Proefschrift Universiteit van Amsterdam. / Met bibliogr, lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Met lit.opg. en samenvatting in het Nederlands.

La legionelosis es una enfermedad respiratoria con origen en sistemas que formen aerosol y que contenga el agente biológico Legionella sp. En las últimas décadas se ha desarrollado en España un marco normativo para su prevención y control. El presente artículo expone la epidemiología de la legionelosis y la importancia del control de la transmisión de la bacteria en la lucha contra la enfermedad. Para ello, se hace patente la revisión de las instalaciones críticas y la inclusión de otras nuevas en la legislación preventiva así como la estimación del riesgo, la mejora en los procesos de diagnóstico y el avance en nuevos protocolos de prevención. / Legionellosis is a respiratory disease originating in systems that produce aerosol and contain Legionella sp. In recent decades, Spain has developed a regulatory framework for prevention and control of legionellosis. This article describes the epidemiology of legionellosis and the importance of controlling the transmission of bacteria in the fight against the disease. In that regard, it becomes clear the role of reviewing critical facilities and the inclusion of new ones in the preventive legislation, the estimation of risk, and the improvement in the diagnostic processes and progress in new prevention protocols.

<p>Although well established that human Vγ9Vδ2 T cells may expand in circulation during intracellular bacterial infections, most underlying studies included only a few cases and only some diseases had been studied so far. In tularemia, a severe invasive disease, only one patient had been described. Legionellosis, including the mild flue-like Pontiac disease caused by Legionella micdadei, had not been studied at all. The aim of the present thesis was to study the circulatory Vγ9Vδ2-T cell response in these two intracellular bacterial diseases. The number of cases included was large enough to draw general conclusions. At various intervals, Vγ9Vδ2-T-cell counts and the capability of the cells to produce proinflammatory cytokines were assayed. Finally, the nature of the stimulating antigens was determined.</p><p>In the acute phase of tularemia, we showed a marked increase of circulatory Vγ9Vδ2 T cells. When 181 samples from 108 patients with ulceroglandular tularemia were assayed, the percentage of Vγ9Vδ2 T cells was found to increase from ~5 to > 20% after the first week of disease. During the ensuing 24 months, levels were normalized. Vaccination with the live attenuated vaccine strain Francisella tularensis LVS, on the other hand, did not cause an increase in numbers of Vγ9Vδ2 T cells.</p><p>Within an outbreak of Pontiac fever, 14 cases were well defined with regard to incubation time and onset of disease. In samples obtained 4 to 6 days after onset of disease, the mean percentage of Vγ9Vδ2 T cells was ~ 1%, i.e., 20% of normal values. Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset of disease, values were ~ 15%. Later, values slowly decreased. In both tularemia and Pontiac fever, the capacity of Vγ9Vδ2 T cells to produce TNF-α in response to phorbol myristate acetate in vitro was transiently decreased, in tularemia up to 6 weeks after onset of disease and in Pontiac fever in samples obtained 5-7 weeks after onset of disease.</p><p>Nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 T cells. Various strains of F. tularensis, including LVS, and a strain of L. micdadei were shown to produce Vγ9Vδ2 T-cell stimulating phosphoantigen. Notably, stimulation with an extract from each agent caused a similar degree of expansion of cells from subjects infected with the homologous and heterologous agent and also of cells from healthy subjects. Thus no immunospecific memory was detected in the Vγ9Vδ2-T cell response.</p><p>Since it had been suggested that homologs of the conserved heat shock protein, chaperon-60, may be recognized by human Vγ9Vδ2 T cells, we determined the subpopulation of T cells responding to this protein as well as to DnaK, another heat-shock protein. Under in vitro conditions allowing a vigorous expansion of Vγ9Vδ2 T in response to a phosphoantigen, no expansion of γδ T cells in response to Cpn60 or DnaK of F. tularensis occurred. αβ T cells of tularemia-primed subjects, on the other hand, responded vigorously to the heat-shock proteins.