Introdução: O Doppler tecidual (DT) surgiu como uma técnica ecocardiográfica para registro das velocidades do miocárdio e evoluiu para a determinação da deformação miocárdica regional com medida do strain rate (SR) e strain longitudinal e radial unidimensionais. Nosso objetivo foi determinar padrões de normalidade em neonatos e analisar seqüencialmente esses índices em períodos de alta e baixa resistência vascular pulmonar. Casuística e Método: Selecionaram-se 55 crianças com idade média de 20,14 ± 14,0 horas, constituindo o Grupo I (G I). Um segundo exame foi realizado em 30 crianças que retornaram após 31,9 ± 2,9 dias de vida, constituindo o Grupo II (G II). As velocidades do DT pulsado foram obtidas em cortes transversais (R) e longitudinais (L) do ventrículo esquerdo (VE) em posições apical 4 e 2 câmaras e no VD (paredes livre e inferior). Foram medidas velocidades sistólicas (onda Sm), diastólica inicial (Em), diastólica final (Am) de pico e a relação Em/Am. Pelo menos três ciclos cardíacos consecutivos com taxa de quadros/s superior a 300 foram digitalmente obtidos nos cortes mencionados e analisados posteriormente utilizando programa específico para medida das curvas de SR/strain e seus componentes sistólico, diastólico inicial e final. Resultados: As velocidades demonstraram gradiente bem definido com redução progressiva na direção base-ápice do coração. As velocidades do VD foram superiores às do VE quando comparadas com as medidas nas paredes septal (S), anterior (A) e lateral (L). No GI verificamos diferença significativa entre as medidas do SR/strain sistólicos do segmento basal da parede S em relação à apical (-1,90 ± 0,61, - 25,90 ± 4,90 vs -1,66 ± 0,25, - 24,23 ± 3,08), p=0,04 e p=0,02 e do segmento médio em relação ao apical (p=0,01 e 0,02). A avaliação regional do VD demonstrou strain sistólico maior no segmento médio em relação ao basal (-33,20 ± 6,34 vs -28,38 ± 4,90, p=0,00) e em relação ao segmento apical (-33,20 ± 6,34 vs -31,95 ± 5,06, p=0,021). Os valores absolutos de SR/strain e todos os seus componentes foram maiores na direção R quando comparados com a L (SR sistólico 2,99 ± 0,78 s-1 vs (-)1,90 ± 0,60 s-1 strain sistólico 49,72% ± 12,86% vs (-) 25,86% ± 4,83 p=0,00). Quando comparamos os GI e GII verificamos redução do strain sistólico do VE na direção R e L nas paredes S, L e A em todos os segmentos. O SR sistólico reduziu apenas na porção basal da parede L (-1,91 ± 0,46 s-1 vs - 1,71 ± 0,33 s-1, p=0,02). O VD apresentou no GII aumento significativo do strain sistólico e diastólico inicial em todos os segmentos e paredes. O SR sistólico também apresentou aumento dos valores nos segmentos basal e médio de sua parede livre e na parede inferior. A correlação entre a onda Sm e SR/strain sistólicos não foi significativa. Conclusão: Os índices regionais de deformação miocárdica constituem técnica clínica reproduzível em neonatos e podem monitorar alterações seqüenciais fisiológicas da circulação neonatal precoce e tardia. São mais robustos que as velocidades na quantificação da função regional. / Background: Color Doppler myocardial imaging (CDMI) has emerged as an echocardiographic technique for determining myocardial velocities and has been further developed to allow the determination of one-dimension regional longitudinal and radial strain rate (SR)and strain. Our goal was to determine normal values in neonates and sequentially analyse these indices in periods of high and low pulmonary vascular resistance. Study population and methods: Fifty-five term newborns with mean age of 20.14 ± 14.0 hours were selected to be part of Group I (GI). A second echo study was performed on 30 children that had returned with 31.9 ± 2.9 days after birth, being Group II (GII). Pulsed wave Doppler tissue velocities (PWDTV) were obtained in short axis (R) and longitudinal (L) axis of the left ventricle (LV) in apical 4 and 2 chamber view and in 4 and 2 chamber of the right ventricle (RV), including RV free lateral and inferior walls. Peak systolic (Sm), peak early diastolic (Em) and peak late diastolic (Am) motion velocities and Em/Am ratio were measured. At least three consecutive cardiac cycles with frame rate more than 300 fps were stored in digital format from the mentioned views for offline analysis by using dedicated software for measuring peak systolic and peak early and late diastolic SR/strain. Results: PWTDV have shown a well-defined gradient with progressive peak reduction from base to apex. RV velocities were higher than those of the LV whem compared