Forty-Eight-Hour Diagnosis of Onychomycosis with Subtyping of Trichophyton rubrum Strains
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微生物临床杂志 2006年第4期
National Center of Infectious and Parasitic Diseases, Department of Microbiology, Sofia, Bulgaria
Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands
Mycological Laboratory, State Hospital
Department of Dermatology, Aristotelian University of Thessaloniki, Thessaloniki, Greece
Institute of Microbiology and Hygiene, Department of Parasitology (Charite), Humboldt University, Berlin, Germany
ABSTRACT
A novel strategy for the molecular identification of fungal agents of onychomycosis (including Trichophyton rubrum) has been designed based on the use of species-specific and universal primers in conjunction with a commercial kit that allows the extraction of DNA directly from the nail specimens. The microsatellite marker T1, which is based on a (GT)n repeat, was applied for the species-specific identification of Trichophyton rubrum. To evaluate how often Scopulariopsis spp. are detected in nail specimens, a second primer pair was designed to amplify specifically a 336-bp DNA fragment of the 28S region of the nuclear rRNA gene of S. brevicaulis and closely related species. Other fungal species were identified using amplification of the internal transcribed spacer (ITS) region of the rRNA gene, followed by restriction fragment length polymorphism analysis or sequencing. In addition, polyacrylamide gel separation of the T1-PCR product allowed subtyping of T. rubrum strains. We studied 195 nail specimens (the "nail sample") and 66 previously collected etiologic strains (the "strain sample") from 261 onychomycosis patients from Bulgaria and Greece. Of the etiologic agents obtained from both samples, T. rubrum was the most common organism, confirmed to be present in 76% of all cases and serving as the sole or (rarely) mixed etiologic agent in 199 of 218 cases (91%) where the identity of the causal organism(s) was confirmed. Other agents seen included molds (6% of cases with identified etiologic agents; mainly S. brevicaulis) and other dermatophyte species (4%; most frequently Trichophyton interdigitale). Simultaneous infections with two fungal species were confirmed in a small percentage of cases (below 1%). The proportion of morphologically identified cultures revealed by molecular study to have been misidentified was 6%. Subtyping revealed that all but five T. rubrum isolates were of the common type B that is prevalent in Europe. In comparison to microscopy and culture, the molecular approach was superior. The PCR was more sensitive (84%) than culture (22%) in the nail sample and was more frequently correct in specifically identifying etiologic agents (100%) than microscopy plus routine culture in either the nail or the strain samples (correct culture identifications in 96% and 94% of cases, respectively). Using the molecular approach, the time for diagnosing the identity of fungi causing onychomycosis could be reduced to 48 h, whereas culture techniques generally require 2 to 4 weeks. The early detection and identification of the infecting species in nails will facilitate prompt and appropriate treatment and may be an aid for the development of new antifungal agents.
INTRODUCTION
Toenail onychomycosis is a fungal infection caused mainly by dermatophytes. Less frequently, yeasts and nondermatophytic molds are also found to be etiological agents of this type of infection. Onychomycosis occurs worldwide and accounts for up to 50% of all nail disorders (2, 7). The prevalence of onychomycosis has been given as 2 to 18% of the whole human population, and it might be even higher (44). The most significant agent is Trichophyton rubrum, with an incidence of more than 90% in Europe (49). Trichophyton interdigitale is less common (17, 18). Nail invasion by nondermatophytic molds is generally considered uncommon. Reported prevalence rates range from 0% to 50%, with studies that carefully investigate all such cases usually showing a prevalence of around 3.5% (21, 26, 54). The fungal species most frequently implicated in onychomycosis caused by molds in temperate parts of the world is Scopulariopsis brevicaulis (15, 16, 18, 50). Most of the nondermatophytic fungi recurrently involved in opportunistic onychomycosis, inclusive of several well-known Scopulariopsis, Fusarium, and Aspergillus species (47), are very common in the environment but remain relatively uncommon as causes of onychomycosis (25).
The incidence of onychomycosis has increased over the last few years in response to the increased population of immunocompromised patients, the widespread use of immunosuppressive chemotherapy and broad-spectrum antibiotics, the expanding number of elderly persons, and the increasing public participation in fitness-related activities (42, 52). Onychomycosis is increasingly recognized as a nontrivial medical disorder that poses various physical, psychosocial, and occupational problems (3).
Treatment of onychomycosis is closely linked to the identity of the causative agent, particularly in terms of whether or not it is a dermatophyte (4, 40, 45). The most commonly used oral antifungals include itraconazole, terbinafine, and fluconazole; these drugs have superseded the traditional therapies based on the use of griseofulvin and ketoconazole. While terbinafine and itraconazole demonstrate efficacy against T. rubrum and T. interdigitale, griseofulvin and fluconazole are ineffective against T. interdigitale and against molds, e.g., S. brevicaulis, that may cause the infection (19, 45).
Current diagnosis and treatment procedures for dermatophyte onychomycosis rely on direct microscopic examination of fungal elements in nail material using 30% KOH, in combination with the identification of the etiological agent based on the examination of its colony and microscopic morphology. In onychomycosis caused by molds, a second, confirmatory sample may also be needed. Patients are often treated as soon as fungal elements are shown to be present microscopically. These methods are low in specificity and may be very time-consuming, because growth, sporulation, and routine physiological testing (e.g., urease enzyme assay) of the fungi involved may take 2 to 4 weeks. In addition, 15 to 50% of microscopically true-positive samples fail to grow a culture (8, 33). Moreover, dermatophyte species show an unusual level of variation in the expression of phenotypic characteristics, e.g., the formation of pigments or conidia (46).
The application of PCR technology directly to clinical specimens would allow early and accurate identification of agents of onychomycosis. This would permit prompt and targeted initiation of antifungal therapy. The present study tested such a direct PCR application with techniques also designed to allow infraspecific subtyping of some of the elucidated etiologic agents.
MATERIALS AND METHODS
Clinical isolates. Two collections of samples were investigated in this study. Nail specimens were collected as subungual scrapings, clippings, or curettings. The first sample (called "strain sample" in Table 1) was a collection of 66 test isolates obtained from serial patients who were microscopically positive and had grown a culture in procedures for diagnosis of onychomycosis. This collection might be biased against T. interdigitale, which is more often associated with microscopy-negative samples than T. rubrum is. The second sample (called "nail sample" in Table 2) was collected from 195 successive patients whose abnormal nails were indicated for dermatologic mycology evaluation. To allow valid statistical testing, specimens from all patients in this series were included in the sample, regardless of whether or not a positive culture or microscopy result was obtained. Of the 261 patients, 101 were from Greece and the remainder were Bulgarian residents. Sixteen patients suffered from suspected fingernail mycosis; the rest were evaluated for toenail onychomycosis.
A single specimen was analyzed from each of the 261 patients. Patients were recalled when a nondermatophyte mold grew in culture, regardless of whether or not a dermatophyte also grew. For those patients, three independent, serial specimens were analyzed.
Routine morphological identification at the test centers. Nail specimens were divided into three portions. The first portion was examined microscopically with 30% KOH for the presence of fungal elements. The second was cultured on Sabouraud glucose agar, containing 10 g peptone, 40 g dextrose, and 15 g agar per liter distilled water, with a final pH of 5.6 and with no antibiotic supplementation (NCIPD Ltd., Sofia, Bulgaria), at 27°C for 4 weeks. Clinical isolates were identified on the basis of phenotypic characteristics of the colony and on the basis of the shapes of macro- and microconidia. Hair perforation testing and tests for urease degradation, which aid distinction of atypical isolates of T. rubrum and T. interdigitale, are not routinely done at the test centers due to economic stringencies. From the third portion of the nail specimen, DNA was extracted as outlined below.
DNA extraction. A minipreparation method to extract DNA from fungal cultures was used, as described previously by Grser et al. (13).
To extract DNA directly from nail material, a commercial kit, the GenomicPrep Cells and Tissue DNA Isolation kit from Amersham Biosciences (Piscataway, NJ), was used. Prior to use of the kit, whole nails and relatively large nail fragments were cut into small pieces with a surgical blade. Nail shavings were processed directly. The manufacturer's instructions were modified as follows. In step 1, to 0.5 to 3 mg of nail material, 300 μl rather than 600 μl of cell lysis solution was added with 5 to 10 mg glass beads (0.5 mm in diameter; BioSpec Products Inc.) was also added. The reaction mixture was vortexed for 1 min. In step 3, nail material was incubated for 1 h in a 65°C water bath following the addition of 3 μl Proteinase K solution (20 mg/ml), and the solution was incubated overnight at 55°C and vortexed again. In step 7, 100 μl of protein precipitation solution was added instead of 200 μl. In step 9, the solution was centrifuged for 5 min instead of 3 min. In step 11, the reaction mixture was left at –20°C overnight. In steps 12 and 14, centrifugation was done for 10 min. The DNA pellet was dissolved in 10 to 30 μl hydration solution, depending on the amount of nail material used at the beginning.
