RESUMO

Background and Objectives: several patients with COVID-19 require hospital admission due to severe respiratory complications and undergo intensive care with mechanical ventilation (MV) support. Associated with this situation, there is an increase in fungal co-infections, which has a negative impact on the outcome of COVID-19. In this regard, this study intended to compare Candida spp. incidence in the respiratory tract of patients admitted in the COVID and General Intensive Care Units (ICU) at a teaching hospital in 2021. Methods: the results of protected tracheal aspirate samples from 556 patients admitted to the COVID ICU and 260 to General ICU as well as the respective records. Results: of the patients analyzed, 38 revealed a positive sample for Candida in the COVID ICU and 10 in the General ICU, with an incidence of 68.3/1000 and 38.5/1000, respectively. Males were predominant in both wards. The most affected age group was the population over 60 years old, and the average hospital admission for the COVID ICU was 22.1 years, and for the General ICU, 24.2. Conclusion: Candida albicans was the most frequently isolated species, and the mortality rate in patients positive for Candida was higher in patients with COVID-19 compared to patients in the General ICU, suggesting that patients infected with SARS-CoV-2, admitted to the ICU under MV, are more predisposed to colonization by Candida spp., which can have a fatal outcome in these patients.(AU)

Justificativa e objetivos: muitos pacientes com COVID-19 necessitam de hospitalização devido às complicações respiratórias graves, e são submetidos a cuidados intensivos com suporte de ventilação mecânica (VM). Associado a esse quadro, verifica-se o aumento de coinfecções fúngicas, que tem impacto negativo no desfecho da COVID-19. Nesse sentido, este estudo pretendeu comparar a incidência de Candida spp. no trato respiratório de pacientes internados nas Unidades de Terapia Intensiva (UTI) COVID e Geral em um hospital escola em 2021. Métodos: foram avaliados os resultados de amostras de aspirado traqueal protegido provenientes de 556 pacientes internados na UTI COVID e 260 na UTI Geral, bem como os respectivos prontuários. Resultados: dos pacientes analisados, 38 revelaram amostra positiva para Candida na UTI COVID e 10 na UTI Geral, com incidência de 68,3/1000 e 38,5/1000, respectivamente. O sexo masculino foi predominante em ambas as alas. A faixa etária mais acometida foi a população acima de 60 anos, e a média de internação para a UTI COVID foi de 22,1 anos, e para a UTI Geral, 24,2. Conclusão: Candida albicans foi a espécie isolada com maior frequência, e a taxa de mortalidade em pacientes com positivos para Candida foi maior em pacientes com COVID-19 em relação aos pacientes da UTI Geral, sugerindo que pacientes infectados com SARS-CoV-2, internados em UTI sob VM, são mais predispostos à colonização por Candida spp., que pode ter um desfecho fatal nesses pacientes.(AU)

Justificación y objetivos: muchos pacientes con COVID-19 requieren hospitalización debido a complicaciones respiratorias graves y se someten a cuidados intensivos con soporte de ventilación mecánica (VM). Asociado a esta situación, hay un aumento de las coinfecciones fúngicas, lo que repercute negativamente en el desenlace de la COVID-19. En este sentido, este estudio pretendió comparar la incidencia de Candida spp. en el tracto respiratorio de pacientes ingresados en las Unidades de Cuidados Intensivos (UCI) COVID y General de un hospital escuela en 2021. Métodos: los resultados de muestras de aspirado traqueal protegidas de 556 pacientes ingresados en la UCI COVID y 260 en el UCI General, así como los respectivos registros. Resultados: de los pacientes analizados, 38 presentaron muestra positiva a Candida en UCI COVID y 10 en UCI General, con una incidencia de 68,3/1000 y 38,5/1000, respectivamente. Los machos predominaban en ambas alas. El grupo de edad más afectado fue la población mayor de 60 años, y la hospitalización promedio en la UCI COVID fue de 22,1 años, y en la UCI General, de 24,2. Conclusiones: Candida albicans fue la especie aislada con mayor frecuencia, y la tasa de mortalidad en pacientes positivos para Candida fue mayor en pacientes con COVID-19 en comparación con los pacientes en la UCI General, lo que sugiere que los pacientes infectados con SARS-CoV-2, ingresados en la UCI bajo VM, están más predispuestos a la colonización por Candida spp., lo que puede tener un desenlace fatal en estos pacientes.(AU)

Assuntos

RESUMO

The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.

RESUMO

Sporotrichosis is the main subcutaneous mycosis worldwide. Several complications, including meningeal forms, can be observed in immunocompromised individuals. The sporotrichosis diagnosis is time-consuming due to the culture's limitations. The low fungal burden in cerebrospinal fluid (CSF) samples is another important drawback in the diagnosis of meningeal sporotrichosis. Molecular and immunological tests can improve the detection of Sporothrix spp. in clinical specimens. Therefore, the following five non-culture-based methods were evaluated for the detection of Sporothrix spp. in 30 CSF samples: (i) species-specific polymerase chain reaction (PCR); (ii) nested PCR; (iii) quantitative PCR; (iv) enzyme-linked immunosorbent assay (ELISA) for IgG detection; and (v) ELISA for IgM detection. The species-specific PCR was unsuccessful in the diagnosis of the meningeal sporotrichosis. The other four methods presented substantial levels of sensitivity (78.6% to 92.9%) and specificity (75% to 100%) for the indirect detection of Sporothrix spp. Both DNA-based methods presented similar accuracy (84.6%). Both ELISA methods were concomitantly positive only for patients with sporotrichosis and clinical signs of meningitis. We suggest that these methods should be implemented in clinical practice to detect Sporothrix spp. in CSF early, which may optimize treatment, augment the chances of a cure, and improve the prognosis of affected individuals.

