SUSCEPTIBILITY OF Candida spp. ISOLATED FROM BLOOD CULTURES AS EVALUATED USING THE M27-A3 AND NEW M27-S4 APPROVED BREAKPOINTS

The high mortality rates associated with candidemia episodes and the emergence of resistance to antifungal agents necessitate the monitoring of the susceptibility of fungal isolates to antifungal treatments. The new, recently approved, species-specific clinical breakpoints (SS-CBPs)(M27-S4) for evaluating susceptibility require careful interpretation and comparison with the former proposals made using the M27-A3 breakpoints, both from CLSI. This study evaluated the susceptibility of the different species of Candida that were isolated from candidemias based on these two clinical breakpoints. Four hundred and twenty-two isolates were identified and, among them, C. parapsilosis comprised 46.68%, followed by C. albicans (35.78%), C. tropicalis (9.71%), C. glabrata (3.55%), C. lusitaniae (1.65%), C. guilliermondii (1.65%) and C. krusei (0.94%). In accordance with the M27-A3 criteria, 33 (7.81%) non-susceptible isolates were identified, of which 16 (3.79%) were resistant to antifungal agents. According to SS-CBPs, 80 (18.95%) isolates were non-susceptible, and 10 (2.36%) of these were drug resistant. When the total number of non-susceptible isolates was considered, the new SS-CBPs detected 2.4 times the number of isolates that were detected using the M27-A3 interpretative criteria. In conclusion, the detection of an elevated number of non-susceptible species has highlighted the relevance of evaluating susceptibility tests using new, species-specific clinical breakpoints (SS-CBPs), which could impact the profile of non-susceptible Candida spp. to antifungal agents that require continuous susceptibility monitoring

Atividade in vitro de plantas condimentares (Rosmarinus officinalis L., Lippia graveolens HBK e Thymus vulgaris L.) contra o calicivírus felino

<p>O calicivírus felino (FCV) é um importante patógeno de gatos que causa lesões ulcerativas orais e infecções respiratórias. O vírus tem sido utilizado como modelo experimental para avaliação de agente antivirais contra norovírus (NoVs). Nesse estudo, investigou-se a ação dos óleos essenciais de alecrim (Rosmarinus officinalis L.), orégano mexicano (Lippia graveolens HBK.) e tomilho (Thymus vulgaris L.) frente ao FCV, in vitro. A toxicidade celular foi testada pelo método de MTT e os ensaios antivirais pelo teste de redução de placas. Três protocolos foram aplicados: a) diferentes concentrações não tóxicas dos óleos essenciais (CNTOE) foram incubadas com o vírus por 1 hora antes da inoculação (ensaio virucida); b) CNTOE foram adicionadas às células CRFK e incubadas por 1 hora antes da adsorção viral (ensaio de pré-tratamento); c) CNTOE foram adicionadas às células após a inoculação do FCV e mantidas por 18 horas (ensaio de pós-tratamento). A CC50 para os óleos de alecrim, orégano mexicano e tomilho foram: 1300,21 μg mL-1; 435,92 μg mL-1 e 675,34 μg mL-1; respectivamente. O óleo essencial de tomilho apresentou índice de seletividade [IS=CC50/CI50] de 8,57 para o ensaio de pré-tratamento e 6,2 no ensaio virucida. O óleo de alecrim mostrou atividade antiviral no ensaio virucida (IS=6,54) e de pós-tratamento (IS=6,86). O orégano mexicano apresentou IS de 5,75 no ensaio virucida e 5,59 no de pós-tratamento. Conclui-se que os óleos essenciais de tomilho e alecrim apresentaram atividade frente ao FCV em diferentes momentos da infecção viral.</p><p><br />Palavras-chave: Norovírus. FCV. óleos essenciais. Citotoxicidade. MTT. ensaio de placa.</p><p> </p><p>ABSTRACT</p><p><br />In vitro activity of plants used as condiments (Rosmarinus officinalis L., Lippia graveolens HBK e Thymus vulgaris L.) against the feline calicivirus</p><p><br />The feline calicivirus (FCV) is an important pathogen of feline causing oral ulcerative lesions and respiratory disease. This virus has been used as a model to evaluate antiviral compounds against Norovirus (NoVs). In this study, the essential oils of rosemary (Rosmarinus officinalis L.), mexican oregano (Lippia graveolens HBK) and thyme (Thymus vulgaris L.) were examined for their activity towards FCV, in vitro. The cytotoxicity was determined by the MTT test and the antiviral assays were performed by the plaque reduction test. Three protocols were applied: a) different non-toxic concentrations of the essential oils (NTCEO) were incubated with the virus for 1 hour before viral inoculation (virucidal assay); b) NTCEO were added to CRFK cells and incubated for 1 hour before viral adsorption (pre-treatment assay); c) NTCEO were added to cells after virus inoculation and maintained for 18 hours (post-treatment assay). The cytotoxic concentration at 50% (CC50) for the essential oils of rosemary, mexican oregano, and thyme were: 1300.21 μg mL-1; 435.92 μg mL-1 and 675.34 μg mL-1; respectively. The essential oil of thyme showed a selectivity index (IS=CC50/CI50) of 8.57 at the cell pre-treatment assay and 6.2 at the virucidal assay. The essential oil of rosemary showed antiviral activity at the virucidal assay (IS=6.54) and, also, at the posttreatment assay (IS=6.86). The mexican oregano showed an IS of 5.75 at the virucidal assay and 5.59 at the post-treatment. Therefore, it can be concluded that the essential oils of thyme and rosemary show antiviral activity against FCV in different times of the infection.</p><p><br />Keywords: Norovirus. FCV. Essential oils. Cytotoxicity. MTT. Plaque assay.</p

