Application of PCR to Dermatophyte Fungi Identification (2000) PDF

J. Med. Microbiol. Ð Vol. 49 (2000), 493±497 # 2000 The Pathological Society of Great Britain and Ireland ISSN 0022-2615 REVIEW ARTICLE Application of PCR to the identi®cation of dermatophyte fungi D. LIU, S. COLOE, R. BAIRD and J. PEDERSEN Melbourne Pathology, 32 Smith Street, Collingwood, Victo...

J. Med. Microbiol. Ð Vol. 49 (2000), 493±497 # 2000 The Pathological Society of Great Britain and Ireland ISSN 0022-2615 REVIEW ARTICLE Application of PCR to the identi®cation of dermatophyte fungi D. LIU, S. COLOE, R. BAIRD and J. PEDERSEN Melbourne Pathology, 32 Smith Street, Collingwood, Victoria 3066, Australia Infection of the keratinised tissues (skin, hair and nails) in man and animals by keratinophilic fungi (dermatophytes) results in dermatophytosis (also known as tinea or ringworm). As conventional laboratory procedures for the identi®cation of dermato- phytes are either slow or lack speci®city, improved diagnostic methods are required. The application of nucleic acid ampli®cation technology has made rapid and precise identi®cation of dermatophytes possible. Recent studies have shown that when one of the four random primers (OPAA11, OPD18, OPAA17 and OPU15) was used in arbitrarily primed PCR (AP-PCR), up to 20 of the 25 dermatophyte species or subspecies under investigation could be distinguished on the basis of characteristic band patterns detected in agarose gel electrophoresis. A combination of two random primers (OPD18 and OPAA17) used in separate reaction tubes identi®ed 23 of the 25 dermatophyte species or subspecies examined. AP-PCR provides a rapid and practical tool for identi®cation of dermatophyte isolates that is independent of morphological and biochemical character- istics and thus enhances laboratory diagnosis of dermatophytosis. Introduction For many years, conventional laboratory methods based on the detection of phenotypic characteristics, such as Dermatophytes comprise three closely related keratin- microscopy and in-vitro culture, have played an ophilic fungal genera (Epidermophyton, Microsporum essential role in dermatophyte identi®cation. However, and Trichophyton) that colonise keratinised tissues these procedures generally suffer from the drawbacks (skin, hair and nails) of man and animals. The of being either slow or non-speci®c. Recent develop- colonisation process is assisted by the release of ments and applications of nucleic acid ampli®cation various proteolytic and other enzymes by dermato- technology have provided the opportunity to enhance phytes, which in turn provoke in¯ammatory responses the quality and speed of dermatophyte diagnosis. This in the host, resulting in dermatophytosis (also known as review presents a brief update on the use of molecular tinea or ringworm). detection methods, in particular, arbitrarily primed PCR (AP-PCR) for rapid identi®cation of dermatophyte Whereas the genus Epidermophyton is represented by a isolates. single species (E. ¯occosum), the genera Microsporum and Trichophyton are complex and made up of multiple species. In particular, besides the presence of.15 Clinical background recognised species in the genus Trichophyton, several different variants are also identi®ed within the species Despite being highly infectious and extremely com- of T. mentagrophytes. Because many Microsporum mon, dermatophytes have frequently been regarded as and Trichophyton species are pathogenic for man and insigni®cant human pathogens, because dermatophyto- the dermatophytosis caused by various dermatophyte sis is generally mild and not life-threatening. The fact species cannot be easily differentiated on the basis of that, historically, many dermatophyte species and clinical manifestations, it has been necessary to strains had relatively restricted geographical distribu- develop improved laboratory diagnostic methods. tion also reinforces this notion. However, signi®cant demographic changes that have taken place in the past few decades have led to some noticeable