Base teórica: A introdução de agentes biológicos, especialmente os bloqueadores do fator de necrose tumoral (anti-TNF), para o tratamento de doenças reumáticas aumentou o risco de desenvolver tuberculose (TB). O rastreio para infecção tuberculosa latente (ILTB) é fortemente recomendado antes de iniciar a terapia com agentes anti-TNF. Os objetivos deste estudo foram identificar a prevalência de ILTB e TB entre pacientes com doenças reumáticas em uso dos medicamentos anti-TNF. Métodos: Estudo transversal. Foram revisados os registros médicos eletrónicos de todos os doentes adultos (≥ 18 anos) em uso da terapia anti-TNF. Todos os pacientes foram submetidos ao teste tuberculínico (TT) antes de iniciar o tratamento com os medicamentos anti-TNF. Resultados: No total, 176 pacientes foram incluídos no estudo. A idade média de todos os pacientes foi de 51,9 ± 12,4 anos, 34,7% eram do sexo masculino e 90,9% eram brancos. As doenças subjacentes mais comuns foram: Artite Reumatóide (AR) em 89 pacientes (50,6%), Espondilite Anquilosante (EA) em 49 (27,8%) e Artrite Psoriática (AP) em 31 (17,6%). A prevalência de TT positivo foi de 29,5%. O contato domiciliar com TB foi significativamente associado com TT positivo (p = 0,020). Os pacientes com AR apresentaram reações TT menores do que os pacientes com EA (p = 0,022). Houve seis casos de TB (3,4%) diagnosticados durante a terapia anti-TNF. Conclusões: Demonstrou-se alta prevalência de TT positivo (29,5%) em pacientes com doenças reumáticas em uma região com alta prevalência de TB. Nossos dados corroboram a recomendação do Colégio Americano de Reumatologia (ACR) de que os pacientes que vivem em configurações de alta incidência de TB devem ser testados anualmente para ILTB. / Background: The introduction of biological agents, especially the blockers of tumor necrosis factor (anti-TNF), for the treatment of rheumatic diseases increased the risk of developing tuberculosis (TB). Screening for latent TB infection (LTBI|) is strongly recommended before starting therapy with anti-TNF agents. The objectives of this study were to identify the prevalence of LTBI and TB among patients with rheumatic diseases on anti-TNF drugs. Methods: Cross-sectional study. The electronic medical records of all adult patients (≥ 18 years old) undergoing anti-TNF treatment were reviewed. Every patient underwent TST test before starting anti-TNF treatment. Results: In total, 176 patients were included in the study. The mean age of all patients was 51.9 ± 12.4 years, 34.7% were males, and 90.9% were white. The most common underlying diseases were: RA in 89 patients (50.6%), AS in 49 (27.8%), and PA in 31 (17.6%). The prevalence of positive TST was 29.5%. Household contact with TB was significantly associated with a positive TST (p=0.020). RA patients had lower TST reactions than AS patients (p=0.022). There were six cases of TB (3.4%) diagnosed during anti-TNF therapy. Conclusions: We demonstrated a high prevalence of positive TST (29.5%) among patients with rheumatic diseases in a region with high TB prevalence. Our data corroborates the ACR’s recommendation that patients who live in high TB incidence settings should be tested annually for LTBI.

