Rapid Identification of Pseudallescheria and Scedosporium Strains by Using Rolling Circle Amplification

Michaela Lackner,a,b,c Mohammad Javad Najafzadeh,d Jiufeng Sun,a,e Qiaoyun Lu,a,f and G. Sybren de Hooga,e,g CBS–KNAW Fungal Biodiversity Centre, Utrecht, The Netherlandsa; Federal Institute for Drugs and Medical Devices (BfArM) Biosafety Laboratory, Bonn, Germanyb; University of Innsbruck, Institute of Microbiology, Innsbruck, Austriac; Department of Parasitology and Mycology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Irand; Department of Dermatology, The Second Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, Chinae; Peking University Health Science Center, Research Center for Medical Mycology, Beijing, Chinaf; and Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlandsg

The Pseudallescheria boydii complex, comprising environmental pathogens with Scedosporium anamorphs, has recently been subdivided into five main species: Scedosporium dehoogii, S. aurantiacum, Pseudallescheria minutispora, P. apiosperma, and P. Downloaded from boydii, while the validity of some other taxa is being debated. Several Pseudallescheria and Scedosporium species are indicator organisms of pollution in soil and water. Scedosporium dehoogii in particular is enriched in soils contaminated by aliphatic hy- drocarbons. In addition, the fungi may cause life-threatening infections involving the central nervous system in severely im- paired patients. For screening purposes, rapid and economic tools for species recognition are needed. Our aim is to establish rolling circle amplification (RCA) as a screening tool for species-specific identification of Pseudallescheria and Scedosporium. With this aim, a set of padlock probes was designed on the basis of the internal transcribed spacer (ITS) region, differing by up to 13 fixed mutations. Padlock probes were unique as judged from sequence comparison by BLAST search in GenBank and in dedi- cated research databases at CBS (Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre). RCA was applied as an in http://aem.asm.org/ vitro tool, tested with pure DNA amplified from cultures. The species-specific padlock probes designed in this study yielded 100% specificity. The method presented here was found to be an attractive alternative to identification by restriction fragment length polymorphism (RFLP) or sequencing. The rapidity (<1 day), specificity, and low costs make RCA a promising screening tool for environmentally and clinically relevant fungi.

uring the last decade, physiological and morphological iden- widely used for identification, i.e., the rRNA gene internal tran- Dtification and screening methods for filamentous fungi have scribed spacer (ITS) region. been supplemented with molecular techniques. Molecular sib- We selected a group of fungal opportunists belonging to the on May 15, 2020 by guest lings and genotypes within a single species are being described, ascomycete genus Pseudallescheria and its anamorph Scedospo- which are morphologically identical but may differ in ecology rium. The P. boydii complex now comprises five species, while the (27), virulence (12), metabolic ability (12), and antifungal suscep- validities of some sibling species are still being debated (3–6). The tibility (1). Strains belonging to siblings within Scedosporium and taxonomic entities differ in ecology and in their ability to cause Pseudallescheria (e.g., Scedosporium dehoogii, Pseudallescheria infections in humans (12). Consistency in these preferences was minutispora) have a “dual ecology,” able to cause human infection abstracted from a large data set representing isolates from hu- on the one hand but usable for bioremediation on the other (22). mans, animals, and the abiotic environment (16). Pseudallesche- Commonly used methods for species distinction today are se- ria/Scedosporium species were chosen as model fungi because the group comprises emerging human opportunists that have been quencing of ribosomal and structural genes, restriction fragment underdiagnosed in patient populations such as those suffering length polymorphism (RFLP), and PCR-based tools. These meth- from cystic fibrosis (CF). At isolation from CF sputum, these fungi ods have been applied to common and clinically important species may be outcompeted by Aspergillus and Candida species, thus re- and genera (10), but for many fungi outside the well-known spe- quiring selective media for their cultural detection (24). Pseudall- cies spectrum, only a limited number of diagnostic tools are avail- escheria species have been recognized as main agents of near- able. In addition, methods may be relatively expensive and time- drowning-related brain infection (13, 31) and are otherwise consuming. Thus, there is a need for techniques that have high involved in a wide array of opportunistic infections in immuno- specificity, reproducibility, and sensitivity, are suitable for high compromised as well as healthy individuals. Strains affiliated with throughput, have low complexity, and are cost-effective. S. dehoogii tend to accumulate in human-dominated environ- A recent method fulfilling these requirements is rolling circle ments, particularly in those with industrial or agricultural pollu- amplification (RCA). RCA is very fast and requires limited infra- structure (33). Thus far, the technique has only occasionally been applied to fungal pathogens (14, 15, 38). Model fungi used in these Received 17 May 2011 Accepted 24 October 2011 papers showed relatively high interspecific diversity, with species Published ahead of print 4 November 2011 being clearly distinct from each other at the molecular level. In the Address correspondence to G. Sybren de Hoog, [email protected]. present paper, we aim to verify whether RCA is applicable for the Copyright © 2012, American Society for Microbiology. All Rights Reserved. distinction of sets of fungal species which differ by only a few doi:10.1128/AEM.05280-11 nucleotides or by a single nucleotide located in a marker gene

