Occurrence of Scopulariopsis and Scedosporium in nails and keratinous skin. A 5-year retrospective multi-center study

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1 Medical Mycology May 2007, 45, Occurrence of Scopulariopsis and Scedosporium in nails and keratinous skin. A 5-year retrospective multi-center study JOUNI ISSAKAINEN*, HANNELE HEIKKILÄ$, EEVA VAINIO%, PIRKKO KOUKILA-KÄHKÖLÄ, MIRJA CASTREN, OILI LIIMATAINEN, TARJA OJANEN$$, MARKKU KOSKELA & OLLI MEURMAN%% *Herbarium, University of Turku, Turku, $Department of Dermatology, Helsinki University Central Hospital, Helsinki, %Department of Dermatology, Turku University Hospital, Turku, Mycology Unit, Division of Clinical Microbiology, Helsinki University Central Hospital Laboratory, Helsinki, Centre for Laboratory Medicine, Pirkanmaa Hospital District, Tampere, $$Kanta-Häme Central Hospital, Hämeenlinna, Clinical Microbiology Laboratory, Oulu University Hospital, Oulu, and %%Clinical Microbiology Laboratory, Turku University Hospital, Turku, Finland A 5-year retrospective multicenter study was performed for microascaceous moulds (Microascaceae, Ascomycetes) in Finnish clinical specimens. The files from of six clinical mycology laboratories in Finland were searched for reports of these fungi, mainly Scopulariopsis and Scedosporium anamorphs in keratinous specimens. From the 521 primary findings, 165 cases were selected for further study based on direct microscopy, colony numbers and accompanying fungi. The clinical records of 148 cases (141 Scopulariopsis, 7Scedosporium) were studied. Of the nail infections from which Scopulariopsis was recovered, 39 cases were further separated which showed clinical or laboratory-based evidence of dermatophytosis. In the remaining 90 non-dermatophyte nail cases, Scopulariopsis spp. were the only documented fungal agents (c. 6 cases/million/year). The patients were mainly elderly, 66% of whom had problems involving their big toe nails. For 74% of them, the nail problem was mentioned as their reason for visiting the physician. However, only 18% had documented benefit from treatment. The Scopulariopsis nail infections seem to be treatment-resistant and the pathogenesis and etiological role of Scopulariopsis remain poorly understood. Keywords Scopulariopsis, Scedosporium, skin, nails Introduction More than 40 species in about 8 genera have been identified in the ascomycetous family Microascaceae [1,2] and more than 20 additional members of this family have thus far been recovered in their mitosporic (asexual) stages [3]. The reservoirs of these Microascaceae include numerous, partly species-specific outdoor niches, such as soil, dead plant material, dung and keratinous substrates [4]. Received 17 June 2006; Accepted 6 November 2006 Correspondence: J. Issakainen, Herbarium, University of Turku, FIN-20014, Turku, Finland. Tel: / ; mobile / jouni.issakainen@kolumbus.fi The main microascacean genus encountered in human keratinous tissues is Microascus in its mitosporic Scopulariopsis stage. Several members of this large genus, especially Microascus (Scopulariopsis) brevicaulis, can grow on nails, which may be confirmed by direct microscopy. In clinical nail samples, Scopulariopsis spp. are regularly seen, although much more seldom than dermatophytes [5,6]. It is widely accepted that identifying Scopulariopsis in a nail specimen is not in itself diagnostic of a fungal infection. This sort of a finding requires careful clinical interpretation, well-based laboratory evidence and exclusion of possible underlying dermatophytosis [6 9]. In each case, various grades of pathogenicity, saprotrophy and external contamination need to be 2007 ISHAM DOI: /

