Antifungal activity of selective serotonin reuptake inhibitors against Cryptococcus spp. and their possible mechanism of action.

Fungal infections caused by Cryptococcus spp. pose a threat to health, especially in immunocompromised individuals. The available arsenal of drugs against cryptococcosis is limited, due to their toxicity and/or lack of accessibility in low-income countries, requiring more therapeutic alternatives. Selective serotonin reuptake inhibitors (SSRIs), through drug repositioning, are a promising alternative to broaden the range of new antifungals against Cryptococcus spp. This study evaluates the antifungal activity of three SSRIs, sertraline, paroxetine, and fluoxetine, against Cryptococcus spp. strains, as well as assesses their possible mechanism of action. Seven strains of Cryptococcus spp. were used. Sensitivity to SSRIs, fluconazole, and itraconazole was evaluated using the broth microdilution assay. The interactions resulting from combinations of SSRIs and azoles were investigated using the checkerboard assay. The possible action mechanism of SSRIs against Cryptococcus spp. was evaluated through flow cytometry assays. The SSRIs exhibited in vitro antifungal activity against Cryptococcus spp. strains, with minimum inhibitory concentrations ranging from 2 to 32 µg/mL, and had synergistic and additive interactions with azoles. The mechanism of action of SSRIs against Cryptococcus spp. involved damage to the mitochondrial membrane and increasing the production of reactive oxygen species, resulting in loss of cellular viability and apoptotic cell death. Fluoxetine also was able to cause significant damage to yeast DNA. These findings demonstrate the in vitro antifungal potential of SSRIs against Cryptococcus spp. strains.

Cryptococcal meningoencephalitis: Risk factors associated to death in a hospital in Northeastern Brazil.

INTRODUCTION: Cryptococcosis is an opportunistic systemic mycosis caused by pathogenic encapsulated yeasts of the genus Cryptococcus. The objective of the present study was to evaluate the risk factors associated with death of patients diagnosed with meningitis due to Cryptococcus spp. METHODS: This retrospective cohort study included patients admitted to the São José Hospital (SJH) with Cryptococcal Meningoencephalitis (CM) who were diagnosed between 2010 and 2018. Data collection was carried out by reviewing the patients' medical records. Death during hospitalization was considered the primary outcome. RESULTS: From 2010 to 2018, 21,519 patients were admitted to the HSJ, 124 of whom were hospitalized due to CM. The CM incidence rate was 5.8 cases/103 hospitalizations. We included 112 patients in the study. Male patients were the most affected (82.1%), and the median age was 37 years [IQR: 29-45]. HIV coinfection occurred in 79.4% of the patients. Fever (65.2%) and headache (88.4%) were the most frequent symptoms. Greater cellularity in the CSF was the most related factor to CM in non-HIV individuals (p < 0.05). Death during hospitalization occurred in 28.6% (n = 32) of the patients. The independent risk factors associated with death during the hospitalization were women (p = 0.009), age > 35 years (p = 0.046), focal neurological deficits (p = 0.013), altered mental status (p = 0.018) and HIV infection (p = 0.040). The twelve-month survival was lower in HIV-positive patients (p < 0.05). CONCLUSION: Early diagnosis, optimal treatment, and clinical follow-up strategies, especially in HIV patients, should be prioritized.

Pituitary cryptococcoma in an immunocompetent patient with panhypopituitarism: illustrative case.

BACKGROUND: Cryptococcosis is the most common mycosis of the central nervous system. It may develop in immunocompetent and immunocompromised patients, the latter representing most cases. The most common presentation of the disease is meningitis, whereas intra-axial lesions in the form of cryptococcoma are less frequent with a greater tendency to present in immunocompetent patients. The presentation of pituitary cryptococcoma is exceptional. To the best of the authors' knowledge, there is only one case published in the medical literature. OBSERVATIONS: The authors present the case of a 30-year-old male without a relevant medical history. He was referred to our center with a pituitary mass on magnetic resonance imaging and panhypopituitarism. The patient underwent endonasal endoscopic transsphenoidal tumor resection, and a histopathological diagnosis of pituitary cryptococcoma was made. Medical management included fluconazole and intravenous amphotericin. LESSONS: This case underscores the neurosurgical and medical management of an exceptional clinical presentation of pituitary cryptococcoma in an immunocompetent patient. To the best of the authors' knowledge, there is only one case published in the medical literature. This case provides an invaluable review of the clinical, imaging, and therapeutic considerations regarding this exceptional clinical entity.

Cryptococcosis in domestic and wild animals: A review.

