Glycopeptides are the so-called last-resort antibiotics in clinical practice used to treat heavier, predominantly nosocomial infections caused by multi-resistant coagulase-negative staphylococci. The origin and genetic basis of resistance to glycopeptide antibiotics has not yet been elucidated within coagulase-negative staphylococci. Research on Staphylococcus aureus has shown, that intermediate resistance to glycopeptide antibiotics is associated with the presence of one or more mutations, rather than being conditioned by the support of a particular genetic element, such as in enterococci. By using various types of in vitro resistant mutant selection, we were able to obtain isogenic pairs of glycopeptide sensitive and resistant strains of Staphylococcus epidermidis and Staphylococcus haemolyticus. By sequencing the genomes of these pairs, one nucleotide polymorphisms were identified and predominantly found in metabolic and cell wall control systems. Phenotypic analysis did not reveal a direct association of glycopeptide resistance with increased biofilm formation. In clinical practice, the cross-resistance of glycopeptides and other antibiotics is problematic. For the non-glycopeptide antibiotics imipenem and rifampicin, the incidence of cross-resistance with glycopeptide antibiotics in S. aureus...

Made available in DSpace on 2016-12-23T13:56:11Z (GMT). No. of bitstreams: 1 Dissertacao Mestrado - Manuela Tedesco.pdf: 2432201 bytes, checksum: c496ccf4cf25c506ce4352f93047f59a (MD5) Previous issue date: 2011-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Staphylococcus aureus is a pathogen that has the ability to colonize approximately half of the patients undergoing hemodialysis and also is the main cause of infections in these patients. The nasal colonization by S. aureus is a risk factor for developing bacteremia and, despite the great importance in determining the colonization status of patients undergoing hemodialysis, there is no currently a standardized methodology to classify such patients. This study was designed in order to: determine the status of nasal colonization of patients undergoing hemodialysis in two dialysis centers; to improve the reference methodology to classify the status of nasal colonization and assess the risk conferred by nasal colonization in the development of bacteriemia. The study included 219 patients of which 22.8% were nasal carriers of S. aureus. All 182 samples of S. aureus isolates were sensitive to oxacillin and vancomycin but 2.7% (5 / 182) samples were heteroresistant to vancomycin. The classification of the nasal carriage status was performed in 178 patients of which 22.5% were nasal carriers of S. aureus [20% (8 / 40) with persistent and 80% (32/40) intermittent carriers] and 77.5% (138/178) non-carriers. Among the types of colonization, only persistent nasal colonization was substantially associated with the development of bacteremia caused by S. aureus conferring a risk of 17.6% (p = 0.05). The use of fistula demonstrated a protective effect featuring 7% (p = 0.00) and 11% (p = 0.01) the risk conferred by the use of the catheter on the development of bacteremia caused by S. aureus or other microorganisms, respectively. Also, the use of a protocol with seven weekly collections showed an excellent correlation with the reference method (k = 0.834) to distinguish the types of nasal carriers and had a PPV and PNV equal to 100% to differentiate patients with persistent and intermittent colonization, therefore it can be used as an alternative to the reference protocol for screening S. aureus nasal carriers and be used as a surveillance measure. Given the high risk conferred by persistent colonization, our results suggest that classification of nasal carriers is a very important measure to minimize the risk for development of bacteraemia in patients undergoing hemodialysis / O Staphylococcus aureus é um patógeno capaz de colonizar aproximadamente metade dos pacientes submetidos à hemodiálise, e é também o principal micro-organismo isolado de bacteriemias nesse grupo de pacientes. A colonização nasal por S. aureus é fator de risco para o desenvolvimento de bacteriemias e, apesar da grande importância em se determinar o status de colonização dos pacientes submetidos à hemodiálise, não existe atualmente uma metodologia padronizada para classificar tais pacientes. O presente estudo foi delineado com o objetivo de: determinar o status de colonização nasal dos pacientes submetidos à hemodiálise em dois centros de diálise, otimizar o protocolo de referência para classificação do status de colonização e avaliar o risco conferido pela colonização nasal no desenvolvimento de bacteriemias . Foram incluídos no estudo 219 pacientes destes, 22,8% eram portadores nasais de S. aureus. Todas as 182 amostras de S. aureus isoladas foram sensíveis a oxacilina e vancomicina e dessas, 2,7% (5/182) das amostras apresentaram heterorresistência a vancomicina. A classificação do status de colonização foi realizada para 178 pacientes sendo que 22,5% eram