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.

Successful Isavuconazole Salvage Therapy for a Cryptococcus deuterogattii (AFLP6/VGII) Disseminated Infection in a European Immunocompetent Patient.

Members of the Cryptococcus gattii species complex are notorious causes of cryptococcosis as they often cause severe, life-threatening infections. Here we describe a case of a severe disseminated C. deuterogattii infection in a previously healthy patient who was initially treated with amphotericin B, 5-fluorocytosine and fluconazole, which led to a good neurological response, but the infection in the lungs remained unaltered and was not completely resolved until switching the antifungal therapy to isavuconazole. The infection was likely acquired during a one-month stay at the Azores Islands, Portugal. Environmental sampling did not yield any cryptococcal isolate; therefore, the source of this apparent autochthonous case could not be determined. Molecular typing showed that the cultured C. deuterogattii isolates were closely related to the Vancouver Island outbreak-genotype.

The Domestic Environment and the Lung Mycobiome.

This study analyzes the relationship between the mycobiome of the Lower Respiratory Tract (LRT) and the fungi in the domestic environment. Samples studied consisted of Broncho-Alveolar Lavage (BAL) from 45 patients who underwent bronchoscopy for different diagnostic purposes, and dust and air from the houses (ENV) of 20 of them (44.4%). Additionally, five bronchoscopes (BS) were also analyzed and negative controls were included for every procedure. All samples were processed for DNA extraction and cultures, which were performed in Sabouraud Dextrose and Potato Dextrose Agar. The fungal Internal Transcribed Spacer (ITS2) was sequenced by the Solexa/Illumina system and sequences were analyzed by QIIME 1.8.0 and compared with the UNITE Database for identification. The similarity between the two fungal communities (BAL and ENV) for a specific patient was assessed via the percentage of coincidence in the detection of specific operational taxonomic units (OTUs), and about 75% of co-occurrence was detected between the mycobiome of the LRT and the houses. Cultures confirmed the presence of the core mycobiome species. However, the low rate of isolation from BAL suggests that most of its mycobiome corresponds to non-culturable cells. This likely depends on the patient's immune system activity and inflammatory status.

Importance of Resolving Fungal Nomenclature: the Case of Multiple Pathogenic Species in the Cryptococcus Genus.

Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature "C. neoformans species complex" and "C. gattii species complex." Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

Environmental sampling of Ceratonia siliqua (carob) trees in Spain reveals the presence of the rare Cryptococcus gattii genotype AFLP7/VGIV / Muestreos medioambientales de algarrobos ( Ceratonia siliqua) españoles revelan la presencia del raro genotipo AFLP7/VGIV de Cryptococcus gattii

Background. Cryptococcus gattii is a pathogenic basidiomycetous yeast that is emerging in temperate climate zones worldwide. C. gattii has repetitively been isolated from numerous tree species. Ongoing environmental sampling and molecular characterization is essential to understand the presence of this primary pathogenic microorganism in the Mediterranean environment. Aims. To report the first isolation of the rare C. gattii genotype AFLP7/VGIV from the environment in Europe. Methods. Samples were collected from woody debris of carob trees (Ceratonia siliqua) and olive trees (Olea europaea) in El Perelló, Tarragona, Spain. Cryptococcus species were further characterized by using URA5-RFLP, MALDI-TOF, AFLP and MLST. The antifungal susceptibility profile to amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, posaconazole and voriconazole was determined using Sensititre Yeast One and E-test. Results. Cultures from one carob tree revealed the presence of ten Cryptococcus-like colonies. One colony was identified as C. gattii, and subsequent molecular characterization showed that it was an α mating-type that belonged to the rare genotype AFLP7/VGIV. Antifungal susceptibility testing showed values within the range of sensitivity described for other isolates of the same genotype and within the epidemiological cutoff values for this species. Conclusions. The isolation of the rare C. gattii genotype AFLP7/VGIV in Spain is the first report in the European environment, implying the possible presence in other regions of the Mediterranean area, and underlines that clinicians must be aware for C. gattii infections in healthy individuals (AU)