Identification using PCR. (i) Identification of T. rubrum. For the identification of T. rubrum, the following oligonucleotides were used as primers in the PCR: 5'-TGG TCT GGC CTT GAC TGA CC (T1.for) and 5'-GTA AGG ATG GCT AGT TAG GGG G (T1.rev). These primers amplify a 280-bp fragment containing a GT-microsatellite repeat specific for strains of the closely related T. rubrum/T. violaceum clade (38). Briefly, an amplification reaction was performed in a total volume of 35 μl. The PCR mixture contained reaction buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2), 200 μM of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech Inc., Piscataway, NJ), 21 pmol of each primer, 1.75 U of Taq polymerase (AmpliTaq; Applied Biosystems), and 35 ng of template DNA. Samples were overlaid with sterile light mineral oil and amplified through 30 cycles in a thermocycler (Thermoblock, version 2.55b; Biometra TRIO) as follows: initial denaturation for 10 min at 95°C, denaturation for 30 s at 95°C, annealing for 30 s at 60°C, and extension for 45 s at 72°C. This was followed by a final extension step for 3 min at 72°C.
For subtyping strains of T. rubrum, 5 to 10 μl of the PCR product was separated on a polyacrylamide gel (8%) for 7.5 h at constant power (45 W) or for 17 h at 22 W. Following electrophoresis, the gels were silver stained and dried for documentation.
(ii) Identification of the remaining fungal agents of onychomycosis. For identification of the remaining fungal agents of onychomycosis, restriction fragment length polymorphism (RFLP) analysis and sequencing of the internal transcribed spacer (ITS) region of the rRNA gene were applied. For ITS-PCR, the universal primers LSU266 (5'-GCA TTC CCA AAC AAC TCG ACT C) and V9D (5'-TTA CGT CCC TGC CCT TTG TA), amplifying a DNA fragment of about 1,000 bp of the gene, were used (9). The PCR mixture contained reaction buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2), 200 μM of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech Inc., Piscataway, NJ), 50 pmol of each primer, 2 U of Taq polymerase (AmpliTaq; Applied Biosystems), and 50 ng of template DNA. Samples were overlaid with sterile light mineral oil and amplified through 35 cycles in a thermocycler (Perkin Elmer 9600) as follows: initial denaturation for 5 min at 94°C, denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1 min at 72°C. This was followed by a final extension step for 7 min at 72°C. Restriction enzyme analysis of the PCR products was performed using 5 U MvaI enzyme (MBI Fermentas, Amherst, NY) per sample, followed by incubation for 4 h at 37°C. The resulting fragments were electrophoresed through 2% MetaPhor agarose gels (BioWhittaker Molecular Applications Inc.) for 2.5 h at 100 V. A set of reference strains was used for comparison.
Sequence analysis was performed using CEQ dye terminator cycle sequencing with the Quick Start kit and the CEQ 8000 Genetic Analysis System from Beckman Coulter (Fullerton, Calif.).
(iii) Identification of S. brevicaulis and related species. In parallel, a primer pair was developed and tested for the detection of S. brevicaulis and related species. Although, based on a BLAST search via the National Center for Biotechnology Information web site, the primers were predicted to amplify a wide range of species within the class Sordariomycetidae, the only one of these fungi commonly causing onychomycosis is S. brevicaulis. Species rarely causing onychomycosis that would be predicted to be amplified are other Scopulariopsis species (some of them best known by Microascus teleomorph names) and Chaetomium globosum. Based on the large subunit of the rRNA gene, a 336-bp fragment was amplified. The SCOP primer pair (5'-GAA TGC TGC TCA AAA TGG GAG-3' and 5'-CAT TAC GCC AGC ATC CTTG C-3') was selected on the basis of sequences published in GenBank (22). The following PCR conditions were applied: amplification was performed with puReTaq Ready-To-Go PCR Beads (Amersham Biosciences, Piscataway, NJ) in reaction volumes of 25 μl containing 2.5 U puReTaq DNA polymerase and buffer (10 mM Tris-HCl, pH 9.0, at room temperature, 50 mM KCl, 1.5 mM MgCl2), 200 μM of each deoxynucleoside triphosphate, and 15 ng of template DNA. The primers were added, each at a final concentration of 20 pmol. Samples were amplified through 35 cycles in a thermocycler (MJ Research PTC 100) as follows: initial denaturation for 3 min at 94°C, denaturation for 45 s at 94°C, annealing for 1 min at 69°C, and extension for 1 min at 72°C. This was followed by a final extension step for 10 min at 72°C. Five microliters of each reaction was loaded on 1.2% agarose gels and electrophoresed for 1.5 h at 100 V in 0.5x buffer (89 mM Tris-borate, 2.5 mM EDTA, pH 8.3). The gels were stained with ethidium bromide and photographed.
The sensitivity of the SCOP primers was tested with different concentrations of DNA extracted from a known strain of S. brevicaulis (J99079). DNA was serially diluted to give concentrations ranging from 15 ng to 1.5 pg in order to determine the lowest limit of detection.
RESULTS
Conventional identification. In the strain sample (Table 1), all 66 isolates came from microscopy-positive specimens, as mentioned above. In the nail sample, fungal filaments were seen in the KOH preparations in 181 out of 195 specimens (93%), while a culture of a potential agent of onychomycosis grew from 44 specimens (22%; Table 2). T. rubrum made up 83% of isolates from the strain sample and 95% of isolates from the nail sample; when both samples were combined, it made up 88% of isolates. The remaining cultures were mainly T. interdigitale, with S. brevicaulis also being identified on two occasions. Of the two S. brevicaulis isolates found in the nail sample, each was confirmed a posteriori as etiologic by means of later studies on repeat specimens, and one was obtained from a specimen also yielding T. rubrum.
DNA extraction from nails. In order to determine the minimum weight of nail material needed to extract DNA using the GenomicPrep Cells and Tissue DNA Isolation kit (Amersham Biosciences, Piscataway, NJ), 10 nail samples were selected. Each sample was divided into three portions, one containing 0.5 mg of material, one containing 1.0 mg, and one containing 1.5 mg. In addition, 24 nail samples with weights ranging from 2 mg to 4 mg were investigated. These studies established that, for PCR based on the use of T1, a minimal amount of 0.5 mg of nail material was needed to obtain a faint PCR product (data not shown).
Sensitivity and performance of the SCOP primers. The SCOP primers were able to detect as little as 150 pg of template DNA. Given the fact that 10 pg of target DNA generally corresponds to circa 25 fungal cells or CFU per clinical specimens (51), the detection limit of the SCOP assay should correspond to 375 or more cells per clinical specimen. Cultured material of all 13 clinical isolates morphologically identified as S. brevicaulis (Tables 1 and 2) amplified with the SCOP primers. In 6 out of 195 nail specimens (specimens 955H2, 887P3, 1372Gr, 585Gr, 1024Gr, and 1213Gr), a positive amplification with the SCOP primers was obtained. In three cases, these positive amplifications were from nails also positive for T. rubrum, and in three cases only Scopulariopsis signal was amplified, confirmed by culture in one case as S. brevicaulis. All S. brevicaulis amplifications were from specimens positive for fungal elements in direct microscopy, but no structures specific for S. brevicaulis, such as masses of conidia produced in situ, were reported. S. brevicaulis was successfully cultured twice from specimens in the "nail specimen" group; on both occasions, the positive culture was from a nail that also gave a positive amplification and that yielded additional S. brevicaulis cultures in later specimens. In four cases there was positive amplification with the SCOP primers in the absence of a positive culture. Sequences derived from the 336-bp PCR products obtained in each case showed 100% identity with three species that are identical in this region, S. brevicaulis, S. koningii, and Microascus manginii (anamorph: Scopulariopsis candida). Technically, then, the positive amplifications obtained with the SCOP primers could indicate any of these three species; however, S. brevicaulis is much more common in association with nails than either of the other two species.
Identification of other agents of onychomycosis using PCR. The polymorphic microsatellite marker T1 containing a GT repeat motif has been used in this study for the identification of strains and clinical specimens of T. rubrum. Of the 66 isolates for which the culture was used as the template for DNA extraction (Table 1), 55 had been conventionally identified as T. rubrum and 11 as S. brevicaulis. The T1 primer pair designed for the species-specific amplification of isolates of the T. rubrum/T. violaceum clade (38) amplified 51 of them. Four strains (7%) failed. Those four were suspected to have been misidentified and thus were subjected to ITS-PCR. Using RFLP analysis, three (strains 3702Gr, 346Gr, and 5162Gr) were reidentified as T. interdigitale, while one (strain 817P) was identified as Arthroderma benhamiae (anamorph: unnamed, formerly part of Trichophyton mentagrophytes complex sensu lato) (Fig. 1). None of the S. brevicaulis strains had been misidentified morphologically; all of them amplified with the SCOP primer pair.
In the 195 clinical nail specimens used directly as the template for DNA extraction (Table 2), 148 (76%) were amplified with the T1 primers, indicating the presence of T. rubrum. By contrast, only 22% of these specimens had grown a culture. The remaining 47 nails were analyzed using ITS-PCR. In four of them, T. interdigitale (specimens 1052Gr, 7112Gr, 431Gr, and 684H2; Fig. 1) was present. Two of these specimens had yielded a culture that was misidentified as T. rubrum, while two were culture negative. Each of the following species was detected by PCR in one microscopy-positive but culture-negative nail specimen: Fusarium solani (GenBank accession number AF178402; a species complex partially listed in GenBank under the teleomorph-related name Nectria hematococca complex; isolated from our specimen 154Gr; 99.4% similarity over 356 bp with strain NRRL 22354 [NRRL Collection, National Center for Agricultural Utilization Research, Peoria, Ill.]); Fusarium proliferatum (accession number U34558; a member of the Gibberella fujikuroi complex; our specimen 1024Gr; 100% similarity over 518 bp with NRRL 22944); and Aspergillus nidulans (GenBank accession number AF455505; our specimen 7962Gr; 99.5% similarity over 475 bp with strain wb 260 [personal collection of Walter Buzina, Medical University Graz, Austria]; Fig. 1). Results for detection of Scopulariopsis spp. with the SCOP primers are given above.