portadores nasais de S. aureus [20% (8/40) portadores persistentes e 80% (32/40) portadores intermitentes] e 77,5%(138/178) não portadores. Dentre os tipos de colonização, apenas a colonização nasal persistente foi fortemente associada ao desenvolvimento de bacteriemias por S. aureus conferindo um risco de 17,6% (p=0,05) para tal. O uso de fístula demonstrou um efeito protetor apresentando 7% (p=0,00) e 11% (p=0,01) do risco conferido pelo uso do cateter para o desenvolvimento de bacteriemias por S. aureus e outros micro-organismos, respectivamente. Além disso, verificamos que o uso de um protocolo com sete coletas de periodicidade semanal exibiu uma correlação excelente com a metodologia de referência (k=0, 834) para distinguir os tipos de portadores nasais e apresentou um VPP e VPN equivalentes a 100% para diferenciar os portadores persistentes dos intermitentes, podendo ser utilizada como alternativa ao protocolo de referência para a triagem de portadores nasais de S. aureus e empregado como medida de vigilância epidemiológica. lugar desta Em vista do alto risco conferido pela colonização persistente, nossos resultados sugerem que a classificação dos portadores nasais é uma medida de extrema importância para monitorar risco para o desenvolvimento de bacteriemias em pacientes submetidos à hemodiálise

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterococcus resistentes à vancomicina (VRE) são reconhecidos como importantes patógenos causadores de infecções nosocomiais, configurando um grave problema de saúde pública, principalmente pela escassez de opção terapêutica eficaz. O fenótipo de resistência VanA é o mais frequente, sendo definido pela resistência a altos níveis de vancomicina e teicoplanina. VanA é caracterizado por um conjunto gênico (vanRSHAXYZ) localizado no elemento genético móvel denominado transposon Tn1546. A diversidade de Tn1546 resulta de alterações estruturais promovidas por deleções ou integração de sequências de inserção (IS) que, exercem papel chave na evolução do elemento VanA, modificando os aspectos relacionados à sua transferência e expressão do fenótipo. O objetivo deste estudo foi caracterizar e avaliar o polimorfismo de elementos Tn1546 presentes em amostras de diferentes espécies de Enterococcus isoladas em instituições hospitalares do Estado do Rio de Janeiro no período de 2000 a 2012. Foram incluídas neste estudo 70 amostras VRE que foram caracterizadas quanto ao gênero, espécies e genótipo de resistência aos glicopeptídeos por métodos convencionais e PCR multiplex. A susceptibilidade a 17 antimicrobianos foi avaliada pelo método de difusão em ágar, e concentração inibitória mínima (CIM) para vancomicina e teicoplanina foi determinada por microdiluição em caldo. O tranposon foi obtido após lise das células bacterianas e amplificação por PCR longo, utilizando-se oligonucleotídeos específicos para a região repetida e invertida que flanqueia este elemento genético. A diversidade dos elementos Tn1546 foi avaliada por um conjunto de métodos moleculares que incluiu a análise do polimorfismo do tamanho de fragmentos de restrição (restriction fragment lenght polymorphism, RFLP), utilizando-se a endonuclease ClaI, amplificação de segmentos internos por PCR de sobreposição de oligonucleotídeos (overlapping PCR) e detecção de sequências de inserção (ISs). A caracterização em espécies considerada para as demais análises foi obtida pela metodologia de PCR de acordo com a seguinte distribuição: E. avium (N=6), E. faecalis (N=12), E. faecium (N=46), E. gallinarum (N=4) e E. raffinosus (N=2). Todas as amostras apresentaram o genótipo vanA. Nos testes de susceptibilidade aos antimicrobianos foi observado que todas as amostras foram multirresistentes, sendo resistente de 6 a 13 dentre os 17 antimicrobianos testados. A presença de elementos semelhantes ao arquétipo de Tn1546 foi observada em 61,5% das amostras; entretanto, 27 amostras apresentaram perfis variantes de Tn1546. Foram identificados nove perfis de RFLP, dentre 66 avaliadas, sendo o perfil I, prevalente e semelhante ao arquétipo de Tn1546. Não foi possível analisar quatro amostras por RFLP. Os produtos de amplificação de Tn1546 alterados, obtidos pela overlapping PCR e pelo rastreamento de IS, levaram à classificação de 15 tipos polimórficos, nomeados de A a O. A maioria dos Tn1546 polimórficos teve suas regiões de ORF1 e/ou ORF2 deletadas; e IS1542 juntamente com IS1216V foram as inserções mais frequentes, que em muitas situações compartilhavam a mesma região de inserção. IS19 foi detectada apenas na região vanS-vanH. Os dados apresentados neste estudo indicam que o polimorfismo de Tn1546 pode ser explorado no rastreamento de rotas de transmissão, acompanhamento da dispersão de elementos VanA e investigação da evolução de amostras VRE. / Vancomycin-resistant Enterococcus (VRE) is a leading cause of nosocomial infections, remaining as a public health concern in the last two decades. VanA phenotype is the most frequently encountered and it is responsible for high-level vancomycin and