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Journal of Clinical Microbiology, October 1999, p. 3175-3178, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Development of a Species-Specific PCR-Restriction Fragment Length Polymorphism Analysis Procedure for Identification of Madurella mycetomatis

Abdalla O. A. Ahmed,1,2 Moawia M. Mukhtar,1 Marly Kools-Sijmons,3 Ahmed H. Fahal,4 Sybren de Hoog,5 Bert Gerrits van den Ende,5 Eduard E. Zijlstra,1,dagger Henri Verbrugh,3 El Sir A. M. Abugroun,2 Ahmed M. Elhassan,1 and Alex van Belkum3,*

Institute of Endemic Diseases,1 Faculty of Medical Laboratory Sciences,2 and Department of Surgery,4 University of Khartoum, Sudan, and Department of Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam (EMCR), Rotterdam,3 and Centraalbureau voor Schimmelcultures (CBS), Baarn,5 The Netherlands

Received 12 May 1999/Returned for modification 10 June 1999/Accepted 8 July 1999

Madurella mycetomatis is the commonest cause of eumycetoma in Sudan and other countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of M. mycetomatis and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed spacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs. The ITS regions were amplified with universal primers and sequenced, and then two sets of species-specific primers were designed which specifically amplify parts of the ITS and the 5.8S ribosomal DNA gene. The new primers were tested for specificity with DNA isolated from human mycetoma lesions and DNA extracted from cultures of M. mycetomatis reference strains and related fungi as well as human DNA. To study the genetic variability of the ITS regions of M. mycetomatis, ITS amplicons were obtained from 25 different clinical isolates and subjected to restriction fragment length polymorphism (RFLP) analysis with CfoI, HaeIII, MspI, Sau3AI, RsaI, and SpeI restriction enzymes. RFLP analysis of the ITS region did not reveal even a single difference, indicating the homogeneity of the isolates analyzed during the current study.

* Corresponding author. Mailing address: Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Phone: 00-31-10-4635813. Fax: 00-31-10-4633875. E-mail: vanbelkum{at}bacl.azr.nl.

dagger Present address: College of Medicine, Chichiri, Blantyre 3, Malawi.

Journal of Clinical Microbiology, October 1999, p. 3175-3178, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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