Resazurin to determine the minimum inhibitory concentration on antifungal susceptibility assays for Fonsecaea sp. using a modified EUCAST protocol.

Chromoblastomycosis is a fungal chronic disease, which affects humans, especially in cutaneous and subcutaneous tissues. There is no standard treatment for Chromoblastomycosis, and it is a therapeutic challenge, due natural resistance of their causative agents, inadequate response of patients and common cases of relapse. Protocols for determination of antifungal drugs susceptibility are not standardized for chromoblastomycosis agents and endpoint definition is usually based on visual inspection, which depends on the analyst, making it sometimes inaccurate. We presented a colorimetric and quantitative methodology based on resazurin reduction to resofurin to determine the metabolic status of viable cells of Fonsecaea sp. Performing antifungal susceptibility assay by a modified EUCAST protocol allied to resazurin, we validated the method to identify the minimum inhibitory concentrations of itraconazole, fluconazole, amphotericin B, and terbinafine for eight Fonsecaea clinical isolates. According to our data, resazurin is a good indicator of metabolic status of viable cells, including those exposed to antifungal drugs. This work aimed to test resazurin as an indicator of the metabolic activity of Fonsecaea species in susceptibility assays to antifungal drugs. Species of this genus are the main causative agents of Chromoblastomycosis, which affects humans.

Prediction of melanin content of Fonsecaea pedrosoi using Fourier transform infrared spectroscopy (FTIR) and chemometrics.

Fungal melanin contributes to the survival and virulence of pathogenic fungi, such as Fonsecaea pedrosoi, which is responsible for causing chromoblastomycosis. The objective of this study was to employ Fourier transform infrared spectroscopy (FTIR) to predict the melanin content of F. pedrosoi. The melanin content, in percentage, was previously determined using gravimetry for twenty-six clinical isolates. Quintuplicate spectra of each isolate were obtained using attenuated total reflection (ATR) within the range of 4000 to 650 cm-1. To predict the melanin content, modeling was performed using partial least squares regression (PLS) in the region 1800 - 750 cm-1. Two models were tested: PLS and successive projections algorithms for interval selection in partial least squares (iSPA-PLS). The best modeling results were achieved using iSPA-PLS with one factor. The calibration set exhibited a determination coefficient (R2) of 0.9745 and a root mean square error of cross-validation (RMSECV) of 0.0977. In the prediction set, the R2 value was 0.9711, and the root mean square error of prediction (RMSEP) was 0.0999. Modeling with FTIR and multivariate calibration provides a valuable means of predicting fungal melanin content, which is simpler and more robust, thereby contributing to the advancement of this field of study.

Cromoblastomicosis: abordaje con terapia combinada / Chromoblastomycosis: a combination therapy approach

La cromoblastomicosis es una micosis de implantación crónica y progresiva causada por diversos hongos de la familia Dematiaceae. En Latinoamérica, las especies en-contradas con más frecuencia son Fonsecaea pedrosoi y Cladophialophora carrionii. El tratamiento de esta micosis puede ser un desafío por la falta de respuesta y la recidiva, en especial en individuos con lesiones crónicas y extensas.Se presenta un individuo con recaída de cromoblastomico-sis (causada por Fonsecaea pedrosoi) en miembro inferior derecho que había realizado tratamiento incompleto con terbinafina e itraconazol. El paciente respondió de mane-ra favorable al retratamiento con itraconazol y terbinafina combinado con resección quirúrgica parcial de la lesión e injerto de piel en sitio quirúrgico

Chromoblastomycosis is a chronic and subcutaneous mycosis caused by various dematiaceous fungi, In Latin America, the most frequently found species are Fonsecaea pedrosoi and Cladophialophora carrionii.Treatment is a challenge because of the lack of response and recurrence in in some cases, especially in patients with extensive and chronic lesions.We report an individual with relapse of chromoblastomycosis (by Fonsecaea pedrosoi) in the right lower limb, who had undergone incomplete treatment with terbinafine and itraconazole. The patient responded favorably to retreatment with itraconazole and terbinafine combined with partial surgical resection of the lesion and skin grafting at the surgical site.

An Experimental Model of Chromoblastomycosis Caused by Fonsecaea sp. Species.

