Pacientes com câncer de mama localmente avançado são submetidas à quimioterapia neoadjuvante na tentativa de reduzir a dimensão do tumor e aumentar a possibilidade da realização de uma cirurgia conservadora. Nosso grupo identificou previamente através da tecnologia de cDNA microarray, trios de genes, incluindo BZRP, CLPTM1, MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2 e XLHSRF-1, cuja expressão era capaz de predizer a resposta à quimioterapia neoadjuvante com doxorrubicina e ciclofosfamida em pacientes com câncer de mama. No presente estudo, avaliamos se a expressão destes genes é reprodutível na identificação de pacientes responsivas e não-responsivas através de RT-PCR em tempo real, que representa uma técnica mais acessível. Avaliamos inicialmente amostras de 28 pacientes anteriormente estudadas (grupo de validação técnica = 23 responsivas e cinco não-responsivas) e a seguir um grupo de 14 novas pacientes (grupo de validação biológica = 11 responsivas e três não-responsivas). Dentre os trios de genes inicialmente identificados, a expressão de RPL37A + XLHSRF-1 + NOTCH1 e RPL37A + XLHSRF-1 + NUP210 classificou corretamente 86% (24/28) das amostras do grupo de validação técnica e 71% (10/14) das amostras do grupo de validação biológica, através de análise de classificação discriminante. Desse modo, esses trios não demonstraram a mesma precisão em comparação com resultados de cDNA microarray. Uma nova análise combinatória foi realizada na procura do melhor modelo preditivo utilizando valores de expressão obtidos por RT-PCR em tempo real. Identificamos então um novo trio, composto pelos genes RPL37A, SMYD2 e MTSS1, cuja expressão classificou corretamente 93% das amostras do grupo de validação técnica (22/23 responsivas e 4/5 não-responsivas) e 79% do grupo de validação biológica (8/11 responsivas e 3/3 não-responsivas). Portanto, o teste apresentou 88% de sensibilidade e especificidade em detectar pacientes responsivas para o total de amostras analisadas. Ao verificarmos o poder de classificação do mesmo grupo de genes, utilizando os valores de expressão pela análise de cDNA microarray, observamos um resultado semelhante (91% de sensibilidade e especificidade em reconhecer as amostras responsivas). Dessa forma, demonstramos que o perfil de expressão gênica obtido com cDNA microarray é reprodutível através do uso de RT-PCR em tempo real. Um estudo integrando um maior número de pacientes e uma plataforma de cDNA microarray mais abrangente pode auxiliar na identificação de um modelo preditivo baseado em grupos de genes mais acurado para antever a resposta ao tratamento com quimioterapia baseada em doxorrubicina. / Patients with locally advanced breast cancer are submitted to primary chemotherapy as an attempt to reduce tumor dimension and increase breast conserving surgery rates. Our group has previously identified through cDNA microarray technology gene trios, including BZRP, CLPTM1, MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2 and XLHSRF-1, whose expression was capable of predicting response to neoadjuvant chemotherapy with doxorubicin and cyclophosphamide in breast cancer patients. In the current study, it was evaluated whether expression of these genes is reproducible in the identification of responsive and non-responsive patients by real time RT-PCR, which represents a more accessible technique. We initially evaluated samples from 28 patients earlier studied (technical validation group = 23 responsive and 5 non-responsive) and subsequent to a new 14 patients set (biological validation group = 11 responsive and three non-responsive). Among the initially identified gene trios, RPL37A + XLHSRF-1 + NOTCH1 and RPL37A + XLHSRF-1 + NUP210 expression correctly classify 86% (24/28) samples from the technical validation group and 71% (10/14) samples from the biological validation group, through discriminant classification analysis. Therefore, these trios didnt demonstrate the same precision as compared with cDNA microarray results. A new combinatorial analysis was also performed in search of the best predictive model using real time RT-PCR expression values. A new trio was identified, represented by RPL37A, SMYD2 and MTSS1 genes, whose expression correctly classified 93% samples from technical validation group (22/23 responsive and 4/5 non-responsive) and 79% samples from biological validation group (8/11 responsive samples and 3/3 non-responsive samples). Therefore, the test presented 88% sensibility and specificity in identifying responsive patients for all samples analyzed. By means of verifying the classification strength of the same gene group, using cDNA microarray expression values, we observed a similar result (91% sensibility and specificity in recognizing responsive samples). Thus, we demonstrated that gene expression profile obtained by cDNA microarray is reproducible through real time RT-PCR. A study integrating a larger number of patients and a more comprehensive cDNA microarray platform may help the identification of a more accurate predictive model based on gene groups to foresee response to doxorubicin-based chemotherapy treatment.

INTRODUÇÃO: A frequência de Candida parapsilosistem apresentado considerável aumento em UTIs neonatais. Embora a taxa de resistência dessa espécie aos azólicos seja baixa, recentemente têm sido relatados surtos de candidemia por isolados resistentes. A capacidade de adesão e formação de biofilme por essa espécie confere maior potencial patogênico e resistência aos antifúngicos. Portanto, a vigilância epidemiológica, tanto da resistência aos antifúngicos como da virulência dos isolados, é fundamental para o controle e prevenção das infecções e surtoshospitalares. A técnica de MALDI-TOF MS pode ser uma ferramenta útil para realizar análises proteômicas das células planctônicas e sésseis de Candida parapsilosis,e identificar possíveis alvos terapêuticos ou biomarcadores, específicos do biofilme. MÉTODOS: Isolados clínicos do complexo Candida parapsilosis de dois hospitais universitários públicos brasileiros, foram submetidos à identificação por RAPD, RFLP e MALDI-TOF MS e aos testes de suscetibilidade aos antifúngicos. Ensaios de formação de biofilme foram realizados para quantificar a biomassa, a atividade metabólica e ainda, avaliar atividade in vitrodos antifúngicos contra as células sésseis dos isolados com alta formação de biofilme. A análise proteômica por MALDI-TOF MS das células planctônicas e sésseis dos isolados com alta formação de biofilme, foi realizada nas plataformas VITEK-MS(TM) e Microflex(TM). Isolados de Candida parapsilosis (sensu stricto) foram genotipados por PFGE e análise de microssatélites. Os genótipos foram correlacionados com dadosclínicos, para investigar a ocorrência de um surto em CTI adulto, e as sequências do gene ERG11dos isolados não suscetíveis aos azólicos (NSA) foram analisadas. RESULTADOS: Foram obtidos 38 isolados do complexo Candida parapsilosis, sendo Candida parapsilosis(sensu stricto) a espécie de maior frequência, superando 80% em ambos os hospitais, seguida de C. orthopsilosis e C. metapsilosis. Embora todos os isolados tenham sido suscetíveis à anfotericina B ( < 2 mg/L) e apresentado