</p><p>In conclusion, two intracellular bacterial diseases with widely varying clinical expression were both associated with expansion of circulating Vγ9Vδ2 T cells. The expansion was prominent, long-lasting, and consistent within large numbers of individuals tested. In Pontiac fever, the expansion of Vγ9Vδ2 T cells was preceded by a depletion of the cells in circulation, implicating a possible extravasal migration into an infected site before the occurrence of rapid expansion and reentrance to blood. Both in tularemia and Pontiac fever, a modulation of the cytokine expression of Vγ9Vδ2 T cells was demonstrated in vitro, suggesting the presence of modulation of the inflammatory response. In extracts from in vitro culture of F. tularensis and L. micdadei, Vγ9Vδ2 T-cell stimulating phosphoantigens were identified and according to cross stimulation experiments, they induced expansion in vitro of Vγ9Vδ2 T cells without regard to immunospecific memory.</p>

Although well established that human Vγ9Vδ2 T cells may expand in circulation during intracellular bacterial infections, most underlying studies included only a few cases and only some diseases had been studied so far. In tularemia, a severe invasive disease, only one patient had been described. Legionellosis, including the mild flue-like Pontiac disease caused by Legionella micdadei, had not been studied at all. The aim of the present thesis was to study the circulatory Vγ9Vδ2-T cell response in these two intracellular bacterial diseases. The number of cases included was large enough to draw general conclusions. At various intervals, Vγ9Vδ2-T-cell counts and the capability of the cells to produce proinflammatory cytokines were assayed. Finally, the nature of the stimulating antigens was determined. In the acute phase of tularemia, we showed a marked increase of circulatory Vγ9Vδ2 T cells. When 181 samples from 108 patients with ulceroglandular tularemia were assayed, the percentage of Vγ9Vδ2 T cells was found to increase from ~5 to &gt; 20% after the first week of disease. During the ensuing 24 months, levels were normalized. Vaccination with the live attenuated vaccine strain Francisella tularensis LVS, on the other hand, did not cause an increase in numbers of Vγ9Vδ2 T cells. Within an outbreak of Pontiac fever, 14 cases were well defined with regard to incubation time and onset of disease. In samples obtained 4 to 6 days after onset of disease, the mean percentage of Vγ9Vδ2 T cells was ~ 1%, i.e., 20% of normal values. Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset of disease, values were ~ 15%. Later, values slowly decreased. In both tularemia and Pontiac fever, the capacity of Vγ9Vδ2 T cells to produce TNF-α in response to phorbol myristate acetate in vitro was transiently decreased, in tularemia up to 6 weeks after onset of disease and in Pontiac fever in samples obtained 5-7 weeks after onset of disease. Nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 T cells. Various strains of F. tularensis, including LVS, and a strain of L. micdadei were shown to produce Vγ9Vδ2 T-cell stimulating phosphoantigen. Notably, stimulation with an extract from each agent caused a similar degree of expansion of cells from subjects infected with the homologous and heterologous agent and also of cells from healthy subjects. Thus no immunospecific memory was detected in the Vγ9Vδ2-T cell response. Since it had been suggested that homologs of the conserved heat shock protein, chaperon-60, may be recognized by human Vγ9Vδ2 T cells, we determined the subpopulation of T cells responding to this protein as well as to DnaK, another heat-shock protein. Under in vitro conditions allowing a vigorous expansion of Vγ9Vδ2 T in response to a phosphoantigen, no expansion of γδ T cells in response to Cpn60 or DnaK of F. tularensis occurred. αβ T cells of tularemia-primed subjects, on the other hand, responded vigorously to the heat-shock proteins. In conclusion, two intracellular bacterial diseases with widely varying clinical expression were both associated with expansion of circulating Vγ9Vδ2 T cells. The expansion was prominent, long-lasting, and consistent within large numbers of individuals tested. In Pontiac fever, the expansion of Vγ9Vδ2 T cells was preceded by a depletion of the cells in circulation, implicating a possible extravasal migration into an infected site before the occurrence of rapid expansion and reentrance to blood. Both in tularemia and Pontiac fever, a modulation of the cytokine expression of Vγ9Vδ2 T cells was demonstrated in vitro, suggesting the presence of modulation of the inflammatory response. In extracts from in vitro culture of F. tularensis and L. micdadei, Vγ9Vδ2 T-cell stimulating phosphoantigens were identified and according to cross stimulation experiments, they induced expansion in vitro of Vγ9Vδ2 T cells without regard to immunospecific memory.

