Molecular Detection of Human Fungal Pathogens As (Except a Few That Have Newly Evolved As Anthropophilic Dermatophytes

36 Microsporum*

Rahul Sharma and Yvonne Gräser

Contents 36.1 Introduction...... 285 36.1.1 Classification, Morphology, Biology, and Epidemiology...... 287 36.1.1.1 Classification...... 287 36.1.1.2 Morphology...... 288 36.1.1.3 Biology and Epidemiology...... 289 36.1.2 Clinical Features and Pathogenesis...... 290 36.1.2.1 Clinical Features...... 290 36.1.2.2 Pathogenesis...... 290 36.1.3 Diagnosis...... 291 36.1.3.1 Conventional Techniques...... 291 36.1.3.2 Molecular Techniques...... 291 36.2 Methods...... 292 36.2.1 Sample Preparation...... 292 36.2.1.1 DNA Extraction from Fungal Colony...... 292 36.2.1.2 DNA Extraction from Clinical Specimen...... 293 36.2.2 Detection Procedures...... 293 36.2.2.1 Species Recognition...... 293 36.2.2.2 Strain Typing...... 293 36.3 Conclusions and Future Perspectives...... 295 Acknowledgments...... 295 References...... 295

36.1 Introduction phytes—plants); the other two are Trichophyton Malmsten5 and Epidermophyton Sabouraud6 first described in 1845 and More has been written about ringworm than any other 1910, respectively. The name Microsporum refers to numer- mycotic disease of animals and man and for no other myco- sis is the older literature more confused. ous micro-arthrospores covering formed during its (ectothrix type) growth on hair surface unlike Trichophyton that grows 1 G.C. Ainsworth and P.K.C. Austwick, 1973 inside hair shaft. The similarity among these dermatophytic genera extends The above quote was written when polymerase chain reac- to their nutritional requirement, which is mainly keratin7 that tion (PCR)-based molecular methods have not yet arrived forms exoskeleton covering in three groups of higher verte- at the fungal taxonomic scene and especially for mycolo- brates, i.e., birds, reptiles, and mammals. Their keratinolytic gists’ studying dermatophytes. The PCR technology as we nature itself suggests that they originated when life came to know now was only available when Kary B. Mullis made land from water because aquatic vertebrates (e.g., whales) a breakthrough of amplifying DNA in vitro,2 and 2 years lack exoskeleton adnexes like hair, feather, hooves, which are later the use of thermostable polymerase3 made the whole primarily made of keratin and we don’t have Microsporum process automated, which revolutionized biological inves- (or other dermatophyte), which is aquatic (marine) in nature tigations. This single method has undoubtedly had more because it has been shown that growth of human pathogenic application in biology today than any other technique in fungi (including dermatophyte M. gypseum) is inhibited by whole of biological sciences including diagnosing human high salt concentrations.8 Also, rhexolytic dehiscence mech- fungal infections. anism is found among fungi that have dry spores as in all The genus Microsporum established by Gruby in 18434 Microsporum species (and not in mucilaginous mass as in was the first genus to be described among the three genera Fusarium spp., which form blastospores). Among the three commonly referred to as dermatophytes (dermal—skin; dermatophytic genera, Microsporum is well adapted to soil

* The authors dedicate this chapter to Prof. Libero Ajello, the man who knew so well the natural history of dermatophytes.

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© 2011 by Taylor & Francis Group, LLC 286 Molecular Detection of Human Fungal Pathogens as (except a few that have newly evolved as anthropophilic dermatophytes. The gene(s) responsible for triggering or species) its entire species produce macroconidia, which have inhibiting the production of macroconidia and the extent of rough surfaces unlike their Trichophyton counterparts. this inactivation in anthropophiles is yet to be investigated. The member of the genus can be grouped on the basis It is seen in agar or hair cultures of dermatophytes that the of ecological preference as geophilic (saprophytes in soil), fungus forms spores once the nutrients are depleted (this is zoophilic (adapted to lower animals), or anthropophilic true for the formation of sexual stages, i.e., sexual repro- (adapted to human).9 These ecological groupings of various duction and formation of ascomata as in Takashio dilute species are reflected in the phylogenetic clusters obtained by Sabouraud agar20) and the phenomenon is not uncommon in mtDNA10 and rDNA studies.11 This suggests that they all were wide range of fungi. Once a fungus gets human skin (which previously soil dwellers and used to thrive on shed keratin. is still attached to human body), it can ideally grow over Close or direct contact with live animals and later humans its whole body, but due to variable microenvironment and made them well adapted to their zoophilic and anthropo- medical arrangements, the fungus is prevented from spread- philic lifestyle, respectively. Also, the anthropophilic mem- ing and mostly controlled; however, severe cases are often bers (M. ferrugineum and M. audouinii) must have evolved reported when no medical treatment is done or due to altered along with Homo sapiens (modern human), which have microclimate (caused by drugs or reduced inherent immu- sparse hairs on their body unlike its earlier hominid viz. nity). Human skin is like a continuous culture with regular Australopithecus afarensis, Homo habilis or related primate supply of fresh keratin from beneath. The fungus anchors its like ancestors. This hypothesis needs further investigation mycelia with the fresh integument by spreading radial in a with right molecular marker and algorithm,12,13 which could ring-like fashion (hence, the popular term ringworm coined precisely date and calibrate the divergence of M. canis into by Anglo-Saxon ancestors in sixteenth century),9 and thus sibling species M. auduoinii in Africa and M. ferrugineum ensuring its survival on host. in East Asia to superimpose precisely with the divergence Africa is thought to be the center of origin of Homo sapi- of Homo sapiens from Africa to East Asia. One theory sug- ens based on the available evidence.21 Ancestral dermato- gests that modern Homo sapiens developed relatively hair- phytes including young anthropophiles also might have their less bodies to evade parasites14 which certainly would have origin in Africa.22 fsI this i true, then the separation of two included dermatophytic fungi. For adaptation to this altered lineage to become distinct species of the M. canis clade after trait of nakedness15 ni humans, the evolving dermatophytes early humans moved “out of Africa” to Southeast Asia23,24 (geophiles > zoophiles > anthropophiles) that produce mac- is probable, thereby causing allopatric speciation25 resulting roconidia for transmission, slowly lost their ability by natural into two closely related anthropophilic species M. auduoi- selection since transmission became mainly through direct nii and M. ferrugineum. A recent trend of cryptic lineages26 contact and propagules like arthroconidia. Macroconidia is witnessed in the distribution of minus (−) mating type, cling to hairs of animals for dispersal and host can be symp- which tend to be well adapted to human but a lineage origi- tomless carriers like the cats,16 snwhich i a indicator that nally occurring on animals.27 This assumption gains support cat dwelling areas are survival grounds of M. canis. Cats’ from the study establishing first domestication of middle loose hairs are shed in large numbers (an attribute familiar eastern wild cat by people of Israel 9500 years ago based to cat owners), enabling the host to dislodge clinging mac- on archeological and historical records.28 Also, examination roconidia and avert infection; however, the macroconidia of of DNA from 1000 wild cats and domestic cats across old M. canis in particular is well adapted to zoophilic nature world showed all modern domestic cats descended from the as its curved and roughened apex help attachment to ani- middle eastern wild cat Felis silvestris lytica.29 Could this mal skin, and the overall shape resemble an arrow head be the time (10,000 years ago) when M. canis first seriously that penetrate through the dense hairs to reach skin apart interacted with humans? from being anti-grazing devise postulated by Summerbell.17 The cosmopolitan distribution of certain species of der- Macroconidia are rare or absent in present anthropophiles matophytic fungi (not associated with birds) was greatly because the transmission also is hardly a problem for the facilitated by humans and the animals he transported (wild fungus when the host are in plenty (considerable rise in and domestic) in recent past, i.e., within 5000 year BP. The human population), and chances of not finding a suitable massive movement of human population during wars in more host are rare because urban population recently reached 3.2 recent time (1–2k years) dispersed dermatophytic fungi, billion (crossing the world rural population of 3.1 billion),18 which otherwise were restricted continently. Unlike certain where more humans live per unit area, a condition favorable rust fungi, which infect huge number of host plants at a time for anthropophilic adaptation and evolution. Therefore, the that could generate unimaginable spore inoculums and due to need for dispersal spores adapted to transfer through furred small size and lighter spores have intercontinental dispersal animals was gradually lost or greatly reduced, leaving the through air current,30 the dermatophytes have comparatively production of resting spores resistant to adverse condi- large and heavy spores (due to which it can hardly reach the tions like arthrospores (chlamydospores). These contagious turbulence layer)31 and are never able to develop such a huge propagules are produced in large numbers and have shown spore inoculum since they infect meager number of hosts successful adhesion to stratum corneum under experimental at a time to have intercontinental dispersal by themselves conditions,19 thereby effectively transmitting anthropophilic and need animal or other aid for long-distance travel. Due

