Tübingen, Univ., Diss., 2009.

Orientador: Paulo Eduardo Neves Ferreira Velho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T03:36:54Z (GMT). No. of bitstreams: 1 Pitassi_LuizaHelenaUrso_D.pdf: 10118032 bytes, checksum: 2717c0557147ebb4a9a080297c902cd6 (MD5) Previous issue date: 2013 / Resumo: O gênero Bartonella constitui um grupo de patógenos zoonóticos re-emergentes e negligenciados que têm sido associados a um amplo espectro de doenças humanas graves. As Bartonella spp. são bacilos gram-negativos que infectam hemácias e células endoteliais, que podem causar infecção crônica ou mesmo fatal. As bartoneloses humanas mais conhecidas são a doença de Carrión, febre das trincheiras, doença da arranhadura do gato e angiomatose bacilar. Como existem espécies de Bartonella intraeritrocitárias e que podem causar infecção persistente e assintomática em humanos, objetivou-se avaliar o risco potencial de transmissão de Bartonella spp. por transfusão sanguínea. No primeiro estudo, os eritrócitos humanos foram infectados com B. henselae em 30 min e 1, 5, 10, e 72 h para a avaliação ultraestrutural por microscopia eletrônica de transmissão (ME). A B. henselae foi vista aderida a eritrócitos humanos 10 h após a inoculação e no interior de eritrócitos após 72 h. A localização intraeritrocitária de B. henselae havia sido mostrada em eritrócitos de gatos, mas nunca antes identificada em eritrócitos humanos. Foi realizado um segundo estudo para determinar as características ultraestruturais da Bartonella spp. nas células do sangue, pois estas informações eram muito limitadas. Este trabalho definiu os parâmetros para se caracterizar a bactéria no interior de células sanguíneas à ME. Um terceiro estudo, utilizando a ME e cultura de bactérias, demonstrou a capacidade da B. henselae sobreviver em uma unidade de concentrado de hemácias armazenada a 4° C durante 35 dias. A viabilidade de B. henselae em unidades de células vermelhas do sangue define o potencial de infecção associada à transfusão de doadores de sangue assintomáticos. Os receptores de transfusão de sangue muitas vezes são ou podem tornar-se imunodeficientes, apresentando o risco de desenvolver formas graves de bartonelose. Utilizando-se parâmetros ultraestruturais da infecção por Bartonella sp., relatou-se um caso de bartonelose como causa de morte 72h após uma transfusão. Um jovem com anemia aplástica, após receber várias transfusões sanguíneas, apresentou estado de choque e morreu após transfusão de um concentrado de hemácias. Os achados da ME permitiram o diagnóstico de infecção por Bartonella sp.. Foi investigada nesta tese a prevalência da Bartonella spp. em uma amostra de 500 doadores de sangue voluntários de Campinas, estado de São Paulo, Brasil, utilizando sorologia, cultura líquida e sólida, PCR e sequenciamento como métodos de identificação destes patógenos. Os anticorpos contra B. henselae e B. quintana foram detectados em 16,2% e 32%, respectivamente. O DNA das Bartonella spp. foi sequenciado e demonstrado em 3,2% do sangue doado (B. henselae em 15 amostras e B. clarridgeiae em uma) e em 1,2% foi documentada, por meio do isolamento do agente, bacteremia por B. henselae. Este estudo revelou a presença de patógenos no sangue de doadores assintomáticos que não estão sendo rastreados no sangue doado e que podem ser responsáveis por doenças não diagnosticadas em pacientes imunodeficientes. Os resultados apresentados estabelecem a necessidade de uma reavaliação por autoridades de saúde pública dos riscos e do impacto da transmissão da Bartonella spp. através de transfusões sanguíneas, especialmente para os pacientes imunodeficientes. Esforços devem ser feitos para a melhoria dos métodos de diagnóstico com sensibilidade suficiente para excluir a Bartonella spp. em doadores de sangue e para proteger os receptores de transfusão de sangue / Abstract: The genus Bartonella is a group of re-emerging zoonotic and neglected pathogens that have been associated with a wide range of serious human diseases. Bartonella spp. are gram-negative bacilli that infect red blood cells and endothelial cells, which can cause chronic infection or even fatal. In humans, Bartonella spp. are known causative agents of Carrion's disease, trench fever, cat scratch disease and bacillary angiomatosis. As they are intra-erythrocytic bacteria and can cause persistent infection and asymptomatic in humans, our objective for this proposal was evaluate the potential risk of transmission of Bartonella spp. by blood transfusion. In the first study, human erythrocytes were infected with B. henselae in 30 min and 1, 5, 10, and 72h for ultrastructural evaluation by transmission electron microscopy (TEM). B. henselae was seen adhering to human erythrocytes 10h after inoculation and inside the erythrocyte after 72 h. Intraerythrocytic localization of B. henselae has been demonstrated in cat erythrocytes, but never before identified in human erythrocytes. A second study was conducted to determine the ultrastructural features of the Bartonella spp. on blood cells because this information was very limited. This paper set the parameters to characterize the bacteria within the blood cell using TEM. A third study using TEM and bacterial culture demonstrated the ability of B. henselae to survive in a unit of red blood cells stored at 4°C for 35 days. The viability of B. henselae in units of red blood cells defines the potential for infection associated with transfusion in asymptomatic blood donors. Recipients of blood transfusions often are or may become immunodeficient, with the risk of developing severe forms of bartonellosis. Using the ultrastructural findings for the diagnosis of Bartonella sp. infection, we reported a case of bartonellosis as cause of death 72 h after transfusion. A young man with aplastic anemia who had received multiple blood transfusions went into shock and died after transfusion of a red blood cell. The findings of TEM allowed the diagnosis of infection with Bartonella sp.. Was investigated in this thesis the prevalence of Bartonella spp. in 500 volunteer blood donors from Campinas, São Paulo State, Brazil, using serology, solid and liquid culture, PCR and sequencing as methods for identifying these pathogens. Antibodies against B. henselae and B. quintana were detected in 16.2% and 32%, respectively. Bartonella sp. DNA was demonstrated in 3,2% of donated blood (15 infected with B. henselae and one with B. clarridgeiae) and 1.2% had B. henselae documented bacteremia by isolation of the agent. This study reveals the presence of pathogens in asymptomatic blood donors who are not being screened in donated blood and may be responsible for undiagnosed diseases in immunodeficient patients. The results presented establish the need for re-evaluation by public health officials of the risks and impact of Bartonella spp. transmission through blood transfusions, especially to immunocompromised patients. Efforts should be made towards improve diagnostic tests with sufficient sensitivity to rule out Bartonella ssp. infection in blood donors to protect transfusion recipients / Doutorado / Clinica Medica / Doutora em Clínica Médica

