Interference of mycobacterium tuberculosis with the endocytic/ antigen presentation pathways on macrophages and dendritic cells

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014

Evaluation of public transportation as a risk factor for active tuberculosis in Lima, Peru

Thesis (Master's)--University of Washington, 2012

The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Infeção por Mycobacterium Tuberculosis em profissionais de saúde : metodologia de avaliação do risco e sua aplicação num hospital central

RESUMO - A Tuberculose surge, de acordo com o último relatório da Organização Mundial da Saúde, como a segunda principal causa de morte em todo o mundo, de entre as doenças infeciosas. Em 2012, 1.3 milhões de pessoas morreram devido a esta patologia e surgiram 8.6 milhões de novos casos. De entre os grupos de risco de infeção, surgem os profissionais de saúde. A dificuldade no diagnóstico da Tuberculose, o contacto próximo com os pacientes, as medidas de controlo de infeção por vezes inadequadas são algumas das razões que explicam o risco mais elevado de contrair Tuberculose no local de trabalho. Esta Dissertação de Mestrado pretende estabelecer uma nova classificação de risco de infeção por M. tuberculosis em estabelecimentos de saúde, com vista a promover a saúde destes profissionais, inovadora nos critérios de avaliação das medidas de controlo de infeção e de análise dos casos de exposição não protegida a Tuberculose ativa. Esta metodologia de avaliação foi o resultado de uma revisão bibliográfica sobre a temática, tendo sido aplicada num hospital para verificar a sua adequabilidade e mais-valia.

Safety and immunogenicity of the M72/AS01 candidate tuberculosis vaccine in HIV-infected adults on combination antiretroviral therapy: a phase I/II, randomized trial.

OBJECTIVE: Tuberculosis (TB) is highly prevalent among HIV-infected people, including those receiving combination antiretroviral therapy (cART), necessitating a well tolerated and efficacious TB vaccine for these populations. We evaluated the safety and immunogenicity of the candidate TB vaccine M72/AS01 in adults with well controlled HIV infection on cART. DESIGN: A randomized, observer-blind, controlled trial (NCT00707967). METHODS: HIV-infected adults on cART in Switzerland were randomized 3 : 1 : 1 to receive two doses, 1 month apart, of M72/AS01, AS01 or 0.9% physiological saline (N = 22, N = 8 and N = 7, respectively) and were followed up to 6 months postdose 2 (D210). Individuals with CD4⁺ cell counts below 200 cells/μl were excluded. Adverse events (AEs) including HIV-specific and laboratory safety parameters were recorded. Cell-mediated (ICS) and humoral (ELISA) responses were evaluated before vaccination, 1 month after each dose (D30, D60) and D210. RESULTS: Thirty-seven individuals [interquartile range (IQR) CD4⁺ cell counts at screening: 438-872 cells/μl; undetectable HIV-1 viremia] were enrolled; 73% of individuals reported previous BCG vaccination, 97.3% tested negative for the QuantiFERON-TB assay. For M72/AS01 recipients, no vaccine-related serious AEs or cART-regimen adjustments were recorded, and there were no clinically relevant effects on laboratory safety parameters, HIV-1 viral loads or CD4⁺ cell counts. M72/AS01 was immunogenic, inducing persistent and polyfunctional M72-specific CD4⁺ T-cell responses [medians 0.70% (IQR 0.37-1.07) at D60] and 0.42% (0.24-0.61) at D210, predominantly CD40L⁺IL-2⁺TNF-α⁺, CD40L⁺IL-2⁺ and CD40L⁺IL-2⁺TNF-α⁺IFN-γ⁺]. All M72/AS01 vaccines were seropositive for anti-M72 IgG after second vaccination until study end. CONCLUSION: M72/AS01 was clinically well tolerated and immunogenic in this population, supporting further clinical evaluation in HIV-infected individuals in TB-endemic settings.

Mycobacterium tuberculosis aortic graft infection with recurrent hemoptysis: a case report.

