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.

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.

Madurella mycetomatis grains within a eumycetoma lesion are clonal.

Eumycetoma is a neglected tropical infection of the subcutaneous tissue, characterized by tumor-like lesions and most commonly caused by the fungus Madurella mycetomatis. In the tissue, M. mycetomatis organizes itself in grains, and within a single lesion, thousands of grains can be present. The current hypothesis is that all these grains originate from a single causative agent, however, this hypothesis was never proven. Here, we used our recently developed MmySTR assay, a highly discriminative typing method, to determine the genotypes of multiple grains within a single lesion. Multiple grains from surgical lesions obtained from 11 patients were isolated and genotyped using the MmySTR panel. Within a single lesion, all tested grains shared the same genotype. Only in one single grain from one patient, a difference of one repeat unit in one MmySTR marker was noted relative to the other grains from that patient. We conclude that within these lesions the grains originate from a single clone and that the inherent unstable nature of the microsatellite markers may lead to small genotypic differences. LAY ABSTRACT: In lesions of the implantation mycosis mycetoma many Madurella mycetomatis grains are noted. It was unknown if grains arose after implantation of a single isolate or a mixture of genetically diverse isolates. By typing the mycetoma grains we showed that all grains within a single lesion were clonal and originated from a single isolate.

In depth search of the Sequence Read Archive database reveals global distribution of the emerging pathogenic fungus Scedosporium aurantiacum.

Scedosporium species are emerging opportunistic fungal pathogens causing various infections mainly in immunocompromised patients, but also in immunocompetent individuals, following traumatic injuries. Clinical manifestations range from local infections, such as subcutaneous mycetoma or bone and joint infections, to pulmonary colonization and severe disseminated diseases. They are commonly found in soil and other environmental sources. To date S. aurantiacum has been reported only from a handful of countries. To identify the worldwide distribution of this species we screened publicly available sequencing data from fungal metabarcoding studies in the Sequence Read Archive (SRA) of The National Centre for Biotechnology Information (NCBI) by multiple BLAST searches. S. aurantiacum was found in 26 countries and two islands, throughout every climatic region. This distribution is like that of other Scedosporium species. Several new environmental sources of S. aurantiacum including human and bovine milk, chicken and canine gut, freshwater, and feces of the giant white-tailed rat (Uromys caudimaculatus) were identified. This study demonstrated that raw sequence data stored in the SRA database can be repurposed using a big data analysis approach to answer biological questions of interest. LAY SUMMARY: To understand the distribution and natural habitat of S. aurantiacum, species-specific DNA sequences were searched in the SRA database. Our large-scale data analysis illustrates that S. aurantiacum is more widely distributed than previously thought and new environmental sources were identified.

Inhibiting DHN- and DOPA-melanin biosynthesis pathway increased the therapeutic value of itraconazole in Madurella mycetomatis infected Galleria mellonella.

Eumycetoma is a neglected tropical disease, and Madurella mycetomatis, the most common causative agent of this disease forms black grains in hosts. Melanin was discovered to be one of the constituents in grains. Melanins are hydrophobic, macromolecular pigments formed by oxidative polymerisation of phenolic or indolic compounds. M. mycetomatis was previously known to produce DHN-melanin and pyomelanin in vitro. These melanin was also discovered to decrease M. mycetomatis's susceptibility to antifungals itraconazole and ketoconazole in vitro. These findings, however, have not been confirmed in vivo. To discover the melanin biosynthesis pathways used by M. mycetomatis in vivo and to determine if inhibiting melanin production would increase M. mycetomatis's susceptibility to itraconazole, inhibitors targeting DHN-, DOPA- and pyomelanin were used. Treatment with DHN-melanin inhibitors tricyclazole, carpropamid, fenoxanil and DOPA-melanin inhibitor glyphosate in M. mycetomatis infected Galleria mellonella larvae resulted in presence of non-melanized grains. Our finding suggested that M. mycetomatis is able to produce DOPA-melanin in vivo. Inhibiting DHN-melanin with carpropamid in combination with the antifungal itraconazole also significantly increased larvae survival. Our results suggested that combination treatment of antifungals and melanin inhibitors can be an alternative treatment strategy that can be further explored. Since the common black-grain eumycetoma causing agents uses similar melanin biosynthesis pathways, this strategy may be applied to them and other eumycetoma causative agents. LAY SUMMARY: Melanin protects fungi from environmental stress and antifungals. We have discovered that Madurella mycetomatis produces DHN-, pyomelanin and DOPA-melanin in vivo. Inhibiting M. mycetomatis DHN-melanin biosynthesis increases therapeutic value of the antifungal itraconazole in vivo.

