The genus Madurella: Molecular identification and epidemiology in Sudan.

Eumycetoma (mycotic mycetoma) is the fungal form of mycetoma, a subcutaneous infection occurring in individuals living in endemic areas of the disease. The Sudan is hyperendemic for mycetoma, with the highest incidence being reported from Gezira State, Central Sudan. The present study was conducted at the Gezira Mycetoma Center and aimed to determine the cause of black-grain eumycetoma in the state and describe its epidemiology. Black-grain specimens were collected during the surgical operation and direct detection of the causative agent was performed using M. mycetomatis species-specific PCR and ITS PCR followed by sequencing. Black-grain was reported from 93.3% of all confirmed mycetoma cases (n = 111/119), with a prevalence in young males. Of the 91 samples subjected to direct PCR, 90.1% (n = 82) gave positive results. The predominant species (88.2%) was Madurella mycetomatis. One sample was identified as M. fahalii, one as M. tropicana, and one matched the phytopathogenic species Sphaerulina rhododendricola. The highest endemic zones were Southern Gezira (76.6%) and Northern Sinnar (23.4%). The study confirmed that direct molecular detection on grains provides rapid and specific diagnosis of agents of eumycetoma.

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

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.

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.

Eumicetoma: actualidades y perspectivas.

The eumycetoma is a severely debilitating chronic progressive fungal cutaneous infection. Classic clinical triad is characterized by painless subcutaneous mass, sinus tracts formation and sero-purulent discharge that contain aggregates of fungal hyphae called grains. Any part of the body can have affected, with extension to muscular or bone, even visceral compromised. The eumycetoma is observed in tropical and subtropical countries; In Latin-America, is reported with less frequency. In endemic areas, antibody presence again etiological agents were higher compared with number of people affected, thus it is supposed that individual genetic susceptibility most by exist. Recently, it was reported specific polymorphism in genes CR1, IL-8, NOS2 and chitriosidase, which were associated with development of eumycetoma. The diagnosis is suggested by clinical presentation; the histopathology and microbiology studies, plus radiologic valuation confirmed diagnosis. Madurella mycetomatis is the most informed etiological agent. Using phylogenetic tools new species in genus Madurella were reported; moreover, Trematosphaeria grisea and Pseudallescheria boydii were reclassified. Etiological agent Identification is important, because differences in antifungal susceptibility exist. Eumycetoma treatment includes surgery plus antifungal drugs. Identification of etiological agents is primordial, because antifungal resistance could exist. To development new pharmacological strategies, comprehension of grain formation physiology and drugs effects are necessary.

Phylogenetic findings suggest possible new habitat and routes of infection of human eumyctoma.

Eumycetoma is a traumatic fungal infection in tropical and subtropical areas that may lead to severe disability. Madurella mycetomatis is one of the prevalent etiologic agents in arid Northeastern Africa. The source of infection has not been clarified. Subcutaneous inoculation from plant thorns has been hypothesized, but attempts to detect the fungus in relevant material have remained unsuccessful. The present study aims to find clues to reveal the natural habitat of Madurella species using a phylogenetic approach, i.e. by comparison of neighboring taxa with known ecology. Four species of Madurella were included in a large data set of species of Chaetomium, Chaetomidium, Thielavia, and Papulaspora (n = 128) using sequences of the universal fungal barcode gene rDNA ITS and the partial LSU gene sequence. Our study demonstrates that Madurella species are nested within the Chaetomiaceae, a family of fungi that mainly inhabit animal dung, enriched soil, and indoor environments. We hypothesize that cattle dung, ubiquitously present in rural East Africa, plays a significant role in the ecology of Madurella. If cow dung is an essential factor in inoculation by Madurella, preventative measures may involve the use of appropriate footwear in addition to restructuring of villages to reduce the frequency of contact with etiologic agents of mycetoma. On the other hand, the Chaetomiaceae possess a hidden clinical potential which needs to be explored.

Phylogenetic analysis of the complete mitochondrial genome of Madurella mycetomatis confirms its taxonomic position within the order Sordariales.