126 aem.asm.org 0099-2240/12/12.00 Applied and Environmental Microbiology p. 126–133 Scedosporium Identification Based on RCA

FIG 1 Design of an RCA padlock probe exemplified by the S. aurantiacum (AuraRCA) probe. RCA primer binding sites are in bold capital letters. Linker regions are in bold lowercase letters. Species-specific sites of the RCA padlock probe are underlined and capitalized. Target DNA of S. aurantiacum ITS amplicon is in gray and capitalized. The numbers 68 and 101 indicate the positions where the padlock probe binds to ITS amplicons of S. aurantiacum target DNA (counted from

the first nucleotide of ITS1, excluding gaps). Downloaded from

tion (12). The species are resistant to most commonly used anti- with a delay at 72°C for 7 min. The BT2 fragment of the ␤-tubulin gene, fungal drugs and therefore are refractory to therapy (30). Some known to be usable for distinction of species in Pseudallescheria and Sce- species are difficult to identify (3, 5, 6). The most common species dosporium (3), was sequenced for a random selection of 359 strains rep- of this group, P. boydii and P. apiosperma, are very close in their resenting all species under study by using Bt2a and Bt2b as primers (In- ITS regions, differing only by a few nucleotides. The molecular vitrogen) (7). The annealing temperature was 58°C; sequencing was screening technique used should therefore perform at a low level performed with a BigDye Terminator cycle sequencing kit (Applied Bio- systems) using an ABI Prism 3730XL sequencer. of diversity. http://aem.asm.org/ Phylogenetic reconstruction. One representative of each recognized MATERIALS AND METHODS ITS genotype was used to reconstruct a maximum parsimony tree using the Cipres portal (26). PAUP (Sinauer Associates, Sunderland, MA) was Strains studied. The set of strains analyzed (677 in total) comprised selected with the following parameters: heuristic search and tree 648 isolates of Pseudallescheria and Scedosporium (P. apiosperma [375 bisection-reconnection (TBR), using Scedosporium prolificans as the out- strains], S. dehoogii [110 strains], P. boydii [90 strains], S. aurantiacum group to root the tree. To obtain a maximum parsimony tree, 200 ratchet [37 strains], P. minutispora [14 strains], P. ellipsoidea [1 strain], P. fusoidea [3 strains], and S. prolificans [18 strains]) and 29 distantly replicates were used. Bootstrap values were calculated. The tree was created related strains belonging to the genera Petriella, Lophotrichus, Parasce- with Mega 4.0, and the bootstrap minimal concordance was set to 80.0%. dosporium, and Graphium. Isolates were taken from the collections of (i) Padlock probe design. Padlock probes were designed on the basis of

