Molecular Detection of Human Fungal Pathogens Olivaceous Green to Olivaceous Gray with Age; Reverse Is Dark

Dongyou Liu

Contents 18.1 Introduction...... 145 18.1.1 Classification and Morphology...... 145 18.1.2 Clinical Features...... 146 18.1.3 Diagnosis...... 147 18.2 Methods...... 148 18.2.1 Sample Preparation...... 148 18.2.1.1 Procedure for Endophthalmitis Samples...... 148 18.2.1.2 Procedure for Keratitis Samples...... 148 18.2.1.3 DNA Extraction...... 148 18.2.2 Detection Procedures...... 149 18.2.2.1 Standard PCR and Sequencing Analysis of ITS Region...... 149 18.2.2.2 Real-Time PCR and Sequencing Analysis of ITS Region...... 149 18.3 Conclusion...... 149 References...... 150

18.1 Introduction Nimbya, Pithomyces, Pyrenochaeta, Stagonospora, Stemphylium, Ulocladium, and Unifilum. In turn, the Pyrenochaeta is dark-walled, coelomycete genus in the order genus Pyrenochaeta contains eight recognized spe- Dothideales that produces asexual fruit bodies during its cies: Pyrenochaeta acicola, Pyrenochaeta gentianicola, life cycle. Members of this genus are widely distributed in Pyrenochaeta inflorescentiae, Pyrenochaeta lycopersici, the environment, soil, wood, and plant debris and are also Pyrenochaeta nobilis, Pyrenochaeta romeroi, Pyrenochaeta encountered as plant pathogens. Several Pyrenochaeta spe- terrestris, and Pyrenochaeta unguis-hominis, in addi- cies such as P. romeroi and P. mackinnonii are involved as tion to six unassigned species [1]. The teleomorphs of the agents of chronic, suppuratives, and subcutaneous infections Pyrenochaeta genus are found in the genus Herpotrichia in immuno-competent patients after traumatic implantation, (obsolete synonyms: Herpotrichiopsis and Lasiophoma) and ultimately leading to mycetoma. In addition, Pyrenochaeta possibly also in the genus Leptosphaeria, both of the family species are occasionally implicated in onychomycosis and Leptosphaeriaceae [2,3]. keratitis as well as deep, non-mycetomatous infections. Pyrenochaeta spp. are saprophytic fungal organisms Although Pyrenochaeta is a relatively infrequent cause that inhabit the soil and plant debris, particularly in tropi- of human diseases in comparison with black yeast genera cal and subtropical areas. Many Pyrenochaeta spp. are Cladophialophora, Exophiala, and Fonsecaea, in the order pathogenic to plants, and several are among the rare causes Chaetothyriales, their similarity in morphological, biologi- of human infections. The human pathogenic Pyrenochaeta cal, and clinical terms make correct identification of these species include Pyrenochaeta keratinophila, Pyrenochaeta organisms essential in order to implement effective control mackinnonii, Pyrenochaeta romeroi (obsolete synonym: and prevention strategies. Phlenodomus avramii), and Pyrenochaeta unguis-hominis [3]. Pyrenochaeta romeroi and Pyrenochaeta mackinnonii have been isolated from mycetoma, containing soft, irregu- 18.1.1 Classification and Morphology lar, and black grains with a subhyaline center. A number of The genus Pyrenochaeta is a dematiaceous (dark- isolates previously recognized as Madurella grisea have also walled) filamentous fungus belonging to the mito- been reidentified as Pyrenochaeta romeroi [4]. Pyrenochaeta sporic Pleosporaceae group, family Pleosporaceae, unguis-hominis has been isolated from the infected nails of order Pleosporales, subclass Pleosporomycetidae, class some cases. Pyrenochaeta keratinophila is a new species Dothideomycetes, subphylum Pezizomycotina, phylum that was recently isolated from corneal scrapings of a case of Ascomycota, kingdom Fungi. The mitosporic Pleosporaceae keratitis in Spain [3]. group covers 13 genera: Alternaria, Dendryphiella, Pyrenochaeta colonies grow moderately rapidly and Dendryphion, Drechslera, Embellisia, Exserohilum, appear flat, woolly to cottony, white initially and becoming