suscetibilidade intermediária à anidulafungina, caspofungina e micafungina ( > 0,002 mg/L), elevada frequência de não suscetibilidade (resistência ou suscetibilidade intermediária) ao fluconazol e voriconazol foi observada entre isolados de um dos hospitais. Alta formação de biofilme foi observada apenas entre os isolados da espécie Candida parapsilosis(sensu stricto). Por outro lado, a maioria dos isolados NSA, apresentou baixa formação de biofilme e baixa atividade metabólica. Apenas anfotericina B apresentou atividade contra os biofilmes de Candida parapsilosis. As duas plataformas de MALDI-TOF MS conseguiram diferenciar os perfis proteômicos das células planctônicas e sésseis dos isolados. A genotipagem de Candida parapsilosis(sensu stricto) revelou a persistência de isolados clonais NSA e a mutação A395T no gene ERG11foi identificada exclusivamente entre os isolados resistentes ao azólicos. O uso de corticosteroide foi associado, estatisticamente, com a ocorrência de isolados clonais NSA. CONCLUSÕES: Candida parapsilosis (sensu stricto) se mantém como a principal espécie do complexo em infecções sanguíneas. Isolados resistentes aos azólicos, com mutações no gene ERG11, ocorreram nos dois hospitais avaliados. A correlação dos genótipos com os dados clínicos evidenciou a ocorrência de um surto envolvendo isolados clonais NSA, com associação estatisticamente significativa, ao uso prévio de corticosteroides. Candida parapsilosis (sensu stricto) foi a única espécie que apresentou alta formação de biofilme, o qual demonstrou elevada resistência às equinocandinas. As duas plataformas de MALDI-TOF MS, diferenciaram os perfis proteômicos, das células planctônicas e sésseis de Candida parapsilosis, demonstrando o potencial emprego dessa tecnologia na identificação de possíveis alvos terapêuticos ou biomarcadores, específicos de biofilmes / INTRODUCTION: The frequency of Candida parapsilosis isolates has increased considerably in neonatal ICUs. Although resistance to azoles is usually low in this species, candidemia outbreaks by resistant isolates have been recently reported. Theability of adhesion and biofilm formation by this species confers higher pathogenic potential and resistance to antifungal agents. Therefore, establishment of profiles of antifungal susceptibility and virulence, besides the epidemiological surveillance ofC. parapsilosisisolates are essential for the control and prevention of nosocomial infections and outbreaks. The MALDI-TOF MS technique can be a useful tool to perform proteomic analyzes of the planktonic and sessile cells of Candida parapsilosis, identifying possible biofilm-specific therapeutic targets or biomarkers. METHODS: Candida parapsilosisclinical isolates from two Brazilian public university hospitals were identified by RAPD, RFLP and MALDI-TOF MS and submitted to antifungal susceptibility tests. Biofilm formation assays were carried out to quantify the biomass and metabolic activity, and to evaluate the in vitroactivity of antifungal drugs against the sessile cells of the isolates with high biofilm formation. Proteomic analysis of the planktonic and sessile cells of the isolates with high biofilm formation was performed in two MALDI-TOF MS platforms, VITEK-MS(TM) and Microflex(TM). Candida parapsilosis(sensu stricto) isolates were genotyped by PFGE and microsatellite analysis. The genotypes were correlated with clinical data to investigate the occurrence of an outbreak in the adult ICU andERG11gene sequences from non-susceptible to azoles (NSA) isolates were also analyzed. RESULTS: 38 clinical isolates of the Candida parapsilosiscomplex were obtained, with Candida parapsilosis(sensu stricto) being the most frequent species (exceeding 80% in both hospitals), followed by C. orthopsilosisand C. metapsilosis. Although all isolates were susceptible to amphotericin B ( < 2 mg/L) and showed intermediate susceptibility to anidulafungin, caspofungin e micafungin ( > 0,002 mg/L), high frequency of non-susceptibility (resistance or intermediate susceptibility) to fluconazole and voriconazole was observed among isolates from one of the hospitals. High biofilm formation was only observed among isolates of the Candida parapsilosis. (sensu stricto) species. On the other hand, most of the NSA isolates presented low biofilm formation and low metabolic activity. Only amphotericin B showed activity against Candida parapsilosisbiofilms. The two MALDI-TOF MS platforms were able to differentiate the proteomic profiles of planktonic and sessile cells of isolates. Candida parapsilosis(sensu stricto) genotyping revealed the persistence of clonal NSA isolates. The A395T mutation in the ERG11gene was identified exclusively among azole resistant isolates. The use of corticosteroid was statistically associated with the occurrence of clonal NSA isolates. CONCLUSIONS: Candida parapsilosis(sensu stricto) remains the main species of the complex in bloodstream infections. Azole-resistant isolates with mutations in the ERG11gene are emerging in the two hospitals evaluated. Additionally, the correlation between the genotypes and the clinical data showed the occurrence of an outbreak involving isolates resistant to azoles, with a statistically significant association with previous use of corticosteroids. Candida parapsilosis(sensu stricto) was the only species that presented high biofilm formation and resistance against echinocandins. The two MALDI-TOF MS platforms differentiated the proteomic profiles of the planktonic and sessile cells of Candida parapsilosis, demonstrating the potential use of this technology to identify possible biofilm-specific therapeutic targets or biomarkers

Made available in DSpace on 2014-07-29T15:25:19Z (GMT). No. of bitstreams: 1 Tese Marlene Andrade Martins.pdf: 838189 bytes, checksum: 6a399180bc5d72130785d31bc207a701 (MD5) Previous issue date: 2012-04-05 / INTRODUCTION: In recent years, there has been the emergence of multidrug-resistant strains from patients seen in primary lesions with venous ulcers. OBJECTIVES: To determine the frequency and susceptibility profile of Staphylococcus sp.; verify the prevalence of methicillin resistant Staphylococcus aureus (MRSA) and minimum inhibitory concentration (MIC) of isolates; detect MLSB resistance in Staphylococcus sp. isolated from venous ulcers; describe the frequency of clinical signs and symptoms indicative of infection of venous ulcers (2005); identify the clinical stage of infection, to determine the relationship between clinical signs and symptoms of infection and culture results found for Staphylococcus sp. METHODS: Were evaluated 69 people with 98 ulcers in the period of October/09 to October/2010. The isolates resistant to cefoxitin and/or oxacillin (disk diffusion) were subjected to confirmatory test for detection of MIC, using tapes of oxacillin (E-test ®). The phenotypic detection of the inducible resistance to the MLSB group was performed by the D-test. The clinical signs and symptoms were investigated in accordance with criteria established by the document Identifying criteria for wound infection (EWMA, 2005). Were used the software Statistics Package for Social Sciences for Windows ® (SPSS 17.0) for data processing. For association analysis were used the Chi-square or Fisher's exact tests, adopting a significance level of 5% (&#945; = 0.05). Legal ethical aspects have been respected. RESULTS: The prevalence of S. aureus was 83% and 15% of CoNS. Were identified 28% of MRSA and 47% MRCoNS. Among S. aureus, 69.6% were resistant to erythromycin, 69.6% to clindamycin, 69.6% to gentamicin and 100% to ciprofloxacin. 