juana.delvalle@upc.edu.pe / Objetivo General: Caracterizar a nivel epidemiológico molecular cepas de Bartonella bacilliformis en las diferentes fases de la infección (Fiebre, Verruga, Bacteriemia asintomática). Objetivos específicos: 1.- Describir la presentación clínica e inmunológica de la enfermedad causada por Bartonella bacilliformis en las diferentes fases de infección: Determinar la etiología y caracterizar la presentación fenotípica y epidemiológica. Caracterizar los parámetros bioquímicos y hematológicos. Cuantificar los niveles de anticuerpos específicos para Bartonella bacilliformis

Bartonella bacilliformis es el agente causal de la enfermedad de Carrión. Actualmente, en el Perú los casos se siguen presentando y se ha reportado fallas en el tratamiento. Hay muy pocas investigaciones acerca de la susceptibilidad a antimicrobianos, y no se tiene estandarizada una prueba de antibiograma para esta bacteria. Asímismo, aún no se conocen los mecanismos de resistencia ni las secuencias de los genes asociados a ellas en cepas nativas circulantes de Bartonella bacilliformis. Por ello, el objetivo fue caracterizar molecularmente los genes asociados a la resistencia antimicrobiana en cepas circulantes en zonas endémicas para evaluar si presentan o no mutaciones en estos genes. Se estandarizó una prueba de antibiograma utilizando la cepa B. bacilliformis CIP 57.18 colocándose los discos de antibióticos comerciales: ciprofloxacina (CIP, 5 µg), rifampicina (RD, 30 µg), eritromicina (E, 15 µg), gentamicina (CN, 15 µg), doxiciclina (DO, 30 µg), cloranfenicol (30 µg), ácido nalidíxico (AN, 5 µg) y levofloxacina (LVX, 5 µg). Todas las cepas ensayadas mostraron resistencia al ácido nalidíxico pero sensibilidad a ciprofloxacina, a excepción de la cepa USM-LMM-002 que presentó susceptibilidad disminuida a ciprofloxacina. El análisis de las proteínas que codifican los genes gyrA y parC implicados en la resistencia a estos antibióticos, determinó que ambos presentan una diferencia aminoacídica (Ser83 por Ala) en la región del QRDR en relación a la GyrA y ParC de E. coli. Interesantemente, esta Ala83 en GyrA y ParC sólo proporciona resistencia a ácido nalidíxico y no a ciprofloxacina, siendo necesaria la acumulación de mutaciones en gyrA para la resistencia a ciprofloxacina. Para los demás antibióticos, las cepas fueron sensibles y en el secuenciamiento de los genes (rpoB, L4 y 16S ribosomal) asociado a dichas resistencia no se encontró ninguna mutación. La resistencia constitutiva al ácido nalidíxico es una propiedad de B. bacilliformis, y sería un buen indicador de la susceptibilidad a las fluoroquinolonas pero su resistencia no está relacionada a la resistencia de otras fluoroquinolonas. Además, la sensibilidad a otros antibióticos como doxiciclina, rifampicina, cloranfenicol y gentamicina podría indicar alternativas en el tratamiento de esta enfermedad aunque ensayos clínicos serian necesarios. / --- Bartonella bacilliformis is the causative agent of Carrion's disease. Currently in Peru are still present cases and are reported failures in treatment. There is very little research on antimicrobial susceptibility, nor has been standardized a test of sensitivity to the bacteria. Also, are not yet known resistance mechanisms and the sequences of genes associated with them in circulating native strains of Bartonella bacilliformis. Therefore, the objective was molecularly characterize the genes associated with antimicrobial resistance in isolates circulating in endemic areas to assess whether