Proefschrift Universiteit van Amsterdam. / Auteursnaam op omslag: J. Wendelien Dorigo-Zetsma. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

No description available.

No description available.

Das hochadaptierte, zellwandlose Bakterium Mycoplasma pneumoniae gehört zu den häufigsten Erregern von ambulant erworbenen Pneumonien. Während den alle 3-7 Jahre auftretenden Epidemien könnten gefährdete Personengruppen bei Verfügbarkeit einer effektiven Vakzinierung vor einer Infektion geschützt werden. Ziel dieser Arbeit war die Erarbeitung von Grundlagen zum Aufbau einer schützenden Immunantwort durch Immunisierung mit rekombinant hergestellten Teilbereichen verschiedener Adhärenzproteine von M. pneumoniae. Da die bereits bekannten Adhäsine des Erregers einerseits unverzichtbare Virulenzfaktoren darstellen und andererseits als dominante Immunogene des Bakteriums beschrieben wurden, war die Feincharakterisierung dieser Proteine im Hinblick auf ihre Eignung als Vakzinekandidat Ausgangspunkt der Untersuchungen. Definierte Teilbereiche des Hauptadhäsins P1 und weiterer adhärenzassoziierter Proteine wurden rekombinant hergestellt und auf ihre Antigenität qualitativ und quantitativ mit Seren experimentell infizierter Tiere und Seren von Patienten mit bestätigter M. pneumoniae-Infektion geprüft. Dabei gingen der C-terminale Bereich des P1-Adhäsins (RP14; AS 1287-1518) und das Protein P30 (RP30; AS 17-274) als hoch antigene Protein(regionen) hervor. Die Oberflächenexposition beider Proteinbereiche wurde experimentell bestätigt. Parallel dazu sollte der Einfluss der monospezifischen Seren gegen die rekombinanten Proteine auf die Adhärenz von M. pneumoniae an verschiedene Zelllinien bewertet werden. Nach Entwicklung eines quantitativen und schnell durchzuführenden in vitro Adhärenzhemmtests wurde gezeigt, dass Antikörper sowohl gegen RP14 als auch gegen RP30 die Adhärenz des Erregers an humane Zellen signifikant reduzieren. Im Hinblick auf die Beeinflussung von mehr als einer an der Adhärenz beteiligten Struktur wurden beide Adhäsinregionen in einem Hybridprotein vereinigt, wodurch die adhärenzhemmende Wirkung des entsprechenden Serums in vitro mehr als additiv verstärkt werden konnte und dem Effekt des polyspezifischen Serums gegen M. pneumoniae entspricht. In Immunisierungs- und Infektionsversuchen mit dem Hybridprotein konnte über eine neu etablierte sehr sensitive real-time PCR basierend auf einer Multicopy-Targetsequenz in M. pneumoniae jedoch nur in einem Teil der Versuchstiere eine signifikante Hemmung der Kolonisierung des Respirationstraktes mit dem Erreger nachgewiesen werden. Eine Steigerung der humoralen Immunantwort durch Zugabe des mukosalen Adjuvans MALP-2 konnte nicht ausreichend belegt werden. Dagegen wurde durch Immunisierung auf intranasalem Wege eine signifikante Erhöhung des sekretorischen IgA-Titers in der BAL auf das Vierfache erreicht. Um einen stabil hohen sekretorischen Antikörperspiegel im Tiermodell zu erreichen, sind weitere Optimierungen des Immunisierungsschemas notwendig. Der Einsatz des Hybridproteins HP14/30, bestehend aus mehreren immunogenen und funktionalen Adhäsinbereichen, hat sich als sehr vielsprechend zur Entwicklung einer effektiven Vakzinierung von Risikopopulationen zur Prophylaxe von Infektionen mit M. pneumoniae erwiesen.

Thèse d'exercice : Médecine. Médecine générale : Paris 12 : 2006. / Titre provenant de l'écran-titre. Bibliogr. f. 83-88.

