The use of nebulization is becoming increasingly frequent in treatment of acute or chronic lung diseases for delivery of topically active drugs and is also an attractive way to deliver systemic drugs. A nebulizer can be defined by the aerodynamic properties of the emitted particles which are directly related to the lung deposition and the clinical response to a nebulized drug. New guidelines elaborated by an European norm (ENFR13544-1) aim to participate to a better control on quality and efficiency of existing devices and inspired the elaboration of the studies of this thesis. The aim of these works was to evaluate the nebulization function of a new kind of modality, the intrapulmonary percussive ventilation which contrarily to classical jet nebulizers nebulizes drugs under superimposed percussion conditions. In vitro measurements were realized by cascade impaction and laser diffraction. Lung deposition was investigated by imagery techniques and pharmacokinetic study. Aerodynamic properties were different between the in vitro methods. When measured by cascade impaction, MMAD and FPF were smaller for IPV comparatively to SST. By laser diffraction, FPF remained lower but MMAD was higher with IPV than with SST. The effect of percussions was greater on MMAD than on FPF. An irregular intrapulmonary deposition and a higher whole body deposition due to a higher extrapulmonary deposition with the IPV were measured by scintigraphy. The pharmacokinetic study highlighted that the drug output and the lung dose were lower when amikacin was delivered by IPV comparatively to SST. All results of these different studies seem unfavourable to the use of intrapulmonary percussive ventilation as modality of administration for nebulized drugs without further investigations. Results presented in this thesis concerning exclusively healthy subjects, we hope that they encouraged to perform complementary analysis and observations in different conditions such as patients with lung disease.

Σκοπός της μελέτης ήταν να καθορισθεί εάν η μονοθεραπεία με σιπροφλοξασίνη σε υψηλές δόσεις (400 mg x 3φορές/ημέρα ενδοφλεβίως), είναι αποδεκτή ως αρχική εμπειρική αντιμικροβιακή θεραπεία σε εμπύρετα επεισόδια ουδετεροπενικών ασθενών με αιματολογικά νοσήματα σε σχέση με τον καθιερωμένο συνδυασμό κεφταζιντίμης+αμικασίνης, και επιπρόσθετα να διερευνηθεί εάν η χορήγηση σιπροφλοξασίνης από του στόματος (750 mg x 2 φορές/ημέρα) μπορεί αποτελεσματικά να υποκαταστήσει την ενδοφλέβια χορήγηση 72 ώρες μετά την έναρξη αυτής. Σε προοπτική τυχαιοποιημένη μελέτη μελετήθηκαν συνολικά 123 εμπύρετα ουδετεροπενικά επεισόδια, σε 61 από τα οποία χορηγήθηκε σιπροφλοξασίνη και σε 62 ο συνδυασμός κεφταζιντίμης+αμικασίνης. Επί του συνόλου σε 96 (78,1%) η υποκείμενη νόσος ήταν οξεία λευχαιμία, σε 24 (19,5%) μη-Hodgkin λέμφωμα και σε 3 (2,4%) απλαστική αναιμία. Επιτυχής κλινική ανταπόκριση στο τέλος της θεραπείας χωρίς τροποποίηση αυτής παρατηρήθηκε σε 30/61 (49,2%) επεισόδια στην ομάδα της σιπροφλοξασίνης και σε 31/62 (50%) στην ομάδα της κεφταζιντίμης+αμικασίνης (p=1,00). Σε 40/61 (65,6%) επεισόδια στην ομάδα της σιπροφλοξασίνης η ενδοφλέβια θεραπεία αντικαταστάθηκε με από του στόματος μετά από 4,3±069 ημέρες, με επιτυχή ανταπόκριση στο 75%(30/40) αυτών. Επί του συνόλου των επεισοδίων στις μικροβιολογικά αποδεδειγμένες λοιμώξεις κυριαρχούσαν οι Gram-θετικοί μικροοργανισμοί σε ποσοστό 56,8%, ενώ οι Gram-αρνητικοί αποτελούσαν το 43,2%. Η ανταπόκριση στην θεραπεία με σιπροφλοξασίνη στις μικροβιολογικά αποδεδειγμένες λοιμώξεις (βακτηριαιμίες και άλλες λοιμώξεις) ήταν 40,0%, στις κλινικά αποδεδειγμένες λοιμώξεις 50% και στα επεισόδια πυρετού αγνώστου αιτιολογίας 55,2%, έναντι 41,2%, 43,8% και 58,6% αντίστοιχα στην δεύτερη ομάδα. Ειδικότερα στις βακτηριαιμίες από gram-θετικά παθογόνα επιτυχής έκβαση παρατηρήθηκε στο 20% των επεισοδίων στην ομάδα της σιπροφλοξασίνης και στο 12,5% στην ομάδα του συνδυασμού. Στη διάρκεια της μελέτης παρατηρήθηκαν 6 επιλοιμώξεις μόνο στην ομάδα της σιπροφλοξασίνης και 4 επαναλοιμώξεις μόνο στην ομάδα του συνδυασμού κεφταζιντίμης/αμικασίνης, (p=0,006). Οι επιλοιμώξεις ήταν δύο μυκητιασικές πνευμονίες, μια λοίμωξη ουροποιητικού από P. aeruginosa, και τρεις βακτηριαιμίες από P. Aeruginosa, St. viridans και Enterococcus spp.. Οι επαναλοιμώξεις αφορούσαν δύο βακτηριαιμίες μία από P. aeruginosa και μια από St. Aureus, μία λοίμωξη ουροποιητικού από E. coli και μία γαστρεντερίτιδα από Ε. faecalis. Ο αριθμός των θανάτων στις πρώτες 72 ώρες ήταν 2 για την ομάδα της σιπροφλοξασίνης και 1 για την ομάδα κεφταζιντίμης+αμικασίνης. Επιπλέον 11 ασθενείς ακόμη (7 vs 4, p=0,363) κατέληξαν στη φάση της ουδετεροπενίας. Επί του συνόλου 12/14 θανάτους οφείλονταν σε λοίμωξη. Από τη στατιστική ανάλυση των δεδομένων προκύπτει ότι η παρατεταμένη και σοβαρή ουδετεροπενία αποτελεί σημαντικό παράγοντα για την ανταπόκριση στη θεραπεία. Στην ομάδα της σιπροφλοξασίνης ουδετεροπενία με ουδετερόφιλα <100/μl για περισσότερο από 14 ημέρες επηρεάζει εξαιρετικά ισχυρά την ανταπόκριση στη θεραπεία (p=0,003), ενώ στην ομάδα του συνδυασμού κεφταζιντίμης+αμικασίνης η διαφορά αυτή, παρότι υπάρχει, δεν εμφανίζεται στατιστικά σημαντική (p=0,07). Με ανάλυση λογαριθμικής παλινδρόμησης καθορίσθηκαν παράγοντες που κατά την έναρξη του εμπυρέτου επεισοδίου και την διάρκεια αυτού ασκούν σημαντική επίδραση στην ανταπόκριση στη θεραπεία, και επομένως είναι δυνατόν να χαρακτηρίσουν τον ασθενή ως “χαμηλού κινδύνου” για την εμφάνιση επιπλοκών. Θετική επίδραση στην ανταπόκριση στη θεραπεία κατά την έναρξη του πυρετού έχουν η απουσία κλινικής λοίμωξης, ο αριθμός των ουδετεροφίλων >100/μl, η αιματολογική κακοήθεια σε ύφεση, το διάστημα από την προηγηθείσα αντινεοπλασματική χημειοθεραπεία να είναι > από 10 ημέρες από την έναρξη αυτής και το περιβάλλον εμφάνισης της λοίμωξης να είναι αυτό της κοινότητας (εξωνοσοκομειακή λοίμωξη). Κατά τη διάρκεια του επεισοδίου οι παράγοντες αυτοί είναι η άνοδος των ουδετεροφίλων >100/μl και >500/μl, η διάρκεια της ουδετεροπενίας <100/μl να είναι <14ημέρες, καθώς και η διάρκεια της ουδετεροπενίας <500/μl να είναι επίσης <14ημέρες. Τέλος αναλύοντας τα επεισόδια με ουδετερόφιλα <100/μl για περισσότερο από 14ημέρες είχαν 2,75 φορές πιθανότητα επιτυχούς έκβασης εάν έπαιρναν το συνδυασμό κεφταζιντίμης+αμικασίνης (p=0,038, odds ratio=2,75). Ανεπιθύμητες ενέργειες παρατηρήθηκαν σε 9/61 (14,7%) επεισόδια της ομάδας της σιπροφλοξασίνης και σε 10/62 (16,1%) της ομάδας κεφταζιντίμης+αμικασίνης (p=1,00), χωρίς να απαιτηθεί σε κανένα διακοπή της θεραπείας. Συμπερασματικά φαίνεται ότι η μονοθεραπεία με υψηλές δόσεις σιπροφλοξασίνης είναι εξ’ ίσου αποτελεσματική και ασφαλής με τον καθιερωμένο συνδυασμό κεφταζιντίμης+αμικασίνης, ως εμπειρική θεραπεία ουδετεροπενικών ασθενών “χαμηλού κινδύνου” με πυρετό. Επιπρόσθετα στους ασθενείς που ανταποκρίθηκαν, έχει το πλεονέκτημα της από του στόματος χορήγησης για τη συμπλήρωση της αγωγής, ενώ συγχρόνως στερείται νεφροτοξικότητας και ωτοτοξικότητας. Παρόλα αυτά ιδιαίτερα σημαντικό είναι κατά την επιλογή της εμπειρικής αντιμικροβιακής θεραπείας, να λαμβάνεται υπόψη το μικροβιακό φάσμα που επικρατεί και η ανθεκτικότητα των μικροοργανισμών που απομονώνονται στο συγκεκριμένο νοσοκομείο. / The aim of the present study was to compare administration of ciprofloxacin, given initially at the higher intravenous dose 400 mg three times a day for at least 72 hours, followed by oral administration 750 mg twice a day, with the standard combination regimen of ceftazidime plus amikacin as empiric treatment in patients with febrile neutropenia. In a prospective study, a total of 123 febrile neutropenic patients were randomized: 61 in the ciprofloxacin group and 62 in the ceftazidime plus amikacin group. In 78,1% of the patients acute leukemia was the underlying disease, another 19,5% of the patients suffered from high-grade non-Hodgkin’s lymphoma, and the remaining 2,4% of the patients suffered from aplastic anemia. The frequency of successful clinical response without modification at the end of therapy was almost identical for ciprofloxacin [49,2% (30/61 patients)] compared with that for ceftazidime plus amikacin [50% (31/62 patients)], (p=1,00). For 40/61 (65,6%) patients, it was possible to switch from parenteral ciprofloxacin to the oral after a mean of 4,3±069 days, and the response was successful for 30/40 (75%) patients. Gram-positive organisms accounted for 56,8% of all organisms isolated. The response to therapy in ciprofloxacin group was 40,0% for the microbiologically documented infections, 50% for the clinically documented infections and 55,2% for the episodes with fever of unknown origin, compared with 41,2%, 43,8% and 58,6% respectively in ceftazidime plus amikacin group. The efficacies of the regimens against gram-positive bacteremias were 20% for the ciprofloxacin group and 12,5% for the combination group. Superinfections were seen in 6 episodes in ciprofloxacin group and 4 episodes of reinfection in ceftazidime plus amikacin group (p=0,006). 3 patients (2 of ciprofloxacin group and 1 of combination group) died within 72 hours of randomization. Another 11 patients (7 vs 4 respectively, p=0,363) died before resolution of neutropenia. Of the total 12/14 patients died because of infection. The analysis of the data shown that the prolonged and severe granulocytopenia is a critical factor for the successful outcome. In ciprofloxacin group the neutropenia with neutrophils <100/μl for 14 days or more is significantly associated with the response to treatment (p=0,003). In the combination group this association is not significant (p=0,07). Logistic regression analyses were performed to estimate the probability of success and to identify “low risk” neutropenic patients. The covariates could be assessed at the onset of fever and during treatment as well. Among the tested covariates, the following variables were significant predictors of outcome at the onset of fever: absence of signs of clinically documented infection, neutrophils >100/μl, primary disease in remission, fever developing more than 10 days from the recent course of chemotherapy and outpatients status before the onset of fever. The significant predictors of outcome during treatment were: increasing neutrophils count >100/μl, increasing neutrophils count >500/μl, neutrophils <100/μl for less than 14 days and neutrophils <500/μl for less than 14 days as well. Furthermore episodes with 14 days or more of neutropenia <100/μl treated with ceftazidime plus amikacin had response rates 2,75 times higher compared to episodes treated with ciprofloxacin (p=0,038, odds ratio=2,75). Adverse events were mostly self-limited and were observed in 9 (14,7%) ciprofloxacintreated patients and 10 (16,1%) patients who were receiving the combination. In summary, this trial suggests that high-dose ciprofloxacin is therapeutically equivalent to the routine regimen of ceftazidime plus amikacin in “low risk” febrile neutropenic patients and has the advantages of intravenous and oral administration, without nepfro- and ototoxicity. However, it is very important that before an empirical therapy is chosen each hospital determine bacteriologic predominance and perform resistance surveillance.