RESUMO

Leishmania (Viannia) braziliensis is the main species responsible for American tegumentary leishmaniasis in Brazil. Nevertheless, the use of this parasite species to study Leishmania infection in the murine model has been less conducted when compared with other Leishmania species. The control of murine infection with Leishmania has been associated with nitric oxide (NO) produced by inducible NO synthase (iNOS) from M1 macrophages, while arginase expressed by M2 macrophages is related to Leishmania proliferation. Here we use three different strains of L. (V.) braziliensis and one strain of L. (L.) major to study a 9-day infection of macrophages in vitro. Wild-type bone marrow-derived macrophages (BMDM) supported the proliferation of L. (L) major amastigotes from the 3rd day after infection, while all strains of L. (V.) braziliensis did not proliferate even inside IL-4-treated or iNOS knockout (KO) macrophages. The arginase activity was higher in iNOS KO than IL-4-treated macrophage showing that the absence of proliferation is independent of arginase. Importantly, L. (V.) braziliensis was able to cause uncontrolled disease in iNOS KO mice in vivo demonstrating that murine macrophages present at the site of infection have additional changes beyond inhibition of NO production or stimulation of arginase activity to support parasite proliferation.

Assuntos

RESUMO

Histoplasma capsulatum is a thermally dimorphic fungus distributed worldwide, but with the highest incidence in the Americas within specific geographic areas, such as the Mississippi River Valley and regions in Latin America. This fungus is the etiologic agent of histoplasmosis, an important life-threatening systemic mycosis. Dimorphism is an important feature for fungal survival in different environments and is related to the virulence of H. capsulatum, and essential to the establishment of infection. Proteomic profiles have made important contributions to the knowledge of metabolism and pathogenicity in several biological models. However, H. capsulatum proteome studies have been underexplored. In the present study, we report the first proteomic comparison between the mycelium and the yeast cells of H. capsulatum. Liquid chromatography coupled to mass spectrometry was used to evaluate the proteomic profile of the two phases of H. capsulatum growth, mycelium, and yeast. In summary, 214 and 225 proteins were only detected/or preferentially abundant in mycelium or yeast cells, respectively. In mycelium, enzymes related to the glycolytic pathway and to the alcoholic fermentation occurred in greater abundance, suggesting a higher use of anaerobic pathways for energy production. In yeast cells, proteins related to the tricarboxylic acid cycle and response to temperature stress were in high abundance. Proteins related to oxidative stress response or involved with cell wall metabolism were identified with differential abundance in both conditions. Proteomic data validation was performed by enzymatic activity determination, Western blot assays, or immunofluorescence microscopy. These experiments corroborated, directly or indirectly, the abundance of isocitrate lyase, 2-methylcitrate synthase, catalase B, and mannosyl-oligosaccharide-1,2-alpha-mannosidase in the mycelium and heat shock protein (HSP) 30, HSP60, glucosamine-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, and N-acetylglucosamine-phosphate mutase in yeast cells. The proteomic profile-associated functional classification analyses of proteins provided new and interesting information regarding the differences in metabolism between the two distinct growth forms of H. capsulatum.

RESUMO

Paracoccidioides spp. are thermally dimorphic fungi that cause paracoccidioidomycosis and can affect both immunocompetent and immunocompromised individuals. The infection can lead to moderate or severe illness and death. Paracoccidioides spp. undergo micronutrients deprivation within the host, including iron. To overcome such cellular stress, this genus of fungi responds in multiple ways, such as the utilization of hemoglobin. A glycosylphosphatidylinositol (GPI)-anchored fungal receptor, Rbt5, has the primary role of acquiring the essential nutrient iron from hemoglobin. Conversely, it is not clear if additional proteins participate in the process of using hemoglobin by the fungus. Therefore, in order to investigate changes in the proteomic level of P. lutzii cell wall, we deprived the fungus of iron and then treated those cells with hemoglobin. Deprived iron cells were used as control. Next, we performed cell wall fractionation and the obtained proteins were submitted to nanoUPLC-MSE. Protein expression levels of the cell wall F1 fraction of cells exposed to hemoglobin were compared with the protein expression of the cell wall F1 fraction of iron-deprived cells. Our results showed that P. lutzii exposure to hemoglobin increased the level of adhesins expression by the fungus, according to the proteomic data. We confirmed that the exposure of the fungus to hemoglobin increased its ability to adhere to macrophages by flow cytometry. In addition, we found that HSP30 of P. lutzii is a novel hemoglobin-binding protein and a possible heme oxygenase. In order to investigate the importance of HSP30 in the Paracoccidioides genus, we developed a Paracoccidioides brasiliensis knockdown strain of HSP30 via Agrobacterium tumefaciens-mediated transformation and demonstrated that silencing this gene decreases the ability of P. brasiliensis to use hemoglobin as a nutrient source. Additional studies are needed to establish HSP30 as a virulence factor, which can support the development of new therapeutic and/or diagnostic approaches.