It was found that 39 specimens did not produce a PCR product with any of the primers used in this study. In 30 of these, structures interpreted as fungal elements were seen in direct microscopy (Table 2). In 7 (23%) of the 30 specimens, the DNA amplification was concluded to be negative due to the presence of substances inhibitory to PCR. PCR inhibition was evidenced for each specimen involved by means of a control reaction in which the DNA from the specimen was spiked with a concentration of control template DNA from a strain known to amplify well. Even serial dilutions of the template DNAs did not change the results (data not shown). Despite the apparent hyphae seen under the microscope, the PCR-inhibited specimens had to be classified as incompletely analyzed.
In the total collection of 195 nail specimens, there were five cases yielding positive PCR evidence for more than one species either occurring in the nail or, for some of the nondermatophytes involved, possibly occurring as a relatively heavy contaminant of the corroded nail surface. A Scopulariopsis sp. was associated with T. rubrum in three cases (and confirmed as S. brevicaulis in one of them) and with F. proliferatum in one, while T. rubrum was associated with T. interdigitale in one.
For the subclassification of T. rubrum isolates into types A and B and for the separation of T. violaceum, PCR products were run on the polyacrylamide gels. The majority of isolates were type B, the type that is predominant in Europe. Only five patients from Greece yielded type A isolates (Fig. 2).
Conventional versus molecular identification and detection. The rate of morphological misidentifications among all cultures studied was 6%. For the most part, T. interdigitale had been misidentified as T. rubrum.
In direct nail analyses, DNA of T. rubrum was detected in 5 (36%) of the 14 specimens that were microscopy- and culture-negative. In 30 of the 138 specimens that were microscopy positive but culture negative (21.7%), no fungal DNA was detected; of the remaining 108 specimens, 103 (74.6% of the 138 specimens considered) yielded positive signal for T. rubrum, 1 yielded signal for T. interdigitale, and 6 yielded a positive amplification for one or more nondermatophytes (one F. proliferatum plus Scopulariopsis sp., one F. solani, one A. nidulans, and three additional Scopulariopsis spp. amplifications, including two from specimens also amplifying for T. rubrum). No specimen yielded a culture without also yielding a positive PCR amplification for the same fungus.
DISCUSSION
In onychomycosis worldwide, two dermatophyte species are most frequently involved as causal agents, namely, T. rubrum and T. interdigitale. The prevalence of these pathogens, however, varies according to geography, climate, and patterns of population migration, and it may differ among regions with similar environmental conditions. The few onychomycosis studies that have been carried out for the Balkan region (27, 39, 41, 48), where the patient population of the present study originated, have shown that the two dermatophyte species predominate, making up 55 to 70% of isolates from nails, while molds and yeasts are less common, growing from 10 to 27% of specimens (but not always rigorously implicated as causal). Variation among countries is evident. For example, in Croatia (48) the most common species was T. interdigitale (55%), whereas in our sample (Bulgaria and Greece) T. rubrum was most frequently isolated (76%) and T. interdigitale made up only 2% of isolates. An earlier study from the Plovdiv region of Bulgaria (where our own Bulgarian collection also originated) detected 83% T. rubrum and 17% T. interdigitale isolates among 130 cases (41). This shows that the incidence of T. interdigitale has dropped since 1965. Our results are therefore in agreement with the global ascendancy of T. rubrum as the predominant etiological agent of onychomycosis. Even using PCR in the present study, nondermatophytic molds (Scopulariopsis spp., Fusarium spp., and A. nidulans) as confirmed or suggestive etiologic agents were detected in only 3% of infections; also, only 3% of infections yielded mixed isolations. Those results are comparable with those of the study done by Saulov in Plovdiv (41), in which 2.5% of onychomycoses were ascribed to molds and 3.3% were classified as mixed infections. Scopulariopsis spp. were not detected in that study. The predominance of the dermatophytes in onychomycosis is very likely due to their broad spectrum of secreted proteases (24) acting as virulence factors enabling the destruction of the nail plate.
In the 13 cases (2 in the nail sample and 11 in the strain sample) where molds such as S. brevicaulis were the only cultures to grow from three serial microscopy-positive specimens, infection by these fungi is very likely. However, all nine cases (including three in which dermatophytosis was confirmed) in which a mold was detected by direct PCR in a case where only a single patient specimen was available are considered not to be authoritatively confirmed. Notwithstanding this cautious interpretation, it should be noted that, as calculated above, probably at least 375 Scopulariopsis sp. cells would need to be present before a positive reaction with the SCOP primers was engendered. At least four out of our nine cases very likely had at least this level of viable Scopulariopsis material present. Thus, though we must refrain from recording these amplifications as fully confirmed cases, the balance of probability suggests that true infection rather than contamination is the most parsimonious explanation for our positive SCOP primer results. This interpretation also fits well with the relatively small number of times the environmentally common S. brevicaulis was detected. Based on the figures given by Gupta et al. (20) in their Table 3, growth in culture of insignificant Scopulariopsis sp. inoculum on nails is nearly 2.5 times as common as growth from a true Scopulariopsis sp. infection, even in an elderly patient population particularly vulnerable to onychomycotic molds. If small numbers of contaminating Scopulariopsis sp. conidia were giving rise to positive amplifications, we would probably have had many more positive SCOP results. Moreover, some of the nononychomycosis-causing molds predicted by BLAST to react with the SCOP primer, such as members of the genera Trichoderma, Lecanicillium, and Coniochaeta, are very common in the indoor environment. If the SCOP primers were detecting background levels of fungal contamination on nail material, the proportion of positive results could be very high indeed.
In terms of firm confirmation, however, our sampling strategy only partly fulfilled the classic evaluation criteria for the acceptance of an infection by a nondermatophytic fungus, in which such a fungus should be isolated from at least two distinct, temporally separated samples taken from the same microscopy-positive nail (47). This criterion, based on the well-supported idea that a pathogen is much more likely than a contaminant to be consistently associated with the nail on separate occasions, works well with conventional studies (47), but the extent to which it is applicable to PCR-based onychomycosis studies remains to be investigated.
Even though PCR may not as yet, pending further testing, allow immediate certainty about whether the molds it detects in microscopy-positive nails are etiologic or contaminating, it may still significantly improve the diagnosis of nondermatophytic onychomycosis. Culture of the initial toenail specimen in proven cases of onychomycosis at best only detects etiologically causal nondermatophytes circa 75% of the time they are present, a rate similar to that obtained with dermatophytes in the initial specimen (47). PCR may be more sensitive than culture in this regard. In the present study, in relation to culture, specific PCR brought about a 200% increase in the detection of Scopulariopsis spp. This may at first seem difficult to explain, since S. brevicaulis grows vigorously in culture and a single viable cell is enough to start a culture, while PCR may require a somewhat larger amount of material as a template. Fungus-killing bacteria, known to occur abundantly in distal areas of onychomycosis-affected nails, may be responsible for the lack of outgrowth in at least some cases (25, 32, 53).
Another benefit of PCR in diagnosis of nondermatophyte mold infections is that it allows better identification of etiologic agents than can be accomplished in culture. For example, the F. solani isolate suggestively associated with positive direct microscopy in one case can be determined through sequencing to be most similar to a rarely isolated genetic subgroup, probably representing an undescribed phylogenetic species, only represented in GenBank by the sequence of an isolate (NRRL 22354) obtained from a teleomorph collected on tree bark in Guyana (36). F. solani is in the process of being broken up into 50 or more phylogenetic species (36); which of the new taxa make up the F. solani complex populations associated with human feet and nails is not yet known. F. proliferatum, recognized here via PCR in a case suggestive of a mixed infection with S. brevicaulis, is also a member of a phylogenetically complex group, the Gibberella fujikoroi complex, containing many anamorphic Fusarium species that are very difficult to identify accurately in culture (37).
The gold standard procedure for diagnosis of fungal nail infections is direct microscopy (KOH preparation) combined with the fungal culture, and the performance of new techniques is generally gauged against this standard. As a "gold standard," KOH and culture is problematic: various studies have shown that 15 to 50% of the microscopy-positive nail specimens do not grow a culture (1, 6, 8, 33). In our nail study (Table 2), the failure of culture was even more severe: a full 72.1% of PCR-confirmed dermatophytosis specimens (i.e., 109 of 151 specimens) did not yield the dermatophyte in culture. This, however, is very unusual, and we note that two studies (11, 47) using long-term patient follow-up as a gold standard to determine the true number of dermatophytosis cases in patient population both found that approximately 75% of initial specimens from true dermatophyte-infected toe onychomycosis grew cultures in the initial sample. When the original data are examined from these studies, our low rate of culture positivity can be confirmed as being strongly statistically different (P < 0.001 by chi-squared test) from the rates seen in these studies. This suggests a source of interference in culture efficiency in our study. Though various factors can contribute to culturing problems, e.g., patients not disclosing recent antimycotic treatment, our tentative conclusion is that the factor most probably responsible was the use of a primary isolation medium free of antibacterial antibiotics. It is important to note that this usage was not part of the study design but rather merely reflected the longstanding and ongoing economic reality of routine isolation at both of the southeastern European institutions involved, as well as at most or all other institutions in the region.