Base teórica: Devido à diminuição do desenvolvimento de novos antimicrobianos nas últimas décadas, terapias combinadas têm sido empregadas contra bactérias multirresistentes como opção à monoterapia no tratamento de infecções graves. Para tratar infecções causadas por Peusdomonas aeruginosa resistente a carbapenêmicos, amicacina e polimixina B têm sido utilizadas em associações com imipenem, pois possuem diferentes mecanismos de ação, o que, teoricamente, indicaria a possibilidade de efeito sinérgico. Objetivo: O objetivo deste trabalho foi verificar a interação, in vitro, de amicacina e polimixina B em associação com imipenem frente a diferentes isolados de P. aeruginosa resistentes a carbapenêmico. Métodos: Foram selecionados isolados de P. aeruginosa resistentes a carbapenêmico oriundos do Hospital de Clínicas de Porto Alegre, coletados no período de janeiro a março de 2015. Foi realizada eletroforese em gel de campo pulsado e detecção do gene blaSPM-1 para selecionar diferentes clones. Seis isolados (três SPM-1 positivos e três negativos para a carbapenemase) foram selecionados para realização da técnica de time-kill, a fim de avaliar a atividade antimicrobiana das combinações de imipenem com amicacina e imipenem com polimixina B. Resultados: Sinergismo ocorreu para combinações de imipenem com amicacina em 3 isolados, dos quais um era SPM-1 positivo e dois eram SPM-1 negativos e todos apresentaram CIMs relativamente baixas a intermediárias. Quanto às combinações de imipenem com polimixina B, houve sinergismo somente para dois isolados, um SPM-1 positivo e um SPM-1 negativo, contudo houve antagonismo em 5 isolados, dois SPM-1 positivos e três SPM-1 negativos. Para 4 isolados, as combinações de imipenem com amicacina tiveram atividade bactericida, enquanto, para todos os isolados, as combinações de imipenem com polimixina B, bem como polimixina B isoladamente, 1x e 2x a CIM apresentaram atividade bactericida. Conclusão: Sinergismo pode ocorrer, para combinações de imipenem mais amicacina, quando os isolados apresentam CIMs relativamente baixas ou intermediárias para imipenem (≤16 μg/mL a 128 μg/mL) e amicacina (≤32 μg/mL). Entretanto, antagonismo aconteceu independentemente de valores altos ou baixos de concentrações inibitórias mínimas para imipenem e polimixina B. Além disso, a presença ou ausência do gene blaSPM-1 não pareceu influenciar nos resultados. / Background: Due to a decrease on new antibiotic development over the last decades, combined therapies have been employed against multigrug-resistant bacteria as an option to monotherapy in severe infection treatment. In order to treat infections caused by carbapenem resistant Pseudomonas aeruginosa, amikacin and polymyxin B have been used in associations with imipenem, because they possess different mechanisms of action, which could, theoretically, indicate the possibility of synergistic effect. Objective: The aim of this study was to verify the interaction, in vitro, of amikacin and polymyxin B in association with imipenem against various carbapenem resistant P. aeruginosa isolates. Methods: Carbapenem resistant P. aeruginosa isolates have been selected from Hospital de Clínicas de Porto Alegre, collected from January to March 2015. Pulsed field gel electrophoresis and detection of blaSPM-1 gene has been performed to select different clones. Six isolates (three SPM-1 positive and three negative for the carbapenemase) were selected for time-kill assay, in order to assess antimicrobial activity of imipenem plus amikacin and imipenem plus polymyxin B combinations. Results: Synergism occurred for combinations of imipenem plus amikacin in three isolates, from which one was SPM-1 positive and two were SPM-1 negative, and all presented relatively low to intermediate minimum inhibitory concentrations. About imipenem plus polymyxin B combinations, synergism occurred in only two isolates, one SPM-1 positive and one SPM-1 negative, however antagonism occurred in five isolates, two SPM-1 positive and three SPM-1 negative. For 4 isolates, imipenem plus amikacin combinations had bactericidal effect, while, for all isolates, combinations of imipenem plus 1x and 2x the MIC of polymyxin B presented bactericidal activity. Conclusions: Synergism can occur, for imipenem plus amikacin combinations, when isolates present relatively low or intermediate MIC of imipenem (≤16 μg/mL to 128 μg/mL) and amikacin (≤32 μg/mL). However, antagonism happened regardless high or low minimum inhibitory concentrations for imipenem and polymyxin B. Also, the presence or absence of blaSPM-1 gene did not seem to influence the results.