SUSCEPTIBILITY OF Candida spp. ISOLATED FROM BLOOD CULTURES AS EVALUATED USING THE M27-A3 AND NEW M27-S4 APPROVED BREAKPOINTS

As elevadas taxas de mortalidade associadas com episódios de candidemia e a emergência da resistência aos antifúngicos, requerem o monitoramento da suscetibilidade de Candida spp., isoladas das candidemias, frente aos agentes antifúngicos. Os novos breakpoints, chamados “espécie-específicos,” foram recentemente aprovados (M27-S4) requerendo, pois, cuidadosa interpretação e comparações com aqueles até agora utilizados (M27-A3); ambos são propostos pelo Clinical Laboratory Standard Institute (CLSI). O presente estudo avaliou a suscetibilidade de espécies de Candida isoladas de candidemias baseando-se nestes dois breakpoints. Quatrocentos e vinte e dois isolados de Candida foram identificados e assim distribuídos: C. parapsilosis (48,68%), C. albicans (35,78%), C. tropicalis (9,71%), C. glabrata (3,55%), C. lusitaniae (1,65%), C. guilliermondii (1,65%), C. krusei (0,94%). Com base nos critérios do M27-A3, um total de 33 (7,81%) isolados foram julgados não-sensíveis, dos quais 16 (3,79%) como resistentes aos antifúngicos. De acordo com os breakpoints espécie-específicos (M27-S4) um total de 80 (18,95%) isolados foram considerados não-sensíveis, dos quais 10 (2,36%) resistentes a algum dos antifúngicos testados. Com base nos novos breakpoints espécie-específicos, o número de isolados não-sensíveis foi 2,4 vezes maior do que o número de não-sensíveis detectado pelos breakpoints do documento M27-A3. A detecção de um elevado número de isolados não-sensíveis através dos breakpoints propostos pelo M27-S4 destaca a importância dos testes de suscetibilidade, os quais trarão impactos no reconhecimento de isolados de Candida spp. não-sensíveis em episódios de candidemias, requerendo, portanto, continua avaliação.The high mortality rates associated with candidemia episodes and the emergence of resistance to antifungal agents necessitate the monitoring of the susceptibility of fungal isolates to antifungal treatments. The new, recently approved, species-specific clinical breakpoints (SS-CBPs)(M27-S4) for evaluating susceptibility require careful interpretation and comparison with the former proposals made using the M27-A3 breakpoints, both from CLSI. This study evaluated the susceptibility of the different species of Candida that were isolated from candidemias based on these two clinical breakpoints. Four hundred and twenty-two isolates were identified and, among them, C. parapsilosis comprised 46.68%, followed by C. albicans (35.78%), C. tropicalis (9.71%), C. glabrata (3.55%), C. lusitaniae (1.65%), C. guilliermondii (1.65%) and C. krusei (0.94%). In accordance with the M27-A3 criteria, 33 (7.81%) non-susceptible isolates were identified, of which 16 (3.79%) were resistant to antifungal agents. According to SS-CBPs, 80 (18.95%) isolates were non-susceptible, and 10 (2.36%) of these were drug resistant. When the total number of non-susceptible isolates was considered, the new SS-CBPs detected 2.4 times the number of isolates that were detected using the M27-A3 interpretative criteria. In conclusion, the detection of an elevated number of non-susceptible species has highlighted the relevance of evaluating susceptibility tests using new, species-specific clinical breakpoints (SS-CBPs), which could impact the profile of non-susceptible Candida spp. to antifungal agents that require continuous susceptibility monitoring