74% of MRSA showed high level resistance to oxacillin, MIC &#8805; 256 &#956;g/mL, and in 65.2% predominated MLSBc constitutive resistance. Of the two MRSA isolates with sensibility to clindamycin, just one was positive for D-test. Signs and symptoms of infection more frequent were: discoloration of the wound and increase the volume of exudate. The stage III of infection was identified in 70 (71.4%) ulcers. An association among friable granulation tissue and cultures positive for Staphylococcus sp. (P = 0.004) and increase the local temperature of the skin and multiresistant Staphylococcus (p = 0.002). CONCLUSIONS: Staphylococcus sp. multidrug-resistant were isolates of venous ulcers in people treated in primary care. The results confirm that the MRSA isolates, beyond resistance to beta-lactams, also exhibit cross-resistance to other antimicrobials such as clindamycin, erythromycin, ciprofloxacin, and gentamicin. Remain a challenge to find indicators of infection for venous ulcers / INTRODUÇÃO: Nos últimos anos, tem sido observada a ocorrência de cepas multirresistentes provenientes de pacientes atendidos na atenção primária com lesões de etiologia venosa. OBJETIVOS: Determinar nos isolados de úlceras venosas a frequência e o perfil de suscetibilidade de Staphylococcus spp. aos antimicrobianos e, assim determinar a concentração inibitória mínima (MIC); detectar a resistência MLSB e correlacionar a positividade das culturas com os sinais e sintomas clássicos de infecção. Adiciona-se a classificação do estágio clínico de infecção das úlceras segundo critérios de EWMA, 2005. MÉTODOS: Foram avaliadas 69 pessoas com 98 úlceras no período de outubro/09 a outubro/2010. Os isolados resistentes a cefoxitina e/ou oxacilina (disco-difusão) foram submetidos ao teste confirmatório para detecção da CIM, empregando fitas de oxacilina (E-test®). Realizou-se a detecção fenotípica da resistência induzível ao grupo MLSB por meio do D-test. Utilizou-se o software Statistics Package for the Social Sciences for Windows® (SPSS 17.0), para processamento dos dados. A análise de associação envolveu os testes Qui-quadrado ou Exato de Fisher s, adotando-se o nível de significância de 5% (&#945;=0,05). Aspectos éticos legais foram atendidos. RESULTADOS: A prevalência de S. aureus foi de 83% e de 15% de CoNS. Identificou-se 28% de MRSA e 47% de MRCoNS. Entre o S. aureus, 69,6% apresentaram resistência a eritromicina, 69,6% a clindamicina, 69,6% a gentamicina e 100% a ciprofloxacina. 74% dos MRSA apresentaram elevado nível de resistência a oxacilina, MIC &#8805; 256 &#956;g/mL, e em 65,2% predominou a resistência constitutiva MLSBc. Dentre os dois isolados MRSA com sensibilidade à clindamicina, apenas um foi positivo para o D-teste. Os sinais e sintomas de infecção mais frequentes foram: descoloração do leito da lesão e aumento do volume do exsudato. O estágio III de infecção representou 70 (71,4%) das úlceras. Verificou-se associação entre tecido de granulação friável e culturas positivas para Staphylococcus spp. (p=0,004) e, aumento da temperatura local da pele com a ocorrência de Staphylococcus multirresistentes (p=0,002). CONCLUSÕES: Staphylococcus multirresistentes foram isolados de úlceras venosas em pessoas atendidas na atenção primária. Os resultados apresentados confirmam que os isolados MRSA, além da resistência aos beta-lactâmicos, também apresentam resistência cruzada a outros antimicrobianos. Cabe a preocupação com o aumento dessa resistência microbiana o que exige além da vigilância epidemiológica, também a priorização das políticas publicas em saúde por meio de programas efetivos de prevenção e controle. Ainda, permanece o desafio acerca dos indicadores de infecção para as úlceras venosas.

<p>Uvod: Infekcija krvi izazvana multirezistentnim bakterijama roda Acinetobacter (MDRA) je praćena značajnim letalitetom i visokim tro&scaron;kovima bolničkog lečenja. Ciljevi istraživanja: Ustanoviti uče&scaron;će izolata Acinetobacter spp. u strukturi pozitivnih hemokultura i kretanje procenta rezistencije na antibiotike u zdravstvenim ustanovama sekundarnog i tercijarnog nivoa na teritoriji AP Vojvodine u periodu 2013-2015. godina; Utvrditi kod kojih pacijenata se najče&scaron;će javljaju infekcije krvi izazvane MDRA; Utvrditi faktore rizika za nastanak bolničke infekcije (BI) krvi izazvane MDRA i uticaj BI krvi izazvane ovim uzročnicima na dužinu trajanja hospitalizacije i na ishod lečenja pacijenata hospitalizovanih u zdravstvenim ustanovama sekundarnog i tercijarnog nivoa u AP Vojvodini. Materijal i metode: Podaci iz protokola mikrobiolo&scaron;ke laboratorije Centra za mikrobiologiju Instituta za javno zdravlje Vojvodine su kori&scaron;teni za retrospektivnu analizu učestalosti izolata Acinetobacter spp. u strukturi hemokultura i za praćenje kretanja procenta rezistentnih izolata Acinetobacter spp. na posmatrane vrste antibiotika u zdravstvenim ustanovama sekundarnog i tercijarnog nivoa u AP Vojvodini u periodu od 01.01.2013. do 31.12.2015. godine. Utvrđivanje faktora rizika za nastanak infekcije krvi izazvane MDRA je sprovedeno kao prospektivna kohortna studija u jedinicama intenzivnih nega (JIN) u zdravstvenim ustanovama u AP Vojvodini u periodu od 01.01.2013. do 31.03.2016. godine. Grupu 1 (n=164), studijsku grupu kohortne studije su činili ispitanici sa BI krvi izazvanom MDRA. Grupu 2 (n=328), kontrolnu grupu kohortne studije, sačinjavali su pacijenti JIN bez izolata Acinetobacter spp. u hemokulturi. Kontrole su bile uključene u istraživanje samo ako je dužina njihovog boravka u JIN (dužina trajanja hospitalizacije do otpusta) bila ista ili duža od dužine boravka para iz studijske grupe do izolacije MDRA iz hemokulture. Kontrole su bile uparene sa slučajem iz studijske grupe u odnosu (1:2) prema: uzrastu (+/-5 godine), vrsti JIN i vremenu (isti kalendarski mesec u kojem je kod para iz studijske grupe izolovana pozitivna hemokultura). U cilju utvrđivanja predisponirajućih faktora za letalni ishod (14-dnevni letalitet) pacijenata u JIN sa infekcijom krvi izazvanom MDRA sprovedena je anamnestička studija. Rezultati: Uče&scaron;će izolata Acinetobacter spp. u strukturi hemokultura pacijenata uzrasta 18 i vi&scaron;e godina hospitalizovanih u zdravstvenim ustanovama u AP Vojvodini u periodu 2013-2015. godina iznosilo je 13,9%. Primoizolati Acinetobacter spp. iz uzoraka hemokultura pacijenata su u 96,1% (198/204) bili multirezistentni. Analizom kretanja rezistencije izolata Acinetobacter spp. na ispitivane antibiotike jedino je na cefepim ustanovljeno statistički značajno smanjenje uče&scaron;ća rezistentnih izolata (od 98,5% u 2014. godini do 83,3% u 2015. godini), (p=0,025). Izolati Acinetobacter spp. su najče&scaron;će registrovani kod pacijenata hospitalizovanih u JIN (71,1% (145/204)). Multivarijantnom