Base teórica: Devido à diminuição do desenvolvimento de novos antimicrobianos nas últimas décadas, terapias combinadas têm sido empregadas contra bactérias multirresistentes como opção à monoterapia no tratamento de infecções graves. Para tratar infecções causadas por Peusdomonas aeruginosa resistente a carbapenêmicos, amicacina e polimixina B têm sido utilizadas em associações com imipenem, pois possuem diferentes mecanismos de ação, o que, teoricamente, indicaria a possibilidade de efeito sinérgico. Objetivo: O objetivo deste trabalho foi verificar a interação, in vitro, de amicacina e polimixina B em associação com imipenem frente a diferentes isolados de P. aeruginosa resistentes a carbapenêmico. Métodos: Foram selecionados isolados de P. aeruginosa resistentes a carbapenêmico oriundos do Hospital de Clínicas de Porto Alegre, coletados no período de janeiro a março de 2015. Foi realizada eletroforese em gel de campo pulsado e detecção do gene blaSPM-1 para selecionar diferentes clones. Seis isolados (três SPM-1 positivos e três negativos para a carbapenemase) foram selecionados para realização da técnica de time-kill, a fim de avaliar a atividade antimicrobiana das combinações de imipenem com amicacina e imipenem com polimixina B. Resultados: Sinergismo ocorreu para combinações de imipenem com amicacina em 3 isolados, dos quais um era SPM-1 positivo e dois eram SPM-1 negativos e todos apresentaram CIMs relativamente baixas a intermediárias. Quanto às combinações de imipenem com polimixina B, houve sinergismo somente para dois isolados, um SPM-1 positivo e um SPM-1 negativo, contudo houve antagonismo em 5 isolados, dois SPM-1 positivos e três SPM-1 negativos. Para 4 isolados, as combinações de imipenem com amicacina tiveram atividade bactericida, enquanto, para todos os isolados, as combinações de imipenem com polimixina B, bem como polimixina B isoladamente, 1x e 2x a CIM apresentaram atividade bactericida. Conclusão: Sinergismo pode ocorrer, para combinações de imipenem mais amicacina, quando os isolados apresentam CIMs relativamente baixas ou intermediárias para imipenem (≤16 μg/mL a 128 μg/mL) e amicacina (≤32 μg/mL). Entretanto, antagonismo aconteceu independentemente de valores altos ou baixos de concentrações inibitórias mínimas para imipenem e polimixina B. Além disso, a presença ou ausência do gene blaSPM-1 não pareceu influenciar nos resultados. / Background: Due to a decrease on new antibiotic development over the last decades, combined therapies have been employed against multigrug-resistant bacteria as an option to monotherapy in severe infection treatment. In order to treat infections caused by carbapenem resistant Pseudomonas aeruginosa, amikacin and polymyxin B have been used in associations with imipenem, because they possess different mechanisms of action, which could, theoretically, indicate the possibility of synergistic effect. Objective: The aim of this study was to verify the interaction, in vitro, of amikacin and polymyxin B in association with imipenem against various carbapenem resistant P. aeruginosa isolates. Methods: Carbapenem resistant P. aeruginosa isolates have been selected from Hospital de Clínicas de Porto Alegre, collected from January to March 2015. Pulsed field gel electrophoresis and detection of blaSPM-1 gene has been performed to select different clones. Six isolates (three SPM-1 positive and three negative for the carbapenemase) were selected for time-kill assay, in order to assess antimicrobial activity of imipenem plus amikacin and imipenem plus polymyxin B combinations. Results: Synergism occurred for combinations of imipenem plus amikacin in three isolates, from which one was SPM-1 positive and two were SPM-1 negative, and all presented relatively low to intermediate minimum inhibitory concentrations. About imipenem plus polymyxin B combinations, synergism occurred in only two isolates, one SPM-1 positive and one SPM-1 negative, however antagonism occurred in five isolates, two SPM-1 positive and three SPM-1 negative. For 4 isolates, imipenem plus amikacin combinations had bactericidal effect, while, for all isolates, combinations of imipenem plus 1x and 2x the MIC of polymyxin B presented bactericidal activity. Conclusions: Synergism can occur, for imipenem plus amikacin combinations, when isolates present relatively low or intermediate MIC of imipenem (≤16 μg/mL to 128 μg/mL) and amikacin (≤32 μg/mL). However, antagonism happened regardless high or low minimum inhibitory concentrations for imipenem and polymyxin B. Also, the presence or absence of blaSPM-1 gene did not seem to influence the results.

abstract: Many therapeutics administered for some of the most devastating illnesses can be toxic and result in unwanted side effects. Recent developments have been made in an alternative treatment method, called gene therapy. Gene therapy has potential to rectify the genetic defects that cause a broad range of diseases. Many diseases, such as cancer, cystic fibrosis, and acquired immunodeficiency (AIDS) already have gene therapy protocols that are currently in clinical trials. Finding a non-toxic and efficient gene transfer method has been a challenge. Viral vectors are effective at transgene delivery however potential for insertion mutagenesis and activation of immune responses raises concern. For this reason, non-viral vectors have been investigated as a safer alternative to viral-mediated gene delivery. Non-viral vectors are also easy to prepare and scalable, but are limited by low transgene delivery efficacies and high cytotoxicity at effective therapeutic dosages. Thus, there is a need for a non-toxic non-viral vector with high transgene efficacies. In addition to the hurdles in finding a material for gene delivery, large-scale production of pharmaceutical grade DNA for gene therapy is needed. Current methods can be labor intensive, time consuming, and use toxic chemicals. For this reason, an efficient and safe method to collect DNA is needed. One material that is currently being explored is the hydrogel. Hydrogels are a useful subclass of biomaterials, with a wide variety of applications. This class of biomaterials can carry up to a thousand times their weight in water, and are biocompatible. At smaller dimensions, referred to as micro- and nanogels, they are very useful for many biomedical applications because of their size and ability to swell. Based on a previously synthesized hydrogel, and due to the advantages of smaller dimension in biomedical applications, we have synthesized aminoglycoside antibiotic based nanogels and microgels. Microgels and nanogels were synthesized following a ring opening polymerization of epoxide-containing crosslinkers and polyamine-containing monomers. The nanogels were screened for their cytocompatibilities and transfection efficacies, and were compared to polyethylenimine (PEI), a current standard for polymer-mediated transgene delivery. Nanogels demonstrated minimal to no toxicity to the cell line used in the study even at high concentrations. Due to the emerging need for large-scale production of DNA, microgels were evaluated for their binding capacity to plasmid DNA. Future work with the aminoglycoside antibiotic-based nanogels and microgels developed in this study will involve optimization of nanogels and microgels to facilitate in better transgene delivery and plasmid DNA binding, respectively. / Dissertation/Thesis / M.S. Chemical Engineering 2014