to septal (S), anterior (A) and lateral (L) walls. In GI there was a significant difference between systolic SR/strain measurement of basal S segment in relation to apical (-1.90 ± 0.61, -25.90 ± 4.90 vs -1.66 ± 0.25, - 24.23 ± 3.08), p=0.04 e p=0.02 and from the mid in relation to the apical segment (p=0.01 e 0.02). Regional RV longitudinal function showed that systolic strain recorded from the mid segment was significantly higher than that recorded from the basal segment (-33.20 ± 6.34 vs -28.38 ± 4.90, p=0.00) and that from the apical segment (-33.20 ± 6.34 vs ?31.95 ± 5.06, p=0.021). The absolute and all components of SR/strain were significantly higher in R direction when compared to the L ones (systolic SR 2.99 ± 0.78 s-1 vs (-)1.90 ± 0.60 s-1 systolic strain 49.72% ± 12.86% vs (-)25.86% ± 4.83 p=0.00). When comparing data from GI and GII, we noticed reduction of LV systolic strain in the second group for both R and L, in all segments of S, L and A walls. Systolic SR showed reduction of the values in GII only in the basal segment of the L wall (-1.91 ± 0.46 s-1 vs ?1.71 ± 0.33s-1, p=0.02). Regional RV function showed systolic and early diastolic strain significantly higher in all segments of wall in GII. Systolic SR also showed higher values in the basal and mid segments of RV free lateral and inferior wall in GII when comparing to GI. The correlation between peak systolic velocity Sm and peak systolic SR/strain was not significant. Conclusion: Regional myocardial deformation indices are reproducible clinical techniques in neonates and can monitor physiological sequential circulatory changes of the early and late neonatal period. They are more robust than velocities in the quantification of the regional myocardial function.

A icterícia por hiperbilirrubinemia indireta afeta mais de 60% dos recém-nascidos a termo. O tratamento, quando necessário, é realizado através da fototerapia. Não existem estudos na literatura avaliando os efeitos da fototerapia na função dos neutrófilos de recém-nascidos. O melhor entendimento da função dos neutrófilos nos recém-nascidos antes e após a fototerapia seria importante para avaliar as possíveis repercussões na expressão dos neutrófilos desencadeadas pelo tratamento fototerápico. O objetivo deste estudo foi avaliar e comparar a função dos neutrófilos, através da mensuração pela citometria de fluxo da expressão dos principais marcadores de superfície em recémnascidos ictéricos, antes e após 24 horas de fototerapia. Metodologia: Foram incluídos recém-nascidos com idade gestacional ≥ 35 semanas e peso de nascimento ≥ 2000g, que possuiam critérios da Academia Americana de Pediatria para tratamento fototerápico. Os critérios de exclusão foram: mal-formações congênitas, síndromes com alterações cromossômicas, erro inato do metabolismo, infecções do grupo STORCH, asfixia neonatal, sepse ou suspeita de sepse, exsanguineotransfusão, transfusão de hemocomponentes e uso de imunoglobulina. Foi realizada a avaliação de expressão da intensidade média de fluorescência (IMF) de CD10, CD11b, CD11c, CD15, CD16, CD18, CD62L, CD64 e CD66, antes do início e após 24 horas do início da fototerapia. Foram utilizados o teste T de Student para análise dos dados. Resultados: Foram incluídos 25 recém-nascidos no estudo, com idade mediana de 53 (27.5-75.5) horas de vida e bilirrubina média de 13.6±2.85 mg/dL. Não houve diferença estatística na expressão de CD11b, CD15, CD18, CD62L, CD64 e percentual de neutrófilos antes e após 24 horas de fototerapia. Ocorreu aumento da expressão de CD10 8 (p=0.038) e CD16 (p=0.017) e redução da expressão de CD11c (p=0.023) e CD66acde (p=0.004) após 24 horas de fototerapia. Conclusão: Os recém-nascidos submetidos ao tratamento fototerápico apresentaram aumento da expressão de CD10 e de CD16 e diminuição da expressão de CD11c e de CD66acde após 24 horas de exposição, que pode estar relacionado a um efeito antiinflamatório da fototerapia nos recém-nascidos expostos a este tratamento. / Jaundice due to indirect hyperbilirubinemia affects more than 60% of term neonates. The treatment when necessary is carried out using phototherapy. There are no studies in the literature evaluating the effect of phototherapy on the function of neonates' neutrophils. A better understanding of the function of neutrophils in neonates before and after phototherapy would be important in order to assess potential effects on the expression of neutrofils triggered by the phototherapy treatment. The aim of this study was to assess and compare the