analizom izdvojili su se nezavisni prediktori za nastanak infekcije krvi izazvane MDRA: prijem iz drugog odeljenja/bolnice, prijemne dijagnoze politrauma i opekotina, prethodna kolonizacija gornjeg respiratornog trakta MDRA, prisustvo dva i vi&scaron;e komorbiditeta, prethodna primena mehaničke ventilacije, vi&scaron;i indeks invazivnih procedura, prethodna primena derivata imidazola i prethodna primena četiri i vi&scaron;e klasa antibiotika. Pacijenti sa infekcijom krvi izazvanom MDRA su statistički značajno duže boravili u JIN (24.5&plusmn;17,5) u odnosu na neinficirane kontrole (19,7&plusmn;12,6), (p=0,001) i statistički značajno če&scaron;će su imali letalan ishod (51,2% (84/164) u odnosu na pacijente bez infekcije krvi izazvane ovim mikroorganizmom (25,0% (82/328), (p&lt;0,0001). Multivarijantnom analizom, kao nezavisni prediktori letalnog ishoda pacijenata, izdvojili su se: starija životna dob, prijemnom dijagnoza akutne respiratorne insuficijencije i primena neadekvatne antimikrobne terapije nakon izolacije uzročnika iz hemokulture. Zaključak: Učestalost i struktura faktora rizika je ukazala da je snižavanje prevalencije i snižavanje letaliteta moguće ostvariti kombinovanom primenom mera koje obuhvataju racionalnu upotrebu antibiotika &scaron;irokog spektra u empirijskoj antimikrobnoj terapiji i striktno po&scaron;tovanje procedura vezanih za primenu invazivnih nastavaka.</p> / <p>Aim: Establish the participation of Acinetobacter spp. isolates in the structure of positive hemocultures and the percentage range of resistance to antibiotics in the health institutions of secondary and tertiary level on the territory of AP of Vojvodina in the period from 2013 to 2015; determine which patients most commonly get BSI caused by MDRA; determine risk factors for the occurrence of healthcare-associated infection (HAI) of blood caused by MDRA and the impact of HAI of blood caused by these pathogens to the duration of hospitalization, and the treatment outcome of patients admitted to the health care institutions of secondary and tertiary levels in the AP of Vojvodina. Material and Methods: Data from the protocol of the microbiological laboratory of the Center for Microbiology, Institute of Public Health of Vojvodina were used for retrospective analysis of the frequency of isolates of Acinetobacter spp. in the structure of positive hemocultures and for monitoring the percentage isolates of Acinetobacter spp. resistant to the observed type of antibiotics in health institutions of secondary and tertiary levels in AP of Vojvodina in the period from January 1, 2013 to December 31, 2015. Determining the risk factor for the occurrence of BSI induced by MDRA was conducted as a prospective cohort study in intensive care units (ICU) in the health institutions in AP of Vojvodina in the period from January 1, 2013 to March 31, 2016. Group 1 (n=164), study group of the cohort study included the patients with HAI of blood induced by MDRA. Group 2 (n=328), control group of the cohort study consisted of ICU patients without isolates of Acinetobacter spp. in the hemoculture. Controls were included in the study only if the length of their stay in the ICU (duration of hospitalization until discharge) was the same or longer than the length of the stay of their study group counterparts until the isolation of MDRA from blood culture. Controls were matched with the cases of the study group in the ratio (1: 2) according to: age (+/- 5 years), type of ICU and time (the same calendar month in which positive hemoculture was isolated in the the study group pair). In order to determine the predisposing factors of lethal outcome (14-day lethality) of patients in the ICU with the BSI caused by MDRA, anamnestic study was conducted. Results: Participation of Acinetobacter spp. isolates in the structure of hemocultures of patients, aged 18 and older, hospitalized in medical institutions in AP of Vojvodina in the period from 2013 to 2015 amounted to 13.9%. Acinetobacter spp. primoisolates from the patients&#39; hemoculture samples were in 96.1% (198/204) multi-drug resistant. Analysing the Acinetobacter spp. isolates resistance to the tested antibiotics, Cefepime was the only to prove to cause statistically significant decrease in the share of resistant isolates (from 98.5% in the year 2014 to 83.3% in 2015), (p=0.025). Isolates of Acinetobacter spp. are most frequently registered in patients hospitalized in ICU (71.1% (145/204)). Multivariate analyses separated independent predictors for the occurrence of blood infection caused by the MDRA: patient transfers from another ward/hospital, admission diagnoses of polytrauma and burns, previous colonization of the upper respiratory tract MDRA, the presence of two or more co-morbidity, previous use of mechanical ventilation, higher index of invasive procedures, previous use of Imidazole derivates and the previous use of four or more classes of antibiotics. Patients with BSI caused by MDRA stayed statistically much longer in the ICU (24.5&plusmn;17.5) as compared to uninfected controls (19.7&plusmn;12.6), (p=0.001) and significantly more likely to have the lethal outcome (51.2% (84/164)) compared to patients without bloodsteram infections caused by this micro-organism (25.0% (82/328) (p&lt;0.0001). Using multivariate analysis, independent predictors of death of patients, were found to be: advanced age, admission diagnosis of acute respiratory insufficiency and the application of inadequate antibiotic therapy after the isolation of pathogens from the hemoculture. Conclusion: The frequency and the structure of the risk factors suggested that the reduction of the prevalence and lowering of lethality can be achieved by combined administration of measures that include the rational use of broad spectrum antibiotics in the empirical antimicrobial treatment and strict compliance with the procedures related to the use of invasive follow-ups.</p>

<p>Uvod: Antimikrobna rezistencija bakterija predstavlja globalni problem. Najvažniji faktor za njen nastanak je neadekvatna primena antibiotika, koja podrazumeva: Upotrebu antibiotika bez odgovarajuće dijagnoze, neadekvatan izbor leka, dužinu<br />primene i doziranje. Zbog specifičnosti populacije vitalno ugroženih bolesnika u jedinicama intenzivne terapije (JIT) i bolničkih infekcija uzrokovanih multirezistentnim bakterijama, primena antibiotika je na ovim odeljenjima učestala. Pokazana je povezanost između razvoja antimikrobne rezistencije i veličine potro&scaron;nje antibiotika u JIT. Cilj: Analiza primene antibiotika prema indikacijama na Klinici za anesteziju i intenzivnu terapiju, KC Vojvodine, zatim analiza stanja antimikrobne rezistencije<br />najče&scaron;ćih uzročnika bolničkih infekcija i analiza korelacije između navedenih uzročnika bolničkih infekcija i empirijski primenjivane antibiotske terapije na Klinici za anesteziju i intenzivnu terapiju. Materijal i metode: Prospektivna, opservaciona studija, sprovedena u jednogodi&scaron;njem period, u JIT, Klinike za anesteziju i intenzivnu terapiju, uključila je 856 ispitanika, oba pola, starijih od 18 godina kod kojih je tokom hospitalizacije u JIT bio primenjen antibiotik. Ispitanici su, radi