Fatores microbiológicos da criptococose e sua importância na carga da doença em pacientes atendidos em um hospital de referência do Estado de São Paulo / Not available

Introdução: A meningite criptocócica causa elevada mortalidade, sobretudo em pacientes acometidos de alguma condição imunossupressora. O objetivo deste estudo foi identificar fenômenos de baixa suscetibilidade a antifúngicos e outros preditores clínicos que possam explicar falha terapêutica e recidiva da neurocriptococose Metodologia: Foram avaliados 96 casos com coleta de dados clínicos epidemiológicos e laboratoriais. Os isolados foram identificados quanto a genótipo molecular, suscetibilidade de anfotericina B (AMB) e fluconazol (FCZ) pela determinação da concentração inibitória mínima (Minimal Inhibitory Concentration, MIC), nível de heteroresistência ao FCZ (NHF) e determinação do tempo de morte frente AMB (Time-Kill, TK). Foram selecionados isolados heterorresistentes para análise quantitativa de DNA por PCR em tempo Real, expressão de bombas de efluxo por citometria de fluxo e isolados tolerantes a AMB para estudo de resistência ao estresse oxidativo. Foi realizada análise univariável e múltipla usando regressão logística para identificar preditores de óbito hospitalar e de um desfecho composto definido pelo óbito, encaminhamento para unidade de terapia intensiva ou recidiva 6 meses após alta hospitalar. Resultados: A maioria dos pacientes eram imunodeprimidos, com CD4 de 2 a 722 cel./mm3 e 96,7% eram portadores do HIV. Foram identificados 93 isolados de C. neoformans, sendo 76 do genótipo VNI e 17 VNII e 3 C. gattii, todos VGII. MIC de AMB variou de 0,012 a 0,94 mg/L e MIC de FCZ estiverem entre 0,12 e 64 mg/L. Resistência a FCZ (MIC>16mg/L) foi maior em VNI do que em VNII (p=0,03). Dentre os isolados VNI, 64,5% sofreu atividade fungicida até as 24h (TK24) de exposição à AMB e 6 cepas VNI não sofreram ação fungicida (TK>72). A maioria dos isolados VNII (64,7%) apresentou TK24. Os 3 isolados VGII sofreram atividade fungicida a partir de TK24. A maioria dos isolados VNI, VNII e todos os isolados VGII apresentaram alto NHF (>32mg/L). Diferença no NHF de acordo com os genótipos foi observada (p=0,005). No modelo múltiplo, as variáveis associadas significativamente ao óbito foram: idade em anos (OR=1,08;IC95%=1,02-1,15), contagem de leveduras no líquido cefalorraquidiano em logaritmo (LCR) (OR=1,66;IC95%=1,21-2,28) e uma variável composta por hipertensão arterial sistêmica ou diagnóstico de edema cerebral ou dilatação ventricular por tomografia (OR=35,68;IC95%=4,97-256,31). Para o desfecho composto, as variáveis associadas foram: contagem de leveduras do 1D em logaritmo (OR=1,50; IC95%=;1,20-1,86; p=<0,001), cultura de sangue positiva para Cryptococcus spp. (OR=3,30; IC95%=0,86-12,59; p=0,08) e descrição de neurotoxoplasmose (OR=18,62; IC95%=1,85-187,5; p=0,01). As associações foram consistentes em modelos de sobrevida. Conclusão: Foi possível descrever genótipos mais frequentes e identificar fatores genéticos, como aumento da expressão de genes e bombas de efluxo, relacionados à resistência aos fármacos. Nenhum dos testes de suscetibilidade esteve associado com os desfechos. Variáveis obtidas nos primeiros dias de internação mostraram utilidade para predizer o prognóstico em pacientes com meningite criptocóccica. Estes preditores podem ajudar a identificar os casos com maior potencial de óbito e que necessitam da otimização dos recursos terapêuticos.