2 202 Issakainen et al. considered. When found together with dermatophytes, Scopulariopsis species are usually considered to be secondary saprotrophs [10]. However, Scopulariopsis spp. do occur alone and may be the causative agent for at least 15% of fungal nail infections [5]. Scopulariopsis brevicaulis can cause both distal and proximal subungual onychomycosis which is difficult to treat [11]. An underlying local or systemic nail disorder is often suspected but seldom proven [6,9]. Scopulariopsis spp. have been reported to be the cause of systemic infections in at least 10 patients, about half of whom died because of fungal resistance to therapy [12,13]. Some of these infections were associated with cutaneous lesions or originated from such lesions. Cases involving otitis [14] and keratitis [15] may also be related to Scopulariopsis infections. Two other microascacean genera, Pseudallescheria and Petriella, are usually observed in culture in their asexual Scedosporium stages. Two species are known to be opportunistic pathogens, namely the Scedosporium apiospermum stage of Pseudallescheria boydii and the Scedosporium prolificans stage of an unknown Petriella spp. [3,16]. These fungi are less common in keratinous sites, although they may, at least when supported by an underlying dermatophyte infection, grow in symptomatic nails for several months [10]. They are notorious for causing serious, treatmentresistant infections in respiratory tract cavities and in deep tissues, in patients who have some local or general debilitating factors [16]. In immunocompromised patients, they may represent disseminating skin pathogens [17,18]. In otherwise healthy individuals they seldom disseminate from the initial site of infection. Traumatic Scedosporium infections of the skin may become chronic and give rise to mycetomas [19]. Rarely, Scedosporium apiospermum may cause toe-web infections which mimic tinea pedis [20]. In this retrospective 5-year study, we have reviewed the occurrence and significance of microascacean fungi in clinical specimens. This was done by reviewing the clinical records of patients with positive laboratory findings. This paper reports the cutaneous part of the study, namely, the findings related to nail and keratinous skin specimens. Deeper infections, including otitis, keratitis and subcutaneous abscesses, will be reported separately. Patients, materials and methods Population The population of Finland during the study period of was about 5 million. Life expectancy at birth was 73 years for males and 80 years for females [21]. Because of a well-functioning public health care system, the data covers a homogenous population, i.e., socioeconomic circumstances did not affect the data in this study. Participating laboratories, numbers of specimens Laboratory data were collected from the six largest public health laboratories in Finland that perform fungal cultures, i.e., Mycology Laboratories of the University Central Hospitals of Helsinki and Turku, Department of Immunology and Bacteriology at the University of Helsinki and Microbiology Laboratories of the University Central Hospitals of Tampere, Kuopio and Oulu. These laboratories studied about 85,000 keratinous specimens (35,000 nail, 50,000 skin) during the 5-year study period, which was about 60% of all keratinous specimens processed in Finland (inferred from Ruutu P. et al., unpublished data, National Public Health Institute of Finland, Diary Nr. 377/06/98). About 23,000 specimens (15,000 nail, 8,000 skin) were positive for dermatophytes (i.e., Arthroderma anamorphs, not Microascaceae), from which 18,000 yielded Trichophyton rubrum (12,000 nail, 6,000 skin). The remaining specimens yielded primarily T. mentagrophytes. Laboratory methods Culture methods varied slightly between the laboratories, but all specimens were studied using standard methods that would reveal at least the most common keratinophilic dermatophytes such as Trichophyton species. As a rule, a part of the specimen was clarified with potassium hydroxide and then directly examined microscopically. General-purpose mycological agars, such as Sabouraud glucose or Malt extract, supplemented with antibacterials were used to culture portions of the specimens. At least one medium without cycloheximide was usually included in culture studies to allow for the growth of opportunistic fungi. For delimitation and identification of fungal genera and species, see de Hoog and Guarro 1995 [22] and later editions. Study logistics Permission to access confidential patient data was obtained from the Ministry of Health (Diary Nr. 21/ 07/2000, Act 621/1999, 28). The permission covered only the public laboratories and did not allow contacting patients.

