Identification and Typing of Isolates of Cyphellophora and Relatives by Use of Amplified Fragment Length Polymorphism and Rolling Circle Amplification

Abstract

The species diversity and identification of black fungi belonging to Cyphellophora and Phialophora, which colonize and infect human skin and nails, were studied using amplified fragment length polymorphism (AFLP). A total of 76 Cyphellophora and Phialophora isolates were evaluated, and their delimitation was compared to earlier studies using multilocus sequencing. The results of the AFLP analysis and sequencing were in complete agreement with each other. Seven species-specific padlock probes for the most prevalent species were designed on the basis of the ribosomal DNA internal transcribed spacer region, and identification of the respective species could easily be achieved with the aid of rolling circle amplification.

INTRODUCTION

Black yeasts and their filamentous relatives are known to be involved in a wide diversity of clinical pictures, ranging from superficial cutaneous to invasive and disseminated, potentially fatal infections in healthy individuals (1, 2). Their environmental counterparts usually live in extreme or toxic environments (3–5). However, only recently we began to realize that black yeast-like fungi are all around in outdoor and indoor, man-made environments, such as railways, bathrooms (6, 7), steam baths (8), and dishwashers (9). They are frequently overlooked because they grow slowly, require dedicated isolation methods, and are difficult to diagnose by morphology.

Cyphellophora and its relatives in Phialophora are filamentous members of the group forming a well-supported clade within the order Chaetothyriales (10, 11). Clinically the group is homogeneous because all species are recovered regularly as superficial colonizers or as causes of local invasive disease of human skin and nails (12). The species Cyphellophora laciniata, C. pluriseptata, and C. suttonii thus far have exclusively been isolated from humans (13–16). Phialophora europaea accounted for 26.9% of positive cultures with black yeasts in dermatological specimens in Denmark (12). The clinical manifestations of P. europaea infections were described as hyperkeratosis (17) or as asymptomatic colonization (18), but in the absence of precise case studies, the role of Cyphellophora and Phialophora species in infection remains unclear. A better understanding of the fungi concerned requires molecular techniques for detection, diagnostics, and epidemiology.

In present study, the diversity of 76 Cyphellophora and Phialophora isolates from the CBS reference collection is evaluated using amplified fragment length polymorphism (AFLP) fingerprinting. AFLP detects genomic restriction fragments by PCR amplification and has been shown to be a powerful method to discriminate between species at a high resolution (19, 20) and supports species delimitations in black yeast-like fungi. Subsequently, rolling circle amplification (RCA) is developed as a sensitive, rapid, and cost-effective technique to identify the cutaneous Cyphellophora and Phialophora species. RCA is an isothermal amplification method which has been applied for molecular diagnosis of microbial human pathogens (21–23). We developed seven RCA padlock probes for the main species in the clade of cutaneous agents of Cyphellophora and Phialophora.

MATERIALS AND METHODS

Isolates.

A total of 76 isolates of Cyphellophora and Phialophora used in this study are summarized in Table 1. Cultures are maintained in the reference collection of the Centraalbureau voor Schimmelcultures (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands). Cultures were grown on oatmeal agar (OA) (Difco, Brunschwig, Amsterdam, The Netherlands) at 24°C for 1 week and identified down to the species level by partial sequencing of the internal transcribed spacer (ITS) region, the DNA-dependent RNA polymerase II largest subunit (RPB1), the partial β-tubulin gene (BT2), and the D1/D2 region of the nuclear large subunit ribosomal DNA (LSU rDNA) gene for a selection of the strains (10).

Table 1.