Background: Cryptococcal meningitis causes high mortality in immunocompromised patients. The objective of this study was to identify the phenomena of low susceptibility to antifungal and other clinical predictors that may explain therapeutic failure and relapse of neurocryptococcosis Methodology: It was analyzed 96 cases with clinical and epidemiological data. The respective isolates were identified for genotype, susceptibility profile by Minimal Inhibitory Concentration (MIC), FCZ heteroresistance level (NHF), and time to death determination against 1 mg / L BMA (Time-Kill, TK). We isolated heteroresistant DNA expression analysis by real-time PCR, expression of efflux pumps by flow cytometry and, some isolates tolerant to AMB were selected to study resistance to oxidative stress. Univariable and multiple analyses using logistic regression were performed to identify predictors of in-hospital mortality and a composed outcome defined by death, referral to the intensive care unit and relapse 6 months after hospital discharge. Results: Most of the patients were immunocompromised, with CD4 range from 2 to 722 cells/mm3 and 96.7% patients HIV-positive. It was analyzed 93 strains of Cryptococcus neoformans of which 76 were genotype VNI and 17 were VNII and 3 were C. gattii, all were VGII. AMB MIC ranged from 0.012 to 0.94 mg/L and FCZ MIC were between 0.12 and 64 mg/L. Resistance to FCZ (MIC>16mg/L) was higher to VNI than VNII (p=0.03). Among the VNI strains, 64.5% had fungicidal activity up to 24h (TK24) of exposure to AMB and 6 VNI did not present this activity until 72h (TK> 72). Most VNII strains (64.7%) had TK24. The 3 VGII strains presented fungicidal activity from TK24. According to the MIC, all strains were susceptible to AMB. The majority of VNI strains (93.4%) and VNII (76.5%) and 3 VGII strains showed high NHF (>32mg/L) and it was observed statistical difference according to the genotypes VNI and VNII (p=0.005). At the multiple analysis, the variables significantly associated with the death were the age in years (OR=1.08,95%CI=1.02-1.15), the cerebrospinal fluid (CSF) yeasts count-log (OR=1.66,95%CI=1.21-2.28), and a variable composed of systemic arterial hypertension or diagnosis of cerebral edema or ventricular dilatation by tomography (OR=35.68,95%CI=4.97-256.31). At the composed outcome, the variables associated were: CSF yeasts count-log (OR=1,50; IC95%=;1,20-1,86; p=<0,001), positive blood culture for Cryptococcus spp. (OR=3,30; IC95%=0,86-12,59; p=0,08) and neurotoxoplasmosis (OR=18,62; IC95%=1,85-187,5; p=0,01). The associations were consistent at survival models. Conclusion: It was possible to describe more frequent genotypes and to identify genetic factors, such as increased gene expression and efflux pumps, related to drug resistance. The antifungal susceptibilities were not associated with the outcomes. were not associated with outcomes. Variables available in the first days of hospitalization showed utility to predict the prognosis in patients with cryptococcal meningitis. These predictors can help to identify the cases with higher potential of death and that require the optimization of the therapeutic resources.

Prevalencia de anticuerpos anti-Saccharomyces cerevisiae en pacientes celíacos / Prevalence of anti-Saccharomyces cerevisiae antibodies in celiac patients / Prevalência de anticorpos anti-Saccharomyces cerevisiae em pacientes celíacos

La enfermedad celíaca (EC) es una condición inflamatoria crónica del intestino delgado causada por intolerancia al gluten. El tratamiento consiste en la dieta libre de gluten (DLG). Los anticuerpos anti Saccharomyces cerevisiae (ASCA) están dirigidos contra la pared celular de la levadura, se asocian a enfermedades autoinmunes, y se propone la permeabilidad intestinal alterada como causa de activación de la inmunidad humoral. El objetivo del trabajo fue determinar la prevalencia de ASCA IgG e IgA en pacientes celíacos bajo tratamiento y evaluar la asociación de ASCA con el grado de adherencia a la DLG. Se analizaron 59 sueros de pacientes adultos celíacos con alta o baja adherencia a la DLG, y se determinó ASCA IgG e IgA. Se halló una prevalencia de ASCA IgG y/o IgA del 44%. Se encontró asociación entre ASCA-IgG y adherencia a DLG (OR 4,04 IC 95%: 1,32-12,38). La prevalencia de ASCA en la población celíaca estudiada es similar a la reportada en la bibliografía. La menor prevalencia de ASCA IgG en pacientes con una estricta DLG respecto de aquellos con baja adherencia, indicaría que su presencia depende del nivel de ingesta de gluten, sugiriéndolos como herramienta complementaria en el seguimiento del paciente celíaco.

Celiac disease (CD) is a chronic inflammatory condition of the small intestine caused by gluten intolerance. The treatment consists of gluten free diet (GFD). Anti Saccharomyces cerevisiae antibodies (ASCA) are directed against the cell wall of yeast, associated with autoimmune diseases, and an altered intestinal permeability is proposed as a cause of activation of humoral immunity. The objective of this work was to determine the prevalence of IgG and IgA ASCA in celiac patients under treatment and to evaluate the association of ASCA with the degree of adherence to GFD. Fifty-nine serum samples from adult celiac patients with high or low adherence to GFD were analyzed, determining IgG and IgA ASCA. A 44% prevalence of IgG and/or IgA ASCA was found. An association was discovered between IgG ASCA and GFD adherence (OR 4.04, 95% CI: 1.32-12.38). The prevalence of ASCA in the studied celiac population is similar to that reported in the literature. The lower prevalence of IgG ASCA in patients with a strict GFD compared to those with low adherence would indicate that their presence depends on the level of gluten intake, suggesting them as a complementary tool in the follow-up of the celiac patient.