INTRODUCTION: Mycobacterium tuberculosis may cause a large variety of clinical presentations due to its ability to disseminate by contiguity or hematogenously. Tuberculosis may remain undiagnosed for years due to the chronic course of the disease, with potentially life-threatening long-term complications. CASE PRESENTATION: In this case report, we describe a tuberculous aortic graft infection in a 72-year-old man documented by polymerase chain reaction and cultures. The patient presented with three episodes of hemoptysis following a remote history of miliary tuberculosis. The infection was treated by graft replacement and prolonged antimycobacterial therapy. CONCLUSION: Tuberculous infection of a vascular graft is an uncommon complication, but should be considered in patients with an intravascular device and a history of previous tuberculosis, especially when hematogenous spread may have occurred a few months after surgery, or when an active mycobacterial infection is present in close proximity to the graft.

Factors associated with latent tuberculosis among asylum seekers in Switzerland: a cross-sectional study in Vaud County.

BACKGROUND: Screening and treatment of latent tuberculosis infection (LTBI) in asylum seekers (AS) may prevent future cases of tuberculosis. As the screening with Interferon Gamma Release Assay (IGRA) is costly, the objective of this study was to assess which factors were associated with LTBI and to define a score allowing the selection of AS with the highest risk of LTBI. METHODS: In across-sectional study, AS seekers recently arrived in Vaud County, after screening for tuberculosis at the border were offered screening for LTBI with T-SPOT.TB and questionnaire on potentially risk factors. The factors associated with LTBI were analyzed by univariate and multivariate regression. RESULTS: Among 393 adult AS, 98 (24.93%) had a positive IGRA response, five of them with active tuberculosis previously undetected. Six factors associated with LTBI were identified in multivariate analysis: origin, travel conditions, marital status, cough, age and prior TB exposure. Their combination leads to a robust LTBI predictive score. CONCLUSIONS: The prevalence of LTBI and active tuberculosis in AS is high. A predictive score integrating six factors could identify the asylum seekers with the highest risk for LTBI.

Contact tracing investigation after professional exposure to tuberculosis in a Swiss hospital using both tuberculin skin test and IGRA.

SETTING: A 950 bed teaching hospital in Switzerland. AIM: To describe the result of a contact investigation among health care workers (HCW) and patients after exposure to a physician with smear-positive pulmonary tuberculosis in a hospital setting using standard tuberculin skin tests (TST) and Interferon-gamma release assay (IGRA). METHOD: HCW with a negative or unknown TST at hiring had a TST two weeks after the last contact with the index case (T0), repeated six weeks later if negative (T6). All exposed HCW had a T-SPOT.TB at T0 and T6. Exposed patients had a TST six weeks after the last contact, and a T-SPOT.TB if the TST was positive. RESULTS: Among 101 HCW, 17/73 (22%) had a positive TST at T0. TST was repeated in 50 at T6 and converted from negative to positive in eight (16%). Twelve HCW had a positive T-SPOT.TB at T0 and ten converted from negative to positive at T6. Seven HCW with a positive T-SPOT.TB reverted to negative at T6 or at later controls, most of them with test values close to the cut-off. Among 27 exposed patients tested at six weeks, ten had a positive TST, five of them confirmed by a positive T-SPOT.TB. CONCLUSIONS: HCW tested twice after exposure to a case of smear-positive pulmonary TB demonstrated a possible conversion in 10% with T-SPOT and 16% with TST. Some T-SPOT.TB reverted from positive to negative during the follow-up, mostly tests with a value close to the cut-off. Due to the variability of the test results, it seems advisable to repeat the test with values close to the cut-off before diagnosing the presence of a tuberculous infection.

Investigación parasitológica de la merluza (merluccius gayii peruanus)

Presenta los resultados obtenidos dentro del marco de cooperación entre el Instituto del Mar del Perú y la Universidad Nacional Federico Villarreal, para investigar la posición taxonómica de los parásitos que pudieran estar presentes en los tejidos musculares de la merluza, así mismo, se averiguo la existencia de alguna relación entre la presencia del o de los parásitos, con el reblandecimiento degenerante de los músculos de la merluza.

Investigación sobre los posibles efectos del represamiento del río Puyango Tumbes en la hidrobiología y pesquerías de la región

Estudio complementario de la primera evaluación sobre los posibles efectos del represamiento del río Puyango - Tumbes. Los datos obtenidos confirman algunas modificaciones de orden limnológico - pesquero en el río y canales de marea, determinando la zona de Cazaderos como la más adecuada para el establecimiento del embalse. Se hace la expresa recomendación acerca del mínimo caudal del río, el cual puede ser similar al flujo de la estación seca. Indica las implicancias en los recursos acuáticos cultivables especialmente langostinos Penaeus.

