Streptococcus pneumoniae: susceptibility to penicillin and moxifloxacin

Objective: To determine the minimum inhibitory concentrations (MICs) of parenteral penicillin and moxifloxacin against Streptococcus pneumoniae strains isolated at a hospital center. Methods: In-vitro, prospective study involving 100 S. pneumoniae isolates collected from patients who had been treated, between October of 2008 and July of 2010, at the Hospital das Clinicas complex of the University of Sao Paulo School of Medicine, located in the city of Sao Paulo, Brazil. The isolates were obtained from respiratory tract cultures or blood samples unrelated to meningeal infections, and they were tested for penicillin and moxifloxacin susceptibility by E-test. The MIC category interpretations were based on updated standards. Results: All isolates were fully susceptible to parenteral penicillin (MIC <= 2 mu g/mL), and, consequently, they were also susceptible to amoxicillin, ampicillin, third/fourth generation cephalosporins, and ertapenem. Of the S. pneumoniae strains, 99% were also susceptible to moxifloxacin, and only one strain showed an MIC = 1.5 mu g/mL (intermediate). Conclusions: Our results showed high susceptibility rates to parenteral penicillin and moxifloxacin among S. pneumoniae isolates unrelated to meningitis, which differs from international reports. Reports on penicillin resistance should be based on updated breakpoints for non-meningitis isolates in order to guide the selection of an antimicrobial therapy and to improve the prediction of the clinical outcomes.

Auditory brainstem implant outcomes and MAP parameters: Report of experiences in adults and children

The auditory brainstem implant (ABI) was first developed to help neurofibromatosis type 2 patients. Recently, its use has been recently extended to adults with non-tumor etiologies and children with profound hearing loss who were not candidates for a cochlear implant (Cl). Although the results has been extensively reported, the stimulation parameters involved behind the outcomes have received less attention. Objective: The aim of this study is to describe the audiologic outcomes and the MAP parameters in ABI adults and children at our center. Methods: Retrospective chart review. Five adults and four children were implanted with the ABI24M from September 2005 to June 2009. In the adult patients, four had Neurofibromatosis type 2, and one had postmeningitic deafness with complete ossification of both cochleae. Three of the children had cochlear malformation or dysplasia, and one had complete ossified cochlea due to meningitis. Map parameters as well as the intraoperative electrical auditory brainstem responses were collected. Evaluation was performed with at least six months of device use and included free-field hearing thresholds, speech perception tests in the adult patients and for the children, the Infant-Toddler Meaningful Auditory Integration Scale (IT-MAIS) and (ESP) were used to evaluate the development of auditory skills, besides the MUSS to evaluate. Results: The number of active electrodes that did not cause any non-auditory sensation varied from three to nineteen. All of them were programmed with SPEAK strategy, and the pulse widths varied from 100 to 300 mu s. Free-field thresholds with warble tones varied from very soft auditory sensation of 70 dBHL at 250 Hz to a pure tone average of 45 dBHL. Speech perception varied from none to 60% open-set recognition of sentences in silence in the adult population and from no auditory sensation at all to a slight improvement in the IT-MAIS/MAIS scores. Conclusion: We observed that ABI may be a good option for offering some hearing attention to both adults and children. In children, the results might not be enough to ensure oral language development. Programming the speech processor in children demands higher care to the audiologist. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Infection and immune-mediated meningococcal-associated arthritis: combination features in the same patient

We present a case of a 16-year-old male patient with sudden-onset, rash, arthritis and meningitis by Neisseria meningitidis one week after an acute upper respiratory infection. On the 10th day of treatment followed by neurological and arthritis clinical improvement, he presented once again a tender and swollen left knee with a moderate effusion, and active and passive range of motion was severely limited secondary to pain, and when he was submitted to surgical drainage and synovial fluid analysis he showed inflammatory characteristics. A non-steroidal anti-inflammatory drug was taken for five days with complete improvement of symptoms. The case is notable for its combination of features of septic and immune-mediated arthritis, which has rarely been reported in the same patient.

