INTRODUCCIÓN: la NAC no complicada en el niño es una infección muy frecuente, cuya epidemiología se encuentra pobremente definida y cuyo diagnóstico microbiológico sigue siendo difícil, dificultando la elección del tratamiento antibiótico, especialmente cuando no puede distinguirse clínicamente entre neumonía típica y atípica.OBJETIVOS: describir la presentación clínico-epidemiológica de la NAC no complicada en el niño y buscar rasgos clínico-epidemiológicos predictivos de infección por M. pneumoniae.MÉTODOS: estudio prospectivo, descriptivo, de seguimiento de una cohorte, desde el 1 de Enero de 2000 hasta el 31 de Diciembre de 2001. Se incluyeron niños de edad > 1 mes, inmunocompetentes, diagnosticados de NAC no complicada en el Servicio de Urgencias de Pediatría del Hospital Vall d'Hebron, cuyos padres aceptaron el protocolo de estudio y seguimiento.RESULTADOS: Se estudiaron 166 pacientes, 54,2% varones, mediana de edad de 48 meses. La duración entre inicio de la sintomatología y diagnóstico tuvo una mediana de 3 días, y la mediana de la duración del tratamiento fue de 8 días. El 83,1% de los pacientes fueron tratados con amoxicilina-clavulánico y 7 días después del inicio del tratamiento el 98,2% de los pacientes estaban asintomáticos. Después de 2 semanas únicamente el 24,3% tenía imágenes pulmonares residuales. Se detectó etiología atípica en el 25,3% de los pacientes, siendo debida a M. pneumoniae en el 85,7%, C. pneumoniae en el 9,5% y coinfección en el 4,8%. La presentación clínico-radiológica no permitió un diagnóstico clínico de neumonía atípica vs no atípica y no se apreciaron diferencias en la evolución, habiendo sido tratados en su gran mayoría y en ambos grupos, con betalactámicos. El análisis de datos multivariable, con regresión logística demostró que la edad >5 años (RR 4,48, IC:2,14-9,39), la presencia de una cifra de neutrófilos<7000 (RR: 7,74, IC 3-20)y un valor de Proteina C Reactiva <7 mg/dL (RR3,99, IC 1,27-12,55), se asociaban a infección atípica.CONCLUSIONES: El recuento de neutrófilos, con un punto de corte de 7000, constituye una variable con capacidad predictiva en el diagnóstico diferencial de neumonía atípica, con una sensibilidad del 76,2%, especificidad de 83,9% y valor predictivo negativo de 91,2%. El 91,5% de nuestros pacientes recibieron un antibiótico beta-lactámico, no existiendo diferencias significativas en ambos grupos ni en cuanto a la clase de antibiótico ni en cuanto a la duración y vía del tratamiento. La evolución clínico-radiológica es buena, aún en ausencia de tratamiento específico, pues el 88,1% de las neumonías atípicas (37 pacientes), fueron tratados exclusivamente con beta-lactámicos. Se podría pues afirmar que el diagnóstico etiológico de la NAC no complicada no resulta imprescindible para su tratamiento y habría por tanto que valorar su costo-efectividad. No obstante sería interesante disponer de más estudios que permitieran elaborar conclusiones extrapolables a toda la población. / INTRODUCTION: Community-acquired childhood pneumonia (CAP) is a common infection but its precise epidemiology remains poorly defined and its treatment is complicated by the difficulty in microbiological diagnosis and the increasing incidence of antibiotic resistance among respiratory pathogens. OBJECTIVES: The purpose of this study is to present the main epidemiologic features of patients with CAP and to compare the clinical, biological, and radiologic features of presentation in atypical pneumonia and in other community-acquired pneumonia, to try to help in early diagnosis of atypical pneumonia. METHODS: Consecutive immunocompetent children with radiographically confirmed lower respiratory infection were enrolled and evaluated in the emergency department of a 400-bed university children's hospital, prospectively from January 2000 through December 2001. Clinical, radiologic and microbiological evaluations were performed at study entry, at 10-15 days, and at 4 weeks post-therapy. Mycoplasmal, chlamydial, Legionella and Coxiella serologic tests were performed on paired samples in order to identifye