In the present study, direct microscopy showed its normal false-negative rate (filaments not detected in confirmed dermatophytosis cases) of circa 5% (47), in this case 3.3% (5 of 151 confirmed dermatophytoses). The five microscopy-negative, PCR-positive specimens involved made up 36% of the 14 specimens in our study that would have been considered completely negative for mycotic agents in conventional studies. All five of these cases involved T. rubrum as the etiologic agent. (An alternative scenario, that the T. rubrum amplified in these five cases came from nearby infected skin rather than the nails themselves, seems highly unlikely, given the onychomycotic symptomatology of the nails combined with the likelihood that any dermatophytosis of skin adjacent to the nails would probably also affect the nails. Also, infections of more distant areas such as toe webs would probably not yield sufficient contaminating material to cause false positives in nail PCR, given the normal dermatological practice of cleaning away the contaminated superficial surface of infected nails before submitting a sample to the laboratory.) In all other cases in which a known or suggestive etiologic agent was detected by PCR alone, the presence of fungal filaments in direct microscopy would have indicated at least that a mycosis was present. The five microscopy-negative, PCR-positive cases, though, mean that PCR increased the number of confirmed mycoses in this study from 181, as would have been recognized in conventional studies, to 186, an increase of 2.8%. Though this may not seem a high percentage, the high numbers of patients seeking relief from onychomycosis must be kept in mind. Scher (43) showed that in one year alone, 662,000 Americans over the age of 65 consulted a physician for handling of suspected onychomycosis. If 2.8% of this population were to have their mycosis missed on the first examination, this would amount to 18,536 inadequately handled patients over 1 year in just that single demographic group.
In the present study, PCR in direct analysis of nails detected a definite or potentially etiologic fungus in 156 of the 186 patients known by any criterion to have a mycosis (assuming all of the 30 recorded microscopy-positive but PCR-negative samples were correctly read microscopically and that these patients all truly had a mycosis). Its sensitivity, then, measured against the ad hoc standard of its own positive results, plus additional positives obtained only via direct microscopy (no additional positives were elucidated only by culturing), was 83.9%. Importantly, PCR was positive in 100% of cases where a culture was positive, and the positive PCR results for a specimen always corroborated the presence of any species that grew in culture (though this was initially confused by morphological misidentification of a small number of cultures) as well as often revealing some additional species. Due strictly to the fact that repeat specimens could not be obtained (except in two cases) to confirm the significance of cases in which only potentially invasive nondermatophyte molds were PCR detected in microscopy-positive specimens, PCR completely settled the identity of at least one definite etiologic agent in a slightly smaller proportion of probable mycosis cases, i.e., 151 of the 186 cases (81.2%). Of these 151 cases of definite etiologic agent confirmation, 109 (72.2%) were achieved only through PCR, not culture. Of course, this to some extent reflects the unanticipated culture problems with this study discussed above, but it does stress that PCR is capable of taking on the burden of identification when culture is unreliable.
Our use of a commercial kit for the extraction of DNA from nail specimens gave us access to an easy, rapid, and well-standardized method. Although a method for DNA extraction from nails had been described previously (51), its reliability may be problematic due to the involvement of the enzyme lyticase, which is often contaminated with genomic DNA of the species it has been purified from (31). In addition, using the Amersham kit, we avoided steps such as mincing and crumbling the nails and homogenization of the material by shearing in a syringe (51). The kit substitutes overnight proteinase K treatment for the usual procedures for mechanical destruction of the cell integument that becomes involved in fungal DNA extraction from keratinized nails. Supplementing general PCR with the use of T1 as a specific marker, we were able to identify the major causative agent of onychomycosis, T. rubrum, within 2 days in all cases where it occurred, even in numerous specimens not yielding a culture. This decreases enormously the time needed for the species identification of fungi (normally 2 to 4 weeks), which can be problematic due to pleomorphic growth, slow generation time, or difficulties with culture. The early and accurate detection and identification of the infecting species in nails will facilitate prompt and appropriate treatment and may be an aid for the evaluation of new antifungal agents.
In general, identification methods based on the genotype of an isolate are considered more reliable than morphological and physiological identification techniques, and a large number have been applied already to differentiate dermatophytes. For example, based on ribosomal small subunit rRNA gene sequences, a primer system specific for the detection of dermatophytic DNA has been described, though this system is not designed to distinguish individual dermatophyte species (5). Restriction fragment length polymorphism analysis (35), PCR fingerprinting (12), arbitrarily primed PCR (29), random amplified polymorphic DNA analysis (30, 34), or sequencing of the ITS region (13, 14, 23) has been stated to be useful for the identification of species in dermatophytes and other fungi. Some of these methods, however, lack reproducibility; in particular, few of the random amplified polymorphic DNA techniques proposed for fungi in the 1990s are in use today. Many of the previous techniques also lack the convenience of allowing use of DNA extracted directly from nail specimens and, thus, may be delayed at least by the number of days necessary to grow a minimal culture for DNA work (which varies by species but is approximately 4 to 10 days). PCR-RFLP techniques require a permanent set of reference strains for species identification, and hybridization techniques are quite laborious. Sequencing of the ITS would be the gold standard and is not more expensive than identifying T. interdigitale using conventional diagnostics (6.60 versus 6.13 for conventional diagnostics; Table 3 ), which is inclusive of urease test and Trichophyton agar. Specific markers, however, are superior in cases where a limited range of etiological agents needs to be considered, and in particular, a single PCR is sufficient for the diagnosis of T. rubrum (T1-PCR). The cost of this test would be only slightly higher than that of conventional diagnostics when the latter are performed according to recommended standard procedures (25) (2.90 versus 2.30 ; Table 3). The specific microsatellite marker applied in the present study also amplified species that are phylogenetically closely related to T. rubrum, such as T. violaceum (38). However, T. violaceum is only rarely found as an agent of onychomycosis and is uncommon in many parts of the world. In addition, using the intrinsic length polymorphisms of the T1 marker by applying polyacrylamide gel analysis, it is possible to differentiate T. violaceum from T. rubrum and even to subtype isolates of T. rubrum into two epidemiologically distinct groups (38). To avoid the coincidental detection of T. violaceum, we have recently designed and tested a primer pair that is specific for T. rubrum. It amplifies a 100-bp region of the T1 fragment where a polymorphism (A/G) at the 3' end can be used as template for a specific forward primer (unpublished data). Recently, a similarly specific DNA marker (600 bp) has been developed by Liu et al. (28). However, the use of this marker for direct amplification from clinical specimens has never been tested, nor has its use for subtyping T. rubrum isolates. When such a large DNA fragment (600 bp) is used, single-nucleotide polymorphisms can only be distinguished by sequencing. In the gene regions studied so far, T. rubrum isolates have proven to have very few polymorphisms, probably due to the known clonal reproduction and probable recent common ancestry of all strains analyzed to date, including types A and B. Therefore, in general, the greater the length of any gene region used in electrophoretic studies, the higher the risk that any strain-level differences occurring will be overwhelmed by the preponderance of similarities.
Subtyping of 204 isolates of T. rubrum (consisting of 93 from Greece and 111 from Bulgaria) using the T1 marker and polyacrylamide gel electrophoresis detected only types A and B. Types C and D, which correspond to the T. violaceum clade, were not detected. This implies that the probability of misidentifying T. rubrum as T. violaceum in patients with onychomycosis is very low using our techniques, even if the polyacrylamide gel step is not done. Even in parts of Africa where T. violaceum is abundantly present, onychomycosis is a very uncommon manifestation.
Recently, Ohst et al. have shown that type A T. rubrum is widespread in Africa, whereas type B is dominant in Europe (38). In the present sample, only five isolates of type A were detected, all of them in specimens from Greece. In northern Greece, Asian and African type strains (e.g., T. rubrum with granular morphology, consistent with that characteristic of most type A strains, previously sometimes assigned the synonymous name T. raubitschekii or T. fluviomuniense) have been often isolated from persons who had never visited Asia or Africa (10). Northern Greece is part of the south Balkan region, which is located at a crossroads between Asia, Africa, and Europe. Historical human settlement patterns may thus explain the distribution of type A isolates found in our sample.
Though molecular techniques for dermatophyte diagnosis are clearly in development with the aim of ultimately providing new methodologies to all relevant medical services worldwide, it is recognized that the techniques described here will not be practical for a large number of laboratories that are either small scale or very tightly budgeted. They may, however, cross the line of practicability for mid- to high-level reference laboratories, laboratories with preexisting molecular facilities, and laboratories undertaking the further development of these methodologies.
ACKNOWLEDGMENTS
We thank Mariela Hitova, Lilia Zissova, Emil Bardarov, and Grigor Mateev for their assistance in collecting nail specimens. We thank the staff of the Laboratory of Molecular Microbiology, National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria, for their technical assistance.
Funding was provided by the Bulgarian Ministry of Education, Science and Technology, grant MU-L-1202/02, given to V. Kardjeva (maiden name, V. Stavrakieva). Molecular experiments were carried out at the Institute of Microbiology and Hygiene, Department of Parasitology, Charite, Humboldt University, Berlin, Germany, by the first author and were financially supported by that institute. A scholarship was provided to V. Kardjeva by the Deutscher Akademischer Austauschdienst (DAAD).