Isolation data of tested Cyphellophora and Phialophora strains^a

Species	CBS no.	Other reference no.	Source	Host	Origin
C. guyanensis	CBS 124764T	CPC 13412	Eucalyptus	Plant	Australia
C. guyanensis	CBS 125751	dH 20458	Roof of patient's house	Environment	Brazil
C. guyanensis	CBS 125756	dH 20483	Roof of patient's house	Environment	Brazil
C. guyanensis	CBS 126014	dH 20465	Tucum palm	Plant	Brazil
C. guyanensis	CBS 126020	dH 20526	Rotting wood	Environment	Brazil
C. guyanensis	CBS 126026	dH 21344	Tucum palm	Plant	Brazil
C. guyanensis	CBS 126728	dH 21164	Wooden fence	Environment	Brazil
C. guyanensis	CBS 126863	dH 21173	Shell of coconut babaçu	Plant	Brazil
C. guyanensis	CBS 126868	dH 21293	Babassu palm stem	Plant	Brazil
C. guyanensis	CBS 126888	dH 21191	Thorn tucum palm	Plant	Brazil
C. guyanensis	CBS 126890	dH 21197	Fence	Environment	Brazil
C. guyanensis	CBS 127018	dH 21171	Roof of house	Environment	Brazil
C. laciniata	CBS 174.79	dH 15469	Skin (foot)	Human	Netherlands
C. laciniata	CBS 190.61T	dH 15498	Skin	Human	Switzerland
C. laciniata	CBS 239.91	dH 15600	Skin	Human	Netherlands
Cyphellophora sp.	CBS 112.94	dH 15288	Human sputum	Human	Netherlands
C. pauciseotata	CBS 284.85T	dH 15679	Skin (hand)	Human	Netherlands
Cyphellophora cf. pluriseptata	CBS 285.85	dH 15681	Toenail	Human	Netherlands
C. pluriseptata	CBS 286.85T	dH 15683	Toenail	Human	Netherlands
C. pluriseptata	CBS 109633	dH 12325	Skin	Human	Germany
C. suttonii	CBS 449.91T	dH 15893	Dog ear	Mammal	USA
C. vermispora	CBS 227.86	dH 15571	Hordeum	Plant	Germany
C. vermispora	CBS 228.86T	dH 15574	Triticum	Plant	Germany
C. vermispora	CBS 122852		Skin (foot)	Human	Netherlands
P. ambigua	CBS 235.93T	dH 15593	Toenail	Human	Netherlands
P. ambigua	CBS 124682	dH 17057	Toenail	Human	Denmark
P. europaea	CBS 124187	dH 20187	Bathroom	Environment	Netherlands
P. europaea	CBS 129.96	dH 10389	Skin (toe)	Human	Germany
P. europaea	CBS 218.78	dH 15563	Nail	Human	Netherlands
P. europaea	CBS 656.82	dH 16135	Nail	Human	France
P. europaea	CBS 831.91	dH 16273	Nail	Human	Netherlands
P. europaea	CBS 100413	dH 11172	Unknown	Unknown	Unknown
P. europaea	CBS 101466T	dH 11284	Skin (foot)	Human	Netherlands
P. europaea	CBS 102391	dH 11436	Skin	Human	Netherlands
P. europaea	CBS 109045	dH 15217	Nail	Human	Canada
P. europaea	CBS 109500	dH 12024	Nail	Human	Unknown
P. europaea	CBS 109793	dH 12399	Toenail	Human	Unknown
P. europaea	CBS 120389	dH 16666	Nail	Human	Denmark
P. europaea	CBS 120392	dH 16697	Skin	Human	Denmark
P. europaea	CBS 120393	dH 16687	Toenail	Human	Denmark
P. europaea	CBS 123226	dH 16655	Unknown	Human	Denmark
P. europaea	CBS 123234	dH 17039	Toenail	Human	Denmark
P. europaea	CBS 123240	dH 17019	Skin (foot)	Human	Denmark
P. europaea	CBS 123256	dH 17013	Nail	Human	Denmark
P. europaea	CBS 123259	dH 17078	Toenail	Human	Denmark
P. europaea	CBS 123412	dH 17489	Toenail	Human	Denmark
P. europaea	CBS 123416	dH 17492	Toenail	Human	Denmark
P. europaea	CBS 123422	dH 17500	Toenail	Human	Denmark
P. europaea	CBS 123425	dH 17478	Toenail	Human	Denmark
P. europaea	CBS 123430	dH 17501	Skin (foot)	Human	Denmark
P. europaea	CBS 123434	dH 17051	Nail	Human	Denmark
P. europaea	CBS 123979	dH 17474	Toenail	Human	Denmark
P. europaea	CBS 124186	dH 20185	Bathroom	Environment	Netherlands
P. europaea	CBS 124273	dH 17037	Nail	Human	Denmark
P. europaea	CBS 124676	dH 16679	Skin (foot)	Human	Denmark
P. europaea		dH 11478	Unknown	Human	Germany
P. europaea		dH 12320	Nail	Human	Germany
P. europaea		dH 12323	Nail	Human	Germany
P. europaea		dH 12327	Nail	Human	Germany
P. europaea		dH 12330	Nail	Human	Germany
P. europaea		dH 13359	Toenail	Human	USA
P. europaea		dH 13370	Skin	Human	USA
P. europaea		dH 13467	Unknown	Human	Unknown
P. europaea		dH 17452	Toenail	Human	Denmark
P. europaea		dH 20210	Bathroom flask	Environment	Netherlands
P. reptans	CBS 113.85T	dH 15292	Food	Environment	Sweden
P. reptans	CBS 152.90	dH 15424	Nail	Human	Netherlands
P. reptans	CBS 458.92	dH 15901	Skin	Human	Netherlands
P. reptans	CBS 101467	dH 11283	Skin (toe)	Human	Netherlands
P. reptans	CBS 110814	dH 15281	Carwash water	Environment	Germany
P. reptans	CBS 120903	dH 12534	Drinking water, biofilm	Environment	Germany
P. reptans	CBS 123271	dH 17088	Toenail	Human	Denmark
P. reptans	CBS 120912	dH 17748	Ice water	Environment	Netherlands
P. oxyspora	CBS 416.89	dH 17023	Skin	Human	Denmark
P. oxyspora	CBS 698.73T	dH 21366	Decaying leaf	Plant	Sri Lanka
P. oxyspora	CBS 124686	dH 17079	Toenail	Human	Denmark

^aCBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC, culture collection of P. W. Crous, housed at CBS; dH, G. S. de Hoog private collection.

DNA extraction and identification.

Approximately 1 cm² mycelium was transferred to a 2-ml Eppendorf tube containing 490 μl 10% cetyltrimethylammonium bromide (CTAB) buffer and 6 to 10 acid-washed glass beads (diameter, 1.5 to 2 mm; Sigma). Ten microliters of proteinase K was added and vortexed for 10 min, and the mixture was incubated at 60°C for 60 min. Subsequently, 500 μl chloroform-isoamylalcohol (24:1) was added, incubated for 30 min on ice water, and centrifuged for 10 min at 14,000 rpm. The supernatant was transferred to a new tube with 225 μl 5 M NH₄-acetate, incubated on ice water for 30 min, and centrifuged again for 10 min at 14,000 rpm. The supernatant was transferred to another Eppendorf tube with 0.55 volume of isopropanol, mixed carefully, and spun for 5 min at 14,000 rpm. The pellet was washed with ice-cold 70% ethanol, and after drying at room temperature, it was resuspended in 50 μl Tris-EDTA (TE) buffer. For some strains, DNA was prepared with an UltraClean microbial DNA isolation kit (MoBio, Carlsbad, CA) according to the manufacturer's instructions. DNA quality and quantity were verified on agarose gel and by a NanoDrop ND-1000 spectrophotometer using ND-1000 v. 3.3.0 software (Coleman Technologies, Wilmington, DE). DNA extracts were stored at −20°C prior to use.

AFLP.