function of neutrophils by measuring the expression of the main surface markers in icteric neonates, using flow cytometry, before and after 24 hours of phototherapy. Methodology: Neonates at a gestational age ≥ 35 weeks and at a birth weight ≥ 2000g who met the criteria of the American Academy of Pediatrics for phototherapy were included. The exclusion criteria were: congenital malformations, syndromes with chromosomal alterations, inborn errors of metabolism, infections of the STORCH group, neonatal asphyxia, sepsis or suspicion of sepsis, exchange transfusion, transfusion of blood components, and use of immunoglobulin. The evaluation of the MFI expression of CD10, CD11b, CD11c, CD15, CD16, CD18, CD62L, CD64 and CD66 was performed before and 24 hours after the initiation of phototherapy. The chi-square and Student T tests were used for data analysis. Results: Twenty-five neonates were included in the study at the mean age of 53 (27.5- 75.5) hours of life and with a mean bilirubin level of 13.6±2.85 mg/dL. There was no statistical difference in the expression of CD11b, CD15, CD18, CD62L, CD64 and percentage of neutrophils before and after 24 hours of phototherapy. There was an increase in the expression of CD10 (p=0.038) and CD16 (p=0.017) and a reduction in 10 the expression of CD11c (p=0.023) and CD66acde (p=0.004) after 24 hours of phototherapy. Conclusion: The newborns submitted to phototherapy had increased expression of CD10 and CD16 and decreased expression of CD11c and CD66acde after 24 hours of exposure, which may be related to an anti-inflammatory effect of phototherapy on the neonates exposed to this treatment.

In dieser retrospektiven Pilotstudie zum Einsatz von rekombinatem, aktivierten Faktor VII bei Frühgeborenen (Schwangerschaftswoche < 32, Geburtsgewicht < 1750 g) mit intraventrikulären Hirnblutungen wurden anhand sonografischer Untersuchungen der Blutungsprogress sowie die Nebenwirkungen der Therapie im Vergleich zur Standarttherapie mit Fresh Frozen Plasma untersucht. Die Auswertung erfolgte in zwei Gruppen: innerhalb des Gesamtkollektives und innerhalb der Matching-Gruppe. Es zeigte sich in beiden Auswertungsgruppen ein signifikant geringerer Blutungsprogress in der Therapiegruppe. Außerdem konnte ein früherer Blutungsstopp in der Therapiegruppe nachgewiesen werden. Ein erhöhtes Nebenwirkungspotential von rekombinantem, aktivierten Faktor VII war in diesem Patientenkollektiv nicht eruierbar. Weiterführende prospektive, randomisierte, multizentische Studien zur Evaluation dieses vielversprechenden Therapieansatzes werden empfohlen.

Background: Maternal perinatal mental health has been shown to be associated with adverse consequences for the mother and the child. However, studies considering the effect of DSM-IV anxiety disorders beyond maternal self-perceived distress during pregnancy and its timing are lacking. Aims: To examine the role of maternal anxiety disorders with an onset before birth and self-perceived distress during pregnancy for unfavourable maternal, obstetric, neonatal and childhood outcomes. Study design: DSM-IV mental disorders and self-perceived distress of 992 mothers as well as obstetric, neonatal and childhood outcomes of their offspring were assessed in a cohort sampled from the community using the Munich-Composite International Diagnostic Interview. Logistic regression analyses revealed associations (odds ratios) between maternal anxiety disorders and self-perceived distress during pregnancy with maternal depression after birth and a range of obstetric, neonatal and childhood psychopathological outcomes. Results: Lifetime maternal anxiety disorders were related to offspring anxiety disorders, but not to offspring externalizing disorders. Analyses focussing on maternal DSM-IV anxiety disorders before birth yielded associations with incident depression after birth. In addition, self-perceived distress during pregnancy was associated with maternal depression after birth, preterm delivery, caesarean section, separation anxiety disorder, ADHD, and conduct disorder in offspring. Conclusion: Findings confirm the transmission of anxiety disorders from mother to offspring. Apart from maternal anxiety, self-perceived distress during pregnancy also emerged as a putative risk factor for adverse outcomes. The finding that maternal anxiety disorders before birth yielded less consistent associations, suggests that self-perceived distress during pregnancy might be seen as a putative moderator/mediator in the familial transmission of anxiety.

