Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Adjuvant Pam3CSk4 does not improve the immunization against Cryptococcus gattii infection in C57BL/6 mice.

Background: Cryptococcosis is a relevant invasive fungal infection that affects immunocompromised and immunocompetent individuals when caused by Cryptococcus gattii. Host innate and adaptive immune responses can be subverted by C. gattii, that blocks the differentiation of T helper (Th) 1 and Th17 cells, which are involved in the protection against cryptococcosis. Moreover, the macrophage polarization is modulated by C. gattii infection that requires a balance in the macrophage subsets to control the C. gattii infection. Toll-like receptor (TLR) 2 agonists are important immunomodulators favoring a pro-inflammatory response with potential fungicidal activity, and TLR2 agonists have been used as adjuvants in vaccines against infections caused by bacteria or viruses. Therefore, this work aimed to evaluate the immunomodulatory effect of the tripalmitoyl lipopeptide S-glycerol cysteine (Pam3CSK4 or P3C4), a TLR2 agonist, as an adjuvant in the vaccination against C. gattii infection. Methods and Results: C57BL/6 mice were immunized with 2 × 107 inactivated yeasts of C. gattii via intranasal route on day 1, 14 and 28 (Immunized group). Immunization was associated with 1µg or 10µg of adjuvant P3C4 (Immunized+P3C4-1µg or Immunized+P3C4-10 µg), followed by C. gattii infection on day 42 after the immunization protocol. Immunized+P3C4-1 µg group had reduced levels of IgG1, IgG2a and IgA and no significant difference in the IgG and IgM anti-GXM antibody titer was detected, compared to the Immunized group. High levels of IL-17 and IL-1ß in lung tissue of mice from the Immunized+P3C4-1µg group did not promote a predominance of Th17 cells, in contrast, the frequency of TLR2+ cells was increased in immunized mice that received 1 µg of P3C4. The reduction in the relative expression of T-bet and high levels of Foxp3 detected in the lungs of the Immunized+P3C4-1µg group suggest a prevalence of regulatory T cells in the tissue, which did not contribute to the control of C. gattii infection. The immunization protocol associated with 10 µg of adjuvant P3C4 induced high levels of IL-17 in the lung tissue, whereas the levels of pro-inflammatory cytokines were downregulated. To evaluate the effect of adjuvant P3C4 in the control of C. gattii infection, quantification of the fungal burden in the lungs was performed by the CFU assay, and the groups with adjuvant P3C4 showed a pulmonary C. gattii burden that was not significantly altered when compared with the immunized group. The mice that received 1 µg of adjuvant P3C4 had a lower percentage of inflammatory infiltrate in the lungs. Conclusion: The immunomodulatory effect of P3C4, associated with the immunization protocol, plays an imbalance between pro- and anti-inflammatory response in the lungs that did not favor a protection against C. gattii infection, which is related to the immune response characterized by a suppressive/regulatory profile in the pulmonary microenvironment after C. gattii infection.

Effects of combination of Cryptococcus gattii and IFN-γ, IL-4 or IL-27 on human bronchial epithelial cells.

BACKGROUND: Airway epithelial cells are crucial for the establishment of cryptococcosis. In experimental cryptococcosis, the Th2 immune response is associated with host susceptibility, while Th1 cells are associated with protection. The absence of IL-27 receptor alpha in mice favor the increase Cryptococcus neoformans burden in the lung. Here, we evaluated the effects of the combination of IL-4, IFN-γ or IL-27 with C. gattii on human bronchial epithelial cells (BEAS-2B). METHODS: BEAS-2B were stimulated with IL-4, IFN-γ or IL-27 (100 ng/mL) and/or live yeast forms of C. gattii (multiplicities of infection (MOI) of 1-100) and vice-versa, as well as with heat-killed cells of C. gattii for 24 h. RESULTS: None of the C. gattii MOIs had cytotoxic effects on BEAS-2B when compared to control. The cells stimulated by cytokines (IL-4, IFN-γ or IL-27) followed by live yeast forms of C. gattii (MOI of 100) infection and vice-versa demonstrated a reduction in IL-6, IL-8 and/or CCL2 production and activation of STAT6 (induced by IL-4) and STAT1 (induced by IL-27 or IFN-γ) when compared to cells stimulated with C. gattii, IL-4, IFN-γ or IL-27. In the combination of cytokines and heat-killed cells of C. gattii, no inhibition of these inflammatory parameters was observed. The growth of C. gattii was increased while the phagocytosis of live yeast forms of C. gattii in the BEAS-2B were reduced in the presence of IL-4, IFN-γ or IL-27. Conclusion The association of live yeast forms, but not heat-killed yeast forms, of C. gattii with IL-4, IFN-γ or IL-27 induced an anti-inflammatory effect.

