Abstract

BACKGROUND: Onychomycosis has become one of the common fungal infection. However, highly reliable and sensitive methods of detecting and identifying causative fungi of onychomycosis are not established yet. Polymerase chain reaction (PCR) analysis of clinical specimens including blood, sputum, urine, and cerebrospinal fluid collected from patient systemically infected fungus is known as a sensitive diagnostic method. But it has been questionable whether PCR analysis is also applicable to onychomycosis.

OBJECTIVE: The purpose of this study was to develop a DNA-based diagnostic method to improve the sensitivity and specificity of detection and identification of pathogenic fungi of onychomycosis.

METHODS: To detect the fungi in the nail, PCR was performed by using 4 sets of primer (TR1 & TR2, NS5 & NS6, B2F & B4R and CA1 & CA2) designed in conserved sequences of the small ribosomal subunit (185-rRNA) genes and restriction enzyme analysis of amplified product by Hae III was done to identify species. Nail specimens were obtained from 19 cases of onychomycosis confirm by fungus culture.

RESULTS:

1. Preparation of nail powder, which is necessary for removal of keratin, and composition of lysis buffer with guanidinium thiocyanate, Tris-HCl, and beta -mercaptoethanol are the most proper modalities for isolation of fungal DNA from fungus-infesting nails.

2. Specific fragments of the 18S-rRNA gene of fungi, 581 bp, 308 bp, 688 bp and 1106 bp were amplified respectively. From sequences of 18S-rRNA gene of fungi by universal primers, dermatophytes (Trichophyton rubrum, Trichophyton mentagrophytes) and yeast (Candida albicans) yielded identical products.

3. Using Hae III endonuclease, digested patterns of fragment of Trichophyton rubrum and Candida albicans resulted in different pattern.

CONCLUSION: This method released enough DNA from fungus-infected nails to result in proper amplification and it can be possible to differentiate dermatophytes, yeasts, and molds using Hae III endonuclease. The present study is the first one to demonstrate the feasibility of this molecular biologic approach to identify fungi in the infected nail. Therefore, precise detection and identification of the causative fungi would be of help in investigating distribution of the causative fungi of onychomycosis as well as appropriate treatment of the disease.

Keywords

Onychomycosis Polymerase chain reaction Restriction enzyme analysis

KJMM 1999 June;4(1):6-14(9). Epub 2016 February 24
Copyright © 1999 by Korean Journal of Medical Mycology

Language

Korean/English

Author

Hee Jae Chae; Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea
Seung Cheol Baek; Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea
Baik Kee Cho; Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea

Corresponding

Hee Jae Chae, Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea.

Publication history

Acknowledgements

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hee Jae Chae

Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea

Seung Cheol Baek

Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea

Baik Kee Cho

Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea

Since epub date 2016 February 24