A doença celíaca (DC) é uma condição inflamatória crônica do intestino delgado causada pela intolerância ao glúten. O tratamento consiste na dieta sem glúten (DSG). Os anticorpos anti Saccharomyces cerevisiae (ASCA) são dirigidos contra a parede celular da levedura, associados a doenças autoimunes, e à permeabilidade intestinal alterada como causa da ativação da imunidade humoral. O objetivo foi determinar a prevalência de ASCA IgG e IgA em pacientes celíacos em tratamento; avaliar a associação de ASCA com o grau de adesão ao DSG. Foram analisados 59 soros de pacientes celíacos adultos com alta ou baixa adesão ao DSG, determinando ASCA IgG e IgA. Foi encontrada uma prevalência de SCA IgG/ou IgA de 44%. Foi encontrada uma associação entre ASCA-IgG e a adesão ao DSG (OR 4,04 IC 95% 1,32-12,38). A prevalência de ASCA na população celíaca estudada é semelhante à relatada na literatura. A menor prevalência de ASCA IgG em pacientes com rigorosa DSG, em comparação àqueles com baixa adesão, indicaria que sua presença depende do nível de ingestão de glúten, sugerindo-os como uma ferramenta complementar no seguimento do paciente celíaco.

Frequência de Cryptococcus spp. e outras leveduras com potencial patogênico em excretas de aves silvestres em três munícipios do estado de São Paulo / Frequency of Cryptococcus spp. and other yeasts with pathogenic potential in excreta of wild birds in three municipalities of the state of São Paulo

Introdução: Nas últimas décadas, as infeções fúngicas invasivas por leveduras tornou-se um importante problema de saúde pública, dado sua incidência crescente relacionada ao aumento da população suscetível. O reconhecimento destes patógenos em aspectos como, distribuicão ambiental e caraterísticas fenotípicas, são pilares essenciais para sua vigilância e controle. Objetivo: Descrever a frequência dos agentes de criptococose e outras leveduras com potencial patogênico e comparar essa frequência em excretas de aves silvestres em três municípios do estado de São Paulo, com vistas a melhor conhecimento da distribuição desses agentes no ambiente, além de determinar o perfil de suscetibilidade in vitro a antifúngicos de uso clínico. Método: No período de 2 anos, aves silvestres foram identificadas em áreas de circulação de 3 municípios de São Paulo (Praia Grande, Santos e Rio Claro) e submetidas à coleta de excretas para isolamento de leveduras com potencial patogênico. Análise microscópica e macroscópica para classificação presuntiva de gênero foram realizados em todas as colônias de leveduras obtidas das amostras de excretas. A suscetibilidade dos isolados de leveduras aos antífúngicos: fluconazol, voriconazol e anfotericina B foi determinada segundo método de referência europeu (AFST-EUCAST). Análise de dados: Foi utilizada a regressão de Poisson com a opção robusta para estimar razões de prevalência e identificar variáveis associadas com os principais isolados identificados, com opção de cluster para agrupar os isolados por excreta. Foi avaliado o nível de concordância entre os dois métodos de identificação (fenotípico e MALDI-TOF), utilizando o coeficiente Kappa. Adicionalmente, foi estimada a correlação entre os MIC´s dos fármacos estudados no total de espécies identificadas, utilizando o coeficiente de correlação de Spearman. Resultados: Das 294 excretas coletadas, 42,2 por cento continham leveduras, incluindo espécies de Candida 62 por cento , seguido por Rhodotorula 16,4 por cento , Cryptococcus 10,4 por cento , Trichosporon 6,6 por cento e Pichia 2,7 por cento . Muitas espécies, verificadas em alta frequência, tem forte potêncial de causar infecção invasiva, como: C. parapsilosis stricto sensu, C. tropicalis, Clavispora lusitaniae, C. krusei, C. orthopsilosis, C. glabrata, C. laurenti, C. albicans, C. metapsilosis, C. nivariensis e Meyerozyma guilliermondii. A resistência ao fluconazol, voriconazol e anfotericina B ocorreu nesses isolados, sendo documentada uma forte correlação entre a susceptibilidade, principalmente entre os azois (fluconazol e voriconazol), no entanto, a correlação mesmo sendo menor também foi significativa entre esses fármacos e a anfotericina. De 13 espécies de aves silvestres dispersoras de leveduras, as de maior frequência foram: Sula leucogaster 26,2 por cento , Turdus leucomelas 17 por cento , Larus dominicanus 15 por cento , Thalasseus maximus 11,2 por cento , Thalasseus acuflavidus 5,4 por cento , Tangara sayaca 4,4 por cento , Turdus amaurochalinus 3,7 por cento , Sterna hirundinacea e Pitangus sulphuratus 2,7 por cento . Os gêneros identificados apresentaram associações entre local, estação do ano e espécies de aves. Conclusões: Dentre as principais espécies de aves estudadas, 3 eram de hábitos migratórios (Thalasseus maximus, Thalasseus acuflavidus e Sterna hirundinacea) o que permite inferir dispersão interamericana de leveduras patogênicas. Diversas espécies resistentes a antifúngicos foram descritas, pela primeira vez, em excretas de aves silvestres conferindo a este estudo o valor de contribuir para o conhecimento da epidemiologia das infecções fúngicas por leveduras

