A scoping study of pulmonary paracoccidioidomycosis: severity classification based on radiographic and tomographic evaluation

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.

Multiparametric assay of screening for the diagnosis of mycoses of interest in Public Health: standardization of methodology / Ensaio multiparamétrico de triagem para diagnóstico de micoses de interesse em Saúde Pública: padronização da metodologia

The standardization and validation of a multiplex assay requires the combination of important parameters such as sensitivity and specificity, acceptable levels of performance, robustness, and reproducibility. We standardized a multiparametric Dot-blot aimed at the serological screening of paracoccidioidomycosis, histoplasmosis, and aspergillosis. A total of 148 serum were evaluated: 10 from healthy subjects, 36 from patients with paracoccidioidomycosis, 62 from patients with histoplasmosis, and 40 from patients with aspergillosis. It was found that the multiparametric Dot-blot showed a high percentage of cross-reactivity. However, when evaluated individually, in the serological screening of histoplasmosis, a good performance was observed when compared to the double immunodiffusion assay, considered the gold standard test, with 100% co-positivity and 83.3% co-negativity. The performance of serological screening for aspergillosis was not satisfactory when compared to double immunodiffusion, showing 71.4% co-positivity and 100% co-negativity. The evaluation of the stability of nitrocellulose membranes showed that membranes sensitized with H. capsulatum antigen remained stable for 90 days and those sensitized with A. fumigatus antigen for 30 days. We conclude that the use of crude antigens was not suitable for the standardization of the multiparametric Dot-blot assay, due to the high cross-reactivity, and that further tests should be performed with purified proteins (AU).

A padronização e validação de um ensaio multiplex requer a combinação de parâmetros importantes, como sensibilidade e especificidade, níveis aceitáveis de desempenho, robustez e reprodutibilidade. Este trabalho padronizou um Dot-blot multiparamétrico visando a triagem sorológica da paracoccidioidomicose, histoplasmose e aspergilose. Foram avaliadas 148 amostras de soro: 10 de indivíduos saudáveis, 36 de pacientes com paracoccidioidomicose, 62 de pacientes com histoplasmose e 40 de pacientes com aspergilose. Verificou-se que o Dot-blot multiparamétrico apresentou elevado percentual de reatividade cruzada. Entretanto, quando avaliado individualmente, na triagem sorológica da histoplasmose observou-se bom desempenho quando comparado ao ensaio de imunodifusão dupla, considerado o teste padrão ouro, com 100% de co-positividade e 83,3% de co-negatividade. O desempenho da triagem sorológica da aspergilose não foi satisfatório quando comparado a imunodifusão dupla, apresentando 71,4% de co-positividade e 100% de co-negatividade. A avaliação da estabilidade das membranas de nitrocelulose mostrou que membranas sensibilizadas com antígeno de H. capsulatum permaneceram estáveis por 90 dias e as sensibilizadas com antígeno de A. fumigatus, por 30 dias. Concluímos que o uso de antígenos brutos não foi adequado para a padronização do ensaio de Dot-blot multiparamétrico, devido ao alto índice de reatividade cruzada, e que novos testes devem ser realizados com proteínas purificadas (AU).

Impact of laryngeal sequelae on voice- and swallowing-related outcomes in paracoccidioidomycosis

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.(AU)

Impact of laryngeal sequelae on voice- and swallowing-related outcomes in paracoccidioidomycosis

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.(AU)

Perfil sorológico dos pacientes com suspeita clínica de paracoccidiodomicose avaliados pelo Instituto Adolfo Lutz no ano de 2016 / Serological profile of patients with clinical suspicion of paracoccidiodomycosis evaluated by Instituto Adolfo Lutz in 2016

Oitenta porcento dos casos de paracoccidioidomicose (PMC) ocorrem no Brasil. As regiões brasileiras com maior número de casos são: sul, sudeste e centro-oeste, sendo emergente no norte e nordeste. A imunodifusão dupla em gel de agarose assume grande importância no diagnóstico, por permitir o monitoramento da doença e por oferecer subsídios para levantamentos soroepidemiológicos. O objetivo deste trabalho foi de avaliar e caracterizar os pacientes atendidos no Laboratório de Imunodiagnóstico das Micoses do Instituto Adolfo Lutz de São Paulo, em 2016. Trata-se de um estudo retrospectivo realizado utilizando-se dados secundários e avaliando-se as seguintes informações: idade, sexo, procedência do pedido médico, resultado e histórico sorológico dos pacientes. Dos 1.408 pacientes, 12,8% apresentaram reatividade sorológica para Paracoccidioides brasiliensis. Destes, 42,5% não possuiam histórico sorológico, sendo considerados como casos novos da doença. A classificação dos pacientes reagentes por gênero demonstrou que 83,4% eram do sexo masculino, com razão de masculinidade de 5:1. A faixa etária variou de um (1) a 92 anos, e aproximadamente 40% dos pacientes eram da faixa etária de 41 a 60 anos. Este estudo demonstra e reforça a importância da implementação dos estudos soroepidemiológicos como ferramenta auxiliar para nortear as ações de vigilância e políticas em saúde na PCM.

Eighty percent of paracoccidioidomycosis (PMC) cases occur in Brazil. The highest numbers occur in south, southeast and center-west region, being emergent in the north and northeast areas. The double immunodiffusion in agarose gel is valuable for its diagnosis, as it allows the monitoring of the disease and offers subsidies for the seroepidemiological surveys. This study evaluated and characterized the patients attended in 2016 at the Mycoses Immunodiagnosis Laboratory of Adolfo Lutz Institute of São Paulo. This retrospective study, based on the secondary data, evaluated the information: age, sex, medical request origin, result and serological history of the patients. Of 1,408 patients, 12.8% presented positive serological reactivity for Paracoccidioides brasiliensis. Of them, 42.5% had no serological history and they were considered as new cases. The classification of reactive patients by gender showed that 83.4% were males, being the masculinity ratio of 5:1. The age range was one (1) to 92 years old, and ±40% of the patients were of age ranging from 41-60 years old. This study reinforces the importance of the implementation of the seroepidemiological studies as to guide the surveillance actions and the public health politics in PCM.