prikupljanja podataka, bili podeljeni u dve grupe u zavisnosti od toga da li su imali bolničku infekciju ili ne. Adekvatnost primene antibiotika je analizirana prema indikacijama (hirur&scaron;ka profilaksa, bolničke infekcije, vanbolničke infekcije i drugo), a u odnosu na izbor antibiotika, dužinu primene, režim doziranja, veličinu pojedinačne doze i način promene terapije (prema preporukama farmakoterapijskog vodiča The Sanford guide to antimicrobial therapy i antimikrobnoj osetljivosti bakterijskih uzročnika bolničkih infekcija u JIT. Za izračunavanje potro&scaron;nje antibiotika u JIT kori&scaron;ćena je ATC/DDD metodologija. Podaci o antimikrobnoj osetljivosti dobijeni su iz rezultata mikrobiolo&scaron;ke obrade uzorkovanog materijala. Statistička analiza je izvr&scaron;ena pomoću statističkog paketa IBM SPSS 21 Statistics. Podaci su predstavljeni tabelarno i grafički, obrađeni su standardnim statističkim testovima, a statistička značajnost određivanja je bila na nivou p&lt; 0,05. Ispitivanje povezanosti između potro&scaron;nje anibiotika i antimikrobne rezistencije urađeno je primenom Pirsonovog koeficijenta korelacije. Rezultati: Izbor antibiotika kod bolesnika u JIT nije bio adekvatan u 52,19% preskripcija. Izbor empirijski indikovanih antibiotika za lečenje bolničkih infekcija nije bio u skladu antimikrobnom osetljivo&scaron;ću izolovanog uzročnika u 78,44% preskripcija. Izbor antibiotika za hirur&scaron;ku profilaksu nije bio adekvatan u 55,6% preskripcija. Antimikrobna rezistencija Acinetobacter spp.na karbapeneme, fluorohinolone i cefalosporine bila je preko 90%, na aminoglikozide preko 70%. Klebsiella pneumoniae bila je rezistentna na fluorohinolone i cefalosporine 80%, dok je na grupu karbapenema bila 18%. Pseudomonas aeruginosa je bio rezistentan na karbapeneme i aminoglikozide preko 50%, na antipseudomonasne cefalosporine preko 40%. Na kolistin nije zabeležena rezistencija ni jedne izolovane bakterijske vrste. Značajna pozitivna korelacija zabeležena je između potro&scaron;nje empirijski indikovanog meropenema i rezistencije Acinetobacter spp. Zaključak: U vise od 50% slučajeva primena antibiotika u JIT nije bila u skladu sa stanjem antimikrobne rezistencije bakterijskih uzročnika bolničkih infekcija i savremenim farmakoterapijskim protokolima. Antimikrobna rezistencija Acinetobacter spp, Klebsiellae pneumoniae и Pseudomonas aeruginosae je iznosila preko 20% na antibiotike preporučene savremenim farmakoterapijskim smernicama, osim u slučaju rezistencije Klebsiellaе pneumoniae na grupu karbapenema. Između pojave rezistencije Acinetobacter spp. i potro&scaron;nje empirijski indikovanog meropenema utvrđena je statistički značajna pozitivna povezanost, dok za druge dve navedene bakterijske vrste ova povezanost nije bila statistički značajna. Na osnovu podataka o najče&scaron;ćim bakterijskim uzročnicima i njihovoj antimikrobnoj osetljivosti za empirijsku<br />terapiju pneumonija mogao bi biti preporučen jedino kolistin, dok bi za lečenje urinarnih infekcija mogao biti preporučen imipenem ili meropenem. Potrebno je promeniti farmakoterapijski pristup u primeni antibiotika u JIT.</p> / <p>Uvod: Antimikrobna rezistencija bakterija predstavlja globalni problem. Najvažniji faktor za njen nastanak je neadekvatna primena antibiotika, koja podrazumeva: Upotrebu antibiotika bez odgovarajuće dijagnoze, neadekvatan izbor leka, dužinu<br />primene i doziranje. Zbog specifičnosti populacije vitalno ugroženih bolesnika u jedinicama intenzivne terapije (JIT) i bolničkih infekcija uzrokovanih multirezistentnim bakterijama, primena antibiotika je na ovim odeljenjima učestala. Pokazana je povezanost između razvoja antimikrobne rezistencije i veličine potro&scaron;nje antibiotika u JIT. Cilj: Analiza primene antibiotika prema indikacijama na Klinici za anesteziju i intenzivnu terapiju, KC Vojvodine, zatim analiza stanja antimikrobne rezistencije<br />najče&scaron;ćih uzročnika bolničkih infekcija i analiza korelacije između navedenih uzročnika bolničkih infekcija i empirijski primenjivane antibiotske terapije na Klinici za anesteziju i intenzivnu terapiju. Materijal i metode: Prospektivna, opservaciona studija, sprovedena u jednogodi&scaron;njem period, u JIT, Klinike za anesteziju i intenzivnu terapiju, uključila je 856 ispitanika, oba pola, starijih od 18 godina kod kojih je tokom hospitalizacije u JIT bio primenjen antibiotik. Ispitanici su, radi prikupljanja podataka, bili podeljeni u dve grupe u zavisnosti od toga da li su imali bolničku infekciju ili ne. Adekvatnost primene antibiotika je analizirana prema indikacijama (hirur&scaron;ka profilaksa, bolničke infekcije, vanbolničke infekcije i drugo), a u odnosu na izbor antibiotika, dužinu primene, režim doziranja, veličinu pojedinačne doze i način promene terapije (prema preporukama farmakoterapijskog vodiča The Sanford guide to antimicrobial therapy i antimikrobnoj osetljivosti bakterijskih uzročnika bolničkih infekcija u JIT. Za izračunavanje potro&scaron;nje antibiotika u JIT kori&scaron;ćena je ATC/DDD metodologija. Podaci o antimikrobnoj osetljivosti dobijeni su iz rezultata mikrobiolo&scaron;ke obrade uzorkovanog materijala. Statistička analiza je izvr&scaron;ena pomoću statističkog paketa IBM SPSS 21 Statistics. Podaci su predstavljeni tabelarno i grafički, obrađeni su standardnim statističkim testovima, a statistička značajnost određivanja je bila na nivou p&lt; 0,05. Ispitivanje povezanosti između potro&scaron;nje anibiotika i antimikrobne rezistencije urađeno je primenom Pirsonovog koeficijenta korelacije. Rezultati: Izbor antibiotika kod bolesnika u JIT nije bio adekvatan u 52,19% preskripcija. Izbor empirijski indikovanih antibiotika za lečenje bolničkih infekcija nije bio u skladu antimikrobnom osetljivo&scaron;ću izolovanog uzročnika u 78,44% preskripcija. Izbor antibiotika za hirur&scaron;ku profilaksu nije bio adekvatan u 55,6% preskripcija. Antimikrobna rezistencija Acinetobacter spp.na karbapeneme, fluorohinolone i cefalosporine bila je preko 90%, na aminoglikozide preko 70%. Klebsiella pneumoniae bila je rezistentna na fluorohinolone i cefalosporine 80%, dok je na grupu karbapenema bila 18%. Pseudomonas aeruginosa je bio rezistentan na karbapeneme i aminoglikozide preko 50%, na antipseudomonasne cefalosporine preko 40%. Na kolistin nije zabeležena rezistencija ni jedne izolovane bakterijske vrste. Značajna pozitivna korelacija zabeležena je između potro&scaron;nje empirijski indikovanog meropenema i rezistencije Acinetobacter spp. Zaključak: U vise od 50% slučajeva primena antibiotika u JIT nije bila u skladu sa stanjem antimikrobne rezistencije bakterijskih uzročnika bolničkih infekcija i savremenim farmakoterapijskim protokolima. Antimikrobna rezistencija Acinetobacter spp, Klebsiellae pneumoniae i Pseudomonas aeruginosae je iznosila preko 20% na antibiotike preporučene savremenim farmakoterapijskim smernicama, osim u slučaju rezistencije Klebsiellae pneumoniae na grupu karbapenema. Između pojave rezistencije Acinetobacter spp. i potro&scaron;nje empirijski indikovanog meropenema utvrđena je statistički značajna pozitivna povezanost, dok za druge dve navedene bakterijske vrste ova povezanost nije bila statistički značajna. Na osnovu podataka o najče&scaron;ćim bakterijskim uzročnicima i njihovoj antimikrobnoj osetljivosti za empirijsku<br />terapiju pneumonija mogao bi biti preporučen jedino kolistin, dok bi za lečenje urinarnih infekcija mogao biti preporučen imipenem ili meropenem. Potrebno je promeniti farmakoterapijski pristup u primeni antibiotika u JIT.