The AFLP procedure was performed using established procedures (24). Analyses were performed with 100 to 200 ng DNA. Five microliters of DNA was added to 15 μl of restriction and ligation mixture containing 1 U of T4 DNA ligase buffer (Promega, Leiden, The Netherlands), 50 pmol of HpyCH4 IV adapter, 50 pmol MseI adapter, 2 U of HpyCH4 IV (New England BioLabs, Beverly, MA), and 2 U of MseI (New England BioLabs). The restriction and ligation mixture was incubated for 1 h at 20°C and diluted five times with 10 mM Tris-HCl (pH 8.3) buffer. Adapters were made by mixing equimolar amounts of complementary oligonucleotides (5′-CTCGTAGACTGCGTACC-3′ and 5′-CGGGTACGCAGTC-3′ for HpyCH4 IV; 5′-GACGATGAGTCCTGAC-3′ and 5′-TAGTCAGGACTCAT-3′ for MseI) and heating to 95°C, subsequently cooling slowly to ambient temperature. One microliter of the diluted restriction and ligation mixture was used for amplification in a volume of 25 μl under the following conditions: 25 pmol HpyCH4 IV primer with one selective residue (underlined) (5′-Flu-GTAGACTGCGTACCCGTC-3′), 25 pmol MseI primer with four selective residues (underlined) (5′-GATGAGTCCTGACTAATGAG-3′), 0.2 mM each deoxynucleoside triphosphate, and 1 U of Taq DNA polymerase (Roche Diagnostics, Almere, The Netherlands) in 1× reaction buffer containing 1.5 mM MgCl₂. Amplification was done as follows. After an initial denaturation step for 4 min at 94°C in the first 20 cycles, a touchdown procedure was applied: 15 s of denaturation at 94°C, 15 s of annealing at 66°C, with the temperature for each successive cycle lowered by 0.5°C, and 1 min of extension at 72°C. Cycling was then continued for a further 30 cycles, with an annealing temperature of 56°C. After completion of the cycles, incubation at 72°C for 10 min was performed before the reaction mixtures were cooled to room temperature. The amplicons were then combined with the ET400-R size standard (GE Healthcare, Diegem, Belgium) and analyzed on a Mega BACE 500 automated DNA platform (GE Healthcare) according to the manufacturer's instructions. Profiles were inspected visually and were also imported in BioNumerics v. 5.1 software (Applied Maths, Sint-Martens-Latem, Belgium) and analyzed by the unweighted-pair group method using average linkages (UPGMA) with the Pearson correlation coefficient. The analysis was restricted to DNA fragments ranging from 80 to 250 bp.

RCA.