Antecedentes. Cryptococcus gattii es una levadura perteneciente a los basidiomicetos y considerada un patógeno emergente en climas templados. C. gattii se ha aislado en un gran número de especies de árboles en todo el mundo. El muestreo medioambiental y la caracterización molecular de C. gattii es esencial para entender la presencia de este patógeno primario en el entorno de la cuenca mediterránea. Objetivos. Comunicar la presencia del raro genotipo AFLP7/VGIV de C. gattii en el medioambiente en Europa. Métodos. Se tomaron muestras de detritus de algarrobos (Ceratonia siliqua) y olivos (Olea europaea) en las afueras de El Perelló (Tarragona, España). La colonia de C. gattii aislada se sometió a análisis mediante URA5-RFLP, MALDI-TOF, AFLP y MLST. Se llevó a cabo un estudio de sensibilidad in vitro a la anfotericina B, 5-fluorocitosina, fluconazol, itraconazol, posaconazol y voriconazol mediante las pruebas comerciales Sensititre Yeast One® y E-test®. Resultados. De una de las muestras de un algarrobo se aislaron 10 colonias susceptibles de ser Cryptococcus. Una de ellas fue estudiada e identificada como C. gattii, y su subsecuente caracterización molecular mostró que se trataba de un tipo sexual α y que pertenecía al raro genotipo AFLP7/VGIV. El estudio de la sensibilidad a los antifúngicos mostró valores similares a los de otras cepas del mismo genotipo y dentro del rango de valores de corte epidemiológicos para la especie. Conclusiones. El aislamiento en España de C. gattii con el genotipo AFLP7/VGIV es el primero descrito en el medioambiente en Europa; podría encontrarse también en otros países de la cuenca mediterránea, donde debería tenerse un especial cuidado por la posibilidad de infección en individuos no inmunodeprimidos (AU)

Environmental sampling of Ceratonia siliqua (carob) trees in Spain reveals the presence of the rare Cryptococcus gattii genotype AFLP7/VGIV.

BACKGROUND: Cryptococcus gattii is a pathogenic basidiomycetous yeast that is emerging in temperate climate zones worldwide. C. gattii has repetitively been isolated from numerous tree species. Ongoing environmental sampling and molecular characterization is essential to understand the presence of this primary pathogenic microorganism in the Mediterranean environment. AIMS: To report the first isolation of the rare C. gattii genotype AFLP7/VGIV from the environment in Europe. METHODS: Samples were collected from woody debris of carob trees (Ceratonia siliqua) and olive trees (Olea europaea) in El Perelló, Tarragona, Spain. Cryptococcus species were further characterized by using URA5-RFLP, MALDI-TOF, AFLP and MLST. The antifungal susceptibility profile to amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, posaconazole and voriconazole was determined using Sensititre Yeast One and E-test. RESULTS: Cultures from one carob tree revealed the presence of ten Cryptococcus-like colonies. One colony was identified as C. gattii, and subsequent molecular characterization showed that it was an α mating-type that belonged to the rare genotype AFLP7/VGIV. Antifungal susceptibility testing showed values within the range of sensitivity described for other isolates of the same genotype and within the epidemiological cutoff values for this species. CONCLUSIONS: The isolation of the rare C. gattii genotype AFLP7/VGIV in Spain is the first report in the European environment, implying the possible presence in other regions of the Mediterranean area, and underlines that clinicians must be aware for C. gattii infections in healthy individuals.

Fusariosis cutánea por una especie del complejo Fusarium dimerum en una paciente con leucemia mieloblástica aguda / Cutaneous fusariosis by a species of the Fusarium dimerum species complex in a patient with acute mieloblastic leukemia

Antecedentes. La fusariosis es una hialohifomicosis oportunista, emergente, producida por hongos pertenecientes al género Fusarium. Estos hongos pueden provocar enfermedades que amenazan la vida en pacientes inmunodeficientes, en especial en portadores de leucemia. También se ha descrito en individuos inmunocompetentes, en los que induce lesiones localizadas, no invasivas. En pacientes inmunodeficientes, la fusariosis se asocia a una elevada morbimortalidad, sobre todo debido a la falta de sensibilidad de estos hongos a los antimicóticos disponibles. Caso clínico. Describimos el caso de una paciente con leucemia mieloblástica aguda que experimentó una fusariosis por una especie del complejo Fusarium dimerum. El diagnóstico precoz se estableció en función de la observación microscópica de muestras de las lesiones cutáneas y se prescribió tratamiento con voriconazol. Más tarde, un estudio completo del aislamiento fúngico por cultivo y métodos moleculares permitió la identificación de F. dimerum, una especie apenas descrita como patógeno en el ser humano. La sensibilidad de la cepa se examinó con el método comercializado Sensititre YeastOne®, que reveló su sensibilidad a voriconazol y posaconazol, al igual que a anfotericina B. La paciente falleció a los 7 días del ingreso debido a una insuficiencia hemodinámica. Conclusiones. La identificación completa de nuevos aislamientos de Fusarium y su patrón de sensibilidad a los antimicóticos suscita un gran interés para incrementar nuestros conocimientos sobre la epidemiología de la enfermedad y el tratamiento óptimo de los pacientes(AU)