RESUMO

Species of the genus Paracoccidioides cause a systemic infection in human patients. Yeast cells of Paracoccidioides spp. produce melanin in the presence of L-dihydroxyphenylalanine and during infection, which may impact the pathogen's survival in the host. To better understand the metabolic changes that occur in melanized Paracoccidioides spp. cells, a proteomic approach was performed to compare melanized and non-melanized Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells. Melanization was induced using L-dihydroxyphenylalanine as a precursor, and quantitative proteomics were performed using reversed-phase nano-chromatography coupled to high-resolution mass spectrometry. When comparing melanized versus non-melanized cells, 1006 and 582 differentially abundant/detected proteins were identified for P. brasiliensis and P. lutzii, respectively. Functional enrichment and comparative analysis revealed 30 important KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways in melanized P. brasiliensis and 18 in P. lutzii, while differentially abundant proteins from non-melanized cells from these species were involved in 21 and 25 enriched pathways, respectively. Melanized cells presented an abundance of additional virulence-associated proteins, such as phospholipase, proteases, superoxide dis-mutases, heat-shock proteins, adhesins, and proteins related to vesicular transport. The results suggest that L-dihydroxyphenylalanine increases the virulence of Paracoccidioides spp. through complex mechanisms involving not only melanin but other virulence factors as well.

RESUMO

Histoplasma capsulatum is a thermodimorphic fungus that causes histoplasmosis, a mycosis of global incidence. The disease is prevalent in temperate and tropical regions such as North America, South America, Europe, and Asia. It is known that during infection macrophages restrict Zn availability to H. capsulatum as a microbicidal mechanism. In this way the present work aimed to study the response of H. capsulatum to zinc deprivation. In silico analyses showed that H. capsulatum has eight genes related to zinc homeostasis ranging from transcription factors to CDF and ZIP family transporters. The transcriptional levels of ZAP1, ZRT1, and ZRT2 were induced under zinc-limiting conditions. The decrease in Zn availability increases fungicidal macrophage activity. Proteomics analysis during zinc deprivation at 24 and 48 h showed 265 proteins differentially expressed at 24 h and 68 at 48 h. Proteins related to energy production pathways, oxidative stress, and cell wall remodeling were regulated. The data also suggested that low metal availability increases the chitin and glycan content in fungal cell wall that results in smoother cell surface. Metal restriction also induces oxidative stress triggered, at least in part, by reduction in pyridoxin synthesis.

Assuntos

RESUMO

Objetivo: avaliar in vitro a ação antimicrobiana do extrato da própolis (EP) e do digluconato de clorexidina (DCHX) na formação de biofilme por Candida albicans em resina acrílica termopolimerizada. Métodos: o efeito do EP e DCHX em biofilmes de C. albicans foi avaliado pela quantificação de Unidades Formadoras de Colônias (UFCs), pela quantificação da biomassa por cristal violeta e de polissacarídeos por safranina. Para tanto, C. albicans foram pré-aderidas em corpos de prova e somente em microplacas de poliestireno, posteriormente foi realizado o tratamento com diferentes concentrações de EP (221 µg/mL e 443 µg/mL) e DCHX (0,25% e 0,5%). Resultados: foi demonstrada uma redução significativa na formação de biofilme por C. albicans com ambas as substâncias testadas e em todas as concentrações. Conclusão: de acordo com os resultados, observou-se que ambos os tratamentos foram eficazes na redução do biofilme e que o EP, por ser um produto natural, de baixo custo e sem afeitos colaterais, representa uma alternativa inovadora para o tratamento da candidose oral em usuários de próteses removíveis.(AU)

Objective: to evaluate in vitro the antimicrobial action of propolis extract (EP) and chlorhexidine digluconate (DCHX) in the biofilm formation by Candida albicans in thermopolymerized acrylic resin. Methods: the effect of EP and DCHX on C. albicans biofilms were evaluated through the quantification of Colony Forming Units (CFU's), the quantification of biomass by violet crystal and polysaccharides by safranin. For this purpose, C. albicans were pre-adhered to specimens and only on polystyrene microplates and subsequently the treatment was performed with different concentrations of EP (221 µg/mL and 443 µg/mL) and DCHX (0.25% and 0,5%). Results: a significant reduction in the biofilm formation by C. albicans was demonstrated, with both substances tested and in all concentrations. Conclusion: according to the results, it was observed that both treatments were effective in reducing biofilm and EP, being a natural product, low cost and without side effects, represents an innovative alternative for the treatment of oral candidiasis in users removable dentures.(AU)

Assuntos

RESUMO

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

Assuntos