teicoplanin resistance. VanA is characterized by a gene cluster (vanRSHAXYZ) located on the mobile genetic element called Tn1546. The diversity of Tn1546 results from structural changes promoted by deletions or additions of insertion sequences (IS) that play a key role in the evolution of VanA element, changing its transferability and expression of phenotype. The aim of this study was to characterize and evaluate the polymorphism of Tn1546 genetic elements belong to VRE isolates obtained from patients attending in hospitals located in the Rio de Janeiro state, during the period from 2000 to 2012. Seventy VRE strains were included in this study. The strains were identified by conventional physiological testes and multiplex PCR, including the vancomycin resistance phenotype and genotype. Antimicrobial susceptibilities testing were carried out by disk diffusion method for 17 antimicrobials; and the minimal inhibitory concentrations (MIC) values to vancomycin and teicoplanin were evaluated by microdilution technique. Tn1546 were amplified by long-PCR using primers to inverted-repeat sequences flanking the transposon. Tn1546 diversity was evaluated by a set of molecular methods including restriction fragment length polymorphism (RFLP), using the endonuclease ClaI, overlapping PCR and detection of insertion sequence elements (IS). Among the 70 strains, 6 E. avium, 12 E. faecalis, 46 E. faecium, 4 E. gallinarum, and 2 E. raffinosus were characterized by multiplex PCR, as well as the vanA glycopeptide resistance determinant. All the strains were multirresistant, being resistant from 6-13 among the 17 antimicrobials tested. The presence of similar elements to the archetype of Tn1546 was observed in 61.5% of samples; however, 27 samples had variant profiles of Tn1546. Nine RFLP profiles were identified among 66 strains, and the profile I was the most frequent and showed to be similar to the archetype of Tn1546. It was not possible to analyze four strains by RFLP. The amplification products of Tn1546, obtained by overlapping PCR, and screening the IS elements led to the characterization of 15 different types, named A through O. Most Tn1546 had its polymorphisms based on deletion of the ORF1 and / or ORF2 regions; and IS1542 as the most frequent insertion element, which in many cases shared the same region of insertion with IS1216V. IS19 was detected only in vanS-vanH region. The data presented in this study indicate that the polymorphism of Tn1546 can be exploited in tracking transmission routes, monitoring the dispersion of VanA elements and investigation of the evolution of VRE strains.

Durante os anos 90 um aumento dramático no isolamento de VRE foi reportado nos estados Unidos, principalmente em unidades de terapia intensiva, esta tendência continua até hoje. No Brasil o primeiro caso de VRE foi documentado em 1996 e desde então muitos casos têm sido descritos em todo país. Uma variedade de medidas tem sido usada para reduzir taxas de VRE, mas a melhor estratégia para o controle e prevenção deste patógeno ainda não está totalmente definida. O Objetivo deste estudo foi avaliar o impacto de um processo multidisciplinar para monitorar a adesão de profissionais às precauções padrão e de contato e o papel do meio ambiente e de equipamentos na transmissão de VRE. Este estudo consistiu de três períodos: um período basal, um período de intervenção e outro de pós-intervenção. O teste qui-quadrado foi usado para comparar os dados dos períodos pré e pós-intervenção e o teste de qui-quadrado para tendência linear foi usado para avaliar a distribuição de VRE e o uso de glicopeptídeos durante o período de estudo, o nível de p<0.05 foi significante. As amostars foram avaliadas pela técnica de PFGE. PCR para detecção do gene de resistência foi realizada para as amostras clínicas. E. faecium foi a espécie mais freqüente tendo sido responsável por 71% das culturas positivas. Foram documentadas 36 infecções e aquelas da corrente sangüínea foram as mais frequentes 17 (47%) A intervenção educacional foi realizada com 136 profissionais. Foram avaliadas 706 oportunidades de precauções padrão e de contato. A adesão às precauções padrão e de contato não aumentou comparando os períodos pré e pós-intervenção. Entretanto, a proporção de culturas ambientais e de equipamentos diminuiu significativamente comparando os períodos pré (23.2%) and pós-intervenção (2.4%) (p<0.001) e foram associadas a diminuição das Infecções por VRE por 1.000 pacientes-dia (p=0.004). O uso de vancomicina (DDD) não mudou significativamente ao longo do estudo (p=0.97) e o uso de teicoplanina aumentou principalmente nos últimos meses de 2007 (p<0.001). O mesmo clone de E. faecium foi encontrado em 90% das amostras. No presente estudo, redução da proporção de contaminação ambiental e de equipamentos esteve associada com diminuição das taxas de infecção por VRE. / During the 1990s a dramatic increase of VRE isolation was reported mostly in the Intensive Care Units (UCI) in the USA and the trend continues. In Brazil, the first VRE