© 2011 by Taylor & Francis Group, LLC Microsporum 287 to preening habit and flight, dispersal by birds is limited to tool(s). Also, we incorporate a portion of morphological detail fungal species that have specialized clinging structures like in the conventional method section to help non-taxonomist, Ctenomyces serratus (that forms ctenoid appendages)32 whose parataxonomist, or even pure molecular taxonomist to appre- intercontinental dispersal through birds might have enabled ciate the elaborate morphology that Microsporum species regular gene flow among its populations across continents represent through their millions of years of evolutionary his- and prevented allopatric speciation (the species is monotypic) tory, something Perkins felt for Neurospora.40 The principle as noted for some of the M. fulvum strains infrequently iso- aim of our article is to help user accurately identify known lated in Central Europe from sand-martin (Riparia riparia Microsporum species and able to recognize newer ones in L.) whose winter migration is in East Africa (with 8000 km their future encounters with the genus. The article also dis- migratory route).33 The species of dermatophytes and kerati- cusses the recent developments in the dermatophyte identifi- nophilic fungi reported by Ajello and Padhye in 197434 from cation like the use of alternate locus to the ribosomal DNA Galapagos Islands (Darwin fame) mostly are bird associated like the chitin synthase gene for rapid detection,41 efus o mul- and no Microsporum species was recorded. The lack of zoo- tiplex RT-PCR,42,43 oligonucleotide array44 along with the sta- philic or geophilic dermatophyte species (either Microsporum tus of genome sequencing in two of the Microsporum species or Trichophyton) pathogenic to humans is understandable viz. M. canis (a zoophile) and M. gypseum (a geophile) pre- in terms of the relative lack of past human disturbance (or viously prioritized among five dermatophytes (include three migration) in the Galapagos before Darwin arrived. Trichophytons—T. rubrum, T. tonsurans, and T. equinum) by Since the inception of molecular methods in disease diag- the Dermatophyte Genome Steering Committee.45,46 nostics, dermatophyte detection, and species identification of clinically relevant (almost all) Microsporum species has 36.1.1 Classification, Morphology, become more accurate and precise in comparison to the less consistent morphology-based methods. However, even today Biology, and Epidemiology morphology is still important in providing preliminary data 36.1.1.1 Classification for starting the molecular analysis for species identification. The genus Microsporum is a mitosporic fungus that multi- In the absence of any supporting morphological details viz. plies by producing mitospores: macro- and microconidia clinical symptoms, source of infection or even preliminary whose sexual state lies in ascomycete genus Arthroderma, slides prepared from clinical specimen, molecular identifi- some species of which produces Chrysosporium and cation becomes blind and sometimes time consuming; how- Trichophyton asexual stages.11,47,48 Table 36.1 lists all known ever, the above morphological attributes, if checked, greatly teleomorphs, i.e., fungi having Microsporum asexual states help apply molecular methods precisely. including those that lack sexual stage in their life cycle, along For molecular identification of Microsporum species, sev- with their natural habitat and distribution, which has been eral approaches are now available, which initially started with greatly altered due to human migration in the last century. 35 the use of DNA homology studies ro mitochondrial DNA The taxonomic positioning of Microsporum as per current 10 analysis. eNow, w have better approaches or loci available scheme of fungal classification49,50 and the reason thereof is that can utilize even fraction of the genomic DNA to cor- given below, with example of Microsporum gypseum, the rectly diagnose a Microsporum species. The present chapter first Microsporum species (in fact the first dermatophyte) to provides account of the preferred method, which is conve- have complete genome sequenced. nient in application by even small laboratories and diagnostic facilities. Although, in recent times, excellent reviews have Superkingdom Eukarya True nucleus 36–38 been published nthat gives a overall view in itself of the Kingdom Eumycota True fungi dermatophyte species definition, strain differentiation or the Subkingdom Dikarya Dikaryotic hyphae molecular methods involved in their identification and men- Phylum Ascomycota Ascus as diagnostic character tioning them all again will be repetition of the already avail- Subphylum Pezizomycotina Asci within fruit bodies enclosed able text. However, we do discuss in some detail a method partially or completely that is widely applied in population-based investigations, Class Eurotiomycetes Mostly heterogenous assemblage i.e., microsatellite markers for strain typing.37 Microsatellite of taxa markers are useful in identifying origin of infection (e.g., a Subclass Eurotiomycetidae Stroma absent, ascomata mostly pet, cattle, or environment), endemic strain introduced into cleistothecial, often brightly a new area, multiple strain infection, or even relapse or rein- colored, evanescent asci fection, all of which can be detected if a good polymorphic Order Onygenales Ascomata from coiled initials, marker is available. Recently developed microsatellite mark- peridium loosely interwoven thick-walled hyphae ers for two Microsporum species were able to detect poly- Family Arthrodermataceae Rough-walled, dumbbell-shaped morphisms first in an otherwise known clonally reproducing ossiform peridial hyphal cells, species (M. canis) and in the second (M. persicolor), to be a ascospores-minute smooth- 27,39 common old species in India and not introduced recently. walled discoid, anamorph either Heree w present the practical approach of “how to” iden- Chrysosporium, Microsporum, tify or detect Microsporum species using modern molecular or Trichophyton

Genus Microsporum Forms echinulate holothallic two macroconidia in Malt agar or Sabouraud agar supplemented to multiseptate macroconidia with horse or human hair except in species in which they are Species Microsporum Macroconidia in large clusters, seldom formed. gypseum rather thin-walled, regularly There exist gross similarities among these fungal forms verrucose, fusiform, 3–6(−8) in their macrospore morphology in that they are multi- celled, 25–60 × 8.5–15 μm septate and have rhexolytic dehiscence mechanism of dis- charge.51 The similarity in their macrospore morphology is probably a result of adaptive evolutionary pressures for the 36.1.1.2 Morphology dermatophytic lifestyle, which is supported by their phylo- Characteristic morphology of Microsporum is the holothallic genetic and morphological closeness to keratin degrading macroconidia the species produce, which is mainly two to Chrysosporium spp. whose aleurioconidia are indistinguish- multi-septate transmission propagules. The number of cells able from microspore morphology of Microsporum spp and in a single macroconidium represents the same number of Trichophyton spp suggesting Chrysosporium to be ances- sub-propagules, all of which can germinate to form indi- tral to both (macroconidia forming) Microsporum and vidual thalli. Microsporum spp. mostly produces abundant Trichophyton.52

Table 36.1 Known Sexual States of Microsporum Species and Their Habitat/Host Preference Teleomorph Anamorph Natural Prevalent Biosafety Reference Strain Sequence S. No. (Sexual State) (Asexual State) Reservoir Host(s) Distribution Levela Number and Statusb Accession 1 Arthroderma Microsporum Soil — Brazil/Africa BSL1 CBS 967.68, ST AJ877220 borelli amazonicum 2 Arthroderma Microsporum Soil — Global BSL1 CBS228.58, AUT AJ970145 cajetanum cookei 3 Arthroderma Microsporum Soil — Global (seems — CBS101.83, HT, MT− AM000035 cookiellum anamorph to be) 4 Arthroderma Microsporum Soil — Africa (rare) BSL1 CBS364.81, HT, MT+ AJ970143 corniculatum anamorph 5 Arthroderma Microsporum Soil — Global BSL1 CBS287.55, T AJ000627 fulvum fulvum 6 Arthroderma Microsporum Soil/animal Fowl Global BSL2 CBS243.66, T, MT+ AJ000612 grubyi gallinae 7 Arthroderma Microsporum Soil — Global BSL1 CBS258.61, NT AJ970141 gypseum gypseum 8 Arthroderma Microsporum Soil — Global BSL1 CBS174.64, T AJ970153 incurvatum gypseum 9 Arthroderma Microsporum Animal Pig Global BSL2 CBS322.61, T, MT− AJ970149 obtusum nannum 10 Arthroderma otae Microsporum Animal Cat, dog, horse, Global BSL2 CBS496.86, ET, MT− AJ000619 canis human 11 Arthroderma Microsporum Soil — Global BSL1 CBS424.74,T,MT− AJ970146 racemosum racemosum 12 Arthroderma Microsporum Soil Vole, rat Global BSL2 CBS468.74, MT− AJ000615 persicolor persicolor 13 — Microsporum Human Human Africa BSL2 CBS545.93, −NT AJ000623 audouinii 14 — Microsporum Human Human Asia, Africa BSL2 CBS497.48, AUT AJ252336 ferrugenium 15 — Microsporum Soil — France and BSL2 CBS288.55, AUT AJ970148 praecox USA (rare)

Source: de Hoog, G.S. et al., Atlas of Clinical Fungi, 2nd edn., Centraalbureau voor Schimmelculturales, Utrecht, the Netherlands, 2000. a BSL1, saprobes occupying non-vertebrate ecological niches; BSL2, species principally occupying non-vertebrate ecological niches, but with a relatively pronounced ability to survive in vertebrate tissue. b AUT, authentic strain; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, HT, holotype strain; MT, mating type; NT, neotype strain; ST, syntype strain; T, type strain.

