Predicting of novel homoserine dehydrogenase inhibitors against Paracoccidioides brasiliensis: integrating in silico and in vitro approaches.

Aim: To search for potential inhibitors to homoserine dehydrogenase (HSD) in Paracoccidioides brasiliensis the causative agent of paracoccidioidomycosis, an infection with a high mortality rate in Brazil.Materials & methods: The enzyme was modeled and used in the virtual screening of the compounds. The library was first screened by the Autodock, in which 66 molecules were better ranked than substrate, and then, also evaluated by the Molegro and Gold programs.Results: The HS23 and HS87 molecules were selected in common by the three programs, and ADME/Tox evaluation indicates they are not toxic. The molecular dynamics of PbHSD bonded to ligands showed stable complexes until 50 ns. To validate the results, compounds were purchased for assays of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), synergic profile with Amphotericin B (AmB) and cytotoxicity. The two molecules presented MIC of 32 µg/ml and MFC of 64 µg/ml against the P. brasiliensis (strain Pb18). They also showed synergistic activity with AmB and a lack of toxicity against Hela and Vero cell lines.Conclusion: These results suggest that the HS23 and HS87 are promising candidates as PbHSD inhibitors and may be used as hits for the development of new drugs against paracoccidioidomycosis.

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Celecoxib exhibits antifungal effect against Paracoccidioides brasiliensis both directly and indirectly by activating neutrophil responses.

BACKGROUND: Celecoxib, an anti-inflammatory drug, combined therapies using antimicrobials and immune modulator drugs are being studied. OBJECTIVE: To assess whether Celecoxib has direct in vitro antifungal effect against the Paracoccidioides brasiliensis, the causative agent of Paracoccidioidomycosis-(PCM) and also if it improves the in vivo activity of neutrophils-(PMN) in an experimental murine subcutaneous-(air pouch) model of the disease. METHODS: The antifungal activity of Celecoxib(6 mg/mL) on P. brasiliensis-(Pb18) was evaluated using the microdilution technique. Splenocytes co-cultured with Pb18 and treated with Celecoxib(6 mg/mL) were co-cultured for 24, 48 and 72-hours. Swiss mice were inoculated with Pb18 and treated with Celecoxib(6 mg/kg) in the subcutaneous air pouch. Neutrophils were collected from the air pouch. Mitochondrial activity, reactive oxygen production, catalase, peroxidase, cytokines and chemokines, nitrogen species, total protein, microbicidal activity of PMNs and viable Pb18 cells numbers were analyzed. RESULTS: Celecoxib had no cytotoxic effect on splenocytes co-cultured with Pb18, but had a marked direct antifungal effect, inhibiting fungal growth both in vitro and in vivo. Celecoxib interaction with immune system cells in the air pouch, it leads to activation of PMNs, as confirmed by several parameters (mitochondrial activity, reactive oxygen species, peroxidase, KC and IL-6 increase, killing constant and phagocytosis). Celecoxib was able to reduce IL-4, IL-10 and IL-12 cytokine production. The number of recovered viable Pb18 decreased dramatically. CONCLUSIONS: This is the first report of the direct antifungal activity of Celecoxib against P. brasiliensis. The use of Celecoxib opens a new possibility for future treatment of PCM.

Talaromyces marneffei, Coccidioides species, and Paracoccidioides species-a systematic review to inform the World Health Organization priority list of fungal pathogens.

The World Health Organization, in response to the growing burden of fungal disease, established a process to develop a fungal pathogen priority list. This systematic review aimed to evaluate the epidemiology and impact of infections caused by Talaromyces marneffei, Coccidioides species, and Paracoccidioides species. PubMed and Web of Sciences databases were searched to identify studies published between 1 January 2011 and 23 February 2021 reporting on mortality, complications and sequelae, antifungal susceptibility, preventability, annual incidence, and trends. Overall, 25, 17, and 6 articles were included for T. marneffei, Coccidioides spp. and Paracoccidioides spp., respectively. Mortality rates were high in those with invasive talaromycosis and paracoccidioidomycosis (up to 21% and 22.7%, respectively). Hospitalization was frequent in those with coccidioidomycosis (up to 84%), and while the duration was short (mean/median 3-7 days), readmission was common (38%). Reduced susceptibility to fluconazole and echinocandins was observed for T. marneffei and Coccidioides spp., whereas >88% of T. marneffei isolates had minimum inhibitory concentration values ≤0.015 µg/ml for itraconazole, posaconazole, and voriconazole. Risk factors for mortality in those with talaromycosis included low CD4 counts (odds ratio 2.90 when CD4 count <200 cells/µl compared with 24.26 when CD4 count <50 cells/µl). Outbreaks of coccidioidomycosis and paracoccidioidomycosis were associated with construction work (relative risk 4.4-210.6 and 5.7-times increase, respectively). In the United States of America, cases of coccidioidomycosis increased between 2014 and 2017 (from 8232 to 14 364/year). National and global surveillance as well as more detailed studies to better define sequelae, risk factors, outcomes, global distribution, and trends are required.

