Ecology and Physiology of the Emerging Opportunistic Fungi Pseudallescheria Boydii and Scedosporium Prolificans

Home , Microascaceae, Pseudallescheria, Scedosporium

MYCOSES 37, 71-78 (1994) ACCEPTED:NOVEMBER 30, 1993

Ecology and physiology of the emerging opportunistic fungi Pseudallescheria boydii and Scedosporium prolificans

Okologie und Physiologie der opportunistischen Pilze Pseudallescheria bcydii und Scedosporiurn prohficans

G. S. de Hoog',2,Femke D. Marvin-Sikkemaj, Greetje A. Lahpoor3,J. C. Gottschal13, R. A. Prins3 and Eveline Gutho4

Key words. Pseudallescheria boydii, Scedosporium prol$can,, Microascaceae, taxonomy, diagnostics, pathogenetic determinants, physiology, anaerobic growth, opportunistic fungi. Schliisselworter. Pseudallescheria bigdii, Sccdo.doJpo7ium prolgcans, Microascaceae, Taxonomie, Diagnostik, Pathogenitatsfaktoren, Physiologic, anaerobes Wachstum, opportunistische Pilze.

Summary. Nutritionally physiological patterns nDNA/DNA Homologie-Werte sowie morpholo- of Pseudallescheria boydii (Microascaceae) and the gische und klinische Characteristika, nicht bestati- relat~dspecies Scedosporium prol$cans were estab- gen. Einige mogliche Virulenz-Faktoren bei den lished. Differences between the two species were oben beschriebenen sowie auch bei einigen ver- found in assimilation of sucrose, ribitol, xylitol and wandten Arten, wurden untersucht. Alle Arten L-arabinitol. In contrast, no physiological distinc- erwiesen sich im gleichen MaR zum anaeroben tion could be made between the three iritraspecific Wachstum befahigt, unterscheiden sich aber in variants of P. boydii which have been recognized ihrem Temperaturverhalten. on the basis of nDNA/DNA homology data and of morphological and clinical differences. Some potential virulence factors werc studied in the Introduction fungi mentioned above and in some related anamorphs. All species were capable of anaerobic The Ascomycete Pseudallescheria boydii (Negroni & growth, but differed in their temperature relations. Fischer) McGinnis et al. (fam. Microascaceae) is found rather commonly in soil and polluted water. Zusammenfassung. Assimilations-Muster von For a long time it escaped attention as a potential Pseudallescheria boydii (Microascaceae) und der ver- human pathogen because it wab known by a wandten Art Scedosporium prol$cans wurden unter- number of different names. This nomenclatural sucht. Dabei wurden Art-Unterschiede in der confusion was caused by its polymorphism, differ- Assimilation von Saccharose, Ribitol, Xylitol und ent strains being potentially capable of producing L-Arabinitol festgestellt. Im Gegensatz dazu Scedosporium- and Graphium-like anamorphs in wurden keine physiologischen Unterschiede addition to Pseudallescheria cleistothecia. zwischen den drei Varianten innerhalb der Art Several types of pathogenicity have been associ- P. hydii nachgewiesen. Damit lassen sich die ated with P. boydii. The species is particularly bisher verwendeten Merkmalsunterschiede, bzw. known to cause human white-grain mycetoma [ 11 and, more recently, it has emerged as an agent of 'Ccntraalbureau voor Schimmelcultures, Baarn, 'Institute for Molecular Cell Riolo~~,Amsterdam, 'Department of systemic and disseminated mycoses [2, 31. It is Microbiology, Haren, The Netherlands; 4UnitP de Mycologie, found subclinically in the lungs of patients with Insritut Pasteur, Paris, France. leukaemia [4] or cystic fibrosis [5]. In addition, it can cause of otitis externa with discharge [6, 71. Correspondence: Dr G. S. dr Hoog, Centraalbureau voor Schimmelcultures, PO Box 273, NL-3740 AG Baarn, The It rarely causes keratitis [8]. Netherlands. In their review of pseudallescheriasis, Warnock 72 G. S. DE Hooc ET AL.

