Quantification of dermatophyte viability for evaluation of antifungal effect by quantitative PCR.
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.
Tomoyuki
T
Iwanaga
R&D Laboratories, Pola Pharma Inc., 560 Kashio-cho, Totuka-ku, Yokohama, 244-0812, Japan, t-iwanaga@pola-pharma.co.jp.
Iwanaga
I
Tomoyuki
Kazushi
K
Anzawa
Anzawa
A
Kazushi
Takashi
T
Mochizuki
Mochizuki
M
Takashi
D000935:Antifungal Agents / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D006801:Humans; D050296:Microbial Viability; D016133:Polymerase Chain Reaction / Q000379:methods*; D014005:Tinea / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*; D014249:Trichophyton / Q000187:drug effects / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification
D000935:Antifungal Agents; D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer