The objective of the work reported in this paper is one of a two-fold nature. The first objective is to develop a disk-plate sensitivity test that is more rapid than that of existing methods. The second requisite is that the materials, techniques, interpretation, and reporting of results be the sane as those required for the disk-plate method described in the Difco Manual.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / In recent years there has been a significant increase in the incidence of fungal infections caused by Candida species. Although C. albicans be considered the principal representing of the genus, other species have been gaining prominence. C. tropicalis, for example, has been associated with serious invasive cadidiases, being the first or second type of non-Candida albicans Candida most commonly isolated in humans with candidemia and candiduria and is frequently isolated from healthy animals and animals with candidiasis. To establish infection, C. tropicalis expresses many virulence factors such as the secretion of enzymes phospholipases and proteases, biofilm production, among others. This study aimed to evaluate the in vitro antifungal susceptibility profile and production of virulence factors in strains of C. tropicalis (n=100) isolated from several animal species. The strains were subjected to in vitro susceptibility testing by broth microdilution test, M27-A3 protocol, standardized by the Clinical and Laboratory Standards Institute against amphotericin B, itraconazole and fluconazole. We also evaluated the virulence attributes, such as proteases and phospholipases production and biofilm formation. Regarding the susceptibility of C. tropicalis strains, 38% were resistant to itraconazole, 40% were resistant to fluconazole and 34% were resistant to both azoles. None of the strains were resistant to amphotericin B. Regarding the production of proteases, 84% of the strains secreted these enzymes in the medium with pH 5.0, whereas only 40% of the strains were active at pH 3.5. Only 8% of the strains produced phospholipases. The strains showed different pattern in biofilm production, which 63,2% were strong producers, 17,6% were moderate producers, and 13,3% were weak producers. In sumary, the C. tropicalis strains isolated from animals showed high rate of resistance to azoles and expressed important virulence factors, indicating a potential threat to human and animal health. / Nos Ãltimos anos houve um aumento significativo na incidÃncia de infecÃÃes fÃngicas causadas por leveduras do gÃnero Candida. Apesar de C. albicans ser considerada a principal representante do gÃnero, outras espÃcies vÃm ganhando destaque. C. tropicalis, por exemplo, tem sido associada à cadidÃases invasivas graves, sendo a primeira ou segunda espÃcie de Candida nÃo-albicans mais comumente isolada em candidemia e candidÃria em humanos, alÃm de ser frequentemente isolada da microbiota de animais saudÃveis e com candidÃase. Para estabelecer a infecÃÃo, C. tropicalis expressa diversos fatores de virulÃncia, como a secreÃÃo de enzimas protease e fosfolipase, a produÃÃo de biofilme, dentre outros. O presente trabalho buscou avaliar o perfil de sensibilidade antifÃngica in vitro e produÃÃo de fatores de virulÃncia de cepas de C. tropicalis (n=100) isoladas de diferentes espÃcies animais. As cepas foram submetidas a teste de sensibilidade in vitro por meio do mÃtodo de microdiluiÃÃo em caldo, protocolo M27-A3, padronizado pelo Clinical and Laboratory Standards Institute, frente anfotericina B, itraconazol e fluconazol. Foram avaliados ainda os atributos de virulÃncia: produÃÃo de enzimas proteases e fosfolipases e produÃÃo de biofilme. Quanto ao perfil de sensibilidade das cepas de C. tropicalis, 38% foram resistentes a itraconazol, 40% resistentes a fluconazol e 34% foram resistentes a ambos os derivados azÃlicos. Nenhuma cepa apresentou resistÃncia a anfotericina B. Quanto a produÃÃo de proteases, 84% das cepas secretaram estas enzimas em meio com pH 5,0, enquanto somente 40% das cepas foram ativas em pH 3,5. Somente 8% das cepas produziram fosfolipases. As cepas apresentaram padrÃo diferenciado na produÃÃo de biofilme, em que 63,2% foram consideradas fortes produtoras, 17,6% foram consideradas moderadas produtoras e 13,3% foram consideradas fracas produtoras. Em suma, os isolados de C. tropicalis provenientes de animais apresentaram resistÃncia a derivados azÃlicos e expressaram fatores de virulÃncia importantes, indicando potencial risco à saÃde humana e animal.

