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JOURNAL OFCLINICALMICROBIOLOGY, Aug. 2010, p. 2754–2761 Vol. 48, No. 8 0095-1137/10/$12.00 doi:10.1128/JCM.00764-10

Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Identification of

Paecilomyces variotii

in Clinical

Samples and Settings

Jos Houbraken,

1

* Paul E. Verweij,

2

Anthonius J. M. M. Rijs,

2

Andrew M. Borman,

3

and Robert A. Samson

1

CBS-KNAW Fungal Biodiversity Centre, Department of Applied and Industrial Mycology, Utrecht, Netherlands1; Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands2;

and HPA Mycology Reference Laboratory, HPA Southwest, Bristol, United Kingdom3 Received 15 April 2010/Returned for modification 10 May 2010/Accepted 21 May 2010

Paecilomyces variotiiis a commonly occurring species in air and food, but it is also associated with many types of human infections and is among the emerging causative agents of opportunistic mycoses in

immunocom-promised hosts. Paecilomyces can cause hyalohyphomycosis, and two species, Paecilomyces lilacinus and P.

variotii, are the most frequently encountered organisms. In the present study, a set of 34 clinical isolates

morphologically identified as P. variotii or P. lilacinus were formally identified by sequencing intergenic

transcribed spacer regions 1 and 2 (including 5.8S rDNA) and a part of the-tubulin gene. Three isolates were

identified asP. lilacinus, and five of the presumptiveP. variotiiisolates did not belong to the genusPaecilomyces

but were identified asTalaromyces eburneus (anamorph,Geosmithia argillacea) or Hamigera avellanea

(ana-morph, Merimbla ingelheimense). Applying the most recent taxonomy, we found that the clinical P. variotii

isolates could be identified asP. variotiisensu stricto (14 strains),P. formosus(11 strains), andP.

dactylethro-morphus(1 strain). These data indicate thatP. formosusoccurs in clinical samples as commonly asP. variotii.

Susceptibility tests showed that the antifungal susceptibility profiles ofP. variotii, P. formosus, andP.

dac-tylethromorphusare similar and that all strains tested were susceptible to amphotericin Bin vitro.P. lilanicus,

T. eburneus, and H. avellaneahad different susceptibility profiles; and flucytosine and voriconazole were the least active of the antifungal drugs tested against these species. Our results indicate that correct species identification is important to help guide appropriate antifungal therapy.

Paecilomyces variotii is a commonly occurring species that has previously been isolated from various substrates, including (pasteurized) foods, soil, indoor air, and wood (23, 36, 41, 42, 43). However, it is also associated with many types of human infections and is listed among the emerging causative agents of

opportunistic mycoses in immunocompromised hosts.

Paecilo-mycescan cause hyalohyphomycosis (1), and two species, Pae-cilomyces lilacinusandP. variotii, are the most frequently en-countered (20, 52). Both species are morphologically similar but can be differentiated on the basis of conidial color and growth rates (41). However, small-subunit ribosomal gene

se-quences showed that the two species are unrelated:P. variotii

belongs to the orderEurotiales, whileP. lilacinusis a member

of the orderHypocreales(27). Although they are uncommon,

Paecilomycesinfections are associated with almost any organ or system of the human body (40). Most cases concern immu-nocompromised patients and are cutaneous or catheter re-lated. However, dissemination, for example, that involving the central nervous system, has been observed in a number of cases (15, 24). Ocular infections associated with prolonged contact lens use or ocular surgery have also been reported (40), as has peritonitis in patients with continuous ambulatory peri-toneal dialysis (24), which apparently responded well to an-tifungal therapy, with cures occurring in 11 of the 13 cases.

However, the majority of these patients were treated for

Pae-cilomyces peritonitis and were not severely

immunocompro-mised. In vitro amphotericin B was active against clinicalP.

variotii isolates but not against P. lilacinus (8). Among the azoles, only itraconazole and posaconazole showed clinically

relevant activity againstP. variotii. The geometric mean MICs

of voriconazole and ravuconazole were above 4 mg/liter,

indi-cating that these azoles will not be effectivein vivo.