Background: In recent decades, invasive yeast fungal infections have become an important public health problem, due to their increasing incidence related to the increase in the susceptible population. The recognition of these pathogens in aspects such as environmental distribution and phenotypic characteristics are essential pillars for their surveillance and control. Objective: To describe the frequency of cryptococcosis agents and other yeasts with pathogenic potential and to compare this frequency in excreta of wild birds in three municipalities of the state of São Paulo, with a view to a better knowledge of the distribution of these agents in the environment, in addition to determining the profile of in vitro susceptibility to antifungals for clinical use. Method: During two years, wild birds were identified in circulation areas of three municipalities of São Paulo (Praia Grande, Santos and Rio Claro) and were screened for yeasts with pathogenic potential. Microscopic and macroscopic analysis for presumptive genus classification were performed in all yeast colonies obtained from excreta samples. Susceptibility of yeast isolates to antifungals: fluconazole, amphotericin B and voriconazole was determined according to the European reference method (AFST-EUCAST). Data analysis: Poisson regression was used with the robust option to estimate prevalence ratios and to identify variables associated with the main isolates identified, with option of cluster to group the isolates by excreta. The level of agreement between the two identification methods (phenotype and MALDI-TOF) was evaluated using the Kappa coefficient. Additionally, the correlation was estimated between MICs of the drugs studied in total of species identified, using Spearman\'s correlation coefficients. Results: Of the 294 excreta collected, half contained yeasts, including Candida species (62 per cent ), followed by Rhodotorula (16.4 per cent ), Cryptococcus (10.4 per cent ), Trichosporon (6.3 per cent ) and Pichia (2.7 per cent ). Many species, verified at high frequency, have a strong potential to cause invasive infection, such as: C. parapsilosis stricto sensu, C. tropicalis, Clavispora lusitaniae, C. krusei, C. orthopsilosis, C. glabrata, C. laurentii, C. albicans, C. metapsilosis, C. nivariensis and Meyerozyma guilliermondii. Resistance to fluconazole, voriconazole and amphotericin B occurred in these isolates and a strong correlation was reported between susceptibility, mainly between azole (fluconazole and voriconazole), however, the correlation, even though it was lower, was also significant between these drugs and amphotericin. From 13 species of wild birds dispersing yeasts, the ones with the highest frequency were: Sula leucogaster 26,2 per cent , Turdus leucomelas 17 per cent , Larus dominicanus 15 per cent , Thalasseus maximus 11,2 per cent , Thalasseus acuflavidus 5,4 per cent , Tangara sayaca 4,4 per cent , Turdus amaurochalinus 3,7 per cent , Sterna hirundinacea e Pitangus sulphuratus 2,7 per cent . The identified genera presented associations between site, season of the year and species of birds. Conclusion: Among the main species of birds studied, 3 were of migratory habits (Thalasseus maximus, Thalasseus acuflavidus and Sterna hirundinacea), which allows inferring the inter - American dispersion of pathogenic yeasts. Several species resistant to antifungal were described for the first time in excreta of wild birds, conferring to this study the value of contributing to the knowledge of the epidemiology of fungal infections by yeasts

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.

Serological monitoring of antibodies for an early diagnosis ofaspergillosis in captive penguins / Monitoramento sorológico com detecção de anticorpos para diagnóstico precoce da aspergilose em pinguins em cativeiro

This study aimed to evaluate the efficacy of detection of anti-Aspergillus fumigatus antibodies in captive penguins by double radial agar gel immunodiffusion (AGID) for the aspergillosis diagnosis. We included 134 Magellanic penguins (Spheniscus magellanicus) in rehabilitation at the Center for Recovery of Marine Animals (CRAM / FURG). All of them were monitored by AGID weekly until its final destination (death or release), totalizing 660 serum samples studied. All animals were clinically accompanied and post-mortem examinations was performed in penguins that died during the studied period. A total of 28% (37/134) of the penguins died, 89.2% (33/37) due to aspergillosis, 11% (4/37) by other causes and 97 were released. From the 33 animals with proven aspergillosis, 21 presented anti- A. fumigatus antibodies by AGID, being the average interval between death and positive AGID 16.4 days. Twelve animals with negative serology died of aspergillosis. The sensitivity and specificity rates were 63.6% and 95% respectively, and the positive and negative predictive values were 80.7% and 88.9% respectively. These data demonstrate that the serological monitoring for detection of antibodies by AGID can be an important tool for the diagnosis of aspergillosis in penguins.

Este estudo teve como objetivo avaliar a eficácia da detecção de anticorpos anti- Aspergillus fumigatus em pinguins em cativeiro por imunodifusão radial dupla em gel de ágar (IDGA) para diagnóstico da aspergilose. Foram incluídos 134 pingüins de Magalhães (Spheniscus magellanicus) em reabilitação no Centro de Recuperação de Animais Marinhos (CRAM/FURG), que foram monitoradas por IDGA, semanalmente, até o seu destino final (morte ou de liberação), totalizando 660 amostras de soro estudadas. Todos os animais foram acompanhados clinicamente e exames post mortem foram realizados em pingüins que vieram a óbito durante o período de estudo. Um total de 28% (37/134) dos pinguins foram a óbito, 89,2% (33/37) de aspergilose, 11% (4/37) de outras causas, e 97 foram liberados. A partir dos 33 animais com aspergilose comprovada, 21 apresentaram anticorpos anti- A. fumigatus por IDGA, sendo o intervalo médio entre a morte e IDGA positivas 16,4 dias. Doze animais com sorologia negativa vieram a óbito por aspergilose. As taxas de sensibilidade e especificidade foram de 63,6% e 95%, respectivamente, e os valores preditivos positivos e negativos foram de 80,7% e 88,9 %, respectivamente. Estes dados demonstram que o monitoramento sorológico para detecção de anticorpos por IDGA pode ser uma ferramenta importante no diagnóstico de aspergilose em pinguins.