Perfil sorológico dos pacientes com suspeita clínica de paracoccidiodomicose avaliados pelo Instituto Adolfo Lutz no ano de 2016 / Serological profile of patients with clinical suspicion of paracoccidiodomycosis evaluated by Instituto Adolfo Lutz in 2016

Oitenta porcento dos casos de paracoccidioidomicose (PMC) ocorrem no Brasil. As regiões brasileiras com maior número de casos são: sul, sudeste e centro-oeste, sendo emergente no norte e nordeste. A imunodifusão dupla em gel de agarose assume grande importância no diagnóstico, por permitir o monitoramento da doença e por oferecer subsídios para levantamentos soroepidemiológicos. O objetivo deste trabalho foi de avaliar e caracterizar os pacientes atendidos no Laboratório de Imunodiagnóstico das Micoses do Instituto Adolfo Lutz de São Paulo, em 2016. Trata-se de um estudo retrospectivo realizado utilizando-se dados secundários e avaliando-se as seguintes informações: idade, sexo, procedência do pedido médico, resultado e histórico sorológico dos pacientes. Dos 1.408 pacientes, 12,8% apresentaram reatividade sorológica para Paracoccidioides brasiliensis. Destes, 42,5% não possuiam histórico sorológico, sendo considerados como casos novos da doença. A classificação dos pacientes reagentes por gênero demonstrou que 83,4% eram do sexo masculino, com razão de masculinidade de 5:1. A faixa etária variou de um (1) a 92 anos, e aproximadamente 40% dos pacientes eram da faixa etária de 41 a 60 anos. Este estudo demonstra e reforça a importância da implementação dos estudos soroepidemiológicos como ferramenta auxiliar para nortear as ações de vigilância e políticas em saúde na PCM. (AU)

Eighty percent of paracoccidioidomycosis (PMC) cases occur in Brazil. The highest numbers occur in south, southeast and center-west region, being emergent in the north and northeast areas. The double immunodiffusion in agarose gel is valuable for its diagnosis, as it allows the monitoring of the disease and offers subsidies for the seroepidemiological surveys. This study evaluated and characterized the patients attended in 2016 at the Mycoses Immunodiagnosis Laboratory of Adolfo Lutz Institute of São Paulo. This retrospective study, based on the secondary data, evaluated the information: age, sex, medical request origin, result and serological history of the patients. Of 1,408 patients, 12.8% presented positive serological reactivity for Paracoccidioides brasiliensis. Of them, 42.5% had no serological history and they were considered as new cases. The classification of reactive patients by gender showed that 83.4% were males, being the masculinity ratio of 5:1. The age range was one (1) to 92 years old, and ±40% of the patients were of age ranging from 41-60 years old. This study reinforces the importance of the implementation of the seroepidemiological studies as to guide the surveillance actions and the public health politics in PCM.(AU)

Evaluation of the "shelf life" of nitrocellulose membranes immobilized with Paracoccidioides brasiliensis antigen for performing the immunodiagnosis of paracoccidioidomycosis / Avaliação do "tempo de prateleira" de membranas de nitrocelulose imobilizadas com antígeno de Paracoccidioides brasiliensis para o imunodiagnóstico da paracoccidioidomicose

The immunoblotting reaction for performing the paracoccidioidomycosis (PCM) immunodiagnosis is an in-house methodology; and being a laborious task involving two previous steps, SDS-PAGE and Western blot, we evaluated the shelf life of nitrocellulose membranes containing the immobilized P. brasiliensis antigens, stored at -20 o C for 7, 15, 30, 45, 60 and 90 days. Twenty-eight serum samples were analyzed on two nitrocellulose membranes groups: (a) membranes previously blocked with PBS-5 % non-fat dry milk and (b) the priory non-blocked membranes. No difference was detected in the reactivity pattern in serum samples evaluated in the both membrane groups, especially for those stored for 7, 15, 30, 45 and 60 days. It might be emphasized that a good stability of P. brasiliensis antigens, immobilized on the nitrocellulose membranes, enable them to be stored up to 60 days at -20 o C. This finding contributes to the rapid diagnosis of PCM, and for sending them to other laboratories without adequate infrastructure for carrying out the steps that precede the immunodetection as the antigen production, SDS-PAGE and Western blot techniques. This scheme contributes substantially to improve the quality of PCM serodiagnosis, as it provides reproducible results in the units of the Laboratory Network.(AU)

Considerando-se que o immunoblotting para o imunodiagnóstico da paracoccidioidomicose (PCM) é uma metodologia in house e laboriosa envolvendo duas etapas iniciais, SDS-PAGE e Western blot, neste estudo foi avaliado o tempo de prateleira das membranas de nitrocelulose sensibilizadas com antígeno de P. brasiliensis, armazenadas a -20 o C durante 7, 15, 30, 45, 60 e 90 dias. Vinte e oito amostras de soro foram analisadas em dois grupos de membranas de nitrocelulose (membranas previamente bloqueadas com PBS-leite 5 % e as não-bloqueadas). Não houve diferença no padrão de reatividade quando os soros foram avaliados frente a ambos os grupos, especialmente para membranas armazenadas por 7, 15, 30, 45 e 60 dias. A boa estabilidade do antígeno utilizado para sensibilizar as membranas fez com que estas pudessem ser armazenadas a -20 C até 60 dias. Estas características contribuem para efetuar o diagnóstico rápido da PCM, bem como as perspectivas dessas membranas sensibilizadas serem encaminhadas para os laboratórios, que não possuam infraestrutura necessária para executar as etapas que antecedem a realização de immunoblotting, como a produção de antígeno, as técnicas de SDS PAGE e Western blot. Este procedimento contribui substancialmente para melhorar o diagnóstico sorológico da PCM, pois poderá fornecer resultados reprodutíveis nas unidades componentes da Rede de Laboratórios.(AU)