Interest-ingly, the echinocandins, especially micafungin and

anidula-fungin, were highly active againstP. variotii, with MIC values

being as low as 0.016 mg/liter. Conversely,P. lilacinuswas not

inhibited by the echinocandins, underscoring the importance of correct species identification (8).

Paecilomyces variotii grows rapidly on standard agar and

forms velvety olive brown colonies. Conidiophores ofP. variotii

are irregularly branched, and the phialides have a broad base

ending in a long and slender neck. Samson (41) noted thatP.

variotiiis a morphologically variable species, and the taxonomy of P. variotiiand the related Byssochlamys teleomorphs has recently been revised (45). This revision is based on

morphol-ogy, extrolites, and molecules and shows thatP. variotiisensu

lato comprises five species, namely, Byssochlamys spectabilis

(the sexual state ofP. variotii),P. brunneolus, P. formosus,P.

divaricatus, and P. dactylethromorphus. The last species was

incorrectly namedP. saturatusbecauseP. dactylethromorphus

was validly described in 1957 and has priority. The aim of the present study was to determine the prevalence of these species in clinical samples and settings. A total of 34 isolates originat-ing from various clinical specimens and settoriginat-ings were identified by sequencing the intergenic transcribed spacer (ITS) regions,

* Corresponding author. Mailing address: CBS-KNAW Fungal Biodi-versity Centre, Department of Applied and Industrial Mycology, Utrecht 3584 CT, Netherlands. Phone: 31 (0)30 2122600. Fax: 31 (0)30 2512097. E-mail: j.houbraken@cbs.knaw.nl.

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including the 5.8S rDNA, and a part of the ␤-tubulin gene. Furthermore, the antifungal susceptibility profiles of these spe-cies and ex-type strains are reported.

MATERIALS AND METHODS

Strains.This study includes 34 strains isolated from clinical specimens and hospital environments. These strains were identified on the basis of macro- and

microscopic characters and were maintained in various culture collections asP. variotiiorP.lilacinus.In additional, ex-type strains and freshly isolated strains were also included in this study.

[image:2.585.44.545.82.587.2]

Morphological examination.Isolates (Table 1) were grown for 3 days on malt extract agar (MEA) and were incubated in the dark at 25, 30, and 37°C. Fur-thermore, three-point inoculations were made on MEA, Czapek yeast agar (CYA), and creatine agar (CREA); and the isolates were incubated for 7 days at 25°C (the medium compositions are described by Samson et al. [43]). After TABLE 1. Isolates used in this study

Strain no.a Species Source

CBS 100.11NT** Byssochlamys nivea Unknown CBS 146.48NT** Byssochlamys fulva Bottled fruit, UK

CBS 373.70T** Byssochlamys lagunculariae Wood ofLaguncularia racemosa(mangue), Brazil CBS 605.74HT** Byssochlamys verrucosa Nesting material ofLeipoa ocellata, Australia

CBS 374.70isoT** Byssochlamys zollerniae Wood ofZollernia ilicifoliaandProtium heptaphyllum, Brazil UMCN V63-56, DTO 63F6 Hamigera avellanea Human, ear swab; Nijmegen, Netherlands (2007)

CBS 295.48isoT* Hamigera avellanea Soil; San Antonio, TX CBS 370.70T** P. brunneolus Nonfat dry milk, Canada

CBS 110430* P. divaricatum Soil, Thailand

CBS 284.48T P. divaricatum Mucilage bottle with library paste, USA UMCN V54-40, DTO 63F3 P. formosus Human, sputum; Nijmegen, Netherlands (2006) CBS 296.93 P. formosus Human, bone marrow of patient; Taskent, Uzbekistan CBS 297.93 P. formosus Human, blood of patient; Taskent, Uzbekistan CBS 298.93 P. formosus Human, breast milk of patient; Taskent, Uzbekistan

