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Previous Article | Next Article 
Journal of Clinical Microbiology, December 2006, p. 4336-4341, Vol. 44, No. 12
0095-1137/06/$08.00+0 doi:10.1128/JCM.00759-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Laboratório de Micologia, Instituto de Higiene e Medicina Tropical/CREM, Universidade Nova de Lisboa, Lisboa, Portugal,1 Serviço de Dermatologia, Hospital Curry-Cabral, Lisboa, Portugal2
Received 11 April 2006/ Returned for modification 3 July 2006/ Accepted 12 September 2006
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ABSTRACT |
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INTRODUCTION |
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Nonetheless, characterization by microscopic examination and in vitro culture of tinea are required for appropriate diagnosis and treatment, as well as for prevention of epidemic radiation of the infection. For example, in tinea capitis the identification of the species is important in the establishment of therapy. In general, infections by Trichophyton species need a longer period of treatment than those caused by Microsporum and Microsporum canis which only respond to terbinafine when higher doses are employed. Although in vitro culture is specific and sensitive, it has two significant drawbacks as a useful method for rapid diagnosis. These drawbacks are namely the long incubation period (2 or 3 weeks) required before diagnostic characteristics are developed in culture media and, secondly, the fact that many dermatophyte strains often develop atypical characteristics (different colony morphologies, pleomorphism). The latter drawback can result in false-negative diagnosis in as many as 15% of cases where there is actual infection (24, 28). Moreover, misdiagnosis may lead to harmful or inadequate treatment.
Molecular biology techniques, such as arbitrarily primed PCR (16), random amplified polymorphic DNA analysis (21), restriction analysis of mitochondrial DNA (22), and microsatellite-primed PCR (MSP-PCR) (10-12, 25) are considered to be generally adequate for species identification of certain fungi. Such techniques have been used to differentiate 26 species of Candida and up to 17 species of dermatophytes, including intraspecific strains of some species. Nevertheless, species identification based on detection of numerous and complex PCR banding patterns generated by these techniques may be technically allusive and inconclusive. A practical and proven approach to overcome these technical deficiencies is to comparatively analyze individual DNA fragment profiles from the original PCR fingerprint in order to identify species- or strain-specific amplicons. Once such specific bands are identified, they can be sequenced and specific PCR primers/probes can be designed (9, 17, 18, 19).
In the present study, the application of PCR fingerprinting was performed using three sets of primers—M13, (GTG)5, and (GACA)4—for the purpose of species identification and strain typing of common dermatophytes and related fungi. During analysis of the fingerprints of 11 species of dermatophytes, a specific DNA fragment was detected to be exclusive to only Microsporum audouinii and specific for all strains examined. Based on the DNA sequence of this amplicon, species-specific PCR primers were designed and tested for rapid and efficient PCR-based detection and to evaluate the diagnostic value of this method.
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MATERIALS AND METHODS |
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A total of 172 dermatophyte isolates representing three Microsporum species, seven Trichophyton species, and Epidermophyton floccosum were included in this study (Table 1). Ten reference strains, including Microsporum canis, M. audouinii, Trichophyton interdigitale, Trichophyton mentagrophytes var. erinacei, T. mentagrophytes var. goetzii, Trichophyton ajelloi, Trichophyton rubrum, Arthroderma vanbreuseghemii (mating types + and –), and Epidermophyton floccosum (Table 2) were cultured for inclusion as positive controls in PCR tests.
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Extraction of genomic DNA for PCR. A rapid DNA extraction technique for filamentous fungi (26) was used with some slight modifications. Briefly, a portion of mycelium from cultures on Sabouraud dextrose broth was added to a 1.5-ml tube containing 500 µl of lysis buffer (50 mM Tris liter–1, pH 8.0, 50 mM EDTA liter–1, pH 8.0, 250 mM NaCl liter–1, 0.3% [wt/vol] sodium dodecyl sulfate [SDS], pH 8.0), and 500 mg of acid-washed 0.4- to 0.6-mm-diameter glass beads (Sigma, St. Louis, MO).