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

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.(AU)

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

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.(AU)

AVALIAÇÃO MORFOMÉTRICA DE LESÕES GRANULOMATOSAS PULMONARES EM CAMUNDONGOS EXPERIMENTALMENTE INFECTADOS COM PARACOCCIDIOIDES BRASILIENSIS E TRATADOS COM NANOPARTÍCULAS DE ITRACONAZOL POR VIA INALATÓRIA

A paracoccidioidomicose é uma enfermidade causada pelo fungo termodimórfico Paracoccidioides brasilienses. Caracteriza-se por uma inflamação granulomatosa que acomete principalmente o pulmão e o tratamento consiste no uso prolongado de antifúngicos por via oral ou intravenosa, que causam muitos efeitos colaterais. Este estudo teve como objetivo avaliar o número e área dos granulomas nos pulmões de camundongos Swiss experimentalmente infectados com Paracoccidioides brasiliensis e tratados com nanopartículas de itraconazol por via inalatória, como um dos quesitos de avaliação da eficácia deste tratamento. Foram utilizados 28 camundongos, Swiss, entre 8 a 12 semanas de idade. Os animais foram aclimatados às condições do laboratório durante duas semanas antes do início do experimento. Para o procedimento de infecção, os animais foram anestesiados por via intraperitoneal com Cetamina 120 mg/kg e Xilazina 8 mg/kg e, em seguida infectados com 50 L do inóculo contendo 5 x 106 células/mL de Paracoccidioides brasiliensis. Em seguida, foram distribuídos em quatro grupos: Grupo 1 não infectados e não tratados, Grupo 2- infectados e não tratados, Grupo 3 infectados e tratados por via oral e Grupo 4 infectados e tratados por via inalatória. Para avaliar os efeitos da terapia proposta, realizou-se análise histopatológica do tecido pulmonar, para determinar a quantidade e área dos granulomas. O número de granulomas no tecido pulmonar dos animais infectados e tratados com itraconazol por via inalatória mostrou uma redução significativa (P < 0,05) em relação aos demais grupos. Em relação a área desses granulomas, não houve diferença estatística significativa entre os grupos. A administração por via inalatória de nanopartículas de itraconazol é uma proposta terapêutica promissora para o tratamento da paracoccidioidomicose.

Paracoccidioidomycosis is a disease caused by the thermodimorphic fungus Paracoccidioides brasilienses. It is characterized by a granulomatous inflammation that mainly affects the lung and the treatment consists of the prolonged use of oral or intravenous antifungal agents, which cause many side effects. This study aimed to evaluate the number and area of granulomas in the lungs of Swiss mice experimentally infected with Paracoccidioides brasiliensis and treated with inhaled itraconazole nanoparticles, as one of the items to evaluate the effectiveness of this treatment. Twenty-eight Swiss mice between 8 and 12 weeks of age were used. The animals were acclimated to laboratory conditions for two weeks before the start of the experiment. For the infection procedure, the animals were anesthetized intraperitoneally with Ketamine 120 mg/kg and Xylazine 8 mg/kg and then infected with 50 L of the inoculum containing 5 x 106 cells/mL of Paracoccidioides brasiliensis. They were then divided into four groups: Group 1 uninfected and untreated, Group 2- infected and untreated, Group 3 infected and treated orally, and Group 4 infected and treated by inhalation. To assess the effects of the proposed therapy, a histopathological analysis of the lung tissue was performed to determine the quantity and area of granulomas. The number of granulomas in the lung tissue of animals infected and treated with itraconazole by inhalation showed a significant reduction (P < 0.05) in relation to the other groups. Regarding the area of these granulomas, there was no statistically significant difference between the groups. The inhaled administration of itraconazole nanoparticles is a promising therapeutic proposal for the treatment of paracoccidioidomycosis.

Seroepidemiology of Paracoccidioides brasiliensis infection in horses from Rio Grande do Sul, Brazil

Paracoccidioides brasiliensis is the etiological agent of the major systemic mycosis in Brazil, called paracoccidioidomycosis. Although the Rio Grande do Sul is considered an endemic area of the disease, there are few studies on the ecology of P. brasiliensis in the state. Therefore, this study aimed to evaluate the infection of P. brasiliensis in horses from the mesoregion of Southwest Riograndense, using these animals as sentinels. Serological techniques, such as double immunodiffusion in agar gel (AGID) and indirect ELISA, were performed to detect the anti-gp43 P. brasiliensis antibody in horses from five different farms in the region of Bagé, RS, Brazil. Serology was performed in 200 Pure Blood English horses up to two years of age that were born and raised exclusively at the farms. Of these horses, 12% had anti-gp43 antibodies according to the ELISA results, with rates ranging from 0 to 30% according to the farm of origin (p < 0.001). Based on the immunodiffusion results, all equine serum samples were negative. These results indicate the presence of the fungus P. brasiliensis in the middle region of the southwestern state of Rio Grande do Sul, Brazil.(AU)

Paracoccidioidomycosis in southern Rio Grande do Sul: a retrospective study of histopathologically diagnosed cases

