Submitted by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-09-20T14:56:11Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO_Egidio_André_FINAL_26122016.pdf: 1444438 bytes, checksum: 846cb4588a5c486580f4c8af04030572 (MD5) / Approved for entry into archive by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-09-20T14:56:45Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO_Egidio_André_FINAL_26122016.pdf: 1444438 bytes, checksum: 846cb4588a5c486580f4c8af04030572 (MD5) / Made available in DSpace on 2017-09-20T14:56:45Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO_Egidio_André_FINAL_26122016.pdf: 1444438 bytes, checksum: 846cb4588a5c486580f4c8af04030572 (MD5) Previous issue date: 2016 / Centro Universitário São José. UNIFSJ / A colonização por Staphylococcus aureus representa o principal risco para infecções, principalmente em crianças, que apresentam elevada morbimortalidade relacionada a este patógeno. Tal microrganismo apresenta grande variabilidade molecular que confere mudanças epidemiológicas constantes. Linhagens com características variadas de resistência e virulência emergem e sucumbem, constituindo-se em um desafio para a saúde pública mundial. Nos últimos anos, alguns estudos têm sugerido uma mudança epidemiológica nas linhagens de S. aureus resistentes a meticilina (MRSA) no Brasil. O objetivo do presente estudo é atualizar o conhecimento acerca da epidemiologia molecular de MRSA colonizando crianças e adolescentes em Niterói – Rio de Janeiro. Trata-se de estudo de corte transversal, realizado com crianças e adolescentes, de zero a 16 anos, em creches, ambulatórios e hospitais na cidade de Niterói-RJ, no período de agosto de 2011 a junho de 2013. Um total de 1500 participantes (500 de creches, 500 de ambulatório e 500 de hospitais) foi submetido à coleta de secreção nasal por meio de swabs para pesquisa de MRSA. Destes, 749 (49,9%) das 1500 amostras foram caracterizados como S. aureus, sendo 288 (57,6%) das 500 amostras de ambulatório, 239 (47,8%) das 500 de hospitais e 222 (44,4%) das 500 das creches desta região. Do total, 144 (9,6%) das 1500 amostras foram caracterizadas como MRSA, sendo 31 (6,2%) das 500 das creches, 45(9%) das 500 de ambulatório e 68 (13,6%) das 500 dos hospitais. As 144 amostras de MRSA foram submetidas às técnicas de genotipificação por PCR dos genes mecA, lukS/lukFPV e SA442 para a identificação do gene de resistência a meticilina, da leucocidina Panton-Valentine (PVL) e confirmação da espécie S. aureus, respectivamente. Foram realizados testes de susceptibilidade antimicrobiana com 14 antimicrobianos selecionados de acordo com o CLSI (2012). Também foram realizados PCR-multiplex para identificação do tipo de cassete mec (SCCmec) e sequenciamento dos genes da proteína A (spa-Typing) e genes constitutivos (MLST), para a caracterização molecular das linhagens de MRSA. Observou-se a prevalência de diferentes clones de MRSA, incluindo os mais frequentes, ST5-MRSA-IV (CC5) e ST30-MRSA-IV (CC30), cujas características, corroboram com a literatura de linhagens de grande relevância, disseminadas em nível pandêmico, “Clone pediátrico” e “Clone SWP”, respectivamente. Nas creches e nos hospitais, foram identificados seis de complexos clonais (CC) diferentes em cada cenário. Já o ambulatório apresentou nove CCs diferentes. O PVL foi observado em 30 (54,5%) das 55 CC30. Das amostras classificadas como CC5 34 (57,6%) de 59 apresentaram resistência ou resistência intermediária à eritromicina. Evidenciou-se variação sazonal de colonização por MRSA, com maior frequência no verão. Nossos resultados confirmam a mudança epidemiológica do até então conhecido Clone Epidêmico Brasileiro para linhagens amplamente descritas pela literatura e disseminadas em nível pandêmico, com grande relevância, conhecidas como “Clone Pediátrico” e “Clone SWP” em crianças e adolescentes no Brasil, com variação sazonal na frequência de colonização por MRSA. Tais achados são relevantes para uma melhor compreensão do comportamento epidemiológico da colonização por MRSA em nível local, com informações que auxiliem nas estratégias de vigilância epidemiológica para o controle desse patógeno. / The colonization by Staphylococcus aureus (S. aureus) is the main risk factor for infections, especially in children who have high morbidity and mortality related to this pathogen. This microorganism has large molecular variability that provides constant epidemiological changes. Lineages with varied characteristics of resistance and virulence emerge and succumb, constituting a challenge to global public health. In recent years, some epidemiologic studies have suggested a change in strains of methicillin-resistant S. aureus (MRSA) in Brazil. The aim of this study is to update the knowledge of the molecular epidemiology of MRSA colonizing children and adolescents in Niterói - Rio de Janeiro. This is a cross-sectional study, conducted with children and adolescents in community day care centers, outpatient clinics and hospitals in Niterói, RJ, from August 2011 to June 2013. A total of 1500 participants (500 of day care centers, 500 of outpatient clinic and 500 of hospitals) was submitted to nasal secretion collection using swabs for MRSA research. Of these, 749/1500 (49.9%) were characterized as S. aureus, being that 288/500 (57.6%) were of outpatient clinic, 239/500 (47.8%) of hospitals and 222/500 (44.4 %) of the day cares centers. Among the samples of S. aureus 144/1500 (9.6%) were characterized as MRSA, with 31/500 (6.2%) from day care centers, 45/500 (9%) from outpatient clinics and 68/500 (13 6%) of hospitals. The 144 samples of MRSA were submitted to genotyping by PCR from the mecA, luks/lukFPV and SA442 genes to identifi of methicillin resistance gene, the Panton-Valentine leukocidin (PVL) toxin and species, respectively. Antimicrobial susceptibility tests were performed with 14 antimicrobials selected according to the CLSI (2012). It was also performed multiplex-PCR to identify the type of cassette mec (SCCmec) and sequencing of the genes of protein A (spa-Typing) and constitutive genes (MLST), for molecular characterization of MRSA lineages. We observed the prevalence of different MRSA, including the most commons, ST5-MRSA-IV (CC5) and ST30-MRSA-IV (CC30), whose characteristics corroborate with the literature of relevant lineages, disseminated on pandemic level, "Pediatric Clone" and "SWP Clone", respectively. In day care centers and hospitals six types of different clonal complexes (CC) have been identified in each environment, and the outpatient clinics had nine different CCs. The CC30 showed a frequency of 54.5% (30/55) for positivity of PVL genes. Samples classified as CC5 showed 57.6% (34/59) of resistance or intermediate resistance to erythromycin. This finding corroborates with literature, which describes about the epidemiological distribution of these strains. Our results confirm the epidemiological change of the Epidemic Brazilian Clone to strains widely described in the literature with pandemic level of disseminated and great relevance, known as "Pediatric Clone" and "SWP Clone" in children and adolescents in Brazil with seasonal variation in the frequency of MRSA colonization. These findings are relevant for a better understanding of the epidemiological changes of MRSA colonization locally, with information that assists in epidemiological surveillance strategies for the control of this pathogen.

