Epidemiological observations and management challenges in extrapedal mycetoma: A three-decade review of 420 cases.

Mycetoma is a serious, destructive, disfiguring chronic granulomatous inflammatory disease affecting the subcutaneous tissues that spread to involve the skin, deep tissues and bone. The disease predominately affects the limbs, and extrapedal mycetoma is rarely reported. The reported extrapedal ones are characterised by high morbidity and mortality. This communication reports on 420 patients with extrapedal mycetoma seen and managed at the Mycetoma Research Centre (MRC), University of Khartoum, between January 1991 and December 2021. In this descriptive, cross-sectional, hospital-based study, the electronic records of all mycetoma-confirmed patients seen during the study period were carefully and meticulously reviewed. The confirmed patients with extrapedal mycetoma were included in this study. The study included 420 patients with extrapedal mycetoma, 298 (70.7%) had eumycetoma, and 122 (29.3%) had actinomycetoma. There were 343 male patients (81.7%) and 77 (18.3%) females, with a male-to-female ratio of 4:1. Their ages ranged between 1.5 and 95 years, with a median of 28 years. Most of the patients were students and farmers. The majority of patients were from El Gezira, North Kordofan, and the White Nile States. Mycetoma was painful in 21%, and a family history of mycetoma was recorded in 11.5% of patients. The buttocks (37.9%) and head and neck (16.9%) were affected most. Less frequently affected sites were the trunk and back (12%) each, abdominal and chest walls (4.5%) each and loin (1%). The prominent clinical presentation findings were multiple sinuses discharging grains (55%), massive swellings (46%), and lymphadenopathy (11.5%). Less commonly observed clinical findings were local hyperhidrosis (5.3%) and dilated tortuous veins close to mycetoma lesions (0.5%). The study showed that 204 patients (48.6%) had clinical improvement in terms of decreased lesion size and healing of sinuses following medical therapy. Sixty-six patients (15.7%) had no noticeable improvement. The lesion continued progressing despite treatment in 44 patients (10.5%). In the study, 118 patients were on regular follow-up, and in this group, a cure was documented in 25 patients (21.1%) with eumycetoma and 23 (19.4%) with actinomycetoma. Post-operative recurrence among eumycetoma patients was 40%, with a 1% mortality rate. The treatment outcome was unsatisfactory, characterised by a low cure rate, high recurrence (40%) and follow-up dropout (57%) rates. This emphasises the importance of early case detection and management, objective health education programmes and thorough patient counselling to urge people to seek treatment early and reduce dropouts.

Sandalwood Oils of Different Origins Are Active In Vitro against Madurella mycetomatis, the Major Fungal Pathogen Responsible for Eumycetoma.

In the search for new bioactive agents against the infectious pathogen responsible for the neglected tropical disease (NTD) mycetoma, we tested a collection of 27 essential oils (EOs) in vitro against Madurella mycetomatis, the primary pathogen responsible for the fungal form of mycetoma, termed eumycetoma. Among this series, the EO of Santalum album (Santalaceae), i.e., East Indian sandalwood oil, stood out prominently with the most potent inhibition in vitro. We, therefore, directed our research toward 15 EOs of Santalum species of different geographical origins, along with two samples of EOs from other plant species often commercialized as "sandalwood oils". Most of these EOs displayed similar strong activity against M. mycetomatis in vitro. All tested oils were thoroughly analyzed by GC-QTOF MS and most of their constituents were identified. Separation of the sandalwood oil into the fractions of sesquiterpene hydrocarbons and alcohols showed that its activity is associated with the sesquiterpene alcohols. The major constituents, the sesquiterpene alcohols (Z)-α- and (Z)-ß-santalol were isolated from the S. album oil by column chromatography on AgNO3-coated silica. They were tested as isolated compounds against the fungus, and (Z)-α-santalol was about two times more active than the ß-isomer.

Naphthylisoquinoline alkaloids: novel agents against the causative pathogens of eumycetoma and actinomycetoma-en route to broad-spectrum antimycetomal drugs.

