Rapid Identification of Black Grain Eumycetoma Causative Agents Using Rolling Circle Amplification

Ahmed, Sarah; Ende, Bert H. G. Gerrits van den; Fahal, Ahmed H.; Sande, Wendy W. J. van de; Hoog, G. Sybren de

doi:10.1371/journal.pntd.0003368

Cited by 38 publications

(38 citation statements)

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“…Although sequencing of the ITS1 region remains the "gold standard" for the identification of many pathogenic fungi, a variety of alternative molecular approaches to the identification of the agents of eumycetoma have been evaluated, including species-specific PCR-restriction fragment length polymorphism analysis (34), rolling circle amplification (35), recombinase polymerase amplification (RPA) (36), and loopmediated isothermal amplification (LAMP) (36). Although several of these approaches have been reported to have a high specificity, their utility in resource-limited settings is reduced by the requirement for skilled laboratory staff, the need for species-specific primer combinations, and high costs (36; reviewed in reference 37).…”

Section: Discussionmentioning

confidence: 99%

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

2017

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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

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Section: Discussionmentioning

confidence: 99%

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

2017

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“…In this study, RPA was evaluated as an alternative isothermal amplification method with potential for use in low-technology laboratory settings. Specific RPA primers were based on the ribosomal ITS region, not only because of the in silico specificity but also because of the high copy number of the ribosomal operon (18). RPA was performed using reagents that were provided as dry pellets, and thus no additional instrumentation was required except for a heating block.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, application of DNA-based diagnostics for direct detection of M. mycetomatis from clinical material is challenging because of the difficulty in obtaining sufficient, purified DNA from the grains. In a previous study, we developed rolling-circle amplification (RCA) for specific identification of agents of blackgrain eumycetoma (18). The technique is highly specific, rapid, and cost-effective, but culturing of the pathogen and PCR ampli-fication are still required (18).…”

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confidence: 99%

“…In a previous study, we developed rolling-circle amplification (RCA) for specific identification of agents of blackgrain eumycetoma (18). The technique is highly specific, rapid, and cost-effective, but culturing of the pathogen and PCR ampli-fication are still required (18). Therefore, the purpose of the present study was to develop more simple isothermal amplification techniques that can be used for direct detection of M. mycetomatis from clinical specimens.…”

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Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

et al. 2015

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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. Madurella mycetomatis is the most common causative agent of human eumycetoma worldwide, as evidenced in a recent review and meta-analysis on the disease (1). This highly morbid and destructive infection mainly affects individuals of low socioeconomic status in arid climate zones in the tropics (2). Although mycetoma was introduced into the medical literature in the 18th century, it still remains neglected and it has recently been recognized by the World Health Organization as a neglected tropical condition (2-4). The prevalence of the infection in villages in Sudan where the condition is endemic is estimated to be 14 per 1,000 inhabitants (5). At the Mycetoma Research Centre, Khartoum, Sudan, the main reference center in the country, the annual incidence of new cases is Ͼ300 (6). Despite this high incidence, appropriate diagnoses and therapies for this disease are still lacking (7).Mycetoma is a subcutaneous infection that mainly affects the extremities and leads to massive disfigurement and disability (8, 9). The infection is thought to occur as a result of transcutaneous trauma, which allows insertion of the causative agent into skin and subcutaneous tissue. A distinctive and unique feature of mycetoma agents is their ability to form compact grains in infected tissue that will be discharged through the sinuses (10). Since mycetoma can be caused by both bacteria (actinomycetoma) and fungi (eumycetoma), the discharged grains are different (11). Black grains are caused by fungi, while actinomycetoma grains are white, yellow, or red (2, 10, 12). Molecular phylogeny of the eumycetoma causative agents has shown that the black-grain agents belong to entirely different fungal species, and these fungi differ in their antifungal susceptibility profiles (13,14). Therefore, precise identification of the causative agent is necessary.Current techniques for mycetoma diagnostics mostly co...

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“…The molecular tests are based on ITS sequences and developed for Falciformispora senegalensis, F. tompkinsii, Madurella fahalii, M. mycetomatis, M. pseudomycetomatis, M. tropicana, Medicopsis romeroi, and Trematosphaeria grisea [12].…”

Section: Molecular Techniques For Diagnosismentioning

confidence: 99%

Black Grain Eumycetoma Revisited: Enhanced Fungal Isolation by Use of a Novel Technique

Hazra¹

2017

JBMOA

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Background: Dematiaceous fungi or brown-black fungi distributed worldwide are being increasingly recognized as pathogens of importance. Found in soil, they have unique pathogenic mechanisms owing to the presence of melanin in their cell walls, which imparts the characteristic dark color to their spores and hyphae. Eumycetoma is a common, chronic, granulomatous infection that is present worldwide and endemic in tropical and subtropical regions. Black grain eumycetoma accounts for about 95% of eumycetomas in India. Isolation of the causative fungus from the coloured grains in the laboratory is an arduous task resulting in empirical treatment. At present, clear evidence based treatment recommendations are unavailable and it becomes pertinent to identify, isolate and speciate the causative fungus so that appropriate treatment can be instituted for complete resolution.Objective: This article revisits black grain mycetoma in the larger spectrum of phaeohyphomycosis, describes four patients with black grain mycetoma and elaborates on a novel technique developed in our laboratory for assured isolation of fungi from eumycotic "grains".Clinical management: All four patients gave a clinical history of mycetoma, having observed intermittent discharge of black grains from lesions. Processing of grains was comprehensively carried out with direct KOH preparation, mycological analysis of the grains and histology. All patients were started on oral itraconazole 200-400 mg twice daily, post wide excision of soft tissue that resulted in near complete regression of the lesion. Summary:Timely identification of the fungal isolate based on grain morphology and culture in a setting where molecular setup and techniques are not available is crucial to ensure guided therapy and best clinical outcomes for the patient.

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Rapid Identification of Black Grain Eumycetoma Causative Agents Using Rolling Circle Amplification

Cited by 38 publications

References 29 publications

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

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

Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

Black Grain Eumycetoma Revisited: Enhanced Fungal Isolation by Use of a Novel Technique

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