Scanning Microscopy

Volume 4 Number 2 Article 15

4-5-1990

Dermatophyte Morphology: A Scanning Electron Microscopy Study

R. Vazquez University of Salamanca

J. M. Riesco University of Salamanca

A. Martin Pascual University of Salamanca

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Recommended Citation Vazquez, R.; Riesco, J. M.; and Pascual, A. Martin (1990) "Dermatophyte Morphology: A Scanning Electron Microscopy Study," Scanning Microscopy: Vol. 4 : No. 2 , Article 15. Available at: https://digitalcommons.usu.edu/microscopy/vol4/iss2/15

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DERMATOPHYTE MORPHOLOGY: A SCANNING ELECTRON MICROSCOPY STUDY

R. Vazquezl,2*, J.M. Riescol, and A. Martin Pascual3

Departments of Morphological Sciences!, Electron Microscopy2, and Dermatology3 University of Salamanca, University Hospital, Faculty of Medicine, Salamanca, Spain

(Received for publication November 28, 1989, and in revised form April 5, 1990)

Abstract Introduction

The Dermatophytes are a broad group of fungi The Dermatophytes belong to a broad group of belonging to the class Fungii imperfecti that are the fungi with pathogenic capacity in man and diverse causative agents of dermophytos1s (rmgworm infec - animal species. They are keratinophilic agents; that tions). The present work offers an overview of the is, they only parasitize keratin or keratinized struc - morphology of these fungi found in cultures accord­ tures (the horny layer, skin, and nails) causing der­ ing to the scanning electron microscope. The fungi matological lesions encompassed within the term der­ were obtained from cultures left to develop over matophytosis (ringworm infections). Speculations variable periods of time that would be sufficient for have been made about the possibility of Dermato­ growth. The morphological features of some derma­ phytes having been derived from a large family of tophytes obtained in artificial cultures are detailed: perfect soil fungi (ascomycetes). With the hair bait M. canis, M. gypseum, M. audouini, M. cookei, T. technique, it has been possible to isolate fungi with mentagrophytes, T. schoulemu, T. verrucosuin, T. morphological features similar to those of the Der­ aJello1, T. prohferans, and E. floccosum. In all cases matophytes but that do not induce pathological states an analysis of the morphology of the reproductive in man or in animals. mycelium developed in the culture was made: hyphae, Al though they belong to the class Fungii im - macroconidia, microconidia, and chlamydospores; de­ perfecti (asexual), some species are also able to tails that serve to distinguish one fungus from an­ develop a perfect state (sexual) when grown on hairs other. In the perfect forms, the morphology of the on the surface of soil . peridial hyphae and of the ascocarps (cleistotethia) Asexual reproduction is achieved through the are described. formation of spores. Upon finding a suitable me­ dium, a spore germinates and emits one or several tubes that grow distally and branch in the form of filaments known as hyphae. The branchings of the hyphae constitute the mycelium which shows two dif­ ferentiated portions: the vegetative mycelium, res­ ponsible for the maintenance and growth of the fun - gus, and the reproductor mycelium that contains spore-bearing hyphae (sporophores). The spores are mainly located on lateral projections of the hyphae and have a bud or finger-like aspect; these are the conidia. When the largest axis of the conidium is less than 5 micrometers and is formed of a single cell it is known as a microconidium. If the evagin­ ation is larger and is composed of two or more cells, the term macroconidium is used. Morphological varieties within the hyphae are the chlamydospores and the pectinate hyphae. The Key Words: Dermatophytes, Scanning Electron former are globose bulges surrounded by a strong Microscopy, Mycosis, Fungus, Tineas, M. canis, M. wall and localized on the distal portion of the hypha gypseum, M. audouini, M. cookei, T. mentagrophytes, or intercalated on its trajectory. Pectinate hyphae T. schouleinii, T. verrucosum, T. ajelloi, T. have a comb-like morphology. proliferans, E. floccosum. In the perfect state, the reproduction of Der­ matophytes is sexual and is characterized in that the * Address for correspondence: spores, in this case called ascospores, are the pro­ Ricardo Vazquez duct of fusion of two gametes of opposite sign (con­ Hospital Clinico Universitario ventionally gamete + and gamete - ) . Out of this Servicio de Microscopia Electronica union arise more or less spherical formations - the Paseo de San Vicente sin ascocarps (cleistotethia) - surrounded by thick and 37007 Salamanca, Spain claw-like hyphae called peridial. In turn, each as­ Phone No.: (923) 263450 cocarp is formed by sacs (asci) in whose interior the