Cryptococcosis is a fungal disease of public health relevance that affects numerous animal species and humans, causing respiratory and neurological impairment. Hence, we conducted a systematic review that included publications from 1975 to 2021 and covered 132 articles that addressed reports of cryptococcosis in domestic and wild animals, its main clinical manifestations, pathological findings, etiology, diagnosis, and therapeutic protocols. We found that the highest number of reports of cryptococcosis is in domestic species, especially cats. Among the wild and/or exotic animals, koalas and ferrets are the most affected, being important carriers of Cryptococcus spp. Pulmonary and neurological involvement is predominant in all species, although nonspecific clinical manifestations have been reported in various species, making clinical suspicion and diagnosis difficult. The countries with the most reports are Australia, the United States, Brazil, and Canada, with C. gattii VGI and VGII standing out. The therapies were based on azoles, amphotericin B, and 5-flucytosine, although there is no standard treatment protocol. Although, several diagnostic methods have been described, in a significant number of reports the diagnosis was made after a necropsy. Professionals are warned about diverse and nonspecific clinical manifestations in different animal species, which underlines the importance of cryptococcosis in the differential diagnosis in clinical practice. Furthermore, it is necessary to encourage the use of laboratory and molecular tools to improve the diagnosis of cryptococcosis. We also emphasize the urgent need for standardized therapeutic protocols to guide veterinary clinicians.

This review compiles studies on cryptococcosis in domestic and wild animals. Most reports occurred in cats and koalas. Pulmonary and neurological involvement was predominant in all affected species, and C. gattii VGI and VGII stood out in the etiology of the disease.

Reposicionamento de fármacos para cryptococose: avaliação experimental do cloridrato de duloxetina em Cryptococcus neoformans e Cryptococcus gattii / Repositioning of drugs for cryptococcosis: experimental evaluation of duloxetine hydrochloride in Cryptococcus neoformans and Cryptococcus gatti

Infecções fúngicas como as causadas por Cryptococcus spp. são de alta mortalidade e morbidade. O reposicionamento de fármaco, ou seja, a utilização de compostos para finalidade diferente da qual esse foi desenvolvido, pode ser uma alternativa para identificar fármacos mais eficazes. Assim, este estudo tem como propósito avaliar a atividade antifúngica, in vitro e in vivo, do fármaco cloridrato de duloxetina (CD), antidepressivo pertencente a classe dos Inibidores Seletivos da Recaptação da Serotonina e Norepinefrina frente a cepas padrões e clínicas de Cryptococcus neoformans e C. gattii. Foi utilizada a técnica de microdiluição de acordo com o European Committee on Antimicrobial Susceptibility Testing (EUCAST) para determinar a Concentração Inibitória Mínima (MIC), e a técnica do "tabuleiro de xadrez" para avaliar o efeito sinérgico de anfotericina B (AmB) em associação com CD. Além disso, foi avaliado o efeito de CD na quantidade do ergosterol. O efeito do CD também foi avaliado em biofilmes de C. neoformans e C. gattii, analisando a biomassa por cristal violeta, a viabilidade celular por XTT e morfologia através das imagens de Microscopia Eletrônica de Varredura (MEV). In vivo, a eficácia de CD foi avaliada por curvas de sobrevivência no modelo invertebrado Galleria mellonella. CD foi ativo frente a todas as cepas clínicas e padrões de C. neoformans e C. gattii, apresentando valores de CIM e CFM na faixa de 15,62 ­ 62,5 µg/mL. A combinação de CD com AmB apresentou uma combinação sinérgica, reduzindo o valor da CIM em 4 vezes tanto para CD quanto para AmB. CD não produziu redução na quantidade de ergosterol presente na membrana de C. gattii ATCC e C. neoformans ATCC. Em biofilmes, foi observada a redução da biomassa do biofilme em até 82,16% e redução de 99,6% na viabilidade celular de C. gattii. Em biofilmes de C. neoformans a redução foi de 81,13% e 99,5% respectivamente para a análise de biomassa e viabilidade. As imagens de MEV corroboraram com os achados dos ensaios realizados para análise do efeito de CD em biofilmes. Em G. mellonella aumentou a sobrevivência da larva quando utilizado na concentração de 3,125 mg/larva. Assim, os ensaios validaram a hipótese de que o cloridrato de duloxetina tem ação antifúngica e antibiofilme in vitro frente a cepas clínicas e cepas padrões de C. neoformans e C. gattii. (AU)