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis and is endemic to Brazil. The aim of this study was to perform a retrospective analysis of the PCM cases in the countryside south of Rio Grande do Sul, Brazil. The files from four histopathology laboratories located in the city of Pelotas were obtained, and all of the epidemiological and clinical data from the PCM diagnosed cases were collected for analysis. A total of 123 PCM cases diagnosed between 1966 and 2009 were selected. Of these patients, 104 (84.5%) were male, and 17 were female. The patients ranged from 02 to 92 years of age. Fifty-two cases (41.9%) were obtained from the oral pathology laboratory, and the remaining 71 cases (58.1%) were obtained from the three general pathology laboratories. Of all of the patients studied, 65.2% lived in rural zones and worked in agriculture or other related fields. Data on the evolution of this disease was available for 43 cases, and the time frame ranged from 20 to 2920 days (mean = 572.3 days). An accurate diagnosis performed in less than 30 days only occurred in 21% of the cases. PCM is endemic to the countryside of Rio Grande do Sul. Therefore, it is recommended that PCM be included as a differential diagnosis, mainly for individuals between 30 and 60 years of age, living in rural zones and who have respiratory signs and associated-oropharyngeal lesions.(AU)

Investigação sorológica e molecular da presença de Paracoccidioides spp. em três mesoregiões do Rio Grande do Sul

A paracoccidioidomicose (PCM) vem sendo descrita no Rio Grande do Sul (RS) desde a década de 40, no entanto, os processos ecológicos que envolvem os seus agentes etiológicos ainda não foram totalmente esclarecidos. O solo é descrito como o habitat do Paracoccidioides spp., no entanto, devido à dificuldade de isolar o agente do ambiente, dados específicos sobre seu nicho ecológico são ainda pouco conhecidos. Isto, associado ao grande período de latência que a doença pode apresentar, dificulta a determinação da via e fonte de infecção dos indivíduos pelo patógeno. Contudo, técnicas sorológicas e moleculares vem sendo empregadas para melhor compreensão da ecoepidemiologia do Paracoccidioides spp. demonstrando que está ocorrendo uma expansão geográfica de suas áreas endêmicas. Sendo assim, este estudo teve por objetivo pesquisar a presença de P. brasiliensis e P. lutzii em distintas mesorregiões do RS como forma de auxiliar na compreensão dos aspectos ecológicos desses agentes no estado. Para tanto foram realizados estudos utilizando animais como sentinelas da presença do fungo, a partir da busca de anticorpos por sorologia, e utilizando a biologia molecular para detecção do DNA fúngico diretamente de amostras de solo. Para sorologia foram incluídas amostras de 481 animais domésticos e silvestres as quais foram submetidas ao teste de ELISA indireto buscando anticorpos anti-P. lutzii. Para o estudo molecular, amostras de solo das cidades de Bagé (n=30) e Rio Grande (n=16) foram devidamente coletadas e submetidas a protocolo de extração de DNA por kit comercial (Norgen Biotek Corp®) e técnicas de PCR e Nested-PCR com primers específicos para o gênero. Dos 481 animais estudados, 51 (10,6%) foram soropositivos para IgG anti-P. lutzii. E, das 46 amostras de solo estudadas, o DNA de Paracoccidioides spp. foi encontrado em 19 (41,3%), sendo 10 em Bagé (52,6%) e 9 no solo de Rio Grande (47,4%). Este estudo trouxe dados importantes sobre a ecologia dos agentes da PCM no RS, demostrando pela primeira vez a presença de P. lutzii no estado e uma alta taxa de detecção de DNA de Paracoccidioides spp. em solos do Bioma Pampa, o que ainda não havia sido descrito na literatura. Esses dados ajudam a compreender a ecologia dos agentes da PCM como um todo, fazendo com que essa doença ainda hoje negligenciada ganhe uma maior visibilidade na região e indiretamente no país.

Paracoccidioidomycosis (PCM) has been described in Rio Grande do Sul (RS) since the decade of 40, however, ecological processes involving their etiological agents have not yet been fully clarified. Soil is described as the habitat of Paracoccidioides spp., however, due to the difficulty in isolate the agent from the environment, specific data about its ecological niche are still unknown. In addition, the long latency period of the disease, makes it difficult to determine the pathway and source of infection of the individuals by the pathogen. On the other hand, serological and molecular techniques have been used to better understand the ecoepidemiology of Paracoccidioides spp. demonstrating that a geographical expansion of their endemic areas is occurring. Thus, this study aimed to investigate the presence of P. brasiliensis and P. lutzii in different mesoregions of RS. We performed studies with animal serology to use it as sentinels of the presence of the fungus, and with molecular biology to detect fungal DNA directly from soil samples. For serology, samples from 481 domestic and wild animals were included, which were submitted to the indirect ELISA test for anti-P.lutzii antibodies. For the molecular study, soil samples from the cities of Bagé (n = 30) and Rio Grande (n = 16) were collected and submitted to DNA extraction protocol by commercial kit (Norgen Biotek Corp®) and PCR and Nested-PCR with primers specific for the genus. Of the 481 animals studied, 51 (10.6%) were seropositive for IgG anti-P.lutzii. And, of the 46 soil samples studied, the DNA of Paracoccidioides spp. was found in 19 (41.3%), 10 in Bagé (52.6%) and 9 in Rio Grande (47.4%). This study provided important data on the ecology of PCM agents in RS, demonstrating for the first time the presence of P. lutzii in the state, and a high DNA detection rate of Paracoccidioides spp. in soils of the Pampa Biome, which had not yet been described in the literature. These data help to understand the ecology of PCM agents as a whole, making this neglected disease even more visible in the region and indirectly in the country.