& Richardson [9 1 distinguished between the mor- Ceritraalbureau voor Schimmelcultures, Baarn bidity of localized and disseminated dccp mycoses (CBS), The Netherlands, and of the Unit6 de by P. Boydii. Disseminated mycoses, in which Mycologie, Institut Pasteur, Paris (IP). Stock rieurotropic involvement is frequent, are mostly cultures wcrc inoculated with conidial suspensions fatal despite aritimyc,otic therapy. The portal of and grown on oatmeal agar (OA) or 4?4n malt entry also varies, being via the sinuses in localized extract agar (MEA) slants at 30 "C. Strains 9.1 mycoses and possibly via the lungs in deep and 1.2 were isolated under 80?h N2/20'%CO, mycoses. Gutho & de Hoog [ 101 suggested a and cultivated on 1% malt extract (ME)/O.4.%> possible molccular basis for the purported differ- yeast extract/0.4% glucose flushed with N2, final ences in pathogenesis. Using nDNA/DNA reas- pH 7.0. For karyolo'gy, strains were grown on sociation techniques, they established three permeable PT cellophane membranes, eventually infraspecific entities with difl'erent sources of iso- covered by a second niembrane. Colony diam- lation, namely lungs, sinuses and soil. As a conse- eters were measured twice weekly on MEA petri quence, human infection by P. boydii might be dishes incubated in the dark at 24, 30, 36 and more complicated than originally supposed. Thc 42 "C until thc stationary growth phase. prcserit paper focuscs on the ecology and nutritional physiolcgy of thc P. bqydii variants and of related species in order to characterize further the clinically relevant taxa of Microascaceae. Anaerobic growth Among these, Scedosporium prol$cnns (Hennebert & Strains were grown in Hungate tubes Desaij Gukho & de Hoog is particularly emerging (16 x 125 mm) fitted with butyl septum stoppers as an opportunist in humans [ 1 1 - 13 and is there- (Bellco Glass, Vineland, NJ, USA), containing fore considered in more detail. 1 O-ml aliquots of I O/O malt cxtract/0.4% yeast extract/0.4% glucose/2% agar medium, flushed with 02-free COP and subsequently autoclaved. Materials and methods Filter-sterilized vitamin and haemin solutions were added to a basal growth medium [ 141. Strain I and i ulture condhns Fungi were grown under 0,-free N2 (80%j/CO2 The strains used, which are htcd in Fig. 1, are (20%) gas phase (pH 7.0) and subcultured every maintained in the culture collections of the 3 4 days.

Group 1 (94-99%) Group 2 (8596%) I 1 Ps Gr CBS 101.22 Mycetoma CBS 297.38 Wood pulp -+ CDC A 495 Knee arthritis CBS 695.70 Sinus, swine -t IP 1251780 Man? CBS 987.73 Otitis -f IP 1448.83 Abscess IP 141 1.82 Mycetoma - - IP 1698.87 Lung, leukaemia 53-72% IP 1907.90 Sinus -+ IP 1945.90 Sputum, CF IP 1946.90 Sinus -+ IP 1947.90 Sputum, CF IP 2003.91 Foot -+ IP 1988.91 Sputum, CF IP 2004.91 Blood -+ IP 1989.91 Sputum, CF IP 2008.91 Bone -+ IP 2002.91 Sputum, lymphoma 1.2 Mud -+ M-79 Lung, leukaemia 9.1 Mud -4- M-175 Lung, aspergillosis IP 3350.91 Lung, CF -+ IP 3359.91 Sputum -+

Group 3 (83%)

CBS 108.54 Soil IP 1742.88 Soil I'" "'I

Figure 1. Chioiriic rclationsliips among isolatrs (11 Pseudoulle.rrhuna bydii and prevalent form ofsporulation in addition to Svdusporiuin anarrrorph. Pr, 1'widullr.v hnu; Gr, fhph'iurn synnrmata; C:DC, Centers kJ1Disease Control (Atlanta, GA, LISA); CBS, Centraalbureau voor Schitnrrit.lrultures, Baarn, 'I'tte Nctherlauds; I P, Institute Pastcur (Franre); Ct;,cystic fibrosis.