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Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands
Mycological Laboratory, State Hospital
Department of Dermatology, Aristotelian University of Thessaloniki, Thessaloniki, Greece
Institute of Microbiology and Hygiene, Department of Parasitology (Charite), Humboldt University, Berlin, Germany
ABSTRACT
A novel strategy for the molecular identification of fungal agents of onychomycosis (including Trichophyton rubrum) has been designed based on the use of species-specific and universal primers in conjunction with a commercial kit that allows the extraction of DNA directly from the nail specimens. The microsatellite marker T1, which is based on a (GT)n repeat, was applied for the species-specific identification of Trichophyton rubrum. To evaluate how often Scopulariopsis spp. are detected in nail specimens, a second primer pair was designed to amplify specifically a 336-bp DNA fragment of the 28S region of the nuclear rRNA gene of S. brevicaulis and closely related species. Other fungal species were identified using amplification of the internal transcribed spacer (ITS) region of the rRNA gene, followed by restriction fragment length polymorphism analysis or sequencing. In addition, polyacrylamide gel separation of the T1-PCR product allowed subtyping of T. rubrum strains. We studied 195 nail specimens (the "nail sample") and 66 previously collected etiologic strains (the "strain sample") from 261 onychomycosis patients from Bulgaria and Greece. Of the etiologic agents obtained from both samples, T. rubrum was the most common organism, confirmed to be present in 76% of all cases and serving as the sole or (rarely) mixed etiologic agent in 199 of 218 cases (91%) where the identity of the causal organism(s) was confirmed. Other agents seen included molds (6% of cases with identified etiologic agents; mainly S. brevicaulis) and other dermatophyte species (4%; most frequently Trichophyton interdigitale). Simultaneous infections with two fungal species were confirmed in a small percentage of cases (below 1%). The proportion of morphologically identified cultures revealed by molecular study to have been misidentified was 6%. Subtyping revealed that all but five T. rubrum isolates were of the common type B that is prevalent in Europe. In comparison to microscopy and culture, the molecular approach was superior. The PCR was more sensitive (84%) than culture (22%) in the nail sample and was more frequently correct in specifically identifying etiologic agents (100%) than microscopy plus routine culture in either the nail or the strain samples (correct culture identifications in 96% and 94% of cases, respectively). Using the molecular approach, the time for diagnosing the identity of fungi causing onychomycosis could be reduced to 48 h, whereas culture techniques generally require 2 to 4 weeks. The early detection and identification of the infecting species in nails will facilitate prompt and appropriate treatment and may be an aid for the development of new antifungal agents.
INTRODUCTION
Toenail onychomycosis is a fungal infection caused mainly by dermatophytes. Less frequently, yeasts and nondermatophytic molds are also found to be etiological agents of this type of infection. Onychomycosis occurs worldwide and accounts for up to 50% of all nail disorders (2, 7). The prevalence of onychomycosis has been given as 2 to 18% of the whole human population, and it might be even higher (44). The most significant agent is Trichophyton rubrum, with an incidence of more than 90% in Europe (49). Trichophyton interdigitale is less common (17, 18). Nail invasion by nondermatophytic molds is generally considered uncommon. Reported prevalence rates range from 0% to 50%, with studies that carefully investigate all such cases usually showing a prevalence of around 3.5% (21, 26, 54). The fungal species most frequently implicated in onychomycosis caused by molds in temperate parts of the world is Scopulariopsis brevicaulis (15, 16, 18, 50). Most of the nondermatophytic fungi recurrently involved in opportunistic onychomycosis, inclusive of several well-known Scopulariopsis, Fusarium, and Aspergillus species (47), are very common in the environment but remain relatively uncommon as causes of onychomycosis (25).
The incidence of onychomycosis has increased over the last few years in response to the increased population of immunocompromised patients, the widespread use of immunosuppressive chemotherapy and broad-spectrum antibiotics, the expanding number of elderly persons, and the increasing public participation in fitness-related activities (42, 52). Onychomycosis is increasingly recognized as a nontrivial medical disorder that poses various physical, psychosocial, and occupational problems (3).
Treatment of onychomycosis is closely linked to the identity of the causative agent, particularly in terms of whether or not it is a dermatophyte (4, 40, 45). The most commonly used oral antifungals include itraconazole, terbinafine, and fluconazole; these drugs have superseded the traditional therapies based on the use of griseofulvin and ketoconazole. While terbinafine and itraconazole demonstrate efficacy against T. rubrum and T. interdigitale, griseofulvin and fluconazole are ineffective against T. interdigitale and against molds, e.g., S. brevicaulis, that may cause the infection (19, 45).
Current diagnosis and treatment procedures for dermatophyte onychomycosis rely on direct microscopic examination of fungal elements in nail material using 30% KOH, in combination with the identification of the etiological agent based on the examination of its colony and microscopic morphology. In onychomycosis caused by molds, a second, confirmatory sample may also be needed. Patients are often treated as soon as fungal elements are shown to be present microscopically. These methods are low in specificity and may be very time-consuming, because growth, sporulation, and routine physiological testing (e.g., urease enzyme assay) of the fungi involved may take 2 to 4 weeks. In addition, 15 to 50% of microscopically true-positive samples fail to grow a culture (8, 33). Moreover, dermatophyte species show an unusual level of variation in the expression of phenotypic characteristics, e.g., the formation of pigments or conidia (46).
The application of PCR technology directly to clinical specimens would allow early and accurate identification of agents of onychomycosis. This would permit prompt and targeted initiation of antifungal therapy. The present study tested such a direct PCR application with techniques also designed to allow infraspecific subtyping of some of the elucidated etiologic agents.
MATERIALS AND METHODS
Clinical isolates. Two collections of samples were investigated in this study. Nail specimens were collected as subungual scrapings, clippings, or curettings. The first sample (called "strain sample" in Table 1) was a collection of 66 test isolates obtained from serial patients who were microscopically positive and had grown a culture in procedures for diagnosis of onychomycosis. This collection might be biased against T. interdigitale, which is more often associated with microscopy-negative samples than T. rubrum is. The second sample (called "nail sample" in Table 2) was collected from 195 successive patients whose abnormal nails were indicated for dermatologic mycology evaluation. To allow valid statistical testing, specimens from all patients in this series were included in the sample, regardless of whether or not a positive culture or microscopy result was obtained. Of the 261 patients, 101 were from Greece and the remainder were Bulgarian residents. Sixteen patients suffered from suspected fingernail mycosis; the rest were evaluated for toenail onychomycosis.
A single specimen was analyzed from each of the 261 patients. Patients were recalled when a nondermatophyte mold grew in culture, regardless of whether or not a dermatophyte also grew. For those patients, three independent, serial specimens were analyzed.
Routine morphological identification at the test centers. Nail specimens were divided into three portions. The first portion was examined microscopically with 30% KOH for the presence of fungal elements. The second was cultured on Sabouraud glucose agar, containing 10 g peptone, 40 g dextrose, and 15 g agar per liter distilled water, with a final pH of 5.6 and with no antibiotic supplementation (NCIPD Ltd., Sofia, Bulgaria), at 27°C for 4 weeks. Clinical isolates were identified on the basis of phenotypic characteristics of the colony and on the basis of the shapes of macro- and microconidia. Hair perforation testing and tests for urease degradation, which aid distinction of atypical isolates of T. rubrum and T. interdigitale, are not routinely done at the test centers due to economic stringencies. From the third portion of the nail specimen, DNA was extracted as outlined below.
DNA extraction. A minipreparation method to extract DNA from fungal cultures was used, as described previously by Grser et al. (13).
To extract DNA directly from nail material, a commercial kit, the GenomicPrep Cells and Tissue DNA Isolation kit from Amersham Biosciences (Piscataway, NJ), was used. Prior to use of the kit, whole nails and relatively large nail fragments were cut into small pieces with a surgical blade. Nail shavings were processed directly. The manufacturer's instructions were modified as follows. In step 1, to 0.5 to 3 mg of nail material, 300 μl rather than 600 μl of cell lysis solution was added with 5 to 10 mg glass beads (0.5 mm in diameter; BioSpec Products Inc.) was also added. The reaction mixture was vortexed for 1 min. In step 3, nail material was incubated for 1 h in a 65°C water bath following the addition of 3 μl Proteinase K solution (20 mg/ml), and the solution was incubated overnight at 55°C and vortexed again. In step 7, 100 μl of protein precipitation solution was added instead of 200 μl. In step 9, the solution was centrifuged for 5 min instead of 3 min. In step 11, the reaction mixture was left at –20°C overnight. In steps 12 and 14, centrifugation was done for 10 min. The DNA pellet was dissolved in 10 to 30 μl hydration solution, depending on the amount of nail material used at the beginning.