CBS 990.73BT P. formosus Unknown

DTO 45H8 P. formosus Pseudo-outbreak in hospital, blood culture, United Kingdom UMCN 1274, DTO 63E3 P. formosus(lecythidis type) Human, bronchoalveolar lavage fluid; Nijmegen, Netherlands UMCN V49-58, DTO 63F1 P. formosus(lecythidis type) Human, sputum; Zwolle, Netherlands (2006)

UMCN V56-25, DTO 63F4 P. formosus(lecythidis type) Human, sputum; Nijmegen, Netherlands (2006)

CBS 372.70 P. formosus(lecythidis type) Type ofP. lecythidis,Lecythis unsitata(Lecythidaceae), wood, Brazil DTO 45I1 P. formosus(lecythidis type) Pseudo-outbreak in hospital, blood culture; UK

NCPF 2825, DTO 49D5 P. formosus(lecythidis type) Brain abscess, United Kingdom (1991)

NCPF 2837, DTO 49D6 P. formosus(lecythidis type) Brain abscess, same patient as NCPF 2825, United Kingdom (1991) CBS 113247* P. formosus(maximus type) Soil, Thailand

CBS 371.70 P. formosus(maximus type) Type ofP. maximus,Annona squamosa, Brazil UMCN 1156,* DTO 63E1 P. lilacinus Unknown source

UMCN 2419,* DTO 63E5 P. lilacinus Human, skin swab; Nijmegen, Netherlands (1994)

CBS 284.36T* P. lilacinus Soil; Ithaca, NY

V52-21,* DTO 63F2 P. lilacinus Human, sputum; Nijmegen, Netherlands (2006)

UMCN V66-47, DTO 63F7 P. dactylethromorphus Human, cornea scrapings from keratomycosis; Nijmegen, Netherlands (2008) CBS 323.34T P. dactylethromorphus Unknown source

CBS 492.84* P. dactylethromorphus Lepidium sativum, Denmark

CBS 110036, DTO 34C8 P. variotii Cerebrospinal fluid of 60-year-old female with diabetes and cancer; Istanbul, Turkey

UMCN 1157*, DTO 63E2 P. variotii Unknown source

UMCN 2266, DTO 63E4 P. variotii Human, feces; Nijmegen, Netherlands (1994) UMCN 3796, DTO 63E6 P. variotii Human, mouthwash; Nijmegen, Netherlands (1995) UMCN 45H9*, DTO 45H9 P. variotii Liver biopsy; London, UK

UMCN 577, DTO 63D6 P. variotii Human, mouthwash; Nijmegen, Netherlands UMCN 654, DTO 63D7 P. variotii Human, feces, Nijmegen; Netherlands

UMCN 730, DTO 63D8 P. variotii Hospital environment, elevator shaft; Nijmegen, Netherlands UMCN 731, DTO 63D9 P. variotii Human, mouthwash; Nijmegen, Netherlands

UMCN 7845, DTO 63E7 P. variotii Human, cerebrospinal fluid; Nijmegen, Netherlands (1998) UMCN 8490, DTO 63E9 P. variotii Human, mouthwash; Nijmegen, Netherlands (1999) UMCN V57-21, DTO 63F5 P. variotii Human, abscess; Zwolle, Netherlands (2007)

CBS 101075 P. variotii Type ofB. spectabilis, heat-processed fruit beverage, Japan

CBS 102.74T P. variotii Unknown source

CBS 124.97 P. variotii Human, vitreous tumor left eye, neutropenic leukemic patient with acute endophthalmitis, Hong Kong

CBS 339.51 P. variotii Human, sputum, Netherlands

DTO 45I3 Talaromyces eburneus Pseudo-outbreak in hospital, blood culture, UK NCPF 2801, DTO 49D4 Talaromyces eburneus Sputum, cystic fibrosis patient, UK (1991) NCPF 7594, DTO 49D7 Talaromyces eburneus Blood culture, patient with peritonitis, UK (2002)