isolated occurred in 1996 and many cases have been described thereafter. A variety of measures have been used to reduce VRE rates, the optimal approach however to control and prevent this pathogen, is not well defined. The aim of this study was to evaluate the impact of a multidisciplinary process to monitor improve healthcare work (HCW) compliance with standard and contact precautions and the role of environment and equipments on the transmission of VRE. This study consisted of three period baseline, intervention and pos-intervention period. Chi-2 test was used to compared data pre and pos intervention and chi-2 test for linear trend was used to evaluate the distribution VRE and use of glycopeptides during the study period, the level of p<0.05 was significant. PFGE was performed. Detection of genes of resistance of VRE isolated from clinical samples was performed by PCR E. faecium was the most frequent species isolated being responsible for 71% of positives cultures. Thirty-six infection were documented, bloodstream infection 17 (47%) was the most frequent site. The educational intervention was given to 136 HCW. 706 opportunities were evaluated, the compliance with standard and contact precautions did not improve comparing pre and pos-intervention period. However, the proportion of environmental and equipments positive cultures decreased significantly comparing pre (23.2%) and pos-intervention (2.4%) period (p<0.001) and was associated with decrease of VRE infection per 1.000 pts-day (p=0.004). The use of vancomycin (DDD) did not change significantly over the study period (p=0.97) and the use of teicoplanin increased (p<0.001). Ninety percent of E. faecium belong to the same type. In the present study, reduction of proportion of positive environmental and equipments cultures was associated with decreased of rates of VRE infections.

Introdução: Vancomicina, considerada o antibiótico de primeira escolha para o tratamento de infecções estafilocócicas, é eliminada por filtração glomerular, e a sua administração deve ser individualizada de acordo com a função renal. As diretrizes atuais recomendam doses e níveis séricos maiores, para aumentar as chances de bons resultados clínicos. Questiona-se se esta estratégia causaria maior nefrotoxicidade. Objetivos: Comparar a frequência de injúria renal aguda (IRA) em pacientes com suspeita de infecção estafilocócica tratados com vancomicina ou com outros antimicrobianos com o mesmo perfil terapêutico em um hospital terciário. Analisar a associação do uso de vancomicina com o desenvolvimento de IRA nestes pacientes. Avaliar os fatores de risco associados ao desenvolvimento de IRA nos pacientes tratados com vancomicina. Identificar os fatores de risco associados à letalidade precoce e tardia nos pacientes com suspeita de infecção estafilocócica tratados com vancomicina ou outros antimicrobianos com o mesmo perfil terapêutico. Métodos: Foram analisados os prontuários dos pacientes com suspeita de infecção estafilocócica que receberam os antimicrobianos vancomicina, teicoplanina, oxacilina, daptomicina ou linezolida por pelo menos três dias nos anos de 2010 e 2011 em um hospital terciário. Analisou-se a frequência de IRA associada ao uso de vancomicina (critério KDIGO) e. por regressão logística, se o uso de vancomicina foi associado ao desenvolvimento de IRA. Avaliou-se por regressão logística os fatores de risco associados ao desenvolvimento de IRA no grupo de pacientes tratados com vancomicina. Analisou-se por regressão de Cox os fatores de risco para letalidades intra-hospitalar, seis meses e até um ano após a internação. Resultados: Foram incluídos 591 pacientes, dos quais 508 foram expostos à vancomicina e 83 foram expostos a teicoplanina, oxacilina, linezolida, ou daptomicina. IRA ocorreu em 28,5% dos pacientes que utilizaram vancomicina e em 14,5% dos que utilizaram outros antimicrobianos (p < 0,001). O grupo de pacientes tratados com vancomicina apresentou parâmetros sugestivos de maior gravidade, como maior frequência de culturas positivas para estafilococos, hipotensão grave, contagem de leucócitos em sangue periférico mais elevada e níveis séricos maiores de lactato, procalcitonina e PCR. Quando pacientes que desenvolveram IRA foram comparados com pacientes que mantiveram a função renal estável, observou-se que o uso de vancomicina, a duração do tratamento e nível sérico de vancomicina foram significativamente maiores entre os primeiros. Vancomicina foi identificada como fator independente para o desenvolvimento de IRA na regressão logística. Os fatores de riscos independentes para o desenvolvimento de IRA no grupo exposto à vancomicina foram uso de medicamentos nefrotóxicos ou que alteram a função renal, uso de medicamento vasopressor e concentração sérica de vancomicina >= 20 mg/L. Vancomicina não se associou a letalidade em nenhum dos períodos estudados, enquanto IRA se associou de forma independente à letalidade precoce e tardia. Conclusões: Estes resultados indicam que a vancomicina apresenta nefrotoxicidade significativa