Made available in DSpace on 2015-04-11T13:38:44Z (GMT). No. of bitstreams: 1 Dissertacao Mirna Miyamoto.pdf: 3227973 bytes, checksum: 7f5d27c4ca1e753ebe05cec66ce51343 (MD5) Previous issue date: 2010-06-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Staphylococcus aureus is a potential pathogen, accounting for 60% of infections in ICU`s and can be found in the nasopharyngeal region and also in the nasal cavity, especially in health care workers who become sources of dissemination of these microorganisms in hospital. However, the indiscriminate use of antibiotics to combat these pathogens, has caused the emergence of bacteria possessing resistance genes such as mecA. Of which are about 30 to 50% of the strains of S. aureus and more than 50% of coagulase-negative staphylococci. The CONS are increasingly becoming the target of concern in hospital environments, increasing the need for screening, prevention and control of resistant strains. Therefore, the objective of this work was the characterization of Staphylococcus sp., looking for the resistance gene and genetic mapping of S. aureus in clinical samples from the cities of Manaus and Porto Velho. The techniques used were biochemical tests, antibiogram, PCR (for 16 rRNA gene and mecA) and MLST. The four strains of CONS were found, three of Staphylococcus sciuri and S. epidermidis, characterized by phenotypic and genotypic tests. They were possessed of the methicillin resistance gene mecA. As for S. aureus studied, failed to detect the presence of the mecA gene, but gene mapping was performed from these samples with the technique of MLST, using five housekeeping genes. In this analysis we observed the formation of two large clusters between the cities of Manaus and Porto Velho, and two samples showed genetic divergence from other, thus demonstrating genetic variability between the cities of northern Brazil. The evidence related in the scientific literature, little research related to knowledge and epidemiological characterization of strains existing in the Amazon region, in this research, we discussed some questions of phenotypic analysis, characterization of new resistant strains and preparation of database for future development of biotechnological tools. / Staphylococcus aureus é um patógeno em potencial, sendo responsável por 60% das infecções nos CTIs, podendo ser encontrado na região da nasofaringe e também nas fossas nasais, principalmente em profissionais da saúde que se tornam fontes de disseminação desses micro-organismos no ambiente hospitalar. No entanto, o uso indiscriminado de antimicrobianos no combate desses patógenos, tem ocasionado o surgimento de bactérias possuidoras de genes de resistência, como o gene mecA, entre as quais, cerca de 30 a 50% das cepas de S. aureus e mais de 50% são de estafilococos coagulase-negativos são portadores deste gene. Os CONS estão cada vez mais se tornando alvo de preocupação nos ambientes hospitalares, aumentando a necessidade de rastreamento, prevenção e controle das cepas resistentes. Sendo assim, o objetivo deste trabalho foi realizar a caracterização de Staphylococcus sp., quanto a presença do gene de resistência e mapeamento genético de S. aureus em amostras clínicas das cidades de Manaus e Porto Velho. As técnicas aplicadas foram testes bioquímicos, antibiograma, PCR (para gene 16S rRNA e gene mecA) e MLST. Neste trabalho, encontramos quatro cepas de CONS, três de Staphylococcus sciuri e um S. epidermidis, caracterizados por testes fenotípicos e genotípicos, sendo estas possuidoras do gene de resistência a meticilina mecA. Quanto aos S. aureus estudados, não se detectou a presença do gene mecA, mas foi realizado o mapeamento gênico destas amostras com a técnica de MLST, utilizando cinco genes constitutivos. Nesta análise, foi observada a formação de dois grandes agrupamentos principais (clusters) entre as cidades de Manaus e Porto Velho, e duas amostras apresentaram divergência gênica das outras, demonstrando assim, variabilidade genética entre essas cidades da Região Norte do Brasil. Diante das evidências científicas na literatura, escassas pesquisas relacionadas ao conhecimento epidemiológico e caracterização das linhagens existentes na região Amazônica, nesta pesquisa, foram discutidas algumas questões de análise fenotípica, caracterizações de novas cepas resistentes e elaboração de banco de dados para futuro desenvolvimento de ferramentas biotecnológicas.

Introdução: Staphylococcus aureus é um patógeno que frequentemente coloniza pacientes cirróticos e transplantados de fígado. A colonização por S. aureus aumenta o risco de infecções invasivas por MRSA (Methicillin Resistant Staphylococcus aureus) o que aumenta a duração da hospitalização, os custos, a morbidade e a mortalidade. Com isso, é de grande importância o desenvolvimento de estratégias para identificação rápida, prevenção e controle de MRSA nesta população de pacientes. Objetivos: detectar MRSA em pacientes hepatopatas e descrever as características fenotípicas e moleculares de isolados MRSA neste grupo de pacientes. Metodologia: swabs nasais e inguinais foram coletados de 126 pacientes pré-transplante e 64 pacientes transplantados de fígado. Os swabs foram destinados para cultura microbiológica e também para extração direta de DNA. Foi determinada a Concentração Inibitória Mínima de 10 antimicrobianos de todos os isolados MRSA os quais também foram submetidos à caracterização de SCCmec, PFGE e spa typing. Amostras de DNA também foram submetidas à PCR 16S, MPCR coA/mecA e caracterização de SCCmec. Resultados: de acordo com a cultura microbiológica 12% dos swabs cultivados apresentaram crescimento de MRSA, resultando em 44 isolados MRSA. Todos os isolados foram resistentes à oxacilina, penicilina e sensíveis à vancomicina; todos amplificaram os genes coA e mecA; SCCmec tipo II e spa t002 predominaram nos dois grupos de estudo; além disso houve um cluster entre os isolados, o qual foi relacionado ao clone New York/Japan. O Clone Endêmico Brasileiro (BEC) foi relacionado somente à 4 isolados póstransplante. 98% das amostras de DNA amplificaram o gene 16S; a M-PCR identificou a presença de MRSA em 71% amostras de DNA e 61% das amostras tiveram a caracterização de SCCmec. O SCCmec tipo II predominou nos dois grupos de estudo. Conclusão: uma alta proporção de pacientes hepatopatas está colonizada com MRSA. Pacientes transplantados de fígado foram mais colonizados que pacientes cirróticos pela cultura microbiológica. A M-PCR coA/mecA diretamente de swabs foi mais sensível que a cultura microbiológica para identificar MRSA. Além disso, o PFGE demonstrou ter maior poder discriminatório que spa typing e não houve uma boa concordância entre os padrões de PFGE e spa types. Este estudo também demonstrou que está havendo uma mudança de clones MRSA em nosso hospital, devido à substituição do clone BEC pelo clone NY/Japan na última década / Introduction: Staphylococcus aureus is a pathogen that frequently colonizes cirrhotic and also liver transplanted patients. The S. aureus colonization increases the risk of invasive infections by MRSA (Methicillin Resistant Staphylococcus aureus) that increases the duration of hospitalization, costs, morbidity and mortality. Thus, it is of great importance to develop strategies for early identification, prevention and control of MRSA in this population of patients. Objectives: to detect MRSA in patients with liver diseases, in addition to observe the phenotypic and molecular characteristics of MRSA isolates in this group of patients. Methods: nasal and groin swabs were collected from 126 patients pre-transplant and 64 liver transplanted patients. The swabs were designed to microbiological culture and also to direct extraction of DNA. Determined the Minimum Inhibitory Concentration of 10 antimicrobials of all MRSA isolates which were also submitted to M-PCR of coA/mecA, characterization of SCCmec, PFGE and spa typing. DNA samples were also submitted to PCR of 16S, M-PCR of coA/mecA and characterization of SCCmec. Results: according to microbiological culture, 12% of swabs showed growth of MRSA, resulting in 44 MRSA isolates. All isolates were resistant to oxacillin, penicillin and vancomycin susceptible; all amplified the coA and mecA genes; SCCmec type II and spa t002 predominated in both groups; in addition there was a cluster among isolates, which was related to clone New York / Japan. Furthermore Brazilian Endemic Clone (BEC) was also related to 4 post transplantation isolates. 98% of DNA samples amplified 16S gene; M-PCR identified the presence of MRSA in 71% of DNA samples and 61% of the samples had the characterization of SCCmec. SCCmec type II was predominant in the two study groups. Conclusion: a high proportion of patients with liver disease are colonized with MRSA. Liver transplant patients are more colonized that cirrhotic patients by culture method. M-PCR CoA / mecA directly from swabs was more sensitive than microbiological culture to identify MRSA. Moreover, the PFGE shown to have a higher discriminatory power than spa typing and there was no good concordance between PFGE patterns and spa types. This study also demonstrated that there is a change of MRSA clones in our hospital, due to the replacement of the clone BEC by NY/Japan clone in the last decade