NCPF 7596, DTO 49D9 Talaromyces eburneus Peritoneal dialysis fluid (same patient source as NCPF 7594)

aStrains indicated with one asterisk are not included in the phylogenetic analysis; these strains are used only in the susceptibility tests. The strains labeled with two asterisks are included in the phylogenetic study but not in the susceptibility tests. NT, neotype; HT, holotype; isoT, isotype. CBS, culture collection of the CBS-Fungal Biodiversity Centre, Utrecht, Netherlands; UMCN, culture collection of Radboud University Nijmegen Medical Center; DTO, internal culture collection of CBS-Fungal Biodiversity Centre.

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incubation, the colony diameters were measured and the reactions on creatine agar recorded.

Phylogeny and molecular identification.Strains were grown on MEA (Oxoid) for 4 to 7 days at 25°C. Genomic DNA was isolated using an Ultraclean microbial DNA isolation kit (MoBio), according to the manufacturer’s instructions. Frag-ments containing ITS region 1 (ITS1) and ITS2, including 5.8S rDNA) and a part of the␤-tubulin gene were amplified and subsequently sequenced and analyzed according to the procedure described previously (22). For parsimony analyses, PAUP (version 4.0) software was used (48) andByssochlamys verrucosaCBS 605.74 was used as the outgroup.

Antifungal susceptibility tests. The susceptibilities of the majority of the strains listed in Table 1 were tested; exceptions were the (ex type) strains of uncommon species in clinical environments, such asByssochlamys nivea,B. fulva,

B. verrucosa,B. lagunculariae,B. zollerniae, andP. brunneolus.Paecilomyces divaricatusis also uncommon, but it is included to provide a representative overview of the susceptibility of the members of thePaecilomyces variotii com-plex. Isolates were revived by subculturing twice on Sabouraud dextrose agar tubes for 5 to 7 days at 35°C. Conidial suspensions were adjusted spectrophoto-metrically and were further diluted in RPMI 1640 medium (withL-glutamine and without bicarbonate; Gibco BRL, Life Technologies, Woerden, Netherlands). Microtiter plates were inoculated with an initial concentration of 1⫻104to 5 104

conidia/ml, as recommended by the CLSI (formerly the NCCLS) for mold testing (30).

The antifungal activities of amphotericin B (Bristol-Myers Squibb, Woerden, Netherlands), flucytosine (5FC; Valeant, Zoetermeer, Netherlands), itracon-azole (Janssen Pharmaceutica BV, Tilburg, Netherlands), voriconitracon-azole (Pfizer, Capelle aan de IJssel, Netherlands), posaconazole (Schering-Plough, Maarssen, Netherlands), terbinafine (Novartis Pharma, Arnhem, Netherlands), and caspo-fungin (Merck, Sharpe, and Dohme, Haarlem, Netherlands) were determinedin vitrousing a broth microdilution method, according to CLSI guidelines (M38-A) (30). The concentration range for amphotericin B, terbinafine, itraconazole, voriconazole, and posaconazole was 0.016 to 16 mg/liter; a range of 0.062 to 64 mg/liter was used for 5FC and caspofungin. MICs were determined after 24 and 48 h of incubation. For amphotericin B and the azoles itraconazole, voriconazole, and posaconazole, the MIC was defined as the lowest concentration that showed no visible growth. For 5FC and terbinafine, the MIC was defined as the lowest concentration at which 50% inhibition of growth compared with that of the control was measured (32). For caspofungin, the minimum effective concentra-tion was determined. All susceptibility tests were performed in duplicate.

Nucleotide sequence accession numbers.The sequences newly generated in the present study are deposited in GenBank under accession numbers GU968650 to GU968703.