Aminoglycosides are a group of broad-spectrum antibiotics that, under neutral pH conditions, carry a positive charge. The net cationic charge arises from the high number of amino groups in the core structure of aminoglycosides. Previous studies performed have shown that negatively charged citrate ligand-capped gold nanoparticles (AuNPs) can interact with various biomolecules such as aminoglycosides. AuNPs bound to biomolecules have been used in conjugation with various assaying techniques to detect and study compounds in vitro and in vivo. AuNPs also have strong light scattering properties that can be used with a wide variety of imaging and assaying techniques. Our laboratory has previously performed experiments on the aminoglycoside antibiotic ribostamycin sulfate. During this experiment, the concentration dependent rod-like assembly of ribostamycin sulfate was characterized. This experiment used three analytical techniques in conjunction with AuNPs: (1) dynamic light scattering (DLS), (2) UV-Vis absorption spectroscopy, and (3) dark field optical microscope imaging (DFM). This suite of techniques was used to analyze mixtures of ribostamycin sulfate at different concentration with different sized AuNPs. The primary objective of this research was to determine if the techniques used to characterize the self-assembly of ribostamycin sulfate could be generalized and applied to other aminoglycoside antibiotics. The secondary objective of this research was to determine if other aminoglycoside antibiotics formed rod-like assemblies. This study demonstrated that AuNPs can be used to detect self-assembled oligomers for different aminoglycoside antibiotics. In addition, this study also revealed that not all aminoglycoside antibiotics will self assemble into rod-like oligomers similar to ribostamycin. It was observed that the aminoglycoside antibiotic amikacin self assembled into rod-like aggregates similar to ribostamycin sulfate but the aminoglycoside antibiotics neomycin sulfate and streptomycin sulfate did not.

Base Teórica: Bacteremias causadas por Klebsiella pneumoniae produtoras de carbapenemase são infecções ameaçadoras da vida e com elevadas taxas de mortalidade. As Enterobacteriaceae são as principais reponsáveis por bacteremias nos hospitais brasileiros. Há limitadas opções terapêuticas e o melhor tratamento para essas infecções ainda não está definido. O racional teórico para a terapia combinada nesse cenário seria o aumento da ação bactericida e a diminuição da indução de resistência. A terapia combinada com colistina parece estar relacionada com maior sobrevida. Com relação a terapia combinada com polimixina B, no entanto, as evidências são exíguas. Objetivo: Avaliar a mortalidade em 30 dias de pacientes com bacteremias por KPC-KP com enfoque na terapia combinada.Métodos: Trata-se de um estudo de coorte retrospectivo e unicêntrico que incluiu pacientes maiores de 18 anos com diagnóstico de bacteremia por KPC-KP. O desfecho primário avaliado foi mortalidade em 30 dias. Bacteremia por KPC-KP foi definida como uma ou mais hemoculturas positivas para esse microorganismo. A identificação bacteriana e os testes de susceptibilidade foram realizados utilizando o sistema automatizado Vitek 2 (bioMérieux, France). A terapia antimicrobiana foi caracterizada como empírica (iniciada nas primeiras 48 horas) e definitiva (esquemas iniciados ou mantidos após 48 horas) e avaliada da seguinte forma: nenhuma droga ativa, monoterapia (apenas um agente ativo), terapia combinada entre uma droga ativa e uma ou mais drogas inativas e terapia combinada com duas ou mais drogas ativas A análise estatística foi realizada com o software SPSS para Windows, versão 18.0. As análises de sobrevivência foram realizadas com curvas de Kaplan-Meier e as diferenças foram avaliadas utilizando o log-rank test. Todos os testes foram bicaudais considerando um nível de significância de 95%. Um modelo de regressão de Cox foi realizado para identificar fatores independentemente relacionados com a mortalidade em 30 dias. Resultados: Foram incluídas 105 bacteremias por KPC-KP. A mortalidade em 30 dias foi de 63 (60%) pacientes. O tempo médio de sobrevida foi de 24 dias (95% IQR, 17-21 dias). A taxa de mortalidade em pacientes tratados com terapia combinada foi significativamente menor (16,5/1000 pacientes-dia) comparada com os pacientes recebendo outros regimes terapêuticos (57,5/1000 pacientes-dia). Terapia combinada (Hazard Ratio [HR]; 0,32; 95% IC, 0,18-0.57; p<0,01) e bacteremia urinária (HR; 0,29; 95% IC, 0,09- 0,95; p=0,04) foram independentemente associados com a sobrevida em 30 dias. Em contrapartida, neoplasias (HR; 1,98; 95% IC, 1,18-3,32; p=0,01), admissão por patologia clínica (HR; 2,91; 95% IC, 1,56-5,42; p<0.01) e necessidade de tratamento com droga vasoativa (HR; 2,94; 95% IC, 1,59- 5,29; p<0,01) foram independentemente associados com o desfecho primário. Conclusão: O presente estudo demonstrou uma mortalidade em 30 dias de 60% nas bacteremias por KPC-KP. A terapia combinada com pelo menos dois agentes ativos in vitro foi consistentemente associada com sobrevida em 30 dias. / Background: BSI for KPC-KP is a life-threatening disease and compared to other sites of infection related to higher mortality rates. Enterobacteriaceae are the leading cause of BSI in Brazilian hospitals. There are limited treatment options and the best available treatment is still unknown. Rationale for combination therapy in this setting would be increasing bactericidal action and decreasing resistance induction. Combination therapy regimens with colistin seemed to be related with higher rates of survival. Polymyxin B combinations were studied only in a few studies. Objective: In this study we aim to evaluate 30-day mortality in KPC-KP bacteremia with particular emphasis on combination therapy. Methods: This is a single center retrospective cohort study that included patients older than 18 years diagnosed with KPC-KP BSI. The primary outcome was 30-day mortality after BSI. KPC-KP BSI was defined as one or more positive blood cultures with recovery of KPC-KP. Bacterial identification and antimicrobial susceptibility tests were performed using Vitek 2 (bioMérieux, France) automatized system. Antimicrobial therapy was defined as empirical (started on first 48 hours) and definitive (schemes initiated or maintained after 48 hours) and evaluated as follows: no active agents, monotherapy (only one active agents), combination therapy between one active agent plus one or more non-active agents and combination with two or more in vitro active agents Statistical analysis was performed using SPSS for Windows, version 18.0. Kaplan-Meier survival estimates were calculated and the difference was evaluated using the log-rank test. All tests were two-tailed and a p value < 0.05 were considered statistically significant. A Cox regression model were performed to identify independent factors related to 30-day mortality. Results: A total of 105 bloodstream infections caused by KPC-KP were included. A total of 63 (60%) patients died in the first 30 days after BSI. Median time to death was 24 days (95% IQR, 17-21 days). The mortality rate in patients treated with the combination of two antibiotics with in vitro activity was significantly lower (16.5/1000 patients-day) compared with that patients receiving other regimens (57.5/1000 patients-day). Combination therapy (Hazard Ratio [HR]; 0.32; 95% CI, 0.18-0.57; p<0.01) and urinary BSI (HR; 0.29; 95% CI, 0.09- 10 0.95; p=0.04) were independently associated to 30-day survival. On the other hand, having Cancer (HR; 1.98; 95% CI, 1.18-3.32; p=0.01), non-surgical admission (HR; 2.91; 95% CI, 1.56-5.42; p<0.01) and requirement of vasoactive drugs (HR; 2.94; 95% CI, 1.59-5.29; p<0.01) were independently associated to 30-day mortality. Conclusion: This study showed a 60% 30-day mortality in KPC-KP BSI. Combination therapy with two in vitro active agents was independently associated with 30-day survival.