Immunomodulatory activity of ß-glucan-containing exopolysaccharides from Auricularia auricular in phagocytes and mice infected with Cryptococcus neoformans.

Current antifungal drugs present poor effectiveness and there is no available vaccine for fungal infections. Thus, novel strategies to treat or prevent invasive mycosis, such as cryptococcosis, are highly desirable. One strategy is the use of immunomodulators of polysaccharide nature isolated from mushrooms. The purpose of the present work was to evaluate the immunostimulatory activity of ß-(1,3)-glucan-containing exopolysaccharides (EPS) from the edible mushrooms Auricularia auricula in phagocytes and mice infected with Cryptococcus neoformans. EPS triggered macrophages and dendritic cell activation upon binding to Dectin-1, a pattern recognition receptor of the C-type lectin receptor family. Engagement of Dectin-1 culminated in pro-inflammatory cytokine production and cell maturation via its canonical Syk-dependent pathway signaling. Furthermore, upon EPS treatment, M2-like phenotype macrophages, known to support intracellular survival and replication of C. neoformans, repolarize to M1 macrophage pattern associated with enhanced production of the microbicidal molecule nitric oxide that results in efficient killing of C. neoformans. Treatment with EPS also upregulated transcript levels of genes encoding products associated with host protection against C. neoformans and Dectin-1 mediated signaling in macrophages. Finally, orally administrated ß-glucan-containing EPS from A. auricular enhanced the survival of mice infected with C. neoformans. In conclusion, the results demonstrate that EPS from A. auricula exert immunostimulatory activity in phagocytes and induce host protection against C. neoformans, suggesting that polysaccharides from this mushroom may be promising as an adjuvant for vaccines or antifungal therapy.

Pulmonary Iron Limitation Induced by Exogenous Type I IFN Protects Mice from Cryptococcus gattii Independently of T Cells.

Cryptococcus neoformans causes deadly mycosis primarily in AIDS patients, whereas Cryptococcus gattii infects mostly non-HIV patients, even in regions with high burdens of HIV/AIDS and an established environmental presence of C. gattii As HIV induces type I IFN (t1IFN), we hypothesized that t1IFN would differentially affect the outcome of C. neoformans and C. gattii infections. Exogenous t1IFN induction using stabilized poly(I·C) (pICLC) improved murine outcomes in either cryptococcal infection. In C. neoformans-infected mice, pICLC activity was associated with C. neoformans containment and classical Th1 immunity. In contrast, pICLC activity against C. gattii did not require any immune factors previously associated with C. neoformans immunity: T, B, and NK cells, IFN-γ, and macrophages were all dispensable. Interestingly, C. gattii pICLC activity depended on ß-2-microglobulin, which impacts iron levels among other functions. Iron supplementation reversed pICLC activity, suggesting C. gattii pICLC activity requires iron limitation. Also, pICLC induced a set of iron control proteins, some of which were directly inhibitory to cryptococcus in vitro, suggesting t1IFN regulates iron availability in the pulmonary air space fluids. Thus, exogenous induction of t1IFN significantly improves the outcome of murine infection by C. gattii and C. neoformans but by distinct mechanisms; the C. gattii effect was mediated by iron limitation, while the effect on C. neoformans infection was through induction of classical T-cell-dependent immunity. Together this difference in types of T-cell-dependent t1IFN immunity for different Cryptococcus species suggests a possible mechanism by which HIV infection may select against C. gattii but not C. neoformansIMPORTANCECryptococcus neoformans and Cryptococcus gattii cause fatal infection in immunodeficient and immunocompetent individuals. While these fungi are sibling species, C. gattii infects very few AIDS patients, while C. neoformans infection is an AIDS-defining illness, suggesting that the host response to HIV selects C. neoformans over C. gattii We used a viral mimic molecule (pICLC) to stimulate the immune response, and pICLC treatment improved mouse outcomes from both species. pICLC-induced action against C. neoformans was due to activation of well-defined immune pathways known to deter C. neoformans, whereas these immune pathways were dispensable for pICLC treatment of C. gattii Since these immune pathways are eventually destroyed by HIV/AIDS, our data help explain why the antiviral immune response in AIDS patients is unable to control C. neoformans infection but is protective against C. gattii Furthermore, pICLC induced tighter control of iron in the lungs of mice, which inhibited C. gattii, thus suggesting an entirely new mode of nutritional immunity activated by viral signals.