RESULTS

Identification.Identification of the strains was performed by

combining phenotypic characteristics and the sequences of the

ITS regions and part of the␤-tubulin gene. The investigated

clinical strains were maintained in various collections as P.

variotii or P. lilacinus. Critical examination of the cultures

showed that oneHamigera avellanea isolate and four

Talaro-myces eburneusisolates were present among the isolates which

had previously been identified asP. variotii. The ITS sequences

of three T. eburneusisolates (NCPF 7594, NCPF 7596, and

DTO 45I3) were identical and had 99.8% homology with the

type strain of T. eburneus(CBS 100538). Isolate DTO 49D4

was more divergent and shared 96.4% homology with the type

strain ofT. eburneusand 98.8% similarity with the type strain

ofGeosmithia argillaea(NRRL 5177). Although this strain is

more closely related toG. argillaea, we identified this strain as

T. eburneus, since both species are claimed to be conspecific

(53). TheT. eburneusstrains were isolated from patient

mate-rial in three separate cases: from the sputum of a patient with cystic fibrosis (NCPF 2801), from a blood culture (DTO 45I3), and from the peritoneal dialysis fluid and blood of a patient

(NCPF 7594 and NCPF 7596).P. variotii superficially

resem-blesT. eburneusin its olive brown conidial colors and

thermo-philic nature. However, it differs in growing very slowly at 25°C (attaining a diameter of between 10 and 25 mm) and having a

Geosmithiaanamorph. Geosmithiaanamorphs are character-ized by cylindrical phialides, ornamented conidiophores and phialides, and cylindrical conidia (Fig. 1). A further

presump-tive isolate ofP. variotiiwas identified asH. avellanea. The ITS

sequence of this strain had a similarity of 98.0% with the type strain of this species (CBS 295.48). This species

macroscopi-cally resembles P. variotii in many respects and also forms

powdery olive brown colonies, and it has a high growth rate at

25°C and 37°C. However,H. avellanea can be distinguished

fromP. variotiiby the presence of aMerimbla-type anamorph

(Fig. 1). Three strains were identified asP. lilacinus, and the

ITS sequences of these strains have 100% homology with the

type strain ofP. lilacinus(CBS 284.36).

In the present study, we have adopted the taxonomy of

Paecilomyces variotiiand related species proposed by Samson et al. (45), which uses morphological characteristics, in combi-nation with extrolite data and sequences. Combined molecular

and morphological examination of the remaining P. variotii

sensu lato isolates showed that three different species were

present, namely,P. variotii,P. formosus, andP.

dactylethromor-phus. P. variotii was the species encountered the most

fre-quently (12), 11 isolates were identified asP. formosus, and 1

isolate was identified as P. dactylethromorphus. These three

species can be differentiated on the basis of morphological

criteria.P. variotiimorphologically resemblesP. formosus, but

the latter produces acid components on creatine agar and

grows faster at 30°C than at 37°C.P. dactylethromorphuscan be

differentiated from the other two species by its cylindrical conidia and regular branched conidiophores (Fig. 1). Figure 2 shows the results of the phylogenetic analysis of the ITS and partial tubulin sequences. Both sequenced regions gave similar

identification results, and all the species of theP. variotii

com-plex can be differentiated by either their ITS or partial tubulin sequences. A high degree of variation was present in the ITS

and tubulin sequences of theP. formosusisolates. Two distinct

groups, with high bootstrap support, were observed. A propor-tion of the isolates (5) formed a group together with the type

strain ofP. formosus, and the other group clustered together

with the type strain of P. lecythidis. These two groups could

represent two cryptic species but were not treated as such since they are morphologically similar and produce the same pattern of extrolites (45). Three strains (CBS 296.93, CBS 297.93, and

CBS 298.93) received asP. variotiivar.zaaminellaand claimed

to be the causal agent of zaaminellosis (11) were identified as

P. formosus.

Susceptibility testing.Susceptibility data, i.e., the geometric

mean (GM) of the MIC and the MIC range, are shown in Table 2. For those isolates that were not inhibited by the highest drug concentration, the next higher concentration was used to calculate the GM MIC; if no growth was observed in the well with the lowest drug concentration, the next lower concentration was used. The activities of most of the antifungal agents differed between the seven different species.