present or not mutations in these genes. Susceptibility testing was standardized using the strain of B. bacilliformis CIP 57.18 placing commercial antibiotic discs: ciprofloxacin (CIP, 5 µg), rifampin (RD, 30 µg), erythromycin (E, 15 µg), gentamicin (CN, 15 µg), doxycycline (DO, 30 µg), chloramphenicol (30 µg), nalidixic acid (NA, 5 µg) and levofloxacin (LVX, 5 µg). All strains tested showed resistance to nalidixic acid but sensitive to ciprofloxacin, except for strain USM-LMM-002 that presented reduced susceptibility to ciprofloxacin. The analysis of the proteins encoded by gyrA and parC genes involved in resistance to these antibiotics, determined that both have an aminoacid difference (Ser83 by Ala) in the QRDR region in relation to the GyrA and ParC of E. coli. Interestingly, the Ala83 in GyrA and ParC only provides resistance to nalidixic acid and not to ciprofloxacin, being necessary the accumulation of mutations in gyrA for ciprofloxacin resistance. For other antibiotics, the strains were sensitive and sequencing of genes (rpoB, and 16S ribosomal L4) associated with such resistance, there was no mutation. Constitutive resistance to nalidixic acid is a property of B. bacilliformis, and would be a good indicator of susceptibility to fluoroquinolones but this resistance is not related to resistance to other fluoroquinolones. In addition, sensitivity to other antibiotics such as doxycycline, rifampicin, chloramphenicol and gentamicin might suggest alternatives in the treatment of this disease, although clinical trials are needed. / Tesis

La Enfermedad de Carrión, es causada por la bacteria Bartonella bacilliformis, que se transmite por varias especies del insecto vector del género Lutzomyia. No se conoce ningún reservorio animal, aparentemente el hombre es el único. Es una enfermedad endémica en el Perú y actualmente es considerada como reemergente debido a que su área geográfica se viene expandiendo. Es importante estudiar marcadores moleculares que sirvan para el diseño de métodos de diagnóstico rápido y efectivos, así como evaluar los antibióticos recomendados como medidas terapéuticas contra esta enfermedad, e indagar más acerca de las proteínas antigénicas de esta bacteria a fin de seleccionar antígenos candidatos para desarrollar, una vacuna contra esta enfermedad o pruebas de diagnóstico para su detección de laboratorio de mayor sensibilidad y especificidad que las actuales. IalB y FlaA son proteínas interesantes debido a su participación en la invasión bacteriana al eritrocito humano. / Carrion’s Disease is caused by Bartonella bacilliformis bacterium, and is transmitted by several species of the vector insect of the Lutzomyia genus. Animal reservoir is not known; seemingly the man is the only one. This disease is endemic in Peru and is actually considered as re-emergent because their geographical distribution comes expanding. Thus it is important to study its molecular markers for established diagnosis methods that are quick and effective. On the treatment aspect, it is necessary to evaluate the antibiotics used against the disease. On the therapeutic topic, more research is required regarding the antigenic proteins from this bacterium; this fact is important to develop future vaccine against this disease or diagnostic assay for their detection in the laboratory or to improve the sensitivity and specificity of the current methods. IalB and FlaA are interesting proteins due to than they are involved in the bacterial invasion to the human erythrocyte. / Tesis