The experimental rodent models for the fungal disease are a handy tool for understanding host-fungus interactions. To Fonsecaea sp., one of the causative agents of chromoblastomycosis, there is an extra challenge because the animals preferably used show a spontaneous cure; so until now, there is no model to reproduce the long-term disease similar to human chronic disease. In this chapter, we described an experimental model using rats and mice with a subcutaneous route, with the checkpoints of acute-like and chronic-like lesion analysis comparable with human lesions, the fungal burden, and the lymphocytes investigation.

Pathogenicity and Growth Conditions Modulate Fonsecaea Extracellular Vesicles' Ability to Interact With Macrophages.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous mycosis caused by black, dimorphic, and filamentous fungi of the Herpothrichiellaceae family, such as species of the genus Fonsecaea. These fungi can switch between the saprophytic forms (conidia and hyphae) and the pathogenic form, the muriform cells (MCs), which is considered an essential mechanism for fungal virulence. Nearly all types of cells can produce membranous structures formed by a lipid bilayer that communicate extracellularly with other cells, known as "extracellular vesicles" (EVs), which may act as virulence factors, as observed for several species of pathogenic fungi. Our findings demonstrated for the first time that F. pedrosoi, F. nubica, and F. erecta produce EVs in response to nutritional conditions. The EVs varied in sterol and protein contents, size, and morphology. Moreover, the EVs induced different cytokine and nitric oxide release patterns by bone marrow-derived macrophages (BMDMs). The EVs activated IL-1ß production, possibly acting as the first signal in inflammasome activation. Unlike the pathogenic species, the EVs isolated from F. erecta did not significantly stimulate TNF and IL-10 production in general. Overall, these results demonstrated that different species of Fonsecaea produce EVs capable of modulating pro- and anti-inflammatory cytokine and nitric oxide production by BMDMs and that growth conditions affected the immunomodulatory capacities of the EVs as well as their size, content, and morphology.

Neutrophil-suppressive activity over T-cell proliferation and fungal clearance in a murine model of Fonsecaea pedrosoi infection.

Neutrophils are essential to control several fungal infections. These cells are commonly known for their pro-inflammatory activities. However, some studies have demonstrated the anti-inflammatory properties of neutrophils during certain infectious diseases, culminating in the inhibition of T cell proliferation. Chromoblastomycosis (CBM) is a deep and progressive mycosis that affects thousands of people worldwide. Although neutrophil infiltrates are observed in the lesion histopathology, the fungus can overtake the immune system response and destroy the host-infected tissue. The present study demonstrated that neutropenic animals had an increase in the IL-6 production in the spleen and liver, followed by a lower fungal burden in these organs up to 14 days of infection. Neutropenic animals also showed a lower F. pedrosoi-specific antibody production 14-days post infection and higher T-cell proliferation in the in vitro experiments after stimulation with F. pedrosoi-purified proteins. Taken together, our results suggest that the presence of regulatory neutrophils in the mouse model of F. pedrosoi infection could act favoring the spread of the fungus and the chronicity of the infection. These findings shed light on the CBM treatment, which might target neutrophil polarization as a new therapy approach to treat CBM lesions.

The global burden of chromoblastomycosis.

BACKGROUND: Chromoblastomycosis (CBM), represents one of the primary implantation mycoses caused by melanized fungi widely found in nature. It is characterized as a Neglected Tropical Disease (NTD) and mainly affects populations living in poverty with significant morbidity, including stigma and discrimination. METHODS AND FINDINGS: In order to estimate the global burden of CBM, we retrospectively reviewed the published literature from 1914 to 2020. Over the 106-year period, a total of 7,740 patients with CBM were identified on all continents except Antarctica. Most of the cases were reported from South America (2,619 cases), followed by Africa (1,875 cases), Central America and Mexico (1,628 cases), Asia (1,390 cases), Oceania (168 cases), Europe (35 cases), and USA and Canada (25 cases). We described 4,022 (81.7%) male and 896 (18.3%) female patients, with the median age of 52.5 years. The average time between the onset of the first lesion and CBM diagnosis was 9.2 years (range between 1 month to 50 years). The main sites involved were the lower limbs (56.7%), followed by the upper limbs (19.9%), head and neck (2.9%), and trunk (2.4%). Itching and pain were reported by 21.5% and 11%, respectively. Malignant transformation was described in 22 cases. A total of 3,817 fungal isolates were cultured, being 3,089 (80.9%) Fonsecaea spp., 552 (14.5%) Cladophialophora spp., and 56 Phialophora spp. (1.5%). CONCLUSIONS AND SIGNIFICANCE: This review represents our current knowledge on the burden of CBM world-wide. The global incidence remains unclear and local epidemiological studies are required to improve these data, especially in Africa, Asia, and Latin America. The recognition of CBM as NTD emphasizes the need for public health efforts to promote support for all local governments interested in developing specific policies and actions for preventing, diagnosing and assisting patients.