e que os seus níveis séricos devem ser obrigatoriamente avaliados. O uso de medicamentos nefrotóxicos ou que alteram a função renal deve ser, quando possível, evitado ou suspenso em pacientes tratados com vancomicina. O desenvolvimento de IRA, mas não o uso de vancomicina, foi fator independente para letalidade, reforçando que este antimicrobiano pode ser utilizado quando indicado, desde que se previna o desenvolvimento de IRA / Introduction: Vancomycin is considered the first choice antibiotic for treatment of staphylococcus infection. Vancomycin is eliminated through glomerular filtration, and so it is administration must be individualized according the renal function. Current treatment guidelines recommend higher doses and blood levels in order to increase the odds for an adequate clinical outcome. On the other hand, this strategy might cause higher vancomycin-associated nephrotoxicity. Objectives: To analyze the frequency of acute kidney injury (AKI) development in patients with suspicion of staphylococcus infection treated with vancomycin, or other antibiotics with the same therapeutic profile in a tertiary hospital. To analyze the association of vancomycin with AKI development in those patients. To analyze the risk factors for AKI development in vancomycin-treated patients. To identify the risk factors associated to early and late mortality in patients with suspicion of staphylococcus infection treated with vancomycin, or other antibiotics with the same therapeutic profile in a tertiary hospital.Methodology:We analyzed the files of all the patients with suspicion of staphylococcus infection treated with vancomycin, teicoplanin, oxacillin, daptomycin, or linezolid antibiotics for at least three days during the years of 2010 and 2011 in a tertiary hospital.The frequency of AKI development (KDIGO criteria) was assessed. Using logistic regression we assessed if vancomycin use was an independent risk factor for AKI development and the risk factors for AKI development in the group of patients treated with vancomycin. We assessed, using Cox regression, the risk factors for in-hospital, six months and one year after hospitalization mortality. Results: We included 591 patients in the final analysis, 508 using vancomycin and 83 using other antibiotics (teicoplanin, oxacillin, daptomycin, or linezolid). AKI developed in 28.5% of the vancomycin group compared with 14.5% in the other antibiotics group (p < 0.001). Patients treated with vancomycin showed parameters suggesting higher clinical severity, such as higher percent of staphylococcus positive cultures, severe hypotension, higher leukocytes blood count, higher serum levels of lactate, procalcitonin and CRP. When patients developing AKI were compared with patients maintaining preserved renal function, the first group showed a statistically significant higher frequency of vancomycin use, longer vancomycin treatment and higher vancomycin through levels. Using logistic regression vancomycin was identified as an independent risk factor for AKI development. The independent risk factors for AKI development in the vancomycin group were simultaneous use of vancomycin and other nephrotoxic drugs or drugs that influence renal function, vasopressor drugs use and blood levels of vancomycin >= 20 mg/L. Vancomycin was not associated with mortality in any studied time, whereas AKI was an independent risk factor for early and late mortality. Conclusions: These results indicate that vancomycin is associated with significative nephrotoxicity and that its blood levels must be mandatorily assessed. The use of drugs that are nephrotoxic or influence renal function must be, when feasible, avoided or halted in vancomycin-treated patients. AKI development, but not vancomycin use, was an independent risk factor for mortality, reinforcing the perception that vancomycin can be used when necessary, since AKI development is prevented

En infectiologie, comme en cancérologie, la médecine personnalisée se développe. Ainsi, les traitements antibiotiques probabilistes cèdent leur place à des traitements adaptés aux pathologies et aux patients. En plus des risques d’échecs liés à une thérapie non adaptée, le traitement probabiliste d’une infection est associé à l’augmentation des résistances acquises chez les bactéries. Cependant, cette orientation nécessite de disposer de tests compagnons, c’est-à-dire des tests diagnostiques sensibles et spécifiques pouvant précocement identifier les bactéries et les marqueurs de résistance à partir des liquides biologiques. A côté des méthodes moléculaires largement développées mais ayant des limites de multiplexage, les techniques protéomiques ont récemment été intégrées dans le diagnostic en infectiologie. Ce travail de thèse a consisté à développer des méthodes de spéctrométrie de masse et à les appliquer à la détection de pathogènes et de marqueurs de résistance. Ce travail s’est focalisé sur trois applications : 1) l’identification, la caractérisation et la quantification précoce de micro-organismes dans des