Fungal infections such as those caused by Cryptococcus spp. are of high mortality and morbidity. Drug repositioning, that is, the use of compounds for a purpose different from the one for which it was developed, can be an alternative to identify more effective drugs. Thus, this study aims to evaluate the antifungal activity, in vitro and in vivo, of the drug duloxetine hydrochloride (CD), an antidepressant belonging to the class of Selective Serotonin and Norepinephrine Reuptake Inhibitors against standard and clinical strains of Cryptococcus neoformans and C. gattii. The microdilution technique according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was used to determine the Minimum Inhibitory Concentration (MIC), and the "chessboard" technique was used to evaluate the synergistic effect of amphotericin B (AmB) on association with CD. In addition, the effect of CD on the amount of ergosterol was evaluated. The effect of CD was also evaluated in C. neoformans and C. gattii biofilms, analyzing the biomass by crystal violet, cell viability by XTT and morphology through Scanning Electron Microscopy (SEM) images. In vivo, the effectiveness of CD was evaluated by survival curves in the Galleria mellonella invertebrate model. CD was active against all clinical strains and patterns of C. neoformans and C. gattii, with MIC and CFM values in the range of 15.62 ­ 62.5 µg/mL. The combination of CD with AmB showed a synergistic combination, reducing the MIC value by 4 times for both CD and AmB. CD did not produce a reduction in the amount of ergosterol present in the membrane of C. gattii ATCC and C. neoformans ATCC. In biofilms, a reduction in biofilm biomass of up to 82.16% and a 99.6% reduction in cell viability of C. gattii were observed. In C. neoformans biofilms the reduction was 81.13% and 99.5% respectively for biomass and viability analysis. The SEM images corroborated the findings of the tests carried out to analyze the effect of CD on biofilms. In G. mellonella, larval survival increased when used at a concentration of 3.125 mg/larvae. Thus, the tests validated the hypothesis that duloxetine hydrochloride has antifungal and antibiofilm action in vitro against clinical strains and standard strains of C. neoformans and C. gattii.(AU)

Modification of Hinge/Transmembrane and Signal Transduction Domains Improves the Expression and Signaling Threshold of GXMR-CAR Specific to Cryptococcus spp.

Chimeric antigen receptors (CARs) redirect T cells to recognize a specific target. CAR components play a pivotal role in antigen specificity, structure stability, expression on cell surface, and induction of cellular activation, which together determine the success of CAR T-cell therapy. CAR products targeting B-cell lymphoma encouraged the development of new CAR applications beyond cancer. For example, our group developed a CAR to specifically target glucuronoxylomannan (GXM) in the capsule of Cryptococcus species, called GXMR-CAR or GXMR-IgG4-28ζ. Cryptococcus are fungi that cause the life-threatening disease cryptococcosis, and GXMR-IgG4-28ζ redirected T cells to target yeast and titan cell forms of Cryptococcus spp. Here, we replaced the IgG4-hinge and CD28-transmembrane domains from GXMR-CAR with a CD8α molecule as the hinge/transmembrane and used CD28 or 4-1BB molecules as co-stimulatory domains, creating GXMR-8-28ζ and GXMR-8-BBζ, respectively. Jurkat cells expressing GXMR-CAR containing CD8α as the hinge/transmembrane improved the CAR expression and induced a tonic signaling. GXMR-8-28ζ and GXMR-8-BBζ induced high levels of IL-2 and up-regulation of CD69 expression in the presence of reference strains of C. neoformans and C. gattii. Moreover, GXMR-8-28ζ and GXMR-8-BBζ showed increased strength in response to incubation with clinical isolates of Cryptococcuss spp., and 4-1BB co-stimulatory domain triggered a more pronounced cellular activation. Dasatinib, a tyrosine kinase inhibitor, attenuated the GXMR-CAR signaling cascade's engagement in the presence or absence of its ligand. This study optimized novel second-generation GXMR-CARs containing the CD8-hinge/transmembrane domain that improved CAR expression, antigen recognition, and signal strength in T-cell activation.

PAMPs and Host Immune Response in Cryptococcal Infection.

Cryptococcus spp. are yeast-type opportunistic fungal pathogens with thick polysaccharide capsules that infect the lungs via airborne routes and frequently cause fatal meningoencephalitis. The cellular immune mechanism plays a central role in controlling cryptococcal infection and is critically regulated by Th1-Th2 immune balance. Pathogens that have invaded the host are recognized by innate immune cells, and appropriate immune responses are initiated. Pathogen-associated molecular patterns (PAMPs) are recognized by macrophages and dendritic cells via pattern recognition receptors (PRRs), which trigger the inflammatory responses as the first line of host defense. Some PRRs, such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs), are involved in the recognition of cryptococcal components, such as glucuronoxylomannan (GXM), mannoproteins (MPs), and nucleic acids. However, some cryptococcal cell components suppress the host immune response. This review will highlight the cryptococcal components involved in host immune responses. Future research is expected to promote the understanding of the mechanism of host immune response to Cryptococcus, which will lead to the development of new vaccines and therapies for cryptococcal infection.

