Abstract Background: Serological evaluation performed by double agar gel immunodiffusion test (DID) is used for diagnosis, evaluation of severity, management of paracoccidioidomycosis patients, and development of new clinical studies. For these reasons, the Botucatu Medical School of UNESP maintains a serum bank at the Experimental Research Unit with patient clinical data. This study aimed to evaluate the influence of the freeze-thaw cycle and different blood matrices on the titration of circulating antibodies. Methods: The study included 207 patients with confirmed (etiology-demonstrated) or probable (serology-demonstrated) paracoccidioidomycosis, and DID was performed with culture filtrate from Paracoccidioides brasiliensis B339 as antigen. First experiment: the antibody levels were determined in serum samples from 160 patients with the chronic form and 20 with the acute/subacute form, stored at -80oC for more than six months. Second experiment: titers of 81 samples of serum and plasma with ethylenediaminetetraacetic acid (EDTA) or heparin, from 27 patients, were compared according to matrix and effect of storage at -20oC for up to six months. Differences of titers higher than one dilution were considered discordant. Results: First experiment: test and retest presented concordant results in serum stored for up to three years, and discordant titers in low incidence in storage for four to six years but high incidence when stored for more than six years, including conversion from reagent test to non-reagent retest. Second experiment: serum, plasma-EDTA and plasma-heparin samples showed concordant titers, presenting direct correlation, with no interference of storage for up to six months. Conclusions: Storage at -80oC for up to six years has no or little influence on the serum titers determined by DID, permitting its safe use in studies depending on this parameter. The concordant titrations in different blood matrices demonstrated that the plasma can be used for immunodiffusion test in paracoccidioidomycosis, with stability for at least six months after storage at -20oC.

Abstract 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.

Abstract The lungs have great importance in patients with paracoccidioidomycosis since they are the portal of entry for the infecting fungi, the site of quiescent foci, and one of the most frequently affected organs. Although they have been the subject of many studies with different approaches, the severity classification of the pulmonary involvement, using imaging procedures, has not been carried out yet. This study aimed to classify the active and the residual pulmonary damage using radiographic and tomographic evaluations, according to the area involved and types of lesions.

Abstract Background: Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. Methods: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. Results: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 μg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 μg.mL-1), including C. krusei (250 μg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 μg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). Conclusion: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.

Abstract Background: The present study was carried out aiming to evaluate the impact of laryngeal sequelae on the quality of life of treated paracoccidioidomycosis (PCM) patients. Methods: This cross-sectional study was conducted at the Otorhinolaryngology Outpatient Clinic of the University Hospital, Federal University of Mato Grosso do Sul, Brazil. Thirty-two PCM patients considered clinically and immunologically cured were included: 16 with laryngeal involvement during the active phase of the disease (laryngeal PCM group) and 16 without laryngeal involvement (control group). They were submitted to structured interview, otorhinolaryngology examination, videolaryngoscopy, videoendoscopic swallowing study, completed two questionnaires for voice self-assessment - Voice-related Quality of Life (V-RQOL) and Voice Handicap Index (VHI) - and were asked to score their voices on a scale from zero to 10 (self-assessment of vocal quality). Results: Dysphonia was present in 50% of the cases. Patients with laryngeal PCM presented worse voice-related quality of life scores on the V-RQOL and poorer vocal quality self-assessment than the control group. No significant differences in the VHI were found between the groups. None of the participants developed dysphagic sequelae, although some minor changes were observed on videoendoscopic examination. Conclusion: There were no dysphagia complaints and only a few mild changes were found on the fiberoptic endoscopic evaluation of swallowing, suggesting that this evaluation should be performed only in specific cases. Patients with laryngeal involvement presented worse V-RQOL and self-assessment voice quality. This study contributes to the current knowledge of the functional assessment of the larynx affected by PCM and the impact of dysphonia on quality of life.

RESUMO Objetivo: Avaliar a adesão ao tratamento de paracoccidioidomicose. Métodos: Estudo conduzido com 188 pacientes com paracoccidioidomicose atendidos em um hospital terciário na Região Centro-Oeste do Brasil, de 2000 a 2010, para avaliar adesão ao tratamento. Foram considerados aderentes pacientes que tiveram o seguinte critério: retiraram os medicamentos na farmácia e autorrelataram a utilização de, no mínimo, 80% de dispensação dos antifúngicos prescritos na última consulta. Resultados: A maioria dos pacientes era homem (95,7%), tinha a forma crônica da doença (94,2%) e foi tratada com sulfametoxazol/trimetropim (86,2%). Apenas 44,6% dos pacientes aderiram ao tratamento. A maior perda de seguimento foi observada nos primeiros 4 meses de tratamento (p < 0,02). Adesão ao tratamento foi maior em pacientes com do que sem envolvimento pulmonar (RC: 2,986; IC95% 1,351-6,6599) e maior para os pacientes com do que sem tuberculose associada (RC 2,763; IC95% 1,004-7,604). Conclusões: A adesão ao tratamento da paracoccidioidomicose foi baixa e os primeiros 4 meses constituíram o período com maior evasão. Pacientes com envolvimento paracocidióidico pulmonar ou tuberculose associada revelaram maior adesão ao tratamento da paracoccidioidomicose.