Posacon-azole and terbinafine showed goodin vitroactivity against all

the species tested. Posaconazole showed the lowest MICs, as

all isolates exceptT. eburneuswere inhibited by a

concentra-tion of 0.25 mg/liter;T. eburneushad slightly higher MIC

val-ues (MIC range, 0.25 to 1 mg/liter). All isolates (except oneP.

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[image:4.585.134.450.46.689.2]

FIG. 1. Macro- and micromorphological features of various species related toP. variotii. Columns, from left to right, MEA, conidiophores, and conidia, respectively; rows, from top to bottom,P. variotii,P. formosus,P. dactylethromorphus,Hamigera avellanea,Talaromyces eburneus, and Paecilomyces lilacinus, respectively. Bars, 10␮m.

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formosusisolate) were inhibited by terbinafine at a concentra-tion of 1 mg/liter or lower. Itraconazole was the second most

active azole, having in vitro activity against all Paecilomyces

species except P. lilanicus and T. eburneus. The P. lilanicus

isolates were not inhibited by itraconazole, which was also true

for three of four of T. eburneus isolates. H. avellanea was

moderately susceptible to itraconazole, but only two isolates were available for testing. Voriconazole was the least active

azole and hadin vitroactivity only againstP. lilanicusandH.

avellanea. Amphotericin B was activein vitroagainst all species

tested with the exception ofP. lilanicusandT. eburneus.

Flucy-tosine was also active against most of the species tested; the

exceptions wereP. lilanicusandH. avellanea. For amphotericin

B, itraconazole, posaconazole, voriconazole, and flucytosine, little intraspecific variation in antifungal susceptibility was noted. This was not the case for caspofungin, where minimal effective concentration values varied between 0.063 and 4 mg/ liter for different isolates of the same species.

DISCUSSION

Of 32 isolates which were identified as P. variotii by their

phenotypic characteristics, 5 were shown here not to belong to

the genusPaecilomycesand instead proved to beTalaromyces

eburneus(anamorph,Geosmithia argillacea) orHamigera avel-lanea(anamorph,Merimbla ingelheimense). These two species

superficially resembleP. variotii, but the micromorphology is

distinct from that of Paecilomyces (35). The occurrence of

these two species in clinical environments might be more com-mon than has been noted to date. Screening of the StrainInfo

bioportal (www.straininfo.net) identified two otherHamigera

isolates that have been reported from clinical environments. One isolate (CBS 128.90) originates from continuous ambulatory peritoneal dialysis liquid of a dialysis patient, and the other

(UAMH 2531), maintained under the anamorphic nameM.

in-gelheimense, was isolated from skin between the toes of a man.

Multiple isolates ofG. argillaceaoriginating from bronchial

wash-ings (UAMH 7717, UAMH 8639, UAMH 9714, UAMH 9854, UAMH 10232), a brain abscess (UAMH 9833), and a dissemi-nated infection in a German shepherd dog (UAMH 10932, UAMH 10933 [21]) are also present in the UAMH culture collection. In addition, this species has recently been proposed to be a potential new pathogen that colonizes patients with cystic fibrosis lung disease (6, 19). The remaining isolates

be-longed to three differentPaecilomycesspecies.P. variotiiandP.

formosuspredominated, although one isolate ofP. dactylethro-morphuswas also detected. The presence of these species in the clinical samples might be explained by the fact that these species occur more commonly in food and indoor

environ-ments than the other members of theP. variotiicomplex (J.

[image:5.585.74.498.70.385.2]

Houbraken, unpublished results).