the Institute for Microbiology, University of Innsbruck, Austria (JK), (ii) 677 ITS sequences of strains of Pseudallescheria and Scedosporium and on May 15, 2020 by guest the Robert Koch-Institute, Berlin, Germany (RKI), (iii) the CBS-KNAW relatives in Petriella, Lophotrichus, Parascedosporium, and Graphium. ITS Fungal Biodiversity Centre, Utrecht, The Netherlands (CBS), (iv) the In- sequences were exported to the automatic alignment package Muscle stitute for Hygiene and Microbiology, Brussels, Belgium (IHEM), and (v) (www.ebi.ac.uk/Tools/muscle/index.html) to identify informative nucle- the Faculty of Medicine, Universitat Rovira i Virgili, Reus, Spain (FMR). otide polymorphisms within and between species, each species containing Strain details are given on the Web page www.scedosporium-ecmm.com its ex-type isolate; the alignment was verified manually. Forty-three geno- under the “Protocol” tab “Supplementary Materials.” The set contained types were detected and trimmed. A single sequence of each genotype was all ex-type strains available from the genera Pseudallescheria and Scedospo- selected to create a genotype alignment file, which was used to design the rium and included reference isolates recommended by Guarro et al. (9). species-specific padlock probes. The ex-type strains of all species analyzed GenBank data were used only if the identity of the isolate could be verified, (see Table 2 below; indicated with T) were among the sequences selected. i.e., when the respective strain was part of the studied set mentioned For each species-specific site, a padlock probe was designed. Padlock above. probes were analyzed for tertiary structure, annealing characteristics, and DNA extraction and identification. DNA extraction and purification palindromes in Primer select (Lasergene software; DNASTAR, Madison, were performed as previously described (20, 29). Briefly, fungal material WI). To enhance specific binding, the 5=-end probe binding arm was ␮ was mechanically disrupted using glass beads (425- to 620- m diameter; constructed with a minimum secondary structure and a melting temper- Sigma, Zwijndrecht, The Netherlands) and vortex adapters for Vortex- ature (T )ofՆ63°C. The ligation temperature was 63°C. Species-specific Genie 2 (MoBio Laboratories, Carlsbad, CA), subsequently purified with m sites are those regions of the padlock probes that bind to the target DNA cetyltrimethyl ammonium bromide (CTAB)-sodium dodecyl sulfate (Fig. 1) (15, 32). To increase specificity, the 3=-end binding arm (Fig. 1) (SDS) buffer (Fluka, Zwijndrecht, The Netherlands). After purification of was designed with a T of between 48 and 53°C (29). More information the lysate, DNA was precipitated with ice-cold isopropanol (Sigma). The m on padlock probe design and structure is provided in Fig. 1 (also see Table DNA was pelleted by centrifugation at room temperature for 10 min at 20,000 ϫ g (Eppendorf C5410, Hamburg, Germany), washed with ice- 2). The 5= terminal end of padlock probe p was phosphorylated (Fig. 1; cold 70% ethanol (Sigma), and air dried. The DNA pellet was eluted in 50 also see Table 2). The species-specific 5= site X (Fig. 1, left, underlined ␮l Tris-EDTA buffer (Sigma) (20, 29). DNA extracts were measured with capital letters) is located downstream of the phosphorylated end, which is a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, connected to the padlock probe core. The 3= end of the padlock probe core Landsmeer, The Netherlands) to determine DNA concentrations. Ribo- is connected to the species-specific site Y (Fig. 1, right, underlined capital somal internal transcribed spacer regions were amplified using the primer letters). Specificity was verified against sequences from the in-house da- pairs ITS4 and ITS5 (Invitrogen, Bleiswijk, The Netherlands) (18, 36). tabase of Pseudallescheria and Scedosporium maintained at CBS for re- Amplification was performed in an ABI PRISM 2720 (Applied Biosys- search purposes and validated by reference to ex-type strains (supplemen- tems, Foster City, CA) thermocycler as follows: 95°C for 4 min, followed tary material, http://www.scedosporium-ecmm.com) and sequences in by 35 cycles consisting of 95°C for 45 s, 52°C for 30 s, and 72°C for 2 min, GenBank.

January 2012 Volume 78 Number 1 aem.asm.org 127 Lackner et al.

