INTRODUÇÃO: Cepas de Pseudomonas aeruginosa (PA) produtoras de Metalo-b- lactamase (MBL) tem sido reportadas como sendo uma importante causa de infecções nosocomiais assim como um grande problema terapêutico mundial. No complexo HC-FMUSP, o maior hospital universitário do Brasil, a prevalência de PA resistente aos carbapenêmicos também se apresenta de forma crítica. No entanto, a prevalência de MBL em nossa instituição e a padronização para detecção fenotípica deste mecanismo de resistência ainda não foram estabelecidos. OBJETIVOS: Determinar a prevalência de MBL em cepas de PA resistentes ao imipenem; comparar métodos fenotípicos e moleculares na detecção de MBL; avaliar a clonalidade dos isolados produtores de MBL e determinar o perfil de suscetibilidade de todos os isolados incluídos no estudo. MÉTODOS: Foi realizado um estudo retrospectivo com 69 cepas de PA resistentes ao imipenem isoladas de hemoculturas de pacientes do HC-FMUSP no ano de 2005. Os isolados foram submetidos ao teste de suscetibilidade pelo método de microdiluição e Etest® para colistina. Estes foram testados para a produção de MBL pelos métodos fenotípicos de Disco Aproximação (DA), Etest® MBL e Teste de Hodge Modificado (THM). A Reação em Cadeia da Polimerase (PCR) foi realizada para detecção dos seguintes genes: (blaSPM-1, blaIMP-1, blaIMP-2, blaVIM-1 e blaVIM-2). A clonalidade dos isolados produtores de MBL foi avaliada por Eletroforese em Campo Pulsado (PFGE). RESULTADOS: Cinqüenta e três cepas (76.8%) foram positivas pelos métodos de DA e Etest® MBL, e 19 cepas (27.5%) foram positivas pelo THM. Vinte e um isolados (30.4%) demonstraram o gene blaMBL por PCR, sendo que 17 (81%) foram positivos para o gene blaSPM-1 e 4 (19%) para o gene blaVIM-2. Os genes blaIMP-1, blaIMP-2 e blaVIM-1 não foram detectados. O inibidor ácido 2-mercaptoacético (MAA) e o THM mostram a melhor concordância com a PCR, com índices de kappa variando de 0.81 a 0.86 e 0.79, respectivamente O ácido etilenodiaminotetracético (EDTA), demonstrou alta sensibilidade (100%), baixa especificidade (33.3%), e pobre concordância com a PCR. Entre os isolados positivos para o gene blaSPM-1, 5 foram indistinguíveis, 11 foram estreitamente relacionados e 1 possivelmente relacionado. Entre os isolados positivos para o gene blaVIM-2, 2 foram indistinguíveis, 1 foi estreitamente relacionado e 1 diferente. Entre os isolados produtores de MBL, a colistina e o aztreonam foram as drogas mais ativas com 90.5% e 85.7% de sensibilidade, respectivamente. CONCLUSÃO: Este é o primeiro relato de PA produtora de MBL no HC-FMUSP, reforçando a epidemiologia brasileira onde a enzima SPM-1 é a mais prevalente entre isolados de PA. Os métodos de DA com o inibidor MAA e o THM mostraram-se boas opções para detecção fenotípica de MBL em laboratórios de microbiologia. Os produtores de MBL demonstraram fenótipo multiresistente com um padrão clonal, enfatizando a necessidade de práticas de controle de infecções em nossa instituição / INTRODUCTION: Pseudomonas aeruginosa (PA) strains Metallo-b-lactamase (MBL) producing have been reported as an important cause of nosocomial infection, also becoming a critical therapeutic problem worldwide. At HC-FMUSP complex, the largest teaching hospital in Brazil, the prevalence of PA resistant to carbapenems is also presented as a critical problem. However, MBL prevalence and the standardization for phenotypical detection of this resistance mechanism still had not been established. PURPOSE: To determine MBL prevalence in PA resistant to imipenem; to compare phenotypic and molecular methods to detect MBL; to evaluate the clonality of the MBL producers strains and to determine the susceptibility profile of all isolates included in this study. METHODS: A retrospective study was carried analyzing 69 PA strains resistant to imipenem isolated from blood cultures of patients at HC-FMUSP during 2005. They were submitted to the susceptibility profile test by microdilution method and Etest® to colistin. The isolates were also tested for MBL production by phenotypic methods like Double Disk