Rapid and Robust Identification of the Agents of Black-Grain Mycetoma by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

Eumycetoma, a chronic fungal infection endemic in India, Indonesia, and parts of Africa and South and Central America, follows traumatic implantation of saprophytic fungi and frequently requires radical surgery or amputation in the absence of appropriate treatment. Fungal species that can cause black-grain mycetomas include Madurella spp., Falciformispora spp., Trematosphaeria grisea, Nigrograna mackinnonii, Pseudochaetosphaeronema larense, Medicopsis romeroi, and Emarellia spp. Rhytidhysteron rufulum and Parathyridaria percutanea cause similar subcutaneous infections, but these infections lack the draining sinuses and fungal grains characteristic of eumycetoma. Accurate identification of the agents of subcutaneous fungal infection is essential to guide appropriate antifungal therapy. Since phenotypic identification of the causative fungi is often difficult, time-consuming molecular approaches are currently required. In the study described here we evaluated whether matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry might allow the accurate identification of eumycetoma agents and related fungi. A panel of 57 organisms corresponding to 10 different species from confirmed cases of eumycetoma and subcutaneous pedal masses, previously formally identified by PCR amplification and sequencing of internal transcribed spacer 1 (ITS1), was employed. Representative isolates of each species were used to create reference MALDI-TOF spectra, which were then used for the identification of the remaining isolates in a user-blinded manner. Here, we demonstrate that MALDI-TOF mass spectrometry accurately identified all of the test isolates, with 100%, 90.4%, and 67.3% of isolates achieving log scores greater than 1.8, 1.9, and 2.0, respectively. KEYWORDS black-grain mycetoma, Madurella, Pleosporales, Trematosphaeriaceae, Emarellia, Biatriospora, Rhytidhysteron, Medicopsis, Falciformispora, Exophiala, MALDI-TOF, Pleosporales B lack-grain eumycetoma is an ancient, debilitating fungal infection that is endemic in India, Indonesia, and parts of Africa and South America (1). Disease progression is protracted and results from the traumatic implantation of pigmented saprophytic fungi, usually on exposed body sites. The disease is characterized by a chronic progressive destruction of soft tissue and adjoining structures with associated tumefaction and the formation of purosanguineous sinuses that drain fungal grains (2-5). Without appropriate treatment, the infection eventually progresses to involve the skeletal system, and clinical and mycological cure is unlikely without extensive debridement/ amputation (2-5). The extruded grains are either pale or dark (black), depending on the etiological agent (4, 5). The principal etiological agents described to date include Madurella spp. (Sordariales); a host of fungi classified in the Pleosporales, including

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

“…Fraser et al [11] in 2017 developed a MALDI-TOF MS library that successfully identified 13 M. mycetomatis and 2 M. fahalii. This method, however, similar to Sanger sequencing, requires a costly purchase and maintenance that prevents their implementation in lowresource countries.…”

“…Both positive and negative biopsies were cultured on Sabouraud's Glucose Agar (SGA) and the identity of the isolates was established by ITS sequencing. These clinical samples were retrospectively collected and in a context of a blind test set they were assigned with numerical code numbers (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The assessor of the qPCR assay was not aware of the identity of DNA samples (for both DNA samples derived from pure cultures and those obtained from clinical samples).…”

“…Due to the lack of sporulation and other in vitro phenotypic characteristics in Madurella species, morphological assays have limited diagnostic value [4]. Alternatively, sequencing of ribosomal internal transcribed spacer (ITS) as the gold standard technique for species identification, and protein-based approaches such as Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) are regarded as highly reliable identification tools [4,11]. However, long turnaround time, high costs and the requirement for skilled laboratory staff interfere with establishment of sequencing and MALDI-TOF MS in developing countries.…”

DNA sequencing to clarify the taxonomical conundrum of the clinical coelomycetes

“…This technique was used previously, mostly for yeasts, but more recently it has been also adapted for identifying molds. A recent study by Fraser et al also demonstrated the usefulness of this technique for coelomycetes that produce black grain mycetoma.…”

“…Recently, the use of matrix‐assisted laser desorption ionization–time of flight (MALDI‐TOF) mass spectrometry for identifying microorganisms in the clinical laboratory has rapidly become important . This technique was used previously, mostly for yeasts, but more recently it has been also adapted for identifying molds.…”