36.1.1.3 Biology and Epidemiology depends largely on hosts’ immunity, pathogen availability, Microsporum is an asexual stage of the sexually reproducing and virulence along with favorable microenvironmental ascomycete genus Arthroderma.47 The fungus is mostly het- conditions (e.g., traumatized skin—wet for long time as in erothallic with two mating types plus (+) and minus (−) that swimming pools, sports person or soldiers during training mates under favorable conditions in soil to produce ascomata with shoes on for long hours, or farmers or farm workers in as in Arthroderma otae.53 sa the phenomenon has been dem- developing world working barefoot) and most importantly onstrated in laboratory on Takashio’s dilute Sabouraud agar reduced hygiene that brings all these conditions together. and soil-keratin culture where compatible mating types fuse This is true for animals also, e.g., cats licking habit (at least to form fertile ascomata, which bear innumerable asci and once a day) and avoidance of water allows it to avert derma- ascospores (the product of meiosis),54 also referred as meio- tophyte spores to reach its skin surface that remain clinging spores. Sexual reproduction is essential for genetic fitness in to its dense but loose body hairs and hence are mostly non- an organism as it involves recombination and production of symptomatic carriers; inability to lick their face might be newer genetic traits for better adaptability to the continuously the reason why cats have inconspicuous lesions around nose, changing environment. Figure 36.1 illustrates a life cycle of eyes, and ears caused by M. canis.57 However, the habit is sexual Microsporum species (Arthroderma sp.); only the uncommon in dogs, which have low rates of infection in pet anamorphic stage is clinically active and acquires greater dogs58; however, this does not hold true in stray dogs (with virulence27 to be able to infect humans. little hygiene and medical care) that have much higher rates Infections by Microsporum species (and other dermato- of dermatophyte infection (personal observation of RS in phytes) affect a considerable part of human body55 and popu- India), thus helping the fungus maintain itself in nature simi- lation worldwide.56 The incidence of dermatophyte infection lar to the mechanism known for obligate plant pathogens like

1 Sexual Arthroderma state (in soil)

7 2

T cap

T corp

6 3 T ped

4.5

+ –

4 5

(a)

(g) (f) (b) (d) Asexual Microsporum state (e) (on host and in soil) (c)

Fge i ur 36.1 Life cycle of Arthroderma species (clockwise, not to scale) having Microsporum anamorph, which cause skin infections in humans. The line separating the two states are crossed when keratin is in plenty or scarcity. 1: Ascoma (peridium) bearing thick-walled dumble-shaped ascomatal hyphae with coiled appendages; 2: Evanescent asci and ascosores shed freely from ascoma after maturity; 3: Ascospores germinating and colonizing hair in soil; 4a: Fungal mycelium with conidiophores bearing young (nonseptate) and mature (septate) macroconidia, b: Clavate sessile microconidia, c: Thick-walled arthroconidia, d: Typical racquet hypha, e: Enlarged single five-celled macroconidium (four septate), f: Germinating macroconidium, g: Liberated microconidium; 4.5: Entry of Microsporum into human niche directly or indirectly through contact with infecting (mitotic) propagule which cause three main clinical manifestations: Tinea capitis, Tinea corporis, or Tinea pedis (first two due to adaptation to active dispersal of macroconidia by flight or through animal contact and third due to passive dispersal by arthroconidia through direct ground contact of humans); 5: Approaching receptive hyphae of opposite mating type (+) and (−); 6: Coiling of ascomatal initials; 7: Nascent asci formed after genetic recombination and meiosis.

© 2011 by Taylor & Francis Group, LLC 290 Molecular Detection of Human Fungal Pathogens the physiological races of wheat rusts (having alternate and time like wet humid skin. Humans get easily infected by zoo- collateral hosts systems)59 for survival in absence of favored philic dermatophyte through contact with infected or nonin- host, i.e., wheat crop. Birds are another example of hygiene fected symptomatic carrier animals57 that may cause severe maintenance as their preening habit meant for removing symptoms in humans because naked human skin has fewer parasites or unwanted objects along with application of oil barriers compared to animals skin (which is densely covered coating on feathers60 prevents them from potential infections. with hair) and, in general, have high levels of immunity. Also, their tough covering on feet makes it difficult for a dermatophyte to colonize and digest, similar to tail hair of lions, 36.1.2 Clinical Features and Pathogenesis which is also relatively unaffected compared to hairs of other animals including human when subjected to Microsporum 36.1.2.1 Clinical Features gypseum treatment.61 Birds also get rid of occasional clinging Clinical features caused by Microsporum apparently visible of propagules onto their feather by flight strokes; however, on human host are mostly characteristic for a dermatophytic weak or ill ones are prone to dermatophytic infections (since infection but not always as the name suggests in the form of they can’t fly). rings. And thus, are commonly but inappropriately referred Tinea capitis in pre-independent India was mostly found in to as ringworm (as no worm is involved) because of a ring- Europeans, Jews, and Anglo-Indians as reported in an exten- like appearance of the advancing infection, which is due sive (50,000 skin samples during 5 years) study of fungal to the radial growth of fungus on the skin (similar to their infections in Calcutta62 possibly due to application of wide growth on artificial medium viz. Sabouraud Dextrose Agar variety of hair oils viz. amla, almond, coconut, and mustard [SDA]). by natives, a custom followed from ages by both males and Principally, clinical features of dermatophytes are recog- females (a similar story in nature can be found among birds nized and named after the part of the human body infected that have natural coating). Garg63 using a combination of bio- along with the word “tinea” that is being used since the time chemical assay and transmission electron microscopy showed of first recorded reference of dermatophyte infection (around the toxicity of certain hair oils used by Indians against four 30 AD).69 tI was used as a generic name for the cloth moth dermatophytes including M. canis and M. gypseum in which (Tinea sp.) that feeds on keratin of woolen garments, thus Amla (Emblica officinalis) oil was most effective. making characteristic holes in them, whose resemblance to Infections are more prevalent in persons with lower ring-like lesion caused by fungal infection on smooth skin socioeconomic conditions but such cases are greatly under made Anglo-Saxon people to coin the term ringworm for reported and unrecorded (reason for this is dermatophytes such fungal infection in sixteenth century.9 The term is now are never life threatening and food is a priority for survival more popular for designating fungal infection types than the than a mere skin infection), unlike in persons with good insect whose generic name is Tinea. The various types of der- socioeconomic conditions and better hygiene (in such cases, matophyte infection or the clinical manifestations in which 90%–95% cases are recorded). On a community scale, this Microsporum spp. are involved (including other dermato- holds good for developed and underdeveloped locations, cit- phytes) are Tinea capitis-infection of head, scalp, eyebrows, ies, or even countries. Lack of basic microbiological knowl- and eyelashes (all Microsporum spp.); Tinea favosa infection edge among common people is one of the foremost causes of of hair called crusty hair (M. gypseum, rare), Tinea barbae increased incidence of dermatophytic infections worldwide. infection of beard (M. canis), and Tinea corporis infection of This includes knowledge that environment is the potential body or smooth skin (M. audouinii, M. canis, or any derma- reservoir of dermatophytes64; stray animals like cats and tophyte).70 All Microsporum species grow as ectothrix, i.e., dogs (with or without visible symptoms); garbage (which is they grow outside the hair shaft and form numerous arthro- dumped indiscriminately in landfills areas, sewage (a big conidia surrounding it while destroying the cuticle. source of shed keratin),65 etc. Also, a fact that a dermatophyte takes a day or two to cause primary athlete’s foot infection in 36.1.2.2 Pathogenesis a human being after adherence of infectious propagules (viz. Pathogenesis of keratinophilic fungi including dermato- microconidia, macroconidia, chlamydospores) on human phytes is due to their ability (they possess from their early skin or its adnexes,66,67 infections in other body parts may origin) to break down keratin,71 the second most abundant take longer due to not so congenial microenvironment like polymeric molecule on earth after cellulose, by releasing feet; not washing oneself for a day or two renders a person extraordinary amounts of enzymes often referred to as kera- susceptible for infection if one is in contact with infectious tinases, which are in true sense proteases.72 sA studies in propagule(s).66 This condition can be with soldiers, hik- M. gypseum using radioactive tracer technique (35S cystine) ers, mountaineers, slum dwellers, or all those persons liv- showed the breakdown of native keratin is by the process ing in water scarcity areas (a considerable portion of human of sulphitolysis,71,73 and production of keratin digesting population does not have enough water).68 Human infec- enzymes as noted in M. canis.74,75 The fungus mainly attack tions are generally caused when they come in contact with keratinous material by surface erosion and radial penetra- propagules(s), which are the overwintering structures formed tion by formation of boring hyphae (similar to appressoria in the life cycle (Figure 36.1) of a dermatophyte and when or haustoria formed by plant pathogenic fungi to absorb favorable environmental conditions are prolonged for some nutrients from host plants59) where most of the secreted