Synergy (DDS), Etest® MBL, Modified Hodge Test (MHT). The Polimerase Chain Reaction (PCR) was used to detect the following genes: (blaSPM-1, blaIMP-1, blaIMP-2, blaVIM-1 and blaVIM-2). The clonality of the MBL producers was evaluated by Pulsed Field Gel Electrophoresis (PFGE). RESULTS: Fifty-three isolates (76.8%) had positive results with DDS and Etest® MBL, and 19 isolates (27.5%) were positive by MHT. Twenty-one isolates (30.4%) had a blaMBL gene by PCR, being 17 (81%) positive for blaSPM-1 and 4 (19%) for blaVIM-2. The blaIMP-1, blaIMP-2 and blaVIM-1 genes had not been detected. Mercaptoacetic acid (MAA) inhibitor and MHT showed the best agreement with PCR, with kappa value ranging from 0.81 to 0.86 and 0.79, respectively. Etilenodiaminotetracetic acid (EDTA) inhibitor showed high sensibility (100%), low specificity (33.3%), and poor agreement with PCR. Among isolates producing blaSPM-1 gene, 5 were indistinguishable, 11 were closely related and 1 was possibly related. Among isolates producing blaVIM-2 gene, 2 were indistinguishable, 1 closely related and 1 was different. Among MBL-producing strains, colistin and aztreonam were the most active drugs with 90.5% and 85.7% of sensitivity, respectively. CONCLUSION: This is the first report of PA MBL producers at HCFMUSP, reinforcing the Brazilian epidemiology where SPM-1 enzyme is the most prevalent among PA isolates. The DDS method with MAA inhibitor and MHT had the best agreement with PCR, showing themselves as good options for MBL phenotypical detection in microbiology laboratories. The MBL producers had shown multiresistant phenotype with a clonal standard, reinforcing the necessity of infection control practices in our institution

Para avaliar a sensibilidade da colistina e polimixina B de isolados de P. aeruginosa multirresistentes foram utilizadas diferentes metodologias, como microdiluição, Etest e disco-difusão, cujos resultados evidenciaram que a microdiluição continua sendo o método de referência. Do total de 109 isolados, não houve resistência para colistina e apenas um isolado mostrou-se resistente para a polimixina B. Para determinar a linhagem clonal destes isolados foi empregada a técnica de eletroforese em campo pulsado, a qual evidenciou a existência de um padrão molecular predominante durante o período de estudo e a presença de 7 grupos de isolados de P. aeruginosa multirresistentes em 2003 / -

Orientador: Fernando Rogério Pavan / Banca: Katiany Rizzieri Caleffi Ferraciolli / Banca: Tais Maria Bauab / Resumo: Tuberculose (TB) é a doença infecciosa que mais mata pessoas no mundo e é causada principalmente pelo bacilo Mycobacterium tuberculosis. Em 2016, 10,4 milhões de pessoas desenvolveram a doença e 1,8 milhão morreu em sua decorrência. Atualmente o principal agravante deste cenário é a resistência do bacilo aos antimicrobianos disponíveis para o tratamento. Entre os principais mecanismos responsáveis pela resistência aos antimicrobianos, as mutações em genes que codificam dos alvos dos fármacos se destacam. Fluoroquinolonas e aminoglicosídeos são duas classes de antimicrobianos de 2ª linha utilizados no tratamento de TB e atuam na proteína DNA girase e no ribossomo bacteriano impedindo o processo de transcrição e síntese proteica respectivamente. Mutações nos genes gyrA e rrs que codificam estes alvos podem ser responsáveis por tal resistência. Para que medidas de saúde pública possam ser tomadas para otimizar o tratamento, é preciso conhecer a que os isolados clínicos são resistentes e qual o mecanismo envolvido neste processo. Para isso, uma biblioteca com 100 isolados clínicos coletados entre 2007 e 2009 no hospital de referência Clemente Ferreira da cidade de São Paulo foi avaliada em relação a resistência a fluoroquinolonas e aminoglicosídeos. A primeira etapa foi a determinação da concentração inibitória mínima (CIM) de três antibióticos da classe das fluoroquinolonas (ofloxacino, moxifloxacino e gatifloxacino) e três da classe dos aminoglicosídeos (amicacina, canamicina e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tuberculosis (TB) is