ABSTRACT Objective: To evaluate the treatment compliance of patients with paracoccidioidomycosis. Methods: We studied 188 patients with paracoccidioidomycosis admitted to a tertiary referral hospital in the Central-West Region of Brazil from 2000 to 2010, to assess their compliance to treatment. In order to be considered compliant, patients needed to present two established criteria: (1) receive medicines from the pharmacy, and (2) achieve a self-reported utilization of at least 80% of the dispensed antifungal compounds prescribed since their previous appointment. Results: Most patients were male (95.7%), had the chronic form of the disease (94.2%), and were treated with cotrimoxazole (86.2%). Only 44.6% of patients were treatment compliant. The highest loss to follow-up was observed in the first 4 months of treatment (p < 0.02). Treatment compliance was higher for patients with than for those without pulmonary involvement (OR: 2.986; 95%CI 1.351-6.599), and higher for patients with than without tuberculosis as co-morbidity (OR: 2.763; 95%CI 1.004-7.604). Conclusions: Compliance to paracoccidioidomycosis treatment was low, and the period with the highest loss to follow-up corresponds to the first four months. Pulmonary paracoccidioidal involvement or tuberculosis comorbidity predicts a higher compliance to paracoccidioidomycosis therapy.

BACKGROUND The elimination of malaria depends on the blocking of transmission and of an effective treatment. In Brazil, artemisinin therapy was introduced in 1991, and here we present a performance overview during implementation outset years. METHODS It is a retrospective cohort (1991 to 2002) of patients treated in a tertiary centre of Manaus, with positive microscopic diagnosis of Plasmodium falciparum malaria, under treatment with using injectable or rectal artemisinin derivatives, and followed over 35-days to evaluate parasite clearance, death and recurrence. FINDINGS This cohort outcome resulted 97.6% (1554/1593) of patients who completed the 35-day follow-up, 0.6% (10/1593) of death and 1.8% (29/1593) of follow-up loss. All patients that died and those that presented parasitaemia recurrence had pure P. falciparum infections and received monotherapy. Considering patients who completed 35-day treatment, 98.2% (1527/1554) presented asexual parasitaemia clearance until D4 and 1.8% (27/1554) between D5-D10. It is important to highlight that had no correlation between the five treatment schemes and the sexual parasite clearance. Finally, it is noteworthy that we were able to observe also gametocytes carriage during all follow-up (D0-D35). MAIN CONCLUSIONS Artemisinin derivatives remained effective in the treatment of falciparum malaria during first 12-years of use in north area of Brazil.

Abstract INTRODUCTION We describe the clinical and laboratorial features of oral candidiasis in 66 HIV-positive patients. METHODS: Polymerase chain reaction-based techniques were performed for differentiation of Candida spp. isolated from patients at a public teaching hospital in Midwest Brazil. RESULTS: Oral lesions, mainly pseudomembranous, were significantly related to higher levels of immunosuppression. Of 45 Candida isolates, 66.7% were C. albicans. Most of the isolates were susceptible to the antifungal drugs tested. CONCLUSIONS: Oral lesions were associated with higher immunosuppression levels. Lower susceptibility to antifungals by non-albicans isolates supports the importance of surveillance studies using susceptibility tests to aid in the treatment.

Resumo A paracoccidioidomicose (PCM) é uma micose sistêmica, relacionada às atividades agrícolas, com incidência e prevalência subestimadas, pela ausência de notificação em várias Unidades da Federação (UFs). A evolução insidiosa do quadro clínico pode ter como consequência sequelas graves se o diagnóstico e o tratamento não forem instituídos precoce e adequadamente. Ao lado do complexo Paracoccidioides brasiliensis (P. brasiliensis), a descrição de nova espécie, Paracoccidioides lutzii (P. lutzii), em Rondônia, onde a doença alcançou níveis epidêmicos, bem como na região Centro-Oeste e no Pará, constituem-se em desafios para a instituição do diagnóstico e a urgente disponibilização de antígenos que tenham reatividade com os soros dos pacientes. Este consenso visa atualizar o primeiro consenso brasileiro em PCM, estabelecendo recomendações para o manejo clínico do paciente, com base nas evidências conhecidas. São apresentados dados de etiologia, epidemiologia, imunopatogenia, diagnóstico, terapêutica e sequelas, enfatizando-se o diagnóstico e a terapêutica, bem como recomendações e desafios atuais nessa área do conhecimento.