© 2011 by Taylor & Francis Group, LLC Microsporum 291 proteolytic enzymes are concentrated.76 Once the mycelia fragments by the brush) and can be brought to the labora- formed from freshly germinated arthroconidia have pene- tory. The brush can then be pressed onto the SDA plate with trated keratin substrate and had established infection, exten- cycloheximide. sive radial colonization under skin follows, which is seen as Microscopic observation: KOH or NaOH mounts (10%– various clinical manifestations like the characteristic rings 30%) plus 5% glycerol (to emulgate lipids) can be prepared on smooth skin. directly from the clinical specimen, gently heated to observe for any hyphae or propagules, the preparation can be appro- priately stained with cotton blue to visualize fungal mate- 36.1.3 Diagnosis rial. Fluorescence stain with Calcoflour,84 Kongo red or Diagnosiss i the key for all successful medical treatment Mycetfluo®79 can improve the visualization. for all types of diseases including dermatophytic infection. Culture preparation on Sabouraud’s dextrose agar or If diagnosis is correct, right medication can prevent fur- mycotic agar: Once microscopy has revealed the presence of ther spread and agony to the patient. A dermatophytic fun- fungal material, a second half of the material can be planted gal diagnosis is primarily important to successfully render aseptically onto SDA or Mycotic agar containing antibac- appropriate therapy and eventually control of the disease. terials (usually gentamicine and/or chloramphenicol plus Diagnosing in early days of infection allows for immediate cycloheximide). antifungal treatment and possible control; however, in the Hair perforation tests: Using human or horse hair (ster- absence of correct diagnosis, the treatment could be false, ilized by tindalization), hair perforation test85 neca b per- which might help the fungus to further establish infection formed, which involves placing the hair on glass beads like in nail infections, which later becomes difficult to treat. kept inside a presterilized Petri dish containing little distil Once infection is deep, the treatment takes long and relapses water. The fungus cultured in pure form can then be inocu- can frequently occur. lated aseptically onto the hair and incubated for 1–2 weeks. Dermatophytes can establish infection faster than we The fungus causing active degradation by means of boring can imagine, i.e., even half day (T. tonsurans),67 which hyphae can be visualized by cotton blue mounts of the inocu- prompt us to find faster and accurate diagnostic procedures. lated hair. The test is particularly useful in differentiating M. Conventional culture-based techniques are time taking and canis (positive) from M. audouinii (negative).86 However, the often do not always give positive results even if sample is horse variant of M. canis cannot be distinguished from M. positive for dermatophytic infection.77 Currently, laboratory audouinii due to being hair-perforation negative for which testing procedures are integrating the two techniques for bet- molecular methods are useful.27 ter diagnoses and understanding of dermatophytes diversity, since culturing a new dermatophyte to reveal its morphology 36.1.3.2 Molecular Techniques is essential in describing a novel species78 and future drug With molecular techniques available, a diagnosis of infec- testing for control therapy. Further section describes briefly tions caused by Microsporum from a clinical sample or the conventional techniques used in dermatophyte diagnoses material has become relatively rapid, exact, and authentic. and molecular techniques in more detail. In a recent study published comparing the conventional (microscopic examination and culture-based method) and 36.1.3.1 Conventional Techniques molecular methods to detect dermatophytes in nail and skin Recently, an excellent review by Robert and Pihet79 has samples clearly showed the contrast in positive rates of PCR appeared on conventional methods for diagnoses of dermato- versus culture (95%–99%—PCR versus culture—67% [skin] phytoses apart from a more comprehensive earlier treatment and 33% [nail]) clearly in favor of PCR method based on by Kane et al.80 that greatly complement the rapid molecular nuclear ribosomal internal transcribed spacer (ITS1) DNA techniques. Some of the conventional techniques mentioned sequences.77 below can be applied as preliminary tests prior to performing However, morphological features are still useful like molecular analysis, all of which starts with the right method clinical symptoms on host or colony appearance on artificial of collecting clinical specimen, i.e., infected material from media (if culture is successful, which is when the samples are an infected host.81 collected from growing portion of the lesion) or the presence Wood’s lamp: It is used to visualize dermatophytic infec- of racquet hyphae in culture; all of which even makes the tion (caused by M. canis and M. audouinii), seen as yellowish molecular identification more focused by excluding most of green fluorescence under dark, however not all fluoresce. the non-dermatophytes from picture and suggesting the use Hair brush technique: Although the method has been of species-specific primers (for M. canis87 and M. audoui- devised for animals,82 tnei ca b used for humans as well.83 nii88)r o probes (for T. rubrum).89 fsI culture i successful, and It includes use of a presterilized hair brush (having dense macroconidia are formed, then their surface feature indicates arrangements of hair bristles) to brush the infected portion whether it is Microsporum (rough walled) or Trichophyton of the hairy body (head of human or body of pet). The brush species (smooth walled). can be placed in sterile, preferably paper bags (since poly- Currently, the technique(s) most commonly applied in rec- thene bags tend to possess static charge, which attracts loose ognizing Microsporum species due to its simplicity (as it can particles and hence can prevent retention of spores or skin be performed even by nonspecialists) and cost effectiveness

(cane b routinely applied by a small diagnostic laboratory) can be collected in fresh paper envelops or sealed plastic bags is—ITS-PCR-RFLP. ITS-PCR-sequencing is able to differ- and brought to the laboratory for processing. Material (skin entiate also close-related species but a method that is not so or hair) can be collected from growing peripheral portion of easy to perform and more expensive. Both techniques involve the lesions. the targeted in vitro amplification of fungal pathogen DNA Most samples can be directly subjected to DNA extraction (Microsporum species) in thermal cycler to enhance its vis- (preferably using a DNA extraction kit if the sample is less ibility (on a gel matrix). The amplified DNA usually from a than 5–10 mg) but a part of the sample can be sent for culture selected site of the genome (viz. best studied is the ITS region (and slide preparation) in appropriate laboratory (if host labo- of the ribosomal DNA) can be cut with restriction enzyme(s) ratory lacks culturing facility) so that bulk DNA extraction to reveal a not always species-specific profile using com- can be made, which could be stored for decades for future parison with the reference strains. The drawback of RFLP is reference studies. Several commercial kits are available for that close-related species like M. canis and M. ferrugineum extracting DNA from clinical sample like infected nail, hair, are not differentiated and a large amount of PCR product is or skin scrapings; however, it depends on the user for prefer- essential. A better alternative is sequencing of the ITS region. ring a particular kit in terms of performance and cost (since Comparison with the reference sequences in the nucleotide kits tend to increase the cost of analysis). databases (e.g., NCBI); better and recommended is the vali- dated CBS database (http://www.cbs.knaw.nl) that helps in 36.2.1.1 DNA Extraction from Fungal Colony identifying the causative fungal species. Other methods, The same kits used for clinical material can be applied when which have now become obsolete, are the mtDNA and DNA– extracting DNA from a grown colony. In this case, colonies DNA hybridization. However, many so-called “in house” not older than 1–2 weeks should be used for the DNA prepa- PCR assays have been developed already, which mainly ration because pigmentation of the culture can cause problem use nested PCR to increase the sensitivity in addition to a and inhibit the following PCR reaction. The cetyl trimethyl species-specific gene probe, which hybridizes in liquid (PCR ammonium bromide (CTAB) method described briefly, is, ELISA) or firm (blot) manner to the amplified and univer- however, not so expensive and yields a larger amount of sal target gene.90–92 Although a few assays using real-time DNA. Mycelia were scraped from the surface of agar col- PCR have been developed preventing contamination of the ony; also portions of colony are cut and placed inverted on sample during the identification process, two out of three are the plain agar. The agar is then scraped off from the surface only able to identify species groups.41,92–94 fAll o these assays till only the mycelial matt remained. Three to four of these involve extra cost but were developed for use in routine diag- mycelial mats is taken in sterile pestle and grinded in liquid nostic laboratories. Here we describe the method that is able nitrogen using a mortar. The finely grinded mycelial powder to recognize almost all currently known Microsporum spe- is then added to 2 mL Eppendorf tube containing 1 mL of cies (except anthropophilic ones that can be recognized by CTAB extraction buffer (100 mM Tris–HCl pH 8.0, 1.4 M sequencing) based on the new species concept.36 The meth- NaCl, 20 mM EDTA, 2% cetylmethylammonium bromide, ods described can be applied by most laboratories and espe- 2% β-mercaptoethanol). The suspension is then subjected to cially sequencing is a tool to identify the causative agent of three rounds of freezing and thawing. Then, proteinase K any fungal infection. is added to give a final concentration of 50 μg/mL and mixture is incubated for 1.5 h at 60°C. After centrifugation at 13,000 × g for 5 min, one volume of phenol/chloroform/isol- 36.2 Methods amyl alcohol (25:24:1) is added to the supernatant and tube is vortexed and centrifuged again for 15 min at 13,000 × g. 36.2.1 Sample Preparation The upper aqueous layer is taken in a fresh tube and the Direct molecular analysis of clinical sample(s) viz. hair, procedure repeated four to five times until the middle layer skin, and nail for diagnosis of fungal infectious agents is clear and shows no further protein precipitation. Finally, makes the detection of dermatophytic species rapid in con- one volume of chloroform was added to the supernatant trast to culture-dependent approach, which at least takes from previous step, mixed and centrifuged at 13,000 × g for a couple of weeks time. In direct approach, total genomic 15 min. Finally, DNA was precipitated with 1/10 volume of DNA can be isolated from clinical sample using DNA sodium acetate (4.0 M, pH 5.2) and 1 mL of cold isopropanol. extraction kits (e.g., Illustra Tissue & Cells GenomicPrep The content of tube is mixed by gentle inversion and incu- Mini Spin Kit, GE Healthcare Life Sciences; QIAamp DNA bating overnight at −20°C. Next day, the precipitated DNA isolation Kit, Quiagen); High Pure-PCR-Template prepa- is centrifuged at 10,000 × g for 10 min and pellet is washed ration Kit, Roche), and pegGOLD Fungal DNA Mini kit, with 70% ethanol and air-dried. DNA is dissolved in 1× TE Peqlab) and KOH. buffer (10 mM Tris–HCl, 1 mM EDTA pH 8.0) and treated Preparation of sample(s) for molecular detection of with RNase with final concentration 100 μg/mL for 15 min Microsporum does not require special care unlike systemic at 37°C. RNase was inactivated by 5 min incubation at 65°C fungi; however, care must be taken while handling since cer- and the pure DNA was stored at 4°C. DNA concentration tain animal origin strains are more virulent. Samples that was estimated by spectrophotometer reading at 280 and include hair, nails, skin scrapings, and even floor dust or soil 260 nm, whose ratio gives the purity of DNA extracted, and

