The development of a novel diagnostic PCR for Madurella mycetomatis using a comparative genome approach

Abstract

Background Eumycetoma is a neglected tropical disease most commonly caused by the fungus Madurella mycetomatis. Identification of eumycetoma causative agents can only be reliably performed by molecular identification, most commonly by species-specific PCR. The current M. mycetomatis specific PCR primers were recently discovered to cross-react with Madurella pseudomycetomatis. Here, we used a comparative genome approach to develop a new M. mycetomatis specific PCR for species identification. Methodology Predicted-protein coding sequences unique to M. mycetomatis were first identified in BLASTCLUST based on E-value, size and presence of orthologues. Primers were then developed for 16 unique sequences and evaluated against 60 M. mycetomatis isolates and other eumycetoma causing agents including the Madurella sibling species. Out of the 16, only one was found to be specific to M. mycetomatis. Conclusion We have discovered a predicted-protein coding sequence unique to M. mycetomatis and have developed a new species-specific PCR to be used as a novel diagnostic marker for M. mycetomatis.

Ultrasound‐guided fine‐needle aspiration cytology significantly improved mycetoma diagnosis

“…The extracted genomic DNA was amplified using pan fungal primers ITS4 and ITS5 and M. mycetomatis specific primers 26.1a (5 0 -AATGAGTTGGGCTTTAACGG-3 0 ) and 28.3a (5 0 -TCCCG GTAGTGTAGTGTCCCT-3 0 ) 17 and Mmy-Fw (5 0 -TCTCCTGTC CTACGACATCTGTGG-3 0 ) and Mmy-Rv (5 0 -TTCCTCACCTC CCAGCCCTTT-3 0 ). 10 In each PCR reaction, 1 lL genomic DNA; 1 lL forward primer (100 pmol/lL); 1 lL reverse primer (100 pmol/ll) and 20 lL distilled water were added to freezedried Maxime TM PCR PreMix (i-Taq) (Intron, South Korea). The PCR reaction for the pan-fungal PCR and the M. mycetomatis specific PCR using primers 26.1a and 28.3a was performed in the Aeris PCR system (Esco scientific, Singapore) with a 4 min primary denaturation step at 95°C followed by 40 cycles consisting of a 30 s denaturation step at 95°C, a 30 s annealing step at 55°C and a 1 min extension step at 72°C.…”

“…Currently, the recommended techniques for the identification of mycetoma causative agents are culturing of grains and histopathological examination of the deep surgical biopsies 8,9 . More recently, in certain specialized centres, molecular identification by PCR is in use 10,11 . However, all these techniques are invasive, require good and safe anaesthesia, are time‐consuming and are associated with certain complications such as bleeding, sepsis and local disease spread 12 .…”

“…8,9 More recently, in certain specialized centres, molecular identification by PCR is in use. 10,11 However, all these techniques are invasive, require good and safe anaesthesia, are time-consuming and are associated with certain complications such as bleeding, sepsis and local disease spread. 12 Additionally, these procedures are not suitable for epidemiological surveys or field-friendly; and hence, patients need to travel to more specialized centres for disease confirmation and treatment.…”

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.…”

Epidemiological cut‐off values for itraconazole and ravuconazole for Madurella mycetomatis, the most common causative agent of mycetoma

“…DNA was extracted using the modified ZR Fungal/Bacterial DNA MicroPrep™ kit (Zymo Research) as previously described 15 . The identity of the isolates was established by amplifying and sequencing the internally transcribed (ITS) spacer region as previously described 16–20 . The genetic diversity was determined by using the Mmy STR typing assay according to the previously published protocol 20 …”