a most deadly infectious disease of people in the world and is mainly caused by the Mycobacterium tuberculosis bacillus. In 2016, 10.4 million people developed disease and 1.8 million died. Currently, the main aggravating factor of this scenario is the resistance of the bacillus to the antimicrobials available for treatment. Among the main mechanisms for resistance to antimicrobials, the mutations in genes that code the targets of the drugs stand out. Fluoroquinolones and aminoglycosides are two classes of antimicrobials of 2nd line for TB treatment and they act on protein DNA gyrase and on the bacterial ribosome impeding the process of transcription and protein synthesis respectively. Mutations in the gyrA and rrs genes encoding these targets may be explain the resistance. Public health guidelines are taken to optimize treatment and for this it is necessary to know what clinical isolates resistant and what mechanism are is involved in the process. For this, a library with 100 clinical isolates collected between 2007 and 2009 at a Clemente Ferreira reference hospital in the city of São Paulo was evaluated for resistance to fluoroquinolones and aminoglycosides. A first stage was the determination of the minimum inhibitory concentration (MIC) of three fluoroquinolone antibiotics (ofloxacin, moxifloxacin and gatifloxacin) and three of the class of aminoglycosides (amikacin, kanamycin and streptomycin) against 100 clinical isolates using a microdilution assay in 96-well plates. According to the MIC, clinical isolates resistant to at least one antimicrobial were selected for the study of the possible mechanism of resistance. Mutations in the gyrA and rrs genes were scree ned using the GenoType MTBDRsl kit because it may explain the resistance... (Complete abstract click electronic access below) / Mestre

Para avaliar a sensibilidade da colistina e polimixina B de isolados de P. aeruginosa multirresistentes foram utilizadas diferentes metodologias, como microdiluição, Etest e disco-difusão, cujos resultados evidenciaram que a microdiluição continua sendo o método de referência. Do total de 109 isolados, não houve resistência para colistina e apenas um isolado mostrou-se resistente para a polimixina B. Para determinar a linhagem clonal destes isolados foi empregada a técnica de eletroforese em campo pulsado, a qual evidenciou a existência de um padrão molecular predominante durante o período de estudo e a presença de 7 grupos de isolados de P. aeruginosa multirresistentes em 2003 / -

Leung, Siu Sing. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 92-97). / Abstracts also in Chinese. / Title from PDF title page (viewed on 25, October, 2016).

Chan, Lap Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 131-147). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / LIST OF ABBREVIATIONS --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- Prevalence of antimicrobial reisitance in gonococcal strains --- p.4 / Chapter 1.2.1 --- Prevalence of penicillin resistant gonococcal strains --- p.5 / Chapter 1.2.2 --- Prevalence of tetracycline resistant gonococcal strains --- p.6 / Chapter 1.2.3 --- Prevalence of quinolone resistant gonococcal strains. --- p.7 / Chapter 1.2.4 --- Emergence of generation cephalosporin reduced susceptible gonococcal strains --- p.8 / Chapter 1.2.5 --- Monitoring the prevalence of gonorrhea --- p.9 / Chapter 1.3 --- Gonococcal Antimicrobial Surveillance Program --- p.10 / Chapter 1.4 --- Antimicrobial Resistance Mechanisms in N. gonorrhoeae --- p.11 / Chapter 1.4.1 --- "Innate Resistance Mechanisms in N, gonorrhoeae" --- p.11 / Chapter 1.4.1.1 --- Natural mechanisms in N. gonorrhoeae against toxic substance --- p.11 / Chapter 1.4.1.2 --- Efflux pump inhibitors --- p.13 / Chapter 1.4.2 --- Development of acquired antimicrobial resistance in N. gonorrhoeae --- p.14 / Chapter 1.4.2.1 --- Penicillin --- p.14 / Chapter 1.4.2.1.1 --- Chromosomal-mediated resistance --- p.14 / Chapter 1.4.2.1.2 --- Plasmid-mediated resistance --- p.16 / Chapter 1.4.2.2 --- Tetracycline --- p.16 / Chapter 1.4.2.2.1 --- Plasmid-mediated resistance --- p.17 / Chapter 1.4.2.2.2 --- Chromosomal mediated resistance --- p.18 / Chapter 1.4.2.3 --- Fluroquinolone --- p.18 / Chapter 1.4.2.3.1 --- Resistant mechanism in quinolone resistant gonococcal strains --- p.19 / Chapter 1.4.2.3.1.1 --- gyrA andparC --- p.19 / Chapter 1.4.2.3.1.2 --- NorM efflux system --- p.21 / Chapter 1.4.2.4 --- 3rd generation cephalosporins --- p.22 / Chapter 1.4.2.4.1 --- Mosaic penA structure in reduced susceptible gonococcal strains --- p.22 / Chapter 1.4.2.4.2 --- Other mechanisms related to reduced susceptibility in gonococcal strains --- p.24 / Chapter 1.5 --- Application of molecular typing methods to study the epidemiology of N. gonorrhoeae --- p.24 / Chapter 1.5.1 --- Opa typing --- p.25 / Chapter 1.5.2 --- K gonorrhoeae Multi-Antigen Sequence Typing --- p.25 / Chapter 1.6 --- Project Objectives --- p.27 / Chapter CHAPTER 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Collecting gonococcal strains --- p.29 / Chapter 2.2 --- Culturing of N. gonorrhoeae --- p.29 / Chapter 2.3 --- Identification --- p.29 / Chapter 2.3.1 --- Gram staining test --- p.30 / Chapter 2.3.2 --- Oxidase test --- p.31 / Chapter 2.3.3 --- Cabohydrate utilization test --- p.31 / Chapter 2.4 --- In-vitro antimicrobial susceptibility testing --- p.32 / Chapter 2.4.1 --- Preparation of cell cultures for MIC tests --- p.32 / Chapter 2.4.2 --- Preparation of antimicrobial agents for MIC tests --- p.32 / Chapter 2.4.3 --- Inoculum preparation and delivering --- p.33 / Chapter 2.5 --- Preparation of genomic DNA for detection of mutations --- p.34 / Chapter 2.6 --- Study of Resistant Mechanism against fluoroquinolone --- p.34 / Chapter 2.6.1 --- PGR detection of mutations in gyrA and parC genes --- p.35 / Chapter 2.6.2 --- Optimization of gyrA and parC genes PGR --- p.35 / Chapter 2.6.3 --- Detection of PGR products for gyrA and parC genes --- p.37 / Chapter 2.6.4 --- Purification of Amplified DNA products --- p.37 / Chapter 2.7 --- Tests of efflux inhibitor on N. gonorrhoeae --- p.38 / Chapter 2.7.1 --- Effect ofCCCP --- p.39 / Chapter 2.7.2 --- Effect of Reserpine --- p.39 / Chapter 2.8 --- Study of Resistant mechanism against β-lactams --- p.40 / Chapter 2.8.1. --- Detection for the presence of β-lactamase --- p.40 / Chapter 2.8.2 --- Mosaic penA patterns --- p.40 / Chapter 2.8.2.1 --- Detection of mutations in penA gene --- p.40 / Chapter 2.8.2.2 --- Optimization of penA gene PGR --- p.41 / Chapter 2.8.2.3 --- Detection of PGR products --- p.43 / Chapter 2.8.2.4 --- Purification of Amplified DNA products --- p.44 / Chapter 2.9 --- Detection of the presence of tetM determinant --- p.45 / Chapter 2.9.1 --- Optimization of tetM determinant PCRs --- p.46 / Chapter 2.9.2 --- Detection of PGR products --- p.47 / Chapter 2.10 --- Detection of different allele types in tbpB andpor genes --- p.48 / Chapter 2.10.1 --- Optimization of PGR for NG-MAST --- p.48 / Chapter 2.10.2 --- Detection of PCR products --- p.49 / Chapter 2.10.3 --- PCR product purification --- p.50 / Chapter 2.11 --- Sequencing of the PCR products --- p.51 / Chapter 2.12 --- Data Analysis --- p.52 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1 --- Gonococcal strains collected --- p.55 / Chapter 3.2 --- Identification of gonococcal strains --- p.55 / Chapter 3.3 --- MIC of Antimicrobial agents --- p.56 / Chapter 3.3.1 --- Interpretive Criteria --- p.56 / Chapter 3.3.2 --- Ciprofloxacin --- p.56 / Chapter 3.3.3 --- Penicillin --- p.57 / Chapter 3.3.4 --- Tetracycline --- p.57 / Chapter 3.3.5 --- Ceftriaxone --- p.58 / Chapter 3.3.6 --- Cefixime --- p.58 / Chapter 3.3.7 --- Cefotaxime --- p.58 / Chapter 3.3.8 --- Spectinomycin --- p.58 / Chapter 3.3.9 --- Levofloxacin --- p.59 / Chapter 3.3.10 --- Ceftibuten --- p.59 / Chapter 3.4 --- Result of PGR --- p.60 / Chapter 3.4.1 --- gyrA andparC genes --- p.60 / Chapter 3.4.2 --- penA gene --- p.61 / Chapter 3.4.3 --- tbpB and por genes --- p.62 / Chapter 3.4.4 --- tetM determinant --- p.62 / Chapter 3.5 --- β-lactamase --- p.63 / Chapter 3.6 --- Efflux pump inhibitor --- p.63 / Chapter 