Na radiologia, válvulas antirrefluxo (VARs) são utilizadas em tubos de infusão para realização de exames de ressonância magnética e tomografia computadorizada. O objetivo desta pesquisa foi investigar procedimentos associados ao uso de VARs em exames radiológicos, a fim de contribuir para a biossegurança, controles da contaminação e do risco de infecção em sistemas de infusão. Uma Revisão Integrativa (RI) a respeito das VARs utilizadas na área da saúde foi realizada no que concerne os aspectos físicos, de funcionalidade e microbiológicos. A pressão máxima, em contra fluxo, dos diafragmas flexíveis (DFs) íntegros e rompidos das VARs foi determinada com auxílio do equipamento Hydraulic Burst-Leak Tester (HB-LT). A funcionalidade das VARs foi verificada por inspeção visual, em contra fluxo, mediante a liberação de bolhas de ar em um recipiente com água, por meio de simulação artificial da pressão da corrente sanguínea humana (SAPCSH). A estrutura e integridade das VARs foram identificadas por inspeção visual. A performance de colunas de ar interpostas por água em conectores com VARs foi analisada por inspeção visual (SAPCSH). A distância da difusão de um contaminante (cristal violeta) através de conectores com VARs foi mensurado por inspeção visual e espectrofotômetro (SAPCSH). A eficácia das VARs, em contra fluxo, como barreira de contaminação bacteriana foi avaliada em um experimento bacteriológico (SAPCSH). Um procedimento operacional padrão (POP) foi elaborado para direcionar a prática adequada do manuseio dos conectores com VARs, visando a biossegurança, controles da contaminação e do risco de infeção em sistemas de infusão em radiologia. Os resultados demostraram que houve diferença entre as pressões máximas suportadas pelos DFs dos Patient-set®: íntegros (média e desvio padrão: 595,44 ± 39,38psi) e rompidos (média e desvio padrão: 90,22 ± 31,26psi), em contra fluxo (p<0,0001). Por outro lado, os Patient-set® com os DFs rompidos não demonstraram falha na funcionalidade das VARs, mediante a ausência da liberação de bolhas de ar. Ainda, as VARs dos Patient-set® demonstraram DFs rompidos, entretanto as outras estruturas continuaram íntegras. As colunas de ar podem ser comprimidas e se deslocar através dos Patient-set® com DFs íntegros e rompidos. Além disso, a maior distância de difusão do cristal violeta foi de 30% (6cm) do comprimento do conector (20cm) do Patient-set®. Outrossim, a técnica de mensuração por espectrofotômetro (absorbância) foi mais sensível do que por inspeção visual para esse propósito. As VARs dos Patient-set® mostraram eficácia como barreira de contaminação bacteriana (Staphylococcus aureus resistente à meticilina, 10psi por 2h30min). Ademais, um POP foi elaborado e validado para a utilização e manuseio dos Patient-set®, visando a biossegurança na prática clínica, controles da contaminação e do risco de infecção em sistemas de infusão em radiologia como recomendação original a ser disponibilizada a todos os profissionais da área. Em suma, a biossegurança dos sistemas de infusão na radiologia depende de vários aspectos físico, de funcionalidade e microbiológico das VARs atrelados à execução adequada na prática clínica. Além disso, pesquisas adicionais são necessárias para elucidar questionamentos futuros sobre o uso seguro das VARs / In radiology, non-return valves (NRV) are used in infusion tubes to perform magnetic resonance imaging and computed tomography scan exams. The objective of this research was to investigate procedures associated to NRV usage in radiologic exams, in order to contribute to biosafety, contamination and infection risk controls in infusion systems. An Integrative Review (IR) about NRVs utilized in health field was performed concerning physical, functionality, and microbiological aspects. The maximum pressure, in backflow, of whole and broken flexible diaphragms (FDs) from NRVs was determined with Hydraulic Burst-Leak Tester (HB-LT) equipment help. The NRVs functionality was verified by visual inspection, in backflow, by means of air bubbles release in a water container, through human bloodstream pressure artificial simulation (HBPAS). The NRVs structure and integrity were identified by visual inspection. The performance of air columns interposed by water in connectors with NRVs was analyzed by visual inspection (HBPAS). Diffusion distance of a contaminant (crystal violet) through connectors with NRVs was measured by visual inspection and spectrophotometer (HBPAS). The NRVs efficacy, in backflow, as barrier to bacterial contamination was evaluated in a bacteriological experiment (HBPAS). A standard operating procedure (SOP) was elaborated to direct the proper handling of connectors with NRVs, aiming at biosafety, contamination and infection risk controls in infusion systems in radiology. The results showed that there was difference among maximum tolerated pressures by FDs of Patient-set®: whole (mean and standard deviation: 595.44 ± 39.38psi) and broken (mean and standard deviation: 90.22 ± 31.26psi), in backflow (p<0.0001). On the other hand, the Patient-set® with broken FDs didn\'t show failure in NRVs functionality, through the lack of air bubbles release. Moreover, NRVs showed broken FDs, but the other structures remained whole. The air columns can be compressed and move through Patient-set® with whole and broken FDs. Besides, the longest diffusion distance of crystal violet was 30% (6cm) of connector length (20cm) of Patient-set®. Furthermore, the measure technique by spectrophotometer (absorbance) was more sensitive than by visual inspection for this purpose. Patientset® NRVs showed efficacy as barrier to bacterial contamination (Methicillin-resistant Staphylococcus aureus, 10psi for 2h30min). Moreover, a SOP was elaborated and validated for Patient-set® utilization and handling, aiming at biosafety in clinical practice, contamination and infection risk controls in infusion systems in radiology as original recommendation to be made available to all professionals in the field. In conclusion, the biosafety depends on various physical, functionality, and microbiological aspects of NRVs coupled with proper practical clinical performance. Besides, further researches are needed to elucidate future questionings about NRVs safe use