Le processus décisionnel est un évènement particulier qui confronte les soignants à leurs doutes et à leurs incertitudes. Chaque décision comporte inéluctablement une part de doute et d’incertitude, sinon elle ne mérite pas d’être appelée « décision », car elle ne serait que le résultat de l’application d’un savoir acquis à l’avance, à une situation connue. C’est cette part d’incertitude qui est le lieu de la réflexion et qui offre à chacun la possibilité de se questionner, c’est-à-dire de se mettre dans la situation où il faut vaincre l’indécidable pour pouvoir décider. Au cours de ce travail, j’aborde le rôle du médecin en tant qu’arbitre entre le certain et l’incertain en situation de fin de vie chez le nouveau-né. Afin de cerner cette problématique, je rapporte six observations cliniques, toutes tirées de ma propre expérience et qui illustrent mieux qu’un long discours les notions du certain et de l’incertain, et permettent de donner à chacune de ces notions son véritable sens pratique de manière concrète.Vouloir aborder la notion de l’incertain au cours du processus décisionnel en situation de fin de vie, consiste à accepter d’aller à l’encontre de la culture médicale dominante, qui valorise la certitude, car l’incertitude véhicule une image négative dans l’imaginaire des soignants. Pourtant, le doute peut avoir un effet salvateur sur le processus décisionnel, car il incite les soignants à se concerter avant de décider. Ainsi, celui qui doute n’est pas un ignorant, mais quelqu’un de prudent au sens aristotélicien, c’est-à-dire qui soumet son choix à la critique des autres, dans le cadre d’une procédure de délibération collégiale. Ainsi, l’incertitude met en tension les convictions qui relèvent de l’éthique déontologique, et la responsabilité qui relève de l’éthique téléologique. C’est la confrontation entre ces deux éthiques qui permet au soignant de décider en ayant la paix dans l’âme. / The end-of-life (EOL) decision-making process remains a particular event that confronts health care teams with their doubts and uncertainties. Indeed, for such a decision to made, there must be some doubt and uncertainty, otherwise, instead of being a conscious “decision”, it becomes a simple application of prior knowledge. EOL decisions are not simply a case of the decision-maker overcoming his or her indecisiveness. In this work, I examine the role of the doctor as an arbitrator in the conflict between the certain and the uncertain in EOL situations in the neonatal period. I have based my work on the analysis of the trajectories of six clinical observations drawn from my own experience, which illustrate, better than a long speech, the notion of the certain and the uncertain, and give these abstract concepts a concrete and practical meaning.To explore the notion of the uncertain during the decision-making process in EOL situations, is to accept to go against the dominant medical culture that values certainty, because uncertainty is negatively perceived amongst caregivers. However, doubt can have a beneficial effect on the decision-making process itself, and on the consequences of the decision taken, since it encourages discussion between caregivers and allows time for decision-making. Thus, he or she who doubts is not ignorant, but cautious in the Aristotelian sense, when trying to deliberate a choice by subjecting it to the criticism of others as part of a procedure of collegial reflection.In this work, I also show that doubt and uncertainty compel the decision-maker to confront his or her personal convictions, which constitutes ethical ethics, with his or her responsibility, which is teleological ethics. It is only in the tension between these two ethics that the caregiver can decide with peace of mind.

OBJECTIVE: This study was conducted to compare the relative influence of the socioeconomic status of both mothers and fathers on feeding method and cosleeping. METHODS: The time and method of feeding and sleeping were recorded in a log during the 4th-week postpartum and analyzed according to the parental Hollingshead Index of Social Position in 33 families with their first newborn. RESULTS: The effect of socioeconomic status on feeding and sleep was parent specific. Low socioeconomic status of the mother, but not the father, was associated with cosleeping (t ≤ 2.39, P < .01); whereas, a low socioeconomic status of the father, but not the mother, was associated with bottle-feeding rather than breast-feeding (t ≤ 1.94, P < .05). CONCLUSIONS: Socioeconomic status of the parents differentially affects neonatal care. Programs to increase breast-feeding rates would be most effective if designed for and aimed at the fathers. Copyright © 2007 Wolters Kluwer Health | Lippincott Williams and Wilkins.