L’accès par voie chimique à des oligosaccharides nécessite souvent le recours à de nombreuses étapes de protection-déprotection. Au cours de ce projet de thèse, une méthodologie pour la protection régiosélective et orthogonale des fonctions alcool de disaccharides dérivant de la biomasse a tout d’abord été développée. Les glycopyranosides protégés ont été préparés par catalyse tandem au FeCl3∙6H2O en réalisant dans le même pot des réactions d’acétalation, d’éthérification réductrice, d’acétylation et/ou d’ouverture réductrice régiosélective d’acétals. Dans un second temps, une stratégie de synthèse flexible, rapide et performante pour accéder à des sulfoglycolipides diacylés et tétraacylés comportant un cœur tréhalose a été mise au point. Ces molécules sont produites par Mycobacterium tuberculosis, l’agent pathogène responsable de la tuberculose, et les sulfoglycolipides diacylés pourraient permettre l’élaboration d’un nouveau vaccin contre cette maladie. Des sulfoglycolipides diacylés et tétraacylés comportant des chaînes monométhylées chirales ont été obtenus. Les précurseurs des acides gras chiraux utilisés au cours de la synthèse ont été analysés par spectroscopie RMN du deutérium en abondance naturelle dans des cristaux liquides chiraux. / The synthesis of oligosaccharides often requires long sequences of protection-deprotection steps. For a rapid access to suitably protected glycopyranosides, we have developed a one-pot regioselective protection strategy based on FeCl3∙6H2O-tandem catalyzed reactions (acetalation, acetylation, reductive etherification, regioselective ring opening of acetal). This procedure was applied to persilylated disaccharides derived from biomass. This methodology allowed the development of a fast, efficient and flexible access to diacylated and tetraacylated sulfoglycolipids based on a trehalose core. These molecules are found in the cell wall of Mycobacterium tuberculosis and the diacylated sulfoglycolipids appear to be promising candidates for the development of a new tuberculosis vaccine. Synthetics diacylated and tetraacylated sulfoglycolipids bearing chiral monomethylated fatty chains were produced. The chiral fatty-acid precursors, used in the procedure, were synthetized and analyzed by NMR spectroscopy of natural abundance deuterium in chiral liquid crystals.

La tuberculose est due à un groupe d’agents infectieux phylogénétiquement proches formant le complexe Mycobacterium tuberculosis, formé de dix espèces. M. tuberculosis est un bacille à croissance lente qui forme des colonies rugueuses. Le complexe M. tuberculosis comporte également des bacilles tuberculeux formant des colonies lisses à croissance rapide, isolées qu’à partir de prélèvements cliniques chez l’homme. Notre revue des articles sur ces souches a montré que les trois premiers isolats ont été obtenus chez des patients en France, à Madagascar et en Polynésie Française par Georges Canetti entre 1968 et 1970. Suite à l'isolement d'une souche lisse à partir d’un ganglion cervical chez un enfant Somali de 2 ans en 1997, ces bacilles tuberculeux ont été nommés "Mycobacterium canettii". Aujourd'hui, moins d'une centaine de ces souches ont été isolées à partir de patients exposés aux pays formant la Corne de l’Afrique, principalement la République de Djibouti, qui présente la plus forte prévalence. Nous avons procédé à l’analyse génotypique de 34 isolats de M. tuberculosis et de 87 isolats de mycobactéries non-tuberculeuses de la Polynésie Française pour voir si des telles souches persistaient.Nous avons pu décrire deux nouvelles lignées de M. tuberculosis et une nouvelle espèce de mycobactérie non-tuberculeuse. Nous avons envisagé une transmission par voie digestive de "M. canettii" et nous avons testé la résistance de "M. canettii" à la chaleur. Nous avons observé la croissance de cette dernière entre 25°C et 45°C. Les données que nous avons obtenues ici pourraient former une base en vue d'élucider les réservoirs et les sources de souches lisses. / Tuberculosis is caused by a group of phylogenetically close infectious agents forming the Mycobacterium tuberculosis complex, consisting of ten species. M. tuberculosis is a slow growing bacterium which form rough colonies. The complex also includes M. tuberculosis tubercle bacilli forming smooth rapidly-growing colonies isolated from clinical specimens in humans. Our review of articles on these strains showed that the first three isolates were obtained from patients in France, Madagascar and French Polynesia by Georges Canetti between 1968 and 1970. Following the isolation of a smooth strain from cervical ganglion of a Somali 2 year-old child in 1997, the tubercle bacilli were named "Mycobacterium canettii". Today, less than a hundred of these strains were isolated from patients exposed to countries forming the Horn of Africa, especially the Republic of Djibouti, which has the highest prevalence. As showed in our revue, one smooth tubercle bacilli was isolated in Frenche Polynesia. To see if such strains persist there, we performed the genotyping of 34 isolates of M. tuberculosis and 87 isolates of non-tuberculous mycobacteria from French Polynesia. We have described two new strains of M. tuberculosis and a new species of mycobacterium tuberculosis not. We considered, on the basis of our data, a transmission through the digestive tract for "M. canettii" and we tested the resistance "Mr. canettii" to heat. We observed the growth of the latter between 25°C and 45°C. The data we got here could form a basis to elucidate the reservoirs and sources of smooth strains.

