Orientador: Paulo Eduardo Neves Ferreira Velho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T05:58:18Z (GMT). No. of bitstreams: 1 Drummond_MarinaRovani_M.pdf: 544765 bytes, checksum: b62366bd6e7873354e418dfef366c14d (MD5) Previous issue date: 2012 / Resumo: Atualmente o genero Bartonella compreende pelo menos 31 especies e subespecies, sendo 15 delas conhecidamente patogenicas ao homem. Tres especies de Bartonella estao associadas ao maior numero de manifestacoes clinicas em seres humanos. Sao elas: Bartonella bacilliformis, Bartonella quintana e Bartonella henselae. A B. henselae e a bacteria mais associada a doencas humanas e sua transmissao e muitas vezes relacionada ao trauma cutaneo causado pelo arranhao de gatos infectados. Em recente estudo, documentou-se que mais de dois por cento dos doadores de sangue da regiao de Campinas testados estavam bacteremicos por Bartonella spp. e o contato com animais foi um fator de risco para a aquisicao da infeccao. Com o objetivo de avaliar a prevalencia de bacteremia por Bartonella spp. e isolar uma cepa regional de B. henselae, e foram analisadas 112 amostras de sangue de gatos, sendo que destes, 84 (75%) eram nao domiciliados. A partir do sangue total coletado durante o procedimento cirurgico para castracao, foi realizada extracao de DNA, seguida de PCR nested que amplifica a regiao FtsZ e e especifica para B. henselae. Este sangue tambem foi inoculado em meio liquido BAPGM (Bartonella Alpha-Proteobacteria Growth liquid Medium). Apos dez dias de incubacao, parte desta cultura liquida de enriquecimento foi semeada em meio solido enriquecido com 30% de sangue de carneiro e parte analisada pela mesma PCR nested e por PCR simples especifica para o genero Bartonella e que amplifica a regiao ITS. As culturas solidas foram incubadas por ate 45 dias e as que apresentaram crescimento foram encaminhadas as duas diferentes reacoes de PCR ja descritas. O DNA de B. henselae foi detectado em 86 (77%) das 112 amostras de sangue e em 56 (50%) das amostras da cultura de liquida de enriquecimento. No total, a bacteremia foi detectada em 90% (101/112) dos gatos deste estudo. Constatou-se maior prevalencia entre gatos nao domiciliados (95%, 80/84) quando comparada com a dos gatos de proprietarios (75%, 21/28). A deteccao da bacteremia por meio de PCR simples, especifica para o genero Bartonella, foi possivel em 31 das 112 (28%) culturas liquidas de enriquecimento. Dezesseis isolados foram obtidos da cultura solida, sendo que 11 foram PCR positivos. As amostras foram sequenciadas e tres destas colonias, que demonstraram 100% de homologia com a cepa de B. henselae Brazil- 1 na regiao ITS analisada, foram depositadas na Colecao de Culturas do Instituto Adolfo Lutz, sendo as primeiras do Brasil. A PCR nested especie especifica foi positiva em, pelo menos, uma amostra de todos os gatos em que a bacteremia foi detectada. Estes resultados mostram que a bacteremia causada por Bartonella spp. e muito frequente nos gatos de Campinas e sugerem que a prevalencia da infeccao por Bartonella spp. nos gatos e suas consequencias para a saude publica tem sido subestimadas. Metodos diagnosticos mais sensiveis precisam ser utilizados na investigação desta infecção / Abstract: Currently, Bartonella genus comprises at least 31 species and subspecies, 15 pathogenic to humans. Three species are associated with the largest number of clinical symptoms in human beings: Bartonella bacilliformis, Bartonella quintana and Bartonella henselae. B. henselae is the one most frequently associated with human diseases. This specie is considered zoonotic and its transmission is usually related with infected cat scratches. In a recent study, bacteremia was detected in more than two percent of blood donors from Campinas area and contact with animals was documented as a risk factor for Bartonella spp. infection. In order to evaluate Bartonella spp. bacteremia prevalence in cats from Campinas and to isolate a B. henselae regional strain, we analyzed blood samples from 112 cats (75% stray cats). The whole blood that was collected during spay surgery was submitted to DNA extraction and tested with specie-specific FtsZ nested PCR for B. henselae. A pre-enrichment culture in liquid medium BAPGM (Bartonella Alpha- Proteobacteria Growth Liquid Medium) was also performed. After ten-day culture, an aliquot was seeded and incubated up to 45 days in a solid medium supplemented with 30% sheep blood and another aliquot was tested for two PCRs: specie-specific FtsZ nested PCR for B. henselae and Bartonella genus-specific ITS single tube PCR. Sample isolates obtained from solid cultures were also tested by the two different PCR reactions described above. B. henselae DNA was detected in 86 (77%) of 112 blood samples and 56 (50%) of pre-enrichment culture samples. In total, bacteremia was detected in 90% (101/112) cats. Higher bacteremia prevalence was detected in stray cats (95%, 80/84 cats) when compared to client-owned subjects (75%, 21/28 cats). When the genus-specific ITS single tube PCR was used, Bartonella spp. bacteremia was detected just in 31/112 (28%) of preenrichment culture. Sixteen Gram-negative isolates were obtained from solid medium culture and eleven of them were PCR positive. Some samples were sequenced and three of these isolates demonstrated a 100% homology with B. henselae Brazil-1 strain at analyzed ITS region. These isolates were the first samples of this strain to be deposited in Brazil, at Adolfo Lutz Institute Culture Collection. The specie-specific FtsZ nested PCR was positive at least at one sample of bacteremic cats. Our results show that Bartonella sp. bacteremia prevalence among cats is very frequent in Campinas and suggest that the prevalence of Bartonella spp. infection among cats and its consequences for public health remains underestimated. More sensitive diagnostic methods must be used in the study of this infection / Mestrado / Clinica Medica / Mestra em Clínica Médica