3.6.1 --- CCCP --- p.64 / Chapter 3.6.2 --- Reserpine --- p.64 / Chapter 3.7 --- Detection of Mutations --- p.66 / Chapter 3.7.1 --- gyrA and parC genes --- p.66 / Chapter 3.7.2 --- penA gene --- p.68 / Chapter 3.8 --- NG-MAST --- p.70 / Chapter 3.8.1 --- tbpB and por gene --- p.71 / Chapter 3.9 --- Porin mutation --- p.72 / Chapter CHAPTER 4. --- DISCUSSION / Chapter 4.1 --- Sampling --- p.74 / Chapter 4.2 --- Methodology --- p.75 / Chapter 4.3 --- MIC distribution of different antimicrobial agents --- p.76 / Chapter 4.4 --- Mechanisms of quinolone resistance --- p.78 / Chapter 4.4.1 --- Mutations at QRDRs --- p.78 / Chapter 4.4.2 --- Association of the number of mutations at parC gene with MIC levels against fluroquinolones --- p.80 / Chapter 4.5 --- Penicillin and Tetracycline Resistant Mechanisms --- p.81 / Chapter 4.6 --- Efflux system --- p.85 / Chapter 4.7 --- NG-MAST --- p.88 / Chapter 4.8 --- Mosaic penA pattern --- p.89 / Chapter 4.9 --- Management of gonorrhea --- p.90 / Chapter CHAPTER 5. --- CONCLUSIONS / REFERENCES

Of the 22.5 million individuals infected with the human immunodeficiency virus (HIV) in sub-Saharan Africa, 62% of patients requiring treatment had access to highly active antiretroviral therapy (HAART) in 2011. The delivery of HAART and the appropriate laboratory monitoring of HIV positive individuals in sub-Saharan African countries has become a public health priority, an intervention which has and will continue to dramatically reduce HIV-related morbidity and mortality. Routine laboratory monitoring of HIV infected individuals should ideally include CD4+ T cell testing to assess when to start ART, viral load monitoring to assess virological failure on ART and when indicated, HIVDR genotyping.However, this is often not implemented in resource limited settings due to challenges such as inadequate infrastructure and laboratory capacity, amongst others. Thus the Affordable Resistance Testing for Africa (ART-A) initiative was established to develop an affordable HIV drug resistance testing (HIVDR) algorithm applicable to Africa. The objective of this study was to evaluate the role of in vitro HIVDR phenotyping in the context of HIV-1 subtype C (the most prevalent circulating subtype in sub-Saharan Africa), genotyping and genotypic interpretation tools using existing algorithms, as well as novel virological failure detection tools for clinical patient management. Current gold standard HIVDR phenotyping technologies use an HIV-1 subtype B backbone to create recombinant viruses with patient-derived polymerase (protease and partial reverse transcriptase). This backbone could impact on the in vitro phenotyping results of non-B subtypes, and therefore it was deemed necessary to establish the applicability of HIVDR phenotypic testing of subtype C polymerase when a commercially available subtype B backbone is used. One hundred and fourteen HIV-1 subtype C samples were HIVDR phenotyped against 17 antiretroviral drugs using both subtype B and C backbones and showed a high level of concordance between the two backbone phenotypic resistance profiles (95.8%; 1590 of 1660 fold change comparisons). Natural assay variability was largely responsible for discordant results. Results confirmed that HIV-1 phenotypic reverse transcriptase inhibitor drug resistance test interpretation is independent of the virus backbone subtype. No conclusions could be made for protease inhibitor resistance since limited samples from 2nd line failure were available. Subsequently, the HIVDR genotypic and phenotypic results of the 114 patient samples were compared to determine whether genotyping is a viable alternative to phenotyping. Results showed a 92.3% concordance between genotyping and phenotyping of individual drug comparisons for a number of HIVDR profiles. Discrepancies were attributed to phenotypic assay variability in addition to the role of mutation mixtures, which impacted genotypic interpretations. Overall, HIVDR genotyping is a reliable tool to detect and interpret antiretroviral drug resistance in HIV-1 subtype C infected