niycoses 37, 7 1 711 (1994) PSEUDALLESCHERIABOYDII AND SCEDOSPORICJMPROLIFICANS 73

Anaiysi c of metabolic products Staining Alcohols arid short-chain fatty acids, both volatile Cell wall glucans and nuclei were fluorescence and non-volatile, were analysed by gas chromatog- stained after fixation in 75% ethanol followed by raphy in cultures grown for 2 weeks at 30 "C in 0.1 O/O Calcofluor white (Sigma), 10% dimethyl- 0.5 x, 1 x, 2 x and 4x concentrations of the sulphoxide (DMSO) and a freshly prepared mix- standard medium. Hydrogen concentrations were ture (1: 1, v/v) of (a) 2.5 mg of ethidium bromide, measured as described by Gerritse et al. [15]. 1.2 g of Tris buffer and 0.6 g of NaCl in 100 ml Protein contents were measured in co-cultures by of H,O (pH 7.4) and (b) 5 mg of mithramycin the method of Lowry et al. [ 161. and 125 nig of MgCI,. Chitin staining was performed using 1 O/O primuline solution. Melanin was stained in a freshly prepared Schmorl's solution Physiology [K,Fe(CN), 1%, 2 ml, FeCI, 1%, 15 ml, aqua dest Comparative nutritional tests were performed in 3 ml], then with 0.1% nuclear fast red (Merck, liquid medium at 24 "C by methods standardized Darmstadt, Germany) in 5% Al,(SO,), and by Van der Walt & Yarrow [17]. Gas production observed in xylol. Ainyloid reactions were in non-agitated liquid 2% glucose/nitrogen base observed in Melzcr's reagent. medium was tested with Durham inserts. Tolerances of NaCl and MgC1, (2'10, 5% and 10%) Single-spore isolations were tested using 5% glucose with nitrogen base Suspensions of conidial cultures were made in 1O/O as growth medium. Cycloheximide tolerance (0.01, Tween 60. Ascomata (cleistothecia) were grown 0.05 and 0.1%) was tested in liquid medium between two cellophane membranes, cut out under according to Van der Walt & Yarrow [ 171, as well a stereomicroscope, vortexed several times in dis- as on commercial Mycosel agar (BBI>;0.040/0), in tilled water and squashed under a microscope which case inhibition was measured relative to slide, series of concentrations of spore suspension growth on Sabouraud's glucose agar (SGA) and a in a.d. being incubated on OA at 30 "C. scored as 'weak' when growth was 50-70%. Diazonium blue B (DBB) reactions were performed according to Hagler & Ahearn [IS!, with Results C'?yptococcus nedormans as control. The presence of urease was tested in slants on Christensen's urea On the basis of DNA/DNA reassociation experi- agar and acid production on chalk agar. Gelatin ments, three variants could be distinguished within liquefaction was tested in slants at 21 "C and read Pseudallescheria boydii. Strains of group 1, which after 10 min incubation at 4 "C. Proteolysis was have an intra-group DNA homology of 94-99%, also tested in Petri dishes with tryptone/yeast reassociate at 53- 72% with members of group 2, extract/gelatin medium (Brocades), using a which have intra-group DNA homoloLgyvalues of HgCl,/HCl solution as indicator. Production of 85- 96%. Most strains of group 1 were isolated siderophores was tested on CAS agar modified from the lungs of human patients; none came after Schwyn & Neilands [ 191 [SGA with Chrome from the oropharynx. In contrast, four strains of Azurol S (Sigma) 60.5 mg, cetyltrimethylammo- group 2 were isolated from human or animal niurnbromide (CTAB) 72.9mg and 1 IIIM FeCl,; oropharynx and only two from human lungs. Both final pH 4.51. Widths of pale halos on a purple groups contain members occurring in nature: a background were measured. Production of alkaline strain isolated from a case of traumatic implant- compounds was measured on the same medium ation was found in group 1 and strains from wood with a Yhilips PW 9410 digital pH-meter with pulp and mud were found in group 2. A third, surface electrodes. Production of phospholipase small group, reassociating at 65-70'?/0 with mem- was tested according to Price et al. [201, using egg bers of groups 1 and 2, contained two isolates yolk (Ilifco) as substrate; Candida albicans was used from soil. as control. Fresh isolates of members of group 1 usually sporulate abundantly with ascomata. A Graphium synanamorph is usually absent, or some small, nDNA/DNA reassociation scattered synnemata are produced. None of the Nuclear DNA was isolated from wet cells obtained members of group 2 was found to produce cleisto- frorn 1 1 of liquid medium after 3 days of growth. thecia. In these cultures, large Gaphiurn synnemata Purification was performed according to GuCho & were usually present in abundance (Table 1). de Hoog [ 101 using a Gilford Response I1 spectro- Analysed members of group 3 lacked a Graphzum photometer with thermoprogrammer. synanamorph. myroses 37, 7 1-78 ( 1994) l'he form of propagation in P. byydii was ana- synnematous microascaceous species not known lyscd in strain TP 1251-80 (group l), which to occur on humans showed no growth at 37 "C:. produces cleistothecia and srriall