Age-Related Macular Degeneration-Associated Genes in Alzheimer Disease

Objectives: Given the clinical and pathological similarities between age-relatedmacular degeneration (AMD) and Alzheimer disease (AD), to assess whether AMDassociatedsingle nucleotide polymorphisms (SNPs), including those from complementrelatedgenes, are associated with AD. 
Design: A case-control association study-typedesign. 
Setting: A UK tertiary care dementia clinic. 
Participants: 322 cognitivelynormal participants and 258 cases with a clinical diagnosis of AD.
Measurements:Polymorphisms in the following genes were studied: CFH, ARMS2, C2/CFB, C3, CFI/PLA2G12a, SERPING1, TLR3, TLR4, CRP, APOE, and TOMM40. Haplotypes were analysedfor CFH, TOMM40, and APOE. Univariate analysis was performed for each geneticchange and case-comparator status, and then correction for multiple testing performed. 
Results: The presence of an ε4 APOE allele was significantly associated with AD. Noassociation was evident between CFH SNPs or haplotypes, or other AMD-associated SNPstested, and AD. The exceptions were TOMM40 SNPs, which were associated with AD evenafter correction for multiple comparisons. The associations disappeared, however, whenentered into a regression model including APOE genotypes. 
Conclusions: The resultsfor most SNPs tested, as well as CFH haplotypes, are novel. The functional effects ofabnormal complement activity in AD’s pathogenesis may be contradictory, butmethodological reasons may underlie the lack of association—for example, geneticchanges other than SNPs being involved.

Predictive Value of Depressive Symptoms for All-Cause Mortality: Findings From the PRIME Belfast Study Examining the Role of Inflammation and Cardiovascular Risk Markers

OBJECTIVES: To improve understanding about the potential underlying biological mechanisms in the link between depression and all-cause mortality and to investigate the role that inflammatory and other cardiovascular risk factors may play in the relationship between depressive symptoms and mortality.

METHODS: Depression and blood-based biological markers were assessed in the Belfast PRIME prospective cohort study (N = 2389 men, aged 50-59 years) in which participants were followed up for 18 years. Depression was measured using the 10-item Welsh Pure Depression Inventory. Inflammation markers (C-reactive protein [CRP], neopterin, interleukin [IL]-1 receptor antagonist [IL-1Ra], and IL-18) and cardiovascular-specific risk factors (N-terminal pro-b-type natriuretic peptide, midregion pro-atrial natriuretic peptide, midregion pro-adrenomedullin, C-terminal pro-endothelin-1 [CT-proET]) were obtained at baseline. We used Cox proportional hazards modeling to examine the association between depression and biological measures in relation to all-cause mortality and explore the mediating effects.

RESULTS: During follow-up, 418 participants died. Higher levels of depressive symptoms were associated with higher levels of CRP, IL-1Ra, and CT-proET. After adjustment for socioeconomic and life-style risk factors, depressive symptoms were significantly associated with all-cause mortality (hazard ratio = 1.10 per scale unit, 95% confidence interval = 1.04-1.16). This association was partly explained by CRP (7.3%) suggesting a minimal mediation effect. IL-1Ra, N-terminal pro-b-type natriuretic peptide, midregion pro-atrial natriuretic peptide, midregion pro-adrenomedullin, and CT-proET contributed marginally to the association between depression and subsequent mortality.

CONCLUSIONS: Inflammatory and cardiovascular risk markers are associated with depression and with increased mortality. However, depression and biological measures show additive effects rather than a pattern of meditation of biological factors in the association between depression and mortality.