Identification using PCR. (i) Identification of T. rubrum. For the identification of T. rubrum, the following oligonucleotides were used as primers in the PCR: 5'-TGG TCT GGC CTT GAC TGA CC (T1.for) and 5'-GTA AGG ATG GCT AGT TAG GGG G (T1.rev). These primers amplify a 280-bp fragment containing a GT-microsatellite repeat specific for strains of the closely related T. rubrum/T. violaceum clade (38). Briefly, an amplification reaction was performed in a total volume of 35 μl. The PCR mixture contained reaction buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2), 200 μM of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech Inc., Piscataway, NJ), 21 pmol of each primer, 1.75 U of Taq polymerase (AmpliTaq; Applied Biosystems), and 35 ng of template DNA. Samples were overlaid with sterile light mineral oil and amplified through 30 cycles in a thermocycler (Thermoblock, version 2.55b; Biometra TRIO) as follows: initial denaturation for 10 min at 95°C, denaturation for 30 s at 95°C, annealing for 30 s at 60°C, and extension for 45 s at 72°C. This was followed by a final extension step for 3 min at 72°C.
For subtyping strains of T. rubrum, 5 to 10 μl of the PCR product was separated on a polyacrylamide gel (8%) for 7.5 h at constant power (45 W) or for 17 h at 22 W. Following electrophoresis, the gels were silver stained and dried for documentation.
(ii) Identification of the remaining fungal agents of onychomycosis. For identification of the remaining fungal agents of onychomycosis, restriction fragment length polymorphism (RFLP) analysis and sequencing of the internal transcribed spacer (ITS) region of the rRNA gene were applied. For ITS-PCR, the universal primers LSU266 (5'-GCA TTC CCA AAC AAC TCG ACT C) and V9D (5'-TTA CGT CCC TGC CCT TTG TA), amplifying a DNA fragment of about 1,000 bp of the gene, were used (9). The PCR mixture contained reaction buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2), 200 μM of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech Inc., Piscataway, NJ), 50 pmol of each primer, 2 U of Taq polymerase (AmpliTaq; Applied Biosystems), and 50 ng of template DNA. Samples were overlaid with sterile light mineral oil and amplified through 35 cycles in a thermocycler (Perkin Elmer 9600) as follows: initial denaturation for 5 min at 94°C, denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1 min at 72°C. This was followed by a final extension step for 7 min at 72°C. Restriction enzyme analysis of the PCR products was performed using 5 U MvaI enzyme (MBI Fermentas, Amherst, NY) per sample, followed by incubation for 4 h at 37°C. The resulting fragments were electrophoresed through 2% MetaPhor agarose gels (BioWhittaker Molecular Applications Inc.) for 2.5 h at 100 V. A set of reference strains was used for comparison.
Sequence analysis was performed using CEQ dye terminator cycle sequencing with the Quick Start kit and the CEQ 8000 Genetic Analysis System from Beckman Coulter (Fullerton, Calif.).
(iii) Identification of S. brevicaulis and related species. In parallel, a primer pair was developed and tested for the detection of S. brevicaulis and related species. Although, based on a BLAST search via the National Center for Biotechnology Information web site, the primers were predicted to amplify a wide range of species within the class Sordariomycetidae, the only one of these fungi commonly causing onychomycosis is S. brevicaulis. Species rarely causing onychomycosis that would be predicted to be amplified are other Scopulariopsis species (some of them best known by Microascus teleomorph names) and Chaetomium globosum. Based on the large subunit of the rRNA gene, a 336-bp fragment was amplified. The SCOP primer pair (5'-GAA TGC TGC TCA AAA TGG GAG-3' and 5'-CAT TAC GCC AGC ATC CTTG C-3') was selected on the basis of sequences published in GenBank (22). The following PCR conditions were applied: amplification was performed with puReTaq Ready-To-Go PCR Beads (Amersham Biosciences, Piscataway, NJ) in reaction volumes of 25 μl containing 2.5 U puReTaq DNA polymerase and buffer (10 mM Tris-HCl, pH 9.0, at room temperature, 50 mM KCl, 1.5 mM MgCl2), 200 μM of each deoxynucleoside triphosphate, and 15 ng of template DNA. The primers were added, each at a final concentration of 20 pmol. Samples were amplified through 35 cycles in a thermocycler (MJ Research PTC 100) as follows: initial denaturation for 3 min at 94°C, denaturation for 45 s at 94°C, annealing for 1 min at 69°C, and extension for 1 min at 72°C. This was followed by a final extension step for 10 min at 72°C. Five microliters of each reaction was loaded on 1.2% agarose gels and electrophoresed for 1.5 h at 100 V in 0.5x buffer (89 mM Tris-borate, 2.5 mM EDTA, pH 8.3). The gels were stained with ethidium bromide and photographed.
The sensitivity of the SCOP primers was tested with different concentrations of DNA extracted from a known strain of S. brevicaulis (J99079). DNA was serially diluted to give concentrations ranging from 15 ng to 1.5 pg in order to determine the lowest limit of detection.
RESULTS
Conventional identification. In the strain sample (Table 1), all 66 isolates came from microscopy-positive specimens, as mentioned above. In the nail sample, fungal filaments were seen in the KOH preparations in 181 out of 195 specimens (93%), while a culture of a potential agent of onychomycosis grew from 44 specimens (22%; Table 2). T. rubrum made up 83% of isolates from the strain sample and 95% of isolates from the nail sample; when both samples were combined, it made up 88% of isolates. The remaining cultures were mainly T. interdigitale, with S. brevicaulis also being identified on two occasions. Of the two S. brevicaulis isolates found in the nail sample, each was confirmed a posteriori as etiologic by means of later studies on repeat specimens, and one was obtained from a specimen also yielding T. rubrum.
DNA extraction from nails. In order to determine the minimum weight of nail material needed to extract DNA using the GenomicPrep Cells and Tissue DNA Isolation kit (Amersham Biosciences, Piscataway, NJ), 10 nail samples were selected. Each sample was divided into three portions, one containing 0.5 mg of material, one containing 1.0 mg, and one containing 1.5 mg. In addition, 24 nail samples with weights ranging from 2 mg to 4 mg were investigated. These studies established that, for PCR based on the use of T1, a minimal amount of 0.5 mg of nail material was needed to obtain a faint PCR product (data not shown).
Sensitivity and performance of the SCOP primers. The SCOP primers were able to detect as little as 150 pg of template DNA. Given the fact that 10 pg of target DNA generally corresponds to circa 25 fungal cells or CFU per clinical specimens (51), the detection limit of the SCOP assay should correspond to 375 or more cells per clinical specimen. Cultured material of all 13 clinical isolates morphologically identified as S. brevicaulis (Tables 1 and 2) amplified with the SCOP primers. In 6 out of 195 nail specimens (specimens 955H2, 887P3, 1372Gr, 585Gr, 1024Gr, and 1213Gr), a positive amplification with the SCOP primers was obtained. In three cases, these positive amplifications were from nails also positive for T. rubrum, and in three cases only Scopulariopsis signal was amplified, confirmed by culture in one case as S. brevicaulis. All S. brevicaulis amplifications were from specimens positive for fungal elements in direct microscopy, but no structures specific for S. brevicaulis, such as masses of conidia produced in situ, were reported. S. brevicaulis was successfully cultured twice from specimens in the "nail specimen" group; on both occasions, the positive culture was from a nail that also gave a positive amplification and that yielded additional S. brevicaulis cultures in later specimens. In four cases there was positive amplification with the SCOP primers in the absence of a positive culture. Sequences derived from the 336-bp PCR products obtained in each case showed 100% identity with three species that are identical in this region, S. brevicaulis, S. koningii, and Microascus manginii (anamorph: Scopulariopsis candida). Technically, then, the positive amplifications obtained with the SCOP primers could indicate any of these three species; however, S. brevicaulis is much more common in association with nails than either of the other two species.
Identification of other agents of onychomycosis using PCR. The polymorphic microsatellite marker T1 containing a GT repeat motif has been used in this study for the identification of strains and clinical specimens of T. rubrum. Of the 66 isolates for which the culture was used as the template for DNA extraction (Table 1), 55 had been conventionally identified as T. rubrum and 11 as S. brevicaulis. The T1 primer pair designed for the species-specific amplification of isolates of the T. rubrum/T. violaceum clade (38) amplified 51 of them. Four strains (7%) failed. Those four were suspected to have been misidentified and thus were subjected to ITS-PCR. Using RFLP analysis, three (strains 3702Gr, 346Gr, and 5162Gr) were reidentified as T. interdigitale, while one (strain 817P) was identified as Arthroderma benhamiae (anamorph: unnamed, formerly part of Trichophyton mentagrophytes complex sensu lato) (Fig. 1). None of the S. brevicaulis strains had been misidentified morphologically; all of them amplified with the SCOP primer pair.
In the 195 clinical nail specimens used directly as the template for DNA extraction (Table 2), 148 (76%) were amplified with the T1 primers, indicating the presence of T. rubrum. By contrast, only 22% of these specimens had grown a culture. The remaining 47 nails were analyzed using ITS-PCR. In four of them, T. interdigitale (specimens 1052Gr, 7112Gr, 431Gr, and 684H2; Fig. 1) was present. Two of these specimens had yielded a culture that was misidentified as T. rubrum, while two were culture negative. Each of the following species was detected by PCR in one microscopy-positive but culture-negative nail specimen: Fusarium solani (GenBank accession number AF178402; a species complex partially listed in GenBank under the teleomorph-related name Nectria hematococca complex; isolated from our specimen 154Gr; 99.4% similarity over 356 bp with strain NRRL 22354 [NRRL Collection, National Center for Agricultural Utilization Research, Peoria, Ill.]); Fusarium proliferatum (accession number U34558; a member of the Gibberella fujikuroi complex; our specimen 1024Gr; 100% similarity over 518 bp with NRRL 22944); and Aspergillus nidulans (GenBank accession number AF455505; our specimen 7962Gr; 99.5% similarity over 475 bp with strain wb 260 [personal collection of Walter Buzina, Medical University Graz, Austria]; Fig. 1). Results for detection of Scopulariopsis spp. with the SCOP primers are given above.