Mycetoma is a devastating neglected tropical infection of the subcutaneous tissues. It is caused by fungal and bacterial pathogens recognized as eumycetoma and actinomycetoma, respectively. Mycetoma treatment involves diagnosing the causative microorganism as a prerequisite to prescribing a proper medication. Current therapy of fungal eumycetoma causative agents, such as Madurella mycetomatis, consists of long-term antifungal medication with itraconazole followed by surgery, yet with usually unsatisfactory clinical outcomes. Actinomycetoma, on the contrary, usually responds to treatment with co-trimoxazole and amikacin. Therefore, there is a pressing need to discover novel broad-spectrum antimicrobial agents to circumvent the time-consuming and costly diagnosis. Using the resazurin assay, a series of 23 naphthylisoquinoline (NIQ) alkaloids and related naphthoquinones were subjected to in vitro screening against two fungal strains of M. mycetomatis and three bacterial strains of Actinomadura madurae and A. syzygii. Seven NIQs, mostly dimers, showed promising in vitro activities against at least one strain of the mycetoma-causative pathogens, while the naphthoquinones did not show any activity. A synthetic NIQ dimer, 8,8'''-O,O-dimethylmichellamine A (18), inhibited all tested fungal and bacterial strains (IC50 = 2.81-12.07 µg/mL). One of the dimeric NIQs, michellamine B (14), inhibited a strain of M. mycetomatis and significantly enhanced the survival rate of Galleria mellonella larvae infected with M. mycetomatis at concentrations of 1 and 4 µg/mL, without being toxic to the uninfected larvae. As a result, broad-spectrum dimeric NIQs like 14 and 18 with antimicrobial activity are considered hit compounds that could be worth further optimization to develop novel lead antimycetomal agents.

Protoplast-mediated transformation of Madurella mycetomatis using hygromycin resistance as a selection marker.

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M. mycetomatis is able to cause this disease. However, the genetic toolbox for M. mycetomatis is limited. To date, no method is available to genetically modify M. mycetomatis. In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M. mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M. mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies.

Actinomycetoma by Cellulosimicrobium cellulans in a Young Man from Guinea-Bissau: Short Literature Review Regarding a Case Report.

Mycetoma is caused by the subcutaneous inoculation of filamentous fungi or aerobic filamentous bacteria. Cellulosimicrobium cellulans is a gram-positive bacterium from the order Actinomycetales that rarely causes human disease. The diagnosis is based on the clinical presentation and identification of the causative microorganism. We present a short literature review regarding the case report of a young man diagnosed with actinomycetoma due to Cellulosimicrobium cellulans and who received treatment with an association of amikacin and sulfamethoxazole/ trimethoprim (Welsh regimen).

Mycetoma case series in Somalia.

Mycetoma is a suppurative chronic bacterial or fungal disease inoculated into the body by minor trauma which may penetrate from subcutaneous tissue to bone. Although the lower extremities are most commonly affected, rare forms can also be seen from time to time. The diagnostic triad of swelling in the affected area, multiple sinus formation, and purulent discharge with grains are typical. Definitive diagnosis is made by isolation of the causative pathogen, radiologic imaging, and histopathologic examination. Antifungal and antibacterial options are applied together with surgery. Our aim in this case series is to report and analyze 10 rare cases of mycetoma.

Invasive Eumycotic mycetoma of the nasal septum in a post-dialysis patient: A case report.

BACKGROUND: Mycetoma is a slowly progressive chronic granulomatous disease of the skin, subcutaneous tissue, and underlying or adjacent cartilage or bone. Most commonly involves the foot region. Other parts such as the knee, arm, leg, head, neck, thigh, perineum, chest, abdominal walls, facial bones, mandible, paranasal sinuses, eyelid, vulva, orbit, and scrotum are seldom affected. METHODS: This is a rare presentation of Eumycotic mycetoma involving the nasal septum. Surgical debridement is done under local anesthesia. Histopathological examination of debrided specimen guided in the diagnosis and treatment. RESULTS: Histopathological examination is the one that confirms the diagnosis and rules out the other granulomatous conditions and fungal rhinitis causing septal perforation. CONCLUSIONS: In an immunocompromised state, we know that mucormycosis and zygomycosis are known to cause aggressive complications like orbital invasion and palatal perforation by vascular route. However, other fungal infections also can lead to septal perforations whenever there is lessened resistance by the mucosal barrier due to trauma (nasal intubations).

A Novel Report of mycetoma with Spinal Spread due to Madurella fahalli from India.

Eumycetoma caused by Madurella fahalii, a drug-resistant fungus, has never been reported in India. Here, we describe a fatal case of eumycetoma with spinal involvement due to M. fahalii for the first time in India.

The combination of manogepix and itraconazole is synergistic and inhibits the growth of Madurella mycetomatis in vitro but not in vivo.