Mycelia were lysed by continuous vortexing for 15 min on a Labinco BV-L46 vortex (Labinco, Breda, The Netherlands) at the highest intensity setting. Vortexed tubes were incubated at 65°C for 1 h and then revortexed for additional 10 min. Lysed suspensions were centrifuged at 13,000 x g for 15 min at 4°C, and supernatants were transferred to 1.5-ml tubes. Proteinase K (Gibco, BRL, Eggenstein, Germany) was added to a final concentration of 500 µl ml–1. Tubes were incubated at 60°C for 60 min (13) and centrifuged at 13,000 x g for 15 min, and the supernatant was transferred to new 1.5-ml tubes and diluted 1:750 in 10 mM Tris-HCl (pH 8.0).
Human DNA to be used in PCR tests was extracted from blood, using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's instructions for 300 µl of whole blood.
Microsatellite-primed PCR. The core sequence of phage M13 (GAGGGTGGCGGTTCT) was used as a single primer in minisatellite-primed PCR experiments (20), and two synthetic oligonucleotides, (GACA)4 and (GTG)5, were tested with microsatellite-primed PCR experiments (20) (MWG-Biotech AG, Ebersberg, Germany). MSP-PCR was performed in a 25-µl reaction mixture containing 10 to 15 ng of genomic DNA (5 µl from Tris-HCl dilution), 16 mM (NH4)2SO4, 67 mM Tris-HCl (pH 9.0), 0.01% Tween 20, 2 mM MgCl2, 250 µM each deoxynucleoside triphosphate (dNTP), 1 U of Taq DNA polymerase (Bioline, London, United Kingdom), and 0.8 µM of PCR primers as previously described (23). PCR amplification was performed using a Tpersonal Combi PCR system (Biometra, Göettigen, Germany) with the following thermocycling program: 1 cycle at 95°C for 5 min; 40 cycles at 93°C for 40 s, 50°C for 60 s, and 72°C for 60 s; and a final cycle at 72°C for 6 min. A tube having no DNA was included as a template control. After completion of amplification, 10 µl of the reaction mixture was added to 3 µl of 10x DNA sample buffer containing bromophenol and glycerol. DNA fragments were separated by electrophoresis in 1% (wt/vol) agarose gels in 0.5x Tris-borate-EDTA buffer at 50 V for 5 h. The gel was stained in ethidium bromide for 10 min and subsequently examined under UV light. Sizes of amplified DNA fragments were estimated using a GeneRuler 100-bp DNA Ladder Plus (MBI, Fermentas, Germany).
DNA cloning, sequencing, and primer design. Fragments generated by MSP-PCR were excised from gels and purified using Gel Extraction Jetquick (Genomed, Oeynhausen, Germany). Purified fragments were then cloned into plasmid vectors (pBluescript II KS [+]) (Stratagene, Integrated Sciences, Melbourne, Australia), according to the manufacturer's instructions. Cloned DNA fragments used for sequencing were amplified by PCR using 50 µM of T3 and T7 primers (Invitrogen, Carlsbad, Calif.), and purified using the Jetquick/PCR purification kit (Genomed, Oeynhausen, Germany), according to the manufacturer's instructions. Sequencing was performed in an automatic Beckam Coulter 2000 XL sequencer using the T3 and T7 primers. PCR primers specific to the M. audouinii fragment were designed with the aid of the online computer program Primer Design Assistant (PDA) (5). Selected primers were then artificially synthesized (MWG-Biotech AG, Ebersberg, Germany).
M. audouinii-specific PCR. PCR using M. audouinii-specific primers was performed as described for MSP-PCR, but with 0.4 µM of each M. audouinii species-specific primer and the following thermal cycle program: one cycle at 95°C for 3 min and 35 cycles at 93°C for 30 s, 62°C for 30 s, and 72°C for 30 s. A tube with no template DNA was included as a negative control. The resultant PCR products were examined in the same fashion as for MSP-PCR.
Multiplex PCR for assessment of reliability of identification. M. audouinii-specific primers were used in a PCR analysis under the same conditions described above, but with 0.2 µM universal primers for fungi TR1 (5'-GTTTCTAGGACCGCCGTA-3') and TR2 (5'-CTCAAACTTCCATCGACT-3') (2) added to the reaction mixture. The PCR thermocycle program was the same as that used for the M. audouinii species-specific PCR. A blank tube having no template served as a negative control.