It was found that 39 specimens did not produce a PCR product with any of the primers used in this study. In 30 of these, structures interpreted as fungal elements were seen in direct microscopy (Table 2). In 7 (23%) of the 30 specimens, the DNA amplification was concluded to be negative due to the presence of substances inhibitory to PCR. PCR inhibition was evidenced for each specimen involved by means of a control reaction in which the DNA from the specimen was spiked with a concentration of control template DNA from a strain known to amplify well. Even serial dilutions of the template DNAs did not change the results (data not shown). Despite the apparent hyphae seen under the microscope, the PCR-inhibited specimens had to be classified as incompletely analyzed.
In the total collection of 195 nail specimens, there were five cases yielding positive PCR evidence for more than one species either occurring in the nail or, for some of the nondermatophytes involved, possibly occurring as a relatively heavy contaminant of the corroded nail surface. A Scopulariopsis sp. was associated with T. rubrum in three cases (and confirmed as S. brevicaulis in one of them) and with F. proliferatum in one, while T. rubrum was associated with T. interdigitale in one.
For the subclassification of T. rubrum isolates into types A and B and for the separation of T. violaceum, PCR products were run on the polyacrylamide gels. The majority of isolates were type B, the type that is predominant in Europe. Only five patients from Greece yielded type A isolates (Fig. 2).
Conventional versus molecular identification and detection. The rate of morphological misidentifications among all cultures studied was 6%. For the most part, T. interdigitale had been misidentified as T. rubrum.
In direct nail analyses, DNA of T. rubrum was detected in 5 (36%) of the 14 specimens that were microscopy- and culture-negative. In 30 of the 138 specimens that were microscopy positive but culture negative (21.7%), no fungal DNA was detected; of the remaining 108 specimens, 103 (74.6% of the 138 specimens considered) yielded positive signal for T. rubrum, 1 yielded signal for T. interdigitale, and 6 yielded a positive amplification for one or more nondermatophytes (one F. proliferatum plus Scopulariopsis sp., one F. solani, one A. nidulans, and three additional Scopulariopsis spp. amplifications, including two from specimens also amplifying for T. rubrum). No specimen yielded a culture without also yielding a positive PCR amplification for the same fungus.
DISCUSSION
In onychomycosis worldwide, two dermatophyte species are most frequently involved as causal agents, namely, T. rubrum and T. interdigitale. The prevalence of these pathogens, however, varies according to geography, climate, and patterns of population migration, and it may differ among regions with similar environmental conditions. The few onychomycosis studies that have been carried out for the Balkan region (27, 39, 41, 48), where the patient population of the present study originated, have shown that the two dermatophyte species predominate, making up 55 to 70% of isolates from nails, while molds and yeasts are less common, growing from 10 to 27% of specimens (but not always rigorously implicated as causal). Variation among countries is evident. For example, in Croatia (48) the most common species was T. interdigitale (55%), whereas in our sample (Bulgaria and Greece) T. rubrum was most frequently isolated (76%) and T. interdigitale made up only 2% of isolates. An earlier study from the Plovdiv region of Bulgaria (where our own Bulgarian collection also originated) detected 83% T. rubrum and 17% T. interdigitale isolates among 130 cases (41). This shows that the incidence of T. interdigitale has dropped since 1965. Our results are therefore in agreement with the global ascendancy of T. rubrum as the predominant etiological agent of onychomycosis. Even using PCR in the present study, nondermatophytic molds (Scopulariopsis spp., Fusarium spp., and A. nidulans) as confirmed or suggestive etiologic agents were detected in only 3% of infections; also, only 3% of infections yielded mixed isolations. Those results are comparable with those of the study done by Saulov in Plovdiv (41), in which 2.5% of onychomycoses were ascribed to molds and 3.3% were classified as mixed infections. Scopulariopsis spp. were not detected in that study. The predominance of the dermatophytes in onychomycosis is very likely due to their broad spectrum of secreted proteases (24) acting as virulence factors enabling the destruction of the nail plate.
In the 13 cases (2 in the nail sample and 11 in the strain sample) where molds such as S. brevicaulis were the only cultures to grow from three serial microscopy-positive specimens, infection by these fungi is very likely. However, all nine cases (including three in which dermatophytosis was confirmed) in which a mold was detected by direct PCR in a case where only a single patient specimen was available are considered not to be authoritatively confirmed. Notwithstanding this cautious interpretation, it should be noted that, as calculated above, probably at least 375 Scopulariopsis sp. cells would need to be present before a positive reaction with the SCOP primers was engendered. At least four out of our nine cases very likely had at least this level of viable Scopulariopsis material present. Thus, though we must refrain from recording these amplifications as fully confirmed cases, the balance of probability suggests that true infection rather than contamination is the most parsimonious explanation for our positive SCOP primer results. This interpretation also fits well with the relatively small number of times the environmentally common S. brevicaulis was detected. Based on the figures given by Gupta et al. (20) in their Table 3, growth in culture of insignificant Scopulariopsis sp. inoculum on nails is nearly 2.5 times as common as growth from a true Scopulariopsis sp. infection, even in an elderly patient population particularly vulnerable to onychomycotic molds. If small numbers of contaminating Scopulariopsis sp. conidia were giving rise to positive amplifications, we would probably have had many more positive SCOP results. Moreover, some of the nononychomycosis-causing molds predicted by BLAST to react with the SCOP primer, such as members of the genera Trichoderma, Lecanicillium, and Coniochaeta, are very common in the indoor environment. If the SCOP primers were detecting background levels of fungal contamination on nail material, the proportion of positive results could be very high indeed.
In terms of firm confirmation, however, our sampling strategy only partly fulfilled the classic evaluation criteria for the acceptance of an infection by a nondermatophytic fungus, in which such a fungus should be isolated from at least two distinct, temporally separated samples taken from the same microscopy-positive nail (47). This criterion, based on the well-supported idea that a pathogen is much more likely than a contaminant to be consistently associated with the nail on separate occasions, works well with conventional studies (47), but the extent to which it is applicable to PCR-based onychomycosis studies remains to be investigated.
Even though PCR may not as yet, pending further testing, allow immediate certainty about whether the molds it detects in microscopy-positive nails are etiologic or contaminating, it may still significantly improve the diagnosis of nondermatophytic onychomycosis. Culture of the initial toenail specimen in proven cases of onychomycosis at best only detects etiologically causal nondermatophytes circa 75% of the time they are present, a rate similar to that obtained with dermatophytes in the initial specimen (47). PCR may be more sensitive than culture in this regard. In the present study, in relation to culture, specific PCR brought about a 200% increase in the detection of Scopulariopsis spp. This may at first seem difficult to explain, since S. brevicaulis grows vigorously in culture and a single viable cell is enough to start a culture, while PCR may require a somewhat larger amount of material as a template. Fungus-killing bacteria, known to occur abundantly in distal areas of onychomycosis-affected nails, may be responsible for the lack of outgrowth in at least some cases (25, 32, 53).
Another benefit of PCR in diagnosis of nondermatophyte mold infections is that it allows better identification of etiologic agents than can be accomplished in culture. For example, the F. solani isolate suggestively associated with positive direct microscopy in one case can be determined through sequencing to be most similar to a rarely isolated genetic subgroup, probably representing an undescribed phylogenetic species, only represented in GenBank by the sequence of an isolate (NRRL 22354) obtained from a teleomorph collected on tree bark in Guyana (36). F. solani is in the process of being broken up into 50 or more phylogenetic species (36); which of the new taxa make up the F. solani complex populations associated with human feet and nails is not yet known. F. proliferatum, recognized here via PCR in a case suggestive of a mixed infection with S. brevicaulis, is also a member of a phylogenetically complex group, the Gibberella fujikoroi complex, containing many anamorphic Fusarium species that are very difficult to identify accurately in culture (37).
The gold standard procedure for diagnosis of fungal nail infections is direct microscopy (KOH preparation) combined with the fungal culture, and the performance of new techniques is generally gauged against this standard. As a "gold standard," KOH and culture is problematic: various studies have shown that 15 to 50% of the microscopy-positive nail specimens do not grow a culture (1, 6, 8, 33). In our nail study (Table 2), the failure of culture was even more severe: a full 72.1% of PCR-confirmed dermatophytosis specimens (i.e., 109 of 151 specimens) did not yield the dermatophyte in culture. This, however, is very unusual, and we note that two studies (11, 47) using long-term patient follow-up as a gold standard to determine the true number of dermatophytosis cases in patient population both found that approximately 75% of initial specimens from true dermatophyte-infected toe onychomycosis grew cultures in the initial sample. When the original data are examined from these studies, our low rate of culture positivity can be confirmed as being strongly statistically different (P < 0.001 by chi-squared test) from the rates seen in these studies. This suggests a source of interference in culture efficiency in our study. Though various factors can contribute to culturing problems, e.g., patients not disclosing recent antimycotic treatment, our tentative conclusion is that the factor most probably responsible was the use of a primary isolation medium free of antibacterial antibiotics. It is important to note that this usage was not part of the study design but rather merely reflected the longstanding and ongoing economic reality of routine isolation at both of the southeastern European institutions involved, as well as at most or all other institutions in the region.