Paracoccidioides brasiliensis infection in dogs from Western Brazilian Amazon / Infecção por Paracoccidioides brasiliensis em cães da Amazônia Ocidental Brasileira

The objective of the study was to evaluate Paracoccidioides brasiliensis infection in urban dogs from the municipality of Monte Negro, Rondonia, Western Brazilian Amazon. The serum samples (n=126) were analyzed by indirect ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. A positivity of 54.8% was observed only in the ELISA test and no statistical difference was observed in the seroprevalence in relation to age or sex. This is the first paracoccidioidomycosis survey carried out with dogs from the Western Brazilian Amazon. The higher positivity rates of P. brasiliensis infection observed in this study suggest that veterinarians must be alert to detect new cases of natural disease in dogs living in paracoccidioidomycosis endemic areas.(AU)

O objetivo deste estudo foi avaliar a infecção por Paracoccidioides brasiliensis em cães urbanos do município de Monte Negro, Rondônia, Amazônia Ocidental Brasileira. As amostras de soro (n=126) foram analisadas por meio dos testes de ELISA indireto e imunodifusão utilizando gp43 de P. brasiliensis e exoantígeno como antígenos, respectivamente. Uma positividade de 54,8% foi observada apé isso mesmoenas no teste de ELISA e nenhuma diferença estatística foi observada na soroprevalência em relação ao sexo ou à idade. Este é o primeiro estudo epidemiológico de paracoccidioidomicose realizado com cães da Amazônia Ocidental Brasileira. A alta positividade de infecção por P. brasiliensis observada neste estudo sugere que os veterinários devem estar atentos para detectar a doença natural em cães de áreas endêmicas para paracoccidioidomicose.(AU)

Retrospective seroepidemiological analysis of patients with suspicion of paracoccidioidomycosis in São Paulo State, Brazil

The geographic distribution of paracoccidioidomycosis (PCM) in the Brazilian state of São Paulo was evaluated in a retrospective study using secondary data from serological analyses, carried out by double immunodiffusion assay of patients with PCM suspicion, from January 1999 to May 2010. Sixty percent of 10,176 patients, from 239 cities, were serologically reactive to P. brasiliensis. The cities that showed the most serological reactivity among patients were São João da Boa Vista (85%), Piracicaba (75%), Sorocaba (73%), Campinas (72%) and São Paulo (62%). São Paulo state has an area of 248,209.4 km²; the climate is tropical and sub-tropical with annual temperatures between 18 and 24ºC, high rainfall (900 to 1800 mm/year), rainy summers and mild winters. It also features large areas composed of acidic soils, and is one of the greatest contributors to Brazilian agricultural production and, separately, the largest producer of orange juice and, the ninth greatest producer of soy and sugar cane and the fourth largest coffee producer. We suggest that the climatic characteristics associated with soil type and development of primary activities can contribute to the endemic potential of PCM in São Paulo state.(AU)

PESQUISA MOLECULAR DE FUNGOS PATOGÊNICOS EM QUIRÓPTEROS DA REGIÃO DE BOTUCATU-SP

Atualmente dentre as doenças emergentes a maioria são zoonoses, e os morcegos desempenham papel importante como fonte de infecção e transmissão de agentes infecciosos. Histoplasmose, criptococose e paracoccidioidomicose são doenças fúngicas relatadas em diversas espécies animais, podendo seus agentes serem isolados de grutas, cavernas, solo e fezes de animais. Desta forma, o objetivo do presente estudo foi realizar a detecção molecular dos fungos Histoplasma capsulatum, Cryptococcus spp. e Paracoccidioides brasiliensis em quirópteros no município de Botucatu e região, SP. O delineamento do estudo foi de série de casos, a partir de morcegos recolhidos e enviados pela Vigilância Epidemiológica municipal de 12 cidades da região Centro-Oeste de São Paulo para o diagnóstico de raiva, no período de abril de 2015 a maio de 2016. Foram coletados pulmão, baço, fígado e intestino de 172 quirópteros, e avaliados pelas técnicas de Nested-PCR e cultivo microbiológico. A prevalência encontrada para H. capsulatum foi de 8,14% (14/172), com um morcego positivo somente no cultivo microbiológico, 12 na técnica de Nested-PCR, e um animal positivo em ambos os métodos. Dois morcegos foram positivos para Cryptococcus spp. e dois para P. brasiliensis pela técnica de Nested-PCR. Como auxílio do software Autocad® foi realizado georreferenciamento dos pontos de recolhimento dos animais, verificando quirópteros positivos em escolas, praças, residências e hospitais. Conclui-se que a presença de morcegos positivos para fungos patogênicos em áreas urbanas, é um risco para a saúde pública, visto que estes animais podem favorecer a manutenção e transmissão de patógenos em ambientes caseiros. Medidas de manejo ambiental devem ser orientadas a população pelo Serviço de Vigilância Epidemiológica Municipal, visando diminuir a população de morcegos em ambientes domésticos e minimizar os riscos de transmissão de fungos patogênicos.

Currently, among the emerging diseases, most are zoonosis, and baths perform an important role as source of infection and transmission of infectious agents. Histoplasmosis, criptococcosis and paracoccidioidomicosis are fungal diseases reported in different kinds of animals, and their agents can be isolated in grottos, caverns, soils and feces of animals.Thus, the objective of the present study was performing the molecular detection of the fungi Histoplasma capsulatum, Cryptococcus spp. Ando Paracoccidioides brasiliensis in bats from the city of Botucatu and region, state of São Paulo.The study was delineated with a series of cases, from bats collected and sent by the Municipal Epidemiologic Surveillance of 12 cities at the Midwest region of São Paulo for the rabies diagnosis, within the time period from April 2015 to May 2016. Lung, spleen, liver and intestine were collected from 172 bats and assessed by Nested-PCR and microbiological cultivation techniques.The prevalence found for H. capsulatum was 8.14% (14/172), with a positive bath only in the microbiological cultivation, 12 in the Nested-PCR technique and a positive animal in both methods.Two bats were positive for Cryptococcus spp. and two for P. brasiliensis by the Nested-PCR technique. With the aid of Autocad® software, the georeferencing of the animalscollection spots was performed, verifying positive bats in schools, squares, residences and hospitals. We conclude that the presence of positive baths for pathogenic fungi in urban areas is a risk to public health, since these animals can help on the maintenance and transmission of pathogens in domestic environments.Environmental management measures shall be taught to the population by the Municipal Service of Epidemiologic Surveillance, with the purpose of decreasing the baths population in domestic environments and minimizing the risk of transmitting pathogenic fungi. ¨¨