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RESULTS |
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50 years, equally from both genders. T. mentagrophytes var. granulare was identified exclusively from skin and nail samples, mostly in the young (18 years) and oldest (>50 years) patients, equally in both genders. T. tonsurans was isolated from skin and nail samples from both male and female patients 18 years of age. These clinical profiles were similar for those of patients from whom Trichophyton soudanense was isolated, except the isolation occurred chiefly from hair. Microsatellite-primed PCR. Identification of certain species of dermatophytes using the microsatellite primers M13, (GTG)5, and (GACA)4 has been reported previously, but under different PCR conditions (11, 12). Figure 1 shows the electrophoretic band patterns obtained with primers M13, (GTG)5, and (GACA)4 under our PCR conditions. All sets of primers generated distinct PCR band profiles of each dermatophyte, with bands varying in intensities and molecular sizes. The PCR fingerprints showed DNA fragments that ranged from 0.2 to 4.9 kb in length for all species. Figure 1 also shows that all 11 species studied could be differentiated with the primer sets used, with the following exceptions: primer (GTG)5 failed to distinguish T. rubrum, T. soudanense, and T. megninii (Fig. 1B), whereas T. soudanense and T. megninii were indistinguishable with primer (GACA)4 (Fig. 1C). On the other hand, T. rubrum and T. soudanense presented similar DNA fragment patterns with primer M13 (Fig. 1A). None of the primers was able to distinguish the mating types of A. vanbreuseghemii (Fig. 1), whose patterns were quite similar to those of T. mentagrophytes, T. mentagrophytes var. goetzii, T. interdigitale, and T. mentagrophytes. var. granulare (Fig. 1). All primers distinguished T. mentagrophytes var. erinacei from the other varieties of T. mentagrophytes of this study. Nevertheless, the remaining varieties could not be differentiated by any of the three primers used.
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Analysis of DNA sequences of unique bands for species-specific identification. The M13 primer produced a clearer distinction between M. canis and M. audouinii than the other primers used. A prominent species-specific fragment, of approximately 0.9 kb, generated from the genomic DNA of M. audouinii (reference strain DSMZ 10649) with the M13 primer (Fig. 1A), was excised from the gel, purified, cloned, and sequenced. The sequence product of the cloned amplicon was 919 bp in length. From this sequence, a forward primer, MA1_F (5'-CGAAGCGAGCCTCTACGGCAATCCAAAGCAG-3'), and a reverse primer, MA1_R (5'-CGAAGCATCTTGCAGGACTCC-3'), were designed according to protocols outlined in Materials and Methods. These primers generated an amplicon of 431 bp. The primers were tested under several PCR conditions and with the genomic DNA of all the strains/isolates used in this study.
Identification reliability assessment of "MA1" primers. The utility of the MA1_F and MA1_R primers as a tool for identifying M. audouinii (4) was compared to that of the microsatellite primers M13, (GTG)5, and (GACA)4. All strains used in this study were tested with these primers in a Multiplex PCR with universal primers. As shown in Fig. 2, the expected band of 431 bp, using the MA1 primers, was generated only with strains of M. audouinii, whereas a band of 600 bp was generated by primers TR1 and TR2 for all fungal species, including M. audouinii. In some cases, the 600-bp fragment was weak or absent for the strain/isolates of M. audouinii. The four isolates that could not be identified by conventional methods, but were identified as M. audouinii by MSP-PCR fingerprinting, produced the 431-bp fragment when tested with the MA1 primers.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the past few years, the number of cases of tinea capitis due to anthropophyllic fungi has significantly increased, particularly in some European city communities (1, 3, 7). In Portugal, recent epidemiological data on dermatophyte infections are not available. In a study of isolates from a clinical laboratory attending to patients from the Lisbon area, T. rubrum was the most frequently isolated agent of dermatophytosis (28.5%), with M. audouinii (22.6%) and T. soudanense (14.3%) being the dermatophytes isolated the second and third most often, respectively. Tinea capitis was the most diagnosed dermatophytosis, followed by tinea pedis. This pattern in the Portuguese clinic is distinct from the one observed throughout other countries in western Europe but is similar to that found in some African countries (P. Viegas, A. Pinto, A. Santos, M. Pinheiro, and R. Vieira, Reun. Centro Soc. Port. Dermat. Vener., abstr.72, 2003). In our laboratory, skin-related dermatophytes were the most frequently identified organisms, followed by tinea capitis. The most isolated agent was T. rubrum, followed by M. audouinii and M. canis (Table 1).
