title: Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma.
abstract: Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide is Madurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of M. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection of M. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples.
authors.affiliation: Faculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Netherlands.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Ahmed
authors.affiliation: Erasmus MC, Department of Medical Microbiology and Infectious Diseases, Rotterdam, Netherlands.
authors.forename: Wendy W J
authors.initials: WW
authors.lastname: van de Sande
authors.affiliation: Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycologie et Antifongiques, Paris, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Desnos-Ollivier
authors.affiliation: Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan.
authors.forename: Ahmed H
authors.initials: AH
authors.lastname: Fahal
authors.affiliation: Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan.
authors.forename: Najwa A
authors.initials: NA
authors.lastname: Mhmoud
authors.affiliation: Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China Basic Pathology Department, Federal University of Paraná State, Curitiba, Paraná, Brazil King Abdulaziz University, Jeddah, Saudi Arabia s.hoog@cbs.knaw.nl.
authors.forename: G S
authors.initials: GS
authors.lastname: de Hoog
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D006801:Humans; D020461:Madurella / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008271:Mycetoma / Q000175:diagnosis* / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer