed to remove low-quality or artifactual nucleotides. First, all nucleotides occurring at the 5' and 3' ends and supported by a Phred quality score of <28 were removed using the program Sickle version 1.33 (GitHub Inc., San Francisco, CA, USA). Second, contaminant oligonucleotides (i.e., library adaptors) were detected and removed using the program AlienTrimmer (33). Third, reads that were below 45 nucleotides in length after performance of the cleaning steps described above were discarded, as were those containing more than 5% of the remaining nucleotides with a Phred score of <28. Finally, the program fqduplicate (http://ftp.pasteur.fr/pub/gensoft/projects/fqtools) was used to discard every duplicate single- or paired-end read.

**Detection of high-quality SNPs among strains.** Whole-genome *de novo* assembly for each strain was performed using the program clc_novo_assemble (CLC Bio, Aarhus, Denmark). A set of 175 conserved and universally distributed fungal protein sequences (34, 35) as well as 18S rRNA regions were extracted from each set of contigs and aligned using MUSCLE (36, 37). As a distinct approach, sequence variation was mined following a read-mapping procedure. To obtain a high-quality reference, all filtered reads from the first batch of 11 strains were pooled and assembled, resulting in 4,618 contigs of >500 nucleotides in length (~3,668-nucleotide length on average, ~17.5 Mb in total). The read sets of each of the 18 isolates were then mapped on the reference using the Burrows-Wheeler Alignment (BWA) tool (38). Information on coverage (the number of reads that map to each reference sequence position) and polymorphisms was extracted using SAMtools (39). To select high-quality SNPs, we retained those that (i) were covered in all strains by at least four reads mapped in each orientation, (ii) had a minimum mapping quality of 30, and (iii) were supported by at least 80% of reads mapped at this position. In complement, SNPs occurring in regions of microrepeats or less than 100 nucleotides from any contig end were discarded.

**Phylogenetic analysis.** All the inferred high-quality SNPs were merged into a set of 312 variable characters. Among these characters, 103 were parsimony informative (i.e., contained more than one type of nucleotides, with at least two of them occurring in at least two isolates, therefore supporting phylogenetic groupings). Phylogenetic inference was performed with the algorithm BioNJ on the pairwise nucleotide p-distances, and confidence supports were estimated for each internal branch with a bootstrap procedure based on 10,000 replicates (40). As expected given that the 312 characters were compatible, the same tree topology was obtained using alternative tree inference methods, including maximum parsimony and maximum likelihood.

**De novo genome assembly of strain CNRMA12.647.** Scaffolded contigs of isolate CNRMA12.647 (belonging to clade A) were pro-

duced from filtered reads by *de novo* assembling with parameters selected to maximize the average scaffold length. This resulted in 339 scaffold sequences of a total length of ~17.5 Mb, which were used to position the specific SNPs of clades A and B identified previously (see Table S3 in the supplemental material).

**Virulence tests in mice.** The mouse experiments had two objectives: (i) to study the potential translocation of *S. clavata* from blood to the digestive tract and (ii) to compare the levels of virulence of *S. clavata* in normal hosts according to host gender and clades of *S. clavata*. For the survival experiments, 6-week-old OF1 male or female mice (Charles River, France) were inoculated intravenously with 100 µl of a suspension of *S. clavata* at 4 × 10⁶ CFU/ml. Groups of 7 animals were inoculated with either clade A (CNRMA12.647) or clade B (CNRMA12.304). Animals were observed daily. Those that appeared moribund or in pain were sacrificed by CO₂ inhalation. Survival rates were compared using the log-rank test and GraphPad Prism version 6 (GraphPad Software, Inc.).

For the gut translocation study, 3 groups of 5 OF1 female mice were infected intravenously with 100 µl of a suspension of *S. clavata* clade A (CNRMA12.647) at 3 × 10⁶ CFU/ml. Five mice were sacrificed on days 2, 5, and 7 postinoculation. Blood (500 µl obtained by cardiac puncture) was cultured on SDA. Feces samples were diluted in 500 µl sterile water on SDA plates containing chloramphenicol and incubated for 5 days at 30°C. The identification of the fungus growing from the feces obtained on day 2 was confirmed by sequencing the ITS. All subsequent colonies were identified by morphology. Animal experiments were approved by the national ethical committee (CETEA 2013-0135) and the local ethical committee (CHSCT 14.135).

**Statistical analysis.** The first descriptive analysis focused on all cases of *S. clavata* recorded during the investigation period. Following the identification of clade A, a case-case study (41) was performed to identify exposure and factors associated with infection with clade A (see above for a description of how clade A was identified) compared to those due to other sublineages. “A” cases were defined as all cases belonging to clade A and “non-A” cases as all cases belonging to other lineages that occurred in France during the investigation period. An index case was defined as the first case infected (positive culture) by either clade A or other lineages in a given HCF. Differences in categorical variables were compared using Fisher’s exact test and differences in quantitative variables using the Mann-Whitney test. Data analysis was performed using Stata 9.2 (Stats Corp., College Station, TX).