Pulmonary cryptococcosis: A review of pathobiology and clinical aspects.

Pulmonary cryptococcosis is an important opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. The main human pathogens are Cryptococcus neoformans and C. gattii, which have a worldwide distribution. In contrast to cryptococcal meningitis, pulmonary cryptococcosis is still underdiagnosed because of limitations in diagnostic tools. It can mimic lung cancer, pulmonary tuberculosis, bacterial pneumonia, and other pulmonary mycoses both clinically and radiologically. Pulmonary nodules are the most common radiological feature, but these are not specific to pulmonary cryptococcosis. The sensitivity of culture of respiratory samples for Cryptococcus is poor and a positive result may also reflect colonisation. Cryptococcal antigen (CrAg) with lateral flow device is a fast and sensitive test and widely used on serum and cerebrospinal fluid, but sera from patients with pulmonary cryptococcosis are rarely positive in the absence of disseminated disease. Detection of CrAg from respiratory specimens might assist the diagnosis of pulmonary cryptococcosis but there are very few data. Molecular detection techniques such as multiplex reverse transcription polymerase chain reaction (RT-PCR) could also provide better sensitivity but these still require validation for respiratory specimens. The first line of treatment for pulmonary cryptococcosis is fluconazole, or amphotericin B and flucytosine for those with central nervous system involvement. Pulmonary cryptococcosis worsens the prognosis of cryptococcal meningitis. In this review, we summarize the biological aspects of Cryptococcus and provide an update on the diagnosis and management of pulmonary cryptococcosis.

Reflexive Laboratory-Based Cryptococcal Antigen Screening and Preemptive Fluconazole Therapy for Cryptococcal Antigenemia in HIV-Infected Individuals With CD4 <100 Cells/µL: A Stepped-Wedge, Cluster-Randomized Trial.

BACKGROUND: HIV-infected persons with cryptococcal antigenemia (CrAg) are at high risk for meningitis or death. We evaluated the effect of CrAg screening and preemptive fluconazole therapy, adjunctive to antiretroviral therapy (ART), on 6-month survival among persons with advanced HIV/AIDS. METHODS: We enrolled HIV-infected, ART-naive participants with <100 CD4 cells/µL, in a stepped-wedge, cluster-randomized trial from July 2012 to December 2014 at 17 Ugandan clinics. Clinics participated in a prospective observational phase, followed by an interventional phase with laboratory-based, reflexive CrAg screening of residual CD4 count plasma. Asymptomatic CrAg+ participants received preemptive fluconazole therapy. We assessed 6-month survival using Cox-regression, adjusting for nadir CD4, calendar time, and stepped-wedge steps. RESULTS: We included 1280 observational and 2108 interventional participants, of whom 9.3% (195/2108) were CrAg+. CD4-, time-, and stepped-wedge-adjusted analyses demonstrated no difference in survival in the observational vs the interventional arms (hazard ratio = 1.34; 95% confidence interval: 0.86 to 2.10; P = 0.20). Fewer participants initiated ART in the interventional (73%) versus the observational phase (82%, P < 0.001). When ART initiation was modeled as a time-dependent covariate or confounder, survival did not differ. However, 6-month mortality of participants with CrAg titers <1:160 and CrAg-negative patients did not differ. Patients with CrAg titers ≥1:160 had 2.6-fold higher 6-month mortality than patients with titers <1:160. CONCLUSIONS: We observed no overall survival benefit of the CrAg screen-and-treat intervention. However, preemptive antifungal therapy for asymptomatic cryptococcosis seemed to be effective in patients with CrAg titer <1:160. A more aggressive approach is required for persons with CrAg titer ≥1:160.

Solving the mystery: Hyalinized cyst wall containing organism-like structures in a lung transplant donor.

A 59-year-old man with non-ischemic cardiomyopathy underwent orthotopic heart transplantation. The donor, a 31-year-old male declared brain dead after a gunshot wound to the head, was considered high risk due to history of incarceration, illicit drug use, and sex with a HIV-positive partner. At organ procurement, the heart, kidneys, pancreas, and liver looked grossly normal. A small right lower lobe nodule was noticed, and lung biopsy was performed. Bronchoscopy showed purulent secretions in the right lower lobe. Images from pathology are presented. Lung biopsy confirmed the presence of hyalinized cyst wall containing organism-like structures. A combination of culture, microscopic morphology, and gene sequencing was used to identify the causative organism. The patient and all other organ recipients received appropriate antifungal prophylaxis and remain asymptomatic 6 months post-transplant.

Treatment of cryptococcosis in non-HIV immunocompromised patients.

PURPOSE OF REVIEW: Cryptococcosis has become a common opportunistic infection among non-HIV immunocompromised hosts. Recent reports have shown the incidence of Cryptococcosis among HIV-negative immunocompromised patients reaches close to half of the overall cases reported in the USA. Management of this infection in this population carries unique challenges. We aim to review relevant and recent research findings to develop treatment recommendations for this type of population. RECENT FINDINGS: Most of the recommendations for the management of non-HIV immunocompromised host are extrapolated from HIV studies. Cryptococcosis among non-HIV patients is common but often overlooked. Some clinical factors, when present, may increase the risk of Cryptococcosis among HIV-negative patients and appropriate screening and assessment for the disease is necessary. Treating clinicians should consider a longer duration of induction with Amphotericin B depending on the type of host, immunocompromised state, antifungal response and presence of neurological complications. Baseline fluconazole resistance can reach up to 12%, which is an important consideration for cryptococcal meningitis relapses or suboptimal responses to therapy. SUMMARY: Cryptococcus spp. conveys a high disease burden among immunocompromised hosts. Clinicians must consider numerous variables and factors in a dynamic way to offer the best possible treatment and to monitor their response to therapy. Due to the high cost and associated toxicities, we still need new affordable therapies and studies among non-HIV immunocompromised patients.

A systematic review of fluconazole resistance in clinical isolates of Cryptococcus species.

Fluconazole is the most commonly used antifungal agent for both the treatment of cryptococcal meningitis, and for prophylaxis against the disease. However, its prolonged use has the potential to exert selection pressure in favour of fluconazole-resistant strains. We evaluated the prevalence of fluconazole resistance in Cryptococcus spp. clinical isolates in 29 studies from 1988 to May 2017 included in EMBASE and MEDLINE databases. A total of 4995 Cryptococcus isolates from 3210 patients constituted this study; 248 (5.0%) of the isolates from relapsed episodes of cryptococcosis were included in this analysis. Eleven (38%) of the studies used minimum inhibitory concentrations (MICs) breakpoints of ≥64 µg/mL to define fluconazole resistance, 6 (21%) used ≥32 µg/mL, 11 (38%) used ≥16 µg/mL and 1 (3%) used ≤20 µg/mL. Overall, mean prevalence of fluconazole resistance was 12.1% (95% confidence interval [CI]: 6.7-17.6) for all isolates (n = 4995). Mean fluconazole resistance was 10.6% (95% CI: 5.5-15.6) for the incident isolates (n = 4747) and 24.1% (95% CI: -3.1-51.2) for the relapse isolates (n = 248). Of the 4995 isolates, 936 (18.7%) had MICs above the ecological cut-off value. Fluconazole resistance appears to be an issue in Cryptococcus isolates from patients with relapses. It remains unclear whether relapses occur due to resistance or other factors. There is an urgent need to establish antifungal breakpoints for Cryptococcus spp.

Introns in Cryptococcus

In Cryptococcus neoformans, nearly all genes are interrupted by small introns. In recent years, genome annotation and genetic analysis have illuminated the major roles these introns play in the biology of this pathogenic yeast. Introns are necessary for gene expression and alternative splicing can regulate gene expression in response to environmental cues. In addition, recent studies have revealed that C. neoformans introns help to prevent transposon dissemination and protect genome integrity. These characteristics of cryptococcal introns are probably not unique to Cryptococcus, and this yeast likely can be considered as a model for intron-related studies in fungi.

The status of cryptococcosis in Latin America

Cryptococcosis is a life-threatening fungal infection caused by the encapsulated yeasts Cryptococcus neoformans and C. gattii, acquired from the environment. In Latin America, as occurring worldwide, C. neoformans causes more than 90% of the cases of cryptococcosis, affecting predominantly patients with HIV, while C. gattii generally affects otherwise healthy individuals. In this region, cryptococcal meningitis is the most common presentation, with amphotericin B and fluconazole being the antifungal drugs of choice. Avian droppings are the predominant environmental reservoir of C. neoformans, while C. gattii is associated with several arboreal species. Importantly, C. gattii has a high prevalence in Latin America and has been proposed to be the likely origin of some C. gattii populations in North America. Thus, in the recent years, significant progress has been made with the study of the basic biology and laboratory identification of cryptococcal strains, in understanding their ecology, population genetics, host-pathogen interactions, and the clinical epidemiology of this important mycosis in Latin America.