synnemata in Most strains of P. boydii were unable. or weakly addition to a Scedosporium anamorph. Under able to assimilate L-sorbose, D-arabinose, methyl- microaerobic conditions, cleistothecium pro- a-glucoside, raffinose, melezitose, inulin, glycerol, duction is stimulated. Optimal production is methanol or higher alcohols and sugar acids obtained at 30 "C using asc,ospore or conidiurn (Table 2). Nitrates were assimilated, with the suspensions in nutritionally poor media. exception of creatine and creatinine. The species Production is stimulated when expansion growth differcd from Scedosporium prol$cans by assimilation is arrcstcd, e.g. at thc edge of the Petri dish. of sucrose, ribitol, xylitol and L-arabinitol and no Ascomata are formed deep in submersion, later or little growth with glycerol. S. prol$clicnns could becoming emcrgent and covered with few setae riot grow in the presence of cycloheximide, while which actively turn away when approached by an the serisitivity of P. bydii was variable. Little inoculation needle. growth of l? boydii was observed in 5% NaCl or Under cellophane the Scedosporium anamorph of 1 O"/O MgC1,. Urease was consistently produced; P. boydii consists of single, lateral conidia on DBB reactions were insignificant. No phospho- hyphac, while in supcrficial colonies differentiated lipases or starch-like cornpounds were detected. In conidingcnous cells producing conidia rcpetitively most strains srriall amounts of siderophores were in slimy heads are observed. Small Graphitm synne- excreted. Alkaline compounds were generally niata are found especially on drying parts of the detected, staining chrome azure SCAS agar deep colo~iy,particularly near the edge of slant cultures. blue; in the two strains of P. bqydii group 3 the Hyphal cells, conidia arid ascospores were entire Petri dish showed a neutral or slightly invariably found to be uninucleate. Ascospore alakaline pH (l'able 2). nuclei stained with difficulty, while conidial nuclei showed intense staining with ethidiurn bromide/ mithramycin. Hyphal cell walls stained well with Discussion Calcofluor, but conidia remained unstained. Extracellular clumps containing melanin-like com- Pseudallescheria bqydii is closely related to Scedosporium pounds were demonstrated alongside hyphae with prol$cans ( = Sc. inzatum Malloch & Salkin), judging Schmorl's solution. No arnyloid reactions were from morpholoby of anamorphs and from simi- noted. larities in nutritional physiology. P. boyrfii is physio- Single-sporc isolations were made from asco- logically distinguished from the latter by being spores arid from Scedo.$oraum and C;ra$hium. conidia ablc to assimilate sucrose, ribitol, xylitol and I>- of P. hoydii, IP 125 1 --80. No significant differences arabinitol, but it is unable or weakly able to utilize were observed between conidium and ascospore glycerol. Salkin et al. [l I] and Dixon & Polak- subcultures, all being able to produce cleistothecia. Wyss [21] found the two species to differ in Sonic isolates fortncd sectors of higher growth cycloheximide tolerance when tested on Mycoscl rate, lacking cleistothecia, or formed some scat- agar. This was confirmed in our study, at least tered cleistothecia only. In the latter case subcul- weak growth being obtained in P. boydii and none tures of thcsc sectors could he restored to their in Sc. prol&ans (Tablc 2). However, when the more origirutl cleistothccial state when asc.ospores werc sensitive test in liquid medium was applied, grow lh transferred. proved to be strain dependent in P. boydii (Table 2). Strains grown in non-agitated test tubes with In contrast to Dixon & Polak-Wyss 1211, the species Durham inserts showed insignificant gas pro- were riot found to differ in urease test results. duction. In anacrobic culture, small amounts of Morphologically, the inflated bases of conidio- carhon dioxide, ethanol, acetate, lactate and succi- genous cells are an important feature to recognize nate were detected (Table 1). Hydrogen and butyr- Sc. prolijicans. Sometimes, however, single lateral ate were often found to be produced in trace conidia are prevalent and therefore the availability amounts; propionate and formate remained of additional physiological tests is useful. Salkiri absent. The quantities of fermcntation products et al. 1221 contested the identity of Lornentospora incrcased linearly with the concentration of the prolijicans Heririebert & Desai and Scedo.$oriurn in@ growth medium. No significant differcnces were tum Malloch & Salkin, as supposed by Gueho & observrd between strains and species from nature de Hoog 1101 using DNA/DNA reassociation atid strains from humans. experiments, on the basis of as yet unpublished All P. bqydii and Scedosporium prol$cans isolates differences in restriction fragment length polymor- grew optirnally betwcen 30 and 37 "C. Some other phisms. However, the physiological patterns of the