modi®cations Received 11 Oct. 1999; revised version received 7 Dec. in dermatophyte pro®les. For instance, in the USA, the 1999; accepted 9 Dec. 1999. most commonly isolated dermatophytes have shifted Corresponding author: Dr D. Liu. from M. audouinii in the 1940s to T. rubrum in the 494 D. LIU ET AL. 1960s and T. tonsurans in the 1990s [3, 4]. Similarly, in Nucleic acid-based techniques Victoria, Australia, while T. mentagrophytes isolations showed a slight decline over the past three decades Nucleic acid-based techniques rely on the detection of (from 29.0% in the 1960s to 26.5% in the 1990s), T. genotypic differences in pathogenic organisms. They rubrum has become more prevalent (from 43.1% to are intrinsically more speci®c and more precise than 69.5%). However, M. canis has decreased dramatically those based on phenotypic features , as genotypic during the same period (from 19.4% to 0.9%). characteristics are less likely to be affected by external in¯uences such as temperature variations and chemo- Furthermore, with an increasingly aging population and therapy. The recent development of nucleic acid the increasing occurance of immunocompromised pa- ampli®cation technology such as PCR, as well as tients, many previously uncommon fungal infections, arbitrarily primed PCR (AP-PCR) or random ampli®ed including dermatophytosis, have emerged as important polymorphic DNA (RAPD) [7±9], has further en- causes of morbidity. The application of chemotherapy hanced the sensitivity and speed of nucleic acid-based has also contributed to the occasional modi®cation and diagnostic procedures. Several groups have successfully alteration of the morphological characteristics of applied PCR technology to the phylogeny and diag- dermatophytes, resulting in atypical colonial growth nosis of pathogenic fungi, including dermatophytes and appearance and complicating laboratory identi®ca- [10±18]. These studies have exploited the differences tion procedures based on phenotypic features. There- of dermatophytes and other fungi in their small (18S) fore, the availability of improved laboratory methods is [10±13] and large (25S) [14, 15] ribosomal RNA genes essential for rapid and accurate detection and differ- and mitochondrial DNA , as well as chitin synthase entiation of the dermatophytes involved, so that 1 gene [17, 18]. This has resulted in a more accurate appropriate treatment and preventative measures can assessment of phylogenic relationships among derma- be applied. tophytes and other fungi [10, 14, 18] and improved detection and identi®cation of some common dermato- phytes [11±13, 15±17]. Nevertheless, as these methods frequently require additional manipulation such as Conventional diagnostic methods restriction endonuclease digestion, hybridisation or sequencing after ampli®cation, they are not easily The current diagnosis of dermatophytosis is based upon adopted for routine use in clinical laboratories. More- microscopic identi®cation of hyphae directly from over, many of these reported methods are capable of lesion materials followed by in-vitro culture. Although distinguishing only a limited number of dermatophyte rapid and economical, microscopical examination is not species and, therefore, are more suited for genus- species-speci®c. Also, this technique is relatively speci®c rather than species-speci®c identi®cation. insensitive, showing false negative results in up to 15% of cases. Therefore, its role in dermatophyte diagnosis is limited to giving an initial, quick screen AP-PCR for hyphae and other mycotic elements, and further follow-up with in-vitro culture is invariably required Random ampli®cation and identi®cation of DNA irrespective of the microscopic observations, as effec- fragments unique to a certain strain or species in AP- tive treatment and prevention of dermatophytosis PCR enables various micro-organisms to be differen- depend on knowledge of the speci®c dermatophyte tiated rapidly and precisely from each other [7±9]. involved. In-vitro culture is capable of providing