Orientador: Angelica Zaninelli Schreiber, Maria Luiza Moretti / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T13:48:27Z (GMT). No. of bitstreams: 1 Loyola_AnaBeatrizAlkmimTeixeira_D.pdf: 5844968 bytes, checksum: 4d27c47d59746710e359ef7d4b61f1b7 (MD5) Previous issue date: 2005 / Resumo: Os fungos filamentosos do gênero Aspergillus são fungos hialinos, saprófitas, encontrados no solo e na vegetação em decomposição e têm sido, crescentemente, relatados em casos de infecções sistêmicas ou disseminadas. A patogenicidade destes fungos oportunistas está na alta concentração dos microconídios no ar, facilitando a sua inalação. Infecções por Fusarium spp são altamente resistentes à terapia disponível, com prognostico desfavorável e taxa de mortalidade em torno de 57% dos casos. Vários relatos têm descrito a tolerância in vitro, ou resistência, de espécies de Aspergillus aos agentes antifúngicos após falha terapêutica com anfotericina B. A infecção por Aspergillus sp em pacientes imunossuprimidos apresenta taxa de mortalidade em torno de 86%, independente do tratamento antifúngico. Assim, testes de suscetibilidade a antifúngicos são de estrema importância. O teste de microdiluição, utilizando suspensão de conídios, foi recentemente padronizado pelo CLSI. No entanto, em regra, as infecções causadas por fungos filamentosos são caracterizadas pela presença de hifas no tecido infectado, sendo portanto, a realização do teste de suscetibilidade com hifas, de grande interesse clinico. A concentração inibitória mínima (CIM) de vários agentes antifúngicos pode ser determinada pela observação da inibição de crescimento de um hifa isolada. Há poucos relatos na literatura sobre a medição automatizada do crescimento de hifas de Aspergillus sp. e nenhum relato para hifas de Fusarium sp. pelo sistema 'BioCell-tracer POT. MARCA REGISTRADA¿... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Aspergillus and Fusarium species are hyaline moulds that are usually found in the soil and plants. These organisms have emerged as a cause of disseminated invasive disease. It has been suggested that airborne conidia might invade the respiratory tract with primary lung or sinus localisation Fusarium species are largely resistant to available chemotherapy and are correlated with poor prognosis and lethal outcome in 57% of the cases. Several reports have described in vitro tolerance or resistance of Aspergillus spp to antifungal infection in immunossupressed patients shows mortality around 86%, independente of antifungal treatment. Antifungal susceptibility testing of filamentous fungi has therefore become more importante. Since most infections caused by filamentous fungi are characterized by the presence of hyphal elements in the tissue, the monitoring of hyphal susceptibility might be of clinical interest. The MICs of several antifungal agents cam be determined based on the growth rate of a single hypha. There are only few reports for automatic growth evaluation for Aspergillus spp and no reports for Fusarium spp using the 'BioCell-tracer POT. MARCA REGISTRADA¿ system. In this work, we selected four clinical isolates of Aspergillus and four clinical isolates of Susarium. For all strains we prodeeded microdilution and hypha susceptibility antifungal evaluation with amphotericin B and itraconazole...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Não consta resumo na publicação. / Abstract not available.

Background Customer satisfaction is important for clinical microbiology laboratories and the most important service aspect is the reliability of responses. One important indicator of the quality of care is turnaround time for a sample referred to a laboratory. Aim This study describes and evaluates an improvement of the blood culture process and evaluates the staff’s experiences of the changes brought by the improvement project. Methods The blood culture process during evenings and nights was re-designed in a cooperation project between the laboratories of clinical microbiology and clinical chemistry in a mid-size Swedish county council. Typing with matrix-assisted laser desorption/ionization time of flight (MALDI ToF) and rapid antibiotic susceptibility testing were also introduced. To describe staff experi-ences semi-structured interviews were performed with twelve of the staff involved. Results The time from sampling to susceptibility testing and typing, for patients with cefotaxime resistant enterobacteriaceae, was before the improvement project on average 55 hours compared to 43 hours after closure of the project. In the qualitative content analysis four categories were found which represented the experience of the staff: patient focus, changed knowledge, cooperation and driving forces. Discussion The study of the implementation of the improvement showed that laboratory staff could handle the change well. The change from traditional biochemical typing, used for over 50 years, to MALDI ToF is indeed a paradigm shift. Nevertheless, nobody was disappointed over the fact that some of the fundamental previous microbiological laboratory work routines were laid to rest.