Mycobacterium tuberculosis, the principal causative agent of tuberculosis (TB) in humans, is considered to be a successful pathogen owing to the elicitation of multidrug resistance, ability to survive inside macrophage phagosomes by taking nutrients from host cell cytoplasm, and the capacity to alternate between proliferating and dormant (nonproliferating) conditions of growth. Thus, whether one looks at tubercle bacillus from the standpoint of regulation of cell division in the host system, or uptake of nutrients from the host cell cytoplasm or elicitation of drug resistance, the requirement for ATP Binding Cassette (ABC) transporter type protein complexes, which might be involved in the transport of drugs, nutrients or proteins, could be of critical importance to the pathogen. Therefore the present study was initiated to characterize ABC transporter type proteins, FtsE and FtsX of M. tuberculosis (MtFtsE and MtFtsX), and their interaction with FtsZ and FtsQ, which are the septation proteins that are recruited respectively before and after the localization of FtsE and FtsX proteins. The study was carried out in 3 parts. 1. Cloning, overexpression and purification of MtFtsE and MtFtsX proteins and elucidation of ATP binding activity of MtFtsE There exists considerable extent of homology between the FtsE and FtsX proteins of M. tuberculosis and E. coli. Therefore, in order to verify whether the structural homology is reflected in functional homology, complementation of growth defect of E. coli ftsE (Ts) by MtFtsE and MtFtsX was carried out. The MtFtsE protein could partially complement growth defect of E. coli ftsE temperature sensitive strain MFT1181, whereas co-expression of MtFtsE and MtFtsX efficiently complemented growth defect, indicating that the MtFtsE and MtFtsX proteins functionally complement E. coli FtsE and FtsX and that the two proteins together might be performing an associated function. Subsequently, in order to biochemically characterize MtFtsE and MtFtsX proteins of M. tuberculosis, MtftsE gene was cloned in pQE30, overexpressed, purified by Ni2+-NTA agarose affinity chromatography under denaturing conditions and refolded. MtFtsX protein, being toxic to E. coli cells, could not be expressed to sufficient amounts. Western blotting with anti-MtFtsE antibody showed that the recombinant 6xHis-MtFtsE protein and the native MtFtsE protein were localized to the membrane of E. coli and M. tuberculosis cells respectively. 6xHis-MtFtsE protein showed ATP binding in vitro, whereas K42R mutation abolished ATP binding. Thus, like in the case of E. coli FtsE, the K42 residue, which is positionally equivalent to K41 in EcFtsE in Walker A motif, was found to be essential for ATP binding. At 1.3 nM concentration of [α32P] ATP,70 molar excess of ATP, ADP, AMP, and GTP competed out respectively 97%, 87%, 73% and 57% of the [α32P] ATP bound to 6xHis-MtFtsE. 2. Biochemical characterization of MtFtsE protein The functional architecture of an ABC transporter consists of two each of nucleotide binding domain (NBD) and transmembrane domain (TMD), which are either part of a single polypeptide chain or individual subunits. The functional NBD is a ‘nucleotide-sandwich dimer’ with ATP flanked by the Walker A and B motifs of one NBD and the signature motif and D-loop of the other. NBD, through ATPase activity, is involved in energizing the transport of substrates namely drugs, proteins, ions, and solutes across the membrane. Since