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© 2011 by Taylor & Francis Group, LLC 146 Molecular Detection of Human Fungal Pathogens olivaceous green to olivaceous gray with age; reverse is dark. cells (12–18 × 2–3.5 μm) are phialidic, mostly cylindri- Hyphae are septate, hyaline to subhyaline. Pycnidia (singular cal. Conidia (2–4 × 1–2 μm) are whitish in mass, ellipsoi- pycnidium, which is a round or flask-shaped fruiting body dal, wide, straight or slightly curved, hyaline, and smooth containing conidia) are globose to flask shaped, ostiolate, walled [2]. brown to black, with setae (rigid hair located on the pycnidia) Although P. keratinophila and P. unguis-hominis have arising from their upper portion. Phialides arise from the similar conidiophores and conidia, they can be distinguished inner lining of the pycnidia. Conidia (2–4 × 1–2 μm) are one by the color of the colonies and the location of the setae on celled, oval to cylindrical, hyaline, and straight or slightly the pycnidia. P. keratinophila colonies on OA are gray oli- curved [5,6]. vaceous to greenish olivaceous, while P. unguis-hominis At the species level, Pyrenochaeta keratinophila colonies colonies are brown vinaceous to fawn. The pycnidial setae of reach a diameter of 24–30 mm on oatmeal agar (OA) at 20°C P. keratinophila are scarce and placed mainly near the ostiole, for 10 days, with even margin, and are colorless, flat; immersed while those of P. unguis-hominis are usually more abundant mycelium is pale gray olivaceous to greenish olivaceous, and more dispersed over the entire surface of the pycnidium. aerial mycelium is concolorous, diffuse, woolly, floccose; P. keratinophila also produces conidia from the mycelium, a reverse is olivaceous gray to olivaceous black. Colonies reach feature unknown in P. unguis-hominis or any other species a diameter of 18–23 mm on malt extract agar at 20°C for 10 of the genus Pyrenochaeta. P. mackinnonii is distinguished days, with even margin and buff color; immersed mycelium from P. keratinophila by having more restricted, raised, or is olivaceous black or brown vinaceous; aerial mycelium is wrinkled colonies. P. romeroi is distinguished by its grayish- dense, felty to woolly, pale to olivaceous gray; reverse is dark sepia to fuscous-black colonies and the presence of discrete hazel. Superficial pycnidia are abundant. On OA, pycnidia conidiogenous cells [3]. (100–400 μm in diameter) are olivaceous brown to almost black, globose or flask shaped, single or confluent, with one 18.1.2 Clinical Features to three ostioles (10–25 μm in diameter), displaying hyaline, thick-walled periphyses; pycnidial walls of textura angula- The term “dematiaceous fungi” refers to a heterogeneous ris comprise cells (4–9 mm in diameter) with dark-brown group of fungal organisms with black filaments (dematiaceae) intercellular material. The outer surface of the pycnidial wall that may cause opportunistic superficial or deep infections displays scarce, brown, slightly roughened, septate setae in humans, producing a diverse range of clinical syndromes (20–35 × 2.5–4 μm), with a blunt apex, mostly positioned (collectively known as phaeohyphomycosis, chromoblasto- near the ostiole. Conidiogenous cells (12–23 × 2–3.5 μm) mycosis, and mycetoma). To date, about 130 species belong- arise from the entire inner surface of the pycnidial wall and ing to 70 dematiaceous fungal genera have been shown to are rarely discrete, ampuliform to doliform, mostly cylindri- induce these clinical diseases. cal, and integrated in conidiophores, which are branched at Mycetoma is a highly debilitating disease that is char- the base, acropleurogenous (i.e., having terminal and lateral acterized by a subcutaneous mass with multiple sinuses apertures), phialidic, with a distinct periclinal thickening and draining pus and grains (<0.5–2 mm in diameter), which sometimes with a short collarette. In older cultures, conidiog- may