the infection by those organisms. Univariate and multivariate analyses were performed to compare epidemiologic and demographic data and clinical, analytical, and radiologic features of presentation in the patients with atypical pneumonia and the patients with CAP by other bacterial etiology. RESULTS: 166 patients with CAP were studied, 54,2% male, mean age 48 months. The mean duration between onset of symptoms and diagnosis was 3 days, and the mean treatment duration was 8 days. 83,1% of the patients were treated with amoxicillin-clavulanate. Seven days after the start of therapy clinical symptoms were absent in 98,2% of patients and two weeks after radiologic infiltrates were only present in 24,3% of patients. Etiologic atypical agents were identified in 42 (25,3%) of 166 patients. Infection was attributed to M. pneumoniae in 85,7% (36 of 42), C. pneumoniae in 9,5% (4 of 42), and coinfections in 4,8% (2 of 42). 74,7% patients were diagnosed of CAP by other bacterial etiology . Clinical findings and chest radiographs did not distinguish patients with a defined atypical etiology from those without a known cause for pneumonia. There were no differences in the clinical responses of patients to the antimicrobial regimen. Multivariate logistic-regression analyses revealed that age> 5 years (odds ratio: 4,48; 95% confidence interval:2,14-9,39 ), the presence of neutrophiles<7000 (odds ratio:7,74 ; 95% confidence interval:3-20 ), and C Reactive Protein value < 7 mg/dL(odds ratio: 3,99 ; 95% confidence interval:1,27-12,55) were significantly associated with atypical pneumonia. CONCLUSION: Atypical etiology was identified in 25,3% of the evaluable patients and multivariate analysis demonstrated that only some independent factors were associated with atypical pneumonia. These factors as found in this study can help the evaluating physician to identify the atypical etiology.

Streptococcus pneumoniae is a major pathogen that causes Otitis Media infections and bacterial meningitis in children as well as community acquired pneumonia in adults. Clinical isolates of S. pneumoniae exhibiting resistance to Beta-lactam antibiotics are being isolated with increased frequency in many countries. Streptococcus pneumoniae strains resistant to Beta-lactam drugs have modified forms of penicillin-binding proteins that exhibit reduced affinity for binding to chemotherapeutic Beta-lactams. Penicillin binding proteins are membrane-bound enzymes that catalyze the terminal step in cell wall synthesis, and are targets for Beta-lactam drugs. Seventeen clinical isolates and six vaccine strains of Streptococcus pneumoniae were characterized using conventional phenotypic methods, susceptibility to antimicrobial agents, capsular serotyping, and by different biochemical and genotyping methods. One strain, Sp D2, was resistant to penicillin and other Beta-lactams used in the study, to erythromycin, and to Trimethoprim/Sulfamethoxazole. Sp D2 exhibited a unique protein profile in 1D SDS-PAGE gels of whole-cell proteins. Cells of Sp D2 were fractionated, and the cytoplasmic membrane fraction was obtained by ultracentrifugation and analyzed using a 1D SDS-PAGE gel. A protein band with a mass of ~50 kDa was excised and subjected to Trypsin In-Gel Digestion, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. The resulting MALDI-TOF-MS data (peptide mass fingerprints) did not produce any significant matches with proteins in any of the published S. pneumoniae genome databases. The 50 kDa protein was further subjected to N-terminal and internal sequence analysis and database searching, and the protein could not be identified by significant matches. Sp D2 did not react with any anti-pneumococcal polysaccharide capsular antibodies, and is designated as a non-typeable strain. Sp D2 exhibited a positive reaction in the Bile Solubility Test, the Optochin Test, and also positive reactions in PCR assays for the presence of the pneumococcal surface protein gene (PsaA), the autolysin gene (LytA), and the pneumolysin gene (Ply); which confirms that Sp D2 is a strain of S. pneumoniae.