Seroprevalence of Encephalitozoon cuniculi and Toxoplasma gondii in domestic rabbits (Oryctolagus cuniculus) in China

The breeding of domestic rabbits (Oryctolagus cuniculus) for human consumption has a long tradition in China. Infections that can affect the production of meat or even be transmitted from animals to humans are important to monitor, especially for public health reasons as well as for their impact on animal health. Thus, a total of 1,132 domestic rabbit sera from 4 regions in China were collected for serological screening for Encephalitozoon cuniculi and for Toxoplasma gondii by ELISA and modified agglutination test (MAT), respectively. Antibodies to E. cuniculi were detected in 248/1,132 (21.9%) sera tested while antibodies against T. gondii revealed a seroprevalence of 51/1,132 (4.5%). We believe that the present results are of epidemiological implications and public health importance due to the acknowledged susceptibility of humans to E. cuniculi and T. gondii infections. Therefore, routine screening tests of domestic rabbits are proposed considering the zoonotic potential of these parasites.

Immunological assays employed for the elucidation of an histoplasmosis outbreak in São Paulo, SP

Several reports showed outbreaks of histoplasmosis acquired while bat-inhabited caves were visited by tourists, miners or researchers. We evaluated the performance of double immunodifusion (DI) and immunoblotting (IB) assays, employed for the histoplasmosis outbreak elucidation occurred in Vale do Paraíba, São Paulo. The existence of epidemiologic link, four patients with clinical signs suggestive of histoplasmosis and mycological confirmation has made that all 35 individuals involved to the cave visit were subjected to serological evaluation. By DI, we observed reactivity against H. capsulatum antigen in a single serum examined nearly 20 days after exposure to fungal propagules. On the other hand, IB showed reactivity against H and M fractions in 50% of samples evaluated. The analysis of the second sample batch, collected two months after the exposure showed that 96.7% were reactive by DI with antibodies titers ranging from 1 to 16 and 100% of reactivity against H and M fractions, by IB, suggesting an acute infection. The analysis of the overall agreement between the methods showed to be reasonable (κ = 0.37). This study confirms the importance and efficacy of more sensitive methodologies, such as IB assay, to early elucidation of disease, especially in cases of patients without mycological information.

Double-antigen sandwich ELISA for detecting Aspergillus fumigatus anti-Afmp1cr and Afmp2cr antibodies / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish two double-antigen sandwich ELISA systems to detect anti-Afmp1cr and Afmp2cr antibodies of Aspergillus fumigatus.</p><p><b>METHODS</b>Recombinant Afmp1cr and Afmp2cr proteins of A.fumigatus expressed in Pichia pastoris were obtained. Double-antigen sandwich ELISA systems for detecting specific anti-Afmp1cr and anti-Afmp2cr antibodies were developed after chessboard titrating to determine the appropriate concentrations of the recombinant proteins and HRP-labeled proteins. The sensitivity of the assay was evaluated using serum samples of rabbits immunized with Afmp1cr and Afmp2cr. The specificity of the assay was evaluated by detecting serum samples from healthy donors and patients with other pathogenic fungal and baterial infections. The performance of the two ELISA kits was furthered evaluated using serum samples from patients with suspected Aspergillus infection.</p><p><b>RESULTS</b>The established ELISA kits were capable of detecting anti-Afmp1cr and anti-Afmp2cr antibodies in immunized rabbit serum at the maximum dilutions of 800 and 3200, respectively. No cross-reactivity was observed in detecting serum from patients with other pathogenic fungal or bactetial infections. Both of the two kits yielded positive results in sera from two established Aspergillus-infected cases and a suspected case.</p><p><b>CONCLUSIONS</b>Two antibody-capture ELISA kits were developed for the laboratory diagnosis of A.fumigatus infection and can be potentially useful in the clinical diagnosis of Aspergillosis infections.</p>

Seroprevalence of Encephalitozoon cuniculi in Pet Rabbits in Korea

Encephalitozoon cuniculi is a microsporidian parasite commonly found in rabbits that can infect humans, causing encephalitozoonosis. The prevalence of encephalitozoonosis is not well documented, even when many clinics suspect pet rabbits as being highly infected. This study investigated the seropositivity of E. cuniculi using ELISA. The examination of 186 rabbits using ELISA showed that 22.6% (42/186) were seropositive against E. cuniculi. In analysis with healthy status, all 42 seropositive sera were collected from clinically normal rabbits. Moreover, the gender and age of pet rabbits did not have anysignificant effect on E. cuniculi infection. To the best of our knowledge, this is the first report to describe the seroprevalence of E. cuniculi in pet rabbits and suggests that pet rabbits could act as an important reservoir of encephalitozoonosis for both pet animals and humans in Korea.