Micro-organismos da subclasse Coccidia: resistência e implicações para o processamento de materiais de assistência à saúde

Este estudo teórico propõe uma reflexão sobre a resistência intrínseca da subclasse Coccidia, particularmente o gênero Cryptosporidium, considerado como um agente potencialmente patogênico para pacientes imunocomprometidos, e suas repercussões na prática assistencial. Atualmente, as diretrizes internacionais e nacionais aprovam como procedimento seguro a desinfecção química de alto nível de endoscópios digestivos, após sua limpeza. No entanto, estudos evidenciaram que micro-organismos da subclasse Coccidia, especificamente o Cryptosporidium, responsável por infecção entérica, são mais resistentes que as micobactérias e não são inativados pelos desinfetantes químicos de alto nível, exceto pelo Peróxido de Hidrogênio a 6% e 7,5%, formulação ainda não disponível no Brasil. Conclui-se que a legislação deve incluir este agente entre os micro-organismos teste para aprovação de desinfetantes químicos de alto nível e que as autoridades sanitárias devem se esforçar para garantir que os estabelecimentos de assistência à saúde tenham acesso a produtos eficazes contra o Cryptosporidium.

Classificação Internacional das Práticas de Enfermagem em Saúde Coletiva - CIPESC®: instrumento pedagógico de investigação epidemiológica

A CIPESC® é um instrumento de trabalho do enfermeiro em Saúde Coletiva, que visa apoiar a sistematização de sua prática assistencial, gerencial e de investigação. É também, instrumental pedagógico potente para a formação e qualificação de enfermeiros comprometidos com o SUS. No ensino das doenças transmissíveis, o uso da CIPESC® auxilia a análise sobre as intervenções, ao estimular o raciocínio clínico e epidemiológico do processo saúde-doença e das necessidades de saúde dos indivíduos, famílias e grupos sociais. Com o propósito de desenvolver recursos didáticos para graduação de enfermagem e estimular a reflexão sobre o processo de trabalho de enfermagem, este artigo apresenta o relato de uma experiência de aplicação da CIPESC®, tomando como exemplo a meningite meningocócica.

Custos hospitalares da meningite causada por Streptococcus pneumoniae na cidade de São José dos Campos, São Paulo, Brasil

O conhecimento dos custos hospitalares é de grande importância para os processos de tomada de decisão em saúde pública. O objetivo deste estudo foi estimar os custos hospitalares diretos relacionados à meningite pneumocócica em crianças com até 13 anos (inclusive), na cidade de São José dos Campos, São Paulo, Brasil, de janeiro de 1999 a dezembro de 2008. Foram obtidos dados de prontuários médicos. O cálculo foi realizado pelo método misto de mensuração das quantidades dos itens de custos e atribuição de valor aos itens consumidos (micro-costing e gross-costing). Os valores monetários referem-se a novembro de 2009, sendo expressos em reais. A análise das frequências e médias foi realizada pelo programa Epi Info versão 3.5.1. Foram notificados 41 casos. Os custos hospitalares diretos variaram de R$ 1.277,90 a R$ 19.887,56 (média = R$ 5.666,43), ou seja, 10 a 20 vezes maiores que o custo médio de internações pago pelo SUS. Os custos dos honorários profissionais foram os mais relevantes, seguidos pelos custos dos medicamentos, procedimentos, materiais e exames laboratoriais.

A doença meningocócica na região de Sorocaba, São Paulo, Brasil, no período de 1999 a 2008

O objetivo do estudo foi descrever a ocorrência da doença meningocócica notificada no Departamento Regional de Saúde XVI, Sorocaba, São Paulo, Brasil, no período de 1999 a 2008. Em Sorocaba, a incidência anual da doença foi de cerca de dois casos por 100 mil habitantes, com aumento entre 2006 e 2008. As incidências anuais foram maiores entre 0 a 4 anos de idade. A letalidade no período foi de 21,8%, maior nas faixas etárias de 0 a 4 anos (26,4%), na qual se deu a maior incidência da doença, e com idade superior a 30 anos (28%). A confirmação diagnóstica foi laboratorial em 71% dos casos (cultura em 45,3%) e por critérios clínico-epidemiológicos em 22%. O sorogrupo B ocorreu em 45,7%; o C, em 47,3%; o W135, em 3,7%; e o Y, em 1,5% dos casos identificados, com predomínio do sorogrupo B, entre 1999 e 2003, e do C, entre 2004 e 2008. Os fenótipos B:4,7:P1.19,15 e C:23:P1.14-6 predominaram. Os resultados reforçam a necessidade de acompanhamento, de forma regional, da tendência da doença para a detecção de surtos precocemente e monitoramento de cepas circulantes.