Antifungal activities of tacrolimus in combination with antifungal agents against fluconazole-susceptible and fluconazole-resistant Trichosporon asahii isolates

The antifungal activity of tacrolimus in combination with antifungal agents against different fungal species has been previously reported. Here we report the in vitro interactions between tacrolimus and amphotericin B, fluconazole, itraconazole, and caspofungin against 30 clinical isolates of both fluconazole-susceptible and fluconazole-resistant Trichosporon asahii. For these analyses, we used the broth microdilution method based on the M27-A3 technique and checkerboard microdilution method. Tacrolimus showed no activity against T. asahii strains (minimal inhibitory concentrations, MICs > 64.0 μg mL−1). However, a larger synergistic interaction was observed by the combinations tacrolimus + amphotericin B (96.67%) and tacrolimus + caspofungin (73.33%) against fluconazole-susceptible isolates. Combinations with azole antifungal agents resulted in low rates of synergism for this group (fluconazole + tacrolimus = 40% and itraconazole + tacrolimus = 10%). Antagonistic interactions were not observed. For the fluconazole-resistant T. asahii group, all tested combinations showed indifferent interactions. The synergism showed against fluconazole-susceptible T. asahii isolates suggests that the potential antifungal activity of tacrolimus deserves in vivo experimental investigation, notably, the combination of tacrolimus with amphotericin B or caspofungin. Keywords: Susceptibility, FK506, Resistance, Synergis

Atividade antiviral do extrato de própolis contra o calicivírus felino, adenovírus canino 2 e vírus da diarréia viral bovina Antiviral activity of propolis extracts against feline calicivirus, canine adenovirus 2, and bovine viral diarrhea virus

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. Neste artigo, descreve-se a atividade antiviral de dois extratos etanólicos de própolis (EP1 e EP2) frente aos vírus: calicivírus felino (FCV), adenovírus canino tipo 2 (CAV-2) e vírus da diarréia viral bovina (BVDV). Um dos extratos (EP1) foi obtido por extração etanólica de própolis obtida da região central do Estado do Rio Grande do Sul e o segundo (EP2), obtido comercialmente de uma empresa de Minas Gerais. A análise dos extratos de própolis através da cromatografia líquida de alta eficiência (CLAE) identificou a presença de flavonóides como: rutina, quercetina e ácido gálico. A atividade antiviral bem como a citotoxicidade dos extratos aos cultivos celulares foram avaliadas através do MTT [3- (4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo]. Ambos os extratos evidenciaram atividade antiviral frente ao BVDV e CAV-2 quando acrescidos ao cultivo celular anteriormente à inoculação viral. Os extratos foram menos efetivos contra o FCV em comparação aos resultados obtidos com os outros vírus, e a atividade antiviral neste caso foi observada apenas quando a própolis estava presente após a inoculação viral. O extrato obtido no laboratório (EP1) apresentou valores mais altos de índice de seletividade (IS=CC50/ CE50), quando comparado à outra amostra (EP2). Em resumo, a própolis apresentou atividade antiviral frente a três diferentes vírus, o que a torna alvo para o desenvolvimento de novos compostos naturais com atividade antiviral.<br>Propolis is a resinous substance produced by bees for which several biological activities have been attributed. In this article, the antiviral activity of two propolis extracts was tested against bovine viral diarrhea virus (BVDV), canine adenovirus type 2 (CAV-2), and feline calicivirus (FCV). One of the extracts was obtained by ethanolic extraction of propolis from the Santa Maria (RS) region, while the other was bought from a Minas Gerais industry. The high efficiency liquid cromatography (HPLC) analysis detected the presence of some flavonoids like rutin, quercetin, and gallic acid. The MTT test was applied in order to detect the citotoxicity and also the antiviral activity. Both extracts showed antiviral activity against BVDV and CAV-2 when incubated with the cell cultures before viral inoculation. The extracts were less effective against FCV comparing to the results for the other viruses and, the antiviral activity was observed only when the própolis was present after virus inoculation The extract obtained in the lab showed the highest selectivity index (SI= CC50/ EC50). Thus, propolis showed antiviral activity against three different viruses, making it a target for the development of new natural compounds with antiviral activity