Antifungal Activity of Amphiphilic Perylene Bisimides.

Perylene-based compounds, either naturally occurring or synthetic, have shown interesting biological activities. In this study, we report on the broad-spectrum antifungal properties of two lead amphiphilic perylene bisimides, compounds 4 and 5, which were synthesized from perylene-3,4,9,10-tetracarboxylic dianhydride by condensation with spermine and an ammonium salt formation. The antifungal activity was evaluated using a collection of fungal strains and clinical isolates from patients with onychomycosis or sporotrichosis. Both molecules displayed an interesting antifungal profile with MIC values in the range of 2-25 µM, being as active as several reference drugs, even more potent in some particular strains. The ammonium trifluoroacetate salt 5 showed the highest activity with a MIC value of 2.1 µM for all tested Candida spp., two Cryptococcus spp., two Fusarium spp., and one Neoscytalidium spp. strain. Therefore, these amphiphilic molecules with the perylene moiety and cationic ammonium side chains represent important structural features for the development of novel antifungals.

Clinical performance of the IMMY cryptococcal antigen lateral flow assay in dogs and cats.

BACKGROUND: Cryptococcal lateral flow antigen assays (CLFAs) have been assessed in comparison to the latex cryptococcal antigen agglutination test but their clinical performance is unknown. OBJECTIVE: Determine clinical performance of IMMY CLFA (Immuno-Mycologics Inc, Oklahoma) using patients with and without cryptococcosis as the reference standard. ANIMALS: One-hundred ninety-seven serum samples from client-owned dogs and cats. METHODS: Review of medical records of a referral population of dogs and cats that had CLFA performed between 2012 and 2020. Animals were classified as cryptococcosis positive (Cr+) or negative (Cr-) based on clinical information. Clinical diagnosis was used to calculate positive and negative percent agreement of the CLFA. RESULTS: Twelve specimens (4 canine, 8 feline) were obtained from Cr+ animals and had positive CLFA results. One-hundred eighty-five specimens (139 canine, 46 feline) were collected from Cr- animals. Negative CLFA results were recorded in 129 canine and 44 feline Cr- samples. Positive CLFA results were noted for 10 canine and 2 feline Cr- samples. Positive percent agreement of CLFA was 100% (confidence interval [CI], 39.8%-100% dogs; 63.1%-100% cats). Negative percent agreements were 92.8% (CI, 87.2%-96.5%) for dogs and 95.7% (CI, 85.2%-99.5%) for cats. CONCLUSIONS AND CLINICAL IMPORTANCE: A negative IMMY CLFA result enables reliable exclusion of cryptococcal infection in dogs and cats. By contrast, a positive result must be interpreted cautiously and further testing should be performed to verify a diagnosis of cryptococcosis.

Antifungal activity of liriodenine on clinical strains of Cryptococcus neoformans and Cryptococcus gattii species complexes.

Background: Cryptoccocal meningitis continues to present high incidence among AIDS patients. The treatment of choice is the synergistic combination of flucytosine (5-FC) with amphotericin B deoxycholate (AmBd) or its lipid formulations. However, 5-FC is unavailable in many countries and AmB demands hospitalization. The combination of AmB with the fungistatic fluconazole (FLC) or the use of high FLC daily doses alone became the choice. Nonetheless, sterilization of cerebrospinal fluid is delayed with FLC monotherapy, mainly with high fungal burden. These findings suggest the search for new antifungal compounds, such as liriodenine. Methods: Liriodenine antifungal activity was evaluated by three procedures: determining the minimum inhibitory concentration (MIC) on 30 strains of the Cryptococcus neoformans (C. neoformans) complex and 30 of the Cryptococcus gattii (C. gattii) complex, using EUCAST methodology and amphotericin B deoxycholate as control; performing the time-kill methodology in two strains of the C. neoformans complex and one of the C. gattii complex; and injury to cryptococcal cells, evaluated by transmission electron microscopy (TEM). Liriodenine absorption and safety at 0.75 and 1.50 mg.kg-1 doses were evaluated in BALB/c mice. Results: Liriodenine MICs ranged from 3.9 to 62.5 µg.mL-1 for both species complexes, with no differences between them. Time-kill methodology confirmed its concentration-dependent fungicidal effect, killing all the strains below the limit of detection (33 CFU.mL-1) at the highest liriodenine concentration (32-fold MIC), with predominant activity during the first 48 hours. Liriodenine induced severe Cryptococcus alterations - cytoplasm with intense rarefaction and/or degradation, injury of organelles, and presence of vacuoles. Liriodenine was better absorbed at lower doses, with no histopathological alterations on the digestive tract. Conclusion: The fungicidal activity confirmed by time-kill methodology, the intense Cryptococcus injury observed by TEM, the absorption after gavage administration, and the safety at the tested doses indicate that the liriodenine molecule is a promising drug lead for development of anticryptococcal agents.