Legionella pneumophila est une bactérie à Gram négatif, intracellulaire facultative, responsable de la légionellose (ou maladie des Légionnaires) chez l'Homme. Les fluoroquinolones et les macrolides sont utilisés en première intention dans le traitement antibiotique de cette maladie. Cependant, les échecs thérapeutiques sont fréquents, et le taux de mortalité demeure élevé (10-15% des cas, plus de 30% chez le patient immunodéprimé). Bien qu'aucune souche de L. pneumophila résistante à ces antibiotiques n'ait été isolée à ce jour, ces échecs peuvent faire évoquer la possibilité d'une sélection in vivo de mutants résistants. Le mécanisme génétique principal d'acquisition de la résistance aux fluoroquinolones correspond à l'accumulation de mutations au niveau des gènes codant pour l'ADN gyrase et la topoisomérase IV ; en particulier celles affectant les codons en positions 83 et 87 du QRDR (quinolone resistance determining region) du gène gyrA entrainent une résistance de haut niveau à ces antibiotiques. Le première aspect de notre projet était d'élaborer un test de PCR en temps réel permettant de détecter chez L. pneumophila des mutants gyrA résistants aux fluoroquinolones et de les différencier des souches sauvages par analyse des températures de fusion des amplifias obtenus. Après optimisation, ce test nommé qPCRgyrALp amplifie spécifiquement une portion du QRDR du gène gyrA de l'espèce L. pneumophila et permet de détecter et de différencier les mutations gyrA83 et gyrA87. Nous avons ensuite utilisé ce test pour la recherche de mutants gyrA directement dans divers prélèvements respiratoires provenant de 82 patients atteints de légionellose, certains en échec thérapeutique après traitement par une fluoroquinolone. Les résultats ont montré pour quatre patients un profil de courbe de fusion semblable à celui du mutant gyrA83. Le séquençage du QRDR de gyrA à partir de ces prélèvements respiratoires a confirmé cette mutation chez deux patients. L'utilisation de la technique de séquençage à haut débit a permis de quantifier ces mutants gyrA83 chez ces deux patients, permettant de montrer un remplacement progressif in vivo de la population de L. pneumophila sensible aux fluoroquinolones par une population résistante à ces antibiotiques. Le deuxième aspect de notre travail a été de développer des tests de PCR quantitative en temps réel (qPCR) permettant de quantifier la charge bactérienne à L. pneumophila dans les prélèvements cliniques des patients infectés, avant et au cours du traitement antibiotique, dans la but de prédire l'évolution clinique et le pronostic final de ces patients. Nous avons utilisé deux tests de qPCR, ciblant soit le gène codant pour l'ARNr16s (qPCR16S) soit le gène mip (qPCRmip) dans des prélèvements respiratoires de 116 patients atteints de légionellose. Chez certains patients, nous avons pu déterminer la cinétique de la charge bactérienne au cours du temps, alors que les patients recevaient une antibiothérapie adaptée. Les premières cinétiques recueillies montrent la possibilité de différencier les patients qui répondent rapidement au traitement antibiotique et évoluent favorablement au cours de la 1ère semaine d'hospitalisation, de ceux qui présentent une réponse modeste au traitement et nécessitent une hospitalisation prolongée, voire décèdent. La PCR en temps réel semble donc représenter un outil pronostique d'intérêt au cours de la légionellose. Le type de cinétique observé chez un patient donné semble pouvoir prédire l'évolution des patients et la nécessité d'ajuster le traitement antibiotique. / Legionella pneumophila is a Gram- negative, facultative intracellular bacterium responsible for legionellosis (or Legionnaires' disease ) in humans. The fluoroquinolones and the macrolides are used as first-line antibiotic treatment of this disease. However, treatment failures are common, and the mortality rates remain high (10-15 % of cases, more than 30% in immunocompromised patients). Although L. pneumophila strain resistant to these antibiotics have never been isolated, treatment failures may suggest the possibility of in vivo selection of resistant mutants. The main genetic mechanisms associated with acquired resistance to fluoroquinolones correspond to the accumulation of mutations in the genes encoding DNA gyrase and topoisomerase IV, especially those affecting codons 83 and 87 of the QRDR (quinolone resistance determining region) of the gyrA gene, which are associated with high level resistance to these antibiotics. The first aspect of our project was to develop a real-time PCR test to detect gyrA QRDR mutants and differentiate them from wild-type strains of L. pneumophila by analysis of melting temperatures of the amplified DNA. After optimization, the qPCRgyrALp test specifically amplified a portion of the gyrA QRDR of L. pneumophila and could detect and differentiate gyrA83 and gyrA87 mutations. Then, we checked the presence of gyrA mutants directly in respiratory samples collected in 82 legionellosis patients, including some after treatment failure with a fluoroquinolone. For four patients, results corresponded to a melting curve profile similar to that of the gyrA83 mutant. Amplification and sequencing of the gyrA QRDR directly from these respiratory samples confirmed this mutation in two patients. The use of high-throughput sequencing technology allowed us to quantify the gyrA83 mutants in these two patients, allowing demonstration of in vivo gradual replacement of the fluoroquinolones susceptible population of L. pneumophila by a resistant one. The second aspect of our work was to develop quantitative real-time PCR tests offering the possibility to quantify the L. pneumophila bacterial load in respiratory specimens before and during antibiotic treatment, in order to predict the clinical course and the final prognosis of these patients. We used two qPCR tests, either targeting the gene encoding 16S rRNA (qPCR16S ) or the mip gene (qPCRmip ) in respiratory samples from 116 patients with Legionnaires' disease. In some patients, we determined the kinetics of bacterial loads over time, while patients received appropriate antibiotic therapy. The kinetics we observed allowed differentiation of patients who respond quickly to antibiotic treatment and were released from hospital within the first week following admission, from those with a modest response to treatment and requiring prolonged hospitalization or finally died. Thus, our real-time PCR tests seem to be good prognostic tools for evaluation of legionellosis prognosis. The type of kinetics observed in a given patient may allow the clinician to predict the evolution of patients and the need to adjust the antibiotic treatment.