3 Scopulariopsis and Scedosporium in nails and skin 203 All reports on all sporulating stages of microascacean fungi cultured from 1 January December 1997 were collected from the files of the six participating laboratories. All recorded instances of sparse, direct microscopy negative and mixed-growth findings were explicitly included in this primary search. Data on hyphae observed on initial microscopy and on the semiquantitative colony counts of fungi in cultures were included. The latter was usually recorded as / for sparse (one or two colonies), // for moderate (several colonies) and /// for profuse growth. Data on co-cultured fungi were also recorded. Because of the large number of cutaneous findings in the primary search, a more focused study was needed. For this purpose, criteria for Stronger Microbiological Evidence (SME, below) were designed for this material to maximize the number of cases with real microascacean growth, either saprobic or pathogenic, rather than as contaminants. The SME criteria are illustrated for nail data in Table 1. A given case could fulfill the SME inclusion criteria if (a) a fair amount of microascaceous colonies (// to ///) was recorded in the primary culture or (b) if the microascaceous fungus was the only fungus recovered in culture from the specimen and direct microscopy of the same specimen revealed a mycelial fungus. In other words, alternative (a) allowed mixedgrowth findings if there was a fair number of microascaceous colonies in culture, while (b) allowed unquantified or sparse culture findings in cases in which the microascaceous fungus grew in pure culture and the findings gained support by hyphal elements in direct microscopy. By these combined criteria we were also able to include cases from laboratories with less detailed data collection practices. During further interpretation of the cases, any missing pieces of laboratory data or missing quantification data were conservatively assumed to represent the least significant result. Patient records of the SME cases were obtained from the local health care centers or hospitals covering at least two years before and after the index case. The exclusion criteria included the appearance of the same patient in two centers, equivocal data, failure of the local health care center to respond after two requests or failure to trace the records, and irrelevant records. We are not aware of any significant bias due to the use of these exclusion factors. The patient records were studied by one of the team s dermatology specialists (HH or EV) who used a preformulated case evaluation form. The data fields included the patient s other mycology results, reasons for the visit to the GP, underlying diseases or compromising factors, clinical symptoms, final clinical diagnosis, details of treatment, clinical response, and evaluation of the role of the fungus. The form had a free text section for additional comments. All microbiologically or clinically available information was subsequently used to exclude patients whose disease was caused by a dermatophyte, such as Trichophyton spp. These cases were collected in the dermatophyte group (Table 2). The exclusion criteria included culture-proven or clinical evidence of tinea of skin (18 cases) or the recovery of dermatophytes in culture as part of laboratory studies. Accompanying moulds and yeasts were identified in a few SME cases but in lower concentrations than Scopulariopsis. These fungi, other than dermatophytes, were not used as a grouping criterion. There was no hard evidence of co-infections by non-miscoascacean opportunists, such as Fusarium spp. or Candida spp. The date on which the specimens were collected was also recorded in an attempt to identify any association with environmental conditions (e.g., snowy winter, Table 1 Primary laboratory findings with Scopulariopis in nail cultures (5 years, ), grouped by microbiological evidence. The groups marked with an asterisk (*) were defined as having Stronger Microbiological Evidence (SME, see text) for the presence of the fungus. Signs: /, //, ///, semi-quantitative recording of colony numbers, as written in the laboratory files; #, positivity defined as fungal elements (septate hyphae or spores), seen directly in the KOH treated nail sample, fitting the morphological range of Scopulariopsis in tissue (some may actually represent suppressed Dermatophyte hyphae); ND, no data (quantity not recorded or microscopy not performed). Accompanying fungi Amount of Scopulariopsis colonies in culture Direct microscopy negative or ND % (n ) Direct microscopy positive # % (n ) Total %(n) Scopulariopsis/ Dermatophyte Sparse (/) ornd Fair (// ///) 2.6 (10) 1.8 (7)* 9.3 (36) 1.0 (4)* 11.9 (46) 2.8 (11) Scopulariopsis/other opportunist Sparse (/) ornd Fair (// ///) 13.7 (53) 2.3 (9)* 10.1 (39) 1.6 (6)* 23.8 (92) 3.9 (15) Scopulariopsis as the only fungus Sparse (/) ornd Fair (// ///) 23.5 (91) 8.3 (32)* 17.8 (69)* 8.0 (31)* 41.3 (160) 16.3 (63) Total % (n) 52.2 (202) 47.8 (185) (387)