Twenty-one isolates of Cyphellophora and its relatives in isolates of Phialophora were studied with RCA, including Cyphellophora guyanensis (n = 3), C. laciniata (n = 3), C. pluriseptata (n = 2), C. cf. pluriseptata (n = 1), C. vermispora (n = 3), Phialophora europaea (n = 3), P. oxyspora (n = 3), and P. reptans (n = 3) (Table 1). For the selection of padlock probes, sequences of ITS regions of 101 strains of Cyphellophora and relatives from the CBS reference collection were aligned and adjusted manually using BioNumerics v. 4.61 (Applied Maths) to identify informative nucleotide polymorphisms. Seven padlock probes targeting the ITS region were designed and ordered from Invitrogen, Inc. (Breda, The Netherlands) (Table 2). In order to optimize binding efficiency to target DNAs, the padlock probes were designed with minimum secondary structure and with the midpoint temperature (T_m) of the 5′-end probe binding arm close to or above ligation temperature (63°C; see below). To increase its discriminative specificity, the 3′-end binding arm was designed with a T_m of 10 to 15°C below ligation temperature. The linker regions of each Cyphellophora species-specific probe were taken from Zhou et al. (23), and the 5′ and 3′ binding arms were designed in this article (Table 2). Sequences of the two primers used for RCA and the oligonucleotide padlock probes are listed in Table 2. Padlock probe ligation was as follows: 1 μl of an ITS amplicon was mixed with 2 U Pfu DNA ligase (Promega, Leiden, The Netherlands) and 0.1 μM padlock probe in 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl₂, 0.1% Igepal, 0.01 mM rATP, and 1 mM dithiothreitol (DTT), with a total reaction volume of 10 μl. Padlock probe ligation was conducted with one cycle of denaturation for 5 min at 94°C, followed by five cycles of 94°C for 30 s and 4 min ligation at 63°C. To remove unligated padlock probe and ITS templates, exonucleolysis was performed in a 20-μl volume containing 10 U each of exonuclease I and III (New England BioLabs, Hitchin, United Kingdom) for 30 min at 37°C, followed by 3 min at 94°C to inactivate the exonuclease reaction. The RCA reaction was performed as follows: 2 μl of ligation product was used as the template for RCA reaction in a 50-μl volume containing 8 U Bst DNA polymerase (New England BioLabs), 400 μM deoxynucleoside triphosphate (dNTP) mix, and 10 pmol of each RCA primer (Table 2). Probe signals were amplified by incubation at 65°C for 60 min, and accumulation of double-stranded DNA products was visualized on a 1% agarose gel to verify the specificity of probe-template binding. Positive reactions showed a ladder-like pattern, whereas negative reactions showed a clean background. For direct visual detection of DNA in the RCA detection mixtures, 1 μl of a 10-fold diluted original SYBR green I (Cambrex BioScience, Workingham, United Kingdom) was added to the reaction tubes and visualized on a UV transilluminator (Vilber Lourmat, Marne-la Vallée, France).

Table 2.

Rolling circle amplification padlock probes and padlock probe-specific primers used in this study

Organism	Padlock probe or RCA primer name	Sequencea
	RCA1	5′-ATGGGCACCGAAGAAGCA-3′
	RCA2	5′-CGCGCAGACACGATA-3′
C. guyanensis	GuyaRCA	5′-P-TACAAGAGTTTGTGGTTGGGGgatcatgcttcttcggtgcccatt acgaggtgcggatagctaccgcgcagacacgatagtctaAACTCAGACGACACGTTTAAG-3′
C. laciniata	LaciRCA	5′-P-TGCGGGGCGGCGGGCCTgatcatgcttcttcggtgcccatt acgaggtgcggatagctaccgcgcagacacgatagtctaCCCGGCGGCCACCCCT-3′
C. pluriseptata	PlurRCA	5′-P-GCGGCGGGCCTGCCGAAGgatcatgcttcttcggtgcccatt acgaggtgcggatagctaccgcgcagacacgatagtctaCCCGGTGAGGGGGAGGTT-3′
C. vermispora	VermRCA	5′-P-GCTCCTCGCGGGGCGGCgatcatgcttcttcggtgcccatt acgaggtgcggatagctaccgcgcagacacgatagtctaGGACCCCCGGCGGCT-3′
P. europaea	EuroRCA	5′-P-GGACCCCCGGCGGCCTCgatcatgcttcttcggtgcccatt acgaggtgcggatagctaccgcgcagacacgatagtctaGGGCCCGGGGTCCTTGC-3′
P. oxyspora	OxysRCA	5′-P-CCAGCGGGCCTGCCGAAGCgatcatgcttcttcggtgcccatt acgaggtgcggatagctaccgcgcagacacgatagtctaCCGGGCCCCAAAGGGACA-3′
P. reptans	ReptRCA	5′-P-GTCCAACAACAAGCCGGGCTgatcatgcttcttcggtgcccatt acgaggtgcggatagctaccgcgcagacacgatagtctaCGCGAAGCTCCCGCCGG-3′

^aP at the 5′ end of the probe indicates 5′ phosphorylation. The underlined uppercase sequences are the species-specific binding arms of the padlock probes. The padlock probe core consisting of the linker region and the RCA primer binding sites is shown in lowercase letters, linking to the 5′ (italic letters) and 3′ (roman letters) binding arms of the probes.