In vitro activity of immunosuppressive agents against Cryptococcus neoformans.

INTRODUCTION: Infections caused by Cryptococcus neoformans are a major cause of fungal mortality in HIV-infected/AIDS patients and in those receiving organ transplants. We evaluated the in vitro activity of tacrolimus and cyclosporine in combination with amphotericin B and fluconazole against C. neoformans. METHODS: MICs were determined against a total of 30 clinical isolates of C. neoformans by the microdilution method following the CLSI M27-A3 guidelines and by the checkerboard method. RESULTS: Tacrolimus and cyclosporine A showed in vitro activity against cryptococcal isolates. The combination of amphotericin B with cyclosporine A or tacrolimus was synergistic against 90% and 30% of isolates, respectively. Synergism was also observed with the combination of fluconazole with cyclosporine A or tacrolimus, against 70% and 20% of isolates, respectively. CONCLUSIONS: The synergistic interactions between the calcineurin inhibitors and antifungal drugs against C. neoformans isolates, could potentially have a role in devising novel therapeutic strategies for this opportunistic mycosis.

In vitro activity of immunosuppressive agents against Cryptococcus neoformans / Actividad in vitro de agentes inmunosupresores frente a Cryptococcus neoformans

IntroductionInfections caused by Cryptococcus neoformans are a major cause of fungal mortality in HIV-infected/AIDS patients and in those receiving organ transplants. We evaluated the in vitro activity of tacrolimus and cyclosporine in combination with amphotericin B and fluconazole against C. neoformans.MethodsMICs were determined against a total of 30 clinical isolates of C. neoformans by the microdilution method following the CLSI M27-A3 guidelines and by the checkerboard method.ResultsIntroductionInfections caused by Cryptococcus neoformans are a major cause of fungal mortality in HIV-infected/AIDS patients and in those receiving organ transplants. We evaluated the in vitro activity of tacrolimus and cyclosporine in combination with amphotericin B and fluconazole against C. neoformans.MethodsMICs were determined against a total of 30 clinical isolates of C. neoformans by the microdilution method following the CLSI M27-A3 guidelines and by the checkerboard method.ResultsTacrolimus and cyclosporine A showed in vitro activity against cryptococcal isolates. The combination of amphotericin B with cyclosporine A or tacrolimus was synergistic against 90% and 30% of isolates, respectively. Synergism was also observed with the combination of fluconazole with cyclosporine A or tacrolimus, against 70% and 20% of isolates, respectively.ConclusionsThe synergistic interactions between the calcineurin inhibitors and antifungal drugs against C. neoformans isolates, could potentially have a role in devising novel therapeutic strategies for this opportunistic mycosis.

IntroducciónLas infecciones causadas por Cryptococcus neoformans son la principal causa de mortalidad por hongos en pacientes con infección por el VIH/SIDA o en pacientes trasplantados. Evaluamos la actividad in vitro de tacrolimus y ciclosporina A en combinación con anfotericina B y fluconazol frente a C. neoformans.MétodosSe determinaron las CMI de ciclosporina A y tacrolimus frente a 30 aislados clínicos de C. neoformans mediante microdilución, según el documento CLSI M27-A3 y por el método del tablero de ajedrez.ResultadosTacrolimus y ciclosporina A mostraron actividad in vitro frente a C. neoformans. La combinación de anfotericina B con ciclosporina A o tacrolimus fue sinérgica frente al 90 y 30% de aislados, respectivamente. Se observó sinergismo con fluconazol y ciclosporina A o tacrolimus, frente al 70 y 20% de aislados, respectivamente.ConclusionesLa actividad sinérgica entre inhibidores de la calcineurina y antimicóticos frente a C. neoformans podría ser una nueva estrategia terapéutica para esta micosis.