FIG. 2. One of the most parsimonious trees from each of the two analyzed loci sequenced. (A) ITS1, ITS2, and 5.8S rDNA (consistency index⫽ 0.796; retention index⫽0.938, rescaled consistency index⫽0.747); (B) partial beta-tubulin data (consistency index⫽0.745; retention index⫽ 0.909; rescaled consistency index⫽0.677).

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Species identification of fungi in the past has primarily been based on morphological features. However, identification solely on the basis of morphology appears to be difficult, and trained staff is required for correct identification. Identification of fungi from clinical samples might even encounter the prob-lem that isolates grow atypically on inappropriate agars or become atypical if antimycotics are used (31). Therefore, mo-lecular-based methods, such as sequencing, appear to be more reliable and are a robust alternative to discriminate fungal species (4, 9, 17, 39). Sequencing data are objective and fast, and reliable identification of uncommon species can be ob-tained. The ITS regions are recommended for use for identi-fication of species in a clinical setting, since they are easy to amplify and large data sets are present in various databases, such as GenBank and European Molecular Biology Labora-tory Nucleotide Sequence Database. These databases will ex-pand dramatically in the near future since the ITS region has become the prime bar coding region (5, 46; U. Eberhardt, personal communication). The disadvantage of the ITS region is that it does not have sufficient discriminatory power in

various genera, for example, the generaAspergillus,

Penicil-lium, and Fusarium (5, 18, 33, 47). In this study, the ITS

regions and part of the␤-tubulin gene were used, and both

loci were shown to exhibit sufficient interspecific variation for identification purposes.

Paecilomyces variotii is a commonly occurring species and has previously been isolated from various substrates. Immuno-suppression is the critical risk factor for infection; and cases of pneumonia (7), sinusitis (13, 34, 50), endophthalmitis (25, 49), otitis media (12), wound infection in a transplant recipient (26), cutaneous hyalohyphomycoses (3, 29), onychomycosis (2), osteomyelitis in a patient with granulomatous disorder (10), and dialysis-related peritonitis (38) have all been re-ported to be caused by this fungus. This species can be con-sidered extremotolerant and is able to grow at high tempera-tures, on decaying wood, and on creosote treated wooden utility poles (E. de Meyere et al., unpublished data). The ability to grow on creosote-treated wooden poles suggests that this species is able to break down aromatics and is able to grow under stressful conditions (very hot conditions, dry conditions,

conditions very low in micronutrients). Additionally,P.

formo-sushas also been isolated from toluene gas biofilters. However,

these isolates were misidentified asP. sinensisandP. variotii,

and the correct name for these isolates isP. formosus(14, 16,

37). The extremotolerant nature is suggested to contribute to

the pathogenic potential of fungi. Prenafeta-Boldu´ et al. (37)

speculated that there might be a link between neurotropism and assimilation of aromatic substrates, and this might be one of the factors that enable fungi to grow in the human brain, with its unique chemical properties. This suggested link is also found in our study, as we have also encountered four strains of three independent cases originating from brain or cerebrospi-nal fluid.

Correlations of species identities with susceptibility profiles.

Major differences in in vitro antifungal susceptibility profiles

were found between the investigated species. In general,

vori-conazole is not active against members of the Paecilomyces

variotiicomplex but is active againstP. lilanicusandH.

avella-nea.Treatment of infections due toP. lilanicusmay be

com-plicated, as amphotericin B also showed no activity in vitro.