Abstract Paracoccidioidomycosis is a systemic fungal disease associated with agricultural activities. Its incidence and prevalence are underestimated because of the lack of reporting in several Brazilian states. If paracoccidiodomycosis is not diagnosed and treated early and adequately, endemic fungal infection may result in serious sequelae. In addition to the Paracoccidioides brasiliensis (P. brasiliensis) complex, the appearance of a new species, Paracoccidioides lutzii (P. lutzii), in Rondônia state, where the disease has reached epidemic levels, and in the country’s Midwest region and Pará state, are challenges to diagnosis and to the urgent availability of antigens that are reactive with patients’ sera. These guidelines aim to update the first Brazilian consensus on paracoccidioidomycosis by providing evidence-based recommendations for bedside patient management. The guidelines provide data on etiology, epidemiology, immunopathogenesis, diagnosis, treatment and sequelae, with emphasis on diagnosis and treatment, as well as current recommendations and challenges in this field of knowledge.

Resumen La paracoccidioidomicosis es una micosis sistémica, relacionada con las actividades agrícolas, con incidencia y prevalencia subestimadas por la ausencia de notificación en varios estados. La evolución insidiosa del cuadro clínico puede tener como consecuencia secuelas graves si el diagnóstico y el tratamiento no se establecen precoz y adecuadamente. Al lado del complejo Paracoccidioides brasiliensis (P. brasiliensis), la descripción de nueva especie, Paracoccidioides lutzii (P. lutzii) en Rondonia, donde la enfermedad alcanzó niveles epidémicos, y en la región Centro Oeste y en Pará, se constituyen en desafíos para la institución del diagnóstico y la urgente puesta a disposición de antígenos que tengan reactividad con los sueros de los pacientes. El presente consenso tiene por objeto actualizar el primer consenso brasileño en paracoccidioidomicosis, estableciendo recomendaciones para el manejo del paciente al borde del lecho, con base en las evidencias conocidas. Se presentan datos de etiología, epidemiología, inmunopatogenia, diagnóstico, terapéutica y secuelas, enfatizando el diagnóstico y terapéutica, así como recomendaciones desafíos y actuales en esta área del conocimiento.

Resumo A paracoccidioidomicose (PCM) é uma micose sistêmica, relacionada às atividades agrícolas, com incidência e prevalência subestimadas, pela ausência de notificação em várias Unidades da Federação (UFs). A evolução insidiosa do quadro clínico pode ter como consequência sequelas graves se o diagnóstico e o tratamento não forem instituídos precoce e adequadamente. Ao lado do complexo Paracoccidioides brasiliensis (P. brasiliensis), a descrição de nova espécie, Paracoccidioides lutzii (P. lutzii), em Rondônia, onde a doença alcançou níveis epidêmicos, bem como na região Centro-Oeste e no Pará, constituem-se em desafios para a instituição do diagnóstico e a urgente disponibilização de antígenos que tenham reatividade com os soros dos pacientes. Este consenso visa atualizar o primeiro consenso brasileiro em PCM, estabelecendo recomendações para o manejo clínico do paciente, com base nas evidências conhecidas. São apresentados dados de etiologia, epidemiologia, imunopatogenia, diagnóstico, terapêutica e sequelas, enfatizando-se o diagnóstico e a terapêutica, bem como recomendações e desafios atuais nessa área do conhecimento.

Abstract Paracoccidioidomycosis is a systemic fungal disease associated with agricultural activities. Its incidence and prevalence are underestimated because of the lack of reporting in several Brazilian states. If paracoccidiodomycosis is not diagnosed and treated early and adequately, endemic fungal infection may result in serious sequelae. In addition to the Paracoccidioides brasiliensis (P. brasiliensis) complex, the appearance of a new species, Paracoccidioides lutzii (P. lutzii), in Rondônia state, where the disease has reached epidemic levels, and in the country’s Midwest region and Pará state, are challenges to diagnosis and to the urgent availability of antigens that are reactive with patients’ sera. These guidelines aim to update the first Brazilian consensus on paracoccidioidomycosis by providing evidence-based recommendations for bedside patient management. The guidelines provide data on etiology, epidemiology, immunopathogenesis, diagnosis, treatment and sequelae, with emphasis on diagnosis and treatment, as well as current recommendations and challenges in this field of knowledge.