© 2011 by Taylor & Francis Group, LLC Microsporum 293 also physically checked by running genomic DNA on 0.8% 55°C for 1 min, 72°C for 1 min; a final extension 72°C for agarose gel along with known concentration of λ DNA. 10 min. Five microliter PCR product is then checked on 1.2% agarose gel for successful amplification. 36.2.1.2 DNA Extraction from Clinical Specimen For extraction of DNA from clinical sample—hair, nail, or 36.2.2.1.2 Restriction Digestion (ITS-PCR-RFLP) skin, 0.5–3 mg of material previously collected is cut into The resulting amplicon can be digested by MvaI restriction small pieces (2 mm) and subjected to commercially available enzyme to give species-specific profile (Figure 36.2). For kit extraction as per manufacturer’s instruction. restriction digestion, 20 μL of fresh PCR product (showing Alternatively, homogenization of tissue for DNA extrac- single clear band on 1.2% agarose) is first transferred to tion, clinical specimen can be placed in a 2 mL screw cap an Eppendorf tube. The DNA is precipitated by 1/10 vol- tube containing CTAB buffer11 M(100 m Tris–HCl pH 8.0, ume of sodium acetate (3 M, pH 4.8–5.2) and 2.5 volume of 1.4 M NaCl, 20 mM EDTA, 2% cetyl methylammonium ethanol (95%) and incubated overnight at 4°C. Precipitated bromide, 2% β-mercaptoethanol), sterile sand and a 5 mm DNA is centrifuged at 13,000 × g for 15 min and pellet solid ceramic bead in an MPBio Fast Prep 24 homogenizer (mostly not visible) washed with ethanol (70%) and again (Biomedicals GmbH, Germany). Further steps remain same centrifuged at 13,000 × g for 15 min. The pellet is dried as in the previous section on extraction from colony. A very in vacuum centrifuge for 10 min at 30°C. The dried pellet rapid method (1 h) with slight optimization can be readily is dissolved in 18 μL of PCR grade water with the help of applied to bulk samples obtained by small laboratories or micropipette. 2 μL of 10× RE buffer (10 mM Tris–HCl pH 94 95 private clinics ryo the one b Brillowska-Dobrowska et al., 8.5, 10 mM MgCl2, M 100 m KCl, and 0.1 mg/mL BSA) is which is mainly for nail but can be applied to other clinical added to the cleaned PCR product along with 5–10 U of specimens as well. MvaI RE (Fermentas) before incubating for 4 h at 37°C. The digested product is separated on 2% Metaphor agarose and stained with ethidium bromide and photographed for 36.2.2 Detection Procedures documentation. The results of characteristic species-specific pattern con- 36.2.2.1 Species Recognition siderably depend on the success of digestion reaction. Partial Identification of species of Microsporum using molecu- or incomplete digestion or even mutation (which is rare) at lar means involves primarily the comparison of rDNA restriction site may give altered pattern. The method is most sequences, some regions of which are highly conserved on suitable for routine bulk samples. Figure 36.2A shows the one hand and variable on the other to reveal interspecies dif- MvaI restriction profile of selected species of Microsporum ferences. To visualize the interspecific variations for diag- generated from nearly 1000 bp amplicon covering ITS1, 2 nosis, rDNA regions being multi-copy are first amplified by flanking the 5.8S region of rDNA. PCR technique and then analyzed in one or the other ways for species identification. 36.2.2.1.3 Sequencing of PCR Products 36.2.2.1.1 PCR Amplification Direct sequencing of PCR product is done by cycling Once the genomic DNA is extracted in sufficient amount and sequencing kits using an automated sequencer (gel or quantified, PCR can then be performed with several universal capillary based) or first cloning them in appropriate vec- fungal primers, e.g., ITS4 (5′TCC TCC GCT TAT TGA TAT tor and then performing cycling sequencing reaction using GC3′-LSU 69-50 of Saccharomyces cerevisiae) and ITS5 M13 universal primer. Direct sequencing of PCR product (5′GGA AGT AAA AGT CGT AAC AA3′),96 ro LSU26697 (obtained by primer pair V9D and LSU266) is done by per- and V9D,98 which were used for Microsporum species to forming sequencing PCR with internal primers ITS4 and 96 amplify the ITS1, 5.8S, and ITS2 regions of rDNA giving ITS5. an amplicon of nearly 1000 bp. PCR conditions-50 μL reac- 36.2.2.1.4 Sequence Alignment and Identification tion mixture contained 10 mM Tris–HCl, 50 mM KCl, 3 mM Sequence obtained in the form of chromatogram from auto- MgCl2,1 2 pmol each primer (V9D and LSU266), 50 μM concentration of each deoxynucleotide triphosphate, 2.5 U mated sequencer has to be manually checked for inconsis- of Taq polymerase, and 50 ng of template DNA. Appropriate tency and accordingly edited for accuracy. Edited sequences controls viz. positive (containing template known to amplify can then be aligned with known (reference) sequences in the in given conditions), negative (reaction mixture lacking any database using CBS database or NCBI-BLAST search to template), and inhibition (reaction mixture containing 25 ng finally give identification results. test template and 25 ng template previously known to amplify under given conditions) are required. In case of non-amplifi- 36.2.2.2 Strain Typing cation, even when there is no inhibition, 10% DMSO can be For epidemiological study including tracing the source of added.99 The reaction mixture is finally overlaid with light infection (like pet) intra-specific markers are needed like mineral oil. Amplification conditions in a thermo cycler con- microsatellites or randomly amplified polymorphic DNA sist of 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 1 min, (RAPD). The choice of the former depends on the availability

1 2 3 5 6 8 9 10 1636 bp 4 7 11 12 13 14 15 16 17 18 19 1018 bp

506 bp 396 bp 344 bp 298 bp 220 bp 201 bp 154 bp 134 bp 75 bp (A)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

M. Ferrugineum M. Canis M. Audouinii

140 bp

108 bp GAABCDE EEEEEEF (B)