a AP-PCR ampli®cation is achieved through the use of species-speci®c determination of dermatophytes on the short random primer(s) (usually 10 nucleotides long) at basis of morphological and biochemical criteria in 10± relatively low temperature (normally ,408C). Although 15 days in.95% of cases. However, for some unusual the exact mechanisms behind AP-PCR are not properly and atypical isolates, identi®cation may require a range understood, it has been hypothesised that by reducing of culture media such as Christensen's urea agar, the stringency of the primer annealing temperature, a Lactritmel agar, Littman Oxgall agar, 1% peptone agar, random primer that shows no complete homology to a Sabouraud's dextrose agar with NaCl 5%, Trichophyton genome may have a perfect match of two-to-three agars (no. 1±7) and the in-vitro hair perforation test. nucleotides from the 39- end of the primer to the These tests are not only costly and time-consuming template strand to allow annealing and priming (some results may take a further 3±4 weeks after complementary strand synthesis by the DNA polymer- primary isolation), but also demand specialist skills. ase, as a putative three-nucleotide sequence can be More importantly, because these conventional methods found in principle once in a 64-nucleotide sequence (43 depend on measurement of the phenotypic character- permutations). When two such annealing and priming istics of dermatophytes, they can be easily in¯uenced events occur within a certain distance of each other and by outside factors (such as temperature variations and in proper orientation, the sequence between the chemotherapy) that may interfere with the metabolic matching sites can be ampli®ed effectively. Subsequent process of the dermatophyte and affect the interpreta- identi®cation of characteristic DNA band patterns in tion of in-vitro culture results. agarose gel electrophoresis enables differentiation of  DERMATOPHYTE IDENTIFICATION BY PCR 495 various micro-organisms. The application of AP-PCR be identi®ed with random primer OPD18 (Tables 1 obviates the necessity to know the detailed sequences and 2); 20 (except among T. gourvillii, T. rubrum and of the gene regions concerned as well as the need for T. soudanense; and between T. mentagrophytes var. further manipulation after ampli®cation. interdigitale and var. nodulare) could be identi®ed with random primer OPAA17 (Tables 1 and 2); and 19 By assessing.90 individual random primers in AP- (except among T. gourvillii, T. rubrum and T. PCR it was possible to select several with potential for soudanense and among T. mentagrophytes var. inter- identi®cation and differentiation of dermatophytes. digitale, var. mentagrophytes and var. nodulare) could These included OPAA11 (59-ACCCGACCTG-39) be distinguished with random primer OPU15 [19, 20], OPD18 (59-GAGAGCCAAC-39) , (Tables 1 and 2). It appears that the usefulness of AP- OPAA17 (59-GAGCCCGACT-39) and OPU15 PCR for the identi®cation of dermatophyte species and (59-ACGGGCCAGT-39). By employing a rapid subspecies is further enhanced by a combination of two DNA puri®cation protocol and, more recently, a random primers with PCR performed in separate further improved mini-preparation procedure , 230 reaction tubes. For instance, the use of OPAA11 isolates from 25 dermatophyte species and subspecies and OPD18 in AP-PCR would result in the differentia- (varieties), comprising 16 Trichophyton species and tion of 21 (except between T. gourvillii and T. rubrum; subspecies (varieties), eight Microsporum species and and between T. mentagrophytes var. interdigitale and E. ¯occosum, were examined with one of these four var. mentagrophytes) among the 25 dermatophyte primers in AP-PCR [19±22] (Table 1). The results species or subspecies examined (Tables 1 and 2). indicated that of the 25 dermatophyte species and More importantly, the use of OPD18 and OPAA17 subspecies examined, 16 (except between T. equinum would help identify 23 (except between T. gourvillii var. autotrophicum and T. tonsurans; among