INTRODUÇÃO: Uma possibilidade de pesquisa em audição é a supressão das emissões otoacústicas, que verifica a diminuição de sua magnitude a partir de estimulação acústica simultânea. Acredita-se que esta diminuição ocorra devido ao papel da via eferente auditiva. OBJETIVO: verificar a atividade do sistema auditivo eferente, por meio da supressão contralateral das emissões otoacústicas, em neonatos com e sem indicadores de risco para perda auditiva. MÉTODO: foram avaliados 21 neonatos com indicadores de risco para perda auditiva (grupo estudo) e 30 neonatos sem indicadores (grupo controle) por meio das emissões otoacústicas evocadas por estímulo de freqüência específica \"tone burst\" em 2000 e em 4000 hertz, na ausência e na presença de ruído branco contralateral. RESULTADOS: a orelha direita e o grupo controle apresentaram vantagem na amplitude das EOA TB. Os valores médios de \"Response\" para 2000 hertz foram 17,73 dB no grupo controle e 16,55 dB no grupo estudo, para o gênero feminino e 16,63 dB no grupo controle e 16,12 dB no grupo estudo, para o gênero masculino. Para 4000 hertz, os valores médios de \"Response\" foram 14,63 dB no grupo controle e 15,09 dB no grupo estudo, para o gênero feminino e 18,57 dB no grupo controle e 15,06 dB no grupo estudo, para o gênero masculino. Na supressão, houve vantagem da orelha direita, do grupo controle para o gênero feminino e do grupo estudo para o gênero masculino, embora sem plena significância estatística. Os valores médios de supressão para a variável \"Response\" em 2000 hertz foram 1,66 dB no grupo controle e 0,84 dB no grupo estudo, para o gênero feminino e 1,41 dB no grupo controle e 2,07 dB no grupo estudo, para o gênero masculino. Para 4000 hertz, os valores médios da supressão em \"Response\" foram 1,29 dB no grupo controle e 0,98 dB no grupo estudo, para o gênero feminino e 1,75 dB no grupo controle e 2,14 dB no grupo estudo, para o gênero masculino. CONCLUSÃO: A partir de estímulos de freqüência específica, em 2000 e em 4000 hertz, foi possível verificar o efeito inibitório da via eferente auditiva sobre a amplitude das EOA, em neonatos. A utilização de estímulos de freqüência específica pode contribuir para a avaliação da função auditiva periférica e central nesta população. / INTRODUCTION: A possible hearing research is the suppression of otoacoustic emissions which verifies a decrease of its magnitude from a simultaneous acoustic stimulation. It is assumed that this decrease occurs because of the role of the auditory efferent pathway. AIM: to verify the activity of the auditory efferent pathway by the contralateral suppression of OAE in neonates with and without risk factors for hearing loss. MEHOD: 21 neonates at risk for hearing loss (study group), and 30 neonates without risk factors for hearing loss (control group) were assessed through otoacoustic emissions evoked by a frequency specific stimulus, \"tone burst\", at 2000 and at 4000 hertz, with and without contralateral white noise. RESULTS: the right ear and the control group presented advantage in the amplitude of OAE TB. The mean values of \"Response\" at 2000 hertz were 17,73 dB in the control group and 16,55 dB in the study group, for female subjects, and 16,63 dB in the control group and 16,12 dB in the study group, for male subjects. At 4000 hertz, the mean values of \"Response\" were 14,63 dB in the control group and 15,09 dB in the study group, for female subjects and 18,57 dB in the control group and 15,06 dB in the study group, for male subjects. In the suppression, there was an advantage of the right ear, of the control group for female subjects, and of the study group for male subjects, although without statistical significance. The mean values of suppression for the variable \"Response\" at 2000 hertz were 1,66 dB in the control group and 0,84 dB in the study group, for female subjects, and 1,41 dB in the control group and 2,07 dB in the study group, for male subjects. At 4000 hertz, the mean values of suppression for \"Response\" were 1,29 dB in the control group and 0,98 dB in the study group, for female subjects, and 1,75 dB in the control group and 2,14 dB in the study group, for male subjects. CONCLUSION: From frequency-specific stimuli at 2000 and at 4000 hertz, it was possible to verify the inhibitory effect of the auditory efferent pathway upon the amplitude of otoacoustic emissions in neonates. The use of frequencyspecific stimuli may contribute for the assessment of the central auditory function in this population.