Base Teórica: Bacteremias causadas por Klebsiella pneumoniae produtoras de carbapenemase são infecções ameaçadoras da vida e com elevadas taxas de mortalidade. As Enterobacteriaceae são as principais reponsáveis por bacteremias nos hospitais brasileiros. Há limitadas opções terapêuticas e o melhor tratamento para essas infecções ainda não está definido. O racional teórico para a terapia combinada nesse cenário seria o aumento da ação bactericida e a diminuição da indução de resistência. A terapia combinada com colistina parece estar relacionada com maior sobrevida. Com relação a terapia combinada com polimixina B, no entanto, as evidências são exíguas. Objetivo: Avaliar a mortalidade em 30 dias de pacientes com bacteremias por KPC-KP com enfoque na terapia combinada.Métodos: Trata-se de um estudo de coorte retrospectivo e unicêntrico que incluiu pacientes maiores de 18 anos com diagnóstico de bacteremia por KPC-KP. O desfecho primário avaliado foi mortalidade em 30 dias. Bacteremia por KPC-KP foi definida como uma ou mais hemoculturas positivas para esse microorganismo. A identificação bacteriana e os testes de susceptibilidade foram realizados utilizando o sistema automatizado Vitek 2 (bioMérieux, France). A terapia antimicrobiana foi caracterizada como empírica (iniciada nas primeiras 48 horas) e definitiva (esquemas iniciados ou mantidos após 48 horas) e avaliada da seguinte forma: nenhuma droga ativa, monoterapia (apenas um agente ativo), terapia combinada entre uma droga ativa e uma ou mais drogas inativas e terapia combinada com duas ou mais drogas ativas A análise estatística foi realizada com o software SPSS para Windows, versão 18.0. As análises de sobrevivência foram realizadas com curvas de Kaplan-Meier e as diferenças foram avaliadas utilizando o log-rank test. Todos os testes foram bicaudais considerando um nível de significância de 95%. Um modelo de regressão de Cox foi realizado para identificar fatores independentemente relacionados com a mortalidade em 30 dias. Resultados: Foram incluídas 105 bacteremias por KPC-KP. A mortalidade em 30 dias foi de 63 (60%) pacientes. O tempo médio de sobrevida foi de 24 dias (95% IQR, 17-21 dias). A taxa de mortalidade em pacientes tratados com terapia combinada foi significativamente menor (16,5/1000 pacientes-dia) comparada com os pacientes recebendo outros regimes terapêuticos (57,5/1000 pacientes-dia). Terapia combinada (Hazard Ratio [HR]; 0,32; 95% IC, 0,18-0.57; p<0,01) e bacteremia urinária (HR; 0,29; 95% IC, 0,09- 0,95; p=0,04) foram independentemente associados com a sobrevida em 30 dias. Em contrapartida, neoplasias (HR; 1,98; 95% IC, 1,18-3,32; p=0,01), admissão por patologia clínica (HR; 2,91; 95% IC, 1,56-5,42; p<0.01) e necessidade de tratamento com droga vasoativa (HR; 2,94; 95% IC, 1,59- 5,29; p<0,01) foram independentemente associados com o desfecho primário. Conclusão: O presente estudo demonstrou uma mortalidade em 30 dias de 60% nas bacteremias por KPC-KP. A terapia combinada com pelo menos dois agentes ativos in vitro foi consistentemente associada com sobrevida em 30 dias. / Background: BSI for KPC-KP is a life-threatening disease and compared to other sites of infection related to higher mortality rates. Enterobacteriaceae are the leading cause of BSI in Brazilian hospitals. There are limited treatment options and the best available treatment is still unknown. Rationale for combination therapy in this setting would be increasing bactericidal action and decreasing resistance induction. Combination therapy regimens with colistin seemed to be related with higher rates of survival. Polymyxin B combinations were studied only in a few studies. Objective: In this study we aim to evaluate 30-day mortality in KPC-KP bacteremia with particular emphasis on combination therapy. Methods: This is a single center retrospective cohort study that included patients older than 18 years diagnosed with KPC-KP BSI. The primary outcome was 30-day mortality after BSI. KPC-KP BSI was defined as one or more positive blood cultures with recovery of KPC-KP. Bacterial identification and antimicrobial susceptibility tests were performed using Vitek 2 (bioMérieux, France) automatized system. Antimicrobial therapy was defined as empirical (started on first 48 hours) and definitive (schemes initiated or maintained after 48 hours) and evaluated as follows: no active agents, monotherapy (only one active agents), combination therapy between one active agent plus one or more non-active agents and combination with two or more in vitro active agents Statistical analysis was performed using SPSS for Windows, version 18.0. Kaplan-Meier survival estimates were calculated and the difference was evaluated using the log-rank test. All tests were two-tailed and a p value < 0.05 were considered statistically significant. A Cox regression model were performed to identify independent factors related to 30-day mortality. Results: A total of 105 bloodstream infections caused by KPC-KP were included. A total of 63 (60%) patients died in the first 30 days after BSI. Median time to death was 24 days (95% IQR, 17-21 days). The mortality rate in patients treated with the combination of two antibiotics with in vitro activity was significantly lower (16.5/1000 patients-day) compared with that patients receiving other regimens (57.5/1000 patients-day). Combination therapy (Hazard Ratio [HR]; 0.32; 95% CI, 0.18-0.57; p<0.01) and urinary BSI (HR; 0.29; 95% CI, 0.09- 10 0.95; p=0.04) were independently associated to 30-day survival. On the other hand, having Cancer (HR; 1.98; 95% CI, 1.18-3.32; p=0.01), non-surgical admission (HR; 2.91; 95% CI, 1.56-5.42; p<0.01) and requirement of vasoactive drugs (HR; 2.94; 95% CI, 1.59-5.29; p<0.01) were independently associated to 30-day mortality. Conclusion: This study showed a 60% 30-day mortality in KPC-KP BSI. Combination therapy with two in vitro active agents was independently associated with 30-day survival.