BACKGROUND: Madurella mycetomatis is the most common cause of human eumycetoma. The genus Madurella has been characterized by overall sterility on mycological media. Due to this sterility and the absence of other reliable morphological and ultrastructural characters, the taxonomic classification of Madurella has long been a challenge. Mitochondria are of monophyletic origin and mitochondrial genomes have been proven to be useful in phylogenetic analyses. RESULTS: The first complete mitochondrial DNA genome of a mycetoma-causative agent was sequenced using 454 sequencing. The mitochondrial genome of M. mycetomatis is a circular DNA molecule with a size of 45,590 bp, encoding for the small and the large subunit rRNAs, 27 tRNAs, 11 genes encoding subunits of respiratory chain complexes, 2 ATP synthase subunits, 5 hypothetical proteins, 6 intronic proteins including the ribosomal protein rps3. In phylogenetic analyses using amino acid sequences of the proteins involved in respiratory chain complexes and the 2 ATP synthases it appeared that M. mycetomatis clustered together with members of the order Sordariales and that it was most closely related to Chaetomium thermophilum. Analyses of the gene order showed that within the order Sordariales a similar gene order is found. Furthermore also the tRNA order seemed mostly conserved. CONCLUSION: Phylogenetic analyses of fungal mitochondrial genomes confirmed that M. mycetomatis belongs to the order of Sordariales and that it was most closely related to Chaetomium thermophilum, with which it also shared a comparable gene and tRNA order.

New species of Madurella, causative agents of black-grain mycetoma.

A new species of nonsporulating fungus, isolated in a case of black-grain mycetoma in Sudan, is described as Madurella fahalii. The species is characterized by phenotypic and molecular criteria. Multigene phylogenies based on the ribosomal DNA (rDNA) internal transcribed spacer (ITS), the partial ß-tubulin gene (BT2), and the RNA polymerase II subunit 2 gene (RPB2) indicate that M. fahalii is closely related to Madurella mycetomatis and M. pseudomycetomatis; the latter name is validated according to the rules of botanical nomenclature. Madurella ikedae was found to be synonymous with M. mycetomatis. An isolate from Indonesia was found to be different from all known species based on multilocus analysis and is described as Madurella tropicana. Madurella is nested within the order Sordariales, with Chaetomium as its nearest neighbor. Madurella fahalii has a relatively low optimum growth temperature (30°C) and is less susceptible to the azoles than other Madurella species, with voriconazole and posaconazole MICs of 1 µg/ml, a ketoconazole MIC of 2 µg/ml, and an itraconazole MIC of >16 µg/ml. Since eumycetoma is still treated only with azoles, correct species identification is important for the optimal choice of antifungal therapy.

Phenotypic and molecular characterization of Madurella pseudomycetomatis sp. nov., a novel opportunistic fungus possibly causing black-grain mycetoma.

A case of black-grain mycetoma occurring on the lower jaw with an odontogenic origin, which to our knowledge is the first case reported in China, is presented here. The clinical manifestation, histopathological morphology, and microbiological features are described. The new species, Madurella pseudomycetomatis, isolated from the black grains discharged by this patient, was analyzed using sequence data of the multiloci of ribosomal DNA (rDNA) and its ability to ferment carbohydrate as well as morphology. The analyses of the internal transcribed spacer (ITS) region and the D1/D2 hypervariable region of the 28S ribosomal gene sequences support a new species designation. Antifungal susceptibility testing was conducted, indicating that Madurella pseudomycetomatis was highly susceptible to itraconazole, voriconazole, and amphotericin B; moderately susceptible to terbinafine; and resistant to fluconazole and flucytosine.

Genotyping of Madurella mycetomatis by selective amplification of restriction fragments (amplified fragment length polymorphism) and subtype correlation with geographical origin and lesion size.