The purpose of this thesis was to quantify the prevalence, measure the severity, and describe treatment patterns in patients who present to medical clinics in Texas with community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) skin and soft-tissue infections (SSTI). Ten clinics participated in this prospective, community-based study. Clinicians consented patients and collected clinical information, pictures, and wound swabs; data were processed centrally. MRSASelect[trademark] was used for identification. Susceptibilities were determined via Etest[registered sign]. The results are as follows. Overall, 73/119 (61%) patients had CA-MRSA. Among these, 49% were male, 79% were Hispanic, and 30% had diabetes. Half (56%) of the lesions were [greater than or equal to] 5 cm in diameter. Most patients had abscesses (82%) and many reported pain scores of [greater than or equal to] 7/10 (67%). Many presented with erythema (85%) or drainage (56%). Most received incision and drainage (I&D) plus an antibiotic (64%). Antibiotic monotherapy was frequently prescribed: sulfamethoxazole/ trimethoprim (SMX/TMP) (78%), clindamycin (4%), doxycycline (2%), and mupirocin (2%). The rest frequently received SMX/TMP in combination with other antibiotics. SMX/TMP was commonly administered as one double-strength tablet twice daily. Isolates were 93% susceptible to clindamycin and 100% susceptible to vancomycin, doxycycline, SMX/TMP, and linezolid. We report a predominance of CA-MRSA SSTIs, favorable susceptibilites, and frequent prescribing of SMX/TMP in primary care clinics located in South Texas. / text

Σκοπός της παρούσας ερευνητικής εργασίας ήταν η επιδημιολογική μελέτη των σταφυλοκοκκικών λοιμώξεων και κυρίως των λοιμώξεων από MRSA, τόσο στο ενδονοσοκομειακό περιβάλλον, όσο και στην κοινότητα, το διάστημα 2001-2006. Κατά τη διάρκεια της μελέτης, συλλέχθηκαν συνολικά 1922 στελέχη Staphylococcus aureus από όλες τις κλινικές και από διαφορετικούς ασθενείς στο Πανεπιστημιακό Γενικό Νοσοκομείο Πατρών (ΠΓΝΠ). Στη συνέχεια τα στελέχη ελέγχθηκαν για την παραγωγή της πρωτεΐνης PBP2α και την ύπαρξη του γονιδίου mecA για τον προσδιορισμό των ανθεκτικών στη methicillin στελεχών S. aureus (Methicillin-Resistant S. aureus, MRSA). Από το σύνολο των 1922 S. aureus, τα 757 (39.4%) χαρακτηρίσθηκαν ως MRSA. Γενικά, παρατηρήθηκε αύξηση του αριθμού των λοιμώξεων από S. aureus, με παράλληλη αύξηση του ποσοστού των MRSA λοιμώξεων, από 23% το 2001 στο 49% το 2006. Οι MRSA αποτελούν σοβαρό πρόβλημα για τα νοσοκομεία, αλλά και γενικότερα σε όλο το πληθυσμό, καθώς απομονώνονται με αυξανόμενη συχνότητα από την κοινότητα. Στο διάστημα που καλύπτει η παρούσα μελέτη, 2001-2006, το ποσοστό των στελεχών που απομονώθηκαν από την κοινότητα (Community-acquired MRSA, CA-MRSA) αυξήθηκε από 3% το 2001, σε 37% το 2006, ενώ εκείνο των ενδονοσοκομειακών στελεχών (Hospital-acquired MRSA, HA-MRSA), είναι ενθαρρυντικό ότι μειώθηκε από 20% το 2001, στο 12% το 2006. Για την επιδημιολογική μελέτη των λοιμώξεων που οφείλονται σε MRSA στελέχη, μέθοδος αναφοράς είναι η PFGE, κυρίως σε συνδυασμό και με υβριδισμό του χρωμοσωμικού DNA με ειδικούς ανιχνευτές. Oι μέθοδοι αυτές παρουσιάζουν δυσκολία στη σύγκριση των κλώνων μεταξύ των χωρών και έτσι αναπτύχθηκε η MLST, που βασίζεται στη εύρεση της νουκλεοτιδικής αλληλουχίας επτά συντηρημένων γονιδίων και επιτρέπει τον προσδιορισμό των κλωνικών συμπλεγμάτων (Clonal Complex, CC) με δυνατότητα άμεσης ταυτοποίησής τους. Τα MRSA που απομονώθηκαν κατατάχθηκαν σε επτά ST/SCCmec κλώνους, τους ST80/IV, ST5/IV, ST377/V, ST30/IVvar, ST239/III, ST225/NT και ST217/NT και πέντε κλωνικά συμπλέγματα (Πίνακας 46). Στα CA-MRSA επικρατεί ο ST80/IV, με μικρό ποσοστό να ανήκει και στον πρόσφατο τύπο ST377/V, ενώ στα HA-MRSA απαντώνται κυρίως οι ST239/III και ST30/IVvar. Η PVL είναι μια τοξίνη που καταστρέφει τα λευκοκύτταρα ανοίγοντας πόρους στην κυτταρική τους μεμβράνη, προκαλεί νέκρωση ιστών και νεκρωτική πνευμονία, κυρίως σε μικρά παιδιά, και κωδικοποιείται από τα γονίδια lukS-PV και lukF-PV. Το 74% των MRSA που μελετήθηκαν το διάστημα 2001-2006, έφεραν τα γονίδια αυτά, καταγράφοντας αύξηση από 33% το 2001, σε 88% το 2006. Τα PVL-θετικά MRSA ανήκουν στους κλώνους ST80/IV και ST377/V. Τα περισσότερα PVL-θετικά CA-MRSA της μελέτης μας απομονώθηκαν από λοιμώξεις δέρματος και μαλακών μορίων, ενώ τα PVL-θετικά HA-MRSA προέρχονταν από χειρουργικά τραύματα, κυρίως σε περιπτώσεις χρήσης προσθετικών υλικών. Ένα PVL-θετικό στέλεχος, που απομονώθηκε από αιματοκαλλιέργεια, προερχόταν από οστεομυελίτιδα. Πρόκειται για πρώτη αναφορά περιστατικών οξείας οστεομυελίτιδας, με αίτιο στελέχη CA-MRSA και MSSA που παράγουν PVL. Φαίνεται ότι η παρουσία των γονιδίων lukS-PV και lukF-PV συνδέεται κυρίως με λοιμώξεις που προκύπτουν δευτερογενώς σε τραύματα και επιπολής λοιμώξεις δέρματος και μαλακών μορίων, ενώ η απομόνωση PVL-θετικού στελέχους από οστεομυελίτιδα αποτελεί ένδειξη για την εμπλοκή της τοξίνης αυτής και στη παθογένεια εν τω βάθει λοιμώξεων. Εκτός από την PVL, σημαντικοί λοιμογόνοι παράγοντες και αίτια σοβαρών κλινικών συνδρόμων στον άνθρωπο θεωρούνται και οι τοξίνες της οικογένειας των υπεραντιγόνων, όπως η τοξίνη του συνδρόμου τοξικής καταπληξίας (TSST-1) και οι εντεροτοξίνες (SEs). Το γονίδιο tst κωδικοποιεί την παραγωγή της TSST-1, ενώ το οπερόνιο egc (enterotoxin gene cluster) την έκφραση πρωτεϊνών που μοιάζουν στη δομή και αλληλουχία με τις εντεροτοξίνες. Στα MRSA της συλλογής μας, τα γονίδια tst και egc2 ανιχνεύθηκαν μόνο στον κλώνο ST30/IV και δε βρέθηκαν ποτέ να συνυπάρχουν με τα γονίδια της PVL. Ανιχνεύθηκαν και συνδυασμοί των γονιδίων tst και του οπερονίου egc στους κλώνους ST80/IV και ST239/III, που σημαίνει ότι τα γονίδια αυτά διασπείρονται με οριζόντια μεταφορά, ενώ τα γονίδια lukS-PV και lukF-PV, που εντοπίστηκαν αποκλειστικά στους κλώνους ST80/IV ST377/V και χωρίς να συνυπάρχουν με γονίδια υπεραντιγόνων, φαίνεται ότι μεταφέρονται πιο ειδικά. Προϊόν της παρούσας ερευνητικής εργασίας ήταν η ανάπτυξη μεθοδολογίας για την ποσοτικοποίηση με αλυσιδωτή αντίδραση πολυμεράσης πραγματικού χρόνου, του γονιδίου tst σε στελέχη S. aureus που ήταν ανθεκτικά στη methicillin, κατατάσσονταν σε διαφορετικούς κλώνους και έφεραν ποικίλα γονίδια τοξινών. Για τη σήμανση και παρακολούθηση των προϊόντων χρησιμοποιήθηκε το SYBR Green I (SG), που είναι μη ειδικός τρόπος σήμανσης. Η χρήση του SYBR Green I κάνει τη μέθοδο εύχρηστη, αφού μπορεί η χρωστική να προστεθεί απλά στο υπόλοιπο μίγμα της αντίδρασης και φθηνή, επειδή δε χρειάζεται να σχεδιαστούν καινούριοι, ειδικοί εκκινητές. Οι αντιδράσεις απόλυτης ποσοτικοποίησης, που πραγματοποιήθηκαν με τη συγκεκριμένη μέθοδο, είχαν υψηλή απόδοση (2.04) και τα αποτελέσματα ήταν συνεχή και επαναλαμβανόμενα, γεγονός που σημαίνει ότι μπορεί να εφαρμοσθεί στη ρουτίνα του κλινικού εργαστηρίου για τη γρήγορη ανίχνευση και ποσοτικοποίηση του γονιδίου tst στα κλινικά στελέχη. Επί πλέον αποδείχθηκε με τη στατιστική ανάλυση, ότι στελέχη που απομονώθηκαν από λοιμώξεις μαλακών μορίων συνέθεταν υψηλότερα ποσά tst. Για τον υπολογισμό των λόγων έκφρασης του γονιδίου tst, στη σχετική ποσοτικοποίηση εφαρμόσθηκαν δυο μαθηματικά μοντέλα (2-ΔΔCt και Pfaffl). Συγκρίνοντας τα αποτελέσματα από τους δυο διαφορετικούς τρόπους υπολογισμού, βρέθηκαν διαφορές στα επίπεδα έκφρασης ίδιων στελεχών και καταλήξαμε στο συμπέρασμα ότι το μαθηματικό μοντέλο του Pfaffl είναι πιο ακριβές και αξιόπιστο, καθώς συνυπολογίζει την απόδοση της αντίδρασης. / The purpose of this study was to establish the clonality and evolution of CA-MRSA (Community-acquired MRSA, CA-MRSA) and HA-MRSA (Hospital-acquired MRSA, HA-MRSA), as well as the epidemiology of MRSA (Methicillin-Resistant S. aureus, MRSA) infections, during 2001-2006. In total 1922 Staphylococcus aureus strains were collected from patients with different pathologies admitted at the University Hospital of Patras. Among them 757 (39.4%) strains were MRSA. The prevalence of MRSA infections rose from 23% in 2001 to 49% in 2006. MRSA is a major problem worldwide in the nosocomial setting and the community. During 2001-2006 CA-MRSA isolated with an increasing rate from 3% in 2001 to 37% in 2006, while HA-MRSA decreased from 20% in 2001 to 12% in 2006. The epidemiological study of MRSA infections was based on PFGE, the “gold standard” of typing methods and hybridization with specific DNA probes. However, for the full characterization of a strain it is recommended the application of the multilocus sequence typing (MLST), since it is a highly discriminatory method and permits to compare the results from different laboratories. MLST represents a major advance since it relates organisms on the basis of the nucleotide sequences of ~450 bp internal fragments of seven conserved housekeeping genes resulting to the determination of Sequence Types (ST) and Clonal Complexes (CC). MRSA of our collection belonged to seven ST/SCCmec clones (ST80/IV, ST5/IV, ST377/V, ST30/IVvar, ST239/III, ST225/NT and ST217/NT) and five Clonal Complexes. Most CA-MRSA isolates belonged to ST80/IV clone and a small percentage to the newly described clone, ST377/V, while HA-MRSA strains were mainly characterized as ST239/III and ST30/IVvar clones. PVL is a bicomponent toxin associated with skin and soft tissue infections, but also with necrotizing pneumonia, especially in children. In total 74% of MRSA were positive for the PVL genes rising from 33% in 2001 to 88% in 2006. PVL-positive MRSA strains belonged to ST80/IV and ST377/V clones. Most PVL-positive CA-MRSA isolated from skin and soft tissue infections, while PVL-positive HA-MRSA isolated from surgical wounds, especially when prosthetic devices were used. In one patient acute staphylococcal osteomyelitis (AO) was diagnosed, due to MRSA carrying the PVL genes. This is the first description of CA-MRSA producing PVL as causative agents of AO suggesting that PVL-positive S. aureus can be isolated from patients with invasive musculo-skeletal infections, including acute childhood osteomyelitis, as well as among patient with skin and soft-tissue infections. Staphylococcal enterotoxins, enterotoxin-like superantigens (enterotoxin gene operon, egc) and the toxic shock syndrome toxin-1 that belong to the pyrogenic toxin superantigens (PTSAgs) are considered major virulence factors. The genotype tst/egc belonged only to ST30/IV clone and never coexisted with the PVL genes. Other combinations of genes were also detected belonging to clones ST80/IV and ST239/III, suggesting the horizontal transfer of those genes. On the contrary, the PVL genes were detected only in ST80/IV and ST377/V clones, meaning a more specific way of spread. A real-time PCR assay was developed for the quantification of tst gene in methicillin-resistant S. aureus using SYBR Green I (SG) chemistry, which is an easy and cost-effective approach to real-time, since it does not require the design of sequence specific probes and new primers. By the developed method of absolute quantification the results were reproducible and constant, meaning that the assay can be applied in the routine laboratory. The statistically significant difference of tst gene expression among strains associated with SSTIs, suggests that such strains may be the cause of TSS among patients. For the calculation of expression ratios in the relative quantification we applied two mathematical models (2-ΔΔCt and Pfaffl). Comparing ratios derived from the two mathematical methods we found variations, allowing us to suggest the use of the Pfaffl model as the more precise and reliable method.

Methicillin-resistenta Staphylococcus aureus (MRSA) som bär på genen mecA, har förekommit och spridit sig över hela världen, främst i sjukhusmiljö, och orsakat utbrott av vårdrelaterade (så kallade nosokomiala) infektioner. Dessa infektioner kan inte behandlas med stafylokock-penicilliner och MRSA-bakterierna är ofta resistenta även mot flera andra grupper av antibiotika vilket medför att infektionerna ofta är påtagligt svårbehandlade. Under senare år har emellertid allt fler fall beskrivits av samhällsförvärvad MRSA infektion, det vill säga uppträdande av MRSA hos personer som tidigare ej har haft kontakt med sjukhusvård eller behandlats med antibiotika. Det har länge varit oklart om de samhällsförvärvade MRSA [community-acquired (CA-MRSA)] representerar spridning av bakterier från sjukhusmiljön ut till samhället eller om dessa MRSA är spontant uppträdande. Många av dessa stammar har dessutom visat sig bära på sjukdomsrelaterade gener som vanligen inte återfinns hos S. aureus, t.ex. Panton Valentine leukocidin (PVL) som associeras med hudinfektioner och allvarlig lunginflammation med hög dödlighet hos unga och annars friska individer. Denna avhandling beskriver den molekylära epidemiologin hos MRSA med fokus på samhällsförvärvade MRSA som utgjorde mer än hälften av samtliga fall av MRSA i Örebro län och som dessutom ofta producerade PVL toxinet, vars funktion vidare analyserades i detalj. Undersökning av ursprung och släktskap hos samtliga MRSA som isolerats i Örebro län, samt karaktärisering av det genetiskt element som kallas staphylococcal cassette chromosome mec (SCCmec) vilket innehåller genen mecA och ibland även andra resistensgener, visade att CA-MRSA inte är relaterade till de nosokomiala MRSA, och att dessa har uppstått oberoende av varandra. Flertalet MRSA visade sig dessutom bära på SCCmec, och resistensmekanismer, som tidigare inte beskrivits. Troligen har dessa MRSA uppstått genom ett genetiskt utbyte av SCCmec mellan methicillin-resistenta koagulas-negativa stafylokocker (MR-KNS), som utgör huvudparten av normalfloran på huden, och methicillin-känsliga S. aureus som därvid erhåller genen mecA och resistensmekanismer mot samtliga stafylokockantibiotika. I den här avhandlingen framläggs bevis för att ett sådant genetiskt utbyte har skett på Barnkliniken på Universitetssjukhuset i Örebro i slutet på 1990-talet, vilket resulterade i uppkomsten av en ny klon av MRSA som därefter orsakade ett allvarligt utbrott. Kartläggning av DNA-sekvensen hos flertalet unika SCCmec från svenska MRSA gav dessutom en bättre förståelse för hur resistens uppkommer och sprider sig, samt mekanismerna bakom detta. Dessa nya kunskaper kan bidra till en förbättrad diagnostik av MRSA. Detta är framför allt av stor betydelse eftersom nya effektiva kloner av MRSA verkar kunna uppstå ute i samhället med potential att orsaka svårbehandlade infektioner men även att sprida sig bland den friska befolkningen. / Material and methods - During a period of 14 years, around 2000 patients with head injuries were admitted to the emergency ward at Lindesberg County Hospital and Örebro Medical Centre Hospital. Six hundred subjects suffered from skull fracture and/or brain contusion and diagnosis was established using a computed tomography scan (CT). The degree of initial brain injury was estimated using the Swedish Reaction Level Scale (RLS). Sixty-six subjects were investigated with pure tone audiometry in close proximity to the trauma, and this gave an opportunity to study the issue of progress. The investigation took place two to 14 years after trauma, and the results were compared to matched control groups. A battery of different audiological methods was used to investigate peripheral and central auditory function, and a specially designed acoustic environmental room was also utilized. Cognition was investigated using a computer-based test-battery, text information process system (TIPS). Self-assessed hearing, cognition and quality of life were explored using different questionnaires. Results - A high percentage of peripheral and central auditory impairments and also cognitive shortcomings were demonstrated. Progress of SNHL was a common finding, and fracture, high age at trauma and large initial hearing loss predicted progress. Antibody-mediated autoimmunity as a mechanism behind posttraumatic progress of SNHL or clear evidence for sympathetic cochleolabyrinthitis could not be demonstrated. Binaural auditory deficits could be demonstrated when tested in a realistic acoustic environment. Tinnitus, vertigo and memory shortcomings proved to be common sequelae, even in a long-term perspectiveCognitive shortcomings were found in several of these well-rehabilitated subjects.On a group level, there was a good correlation between self-assessments and audiometric results, even if some individuals had a tendency to over- or underestimate their abilities. Conclusion - Auditory and cognitive long-term sequelae of CHI are a common finding even in well-rehabilitated and socially well-functioning subjects, as are vertigo and tinnitus. Vertigo and tinnitus are also common sequelae after CHI, therefore a basic audiovestibular investigation after CHI is recommended, at least in selected cases.Early awareness of the risk for hearing and cognitive sequelae after CHI could lead to measurements taken to prevent tension-related symptoms.Early detection of HI offers an opportunity to try immunosuppressive treatment in cases with a large initial SNHL.