The increasing infection of Cryptococcus species and Tricosporon asahii emerged in clinical patients who were immunocompromised. They usually induce lung, skin, brain and systemic infection. Morbidity and mortality of immunocompromised patients are higher than normal healthy people. Cryptococcus neoformans var. grubii ¡]serotype A¡^ infections were reported in clinical cases predominantly and they were isolated from birds¡¦ droppings in large amount. Cryptococcus neoformans var. gattii ¡]serotype B, C¡^ had a natural life in plants, especially Eucalypticus trees. Isolations from other trees were reported increasingly in the tropical and subtropical areas. Comparing to Cryptococcus species, Tricosporon asahii is the normal mycoses of soil. In this study, we performed an environmental investigation concerning Cryptococcus species and Tricosporon asahii in Southern Taiwan. 120 droppings of racing pigeons and 114 samples from Eucalypticus trees were obtained. The results revealed that 30 Cryptococcus neoformans were isolated from racing pigeons¡¦ droppings ¡]25%¡^, as well as 4 Cryptococcus laurentii ¡]3.3%¡^ and 2 Cryptococcus albidus ¡]1.7%¡^. In addition, 25 Tricosporon asahii ( 20.8% ) were isolated from droppings of racing pigeons. But, none of Cryptococcus species or Tricosporon asahii is isolated from Eucalypticus trees ¡]0%¡^. All of Cryptococcus neoformans isolated from pigeons¡¦ droppings were var. grubii ¡]serotype A¡^ and their drug susceptibility tests showed sensitive to Amphotericin B ¡]minimal inhibitory concentration ¡Ø0.25£gg/ml¡^ and Fluconazole ¡]minimal inhibitory concentration 2£gg/ml¡^ and Flucytosine ¡]minimal inhibitory concentration ¡Ø1£gg/ml¡^. To sum up, both Cryptococcus species and Tricosporon asahii were isolated from droppings of racing pigeons in our study, especially Tricosporon asahii in large amount. Opportunistic infection caused by these species should be given more attention to racing pigeons which have close contact with human . Intensive investigation and surveillance should be carried out in the future to provide an information for the control and prevention of diseases.

BACKGROUND: Multi drug resistant Tuberculosis (MDR-TB) is a serious public health concern in many parts of the world. As per the WHO- 2010 global report on Surveillance and response 3.6% of all incident TB cases globally are multidrug resistant. In this regard, there is an increasing demand for timely, reliable and comprehensive drug susceptibility testing (DST) as MDR-TB surveillance is being geared up. The intent of this analysis is to determine whether there is a need to continue routine confirmatory DST testing at CDC in addition to just sending the isolates for genotyping. Analysis is done by measuring the discordance between the results of laboratory DST at CDC and the local labs drug type, drug testing concentrations, and study sites. METHODS: The data for this analysis was provided by the Tuberculosis Trials Consortium (TBTC), CDC. Data for this analysis was collected over nearly two decades (1993-2011), gathered from 7 clinical trials. Discordance between the local and CDC lab DST results was measured using Kappa statistic. Sensitivity and specificity analysis was done by taking the CDC DST lab results as the gold standard. Discordance levels were calculated by local sites and baseline drug resistance for each antibiotic in each study was measured. RESULTS: Average Kappa values for inter rater agreement for all the studies was 0.6444 whereas the overall level of discordance across all studies is 7.786%. Drug resistance at baseline was highest for Isoniazid and Streptomycin (except Study 23 and 22). CONCLUSION: Though the current results show few DST result discordances between local and CDC labs, it is better to continue to send isolates to the centralized lab (CDC) in view of the worldwide threat of drug resistant TB epidemic, the recommendations of the current literature and the benefits of reliable confirmatory testing services and availability of other molecular diagnostic methods.