mycoses 37, 7 1-78 (1994) PSEUDALLESCHERIA BOYDII AND SCEDOSPORIUM PROLIFICANS 75

Table 1. Fcrmentation products (in mM) of Pseudullesrh~riu, Gruphiurn and Srrdosporium spp. after 14 days of anaerohir growth, avcragc of’ duplicatc tests proportiorial to pg of protciii produced

--~~ ~ ~ Strnlil H, co, Ethniiol Acrtatr Butyrate Lactate Succinate

P. hoyd~zvariant 1 IP l(i98.87 tr 0.29 0.27 U.05 tr 0.13 0.02 I€’ 1945.90 tr 0.27 0.27 0.05 tr 0.10 0.02 CHS 101.22 0.17 0.16 0.07 tr 0.15 0.02 cl)(: ;1-49.5 tr 0.22 0.19 0.06 tr 0.42 0.07 1’. b(!ydii variant 2 CKS 987.73 0.20 0.17 0.10 tr 0.05 0.0 I C:HS 695.70 0.26 0.21 0.09 0.01 0.27 0.03 CKS 207.38 tr 0.35 0.35 n.o3 tr 0.16 0.02 !). 1 tr 0.23 0.20 om tr 0.14 0.02 I .2 (r 0.19 0.19 0.08 tr 0.13 tr P. bqdii variant 3

CKS 108.54 ~ 0.23 0.24 0.07 tr 0.13 0.01 11’ 1712.88 tr 0.16 0.15 0.04 tr 0.04 tr

,s/. /JTff/t/iffIi\ 467.74 tr 0.13 0.15 0.06 tr 0.12 0.01 1 14.90 tr 0.30 0.26 0.07 tr 0.11 tr

G. ( i~ii~;ijeri~in 779.85 tr 0.26 0.29 0.04 tr 0.14 0.03 s”. rhmtoikoon 111.1-1 tr 0.15 0. I4 0.08 tr 0.13 tr 3.5 7.31 i 0.37 0.20 0.29 tr 0.11 0.01

