1. Abstract Identification of uncommon species of the clinical isolates of the yeasts by ITS-sequencing Hossein Khodadadi 1, Ladan Karimi 2, Hossein Mirhendi 3 1 Department of Medical Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 2 Dr.Beheshti Hospital, Social security organization, Shiraz, Iran. 3 Department of Medical Mycology and Parasitology, School of public health, Tehran university of medical sciences, Tehran, Iran. By using advanced detection/identification methods, the list of emerging uncommon opportunistic yeast infections is rapidly expanding worldwide. In the present study, our aim was identifying the rare clinically yeast isolates. Forty nine out of 855 (5.7%) yeast isolates which formerly remained unidentified by PCR-RFLP method were subjected to sequence analysis of internal transcribed spacers (ITS) regions. These species include: Hanseniaspora uvarum, Saccharomyces cerevisiae, Sporidiobolus salmonicolor, Pichia fabianii, Pichia fermentans, Candida famata, Candida inconspicua, Candida maqnoliae, Candida guilliermondii, Candida kefyr, Candida rugosa, Candida lusitaniae, Candida orthopsilsis, and Candida viswanathii. Opportunistic infections caused by rare yeasts have increased in recent decade and some of these yeasts may resistant to some antifungals. Conventional methods could not definitely identify all yeast species, thus correct identification of yeasts using modern and reliable methods is essential. Introduction Uncommon yeast species have been emerged as new etiologic agents of fungal infections. Due to limitations of conventional yeast identification methods, these yeasts often misidentified or report only as unknown yeast species. DNA-sequencing of internal transcribed spacer (ITS) region has allowed us a better discrimination of rare yeast species. We have collected and identified several hundred of yeasts isolated from various clinical forms of infections, during which the precise identity of some isolate remained undetermined. Our aim in this study was to identify these species by PCR-sequencing. Surprisingly we found some rare yeast that had not previously been reported in Iran. Material and methods Organisms: Among the 855 viable yeast isolates submitted to diagnostic clinical microbiology laboratories from Tehran, Esfahan, Alborz and Mazandaran provinces of Iran, a total of 49 yeast strain which was remained unidentified by PCR-RFLP, were included in the present study. Results Among 855 yeast strains isolated from patients with various forms of fungal infections and identified by PCR-RFLP, a total of 49 yeasts were classified as uncommon yeast species, and none of them showed a specific color of dominant yeasts on chromogenic plates. These isolates were subjected to ITS sequencing and subsequently analyzed for homology with other reliable sequences deposited in GenBank. Among the isolates, we found six new yeast species which have not already reported in Iran, including two strains of Hanseniaspora uvarum, one strain of Pichia fabianii, one Sporidiobolus salmonicolor, one Pichia fermentans and one Candida magnolia. Eight out of 49 (16.3%) isolates were misidentified by PCR-RFLP method (two Candida parapsilosis complex, 5 Candida lusitaniae and 1 Pichia fabianii), the method could not discriminate 41 remaining isolates and reported them as unidentifiable (Table1). Conclusion Some nucleic acid-based methods like PCR-RFLP have improved identification of clinical yeast isolates but this method showed poor ability to straight-forward identifying of some uncommon yeasts. The evidence of inability of this method for discriminating rare yeasts is the lower concordance between PCR-RFLP and sequencing results (16.3%) among 49 uncommon isolates identified by both methods. We identified several rare species by ITS-sequencing that some of them had not been isolated or reported from clinical samples previously. Our findings indicate the existence of newly emerging yeast infection in at risk Iranian patients, even though these infections often are neglecting as a result of their misidentification. DNA Sequencing Methods: Genomic DNA of each fresh colony was isolated using FTA Elute MicroCards (Whatman Inc., Clifton, NJ, USA) as described elsewhere. As the DNA sequence of different parts of ribosomal DNA (rDNA), particularly internal transcribed spacer (ITS1-5.8S-ITS2) region have been proved as the valuable molecular markers for sequence identification of yeast species, all isolates were conducted to ITS-PCR-sequencing. PCR products were purified using a PCR purification kit (Bioneer, Korea) and directed to an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems), and sequenced using an automated DNA Sequencer (ABI Prism 3730 Genetic Analyzer; Applied Biosystems). Subsequently, final designation for species was based on analysis of reliable sequences with BLASTn algorithm in comparison with relevant reliable sequences deposited in GenBank (http://www.ncbi.nlm.nih.gov/BLAST).http://www.ncbi.nlm.nih.gov/BLAST References 1. Hierro N, Gonzalez A, Mas A, Guillamon JM. New PCR-based methods for yeast identification. J Appl Microbiol. 2004;97:792–801. 2. Mirhendi H, Makimura K, Zomorodian K, Maeda N, Ohshima T, Yamaguchi H. Differentiation of Candida albicans and Candida dubliniensis using a single-enzyme PCR-RFLP method. Jpn J Infect Dis. 2005;58(4):235-7. 3. Mirhendi H, Makimura K, Khoramizadeh M, Yamaguchi H. A one-enzyme PCR- RFLP assay for identification of six medically important Candida species. Nihon Ishinkin Gakkai Zasshi. 2006;47(3):225-9. Table 1. Comparative results for DNA sequencing method and PCR-RFLP for uncommon yeasts that displayed discordant results for these two methodologies. Species identified by Sequencing Species identified by PCR-RFLP Clinical Specimen (no. of isolates) Total Candida kefyr Unidentified Oropharngeal(2), Vulva-vagina(3), BAL(2), Blood(2), Urine(3), Nail(6) Sputum(4) 22 Candida rugosa UnidentifiedNail(2), Skin(1)3 Candida lusitaniae C. lusitaniae/intermdiaNail(3), Blood(2)5 Candida orthopsilsis UnidentifiedNail(4)4 Candida parapsilosis Candida Opsilosis ComplexNail(1), Vulva-vagina(1)2 Candida maqnoliae UnidentifiedSkin(1)1 Candida inconspicua UnidentifiedNail(1)1 Candida guilliermondii UnidentifiedNail(2)2 Hanseniaspora uvarum UnidentifiedNail(2)2 Saccharomyces cerevisiae UnidentifiedNail(3)3 Pichia fabianii Candida tropicalisSputum(1)1 Sporidiobolus salmonicolor UnidentifiedSputum(1)1 Pichia fermentans UnidentifiedBAL(1)1 Total 49 58th Annual Meeting of JSMM, 2014 P-21 Basic1 h_khodadadi@sums.ac.ir