T. and T. rubrum) of the 25 dermatophyte species or gourvillii, T. rubrum, T. soudanense and T. violaceum; subspecies examined (Tables 1 and 2). It is of interest and among T. mentagrophytes var. interdigitale, var. to note that although DNA from non-dermatophyte mentagrophytes and var. nodulare) displayed distinct fungi such as Scytalidium, Fusarium, Aspergillus and DNA band patterns after ampli®cation with random Candida spp. also generated DNA products in AP-PCR primer OPAA11 [19, 20] (Tables 1 and 2). Similarly, 19 with these random primers, the band patterns formed of the 25 dermatophyte species or subspecies (no by these fungi were distinct from those formed by differentiation between T. equinum var. autotrophicum dermatophytes. Therefore, they could be differentiated and T. mentagrophytes var. nodulare; between T. from dermatophyte species and subspecies. gourvillii and T. rubrum; and between T. mentagro- phytes var. interdigitale and var. mentagrophytes) could In terms of the time required for testing, AP-PCR has a Table 1. Examination of DNA products from dermatophyte fungi by AP-PCR DNA products (kb) obtained with Number Dermatophyte species and varieties tested OPAA11 OPD18 OPAA17 OPU15 T. ajelloi 1 1.7, 0.8 2.8, 1.9 1.9, 0.8, 0.5 2.2, 1.4, 1.0, 0.7 T. concentricum 1 1.5, 0.9 3.4, 2.1, 2.0, 1.2 2.5, 0.4 2.7, 1.1 T. equinum var. autotraphicum 1 2.5, 1.5, 0.9 3.3, 2.1, 1.0 3.5, 2.9, 1.0 0.9 T. gourvillii 1 1.9, 1.7, 0.5 3.1, 1.2 3.4, 2.5, 0.6, 0.4 2.6, 2.0, 1.8, 0.5 T. mentagrophytes var. erinacei 2 1.8, 1.0 3.0, 1.9, 1.3 1.5, 0.4 2.4, 1.4 T. mentagrophytes var. interdigitale 50 2.5, 1.9, 0.9 3.2, 2.0, 0.9 3.5, 2.8, 1.3 2.5, 2.0, 1.4, 0.8 T. mentagrophytes var. mentagrophytes 7 2.5, 1.9, 0.9 3.2, 2.0, 0.9 3.5, 2.8 2.5, 2.0, 1.4, 0.8 T. mentagrophytes var. nodulare 4 2.5, 1.9, 0.9 3.3, 2.1, 1.0 3.5, 2.8, 1.3 2.5, 2.0, 1.4, 0.8 T. mentagrophytes var. quinckeanum 9 1.8, 1.2 3.2, 2.1, 1.1, 0.8 3.5, 1.0 2.7, 1.8, 1.2, 0.7 T. rubrum 76 1.9, 1.7, 0.5 3.1, 1.2 3.4, 2.5, 0.6, 0.4 2.6, 2.0, 1.8, 0.5 T. schoenleinii 1 2.0, 1.3 3.2, 2.0 3.4, 0.9 2.4, 1.8, 1.2, 0.6 T. soudanense 7 1.9, 1.7, 0.5 3.2, 1.2 3.4, 2.5, 0.6, 0.4 2.6, 2.0, 1.8, 0.5 T. tonsurans 21 2.5, 1.5, 0.9 3.2, 1.8, 1.2 3.4, 2.8, 0.9 2.6, 0.8 T. terrestre 2 2.6 3.4, 2.8, 1.5 2.8, 1.2, 0.9, 0.7 2.0, 1.4, 0.8, 0.5 T. verrucosum 5 2.7, 1.9, 1.5, 0.7 3.3, 1.9, 1.2, 0.5 0.5 2.5, 1.9, 1.0 T. violaceum 11 1.9, 1.7, 0.5 3.2, 2.5, 1.9, 1.2 3.5, 2.6, 1.1, 0.4 2.7, 2.2, 1.9, 0.5 M. audouinii 3 2.8, 1.3, 0.2 2.8, 1.6, 1.4, 1.1 3.0, 2.0, 1.3 2.3, 1.3, 0.8 M. canis 10 2.6, 1.2, 0.8, 0.4 1.2 3.0, 1.2, 0.7 2.2, 1.0, 0.5 M. cookei 2 1.4, 1.2, 0.7, 0.3 2.5, 1.5, 0.9, 0.7 3.0, 2.7, 1.3 1.5, 1.1, 0.8 M. ferrugineum 2 2.6, 1.2, 0.4 1.5 3.0, 1.3 2.3, 1.0, 0.6 M. fulvum 1 1.9, 1.1, 0.8, 0.6 3.3, 2.1, 0.8, 0.3 2.7, 1.8 2.3, 1.8, 0.9, 0.4 M. gypseum 3 1.8, 0.8 3.2, 2.1, 1.6, 1.0 3.0, 2.7, 1.1, 0.9 2.6, 1.5, 0.8 M. nanum 1 2.0, 1.1, 0.7, 0.2 0.9 1.2, 0.9 2.2, 0.6 M. persicolor 2 1.8, 1.0, 0.5 3.0, 1.9, 1.0, 0.3 2.6, 1.3, 1.0 1.3, 0.9 E. ¯occosum 7 2.8, 2.2, 1.7, 0.9 1.8, 1.0 0.8 3.0, 2.5, 1.3 Total 230  The DNA products were ampli®ed in AP-PCR from each of the 25 dermatophyte species and varieties with one of the four random primers (OPAA11, OPD18, OPAA17 and OPU15), and their sizes (in kb) determined by agarose 1.5% gel electrophoresis with ëHindIII ‡ EcoRI as molecular markers [18±21]. 496 D. LIU ET AL. Table 2. Summary of dermatophyte identi®cation by AP-PCR Identi®able by Dermatophyte species and varieties OPAA11 OPD18 OPAA17 OPU15 T. ajelloi Yes Yes Yes Yes T. concentricum Yes Yes Yes Yes T. equinum var. autotrophicum No (1) No (4) Yes Yes T. gourvillii No (2) No (5) No (7) No (9) T. mentagrophytes var. erinacei Yes Yes Yes Yes T. mentagrophytes var. interdigitale No (3) No (6) No (8) No (10) T. mentagrophytes var. mentagrophytes No (3) No (6) Yes No (10) T. mentagrophytes var. nodulare No (3) No (4) No (8) No (10) T. mentagrophytes var. quinckeanum Yes Yes Yes Yes T. rubrum No (2) No (5) No (7) No (9) T. schoenleinii Yes Yes Yes