Base Teórica: Bacteremias causadas por Klebsiella pneumoniae produtoras de carbapenemase são infecções ameaçadoras da vida e com elevadas taxas de mortalidade. As Enterobacteriaceae são as principais reponsáveis por bacteremias nos hospitais brasileiros. Há limitadas opções terapêuticas e o melhor tratamento para essas infecções ainda não está definido. O racional teórico para a terapia combinada nesse cenário seria o aumento da ação bactericida e a diminuição da indução de resistência. A terapia combinada com colistina parece estar relacionada com maior sobrevida. Com relação a terapia combinada com polimixina B, no entanto, as evidências são exíguas. Objetivo: Avaliar a mortalidade em 30 dias de pacientes com bacteremias por KPC-KP com enfoque na terapia combinada.Métodos: Trata-se de um estudo de coorte retrospectivo e unicêntrico que incluiu pacientes maiores de 18 anos com diagnóstico de bacteremia por KPC-KP. O desfecho primário avaliado foi mortalidade em 30 dias. Bacteremia por KPC-KP foi definida como uma ou mais hemoculturas positivas para esse microorganismo. A identificação bacteriana e os testes de susceptibilidade foram realizados utilizando o sistema automatizado Vitek 2 (bioMérieux, France). A terapia antimicrobiana foi caracterizada como empírica (iniciada nas primeiras 48 horas) e definitiva (esquemas iniciados ou mantidos após 48 horas) e avaliada da seguinte forma: nenhuma droga ativa, monoterapia (apenas um agente ativo), terapia combinada entre uma droga ativa e uma ou mais drogas inativas e terapia combinada com duas ou mais drogas ativas A análise estatística foi realizada com o software SPSS para Windows, versão 18.0. As análises de sobrevivência foram realizadas com curvas de Kaplan-Meier e as diferenças foram avaliadas utilizando o log-rank test. Todos os testes foram bicaudais considerando um nível de significância de 95%. Um modelo de regressão de Cox foi realizado para identificar fatores independentemente relacionados com a mortalidade em 30 dias. Resultados: Foram incluídas 105 bacteremias por KPC-KP. A mortalidade em 30 dias foi de 63 (60%) pacientes. O tempo médio de sobrevida foi de 24 dias (95% IQR, 17-21 dias). A taxa de mortalidade em pacientes tratados com terapia combinada foi significativamente menor (16,5/1000 pacientes-dia) comparada com os pacientes recebendo outros regimes terapêuticos (57,5/1000 pacientes-dia). Terapia combinada (Hazard Ratio [HR]; 0,32; 95% IC, 0,18-0.57; p<0,01) e bacteremia urinária (HR; 0,29; 95% IC, 0,09- 0,95; p=0,04) foram independentemente associados com a sobrevida em 30 dias. Em contrapartida, neoplasias (HR; 1,98; 95% IC, 1,18-3,32; p=0,01), admissão por patologia clínica (HR; 2,91; 95% IC, 1,56-5,42; p<0.01) e necessidade de tratamento com droga vasoativa (HR; 2,94; 95% IC, 1,59- 5,29; p<0,01) foram independentemente associados com o desfecho primário. Conclusão: O presente estudo demonstrou uma mortalidade em 30 dias de 60% nas bacteremias por KPC-KP. A terapia combinada com pelo menos dois agentes ativos in vitro foi consistentemente associada com sobrevida em 30 dias. / Background: BSI for KPC-KP is a life-threatening disease and compared to other sites of infection related to higher mortality rates. Enterobacteriaceae are the leading cause of BSI in Brazilian hospitals. There are limited treatment options and the best available treatment is still unknown. Rationale for combination therapy in this setting would be increasing bactericidal action and decreasing resistance induction. Combination therapy regimens with colistin seemed to be related with higher rates of survival. Polymyxin B combinations were studied only in a few studies. Objective: In this study we aim to evaluate 30-day mortality in KPC-KP bacteremia with particular emphasis on combination therapy. Methods: This is a single center retrospective cohort study that included patients older than 18 years diagnosed with KPC-KP BSI. The primary outcome was 30-day mortality after BSI. KPC-KP BSI was defined as one or more positive blood cultures with recovery of KPC-KP. Bacterial identification and antimicrobial susceptibility tests were performed using Vitek 2 (bioMérieux, France) automatized system. Antimicrobial therapy was defined as empirical (started on first 48 hours) and definitive (schemes initiated or maintained after 48 hours) and evaluated as follows: no active agents, monotherapy (only one active agents), combination therapy between one active agent plus one or more non-active agents and combination with two or more in vitro active agents Statistical analysis was performed using SPSS for Windows, version 18.0. Kaplan-Meier survival estimates were calculated and the difference was evaluated using the log-rank test. All tests were two-tailed and a p value < 0.05 were considered statistically significant. A Cox regression model were performed to identify independent factors related to 30-day mortality. Results: A total of 105 bloodstream infections caused by KPC-KP were included. A total of 63 (60%) patients died in the first 30 days after BSI. Median time to death was 24 days (95% IQR, 17-21 days). The mortality rate in patients treated with the combination of two antibiotics with in vitro activity was significantly lower (16.5/1000 patients-day) compared with that patients receiving other regimens (57.5/1000 patients-day). Combination therapy (Hazard Ratio [HR]; 0.32; 95% CI, 0.18-0.57; p<0.01) and urinary BSI (HR; 0.29; 95% CI, 0.09- 10 0.95; p=0.04) were independently associated to 30-day survival. On the other hand, having Cancer (HR; 1.98; 95% CI, 1.18-3.32; p=0.01), non-surgical admission (HR; 2.91; 95% CI, 1.56-5.42; p<0.01) and requirement of vasoactive drugs (HR; 2.94; 95% CI, 1.59-5.29; p<0.01) were independently associated to 30-day mortality. Conclusion: This study showed a 60% 30-day mortality in KPC-KP BSI. Combination therapy with two in vitro active agents was independently associated with 30-day survival.