Clinical response to prednisone in a severe case of infant-juvenile paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis is a neglected tropical disease caused by fungi of the genus Paracoccidioides. A wide range of symptoms is related to the disease; however, lungs and skin are the sites predominantly affected. The disease is mostly seen in people living in rural areas in Latin America. CASE REPORT: We present a pediatric case of severe disseminated paracoccidioidomycosis that slowly responded to the antifungal treatment. Within three months, symptoms evolved into hepatosplenomegaly, necrotic cervical and abdominal lymph nodes, and splenic abscess. Clinical response to amphotericin B deoxycholate and itraconazole was slow, resulting in pleural and peritoneal cavity effusions, heart failure and shock. Amphotericin B deoxycholate was replaced by the liposomal formulation, with no response. Subsequently, prednisone was added to the treatment, which led to improvement in the clinical response. Serological Paracoccidioides antibody titers were atypical, with very low titers in the critical phase and significant increase during the convalescence phase. The infection was finally cleared up with amphotericin B deoxycholate, liposomal amphotericin B and the use of corticosteroids. Paracoccidioidomycosis serology was non-reactive two years post-discharge. CONCLUSIONS: Due to the intense inflammatory response triggered by Paracoccidioides cells, giving low-dose prednisone for a short period of time modulated the inflammatory response and supported antifungal treatment.

Neuroparacoccidioidomycosis.

Paracoccidioidomycosis (PCM) is caused by a dimorphic fungus Paracoccidioides Brasiliensis and is endemic to subtropical areas of Central and South America. CNS involvement of PCM is extremely variable. NeuroPCM is found in 9.65% to 27.18% of PCM cases .Most neuro PCM patients presents with simultaneous involvement of other organ but isolated or initial CNS involvement may be a feature in 21%. Here we report a case of CNS PCM and this appears to be first reported case of PCM and CNS PCM in India.

Autochtonal case of chronic, unifocal, pulmonary paracoccidioidomycosis with methotrexate use, in Salvador ‒ Brazil.

We report an autochthonous case of mild unifocal chronic pulmonary paracoccidioidomycosis in a 48-year-old previously healthy woman with no history of possible environmental exposures in endemic rural areas, supposedly resulting from reactivation of a latent pulmonary focus secondary to the use of methotrexate for the control of Chikungunya arthropathy. Laboratory investigation ruled out other immunosuppression. Her only symptoms were a dry cough and chest pain. Diagnosis confirmed by needle lung biopsy. There were no abnormalities on physical examination nor evidence of central nervous system involvement. MRI of the total abdomen showed no involvement of other organs. Computed chest tomography showed a favorable evolution under the use of itraconazole (200 mg/day). Different tomographic presentations findings are highlighted when performed before and after treatment. CONCLUSIONS: PCM should be considered even in a woman without a history of consistent environmental exposure and in a non-endemic geographic area.

Case Report: Disseminated Paracoccidioidomycosis and Strongyloides Hyperinfection in a Patient with Human T-Lymphotropic Virus Type 1/2 Infection.