Primary Prophylaxis for Cryptococcosis With Fluconazole in Human Immunodeficiency Virus-Infected Patients With CD4 T-Cell Counts <100 Cells/µL and Receiving Antiretroviral Therapy.

A prospective observational cohort study was conducted in 302 human immunodeficiency virus-infected patients who had a CD4 T-cell count <100 cells/µL and negative serum cryptococcal antigen initiating antiretroviral therapy in a resource-limited setting. During 2-year follow-up, there were no differences of survival rates and occurrences of newly diagnosed cryptococcosis between patients with and without fluconazole for primary prophylaxis of cryptococcosis.

Disruption of Early Tumor Necrosis Factor Alpha Signaling Prevents Classical Activation of Dendritic Cells in Lung-Associated Lymph Nodes and Development of Protective Immunity against Cryptococcal Infection.

UNLABELLED: Anti-tumor necrosis factor alpha (anti-TNF-α) therapies have been increasingly used to treat inflammatory diseases and are associated with increased risk of invasive fungal infections, including Cryptococcus neoformans infection. Using a mouse model of cryptococcal infection, we investigated the mechanism by which disruption of early TNF-α signaling results in the development of nonprotective immunity against C. neoformans We found that transient depletion of TNF-α inhibited pulmonary fungal clearance and enhanced extrapulmonary dissemination of C. neoformans during the adaptive phase of the immune response. Higher fungal burdens in TNF-α-depleted mice were accompanied by markedly impaired Th1 and Th17 responses in the infected lungs. Furthermore, early TNF-α depletion also resulted in disrupted transcriptional initiation of the Th17 polarization program and subsequent upregulation of Th1 genes in CD4(+) T cells in the lung-associated lymph nodes (LALN) of C. neoformans-infected mice. These defects in LALN T cell responses were preceded by a dramatic shift from a classical toward an alternative activation of dendritic cells (DC) in the LALN of TNF-α-depleted mice. Taken together, our results indicate that early TNF-α signaling is required for optimal DC activation, and the initial Th17 response followed by Th1 transcriptional prepolarization of T cells in the LALN, which further drives the development of protective immunity against cryptococcal infection in the lungs. Thus, administration of anti-TNF-α may introduce a particularly greater risk for newly acquired fungal infections that require generation of protective Th1/Th17 responses for their containment and clearance. IMPORTANCE: Increased susceptibility to invasive fungal infections in patients on anti-TNF-α therapies underlines the need for understanding the cellular effects of TNF-α signaling in promoting protective immunity to fungal pathogens. Here, we demonstrate that early TNF-α signaling is required for classical activation and accumulation of DC in LALN of C. neoformans-infected mice. Subsequent transcriptional initiation of Th17 followed by Th1 programming in LALN results in pulmonary accumulation of gamma interferon- and interleukin-17A-producing T cells and effective fungal clearance. All of these crucial steps are severely impaired in mice that undergo anti-TNF-α treatment, consistent with their inability to clear C. neoformans This study identified critical interactions between cells of the innate immune system (DC), the emerging T cell responses, and cytokine networks with a central role for TNF-α which orchestrate the development of the immune protection against cryptococcal infection. This information will be important in aiding development and understanding the potential side effects of immunotherapies.

Biofilm Formation by Cryptococcus neoformans.

The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. The increasing use of ventriculoperitoneal shunts to manage intracranial hypertension associated with cryptococcal meningoencephalitis highlights the importance of investigating the biofilm-forming properties of this organism. Like other microbe-forming biofilms, C. neoformans biofilms are resistant to antimicrobial agents and host defense mechanisms, causing significant morbidity and mortality. This chapter discusses the recent advances in the understanding of cryptococcal biofilms, including the role of its polysaccharide capsule in adherence, gene expression, and quorum sensing in biofilm formation. We describe novel strategies for the prevention or eradication of cryptococcal colonization of medical prosthetic devices. Finally, we provide fresh thoughts on the diverse but interesting directions of research in this field that may result in new insights into C. neoformans biology.