appear white (i.e., white-grain mycetoma), black (black- enous cells often show several distinct percurrent prolifera- grain mycetoma), yellow, or red. This disease can be caused tions. Conidia (2–4 × 1–2 μm) are whitish in mass; ellipsoid, by fungi (eumycetoma) or actinomycetes (actinomycetoma). straight, or slightly curved; hyaline, continuous, smooth, While eumycetoma usually produces either white or black with granular contents or few guttules; and rounded at both grains, actinomycetoma may show white, yellow, or red ends. Phialoconidia arising directly from solitary apertures grains [7]. in the aerial mycelium in 3-week-old cultures are initially The most common fungal species involved in white-grain indistinguishable from conidia originating from pycnidia but eumycetoma include Acremonium (Fusarium) falciforme, later become inflated unilaterally, globose or subglobose, Acremonium kiliense, Acremonium recifei, Cylindrocarpon 3–4 μm in diameter, pale brown, and slightly thick walled. destructans, Fusarium moniliforme, Fusarium solani, Chlamydospores-like structures (8–10 μm in diameter) also Neotestudina rosatii, and Pseudallescheria boydii, whereas form on old OA cultures and are terminal, hyaline, globose, common fungal species responsible for black-grain eumyce- smooth, and thick walled. Molecular data suggest that the toma are Exophiala jeanselmei, Madurella grisea, Madurella closest sexual state of P. keratinophila is Leptosphaeria mycetomatis, Leptosphaeria tomkinsii, Leptosphaeria sene- (family Leptosphaeriaceae) [3]. galensis, Pyrenochaeta mackinnonii, Pyrenochaeta romeroi, Pyrenochaeta unguis-hominis pycnidia (100–400 μm and Phlenodomus avramii. in diameter) are olivaceous brown to black, globose or As saprophytes commonly occurring in soil and plants, flask shaped, with one to three ostioles, and with several Pyrenochaeta spp. may gain entry to human hosts through setae positioned near the ostiole. The pycnidial wall is of direct traumas by a plant or a soiled object. With kerato- textura angularis with dark brown intercellular material. mycosis, a number of factors may predispose the hosts to The conidiophores emerging from all over the inner sur- Pyrenochaeta and other fungal infections. These include face of the pycnidial wall are branched at the base, bearing chronic ocular surface disease, dry eye, contact lens wearing, terminal and lateral conidiogenous cells. Conidiogenous atopic disease, topical steroid use, long-term treatment with

© 2011 by Taylor & Francis Group, LLC Pyrenochaeta 147 broad-spectrum antibiotics, and trauma (particularly with Ferrer et al. [2] described a Pyrenochaeta unguis- vegetable material or soil) [8]. In addition, patients with sup- hominis-related case of keratitis in a 77-year-old diabetic pressed immune functions are vulnerable to Pyrenochaeta woman with pain and decreased vision in her left eye. Slit and other fungal diseases [9,10]. lamp examination showed conjunctival hyperemia, periph- First identified in 1959, Pyrenochaeta romeroi has been eral corneal pannus, corneal edema, and a central corneal reported in mycetoma cases involving mainly immunosup- ulcer (4 mm in diameter) with white stromal infiltrates pressed individuals [11–13]. In recent years, it has been inferiorly. Corneal scrapings were plated on Columbia agar shown that besides eumycetoma, P. romeroi may also be plates supplemented with 5% sheep blood, Columbia choc- responsible for non-mycetoma phaeohyphomycosis. Girard olate agar, MacConkey agar, thioglycolate broth, and brain et al. [14] reported a case of non-mycetoma deep cutaneous heart infusion broth (BioMérieux); and incubated at 37°C. infection due to Pyrenochaeta romeroi in an immunocom- Corneal scraping and biopsy taken from the ulcer edge promised 45-year-old man who originated from Senegal. were also inoculated onto Sabouraud dextrose agar with The patient