Yes T. soudanense No (2) Yes No (7) No (9) T. tonsurans No (1) Yes Yes Yes T. terrestre Yes Yes Yes Yes T. verrucosum Yes Yes Yes Yes T. violaceum No (2) Yes Yes Yes M. audouinii Yes Yes Yes Yes M. canis Yes Yes Yes Yes M. cookei Yes Yes Yes Yes M. ferrugineum Yes Yes Yes Yes M. fulvum Yes Yes Yes Yes M. gypseum Yes Yes Yes Yes M. nanum Yes Yes Yes Yes M. persicolor Yes Yes Yes Yes E. ¯occosum Yes Yes Yes Yes (1)±(10) indicate that dermatophyte species or varieties with the same number in the brackets displayed identical band patterns and therefore were not separable with the random primers in AP-PCR. clear advantage over conventional techniques based on consistently between different runs [26±28]. A further morphological and biochemical criteria (such as in- advantage of AP-PCR is that it does not require viable vitro culture). Although most dermatophytes can be organisms for testing. Indeed, this laboratory has been identi®ed after primary isolation (10±15 days), a few able to con®rm the identities of 20-year-old non-viable may require secondary culture on specialised media dermatophyte isolates by AP-PCR. (10±15 days) (with some atypical, unusual or slow- growing isolates, the identi®cation process may take To summarise, AP-PCR provides a practical solution to even longer). With a rapid DNA mini-preparation the dif®culties encountered in the identi®cation of method , genomic DNA from dermatophytes can dermatophyte isolates with varied morphological be obtained from dermatophyte cultures within 1 h. characteristics in conventional procedures, and repre- AP-PCR ampli®cation and product detection are sents a technological advance in the laboratory diag- completed within 4 h. Therefore, a de®nitive result nosis of dermatophytosis. The future development of a can be generated easily from primary dermatophyte procedure for the isolation of DNA from clinical cultures within 1 day by AP-PCR. Besides being rapid, materials such as skin, hair and nails would further AP-PCR is also more sensitive, as the dermatophyte enhance the potential of AP-PCR for identi®cation of DNA sequence is ampli®ed to one billion-fold within dermatophytes. 30 cycles of denaturing, annealing and extension processes. Furthermore, AP-PCR is inherently more precise than the conventional methods, as it measures Future perspectives the genotypic rather than phenotypic characteristics of dermatophytes. In fact, of the 230 dermatophyte The fact that various DNA fragments can be ampli®ed isolates examined, some isolates of T. rubrum, T. from dermatophytes and other fungi with one of four mentagrophytes var. interdigitale, T. tonsurans and T. random primers in AP-PCR suggests that the gene violaceum displayed unusual colony morphology, yet regions recognised by these primers are probably they formed identical band patterns to those with evolutionarily variable. The elucidation of the precise typical cultural characteristics. For example, the structures and functions of the gene regions involved identi®cation by AP-PCR in this laboratory of two will require nucleotide sequence analysis of the non-pigmented (i.e., white colony) T. violaceum characteristic bands ampli®ed. A detailed knowledge isolates, which were later con®rmed by an external of these gene regions will provide insight into the reference laboratory, highlights the usefulness of the molecular basis and genetic relationships of various technique. Moreover, AP-PCR is also robust and dermatophytes. Moreover, the availability of nucleotide reproducible, and major bands can be obtained sequence data of the distinct DNA fragments obtained  DERMATOPHYTE IDENTIFICATION BY PCR 497 will help design primers speci®c for individual and differentiation of causative fungi of onychomycosis using dermatophyte species and subspecies. The subsequent PCR ampli®cation and restriction enzyme analysis. Int J Dermatol 1998; 37: 682±686. application of these speci®c primers in colorimetric 14. Leclerc MC, Philippe H, Gueho E. 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