RESULTS

Distinction of species within the cutaneous group of agents in Cyphellophora and Phialophora (“europaea clade”) (11) is based on multilocus sequence typing (MLST) data provided previously by Feng et al. (10). Dendrograms derived from the AFLP banding patterns of strains analyzed were generated by the UPGMA algorithm (Fig. 1). All profiles contained multiple visibly strong and weak bands between 80 and 250 bp. The AFLP fingerprints of the strains studied were divided into 12 clusters. At a 40% similarity cutoff level, the profiles matched with existing species as previously delimited on the basis of MLST data. At this cutoff level, AFLP profiles deviated significantly between species. Within most species, the AFLP banding patterns were relatively uniform, with subclusters being formed by the presence or absence of a limited number of bands (Fig. 1). The clusters of C. guyanensis, C. laciniata, and P. reptans were, however, heterogeneous; all strains being different from each other without recognition of a substructure. Strains of C. guyanensis were united at a similarity level of 40%, and those of C. laciniata were at a level of 70%. Strain CBS 285.85, doubtfully assigned to C. pluriseptata by Feng et al. (10), had a profile deviating from all strains analyzed, and the same holds true for an unidentified Cyphellophora strain, CBS 112.94.

Fig 1.

Dendrogram of AFLP fingerprints on a collection of 76 Cyphellophora and Phialophora isolates. Strain names and CBS numbers are shown on the right. Each species and cluster is marked with different color boxes. The scale bar on the left indicates the percentage similarity.

Products of the RCA reaction were visualized by electrophoresis on 1% agarose gels. Positive responses showed ladder-like patterns, whereas with negative results, the background remained clean. Positive responses of RCA proved to be highly specific for all species defined by sequence data. Species-specific probes were designed for seven Cyphellophora and Phialophora species, and these were all correctly identified. No cross-reaction was observed between the species studied (Fig. 2 and 3). The concordance of RCA results and identification with MLST and AFLP was 100%. Three available single strains representing the species Cyphellophora pauciseptata (CBS 284.85), C. suttonii (CBS 449.91), and Phialophora ambigua (CBS 235.39), as well as a more distantly related, cutaneous agent of black yeast-like fungi, Cladophialophora immunda (CBS 834.96), were used for comparison and yielded negative results with the seven padlock probes designed in this study (data not shown).

Fig 2.

Gel electrophoresis of species specificity of RCA padlock probes. Amplification and subsequent fluorescent banding were seen only with appropriate template-probe mixtures (empty lanes denote the absence of signals with unmatched template-probe mixtures). The species-specific probes are labeled as listed in Table 2 (GuyaRCA, C. guyanensis; LaciRCA, C. laciniata; PlurRCA, C. pluriseptata; VermRCA, C. vermispora; EuroRCA, P. europaea; OxysRCA, P. oxyspora; and ReptRCA, P. reptans). Lane M, DNA smart ladder; lanes 1 to 7, C. laciniata CBS 190.61^T, C. vermispora CBS 228.86^T, C. pluriseptata CBS 286.85^T, C. guyanensis CBS 124764^T, P. reptans CBS 113.85^T, P. oxyspora CBS 698.73^T, and P. europaea CBS 101466^T, respectively; and lane 8, negative control (reaction without DNA).

Fig 3.

Gel electrophoresis of intraspecific variation of RCA response. Lane M, DNA smart ladder; lanes 1 to 3, positive RCA reactions with corresponding probes; and lane 4, negative control (reaction without DNA). C. laciniata, lanes 1 to 3 are CBS 190.61^T, CBS 174.79, and CBS 239.91, respectively. C. vermispora, lanes 1 to 3 are CBS 228.86^T, CBS 227.86, and CBS 122852, respectively. C. pluriseptata, lanes 1 to 3 are CBS 286.85^T, CBS 109633, and CBS 285.85, respectively. P. europaea, lanes 1 to 3 are CBS 101466^T, CBS 129.96, and CBS 120392, respectively. P. reptans, lanes 1 to 3 are CBS 113.85^T, CBS 120903, and CBS 152.90, respectively. P. oxyspora, lanes 1 to 3 are CBS 698.73^T, CBS 416.89, and CBS 124686, respectively. C. guyanensis, lanes 1 to 3 are CBS 124764^T, CBS 125756, and CBS 126014, respectively.