363 R. Vazquez, J.M. Riesco, and A. Martin Pascual

ascospores are located. Essentially, two genera have Results been described: Mannizzia as the perfect state of the genus Microsporum and Arthroderma as the perfect In artificial cultures, the Dermatophytes de­ state of the genus Trichophyton. veloped a reproductive mycelium with an enormously The Dermatophytes exhibit different morpholo­ rich structure; this permitted their differentiation gies according to whether they are viewed: a) as into three genera: Mycrosporum (M); Trichophyton pathogens, obtained directly from lesions (they would (t); and Epidermophyton (E). The former two genera be seen simply as unicellular and sporulated forms), include several species that are pathogenic for man b) in artificial culture media (where they develop a and animals. reproductor mycelium formed of macro- and micro­ Since a study of all the species would be be­ conidia, special hyphae, etc., which have served as yond the scope of the present work, we selected the basis for their taxonomic classification), and c) only some of those most commonly present in our when they are grown with the hair bait technique, environment (M. canis, M. gypseum, M. audouini, M. where some of them are able to develop a perfect cookei, T. menTagropfiytes, T. schoulemu, T. verru­ stage (for more details, see 1, 2, 5, 6, 9-12, 16, 17, cosum, E-:- floccosum) together with those exhibiting 20, 21, 37, 43, 45, 46, 53). strikmgcharacteristics ( M. audouini), new features The present work offers a scanning electron (T. proliferans), or representing the three epidemio­ microscope (SEM) analysis of the morphological char­ logical groups. acteristics of some Dermatophytes belonging to the Genus Microsporum three existing epidemiological groups (anthropophilic, M. Canis: This is a zoophilic fungus, mainly zoophilic, and geophilic). The morphology described harbored by the cat and, to a lesser extent, the dog. relates to that found in cultures, not in infected Its perfect or sexual state is unknown. It is the integument and/or its appendices. most important causative agent of tinea capitis in our environment ( Salamanca, Spain). It has been Materials and Methods studied by several authors with light, transmission, and scanning electron microscopes (26, 27, 37, 42, 46, The Dermatophytes analyzed were from three and 50). sources: a) clinical lesions of patients attending the Upon examination with the scanning electron University Hospital ( University of Salamanca, Spain); microscope a mycelium with the following character­ b) from lesions (ringworm) in several animal species istics was observed: when the colony was young (rabbits, calves, rats, etc.); c) from different soil (after a short period of time, about eight days of samples, according to the techniques proposed by growth), the hyphae were thin (Figs. 1 and 2), with Dwivedi et al. (13) and Vanbreuseghem (44). some swellings, occasionally showing formations re­ The Dermatophytes were cultured in agar con­ sembling smooth surfaced buds (Fig. 1). The macro­ taining glucose and neopeptone (Saboureaud's me­ conidia were elongate, spindle-shaped, with a sharp dium); in order to prevent contamination and the distal end (Figs. 1, 2, 3); their surface, in the case presence of saprophytic mold, chloramphenicol and of young colonies, was almost smooth (Fig. 1), with a cycloheximide were added (14, 37, and 39). Identification of Trichophyton verrucosum was carried out by the nutritional tests (addmg nutrient substances such as thiamine and inositol) proposed by Fig. 1. Macroconidium (ma) of M. can is belonging to Georg (18). For differentiating Trichophyton menta­ a young colony of short evoluno~ It is fairly grophytes from '!:..:_ rubrum the perforating hair tests smooth and only exhibits a fine granulation. The re­ of Ajello and Georg (3) and the urease test of lief of a septum can be seen (arrow). The hyphae Philpot (34) were used. (hy) shows bud formations with a smooth surface Perfect forms were obtained by crossing iso­ (arrowheads). lated strains with two other strains of different Fig. 2. Macroconidium (ma) belonging to an older signs (37) of the corresponding perfect form, pro­ colony; note that the whole surface is covered with vided by Doctors Ajello (at Communicable Disease buds. Center, Atlanta, Georgia) and Mackenzie (at the Fig. 3. Detail of surface of macroconidium shown in London School of Hygiene and Tropical Medicine). Fig. 2; note buds which have cotton-like appearance. The protocols used for analysis with the scan­ Fig. 4. M. gypseum. Long macroconidia (ma) with ning electron microscope were as follows: fixation few knobson surface. Lateral microconidia can be was in 6% glutaraldehyde in 0.lM phosphate buffer, seen emerging from hyphae (arrows). pH 7 .4, temperature 4°C over 3-6 hours. The prepa­ Fig. 5. Macroconidia of M. gypseum belonging to an rations were then washed in 0 .1 M phosphate buffer, older culture than those shown m Fig. 4; their sur­ pH 7 .4 plus 0.02 M sucrose for 2 hours. The prepa­ face is covered by numerous knobs of different sizes. rations were then post- fixed in 1% osmium tetroxide Fig. 6. Macroconidia (ma) of M. cookei. They are in 0.1 M phosphate buffer, pH 7 .4, for 2 hours at ovoid shaped and have abundantknobs. 4°C and then washed. They were dehydrated in a Fig. 7. Typical terminal chlamydospore (tc) highly graded acetone series (30%, 50%, 70%, 80%, 90%, and characteristic of M. audouini. Lateral microconidia absolute acetone) and then dried by the critical point are seen on some hyphae (arrows). method using acetone as the vehicle. A fine layer of Fig. 8. Pectinate hyphae (ph) of M. audouini. This gold was sputtered onto the pieces for 2-3 minutes is a characteristic of the variety rivaher1. (thickness 20 nm). They were observed with a Philips scanning electron microscope (SEM-500) using Bar lengths in micrometers: 12 and 25 kV as acceleration voltage. Figs. 1, 5 and 8 5; Figs. 2, 4, 6 and 7 10, and Fig. 3 1.