Ligation. The ligation master mix consisted of 1 ␮l of purified ITS (Table 2, bold) were detected by RCA; the remaining species, dif- amplicon (2 to 5 pmol/␮l) mixed with 2 U Pfu DNA ligase (Promega, ferentiated by combinations of mutations (Table 2), were also Leiden, The Netherlands) and 10 ␮l of padlock probe solution (200 invariably recognized. All ITS padlock probes thus were found to ␮ pmol/ l padlock probe [Invitrogen] in 20 mM Tris-HCl [pH 7.5] be species specific, detecting all species based on molecular anal- [Sigma], 20 mM KCl [Sigma], 10 mM MgCl [Sigma], 0.1% IGEPAL 2 ysis within Pseudallescheria and Scedosporium, despite low inter- [(octylphenoxy-)polyethoxyethanol] [Sigma], 0.01 mM rATP [Sigma], 1 species diversity and high intraspecies variability. Positive RCA mM dithiothreitol [DTT] [Sigma]). Ligation started with 5 min of prede- naturation at 94°C and then five cycles as follows: 94°C for 30 s, 4 min at reactions were visualized by UV irradiation as ladderlike, strongly 60°C. Normally the RCA reaction can be performed with one denatur- illuminating smears on the gel (Fig. 3). Negative controls were ation cycle followed with one ligation cycle, but 5 cycles were used to strictly without fluorescence. RCA reactions were subsequently improve the yield of RCA products according to the work of Kong et al. performed without a preceding exonucleolytic step. Positive reac- (15). RCA reaction products were visualized on common agarose gels. tions were analogous to those obtained with exonucleolysis. The Exonucleolysis. Unbound padlock probes and ITS templates were concordance of RCA results with expected species identification removed by exonucleolysis in 20-␮l volumes containing 10 U exonuclease based on ITS sequence data was found to be 100%. No false- I and 10 U exonuclease III (New England BioLabs, Leusden, The Nether- negative or false-positive reactions were found with any of the lands) for 30 min at 37°C. Lytic activity was stopped by incubation at 94°C padlock probes; nor were they found with any Pseudallescheria Downloaded from for 30 min. and Scedosporium species (Fig. 3) or any more distantly related RCA reaction. Rolling circle reactions were run in triplicate in total volumes of 25 ␮l with 2 ␮l of the enzyme-treated ligation product as the fungus. The lower detection limit of fungal RCA was established to 7 template. RCA mix contained 4 U Bst DNA polymerase (Promega), 200 be 10 copies when RCA products were visualized on agarose gel. ␮M deoxynucleoside triphosphate mix (Gentaur, Brussels, Belgium), 5 pmol RCA-primer 1b (Invitrogen), and 5 pmol RCA-primer 2c (Invitro- DISCUSSION gen) dissolved in demineralized water. The RCA reaction mix was incu- Modern molecular taxonomy increasingly leads to the recogni- bated for 60 min at 65°C. RCA products were run on 1.0% agarose gels for tion of sibling species which had been indistinguishable with clas- 5 min at 50 V and then for 40 min at 100 V. A reaction without DNA sical tools. Molecular entities deviate from each other by concor- http://aem.asm.org/ served as the negative control, and as the speed marker we used DNA dant mutations in independent genes and may be regarded as Smart Ladder (Eurogentec, Maastricht, The Netherlands). Double- valid species when they have, e.g., different ecological preferences stranded DNA products were visualized on 1.0% agarose (Invitrogen) gels or different physiological abilities (12). Significant taxonomic de- containing ethidium bromide (Sigma) as a smear illuminating under UV exposure, while negative reactions showed no illumination. velopments have taken place in Pseudallescheria during the past 5 Sensitivity and detection limit. ITS amplicons were compared with years. Scedosporium apiospermum was for a long time considered Smart Ladder (Eurogentec, Maastricht, The Netherlands) to determine to be the anamorph of Pseudallescheria boydii. At present, P. boydii DNA concentrations. Copy numbers were calculated with an online tool (anamorph S. boydii) and S. apiospermum (teleomorph P. apio- based on Avogadro’s number (http://www.uri.edu/research/gsc sperma) are judged to represent two different species (5). Pseudall- /resources/cndna.html). For calculation in general, an amplicon length of escheria apiosperma appears to be the prevalent clinical species. 560 bp was assumed. Various genetic clusters close to P. apiosperma have subsequently on May 15, 2020 by guest been described as separate taxa on the basis of multilocus analyses, RESULTS mating experiments, morphology, and physiology (3, 5, 6, 25). Forty-three ITS genotypes were recognized, and one representa- Scedosporium aurantiacum was segregated from P. apiosperma on tive of each (Table 1) was used to create a parsimony tree (Fig. 2). the basis of mating experiments and of marked physiological dif- All species except P. boydii and P. apiosperma were statistically ferences such as the production of an extracellular, canary yellow- supported (bootstrap values of Ͼ80%). Two supported sub- to-reddish pigment on nutrient-rich agar media (3). The molec- groups (␣ and ␤) within S. dehoogii differed by a maximum of 6 ular species Scedosporium dehoogii is particularly found in nucleotides (nt) in ITS, but bilocus analysis (ITS/BT2) suggested aliphatic hydrocarbon-polluted environments (11, 23) (M. Egg- occasional recombination (7 strains [JK291, JK298, JK316, JK162, ertsberger, unpublished data). Pseudallescheria minutispora dif- JK310, FMR 8532, and JK125] out of a total of 69). Within the P. fered from other Pseudallescheria species by morphology and se- apiosperma ITS, several supported subgroups (␣, ␤, and ␥) (Fig. 2) quence data (3). were found, but these were not confirmed in bilocus analysis (ITS/ Given the near absence of classical criteria and the fact that BT2). Hence, the total number of species recognized in this data some of the species, such as P. boydii and P. apiosperma, also have set on the P. boydii complex was five. closely similar ITS sequences, recognition of these species neces- Species-specific padlock probes were designed for all five spe- sitates the use of molecular techniques with high resolution cies above, which were affiliated with Pseudallescheria and Sce- power. In the present study, we used ITS sequences where possible dosporium (Table 2). For P. minutispora, the padlock probe (Min- as a basis for RCA, because in Pseudallescheria and Scedosporium uRCA) was based on a single, unique transversion, while for P. this region can reliably be amplified. Moreover, no paralogues are boydii (BoyRCA), a 1-nucleotide indel was the unique feature (Ta- amplified with this region. All accepted Pseudallescheria and Sce- ble 2, bold). All remaining padlock probes (AuraRCA, dosporium species can be distinguished by ITS sequencing, al- Deho␣RCA, Deho␤RCA, ProRCA, and ApioRCA) were based on though the difference between P. boydii and P. apiosperma is very two or more nucleotides. The high intraspecific ITS sequence vari- small, with a minimum ITS distance of 0.3%. Two species (P. ability of S. dehoogii required two padlock probes. RCA reactions minutispora, P. boydii) were recognizable by one conserved nucle- were performed with each species listed in Table 3: P. boydii, P. otide difference, whereas the remaining taxa were distinguishable apiosperma, S. dehoogii, P. minutispora, and S. aurantiacum, with by combinations of mutations (Table 2). S. prolificans included for comparison. Rolling circle amplification is very economical since it can be The nucleotides characterizing P. boydii and P. minutispora performed under isothermal conditions in a water bath. Because

128 aem.asm.org Applied and Environmental Microbiology Scedosporium Identification Based on RCA