4 204 Issakainen et al. Table 2 Characteristics of Scopulariopsis-positive nail patients. Data have been accumulated from all obtained Stronger Microbiological Evidence (SME) nail cases of 5 years (See Table 1 and text). The cases have been grouped according to different types of microbiological and clinical evidence (first rows of table). The grouping generally corresponds to that of Table 1. In addition to simultaneous Dermatophyte findings, any clinical or historical evidence of dermatophytosis has caused the transfer of a case from groups A, B or C to the Dermatophyte Group D. For more details, see text. The terms cure and improvement refer to clinical cure and clinical improvement. Group A B C A/B/C Non-Dermatophyte cases, total D Dermatophyte cases Group definitions Any evidence of Dermatophyte No No No No Yes Scopulariopsis amount Fair Fair, Sparse/ND, Variable Fair (pure or not) (pure or not) (pure) Direct microscopy Positive Negative/ND Positive Variable Variable Age and sex distribution No. of cases; males, females n/28 M8,F20 n/27 M 10, F 17 n/35 M 17, F 18 n/90 M 35, F 55 n/39 M 22, F 17 Age range, years, mean age; SD 4384, 61.8; SD-1/ , 52.7; SD-1/ , 53.9; SD-1/ ; SD-1/ , 52.4; SD-1/16.4 Site of nail specimen, invasive specimen taking % (n ) Big toe nail only 71 (20) 63 (17) 63 (22) 66 (59) 23 (9) Other/unspec. toe 29 (8) 33 (9) 29 (10) 30 (27) 72 (28) Fingernail 0 (0) 4 (1) 9 (3) 4 (4) 5 (2) Taken by nail removal 29 (8) 19 (5) 14 (5) 20 (18) 5 (2) Nail symptoms% (n ) Fungus nail 39 (11) 44 (12) 40 (14) 41 (37) 59 (23) Thickened 43 (12) 33 (9) 39 (17) 42 (38) 36 (14) Deformed 21 (6) 19 (5) 20 (7) 20 (18) 15 (6) Yellowish 18 (5) 7 (2) 11 (4) 12 (11) 10 (4) Fragile 11 (3) 11 (3) 9 (3) 10 (9) 10 (4) Discoloured 0 (0) 7 (2) 14 (5) 8 (7) 8 (3) Loosened 11 (3) 11 (3) 0 (0) 7 (6) 0 (0) Underlying diseases, potential predisposing factors % (n ) No mentioned 18 (5) 19 (5) 23 (8) 20 (18) 8 (3) Hypertonia 36 (10) 22 (6) 20 (7) 26 (23) 18 (7) Skin disease not tinea 14 (4) 26 (7) 9 (3) 16 (14) 31 (12) Joint or bone disease 18 (5) 19 (5) 20 (7) 19 (17) 10 (4) Obesity 21 (6) 19 (5) 11 (4) 17 (15) 13 (5) Nail trauma/sport 14 (4) 15 (4) 11 (4) 13 (12) 13 (5) Mental disorder 14 (4) 7 (2) 14 (5) 12 (11) 10 (4) Cardiovascular 14 (4) 7 (2) 9 (3) 10 (9) 3 (1) Diabetes 0 (0) 7 (2) 9 (3) 6 (5) 15 (6) Previous nail removal 4 (1) 4 (1) 9 (3) 6 (5) 0 (0) Thyroid disease 7 (2) 0 (0) 6 (2) 4 (4) 3 (1) Alcoholism 4 (1) 0 (0) 6 (2) 3 (3) 3 (1) Treatments administered % (n ) Not mentioned 4 (1) 7 (2) 23 (8) 12 (11) 3 (1) Not treated 11 (3) 26 (7) 20 (7) 19 (17) 15 (6) Terbinafin PO 21 (6) 4 (1) 17 (6) 14 (13) 38 (15) Surgical removal 32 (9) 30 (8) 11 (4) 23 (21) 3 (1) Itraconazole PO 18 (5) 15 (4) 20 (7) 18 (16) 18 (7) Amorolfine topical 21 (6) 15 (4) 14 (5) 17 (15) 10 (4) Imidazoles topical 14 (4) 7 (2) 3 (1) 8 (7) 15 (6) Fluconazole PO 4 (1) 7 (2) 0 (0) 3 (3) 10 (4) Thinning mech/chem. 0 (0) 4 (1) 6 (2) 6 (3) 8 (3) Griseofulvin PO 4 (1) 0 (0) 0 (0) 1 (1) 5 (2) Ketokonazole PO 0 (0) 0 (0) 0 (0) 0 (0) 5 (2) Subjective need for treatment, stated cure or improvement % (n ) Nail is a reason to GP visit 82 (23) 67 (18) 74 (26) 74 (67) 54 (21) Stated cure or improvement 21 (6) 19 (5) 14 (5) 18 (16) 36 (14)

5 Scopulariopsis and Scedosporium in nails and skin 205 rainy autumn etc.) which might alter the frequency of culture contaminants. Results In the primary search of laboratory records for the period (5 years), there were 521 cutaneous cases which were found to be positive for any microascaceous fungi. Out of these, 456 involved Scopulariopsis spp. (387 nails, 69 skin specimens) and 65 involved Scedosporium spp. (46 nails, 19 skin). After applying the SME and other first-stage reduction criteria, the clinical records were reduced to 158 patients whose cases were associated with Scopulariopsis spp.(144 nail, 14 skin) and seven cases that involved Scedosporium spp. (6 nail, 1 skin). After the second stage exclusions, the clinical files examined concerned 137 Scopulariopsis patients (129 nail, 8 skin) and all seven yeilding Scedosporium spp. Not all laboratories identified the fungal strains to the species level. Even in those facilities that could do so, genus-level identifications were often used, especially if isolates sporulated slowly and findings were suggestive of contamination. On the basis of data obtained from two mycologist-led laboratories, about 80% of Scopulariopsis cases involved S. brevicaulis, 10% S. candida, 5% S. fusca and the remaining 5% were caused by other species, including S. acremonium, S. brumptii and strains forming Microascus