364 SEM Study of Dermatophytes

365 R. Vazquez, J.M. Riesco, and A. Martin Pascual

fine, uniformly distributed granulation. In them the known perfect state is Arthroderma Benhamiae. septa were prominent. In older cultures (at 15 days Among other reports, it has been described in refer­ after the start of growth), the surface of the macro­ ences 4, 8, 23, 26, 28, 30, 32, and 35. conidium exhibited downy knobs starting on the dis­ The species was characterized by displaying ab­ tal end and extending across the whole of the sur­ undant mycelia. The hyphae were thin and regular face of the conidium (Figs. 2 and 3). and contained numerous microconidia grouped in M. gypseum: This is a geophilic fungus that is clusters resembling unripe grapes (Fig. 9). At abundant in soils rich in organic matter (gardens, greater magnification, these microconidia were spher­ orchards, etc.). It may affect humans, producing ical or ovoid and had a smooth surface (Fig. 10). circinate herpes and tinae capitis. In this species Although this was the typical arrangement of two perfect forms are known: Nannizzia incurvata the microconidia, they were also seen to be arranged and Nannizzia gypsea. The species has been studied laterally; these microconidia had different shapes, by several authors using different techniques (5, 9, either bud like (Fig. 9) or spherical or pyriform, 25, 26, 31, 46, 47, 48, 51). with a short anchoring stalk (Fig. 11). The Mycelia of this species were characterized Another feature was the existence of spiral by their abundant "cigar-shaped" macroconidia (Figs. hyphae arranged in spiral coils (Fig. 12). These 4 and 5); they were often found in groups. As the formations, although not exclusive to '!:..:_ mentagro­ age of the culture progressed, the surfaces of these phytes, were very characteristic with regards to macroconidia began to show an increasing number of their high numbers. Occasionally, it was possible to knobs. As an example, Fig. 4 shows macroconidia find terminal and even intermediate chlamydospores. after a short period of development in culture, there The macroconidia were ovoid or spindle-shaped, with are few knobs; by contrast, in Fig. 5, the macro­ a sharp distal end in which some septa were seen. conidia are older and have more knobs. Unlike the T. schouleinii: This fungus is anthropophilic and macroconidia, the microconidia were rare and were is the causative agent of tinea favus. Its develop­ located laterally on the hyphae (Fig. 4). ment is chronic and affects the scalp, where it indu - M. cookei: This species belongs to the group of ces crusty lesions centered around a hair and leading geophilic fungi and are mainly found in the soil; the to scar-due alopecia. It may give rise to onychomy­ fungus is rarely pathogenic for man and even for an­ cosis. Its sexual stage is unknown (26, 33, 37). imals. Its perfect form is known: Nannizzia cajetani The species was characterized by the abundant (24, 26, 29, 46). presence of mycelia, with hyphae varying in thick­ The species was characterized by the presence ness. It displayed terminal hyphae, arranged in the of low numbers of hyphae and abundant macroconidia form of nail-head hyphae (Fig. 13). Macroconidia in its mycelium (Fig. 6). Its characteristics were were almost absent and microconidia were also un - very similar to those of other fungi belonging to the common. genus Microsporum; they were gherkin-like with T. verrucosum: This is a fungus affecting ani­ spherical buds on the surface. There were few mi­ mals and its pr1nc1pal reservoir is cattle, from where croconidia. They never had characteristics differing it may pass to man, inducing lesions with an intense from those of other species. inflammatory reaction (circinate herpes, kerion). Its M. audouini: This is an anthropophilic fungus, perfect stage is not known (7, 26, 46). the causative agent to tinea capitis. It is abundant in Africa and the United States but rare in Europe. Fig. 9. T. mentagrophytes (variety granulosum). It may mutate such that certain varieties are known, Note lateraT m1crocon1dxa (arrows) and other, more e.g., lengeronii and rivalieri (26, 46, 52). abundant ones, that are spherical or ovoid, with a Analyzed with SEM, the species was character­ smooth surface, resembling a cluster of unripe grapes ized by exhibiting macroconidia and abundant myce­ (mi). lia, with certain essential features such as the pre­ Fig. 10. Detail of spherical or ovoid microconidia sence of pectinate hyphae and terminal chlamydo­ (mi) of T. mentagrophytes. Note short anchoring spores. stalk. - The hyphae were thin and abundant with some Fig. 11. Lateral microconidia of '!:..:_ mentagrophytes swelling and septa (Fig. 7) . They sometimes dis - (arrows). played lateral microconidia (Fig. 7) . The variety Fig. 12. Typical filamentous hyphae in spiral coils rivalieri had pectinate hyphae and was characterized (sc) of T. mentagrophytes (variety granulosum). by displaying a zone that broadened on its distal Fig. 13.-Nail-head of T. schouleinii (arrow). end, emitting globose elongations of different sizes Fig. 14. Mycelium of 'f: verrucosum. Some of the (Fig. 8). hyphae have only a few-m1crocomdxa (arrows). A A definitive feature in the characterization of spherical terminal chlamydospores (tc) is clearly seen. this species was the presence of terminal chlamydo­ The branching of the hyphae often occurs at right spores (Fig. 7), spherical in shape with a smooth angles ( *). surface. Occasionally, it was possible to observe the Fig. 15. Intermediate chlamydospores of T. presence of intermediate chlamydospores like simple verrucosum arranged in bead-like formations (cl)-:­ swellings of a hypha. Fxg. 16. Macroconidia (ma) of T. ajelloi. Their Genus Trichophyton surface is smooth, finely folded;-and transversal T. mentagrophytes: There are two varieties: septa (small arrows) are seen. The long arrows show T. M. granulosum, a zoophilic fungus, and T. M. inter­ lateral microconidia. digitale, anthropophilic fungi affecting man. The variety granulosum is the most frequent Bar lengths in micrometers: causative agent of inflammatory tinea ( Kerion), and Figs. 9, 14, 16 10; the variety interdigitale produces circinate herpes, Figs. 10, 11 1, and especially athlete's foot and onychomycosis. Its Figs. 12, 13, 15 5.