Fusariosis is an emergent opportunistic hyalohyphomycosis produced by fungi belonging to the genus Fusarium. These molds are capable of producing life-threatening diseases in immunocompromised hosts, especially in those suffering from leukemia. It has also been described in immunocompetent patients, where it usually causes non-invasive localized lesions. Fusariosis in immunocompromised individuals has a high morbidity and mortality mainly because of the low sensitivity of these fungi to the antifungal drugs available. Case report. We describe here the case of a patient with acute mieloblastic leukemia who developed fusariosis by a species of the Fusarium dimerum species complex. The early diagnosis was made on the basis of microscopic observation of samples from cutaneous lesions, and voriconazole treatment was prescribed. A subsequent complete study of the fungal isolate by culture and molecular methods allowed the identification of F. dimerum, a species rarely described as a human pathogen. The sensitivity of the strain was tested using the Sensititre YeastOne® commercial system, which showed sensitivity to voriconazole and posaconazole, as well as to amphotericin B. The patient died after 7 days at hospital due to an hemodynamic failure. Conclusions. Complete identification of new isolates of Fusarium and their antifungal susceptibility patterns is of high interest to improve our knowledge about the epidemiology of the disease and how to best manage patients(AU)

Cutaneous fusariosis by a species of the Fusarium dimerum species complex in a patient with acute myeloblastic leukemia.

BACKGROUND: Fusariosis is an emergent opportunistic hyalohyphomycosis produced by fungi belonging to the genus Fusarium. These molds are capable of producing life-threatening diseases in immunocompromised hosts, especially in those suffering from leukemia. It has also been described in immunocompetent patients, where it usually causes non-invasive localized lesions. Fusariosis in immunocompromised individuals has a high morbidity and mortality mainly because of the low sensitivity of these fungi to the antifungal drugs available. CASE REPORT: We describe here the case of a patient with acute mieloblastic leukemia who developed fusariosis by a species of the Fusarium dimerum species complex. The early diagnosis was made on the basis of microscopic observation of samples from cutaneous lesions, and voriconazole treatment was prescribed. A subsequent complete study of the fungal isolate by culture and molecular methods allowed the identification of F. dimerum, a species rarely described as a human pathogen. The sensitivity of the strain was tested using the Sensititre YeastOne(®) commercial system, which showed sensitivity to voriconazole and posaconazole, as well as to amphotericin B. The patient died after 7 days at hospital due to an hemodynamic failure. CONCLUSIONS: Complete identification of new isolates of Fusarium and their antifungal susceptibility patterns is of high interest to improve our knowledge about the epidemiology of the disease and how to best manage patients.

Ceratonia siliqua (carob) trees as natural habitat and source of infection by Cryptococcus gattii in the Mediterranean environment.

Recent Cryptococcus gattii infections in humans and animals, including several outbreaks in goats, were the basis of this environmental survey in six provinces of Spain. A total of 479 samples from 20 tree species were studied. Cryptococcus gattii was found for the first time in autochthonous Mediterranean trees in Spain. Fourteen isolates of this pathogen were obtained from seven trees of three different species: 12 from carob (Ceratonia siliqua), one from Mediterranean stone pine (Pinus halepensis) and another from eucalyptus (Eucalyptus camaldulensis). All C. gattii isolates were genotype AFLP4/VGI and mating type alpha, and were found to be genetically identical with C. gattii strains isolated from humans and animals in Spain. This supports the hypothesis that these trees may be a natural source for infection of humans and mammals in the Mediterranean area.

[Molecular biology in the diagnosis of deep-seated candidiasis in the critically ill non-neutropenic patient]. / Biología molecular en el diagnóstico de la candidiasis profunda en el paciente crítico no neutropénico.

Candida infections are an important cause of morbidity and mortality in critically ill patients. Rapid detection of the yeast in blood and other tissues by molecular biology methods has been the goal of some recent studies. An analysis of the sensitivity and specificity of these methods assayed in clinical specimens from critically ill and other patients is carried out. PCR amplification of ribosomal genes and their internal spacers showed a higher sensitivity than culture based methods. A standardization of most of the methodological steps in molecular methods is needed. Real time PCR with fluorescent probes seems to be the most interesting proposal. It has the advantage of the possible quantification of fungal presence in tissues and minimizes the samples' contamination risk.