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[DNA extraction and identification of Trichophyton rubrum by real-time polymerase chain reaction from direct nail scraping specimens of patients with onycomycosis].
Mikrobiyol Bul. 2011 Jan; 45(1):150-8.MB

Abstract

Trichophyton rubrum is the most frequently encountered dermatophyte species causing onichomycosis. The routine diagnosis of dermatophytes depends on the direct microscopic examination (DME) and culture methods, however due to the phenotypic identification problems related to those agents, the molecular methods come into question. The aim of this study was to evaluate the diagnostic performance of real-time polymerase chain reaction (RT-PCR) for the identification of T.rubrum by comparing to DME and culture methods, from nail samples of patients with the complaints of onychomycosis. A total of 90 patients of whom 58 were male who were admitted to the dermatology outpatients clinics of our hospital with the complaints of color/shape changes in the nails and thickening of the nail, were included in the study, together with the 20 healthy volunteer subjects as controls. The nail scraping samples obtained from the patients and controls were examined with direct microscopy using 15% potassium hydroxide, dimethyl sulphoxide and chlorazole black mixture and cultivated onto Sabouraud dextrose agar with and without cycloheximide. For DNA isolation, after the disruption of nail samples with a steel tool, phenol-chloroform-isoamyl alcohol purification method were used. The amplification and demonstration of the T.rubrum DNA have been performed by using specific primers and probes following TaqMan protocol of RT-PCR (LightCycler-Roche, USA) method. Seventy-two of the patients yielded positive and 18 yielded negative results with DME. Growth of molds was detected in the cultures of 20 (27.8%) of the 72 DME positive patients and all of the isolates were identified as T.rubrum. No fungal growth was seen in the samples of 18 patients who were DME negative. In DME positive group, 67 (93%) patients were found to be positive in RT-PCR, while 8 (44.4%) patients were RT-PCR positive in DME negative group. All of the culture positive samples (n= 20) were also found positive in RT-PCR. All of the samples from the control group with healthy nails yielded negative results in DME, culture and RT-PCR methods. The performance of PCR method were compared to direct microscopy that had higher sensitivity than culture and the sensitivity, specificity, positive and negative predictive values of RTPCR assay were estimated as 93%, 56%, 89% and 67%, respectively. In conclusion RT-PCR was thought to be an efficient and rapid assay in the diagnosis of onichomycosis. Although RT-PCR seems more expensive than culture, for the centres which already have support for the molecular methods, the difference in total cost doesn't count much. In conclusion, by the use of molecular methods DNA isolation was successfully done from a relatively difficult clinical specimen, namely nail scraping, a protocole that could easily be applied in routine laboratory was established and species-level identification in a short time was accomplished in this study.

Authors+Show Affiliations

Kayseri Research and Training Hospital, Microbiology Clinics, Kayseri, Turkey.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article

Language

tur

PubMed ID

21341169

Citation

Berk, Elife, et al. "[DNA Extraction and Identification of Trichophyton Rubrum By Real-time Polymerase Chain Reaction From Direct Nail Scraping Specimens of Patients With Onycomycosis]." Mikrobiyoloji Bulteni, vol. 45, no. 1, 2011, pp. 150-8.
Berk E, Kuştimur S, Kalkancı A, et al. [DNA extraction and identification of Trichophyton rubrum by real-time polymerase chain reaction from direct nail scraping specimens of patients with onycomycosis]. Mikrobiyol Bul. 2011;45(1):150-8.
Berk, E., Kuştimur, S., Kalkancı, A., & Oztaş, O. M. (2011). [DNA extraction and identification of Trichophyton rubrum by real-time polymerase chain reaction from direct nail scraping specimens of patients with onycomycosis]. Mikrobiyoloji Bulteni, 45(1), 150-8.
Berk E, et al. [DNA Extraction and Identification of Trichophyton Rubrum By Real-time Polymerase Chain Reaction From Direct Nail Scraping Specimens of Patients With Onycomycosis]. Mikrobiyol Bul. 2011;45(1):150-8. PubMed PMID: 21341169.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [DNA extraction and identification of Trichophyton rubrum by real-time polymerase chain reaction from direct nail scraping specimens of patients with onycomycosis]. AU - Berk,Elife, AU - Kuştimur,Semra, AU - Kalkancı,Ayşe, AU - Oztaş,O Murat, PY - 2011/2/23/entrez PY - 2011/2/23/pubmed PY - 2011/8/17/medline SP - 150 EP - 8 JF - Mikrobiyoloji bulteni JO - Mikrobiyol Bul VL - 45 IS - 1 N2 - Trichophyton rubrum is the most frequently encountered dermatophyte species causing onichomycosis. The routine diagnosis of dermatophytes depends on the direct microscopic examination (DME) and culture methods, however due to the phenotypic identification problems related to those agents, the molecular methods come into question. The aim of this study was to evaluate the diagnostic performance of real-time polymerase chain reaction (RT-PCR) for the identification of T.rubrum by comparing to DME and culture methods, from nail samples of patients with the complaints of onychomycosis. A total of 90 patients of whom 58 were male who were admitted to the dermatology outpatients clinics of our hospital with the complaints of color/shape changes in the nails and thickening of the nail, were included in the study, together with the 20 healthy volunteer subjects as controls. The nail scraping samples obtained from the patients and controls were examined with direct microscopy using 15% potassium hydroxide, dimethyl sulphoxide and chlorazole black mixture and cultivated onto Sabouraud dextrose agar with and without cycloheximide. For DNA isolation, after the disruption of nail samples with a steel tool, phenol-chloroform-isoamyl alcohol purification method were used. The amplification and demonstration of the T.rubrum DNA have been performed by using specific primers and probes following TaqMan protocol of RT-PCR (LightCycler-Roche, USA) method. Seventy-two of the patients yielded positive and 18 yielded negative results with DME. Growth of molds was detected in the cultures of 20 (27.8%) of the 72 DME positive patients and all of the isolates were identified as T.rubrum. No fungal growth was seen in the samples of 18 patients who were DME negative. In DME positive group, 67 (93%) patients were found to be positive in RT-PCR, while 8 (44.4%) patients were RT-PCR positive in DME negative group. All of the culture positive samples (n= 20) were also found positive in RT-PCR. All of the samples from the control group with healthy nails yielded negative results in DME, culture and RT-PCR methods. The performance of PCR method were compared to direct microscopy that had higher sensitivity than culture and the sensitivity, specificity, positive and negative predictive values of RTPCR assay were estimated as 93%, 56%, 89% and 67%, respectively. In conclusion RT-PCR was thought to be an efficient and rapid assay in the diagnosis of onichomycosis. Although RT-PCR seems more expensive than culture, for the centres which already have support for the molecular methods, the difference in total cost doesn't count much. In conclusion, by the use of molecular methods DNA isolation was successfully done from a relatively difficult clinical specimen, namely nail scraping, a protocole that could easily be applied in routine laboratory was established and species-level identification in a short time was accomplished in this study. SN - 0374-9096 UR - https://www.unboundmedicine.com/medline/citation/21341169/[DNA_extraction_and_identification_of_Trichophyton_rubrum_by_real_time_polymerase_chain_reaction_from_direct_nail_scraping_specimens_of_patients_with_onycomycosis]_ L2 - https://medlineplus.gov/tineainfections.html DB - PRIME DP - Unbound Medicine ER -