366 SE M Study of Dermatophytes

367 R. Vazquez, J.M. Riesco, and A. Martin Pascual

The essential characteristic of this trichophyton pect to the hypha. No microconidia were observed. was that it had thick, 90° branched hyphae (Fig. 14), Sexual Forms that sometimes adopted an antler-like disposition. When some species are cultivated on the surface Some terminal or intermediate chlamydospores were of the soil, they develop their perfect or sexual form observed (Fig. 14), occasionally forming chains (Fig. and are characterized by exhibiting more or less 15); the surface of these was smooth or slightly spherical formations known as ascocarps (cleisto­ folded. thecia), surrounded by peridial hyphae. Each asco­ Only on some occasions, and then in enriched carp is in turn formed on asci, within which the so­ media, was it possible to observe some macro- and called ascospores are located. Some genera of per­ microconidia; the latter are seen leaving the hyphae fect forms are known: among them those correspond­ laterally in Fig. 14. ing to T. mentagrophytes (Arthroderma benjamiae) T. ajelloi: This is a geophilic fungus that is and to M---:- gypseum ( Nanniz zia incurvata and Nanniz­ only rarely pathogenic for man. It is found in soil zia gypsea) (3, 8, 26, 31, 32). rich in organic matter. Its sexual form, Arthroderma Arthroderma benj amiae: The Ascocarp (Fig. 2 7) uncinatum, is known (26, 31, 46). The ultrastructural was characterized by its spherical shape and was characteristics of this species were: numerous large, surrounded by peridial hyphae that appeared as spic - elongated fusiform macroconidia with a smooth or ulated surface elements with periodic, regularly slightly surface, and well-defined septa (Fig. 16). interspersed, strangulations conferring them with a Microconidia were sometimes found (Fig. 16). bead-like appearance (Fig. 28). In peripheral zones, T. proliferans: Until recently, there has been it was also possible to see spiral coils that are not considerable discussion as to whether this fungus is true attributes of this form since they also appear in an autochthonous species or simply a variant of T. other species. mentagrophytes, since until now few species have The asci containing the ascospores, generally 8 been isolated (15, 37). Under SEM, its character­ in number (26), were easily broken, revealing the istics were elucidated in detail and it was possible to ascospores. These were seen as small lentil -shaped clearly differentiate it from T. mentagrophytes. formations with a smooth or slightly folded surface The most striking differential feature was the (Fig. 29). presence on the mycelia of thick hyphae with forma­ Nannizzia incurvata and Nannizzia gypsea: tions known as propagules; these were seen as dila­ These are perfect forms of M. gypseum. Their asco­ tions on the hyphae (Figs. 17-22) and have never carp appeared surrounded by peridial hyphae. The been observed in other Dermatophytes. They varied segments were quite long and regular and sometimes in size and shape. Sometimes, they were isolated, branched to form a cactus-like image (compare the while others formed chains, as observed in Figs. 17- difference between Figs. 28 and 30 corresponding to 22. Some were fusiform, while others were more or peridial hyphae of the two perfect forms referred less spherical, with a smooth surface in some cases to). As in the case of A. benjamiae, there were spi­ (Figs. 17, 19) and a rough surface in others (Fig. ral coils and also some macroconidia located periph­ 20). They sometimes displayed granulations, as is erally. The asci were identical to those described the case of those shown in Fig. 2 2. Their mycelia (as in Fig. 29). also had macroconidia (Fig. 23) very similar to those corresponding to'!:_:_ mentagrophytes, with a spherical General Comment free end displaying a rough surface. The image was very similar to that of propagules observed in Fig. In clinical practice, when there is a suspicion 22, suggesting that these were developmental stages of dermophytosis, confirmation as to whether there of a single element. is or there is not a fungus causing the condition The macroconidia were joined to the hyphae by (pathogenic fungus) can be obtained from samples of a short anchoring stalk, as observed in Fig. 23 and, the lesions (scales, hairs, etc.), which upon addition in greater detail, in Fig. 24, where two concentric of a few drops of 10-20% KOH show the presence of layers and a central zone without a clear content fungus under the light microscope. However, this can be seen. This Dermatophyte did not have micro­ procedure is rudimentary and for a surer diagnosis it conidia. Genus Epidermophyton E. floccosum: This is the only species of this genus. The fungus affects man and is widespread; in Figs. 17-24. T. proliferans. Note highly developed Spain it is most often responsible for tinea cruris and irregular hyphae on which there are prominent (19, 24, 26, 49). Under the scanning electron micro­ swellings or "propagules" (p in Figs. 17, 18, 19 and scope it displayed numerous macroconidia either lat­ 20). Some times they are isolated (Fig. 20), although erally or on the distal end of the hypha near it (Fig. they normally form chains (Figs. 17 and 18). Their 25). These were club shaped with a spherical free surface may be smooth (Figs. 17 and 19), slightly end; overall, they constituted the so-called racket­ rough (Fig. 20), or may show granulations (Figs. 21 hyphae. Their surface was rough, with irregular and 22). Macroconidia (ma) can be seen, some with folds separated by furrows giving them a brain-like a smooth or slightly thicker folded surface (Fig. 23). appearance. Annular reliefs, corresponding to the Fig. 2 4 shows the surface of the junction between a septa, were visible (Fig. 25). The hyphae were thick propagule (arrow) similar to the insertive stalk of and septate; in some it was possible to observe small the macroconidium shown in Fig. 23 (arrow). spherical knobs (Fig. 25). Terminal or intermediate chlamydospores were Bar lengths in micrometers: common (Fig. 26). The surface of these was smooth Figs. 17, 19 and 20 10; or finely granulated. Sometimes the chlamydospores Figs. 18, 20, 22 and 24 = 5, and were so abundant that they conferred a bead like as- Fig. 23 1.