One of the causative organisms of mycetoma is the fungus Madurella mycetomatis. Previously, extensive molecular typing studies identified Sudanese isolates of this fungus as clonal, but polymorphic genetic markers have not yet been identified. Here, we report on the selective amplification of restriction fragment (AFLP) analysis of 37 Sudanese clinical isolates of M. mycetomatis. Of 93 AFLP fragments generated, 25 were polymorphic, and 12 of these 25 polymorphic fragments were found in a large fraction of the strains. Comparative analysis resulted into a tree, composed of two main (clusters I and II) and one minor cluster (cluster III). Seventy-five percent of the strains found in cluster I originated from central Sudan, while the origin of the strains in cluster II was more heterogeneous. Furthermore, the strains found in cluster I were generally obtained from lesions larger than those from which the strains found in cluster II were obtained (chi-square test for trend, P = 0.03). Among the 12 more commonly found polymorphisms, 4 showed sequence homology with known genes. Marker A7 was homologous to an endo-1,4-beta-glucanase from Aspergillus oryzae, 97% identical markers A12 and B3 matched a hypothetical protein from Gibberella zeae, and marker B4 was homologous to casein kinase I from Danio rerio. The last marker seemed to be associated with strains originating from central Sudan (P = 0.001). This is the first report on a genotypic study where genetic markers which may be used to study pathogenicity in M. mycetomatis were obtained.

Phylogeny and typification of Madurella mycetomatis, with a comparison of other agents of eumycetoma.

The genus Madurella, described for non-sporulating agents of human mycetoma, is proven to be heterogeneous on the basis of rDNA small subunit (SSU) and Internal Transcribed Spacer (ITS) sequencing data. Madurella mycetomatis, the main agent of mycetoma in arid zones of Central and East Africa, probably belongs to the ascomycete order Sordariales. Madurella mycetomatis, the generic type species, is neotypified. Madurella grisea, with worldwide occurrence, is likely to be a member of the order Pleosporales, just as the mycetoma agents of Leptosphaeria, Pseudochaetosphaeronema, and Pyrenochaeta. Neotestudina rosatii belongs to the order Dothideales. Judging from ITS data, M. mycetomatis and N. rosatii are species complexes. The ex-type strain of N. rosatii, from a human mycetome, has an ITS sequence that deviates from that of environmental strains of the species.

Madurella mycetomatis strains from mycetoma lesions in Sudanese patients are clonal.

Molecular diversity among clinical isolates of Madurella mycetomatis, the prime fungal agent of human mycetoma in Sudan, could possibly explain the diverse clinical presentations of this severely debilitating infectious disease. In addition, culture-independent DNA-mediated typing tests need to be developed for this organism, since M. mycetomatis DNA, but not the organism itself, can be identified in soil, the material from which infections are thought to originate. A collection of 38 different clinical M. mycetomatis isolates was characterized by large-scale random amplification of polymorphic DNA using 20 different primer species. These analyses, involving at least 2,600 annealing sites, showed a complete lack of DNA fingerprint variation among the various isolates. From the resulting homogeneous DNA fingerprints, seven fragments were cloned and sequenced, and novel, species-specific PCR restriction fragment length polymorphism (RFLP) tests were designed. The seven PCR RFLP tests were successfully performed on the 38 different M. mycetomatis strains. However, again all M. mycetomatis DNA patterns obtained appeared to be identical, whereas patterns produced using DNAs from other fungal species were clearly discriminatory. These results suggest that there is little genetic variation among clinically relevant M. mycetomatis strains from Sudan. The data tentatively imply that different manifestations of mycetoma are due to differences in host susceptibility rather than differential virulence of the causative agent.

Isolation and identification of Madurella mycetomatis from two cases of black grain mycetoma in Libya.

Two cases of mycetoma of the foot caused by Madurella mycetomatis are reported in which identification of the etiologic agents was confirmed by culture. A Libyan male aged 40 years and a Chadian female aged 30 years, exhibited eumycetoma of the foot 1 and 2 years, respectively, after a local injury. The diagnoses were based on the clinical presentation and confirmed by mycologic analysis of the grains and culture isolation of the etiologic agent. Prior to this report M. mycetomatis has not been recorded in Libya as a cause of eumycetoma.

Potential utility of random amplified polymorphic DNA (RAPD) and restriction endonuclease assay (REA) as typing systems for Madurella mycetomatis.

Two molecular methods were compared, random amplified polymorphic DNA (RAPD) and restriction endonuclease analysis (REA), in order to evaluate their ability to discriminate, and to characterize Madurella mycetomatis strains isolated from human mycetomas in different parts of the world. Both methods were able to cluster the Madurella mycetomatis isolates into the same number of distinct typing groups. However, RAPD, presenting several advantages over REA such as its rapidity, simplicity, and the accessibility for implementation in the laboratory, is a more sensitive and reproducible tool for the study of Madurella mycetomatis epidemiology than REA.