In Portugal, there are recent data suggesting M. audouinii and T. soudanense as the dermatophytes isolated most frequently from tinea capitis. These fungi, prevalent in African countries, are now infecting European children and are increasingly responsible for outbreaks in European schools (27). Our records are congruent with these data. Still, M. canis is a major tinea capitis etiologic agent. Its widespread occurrence and capability of infecting distinct tissues account for the high number of hair infections detected (Table 1).
In this study, differentiation of species involved in tinea capitis was possible by MSP-PCR using three different primers. As previously described, it was possible to discriminate among all the species with primers (GACA)4 (11) and M13 and (GTG)5 (12). Variability among varieties of T. mentagrophytes was observed for T. mentagrophytes var. erinacei (CECT 2956) for all three primers. Slight differences were observed among the remaining varieties of T. mentagrophytes with these primers as well. Similarity in fingerprinting patterns between A. vanbreuseghemii (both mating types) and varieties of T. mentagrophytes (with the exception of T. mentagrophytes. var. erinacei) reflects the close phylogenetic relationship among them (14). The failure of primers (GTG)5 and (GACA)4 to discriminate T. soudanense and T. megninii is evidence of their close phylogenetic proximity. In fact, in a previous report (15), T. soudanense was reclassified as Trichophyton violaceum and T. megninii was considered an ambiguous taxonomic classification. These three species belong to the T. rubrum complex, as shown by the high degree of similarity in their DNA fragment patterns in our study.
Application of MSP-PCR has enabled rapid identification of several dermatophyte species/varieties (10-12). Nevertheless, development of specific primers for different species would improve specificity of PCR-based tests and eliminate problems of ambiguities in band pattern analysis. Development of a T. rubrum-specific probe (9) and, more recently, a specific M. canis primer set (18) has demonstrated the diagnostic importance of such primer-specific approaches.
Difficulty in identifying M. audouinii by macro- and microscopic features, especially in distinguishing it from M. canis, and time-consuming laboratory diagnosis led us to design a specific primer for identification of M. audouinii. To ensure that no false-negative results were obtained from the PCRs using our MA1 primers, a universal primer set generating an amplicon of 600 bp was added to serve as a positive control of the PCR. This mix of primer sets produced two fragments (of 600 bp and 431 bp; Fig. 2) in the strains/isolates of M. audouinii and just one fragment of 600 bp in the non-M. audouinii strains/isolates (Fig. 2).
The reliability of primers MA1_F and MA1_R as a diagnostic tool showed the MA1 primers had high specificity, sensibility, PPV, and PVN rates (all values near 100%) for the strains/isolates tested (Table 3). It is worth mentioning that two of the false-positive tests obtained with primers M13 and (GTG)5 were attributed to two isolates of M. audouinii that were identified as M. canis by M13 and (GTG)5 and as M. audouinii by (GACA)4 and MA1. Considering these findings, we conclude the two isolates are M. audouinii. Also, the one false-negative resulting in all primer tests was attributed to one isolate identified as M. audouinii by morphological features. However, the MSP-PCR primers identified this isolate as T. interdigitale. Moreover, this isolate also did not produce the 431-bp band expected for M. audouinii with MA1 primers. This shows that MA1 primers had 100% accuracy in the study sample.
In conclusion, the results of our study strongly support use of the MA1 M. audouinii-specific-primers for the identification of M. audouinii in the diagnosis of tinea capitis. In the future, PCR-based identification, as outlined in this study, can be applied directly to skin and hair samples. This has the advantage relative to conventional culture methods of making identifications in a shorter time-frame (1 to 4 days instead of 2 to 3 weeks) and eliminating problems of culture failure and contamination. The PCR approach does rely, however, on efficient extraction of nucleic acids that avoids possible degradation of DNA, which could lead to false-negative results. In our laboratory, we have already started to use this technique as a diagnostic tool in parallel with the conventional methods. So far, results have been 100% compatible. Moreover, detection of dermatophytes by the PCR technique instead of culture will allow rapid diagnosis of the species that will lead to a better management of infections caused by these fungi.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 27 September 2006.
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REFERENCES |
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