In the present study, direct microscopy showed its normal false-negative rate (filaments not detected in confirmed dermatophytosis cases) of circa 5% (47), in this case 3.3% (5 of 151 confirmed dermatophytoses). The five microscopy-negative, PCR-positive specimens involved made up 36% of the 14 specimens in our study that would have been considered completely negative for mycotic agents in conventional studies. All five of these cases involved T. rubrum as the etiologic agent. (An alternative scenario, that the T. rubrum amplified in these five cases came from nearby infected skin rather than the nails themselves, seems highly unlikely, given the onychomycotic symptomatology of the nails combined with the likelihood that any dermatophytosis of skin adjacent to the nails would probably also affect the nails. Also, infections of more distant areas such as toe webs would probably not yield sufficient contaminating material to cause false positives in nail PCR, given the normal dermatological practice of cleaning away the contaminated superficial surface of infected nails before submitting a sample to the laboratory.) In all other cases in which a known or suggestive etiologic agent was detected by PCR alone, the presence of fungal filaments in direct microscopy would have indicated at least that a mycosis was present. The five microscopy-negative, PCR-positive cases, though, mean that PCR increased the number of confirmed mycoses in this study from 181, as would have been recognized in conventional studies, to 186, an increase of 2.8%. Though this may not seem a high percentage, the high numbers of patients seeking relief from onychomycosis must be kept in mind. Scher (43) showed that in one year alone, 662,000 Americans over the age of 65 consulted a physician for handling of suspected onychomycosis. If 2.8% of this population were to have their mycosis missed on the first examination, this would amount to 18,536 inadequately handled patients over 1 year in just that single demographic group.
In the present study, PCR in direct analysis of nails detected a definite or potentially etiologic fungus in 156 of the 186 patients known by any criterion to have a mycosis (assuming all of the 30 recorded microscopy-positive but PCR-negative samples were correctly read microscopically and that these patients all truly had a mycosis). Its sensitivity, then, measured against the ad hoc standard of its own positive results, plus additional positives obtained only via direct microscopy (no additional positives were elucidated only by culturing), was 83.9%. Importantly, PCR was positive in 100% of cases where a culture was positive, and the positive PCR results for a specimen always corroborated the presence of any species that grew in culture (though this was initially confused by morphological misidentification of a small number of cultures) as well as often revealing some additional species. Due strictly to the fact that repeat specimens could not be obtained (except in two cases) to confirm the significance of cases in which only potentially invasive nondermatophyte molds were PCR detected in microscopy-positive specimens, PCR completely settled the identity of at least one definite etiologic agent in a slightly smaller proportion of probable mycosis cases, i.e., 151 of the 186 cases (81.2%). Of these 151 cases of definite etiologic agent confirmation, 109 (72.2%) were achieved only through PCR, not culture. Of course, this to some extent reflects the unanticipated culture problems with this study discussed above, but it does stress that PCR is capable of taking on the burden of identification when culture is unreliable.
Our use of a commercial kit for the extraction of DNA from nail specimens gave us access to an easy, rapid, and well-standardized method. Although a method for DNA extraction from nails had been described previously (51), its reliability may be problematic due to the involvement of the enzyme lyticase, which is often contaminated with genomic DNA of the species it has been purified from (31). In addition, using the Amersham kit, we avoided steps such as mincing and crumbling the nails and homogenization of the material by shearing in a syringe (51). The kit substitutes overnight proteinase K treatment for the usual procedures for mechanical destruction of the cell integument that becomes involved in fungal DNA extraction from keratinized nails. Supplementing general PCR with the use of T1 as a specific marker, we were able to identify the major causative agent of onychomycosis, T. rubrum, within 2 days in all cases where it occurred, even in numerous specimens not yielding a culture. This decreases enormously the time needed for the species identification of fungi (normally 2 to 4 weeks), which can be problematic due to pleomorphic growth, slow generation time, or difficulties with culture. The early and accurate detection and identification of the infecting species in nails will facilitate prompt and appropriate treatment and may be an aid for the evaluation of new antifungal agents.
In general, identification methods based on the genotype of an isolate are considered more reliable than morphological and physiological identification techniques, and a large number have been applied already to differentiate dermatophytes. For example, based on ribosomal small subunit rRNA gene sequences, a primer system specific for the detection of dermatophytic DNA has been described, though this system is not designed to distinguish individual dermatophyte species (5). Restriction fragment length polymorphism analysis (35), PCR fingerprinting (12), arbitrarily primed PCR (29), random amplified polymorphic DNA analysis (30, 34), or sequencing of the ITS region (13, 14, 23) has been stated to be useful for the identification of species in dermatophytes and other fungi. Some of these methods, however, lack reproducibility; in particular, few of the random amplified polymorphic DNA techniques proposed for fungi in the 1990s are in use today. Many of the previous techniques also lack the convenience of allowing use of DNA extracted directly from nail specimens and, thus, may be delayed at least by the number of days necessary to grow a minimal culture for DNA work (which varies by species but is approximately 4 to 10 days). PCR-RFLP techniques require a permanent set of reference strains for species identification, and hybridization techniques are quite laborious. Sequencing of the ITS would be the gold standard and is not more expensive than identifying T. interdigitale using conventional diagnostics (6.60 versus 6.13 for conventional diagnostics; Table 3 ), which is inclusive of urease test and Trichophyton agar. Specific markers, however, are superior in cases where a limited range of etiological agents needs to be considered, and in particular, a single PCR is sufficient for the diagnosis of T. rubrum (T1-PCR). The cost of this test would be only slightly higher than that of conventional diagnostics when the latter are performed according to recommended standard procedures (25) (2.90 versus 2.30 ; Table 3). The specific microsatellite marker applied in the present study also amplified species that are phylogenetically closely related to T. rubrum, such as T. violaceum (38). However, T. violaceum is only rarely found as an agent of onychomycosis and is uncommon in many parts of the world. In addition, using the intrinsic length polymorphisms of the T1 marker by applying polyacrylamide gel analysis, it is possible to differentiate T. violaceum from T. rubrum and even to subtype isolates of T. rubrum into two epidemiologically distinct groups (38). To avoid the coincidental detection of T. violaceum, we have recently designed and tested a primer pair that is specific for T. rubrum. It amplifies a 100-bp region of the T1 fragment where a polymorphism (A/G) at the 3' end can be used as template for a specific forward primer (unpublished data). Recently, a similarly specific DNA marker (600 bp) has been developed by Liu et al. (28). However, the use of this marker for direct amplification from clinical specimens has never been tested, nor has its use for subtyping T. rubrum isolates. When such a large DNA fragment (600 bp) is used, single-nucleotide polymorphisms can only be distinguished by sequencing. In the gene regions studied so far, T. rubrum isolates have proven to have very few polymorphisms, probably due to the known clonal reproduction and probable recent common ancestry of all strains analyzed to date, including types A and B. Therefore, in general, the greater the length of any gene region used in electrophoretic studies, the higher the risk that any strain-level differences occurring will be overwhelmed by the preponderance of similarities.
Subtyping of 204 isolates of T. rubrum (consisting of 93 from Greece and 111 from Bulgaria) using the T1 marker and polyacrylamide gel electrophoresis detected only types A and B. Types C and D, which correspond to the T. violaceum clade, were not detected. This implies that the probability of misidentifying T. rubrum as T. violaceum in patients with onychomycosis is very low using our techniques, even if the polyacrylamide gel step is not done. Even in parts of Africa where T. violaceum is abundantly present, onychomycosis is a very uncommon manifestation.
Recently, Ohst et al. have shown that type A T. rubrum is widespread in Africa, whereas type B is dominant in Europe (38). In the present sample, only five isolates of type A were detected, all of them in specimens from Greece. In northern Greece, Asian and African type strains (e.g., T. rubrum with granular morphology, consistent with that characteristic of most type A strains, previously sometimes assigned the synonymous name T. raubitschekii or T. fluviomuniense) have been often isolated from persons who had never visited Asia or Africa (10). Northern Greece is part of the south Balkan region, which is located at a crossroads between Asia, Africa, and Europe. Historical human settlement patterns may thus explain the distribution of type A isolates found in our sample.
Though molecular techniques for dermatophyte diagnosis are clearly in development with the aim of ultimately providing new methodologies to all relevant medical services worldwide, it is recognized that the techniques described here will not be practical for a large number of laboratories that are either small scale or very tightly budgeted. They may, however, cross the line of practicability for mid- to high-level reference laboratories, laboratories with preexisting molecular facilities, and laboratories undertaking the further development of these methodologies.
ACKNOWLEDGMENTS
We thank Mariela Hitova, Lilia Zissova, Emil Bardarov, and Grigor Mateev for their assistance in collecting nail specimens. We thank the staff of the Laboratory of Molecular Microbiology, National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria, for their technical assistance.
Funding was provided by the Bulgarian Ministry of Education, Science and Technology, grant MU-L-1202/02, given to V. Kardjeva (maiden name, V. Stavrakieva). Molecular experiments were carried out at the Institute of Microbiology and Hygiene, Department of Parasitology, Charite, Humboldt University, Berlin, Germany, by the first author and were financially supported by that institute. A scholarship was provided to V. Kardjeva by the Deutscher Akademischer Austauschdienst (DAAD).
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