TABLE 1 List of 43 strains representative of ITS genotypesa Species Strain name Origin Source P. angusta CBS 116894 Thailand Soil CBS 254.72 T, Petriellidium angusta USA Sewage IFM 52860 China Clinical isolate P. apiosperma JK254 Austria Soil JK32 Austria Soil 335 B Unknown Unknown JK71 Austria Soil 97 compost Unknown Compost CBS 100395 Germany BAL fluid CBS 117407 T, P. apiosperma Brazil Keratitis sample CBS 118233 Austria Near-drowning patient IFM 52928 China Necrosis tissue of eye

IHEM 14754 France CF sputum Downloaded from IP 141182 Unknown Mycetoma IHEM 21890 Austria Soil Wb 300 France Clinical isolate P. boydii CBS 695.70 T, Acremonium suis Ukraine Nasal cavity of a pig CBS 101.22 T, A. boydii USA Human mycetoma CBS 116423 Canada HC soil CBS 116895 Spain Cerebral abscess CBS 116897 Spain Otitis sample CBS 117390 Zaire Forest soil

CBS 117412 Spain Pleural liquid http://aem.asm.org/ CBS 119707 China Necrosis tissue of eye CBS 501.98 Unknown Unknown IFM 52864 Unknown Unknown P. minutispora JK191 Austria Soil JK305 Austria Soil CBS 116595 Belgium HC soil CBS 116911 T, P. minutispora Spain River sediment S. aurantiacum JK34 Austria Soil CBS 101726 The Netherlands Mud

CBS 117423 France CF sputum on May 15, 2020 by guest IHEM 116910 T, S. aurantiacum Spain Ulcer of ankle IHEM 21895 Austria Soil S. dehoogii JK190 Austria Soil CBS 101721 The Netherlands Unknown CBS 101724 The Netherlands Unknown CBS 117406 T, S. dehoogii Spain Garden soil CBS 499.90 Unknown Mud S. prolificans CBS 116907 France CF sputum CBS 452.89 France Human blood CBS 467.74 T, L. prolificans Belgium Soil a Abbreviations: T, ex-type strain; BAL, bronchoalveolar lavage; CF, cystic fibrosis; HC, hydrocarbon-contaminated. of its simplicity, speed, robustness, cost-effectiveness, and speci- RCA has been applied to the identification of clinically relevant ficity, the technique is promising for epidemiological high- fungi (14, 15, 19, 38). Circularized oligonucleotides, also called throughput investigations. In the present study, RCA was proven padlock probes, have been demonstrated to be more sensitive for to provide reproducible results down to single nucleotides (Table the detection of target sequences than conventional primers (10), 2, P. minutispora and P. boydii, bold). Functional padlock probes although gel-based detection of RCA still needs some detection were designed on the basis of alignments validated by inclusion of optimalization (28). Zhou et al. (38) published primers for the sequences derived from ex-type material of each species and ver- identification of S. prolificans and the former S. apiospermum ified against all currently known genotypes. Each species was char- (sensu lato) based on sequences deposited in GenBank. Their S. acterized by one or more fixed mutations that were absent from prolificans padlock was confirmed in our validated database. It is related organisms. The underlying database contained sequences predictive, since this species has a low intraspecific variability (30, with a wide geographical representation and with a wide range of 31). However, with P. boydii/S. apiospermum, Zhou et al. (38) sources of isolation. Our alignment contained 677 ITS sequences disregarded the recent molecular subdivision of this complex (3, derived from strains of Pseudallescheria and Scedosporium (http: 5, 6). The padlock probes of their “S. apiospermum” (sensu lato) //www.scedosporium-ecmm.com), as well as from more distantly proved to be specific neither for the newly circumscribed species related taxa belonging to the genera Petriella, Graphium, Lopho- P. apiosperma (anamorph S. apiospermum) nor for P. boydii (ana- trichus, Parascedosporium, and Petriellopsis. morph S. boydii). This leads us to the conclusion that for reliable

January 2012 Volume 78 Number 1 aem.asm.org 129 Lackner et al. Downloaded from http://aem.asm.org/

FIG 2 Parsimony tree constructed using the ITS genotype set of Pseudallescheria and Scedosporium species (n ϭ 43). Intraspecific ITS variability is reflected by the number of genotypes per species and their mutual distance. Bootstrap-supported subgroups of S. dehoogii are marked with ␣ and ␤, while supported ␣ ␤ ␥ subgroups within P. apiosperma are marked with , , and . on May 15, 2020 by guest diagnostics of less common fungi, such as Scedosporium species, tilocus analyses rather than on rRNA gene ITS alone. After com- GenBank is insufficient because the majority of accessions have parison with BT2 data, all species of Pseudallescheria and Scedospo- not been updated according to the latest taxonomic standards. rium proved to be recognizable with ITS. Internal transcribed Molecular species are currently segregated on the basis of mul- spacer is more convenient for high-throughput purposes than