Mycetoma is a neglected tropical disease commonly caused by the fungus Madurella mycetomatis. Standard treatment consists of extensive treatment with itraconazole in combination with surgical excision of the infected tissue, but has a low success rate. To improve treatment outcomes, novel treatment strategies are needed. Here, we determined the potential of manogepix, a novel antifungal agent that targets the GPI-anchor biosynthesis pathway by inhibition of the GWT1 enzyme. Manogepix was evaluated by determining the minimal inhibitory concentrations (MICs) according to the CLSI-based in vitro susceptibility assay for 22 M. mycetomatis strains and by in silico protein comparison of the target protein. The synergy between manogepix and itraconazole was determined using a checkerboard assay. The efficacy of clinically relevant dosages was assessed in an in vivo grain model in Galleria mellonella larvae. MICs for manogepix ranged from <0.008 to >8 mg/l and 16/22 M. mycetomatis strains had an MIC ≥4 mg/ml. Differences in MICs were not related to differences observed in the GWT1 protein sequence. For 70% of the tested isolates, synergism was found between manogepix and itraconazole in vitro. In vivo, enhanced survival was not observed upon admission of 8.6 mg/kg manogepix, nor in combination treatment with 5.7 mg/kg itraconazole. MICs of manogepix were high, but the in vitro antifungal activity of itraconazole was enhanced in combination therapy. However, no efficacy of manogepix was found in an in vivo grain model using clinically relevant dosages. Therefore, the therapeutic potential of manogepix in mycetoma caused by M. mycetomatis seems limited.

Treatment of Madurella mycetomatis-caused mycetoma consists of extensive exposure to antifungals and surgery. To improve therapy, we evaluated manogepix, a novel antifungal agent, as a therapeutic option against M. mycetomatis. Our findings suggest limited therapeutic potential for manogepix.

Case report: Mycetoma caused by Gordonia soli.

Mycetoma is a neglected tropical disease caused by fungi (eumycetoma) or bacteria (actinomycetoma), with high morbidity. Gordonia spp. are gram-positive bacteria that have previously been reported to cause mycetoma. Here, we report a case of Gordonia soli (initially misidentified as Nocardia spp.) as the etiological agent of actinomycetoma in a 64-year-old patient. After a literature search in the Cochrane Library, LILACS, SciELO, MEDLINE, PubMed, and PubMed Central databases, we concluded that this is the first case report of mycetoma caused by Gordonia soli. The current case highlights the importance of microbiological diagnosis of mycetoma and the challenges in its management.

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent.

BACKGROUND: Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma. PRINCIPAL FINDINGS: We successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas. CONCLUSION: In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.

First report on mycetoma in Turkana County-North-western Kenya.

Mycetoma is one of the six Neglected Tropical Diseases that are prevalent in Turkana County (northwest Kenya). The aim of the study was to estimate the prevalence of mycetoma in the county, as well as to describe the main causative agents involved in the disease using methods affordable locally. Based on the data collected by the team of cooperative medicine Cirugia en Turkana (Surgery in Turkana), a specific study for mycetoma was started during the 16th humanitarian medicine campaign in February 2019. Patients with suspected mycetoma were studied at the Lodwar County Referral Hospital (LCRH). After informing the patient and getting their consent, the lesions were examined and sampled (mainly by biopsy) and clinical data were recorded. Samples were washed in sterile saline solution and cut in fragments. Some of these were inoculated on Sabouraud Dextrose Agar, Malt Extract Agar, and diluted Nutrient Agar plates. One fragment of each sample was used for DNA extraction. The DNA and the rest of the fragments of samples were kept at -20°C. All cultures were incubated at room temperature at the LCRH laboratory. The DNA obtained from clinical samples was submitted to PCR amplification of the ITS-5.8S and the V4-V5 16S rRNA gene region, for the detection and identification of fungi and bacteria respectively. From February 2019 till February 2022, 60 patients were studied. Most of them were men (43, 74,1%) between 13 and 78 y.o. (mean age 37). Half of the patients were herdsmen but, among women 40% (6) were housewives and 26.7% (4) charcoal burners. Lesions were mainly located at the feet (87.9%) and most of the patients (54; 93.1%) reported discharge of grains in the exudate, being 27 (46.6%) yellow or pale colored and 19 (32.8%) of them dark grains. Culture of clinical samples yielded 35 fungal and bacterial putative causative agents. Culture and molecular methods allowed the identification of a total of 21 causative agents of mycetoma (39.6% of cases studied). Most of them (17) corresponded to fungi causing eumycetoma (80.9%) being the most prevalent the genus Madurella (7; 41.2%), with two species involved (M. mycetomatis and M. fahalii), followed by Aspergillus (2; 11.8%). Other minority genera detected were Cladosporium, Fusarium, Acremonium, Penicillium, and Trichophyton (5.9% each of them). Actinobacteria were detected in 19.1% of samples, but only Streptomyces somaliensis was identified as a known agent of mycetoma, the rest being actinobacteria not previously described as causative agents of the disease, such as Cellulosimicrobium cellulans detected in two of the patients. Although Kenya is geographically located in the mycetoma belt, to our knowledge this is the first report on mycetoma in this country from 1973, and the first one for Turkana County.