Research and characterization of selected pathogens of cutaneous and mucocutaneous lesions in cetaceans from the Brazilian coast

Cetáceos são sentinelas do ambiente marinho, atualmente ameaçados por diversos fatores, principalmente antropogênicos. Os processos de pele e mucosas externas são os mais facilmente identificados, bons indicadores do estado de saúde em cetáceos. Lesões cutâneas e mucocutâneas já foram amplamente relatadas em cetáceos de vida livre e de cativeiro, mas pouco se sabe a respeito dos fatores etiológicos envolvidos, evolução das lesões dermatológicas e suas consequências sistêmicas. Vírus são os agentes mais comumente envolvidos em lesões cutâneas e mucocutâneas, especialmente os herpesvírus (HV), associados a lesões de morfologias variáveis em mucosas, e poxvírus dos cetáceos (Cetacean Poxvirus CePV), principalmente associados a lesões de pele tatuagem ou anel características. Agentes fúngicos também podem causar doenças dermatológicas em cetáceos, como por exemplo a paracoccidioidomicose ceti, caracterizada por lesões esbranquiçadas elevadas e proliferativas, causada por leveduras nãocultiváveis de Paracoccidioides brasiliensis (ordem Onygenales). Apesar de mundialmente reportados, a ocorrência desses agentes etiológicos em cetáceos do Atlântico Sul ainda é pouco compreendida. O objetivo desse estudo foi identificar e caracterizar patógenos cutâneos e mucocutâneos selecionados (HV, CePV e P. brasiliensis) de cetáceos brasileiros de vida livre e desenhar métodos diagnósticos mais sensíveis para sua detecção. Todos os animais estudados encalharam ao longo da costa brasileira, entre 2005 e 2015, exceto por três botos-cor-de-rosa que foram fisicamente imobilizados e liberados após a coleta de amostras. Para atingir tais objetivos, empregamos técnicas moleculares e histológicas, e ocasionalmente de imunohistoquímica e microscopia eletrônica. A presença de HV e CePV foi avaliada, respectivamente, em amostras cutâneas e de mucosa oral e genital de 115 espécimes, e amostras de pele de 113 indivíduos; enquanto a presença de membros da ordem Onygenales foi avaliada em quatro espécimes que apresentavam lesões macroscópicas compatíveis. Amostras de pele ou de mucosa oral de quatro animais foram positivas para a PCR de HV: uma lesão ulcerada de pele de coloração esbranquiçada de um boto-cinza (Sotalia guianensis), uma amostra de tecido lingual de um golfinho-pintado-do-Atlântico (Stenella frontalis), lesões ulcerativas e amostras de pele saudável de um cachalote-anão (Kogia sima), e uma lesão proliferativa de pele em boto-vermelho-boliviano (Inia boliviensis). Os primeiros três animais estavam infectados com alphaherpesvirus. Uma sequência mais similar com gammaherpesvírus foi obtida da lesão proliferativa de pele do boto-vermelhoboliviano. A sequência do boto-vermelho-boliviano possivelmente pertence a um novo gênero de gammaherpesvírus. Ademais, todas as outras amostras de tecidos disponíveis dos espécimes HV-positivos, à parte de pele e mucosa oral, também foram avaliadas por técnicas de PCR e histológicas. Uma sequência diferente de alphaherpesvírus foi encontrada no estômago e em um linfonodo mesentérico do cachalote-anão. Achados microscópicos em dois animais HV-positivos (dermatites proliferativas em boto-vermelho-boliviano e boto-cinza) eram compatíveis com HV. CePV foi identificado em lesões de pele do tipo tattoo de um golfinho-nariz-degarrafa (Tursiops truncatus) e de um boto-cinza por meio de técnicas moleculares estabelecidas, e observação de partículas de poxvírus por microscopia eletrônica. Animais CePV-positivos apresentavam degeneração balonosa epidérmica e ocasionais inclusões intracitoplasmáticas anfofílicas ou eosinofílicas compatíveis com CePV. Motivos aminoácidos específicos para todos os CePVs também foram identificados, reforçando a sugestão de um novo gênero, chamado Cetaceanpoxvirus. Nesse estudo também foram desenvolvidas novas técnicas de PCR convencional e real-time com SYBR® Green, significativamente mais sensíveis do que os métodos atualmente disponíveis em literatura. Um boto-cinza, inicialmente negativo segundo os métodos de PCR previamente conhecidos foi diagnosticado positivo para CePV por meio das novas técnicas aqui descritas. Leveduras refratáveis (49 m de diâmetro) foram observadas á microscopia sob a forma de lesões de pele granulomatosas moderadas e necróticas em quatro golfinhos-narizde- garrafa, e pela primeira vez, em um abscesso muscular (esse último um indício do potencial invasivo desse agente). Leveduras de Onygenales sp. foram identificadas em lesões de pele por meio de imunohistoquímica e uma sequência de P. brasiliensis mais semelhante (100% de identidade de nucleotídeos) áquela descrita em um gofinho-nariz-de-garrafa de Cuba do que a casos de humanos e mamíferos descritos no Brasil, foi encontrada em lesões de pele de um dos espécimes. Esse estudo relata a primeira identificação molecular de HV em cetáceos da América do Sul e em golfinhos de rio no mundo. Além disso, descrevemos a primeira amplificação de CePV e P. brasiliensis em odontocetes da América do Sul, confirmando a etiologia desse tipo de lesões. Quatro das cinco novas sequências de HV identificadas são possivelmente novas espécies, provisoriamente chamadas Delphinid HV-10, Kogiid HV-2, Kogiid HV-3 e Iniid HV-1.