An 11 year old, hispanic girl with a history of B-cell acute lymphoblastic leukemia was admitted to the hospital for symptoms compatible with Bartonella henselae infection. The first molecularly diagnosed case of disseminated Bartonella henselae infection was reported in an immunocompromised patient in Lima, Peru. The analysis was confirmed by Polymerase Chain Reaction and automated sequencing of a liver biopsy sample, even though the serologic tests were negative. In conclusion, Bartonella spp. infection should have a particular diagnostic consideration in immunocompromised patients with fever of unknown origin and further investigation regarding the patient's past exposures with cats should also be elicited.

Pas de résumé français / Each Bartonella species appears to be highly adapted to one or a limited number of reservoir hosts, in which it establishes a long-lasting intraerythrocytic bacteremia as the hallmark of infection. Although the course of Bartonella infection has been precisely described, the molecular mechanisms of host specific erythrocyte infection and the stages of precedent the arrival in the bloodstream are poorly understood. In this thesis we purposed to identify the mechanisms of erythrocyte infection by Bartonella and characterize the possible locations of Bartonella during the days before the intraerythrocytic stage.By the establishment of an in vitro model of adhesion and invasion of erythrocytes by Bartonella spp., we demonstrated that host specificity was determined by the interaction between bacteria and erythrocytes. By screening signature-tagged mutagenesis (STM) library of B. birtlesii in vivo and in vitro and ectopic expression, we revealed that type IV Trw locus was required for host-restricted adhesion to erythrocytes in a wide range of mammals. After that, we further characterized that only TrwJ1 and TrwJ2 were expressed and present on the surface of the bacteria and had the ability to bind to mouse erythrocytes, and the receptor of them was erythrocyte band3 by different technology (phage display, electron microscopy, far western blot and adherence and invasion inhibition assay). By the model of experimental infection of laboratory normal Balb/C mice and splenectomized mice with B. birtlesii, we showed that during the first 7 days, no bacteria were recovered from lymph nodes, bone marrow and brain, but in the spleen, transient in the liver, And bacteremia was the same in both infection models during the first 7 days, thereafter, bacteremia was 10 fold higher in splenectomized mice than in normal mice and lasted 2 weeks longer. This suggested that the spleen was able to retain Bartonella.In conclusion, the host specific adhesion between Bartonella and erythrocyte was mediated by Trw and erythrocytic band 3, and spleen had a role in retention Bartonella.

Bartonella henselae ist der Erreger der Katzenkratzkrankheit, einer wenig beachteten, weil meist selbstlimitierend verlaufenden Zoonose. Allerdings kann B. henselae bei Risikogruppen (Kindern, immunsupprimierten Personen) Septikämien, Peliosis hepatis, Bazilläre Angiomatose und andere systemische Erkrankungen verursachen. Mit der vorliegenden Arbeit sollte versucht werden, die Prävalenz von B. henselae in unserer Hauskatzenpopulation abzuschätzen. Dazu wurden 930 EDTA-Katzenblutproben aus dem gesamten Bundesgebiet kulturell untersucht. Die Anzüchtung erfolgte auf doppelt dick gegossenem Hammelblutagar (7,5%) unter mikroaerophilen Bedingungen (5%CO2) über 4 Wochen. Es konnten 15 Stämme isoliert werden (1,61%). Ein Zusammenhang zwischen Alter der Katzen und kulturellem Ergebnis konnte nachgewiesen werden. 302 Katzenseren wurden auf Antikörper untersucht. Die Seroprävalenz betrug 37%. Dieses Ergebnis korreliert mit kulturellem Befund, Haltungsform, Flohbefall und geographischer Herkunft der Katzen. Mit den Isolaten wurden weitere Untersuchungen (PCR, PFGE, SDS-PAGE) zur Charakterisierung durchgeführt. Die Virulenz des Erregers wurde anhand verschiedener Untersuchungen des Referenzstammes untersucht. B. henselae kann nicht ohne schützendes Medium auf dem Fell des Wirtstieres überleben, benötigt bluthaltige Medien zur Isolierung und läßt sich auch nach längerer Aufbewahrung im Kühlschrank oder nach Einfrieren anzüchten. Aufgrund der geringen Anzahl kulturell positiver Befunde ist es schwierig, eine sichere Aussage zum Vorkommen von B. henselae zu treffen. Allerdings läßt die Seroprävalenz von 37% den Schluss zu, dass der Erreger zu einem nicht geringen Anteil unter den Katzen verbreitet ist. Eine Gefährdung gesunder, erwachsener Menschen besteht dabei nicht, aber für Kinder und immunschwache Personen bergen insbesondere verwilderte oder junge Katzen ein gewisses Risiko. Die Infektkette kann allerdings durch konsequente Flohbekämpfung unterbrochen werden.