Objective: To find out the spatial autocorrelation of antibiotic resistance of S. pneumonia and test the significance of distance as a risk factor. Methods: Descriptions of penicillin and macrolide resistance in EARS-Net countries from 2006 to 2010 were given. Global moran’s I and Anselin moran’s I were used to assess the spatial autocorrelation and gravity model was used to test the significance of distance and other socio – economic factors. Results: The trend of resistance in Europe was stable. Positive spatial autocorrelation existed from 2006 to 2010 for penicillin (Z(I): 0.16-0.2) and 2009 to 2010 for macrolide (Z(I): 0.11 -0.13). Some clusters (hotspots) were identified; they were Cyprus (2006-2010 for penicillin and 2009 to 2010 for macrolide), Spain (2006 for penicillin), France (2006 for penicillin), Romania (2009 for penicillin and macrolide) and Bulgaria (2009 for penicillin and macrolide). The result of gravity model showed that only parameters of population in 2007 for penicillin (p<0.05) and parameter of distance in 2009 for penicillin (p<0.05) in Cyprus were statistically significant. Conclusion: Distance was not a risk factor of high prevalence of antibiotic resistance of S. pneumoniae although there was a positive spatial autocorrelation. Improvement in surveillance system and appropriate public action were recommended for controlling the spread of resistant strain of S. pneumoniae. / published_or_final_version / Public Health / Master / Master of Public Health

Influenza is a leading cause of respiratory disease worldwide. During pandemic and seasonal influenza, secondary Streptococcus pneumoniae infection is a severe complication that contributes to morbidity and mortality. With the clinical significance of this co-infection, it is imperative to understand the disease mechanisms and how our immune system would be modulated in dealing with the dual infection. First, in vivo co-infection model was established. Mice were sequentially infected with influenza virus and then Streptococcus pneumoniae. Co-infected mice lost their body weight significantly and had 100% mortality, whereas mice infected with either influenza virus or pneumococcus alone lost their body weight transiently and all recovered from the infection. Then, lung inflammatory response during the co-infection was examined. Although it is a common phenomenon that co-infection enhances inflammation, the kinetic of, and the relative contribution of influenza virus or pneumococcus to the lung inflammation is not well defined. Therefore, this study characterized the general lung inflammatory environment after co-infection. It was found that influenza virus and pneumococcus differentially modulated inflammatory response in terms of kinetics, leukocyte infiltration and cytokine production. At the early time point after co-infection, pneumococcal infection contributed more than the influenza virus infection to enhance inflammatory cytokine and neutrophil infiltrating the lung. At the later time point after co-infection, both influenza virus and pneumococcus contributed to synergistically increase inflammatory cytokine and macrophage infiltrating the lung. Influenza virus infection induced IFN-γ that contributed to the elevated IFN-γ level in co-infected mice. Influenza virus and pneumococcus synergistically increased Th2 associated cytokine including IL-4, IL-5, and IL-10. These up-regulated immune responses might contribute to the severe lung pathology. Next, adaptive immunity to co-infection was examined. Literature studying co-infection often reports how prior influenza virus infection impairs the immune response against subsequent bacterial infection. However, whether and how secondary pneumococcal infection would affect the immunity to the initial influenza virus is unknown. Therefore this study investigated the modulation of immunity to influenza virus by secondary pneumococcal infection. It was found that co-infection significantly enhanced virus titer in lung and depleted the number of cell in spleen. Secondary pneumococcal infection after influenza decreased influenza virus specific IgG in the lung and peripheral blood. The reduced level of virus specific IgG was associated with the decrease in the number and the percentage of follicular B cell and CD4 T follicular helper cell through both pneumococcal capsular polysaccharide dependent and independent manner. Treating co-infected mice with immune serum containing influenza virus specific IgG successfully improved survival, which suggested the important protective function of virus specific IgG to the co-infection. Taken together, these data suggested that secondary pneumococcal infection impairs the antibody response to influenza virus, which might enhance mortality after co-infection. In conclusion, this study provides new insight to understand the pathogenesis of co-infection, reveals the general lung inflammatory environment, highlights the negative role of pneumococcus to impair virus control and explores novel treatment for the co-infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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Introduction: Streptococcus pneumoniae is the main bacterial cause of pneumonia, bacteremia and meningitis. Incidence rates have decreased since the initiation of pneumococcal vaccines, but antibiotic resistant strains continue to emerge and place a heavy burden on healthcare systems to treat such serious resistant infections. This study looks at risk factors that increase a patients probability of contracting a drug resistant