A Falciformispora senegalensis grain model in Galleria mellonella larvae.

Eumycetoma is a subcutaneous implantation mycosis often found in the foot. One of the hallmarks of eumycetoma is the formation of grains. These grains are either black or white, and the consistency and morphology differs per causative agent. The two most common causative agents of black-grain eumycetoma are Madurella mycetomatis and Falciformispora senegalensis. Since grains cannot be formed in vitro, in vivo models are needed to study grain formation. Here, we used the invertebrate Galleria mellonella to establish an in vivo grain model for F. senegalensis. Three different F. senegalensis strains were selected, and four different inocula were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 10 mg/larvae. Larval survival was monitored for 10 days. Grain formation was studied macroscopically and histologically. The efficacy of antifungal therapy was determined for itraconazole, amphotericin B, and terbinafine. A concentration of 10 mg F. senegalensis per larva was lethal for the majority of the larvae within 10 days. At this inoculum, grains were formed within 24 h after infection. The grains produced in the larvae resembled those formed in human patients. Amphotericin B given at 1 mg/kg 4 h, 28 h, and 52 h after infection prolonged larval survival. No enhanced survival was noted for itraconazole or terbinafine. In conclusion, we developed a F. senegalensis grain model in G. mellonella larvae in which grains were formed that were similar to those formed in patients. This model can be used to monitor grain formation over time and study antifungal efficacy.

Within eumycetoma lesions, the causative agents are embedded in grains. However, the grains differ per causative agent. In this study, we developed a grain model of Falciformispora senegalensis in the larvae of Galleria mellonella. This model can be used in the future to study the efficacy of novel antifungal agents.

Linezolid: a safer and effective substitute to aminoglycoside in the treatment of actinomycetoma by Nocardia species.

Actinomycetoma is chronic, suppurative, granulomatous infection caused by bacteria and requires prolonged antibiotic therapy preferrably in combinations. Nephrotoxicity is a common side effect of aminoglycosides used in the management of actinomycetoma. We report here two cases of actinomycetoma due to Nocardia species who received linezolid as a substitute to aminoglycosides after developing nephrotoxicity.

Fatal dermatophytic pseudomycetoma in a patient with non-HIV CD4 lymphocytopenia.

Dermatophytic pseudomycetoma is a rare invasive infection, involving both immunocompetent and immunocompromised individuals. Since the discovery of inherited immune disorders such as the impairment of CARD9 gene, extended dermatophyte infections are mostly ascribed to any of these host factors. This study is to present and explore the potential causes in a fatal dermatophytic pseudomycetoma patient. We present a chronic and deep pseudomycetoma caused by the common dermatophyte Microsporum canis which ultimately led to the death of the patient. Mycological examination, genetic studies and host immune responses against fungi were performed to explore the potential factors. The patient had decreased lymphocyte counts with significantly reduced CD4+ T cells, although all currently known genetic parameters proved to be normal. Through functional studies, we demonstrated that peripheral blood mononuclear cells from the patient showed severe impairment of adaptive cytokine production upon fungus-specific stimulation, whereas innate immune responses were partially defective. This is, to our knowledge, the first report of fatal dermatophytic pseudomycetoma in a patient with non-HIV CD4 lymphocytopenia, which highlights the importance of screening for immune deficiencies in patients with deep dermatophytosis.

Antifungal Activity of Natural Naphthoquinones and Anthraquinones against Madurella mycetomatis.