Fge i ur 36.2 (A) Restriction digestion profile (MvaI) of reference strains (of seven Microsporum sp.) Microsporum gypseum var. gypseum (CBS161.69) (lane 2), same strain as previous (lane 3), Arthroderma incurvatum (M. gypseum) (CBS173.64, MT+) (lane 4), A. incurvatum (CBS 174.64, T) (lane 5), A. gypsea (CBS 258.61, NT) (lane 6), A. fulvum (CBS 167.64, MT+) (lane 7), M. fulvum (CBS 287.55, T) (lane 8), A. persicolor (CBS 468.74, MT–) (lane 9), A. persicolor (CBS 469.74, MT+) (lane 10), M. canis (CBS 132.88) (lane 15), A. borelli (M. amazonicum) (CBS 967.68, ST) (lane 17), A. cookiella (CBS 102.83) (lane 18), Epidermophyton floccosum (CBS 358.93) (lane 19). Test Microsporum species profile (lanes 11–13) corresponds to reference profile of M. persicolor, M. fulvum, and M. gypseum (lane 10, 8, and 6 respectively). Abbreviations: CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, MT, mating type; NT, neotype strain; ST, syntype strain; T, type strain. (Reproduced from Sharma, R. et al., Med. Mycol., 46, 67, 2008. With permission.) (B) Polyacrylamide gel (12%) showing seven different alleles among strains of M. canis (G-10, A-12, B-13, and C-17 repeats), M. ferrugenium (D-25 repeats), and M. audouinii (F-7 and E-9 repeats) analyzed by marker Mc(CA)13. of already developed markers (e.g., M. canis and M. persi- marker (i.e., m arkers 300_27ga [For: 5′-GGCTTGAGTG color),27,39 otherwise they have to be generated afresh in the GCGTCTTC-3′ Rev: 5′-AGCAAACGAACCGCTGAG-3′] species under investigation by one of the several methods 196_23ca [For: 5′-TCGGCCTCCTCATCCTTC-3′ Rev: available.100,101 With the availability of the genome project of 5′-TCGGGATGTAAGTAAAGG-3′] and 300_17ct M. canis and M. gypseum, the design of such markers at least [For: 5′-GGGCAATTCTATGGGCAAG-3′3′ Rev: for these two species became easy. 5′-CTTCTTCCAAGCTCTGCCTG-3′]) had maximum 38, 26, and 18 dinucleotide repeats, respectively. The high 36.2.2.2.1 Microsatellites number of multilocus genotypes (26) obtained in small Microsatellites or simple sequence repeats (SSRs) are short population size (56 strains) suggested a fairly high level stretches of di-, tri-, tetra-, penta-, or hexa-sequences repeated of recombination in the population and its presence in the more than five times. The more the number of repetitions are region to be much older than its first isolation from India.39 present, the better the marker. The flanking regions of the mic- Misidentification of this species in earlier works on Indian rosatellite repeats are conserved (where primer binds), while soils is probable because they all were morphology based variations in the number of repeat results in polymorphisms and M. persicolor is known to be misinterpreted by morpho- among individual of a population, which can be visually logical features.102,103 nI zoophilic species M. canis, 11 mul- observed on PAGE gel or fragment analyzer. In the geophilic tilocus genotypes were obtained with 2 microsatellite loci 27 species M. persicolor, Sharma et al. 6detected 2 genotypes Mc(GT)13 (forward: 5′-GATCGGAGCATGCCATACAG3′; among 56 nonclinical strains analyzed, which were isolated reverse: 5′-TCTTCCCACCCTTCTCAATG-3′) and Mc(GT)17 from various soil locations in Central India viz. public places, (forward: 5′-GCTCTGGGATAAGGTGTTTG-3′; reverse: hair salon, burrow, cattle shed, garbage, cave, and rat carcass. 5′-GTAGCAGTAAAGCCAAGAGGG-3′) among 101 global The number of genotypes obtained with each of the three mic- strains analyzed.27 The markers were also able to differenti- rosatellite marker (with 13, 9, and 5 genotypes) corresponded ate M. canis from close-related anthropophiles M. audoui- with the increase in repeat length of the microsatellite nii and M. ferrugenium, which represented three additional

© 2011 by Taylor & Francis Group, LLC Microsporum 295 genotypes and thus can be differentiated by gel electrophore- to a highly specialized life style (like rusts in plants) that will sis (Figure 36.2B). lead them to become future obligate parasites and their (evolutionary) survival will last until its host species. 36.2.2.2.2 RAPD Markers, AP-PCR, and Apart from detecting fungal pathogens in clinical speci- PCR-Fingerprinting mens, molecular methods have proved useful in more than A number of techniques are available like AP-PCR (arbi- three ways for the study of dermatophytes, thus having direct trary primed-PCR), PCR-fingerprinting, and RAPD, which implication on the clinicians’ knowledge of dermatophytes. employ a single short primer to amplify fragments scattered First, in detecting species in location where they are unre- on the chromosomes for strain typing and population genetic corded,39 second validating the taxonomic status of a closely studies. DNA polymorphisms, in terms of different band pro- related or cryptic species114 ro atypical isolates,115 and third file, are obtained (presence or absence of band(s) that may in providing phylogenetic positioning of newly found spe- be of variable intensities). The reliability of these methods cies78,116 for nomenclatural and taxonomic considerations. We greatly depends on standard conditions used (including make now have numerous markers and methods with us like never of thermal cycler); however, reproducibility is a problem104 before; non-detection of dermatophytes in clinical specimen among laboratories. Although the methods are for assessing or the environmental samples becomes relatively rare, the intraspecific variability, they are of little use in case of der- only impetus that remains is how faster we can diagnose their matophytes that lack variability due to their recent adaptive presence for appropriate therapy. Although dermatophytes radiation; and hence reveal only interspecific differences105,106 are not life threatening, they can influence the quality of life unlike seen in other older fungal lineages. drastically.

36.3 Conclusions and Acko n wledgments Future Perspectives Drs. e G.S. d Hoog and N.D. Sharma are greatly acknowl- Diagnosing fungal pathogen is the first step in treatment of a edged for helpful discussions on dermatophytes and fungi dermatophytic infection, which is one of the most common during several years of their association with Y.G. and R.S. fungal infections worldwide infecting almost 20%–25% of respectively and Dr. S.N. Kulkarni, ARI Library, for access population.107 Molecular methods have proved greatly ben- to current literature. R.S. thanks Department of Science and eficial and have revolutionized detection of pathogens due Technology (DST), Govt. of India, for Fast Track grant SR/ to sensitivity of techniques involved (even in cases where FT/L-36/2005 supporting the work on keratinophilic fungi symptoms are unapparent) and the rapidity in relation to the of national parks of Central India, which led to many con- conventional techniques like hair perforation, mating com- cepts mentioned in this chapter and Deutscher Akademischer petency tests, and even nutritional tests. Apart from the cur- Austausch Dienst (DAAD), Bonn, for research fellowship rently available molecular methods, newer improved ones are that enabled collaboration between the two authors to study still being regularly developed like oligonucleotide array44,108 and generate new information on Microsporum. with increased rapidity95,109 mainly due to the reduced time required for DNA extraction.95 With the increase in global temperature, which is all the References more evident at present like never before, there will be more 1. Ainsworth, G.C. and Austwick, P.K.S., Fungal Diseases cases of dermatophytoses, especially in temperate regions of Animals, Review Series 6, Commonwealth Agricultural of the world, which already have a high rate (35%–40%) of Bureaux, England, 1973, p. 10. fungal foot disease110 due to, e.g., specialized microclimate 2. Mullis, K.B. et al., Specific amplification of DNA in vitro: built by footwear. The relative incidence of dermatophytic The polymerase chain reaction, Cold Harb. Spring Symp. infection varies with the climatic (most important microcli- Quant. Biol., 51, 260, 1986. mate) conditions111 prevailing in a region and on the inherent 3. Saiki, R.K. et al., Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science, 239, immunity of hosts involved. Warm and humid environment 487, 1988. previously lacking in temperate regions of the world is now 4. Gruby, D., Recherches sur la nature, le siege et le devel- available for longer period during the year, which will facili- oppment du porrigo decalvans ou phytoalopecie, C.R. Acad. tate survival of species previously unrecorded in such region. Sci., 17, 301, 1843. This altered environment has a direct affect on the survival 5. Malmstem, P.H., Trichophyton tonsurans, harkarande Mogel, of migrated species including anthropophilic dermatophytes. Bidrag till utredande af de ajukdomar som valla harets affal, Also, the parasexual mechanism112 fo recombination to gener- Stockholm, translated by Creplen F.C.H., and published as ate genetic diversity and fitness (virulence)113 emight b active Trichophyton tonsurans der haarscheerende Schimmel, Ein betrag zur Auseinandersetzung der Krankheiten, welche das in asexual forms like the anthropophilic dermatophytes due Abfalle des Haars bewirken, Arch. Anat. Physiol. Wiss. Med., to its immense utility for species survival. Thus, anthropo- 1848, 19, 1848. philic nature of some dermatophytes not necessarily seems 6. Sabouraud, R., Les Teignes, Masson et Cie., Paris, 1910. to be evolutionary dead ends for them but instead adaptation 7. Fraser, R.D.B., Keratins. Sci. Am., 221, 164, 1969.