teleomorphs. Most Scedosporium strains were identified morphologically as Scedosporium apiospermum anamorphs of Pseudallescheria boydii. Atypical strains, suggestive of Petriella anamorphs, were relatively common in nail specimens, but could not be systematically separated from other Scedosporium results. As to the clinical patient files, a major limitation of the study proved to be the extremely brief and scattered nature of the records pertaining to cutaneous infections. Usually, clinical conditions were described by only a few free words. Information on earlier results and follow-up were often missing. Therefore, the data allowed characterizing the patients with cutaneous lesions only in general terms. Scopulariopsis in nails The primary laboratory search results of nail cases involving Scopulariopsis spp. are shown in Table 1 and descriptive data on the studied SME Scopulariopsis nail cases in Table 2. Figures on symptoms, treatments and responses shown in Table 2 are concerned with those patients whose clinical records included an explicit statement of the given feature. Thus, the statement that 21% of cases were cured or showed improvement does not mean that 79% were treatment failure but rather for 79% of the cases there were no follow-up data. In cases of several recorded features or treatments per patient, each feature or treatment is counted separately. In all groups, the patients were, on average, older than 50 years and the symptoms were nearly always related to toe nails. The non-dermatophyte groups contained relatively more cases with exclusively big toe nail involvement (66% vs. 23% in the dermatophyte group). The non-dermatophyte patients were more often females (61%) than males. The nail problems were more often among the stated reasons for the physician visits in the non-dermatophyte groups than in the dermatophyte group (74% vs. 54%). The clinical findings relative to the nails were similar and overlapped between the groups. Loosened nails were rare but, when present, they occurred only in the non-dermatophyte groups (7%). In the latter, the index specimen was more commonly the entire excised nail (20%) than in the dermatophyte group (5%). No systemic predisposing diseases were identified. Skin infections other than tinea were, however, more common in the dermatophyte group. The frequency of activities suggestive of nail traumas, such as regular jogging which might predispose the nail mechanically to fungal infection, were similar in all groups (11 15%). A history of surgical excisions of the nails was also rare (4%). Patients with clinical or cultureproven tinea of skin were by definition included in the dermatophyte group. They formed nearly half of that group (46%), but only 14% of all groups with Scopulariopsis SME results. Eighteen percent of the nail problems were never treated. If treatment did occur, surgical excision was reported more often in the non-dermatophyte groups (23%) than in the dermatophyte group (3%). Other treatment strategies included oral antifungals (usually terbinafine or itraconazole) or topical antifungals (often amorolfine lacquer). The use of oral terbinafine was more common in the dermatophyte group than the non-dermatophyte groups (38% versus 14%), but the use of oral itraconazole (15 20%) and topical amorolfine (1021%) was similar in both of these groups. Many combinations of drugs were used, either simultaneously or in succession. The infections of the patients of the non-dermatophyte groups seemed to be more resistant to treatment cure or improvement was recorded in only 18% of cases, but in the dermatophyte group the corresponding rate was 36%. The scanty records did not allow for proper comparisons of the effects of individual treatments, but

6 206 Issakainen et al. the following observations could be made concerning the cases. There were almost no cases of complete, documented clinical cures. There was a total of 4 cases (3%) of recorded cure, but in one of these, the nail alteration recurred within three years. These clinical cures were primarily seen among patients who had had combined treatment. The combined treatments involved oral fluconazole followed by oral itraconazole (group B), oral terbinafine combined with topical tioconaole (Group D), surgical removal combined with topical amorolfine (group B) and oral itraconazole only (group C, the patient who had a recurrence). Clinical improvement was recorded more often than cure. These cases were relatively more common in the dermatophyte group, in that there was a cluster of 7 cases treated with only oral terbinafine that were recorded as clinically improved. The outcome was the same in 6 other dermatophyte group patients who had taken either oral itraconazole (2 cases), oral itraconazole with urea avulsion (1), oral