Pythium insidiosum, classificado no Reino Stramenopila e Classe Oomycetes, é o agente etiológico da pitiose, uma doença diagnosticada principalmente em eqüinos, caninos e humanos. As atividades in vitro do voriconazol, itraconazol e terbinafina, sozinhos ou em combinação, foram estudadas empregando uma metodologia de macrodiluição baseada na técnica M 38-A (CLSI, 2002) contra 30 isolados clínicos de Pythium insidiosum, com o objetivo de avaliar novas medidas para tratar infecções causadas por este agente utilizando quimioterapia. As combinações (terbinafina e voriconazol; terbinafina e itraconazol) tiveram suas interações avaliadas utilizando a técnica de checkerboard seguindo o modelo da macrodiluição em caldo. Terbinafina foi ativa quando testada sozinha, mostrando uma concentração inibitória mínima (CIM) e concentração fungicida mínima ≤ 8,0 mg/L para todos os isolados. Voriconazol e itraconazol foram inativos contra os isolados testados quando usados sozinhos. A combinação de voriconazol com terbinafina foi sinérgica contra 17% das amostras e indiferente em 25 (83%) amostras. O uso concomitante de terbinafina e itraconazol foi sinérgico contra 17% e indiferente contra 83% das amostras. As interpretações de ambas as interações foram equivalentes para 26 isolados (87%): vinte e três foram indiferentes e três mostraram efeito sinérgico. Antagonismo não foi observado. A terapia combinada é uma alternativa à monoterapia, especialmente para pacientes com infecções invasivas que são difíceis de tratar. Estes resultados precisam ser correlacionados com relatos clínicos; e ensaios in vivo, usando modelos animais, podem preencher a lacuna entre avaliações clínicas e avaliações in vitro das drogas. / Pythium insidiosum is classified in the kingdom Stramenopila, class Oomycetes. It causes pythiosis, a disease mainly diagnosed in horses, dogs, and humans. In order to evaluate new approaches to treat infections caused by Pythium insidiosum using chemotherapy, the in vitro activities of voriconazole, itraconazole and terbinafine, alone or in combination, against 30 clinical isolates were studied employing a macrodilution methodology based on the M 38-A technique (CLSI, 2002). The combinations (terbinafine plus voriconazole and terbinafine plus itraconazole) had their interactions evaluated using the checkerboard technique following the broth macrodilution design. Terbinafine was active when used as a single drug, showing minimal inhibitory concentration (MIC) and minimal fungicidal concentration ≤ 8,0 mg/L for all the isolates. Voriconazole and itraconazole were inactive against the isolates tested when used alone. The combination of voriconazole with terbinafine was synergic against 17% of the strains and indifferent on 25 (83%) strains. The concomitant use of terbinafine and itraconazole was synergic against 17% and indifferent against 83% of the strains. The interpretations of both interactions were equivalent for 26 isolates (87%): twenty three were indifferent and three showed synergistic effect. Antagonism was not observed. Combination therapy provides an alternative to monotherapy, especially for patients with invasive infections that are difficult to treat. These results need to be correlated with clinical outcomes, and in vivo assays using experimental animal models could bridge the gap between in vitro and clinical evaluation of drugs.

Pythium insidiosum, classificado no Reino Stramenopila e Classe Oomycetes, é o agente etiológico da pitiose, uma doença diagnosticada principalmente em eqüinos, caninos e humanos. As atividades in vitro do voriconazol, itraconazol e terbinafina, sozinhos ou em combinação, foram estudadas empregando uma metodologia de macrodiluição baseada na técnica M 38-A (CLSI, 2002) contra 30 isolados clínicos de Pythium insidiosum, com o objetivo de avaliar novas medidas para tratar infecções causadas por este agente utilizando quimioterapia. As combinações (terbinafina e voriconazol; terbinafina e itraconazol) tiveram suas interações avaliadas utilizando a técnica de checkerboard seguindo o modelo da macrodiluição em caldo. Terbinafina foi ativa quando testada sozinha, mostrando uma concentração inibitória mínima (CIM) e concentração fungicida mínima ≤ 8,0 mg/L para todos os isolados. Voriconazol e itraconazol foram inativos contra os isolados testados quando usados sozinhos. A combinação de voriconazol com terbinafina foi sinérgica contra 17% das amostras e indiferente em 25 (83%) amostras. O uso concomitante de terbinafina e itraconazol foi sinérgico contra 17% e indiferente contra 83% das amostras. As interpretações de ambas as interações foram equivalentes para 26 isolados (87%): vinte e três foram indiferentes e três mostraram efeito sinérgico. Antagonismo não foi observado. A terapia combinada é uma alternativa à monoterapia, especialmente para pacientes com infecções invasivas que são difíceis de tratar. Estes resultados precisam ser correlacionados com relatos clínicos; e ensaios in vivo, usando modelos animais, podem preencher a lacuna entre avaliações clínicas e avaliações in vitro das drogas. / Pythium insidiosum is classified in the kingdom Stramenopila, class Oomycetes. It causes pythiosis, a disease mainly diagnosed in horses, dogs, and humans. In order to evaluate new approaches to treat infections caused by Pythium insidiosum using chemotherapy, the in vitro activities of voriconazole, itraconazole and terbinafine, alone or in combination, against 30 clinical isolates were studied employing a macrodilution methodology based on the M 38-A technique (CLSI, 2002). The combinations (terbinafine plus voriconazole and terbinafine plus itraconazole) had their interactions evaluated using the checkerboard technique following the broth macrodilution design. Terbinafine was active when used as a single drug, showing minimal inhibitory concentration (MIC) and minimal fungicidal concentration ≤ 8,0 mg/L for all the isolates. Voriconazole and itraconazole were inactive against the isolates tested when used alone. The combination of voriconazole with terbinafine was synergic against 17% of the strains and indifferent on 25 (83%) strains. The concomitant use of terbinafine and itraconazole was synergic against 17% and indifferent against 83% of the strains. The interpretations of both interactions were equivalent for 26 isolates (87%): twenty three were indifferent and three showed synergistic effect. Antagonism was not observed. Combination therapy provides an alternative to monotherapy, especially for patients with invasive infections that are difficult to treat. These results need to be correlated with clinical outcomes, and in vivo assays using experimental animal models could bridge the gap between in vitro and clinical evaluation of drugs.