A double-antigen sandwich ELISA for detecting Penicillium marneffei Mp1p-specific antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an immunological method for detecting antibodies of Penicillium marneffei.</p><p><b>METHODS</b>The recombinant Mp1p protein of Penicillium marneffei was expressed in Pichia pastoris and labeled with HRP (Mp1p-HRP) with a modified sodium periodate method. A double-antigen sandwich enzyme-linked immunosorbant assay (ELISA) was established by determining the optimal coating concentration of Mp1p protein and the concentration of the detecting protein Mp1p-HRP. The sensitivity and specificity of the assay was evaluated by detecting Mp1p antibodies in 100 serum samples from healthy donors, 15 samples from culture-confirmed penicilliosis patients, and 21 samples from patients with culture-confirmed other fungal infections.</p><p><b>RESULTS</b>A double-antigen sandwich ELISA was successfully established for detecting Mp1p-specific antibody. The specificity of the assay was 100% (121/121) for detecting Mp1p-specific antibody in the sera from healthy donors and patients with other fungal infection. The detection results of the 15 serum samples from patients with culture-confirmed penicilliosis showed positivity for Mp1p antibody in 2 samples and Mp1p antigen positivity in 12 samples; combining the detection results of Mp1p antigen and antibody obviously increased the diagnostic sensitivity to 93.3% (14/15).</p><p><b>CONCLUSION</b>The double-antigen sandwich ELISA shows a high specificity in detecting Mp1p-specific antibody, and simultaneous detection of Mp1p antigen and antibody can increase the diagnostic sensitivity for penicilliosis.</p>

Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10(9)TCID50/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-gamma and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.

Development of a monoclonal antibody against deoxynivalenol for magnetic nanoparticle-based extraction and an enzyme-linked immunosorbent assay

Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 microg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 microg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.

Atividade antifúngica e alterações morfológicas induzidas pelo óleo essencial de Cinnamomum cassia frente cepas de Candida albicans isoladas de pacientes HIV positivos / Antifungal activity and morphological alterations induced by Cinnamomum cassia essential oil against Candida albicans strains isolated from HIV-positive patients

Objetivo: Avaliar a atividade antifúngica, o efeito sobre a cinética de morte microbiana e as alterações morfológicas do óleo essencial de Cinnamomum cassia (canela) sobre cepas de Candida albicans isoladas de pacientes HIV positivos e cepa padrão (ATCC 76485). Método: Suspensões fúngicas (106UFC/mL) foram preparadas a partir de amostras clínicas (n=15) e padrão (n=1) de C. albicans. Emulsões do óleo essencial foram avaliadas em concentrações que variaram entre 1024µg/mL e 4µg/mL. A ação antifúngica foi determinada pela Concentração Inibitória Mínima (CIM), por meio da técnica da microdiluição. Realizou-se o ensaio de cinética sobre a morte das leveduras (tempos 0, 30, 60, 180 minutos e 24h), bem como a avaliação da interferência do óleo essencial sobre a micromorfologia das cepas. O miconazol (50 µg/mL) foi utilizado como controle e a análise estatística se deu pelos testes Kruska-Wallis e Dunn (p menor que 0,05). Resultados: A CIM variou entre 64 e 128 µg/mL, frente às cepas testadas. Para o teste de cinética, verificou-se ação antifúngica de C. cassia nas concentrações CIM, 2xCIM e 4xCIM, em todos os tempos analisados. O controle de crescimento foi estatisticamente diferente do miconazol e do óleo essencial (p menor que 0,01), não sendo observada diferença estatística entre o efeito do miconazol e do produto natural (p maior que 0,05). Alterações na micromorforlogia das cepas (ausência de pseudohifas e clamidoconídeos) foram verificadas na CIM. Conclusão: O óleo essencial de C. cassia, semelhante ao miconazol, apresentou atividade antifúngica e efeito sobre a cinética de morte microbiana. Os produtos avaliados provocaram alterações sobre a micromorfologia das cepas testadas.

Objective: To evaluate the antifungal activity, the effect on microbial death kinetics and the morphological alterations caused by Cinnamomum cassia (canela) essential oil against Candida albicans strains isolated from HIV-positive patients and a reference strain (ATCC 76485). Method: Fungal suspensions (106CFU/mL) were prepared from clinical (n=15) and reference (n=1) C. albicans samples. Essential oil emulsions were evaluated at concentrations between 1024 µg/mL and 4 µg/mL. The antifungal action was determined by the minimal inhibitory concentration (MIC) using the microdiluition technique. The kinetics assay for yeast death was performed (times: 0, 30, 60, 180 minutes and 24 h) as well as evaluation of the interference of the essential oil on the micromorphology of the strains. Miconazol (50 µg/mL) was used as a control. The statistical analysis was performed by the Kruskal-Wallis and Dunn tests (p less than 0.05). Results: The MIC varied from 64 to 128 µg/mL for the tested strains. The kinetics assay revealed antifungal action of the C. cassia at the concentrations MIC, 2xMIC and 4xMIC, at all analyzed times. Growth control was significantly different for miconazol and for the essential oil (p less than 0.01), with no statistically significant difference between the effect of miconazol and the natural product (p greater than 0.05). Alterations in the micromorphology of the strains (absence de pseudohifas and chlamydoconidias) were verified in the MIC. Conclusion: In the same way as miconazol, C. cassia essential oil presented antifungal activity and effect on microbial death kinetics. The tested products caused alterations on the micromorphology of the tested strains.