patients, and can thus be used for clinical patient management. Once the accuracy of HIVDR genotyping was established, the development, validation and evaluation of a potential virological failure assay (ARTA-VFA) and a simplified HIVDR (ARTA-HIVDRultralight) assay was undertaken. A simplified and conceptually novel approach using a qualitative viral load assay with a pre-determined cut-off that gives a threshold above which virological failure (VF) could be confirmed and below which treatment success was likely, was tested. A real-time PCR (ARTA-VFA) assay was developed which involved the amplification of a short sequence of the HIV-1 LTR region from RNA extracted either from plasma and/or dried blood spots (DBS). The ARTA-VFA was tested on 409 patient samples,and successfully amplified samples from all major HIV-1 group M subtypes with equal specificity. The VF was qualitatively classified as a viral load >1000 RNA copies/ml in plasma samples, and >5000 RNA copies/ml in DBS samples. Comparative testing yielded accurate VF determination for therapy-switching in approximately 93% of clinical cases tested, compared to current gold standard quantitative viral load assays. A simplified HIVDR genotyping assay (ARTA-HIVDRultralight) targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line regimen failures was developed and assessed. The ARTAHIVDRultralight assay was designed to be practical, faster, and more affordable, show flexibility with respect to equipment (open platform), use DBS or plasma as starting material and amplify and sequence a smaller amplicon (RT). The assay performed well when compared to the in-house assay used in the laboratory at the time for both 212 plasma and 25 DBS samples, yielding identical mutations and subsequent resistant profiles. Furthermore, a theoretical in silico exercise to investigate the consequences of using 125,329 shortened RT genotype (ARTA-HIVDRultralight) as compared to full-length RT sequences showed >95% and >90% concordance when using the Stanford HIVdb algorithm and the virco®TYPE tool, respectively. Differences noted were minor and unlikely to have any impact on clinical decision-making. Overall, this study illustrated that the short RT sequences can be reliably used to generate HIVDR genotypes using the Stanford HIVdb and virco®TYPE algorithms and reduce sequencing costs substantially. A field evaluation using the ARTA-VFA and ARTA-HIVDRultralight on 288 clinical samples was conducted, showing that the accuracy and precision of both assays (using 248 plasma or 40 DBS sampling methods) compared well to the reference methodology, thereby extending access of testing to more remote settings.These assays were designed to either be used as a testing strategy of initially assessing VF,and once confirmed performing an HIVDR assay, or alternatively to be used separately as stand-alone, or within different laboratory tiers in resource limited settings. It is envisaged that the ARTA-VFA could be used in the middle laboratory tier, and if confirmatory, patient samples can be referred to a reference laboratory with the available infrastructure for HIVDR testing using the ARTA-HIVDRultralight. Lastly, an automated sequence analysis and editing software for use in correct base calling of nucleotide/mutation mixtures in HIVDR genotyping was validated on 1624 sequences. Compared to reference software, where interpretation is often operator dependent, this software performed extremely well, with minor discrepancies noted. The automated software can be used to reduce subjectivity, time taken for analysis which is often the rate-limiting step and thus improving the turn-around time and clinical relevance of HIVDR genotyping. Overall, the results obtained describe the validation of using HIVDR genotyping as an alternative tool to phenotyping, and the subsequent development and validation of simple, affordable, "open-platform" alternatives to currently used methods for virological failure monitoring, and accommodate a centralized approach to HIVDR with DBS testing in resource limited settings.