</p> / <p>Introduction: Antimicrobial resistance is a global health problem.The most important factor in the development of antimicrobial resistance is inadequate use of antibiotics, which means: inadequate diagnosis of bacterial infection, inadequate antibiotic choice, dosage and duration of therapy. Specificities of critically ill patients and nosocomial infections caused by multidrug-resistant pathogens are important reasons for large antibiotic consumption in ICU settings. Many studies have confirmed a positive correlation between antibiotic use and antimicrobial resistance. Aims: The aims of this study were: to analyze the use of antibiotics at the ICU of the Clinic for anesthesia and intensive care at the Clinical Centre of Vojvodina, according to indications for antibiotic treatment; to analyze the pattern of antimicrobial resistance ofthe most common bacteria causing hospital acquired infections in our participants and to analyze the correlation between the consumption of empirically indicated antibiotics and antimicrobial resistance pattern. Methodology: Prospective observational study was conducted during a one-year period at the Clinic for anesthesia and intensive care, Clinical Centre of Vojvodina. The study included 856 participatns, aged over 18 years and of both genders. The participants were divided into two cohorts, depending on whether they showed symptoms of hospital-acquired infection or not. Adequacy of antibiotic use was analyzed with regard to indication for antibiotic treatment (surgical prophylaxis, treatment of hospital acquired infection, outpatient infection or other) and with regard to antibiotic choice, dosage and duration of treatment. An adequate antibiotic choice was compared to the resistance pattern of positive bacterial isolates as outlined by The Sanford guide to antimicrobial therapy). To calculate the consumption of antibiotics in ICU we used ATC/DDD methodology. Data on antibacterial sensitivity was obtained from the results of microbiological analysis of sample materials. IBM SPSS version 21 was used for statistical analysis, standard statistical tests were applied. The results were presented in tables and graphs. Statistically significant correlation was set at the value of p˂0.05. Pearson correlation coefficient was used to measure the strength between variables. Results: Antibiotic choice was inadequate in 52,19% of all antibiotic prescriptions for all indications. Antibiotic choice in surgical prophylaxis was inadequate in 55,59% of prescriptions for this indication. Inadequate choice of empirically indicated antibiotics (for treatment of hospital-acquired infections) according to antimicrobial resistance pattern occurred in 78,44% of all prescription for this indication. The three the most important bacterial causative agents of hospital acquired infections in ICU were: Acinetobacter spp, Klebsiella pneumonia and Pseudomonas aeruginosa. The resistance of Acinetobacter spp. to antibiotic groups was as follows: to carbapenems, fluoroquinolones and cephalosporins over 90% and to aminoglycosides over 70%. The antimicrobial resistance of Klebsiella pneumoniae was: to fluoroquinolones and cephalosporins over 80% and to carbapenems up to 20%. The resistance pattern of Pseudomonas aeruginosa was as follows: to carbapenems and aminoglykozides over 50%, and to antipseudomonal cephalosporins over 40%. Statistically significant correlation was found between the consumption of empirically prescribed meropenem and antimicrobial resistance of Acinetobacter spp. Conclusion:In more than 50% of antibiotic prescriptions at ICU, regardless of indication, the choice of prescribed antibiotics was inadequate. Antimicrobial resistance pattern of Acinetobacter spp, Klebsiella pneumoniae and Pseudomonas aeruginosa to antibiotics recomennded by contemporary guidelines for antimicrobial therapy was over 20%, except in the case of the resistance of Klebsiellae peneumoniae to carbapenems. Statistically significant correlation was found between the consumption of empirically prescribed meropenem and antimicrobial resistance of Acinetobacter spp. No statistically significant correlation was observed in the other two bacterial strains. Initial, empiric therapy for nosocomial pneumonia in our ICU, should be colistin, and for urinary tract infection imipenem or meropenem. It is important to change antibiotic prescribing praxis in ICU.</p>

INTRODUÇÃO: A hepatite C crônica é uma das principais causas de hepatopatia em todo mundo. A coinfecção pelo vírus C (VHC) e o HIV não é incomum, pois ambos compartilham vias similares de transmissão. Recentemente, a terapêutica da hepatite C crônica foi radicalmente modificada com o advento das drogas antivirais de ação direta (DAAs), elevando as taxas de RVS mesmo na população coinfectada. O VHC é caracterizado pela sua alta taxa replicativa e por grande diversidade populacional. Substituições de ocorrência natural na protease viral associadas a resistência podem comprometer a terapêutica em alguns regimes baseados no uso de inibidores de protease (IPs). OBJETIVOS: Estimar a prevalência de polimorfismos e mutações de ocorrência natural associadas a resistência aos IPs em pacientes monoinfectados e coinfectados pelo VHC e HIV e identificar fatores clínicos e virológicos associados a presença de tais substituições. MATERIAIS E MÉTODOS: Dados epidemiológicos e clínicos foram obtidos de 247 pacientes (135 monoinfectados e 112 coinfectados pelo VHC e HIV). VHC RNA foi extraído do plasma dos indivíduos participantes e um fragmento de 765 pares de base da região NS3 foi amplificado e sequenciado por metodologia populacional (técnica de Sanger). O estadiamento da fibrose hepática foi realizado pelo escore não invasivo FIB- 4. RESULTADOS: 54 indivíduos (21,9%) apresentaram pelo menos uma substituição na região NS3/NS4A do VHC. Somente 14 pacientes (5,7%) apresentaram pelo menos uma mutação de resistência aos IPs (T54S, V55A ou Q80R). A Q80K não foi identificada em nenhuma das amostras. Não houve diferença entre monoinfectados e coinfectados quanto à ocorrência de polimorfismos ou mutações associadas a resistência. As variáveis independentemente associadas com substituições na região da protease foram infecção pelo VHC genótipo 1b, bilirrubinas totais > 1,5 vezes o LSN e níveis de albumina < 3,5 g/dL. Fibrose hepática avançada (FIB-4 > 3.25) não esteve associada a presença de substituições. A análise de diversidade nucleotídica na protease viral revelou maior heterogeneidade do VHC genótipo 1b em relação ao 1a. Contudo, a análise de pressão seletiva não demonstrou maior variabilidade de quasiespécies no grupo de hepatopatia avançada, achado este compatível com uma sequência genômica relativamente conservada. CONCLUSÕES: As substituições na região NS3/NS4 do VHC consistiram majoritariamente por polimorfismos