had a multibacillary leprosy treated by sequential chloramphenicol and incubated at 30°C. Direct examina- antibiotics for more than 20 years, and presented with non- tion of the corneal scrapings and biopsy using Gram and inflammatory, painful and deep lesions of both lower limbs calcofluor white stains showed the presence of numerous reminiscent of cold abscesses. Clinical examination revealed septate and branched hyphae. After 6 days, a filamentous a large subcutaneous, flaccid lesion of the lateral side of the fungus grew on chocolate agar, and a panfungal polymerase left leg, with an occasional purulent white-yellow discharge chain reaction (PCR) generated a 532 bp amplicon, which through superficial sinus tracts, a 2 cm large nodule of the showed 86% homology to Leptosphaeria strain. The fun- right tibial crest and two crusted papulo-nodules of the left gus was subcultured on malt extract agar and oatmeal agar foot. X-rays showed no bone alteration underlying the cuta- (30 g oat flakes, 1 g MgSO4, H 1.5 g K 2PO4,5 1 g agar, 1 L neous lesions, while ultrasound examination of the left leg tap water) at 20°C in the dark. After 1 week of incubation, uncovered a 20 mm long, 5 mm large, and 6 mm thick cav- pycnidia typical of the coelomycetous genus Pyrenochaeta ity with multiple internal septa, extending to the subcutane- developed, and the fungus was identified as Pyrenochaeta ous fat but without obvious involvement of the underlying unguis-hominis. muscle or bone. A deep biopsy sample from the larger cuta- Recently, Verkley [3] described the first case of neous lesion showed a chronic granulomatous dermal infil- Pyrenochaeta keratinophila-associated keratitis in a trate with giant cells and microabscesses and the presence of 77-year-old diabetic woman in Spain. The patient presented septate filaments scattered within the infiltrate upon periodic with conjunctival hyperaemia, peripheral corneal pannus, acid-Schiff (PAS) staining. Direct examination of purulent corneal edema, and a central ulcer with white infiltrates in her material from leg sinus tract showed no granules but fun- left eye. Calcofluor white staining of corneal biopsy revealed gal, septate filaments. Culture of the lesion material grew a fungi invading the cornea. Culture of the corneal biopsy on fungus, which was identified as P. romeroi. After surgical several media grew a fungus that was identified morpho- excision, drainage of the largest abscess, treatment with oral logically as a Pyrenochaeta species. Sequencing analysis of itraconazole (100 mg daily) and anti-bacillary antibiotics (clo- the ITS region in the rRNA gene showed it belonged to an fazimine, rifampicine, dapsone), all cutaneous lesions slowly unknown new species Pyrenochaeta keratinophila. Despite resolved within 1 month with minimal scarring. Itraconazole treatment with topical natamycin and oral ketoconazole for was interrupted after 4 months, and no relapse occurred dur- 1 month with considerable lesion improvement, the cornea ing a 1-year follow-up. could not be saved and the patient underwent an optical pen- In a separate study, Badali et al. [15] documented another etrating keratoplasty. case of non-mycetomatous infection (subcutaneous phaeohyphomycotic cyst) due to Pyrenochaeta romeroi in a 18.1.3 Diagnosis 45-year-old Indian female. The patient presented with a ver- rucous plaque and a swelling (30 mm in diameter) on the Over0 7 dematiaceous fungal genera containing 130 or so right forearm that gradually increased in size over a period distinct species have been implicated in human phaeohypho- of 3 months. Direct microscopic examination with 10% KOH mycosis, chromoblastomycosis, and mycetoma. Given that and histopathological investigation of exudates showed sep- the clinical symptoms caused by these fungi are largely indis- tate hyphae without granules. A fungus was isolated from tinguishable and nonspecific, and that different fungal taxa the exudates in culture and identified as Pyrenochaeta often display varied sensitivity to antifungal drugs, there is romeroi on the basis of morphologic features and sequence a necessity to precisely determine the species identity of the homology in the internal transcribed spacer (ITS) regions suspected fungal organisms for tailor-made, cost-effective of ribosomal RNA (rRNA). Treatment consisted of surgi- treatment. cal excising of the cyst without any antifungal therapy, with Together with clinical and epidemiological information, no relapse during a 1-year follow-up. It is possible that the identification of dematiaceous fungi including Pyrenochaeta apparent immune dysfunction in this patient might contrib- species relies on the observation of mycotic elements in ute partially to the lack of granule formation characteristic biopsy and other specimens by microscopy. Subsequent of mycetoma. isolation of suspected fungal organisms in culture media

© 2011 by Taylor & Francis Group, LLC 148 Molecular Detection of Human Fungal Pathogens facilitates detection of characteristic colonial and micro- 18.2.1.3 DNA Extraction scopic features, leading to the confirmation of the species Pyrenochaeta isolates are grown on potato dextrose agar identity. Unfortunately, this testing scheme can take up to 12 on 9 cm diameter petri dishes at 25°C until mycelium com- weeks to complete. With cultures that are negative or con- pletely covers the agar surface. Mycelia are collected by add- taminated with bacteria, culturing with fresh samples can ing sterile distilled water containing 0.05% (v/v) Tween 80 to further delay the diagnosis. the surface of the culture and gently scrubbing with a sterile To improve the efficiency and specificity of laboratory spatula. The mycelial suspension is transferred to a 1.5 mL diagnosis of fungal pathogens, molecular methods have tube and centrifuged at 3000 × g at 4°C for 5 min. The super- been developed and applied in recent years [16–21]. PCR natant is discarded and the pellet (100 mg mycelia) is stored amplification and sequencing analysis of the ITS1–5.8S– at −80°C until further use. ITS2 rRNA have enabled precise identification of the fungi Extraction buffer (0.2 mL) (3% sodium dodecyl sulfate responsible for black-grain mycetoma [4,22,23]. In addition, [SDS] [w/v] containing 0.5 mM ethylenediaminetetraace- sequencing examination of the D1–D2 regions in the large tic acid [EDTA], 1.0 M NaCl, and 0.1 mM hydroxymethyl- subunit (LSU) rRNA D1–D2 region offers another poten- hydrochloride Tris–HCl pH 8.0) is added to 10 mg of each tial avenue for taxonomical determination of dematiaceous fungal mycelium and the suspension shaken vigorously for fungi. Furthermore, combination of a modified FTA extrac- 15 s. Next, 0.2 mL chloroform-phenol mix (1:1) is slowly tion protocol with pyrosequencing of ITS2 region makes added and incubated at 65°C for 5 min. The mixture is cooled it possible to identity a fungal culture within half a day to room temperature and centrifuged at 10,000 × g at 4°C [24,25]. for 5 min. The supernatant is transferred to a new microtube, and an equal volume of cold absolute isopropanol or ethanol is added and the contents mixed thoroughly for precipitating 18.2 Methods total DNA at −20°C for 20 min; the mixture is then centri- g 18.2.1 Sample Preparation fuged at 10,000 × for 10 min. The pellet is washed twice with 75% ethanol and centrifuged at 10,000 × g at 4°C for 18.2.1.1 Procedure for Endophthalmitis Samples 5 min. The extraocular environment is sterilized with 5% povi- The supernatant is discarded and the pellet is resuspended done iodine solution before surgery. About 100–200 μL of in 0.03 mL MiniQuantum (deionized) water, and stored at aqueous fluid is withdrawn using a 30 gauge needle with a −80°C until further use. Concentration, yield, and quality