Isolamento ambiental e identificação bioquímica de Cryptococcus spp / Environmental isolation and biochemical identification of Cryptococcus spp

A Cryptococose é uma doença fúngica, oportunista, de caráter ubiquitária e crescente em número de casos nos últimos anos. Considerada atualmente como um problema de saúde pública, pode ocasionar doença em muitos hospedeiros, inclusive no homem. A infecção ocorre em decorrência da inalação de basidiósporos ou leveduras desidratadas infectantes presentes no ambiente. O gênero Cryptococcus caracteriza-se por ser uma levedura normalmente encapsulada, responsável por infecção sistêmica, causada principalmente pelas espécies, Cryptococcus neoformans e Cryptococcus gattii, comumente encontrada em substratos de origem animal, como excretas de aves, vegetais, e em troncos ocos de árvores, embora, com o avanço dos estudos, o envolvimento de outras espécies nas infecções humanas venha sendo cada vez mais relatada.Com o objetivo de conhecer a ecoepidemiologia do Cryptococcus spp em 30 amostras de material putrefado de árvores e 30 amostras excretas de aves de 10 locais representativos no perímetro do Instituto Lauro de Souza Lima-Bauru, buscamos conhecer a ecoepidemiologia desses agentes e posterior, profilaxia, minimizando riscos de contágio. As amostras foram processadas e semeadas em placas contendo ágar semente de níger e ágar Sabouraud Dextrose com cloranfenicol, incubadas a temperatura de 30°C e observadas diariamente por dez dias. O crescimento de leveduras com características de Cryptococcus spp foram avaliadas pelo aspecto colonial nos meios de cultivo e pela realização de exames diretos com tinta da China e com lactofenol azul de algodão através da análise dos aspectos micromorfológicos. As leveduras analisadas foram submetidas as provas bioquímicas como; produção de urease, produção de fenoloxidase no ágar semente de níger, termotolerância a 37°C e quimiotipagem em ágar CGB (L-canavanina-glicina-azul de bromotimol). Os dados obtidos foram avaliados estatisticamente pelo Teste exato de Fisher e pelo teste qui-quadrado, adotando o nível de significância de 5% e demonstrando crescimento de C.neoformans, C.gattii e C. Laurentii (Papiliotrema laurentii), concluindo que esses fungos podem infectar o ambiente.

Cryptococcosis is a fungal, opportunistic and ubiquitous disease which is increasing in number of cases in recent years. Currently considered as a public health problem, it can cause disease in many hosts, including humans. The infection occurs as a result of inhalation of basidiospores or dehydrated infective yeasts present in the environment. The genus Cryptococcus is characterized as a normally encapsulated yeast, responsible for systemic infection, mainly caused by the species, Cryptococcus neoformans and Cryptococcus gattii, commonly found in substrates of animal origin, such as Bird droppings, vegetables, and in tree hollows, although, with the advancement of studies, the involvement of other species in human infections has been increasingly reported. With the aim of knowing the ecoepidemiology of Cryptococcus spp in 30 samples of putrefying material from trees and 30 samples of bird droppings from 10 representative sites in the Instituto Lauro de Souza Lima-Bauru (ILSL), we sought to know the ecoepidemiology of these agents and later, prophylaxis, minimizing the risk of contagion. The samples were processed and seeded on plates containing niger seed agar and Sabouraud Dextrose agar with chloramphenicol, incubated at 30°C and observed daily for ten days. The growth of yeasts with characteristics of Cryptococcus spp were evaluated by the colonial appearance in the fungus culture and by performing direct examinations with China ink and cotton blue lactophenol through the analysis of micromorphological aspects. The analyzed yeasts were submitted to biochemical tests such as; urease production, phenoloxidase production on niger seed agar, thermotolerance at 37°C and chemotyping on CGB agar (L-canavanine-glycine-bromothymol blue). The data obtained were statistically evaluated using Fisher's exact test and the chi-square test, adopting a significance level of 5%. Cryptococcus neoformans, Cryptococcus gattii and Cryptococcus laurentii were isolated.

Consensus guidelines for the diagnosis and management of cryptococcosis and rare yeast infections in the haematology/oncology setting, 2021.

Cryptococcosis caused by the Cryptococcus neoformans-Cryptococcus gattii complex is an important opportunistic infection in people with immunodeficiency, including in the haematology/oncology setting. This may manifest clinically as cryptococcal meningitis or pulmonary cryptococcosis, or be detected incidentally by cryptococcal antigenemia, a positive sputum culture or radiological imaging. Non-Candida, non-Cryptococcus spp. rare yeast fungaemia are increasingly common in this population. These consensus guidelines aim to provide clinicians working in the Australian and New Zealand haematology/oncology setting with clear guiding principles and practical recommendations for the management of cryptococcosis, while also highlighting important and emerging rare yeast infections and their recommended management.