ERIC-PCR genotypic characterization of Haemophilus parasuis isolated from Brazilian swine

Haemophilus parasuis infection, known as Glässer’s disease, is characterized by fibrinous polyserositis, arthritis and meningitis in piglets. Although traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, the molecular-based methods are alternatives for species-specific tests and epidemiologic study. The aim of this study was to characterize H. parasuis strains isolated from different states of Brazil by serotyping, PCR and ERIC-PCR. Serotyping revealed serovar 4 as the most prevalent (24 %), followed by serovars 14 (14 %), 5 (12 %), 13 (8 %) and 2 (2 %), whereas 40 % of the strains were considered as non-typeable. From 50 strains tested 43 (86%) were positive to Group 1 vtaA gene that have been related to virulent strains of H.parasuis. ERIC-PCR was able to type isolates tested among 23 different patterns, including non-typeable strains. ERIC-PCR patterns were very heterogeneous and presented high similarity between strains of the same animal or farm origin. The results indicated ERIC-PCR as a valuable tool for typing H. parasuis isolates collected in Brazil.

Axenic cell culture of the seagrass Cymodocea nodosa from cotyledonary tissue

[EN] Plant Tissue Culture, also called “micropropagation”, is the propagation of plants from different tissues (or explants) in a shorter time than conventional propagation, making use of the ability that many plant cells have to regenerate a whole plant (totipotency).There are two alternative mechanisms by which an explant can regenerate an entire plant, namely organogenesis and somatic embryogenesis. Since the last decades, the number of higher terrestrial plants species from which these techniques have been successfully applied has continually increased. However, few attempts have been carried out in marine plants. Previous seagrasses authors have focused their studies on i) vegetative propagation of rhizome fragments as explants in Ruppia maritima, Halophila engelmannii, Cymodocea nodosa and Posidonia oceanica; ii) culture of meristems in Heterozostera tasmanica, C. nodosa or P. oceanica; and iii) culture of germinated seeds on aseptic conditions, in Thalassia testudinum, H. ovalis, P. coriacea, P. oceanica, and H. decipiens. All these studies determine the most adequate culture medium for each species (seawater, nutrients, vitamins, carbon sources, etc...), often supplemented with different plant growth regulators and the necessary conditions for the culture maintenance, such as light and temperature. On the other hand, several studies have previously established protocols for cell or protoplast isolation in the species Zostera marina, Z. muelleri, P. oceanica, and C. nodosa, using shoots collected from natural meadows as original vegetal source, but further cell growth was never accomplished. Due to the absence of somatic embryogenesis or organogenetic studies in seagrasses we wonder: IS THE SUCCESSFUL APPLICATION OF TISSUE CULTURE TECHNIQUES POSSIBLE IN SEAGRASSES?