échantillons primaires (aspirats endotrachéaux (AET)) de patients atteints de pneumopathies acquises sous ventilation mécanique (PAVM), 2) la détection d’éléments génétiques de la résistance aux antibiotiques : les intégrons, 3) la détection de phénotypes de résistance aux antibiotiques chez Staphylococcus aureus. / Personalized medicine for infectious diseases or cancer becomes more and more important in modern therapy. Furthermore, probabilistic treatment has been associated with the development of resistant bacteria causing infectious diseases. As a result, probabilistic treatments are replaced by adapted treatment for pathologies and patients. However, this new approach needs available companion diagnosis tests that are sensitive but also specific tests able to provide rapid pathogen and resistance markers identification in biological fluids. Beside molecular methods, widely developed but with multiplex limits, proteomic technics have recently joined the infectious diagnosis. This work consisted in developing mass spectrometry technics for bacteria and resistance marker identifications. This work focused on 3 applications: 1) identification and quantitation of microorganisms in crude samples (endotracheal aspirates (ETA)) from patient suffering of ventilator associated pneumonia (VAP), 2) detection of genetic elements involved in antibiotic resistance : the integrons, 3) detection of antibiotic resistance phenotypes in S. aureus.

>Magister Scientiae - MSc / The aim of this study was to compare the trough plasma levels of Aspen-Vancomycin® (AV); and Sandoz-Vancocin CP® (SV) in premature infants with suspected Methicillin Resistant Staphylococcus aureus (MRSA) infection. The study was designed as a prospective, double blind, randomised trial involving male and female premature infants admitted in the Neonatal Intensive care Unit (NICU) at Netcare Blaauwberg and N1-city Hospitals for treatment of suspected MRSA-infection between April 2012 and June 2013. The inclusion criteria were: 29-35 weeks postmenstrual age (PMA), informed and written consent from parents of each premature infant enrolled in the study. Blood samples (0.3-0.4ml) were collected for renal function test and vancomycin trough levels determination. Blood samples for vancomycin trough level assay were collected thirty minutes prior to the administration of the third dose of vancomycin. Statistical analysis was performed and estimation was made giving an indication of how many infants will be needed to make the study statistically significant. Wilcoxon Two-Sample test was performed to determine the p-values and Spearman correlation coefficients were used to determine the correlation between trough levels and variables. P-values < 0.05 were considered significant. A total of 19 premature infants met with study criteria, 10 (5 females and 5 males) received AV and 9 (6 females and 3 males) receive d SV. There was no statistical significant difference between the demographic (GA, BW, PMA, PNA, weight at trial entry, height at trial entry) and biological (albumin, serum creatinine concentration and glomerular filtration rate) parameters of the premature infants in the AV and SV group. There were no statistical significant difference between trough level 1 of AV and SV, although trough level 1 had a lower trend in the SV group (p=0.118). No AV trough level 1 was below the minimum effective concentration (<5μg/ml). It was found that 30% of AV trough level 1 was within the therapeutic range (5-10μg/ml) and 70% of AV trough level 1, were above minimum toxic concentration (>10mg/l). It was found that 22.2% of SV trough level 1 was below minimum effective concentration, 44.4% of SV trough level 1 was within therapeutic range and 33.3% of trough level 1 was above minimum toxic concentration. No correlation was found between trough level 1 and the demographic and biological parameters of the premature infants in the AV group. SV had a positive correlation with GA, BBW, PMA and a negative correlation with PNA

Les infections de prothèses vasculaires (IPV) sont des maladies particulièrement graves. Malgré une fréquence finalement assez importante, elles demeurent mal connues. Staphylococcus aureus en est l’agent responsable principal. Les données concernant le traitement antibiotique à administrer pour ces infections sont excessivement pauvres. L’objectif de notre travail était donc de comparer l’efficacité de différents protocoles d’antibiothérapie à l’aide de divers modèles expérimentaux d’IPV. Six souches différentes de S. aureus ont été évaluées : 3 sensibles (SAMS) et 3 résistants à la méticilline (SARM). Nous avons comparé les concentrations minimales inhibitrices et éradicatrices (CMIB et CMEB) au sein du biofilm obtenues avec des techniques classiques sur polystyrène à ceux obtenus à l’aide d’un modèle original in vitro sur Dacron® (dCMIB et dCMEB) ®. Nous avons ensuite utilisé un modèle original d’infection de Dacron chez la souris pour comparer l’efficacité de différents protocoles thérapeutiques. Enfin