Effect of serum sample inactivation on the performance of latex agglutination test for paracoccidioidomycosis serodiagnosis

Paracoccidioidomycosis is diagnosed from the direct observation of the causative agent, but serology can facilitate and decrease the time required for diagnosis. The objective of this study was to determine the influence of serum sample inactivation on the performance of the latex agglutination test (LAT) for detecting antibodies against Paracoccidioides brasiliensis. The sensitivity of LAT from inactivated or non-inactivated samples was 73% and 83%, respectively and the LAT selectivity was 79% and 90%, respectively. The LAT evaluated here was no more specific than the double-immunodiffusion assay. We suggest the investigation of other methods for improving the LAT, such as the use of deglycosylated antigen.

Carbohydrate-rich high-molecular-mass antigens are strongly recognized during experimental Histoplasma capsulatum infection / Antígenos de alta massa molecular ricos em carboidratos são fortemente reconhecidos durante infecção experimental por Histoplasma capsulatum

INTRODUCTION: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction. METHODS: Mice were infected with 2.2x10(4) H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection. RESULTS: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components. CONCLUSIONS: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

INTRODUÇÃO: Durante a histoplasmose, os antígenos solúveis de Histoplasma capsulatum (CFAg) podem ser liberados naturalmente pelas células leveduriformes. Considerando que os CFAg constituem um alvo específico durante a infecção, no presente estudo nós investigamos a liberação de CFAg durante a histoplasmose murina experimental, e avaliamos a resposta imune humoral do hospedeiro contra antígenos de alta MM (hMMAg; >150 kDa), altamente imunogênicos. MÉTODOS: Camundongos foram infectados com 2.2x10(4) leveduras de H. capsulatum, cepa IMT/HC128. Os níveis de CFAg solúveis, IgG anti-CFAg, IgG anti-hMMAg, e também de imunocomplexos circulantes (CIC) IgG-hMMAgs foram determinados por ELISA nos dias 0, 7, 14 e 28 após a infecção. RESULTADOS: Após a infecção por H. capsulatum, observamos um aumento progessivo de CFAg circulantes, IgG anti-CFAg, IgG anti-hMMAg, e também de CIC IgG-hMMAgs. Os hMMAg apresentaram alta porcentagem de carboidratos e, pelo menos, dois componentes imunogênicos. CONCLUSÕES: Mostramos pela primeira vez que os hMMAg de H. capsulatum cepa IMT/HC128 induzem resposta imune humoral e levam à formação de CIC durante a histoplasmose experimental.

Métodos diagnósticos en candidemia: una revisión sistemática de la literatura con meta-análisis / Diagnostic methods in candidemia: a systematic review of literature with meta-analysis

Candidemia is a disease with high morbidity and mortality especially in critical care patients. Early diagnosis enables early treatment. Objectives: To conduct a systematic review of the literature in order to establish the best laboratory tests for the diagnosis of candidemia in critical patients. Materials and Methods: We conducted a systematic review of available literature in PubMed. Serological studies were subjected to meta-analysis in metadisk-Beta 1.1.1. Results: 4 studies of 1286 reviewed were included. Three were about serological tests and one about molecular testing (RT-PCR). The sensitivity and specificity for RT-PCR, antibody testing and antigen and antibody tests were 87 percent and 100 percent, 47.5 percent and 82.6 percent, 96 percent and 81 percent, respectively. Diagnostic Odds Ratio of antigenemia was 1.51 (95 percent CI = 0,032-70,964, p = 0.001). Conclusions: RT-PCR has better diagnostic performance, measuring antigenemia plus antibodies improves sensitivity, specificity, LR + and LR-- . There is insufficient evidence to support this.

La candidemia es una patología con alta morbilidad y mortalidad, especialmente en los pacientes sometidos a servicios de cuidado crítico. El diagnóstico precoz permite realizar tratamiento temprano. Objetivos: Realizar una revisión sistemática de la literatura para establecer cuáles son las pruebas de laboratorio con mejor rendimiento diagnóstico y operativo para el diagnóstico de candidemia en cuidado intensivo. Materiales y Métodos: Se realizó una revisión sistemática de la literatura disponible en PubMed, se sometieron a meta-análisis estudios de pruebas serológicas en MetaDisc-Beta 1.1.1. Resultados: Se incluyeron 4 estudios de 1.286 revisados, 3 de pruebas serológicas y 1 de RPC-RT. La sensibilidad y especificidad fue de 87 y 100 por ciento para RPC-RT, 47,5 y 82,6 por ciento para pruebas de anticuerpos, 96 y 81 por ciento para pruebas de antígeno y anticuerpo. La ORD de antigenemia 1,51(IC95 por ciento = 0,03270,964; p = 0,001). Conclusiones: RPC-RT tiene mejor rendimiento diagnóstico, la medición de antigenemia más anticuerpos mejora la sensibilidad, especificidad, LR+ y LR-. No hay suficiente evidencia que soporte esto.