Η χρήση αντιμικροβιακών παραγόντων συνολικά καθώς και ειδικές κατηγορίες αντιμικροβιακών έχουν ενοχοποιηθεί ως παράγοντες κινδύνου για νοσοκομειακές λοιμώξεις (ΝΛ) από ανθεκτικό στη μεθικιλλίνη χρυσίζοντα σταφυλόκοκκο (methicillin-sensitive Staphylococcus aureus, MRSA). Οι στόχοι της μελέτης ήταν: 1. η διερεύνηση προβλεπτικών παραγόντων για νοσοκομειακή λοίμωξη από MRSA, 2. η σε βάθος αξιολόγηση του ρόλου της πρόσφατης χρήσης αντιμικροβιακών από ποιοτική και ποσοτική άποψη. Υλικό και μέθοδοι. Η χρονική περίοδος της μελέτης ήταν από Οκτώβριο1997 έως και τον Σεπτέμβριο του 2001. Οι ασθενείς με ΝΛ από MRSA σε κρατικό νοσοκομείο φροντίδας βετεράνων πολέμου των ΗΠΑ συγκρίθηκαν με αντίστοιχη ομάδα με ΝΛ από ευαίσθητο στη μεθικιλλίνη Staphylococcus aureus (MSSA). Οι λοιμώξεις στις 2 ομάδες καταγράφηκαν και τεκμηριώθηκαν μα βάση αυστηρά κριτήρια. Καταγράφηκαν δεδομένα σχετικά με παράγοντες κινδύνου (συμπεριλαμβανομένης της ποιοτικής και ποσοτικής χρήσης αντιμικροβιακών) για λοίμωξη από MRSA και ελήφθησαν υπόψη γεγονότα μέχρι 30 ημέρες πριν την ημερομηνία της θετικής καλλιέργειας. Αποτελέσματα. Εκατόν είκοσι επτά ασθενείς (127) με ΝΛ από MRSA και 70 ασθενείς με ΝΛ από MSSA αναλύθηκαν περαιτέρω. Σε μονοπαραγοντική ανάλυση, οι 2 ομάδες διέφεραν σημαντικά στην ηλικία, ιστορικό αιμοκάθαρσης και χρονίων ελκών, είδος κλινικής όπου ελήφθη το δείγμα, διάρκεια προηγούμενης νοσηλείας και παραμονής σε γενική ή καρδιολογική ΜΕΘ, πρόσφατη χειρουργική επέμβαση, άλλες επεμβατικές πράξεις, διασωλήνωση και παρουσία καθετήρα κύστεως για πάνω από 24 ώρες. Επίσης διέφεραν σημαντικά στη χρήση ή όχι τουλάχιστον 1,2 ή 3 αντιμικροβιακών, στο μέσο αριθμό αντιμικροβιακών που είχαν χρησιμοποιηθεί, μέσο αριθμό αντιμικροβιακών-ημερών για κάθε ασθενή, καθώς και ποιοτική και ποσοτική χρήση συγκεκριμένων κατηγοριών αντιμικροβιακών. Σε 2 μοντέλα πολυπαραγοντικής ανάλυσης, εξετάζοντας είτε την ποιοτική (μοντέλο Ι) είτε την ποσοτική (μοντέλο ΙΙ) χρήση αντιμικροβιακών, η διάρκεια παραμονής στο νοσοκομείο τον τελευταίο μήνα αναδείχθηκε ο ισχυρότερος προβλεπτικός παράγοντας λοίμωξης από MRSA (p 0.000), ενώ ιστορικό διασωλήνωσης έδειξε σημαντικότητα στο μοντέλο Ι και οριακή σημαντικότητα στο ΙΙ. Η χρήση αμινογλυκοσιδών και η παρουσία χρονίων ελκών έδειξαν τάση για σημαντικότητα στο μοντέλο Ι. Δεν παρατηρήθηκαν σημαντικές διαφορές στην έκβαση. Συμπεράσματα. Καταλήγουμε στο συμπέρασμα ότι η διάρκεια παραμονής στο νοσοκομείο τις τελευταίες 30 ημέρες αποτέλεσε τον ισχυρότερο προγνωστικό παράγοντα μεταγενέστερης ΝΛ από MRSA στον πληθυσμό των ασθενών μας. Παρά τη σαφώς μεγαλύτερη χρήση αντιμικροβιακών στην ομάδα MRSA, η χρήση αντιμικροβιακών δεν αποτέλεσε ανεξάρτητο προγνωστικό παράγοντα κινδύνου. Όσον αφορά την υπομελέτη, σκοπός ήταν η χρήση αντιμικροβιακών παραγόντων συνολικά καθώς και ειδικές κατηγορίες αντιμικροβιακών έχουν ενοχοποιηθεί ως παράγοντες κινδύνου για νοσοκομειακή βακτηριαιμία (ΝΒ) από ανθεκτικό στη μεθικιλλίνη χρυσίζοντα σταφυλόκοκκο (methicillin-resistant Staphylococcus aureus, MRSA). Από τα δεδομένα της κύριας μελέτης, που ήδη έχει περιγραφεί περιληπτικά παραπάνω, απομονώθηκαν και αναλύθηκαν σε ξεχωριστή υπομελέτη αυτά που αφορούσαν σε ασθενείς που ανέπτυξαν νοσοκομειακή βακτηριαιμία. Οι στόχοι της υπομελέτης ήταν: 1. η διερεύνηση προβλεπτικών παραγόντων για νοσοκομειακή βακτηριαιμία από MRSA, 2. η σε βάθος αξιολόγηση του ρόλου της πρόσφατης χρήσης αντιμικροβιακών από ποιοτική και ποσοτική άποψη. Υλικό και μέθοδοι. Η χρονική περίοδος της μελέτης ήταν από Οκτώβριο1997 έως και τον Σεπτέμβριο του 2001. Οι ασθενείς με ΝΒ από MRSA σε κρατικό νοσοκομείο φροντίδας βετεράνων πολέμου των ΗΠΑ συγκρίθηκαν με αντίστοιχη ομάδα με ΝΒ από ευαίσθητο στη μεθικιλλίνη Staphylococcus aureus (MSSA). Καταγράφηκαν δεδομένα σχετικά με παράγοντες κινδύνου (συμπεριλαμβανομένης της ποιοτικής και ποσοτικής χρήσης αντιμικροβιακών) για λοίμωξη από MRSA και ελήφθησαν υπόψη γεγονότα μέχρι 30 ημέρες πριν την ημερομηνία της θετικής καλλιέργειας Αποτελέσματα. Είκοσι οκτώ ασθενείς (28) με ΝΒ από MRSA και 32 ασθενείς με ΝΒ από MSSA αναλύθηκαν περαιτέρω. Σε μονοπαραγοντική ανάλυση, οι 2 ομάδες διέφεραν σημαντικά στην ηλικία, ιστορικό αιμοκάθαρσης και χρονίων ελκών, διάρκεια προηγούμενης νοσηλείας, ιστορικό και διάρκεια παραμονής σε γενική ή καρδιολογική ΜΕΘ, πρόσφατη χειρουργική επέμβαση, διασωλήνωση και παρουσία καθετήρα κύστεως για πάνω από 24 ώρες. Επίσης διέφεραν σημαντικά στη χρήση ή όχι τουλάχιστον 1,2 ή 3 αντιμικροβιακών, το μέσο αριθμό αντιμικροβιακών που είχαν χρησιμοποιηθεί, μέσο αριθμό αντιμικροβιακών-ημερών για κάθε ασθενή, καθώς και ποιοτική και ποσοτική χρήση συγκεκριμένων κατηγοριών αντιμικροβιακών. Στο μοντέλο πολυπαραγοντικής ανάλυσης Ι (ποιοτική χρήση αντιμικροβιακών), παρατηρήθηκε τάση (trend) για τη διάρκεια της προηγούμενης παραμονή στο νοσοκομείο ως προβλεπτικού παράγοντα (p 0,088), ενώ, από το μοντέλο ΙΙ (ποσοτική χρήση αντιμικροβιακών), ο μέσος αριθμός αντιμικροβιακών-ημερών ανά ασθενή αναδείχθηκε ως ο μόνος ανεξάρτητος προγνωστικός δείκτης ΝΒ από MRSA (p 0,03). Δεν παρατηρήθηκαν σημαντικές διαφορές στην έκβαση. Συμπεράσματα. Καταλήγουμε στο συμπέρασμα ότι η ποσοτική χρήση αντιμικροβιακών, υπό τη μορφή του μέσου αριθμού αντιμικροβιακών-ημερών ανά ασθενή κατά τη διάρκεια του περασμένου μήνα, ήταν ο ισχυρότερος προγνωστικός δείκτης της μεταγενέστερης ΝΒ από MRSA στον πληθυσμό των ασθενών μας, περισσότερο από άλλους παραδοσιακούς παράγοντες κινδύνου. / Both total antimicrobial use and specific antimicrobials have been implicated as risk factors for healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) infection. Aims. The aims of the study were: 1.to explore predictors of a new HA-MRSA infection in comparison with a new healthcare-associated methicillin-sensitive Staphylococcus aureus (HA-MSSA), 2. to thoroughly assess the role of recent antibiotic use qualitatively and quantitatively. Methods. The time-period for our study was from October 1997 through September 2001. Applying strict criteria, we identified two groups of inpatients, one with a new HA-MRSA infection and one with a new HA-MSSA infection. We recorded demographic, clinical and antibiotic use – related data up to 30 days before the positive culture date. Results.We identified 127 and 70 patients for each group respectively. Two logistic regression models were carried out to assess the role of antimicrobial use (qualitatively and qualitatively). In model I, duration of hospital stay, presence of chronic wounds, aminoglycoside and fluoroquinolone use retained statistical significance. In model II, duration of hospital stay and history of intubation during the last month stood out as the only significant predictors of a subsequent HA-MRSA infection. No siginificant differences in outcome were noted. Conclusions. The length of exposure to the hospital environment may be the best predictor of a new HA-MRSA infection. Use of aminoglycosides and fluoroquinolones may also stand independently along with presence of chronic ulcers and exposure to surgical procedures. No independent association between quantitative antibiotic use and subsequent HA-MRSA infection was documented. As for the substudy, the antimicrobial use has been implicated as risk factor for healthcare-associated methicilin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI). The aims of the study were: 1. to explore predictors of a new MRSA BSI, 2. to thoroughly assess recent antibiotic use qualitatively and quantitatively. Methods. Patients in contact with the healthcare system and MRSA BSI were compared with a methicillin-sensitive BSI group, considering events and risk factors up to 30 days before the positive culture date. Results. Twenty eight patients with MRSA BSI and 32 patients with MSSA BSI were further analyzed. In univariate analysis, significant differences were noted in specific demographic, comorbidity and hospital event-related parameters and also in mean number of antibiotics used, mean number of antibiotic-days per patient, and qualitative and quantitative use of specific classes of antibiotics. From logistic regression model I (qualitative antibiotic use), a trend was noted for duration of prior hospital stay, while, from model II (quantitative antibiotic use), mean antibiotic-days per patient emerged as the single independent predictor of HA-BSI by MRSA (p 0.03). No significant differences in outcome were noted. Conclusion. We conclude that the mean number of antibiotic-days per patient during the last month was the strongest predictor of a subsequent MRSA BSI in our patient population, more than other traditional risk factors.

