Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens

详细信息    查看全文

• 作者：Jie Gong ; Menglong Ran ; Xiaowen Wang ; Zhe Wan ; Ruoyu Li
• 关键词：Pan ; dermatophyte ; Tinea unguium ; Fungal infection ; Dermatophyte PCR
• 刊名：Mycopathologia
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：181
• 期：1-2
• 页码：51-57
• 全文大小：366 KB
• 参考文献：1.Ghannoum M, Hajjeh R, Scher R, Konnikov N, Gupta A, Summerbell R, et al. A large-scale North American study of fungal isolates from nails: the frequency of onychomycosis, fungal distribution, and antifungal susceptibility patterns. J Am Acad Dermatol. 2000;43:641鈥?.CrossRef PubMed
  2.Uchida T, Makimura K, Ishihara K, Goto H, Tajiri Y, Okuma M, et al. Comparative study of direct polymerase chain reaction, microscopic examination and culture-based morphological methods for detection and identification of dermatophytes in nail and skin samples. J Dermatol. 2009;36:202鈥?.CrossRef PubMed
  3.Garg J, Tilak R, Singh S, Gulati AK, Garg A, Prakash P, et al. Evaluation of pan-dermatophyte nested PCR in diagnosis of onychomycosis. J Clin Microbiol. 2007;45:3443鈥?.PubMedCentral CrossRef PubMed
  4.Summerbell R, Kane J, Krajden S. Onychomycosis, Tinea Pedis and Tinea Manuum caused by non-dermatophytic filamentous fungi Nicht-Dermatophyten-Fadenpilze als Erreger von Onychomykosen. Tinea pedis und Tinea manuum. Mycoses. 1989;32:609鈥?9.CrossRef
  5.Hainer BL. Dermatophyte infections. Am Fam Physician. 2003;67:101鈥?0.PubMed
  6.Weinberg JM, Koestenblatt EK, Tutrone WD, Tishler HR, Najarian L. Comparison of diagnostic methods in the evaluation of onychomycosis. J Am Acad Dermatol. 2003;49:193鈥?.CrossRef PubMed
  7.Dhib I, Fathallah A, Yaacoub A, Hadj Slama F, Said M, Zemni R. Multiplex PCR assay for the detection of common dermatophyte nail infections. Mycoses. 2014;57:19鈥?6.CrossRef PubMed
  8.Garg J, Tilak R, Garg A, Prakash P, Gulati AK, Nath G. Rapid detection of dermatophytes from skin and hair. BMC Res Notes. 2009;2:60.PubMedCentral CrossRef PubMed
  9.Petrini B, Von Rosen M. Optimal dermatophyte diagnosis requires both microscopy and culture. Lakartidningen. 2002;99:4084.PubMed
  10.Brillowska-D膮browska A, Saunte DM, Arendrup MC. Five-hour diagnosis of dermatophyte nail infections with specific detection of Trichophyton rubrum. J Clin Microbiol. 2007;45:1200鈥?.PubMedCentral CrossRef PubMed
  11.Dubljanin E, Calovski I, Vujcic I, Dzamic A, Arendrup M, Petersen R, et al. Clinical evaluation of a T. rubrum-specific polymerase chain reaction and pandermatophyte polymerase chain reaction in the diagnosis of suspected onychomycosis in 183 Serbian patients. Br J Dermatol. 2014;171:1593鈥?.CrossRef PubMed
  12.Alexander C, Shankland G, Carman W, Williams C. Introduction of a dermatophyte polymerase chain reaction assay to the diagnostic mycology service in Scotland. Br J Dermatol. 2011;164:966鈥?2.CrossRef PubMed
  13.Bergman A, Heimer D, Kondori N, Enroth H. Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR. Clin Microbiol Infect. 2013;19:E205鈥?1.CrossRef PubMed
  14.Verrier J, Pronina M, Peter C, Bontems O, Fratti M, Salamin K, et al. Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism. J Clin Microbiol. 2012;50:553鈥?1.PubMedCentral CrossRef PubMed
  15.Luk N, Hui M, Cheng T, Tang L, Ho K. Evaluation of PCR for the diagnosis of dermatophytes in nail specimens from patients with suspected onychomycosis. Clin Exp Dermatol. 2012;37:230鈥?.CrossRef PubMed
  16.Wisselink G, Van Zanten E, Kooistra-Smid A. Trapped in keratin: a comparison of dermatophyte detection in nail, skin and hair samples directly from clinical samples using culture and real-time PCR. J Microbiol Methods. 2011;85:62鈥?.CrossRef PubMed
  17.Bergmans A, Van der Ent M, Klaassen A, B枚hm N, Andriesse G, Wintermans R. Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material. Clin Microbiol Infect. 2010;16:704鈥?0.CrossRef PubMed
  18.Godornes C, Leader BT, Molini BJ, Centurion-Lara A, Lukehart SA. Quantitation of rabbit cytokine mRNA by real-time RT-PCR. Cytokine. 2007;38:1鈥?.PubMedCentral CrossRef PubMed
  19.Brillowska-Dabrowska A, Nielsen SS, Nielsen HV, Arendrup MC. Optimized 5-hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory. Med Mycol. 2010;48:828鈥?1.CrossRef PubMed
  20.Lanza ST, Dziak JJ, Huang L, Wagner AT, Collins LM. Proc LCA and Proc LTA users鈥?guide (Version 1.3.2). University Park: The Methodology Center, Penn State; 2015.
  21.Kondori N, Tehrani PA, Str枚mbeck L, Faergemann J. Comparison of dermatophyte PCR kit with conventional methods for detection of dermatophytes in skin specimens. Mycopathologia. 2013;176:237鈥?1.CrossRef PubMed
  22.Khot PD, Ko DL, Hackman RC, Fredricks DN. Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. BMC Infect Dis. 2008;8:73.PubMedCentral CrossRef PubMed
  23.de Boer R, Peters R, Gierveld S, Schuurman T, Kooistra-Smid M, Savelkoul P. Improved detection of microbial DNA after bead-beating before DNA isolation. J Microbiol Methods. 2010;80:209鈥?1.CrossRef PubMed
  