368 SEM Study of Dermatophytes

369 R. Vazquez, J.M. Riesco, and A. Martin Pascual is necessary to obtain a sample of the lesion and tant for analyzing the morphological characteristics then analyze histological sections with appropriate of the fungus . In cultures of only a few days evolu­ techniques, among them the Periodic-Acid Schiff re­ tion the mycelium is young and displays certain spe­ action (PAS) and the Gomori methenamine-silver ni­ cific characteristics (macroconidia without the relief trate stain (14). of the septa, a smooth surface, fewer verrucosities, Both in direct visualization and in histological etc.); in cultures with a long evolution the charac­ sections, the pathogenic fungus appears either in my­ teristics of the mycelium are different (very visible celium form or in sporulated form. However, in no reliefs of the septa, numerous verrucosities, etc. ). In case is it possible to observe morphological charac - this sense, it is necessary to take into account that teristics that differentiate one fungus from another the different kinds of Dermatophytes have different and thus reach a diagnosis of the causative agent. periods in which the colony begins to develop; this If the scales from the lesion are cultured in ar­ depends on the type of Dermatophyte itself, the cul­ tificial media and the culture is examined after some ture medium employed, and on the environment in time, it is possible to arrive at a diagnosis of the which it develops, among other causes. Once it has agent responsible for the condition because it has begun to develop, the colony is considered to be developed its reproductive mycelium. young during the first 8 days. Between 8 days and Cultured dermatophytes have been studied in the end of the first month the colony is considered several works using different morphological tech­ to be mature, and thereafter the phenomena of niques. The macroscopic aspect of the thallus (colo­ pleomorphism begin to appear, making it difficult to ny) such as more or less rapid growth, its smooth or study the mycelium. Naturally, these are only rough aspect, consistency, colour, etc., orients diag­ approximate times and are in fact standardized in nosis of the causative agent although several fungi any mycological laboratory. For us, between 8 and have similar characteristics (37). 15 days is the ideal time for microscopic observation Using samples obtained in culture, light micros­ of the fungi. copy analyzes many details of these fungi and many In this sense, for example, Akin and Michaelis years ago this permitted their taxonomic classifica­ (5) have reported that at 8 days of evolution of the tion. Among these details one could cite, for exam­ culture the surface of the macroconidia of ~ gypse­ ple, the fact that the macroconidia of the genus um shows polypoid projections that cannot be seen Microsporum have rough walls, whereas those of the before this time; in a previous work (26) we have genera Trichophyton and Epidermophyton have observed similar details in several Dermatophytes. smooth walls. Other differential features are whe­ Epidemiologically, Dermatophytes are classified ther they have microconidia or not, their form and in three groups: anthropophilic , zoophilic, or number; or whether the mycelium has pectinate hy­ geophilic; all three can produce lesions in man, phae (Characteristic of M. audouini), nail head hy­ although essentially the former two are responsible phae (typical of T. schocleinn), abundant spherical for these. Lesions caused by zoophilic or geophilic microconidia (T. mentagrophytes), etc. (14, 37, 40). Dermatophytes are more eczematous and inflammatory Currently7n most cases with such observations and less chronic than those produced by anthropo­ it is possible to arrive at an exact diagnosis of the philic species. In this review, we have attended to causative agent of ringworm. fungi belonging to three epidemiological groups in a Transmission electron microscopy sheds further fairly proportional number of species, some of them light on the morphology of the Dermatophytes and being more pathogenic than others. Among the affords a better understanding of their walls, septa, geophilic species (less pathogenic), we have chosen and organelles, yielding interesting data for research two from the genus Microsporum (M. gypseum and M. into these fungi. Descriptions have been offered of cookei) and one from the genus Trichophyton (T. the details of the walls of hyphae and macroconidia ajelloi); the former two because one is pathogenic (22, 36, 50, 52); of the structure of septa (35), of and the other only rarely pathogenic, and the latter the existence of pores in the septa (52), and of the because it is the only geophilic species Trichophyton existence of Woronin bodies (38), etc. that we have studied. Use of the scanning electron microscope fur­ Along similar lines, with the SEM it has been nishes a detailed analysis of the morphology of the possible to determine the morphological character­ surface of these fungi and helps in their classifica­ istics of the Dermatophytes mentioned in the present tion. Its resolution and depth of field yields good work: T. proliferans. This species has not received and easily interpretable images for a better analysis much attention and is thus not very well known. of the surface details of the fungi. In a previous The characteristics of the most significant elements work we undertook a study of the morphology of the of this fungus, the propagules, have been described Dermatophytes, combining scanning and transmission in this overview and we are in no doubt that they electron microscopy and compiling an Atlas with the are exclusive to this Trychophyton and are not pre­ results found (26). sent in T. mentagrophytes or in the other species of SEM confirms the findings of the light micro­ Trichophyton referred to. For the first time, using scope; the surface of the macroconidia corresponding the SE M it has been possible to describe the micro­ to the genera Trichophyton and Epidermophyton are scopic morphology of T. proliferans, assigning it as smooth and those of the species of Microsporum are an autochthonous species different from T. menta­ rough (24, 41, 48). However, the richness of inform ­ grophytes. - --- ation is much greater owing to the stereoscopic visu­ This kind of insight is exclusive to the scanning alization of the fungus, the greater resolution of the electron microscope. However, currently there are microscope, allowing one to clearly define the shape few advantages of this (regarding time, costs and of the rugosities, their number, the way in which the material required) compared with less sophisticated macroconidia are implanted, etc. methods. It would be difficult to substitute the The time of evolution of the cultures is impor- classical method of diagnosis (light microscopy) ,

370 SEM Study of Dermatophytes

Figure 25. E. floccosum. Thick hyphae (hy), with a smooth and I or rough surface showing spherical knobs. The ro acrocomdia (ma) are uniform in thickness and their distal end is blunt; their surface is smooth, and relief3 of the septa can be seen (arrows). Figure 26. Clamydospores of ~:.... floccosum (cl). Figure 27. Ascocarp (cleistothecium) of A. benhamiae at 25 days of culture on hair bait. Peripherally, the perid'.al hyphae (peh) surround and obscure the asc1. Figure 28. Peridial hyphae (peh) of A. benhamiae. Their surface is spiculated and they exhibit regular strangulations conferring them with a c-haracter1st1c bead-like aspect. Figure 29. Ascospores (A) of A. benhamiae. Upon breaking the sac containing them, they are seen as small lentil-shaped bodies, with a smooth or slightly folded surface. Figure 30 . Peridial hyphae (peh) of ~. incurvata. They resemble cacti. Bar lengths: Figs. 25, 26, 28 and 30 = 5; Figs. 27 = 100; Fig. 29 1 micrometers.

371 R. Vazquez, J.M. Riesco, and A. Martin Pascual although the SEM method would be a useful comple­ sion and epidemicity. Ann New York Acad Sci, 89: mentary tool and would be of great use in the field 69-77 of research into the different species of the Derma­ 18. Georg LK, Camp LB (1957) Routine nutri­ tophytes. tional tests for the identification of dermatophytes. J Bact 74: 113-121 19.- Gueho E, Castro I, Badillet G (1985) References Existence of ornamentations on macroconidia and hyphae of Epidermophyton floccosum. Ann Inst 1. Ajello L (1956) Soil as natural reservoir of Pasteur Microbial, 136B: 195-207 human pathogenic fungi. Science, 123: 876-879 20. Hall FR (1966) Ringworm contracted from 2. Ajello L (1968) A taxonoimc review of the cattle in Western New York State. Arch Derm, 94: dermatophytes, and related species. Sabouraudia, 6: 35-37 147-159 - 21. Hildick-Smith G, Blank H, Sarcany I (1964) 3. Ajello L, Georg LK (1957) In vitro hair Fungus diseases and their treatment. Little Brown cultures for differentiating between atypical isolates and Company, Boston of Trichophyton mentagrophytes and Trichophyton 22. Ito Y, Setoguti T, Nozowa Y, Sakurai S rubrum. Mycopathol Mycol Appl, 8: 3-17 (1967) An electron microscopic observation of 4. Ajello L, Cheng SL (1967) The perfect Trichophyton violaceum. J Invest Dermat, 48: 124- 127 state of Trichophyton mentagrophytes. Sabouraudia, 23. Ito Y, Setoguti T, Nozowa Y (T969) Cell 5: 2 30-234 walls of Trichophyton mentagrophytes: Isolation and 5. Akin DE, Michaels GE (1972) Microsporum fine structure. Jap J Med Mycol, 9: 171-173 gypseum macroconidal development revealed by trans­ 24. Ito Y, Nozowa Y, Suzuki H, Setoguti T mission and scanning electron microscopy. (1970) Surface structure of dermatophytes as seen by Sabouraudia, 10: 52-55 the scanning electron microscope. Sabouraudia, 7: 6. Barlow AJE, English MP (1977) Infecciones 270-272 - micoticas. In: Recientes avances en Dermatologia 25. Keddie FM (1967) Further investigations of (Mycotic infections. In: Recent advances in Dermatol­ the electron microscopic examination of human hair ogy). A Rook (ed.), Editorial Jims, Barcelona, Spain, naturally infected by Microsporum gypseum. p. 124-152 Sabouraudia, 6: 1-4 7. Blank H. Taplin D, Roth FJ (1960) Electron 26. Marfin- Pascual A, Vazquez R (1979) Der­ microscopic observations of the effects of Griseo­ matofitos (Aspectos Clinicos y morfologia ultraestruc - fulvin in dermatophytes. Arch Derm, 81: 667-680 tural). Atlas (Dermatophytes (Clinical aspects and 8. Carbonell LM, Grill R (1971) Ultrastructure ultrastructure). Atlas). Ediciones Universidad de of sexual reproduction in Arthroderma benhamiae: In: Salamanca, Spain Recent Advantages in Microbiology, A Perez-Miravete 27. Mazabrey D, Nadal J, Seguela JP, Linas and D Pelaez (eds), Asociacion Mexicana de Micro­ MD (1985) Scanning and transmission electron biologia, Mexico, p. 481-492 microscopy: study of effects of econazole on 9. Chmel L, Buchvald J (1970) Ecology and Microsporum canis. Mycopathologia, 91: 151-157 transmission of Microsporum gypseum from soil to 28. Niimi K, Niimi M, Harada ~ Tokunaga M, man. Sabouraudia, 8: 149-156 Tokunaga J (1988) Macroconidial development and 10. Connole MD (1965) Keratinophilic fungi on germination in Trichophyton mentagrophytes. J Invest cats and dogs. Sabouraudia, 4: 45-48 Dermat, 90: 165-169 11. Dawson CO, Gentles JC, Brown EM (1964) 29. -Okoshi S, Takashio M, Hasegawa A (1963) Environmental conditions affecting sexual reproduc­ Isolation of Microsporum cookei from soil attaching tion in species of Arthroderma and Nannizzia. to animal hooves. Jap J Med Mycol, 4: 16-21 Sabouraudia, 3: 245-250 30. Osumi M, Yamada N, YamadaY, Yamaguchi 12. De Vroey C (1964) Formes sexuees des H (1984) The effect of Bifonazole on the structure of dermatophytes. Production de cleistotheces de Micro­ Trichophyton mentagrophytes. Dermatologica, 196: 19 - sporum gypseum (Bodin) Guiart et Grigoraki sur 22 - divers milieux steriles (Sexual forms of Dermato­ 31. Padhye AA, Carmichael JW (1972) Asco­ phytes. Production of cleistotheces of Microsporum spore morphology of Nannizzia and Arthroderma gypseum (Bodin) Guiart and Grigoraki in various species by scanning electron microscopy. Sabouraudia, sterile media) . Annales de la Societe Belge de 10 : 313-314 Medecine Tropicale (Brux elles), 44: 831-840 32. Padhye AA, Sekhon AS, Carmichael JW 13. Dwivedi AK, Kulshreshtha K, Ahmad KJ (1973) Ascocarp production by Nannizzia and Arthro­ (1985) A new technique for removal of fungus from derma on keratinous and non-keratinous media. leaf surfaces for scanning electron microscopic Sabouraudia, 11: 109-114 studies. Stain Technol, 60: 345-346 33. PettTI JHS (1960) Griseofulvin and favus. 14. Emmons CW, ITTnfor CH, Utz JP (1970) Brit J Dermat, 72 : 179-184 Medical mycology. 2nd Ed. Lea and Febiger. 34. PhilpoT C (1967) The differentiation of Philadelphia Trichophyton mentagrophytes from Trichophyton ru­ 15. English MP, Stockdale PM (1968) Tricho­ brum by simple urase tests. Sabouraudia, 5: 189-193 phyton proliferans sp. nov., A human pathogen. 35. Pock-Steen B, Kobayashi T (1970) Ultra­ Sabouraudia, 6: 267-270 structure of the hyphal wall and septum of Tricho­ 16. English MP (1972) The epidemiology of phyton mentagrophytes. J Invest Dermat, 55: 404-409 animal ringworm in man. Brit. J. Derm., 86, suppl. 8: 36. Poulain D, Biguet J, Deblock s;-vernes A 78-87 (1975) La paroi mycelienne de Trichophyton menta­ 17. Georg LK (1960) Epidemiology of the der­ grophytes. Etude ultrastructural de ses constitutives matophytes: sources of infection, model of transmis- apres application d 'une methode de mise en evidence