Serum Cytokines and Adipokines in Periodontitis

Objective: To investigate whether there was an association between serum levels of pro-inflammatory cytokines, adipokines and periodontitis in 60-70 year-old men in Northern Ireland. Methods: Test subjects (n=61) with established periodontitis, categorised by loss of periodontal attachment (PAL) and pocketing of at least 5mm, were matched for age, smoking and BMI with controls (n=60) who were periodontitis resistant (no PAL or pocketing over 3mm). Serum levels of IL 1 alpha, TNF alpha, IL 6, adiponectin, resistin, leptin and C-reactive protein (CRP) were measured from serum using a multiplex array (Randox, UK). Mann Whitney analysis was used with the level of significance set at p<0.05. Results: There was considerable inter-individual variability in all the analytes measured. There was a significantly higher level of IL 6 in men with periodontitis (3.25 pg/ml, SD 2.29) than in those with no periodontitis (2.38 pg/ml, SD1.62), p=0.0041 (after Bonferroni correction for multiple testing p=0.029). Serum IL 6 was strongly correlated with CRP: r=0.52 (95% CI 0.38-0.64), p<0.0001. There was a lower serum level of adiponectin in men with periodontitis (3662.1 ng/ml, SD 2383.4) compared with those who were periodontitis resistant (4265.9 ng/ml, SD 2266.0), which just escaped statistical significance at p=0.068. There were no significant differences in the serum levels of any of the other analytes measured in relation to periodontal status. Conclusions: There was evidence in the 60-70 year-old male population investigated that periodontitis was associated with an increased serum level of IL 6.

Glycosylation of Vitamin D Binding Protein Reduced in Juvenile Idiopathic Arthritis Patients At Risk of Disease Extension.

Background/Purpose:Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish the subset of patients in whom inflammation extends to affect a large number of joints, early in the disease process. The post-translational modifications to candidate protein markers were verified by a novel deglycosylation strategy.Methods:SF samples from 57 patients were obtained around time of initial diagnosis of JIA. At 1 year from inclusion patients were categorized according to ILAR criteria as oligoarticular arthritis (n=26), extended oligoarticular (n=8) and polyarticular disease (n=18). SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with vitamin D binding protein (VDBP) expression and siaylation further verified by immunohistochemistry, ELISA test and immunoprecipitation. Candidate biomarkers were compared to conventional inflammation measure C-reactive protein (CRP). Sialic acid residues were enzymatically cleaved from immunopurified SF VDBP, enriched by hydrophilic interaction liquid chromatography (HILIC) and analysed by mass spectrometry.Results:Hierarchical clustering based on the expression levels of a set of 23 proteins segregated the extended-to-be oligoarticular from the oligoarticular patients. A cleaved isoform of VDBP, spot 873, is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Conversely total levels of vitamin D binding protein are elevated in plasma and ROC curves indicate an improved diagnostic sensitivity to detect patients at risk of disease extension, over both spot 873 and CRP levels. Sialysed forms of intact immunopurified VDBP were more prevalent in persistent oligoarticular patient synovial fluids.Conclusion:The data indicate that a subset of the synovial fluid proteome may be used to stratify patients to determine risk of disease extension. Reduced conversion of VDBP to a macrophage activation factor may represent a novel pathway contributing to increased risk of disease extension in JIA patients.

Nanosensor for assessing the risk of a cardiovascular disease

The incidence of cardiovascular diseases (CVD) has been increasing according to the European and global statistics. Thus, the development of new analytical devices, such as biosensors for assessing the risk of CVD could become a valuable approach for the improvement of healthcare service. In latest years, the nanotechnology has provided new materials with improved electronic properties which have an important contribution in the transduction mechanism of biosensors. Thus, in this thesis, biosensors based on field effect transistors with single-walled carbon nanotubes (NTFET) were developed for the detection of C-reactive protein (CRP) in clinical samples, that is, blood serum and saliva from a group of control patients and a group of CVD risk patients. CRP is an acute-phase protein, which is commonly known as the best validated biomarker for the assessment of CVD, the single-walled carbon nanotubes (SWCNT) were applied as transduction components, and the immunoreaction (interaction between the CRP antigen and the antibodies specific to CRP) was used as the mechanism of molecular recognition for the label-free detection of CRP. After the microfabrication of field effect transistors (FET), the screening of the most important variables for the dispersion of SWCNT, the assemblage of NTFET, and their application on standard solutions of CRP, it was found that NTFET respond accurately to CRP both in saliva and in serum samples, since similar CRP levels were found with the NTFET and the traditional methodology (ELISA technique). On the other hand, a strong correlation between salivary and serum CRP was found with NTFET, which means that saliva could be used, based on non-invasive sampling, as an alternative fluid to blood serum. It was also shown that NTFET could discriminate control patients from CVD risk patients, allowing the determination of a cut-off value for salivary CRP of 1900 ng L-1, which corresponds to the well established cut-off of 3 mg L-1 for CRP in serum, constituting an important finding for the possible establishment of a new range of CRP levels based on saliva. According to the data provided from the volunteer patients regarding their lipoprotein profile and lifestyle factors, it was concluded that the control and the CVD risk patients could be separated taking into account the various risk factors established in literature as strong contributors for developing a CVD, such as triglycerides, serum CRP, total cholesterol, LDL cholesterol, body mass index, Framingham risk score, hypertension, dyslipidemia, and diabetes mellitus. Thus, this work could provide an additional contribution to the understanding of the association of biomarkers levels in serum and saliva samples, and above all, cost-effective, rapid, label-free, and disposable NTFET were developed, based on a noninvasive sampling, for the assessment of CVD risk, thus constituting a potential point-of-care technology.