Investigación del recurso potencial langostino rojo de profundidad a bordo del BIC Fridtjof Nansen (25 de abril - 25 de mayo, 1990)

Describe las actividades desplegadas en el crucero 9004-05 del BIC "Fridtjof Nansen", se realizaron 24 arrastres en el área comprendida entre 03°41,8 '-03°49,4´S y 81°24,7 'W, en profundidades de 540 a 880 m, con la finalidad de seleccionar las especies de crustáceos con posibilidades de explotación a nivel comercial, así como determinar sus índices de abundancia, biomasa, proporción sexual, relación longitud-ancho del cefalotórax, relación longitud-peso y algunas variaciones morfométricas. Se analizaron también, las condiciones oceanográficas del hábitat.

Los efectos de "El Niño" 1982-83 y los mecanismos internacionales para la investigación

Durante 1982 -83 se presentó, frente a las costas sudamericanas del Pacífico, el fenómeno de «El Niño» (EN) catalogado como el más intenso del siglo. Inesperadamente, el régimen de temperaturas superficiales del mar se incrementó rápidamente a partir de octubre de 1982, superando más de 7 ºC por encima del promedio. Las precipitaciones normalmente altas en el Ecuador, sobrepasaron al promedio anual en más de 600 % y mucho más en las zonas semiáridas del norte del Perú. Las perturbaciones climáticas y oceánicas se manifestaron durante 12meses por lo menos; las lluvias se propagaron hasta la zona central del Perú y los cambios oceánicos a todo el Pacífico Sudeste. Las reuniones científicas realizadas en la región del Pacífico Sudeste y fuera de esta, dieron a conocer notables cambios tanto físicos como biológicos en toda la Cuenca del Pacífico. Estas alteraciones afectaron en el mar la actividad pesquera y en el continente la agricultura, infraestructura vial, industrial y viviendas, así como la educación y la salud. Los gobiernos de Ecuador y Perú se vieron precisados a declarar en emergencia las áreas afectadas. Los daños que se consignan en el presente informe se consideran sólo parciales por tener como base evaluaciones realizadas hasta febrero de 1983. Las lluvias continuaron aún con mayor fuerza en los meses siguientes. Los gobiernos y las instituciones de la región reaccionaron intensificando la vigilancia de los cambios climáticos y oceánicos en relación con los recursos. La Comisión Permanente del Pacífico Sur (CPPS), coor dinó las actividades del Grupo ERFEN y organizó reuniones científicas con apoyo de la COI-UNESCO y la OMM. Estos organismos así como la FAO propiciaron y estimularon por su parte programas globales. A pesar de lo anterior, la importancia socio-económica de EN requiere de una mayor coordinación y cooperación internacional. La meta de los programas es lograr una adecuada predicción de EN.

Investigación sobre el calamar gigante o pota en primavera 1996 en el área de Puerto Pizarro a Paita. Crucero BIC SNP-1 9609-10

Presenta los resultados de la prospección acústica pesquera, en la zona de Puerto Pizarro a Paita, hasta las 320 millas náuticas de distancia a la costa, del 25 de setiembre al 20 de Octubre 1996. El recurso se encontró muy disperso en casi toda el área prospectada, asociado a temperaturas superficiales y salinidades.

Crucero de investigación del calamar gigante BIC SNP-2 9903-04, 23 marzo al 12 abril 1999

Presenta los resultados del crucero de Investigación del calamar gigante, realizado a bordo del BIC SNP-2, del 23 de marzo al 12 de abril de 1999, de Puerto Pizarro al Callao, con el objeto de recopilar información actualizada sobre el estado biológico poblacional del recurso y las condiciones oceanográficas en el área y período de estudio.

Informe del crucero Krill III, a bordo del barco de investigación científica Alexander Agassiz de la Universidad de California y estadía en el departamento de oceanología de Montemar-Chile

Expone los resultados del análisis de las muestras de zooplancton colectados frente al Perú, para cumplir uno de los objetivos, la obtención de huevos y larvas de peces y Myctophidoe en diversas fases de desarrollo.