Contexto: As infecções causadas pelo Staphylococcus aureus resistente a meticilina tem conseqüências graves aos pacientes e aos serviços de saúde. As medidas gerais de controle de infecção são fundamentais para conter a disseminação desta bactéria, mas não são sempre suficientes, e estratégias mais agressivas são empregadas muitas vezes para obter-se uma diminuição das taxas. Os estudos publicados para a avaliação da efetividade das medidas tem recebido criticas pela sua qualidade, e ainda não ha consenso sobre o assunto. Objetivos: Quantificar a prevalência de pacientes colonizados pelo Staphylococcus aureus resistentes a meticilina admitidos em um hospital terciário universitário, avaliar aspectos associados a transmissão da bactéria e estimar, através de modelo matemático, o efeito de medidas de isolamento de pacientes com Staphylococcus aureus resistentes meticilina na aquisic5o de colonização. Métodos: Um estudo de coorte prospectivo, com pacientes clínicos admitidos no hospital selecionados aleatoriamente na sua admissão. Os pacientes foram entrevistados na admissão e a cada semana e houve coleta de swab nasal para identificação de colonização pelo MRSA na admissão e a cada semana de internação. Os fatores de risco associados com a presença de colonização na admissão e sua aquisição durante a hospitalização foram investigados. Construiu-se um modelo determinístico de aquisição de MRSA considerando-se o impacto da prevalência de colonização basal pelo MRSA, a adesão a higienização das mãos e o use de isolamento na carga de trabalhos de enfermeiros e nas taxas de infecção pelo MRSA. Resultados: Foram selecionados 301 pacientes adultos e 189 pediátricos. A prevalência de MRSA na admissão foi de 5,3% (IC 95%: 3,1% a 8,5%) na população adulta, e 1,6% (1C95%: 0,3% a 4,6%) entre as crianças. A analise multivariável identificou os seguintes fatores de risco associados com a colonização em adultos na admissão: idade acima de 60 anos (RP = 2,9, IC 95%: 1,1 a 7,6), e internação no ano anterior (RP = 5,4, IC 95%: 1,3 a 23,4). A analise da incidência foi realizada em 285 pacientes que não eram portadores de MRSA na admissão, e que tiveram uma segunda amostra coletada. Entre estes, 9,5% (1C95%: 8,1 a 11,1) adquiriram colonização ou infecção durante a hospitalização. A taxa de colonização foi de 5,5/1.000 pacientes-dia entre os adultos (1C95%: 3,36 a 8,49), e de 1,6/1.000 pacientes-dia entre as crianças (1C95%: 0,33 a 4,61). 0 risco de aquisição aumentou conforme aumentou o tempo de internação, e este aumento foi estatisticamente significativo. No modelo proposto, se isolamento de pacientes aumenta o volume de trabalho dos trabalhadores da saúde, reduzindo a adesão aos protocolos básicos de prevenção de infecção, a execução das medidas de isolamento podem provocar o aumento da prevalência de MRSA. Entretanto, a aplicação de medidas de isolamento pode ter resultados diferentes dependendo da prevalência inicial MRSA e da carga de trabalho dos trabalhadores. A redução da carga de trabalho é mais custo-efetivo quando implementada para impedir a transição de um estado de baixa endemicidade para de alta. Conclusões: A identificação de portadores nasais de MRSA poderia ajudar a diminuir a pressão de colonização dentro do hospital e reduzir o alto índice de aquisição nesses pacientes. Entretanto, as medidas de controle devem ser cuidadosamente avaliadas num cenário com altas taxas de infecção, pois o grande numero de pacientes em isolamento pode comprometer a adesão a higienização de mãos ao aumentar a carga de trabalho dos enfermeiros, tendo efeito contrario ao desejado na prevenção de infecções. / Background: Infections caused by methicillin-resistant Staphylococcus aureus have serious consequences for patients and healthcare services. Basic infection control measures are essential to contain the spread of the bacteria, but are not always sufficient, and more aggressive strategies are often employed to obtain a reduction of rates. Studies published to assess the effectiveness of the measures have received criticism for its quality, and there is no consensus if the search and destroy policy is necessary Objectives: To quantify the prevalence of patients colonized by methicillin-resistant Staphylococcus aureus admitted in a tertiary university hospital, evaluate factors associated with transmission of the bacteria and to estimate, through a deterministic mathematical model, the effect of isolation measures of patients with the bacteria in the acquisition of colonization. Methods: A prospective cohort with clinical patients randomly selected in their entry to a hospital. Patients were interviewed on admission and weekly, with collection of nasal swab for identification of MRSA colonization at admission and every week of hospitalization. Risk factors associated with the presence of colonization on admission, and associated to its acquisition during hospitalization were investigated. A deterministic model for acquisition of MRSA was built, considering the impact of the baseline prevalence of MRSA colonization, adherence to hand hygiene and use of isolation measures in the workload healthcare workers and on the rates of MRSA infection. Results: We randomly selected 301 adult and 189 pediatric patients. The prevalence of MRSA at admission was 5.3% (95% CI, 3.1% to 8.5%) in adults and 1.6% (95% CI, 0.3% to 4.6%) among children. Multivariate analysis identified the following risk factors associated with colonization of adults at admission: age over 60 years (PR = 2.9 95% CI 1.1 10 7.6) and hospitalization in the previous year (PR = 5.4, 95% CI 1.3 to 23.4). The analysis of incidence was performed in 285 patients who were not carriers of MRSA on admission, and who had a second sample collected. Of these, 9.5% (95% CI 8.1 to 11.1) acquired colonization or infection during hospitalization. The colonization rate was 5.5 / 1,000 patient-days among adults (1C95%, from 3.36 to 8.49) and 1.6 / 1,000 patient-days among children (95% Cl, 0.33 to 4.61). The risk of acquisition increases as the length of stay increases, and this increase is statistically significant. In the proposed model, if isolation of patients increases the workload of health workers, and reduces adherence to the basic protocols for infection prevention, the implementation isolation can increase the prevalence of MRSA. However, the application of isolation can have different results depending on the initial prevalence of MRSA and the effect on healthcare workers workload. The reduction in workload is more cost-effective when implemented to prevent the transition from a state of low to hitzh. endemicity. Conclusions: The identification of nasal carriers of MRSA could help reduce the colonization pressure within the hospital and reduce the high rate of acquisition in these patients, but the measures of control should be carefully evaluated in a setting with high rates of infection, because the great number of patients in isolation can compromise adherence to hand hygiene of increasing the workload of the nurses, and result in the opposite effect in preventing infections.