Optimisation of an aptamer-biosensor to detect C-reactive protein

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009

Platelets induce Pai-1 mRNA expression in monocytes through TGF-BETA

Introduction: Plasminogen activator inhibitor type-1 (PAI-1) is a physiological modulator of fibrinolysis. High plasma PAI-1 is associated with the 4G/5G promoter polymorphism and with increased cardiovascular risk. Here we explored the role of platelets in regulating expression of the PAI-1 gene in monocytes. Methods: Blood from PAI-1 4G/5G genotyped volunteers (n=6) was incubated with the platelet GPVI-specific agonist, cross-linked collagen related peptide (CRP-XL), in the presence or absence of Mab 9E1 that blocks the binding of P-selectin to PSGL1. Monocytes were isolated by +ve selection on CD14 beads and monocyte PAI-1 mRNA expression was measured by real-time PCR. Results: Activation of platelets with CRP-XL resulted in platelets binding to >70% of monocytes and was accompanied by >5000-fold induction of PAI-1 mRNA, peaking at 4hrs. PAI-1 expression was independent of the 4G/5G genotype. Blocking the binding of platelets to monocytes enhanced PAI-1 induction (p<0.05 at 4 hrs). Incubation of isolated monocytes with the releasate from CRP-XL stimulated platelets also led to PAI-1 mRNA expression. The platelet secretome contains >100 different proteins. To identify the soluble factor(s) responsible for induction of PAI-1, neutralizing antibodies to likely candidates were added to monocytes incubated with the platelet releasate. Anti- TGF-beta inhibited platelet releasate-mediated PAI-1 mRNA induction by >80%. Monocyte PAI-1 was also induced by stimulation of PSGL-1 with a P-selectin-Fc chimera, in the absence of platelets, which was also blocked by the TGF-beta antibody. Conclusions: These results suggest that platelets induce PAI-1 mRNA in monocytes predominantly via TGF-beta, released from both platelets, and monocytes via activation by PSGL-1 signalling.This stimulation is independent of 4G/5G genotype

A ansiedade antes, durante e após a performance: relatório de estágio

I (Prática Pedagógica) - Neste relatório de estágio apresenta-se uma caracterização do CRP, contextualizando um pouco da sua história, o seu funcionamento e os seus objetivos pedagógicos. Caracterizam-se, também, os alunos que participaram no estágio, destacando o seu percurso académico, as suas influências e motivações musicais. Nas práticas educativas desenvolvidas apresentam-se os princípios pedagógicos, segundo o portal Ponazapino, e os métodos de ensino lecionados durante o ano letivo que tiveram em conta o processo integrado de Ensino/Aprendizagem (Teaching and Learning). Por último apresentam-se os objetivos pedagógicos propostos para cada aluno do estágio. No final efetua-se uma análise crítica da atividade docente destacando o processo ensino/aprendizagem, a sua aplicação e benefícios no desenvolvimento integral do indivíduo.

O Poder Tributário das Autarquias

Dissertação de Mestrado em Solicitadoria

Belém e São Bento nas Relações Externas Pós-Coloniais Portuguesas (1976-1982)

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Ciência Política e Relações Internacionais

A admissibilidade do despedimento com justa causa devido ao publicado nas redes sociais

