Madurella real-time PCR, a novel approach for eumycetoma diagnosis

Abstract

The genus Madurella comprising four species, M. fahalii, M. mycetomatis, M. pseudomycetomatis, and M. tropicana, represents the prevalent cause of eumycetoma worldwide. The four species are phenotypically similar and cause an invariable clinical picture, but differ markedly in their susceptibility to antifungal drugs, and epidemiological pattern. Therefore, specific identification is required for optimal management of Madurella infection and to reveal proper epidemiology of the species. In this study, a novel multiplex real-time PCR targeting the four Madurella species was developed and standardized. Evaluation of the assay using reference strains of the target and non-target species resulted in 100% specificity, high analytical reproducibility (R2 values >0.99) and a lowest detection limit of 3 pg target DNA. The accuracy of the real-time PCR was further assessed using biopsies from eumycetoma suspected patients. Unlike culture and DNA sequencing as gold standard diagnostic methods, the real-time PCR yielded accurate diagnosis with specific identification of the causative species in three hours compared to one or two weeks required for culture. The novel method reduces turnaround time as well as labor intensity and high costs associated with current reference methods.

Identification of Mycoses in Developing Countries

“…In addition to relatively low sensitivity, simple PCR assays depend on fragment visualization by gel electrophoresis, which can increase the turn-around time and the risk of carry-over contamination. [126][127][128][129][130] TAT, turn-around time; pg, picogram; fg, femtogram; ag, atomgram; NI, Not-indicated. A,B , Sensitivity and specificity was high except for minor cases.…”

Genomics and metagenomics of Madurella mycetomatis, a causative agent of black grain mycetoma in Sudan

“…Better understanding of the genetic structure and diversity of mycetoma causative agents is essential for the development of diagnostic methods and identification of potential drug targets. Whole genome sequencing (WGS) provides a useful tool for this purpose as it provides numerous genetic markers that can be mined for developing diagnostic methods and identifying potential drug markers [24][25][26]. Here, we describe a high-quality genomic assembly of M. mycetomatis, describe genomic diversity of this species in Sudan and perform detailed metagenomic analysis of mycetoma grains from Sudanese patients.…”

Complete Genome Sequence of the Itraconazole Decreased Susceptible Madurella fahalii Type-Strain CBS 129176

“…Madurella species share similar morphology and are non-sporulating. They can only be differentiated to species level by molecular identification methods [ 3 ]. These molecular tools are not widely available in endemic regions and as a result the epidemiology of the different Madurella species remains widely unknown [ 4 , 5 ].…”