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Identification Problems of Sterile Fungi from Clinical Samples: Description of Six Novel Species

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Identification Problems of Sterile Fungi from Clinical Samples: Description of Six Novel Species Identifcation problems of sterile fungi from clinical samples: Description of six novel species CBS-KNAW Fungal Biodiversity Centre University of Khartoum Sarah A. Ahmed 1, Sybren de Hoog 2, 3, 4, 5 1Faculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan, 2CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, 3Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands, 4Basic Pathology Department, Federal University of Paraná, Curitiba, Brazil, 5Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia 1 Subramaniula sp.1 100 Calosphaeriales 87 Introduction and purpose Results 98 2 Subramaniula sp.1 88 93 3 Subramaniula sp.1 71 Diaporthales 98 77 4 Subramaniula sp.1 68 5 Subramaniula sp.1 100 100 Togniniaceae 100 In the routine clinical laboratories identifcation of flamentous molds is still based on microscopic The clinical isolates were from patiens with cutaneous or subcutaneous infections (6 cases) (Fig. 1), 6 Subramaniula sp.1 62 Coniochaetales 7 Subramaniula sp.1 examination of sporulating colonies. ocular infections (4 cases), and one case of peritonitis in a kidney transplant patient. 100 100 CBS 122.78 Subramaniula thielavioides 100 100 Magnaporthaceae 100 CBS 560.84 Subramaniula thielavioides From many cases of opportunistic mycoses which have emerged in patients with immunosuppression The phylogenetic analysis revealed that the unknown isolates clustered into two fungal orders, Sordariales 95 100 Ophiostomatales 8 Subramaniula sp.2 or severe underlying illness, many isolates lack of characteristic sporulation. and Pleosporales (Fig. 2, 3). CBS 121.57 Chaetomium cuniculorum 100 CBS 156.52 Chaetomium cuniculorum For such sterile fungi phenotypic identifcation methods are insuffcient and hence DNA analysis is only Six novel species were discovered, three belong to the Sordariales family Chaetomiaceae and 3 to the 79 Sordariales 100 CBS 227.82 Chaetomium irregulare way to ascertain the identity of the isolates. Pleosporales (Fig. 2, 4). CBS 446.66 Chaetomium irregulare CBS 199.84 Chaetomium fusisporum 100 84 Boliniales Sequencing of the universal fungal barcode markers (ITS) or (LSU) followed by homology search in the The Pleosporales species were found to belong to three different families, Macrodiplodiopsidaceae, 9 Chaetomium sp.1 Helotiales public repositories of DNA sequences like GenBank is the most common molecular approach been used. Trematospheriaceae, and Roussoellaceae. 100 CBS 265.79 Papulaspora sepedonioides 0.02 0.02 Sequences of clinical isolates that could not be matched with known taxa in the public databases are Sordariales species were mostly associated with superfcial and ocular infections (Fig. 5) in addition therefore remains unknown. to systemic infection in severely immunocompromised patient whereas all pleosporalean species were Figure 3: Phylogram of Sordariales. Figure 4: Phylogram of clinical species of Sordariales. Here we studied strains from clinical cases of which their ITS sequences did not resulted in identity associated with traumatic subcutaneous cases. • Higher-level phylogeny of the clinical isolates obtained by • Deeper-level phylogeny of the Sordariale clinical isolates obtained by with known taxa in GenBank. maximum likelihood analysis of LSU. maximum likelihood analysis of Btub, rPB1, rPB2, and TEF1. • Values in the nodes are maximum-likelihood bootstrap supports. • Values in the nodes are maximum-likelihood bootstrap supports. 100 KT 2313 Kalmusia brevispora • Eight clinical isolates culstered in the order Sordariales. • The clinical isolates are shown in red and three species are resolved. 76 KT 1466 Kalmusia brevispora KT 1023 Kalmusia scabrispora 100 Methods 91 KT 2202 Kalmusia scabrispora CBS 107.79 Bimuria novae-zelandiae CBS 438.65 Didymocrea sadasivanii Montagnulaceae CBS 690.94 Karstenula rhodostoma 98 Figure 2: Phylogenetic analysis of CBS 652.86 Paraphaeosphaeria michotii Figure 5: Clinical and strain CBS 884.85 Letendraea helminthicola Pleosporales. 