Com a crescente utilização mundial das novas tecnologias, como o uso de determinadas redes sociais, Facebook, Twitter, MSN, My Space, Blogs, Youtube, Instagram, entre muitos outros programas ao nosso alcance, cresceu também um o problema. A forma como os utilizamos comunicarmos com familiares, amigos e colegas de trabalho no âmbito da nossa esfera privada No entanto, muitas vezes não temos consciência de que aquilo que divulgamos no meio virtual nos pode trazer consequências a nível profissional. Implicações essas, que podem levar mesmo ao despedimento com justa causa, ou seja, sem direito a qualquer tipo de indeminização. Exemplo disso mesmo, é o caso do trabalhador da empresa Esegur que foi despedido, devido a comentários realizados no Facebook, a rede social mais utilizada em Portugal, que colocavam em causa os direitos de personalidade de colegas e superiores hierárquicos. Todavia, para que esta forma de despedimento tão gravosa possa vir a ocorrer temos de ter sempre em mente, o seguinte: - Se fotos, publicações ou comentários publicados causam danos não patrimoniais ou patrimoniais nos direitos da empresa/empregador; -Se a Quebra do elo de Confiança entre o trabalhador e o empregador é tão gravosa que justifique o despedimento; - Se Lesões patrimoniais na esfera da entidade empregadora. Assim, há que ter sempre em conta, se na situação em concreto, o comportamento foi tão grave que destruiu ou abalou de tal modo a confiança da parte do empregador, que justifique sanção tão gravosa. Tendo em conta que naquela situação um trabalhador normal teria tomado uma opção completamente diferente, que não colocaria em causa a relação laboral. Facilmente se compreende que o princípio da proporcionalidade (art.18º CRP) é fundamental apreciação deste tipo de casos. Uma vez que, muitas vezes é bastante difícil aplicar a medida disciplinar, das que o empregador tem á sua disposição (artº328CT), que mais se adequa á gravidade do ato cometido pelo trabalhador, o que nem sempre será o despedimento do trabalhador cujo comportamento resultou nas lesões. O que no âmbito deste trabalho é a divulgação de conteúdos nas redes sociais, claro que uns serão mais graves, que outros.

Assessment by doppler ultrasound of entheseal lesions in spondyloarthritis : a longitudial study to determine structural damage and disease activity lesions

RESUMO: Enthesitis is the hallmark of spondyloarthritis (SpA), and is observed in all subtypes. Wide information on SpA abnormalities, including synovitis, tendinitis and enthesitis, can be efficiently perceived by Doppler ultrasound. Furthermore, several studies on imaging of enthesis showed that imaging techniques are better than clinical examination to detect enthesis alterations; and vascularized enthesitis detected by Doppler ultrasound appears to be a valuable diagnostic tool to confirm SpA diagnosis. However, data published until now concerning entheseal elementary alterations that characterize SpA enthesitis (enthesis inflammatory activity) or enthesopathy (permanent structural changes) reflect rather the authors’ empiric opinion than a methodological validation process. In this sense it seems crucial to identify elementary entheseal lesions associated with activity or damage, in order to improve monitoring and treatment response in SpA patients. The development of better assessment tools is today a challenge and a need in SpA. The first study of this thesis focused on the analysis of the reliability of inter-lector and inter-ultrasonography equipment of Madrid sonography enthesitis index (MASEI). Fundamental data for the remaining unrolling project validity. In the second and third studies we concerned about two entheseal elemental lesions: erosions and bursa. In literature erosions represent a permanent structural damage, being useful for monitoring joint injury, disease activity and therapeutic response in many rheumatic diseases; and to date, this concept has been mostly applied in rheumatoid arthritis (RA). Unquestionably, erosion is a tissue-related damage and a structural change. However, the hypothesis that we decided to test was if erosions represent a permanent structural change that can only grow and worsen over time, as occurs in RA, or a transitory alteration. A longitudinal study of early SpA patients was undertaken, and the Achilles enthesis was used as a model. Our results strongly suggested that previously detected erosions could disappear during the course of the disease, being consistent with the dynamic behavior of erosion over time. Based on these striking results it seems reasonable to suggest that the new-bone formation process in SpA could be associated with the resolution of cortical entheseal erosion over time. These results could also be in agreement with the apparent failure of anti-tumor necrosis factor (TNF) therapies to control bone proliferation in SpA; and with the relation of TNF-α, Dickkopf-related protein 1 (Dkk-1) and the regulatory molecule of the Wnt signaling pathway in the bone proliferation in SpA. In the same model, we then proceeded to study the enthesis bursa. Interestingly, the Outcome Measures in Rheumatology Clinical Trials (OMERACT) enthesopathy definition does not include bursa as an elementary entheseal lesion. Nonetheless, bursa was included in 46% of the enthesis studies in a recently systematic literature review, being in agreement with the concept of “synovio-entheseal complex” that includes the link between enthesitis and osteitis in SpA. It has been clarified in recent data that there is not only a close functional integration of the enthesis with the neighboring bone, but also a connection between enthesitis and synovitis. Therefore, we tried to assess the prevalence and relevance of the bursa-synovial lesion in SpA. Our findings showed a significant increase of Achilles bursa presence and thickness in SpA patients compared to controls (healthy/mechanical controls and RA controls). These results raise awareness to the need to improve the enthesopathy ultrasonographic definition. In the final work of this thesis, we have explored new perspectives, not previously reported, about construct validity of enthesis ultrasound as a possible activity outcome in SpA. We performed a longitudinal Achilles enthesis ultrasound study in patients with early SpA. Achilles ultrasound examinations were performed at baseline, six- and twelve-month time periods and compared with clinical outcome measures collected at basal visit. Our results showed that basal erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are higher in patients with Doppler signal in enthesis, and even that higher basal ESR, CRP and Ankylosing Spondylitis Disease Activity Score (ASDAS) predicted a higher Doppler signal (an ultrasound alteration accepted as representative of inflammation) six months later. Patients with very high disease activity assessed by ASDAS (>3.5) at baseline had significantly higher Achilles total ultrasound score verified at the same time; and ASDAS <1.3 predicted no Doppler signal at six and twelve months. This seems to represent a connection between classical biomarkers and clinical outcomes associated with SpA activity and Doppler signal, not only at the same time, but also for the following months. Remarkably, patients with inactive disease (ASDAS < 1.3) at baseline had no Doppler signal at six and twelve months. These findings reinforce the potential use of ultrasound related techniques for disease progression assessment and prognosis purposes. Intriguingly, Ankylosing Spondylitis Disease Activity Index (BASDAI) didn’t show significant differences between different cut-offs concerning ultrasound lesions or Doppler signal, while verified with ASDAS. These results seem to indicate that ASDAS reflects better than BASDAI what happens in the enthesis. The work herein discussed clearly shows the potential utility of ultrasound in enthesis assessment in SpA patients, and can be important for the development of ultrasound activity and structural damage scores for diagnosis and monitoring purposes. Therefore, local promotion of this technique constitutes a medical intervention that is worth being tested in SpA patients for diagnosis, monitoring and prognosis purposes.