naturais sem impacto clínico num eventual tratamento que envolva o uso de IPs. A prevalência de substituições associadas a resistência foi baixa e compatível com os valores informados pela maioria dos estudos nacionais e internacionais. A coinfecção pelo HIV não parece elevar a frequência de substituições na protease do VHC. A região NS3 do genótipo 1b foi altamente variável em relação ao genótipo 1a, reforçando o conceito de possíveis diferenças geográficas em relação ao perfil genético deste vírus / INTRODUCTION: Chronic hepatitis C is a major cause of liver disease worldwide. Hepatitis C vírus (HCV) and HIV coinfection is not uncommon due to similar transmission routes. Recently developed direct-acting antivirals drugs (DAAs) have increased the rate of SVR even in coinfected patients. HCV has a high replication rate and a lack of proofreading activity, leading to a greatly diverse viral population. Baseline spontaneously occurring resistance substitutions in the protease region may impair the rate of success in some protease inhibitors (PI) based regimens. OBJECTIVE: to determine the prevalence of naturally occurring polymorphisms and resistance associated variants to HCV PIs in mono and coinfected HCV HIV patients and to evaluate potential associations between amino acid substitutions in protease domain and clinical / virological features of those patients. METHODS: Clinical and epidemiological data were retrieved from medical records of 247 subjects in Brazil (135 HCV monoinfected and 112 HIV HCV coinfected patients). HCV-RNA was extracted from plasma and a fragment of 765 base pairs from the NS3 region was amplified and sequenced with Sanger-based technology. Fibrosis staging was assessed by non invasive score (FIB-4). RESULTS: Overall, 54 patients (21.9%) had at least one amino acid substitution in the NS3 region; only 14 patients (5.7%) harboured at least one resistance mutation (T54S, V55A, Q80R). Q80K mutation was not found in any sample. There was no difference between monoinfected and coinfected patients regarding the frequency of natural polymorphisms and resistance mutations. Variables independently associated with amino acid substitution were HCV subtype 1b, total bilirubin level > 1.5 ULN and albumin level < 3.5 g/dL. Advanced liver fibrosis (FIB-4 > 3.25) was not related to NS3 polymorphisms nor resistance associated variants. Examination of HCV protease nucleotide diversity revealed greater heterogeneity in subtype 1b than subtype 1a. Analysis of selective pressure did not reveal a greater quasispecies variability in advanced liver fibrosis group, being such finding consistent with a relatively conserved gene in this setting. CONCLUSION: Baseline HCV NS3 amino acid substitutions depicted herein were considered mostly natural polymorphisms with no clinical impact in a PI based therapy. The prevalence of resistance-associated substitutions was low and compatible with values reported by most national and international studies. HIV coinfection was not associated with a greater frequency of such substitutions in the studied sample. The NS3 region of genotype 1b was highly variable in relation to genotype 1a, highlighting geographic differences concerning HCV genetic profile

Introduction: Emergence of resistance to artemisinin-based combination therapy (ACT) is a major threat to combat Plasmodium falciparum malaria. Regular therapeutic studies to monitor treatment efficacy is essential, and genotyping of molecular makers is useful for mapping development and spread of resistance. Aims: The study aims are to assess efficacy of artesunate-amodiquine (ASAQ) and prevalence of molecular markers of drug resistance in Zanzibar in 2017. Methods: Treatment efficacy of the clinical trial conducted in 2017 was compared with efficacies in 2002 and 2005. A total of 142 samples were genotyped for single nucleotide polymorphisms (SNPs) in the P. falciparum chloroquine resistance transporter gene (pfcrt) gene, the P. falciparum multi drug resistance 1 (pfmdr1) gene, and in the P. falciparum Kelch 13 (PfK13) propeller region. Prevalence of SNPs were assessed during the period 2002-2017. Results: Cure rate was 100% in 2017, compared to 94% and 96%, in 2002-2003 and 2005, respectively. Day 3 fever clearance rate were also high 93% (2002-3), 99% (2005) and 98% (2017) in all studies. Prevalence of pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y and pfmdr1 (86Y, 184Y and 1246Y) YYY haplotypes were significantly decreased between 2002-3 and 2017 (p &lt; 0.001). No SNP in the PfK13 gene related to artemisinin resistance was identified. Conclusion: Efficacy of ASAQ remains high after fourteen years as first-line treatment, despite the wide-scale use of ASAQ, and there is no evidence of selection of resistance markers in Zanzibar. Continuous monitoring of drug efficacy and resistance markers is recommended. / <p>This master thesis is a collaboration project between Institutionen för kvinnors och barns hälsa, Department of Women's and Children's Health, Uppsala Universtiy and Anders Björkman group, Department of Microbiology, Tumor and Cell Biology (MTC), C1, Karolinska Institutet. Laboratory examinations were mainly conducted at MTC house, Karolinska Institutet.</p>

Background Coagulase-negative staphylococci (CoNS) and in particular Staphylococcus epidermidis have emerged as major pathogens primarily causing nosocomial infections in patients with indwelling medical devices. These infections are often caused by multidrug-resistant strains of S. epidermidis (MDRSE). Other clinical entities due to CoNS are lower urinary tract infections (UTI) in women and native valve endocarditis. The purpose of this work was to investigate the frequency of antibiotic resistance and the molecular epidemiology of both hospital and community-associated isolates of S. epidermidis in order to examine if certain clones are related to MDRSE infections. Furthermore, we aimed to explore if specific clones of S. saprophyticus are associated with UTI in women. Methods A total of 359 hospital-associated methicillin-resistant isolates of CoNS obtained from 11 hospitals in northern Europe and 223 community-associated staphylococcal isolates were examined. Furthermore, 126 isolates of S. saprophyticus isolated from women with uncomplicated UTI from five different locations in northern Europe were analyzed. Pulsed-field gel electrophoresis (PFGE) was used for genotyping. Additionally, some of the S. epidermidis isolates were analyzed with multilocus sequence typing (MLST). Antibiotic susceptibility was determined for all isolates by the disc diffusion test. Results 293 of the 359 (82%) hospital-associated and 124 of the 223 (56%) community-associated isolates belonged to the species S. epidermidis. Among the hospital-associated S. epidermidis isolates, two dominating PFGE types (type A and B) were distinguished, comprising 78 (27%) and 51 (17%) isolates, respectively. Type A, which was detected in a Norwegian and eight Swedish hospitals, corresponded with a novel sequence type (ST215). Type B was discovered in a German, a Danish and seven Swedish hospitals and corresponded with ST2. In contrast, community-associated