Characterization of specific monoclonal antibodies to Aspergillus conidia by flow cytometry / 南方医科大学学报

<p><b>OBJECTIVE</b>To characterize the specific monoclonal antibodies to Aspergillus conidia.</p><p><b>METHODS</b>Flow cytometry was used to examine the reactivity of the specific monoclonal antibodies to Aspergillus conidia.</p><p><b>RESULTS</b>Both the monoclonal antibodies MA3 and Con2 showed specific reactivity to Aspergillus conidia suspensions. MA3 was capable of binding to the conidia of A.fumigatus, A.flavus, A.niger and A.terreus, while Con2 was reactive only to the conidia of A.fumigatus.</p><p><b>CONCLUSION</b>Two specific monoclonal antibodies (MA3 and Con2) to Aspergillus conidia have been obtained.</p>

Serum Anti-Saccharomyces Cerevisiae Antibodies in Greek Patients with Behcet's Disease

We tested 59 Greek patients with Behcet's Disease (BD) for serum anti-Saccharomyces cerevisiae antibodies. No increase of these antibodies was detected in the cases compared to 55 healthy unrelated blood donors from the same population. This finding is in contrast with the correlation between Saccharomyces cerevisiae antibodies and BD as reported in other populations. It seems that environmental factors may contribute to disease expression in different populations, producing different effects according to the individual's genetic predisposition. Saccharomyces cerevisiae antibodies do not seem to be of any significance in the Greek population.

Serological diagnosis of paracoccidioidomycosis in HIV-coinfected patients

Paracoccidioidomycosis should be differentiated from other opportunistic diseases in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients who live in Latin America. Laboratory investigation can begin with serological tests, which are rapid and efficient. In the present study, double immunodiffusion (DID), counterimmunoelectrophoresis (CIEP) and an enzyme linked immunosorbent assay (ELISA) tests were assessed for the detection of anti-Paracoccidioides brasiliensis antibodies in 40 patients coinfected with HIV. The results were compared to those obtained for 75 non-HIV-infected patients with endemic paracoccidioidomycosis. Anti-P. brasiliensis antibodies were detected in 65 percent (DID), 79 percent (CIEP) and 95 percent (ELISA) of the patients with HIV/AIDS, significantly lower rates than those detected in cases of endemic paracoccidioidomycosis, which were 89 percent, 99 percent and 100 percent, respectively. The reactive sera of HIV-infected patients also showed lower anti-P. brasiliensis antibody titres than those of non-HIV-infected patients. Despite the lower intensity of the specific humoral response, serological tests are useful for the diagnosis of opportunistic paracoccidioidomycosis in the HIV/AIDS population. We suggest optimization of the laboratory diagnosis by combining the ELISA test with CIEP or DID.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis / Fração de alta massa molecular em isolados clínicos de Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

INTRODUÇÃO: Diferentes níveis sorológicos de IgG/IgE contra a fração de alta massa molecular (hMM) (~366kDa) de Paracoccidioides brasiliensis têm sido encontrados na PCM aguda e crônica. Considerando a inexistência de investigação sobre esta fração em isolados clínicos de P. brasiliensis, o objetivo deste estudo foi investigar a presença da fração hMM (~366kDa) no preparado livre de células (CFA) de P. brasiliensis obtidos de isolados clínicos. MÉTODOS: CFA de 10 isolados e de cepa de referência (Pb18) foram submetidas à eletroforese em gel de SDS-poliacrilamida (SDS-PAGE) seguida de captura de imagem e análise por densitometria. Adicionalmente, CFA de 20 isolados e de Pb18 foram analisados por ELISA captura (cELISA) utilizando anticorpos policlonal (polAb) ou monoclonal (mAb) para fração hMM. RESULTADOS: A presença do componente de hMM foi observada em todas as amostras analisadas por SDS-PAGE/densitometria e por cELISA. Adicionalmente, o teste de correlação de Pearson demonstrou forte relação entre os níveis de fração hMM usando pAb e mAb (R = 0.853) no cELISA. CONCLUSÕES: Conclui-se que a fração hMM está presente em todos os isolados clínicos de P. brasiliensis analisados e no isolado referencial, sugerindo a possibilidade dos mesmos serem utilizados como fonte desta fração antigênica. Este trabalho também introduz pela primeira vez o método de cELISA para detecção da fração hMM de P. brasiliensis, sugerindo que detecção utilizando anticorpos polAb ou mAb é viável e essa metodologia poderá ser útil para investigação futura desta fração solúvel (~366kDa) em soros de pacientes com PCM.