O âmbito de aplicação material da legislação de proteção de dados pessoais e o tratamento de imagem por pessoa singular

Images have gained a never before seen importance. Technological changes have given the Information Society extraordinary means to capture, treat and transmit images, wheter your own or those of others, with or without a commercial purpose, with no boundaries of time or country, without “any kind of eraser”. From the several different ways natural persons may engage in image processing with no commercial purpose, the cases of sharing pictures through social networks and video surveillance assume particular relevance. Consequently there are growing legitimate concerns with the protection of one's image, since its processing may sometimes generate situations of privacy invasion or put at risk other fundamental rights. With this in mind, the present thesis arises from the question: what are the existent legal instruments in Portuguese Law that enable citizens to protect themselves from the abusive usage of their own pictures, whether because that image have been captured by a smartphone or some video surveillance camera, whether because it was massively shared through a blog or some social network? There is no question the one's right to not having his or her image used in an abusive way is protected by the Portuguese constitution, through the article 26th CRP, as well as personally right, under the article 79th of the Civil Code, and finally through criminal law, articles 192nd and 193rd of the Criminal Code. The question arises in the personal data protection context, considering that one's picture, given certain conditions, is personal data. Both the Directive 95/46/CE dated from 1995 as well as the LPD from 1998 are applicable to the processing of personal data, but both exclude situations of natural persons doing so in the pursuit of activities strictly personal or family-related. These laws demand complex procedures to natural persons, such as the preemptive formal authorisation request to the Data Protection National Commission. Failing to do so a natural person may result in the application of fines as high as €2.500,00 or even criminal charges. Consequently, the present thesis aims to study if the image processing with no commercial purposes by a natural person in the context of social networks or through video surveillance belongs to the domain of the existent personal data protection law. To that effect, it was made general considerations regarding the concept of video surveillance, what is its regimen, in a way that it may be distinguishable from Steve Mann's definition of sousveillance, and what are the associated obligations in order to better understand the concept's essence. The application of the existent laws on personal data protection to images processing by natural persons has been analysed taking into account the Directive 95/46/CE, the LPD and the General Regulation. From this analysis it is concluded that the regimen from 1995 to 1998 is out of touch with reality creating an absence of legal shielding in the personal data protection law, a flaw that doesn't exist because compensated by the right to image as a right to personality, that anyway reveals the inability of the Portuguese legislator to face the new technological challenges. It is urgent to legislate. A contrary interpretation will evidence the unconstitutionality of several rules on the LPD due to the obligations natural persons are bound to that violate the right to the freedom of speech and information, which would be inadequate and disproportionate. Considering the recently approved General Regulation and in the case it becomes the final version, the use for natural person of video surveillance of private spaces, Google Glass (in public and private places) and other similar gadgets used to recreational purposes, as well as social networks are subject to its regulation only if the images are shared without limits or existing commercial purposes. Video surveillance of public spaces in all situations is subject to General Regulation provisions.