MtFtsE possesses Walker A and Walker B motifs that constitute NBD, and MtFtsX possesses TMD (four transmembrane segments), the two proteins together might constitute an ABC transporter type complex. Therefore, we wanted to know whether MtFtsE could hydrolyze ATP. MtFtsE not only could bind ATP with high affinity but could hydrolyse it also (Km, 1.5 µM; Vmax, 0.87 nmole/mg/min). It could bind and hydrolyse GTP as well, but not CTP, albeit with lower affinity and rate (Km, 25 µM; Vmax, 0.54 nmole/mg/min). The ATPase activity is strongly dependent on Mg2+ or Mn2+, with a pH optimum of 6.5 – 8.0 and temperature range of 27oC - 40oC. Kinetic analysis of ATPase and GTPase activities indicated nucleotide- dependent cooperativity (Hill coefficient for ATP is 1.7 and for GTP, 2.1). Inhibition of ATPase activity, to almost similar extent, in the presence of 10-fold excess of ATPγS, ADP, AMP, GTP, and CTP, but not TTP, indicated that nucleotide binding is through nitrogenous base of the nucleotide. Inhibition of MtFtsE by orthovanadate classified the enzyme as a P-type ATPase. Partially purified MtFtsE in soluble fraction also showed ATPase activity. The ATPase-active form of MtFtsE is a dimer with the sole cysteine (C84) at the dimer interface. Homology modeling of MtFtsE, using MalK (the NBD component of an ABC transporter for maltose) as the template, supported this observation. Stabilization of the dimer through cys-cys disulphide bond increased ATPase activity by 3.7-fold, although C84 does not have any role in ATPase activity. 3. Identification and elucidation of interaction among cell division proteins FtsE, FtsX, FtsQ and FtsZ of Mycobacterium tuberculosis Septum synthesis in E. coli is mediated by a dozen of proteins, among which the bacterial cytoskeletal protein FtsZ is the first molecule to localise to the mid-cell site, where it forms a scaffold for the localization of downstream cell division proteins namely, FtsA /ZipA < FtsE / FtsX < FtsK < FtsQ < FtsL < FtsB < FtsW < FtsI < FtsN and AmiC. If the above order of recruitment of proteins holds true for M. tuberculosis as well, the immediate proteins recruited to the mid-cell site after MtFtsZ in M. tuberculosis would be MtFtsE and MtFtsX, followed with MtFtsK and MtFtsQ. Thus it is possible that MtFtsE and MtFtsX could be interacting with MtFtsZ and MtFtsQ. Therefore attempts were made to delineate the interaction network among MtFtsE, MtFtsX, MtFtsQ and MtFtsZ of M. tuberculosis. Ni2+-NTA agarose pulldown, co-immunoprecipitation and bacterial two-hybrid assays using wild type and deletion mutants of the proteins showed that MtFtsE interacts with MtFtsQ and MtFtsX through its C-terminus. In addition, MtFtsX could interact with MtFtsZ and MtFtsQ. MtFtsX was found to homodimerise and interact with MtFtsQ in vivo. The ATPase-active of MtFtsE in vivo being a dimer, a hypothetical model for the translocation of MtFtsQ into the membrane at mid-cell site was proposed. According to this model, MtFtsQ might be inserted into the membrane at the mid-cell site by (MtFtsX)2 functioning as the membrane channel for the transport, which could be energized by the ATPase subunit (MtFtsE)2 of the (MtFtsE)2(MtFtsX)2 complex. MtFtsX might have a role in tethering the FtsZ-ring with the membrane at the mid-cell site. An altogether different possibility could be that the (FtsE)2(FtsX)2 complex might have a role in the stabilization or constriction of FtsZ-ring during the inward growth of septum.