Cetaceans are sentinels of the marine environment, currently threatened by many factors, mainly anthropogenic. The most easily identified compromising conditions are those affecting the skin and external mucosae - good indicators of the cetaceans health status. Cutaneous and mucocutaneous lesions have been extensively reported in wild and captive cetaceans, but little is known about the involved etiological factors, evolution of the dermatological lesions and their systemic consequences. Viruses are the most commonly involved agents in cutaneous and mucocutaneous lesions, especially herpesviruses (HV) and, associated with skin and mucosal lesions with varying morphologies, and cetacean poxviruses (CePV), mainly associated with characteristic tattoo or ring skin lesions. In addition, fungal agents are also recognized as causative agents of dermatological disease in cetaceans, especially in the process known as paracoccidioidomycosis ceti, observed as raised proliferative whitish lesions, caused by non cultivable yeast of Paracoccidioides brasiliensis (order Onygenales). Despite being reported worldwide, the occurrence of these etiological agents in southern Atlantic cetaceans is still poorly understood. The goal of this study was to identify and characterize selected cutaneous and mucocutaneous pathogens (HV, CePV and P. brasiliensis) of free-ranging cetaceans from Brazil, and to design more sensitive diagnostic methods for their detection. All the studied cetaceans stranded along the coast of Brazil, between 2005 and 2015, except three wild riverine dolphins that were physically contained and released after sample collection. In order to achieve our goals, we employed molecular, histopathological, and occasionally immunohistochemical and electron microscopy techniques. The presence of HV and CePV was evaluated in cutaneous, and oral and genital mucosal samples from 115 specimens and skin samples from 113 individuals, respectively; whereas the presence of members of the genus Onygenales sp. was evaluated in four specimens presenting macroscopic compatible lesions. Skin or oral mucosal samples from four animals were HV PCR-positive: a whitish ulcerated skin lesion from a Guiana dolphin (Sotalia guianensis), a lingual sample from an Atlantic spotted dolphin (Stenella frontalis), ulcerative lesions and healthy skin samples from a dwarf sperm whale (Kogia sima), and a proliferative skin lesion from a Bolivian river dolphin (Inia boliviensis). The tree first animals were infected with alphaherpesvirus. A sequence more similar to gammaherpesvirus was obtained from the Bolivian river dolphins proliferative skin lesion. The Bolivian river dolphin sequence could possibly be a member of a new gammaherpesvirus genus. Additionally, all other available tissue samples from HV-positive specimens, aside from skin and oral mucosa, were also tested by PCR and histologically evaluated. A different alphaherpesvirus sequence was found in the stomach and in a mesenteric lymph node of the dwarf sperm whale. Microscopic findings in two HV-positive animals (chronic proliferative dermatitis in Bolivian river dolphin and Guiana dolphin) were compatible with HV. CePV was identified in tattoo skin lesions of an Atlantic bottlenose dolphin and a Guiana dolphin by established molecular methods, and poxviral particles were observed by electron microscopy. CePV-positive animals presented epidermal ballooning degeneration and occasionally small, pale eosinophilic or amphophilic intracytoplasmic inclusions, compatible with CePV. Specific amino acid motifs for all CePV were also identified, reinforcing the suggestion of the new Cetaceanpoxvirus genus. We also designed novel SYBR® Green real-time and conventional CePV PCR methods significantly more sensitive than those currently available in the literature. An additional Guiana dolphin, previously negative based in established PCR methods was diagnosed positive for CePV through these new techniques. Refractile yeasts (49 m in diameter) were observed under light microscopy in mild granulomatous and necrotic skin lesions of four Atlantic bottlenose dolphin, and for the first time, in a skeletal muscle abscess (the former possibly indicating the invasive potential of the agent).Onygenales sp. yeasts were identified in skin lesions by immunohistochemistry and a sequence of P. brasiliensis, more similar (100% nucleotide identity) to the one described in an Atlantic bottlenose dolphin from Cuba than to human or any other terrestrial mammals cases in Brazil, was obtained from the skin lesion of one of the specimens, confirming the etiological agent of these type of lesions. Herein we report the first molecular identification of HV in South American cetaceans and in riverine dolphins worldwide. This study also describes the first amplification of CePV and P. brasiliensis in odontocetes from South America. Four of the five novel herpesvirus sequences herein identified are possibly novel species, tentatively named Delphinid HV-10, Kogiid HV-2, Kogiid HV-3 and Iniid HV-1.

Estudo soroepidemiológico da infecção pelo Paracoccidioides brasiliensis em caninos domésticos no sul do Rio Grande do Sul, Brasil