limbal paracentesis. Vitreous samples (200 μL) are taken at control indices based on absorbance readings at 230, 260, the time of three-port pars plana vitrectomy. The samples and 280 nm (A260/280 and A260/230 ratios) are carried out are divided into two aliquots and transported at 4°C. One with 2 μL resuspended total DNA [26]. portion is immediately examined by conventional microbio- Alternatively, fungal strains are cultured in 20 mL of logical diagnostic tests and the other is frozen at −20°C until RPMI 1640 medium with l-glutamine but without sodium processed by PCR. bicarbonate (Sigma-Aldrich) buffered to pH 7.0 with For the microbiological diagnostic test, 50 μL of aqueous 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma- humor or 50 μL of vitreous is cultured at 30°C in Sabouraud’s Aldrich). After 3–14 days of growth at 30°C under agitation dextrose agar or at 37°C in thioglycolate broth, blood agar, (100 rpm), mycelium is transferred into a tube and washed chocolate agar, cystine lactose electrolyte deficient (CLED) in 40 mL of sterile distilled water. Mycelium is then stored agar, or MacConkey agar. Filamentous fungi are differenti- at −20°C until use. DNA extraction is carried out by a glass ated by isolation in Sabouraud dextrose agar plus chloram- bead lysis method. Approximately 100 mg of mycelium is phenicol and morphological examination of macroscopic and homogenized for 1 min in a tube containing 1 mL of lysis microscopic characteristics. Microscopic structures may also buffer (2% Triton X-100, 1% SDS, 10 mM Tris–HCl pH 8, be observed on tease or tape preparations and slide cultures 100 mM NaCl, 1 mM EDTA pH 8), three 0.5 cm diameter for up to 21 days [18]. glass beads (Sigma), and approximately 500 mg of 425– 600 μm glass beads (Sigma). The homogenized mycelia are 18.2.1.2 Procedure for Keratitis Samples then snap-frozen in liquid nitrogen, thawed, and refrozen Upon completion of the ocular examination and after instil- once. DNA extraction is then done with the DNeasy plant lation of topical anesthetic, a sterile Kimura spatula is used kit (QIAGEN) [4]. to scrape the area of infection. Scrapings are inoculated into Furthermore, Whatman FTA filter may be used for DNA thioglycolate broth, Roiron broth, and Löwenstein-Jensen extraction. Whatman FTA filter matrices are fibrous cards medium and are placed onto glass slides for staining with pretreated with chelators and denaturants that lyse and inac- Gram and Giemsa stains. The PCR sample is obtained by tivate most microorganisms on contact. The large nucleic scraping and stirring the spatula for a few seconds in 100 μL acids released after lysis become physically entangled in the of sterile water in a 1.5 mL sterile Eppendorf tube. Two ali- fibers of the FTA matrix, whereas cellular debris is rapidly quots of 50 μL are taken from each sample and stored at removed by washing the inoculated card. Fungal hyphae −20°C [18]. fragments and conidia (in the case of moulds) are added

© 2011 by Taylor & Francis Group, LLC Pyrenochaeta 149 to dry FTA filters, followed by brief microwave treatment, mixture (Roche Applied Science) (containing allowed liberation of amplifiable DNA with a total prepara- deoxynucleoside triphosphates, FastStart Taq DNA tion time of ∼15 min [25]. polymerase, and 1 mM MgCl2, additional MgCl2 si added to a final concentration of 4.6 mM), 0.4 μM each of ITS1 forward and ITS4 reverse primers, 1× 18.2.2 Detection Procedures SYBR green (Molecular Probes), and 3 μL template 18.2.2.1 S tandard PCR and Sequencing DNA. Analysis of ITS Region 2. Thermal cycling parameters include 95°C for 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, Ferrer et al. [18] utilized universal primers ITS1 (5′-TCC and 76°C for 30 s; and a final extension at 72°C for GTA GGT GAA CCT GCG G-3′, which hybridizes at the 2 min. end of 18S rRNA), and ITS4 (5′-TCC TCC GCT TAT TGA 3. The quality of the amplicon is determined using the TAT GC-3’, which hybridizes at the beginning of 28S rRNA) derivative of the melt analysis curve (55°C–99°C, [27] to amplify a region covering ITS/5.8S rRNA for identifi- 45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 cation of pathogenic fungi. (Corbett Robotics, Inc). Procedure 4. The amplified product is purified for bidirectional sequencing using ExoSAP-IT (USB Corp). Five 1. PCR mixture (50 μL) is made up of 10 μL of DNA microliters of Big Dye Terminator Ready Reaction