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.

Objetivos: Describir las características clínico epidemiológicas de la Enfermedad de Carrión en pacientes que concurrieron al Instituto de Medicina Tropical Daniel Alcides Carrión durante el periodo 2010 al 2014. Material y métodos: Se analizó las historias clínicas de los pacientes que acudieron al IMT- UNMSM por sospecha de bartonelosis. Presenta un diseño no experimental tipo serie de casos, de modalidad retrospectiva, nivel de profundidad exploratoria, teniendo enfoque mixto (cuali-cuantitativo). Resultados: De 67 pacientes con sospecha de Bartonelosis el 90 % resultó con hemocultivo positivo. Del total de pacientes que acudieron al IMT – UNMSM el 42% fueron mujeres y 58% fueron hombres.La mayor cantidad de casos correspondieron a la fase aguda, encontrándose solo un caso en forma Verrucosa. Hubo 6 pacientes con frotis sanguíneo positivo, (3 mujeres y 3 hombres). El tiempo de enfermedad hasta que acuden al IMT-UNMSM fue: menor a 30 dias (28%), 1-6 meses (10%), mayor a 6 meses (18%), sin especificar (43%).El origende procedencia de contagio fue un 27% de Lima, 15% de Ancash, 8% de Junín, 7 % La Libertad, 5% Ucayali, 5% Piura y otros 33%. Los síntomas y signos más frecuentes que se encontraron en los pacientes con Bartonelosis fueron la fiebre en 48%, cefalea 34%, dolor articular 34%, palidez 21% y otros 37%. Conclusiones: Los pacientes que concurrieron al IMT- UNMSM para descartar, diagnosticar la Enfermedad de Carrión, al final representaron un gran porcentaje (90%) confirmados por hemocultivo y frotis sanguíneo. Existieron mayor porcentaje de hombres con Bartonelosis con respecto a mujeres. Los síntomas más frecuentes son fiebre 48%, cefalea 34%, dolor articular 34%.

Bartonella bacilliformis is a pathogen that is endemic in some areas of the Andean region of Peru, southern Ecuador and southern Colombia. This pathogen causes so-called Carrion's disease, a biphasic disease with acute and chronic phases (called Oroya fever and "Peruvian wart" respectively1-3). In the absence or delay of antibiotic treatment, the mortality rate in the acute phase is up to 88%1. The acute phase is characterised by fever and severe anaemia and may be followed, several weeks or months later, by the chronic eruptive phase due to endothelial cell proliferation2. No animal reservoir has been identified to date and it is considered that healthy carriers act as a pathogen reservoir in endemic areas