nous avons visualisé l’effet de ces antibiotiques in vivo par microscopie confocale. Nous avons montré que les mesures classiques de CMIB et CMEB obtenues sur polystyrène pouvaient surestimer la baisse d’efficacité des antibiotiques dans le biofilm et que des mesures sur le matériel d’intérêt pouvaient être plus pertinentes. Dans notre modèle in vivo, la daptomycine pouvait être supérieure que les comparateurs pour certaines souches de SARM et de SAMS mais pas pour toutes. Par contre, si l’ajout de rifampicine était bénéfique pour la cloxacilline et la vancomycine, cela n’était pas le cas pour la daptomycine. Enfin, nous avons visualisés des effets totalement différents sur le biofilm selon les antibiotiques utilisés mais également selon les souches testées. Nos modèles ont permis d’obtenir des informations nouvelles concernant l’antibiothérapie des IPV qui, nous l’espérons, permettront d’aider à la prise en charge des patients. / Prosthetic vascular graft infection (PVGI) is an emerging disease, mostly due to staphylococci, with limited data regarding efficacy of current antistaphylococcal agents. We aimed to assess the efficacy of different antibiotic regimens. Six different strains of methicillin-susceptible (MSSA) and methicillin-resistant S. aureus (MRSA) were used. We compared results of minimal biofilm inhibitory and eradicating concentrations (MBICs and MBECs) obtained with a Calgary Biofilm Pin lid Device (CBPD) to those yielded by an original Dacron®-related minimal inhibitory and eradicating concentrations measure model. We then used an original murine model of Staphylococcus aureus vascular material infection to evaluate efficacy of different antibiotic regimens. We finally visualized the effect of antibiotics on biofilm by confocal microscopy. We demonstrated that classical measures of MBICs and MBECs with CPBD could overestimate the decrease of antibiotic susceptibility in material related infections and that the nature of the support used to measure biofilm susceptibility might be influent since results yielded by our Dacron®-related minimal eradicating assay were lower than those found on a plastic device. In our in vivo model, we shown that daptomycin was significantly more bactericidal than comparators for some strains of MRSA or MSSA but not for all. For the majority of strains, it was as efficient as comparators. The addition of rifampicin to daptomycin did not enhance daptomycin efficacy in our model. Finally, we highlighted an in vivo differential effect on biofilm depending on the antibiotic used but also on the bacterial strain evaluated. Our models represent an option to better define the best antibiotic options for PVGIs.

Primary sclerosing cholangitis (PSC) is a rare chronic liver disease characterized by inflammation and fibrosis of the biliary ducts, resulting in cholestasis and eventually liver failure. No effective treatment is currently available and most patients ultimately require liver transplantation in order to survive. The underlying mechanisms of the disease is poorly understood but a range of hypotheses exist, many of which recognize and grapple with PSC's close relationship with inflammatory bowel disease. Most agree genetics is involved, predisposing for an imbalance in 1) bile acid metabolism, 2) immune response and/or 3) gut microbiota. This literature study aims to describe and elucidate recent progress in the field of pharmacotherapy, as it relates to PSC and our current understanding of the disease. Covered in this study is a total of seven randomized, controlled trials, published between 2015-2022, and available through the medical database/search engine PubMed. Endpoints of particular note are ALP and ELF. ALP, or alkaline phosphatase, is an enzyme found in the liver. Rising levels of ALP in the blood stream is indicative of liver damage. ELF, or Enhanced Liver Fibrosis, is a blood test measuring markers of fibrosis, useful in assessing and staging fibrosis in chronic liver disease. Drugs included in this literature study are aldafermin, cilofexor, fenofibrate, norUrsodeoxicholic acid, obeticholic acid, simtuzumab and vancomycin. With the exception of aldafermin and simtuzumab, all showed promise as ALP reducing agents, in general lowering levels with 15-40 percent. In the case of fenofibrate, a reduction of 65 percent was observed. Of the drugs measured against ELF, only aldafermin produced a statistically significant reduction in fibrosis markers. At the time being it is not entirely clear what to make of the results, due to uncertainties surrounding ALP as a prognostic marker. To what extent ALP predicts transplantation free survival is still a matter of debate. Although considerable efforts have been made to further our understanding of PSC, much is yet to be solved. With regards to pharmacotherapy, the field is experiencing somewhat of a renaissance, showcased by the dozen on-going randomized, controlled trials on a plethora of potential PSC substances. Thus, the search for an effective therapy against PSC goes on.