terbinafine with oral fluconazole (1), oral fluconazole with topical tioconazole (1), or oral griseofulvin only (1). In the non-dermatophyte groups, the treatment options and outcomes were too scattered and records too scanty for any general conclusions. In these groups, improvement followed surgical removal (3 cases), oral terbinafine (3), oral itraconazole (2), topical amorolfine (1) or no treatment at all (1). In one case, the patient had tolerated the situation for years, by filing the nail regularly. In the dermatophyte group, lack of clinical effect was recorded in practically all cases treated exclusively with a topical antifungal. These involved the use of amorolfine (2 cases), tioconazole (1), miconazole (1) and econazole (1) and oral griseofulvin (1 case). In the non-dermatophyte groups, a lack of clinical response was explicitly recorded with oral terbinafine (two weeks of treatment only; 1 case), oral itraconazole (1) or oral fluconazole (1). In one additional case, the disorder had improved through the use of oral itraconazole but recurred during subsequent oral treatment with fluconazole. Cases treated with topical amorolfine (2), surgical removal with amorolfine (1) and no treatment at all (2), showed no improvement. Seasonal occurrence of Scopulariopsis spp. in nails These cases were generally randomly distributed throughout the year (data not shown). However, in some small subgroups, relatively more cases were observed during MayJune which may suggest a springtime growth peak or may reflect the social motivation to treat symptomatic nails before the summer when the feet are more often exposed. Scopulariopsis in the skin Of the 14 skin cases involving Scopulariopsis spp. which fulfilled the SME criteria, informative records for 8 of the patients were received for examination. Scopulariopsis infections of the ear are not included. In two cases, fair numbers of Scopulariopsis brevicaulis colonies were recovered together with Trichophyton mentagrophytes. One of these patients who had a crusty leg infection was an 88-year-old female with anemia and in poor general condition. The other patient, a 62-year-old with terminal cancer of the esophagus had toe-web tinea pedis. We interpret these findings as indicating that in debilitated patients, S. brevicaulis can accompany tinea of skin. In two other cases, fair numbers of Scopulariopsis brevicaulis colonies were isolated from skin lesions in association with sparse numbers of Candida species. The patients had some debilitating factors. One of the patients was a 75-year-old diabetic female whose lesions had dispersed from skin folds to her back and feet. Her lesions responded to fluconazole. The other patient was an obese, alcoholic 72-year-old male from whom Scopulariopsis was recovered from lesions on his upper body. Follow-up data were missing. In another two cases, fair numbers of Scopulariopsis (S. brevicaulis and unidentified Scopulariopsis sp.) colonies were found in lesions of chronic allergic eczemas (arms, feet) of otherwise healthy females aged 25 and 46 years, respectively. We interpret these as colonization of allergic skin defects. Finally, in two cases, fair numbers of Scopulariopsis (S. brevicaulis and unidentified Scopulariopsis sp.) colonies grew in cultures inoculated with portions of unilateral lesions of foot soles. The patients, a 53- year-old female and a 59-year-old male had no dermatophytic infections or direct predisposing factors. We interpret that the foot sole is a potential site for genuine Scopulariopsis growth but the data were insufficient to make any conclusions as to its causative role in the infections. Scedosporium in nails There were 6 cases of nail infections caused by or from which Scedosporium was recovered. In two, (females aged 44 and 50 years) there was evidence of an underlying dermatophyte infection in that Trichophyton rubrum was isolated in culture 1 year after the initial recovery of Scedosporium and both had earlier symptoms suggestive of tinea pedis. Both cases responded

7 Scopulariopsis and Scedosporium in nails and skin 207 favorably to oral antifungals (itraconazole and terbinafine). We interpret these cases similar to the dermatophyte group of Scopulariopsis, i.e., Scedosporium species can accompany dermatophytes in cases of tinea unguium. The four other cases had no evidence of dermatophyte involvement or specific predisposing conditions. Three were females (5674 years) and one male (18 years). While two of the female patients had only one big toe nail affected, in the other two cases the fungus was associated with unspecified toe nails. We consider that these findings indicate that Scedosporium spp. can, on rare occasions, be associated with symptomatic toe nails, perhaps in a situation similar to the non-dermatophyte group