• 作者单位：Jie Gong (1) (2) (3)
  Menglong Ran (1)
  Xiaowen Wang (1) (2) (3)
  Zhe Wan (1) (2) (3)
  Ruoyu Li (1) (2) (3)
  
  1. Department of Dermatology, Peking University First Hospital, No. 8 Xishiku St., West District, Beijing, 100034, People鈥檚 Republic of China
  2. Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, 100034, People鈥檚 Republic of China
  3. Research Center for Medical Mycology, Peking University, Beijing, 100034, People鈥檚 Republic of China
  
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Microbiology
  Medical Microbiology
  Plant Sciences
  Microbial Ecology
  
• 出版者：Springer Netherlands
• ISSN：1573-0832

文摘

An accurate diagnosis of tinea unguium is necessary for the selection of antimycotics and successful treatment. To rapidly and accurately identify the aetiological agents causing tinea unguium, we improved upon the conventional boiling method for DNA extraction and developed a novel real-time PCR detection system that includes two assays. The two assays, based on the amplification of ribosomal internal transcribed spacer regions and 28S rDNA, were designed to detect pan-dermatophyte and Trichophyton rubrum, respectively. The analytical sensitivities of both assays permitted the detection of ten copies of plasmid DNA template. The analytical specificity of the detection system was confirmed using 11 dermatophyte strains and 25 non-dermatophyte strains. In total, 165 nail specimens were examined by microscopy, culture, conventional PCR, and the novel real-time PCR method. Real-time PCR gave positive results in 47.3 % of the specimens (78), a rate exceeding those obtained using microscopy (72, 43.6 %), conventional PCR (69, 41.8 %), and culture (49, 29.7 %). All conventional PCR-positive specimens were detected by real-time PCR, and real-time PCR detected nine specimens that were missed by conventional PCR. The results from latent class analysis, and further calculations, showed that real-time PCR was the most sensitive method, but the diagnostic specificity of the four approaches was equivalent. In particular, molecular approaches may be more effective than microscopy and culture when the clinical symptoms of tinea unguium are not evident. Keywords Pan-dermatophyte Tinea unguium Fungal infection Dermatophyte PCR