Pythium insidiosum, classificado no Reino Stramenopila e Classe Oomycetes, é o agente etiológico da pitiose, uma doença diagnosticada principalmente em eqüinos, caninos e humanos. As atividades in vitro do voriconazol, itraconazol e terbinafina, sozinhos ou em combinação, foram estudadas empregando uma metodologia de macrodiluição baseada na técnica M 38-A (CLSI, 2002) contra 30 isolados clínicos de Pythium insidiosum, com o objetivo de avaliar novas medidas para tratar infecções causadas por este agente utilizando quimioterapia. As combinações (terbinafina e voriconazol; terbinafina e itraconazol) tiveram suas interações avaliadas utilizando a técnica de checkerboard seguindo o modelo da macrodiluição em caldo. Terbinafina foi ativa quando testada sozinha, mostrando uma concentração inibitória mínima (CIM) e concentração fungicida mínima ≤ 8,0 mg/L para todos os isolados. Voriconazol e itraconazol foram inativos contra os isolados testados quando usados sozinhos. A combinação de voriconazol com terbinafina foi sinérgica contra 17% das amostras e indiferente em 25 (83%) amostras. O uso concomitante de terbinafina e itraconazol foi sinérgico contra 17% e indiferente contra 83% das amostras. As interpretações de ambas as interações foram equivalentes para 26 isolados (87%): vinte e três foram indiferentes e três mostraram efeito sinérgico. Antagonismo não foi observado. A terapia combinada é uma alternativa à monoterapia, especialmente para pacientes com infecções invasivas que são difíceis de tratar. Estes resultados precisam ser correlacionados com relatos clínicos; e ensaios in vivo, usando modelos animais, podem preencher a lacuna entre avaliações clínicas e avaliações in vitro das drogas. / Pythium insidiosum is classified in the kingdom Stramenopila, class Oomycetes. It causes pythiosis, a disease mainly diagnosed in horses, dogs, and humans. In order to evaluate new approaches to treat infections caused by Pythium insidiosum using chemotherapy, the in vitro activities of voriconazole, itraconazole and terbinafine, alone or in combination, against 30 clinical isolates were studied employing a macrodilution methodology based on the M 38-A technique (CLSI, 2002). The combinations (terbinafine plus voriconazole and terbinafine plus itraconazole) had their interactions evaluated using the checkerboard technique following the broth macrodilution design. Terbinafine was active when used as a single drug, showing minimal inhibitory concentration (MIC) and minimal fungicidal concentration ≤ 8,0 mg/L for all the isolates. Voriconazole and itraconazole were inactive against the isolates tested when used alone. The combination of voriconazole with terbinafine was synergic against 17% of the strains and indifferent on 25 (83%) strains. The concomitant use of terbinafine and itraconazole was synergic against 17% and indifferent against 83% of the strains. The interpretations of both interactions were equivalent for 26 isolates (87%): twenty three were indifferent and three showed synergistic effect. Antagonism was not observed. Combination therapy provides an alternative to monotherapy, especially for patients with invasive infections that are difficult to treat. These results need to be correlated with clinical outcomes, and in vivo assays using experimental animal models could bridge the gap between in vitro and clinical evaluation of drugs.