TABLE 2 Details of species-specific sites and padlock probe core of Pseudallescheria and Scedosporium RCA probesa Species-specific site of padlock probe (length)b Padlock probe name or primer Organism or RCA primer sequence Site X at 5= end (18-22 nt) Site Y at 3= end (13-16 nt) Organisms S. aurantiacum AuraRCA p-TCCTCGCGGAGAGGGGGCG GTCCATCGTCTCCCC P. minutispora MinuRCA p-GAAACAGAGGTTCGAGGGTTG CTGAGCTTCGCTAAC S. dehoogii ␣ Deho␣RCA p-CGCTGACCGCGCCGCCGAG CGTCGTCCGTGGGGA S. dehoogii ␤ Deho␤RCA p-GGGCGCTGACCGCGCCGC GGCCGTCGTCCTTTC S. prolificans ProRCA p-AAACCTAGGCTCGAGGGTTGAA TCCTTGGGCTTGTAA P. apiosperma ApioRCA p-CATCGTCCTCTTYTCAGAGGGG CCGGCGGGAGGGG P. boydii BoyRCA p-GGGTCGCGAAGACTCGCCGTA TTTCAGGGCCTACGGA RCA primers RCA primer 1 ATGGGCACCGAAGAAGCA RCA primer 2 CGCGCAGACACGATA

Padlock probe corec: X-gatcaTGCTTCTTCGGTGCCCATtaccggtgcggatagctacCGCGCAGACACGATAgtcta-Y a Fig. 1 shows a schematic representation of the AuraRCA probe. Bold bases represent single-nucleotide polymorphisms (SNPs). b The species-specific sites of padlock probes hybridize with the target DNA. The bold nucleotides are conserved, unique single-nucleotide mutations. The species-specific sites were 36 nt long or shorter. c The padlock probe core consists of the linker region (lowercase bold letters) and the RCA primer binding sites (capital letters). The padlock probe core comprises 63 nt and represents the backbone of the probe that inhibits interlooping of DNA.

130 aem.asm.org Applied and Environmental Microbiology Scedosporium Identification Based on RCA

TABLE 3 Evaluation of specificity of padlock probesa Result for indicated padlock probe Positive Deho␣RCA- Amplicon source species control AuraRCA MinuRCA Deho␤RCA mix ProRCA ApioRCA BoyRCA S. aurantiacum (n ϭ 10) ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ P. minutispora (n ϭ 4) ϩϪ ϩ Ϫ Ϫ Ϫ Ϫ S. dehoogii (n ϭ 12) ϩϪ Ϫ ϩ Ϫ Ϫ Ϫ S. prolificans (n ϭ 8) ϩϪ Ϫ Ϫ ϩ Ϫ Ϫ P. apiosperma (n ϭ 8) ϩϪ Ϫ Ϫ Ϫ ϩ Ϫ P. boydii (n ϭ 7) ϩϪ Ϫ Ϫ Ϫ Ϫ ϩ a Padlock probes shown are AuraRCA, MinuRCA, Deho␣RCA-Deho␤RCA Mix, ProRCA, ApioRCA, and BoyRCA. Positive results (ϩ) are padlock probes amplifying target DNA; negative results (Ϫ) are combinations of padlock probes and nontarget amplicons. Positive controls were generated with primers ITS4 and ITS5. Downloaded from BT2, because it is amplified unambiguously. In the present study, tients). Given the differential ecological preferences of species we therefore maintained a preference for ITS as a diagnostic (12), these differences are likely to exist. marker. Scedosporium strains are slow-growing fungi compared to The design of a generic ITS primer for the entire genus Pseu- other saprobic opportunists such as Aspergillus (7 to 10 days ver- dallescheria proved to be impossible, but the large-subunit (LSU) sus 2 to 4 days, respectively, to produce sporulating colonies). This primers proposed by Wedde et al. (34) were verified and found to is a possible explanation of the underdiagnosis of Scedosporium be usable. LSU primers were checked in the CBS in-house research species in patients with cystic fibrosis, where they colonize the database of 677 sequences, with the result that all Pseudallescheria lungs concomitantly with A. fumigatus (17). Culture-independent and Scedosporium strains could be detected and no mismatches detection is needed since Scedosporium strains have reduced sus- with other fungi were found. http://aem.asm.org/ ceptibilities to many antifungal agents. Early diagnosis is the key to For screening and epidemiological purposes, time- and cost- successful antifungal treatment (8, 21, 35). At present, no compre- effectiveness are essential. Thus far, RCA has not been tested di- hensive data exist on mortality rates attributable to the newly cir- rectly on clinical specimens. To assess the sensitivity in clinical cumscribed Pseudallescheria and Scedosporium species in different practice, further studies are needed. RCA in Pseudallescheria com- patient groups (e.g., transplant recipients or cystic fibrosis pa- prises the following workload: (i) DNA extraction from sample on May 15, 2020 by guest