**Nucleotide sequence accession numbers.** Assembled sequences of isolate CNRMA 12.647 were submitted to the EMBL database and are accessible under accession numbers CBXB010000001 to CBXB010000339.

## SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mbio.asm.org/lookup/suppl/doi:10.1128/mBio.02309-14/-/DCSupplemental.

Figure S1, JPG file, 0.1 MB.

Table S1, PDF file, .01 MB.

Table S2, PDF file, 0.02 MB.

Table S3, PDF file, 0.01 MB.

## ACKNOWLEDGMENTS

The members of the Geotrichum Investigation Group who contributed data are as follows: Dea Garcia-Hermoso, Catherine Blanc, Damien Hoiward, Olivier Lortholary, and Stéphane Bretagne (Center National de Référence Mycoses Invasives et Antifongiques, Institut Pasteur, Paris); Jean-Michel Thiolet and Henriette de Valk (Institut de Veille Sanitaire, Saint-Maurice); Rémi Courbil, Anne Chabanel, Marion Simonet, Francoise Maire, Saadia Jbilou, and Pierre Tiberghien (Etablissement Français du Sang, Paris); Hervé Blanchard (CClin Paris-Nord), Anne-Gaëlle Venier (CClin Sud-Ouest), Claude Bernet (CClin Sud-Est, Loïc Simon (CClin Est), Hélène Sénéchal (CClin Ouest); Elodie Pouchet and Christiane Angot (AnSM); Patricia Ribaud, G. Socié, and M. Flèche (Société Française d'hématologie); Nathalie Brieu, Evelyne Lagier, Vanessa Chartier, Thierry
Allegre, and Laurence Maulin (Aix-en-Provence); Hélène Lanic and Hervé Tilly (Amiens); Jean-Philippe Bouchara, Marc Pihet, Aline Schmidt, Achille Kouatchet, Yves-Marie Vandamme, Marcort Ifrah, and Alain Mercat (Angers); Isabelle Accoceberry, Olivier Albert, Thibaut Le-guay, and Anne-Marie Rogues (Bordeaux); Julie Bonhomme, Oumédaly Reman, and Claire Lesteven (Caen); Philippe Poirier, Cécile Molucon Chabrot, Laure Calvet, Olivier Baud, and Monique Cambon (Clermont-Ferrand); Jean Chistophe Farkas and Bruno Lafon (Courlancy); Frédéric Dalle, Denis Caillot, Aline Lazzarotti, Serge Aho, and Sandrine Combret (Dijon); Thierry Façon, Boualem Sendid, Séverine Loridant, Terriou Louis, Bruno Cazin, and Bruno Grandbastien (Lille); Nathalie Bourgeois, Anne Lotthé, Guillaume Cartron, Christophe Ravel, Pascal Colson, and Philippe Gaudard (Montpellier); Caroline Bonmati, Loïc Simon, Christian Rabaud, and Marie Machouart (Nancy); Didier Poisson, Diana Carp, and Jérôme Meunier (Orléans); Anne Gaschet, Chantal Miquel, Laurence Sanhes, Milagros Ferreyra, Franck Leibinger, and Philippe Geudet (Pépignan); Dominique Toubas, Chantal Himberlin, Florence Bureau-Chalot, and Alain Delmer (Reims); Loïc Favençon, Gilles Gargala, Jean-Baptiste Michot, Christophe Girault, Marion David, Stéphane Leprétre, Fabrice and Jardin (Rouen); Pierre Honderlick, Vincent Caille, and Charles Cerf (Suresnes); Sophie Cassaing, Christian Recher, Muriel Picard, Caroline Protin, Françoise Huguet, Anne Huynh, Jean Ruiz, Béatrice Riu-Poulenc, Philippe Letocat, Bruno Marchou, Xavier Verdeil, Laurent Cavalié, Pamela Chauvin, Xavier Iriart, Alexis Valentin, and Emmanuelle Bouvet (Toulouse); and Béatrice Delmas-Marsalet, Asma Jéblaoui, and Najiby Kassis-Chikhani (Villejuif) (Health Care Facilities of the cities listed above); and Konrad Mühlethaler and Stefan Zimmerli (Institute for Infectious Diseases, University of Bern, Bern, Switzerland); Polona Zalar (Biology Department, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia); and Ferran Sánchez-Reus and Merce Gurguí (Hospital de la Santa Creu i Sant, Pau, Barcelona, Spain).

This work was supported by Institut Pasteur and Institut de veille sanitaire. No other funding was received for this work.

There is no conflict of interest.

We thank Cecile Neuvéglise (INRA, Grignon) for helpful discussions and Judith Mueller (École des Hautes Etudes en Santé Publique, Rennes) for advice and discussions.

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