8. Dzawachiszwili, N. et al., The effect of sea water and sodium 31. Burnet, J. (ed), The generation of variation-I Mutation and chloride on the growth of fungi pathogenic to man, J. Invest. migration, in Fungal Population and Species, Burmett, J., Ed., Dermatol., 103, 1964. Oxford University Press, New York, 2003, p. 121. 9. Ajello, L., Natural history of the dermatophytes and related 32. Webster, J. and Weber, R.W.S., Introduction to Fungi, 3rd edn., fungi, Mycopath. Mycol. Appl., 53, 93, 1974. Cambridge University Press, Cambridge, U.K. 2007, p. 294. 10. Kawasaki, M., Aoki, M., and Ishizaki H., Phylogenetic rela- 33. Hubalek, Z. and Rush-Munro, F.M., A dermatophyte from tionships of some Microsporum and Arthroderma species birds: Microsporum ripariae sp.nov., Sabouraudia, 11, 287, inferred from mitochondrial DNA analysis, Mycopathologia, 1973. 130, 11, 1995. 34. Ajello, L. and Padhye, A., Keratinophilic fungi of the 11. Gräser, Y. et al., Phylogeny and taxonomy of family Galapagos Islands, Mykosen, 17, 239, 1974. Arthrodermataceae (dermatophytes) using sequence analysis 35. Davidson, F.D.D., Mackenzie, D.W.R., and Owen, R.J., of the ribosomal ITS region, Med. Mycol., 37, 105, 1999. Deoxyribonucleic acid base composition of dermatophytes, 12. Magallon, S.A., Dating lineages molecular and paleontologi- J. Gen. Microbiol., 118, 465, 1980. cal approaches to the temporal framework of clades, Int. J. 36. Gräser, Y., Scott, J., and Summerbell R.C., The new spe- Plant. Sci., 165, S7, 2004. cies concept in dermatophytes—A polyphasic approach, 13. Taylor, J.W. and Berbee, M.L., Dating divergences in the fun- Mycopathologia, 166, 239, 2008. gal tree of life: Review and new analysis, Mycologia, 98, 838, 37. Abdel-Rahman, S.M., Strain differentiation of dermatophytes, 2006. Mycopathologia, 166, 319, 2008. 14. Pagel, M. and Bodmer, W., A naked ape would have fewer 38. Kanbe, T., Molecular approaches in the diagnosis of dermato- parasites, Proc. R. Soc. Lond. B, 270, S117, 2003. phytes, Mycopathologia, 166, 307, 2008. 15. Schwartz, G.G. and Rosenblum, L.A., Allometry of primate 39. Sharma, R. et al., Molecular detection of Microsporum persi- hair density and the evolution of human hairlessness, Am. J. color in soil suggesting widespread dispersal in central India, Phys. Anthropol., 55, 9, 1981. Med. Mycol., 46, 67, 2008. 16. Romano, C., Valenti, L., and Barbara, R., Dermatophytes iso- 40. Perkins, D.D., In praise of diversity, in More Gene lated from asymptomatic stray cats, Mycoses, 40, 471, 1997. Manipulations in Fungi, Bennet, J.W. and Lasure, L.L. Eds., 17. Summerbell, R., Form and function in the evolution of Academic Press, New York, 1991, p. 3. dermatophytes, in Biology of Dermatophytes and Other 41. Garg, J. et al., Rapid detection of dermatophytes from skin Keratinophilic Fungi, Kushwaha, R.K.S. and Guarro, J., Eds., and hair, BMC Res. Notes, 2, 60, 2009. Rev. Iberoam. Micol., Bilbao, Spain, 2000, p. 30. 42. Arabatzis, M. et al., Diagnosis of common dermatophyte 18. Rugmer, C., The fragility of gigantism. Research*eu, Special infections by a novel multiplex real time polymerase chain issue 22, September 2008. reaction detection/identification scheme, Brit. J. Dermatol., 19. Richardson, M. and Edward, M., Model systems for the 157, 681, 2007. study of dermatophyte and non-dermatophyte invasion of 43. Vollmer, T. et al., Evaluation of novel broad-range real-time human keratin, in Biology of Dermatophytes and Other PCR assay for rapid detection of human pathogenic fungi Keratinophilic Fungi, Kushwaha, R.K.S. and Guarro, J., Eds., in various clinical specimens, J. Clin. Microbiol., 46, 1919, Rev. Iberoam. Micol., Bilbao, Spain, 2000, p. 115. 2008. 20. Takashio, M., Sexual reproduction of some Arthroderma and 44. Li, H.C. et al., Identification of dermatophytes by an oligo- Nannizzia on dilute Sabouraud agar with or without salts, nucleotide array, J. Clin. Microbiol., 45, 3160, 2007. Mykosen, 15, 11, 1972. 45. White, T.C. and Henn, M.R., Genomic determinants of infec- 21. Wong, K., The human pedigree: A timeline of hominid evolution competence in dermatophyte fungi. A white paper for the tion, Sci. Am. India, 4, 60, 2009. Fungal Genome Initiative, http://www.genome.gov/Pages/ 22. Kaszubiak, A. et al., Population structure and evolutionary Research/Sequencing/Seq/Proposals/Dermatophytes_WP_ origins of Microsporum canis, M. ferrugineum and M. aud- Seq.pdf, pp12. ouinii, Infect. Genet. Evol., 4, 179, 2004. 46. White, T. et al., Generating and testing molecular hypothesis, 23. Dixon, D. et al., Cassell’s Atlas of Evolution, Cassell & Co. Eukar. Cell, 7, 1238, 2008. London, U.K., 2001, p. 312. 47. Padhye, A.A. and Carmichael, J.W., The genus Arthroderma 24. Bettis III, E.A., Way out of Africa: Early Pleistocene paleoen- Berkeley, Can. J. Bot., 49, 1525, 1971. vironments inhabited by Homo erectus in Sangiran, Java, 48. Weitzman, I. et al., The genus Arthoderma and its later syn- J. Hum. Evol., 56, 11, 2009. onym Nannizia, Mycotaxon, 25, 505, 1986. 25. Giraud, T. et al., Speciation in fungi, Fung. Genet. Biol., 45, 49. James, T.Y. et al., Reconstructing the early evolution of fungi 791, 2008. using a six-gene phylogeny, Nature, 443, 818, 2006. 26. Summerbell, R.C., Li, A., and Haugland, R., What constitute 50. Hibbett, D.S. et al., A higher-level phylogenetic classification a functional species in the asexual dermatophytes? Microbiol. of the fungi, Mycol. Res., 111, 509, 2007. Cult. Coll., 13, 29, 1997. 51. Cole, G.T. and Samson R.A., Patterns of Development in 27. Sharma, R. et al., A virulent genotype of Microsporum canis Conidial Fungi, Pitman Publishing Ltd., London, U.K., 1979. is responsible for the majority of human infections, J. Med. 52. Van Oorschot, C.A.N., A revision of Chrysosporium and Microbiol., 56, 1377, 2007. allied genera, Stud. Mycol., 20, 1, 1980. 28. Dirscoll, C.A. et al., The near eastern origin of cat domestica- 53. Kubo, H., Survey of keratinophilic fungi from soils of Hokkaido tion, Science, 317, 519, 2007. prefecture of Japan—Especially on the isolation of Nannizzia 29. Dirscoll, C.A. et al., The taming of the cat, Sci. Am. India, 4, otae from the soils, Jpn. J. Med. Mycol., 27, 230, 1986. 54, 2009. 54. Hironga, M., Nozaki, K., and Watanabe, S., Ascocarp produc- 30. Brown, J.K.M. and Hovmoller, M.S., Epidemiology—Aerial tion by Nannizzia otae on keratinous and non-keratinous agar dispersal of pathogens on the global and continental scales media and mating behavior of N. otae and 123 Japanese iso- and its impact on plant disease, Science, 297, 537, 2002. lates of Microsporum canis, Mycopathologia, 72, 135, 1980.