Chromoblastomycosis caused by Fonsecaea monophora in Mexico.

Chromoblastomycosis is a chronic subcutaneous fungal infection caused by melanized fungi. It is usually an occupational mycosis affecting people in rural areas in tropical and subtropical regions. We present two cases of chromoblastomycosis in Mexican farmers, characterized by skin verrucous plaques. Direct examination with KOH 10% showed the presence of muriform cells. The fungal isolation was carried out in Sabouraud dextrose agar and molecular identification was achieved by 18S-ITS1-5.8S-ITS2-28S rRNA gene amplification and sequencing. Fonsecaeamonophora was identified in both cases. A therapy with itraconazole and terbinafine was used with a partial favorable response. However, patients did not return for medical examination after 4 months. The current status of the patients is unknown. We reported the first two cases of chromoblastomycosis caused by F. monophora in Mexico.

Prediction of itraconazole minimum inhibitory concentration for Fonsecaea pedrosoi using Fourier Transform Infrared Spectroscopy (FTIR) and chemometrics.

Fonsecaea pedrosoi is one of the main agents of chromoblastomycosis, a chronic subcutaneous mycosis. Itraconazole (ITC) is the most used antifungal in its treatment, however, in vitro antifungal susceptibility tests are important to define the best therapy. These tests are standardized by the Clinical and Laboratory Standards Institute (CLSI), but these protocols have limitations such as the high complexity, cost and time to conduct. An alternative to in vitro susceptibility test, which overcomes these limitations, is FTIR. This study determined the minimum inhibitory concentration (MIC) of itraconazole for F. pedrosoi, using FTIR and chemometrics. The susceptibility to ITC of 36 strains of F. pedrosoi was determined according to CLSI and with the addition of tricyclazole (TCZ), to inhibit 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Strains were grown in Sabouraud agar and prepared for Attenuated Total Reflection (ATR)/FTIR. Partial least squares (PLS) regression was performed using leave-one-out cross-validation (by steps of quintuplicates), then tested on an external validation set. A coefficient of determination (R²) higher than 0.99 was obtained for both the MIC-ITC and MIC-ITC+TCZ ATR/PLS models, confirming a high correlation of the reference values with the ones predicted using the FTIR spectra. This is the first study to propose the use of FTIR and chemometric analyses according to the M38-A2 CLSI protocol to predict ITC MICs of F. pedrosoi. Considering the limitations of the conventional methods to test in vitro susceptibility, this is a promising methodology to be used for other microorganisms and drugs.

Fonsecaeapedrosoi Conidia and Hyphae Activate Neutrophils Distinctly: Requirement of TLR-2 and TLR-4 in Neutrophil Effector Functions.

Chromoblastomycosis is a chronic and progressive subcutaneous mycosis caused mainly by the fungus Fonsecaea pedrosoi. The infection is characterized by erythematous papules and histological sections demonstrating an external layer of fibrous tissue and an internal layer of thick granulomatous inflammatory tissue containing mainly macrophages and neutrophils. Several groups are studying the roles of the innate and adaptive immune systems in F. pedrosoi infection; however, few studies have focused on the role of neutrophils in this infection. In the current study, we verify the importance of murine neutrophils in the killing of F. pedrosoi conidia and hyphae. We demonstrate that phagocytosis and reactive oxygen species during infection with conidia are TLR-2- and TLR-4-dependent and are essential for conidial killing. Meanwhile, hyphal killing occurs by NET formation in a TLR-2-, TLR-4-, and ROS-independent manner. In vivo experiments show that TLR-2 and TLR-4 are also important in chromoblastomycosis infection. TLR-2KO and TLR-4KO animals had lower levels of CCL3 and CXCL1 chemokines and impaired neutrophil migration to the infected site. These animals also had higher fungal loads during infection with F. pedrosoi conidia, confirming that TLR-2 and TLR-4 are essential receptors for F. pedrosoi recognition and immune system activation. Therefore, this study demonstrates for the first time that neutrophil activation during F. pedrosoi is conidial or hyphal-specific with TLR-2 and TLR-4 being essential during conidial infection but unnecessary for hyphal killing by neutrophils.