A ocorrência de candidemias em pacientes internados em unidades de cuidados intensivos é um grave problema por estar relacionada a elevadas taxas de mortalidade. Sinais e sintomas de infecção hematogênica causada por Candida sp. são inespecíficos, dificultando o diagnóstico e representando um desafio para o desenvolvimento de métodos diagnósticos que identifiquem precocemente o agente em amostras clínicas. As metodologias tradicionais (hemocultura e identificação fenotípica) e sorológicas (detecção de beta-D-glucana, manana e anticorpos anti-manana) apresentam limitações de sensibilidade e especificidade, sendo os métodos moleculares uma alternativa para o diagnóstico dessas infecções. Neste trabalho foi desenvolvida uma técnica de nested PCR multiplex capaz de identificar sete espécies de Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae e C. pelliculosa) diretamente em amostras sanguíneas de pacientes com elevado risco de desenvolver candidemias. Na primeira etapa de amplificação foram utilizados primers universais para fungos (ITS1 e ITS4); e na segunda, os primers espécie-específicos foram distribuídos em dois sistemas de amplificação, a saber: um com primers para C. albicans, C. glabrata, C. tropicalis e C. lusitaniae; e outro com primers para C. parapsilosis, C. krusei e C. pelliculosa. A nested PCR multiplex foi capaz de detectar cerca de 4UFC/mL das espécies envolvidas no estudo, não apresentando reatividade cruzada quando avaliada com amostras de DNA de outros fungos e bactérias envolvidos em episódios de infecção hematogênica. Foram analisadas 55 amostras obtidas de pacientes pediátricos internados em unidades de cuidados intensivos do Instituto da Criança do HC-FMUSP e do Hospital de Base da Faculdade de Medicina de São José do Rio Preto - UNESP, com idades variando de seis dias a 16 anos, apresentando Síndrome da Resposta Inflamatória Sistêmica e/ou dois ou mais fatores de risco para infecção fúngica. Também foi avaliado no estudo um grupo controle, composto por 28 crianças sem risco de desenvolver fungemias, para verificação da ocorrência de possíveis reações inespecíficas pela técnica molecular. As hemoculturas foram positivas em oito pacientes (14,8%), tendo sido identificadas cinco C. albicans, uma C. tropicalis, uma C. parapsilosis e uma C. krusei. Por outro lado, a técnica de nested PCR multiplex detectou DNA de Candida em 13 pacientes (23,6%), tendo havido concordância na identificação das espécies em 100% dos casos. Entretanto, não foi observada discordância estatisticamente significativa (p=0,063) entre as duas metodologias. Dos cinco pacientes com hemoculturas negativas, a técnica molecular foi capaz de detectar dupla candidemia em três casos, identificando nessas amostras C. tropicalis e C. parapsilosis simultaneamente. Nos cinco casos em que apenas a técnica de PCR foi positiva, as amostras amplificadas foram sequenciadas, apresentando similaridades de 99 a 100%, quando comparadas às sequencias de cepas de Candida depositadas no GenBank. O tempo para execução da técnica e liberação de resultados foi de aproximadamente 24 horas. O custo relativo aos procedimentos realizados para execução da PCR foi calculado e comparado ao custo dos procedimentos executados nas hemoculturas, tendo sido mais baixo. Os resultados demonstram que a nested PCR multiplex é técnica alternativa para a detecção e identificação de espécies de Candida em pacientes com risco de desenvolver candidemias, apresentando limiar de detecção adequado e capacidade de identificar mais de uma espécie de Candida simultaneamente em amostras clínicas. A técnica é ainda capaz de permitir a liberação de resultados em menor tempo, além de apresentar menor custo, quando comparada à metodologia convencional de cultivo / The incidence of candidemia among intensive care unit patients is associated with high mortality rates. The non-specific nature of the signs and symptoms of this infection difficult the diagnosis. Development of methods that allow early identification of the agent in clinical samples still represents a challenge. Classical methodologies (blood cultures and phenotypic identification) and serological tests (beta-D-glucan and mannan detection; anti-mannan antibodies) to diagnose candidemia lack sensitivity and specificity. Molecular methods are an alternative approach to the diagnosis of candidemia. In this work we developed a nested PCR multiplex assay able to identify seven Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae and C. pelliculosa) directly in blood samples of patients at high risk of candidemia. In the first stage of amplification universal fungal primers (ITS1 and ITS4) were used and in the second stage, the species-specific primers were divided into two amplification systems, one with primers for C. albicans, C. glabrata, C. tropicalis and C. lusitaniae, and another with primers for