Resumen La paracoccidioidomicosis es una micosis sistémica, relacionada con las actividades agrícolas, con incidencia y prevalencia subestimadas por la ausencia de notificación en varios estados. La evolución insidiosa del cuadro clínico puede tener como consecuencia secuelas graves si el diagnóstico y el tratamiento no se establecen precoz y adecuadamente. Al lado del complejo Paracoccidioides brasiliensis (P. brasiliensis), la descripción de nueva especie, Paracoccidioides lutzii (P. lutzii) en Rondonia, donde la enfermedad alcanzó niveles epidémicos, y en la región Centro Oeste y en Pará, se constituyen en desafíos para la institución del diagnóstico y la urgente puesta a disposición de antígenos que tengan reactividad con los sueros de los pacientes. El presente consenso tiene por objeto actualizar el primer consenso brasileño en paracoccidioidomicosis, estableciendo recomendaciones para el manejo del paciente al borde del lecho, con base en las evidencias conocidas. Se presentan datos de etiología, epidemiología, inmunopatogenia, diagnóstico, terapéutica y secuelas, enfatizando el diagnóstico y terapéutica, así como recomendaciones desafíos y actuales en esta área del conocimiento.

BACKGROUND The main clinical forms of paracoccidioidomycosis (PCM) are the acute/subacute form (AF) and the chronic form (CF), and they both display considerable clinical variability. The immune responses of PCM patients, during and after treatment, remain neglected, mainly in the case of CF patients, due to the high prevalence of pulmonary sequelae. OBJECTIVE To evaluate the distribution of whole blood T cell subsets, serum cytokines, and biomarkers of pulmonary fibrosis in PCM patients, according to the clinical form and at different time points, during the antifungal therapy. METHODS Eighty-seven PCM patients, from an endemic area in Brazil, were categorised into groups, according to the clinical form (AF or CF) and the moment of treatment. The peripheral blood T lymphocyte subsets of these patients were analysed using fluorescence-activated cell sorting. The serum levels of cytokines, basic fibroblast growth factor and surfactant protein-D (SP-D) were also analysed. FINDINGS In the CF patients, an expansion of the peripheral blood TCD4+ cells was observed during the treatment, and this persisted even after two years of antifungal treatment. In addition, these patients showed high serum levels of SP-D. CONCLUSION Our findings highlight the immunological changes CF patients undergo, during and after treatment, possibly due to the hypoxia triggered by pulmonary fibrosis and emphysema.

Abstract Paracoccidioidomycosis is a systemic fungal disease occurring in Latin America that is associated with rural environments and agricultural activities. However, the incidence and prevalence of paracoccidiodomycosis is underestimated because of the lack of compulsory notification. If paracoccidiodomycosis is not diagnosed and treated early and adequately, the endemic fungal infection could result in serious sequelae. While the Paracoccidioides brasiliensis ( P. brasiliensis ) complex has been known to be the causal agent of paracoccidiodomycosis, a new species, Paracoccidioides lutzii ( P. lutzii ), has been reported in Rondônia, where the disease has reached epidemic levels, and in the Central West and Pará. Accurate diagnoses and availability of antigens that are reactive with the patients’ sera remain significant challenges. Therefore, the present guidelines aims to update the first Brazilian consensus on paracoccidioidomycosis by providing evidence-based recommendations for bedside patient management. This consensus summarizes etiological, ecoepidemiological, molecular epidemiological, and immunopathological data, with emphasis on clinical, microbiological, and serological diagnosis and management of clinical forms and sequelae, as well as in patients with comorbidities and immunosuppression. The consensus also includes discussion of outpatient treatments, severe disease forms, disease prevalence among special populations and resource-poor settings, a brief review of prevention and control measures, current challenges and recommendations.

Abstract Background Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life – membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories – and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.

ABSTRACT The aim of this study was to identify Candida spp. isolated from candiduria episodes at a tertiary hospital in the Midwest region of Brazil, and to determine their susceptibility profiles to antifungal compounds. From May 2011 to April 2012, Candida spp. isolated from 106 adult patients with candiduria admitted to the University Hospital of the Federal University of Mato Grosso do Sul were evaluated. Both, species identification and susceptibility testing with fluconazole-FLC, voriconazole-VRC, and amphotericin B-AmB were carried out using the Vitek 2. To discriminate species of the C. parapsilosis complex, a RAPD-PCR technique using the RPO2 primer was performed. From the total of 106 isolates, 42 (39.6%) C. albicans and 64 (60.4%) Candida non-albicans (CNA) - 33 C. tropicalis, 18 C. glabrata, 5 C. krusei, 4 C. parapsilosis sensu stricto, 2 C. kefyr, 1 C. lusitaniae, and 1 C. guilliermondii were identified. All isolates were susceptible to AmB and VRC, whereas all C. glabrata isolates presented either resistance (5.6%) or dose-dependent susceptibility (94.4%) to FLC. The study of Candida spp. and their resistance profiles may help in tailoring more efficient therapeutic strategies for candiduria.

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.