Epidemiological and functional characterization of Streptococcus pneumoniae pili

Streptococcus pneumoniae is an important life threatening human pathogen causing agent of invasive diseases such as otitis media, pneumonia, sepsis and meningitis, but is also a common inhabitant of the respiratory tract of children and healthy adults. Likewise most streptococci, S. pneumoniae decorates its surface with adhesive pili, composed of covalently linked subunits and involved in the attachment to epithelial cells and virulence. The pneumococcal pili are encoded by two genomic regions, pilus islet 1 (PI-1), and pilus islet-2 (PI-2), which are present in about 30% and 16% of the pneumococcal strains, respectively. PI-1 exists in three clonally related variants, whereas PI-2 is highly conserved. The presence of the islets does not correlate with the serotype of the strains, but with the genotype (as determined by Multi Locus Sequence Typing). The prevalence of PI-1 and PI-2 positive strains is similar in isolates from invasive disease and carriage. To better dissect a possible association between PIs presence and disease we evaluated the distribution of the two PIs in a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). We found that 50% of the PI-1 positive isolates belonged to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), and that PI-2 was not present in the absence of Pl-1. In conclusion, there was no correlation between PIs presence and AOM, and, in general, the observed differences in PIs prevalence are strictly dependent upon regional differences in the distribution of the clones. Finally, in the AOM collection the prevalence of PI-1 was higher among antibiotic resistant isolates, confirming previous indications obtained by the in silico analysis of the MLST database collection. Since the pilus-1 subunits were shown to confer protection in mouse models of infection both in active and passive immunization studies, and were regarded as potential candidates for a new generation of protein-based vaccines, the functional characterization was mainly focused on S. pneumoniae pilus -1 components. The pneumococcal pilus-1 is composed of three subunits, RrgA, RrgB and RrgC, each stabilized by intra-molecular isopeptide bonds and covalently polymerized by means of inter-molecular isopeptide bonds to form an extended fibre. The pilus shaft is a multimeric structure mainly composed by the RrgB backbone subunit. The minor ancillary proteins are located at the tip and at the base of the pilus, where they have been proposed to act as the major adhesin (RrgA) and as the pilus anchor (RrgC), respectively. RrgA is protective in in vivo mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Entwicklung und Charakterisierung von Gassterilisationsprozessen am Beispiel der Oberflächensterilisation mittels Wasserstoffperoxid und Ethylenoxid

Bei der Auswahl einer Sterilisationsmethode stehen, gemäß den Vorgaben des Europäischen Arzneibuches, die Sterilisation mittels Hitze (Dampf bzw. Heißluft) oder ionisierender Strahlung im Vordergrund. Diese so genannten Referenzverfahren verfügen neben einem breiten Wirkungsspektrum über eine hohe Effektivität und zeichnen sich durch eine gute Reproduzierbarkeit aus. Häufig jedoch verbieten die physikalisch – chemischen Eigenschaften des zu sterilisierenden Gutes eine Anwendung der Referenzverfahren. In derartigen Fällen muss nach geeigneten Alternativverfahren gesucht werden, will man eine aseptische Herstellung vermeiden. Besonders bei thermolabilen Gütern hat sich der Einsatz von gasförmigen Agenzien mit keimtötender Wirkung bewährt. Bei entsprechenden Rahmenbedingungen ist hier eine Sterilisation unterhalb der Raumtemperatur möglich. Die vorliegende Arbeit untersucht die Anwendbarkeit der Ethylenoxidsterilisation bzw. der Sterilisation mittels gasförmigen Wasserstoffperoxids (Vapour Phase Hydrogen Peroxide) auf verschiedene Materialoberflächen am Beispiel zweier aktueller Anwendungen aus der pharmazeutischen Industrie. Dabei werden die Vor- und Nachteile der beiden Verfahren aufgezeigt und in Relation zu den Referenzverfahren gestellt. Im Rahmen der abschließenden mikrobiologischen Leistungsbeurteilung beider Verfahren werden verschiedene methodische Ansätze, wie Halbzyklus und Fraction Negative Analysis zur Anwendung gebracht und bewertet.

Diet Effects on Activation and Maturation of Feed Control over the Gastrointestinal Defence Barrier in Piglets