Co-occurrence of paracoccidioidomycosis and strongyloidiasis in immunosuppressed patients, particularly those infected with human T-lymphotropic virus type 1/2, is infrequent. We describe the case of a Peruvian farmer from the central jungle with human T-lymphotropic virus type 1/2 infection, with 2 months of illness characterized by respiratory and gastrointestinal symptoms associated with fever, weight loss, and enlarged lymph nodes. Strongyloides stercoralis and Paracoccidioides brasiliensis were isolated in sputum and bronchoalveolar lavage samples, respectively. The clinical evolution was favorable after the patient received ivermectin and amphotericin B. We hypothesize that autoinfestation by S. stercoralis in human T-lymphotropic virus type 1/2-infected patients may contribute to the disseminated presentation of Paracoccidioides spp. Understanding epidemiological context is crucial for suspecting opportunistic regional infections, particularly those that may coexist in immunosuppressed patients.

Synthesis of 1,3,4-oxadiazoles with 4-methoxynaphthalene ring: discovering new compounds with antimicrobial activity.

Background: Paracoccidioidomycosis (PCM) is a systemic infection caused by Paracoccidioides spp. (Pb). PCM can be associated or clinically confused with tuberculosis (TB), another pulmonary infection, caused by Mycobacterium tuberculosis (Mtb). Futhermore, the long treatment time of TB and PCM and the cases of TB drug resistance impose difficulties for the cure of these diseases. Results: New 1,3,4-oxadiazoles containing the 4-methoxynaphthalene ring were synthesized and their antimicrobial activity was evaluated against Pb and Mtb. The derivative 6n (with 2-hydroxy-5-nitrophenyl subunit) is the most promising of the series. Conclusion: The 1,3,4-oxadiazole 6n can be used as a prototype drug candidate, with anti-Pb and anti-MTb activities, showing a broad-spectrum profile for the treatment of both pulmonary infections.

Cheminformatics-driven discovery of hit compounds against Paracoccidioides spp.

Aims: The development of safe and effective therapies for treating paracoccidioidomycosis using computational strategies were employed to discover anti-Paracoccidioides compounds. Materials & methods: We 1) collected, curated and integrated the largest library of compounds tested against Paracoccidioides spp.; 2) employed a similarity search to virtually screen the ChemBridge database and select nine compounds for experimental evaluation; 3) performed an experimental evaluation to determine the minimum inhibitory concentration and minimum fungicidal concentration as well as cytotoxicity; and 4) employed computational tools to identify potential targets for the most active compounds. Seven compounds presented activity against Paracoccidioides spp. Conclusion: These compounds are new hits with a predicted mechanisms of action, making them potentially attractive to develop new compounds.

Paracoccidioidomycosis and pregnancy: A 40-year single-center cohort study in the endemic area of Rio de Janeiro, Brazil.

The occurrence of acute paracoccidioidomycosis (PCM) in urban areas of the Rio de Janeiro state, Brazil, has emerged in recent years. Therefore, young populations, including pregnant women, are at a higher risk of infection. Furthermore, young women undergoing itraconazole treatment for PCM have increased chances to get pregnant because this medication may reduce the effectiveness of contraceptives. Acute PCM is invasive, reaching abdominal organs, posing a maternal-fetal risk. PCM treatment in pregnant women is also challenging due to the teratogenicity associated with the currently available oral drugs. There are scarce studies on PCM and pregnancy, mainly consisting of case reports and experimental murine models that highlight the severity of this association. We conducted a database research at a PCM reference center in Rio de Janeiro state from 1980 to 2020. We included patients diagnosed with PCM who were pregnant shortly before, at admission, or at any moment of their PCM follow-up care. Data related to pregnancy, childbirth, and the newborn were obtained from the Brazilian official public databases. We also reviewed the epidemiological and clinical features of these patients. During the study period, we identified 18 pregnant patients, with a median age of 26 years (range: 16-38). Among these cases, six (33.3%) were detected in the last 5 years, and 14 (77.8%) presented acute PCM, supporting the recent shift in the epidemiological profile towards acute PCM. Most pregnancies occurred during PCM treatment (n = 11, 61.1%), which led to challenges in the therapeutic management. Maternal-fetal complications occurred in some of these cases, including vaginal bleeding (n = 1), preeclampsia (n = 1), prematurity (n = 2), low birth weight (n = 4), and fetal deaths (n = 2). PCM during pregnancy presents a significant public health concern in the context of the emergence of acute PCM in urban areas.

Endemic Systemic Mycoses in Italy: A Systematic Review of Literature and a Practical Update.