FIG 3 Proof of species specificity of RCA padlock probes and intraspecific variation of RCA response. 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 (ProRCA, S. prolificans; MinuRCA, S. minutispora; AuraRCA, P. aurantiacum; Deho␣RCA, S. dehoogii ␣; Deho␤RCA, S. dehoogii ␤; BoyRCA, S. boydii). Lanes: L, DNA Smart Ladder; 1 to 7, RCA reaction with DNA of P. aurantiacum (JK226) (lane 1), P. minutispora (JK305) (lane 2), S. dehoogii ␣ (JK16) (lane 3), S. dehoogii ␤ (JK63) (lane 4), S. prolificans (CBS 114.90) (lane 5), P. apiosperma (CBS 117407) (lane 6), and P. boydii (JK36) (lane 7); 8, negative control (reaction without DNA). Reactions for testing intraspecificity within P. apiosperma and ITS RCA padlock probe ApioRCA: lane A, P. apiosperma (CBS 116899); lane B, P. apiosperma (CBS 119697); lane C, P. apiosperma (CBS 101718); lane D, P. apiosperma (dH 18395); lane E, P. apiosperma (IHEM 21799); lane F, P. apiosperma (CBS 118234); lane G, P. apiosperma (CBS 116779); lane H, negative control (reaction without DNA).

January 2012 Volume 78 Number 1 aem.asm.org 131 Lackner et al. pure cultures (60 to 180 min depending on extraction method), iospermum and Pseudallescheria boydii and the proposed new species Sce- (ii) ITS amplification (60 to 120 min, depending on number of dosporium dehoogii. J. Clin. Microbiol. 46:766–771. amplification cycles), (iii) ligation of padlock probes (less than 15 6. Gilgado F, Gené J, Cano J, Guarro J. 2007. Reclassification of Graphium tectonae as Parascedosporium tectonae gen. nov., comb. nov., Pseu- min), (iv) exonucleolytic reaction (60 min), (v) RCA reaction (60 dallescheria africana as Petriellopsis africana gen. nov., comb. nov. and min), and (vi) electrophoresis (15 to 45 min). RCA thus takes Pseudallescheria fimeti as Lophotrichus fimeti comb. nov. Int. J. Syst. Evol. between 4.5 h and 8 h. We proved that exonucleolysis can be Microbiol. 57:2171–2178. omitted because background signals caused by nonligated DNA 7. Glass NL, Donaldson GC. 1995. Development of primer sets designed for were insignificantly higher than with inclusion of the lytic re- use with the PCR to amplify conserved genes from filamentous ascomy- cetes. Appl. Environ. Microbiol. 61:1323–1330. action, as was also found by Tong et al. (32). The exclusion of 8. Gonzalez GM, et al. 2003. Activity of posaconazole against Pseudallesche- an exonuclease reaction shortens the procedure by a further 60 ria boydii: in vitro and in vivo assays. Antimicrob. Agents Chemother. min. 47:1436–1438. Concentrations of amplicons were not determined quantitatively 9. Guarro J, et al. 2006. Scedosporium apiospermum: changing clinical spec- prior to RCA reaction. This influenced yield of RCA products as can trum of a therapy-refractory opportunist. Med. Mycol. 44:295–327. 10. Harun A, et al. 2009. Genotyping of Scedosporium species: a review of be seen on agarose gel (Fig. 3, lanes B, F, and G). In these lanes, the

molecular approaches. Med. Mycol. 47:406–414. Downloaded from yield was lower than expected, but the reaction was clearly different 11. Janda-Ulfig K, Ulfig K, Cano J, Guarro J. 2008. A study of the growth of from that seen for the negative control (lane H). The intensity of Pseudallescheria boydii isolates from sewage sludge and clinical sources on reactions can also be influenced by the quality of the padlock probes, tributyrin, rapeseed oil, biodiesel oil and diesel oil. Ann. Agric. Environ. e.g., the presence of nonphosphorylated probe material competing Med. 15:45–49. 12. Kaltseis J, Rainer J, De Hoog GS. 2009. Ecology of Pseudallescheria and with phosphorylated probes. Since RCA is based on the principle of Scedosporium species in human-dominated and natural environments hybridization and ligation, limitations similar to those encountered and their distribution in clinical samples. Med. Mycol. 47:398–405. in using these techniques may be expected. 13. Kantarcioglu AS, Guarro J, De Hoog GS. 2008. Central nervous system In conclusion, RCA is a very fast (less than 1 working day), infections by members of the Pseudallescheria boydii species complex in specific (down to the single-nucleotide level), and economical (no healthy and immunocompromised hosts: epidemiology, clinical charac- teristics and outcome. Mycoses 51:275–290. http://aem.asm.org/ additional equipment required) tool for fungal screening (2, 37), 14. Kaocharoen S, et al. 2008. Hyperbranched rolling circle amplification as but it is less useful for the identification of single strains when a rapid and sensitive method for species identification within the Crypto- padlock probes are not in stock. The method may be an attractive coccus species complex. Electrophoresis 29:3183–3191. alternative to currently available PCR- and RFLP-based screening. 15. Kong F, et al. 2008. Rapid identification and differentiation of Trichophy- Potential areas of application are in clinical microbiological assess- ton species, based on sequence polymorphisms of the ribosomal internal transcribed spacer regions, by rolling-circle amplification. J. Clin. Micro- ment of CF patients whose lungs are recurrently colonized by Sce- biol. 46:1192–1199. dosporium species, but also in environmental microbiology, where 16. Lackner M, Poeder R, Horré R. 2008. Culture characteristics of Scedosporium species are differentially associated with environments Pseudallescheria/Scedosporium strains. Mycoses 52:402–403. polluted by aliphatic and aromatic hydrocarbons. Another advantage 17. Nagano Y, et al. 2008. Comparision of techniques to examine the diver-