O Staphylococcus aureus é bactéria espécie não específica que, além de potencial patogenicidade, evoluiu em mecanismos de resistência a antimicrobianos. O S. aureus meticilina resistente (MRSA) antes restrito a infecções nosocomiais, dispersou-se na comunidade e nos animais de companhia e para produção de alimento. Na conduta para o controle da transmissão, além do uso de antibióticos, a ação sobre os agentes causais nas fontes de contaminação exige atenção, sendo decisiva e crítica a escolha de desinfetantes e antisépticos. A busca por recursos frente a agentes patogênicos resistentes a antimicrobianos convencionais e a demanda por insumos sanitários aplicáveis em modelos sustentáveis de produção agropecuária, motivam a investigação de extrações vegetais que apresentem atividade antibacteriana. O objetivo deste estudo foi avaliar a atividade bactericida de desinfetantes convencionais sobre isolados MRSA, testar a hipótese da possibilidade de resistência cruzada entre grupos químicos antibióticos (beta-lactâmicos) e desinfetantes e também avaliar a atividade bactericida de extrações das inflorescências de Achyrocline satureioides (Lam.) DC. (Asteraceae) (“macela”), planta medicinal, de uso popular e tradicional, nativa na região sul do Brasil, sobre os mesmos inóculos. A técnica de referência foi o “Teste de Suspensão na Avaliação Quantitativa da Atividade Bactericida de Desinfetantes e Antissépticos Químicos”. Nos testes com os desinfetantes hipoclorito de sódio (HS), iodofór (I) e quaternário de amônio (QAC - cloreto de cetil trimetilamônio), quatro concentrações de cada grupo químico foram confrontadas com 21 MRSA, em tempos de contato de cinco, 15 e 30 minutos e densidade populacional inicial dos inóculos de 107UFC/mL. Observou-se que os grupos químicos nas menores concentrações HS 25 ppm, I 12,5 ppm e QAC 125 ppm, apresentaram atividade bactericida frente a todos os isolados no menor tempo de contato. A proporção usada da A. satureioides foi de 5 g:100 mL de solvente e as densidades iniciais dos inóculos confrontados foram 107, 106 e 105 UFC/mL. A atividade da forma decocto foi verificada frente a 51 isolados MRSA, em tempos de contato de uma, oito e 24 h. Vinte e um deles também foram submetidos ao extrato hidroetanólico hidratado (EH), obtido de maceração hidroetanólica 70º GL, desalcoolizada e hidratada ao volume inicial, nos tempos de contato de cinco e 30 minutos e de uma até quatro horas. Por Cromatografia Liquida de Alta Eficiência (CLAE) foi confirmada, no acesso da planta, a presença dos marcadores fitoquímicos quercetina, luteolina e 3-Ometilquercetina. As extrações da A. satureioides apresentaram atividade antibacteriana frente a todos os isolados MRSA. O EH mostrou atividade de inativação em menor tempo. Tomando como exemplo a 1 h de contato, na maior densidade do inóculo, 19% dos isolados estavam inativados, enquanto que no decocto não foi observada inativação. Em 4 horas de contato com o EH 85,7% dos isolados, na maior densidade desafio, estavam inativados e 100% dos isolados sofreram redução da densidade populacional. O decocto demonstrou maior atividade bactericida entre 8h e 24 horas, inativando 100% dos isolados até as 24 h. Concluiu-se que, controlados os conhecidos fatores limitantes da atividade bactericida, o hipoclorito de sódio o iodofor e o quaternário de amônio são adequados para controlar os MRSA nas fontes de contaminação em ambientes de saúde humana ou nos de saúde e de produção animal. Para os isolados resistentes aos antibióticos beta-lactâmicos confrontados não foi observada relação de resistência com os desinfetantes. A atividade bactericida das soluções de Achyrocline satureioides frente aos isolados MRSA e ao microrganismo de referência Staphylococcus aureus ATCC 6538 sugere seu potencial uso, diretamente nas formas avaliadas ou em formulações, em procedimentos de higiene, tanto nas fontes de infecção de ambientes de saúde humana quanto nos de saúde e de produção animal. / The bacterium Staphylococcus aureus is a speciea not specific and potential pathogenicity, which has evolved mechanisms for antimicrobial resistance. S. aureus methicillin resistant (MRSA) once restricted to nosocomial infections, dispersed in the community and in companion animals and production. In order to control the transmission, besides the use of antibiotics, the action on the causative agents in the sources of contamination requires attention, being decisive and critical the choice of disinfectants and antiseptics. The search for resources against pathogens resistant to conventional antibiotics and the demand for health inputs applicable in sustainable agriculture production motivated the investigation of plant extractions that have antibacterial activity. The aim of this study was to evaluate the bactericidal activity of conventional disinfectants on MRSA isolates, testing the hypothesis of the possibility of cross-resistance between chemical groups antibiotics (beta-lactams) and disinfectants and also assess the bactericidal activity of extractions of inflorescences Achyrocline satureioides (Lam.) Dc. (Asteraceae) ("macela"), a medicinal plant with a popular and traditional use, native from southern Brazil on the same isolates. The reference technique was the "Suspension Test in Quantitative Evaluation of Bactericidal Activity of Chemical Disinfectants and Antiseptics." In tests with disinfectant sodium hypochlorite (HS), iodophor (I) and quaternary ammonium (QAC-cetyl trimethylammonium chloride), four concentrations of each chemical group were confronted with 21 MRSA on contact time of five, 15 and 30 minutes. The population density of the initial inoculum was 107UFC/mL. It was observed that the chemical groups at lower concentrations HS 25 ppm, and 12.5 ppm I 125 ppm QAC showed bactericidal activity against all isolates in less contact time. The proportion used of A. satureioides was 5 g: 100 mL of solvent and the initial densities of the inocula confronted were 107, 106 and 105 CFU / mL. The activity of decoction form was checked against 51 MRSA isolates, in times of a contact, eight and 24 h. Twenty-one of them also underwent hydroethanolic extract hydrate (EH) obtained by maceration hydroethanol 70 º GL, dealcoholised hydrated and the initial volume, the contact time of five and 30 minutes and one to four hours. By High performance liquid chromatography (HPLC) was confirmed in the access plan, the presence of markers phytochemicals quercetin, luteolin and 3-O-methylquercetin. The extractions of A. satureioides showed antibacterial activity against all MRSA isolates. The EH showed activity inactivation in less time. Taking as an example the 1 hr of contact, the greater density of the inoculum, 19% of the isolates were inactive, whereas the decoction did not show inactivation. In 4 hours of contact with the EH 85.7% of isolates in higher density challenge were inactivated and 100% of isolates reduced population density. The decoction showed greater bactericidal activity between 8 and 24 hours, inactivating 100% of isolates until 24 h. It was concluded that, controlling for known factors affecting the bactericidal activity, the sodium hypochlorite and quaternary ammonium iodophor are suitable for controlling MRSA in the sources of contamination in healthcare environments or in human health and animal production. For the isolates resistant to beta-lactam antibiotics confronted no relationship was observed resistance to disinfectants. The bactericidal activity of solutions Achyrocline satureioides against of MRSA isolates and reference microorganism Staphylococcus aureus ATCC 6538 suggests its potential use, directly into forms or formulations evaluated in hygiene procedures, both in the sources of infection in healthcare environments as in human health and animal production.