Bartonella henselae es un microorganismo Gram negativo, zoonótico, oportunista y cosmopolita, causante de diversas patologías humanas emergentes y reemergentes, que varían desde infecciones autolimitantes hasta fallas multiorgánicas y muerte dependiendo del estado inmunitario del afectado. El presente estudio analítico-descriptivo fue desarrollado en dos etapas; en la primera, se estandarizó la prueba de aislamiento de Bartonella henselae cepa ATCC 49882 por el método de Shell Vial en líneas celulares continuas Vero y Hep-2, determinándose la línea celular más susceptible a la infección en un tiempo determinado de incubación. En la segunda etapa, se realizó el aislamiento primario empleando la técnica estandarizada, evaluándose 60 muestras de sangre total de pacientes procedentes del sistema de vigilancia de enfermedades febriles metaxénicas a nivel nacional (INSCNSP) asociadas a factores epidemiológicos favorables a la infección por Bartonella henselae. Las muestras seleccionadas fueron reportadas como negativas a rickettsiosis y Enfermedad de Carrión. En ambas etapas, se determinó la presencia o ausencia del microorganismo de interés mediante la técnica de Inmunofluoresecencia Indirecta (IFI-Bartonellosis), coloración Giménez y la valoración cualitativa del efecto citopático. Los resultados obtenidos aplicando la técnica estandarizada fueron, 5 aislamientos primarios a partir de 60 muestras de sangre total (3 en células Vero y 5 en células Hep-2) en un periodo de incubación de 12 días a 37°C. De los resultados obtenidos en el desarrollo de la primera y segunda etapa se concluyó que Hep-2 es la línea celular más susceptible a la infección por Bartonella henselae. Palabras claves: Bartonella henselae, Ctenocephalides felis, cultivo celular, Shell Vial, efecto citopático. / --- Bartonella henselae is a Gram-negative, zoonotic, opportunist and Cosmopolitan microorganism, responsible of diverse emergent and reemergents human pathologies that vary from autolimitant infeccions to multiorganic failures and death, depending of the inmunitarian status of the affected individual. The present study descriptive-analytic was developed in two stages. In the first, the isolation test of Bartonella henselae strain ATCC 49882 was standardized with the Shell Vial method in continuous cell strains, Vero y Hep-2, determining the most susceptible cell line in an established incubation period. In the second stage, the primary isolation was realized applying the standardized technique, with 60 total blood samples evaluated from patients belonging to the surveillance system of metaxenic febrile diseases nationwide (INS-CNSP) associated to favourable epidemiological factors to infections with Bartonella henselae. The selected samples were reported as negative to rickettsiosis and Carrion disease. In both stages, the presence or absence of the analyzed microorganism was determined through the indirect immunofluorescence technique (IFI-Bartonellosis), Giménez coloration and the qualitative assessment of the cytopathic effect. The results obtained applying the standardized technique came, 5 primary isolations from 60 samples of total blood (3 in Vero cells and 5 in Hep-2 cells) during a period of incubation of 12 days at 37 °C. From the results obtained in the development of the firstand second stage, it was concluded that Hep-2 is the most susceptible cell line to the infection by Bartonella henselae. Key words: Bartonella henselae, Ctenocephalides felis, cell culture, Shell Vial, cytophatic effect

Memoria para optar al Título Profesional de Médico Veterinario / En el presente estudio se analizaron 80 sueros de gatos con el propósito de detectar anticuerpos contra la bacteria Bartonella henselae, principal agente de la enfermedad del arañazo del gato. El diagnóstico serológico se realizó a través de inmunofluorescencia indirecta, obteniéndose un 70% de gatos con anticuerpos contra B. henselae. No se encontraron diferencias estadísticamente significativas, considerando las características individuales (edad, sexo, contacto con otros gatos, positividad al virus de la leucemia felina, total de gatos por casa y visita periódica al médico veterinario) de los gatos estudiados.

Glucose-6-phosphate dehydrogenase (G6PD) is a cytoplasmic enzyme with an important function in cell oxidative damage prevention. Erythrocytes have a predisposition towards oxidized environments due to their lack of mitochondria, giving G6PD a major role in its stability. G6PD deficiency (G6PDd) is the most common enzyme deficiency in humans; it affects approximately 400 million individuals worldwide. The overall G6PDd allele frequency across malaria endemic countries is estimated to be 8%, corresponding to approximately 220 million males and 133 million females. However, there are no reports on the prevalence of G6PDd in Andean communities where bartonellosis is prevalent.

Evalúa in silico y serológicamente el potencial antigénico del dominio extracelular recombinante de la proteína de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis. Por medio del análisis in silico se escogió a la proteína LptD por estar localizado en la membrana externa, poseer una alta probabilidad de ser antigénica, no presentar similitud con proteínas humanas, de ratón o de cerdo, ser una proteína de mediano peso molecular (86.80 kDa), presentar epítopes con alta afinidad a HLA clase I y II, los cuales, presentaron un alto índice de cobertura poblacional (Perú 100%, Sudamérica 93.8% y a nivel Mundial 99.28%). Mediante el uso de la tecnología de ADN recombinante se clonó en Escherichia coli TOP10 el gen del dominio extracelular de LptD fusionada a una cola de seis histidinas y se expresó en E. coli BL21 (DE3) pLysS. Las condiciones para inducción de la proteína recombinante fueron 0.5 mM IPTG, 16 horas, medio TB etanol al 3% (v/v), 28 0C, OD600: 1-1.5 y 200 r.p.m. La purificación se realizó en condiciones denaturantes con resina Ni-IDA (His60 Ni Superflow) a pequeña escala y se obtuvo 2.6 μg de LptD recombinante parcialmente purificada por cada 1 mL de cultivo bacteriano inducido (OD600: 1 - 1.5). El resultado positivo del ensayo de Western Blot permitió evidenciar la antigenicidad de la proteína LptD recombinante ante sueros de pacientes que sufrieron la enfermedad de Carrión. En conclusión, la proteína LptD recombinante muestra antigenicidad tanto in silico y serológicamente, estos resultados son base para posteriores estudios de la proteína LptD en la línea de investigación de candidatos vacunales contra la enfermedad de Carrión. / Perú. Ministerio de la Producción. Programa Nacional de Innovación para la Competitividad y Productividad (Innóvate Perú). Universidad Nacional Mayor de San Marcos (Lima). Vicerrectorado de Investigación y Posgrado / Tesis

