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Quantification of Dermatophyte Viability for Evaluation of Antifungal Effect by Quantitative PCR

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An Erratum to this article was published on 09 December 2014

Abstract

Dermatophytosis is a common disease caused by dermatophyte fungi such as Trichophyton rubrum and Trichophyton mentagrophytes. A method of quantifying fungal viability in the lesions of dermatophytosis is indispensable for understanding the therapeutic process and outcome; however, no such method has yet been developed. The aim of this study was to develop a method for quantifying dermatophyte viability by quantitative polymerase chain reaction (qPCR). The internal transcribed spacer (ITS) and D1/D2 regions, including each of rRNA and rDNA, were chosen as the targets, and dermatophyte-specific primer pairs were designed corresponding to ITS and D1/D2 regions. The amounts of target RNA and DNA after heat or antifungal treatment were measured by qPCR and compared with colony-forming unit (CFU) counts. RNA and DNA could extract from dermatophytes by mechanical pulverization of conidia using a Multi-Beads Shocker cell disruptor. Our method was sufficiently sensitive to detect 10 copies by qPCR using both ITS and D1/D2 primer pairs. The most sensitive target was ITS-cDNA after heat or antifungal treatment, and essentially consistent with CFU counts. On the other hands, ITS-DNA and D1/D2-DNA were not decreased soon after heat or antifungal treatment, but those were decreased significantly and reflected the CFU counts after 48 h of antifungal treatment. We conclude that ITS-cDNA is useful mainly for quantifying dermatophyte viability at early responses, but ITS-DNA and D1/D2-DNA are also available for evaluation, which does not need an early response.

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Authors and Affiliations

  1. R&D Laboratories, Pola Pharma Inc., 560 Kashio-cho, Totuka-ku, Yokohama, 244-0812, Japan

    Iwanaga Tomoyuki

  2. Department of Dermatology, Kanazawa Medical University, Uchinada, Ishikawa, Japan

    Iwanaga Tomoyuki, Anzawa Kazushi & Mochizuki Takashi

Authors
  1. Iwanaga Tomoyuki
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  2. Anzawa Kazushi
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  3. Mochizuki Takashi
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Correspondence to Iwanaga Tomoyuki.

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Tomoyuki, I., Kazushi, A. & Takashi, M. Quantification of Dermatophyte Viability for Evaluation of Antifungal Effect by Quantitative PCR. Mycopathologia 177, 241–249 (2014). https://doi.org/10.1007/s11046-014-9745-5

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  • DOI: https://doi.org/10.1007/s11046-014-9745-5

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