strain of S. pneumo. Methods: Confirmed cases of S. pneumo were acquired through the Active Bacterial Core Surveillance program from 2009-2012 for the state of Georgia. Cumulative incidence rates, odds ratios and Pearson’s chi square were calculated to test for trends. Multi-logistic regression model was designed to control for covariates. Antibiotic Susceptibility results were analyzed by resistant profiles through WHONET. Results: Cumulative incidence rates have decreased significantly, however antibiotic resistant and multidrug resistant strains have increased. Incidence rates for children less than five and adults over 65 have decreased, however, the burden of disease remains in young to middle adults. Antibiotic resistant strains have shifted from penicillin to erythromycin and cefotaxime. Discussion: Interventions need to be targeted towards young to middle aged adults. Antibiotic stewardship programs should seek uniform guidelines to battle the increasing emergence of multidrug resistant strains.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Streptococcus pneumoniae (the pneumococcus) remains a major cause of morbidity and mortality worldwide, particularly in young children and the elderly. It is responsible for a spectrum of diseases ranging from otitis media, to potentially fatal conditions such as pneumonia and meningitis, and is estimated to cost health services billions of dollars each year. The interaction of S. pneumoniae with the host generally begins in the nasopharynx, and invasive disease is almost invariably preceded by nasopharyngeal colonisation. In some circumstances, S. pneumoniae may translocate from the nasopharynx to the lungs where pneumonia can develop, and inflammation is believed to play a role in this process. The presence of pneumococci in the lungs also triggers an inflammatory response, which is important for clearance of the bacteria. However, a prolonged inflammatory response leads to tissue damage, and is linked with a poor prognosis of disease. It has been shown that respiratory epithelial cells are able to play an active part in the response to respiratory pathogens by releasing chemokines that are responsible for neutrophil recruitment, and it has recently been shown that infection of type II pneumocytes with S. pneumoniae leads to the release of interleukin (IL)-8. In order to determine the role of specific pneumococcal factors in eliciting a CXC chemokine response from type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562), monolayers of these cells were infected with wild type (WT) S. pneumoniae 039, or mutants deficient in choline binding protein A (CbpA), pneumococcal surface protein A (PspA), or pneumolysin (Ply), and the CXC chemokine mRNA response was measured by real-time RT-PCR. Release of IL-8 was also measured by ELISA. In response to WT D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA, and IL-8 protein. This response was increased 2-fold when a CbpA-negative (ACbpA) mutant was used to infect cells, suggesting that CbpA may have an inhibitory effect on the CXC chemokine response of these cells. Further investigatiDn demonstrated that this activity is dependent on the N-terminal region of CbpA and that all three N-terminal domains are required for this effect, as deletion of any one of these domains had the same effect on the CXC chemokine response as removing CbpA altogether. Infection with a PspA-negative mutant (APspA) led to a 2-fold decrease in the CXC chemokine response of A549 cells, compared to infection with WT D39 at 2 h, but no difference was seen in the response of Detroit-562 cells to this mutant compared to WT D39. Thus, PspA appears to have the ability to stimulate an early CXC chemokine release from A549 cells. Deletion of the first of 2 regions of the N-terminal a-helical domain of PspA reduced the ability of S pneumoniae to elicit a chemokine response to the same degree as removing PspA altogether, indicating that it is this region that is responsible for the chemokine inducing ability of PspA. Ply appeared to have no effect on the CXC chemokine response of A549 cells with no obvious difference seen in the response of these cells to APly compared to WT D39. However, infection of Detroit-562 cells with APly led to a 2-fold decrease in IL-8 mRNA and protein release compared to WT D39. Using D39 strains producing mutant forms of Ply with reduced cytotoxicity and/or complement activating abilities, the role of the cytotoxic activity of Ply was demonstrated to be important in generation of a chemokine response from both cell lines. Infection of A549 or Detroit-562 cells with mutants producing Ply with only 0.02% or 0.1% haemolytic activity led to a 2-fold decrease in IL-8 release compared to that elicited by WT D39. The complement activating ability of Ply also appeared to be important in the generation of a CXC chemokine response from A549 cells. Cells infected with a mutant that produced Ply with no complement activating ability released significantly less IL-8 than cells infected with WT D39. This activity of Ply did not appear to have an effect on the CXC chemokine release of Detroit-562 cells. Thus all three virulence factors investigated had some role in the ability of S. pneumoniae to generate a CXC chemokine response from respiratory epithehal cells, although their roles and the cell lines that were affected differed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1225410 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2005

Proefschrift Maastricht. / Lit. opg. - Met een samenvatting in het Nederlands.