Eumycetoma and disseminated phaeohyphomycosis in a Sumatran tiger.

A 16-y-old female Sumatran tiger (Panthera tigris sumatrae) was evaluated for hyporexia. Examination revealed chronic kidney disease and a large subcutaneous axillary mass with draining tracts that contained numerous small black grains. Histologic examination revealed the presence of intralesional fungal hyphae. Persistent hyporexia and pyogranulomatous disease, as well as progressive cachexia and azotemia occurred despite treatment, and euthanasia was performed. Disseminated phaeohyphomycosis was diagnosed on postmortem examination, additionally affecting various lymph nodes, the nasal cavity, mesenteric adipose tissue, abdominal aorta, pericardium, and kidney. Fungal culture from a deep-tissue sample isolated a pure growth of Curvularia sp., a dematiaceous opportunistic fungus able to cause eumycetomas and/or phaeohyphomycosis. Phaeohyphomycosis is a rare but emerging condition, not previously reported as disseminated disease in an exotic carnivore, to our knowledge. Aggressive systemic antifungal treatment was unsuccessful, likely complicated by diagnostic challenges and concurrent renal disease. The presence of a swelling with abundant grains exiting draining tracts should direct clinicians to the diagnosis of a mycetoma, warranting early and aggressive treatment.

Successful medical treatment of an Aspergillus terreus mycetoma of the nostril/lip in a 16-year-old Fjord pony gelding with pituitary pars intermedia dysfunction.

BACKGROUND: Mycetoma is a chronic, proliferative lesion of cutaneous/subcutaneous tissue characterized by draining tracts and granules in the discharge caused by actinomycetes (actinomycetoma) or filamentous fungi (eumycotic mycetoma). OBJECTIVES: This case report describes the unusual finding of a cutaneous mycetoma of the lateral wing of the right nostril in a gelding. ANIMAL: A 16-year-old Fjord gelding with suspected pituitary pars intermedia dysfunction (PPID) was presented for evaluation of a nonpainful, firm and raised mass involving the lateral wing of the right nostril and the lip. METHODS AND RESULTS: Cytological examination of the mass showed marked pyogranulomatous inflammation and histopathological examination revealed a fungal mycetoma. Fungal culture identified the causative organism as Aspergillus terreus, which is not known for its propensity to cause either dermal granulomas or mycetoma in domestic animals. Further investigation, including a TRH stimulation test, led to a diagnosis of PPID (Cushing's disease), which may have led to immunosuppression of the animal and increased susceptibility to infection. CONCLUSIONS AND CLINICAL IMPORTANCE: The horse was treated medically with pergolide for the PPID and oral potassium iodide for the fungal infection, with good therapeutic response and no relapse after five months. Surgical debridement or excision was not performed. To the best of the authors' knowledge, this is the first case report of a cutaneous mycetoma caused by A. terreus in a horse.

Successful treatment of intra-abdominal eumycotic mycetoma caused by Penicillium duponti in a dog.

A 2-year-old female neutered golden retriever was presented for investigation of an intra-abdominal mass. Computed tomography revealed a mass associated with the caudal pole of the right kidney. Incisional biopsy findings were consistent with eumycotic mycetoma. The mass was subsequently removed in conjunction with right ureteronephrectomy. Two years later, the dog re-presented with a splenic mass and fungal plaques located throughout the peritoneum. Splenectomy was performed and the mass was diagnosed as eumycotic mycetoma caused by Penicillium duponti. Indefinite systemic treatment with 10 mg/kg itraconazole orally once a day was initiated. Thirty-two months after the last surgery, there were no clinical signs apart from mild polydipsia. Haematology and biochemistry results were unremarkable. To the authors' knowledge, this is the first report of successful treatment of intra-abdominal eumycotic mycetoma with a combination of surgery and systemic antifungal therapy in the dog. Penicillium duponti has not apparently been reported to cause disease in animals or humans.

Variable antibiotic susceptibility patterns among Streptomyces species causing actinomycetoma in man and animals.