<p>Characteristically, in infections that are caused by the zoonotic pathogen <i>Bartonella</i> naturally infected reservoir hosts are asymptomatic, where infected incidental, non-natural, hosts develop symptomatic disease. Cat-scratch disease (CSD) is a well known example. <i>Bartonella </i>infections in humans may be self-limiting or fulminant and affect different organ systems. </p><p>The objectives of the present thesis were to (1) identify and characterise <i>Bartonella </i>infection cases in Sweden, (2) to investigate certain human populations regarding <i>Bartonella </i>infections, and (3) compare natural populations of different <i>Bartonella </i>species.</p><p>Cases with typical and atypical CSD were recognised by using a combination of PCR and serology. Gene sequence comparisons of different genes in <i>B. henselae</i> isolates from the United States and Europe showed that<i> fts</i>Z gene variation is a useful tool for <i>Bartonella</i> genotyping. </p><p>Myocarditis was a common finding among Swedish elite orienteers succumbing to sudden unexpected cardiac death (SUCD). The natural cycle of <i>Bartonella</i> spp., the life style of orienteers, elevated antibody titres to <i>Bartonella</i> antigens, <i>Bartonella</i> DNA amplified from myocardium and the lack of another feasible explanation make <i>Bartonella</i> a plausible aetiological factor.</p><p>The first reported case of <i>Bartonella</i> endocarditis (<i>B. quintana</i>) was identified in an immunocompromised patient who underwent heart valve replacement. The patient had been body louse-infested during his childhood. It is hypothesised that a chronic <i>B. quintana</i> infection was activated by the immunosuppression.</p><p>There was no evidence of an ongoing trench fever (TF) epidemic in a Swedish homeless population, although an increased risk for exposure to <i>Bartonella</i> antigens was demonstrated. The lack of louse infestation might explain the absence of <i>B. quintana</i> bacteremia and low <i>B. quintana</i> antibody titres. </p><p>Comparisons of genetic loci and the whole genomes of environmental <i>B. grahamii</i> isolates from the Uppsala region, Sweden displayed variants that were not related to specific host species but to geographic locality. Natural boundaries seemed to restrict gene flow.</p>

B. henselae, B. bacilliformis and B. quintana are capable of causing vasoproliferative diseases in humans by modulating apoptosis and proliferation of endothelial cells. Bartonella clarridgeiae, a close relative of the pathogenic Bartonellae, has been implicated in human disease but has not yet been isolated from a human patient. Both B. bacilliformis and B. clarridgeiae have flagella and a flagellar type 3 secretion system, while B. henselae and B. quintana do not. We created 2 non-motile mutants of B. clarridgeiae by interrupting the flagellin gene, flaA, or the flagellar motor genes, motBC. We investigated whether B. clarridgeiae could invade human endothelial cells (HMECs) and if functional flagella were important for invasion. The non-motile mutants and the wild-type strain were capable of entering HMECs in vitro. The flaA mutant was deficient in attachment, but the HMECs in culture with the flaA mutant demonstrated increased proliferation. The motBC mutant showed enhanced invasion. Differential secretion of proteins was revealed by 2-D electrophoresis and MALDI-TOF analysis of secretomes from the co-cultures compared to uninfected HMECs. HMECS infected with wild-type B. clarridgeiae secreted proteins indicative of proliferation. The flaA mutant induced the secretion of proteins involved in cytoskeletal rearrangement, cell migration, and proliferation. The motBC-infected HMECs showed signs of hypoxia. The co-chaperonin GroES was found in higher concentration in the supernatant of the hyper-invasive motBC strain/HMEC co-culture than the wild-type co-culture and was found at a very low concentration in the flaA culture supernatant. Cross-talk between secretion systems is suggested.