Eumycetoma, the fungal form of the neglected tropical disease mycetoma, is a crippling infectious disease with low response rates to currently available antifungal drugs. In this study, a series of natural naphthoquinones and anthraquinones was evaluated for their activity against Madurella mycetomatis, which is the most common causative agent of eumycetoma. The metabolic activity of Madurella mycetomatis as well as the viability of Galleria mellonella larvae upon treatment with quinones was investigated. Several hydroxy-substituted naphthoquinones exhibited activity against Madurella mycetomatis. In particular, naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) was identified as a considerably active antifungal compound against Madurella mycetomatis (IC50 =1.4 µM), while it showed reduced toxicity to Galleria mellonella larvae, which is a well-established in vivo invertebrate model for mycetoma drug studies.

Genetic variability of the 16S rRNA gene of Nocardia brasiliensis, the most common causative agent of actinomycetoma in Latin America and the Caribbean.

Mycetoma is a neglected tropical disease (NTD) declared by the World Health Organization (WHO) in 2016. It is characterized by the progressive growth of nodules and granulomatous lesions on the legs, arms, and trunk. It is potentially disfiguring and causes disability or amputations in working-age people from marginalized areas. The causative agents can be fungi (eumycetoma) or actinobacteria (actinomycetoma), the latter being the most common in America and Asia. Nocardia brasiliensis is the most important causal agent of actinomycetoma in the Americas. Taxonomic problems have been reported when identifying this species, so this study aimed to detect the 16S rRNA gene variations in N. brasiliensis strains using an in silico enzymatic restriction technique. The study included strains from clinical cases of actinomycetoma in Mexico, isolated from humans and previously identified as N. brasiliensis by traditional methods. The strains were characterized microscopically and macroscopically, then subjected to DNA extraction and amplification of the 16S rRNA gene by PCR. The amplification products were sequenced, and consensus sequences were constructed and used for genetic identification and in silico restriction enzyme analysis with the New England BioLabs® NEBcutter program. All study strains were molecularly identified as N. brasiliensis; however, in silico restriction analysis detected a diversity in the restriction patterns that were finally grouped and subclassified into 7 ribotypes. This finding confirms the existence of subgroups within N. brasiliensis. The results support the need to consider N. brasiliensis as a complex species.

Chronic Painless, Multiple Papulo-Nodular Skin Lesion at Foot Resembles Mycetoma Infection (Madura Foot): A Case Report.

Madura foot is a chronic, progressively destructive bacterial or fungal infection of the subcutaneous tissues, which may affect skin, muscle, and bone. It is endemic in tropical and subtropical areas known as the "Mycetoma belt" between latitude 30°N and 15°S. Mycetoma is prevalent in the poor population living in remote areas of developing countries that lack proper reporting system and management. The World Health Organization recognized mycetoma as a neglected tropical disease back in 2016. Though the diagnosis is challenging, early detection and proper treatment can reduce morbidity and provide a promising outcome. We report a case of chronic painless, multiple papulo-nodular skin lesions at the foot that resembles mycetoma infection which was detected early and recovered with a satisfactory outcome with proper treatment.

Clinical triad with multi-biopsy histopathology as a reliable diagnostic marker of mycetomas: a retrospective review from a tertiary care center.

BACKGROUND: Mycetoma is a neglected tropical infectious disease which runs a prolonged and protracted course. Microbiological confirmation is diagnostic yet unreliable due to poor sensitivity and variable availability of culture facilities in resource poor settings. METHODS: A retrospective review was performed on electronic records (histopathology, microbiology, and radiology) of all patients who underwent skin biopsies with mycetoma as one of the clinical differential diagnoses from year 2016 to 2020. RESULTS: Out of 73 patients biopsied with a differential of mycetoma, 42 fit the clinical triad of swelling-sinuses-granules. After clinical, microbiological, pathological, and radiological correlation, 31 cases were of eumycetoma and seven were of actinomycetoma. Mean patient age was 37.58 ± 13.8 years with a male to female ratio 2.45 : 1 and mean disease duration of 11.31 ± 10.9 years. Histopathological findings revealed fungal hyphae in 18 cases and gram-positive bacteria in six cases. Fungal culture was positive in 13 cases with the three commonest organisms being Madurella mycetomatis in five cases, Fusarium and Aspergillus nidulans in two cases each. X-ray changes of soft tissue, bones, and joints were seen in 25 cases, and "dot-in-circle" sign was seen in eight of nine MRIs. CONCLUSION: Eumycetoma was more common than actinomycetoma in our setup, ratio being 4.43 : 1. A clinical triad of swelling, multiple sinuses and grainy discharge with any one diagnostic support (histopathology/radiology) is sufficient to make a definitive diagnosis of mycetoma in the absence of microbiological identification.