template, 6 μL of 25 mM MgCl2,fCR 5 μL o P buffer Mix v. 1.1 (Applied Biosystems) is added to 4 μL of without MgCl2, 200 μM each deoxynucleoside tri- each primer (0.8 pmol/μL) and 3 μL of purified PCR phosphate, 25 pmol of each primer, and 1 U of Taq product. Cycle sequencing is performed with a 9700 DNA polymerase (Biotools B&M Labs). thermal cycler (ABI), using 25 cycles of 96°C for 2. Amplification is performed with 1 cycle at 95°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing 5 min; 35 cycles of 95°C for 30 s, 55°C for 1 min, and reaction products are passed through a Sephadex 72°C for 1 min; and 1 cycle at 72°C for 6 min. G-50 fine column to remove unincorporated dye ter- 3. Aliquots (10 μL) of each amplified product are minators. Purified sequencing reaction products are electrophoretically separated in a 2% agarose gel in run on an ABI Prism 3100 Genetic Analyzer with a 1× Tris–borate–EDTA buffer and visualized using 50 cm capillary array. ethidium bromide under UV illumination. 5. Sequences are analyzed with the SmartGene 4. PCR amplicon is purified using the GeneClean II Integrated Database Network software version kit (Bio 101) and directly cycle sequenced in both 3.2.3 vr. SmartGene is a web-based software and directions using the BigDye terminators Ready database system with reference sequences derived Reaction Kit (PE Applied Biosystems) on an ABI from the National Center for Biological Information Prism automated DNA sequencer (model 377, ver- (NCBI) GenBank repository. sion 2.1.1; Applied Biosystems), using primers ITS4 and ITS86. Note. Sequence-based identifications are defined by percent 5. The 550 bp ITS2/5.8S rRNA sequences are ana- identity: species, ≥99%; genus, 93%–99%; and inconclusive, lyzed by using the BLAST alignment program of the ≤93%. GenBank database. Multiple-sequence alignment is For strains producing discrepant identification carried out using ClustalW 1.8. Phylogenetic trees between the methods based on phenotypic character- are constructed by the neighbor-joining method using istics and ITS sequence analysis, the D1–D2 region the Phylip package and visualized using Treeview. of the LSU RNA gene is amplified with primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5 -GGTCCGTGTTTCAAGACGG-3 ) and sequenced for 18.2.2.2 R eal-Time PCR and Sequencing ′ ′ species clarification. Analysis of ITS Region Pounder et al. [23] described a real-time PCR with SYBR green DNA binding dye and amplicon melting temperature analysis 18.3 Conclusion for fungal detection also using panfungal primers ITS1 for- The genus Pyrenochaeta consists of eight dematiaceous fun- ward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 reverse gal species that are soil saprophytes with potential to cause (5′-TCCTCCGCTTATTGATATGC-3′). The identity of the fungi is verified by subsequent sequencing analysis. infections in plants. Four Pyrenochaeta species have been associated with occasional infections in humans, producing Procedure clinical presentations ranging from mycetoma, subcutaneous infections, keratitis, and other diseases, especially in 1. PCR mixture is composed of 1× Lightcycler immunocompromised individuals. Because clinical diseases FastStart DNA Master Hybridization Probes induced by Pyrenochaeta spp. are indistinguishable from

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