C. parapsilosis, C. krusei and C. pelliculosa. The multiplex nested PCR was able to detect approximately 4 CFU/mL of the seven Candida species, and showed no cross reactivity when evaluated with samples of DNA from other fungi and bacteria causing hematogenous infection episodes. We analyzed 55 samples obtained from patients admitted to pediatric intensive care units of the Instituto da Criança do HCFMUSP and Hospital de Base of Medical School from São José do Rio Preto - UNESP, with ages ranging from six days to 16 years, presenting the Systemic Inflammatory Response Syndrome and/or two or more risk factors of fungal infection. A control group of 28 children without risk of fungemia, to check for occurrence of nonspecific reactions in the molecular technique, was also evaluated. Blood cultures were positive in eight patients (14.8%), five were identified as C. albicans, and the remaining as C. tropicalis, C. parapsilosis and C. krusei. The nested multiplex PCR assay detected Candida DNA in 13 patients (23.6%), and there was concordance in species identification in 100% of the cases. In five patients with negative blood cultures, the molecular technique was capable of detecting dual candidemia in three patients\' samples, identifying C. tropicalis and C. parapsilosis simultaneously. However, there was no statistically significant disagreement (p = 0.063) between the two methods. In these five cases for whom only the PCR was positive, the amplified samples were sequenced, showing similarities from 99 to 100% when compared with sequences of Candida strains deposited in GenBank. Time for execution of the technique and release of results was about 24 hours. The cost of PCR assay was compared with the cost of the conventional blood culture tests and was lower. In conclusion, we demonstrated that the nested PCR multiplex developed in our laboratory is an alternative technique for the detection and identification of Candida species in patients at risk of candidemia, showing appropriate detection threshold and the ability to identify simultaneously more than one Candida species in clinical samples. The technique is also rapid, giving results in 24 h and exhibiting lower cost when compared with the conventional culture methodology

O habitat natural da Neisseria meningitis é a nasofaringe humana e a transmissão da bactéria é por contato direto ou por inalação de partículas durante a fase de transmissão N. meningitis é uma bactéria Gram-negativa responsável por uma significante mortalidade em todo o mundo. Embora existam vacinas polissacárides contras os sorogrupos A, C, W135 e Y , não há uma vacina adequada para crianças menores de 4 anos para o sorogrupo B. Estudos estão sendo direcionadas para pesquisa de antígenos vacinais que são derivados da proteínas de membrana externa(NOMV). Entretanto vacinas baseadas em NOMV são consideradas pouco imunogênicas , fazendo com que o uso de adjuvantes seja necessário. Este estudo investiga a imunogenicidade da NOMV de N. meningitidis administrada pela via intranasal/intramuscular em camundongos neonatos BALC/c, usando proteína de membrana externa (NOMC) obtido de uma cepa epidêmica de N. meningitidis B:4:P1:15. As cepas usadas para imunização dos camundongos foram selecionadas por colony-blot, usando anticorpo monoclonal anti L3,7,9 para maior expressão do LPS contra o imunotipo L3,7,9 presente na cepa (B:4:P1:15 3,7,9). Como adjuvantes de mucosa foram utilizados Bordetella pertussis (células íntegras) ou sobrenadante de cultura com 48 horas ou hidróxido de alumínio [Al(OH)3]. O soro dos camundongos imunizados foram analisados pelo método de ELISA à fim de se comparar os diferentes adjuvantes utilisados. O índice de avidez também foi determinado. IgG e IgM foram detectados nos soros dos camundongos após imunização, com índices de intermediária e alta avidez. Todos os adjuvantes foram capazes de aumentar a resposta imune contra NOMV de N.meningitidis. A via intranasal foi adequada para sensibilizar as células do sistema imune que foram rapidamente estimuladas pela via intramuscular usando os adjuvantes utilizados na presente investigação. Dados sugerem que o estudo da NOMV é importante na indução da imunidade de mucosa para N. meningitidis B, e que a qualidade e magnitude da resposta imune gerada pelas vacinas de mucosa são influenciadas tanto pelo adjuvante como pelo antígeno. Concluímos que NOMV juntamente com adjuvantes de mucosa tem considerável potencial no desenvolvimento de vacinas contra o meningococo do sorogrupo B. / The natural habitat of Neisseria meningitidis is the human nasopharynx, and the bacterium is transmitted by direct mouth-to-mouth contact or by the inhalation of released mucous particles during close contact. N meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharide-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Current studies are searching for vaccinal antigens