BACKGROUND: Drug therapy is recommended in conjunction with surgery in treatment of actinomycetoma. The specific prescription depends on the type of bacteria (actinomycetoma) or fungi (eumycetoma) causing the disease and their in vitro antimicrobial susceptibility. OBJECTIVES: To investigate the antimicrobial susceptibility among isolates of Streptomyces spp. isolated from cases of actinomycetoma in man and animals in Sudan. METHODS: Streptomyces strains (n = 18) isolated from cases of actinomycetoma were tested in vitro against 15 commonly prescribed antibacterial agents using MIC agar dilution method as per standard guidelines. RESULTS: Streptomyces strains isolated from actinomycetoma fall into various phenotypic groups. All of the strains were inhibited by novobiocin (8 µg/mL), gentamycin (8, 32 µg/mL) and doxycycline (32 µg/mL). Fusidic acid (64 µg/mL) inhibited 94.4% of the strains; bacitracin, streptomycin, cephaloridine, clindamycin, ampicillin, rifampicin and tetracycline (64 µg/mL) inhibited between 61.1 and 77.8% of the strains. All strains were found resistant to amphotericin B (64 µg/mL), penicillin (20 µg/mL) and sulphamethoxazole (64 µg/mL). CONCLUSIONS: Saprophytic Streptomyces spp. cause actinomycetoma in man and animal belong to separate phenotypes and have a wide range of susceptibility patterns to antimicrobial agents, which pose a lot of difficulties in selecting effective in vivo treatment for actinomycetoma.

Subconjunctival mycetoma caused by Scedosporium apiospermum infection in a horse.

An 11-year-old American Saddlebred gelding was presented for evaluation of a nonpainful subconjunctival mass involving the lateral canthus of the left eye. Other findings included a central corneal scar and a small central cataract of the lens in the left eye. Fine-needle aspiration of the mass was performed and cytologic examination revealed marked pyogranulomatous inflammation with intralesional fungal hyphae, consistent with mycetoma. The fungal structures were elongated and characterized by nonstaining walls; several bulbous yeast-like structures were also observed. The mycetoma was surgically removed and submitted for histopathologic examination and fungal culture. The histopathologic diagnosis was subconjunctival phaeohyphomycosis. Scedosporium apiospermum was identified based on macroscopic and microscopic features of the organism in culture. Scedosporium spp. have been reported as causes of mycetomatous and nonmycetomatous infections in both immunocompromised and immunocompetent people and animals. S. apiospermum and Pseudallescheria boydii, which is its teleomorphic counterpart, have been implicated as potentially emerging human and veterinary pathogens. Timely diagnosis is essential as the organism is often resistant to commonly used antifungal drugs. This report provides a detailed cytologic description of the organism and recent information on the taxonomy of this fungus and the diagnostic peculiarities of this particular infection.

Infections caused by fungi of the Scedosporium/Pseudallescheria complex in veterinary species.

Fungi belonging to the Scedosporium/Pseudallescheria complex (SPCF) have been known to cause human infections for nearly a century and are important human pathogens, with an increasing frequency of infection in patients with underlying conditions. There appears to be a lower incidence of infections with SPCF in veterinary species, although this may be related to a lack of awareness of these diseases. Important recent taxonomic changes in this group of fungi include the classification of Pseudallescheria boydii and Scedosporium apiospermum as two distinct species and the identification of new pathogenic species of SPCF. In this article, the literature on natural and experimental infections caused by SPCF in veterinary species is reviewed. The importance of an accurate identification of veterinary isolates by molecular methods is stressed, especially since virulence and susceptibility to antimycotic drugs of different species may vary.

Disseminated pseudallescheriosis in a dog.

A case of disseminated pseudallescheriasis in a German Shepherd bitch is presented. Bones (ilium, a rib and phalanges), joints (elbow and acetabulum) and the surrounding tissues were the principal organs affected. In addition, Pseudallescheria boydii was isolated, in lower numbers, from the eye, kidney, lymph nodes draining the affected regions and urine. The dog was euthanized. P. boydii was identified by morphologic characteristics and molecular techniques (beta tubulin sequence). In addition, an ITS nucleotide sequence analysis showed that this strain differed from another isolate identified as Scedosporium apiospermum that had caused a disseminated infection in another German Shepherd. The importance of the molecular characterization of fungi belonging to the Pseudallescheria/Scedosporium complex, isolated from animals is stressed in light of the ongoing attempts to recharacterize these fungi.

Eumycetoma caused by Madurella mycetomatis in a mare.