of Scopulariopsis. However once again the limited information in the records does not permit any definitive conclusions as to the role of Scedosporium sp. in these cases. Scedosporium in the skin There was one case in which Scedosporium ssp. was reovered from skin lesions. Direct microscopy of specimens from the toe-webs of this 52-year-old female, yeilded hyphae that were in the morphological range of Scedosporium apiospermum. While no dermatophytes were recovered in culture on several occasions, she had a 2-year clinical history of affected toe-webs, foot sole and nails suggestive of tinea pedis. She responded favorably to 7.5 months of treatment with oral antifungals (itraconazole followed by terbinafine), supplemented with topical and mechanical treatments. We believe that this indicates that Scedosporium may accompany an underlying dermatophyte infection. Discussion Skin and nails are heterogeneous substrates for fungal growth. Lesions may reside in different anatomical parts, crevices and regions of accumulated debris. The microbial flora also fluctuates over time. Therefore, growth of opportunistic microbes should be interpreted with caution and repeated specimens need to be taken to substantiate their roles as pathogens [6,7,10]. Histological examination of biopsy material is also highly recommended to identify the exact site of fungal growth in the tissue [6,8]. Our data show that benign skin and nail problems are often handled in a hasty manner in a general clinical practice. Because of the scattered nature of our data, the lack of histological specimens and few repeat cultures, the present retrospective study could not meet the criteria of proving or disproving the causative role of Microascaceae in the superficial infections. We found it useful, however, to reconstruct an overall picture of the patients harboring Scopulariopsis spp. in their nails (Table 2). Interpretation of the data should be cautiously made as they merely describe the ecological spectrum of Microascales in the cutaneous environment. However, some of the information certainly refers to genuine infections. To minimize speculation as to air-borne contamination, we established Stronger Microbiological Evidence (SME) criteria to expand the number of cases involving genuine microascalean growth, either as pathogenes or saprotrophytes, in the specimen. In addition to limited clinical data, tens of results were omitted or classified as poor evidence, due to less detailed record keeping of a given laboratory. The material probably includes some masked dermatophyte cases. However, our material is essentially different from that of Ellis et al. [10] which included only proven dermatophytosis cases (representing, perhaps, a wider variety of case than presented in column D of Table 2). Their paper also included obvious air-borne contaminants (non-sme cases involving various mold genera) among the better documented mold growths. A clear majority of our cases could not be positively associated with dermatophyte infections (70% of all SME cases were in non-dermatophyte groups) or with mechanical injury (traumatic history was recorded in only 13% of cases). Obviously, all predisposing factors could not be considered since minor traumas may be overlooked [6] and the microscopic morphologies of members of the Microascaceae and dermatophytes [5] can overlap. The results of our studies do not permit us to relate our case numbers to the general Finnish population. However, considering that the laboratories participating in these studies processed about 60% of keratinous fungal specimens of Finland, then the 90 Scopulariopsis SME cases involving the nails would represent an incidence of 6 per 1,000,000 per year. The number of these cases was equal to about 1% of all dermatophyte nail findings at these same laboratories at the same period of time. These results are similar to those reported in Canada by Summerbell et al. [5], but using different criteria. The authors noted that authentic Scopulariopsis infections comprised 1.6% of all cases of onychomycoses. Higher numbers (up to 6%) have been presented elsewhere [5,9]. Symptoms associated with the growth of Scopulariopsis in nails (non-dermatophyte SME cases) were considered clinical problems by three of every four patients. The condition was chronic though benign, but

