Diagnostic implications of mycetoma derived from Madurella pseudomycetomatis isolates from Mexico.

BACKGROUND: At the dermatology service of the General Hospital of Mexico City, Mexico, two patients, father and son, with black-grain mycetoma were seen. The grains were isolated, and the cultured fungi were identified as Madurella mycetomatis based on morphology. Using the M. mycetomatis specific PCR, amplicons of a different size than that of the M. mycetomatis type strain were obtained. OBJECTIVE: To determine the causative agent of the two black-grain mycetoma cases and develop non-culture-based diagnostic tools to identify them to the species level. METHODS: The M. mycetomatis specific, the internal transcribed spacer (ITS) region, ß-tubulin (BT) and ribosomal binding protein 2 (RBP2) PCRs were used to confirm the identity of the isolates. Genetic variation was established by amplification fragment length polymorphisms. To determine the antifungal susceptibility profile, the Sensititre™ YeastOne™ assay was used. To develop a species-specific PCR primers were designed on the sequenced PCR amplicon from the M. mycetomatis specific PCR. RESULTS: By analyzing the ITS, BT and RBP2 regions the isolates were identified as Madurella pseudomycetomatis. The isolates from father and son were similar but not identical to M. pseudomycetomatis from Venezuela and one from an unknown origin. Madurella pseudomycetomatis isolates were inhibited by itraconazole, posaconazole and voriconazole but showed increased MIC values for amphotericin B and fluconazole. They were not inhibited by the echinocandins and five flucytosine. The two patients were treated with itraconazole resulting in cure for the father while the son was lost to follow-up. The species-specific PCR developed for M. pseudomyceotmatis was discriminative and specific. CONCLUSION: Madurella pseudomycetomatis is genetically diverse with same susceptibility profile as M. mycetomatis and causes eumycetoma in Latin America. The M. pseudomycetomatis specific PCR can be used to identify this causative agent to the species level; however, this needs to be validated in an endemic setting.

VNTR confirms the heterogeneity of Madurella mycetomatis and is a promising typing tool for this mycetoma causing agent.

The neglected tropical disease mycetoma is a chronic granulomatous inflammatory and infectious disease affecting various body parts. The most common causative agent is the fungus Madurella mycetomatis. In order to study the genetic diversity of this fungus and to monitor any potential outbreaks, a good typing method that can be used in endemic settings is needed. Previous typing methods developed were not discriminative and not easy to perform in resource-limited laboratories. Variable-Number-Tandem-Repeat (VNTR) typing overcomes these difficulties and further enables interlaboratory data comparison. Therefore, in this study we developed a VNTR method for typing M. mycetomatis. Six tandem-repeats were identified in the genome of M. mycetomatis isolate MM55 using an online tandem repeats software. The variation in these repeats was determined by PCR and gel-electrophoresis on DNA obtained from 81 M. mycetomatis isolates obtained from patients. These patients originated from Sudan, Mali, Peru, and India. The 81 isolates were divided into 14 genotypes which separated into two main clusters with seven and five subdivisions, respectively. VNTR typing confirms the heterogeneity of M. mycetomatis strains and can be used to study the epidemiology of M. mycetomatis. The results presented in this article are made fully available to the scientific community on request from the Eumycetoma Working Group. We hope that this open resource approach will bridge scientific community working with mycetoma from all around the world and lead to a deeper understanding of M. mycetomatis.

Nigrograna mackinnonii, Not Trematosphaeria grisea (syn., Madurella grisea), Is the Main Agent of Black Grain Eumycetoma in Latin America.

Mycetoma, a chronic and mutilating subcutaneous infection recognized by the WHO as a neglected tropical disease, has been reported in >25 countries in Africa, Asia, and South America. In Latin America, Trematosphaeria grisea is assumed to be the prevalent fungal agent. Recent molecular studies have shown that this is an environmental saprobe in Europe, where it is rarely implicated in human diseases. The aim of the present paper is to establish the identity of Latin American cases ascribed to Trematosphaeria grisea Three cases analyzed were caused by Nigrograna mackinnonii Data on an additional 21 strains in the literature revealed that N. mackinnonii rather than T. grisea is responsible for most cases of black grain eumycetoma in Latin America.

Molecular identification of unusual Mycetoma agents isolated from patients in Venezuela.

Mycetoma is a chronic granulomatous, subcutaneous disease endemic in tropical and subtropical countries. It is currently a health problem in rural areas of Africa, Asia and South America. Nine cases of mycetoma were analysed in a retrospective study. All isolates were identified by morphological features. The level of species identification was reached by molecular tools. Definitive identification of fungi was performed using sequence analysis of the ITS of the ribosomal DNA region and the ribosomal large-subunit D1/D2. Identification of actinomycetes was accomplished by the 16S rRNA gene sequence. Six unusual clinical isolates were identified: Aspergillus ustus, Cyphellophora oxyspora, Exophiala oligosperma, Madurella pseudomycetomatis, Nocardia farcinica and Nocardia wallacei. The prevalence of mycetoma in Venezuela remains unknown. This study represents the first report in the literature of mycetoma caused by unusual pathogens identified by molecular techniques.