Introduction: Mycoplasma pneumoniae(M. pneumoniae) causes 10% to 30% of community-acquired pneumonia (CAP). The commonly used first-line antibiotic macrolide (ML) against respiratory tract infection may lead to the increase of ML-resistant M. pneumoniaeinfection. To resolve the problem, a rapid and accurate method for detection of ML-resistant M. pneumoniaeis necessary for treatment adjustment. Aims: The study aims to (1) develop a rapid method for diagnosis of ML-resistance of M. pneumoniaedirectly from clinical specimens; and (2) investigate the prevalence of M. pneumoniaeand ML-resistant M. pneumoniae. Methods: The M. pneumoniaeqPCR results of 689 respiratory tract samples from Queen Mary Hospital collected during April 2010 to May of 2012 were analyzed. Positive nucleic acid from M. pneumoniaeqPCR samples were tested with SimpleProbe real-time PCR coupled to melting curve analysis (SimpleProbe PCR), pyrosequencing and 23S rRNA gene sequencing(23S sequencing) for detection of ML-resistance. Results: A total of 111 samples (16.11%) in 689respiratory tract samples were found M. pneumoniaepositive by qPCR. Of 111, 96 positive nucleic acids were available for this study. Overall, 29 (30.21%, n=96) of ML-resistant M. pneumoniaewere found. 23S sequencing identified 28 mutants (29.17%) and 62 wild–type (64.58%), while 6 (6.25%) of them are failed to be identified. Pyrosequencing identified 28 mutants (29.17%) and 63 wild–type (65.63%), while 5 (5.21%) of them are failed to be identified. The SimpleProbe PCR identified 29 mutants (30.21%) and 65 wild–type (67.71%), while 2 (2.08%) of them are failed to be identified. All ML-resistant M. pneumoniaepositives were found to have A2063G mutation either by 23S sequencing or pyrosequencing. Conclusion: From this study, SimpleProbe PCR is the most sensitive and simple to perform. Therefore, it is highly recommended to be included in the routine testing with positive M. pneumoniaesamples for diagnosis of ML-resistant strain. 23S sequencing or pyrosequencing is recommended to use as a confirmatory test if necessary. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Introduction: Mycoplasma pneumoniae (M. pneumoniae) has been a major cause of community-acquired pneumonia (CAP), accounting for about 10-30% of the cases. Previously, a local study revealed that more than 60% of clinical isolates of M. pneumoniae exerted A2063G mutation, which confers a high level of macrolide drug resistance and results in treatment failure. While A2063G is the only mutation identified locally, a rapid diagnostic assay for detection of this single point mutation is urgently needed for switching the drug of choice. Aims: This study aims to develop a rapid PCR assay for detection of A2063G mutation of M. pneumoniae isolates for our locality, to compare with other commercially available assays and to further confirm the prevalence of A2063G mutation in macrolide-resistance M. pneumoniae (MRMP) in Hong Kong. Methods: A total of 110 respiratory tract samples were collected from 102 patients in Hong Kong Sanatorium and Hospital during April 2013 to April 2014. They were analyzed by an in-house hybridization-probe real-time PCR assay coupled with melting curve analysis to detect the presence of M. pneumoniae and the target A2063G point mutation. Results were compared with a commercial real-time PCR assay and the A2063G point mutation was further confirmed by 23S rRNA gene sequencing. The limit of detection (LOD), mutation threshold determination and cross reactivity of the in-house assay were also evaluated. Results: Over 40% (47/110) of the respiratory tract samples were tested positive for M. pneumoniae by the in-house assay and 36.2% (17/47) of the positive samples exerted A2063G mutation. The limit of detection was 500 copies/ml as evaluated using external quality control samples. Twenty well-characterized clinical isolates of M. pneumoniae were used to evaluate the A2063G mutation threshold. The mutation threshold for A2063G mutant detection was above 60%. This assay did not show any cross-reactivity with common clinical isolates from the respiratory tract samples. Conclusion: In this study, an in-house real-time PCR assay was evaluated and demonstrated its great potential as a rapid clinical diagnostic tool. The assay was highly sensitive and specific in detecting M. pneumoniae and its A2063G mutation from clinical samples in Hong Kong. The results were almost concordant to the current routine testing, with the advantage of lower cost and shorter turnaround time for rapid detection. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

No description available.

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.

No description available.