O fungo termodimórfico Paracoccidioides brasiliensis é o principal causador da paracoccidioidomicose, micose sistêmica restrita à América Latina e endêmica no Brasil. No extremo sul do país a enfermidade é diagnosticada em humanos desde a década de 60 com crescimento do número de casos a partir dos anos 90. Com o propósito de detectar a presença do agente em áreas urbanas, alertando para a possibilidade da exposição de indivíduos suscetíveis à doença, este estudo teve como objetivo investigar a infecção por P. brasiliensis em cães no sul do Rio Grande do Sul, Brasil. Foram avaliados 196 caninos, errantes e semidomiciliados, participantes de um programa de controle populacional dos municípios de Pelotas e Capão do Leão localizados na zona sul do Rio Grande do Sul. Utilizou-se a técnica de ELISA indireto para detecção de anticorpos anti-gp43 de P. brasiliensis no soro dos animais. Na técnica sorológica foram testados e comparados dois conjugados diferentes, anti-IgG canina-peroxidase e proteína G-peroxidase, estes foram avaliados quanto à concordância dos resultados nos testes (índice Kappa) e a sensibilidade, especificidade, valor preditivo positivo e negativo do conjugado porteína G-peroxidase foram calculados considerando o conjugado específico para cães como padrão ouro. A infecção fúngica foi constatada em 58 animais (29,6%), sem diferença significativa quanto ao sexo, idade e raça dos cães (P>0,05). Foram detectados animais soropositivos procedentes de todos os bairros do Distrito Sede de Pelotas, bem como do município vizinho Capão do Leão. Os resultados dos testes com os dois conjugados diferentes resultaram em fraca concordância (Kappa=0,140; P< 0,001). A proteína G-peroxidase apresentou baixa sensibilidade e ocorrência de falsos negativos em relação ao conjugado específico, demonstrando não ser uma alternativa viável para realização do imunoensaio na espécie canina. A detecção de anticorpos específicos nos cães do presente estudo comprova a presença e ampla distribuição de P. brasiliensis na área urbana da cidade de Pelotas e no município Capão do Leão, e alerta para ocorrência da PCM em indivíduos suscetíveis, bem como a possibilidade da doença em pequenos animais nesta região.

The thermally dimorphic fungus Paracoccidioides brasiliensisis the main agent that triggers the paracoccidioidomycosis (PCM), a systemic infectious disease restricted to Latin America and endemic in Brazil. In southern Brazil, this disease has been diagnosed in humans since the 60s and the number of cases has increased since the 90s. In order to detect the presence of the agent in urban areas and warn about the possibility of the exposion of susceptibles individuals to the disease, this study aimed to investigate the infection by P. brasiliensis in dogs in Rio Grande do Sul, southern state of Brazil. 196 dogs which participated in a program of animal population control in the cities of Pelotas and Capão do Leão were evaluated. The indirect ELISA technique was used in order to detect the presence of anti-gp43 antibodies in the animal serum. Two different conjugates were tested and compared in this serological technique, the dog anti-IgG peroxidase and the protein Gperoxidase, that were evaluated for the concordance of the test results (Kappa index). The sensitivity, specificity, positive and negative predictive value of protein Gperoxidase were calculated considering the specific conjugate of dog as a gold standard. The fungal infection was confirmed in 58 animals (29,6%), and no significant difference was found between the gender, age and breed of the dogs (P>0,05). Seropositive animals could be detected coming from the two cities, both Pelotas and Capão do Leão. The result of the tests made with the different conjugate showed poor concordance (Kappa=0,140; P< 0,001). The protein G-peroxidase presented low sensitivity and occurrence of false negatives regarding to the specific conjugate, what demonstrated not being a viable alternative to realize an immunoassay in dogs. The detection of specific antibodies in the dogs serum in this study proved the presence and a wide distribution of P. brasiliensis in the urban areas of Pelotas and Capão do Leão city and alerted to the occurrence of PCM in susceptible individuals, as well as the possibility of the disease in small animals in the region.

Activation of the alternative complement pathway in canine normal serum by Paracoccidioides brasiliensis

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis.

O fungo dimórfico Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose, uma doença granulomatosa humana. Recentemente, foi relatado o primeiro caso da doença natural em cães. O sistema complemento é um importante componente efetor da imunidade humoral contra agentes infecciosos. Portanto, o objetivo deste trabalho foi avaliar a ativação da via alternativa do complemento canina pelo P. brasiliensis. Inicialmente, foi avaliada a capacidade de eritrócitos de cobaia, coelho, carneiro, galinha e suíno ativarem a via alternativa do complemento canino. Os eritrócitos de cobaia apresentaram maior capacidade de ativar a via alternative do complemento canino. A atividade hemolítica da via alternativa (AH50) foi avaliada em 27 amostras de soro de cães saldáveis e os valores médios observados foram de 87,2 AH50/ml. Não foi observada diferença significativa ao sexo e idade. A ativação da via alternativa pelo P. brasiliensis foi maior nas amostras de soro de cães adultos quando comparada aos cães filhotes e idosos (p 0.05). Este é o primeiro relato da ativação da via alternative do complemento canino pelo fungo P. brasiliensis e sugere que pode ter um papel protetor na paracoccidioidomicose canina.

Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin b and itraconazole

In vitro tests employing microdilution to evaluate fungal susceptibility to antifungal drugs are already standardized for fermentative yeasts. However, studies on the susceptibility of dimorphic fungi such as Paracoccidioides brasiliensis employing this method are scarce. The present work introduced some modifications into antifungal susceptibility testing from the European Committee on Antimicrobial Susceptibility Testing (EUCAST), concerning broth medium and reading time, to determine minimal inhibitory concentration (MIC) of amphotericin B and itraconazole against Paracoccidioides brasiliensis. Yeast-like cells of P. brasiliensis (Pb18 strain) were tested for susceptibility to amphotericin B and itraconazole in RPMI 1640 medium, supplemented with 2 percent glucose and nitrogen source and incubated at 35ºC. The MIC of amphotericin B and itraconazole against Pb18 were respectively 0.25 µg/mL and 0.002 µg/mL. The results of minimal fungicidal concentration (MFC) showed that amphotericin B at 0.25 µg/mL or higher concentrations displayed fungicidal activity against Pb18 while itraconazole at least 0.002 µg/mL has a fungistatic effect on P. brasiliensis. In conclusion, our results showed that the method employed in the present study is reproducible and reliable for testing the susceptibility of P. brasiliensis to antifungal drugs.(AU)