Dendritic cell-based immunization ameliorates pulmonary infection with highly virulent Cryptococcus gattii.

Cryptococcosis due to a highly virulent fungus, Cryptococcus gattii, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that C. gattii strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against C. gattii has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against C. gattii infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular C. gattii (Δcap60 strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Δcap60 strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-γ)-, and tumor necrosis factor alpha (TNF-α)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-γ knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent C. gattii and suggested that IFN-γ may have been an important mediator for this vaccine-induced protection.

Consensus guidelines for the treatment of yeast infections in the haematology, oncology and intensive care setting, 2014.

Pathogenic yeast forms are commonly associated with invasive fungal disease in the immunocompromised host, including patients with haematological malignancies and patients of haemopoietic stem cell transplants. Yeasts include the Candida spp., Cryptococcus spp., Pneumocystis jirovecii and some lesser-known pathogens. Candida species remain the most common cause of invasive yeast infections (and the most common human pathogenic fungi). These guidelines present evidence-based recommendations for the antifungal management of established, invasive yeast infections in adult and paediatric patients in the haematology/oncology setting. Consideration is also given to the critically ill patient in intensive care units, including the neonatal intensive care unit. Evidence for 'pre-emptive' or 'diagnostic-driven antifungal therapy' is also discussed. For the purposes of this paper, invasive yeast diseases are categorised under the headings of invasive candidiasis, cryptococcosis and uncommon yeast infections. Specific recommendations for the management of Pneumocystis jirovecii are presented in an accompanying article (see consensus guidelines by Cooley et al. appearing elsewhere in this supplement).

The role of exosomes in infectious diseases.

An exosome is a nano vesicle that buds from the endosomal compartment; it is produced and released by all kinds of mammalian cells. This vesicle contains a variety of proteins, lipids, mRNAs and miRNAs. These components are specific to the origin of the exosomes and contribute to cell-cell communications. Recently, it has been reported that a few single cell eukaryotic pathogens such as Cryptoccoccus neoformance and Leishmania major and donovanican secrete an exosome and influence the host immune system. In addition, it has been observed that cells infected by intracellular pathogens are capable of secreting an exosome which is involved in the fate of the infection. Furthermore, retroviruses recruit the host`s endosomal compartments in order to generate viral vesicles which are similar to the exosome. Most of the exosomes involved in infectious biology can either spread or limit an infection based on the type of pathogen and its target cells. Hence, an exosome may be an appropriate candidate for a vaccine therapy in prophylaxis and treatment.

Metodologia científica na formação do médico veterinário: experiênciateórico-prática na análise de fontes urbanas de Cryptococcus spp – Jaboticabal, SP – Brasil / Scientific methodology in veterinary medical training: a theoretical andpractical experience in the analysis of urban sources of Cryptococcus spp in Jaboticabal, SP – Brazil / Metodología científica en la formación médica veterinaria: la experienciateórica y práctica en el análisis de fuentes urbanas de Cryptococcus spp de Jaboticabal, SP – Brasil

O artigo apresenta resultados de um projeto de ensino de Metodologia Científica em Curso de Graduação deMedicina Veterinária. Pautou-se por uma reflexão crítica da necessidade de interação entre as dimensões teórica eprática na formação do profissional e realizou-se análise de fontes urbanas de Cryptococcus spp em fezes de pombos(Columbia livia), evidenciando a importância de orientação à população sobre os riscos de contágio, caracterizando a patogenicidade dos agentes identificados em dez ambientes públicos.

This article presents the results of an educational project for the Scientific Methodology degree course for veterinary medicine. The project was guided by the critical reflection on the necessity for the interaction between the theoretical and practical dimensions of professional training. An analysis was performed of urban sources of Cryptococcus spp in excrements of pigeons (Columbia livia), indicating the need for education of the population about the risks factors of transmission and characterizing the pathogenicity of the identified agents in ten public places.

El artículo presenta los resultados de un proyecto de enseñanza de Metodología Científica en el Curso de Graduaciónde Medicina Veterinaria. Se marcó por una reflexión crítica da la necesidad de la interacción entre las dimensionesteóricas y prácticas en la formación del profesional y se realizó un análisis de fuentes urbanas de Cryptococcus sppen excremento de palomas (Columbia livia), mostrando la importancia de la orientación a la población sobre losriesgos del contagio, caracterizando la patogenicidad de los agentes identificados en diez ambientes públicos.