372 SEM Study of Dermatophytes des polysaccharides (The mycelium wall of Trichophy­ Discussion with Reviewers ton mentagrophytes. Ultrastructural study of their coats after a method to study polysaccharides). B. Persky: What is meant by the term "enriched Sabouraudia, 13: 244-254 media"? Do enriched media have a direct effect on 37. Rebel G, Taplin D (1974) Dermatophytes. the production of macro- and microconidia? Their recognition and identification. University of Authors: Some dermatophytes require special nutri­ Miami Press, 2nd edition, Coral Gables, Florida ent media to help in their identification or develop­ 38. Reichle RE, Alexander JV (1965) Multiper­ ment. For example, T. verrucosum needs thiamine forante septations, Woronin bodies and septa! plugs and inositol to develop macro- and microconidia; T. in Fusarium. J Cell Biol, 24: 489-496 violaceum and T. tonsurans require thiamine, etc. 39. Ripon SW (1982)Medical Mycology. 2nd edition, Saunders, Philadelphia B. Persky: Is the age of the fungal colony, i.e., 40. Roberts SOB, Mackenzie DWR (1986) My­ young versus old, important in the diagnosis of the cology. In: Textbook of Dermatology. 4th edition, A fungal species? Rook, DS Wilkinson, FJG Ebling, JLB Champion Authors: Yes, it is important. Very young colonies (eds.). Blackwell Scientific Publications, London, might not develop the macro and/or microconidia. pages 885-986 Old colonies may become pleomorphic, making it dif­ 41. Rush-Munro FM, Smith JMB (1971) Further ficult to identify the structures. observations on Trichophyton erinacei and T. pro­ liferans. Sabouraudia, 9: 61-64 B. Persky: Is there a correlation between the in 42. Shelley WB, -Shelley ED, Burmeister V vitro age of Trichophyton mentagrophytes interdigif= (1987) The infected hairs of tinea capitis due to ale colonies and the m vivo chmcal manifestation: Microsporum canis: demonstration of uniqueness of athlete's foot? - -- the hair cuticle by scanning electron microscopy. J Authors: We believe there is no correlation since in Am Acad Dermatol, 16: 354-361 the lesion the fungus never develops the morpholog­ 43. Smith JMB, Sandler JU (1971) The ical characteristics observed in the colony. structure of saprophytic and parasitic dermatophyte spores. Mycopathol Mycol Appl, 43: 153-159 B. Forslind: In your opinion, how can the TEM and 44. Vanbreuseghem R (1952)Techniquebiologi­ SE M, respectively, be used for analysis of dermato­ que pour l 'isolement des dermatophytes du sol (Bio­ phytes in culture and in infected integument and its logical technique to isolate soil dermatophytes). appendages? Annales de la Societe Beige de Medecine Tropicale, Authors: As explained in the overview, the samples 32: 173-178 (scales, hair, etc.) are obtained directly from the 45. Vanbreuseghem R (1966) Guide pratique de lesion; when these are suitably prepared they can be Mycologie Medicate de Veterinaire (Guidebook of observed with the light microscope, SEM and TEM. Medical and veterinary mycology). 1st edition, This does not permit one to differentiate the causa­ Masson and Cie (eds). Paris tive agent but it does allow one to know whether 46. Velasco-Benito JA, Martin-Pascual A, the lesion has in fact been produced by fungi. Garcia-Perez A (1979) Epidemiologic study of Derma­ tophytoses in Salamanca (Spain). Sabouraudia, 17: B. Forslind: It appears, at least in ~ gypseum, that 113-123 - the development (number) of knobs on macroconidia 47. Vismer HF, Findlay GH, Eicker A (1987) is related to the age of the culture. Can you de­ The septa! ontogeny, germination and electron mi­ scribe this in relation to a time scale of culture? croscopy of Microsporum gypseum macroaleurioconi­ Are corresponding phenomena also observed in other dia. Mycopathologia, 98: 149-164 pathogenic organisms? Do you see corresponding 48. Visset MF 0972) Les formes conidiennes events in infected skin? Can you comment on the du complexe Microsporum gypseum observees en mi­ possible important growing condition differences croscopie electronique a balayage (Conidia forms of between cultures and actual infected tissue? Microsporum gypseum complex studied by scanning Authors: The time of development of a colony of electron microscopy). Sabouraudia, 10 : 191 - 192 dermatophytes varies depending to several different 49. Werner HJ, Jolly HW, LeeJH (1964) Elec­ factors (temperature, the amount of fungi in the cul­ tron microscopic observations of Epidermophyton ture media, etc.). In the case of M. gypseum, when floccosum. J Invest Dermat, 43: 139-143 the colony begins to develop (3-5 days) young ma­ 50. Werner HJ, Jolly HW, Spurloch BO (1966) croconidia with few knobs are seen. When the colo­ Electron microscopic observations of the fine struc - ny is well developed (7-10 days) the number of knobs ture of Microsporum canis. J Invest Dermat, 46: 130- is much larger. This latter fact can also be seen in 134 - other dermatophytes such as M. canis. 51. Werner HJ, Catsulis C, Jolly HW, Carpenter When dermatophytes are seen on infected skin CL (1967) Electron microscopic observations of the (lesions), they do not show any features with which fine structure of Microsporum gypseum. J Invest Der­ they can be differentiated amongst one another. mat, 48: 481-484 Their morphology is completely different from that 52. Werner HJ, Jolly HW, Carpenter CL (1968) observed in artificial culture media. Thus, on infec­ Observations on the fine structure of Microsporum ted skin they do not develop either macro- or micro­ audouinii. J Invest Dermat, 50: 276-279 conidia; their reproduction under such circumstances 53. Wilson JW, Plunkett OA (1965) The fun­ is by spores. gous diseases of man. University of California Press, It is important to note that the development of Berkeley the fungus, its morphology, and form of reproduction are different according to the medium in which it is observed (on infected skin, in artificial culture media