Titan Cells and Yeast Forms of Cryptococcus neoformans and Cryptococcus gattii Are Recognized by GXMR-CAR.

Cryptococcosis, a systemic mycosis that affects both the immunocompromised and immunocompetent, is caused by the inhalation of dehydrated yeasts or fungal spores of Cryptococcus gattii or Cryptococcus neoformans. The Cryptococcus spp. polysaccharide capsule is composed mainly of glucuronoxylomannan-GXM, its major virulence factor. The capsule thickness increases to more than 15 µm during titanization, favoring the pathogenesis of cryptococcosis. Previous studies demonstrated that cytotoxic T cells that had been bioengineered with GXM-targeting chimeric antigen receptor (GXMR-CAR) were able to recognize C. neoformans by promoting the control of titanization. GXMR-CAR, a second-generation CAR, contains a single-chain variable fragment that originates from a 18B7 clone: a human IgG4 hinge, followed by a human CD28 (transmembrane/cytoplasmic domains) and a CD3ς chain. In the current study, we redirected T cells to target distinct C. neoformans and C. gattii cell types by GXMR-CAR. Lentiviral particles carrying the GXMR-CAR sequence were used to transduce Jurkat cells, and these modified cells interacted with the GXM of the C. gattii R265 strain. Moreover, GXMR-CAR mediated the recognition of C. gattii and C. neoformans yeasts with both thin and thick polysaccharide capsules, and GXMR-CAR Jurkat cells interacted with titan cells sourced from both Cryptococcus spp. Thus, bioengineered cells using CAR can improve the treatment of cryptococcosis.

Comprehensive Analysis and Risk Identification of Pulmonary Cryptococcosis in Non-HIV Patients.

Pulmonary cryptococcosis in the non-human immunodeficiency virus-infected population is uncommon. We aimed to explore the relevance between clinical presentations, radiological findings, and comorbidities and identify the outcome predictors. A total of 321 patients at Taichung Veterans General Hospital between 2005 and 2019 were included; of them, 204 (63.6%) had at least one comorbidity, while 67 (20.9%) had two or more. The most common comorbidities were diabetes mellitus (27.4%), malignant solid tumor (19.6%), autoimmune disease (15.6%), and chronic kidney disease (8.4%). Patients experiencing comorbidity, particularly those with multiple comorbidities, had a higher multilobar and extrapulmonary involvement, which could explain these patients being more symptomatic. In the overall population, extrapulmonary involvement independently predicted disease recurrence and death. Amongst patients with isolated pulmonary cryptococcosis, age, cryptococcal antigen (CrAg) titer in blood, and comorbidities not only predicted the extent of disease, but also its outcome. Of note, patients simultaneously with age ≥ 65 years, CrAg test ≥ 1:128, and multiple comorbidities had the lowest disease control of antifungal treatment (76.9%) and the highest rate of disease recurrence or death from any cause (40.0%). In conclusion, approximately two-thirds of patients had at least one underlying comorbidity. In addition to extrapulmonary involvement, old age, high CrAg titer in blood, and multiple comorbidities could act as risk factors for predicting the extent of disease and outcome.

Fungal Biofilms as a Valuable Target for the Discovery of Natural Products That Cope with the Resistance of Medically Important Fungi-Latest Findings.

The development of new antifungal agents that target biofilms is an urgent need. Natural products, mainly from the plant kingdom, represent an invaluable source of these entities. The present review provides an update (2017-May 2021) on the available information on essential oils, propolis, extracts from plants, algae, lichens and microorganisms, compounds from different natural sources and nanosystems containing natural products with the capacity to in vitro or in vivo modulate fungal biofilms. The search yielded 42 articles; seven involved essential oils, two Brazilian propolis, six plant extracts and one of each, extracts from lichens and algae/cyanobacteria. Twenty articles deal with the antibiofilm effect of pure natural compounds, with 10 of them including studies of the mechanism of action and five dealing with natural compounds included in nanosystems. Thirty-seven manuscripts evaluated Candida spp. biofilms and two tested Fusarium and Cryptococcus spp. Only one manuscript involved Aspergillus fumigatus. From the data presented here, it is clear that the search of natural products with activity against fungal biofilms has been a highly active area of research in recent years. However, it also reveals the necessity of deepening the studies by (i) evaluating the effect of natural products on biofilms formed by the newly emerged and worrisome health-care associated fungi, C. auris, as well as on other non-albicans Candida spp., Cryptococcus sp. and filamentous fungi; (ii) elucidating the mechanisms of action of the most active natural products; (iii) increasing the in vivo testing.