isolates of S. epidermidis were genetically extremely diverse with no predominating genotype, and showed a low rate of antibiotic resistance; only two (1.6%) methicillin-resistant strains were detected. Among 126 analyzed isolates of S. saprophyticus, 47 different PFGE profiles were identified. Several clusters of genetically highly related isolates were detected among isolates obtained from different locations and periods of time. Conclusion We have demonstrated the occurrence, persistence and potential dissemination of two multidrug-resistant S. epidermidis (MDRSE) genotypes, including a novel sequence type (ST215), within hospitals in northern Europe. Community-associated isolates of S. epidermidis showed a low rate of methicillin-resistance and were genetically heterogeneous. These results indicate that MDRSE by large are confined to the hospital setting in our region. Moreover, although the S. saprophyticus population was quite heterogeneous, indistinguishable isolates of S. saprophyticus causing lower UTI in women were identified in different countries 11 years apart, indicating the persistence and geographical spread of some clones of S. saprophyticus.

Systems biology adopts an integrated approach to study and understand the function of biological systems, particularly, the response of such systems to perturbations, such as the inhibition of a reaction in a pathway, or the administration of a drug. The complexity and large scale of biological systems make modelling and simulation an essential and critical part of systems-level studies. Systems-level modelling of pathogenic organisms has the potential to significantly enhance drug discovery programmes. In this thesis, we show how systems--level models can positively impact anti-tubercular drug target identification. *Mycobacterium tuberculosis*, the principal aetiological agent of tuberculosis in humans, is estimated to cause two million deaths every year. The existing drugs, although of immense value in controlling the disease to some extent, have several shortcomings, the most important of them being the emergence of drug resistance rendering even the front-line drugs inactive. As drug discovery efforts are increasingly becoming rational, focussing at a molecular level, the identification of appropriate targets becomes a fundamental pre-requisite. We have constructed many system-level models, to identify drug targets for tuberculosis. We construct a constraint-based stoichiometric model of mycolic acid biosynthesis, and simulate it using flux balance analysis, to identify critical points in mycobacterial metabolism for targeting drugs. We then analyse protein--protein functional linkage networks to identify influential hubs, which can be targeted to disrupt bacterial metabolism. An important aspect of tuberculosis is the emergence of drug resistance. A network analysis of potential information pathways in the cell helps to identify important proteins as co-targets, targeting which could counter the emergence of resistance. We integrate analyses of metabolism, protein--protein interactions and protein structures to develop a generic drug target identification pipeline, for identifying most suitable drug targets. Finally, we model the interplay between the pathogen and the human immune system, using Boolean networks, to elucidate critical factors influencing the outcome of infection. The strategies described can be applied to understand various pathogens and can impact many drug discovery programmes.

As international travel is rapidly increasing, more people are being exposed to potentially more antibiotic resistant bacteria, a changed infectious disease epidemiology, and an increased risk of accidents and crime. Research-based advice is needed to adequately inform travellers about these risks. We studied travellers who sought advice from the Travel Medicine Clinic at the Department of Infectious Diseases, Umeå University Hospital, as well as university students from Umeå, Stockholm, and Gothenburg travelling abroad for study, research, and clinical exchange programs. From retrospective data at the Travel Medicine Clinic, we found that pre-existing health problems were rare among travellers from Umeå seeking pre- travel health advice and vaccinations. In addition, we found that the travel destination and the sex of the traveller affected vaccination levels. Although hepatitis A is endemic to both Thailand and Turkey, compared to travellers to Thailand few travellers to Turkey visited the clinic for hepatitis A vaccination. The data also revealed that more women than men were vaccinated against Japanese encephalitis despite comparable trips. A prospective survey study showed that travellers felt that the pre-travel health advice they received was helpful. Two-thirds of the travellers followed the advice given although they still fell ill to the same extent as those who were not compliant with the advice. Factors outside the control of travellers likely affect the travel-related morbidity. Compared to older travellers, younger travellers were less compliant with advice, fell ill to a greater extent, and took greater risks during travel. In a prospective survey study, we found that healthcare students had higher illness rates and risk exposure when abroad compared to students from other disciplines. This difference was mainly due to the fact that healthcare students more often travelled to developing regions during their study period abroad. When abroad, half of all students increased their alcohol consumption and this was linked to an increased risk of theft and higher likelihood of meeting a new sex partner. The healthcare students participating in the survey study also submitted stool samples before and after travel. These samples were tested for the presence of antibiotic resistance, both by selective culturing for ESBL-PE (Extended-Spectrum Beta-Lactamase Producing Enterobacteriaceae) as well as by metagenomic sequencing. About one-third (35%) of the students became colonised by ESBL-PE following their study abroad. The strongest risk factor for colonisation was travel destination; for example, 70% of students who had travelled to India became colonised. Antibiotic treatment during travel was also a significant risk factor for colonisation. The stool samples from a subset of study subjects were analysed using metagenomic sequencing. From this we learned that although the majority of resistance genes in the gut microbiome remained unchanged following travel, several clinically important resistance genes increased, most prominently genes encoding resistance to sulphonamide, trimethoprim, and beta-lactams. Overall, taxonomic changes associated with travel were small but the proportion of Proteobacteria, which includes several clinically important bacteria (e.g., Enterobacteriaceae), increased in a majority of the study subjects. Clearly, there are risks associated with international travel and these risks include outside factors as well as the personal behaviour of travellers. We believe our results can be used to develop better pre-travel advice for tourists as well as university students studying abroad resulting in safer travel.