(;. IfYflJlZU? 127.8,1 tr 0.44 0.49 0.08 tr 0.04 tr

ti. tra e. two type strains, CBS 114.90 and CBS 467.74 ium formation is repetitive, conidia being pro- respectively, are remarkably similar (Table 2). duced in heads [27]. The submerged conidiation ‘l’he molecular variants of P. boydii found by possibly enables P. boydii to disseminate haemato- GuCho & de Hoog [ 101 are physiologically indis- gcnously. The fungus often rapidly invades the tinguishable. Most strains tolerate 2% NaCl and central nervous system [3]. Judging from its sidero- 5’10 MgC1,. Kirk [ 231 obtained optimal growth in phore activity, it is iron dependent. In contrast to seawater (1.7-2.8’h salinity). He found that cleisto- serum, CNS tissue contains free iron, which may thecium formation was hampered and synnema partly explain the neurotropic behaviour of the production stimulated at higher salt concen- fungus. Siderophores are also found in the equally trations. This underlines the slightly osmophilic neurotropic black yeast Exophiala dermatitidis [28] nature of the synnematal morph, which develops and are insignificant in the superficial agent Hortaea particularly on drying parts of colonies. werneckii [ 2 91. All tested microascaceous species are able to Single-spore isolations, whether from ascospores grow anaerobically and produce similar small or from the two types of conidia, resulted in amounts of fermentation products (Table 1). No identical, ascosporulating cultures. Lingappa & or only slight fermentation is observed in standing Lingappa [ 301 presented evidence that ascospores text tubes with Durham inserts (Table 2). are more heat shock resistant than conidia. The Submerged growth is strictly hyphal arid mostly entire thallus of the fungus is uninucleate. Croziers without any macroscopical trace of melanin; were not observed, asci being directly produced sporulation is relatively abundant. P. boydii and Sc. from ascogonia. The fungus is thus truely homo- prol$cans are able to produce conidia in submersion thallic, each uninucleate cell being able to produce in riitro, as well as in host tissue [ 12, 24, 251, which the sexual form of propagation. Cleistothecial is uncommon in human pathogenic hyphomycetes cultures showed recurrent production of sectors [26]. A single conidium is formed on each coni- with different expansion growth and lacking cleis- diogenous cell, as observed in agar cultures under tothecia, confirming earlier observations by El-Ani cellophane membranes. In aerobic culture, conid- [3 11. Occasionally some scattered cleistothecia m)roses 37, 71-78 (1994) 76 G. S. DE Hooc ET AT..

Table 2. physiological patterns of I2 strains of PmdulleJchtria hnydii arid .Sxdn.cpnriiim prul$can.r

1'. hnydzi Sc. prolijcam

Variant I Variant I1 Variant 111 1251.80 1698.87 101.22 297.38 695.70 1.2 9.1 108.54 1742.88 1919.90 114.90 467.74

+ + t + - - + - + + t t + - W - w w + + W + u

~ - + + t + + + + __ W M' W - - W

- - + +

- ~

- - + + W W + + W W + - - -

W - + - + W

- ~

- -

- - + W + + + t t + + t t + - - - -

2 3* 2.4 39 4 .0 2.8 3.9 0.3 0.1 + \h u - - + + + W

rnycoses 37, 71 78 (1994) PSEUDALLESCHERIA UOYDII AND SCEDOSPORIUM PROLIFICANS 77

Table 2. (cvntinued

P. boydii Sc, prvlificans

Variant 1 Variant I1 Variant I11 1251.80 1698.87 101.22 297.38 695.70 1.2 9.1 108.54 1742.88 1913.90 114.90 467.74

10"b RlgC:1, W - w- - - - W W - W W W 0.(15"'n Cycloheximide - - +- + -- + + - - - 0. I '%I Cycloheximide - - +- W -- + + - - - Mycostl W W ++ +WW + + - - - Starch production ------Urcahe W + ++ + +W + + + + + 11 13 B - - - - - WW - - - W - Phospholipase ------Grlat in liquefaction + + ++ + ++ + + + + + Acid production ------Sidrt-ophon- halu 1 3 5 2 2 ent II 5 2 en1 2 ND pH UII CAS margin 5.6 4.5 6.7 4.6 7.9 8.7 8.2 8.0 7.3 8.6 5.2 ND pH on <:AS centre 5.6 7.5 6.7 7.9 8.0 8.7 8.3 8.0 7.9 8.6 5.2 ND