373 R. Vazquez, J.M. Riesco, and A. Martin Pascual

- Sabaureaud's agar - or in the case of some derma­ J .T. Sibley: Candida albicans is a fungus with tophytes, on hair on soil surfaces). keratmolytlc ability that infects man. Can you compare the morphology of C. albicans with the J. T. Sibley: Do Dermatophytes contain keratinolytic morphology of dermatophytes by emphasizing the enzymes? most salient differences and their functional Authors: Yes they do, and that is why they are able importance? to digest keratin. Authors: C. Albicans may exhibit two different mor­ phological aspects: 1) the yeast form, when it is J. T. Sibley: By what mechanisms do dermatophytes found as a saprophyte, and 2) the mycelium form invade the skin? when it acts as a pathogen. These morphological Authors: As in the case of any other infectious differences in both the saprophyte and pathogenic agents, the reasons why a dermatophyte can or can­ states are not exclusive to candida albicans; they can not invade skin are not well known. An example of also be seen in other yeasts such as Phytosporum this is to be found in the classical cases of Kligman orbicularis or obalis (the agent responsible for with experimental inoculation of humans with M. versicolor pitiriasis). canis and other species. In these instances, only m On lesions, dermatophytes can be observed as some cases was it possible to achieve infection, mycelia, more or less septate, and as spores, or even regardless of whether the receivers had or had not in both forms; however, they are always seen as had previous infections. In the work of this author pathogens. information was given as to how parasitization occurs but not through which mechanisms.

J. T. Sibley: How do antifungal agents act? Authors: Antifungal agents may act through two mechamsms: fungistatic or fungicidal. In many cases, such as with griseofulvin, it is difficult to say which of these two mechanisms operates.

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