98 100 morphology of Subramaniula A total of 14 sterile clinical isolates were studied, of which their ITS sequences do not matched any taxa CBS 485.70 Letendraea padouk CBS 168.34 Montagnula opulenta • Maximum-likelihood tree of the asteroides sp. nov. CBS 615.86 Montagnula anthostomoides in the public databases. CBS 845.96 Massarina igniaria combined data of LSU, SSU, RPB2, 98 CBS 675.92 Byssothecium circinans and TEF1. A. Patient with keratitis. Multi-locus sequence analysis and phylogenetic studies were applied to determine the taxonomic position 75 CBS 331.37 Neottiosporina paspali Massarinaceae 100 CBS 473.64 Massarina eburnea • Values in the nodes are maximum- B. Isolate growth on malt extract agar. 96 CBS 266.62 Massarina cisti C. Isolate growth on oatmeal agar. of the unknown isolates. 99 CBS 619.86 Lentithecium arundinaceum 98 likelihood bootstrap supports. CBS 123131 Lentithecium arundinaceum D. Clumps. 99 A The analyses were performed for the nuclear ribosomal DNA regions, i.e. small subunit (18S rDNA), CBS 114202 Stagonospora macropycnidia • The tree is rooted to Dothidea sambuci 84 CBS 618.86 Wettsteinina lacustris E. Chlamydospores-like structures in KT 1706 Ophiosphaerella sasicola and Phaeosclera dematioides. large subunit (28S rDNA), Internal Transcribed Spacer (ITS). 94 chain. KT 1517a Katumotoa bambusicola Lentitheciaceae 100 CBS 123099 Lentithecium aquaticum • Three clinical species are resolved F. minute conidia. KH68 Tingoldiago graminicola Protein-coding loci namely, β-tubulin (Btub), actin (ACT1), translation elongation factor 1-α (TEF1), 98 B C KT891 Tingoldiago graminicola and shown in red. — Scale bars, 10 μm. and DNA-dependent RNA polymerase II largest subunit (RPB1) and second largest subunit (RPB2) were KH155 Tingoldiago graminicola CBS 116668 Pleurophoma pleurospora 95 CBS 104.55 Keissleriella cladophila also analyzed. 54 CBS 113798 Keissleriella genistae 96 CBS 130329 Pleurophoma pleurospora 100 CBS 221.37 Splanchnonema platani CBS 222.37 Splanchnonema platani 100 Pleosporales species 1 Macrodiplodiopsidaceae 55 CBS 639.94 Pseudochaetosphaeronema larense 77 Figure 1: Clinical and strain morphology 100 CBS 640.73 Pseudochaetosphaeronema larense 100 JK 5304B Morosphaeria ramunculicola of Pseudochaetosphaeronema martinelli 95 BCC 18405 Morosphaeria ramunculicola BCC 17059 Morosphaeria velataspora 100 sp. vo. 100 BCC 17058 Morosphaeria velataspora Morosphaeriaceae 99 BCC 36751 Helicascus nypae 100 BCC 36752 Helicascus nypae A. Clinical presentation of the patient CBS 124082 Asteromassaria pulchra 100 BCC 21117 Falciformispora lignatilis showing subcutaneous nodules. 96 BCC2 1118 Falciformispora lignatilis D E F CBS 201.79 Falciformispora tompkinsii 100 B. Colony on malt extract agar. 80 100 CBS 200.79 Falciformispora tompkinsii CBS 196.79 Falciformispora senegalensis C. Colony on oatmeal agar. 100 CBS 198.79 Falciformispora senegalensis 97 CBS1 99.79 Falciformispora senegalensis Trematosphaeriaceae Conclusions D. Hyphae and chlamydospores. CBS 332.50 Trematosphaeria grisea CBS 246.66 Trematosphaeria grisea E. Thick-walled hyphae. 99 100 A B C CBS 136543 Trematosphaeria grisea 100 F. Coiled hyphae. CBS 135983 Trematosphaeria grisea 67 CBS 122368 Trematosphaeria pertusa 100 — CBS 122371 Trematosphaeria pertusa The non-sporulating isolates can be of high diversity and are associated with wide range of medical 88 BCC 17054 Halomassarina thalassiae 1 Pleosporales species 2 conditions. 100 2 Pleosporales species 2 100 KT 1651 Roussoella hysterioides 100 Infections are in both immunocompetent and immunocompromised individuals and ranged from CBS 546.94 Roussoella hysterioides 70 CBS 125434 Roussoella hysterioides 100 KT 1709 Roussoella pustulans superfcial cutaneous to deep or disseminated infections. 82 KT 1659 Roussoellopsis tosaensis Roussoellaceae CBS 170.96 Roussoella intermedia 100 Medical mycologists and infectious diseases specialist need to be aware of such sterile opportunists. 100 CBS 368.94 Arthopyrenia salicis 1 Pleosporales species 3 100 2 Pleosporales species 3 Proper identifcation and taxonomic description of the unknown clinical species is crucial for both CBS 157.81 Phaeosclera dematioides 100 AFTOL-ID 274 Dothidea sambuci diagnostic and epidemiological studies of fungal diseases. D E F 0.05 .
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