8 208 Issakainen et al. seemed to respond more poorly to a variety of treatment methods than dermatophyte cases. Scopulariopsis growth was most common in the big toe nails of elderly females. This contrasts with usual dermatophyte infections, which predominate among males [23]. In the present study the cases with underlying dermatophyte infections (Table 2, Group D) were male-dominated. The Scopulariopsis infections of the nails are characterized by different predisposing factors and etiologies. For instance, the loosened nail in 11% of Groups A and B cases may support saprophytic growth, while some of the discolored nails (14% of Group C) may include a bacterial component, e.g., Pseudomonas. Experimental work suggests that Scopulariopsis is keratinophilic but does not readily infect healthy nail tissue [24]. More work, including histology [6], is needed to elucidate the pathogenesis of fungal infections in different settings. In addition to overlooked traumas, attention should be paid to the role of retarded nail growth and hormonal variations in Scopulariopsis nail infections [9]. Since we found numerous cutaneous cases throughout all seasons of the year, it is highly unlikely that direct spore contamination of sites from the outdoor air could account for our Scopulariopsis findings. We believe that, as a rule, the SME cases represent longterm growth of Microascaceae in the diseased tissues. However, the clinical role of the fungus may vary depending on the case and the stage of infection. As to sources of air-borne contamination of specimens (enriched in the rejected non-sme cases), several potential indoor reservoirs may be considered. These may help to understand the general ecology of Scopulariopsis. In Finland, Scopulariopis brevicaulis often forms spores in cowhouses, especially on dried grass or straw which may have become wet prior to use as cattle feed or litter. The fungus can also be isolated from the fur of pet dogs (R. Aho, personal communications). According to Finnish aerobiological studies, S. brevicaulis is found more commonly in waterdamaged residential buildings than in the outdoor air (A-M. Pessi, personal communication). Together with our study of systemic specimens, the data allowed some comparisons between the ecology of Scopulariopsis and Scedosporium in the human body. Scopulariopsis spp. are regularly associated with the nails, weak skin, and ears, they are able to better cope with drier, keratinous environments. In contrast, Scedosporium spp., were associated with moist conditions such as the sinuses and lungs. Mechanical tissue injuries may also be here considered as moist environments. The labels of moistness and dryness naturally correlate with other factors, such as nutrient availability and leukocyte counts. The feet of immunocompromised individuals may be a portal of entry for Microascaceae. A fatal Scopulariopsis infection originating from a toenail has been reported [12]. Deep Scedosporium infections may occur adjacent to toe-webs [18] and on lower surfaces of the feet [25]. The latter areas accumulate weakened keratinous tissue and may harbor unnoticed dermatophytes. Among the deep infections in our study (data not shown), there were 2 new Scedosporium cases originating from the big toe. For epidemiological discussion, we propose here the informal term keratinous waiting room for hypothetical cutaneous sites which support innocuous long-term colonization by keratin-associated microbes and may later give rise to deep infections by the same organisms. The hypothesis of keratinous waiting rooms is supported by the results of the present studies. Scedosporium spp. were found in several patients with symptomatic toe nails. Scopulariopsis spp. were also found in various skin lesions of the feet. Even in case of saprobic colonization, facultatively keratinophilic Microascaceae can be present for long periods of time [10]. If these keratinous sites function as long-term reservoirs of deep infections, some serious infections might be avoided by improving the general foot hygiene of immunocompromised patients, including careful treatment of innocuous dermatophyte infections. Lastly, we would like to mention two recent lines of study which may prove very fruitful in solving the clinical roles of microascaceous opportunists. Summerbell and Gupta, with co-workers, have shown by painstaking research that Scopulariopsis and other opportunistic moulds do cause nail infections but that the genuine cases cannot be reliably diagnosed on the basis of a single isolation [26, 27]. The direct microscopy results and colony counts do correlate with true infections but not enough to omit repetition of selected cultures. On the other hand, Filipello Marchisio and co-workers [28] have shown that some Scopulariopsis brevicaulis strains are slowly but genuinely keratinolytic while other strains of the same species are not. Intraspecific variation in enzymatic activity could explain, at least partly, the decades-long ambiguity about Scopulariopsis findings of nails. Acknowledgements We thank the personnel of all the participating hospitals and laboratories for their kind assistance, especially Mr Niko Sahlberg for his help in data

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