Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. In the present study, the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus Cryptococcus gattii were examined. We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by these drugs in C. gattii cells: lipid peroxidation, ergosterol content, ROS and peroxynitrite production, enzymatic activity of the antioxidant system and the in vitro interaction of antifungal drugs with a peroxidase inhibitor, a superoxide dismutase inhibitor and a peroxynitrite scavenger. The data demonstrated that itraconazole led to ROS formation and lipid peroxidation in C. gattii cells in the early stages of the treatment; this did not occur with fluconazole. This phenomenon strongly increased the activities of enzymes of the antioxidant system. These results were confirmed by synergism observed between the catalase inhibitor and itraconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased peroxidase activity. These data were confirmed by the synergism between the catalase/superoxide dismutase inhibitors and amphotericin B. In addition, the effect of this antifungal was antagonized by the peroxynitrite scavenger. Oxidative and nitrosative bursts play an important role in the antifungal activity of itraconazole and amphotericin B against C. gattii.

The role of different cytokines in the peripheral blood mononuclear cell (PBMC) proliferative response and in in vitro granuloma formation was evaluated in a cross-sectional study with patients with the different clinical forms and phases of Schistosoma mansoni infection, as well as a group of individuals "naturally" resistant to infection named normal endemic (NE). The blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL-5 antibodies significantly reduced the PBMC proliferative response to soluble egg (SEA) and adult worm (SWAP) antigens in acute (ACT), chronic intestinal (INT) and hepatosplenic (HS) patients. Similar results were obtained in the in vitro granuloma formation. Blockage of IL-10 had no significant effect on either assay using PBMC from ACT or HS. In contrast, the addition of anti-IL-10 antibodies to PBMC cultures from INT patients significantly increased the proliferative response to SEA and SWAP as well as the in vitro granuloma formation. Interestingly, association of anti-IL-4 and anti-IL-10 antibodies did not increase the PBMC proliferative response of these patients, suggesting that IL-10 may act by modulating IL-4 and IL-5 secretion. Addition of recombinant IL-10 decreased the proliferative response to undetectable levels when PBMC from patients with the different clinical forms were used. Analysis of IFN-g in the supernatants showed that PBMC from INT patients secreted low levels of IFN-g upon antigenic stimulation. In contrast, PBMC from NE secreted high levels of IFN-g. These data suggest that IL-10 is an important cytokine in regulating the immune response and possibly controlling morbidity in human schistosomiasis mansoni, and that the production of IFN-g may be associated with resistance to infection.

During natural schistosome infection, the induction of T helper type 2 (Th2) responses has been ascribed to parasite eggs, because exposure of the host to this life-cycle stage elicits a polarized Th2 response to egg antigens. In the present study, we show that schistosome worms also elicit systemic, antigen-specific type 2 responses during prepatent infection, before egg deposition begins. CD4(+) T cells producing interleukin (IL)-4 were induced by both male and female worms during single-sex infections, demonstrating that this response is independent of exposure to eggs. The Th2 response was accompanied by production of immunoglobulin E and the sensitization of circulating basophils to produce additional IL-4 in response to schistosome antigens. Together, our data show that schistosome worms establish an immunologic milieu where CD4(+) T cells and basophils are both primed to produce IL-4 before eggs are laid, suggesting that worms play a role in establishment of the Th2 response that is critical for host survival and parasite transmission.

Background: During the pre-patent stage of infection, juvenileSchistosomablood flukes co-opt signals from the adaptive immune system to facilitate parasite development, but the types of responses that are induced at this early stage of infection, and the parasite antigens they target, have not been characterized.Results: Through analysis of experimental pre-patent infections, we show that theS. mansonicysteine protease SmCB1 is rapidly targeted by an antigen-specific IgE response. The induction of this response is independent of schistosome eggs as infection with male or female worms alone also induced SmCB1-specific IgE. We also show that the SmCB1-specific IgE response is dependent on cognate CD4⁺T cell help and IL-4, suggesting that pre-patent Th2 responses provide T cell help for the SmCB1-specific IgE response. Finally, exposed human subjects also produced IgE against SmCB1.Conclusions: Our data demonstrate that, like eggs, schistosome worms also induce functional type 2 responses and that a parasite cysteine protease is an inducer of type 2 responses during the early stages of schistosome infection.

Background: According to the World Health Organization, achieving targets for control of leprosy by 2030 will require disease elimination and interruption of transmission at the national or regional level. India and Brazil have reported the highest leprosy burden in the last few decades, revealing the need for strategies and tools to help health professionals correctly manage and control the disease. Objective: The main objective of this study was to develop a cross-platform app for leprosy screening based on artificial intelligence (AI) with the goal of increasing accessibility of an accurate method of classifying leprosy treatment for health professionals, especially for communities further away from major diagnostic centers. Toward this end, we analyzed the quality of leprosy data in Brazil on the National Notifiable Diseases Information System (SINAN). Methods: Leprosy data were extracted from the SINAN database, carefully cleaned, and used to build AI decision models based on the random forest algorithm to predict operational classification in paucibacillary or multibacillary leprosy. We used Python programming language to extract and clean the data, and R programming language to train and test the AI model via cross-validation. To allow broad access, we deployed the final random forest classification model in a web app via shinyApp using data available from the Brazilian Institute of Geography and Statistics and the Department of Informatics of the Unified Health System. Results: We mapped the dispersion of leprosy incidence in Brazil from 2014 to 2018, and found a particularly high number of cases in central Brazil in 2014 that further increased in 2018 in the state of Mato Grosso. For some municipalities, up to 80% of cases showed some data discrepancy. Of a total of 21,047 discrepancies detected, the most common was “operational classification does not match the clinical form.” After data processing, we identified a total of 77,628 cases with missing data. The sensitivity and specificity of the AI model applied for the operational classification of leprosy was 93.97% and 87.09%, respectively. Conclusions: The proposed app was able to recognize patterns in leprosy cases registered in the SINAN database and to classify new patients with paucibacillary or multibacillary leprosy, thereby reducing the probability of incorrect assignment by health centers. The collection and notification of data on leprosy in Brazil seem to lack specific validation to increase the quality of the data for implementations via AI. The AI models implemented in this work had satisfactory accuracy across Brazilian states and could be a complementary diagnosis tool, especially in remote areas with few specialist physicians.