Cinnamyl Schiff bases: synthesis, cytotoxic effects and antifungal activity of clinical interest.

The aim of this study was to synthesize and investigate the in vitro antifungal properties of 23 cinnamyl Schiff bases. In addition, cytotoxic effects of such cinnamyl Schiff bases against human lung, kidney or red blood cells were also checked. The compounds were synthesized in a single-step, 2 min of reaction under microwave irradiation produced up to 97% yield. Six of the 23 cinnamyl Schiff bases possessed antifungal activities against strains of Candida, Aspergillus, Fonsecaea and, particularly, Cryptococcus species. Indeed, cinnamyl Schiff bases 1 and 23 exhibited minimum inhibitory concentration (MIC) values more than twofold lower than fluconazole (FCZ) against all the Cryptococcus neoformans strains (MIC = 1·33, 1·4 and 5·2 µg ml-1 , respectively) and Cryptococcus gattii strains (MIC = 5·3, 2·8 and 9·2 µg ml-1 , respectively) (12 strains of each species) while cinnamyl Schiff base 11 was as potent as FCZ against all strains from both Cryptococcus species. No significant cytotoxic effects were observed for Schiff bases against human lung, kidney or red blood cells, all presenting selective indexes higher than 10. In conclusion, this study revealed cinnamyl Schiff bases, especially 1 and 23, as new lead anticryptococcal agents for the discovery of novel antifungal drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: The occurrence and severity of fungal infections have increased in recent decades due to resistance to available antifungal drugs and the appearance of new emerging pathogens. Thus, the search for new antifungal agents is mandatory. From a series of 23 cinnamyl Schiff bases, two compounds (1 and 23) were interrogated as new anticryptococcal agents without significant cytotoxicity against human lung, kidney or red blood cells. In turns, these new Schiff bases are lead compounds for the discovery of novel antifungal drugs.

Fonsecaea pedrosoi Conidia Induces Activation of Dendritic Cells and Increases CD11c+ Cells in Regional Lymph Nodes During Experimental Chromoblastomycosis.

The chromoblastomycosis is a subcutaneous mycosis with a high morbidity rate, Fonsecaea pedrosoi being the largest etiologic agent of this mycosis, usually confined to the skin and subcutaneous tissues. Rarely people get the cure, because the therapies shown to be deficient and few studies report the host-parasite relationship. Dendritic cells (DCs) are specialized in presenting antigens to naïve T lymphocytes inducing primary immune responses. Therefore, we propose to study the migratory capacity of DCs after infection with conidia of F. pedrosoi. The phenotype of DCs was evaluated using cells obtained from footpad and lymph nodes of BALB/c mice after 12, 24 and 72 h of infection. After 24 and 72 h of infection, we found a significant decrease in DCs in footpad and a significant increase in the lymph nodes after 72 h. The expression of surface markers and co-stimulatory molecules were reduced in cells obtained from footpad. To better assess the migratory capacity of DCs migration from footpad, CFSE-stained conidia were injected subcutaneously. We found that after 12 and 72 h, CD11c+ cells were increased in regional lymph nodes, leading us to believe that DCs (CD11c+) were able to phagocytic conidia present in footpad and migrated to regional lymph nodes.

Successful treatment of chromoblastomycosis caused by Fonsecaea pedrosoi using imiquimod.

Chromoblastomycosis (CBM) is a fungal infection caused by fungi belonging to the order Chaetothyriales, and caused mainly by Fonsecaea pedrosoi. The classic treatment, based on itraconazole and/or terbinafine as well as physical approaches, is considered complex and ineffective due to the high rate of relapses. Thus, new strategies are needed to manage CBM; in this regard, the present work reports the evolution of lesions in patients successfully treated with imiquimod. Of note, classic treatment was not effective in healing the lesions of two of them, but single topical treatment with imiquimod healed the lesions.