INTRODUÇÃO: Os aminoglicosídeos são freqüentemente usados no berçário e podem ser tóxicos para as células ciliadas cócleo-vestibulares, especialmente para as células ciliadas externas da base da cóclea. As emissões ototacústicas produto de distorção permitem avaliar porções específicas da cóclea, antes mesmo que a sensação auditiva seja alterada, sendo ideais para análise precoce da integridade dessa estrutura auditiva, além de ser indicada para recém-nascidos por serem objetivas. O objetivo deste estudo prospectivo longitudinal é pesquisar a amplitude das emissões otoacústicas produto de distorção, causadas pelo uso de drogas ototóxicas, entre o término da administração e de 15 a 40 dias após seu uso. MÉTODOS: A população foi de recém-nascidos a termo e pré-termo provenientes de berçário e maternidade de um hospital público em Santo André, no período de julho de 2003 a setembro de 2004. A primeira avaliação ocorreu no dia da alta hospitalar. Foram avaliados três grupos: grupo controle com 33 recém-nascidos saudáveis e de termo; grupo de estudo a termo exposto a amicacina e /ou vancomicina com 19 recém-nascidos com mais de 37 semanas e grupo de estudo pré-termo exposto aos mesmos ototóxicos, com 15 recém-nascidos de 32 a 37 semanas. Os recém-nascidos não apresentavam indicadores de risco para deficiência auditiva preconizados pelo JCIH,2000 concomitante à infecção neonatal. Todos os recém-nascidos foram avaliados com idade gestacional corrigida maior que 37 semanas. As amplitudes das emissões otoacústicas dos recém-nascidos em fase de alta hospitalar foram comparadas às obtidas de 15 a 40 dias após a alta. RESULTADOS: As amplitudes das emissões otoacústicas dos recém-nascidos do grupo de estudo pré-termo foram menores que as amplitudes do grupo controle e do grupo de estudo a termo nos dois momentos de teste. As amplitudes das emissões dos recém-nascidos dos três grupos aumentaram no segundo momento de teste. As amplitudes das emissões dos recém-nascidos foram maiores na freqüência de 6.000 Hz e na orelha direita para a freqüência f2 3.000 Hz. As amplitude das emissões do grupo controle no segundo momento de teste foram semelhantes as do grupo de estudo à termo no primeiro momento da pesquisa. CONCLUSÔES: Houve aumento da amplitude das emissões otoacústicas produto de distorção desde a fase de alta até 15 a 40 dias após. A exposição a amicacina e vancomicina nas doses preconizadas pelo Neofax®, 2003/2004 não alterou as amplitudes das emissões nos recém-nascidos sem indicadores de risco concomitante à infecção neonatal. / The amynoglicosides are frequently used in nurseries and may be toxic for the cochleo-vestibular hair cells, specially for the outer cells of the cochlear base. The distortion product otoacoustic emissions allow to evaluate specific portions of the cochlea even before the hearing sensation is altered, and are ideal for the early analysis of this auditory structure integrity, besides being indicated for newborns once they are objective. The aim of this prospective longitudinal study is to research the amplitude of distortion product otoacoustic emissions caused by the ototoxic drugs use, between the end of the administration and from 15 to 40 days after its use. The population studied was composed by term and preterm newborns from a nursery and maternity of a Santo André city hospital, in the period from July 2003 to September 2004. The first evaluation occurred on the hospital discharge day. Three groups were evaluated: control group with 33 term and healthy newborns; term study group with 19 term newborns with more than 37 weeks exposed to amicacin and/or vancomycin; and preterm study group with 15 preterm newborns from 32 to 37 weeks exposed to the same ototoxic. The newborns did not present risk factors for hearing loss according to the JCIH, 2000 concomitant to the neonatal infection. All newborns were evaluated at a corrected gestational age greater than 37 weeks. The otoacoustic emissions amplitudes obtained at the hospital discharge were compared to the ones obtained from 15 to 40 days after the discharge. The results showed that the otoacoustic emissions amplitudes of the preterm study group were smaller than the amplitudes of the control group and the term study group in both moments of the test. The amplitude of the newborns’ otoacoustic emissions increased in the second moment of the test. The amplitudes were higher in the frequency of 6.000Hz and, in the right ear in the frequency f2 3.000 Hz. The otoacoustic emissions amplitudes of the control group in the second moment of the test were similar to the term study group in the first moment of the research. It was concluded that there is an increase of the distortion product otoacoustic emissions amplitude from the discharge moment until 15 to 40 days after, suggesting a maturation of the cochlear structures in the post-natal period, and that the exposure to amicacin and vancomycin on the recommended dose by Neofax®, 2003/2004 did not alter the amplitude of the emissions in the newborns without risk indicators concomitant with neonatal infection.