55. Degreef, H., Clinical forms of dermatophytosis (ringworm 78. Brasch, J. and Graeser, Y., Trichophyton eboreum sp.nov. iso- infection), Mycopathologia, 166, 257, 2008. lated from human skin, J. Clin. Microbiol., 43, 5230, 2005. 56. Havlickova, B., Czaika, V.A., and Friedrich, M., 79. Robert, R. and Pihet, M., Conventional methods for the diag- Epidemiological trends in skin mycoses worldwide, Mycoses, nosis of dermatophytosis, Mycopathologia, 166, 295, 2008. 51, 2, 2008. 80. Kane et al., Laboratory Handbook of Dermatophytes, Star 57. Chermett, R., Ferreiro, L., and Guillot, J., Dermatophytoses in Publishing Company, Belmont, CA, 1997. animals, Mycopathologia, 166, 385, 2008. 81. Robinson, B.E. and Padhye, A.A., Collection, transport and 58. Carlotti D.N. and Bensignor, E., Dermatophytosis due to processing of clinical specimens, in Diagnostic Procedures Microsporum persicolor (13 cases) or Microsporum gypseum for Mycotic and Parasitic Infections, Wentworth, B.B., Ed., (20 cases) in dogs, Vet. Dermatol., 10, 17, 1999. American Public Health Association, Washington, DC, 1988, 59. Agrios, G.N., Introduction to Plant Pathology, Elsevier p. 11. Academic Press, New Delhi, 2006. 82. Mackenzie, D.W.R., “Hairbrush diagnosis” in detection and 60. Moss, S., Understanding Bird Behavior, New Holland eradication of non-fluorescent scalp ringworm, Br. Med. J., 2, Publishers Ltd., U.K., 2006, p. 70. 363, 1963. 61. Sharma, R., Taxonomic studies on keratinolytic fungal diver- 83. Klokke, A.H.A., Purushotham, A.G., and Sundaraju, D., Mass sity of central India, PhD thesis, Rani Durgavati University, treatment of non-fluorescent tinea-capitis in an orphanage Jabalpur, India, 2002. in India. Trial with a low griseofulvin dose using a modified 62. Ghosh, L.N., An analysis of 50,000 skin cases as seen in brush sampling technique, Trop. Georgr. Med., 18, 305, 1966. the outpatient department of school of tropical medicine, 84. Johnson, L., Dermatophytes-the skin eaters, Mycologist, 17, Calcutta, during 5 years from 1942–1946, Ind. Med. Gaz., 83, 147, 2003. 493, 1948. 85. Ajello, L. and George, L.K., In vitro cultures for differentiat- 63. Garg, A.P., Indian hair oils and protection of human hair ing between atypical isolates of Trichophyton mentagrophytes against fungal infections, J. Ind. Bot. Soc., 71, 251, 1992. and Trichophyton rubrum, Mycopath. Mycol. Appl., 8, 3, 1957. 64. Katoh, T., Dermatomycosis and environment, Jpn. J. Med. 86. Padhye, A.A., Young, C.N., and Ajello, L., Hair perforation as Mycol., 47, 63, 2006. a diagnostic criterion in the identification of Epidermophyton, 65. Ulfig, K., The occurrence of keratinolytic fungi in water and Microsporum and Trichophyton species, Pan. Am. Health waste-contaminated habitats, in Biology of Dermatophytes and Org. Sci. Publ., 396, 115, 1980. Other Keratinophilic Fungi, Kushwaha, R.K.S. and Guarro, J., 87. Liu, D. et al., A specific PCR assay for the dermatophyte fun- Eds., Rev. Iberoam. Micol. Bilbao, Spain, 2000, p. 44. gus Microsporum canis, Med. Mycol., 39, 215, 2001. 66. Morishita, N. et al., Effects of temperature, humidity, minor 88. Roque, H.D. et al., Specific primers for rapid detection of injury and washing on penetration of dermatophytes into Microsporum audouinii by PCR in clinical samples, J. Clin. human stratum corneum, Jpn. J. Med. Mycol., 44, 269, 2003. Microbiol., 44, 4336, 2006. 67. Morishita, N. et al., Experimental studies on the penetration 89. El Fari, M. et al., Development of an oligonucleotide probe of dermatophytes into the human stratum corneum and the specific for Trichophyton rubrum, Brit. J. Dermatol., 141, effects of washing, Jpn. J. Med. Mycol., 45, 247, 2004. 240, 1999. 68. May, R.M., Global problems and global science, Notes Rec. 90. Savin, C. et al., Multicenter evaluation of a commercial R. Soc. Lond., 59, 105, 2005. PCR-enzyme-linked immunosorbent assay diagnostic kit 69. Rosenthal, T., Aulus Cornelium Celsus, Arch. Derm., 84, 613, (Onychodiag) for diagnosis of dermatophytic onychomycosis, 1961. J. Clin. Microbiol., 45, 1205, 2007. 70. Rippon, J.W., Clinical aspects of medically important conidial 91. Bergmans, A.M.C. et al., Validation of PCR-reverse line blot, fungi, in Biology of Conidial Fungi, Vol. 2, Cole, G.T. and a method for rapid detection and identification of nine derma- Kendrick, W., Ed., Academic Press Inc., New York, 1981, p. 13. tophyte species in nail, skin and hair samples, Clin. Microbiol. 71. Kunert, J., Keratin decomposition by dermatophytes: Infect., 14, 778, 2008. Evidence of the sulphitolysis of protein, Experimentia, 28, 92. Gutzmer, R. et al., Rapid identification and differentiation 1025, 1972. of fungal DNA in dermatological specimens by LightCycler 72. Kunert, J., Personal communication, 2001. PCR, J. Med. Microbiol., 53, 1207, 2004. 73. Kunert, J. and Trüper, H.G., Cystine catabolism in myce- 93. Bergmans, A.M.C. et al., Evaluation of a single-tube real- lia of Microsporum gypseum a dermatophyte fungus, Arch. time PCR for detection and identification of 11 dermatophyte Microbiol., 145, 181, 1986. species in clinical material, Clin. Microbiol. Infect., 16, 704, 74. Sei, Y. and Takiuchi, I., Studies on the effect of keratinase on 2009. the keratinized structure, and localization of keratinase, Jpn. 94. Liu, D. et al., Rapid mini-preparation of fungal DNA for PCR. J. Med. Mycol., 23, 308, 1982. J. Clin. Microbiol., 38, 471, 2000. 75. Takiuchi, I. et al., Isolation of an extracellular proteinase (kera- 95. Brillowska-Dobrowska, A., Saunte, D.M., and Arendrup, tinase) from Microsporum canis, Sabouraudia, 20, 281, 1982. M.C., Five hour diagnosis of dermatophyte nail infection with 76. Marchisio, V.P., Keratinophilic fungi: Their role in nature specific detection of Trichophyton rubrum, J. Clin. Microbiol., and degradation of keratinic substrates, in Biology of 45, 1200, 2007. Dermatophytes and Other Keratinophilic Fungi, Kushwaha, 96. White, T.J. et al., Amplification and direct sequencing of R.K.S. and Guarro, J., Eds., Rev. Iberoam. Micol. Bilbao, fungal ribosomal RNA genes for phylogenetics, in PCR Spain, 2000, p. 86. Protocols: A Guide to Methods and Applications, Innis, M.A. 77. Uchida, T. et al., Comparative study of direct polymerase et al. Eds., Academic Press, New York, 1990, p. 315. chain reaction, microscopic examination and culture-based 97. Masclaux, F. et al., Phylogenetic relationships of human morphological methods for detection and identification of pathogenic Cladosporium (Xylohypha) species inferred from dermatophytes in nail and skin samples, J. Dermatol., 36, 202, partial LS rRNA sequences, J. Med. Vet. Mycol., 33, 327, 2009. 1995.

98. de Hoog, G.S. and Gerrits van den Ende, A.H.G., Molecular 108. Champa, D. et al., DNA microarray based on arrayed-primer diagnostics of clinical strains of filamentous Basidiomycetes, extension technique for identification of pathogenic fungi Mycoses, 41, 183, 1998. responsible for invasive and superficial mycoses, J. Clin. 99. Dieffenbach, C.W. and Dveksler, G.S., PCR Primer—A Microbiol., 46, 909, 2008. Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory 109. Kardjeva, V. et al., Forty-eight-hour diagnosis of onychomy- Press, Cold Spring Harbor, New York, 2003. cosis with subtyping of Trichophyton rubrum strains, J. Clin. 100. Zane, L., Bargelloni, L., and Patarnello, T., Strategies for mic- Micrbiol., 44, 1419, 2006. rosatellite isolation: A review, Mol. Ecol., 11, 1, 2002. 110. Burzykowski, T. et al., High prevalence of foot diseases in 101. Dutech, C. et al., Challenges of microsatellite isolation in Europe: Results of the Achilles Project, Mycoses, 46, 496, 2003. fungi, Fung, Genet. Biol., 44, 933, 2007. 111. Marsella, R., Fungal skin diseases. In Current Therapy in 102. Badillet, G., Microsporum persicolor, an easily misinter- Equine Medicine, Robinson, E.L. and Strayberry, K.A. Eds., preted dermatophyte, Hautartz, 29, 10, 1987. Saunders, Elsevier, Philadelphia, PA, 2009, p. 687. 103. Kane, J., Sigler, L., and Summerbell, R.C., Improved pro- 112. Pontecorvo, G., The parasexual cycle in fungi, Annu. Rev. cedure for differentiating Microsporum persicolor, an easily Microbiol. 10, 393, 1956. misinterpreted dermatophyte, J. Clin. Microbiol., 25, 2449, 113. Heitman, J., Sexual reproduction and the evolution of micro- 1987. bial pathogens, Curr. Biol., 16, R711, 2006. 104. Hansen, M., Hallden, C., and Sall T., Error rates and polymor- 114. Graeser, Y. et al., Molecular and conventional taxonomy of phism frequencies for three RAPD protocols, Plant Mol. Biol. Microsporum canis complex, Med. Mycol., 38, 143, 2000. Reptr., 16, 139, 1998. 115. Sharma, R. et al., Internal transcribed spacer (ITS) of rDNA 105. Kano, R. et al., Differentiation of Microsporum species by of appendaged and non-appendaged strains of Microsporum random amplification of polymorphic DNA (RAPD) and gypseum reveals Microsporum appendiculatum as its syn- southern hybridization analyses, Mycoses, 41, 229, 1998. onym, Antonie Leeuwenhoek, 20, 197, 2006. 106. Graeser, Y. et al., Identification of common dermatophytes 116. Campbell, C.K. et al., Arthroderma olidum, sp.nov. a new (Trichophyton, Microsporum, Epidermophyton) using poly- addition to the Trichophyton terrestre complex, Med. Mycol., merase chain reactions, Br. J. Dermatol., 138, 576, 1998. 44, 451, 2006. 107. Male, O., The significance of mycology in medicine, 117. de Hoog, G.S. et al., Atlas of Clinical Fungi, 2nd edn., in Frontiers in Mycology, Hawksworth, D.L. Ed., CAB Centraalbureau voor Schimmelculturales, Utrecht, the International, Wallingford, 1990, p. 131. Netherlands, 2000.