Panther Fusion® system är ett fullständigt automatiserat in vitro diagnostiksystem som har högt flöde av prov-till-resultat och tillåter utförandet av in vitro diagnostiska tester genom användning av realtids Polymerase Chain Reaction (realtids-PCR). Syftet med detta projekt är att validera om det finns möjlighet att använda ett kit från Amplidiag på Panther fusion open access för att diagnostisera vankomycin resistenta enterokocker (VRE) samt att utvärdera analys direkt på E-swab prover istället för anrikningsbuljong. Det är viktigt att kunna detektera vanA-och vanB-generna i prover för att VRE hyser dessa gener. Sensitivitet och effektivitet av Panther fusion open access undersöktes genom att analysera proverna parallellt med Bio-Rad CFX384/96-PCR som är rutinmetoden för VRE-diagnostik på Klinisk mikrobiologi i Lund. Specificitet av en tidigare publicerad PCR-mix (PCR-mix) som kan detektera vanA och vanB i prover jämfördes med Amplidiag assay mix 2 som används i rutinmetoden. För att kunna utvärdera skillnaden av specificiteten och känsligheten mellan E-swab och anrikningsbuljong utfördes analysen på både E-swab och anrikningsbulong med både Panther fusion open access och Bio-Rad CFX384/96-PCR. MgCl2- koncentration optimerades till 3,0 mM. Sensitivitet och effektivitet av Panther fusion open access var högre med PCR-mix än Amplidiag assay mix 2 och uppvisade optimalt linjäritet. Specificitetstest av PCR-mixen visade att den bara kunde detektera vanA och vanB. Resultaten av spikade VRE-negativa patientprover (E-swab) visade sig vara orimliga då flera av proverna och kontrollerna gav resultat för båda generna. För att ersätta VRE-diagnostik på Bio-Rad CFX384/96 med Panther fusion open access behöver ytterligare experiment genomföras direkt på E-swab och vankomycine variabla enterokocker (VVE)-prover på Panther fusion open access. / Panther Fusion® system is a fully automated in vitro diagnostic system that has a high flow of sample-to-results and allows the performance of in vitro diagnostics tests using real-time Polymerase Chain Reaction (real-time PCR). The purpose of this project is to validate whether it is possible to use a kit from Amplidiag on Panther fusion open access to diagnose vancomycin resistant enterococcus (VRE). In addition, this project aims to evaluate and analyze E-swab samples directly instead of enrichment broth. It is important to be able to detect VanA and VanB genes in samples because VRE harbors these genes. Sensitivity and efficiency of Panther fusion open access were tested by analyzing the samples simultaneously with Bio-Rad CFX384/96 which is the routine method for VRE diagnostic at Clinical Microbiology in Lund. Specificity of a previously published PCR-mix (PCR-mix) that can detect VanA and VanB genes in samples was compared with Amplidiag assay mix 2 used in the routine method. In order to evaluate the difference in specificity and sensitivity between E-swab and enrichment broth, the analysis was performed on both E-swab and enrichment broth with both Panther fusion open access and Bio-Rad CFX384/96-PCR. MgCl2 concentration was optimized to 3,0 mM. Both sensitivity and efficiency of Panther fusion open access were higher with PCR-mix than those with Amplidiag assay mix 2 and it showed optimal linearity. Specificity test of PCR-mix detected only VanA and VanB genes. The results of inoculated the negative VRE-patient samples (E-swab) indicated that they were unreasonable because some of the samples and controls gave results of both genes. Replacement of the VRE-diagnostic method on Bio-Rad CFX384/96 with Panther fusion open access requires performing additional experiments directly on E-swab and vancomycin variable enterococcus (VVE) samples using Panther fusion open access.