A case of equine eumycotic mycetoma caused by Madurella mycetomatis is described. This is the first report of M. mycetomatis as the etiologic agent of human or animal infections in Israel. The definitive identification of the isolate was established by molecular methods. In addition the mold was cultured on various media through which we found that its growth rate was significantly augmented on incubation on trypticase soy agar, more so if horse serum was added to this medium. Sixteen previously published cases defined by the authors as mycetoma in horses are briefly reviewed. Among these, only one indicated that the infection was caused by M. mycetomatis.

Intra-abdominal actinomycetoma in a cat.

A 5-year-old, female Ragdoll cat was diagnosed with an intra-abdominal mycetoma involving the ileocaecal region. Diagnosis was obtained via histopathological examination following surgical resection of the mass and an ileocolic anastomosis. The initial surgery was complicated by lymphangiectasia, chylous abdominal effusion and mild bacterial leakage from the anastomosis site. A second, exploratory laparotomy was performed to augment the anastomosis with serosal patching and omentalisation and to investigate a cystic structure observed on follow-up abdominal ultrasound. Initial amoxycillin clavulanate (Clavulox; Pfizer Animal Health) therapy was ineffective, but clindamycin (Antirobe; Pfizer Animal Health) proved successful in resolving the infection. Abdominal actinomycetoma in the cat may be an under-diagnosed condition due to its close resemblance to neoplastic disease. Standard diagnostic and therapeutic regimens are commonly ineffective in Actinomyces species infections. Surgical resection along with adjunctive, long-term, selective antimicrobial therapy is effective and prognosis is good for localised lesions.

Confirmed case of feline mycetoma due to Microsporum canis.

The first case of feline true mycetoma because of a dermatophyte has been reported in this manuscript, although pseudomycetoma has been described in dogs and cats. The patient was a 9-year-old castrated male Persian cat weighing 4.2 kg with subcutaneous nodules on the dorsal trunk. Physical examination revealed two subcutaneous nodules (2.5 x 5.0 cm and 3.5 x 5.0 cm in size) that drained purulent exudates with cement-like substances containing yellowish granules. The impression smear of the yellowish granules demonstrated by PAS staining that they were masses of fungal septated hyphae (3-4 mum of width). From the nodular inflammation with fibrosis, fistulae draining from deep tissue and many grains containing abundant hyphal filaments, the case was diagnosed as mycetoma, complying with the definitive criteria of mycetoma. The etiologic fungus was molecularly as well as morphologically identified as Microsporum canis.

Successful resolution of dermatophyte mycetoma following terbinafine treatment in two cats.

Microsporum canis sensitive to itraconazole and terbinafine was isolated from two cats presented with generalized dermatophytosis and dermatophyte mycetoma. Itraconazole therapy was withdrawn through lack of efficacy in one cat (a Persian) and unacceptable adverse effects in the other (a Maine Coon). Both cats achieved clinical and mycological cure after 12-14 weeks therapy with 26-31 mg kg(-1) terbinafine every 24 h per os (PO). Clinical signs in the Maine Coon resolved completely after 7 weeks treatment. Four weeks of therapy with additional weekly washes with a 2% chlorhexidine/2% miconazole shampoo following clipping produced a 98% reduction in the Persian cat's mycetoma, which was then surgically excised. Recurrent generalized dermatophytosis in the Persian cat has been managed with pulse therapy with 26 mg kg(-1) terbinafine every 24 h PO for 1 week in every month. No underlying conditions predisposing to dermatophytosis were found in either cat despite extensive investigation. Terbinafine administration was associated with mild to moderate lethargy in the Persian cat, but no other adverse effects or changes in blood parameters were seen. To the best of the authors' knowledge this is the first report of a dermatophyte mycetoma in a Maine Coon and of successful resolution of this condition in cats following terbinafine therapy.

Imaging diagnosis--sublumbar pseudomycetoma in a Persian cat.

A 6-year-old Persian cat was examined for constipation, anorexia, and vomiting that was subsequently found to be due to a pseudomycetoma originating from the descending colon and sublumbar region, and causing mechanical obstruction of the colon and rectum. Multiple discrete hyperechoic foci likely representing fungal grains within the lesion gave the mass a coarse echotexture on ultrasound and was supportive of the diagnosis and computed tomography allowed delineation the extent of the mass. A pseudomycetoma is a granulomatous/pyogranulomatous reaction that surrounds dermatophytic fungal hyphae. Definitive diagnosis of a dermatophytic pseudomycetoma requires identification of the etiologic agent by cultivation or immunohistochemical staining. A pseudomycetoma should be included in the differential diagnosis for an abdominal mass in a Persian cat, especially is accompanied by the sonographic findings noted above.