Orientador: Paulo Eduardo Neves Ferreira Velho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T22:53:34Z (GMT). No. of bitstreams: 1 Pitassi_LuizaHelenaUrso_M.pdf: 12609657 bytes, checksum: 75cfaf4f2545cde781142bf97752e052 (MD5) Previous issue date: 2007 / Resumo: A Bartonella henselae, uma bactéria intracelular facultativa, é conhecida como agente da doença da arranhadura do gato, da angiomatose bacilar, da peliose bacilar hepática, de endocardites e de síndromes bacterêmicas em humanos. As espécies de Bartonella podem causar infecções intra-eritrocitárias e têm sido isoladas do sangue de pacientes por vários métodos. Foi demonstrado que a B. bacilliformis e a B. quintana infectam células endoteliais e eritrócitos humanos e que a B. henselae infecta células humanas (células endoteliais, células epiteliais, monócitos, macrófagos) e eritrócitos de gatos. O objetivo deste estudo foi investigar, através da microscopia eletrônica de transmissão, se a B. henselae infecta eritrócitos humanos maduros. Uma amostra de um concentrado de hemácias foi infectada experimentalmente com uma cepa-padrão de B. henselae. Alíquotas de sangue foram coletadas da amostra imediatamente após a infecção experimental, no minuto 30 e nas horas 1, 5, 10 e 72 para avaliação ultra-estrutural. A B. henselae foi vista aderida a eritrócitos humanos dez horas após a inoculação e no interior de eritrócitos após setenta e duas horas. Este estudo demonstra que a B. henselae se adere a e invade eritrócitos humanos maduros. Nossos resultados favorecem a possibilidade de que os eritrócitos possam servir como um nicho primário nas infecções causadas por Bartonella spp.. Diante desta observação, novos estudos deverão ser realizados para prevenir a transmissão transfusional da Bartonella sp / Abstract: Bartonella henselae, a facultative intracellular bacterium, has been known as the agent of cat scratch disease, bacillary angiomatosis, peliosis hepatis, endocarditis and bacteraemic syndrome in humans. Bartonella species can cause intraerythrocytic infections and have been isolated from patients blood stream by several methods. It was demonstrated that B. bacilliformis and B. quintana infect human endothelial cells and human erythrocytes and B. henselae infects erythrocytes of cats. The aim of our study was to investigate through transmission electron microscopy whether B. henselae infects mature human erythrocytes. One sample of red blood cell (RBC) unit received experimentally standard strain of B. henselae. Blood aliquots were collected from infected unit immediately after inoculation, on minute 30 and on hours 1, 5, 10 and 72 for ultrastructural evaluation. B. henselae was seen adhered to human erythrocytes ten hours after inoculation and inside the erythrocyte after seventy two hours. This study demonstrates that B. henselae adhere to and invades mature human erythrocytes. Our results favor the possibility that erythrocytes can serve as a primary target in Bartonella spp. infections. From this observation, further studies are warranted to prevent Bartonella transfusional transmission / Mestrado / Clinica Medica / Mestre em Clinica Medica

Bartonella bacilliformis is a facultative, intracellular, aerobic, Gram-negative coccobacillus causing the so-called Carrión's disease, a human infection endemic to specific areas mainly inhabited by low-income communities of Peru but also present in other Andean communities. It is considered a truly neglected tropical disease and is transmitted through the bite of female sandflies of the genus Lutzomyia [1]. Carrión's disease has two different clinical presentations; an initial febrile and haemolytic anaemia phase, known as Oroya fever, which has a mortality rate ranging from 44% to 88% in untreated patients; and a second phase characterised by the development of dermal eruptions known as Peruvian wart [1,2]. / The study was supported by internal funds from the Universidad Peruana de Ciencias Aplicadas (Lima, Peru); by a grant from the Instituto de Salud Carlos III, Spain [PI11/00983], which included FEDER funds; by the UBS Optimus Foundation; and by Generalitat de Catalunya, Departament d’Universitats, Recerca i Societat de la Informació [2014 SGR 26]. MJP has received a postdoctoral fellowship from CONCYTEC/FONDECYT. JR has received a fellowship from the program I3 of the ISCIII [grant no. CES11/012]. / Revisión por pares