that are derived from the native outer membrane (NOMV). However, vaccines based on NOMV are considered weak, making the use of adjuvants necessary. This study investigated the immunogenicity of NOMV of N. meningitidis administered intranasal/intramuscular in neonate BALB/c mice, using the native outer membrane complex (NOMC) obtained from an epidemic strain of N. meningitidis B:4:P1.15. The strains used for immunization of mice were selected by colony-blot, using anti L3,7,9 monoclonal antibodies, for the highest expression of LPS among the immunotypes (B:4:P1:15 L9á). As mucosal adjuvants, we used Bordetella pertussis (whole cells) or the supernatant of 48 h culture of this bacterium, followed by an intramuscular dose of the same protein adsorbed onto , B. pertussis (whole cells) or 48-h B. pertussis culture supernatant or aluminum hydroxide [Al(OH)3]. Sera of immunized mice were evaluated by ELISA in order to compare the different adjuvants used. We also determined their avidity index. IgG and IgM were detected in the serum of mice after immunization, with avidity indices that ranged from intermediate to high. All adjuvants were capable of increasing the immune response against NOMV of N. meningitidis in the homologous prime/boost schedule used. The intranasal route was suitable for sensitizing the cells of the immune system which were quickly stimulated by the intramuscular route using the adjuvants analysed in the present invertigation. Data suggest that the NOMV studied is important in the induction of mucosal immunity to N. meningitidis B, and that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the adjuvant as well as the antigen. In conclusion, nasal delivery of NoMV with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.

Le recours aux prescriptions sans ou hors AMM, dû principalement au manque d’essais cliniques, est très largement répandu en pédiatrie en particulier chez le nouveau-né. Les prescriptions hors AMM constituent un facteur de risque dans la survenue des effets indésirables médicamenteux (EIM). La pharmacovigilance est indispensable mais insuffisante afin d’évaluer le rapport bénéfice/risque des traitements en particulier lorsque les prescriptions hors AMM sont fréquentes. En combinant plusieurs approches pharmacoépidémiologiques, méta-analytiques et pharmacométriques, ce travail de thèse a pour objectifs de décrire les prescriptions hors AMM en néonatologie et explorer des approches qui permettront d’aboutir à mieux estimer le rapport bénéfice/risque des médicaments pour les enfants. Nous avons confirmé que le taux de prescription hors AMM en néonatologie est important (46%). Même en absence de soins de réanimation, plus de deux tiers des nouveaux-nés reçoivent au moins un médicament hors AMM pendant leur hospitalisation. En utilisant une approche méta-analytique explorant le rapport bénéfice risque du fluconazole en néonatologie en fonction du risque de base dans chaque étude (taux d’infection <10%), nous avons montré que la prophylaxie ciblée sur le risque de base serait plus bénéfique que la prophylaxie systématique des infections fongiques invasives. Nos travaux en pharmacovigilance pédiatrique, ont montré que les deux méthodes de détection actives (collaboration entre pharmaciens et cliniciens et le « trigger tool »), utilisant les dossiers patients informatisés, sont faisables et plus efficaces que la notification spontanée / Many drugs used to treat children in hospitals are either not licensed for use in children or are prescribed outside the terms of their product license (off-label prescribing) because of the lack of clinical trials in this population, and practical difficulties to involve children in clinical research. Drugs used within the specifications of the product license should therefore be less likely to cause ADRs compared to drugs that are either unlicensed or off-label for use in children. With different approaches such as pharmacoepidemiologic, meta-analytic and pharmacometrics, we sought to improve the estimate of the benefit risk ratio of medicines used for treating children. Our observational prospective study in a neonatal unit demonstrated that the prescriptions of unlicensed or off-label drugs in neonatal unit is common (46%). We also used meta-analytic approach in order to estimate the benefit risk ratio of the prophylactic use of fluconazole as a function of the baseline risk. Our results suggest that “systematic fluconazole prophylaxis for all VLBW in NICUs is not warranted by the available evidence and should be adapted to the baseline risk. We also showed that active drug monitoring by using electronic patient files by targeting review chart with a trigger tool in neonates and with close collaboration between the pharmacovigilance center, pharmacologists, and clinicians was necessary and feasible for improving the detection of ADRs in children