Endemic systemic mycoses such as blastomycosis, coccidioidomycosis, histoplasmosis, talaromycosis, paracoccidioidomycosis are emerging as an important cause of morbidity and mortality worldwide. We conducted a systematic review on endemic systemic mycoses reported in Italy from 1914 to nowadays. We found out: 105 cases of histoplasmosis, 15 of paracoccidioidomycosis, 10 of coccidioidomycosis, 10 of blastomycosis and 3 of talaromycosis. Most cases have been reported in returning travelers and expatriates or immigrants. Thirtytwo patients did not have a story of traveling to an endemic area. Fortysix subjects had HIV/AIDS. Immunosuppression was the major risk factor for getting these infections and for severe outcomes. We provided an overview on microbiological characteristics and clinical management principles of systemic endemic mycoses with a focus on the cases reported in Italy.

3-phenacylideneoxindoles as a new class of antifungal compounds against Paracoccidioides spp.

Aims: Considering the need to identify new compounds with antifungal action, the activity of five 3-phenacylideneoxindoles compounds was evaluated. Materials & methods: The compounds were synthesized, and their antifungal activity was elucidated through minimum inhibitory concentration tests and interaction assay with other antifungals. Potential targets of compounds were predicted in silico. Results: 3-phenacylideneoxindoles compounds inhibited fungal growth with minimum inhibitory concentration and minimum fungicidal concentration ranging from 3.05 to 12.26 µM. The compounds demonstrated high selectivity index and presented a synergistic effect with itraconazole. In silico prediction revealed the pentafunctional AROM polypeptide, enolase, superoxide dismutase, catalase and kinases as proteins targets of the compound 4a. Conclusion: The results demonstrate that 3-phenacylideneoxindoles is a potential new class of antifungal compounds for paracoccidioidomycosis treatment.

Patients affected by paracoccidioidomycosis (PCM) require long-term treatment, which commonly influences their adherence. In addition, only three drugs are in clinical use, which indicates the relevance of research in identifying new drugs for treating PCM. Thus, five drugs were tested in the laboratory to verify whether they could prevent the growth of the fungus without being toxic to humans. In addition, whether these compounds in combination with drugs used to treat PCM could be even more potent was evaluated. All compounds tested efficiently inhibited the growth of Paracoccidioides, the fungus that causes PCM. One drug was identified that, combined with itraconazole, decreased the required dose of both the discovered compound and itraconazole needed to inhibit fungal growth. Using computational tools, this work suggests how the new drug could act against the fungus. The results demonstrate a potential new treatment option, but more studies are needed to confirm the safety of these drugs.

Itraconazole and neutrophil interactions in the immune-inflammatory response of paracoccidioidomycosis using a murine air pouch infection model.

Paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis (Pb), is a severe mycosis, prevalent in tropical countries. The presence of polymorphonuclear neutrophils (PMN) in lesions is conspicuous, indicating their central role in innate immunity through the direct killing of Pb and the production of cytokines that activate acquired immunity in the presence of itraconazole (Itra). The toxicity and direct antifungal activity of Itra on Pb in splenocyte co-cultures were evaluated in vitro. Itra showed no toxic effect but marked antifungal activity against Pb. Purified PMN were obtained by the subcutaneous (SC) injection of Pb into mice. Results showed the effect of Itra on the size of the air pouch produced, the cellular population that migrated to the infection site, protein, and mitochondrial metabolism patterns, production of ROS an NO, and the number of cytokines synthesized. Lower doses (3 and 10 mg/kg) of Itra did not affect the cellular profile but led to a lower influx of viable more active PMN, and increased production of ROS and proteins. At a dose of 50 mg/kg the PMN profile remained unchanged along with all other parameters analyzed remained unaltered. Decreases in most cytokine levels were inversely proportional to the Itra concentration. Lower Itra concentrations may elicit activation of the immune response because the combined effects of therapy and immune response are needed, while the higher dose does not require it. Itra also promotes the activation of the cytokines which elicit PMN activation and consequently the resolution of Pb18 infection in the air pouch.

Paracoccidioidomycosis and Addison's syndrome - case report.