TABLE 2. Susceptibility results for Paecilomyces species, Hamigera avellanea , and Talaromcyes eburneus strains, by species and antifungal agent Species No. of isolates Geometric mean (range) MIC (mg/liter) a AMB 5FC ITZ VCZ POS TB CAS P. variotii 16 0.11 (0.03–0.5) 0.03 0.04 (0.008–4) 11.3 (1–32) 0.02 (0.008–0.125) 0.08 (0.031–0.5) 0.52 (0.063–4) P. lilanicus 4 32 128 5.7 (0.063–32) 0.15 (0.063–0.25) 0.2 (0.008–0.25) 0.04 (0.031–0.063) 0.59 (0.5–1) P. formosus 14 0.13 (0.063–0.25) 0.04 (0.031–0.25) 0.13 (0.063–1) 26.25 (16–32) 0.08 (0.031–0.25) 0.12 (0.031–2) 1.16 (0.063–4) P. dactylethromorphus 3 0.16 (0.125–0.25) 0.05 (0.031–0.125) 0.08 (0.031–0.125) 32 0.03 0.31 (0.125–1) 0.25 (0.125–0.5) P. divaricatus 2 0.25 (0.125–0.5) 0.008 0.5 (0.25–1) 32 0.18 (0.125–0.25) 0.06 (0.031–0.125) 0.71 (0.25–2) H. avellanea 2 0.5 2 (1–4) 0.031 0.09 (0.063–0.125) 0.031 0.04 (0.031–0.063) 0.063 T. eburneus 4 3.3 (1–8) 0.032 12.5 (1–32) 28 (16–32) 0.69 (0.25–1) 0.032 0.31 (0.25–0.5) a For caspofungin, the minimum ef fective concentration was determined. AMB, amphotericin B; 5FC, flucytosine; ITZ, itraconazole; VCZ, voriconazole ; POS, posaconazole; TB, terbinafine; CAS, caspofungin.

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Posaconazole may be the only appropriate alternative agent, although the lack of an intravenous formulation and limited penetration into the cerebrospinal fluid might limit its use. Amphotericin B showed good activity against all other species tested, as was also the case for flucytosine. The combination of amphotericin B and flucytosine may therefore be an option in

complicated infections due toPaecilomycesspecies other than

P. lilanicus. Flucytosine was recently shown to be activein vitro

andin vivoagainstA. fumigatus, with the MIC measured at pH 5.0 being found to correlate better with the outcome in a murine model of disseminated aspergillosis than that deter-mined at pH 7.0 (51). It would be of interest to determine the activity of flucytosine at pH 5.0 against other molds, including

Paecilomycesspecies. As published previously, terbinafine also shows potent activity against all species tested (8). However, the clinical use of this drug for the treatment of invasive fungal infections remains limited due to its pharmacological

proper-ties. The role of the echinocandins remains unclear, as thein

vitroactivity of caspofungin was variable, with the MIC ranges within species being broad. This indicates that the activity of the drug is not easily predictable, thereby precluding a

prom-inent role in the first-line therapy ofPaecilomycesinfections. In

general, the antifungal susceptibility profiles ofP. variotii,P.

formosus,P. dactylethromorphus, andP. divaricatusappeared to be similar, although a limited number of species have been

tested. The profiles ofP. lilanicus,T. eburneus, andH. avellanea

are different. This is in agreement with the phylogeny, sinceT.

eburneusandH. avellanea are not related toPaecilomycesor

Byssochlamys (45, 53) andP. lilacinus will shortly be accom-modated in a new genus because it is only distantly related to

P. variotiiand the other species hitherto placed in the genus

Paecilomyces(27, 28). In summary, it is clear that correct

spe-cies identification ofPaecilomycesisolates is important to help

guide appropriate antifungal therapy. The correlation between the in vitro activity and the in vivo efficacy of these agents

againstPaecilomyces species remains to be investigated

fur-ther.

ACKNOWLEDGMENT

Andrew Borman thanks Elizabeth Johnson for her interest in the study and permitting him to collaborate in this study.

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Figure

TABLE 1. Isolates used in this study
FIG. 1. Macro- and micromorphological features of various species related to P. variotiiPaecilomyces lilacinusconidia, respectively; rows, from top to bottom,
FIG. 2. One of the most parsimonious trees from each of the two analyzed loci sequenced
TABLE 2. Susceptibility results for Paecilomyces species, Hamigera avellanea, and Talaromcyes eburneus strains, by species and antifungal agent

References

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