Comparison of Cryptococcus gattii/neoformans Species Complex to Related Genera (Papiliotrema and Naganishia) Reveal Variances in Virulence Associated Factors and Antifungal Susceptibility.

Cryptococcosis is an infectious disease of worldwide distribution, caused by encapsulated yeasts belonging to the phylum Basidiomycota. The genus Cryptococcus includes several species distributed around the world. The C. gattii/neoformans species complex is largely responsible for most cases of cryptococcosis. However, clinical series have been published of infections caused by Papiliotrema (Cryptococcus) laurentii and Naganishia albida (Cryptococcus albidus), among other related genera. Here, we examined the pathogenic potential and antifungal susceptibility of C. gattii/neoformans species complex (clades I and II) and related genera (Papiliotrema and Naganishia) isolated from environmental and clinical samples. P. laurentii (clade III), N. liquefasciens/N. albidosimilis (clade IV); and N. adeliensis/N. albida (clade V) strains produced higher levels of phospholipase and hemolysins, whereas the C. gattii/neoformans species complex strains (clades I and II) had markedly thicker capsules, produced more biofilm biomass and melanin, which are known virulence attributes. Interestingly, 40% of C. neoformans strains (clade II) had MICs above the ECV established for this species to amphotericin B. Several non-C. gattii/neoformans species complex (clades III to V) had MICs equal to or above the ECVs established for C. deuterogattii and C. neoformans for all the three antifungal drugs tested. Finally, all the non-C. gattii/neoformans clinical isolates (clades III to V) produced more melanin than the environmental isolates might reflect their particularly enhanced need for melanin during in vivo protection. It is very clear that C. gattii/neoformans species complex (clades I and II) strains, in general, show more similar virulence phenotypes between each other when compared to non-C. gattii/neoformans species complex (clades III to V) isolates. These observations together with the fact that P. laurentii and Naganishia spp. (clades III to V) strains were collected from the outside of a University Hospital, identify features of these yeasts important for environmental and patient colonization and furthermore, define mechanisms for infections with these uncommon pathogens.

Disseminated Cryptococcosis in an Immunocompetent Child.

Disseminated cryptococcosis in children is a classic affliction associated with human immunodeficiency virus (HIV) infection or primary inherited immunodeficiency disorders (PID) with central nervous system being the most common site of dissemination. We report a rare case of disseminated cryptococcosis in an 11-year-old girl who presented with pulmonary involvement, hepatosplenomegaly, and generalized lymphadenopathy. No known inherited or acquired immune deficiencies were identified after a comprehensive laboratory work-up including genetic sequencing. She responded well to anti-fungal therapy (flucytosine and amphotericin followed by fluconazole) and is on regular follow-up.

The role of Cryptococcus neoformans histone deacetylase genes in the response to antifungal drugs, epigenetic modulators and to photodynamic therapy mediated by an aluminium phthalocyanine chloride nanoemulsion in vitro.

Cryptococcus is a globally distributed fungal pathogen that primarily afflicts immunocompromised individuals. The therapeutic options are limited and include mostly amphotericin B or fluconazole, alone or in combination. The extensive usage of antifungals allowed the selection of resistant pathogens posing threats to global public health. Histone deacetylase genes are involved in Cryptococcus virulence, and in pathogenicity and resistance to azoles in Candida albicans. Aiming to assess whether histone deacetylase genes are involved in antifungal response and in synergistic drug interactions, we evaluated the activity of amphotericin B, fluconazole, sulfamethoxazole, sodium butyrate or trichostatin A (histone deacetylase inhibitors), and hydralazine or 5- aza-2'-deoxycytidine (DNA methyl-transferase inhibitors) against different Cryptococcus neoformans strains, C. neoformans histone deacetylase null mutants and Cryptococcus gattii NIH198. The drugs were employed alone or in different combinations. Fungal growth after photodynamic therapy mediated by an aluminium phthalocyanine chloride nanoemulsion, alone or in combination with the aforementioned drugs, was assessed for the C. neoformans HDAC null mutant strains. Our results showed that fluconazole was synergistic with sodium butyrate or with trichostatin A for the hda1Δ/hos2Δ double mutant strain. Sulfamethoxazole was synergistic with sodium butyrate or with hydralazine also for hda1Δ/hos2Δ. These results clearly indicate a link between HDAC impairment and drug sensitivity. Photodynamic therapy efficacy on controlling the growth of the HDAC mutant strains was increased by amphotericin B, fluconazole, sodium butyrate or hydralazine. This is the first study in Cryptococcus highlighting the combined effects of antifungal drugs, histone deacetylase or DNA methyltransferase inhibitors and photodynamic therapy in vitro.