Orientador: Luciane Dias de Oliveira / Banca: Marianne Spalding / Banca: Vivian Cristina Costa Castilho Hyodo / Resumo: Pseudomonas aeruginosa e Klebsiella pneumoniae são enterobactérias que acometem especialmente indivíduos imunologicamente comprometidos com grande importância por sua resistência a antibióticos, dessa forma, o objetivo do estudo foi avaliar a atividade antimicrobiana de extratos de própolis (glicólico e aquoso) distribuídos comercialmente em cepas ATCC e clínicas multirresistentes das espécies. Inicialmente foram determinados os valores de Concentração Microbicida Mínima (CMM) sobre cultura planctônica por microdiluição em caldo, segundo Clinical and Laboratory Standards Institute (CLSI), seguido por semeadura em ágar. A concentração correspondente ao quádruplo da CMM foi utilizada para testes em biofilmes monotípicos (contato de 5 min). Foi usada como controle positivo a solução de clorexidina (0,12%) e como controle negativo caldo BHI. Após o tratamento foi verificada a viabilidade dos micro-organismos pelo teste MTT, com leitura em espectrofotômetro de microplacas. Os dados de densidade óptica foram convertidos em porcentagem de redução microbiana e foi realizada a análise estatística com 5% de significância em todos os testes. Os extratos apresentaram ação contra forma planctônica e biofilmes de cepas multirresistentes de ambas as espécies. Para culturas planctônicas, P. aeruginosa apresentou CMM de 6,25 e 12,5 mg/mL para o extrato glicólico e de 13,75 e 55 mg/mL para o extrato aquoso, enquanto K. pneumoniae apresentou CMM de 6,25 a 25 mg/mL para o extrato glicólico e de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pseudomonas aeruginosa and Klebsiella pneumoniae are enterobacteria that especially affect immunologically compromised individuals with great importance for their resistance to antibiotics. Therefore, the objective of this study was to evaluate the antimicrobial activity of propolis extracts (glycolic and aqueous) commercially distributed in ATCC and multiresistant clinics strains of these species. The values of Minimum Microbicidal Concentration (MMC) on plankton culture were determined by microdilution in broth, according to Clinical and Laboratory Standards Institute (CLSI), followed by sowing in agar. The concentration corresponding to the quadruple MMC was used for monotypic biofilm tests (5-minute contact). A positive control solution of chlorhexidine (0.12%) and as a negative control BHI broth were used. After the treatment, the viability of the microorganisms was verified by the MTT test, with microplate spectrophotometer reading. Optical density data were converted to microbial reduction percentage and statistical analysis was performed with 5% significance in all tests. The extracts showed action against planktonic form and biofilms of multiresistant strains of both species. For planktonic cultures, P. aeruginosa presented MMC of 6.25 and 12.5 mg / mL for the glycolic extract and 13.75 and 55 mg / mL for the aqueous extract, while K. pneumoniae had MMC of 6.25 to 25 mg / mL for the glycolic extract and 55 mg / mL for the aqueous extract. The reduction of viability in biofilms of P. aeruginosa reached 54.42% for the glycolic extract, and 64.66% for the aqueous extract. K. pneumoniae presented a maximum reduction of 64.24% with glycolic extract and 65.13% with aqueous extract, these reductions being statistically significant in relation to the negative control (p <0.05). Therefore, it can be concluded that propolis extracts present an important ...(Complete abstract click electronic access below) / Mestre

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.