*Daily c.xpansion growth in mm. ent. cntire dish homogmeously discoluurcd. were formed in a very late stage. A possible tidal waste also fit this category [23, 40, 411. explanation is that cells are normally diploid. Patients who have nearly drowned in such waters Local spontaneous haploidization may occur, frequently develop fatal, neurotropic, disseminated growing out with anamorph sectors. Later the mycoses 1421. original condition may be restored by local somatic In nature, Pseudallexheria boydii occurs in nutri- diploidization. In contrast, Corlett [ 321 supposed ent-rich, poorly aerated environments, such as that the fungus would be a haplont; mitosis, polluted water. From these environments it may karyogamy and meiosis would all take place within colonize cavities inside animal hosts, as is proven a single cell, the ascogonium. by its asymptomatic occurrence, e.g. in lizard DNA/DNA reassociations confirmed the exist- lungs [43], where it lives on epithelial or inhaled ence of several inolecular variants within the debris. In patients with leukaemia or cystic fibrosis, specks P. boydii [ 101. Isolates with intermediate biomass production may reach detectable levels. DNA homology broadly correspond with sources The synnematous variant is slightly osmophilic of isolation and with morphology. Members of the and is hence relatively often found in debris cleistothecial variant (1) have mainly been isolated accumulated in the oropharynx. from deep locations in patients with impaired, innate cellular immunity, while the synnematal variant (2) is particularly involved in otitis or Acknowledgements sinusitis of various species of warm-blooded ani- mals. In present-day clinical practice, the two M. Scholz, I. van Oosterom and W. Zijlstra are variants are encountered with equal frequencies. thanked for technical assistance. In older literature, however [24], the cleistothecial variant was much less common. Gordon [33] References isolated mainly cleistothecial strains from soil, possibly corresponding to our variant 3, while 1 Shear, C. L. (1922). Life history of an undcscribed strains with synnemata were rare. It may thus be Ascomycete isolated from a granular mycetoma of man. supposed that variant 1 in particular, taking advan- hlycologiu 14, 239 243. tage of human natural immunodeficiency, is 2 Fisher, J. F., Shadomy, S., Teabeaut, J. R., Woodward, J., Michaels, G. E., Newman, M. A,, White, E., Cook, P., emerging. Seagraves, A,, Yaghmai; F. Cir Rising, P. (1982). Near- In the literature, P. bydii is reported from drowning complicated by brain abscess due to Petriellidium substrates rich in nutrients, mostly soil [24, 341, boydii. Arch. Neurol. 39, 5 1 1-5 13. soil enriched by manure [GI, agricultural and 3 Brrengucr, J., Diaz-Mediavilla, J., Urra, D. & Mufioz, P, garden soil 123, 351, potted plant soil [36] or (1989). Central nervous system infection caused by Pseudallescheria bcydii: case report and review. Rev. hjct. manure [37, 381. The isolates from water mostly Dis. 11, 890-896. came from sewer [26] or heavily polluted water 4 Piens, M. A,, Jimenez, J. L., Guyotat, D., Bouvier, R. & or mud in stagnant ponds [Z, 391. Locations under Mojon, M. (1991). A propos de trois observations d'infec- mycosrs 37, 7 1-78 ( 1994) 78 G. S. DE HOOGEr AL.

tiorr hurnaiiic a Srfdosfinrium apzo~sprmzirr2.IiitkrBt dn traite- sporiilation in niarinc and clinical isolates of dllt.scherin nient par itraconazolc. J. htycol. Mbd. 1, 156-159. hq~diiShear. hlyropnth. h@~col.Apbl. 33, 65- 75. 5 Ctialxissc, D., Houchara, J. P., Chnzalettr, ,J. P., 21 Ajello, I,. (1952). The isolation of Allc.rchcrici bqyrlii Shear, (hrr+rr, ,J., Giriics, J. I,,, Gentile, L. dc & Cinion, H. an etiologic agent of mycctonias, firom soil. Am. ,7.7rol. (1991). Mucoviscitiosc et colonisation fongique ;i ScedoJpor- Mfd. Hyg. 1, 227-238. ium (ifiiospermum.J. itlyrol. Mid, 1, 152- 155. 25 Bcll, R. G. (1978). Coniparative virulrnce and iinrriuno- 6 Rippon, .J. W. & Carmichacl, J. W. (1 976). Petriellidiosis diffusion analysis of Pttripllidium bqydii (Shear) Malloch (Allcscheriosis): four unusual cases and review of thc strains isolated from fccdlot manure and a 1iiunai-i literature. h~yeoputhologia58, 117 124. mycctonia. Can. J. hficrobiol. 24, 856-863. 7 C;[ignani, H. 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mycoses 37, 7 1- 78 ( 1994)