DISCUSSION

Identification of black yeast-like fungi is presently based on sequencing rDNA ITS, since most species are indistinguishable morphologically and physiologically. The taxonomy of the genus Cyphellophora, and related Phialophora species composing the “europaea clade” (11) within the Chaetothyriales, has recently been reviewed (10). Eleven species were confirmed by MLST using ITS, LSU, BT2, and RPB1 as markers. The delimitations matched with AFLP profiles, which were united in clearly circumscribed clusters with very little corresponding bands between species. Species distinguished by MLST and AFLP were Cyphellophora laciniata, C. vermispora, C. pluriseptata, C. suttonii, C. fusarioides, C. pauciseptata, C. guyanensis, Phialophora ambigua, P. oxyspora, P. reptans, and P. europaea. There was good agreement between the MLST and AFLP genotypes, and both methods allowed unambiguous discrimination among related species.

Population genetic studies of medically important fungi commonly manifest evidence of both clonal propagation and recombination (25–27). Fingerprints obtained in the species most commonly isolated from human skin within the group under study, Phialophora europaea, showed a profile with nearly monomorphic bands. A small number of variable bands composed subgroups (Fig. 1). The level of uniformity and the partitioning of genetic diversity suggest a clonal reproduction in P. europaea. The spread of pathogenic fungi with a prevalently clonal reproduction structure has been attributed to the migration of infected human hosts (28). For P. europaea, it has been hypothesized that environmental isolates are taken up by humans on moisturized skin in the bathroom (7). Three strains from bathrooms, CBS 124186, CBS 124187, and dH 20210, have AFLP patterns similar to those of clinical isolates, confirming this hypothesis. Water may contribute to the dispersal of clonal lineages (26, 28). In contrast, the molecular variation of the plant-associated species C. guyanensis yielded highly polymorphic bands, suggesting a high evolutionary rate.

Though AFLP and MLST are useful techniques for taxonomy and population genetics, they have limited applicability in an epidemiological investigation because of the relatively high cost of multilocus sequencing (29). Furthermore, the results of AFLP fingerprints are difficult to objectively interpret due to minor band variations which cause problems with long-term reproducibility and interlaboratory comparisons (24). For clinical practice, a simple and robust diagnostic method is required. RCA has been applied to the identification of diverse human-pathogenic fungi (21, 30, 31). According to previous studies, the method proved to be easily operated, rapid, cost-effective, sensitive, and specific. In the present study, we evaluated an identification method based on RCA enabling rapid detection, with specificity down to single nucleotide differences. We developed seven padlock probes on the basis of the ITS region to identify the most frequent species of cutaneous black fungi, viz., Cyphellophora laciniata, C. vermispora, C. pluriseptata, C. guyanensis, Phialophora oxyspora, P. reptans, and P. europaea. Several of these species have also been found in indoor wet cells (6, 7). The probes allowed species-specific detection of the target groups, while cross-reactivity was observed neither between tested species nor with more distantly related species of the genus Cladophialophora. The amplification product can be visualized by agarose gel electrophoresis but can also be visualized in gel-free systems using fluorescence staining of the amplified product by SYBR green in combination with a UV transilluminator (32). Judging from these results and given the simplicity of the method, it is our impression that RCA is well suited for screening large numbers of samples for routine testing in laboratories. This may help to underline the clinical significance of black fungi, which currently are often discarded as supposed contaminants (12), leaving the etiology in symptomatic patients unresolved.