Inflammatory markers and incident heart failure risk in older adults: the Health ABC (Health, Aging, and Body Composition) study.

OBJECTIVES: The purpose of this study was to evaluate the association between inflammation and heart failure (HF) risk in older adults. BACKGROUND: Inflammation is associated with HF risk factors and also directly affects myocardial function. METHODS: The association of baseline serum concentrations of interleukin (IL)-6, tumor necrosis factor-alpha, and C-reactive protein (CRP) with incident HF was assessed with Cox models among 2,610 older persons without prevalent HF enrolled in the Health ABC (Health, Aging, and Body Composition) study (age 73.6 +/- 2.9 years; 48.3% men; 59.6% white). RESULTS: During follow-up (median 9.4 years), HF developed in 311 (11.9%) participants. In models controlling for clinical characteristics, ankle-arm index, and incident coronary heart disease, doubling of IL-6, tumor necrosis factor-alpha, and CRP concentrations was associated with 29% (95% confidence interval: 13% to 47%; p < 0.001), 46% (95% confidence interval: 17% to 84%; p = 0.001), and 9% (95% confidence interval: -1% to 24%; p = 0.087) increase in HF risk, respectively. In models including all 3 markers, IL-6, and tumor necrosis factor-alpha, but not CRP, remained significant. These associations were similar across sex and race and persisted in models accounting for death as a competing event. Post-HF ejection fraction was available in 239 (76.8%) cases; inflammatory markers had stronger association with HF with preserved ejection fraction. Repeat IL-6 and CRP determinations at 1-year follow-up did not provide incremental information. Addition of IL-6 to the clinical Health ABC HF model improved model discrimination (C index from 0.717 to 0.734; p = 0.001) and fit (decreased Bayes information criterion by 17.8; p < 0.001). CONCLUSIONS: Inflammatory markers are associated with HF risk among older adults and may improve HF risk stratification.

Reduction in the use of diagnostic tests in infants with risk factors for early-onset neonatal sepsis does not delay antibiotic treatment.

Despite a low positive predictive value, diagnostic tests such as complete blood count (CBC) and C-reactive protein (CRP) are commonly used to evaluate whether infants with risk factors for early-onset neonatal sepsis (EOS) should be treated with antibiotics. We investigated the impact of implementing a protocol aiming at reducing the number of diagnostic tests in infants with risk factors for EOS in order to compare the diagnostic performance of repeated clinical examination with CBC and CRP measurement. The primary outcome was the time between birth and the first dose of antibiotics in infants treated for suspected EOS. Among the 11,503 infants born at ≥35 weeks during the study period, 222 were treated with antibiotics for suspected EOS. The proportion of infants receiving antibiotics for suspected EOS was 2.1% and 1.7% before and after the change of protocol (p = 0.09). Reduction of diagnostic tests was associated with earlier antibiotic treatment in infants treated for suspected EOS (hazard ratio 1.58; 95% confidence interval [CI] 1.20-2.07; p <0.001), and in infants with neonatal infection (hazard ratio 2.20; 95% CI 1.19-4.06; p = 0.01). There was no difference in the duration of hospital stay nor in the proportion of infants requiring respiratory or cardiovascular support before and after the change of protocol. Reduction of diagnostic tests such as CBC and CRP does not delay initiation of antibiotic treatment in infants with suspected EOS. The importance of clinical examination in infants with risk factors for EOS should be emphasised.