Weaning is an important and complex step involving many stresses that interfere deeply with feed intake, gastro-intestinal tract (GIT) development and adaptation to the weaning diet in young pigs. The health of the pig at weaning, its nutrition in the immediate post-weaning period, and the physical, microbiological and psychological environment are all factors that interact to determine food intake and subsequent growth. GIT disorders, infections and diarrhoea increase at the time of weaning, in fact pathogens such as enterotoxigenic Escherichia coli (ETEC) are major causes of mucosal damage in post-weaning disease contributing to diarrhoea in suckling and post-weaned pigs. The European ban in 2006 put on antibiotic growth promoters (AGP) has stimulated research on the mechanisms of GIT disorders and on nutritional approaches for preventing or reducing such disturbances avoiding AGPs. Concerning these aspects here are presented five studies based on the interplay among nutrition, genomic, immunity and physiology with the aim to clarify some of these problematic issues around weaning period in piglets. The first three evaluate the effects of diets threonine or tryptophan enriched on gut defence and health as possible alternatives to AGP in the gut. The fourth is focused on the possible immunological function related with the development of the stomach. The fifth is a pilot study on the gastric sensing and orexygenic signal given by fasting or re-feeding conditions. Although some results are controversial, it appears that both tryptophan and threonine supplementation in weaning diets have a preventive role in E.coli PWD and favorable effects in the gut especially in relation to ETEC susceptible genotype. While the stomach is believed as almost aseptic organ, it shows an immune activity related with the mucosal maturation. Moreover it shows an orexygenic role of both oxyntic mucosa and pyloric mucosa, and its possible relation with nutrient sensing stimuli.

Trascriptome analysis and functional genomics of Neisseria meningitidis in human blood

Neisseria meningitidis (Nm) is the major cause of septicemia and meningococcal meningitis. During the course of infection, it must adapt to different host environments as a crucial factor for survival. Despite the severity of meningococcal sepsis, little is known about how Nm adapts to permit survival and growth in human blood. A previous time-course transcriptome analysis, using an ex vivo model of human whole blood infection, showed that Nm alters the expression of nearly 30% of ORFs of the genome: major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. Starting from these data, mutagenesis studies of a subset of up-regulated genes were performed and the mutants were tested for the ability to survive in human whole blood; Nm mutant strains lacking the genes encoding NMB1483, NalP, Mip, NspA, Fur, TbpB, and LctP were sensitive to killing by human blood. Then, the analysis was extended to the whole Nm transcriptome in human blood, using a customized 60-mer oligonucleotide tiling microarray. The application of specifically developed software combined with this new tiling array allowed the identification of different types of regulated transcripts: small intergenic RNAs, antisense RNAs, 5’ and 3’ untranslated regions and operons. The expression of these RNA molecules was confirmed by 5’-3’RACE protocol and specific RT-PCR. Here we describe the complete transcriptome of Nm during incubation in human blood; we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. In addition the tiling array analysis demonstrated that Nm expresses a set of new transcripts, not previously identified, and suggests the presence of a circuit of regulatory RNA elements used by Nm to adapt to proliferate in human blood.

The role of the NadR regulator during infection and its implication for the coverage of a new Meningococcus B vaccine

Background: Neisseria meningitides represents a major cause of meningitis and sepsis. The meningococcal regulator NadR was previously shown to repress the expression of the Neisserial Adhesin A (NadA) and play a major role in its phase-variation. NadA is a surface exposed protein involved in epithelial cell adhesion and colonization and a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B infection. The NadR mediated repression of NadA is attenuated by 4-HPA, a natural molecule released in human saliva. Results: In this thesis we investigated the global role of NadR during meningogoccal infection, identifying through microarray analysis the NadR regulon. Two distinct types of NadR targets were identified, differing in their promoter architectures and 4HPA responsive activities: type I are induced, while type II are co-repressed in response to the same 4HPA signal. We then investigate the mechanism of regulation of NadR by 4-HPA, generating NadR mutants and identifying classes or residues involved in either NadR DNA binding or 4HPA responsive activities. Finally, we studied the impact of NadR mediated repression of NadA on the vaccine coverage of 4CMenB. A selected MenB strains is not killed by sera from immunized infants when the strain is grown in vitro, however, in an in vivo passive protection model, the same sera protected infant rats from bacteremia. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h post-infection. Conclusions: Our results suggest that NadR coordinates a broad transcriptional response to signals present in the human host, enabling the meningococcus to adapt to the relevant host niche. During infectious disease the effect of the same signal on NadR changes between different targets. In particular NadA expression is induced in vivo, leading to efficient killing of meningococcus by anti-NadA antibodies elicited by the 4CMenB vaccine.