Paracoccidioidomycosis is caused by a fungus (Paracoccidioides brasiliensis), which is endemic to Brazil. It is most frequently found in the lungs, with haematogenous and lymphatic spread. The condition is more prevalent in men, between 30 and 60 years old, commonly rural workers. It is the third leading cause of death among chronic infectious diseases today. The systemic disease has an insidious and nonspecific course, with adrenal involvement being observed in 5% of cases and requiring the destruction of 80% of the glands for symptoms of adrenal insufficiency to appear. Isolated involvement of this gland is quite rare. In this case report, however, our patient presented wasting and adrenal insufficiency with isolated adrenal involvement by the fungus.

Paracoccidioidomycosis: Current Status and Future Trends.

Paracoccidioidomycosis (PCM), initially reported in 1908 in the city of São Paulo, Brazil, by Adolpho Lutz, is primarily a systemic and neglected tropical mycosis that may affect individuals with certain risk factors around Latin America, especially Brazil. Paracoccidioides brasiliensis sensu stricto, a classical thermodimorphic fungus associated with PCM, was long considered to represent a monotypic taxon. However, advances in molecular taxonomy revealed several cryptic species, including Paracoccidioides americana, P. restrepiensis, P. venezuelensis, and P. lutzii, that show a preference for skin and mucous membranes, lymph nodes, and respiratory organs but can also affect many other organs. The classical diagnosis of PCM benefits from direct microscopy culture-based, biochemical, and immunological assays in a general microbiology laboratory practice providing a generic identification of the agents. However, molecular assays should be employed to identify Paracoccidioides isolates to the species level, data that would be complemented by epidemiological investigations. From a clinical perspective, all probable and confirmed cases should be treated. The choice of treatment and its duration must be considered, along with the affected organs, process severity, history of previous treatment failure, possibility of administering oral medication, associated diseases, pregnancy, and patient compliance with the proposed treatment regimen. Nevertheless, even after appropriate treatment, there may be relapses, which generally occur 5 years after the apparent cure following treatment, and also, the mycosis may be confused with other diseases. This review provides a comprehensive and critical overview of the immunopathology, laboratory diagnosis, clinical aspects, and current treatment of PCM, highlighting current issues in the identification, treatment, and patient follow-up in light of recent Paracoccidioides species taxonomic developments.

Proteomic alterations in Paracoccidioides brasiliensis caused by exposure to curcumin.

Paracoccidioides spp. are the etiological agent of paracoccidioidomycosis, a disease that causes skin lesions and affect the lungs and other organs. The current management of the disease is long and has several side effects that often lead the patient to give up the treatment, sequelae and even death. The search for new forms of treatment that minimize these drawbacks is very important. Thus, natural compounds are targets of great interest. Curcumin is one of the main components of the tubers of Curcuma longa, presenting medicinal effects well described in the literature, including the antifungal effect on Paracocidioides brasiliensis. Nevertheless, the mechanisms related to the antifungal effect of such compound are still unknown, so the objective of the present research is to understand what changes occur in the metabolism of P. brasiliensis after exposure to curcumin and to identify the main targets of the compound. Proteomic analysis as based on nanoUPLC-MS analysis and the functional classification of the identified proteins. The main metabolic processes that were being regulated were biologically validated through assays such as fluorescence microscopy, EPR and phagocytosis. Proteomic analysis revealed that curcumin regulates several metabolic processes of the fungus, including important pathways for energy production, such as the glycolytic pathway, beta oxidation and the glyoxylate cycle. Protein synthesis was down-regulated in fungi exposed to curcumin. The electron transport chain and the tricarboxylic acid cycle were also down-regulated, indicating that both the mitochondrial membrane and the mitochondrial activity were compromised. Plasma membrane and cell wall structure were altered following exposure to the compound. The fungus' ability to survive the phagocytosis process by alveolar macrophages was reduced. Thus, curcumin interferes with several metabolic pathways in the fungus that causes paracoccidioidomycosis. BIOLOGICAL SIGNIFICANCE: The challenges presented by the current treatment of paracoccidioidomycosis often contributing to patients' withdrawal from treatment, leading to sequelae or even death. Thus, the search for new treatment options against this disease is growing. The discovery that curcumin is active against Paracoccidioides was previously reported by our study group. Here, we clarify how the compound acts on the fungus causing its growth inhibition and decreased viability. Understanding the mechanisms of action of curcumin on P. brasiliensis elucidates how we can seek new alternatives and which metabolic pathways and molecular targets we should focus on in this incessant search to bring the patient a treatment with fewer adverse effects.