sity of fungi in adult patients with cystic fibrosis. Med. Mycol. 48:116–176. on May 15, 2020 by guest of RCA is that it can be performed under isothermal conditions with 18. Najafzadeh MJ, et al. 2009. Genetic diversity and species delimitation in minimal reagents and no generation of false-positive results, which the opportunistic genus Fonsecaea. Med. Mycol. 47:17–25. may be encountered in PCR-based assays (33). 19. Najafzadeh MJ, Sun J, Vicente VA, De Hoog GS. 2011. Rapid identifi- cation of fungal pathogens by rolling circle amplification (RCA) using ACKNOWLEDGMENTS Fonsecaea as a model. Mycoses. 54:e577–e582. 20. Najafzadeh MJ, et al. 2010. Fonsecaea nubica sp nov., a new agent of We thank Bert Gerrits van den Ende and Regine Horré for their sugges- human chromoblastomycosis revealed using molecular data. Med. Mycol. tions and comments on the manuscript. We thank the reviewers for their 48:800–806. suggestions and comments to improve the manuscript. We are indebted 21. Odds FC, et al. 1998. Evaluation of possible correlations between anti- to Ferry Hagen, Rolf Boeston, Corné Klaassen, and Jacques Meis for pro- fungal susceptibilities of filamentous fungi in vitro and antifungal treat- viding DNA extracts. ment outcomes in animal infection models. Antimicrob. Agents Che- The work of Mohammad Javad Najafzadeh was financially supported mother. 42:282–288. by Mashhad University of Medical Sciences, Mashhad, Iran. 22. Prenafeta-Boldú FX, Summerbell R, De Hoog GS. 2006. Fungi growing All experiments on living material of Scedosporium and Pseudallesche- on aromatic hydrocarbons: biotechnology’s unexpected encounter with biohazard? FEMS Microbiol. Rev. 30:109–130. ria were performed according to the International Biosafety policies (bio- 23. Rainer J, De Hoog GS. 2006. Molecular taxonomy and ecology of Pseu- safety level 2). The authors report no conflicts of interest. The authors dallescheria, Petriella and Scedosporium prolificans (Microascaceae) con- alone are responsible for the content and writing of the paper. taining opportunistic agents on humans. Mycol. Res. 110:151–160. 24. Rainer J, Kaltseis J, De Hoog GS, Summerbell RC. 2008. Efficacy of a REFERENCES selective isolation procedure for members of the Pseudallescheria boydii 1. Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA. 2005. Aspergillus complex. Antonie Van Leeuwenhoek 93:315–322. lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot. Cell 25. Santos AL, Bittencourt VC, Pinto MR, Silva BA, Barreto-Bergter E. 4:625–632. 2009. Biochemical characterization of potential virulence markers in the 2. Demidov VV. 2002. Rolling-circle amplification in DNA diagnostics: the human fungal pathogen Pseudallescheria boydii. Med. Mycol. 47:375–386. power of simplicity. Expert Rev. Mol. Diagn. 2:542–548. 26. Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algo- 3. Gilgado F, Cano J, Gené J, Guarro J. 2005. Molecular phylogeny of the rithm for the RAxML Web servers. Syst. Biol. 57:758–771. Pseudallescheria boydii species complex: proposal of two new species. J. 27. Sudhadham M, et al. 2008. The neurotropic black yeast Exophiala der- Clin. Microbiol. 43:4930–4942. matitidis has a possible origin in the tropical rain forest. Stud. Mycol. 4. Gilgado F, Cano J, Gené J, Serena C, Guarro J. 2009. Different virulence 61:145–155. of the species of the Pseudallescheria boydii complex. Med. Mycol. 47: 28. Sun J, et al. 2011. Single-nucleotide polymorphism (SNP) based diagnos- 371–374. tics of Penicillium marneffei by rolling circle amplification (RCA). 5. Gilgado F, Cano J, Gené J, Sutton DA, Guarro J. 2008. Molecular and Mycoses. 54:e751–e759. phenotypic data supporting distinct species statuses for Scedosporium ap- 29. Sun J, Najafzadeh MJ, Vicente VA, Xi L, De Hoog GS. 2010. Rapid

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