In bacteria, nucleoid associated proteins (NAPs) represent a prominent group of global regulators that perform the tasks of genome compaction, establishing chromosomal architecture and regulation of various DNA transactions like replication, transcription, recombination and repair. HU, a basic histone like protein, is one of the most important NAPs in Eubacteria. Mycobacterium tuberculosis produces a homodimeric HU (MtbHU), which interacts with DNA non-specifically through minor groove binding. Exploration for essential genes in Mtb (H37Rv) through transposon insertion has identified HU coding gene [Rv2986c, hupB; Gene Id: 15610123; Swiss-Prot ID: P95109)] to be vital for the survival and growth of this pathogen. MtbHU contains two domains, the N-terminal domain which is considerably conserved among the HU proteins of the prokaryotic world, and a C–terminal domain consisting of Lys-Ala rich multiple repeat degenerate motifs. Sequence analysis carried out by the thesis candidate showed that MtbHU exhibits 86 to 100 percent identity within the N-term region among all the mycobacterium species and some of the members of actinobacteria, including important pathogens like M. tuberculosis, M. leprae, M. ulcerans, M. bovis, Nocardia; while C term repeat region varies relatively more. This strikingly high cross species identity establishes the MtbHU N-terminal domain (MtbHUN) as an important representative structural model for the above mentioned group of pathogens. The thesis candidate has solved the X-ray crystal structure of MtbHUN, crystallized in two different forms, P2 and P21. The crystal structures in combination with computational analyses elucidate the structural details of MtbHU interaction with DNA. Moreover, the similar mode of self assembly of MtbHUN observed in two different crystal forms reveals that the same DNA binding interface of the protein can also be utilized to form higher order oligomers, that HU is known to form at higher concentrations. Though the bifunctional interface involved in both DNA binding and self assembly is not akin to a typical enzyme active site, the structural analysis identified key interacting residues involved in macromolecular interactions, allowing us to develop a rationale for inhibitor design. Further, the candidate has performed virtual screening against a vast library of compounds, and design of small molecules to target MtbHU and disrupt its binding to DNA. Various biochemical, mutational and biological studies were performed in the laboratory of our collaborator Prof. V. Nagaraja, MCBL, IISc., to investigate these aspects. After a series of iterations including design, synthesis and validation, we have identified novel candidate molecules, which bind to MtbHU, disrupt chromosomal architecture and arrest M. tuberculosis growth. Thus, the study suggests that, these molecules can serve as leads for a new class of DNA-interaction inhibitors and HU as a druggable target, more so because HU is essential to Mtb, but absent in human. Our study proposes that, targeting the nucleoid associated protein HU in Mtb can strategize design of new anti-mycobacterial therapeutics. Perturbation of MtbHU-DNA binding through the identified compounds provides the first instance of medium to small molecular inhibitors of NAP, and augurs well for the development of chemical probe(s) to perturb HU functions, and can be used as a fundamental chemical tool for the system level studies of HU-interactome. Section I: “Crystal structure of Mycobacterium tuberculosis histone like protein HU and structure based design of molecules to inhibit MtbHU-DNA interaction: Leads for a new target.” of this thesis presents an elaborate elucidation of the above mentioned work. The candidate has additionally carried out structure based computational and theoretical work to elucidate the interaction of amino acid based metal complexes which efficiently bind to DNA via minor-groove, major-groove or base intercalation interaction and display DNA cleavage activity on photo-irradiation. This understanding is crucial for the design of molecules towards Photodynamic Therapy (PDT). PDT is an emerging method of non-invasive treatment of cancer in which drugs like Photofrin show localized toxicity on photoactivation at the tumor cells leaving the healthy cells unaffected. The work carried out in our group in close collaboration with Prof. A.R. Chakravarty of Inorganic and Physical Chemistry Department elaborates the structure based design of Amino acid complexes containing single Cu (II), such as [Cu(L-trp)(dpq)(H2O)]+ , [Cu (L-arg) 2](NO3)2 , Amino acid complexes containing oxobridged diiron Fe(III), such as [{Fe(L-his)(bpy)}2(μ-O)](ClO4)2 , [{Fe(L-his)(phen)}2(μ-O)](ClO4)2 , and Complexes containing Binuclear Cu(II) coordinated organic moiety, such as [{(dpq) CuII}2(μ-dtdp)2], which bind to DNA through minor groove/major groove/base intercalation interactions. Docking analysis was performed with the X-ray crystallographic structure of DNA as receptor and the metal complexes as ligands, to study the mode of binding to DNA and to understand the possible mode of DNA cleavage (single/double strand) when activated with laser. Section II: “Structure based computational and theoretical analysis of metal coordinated complexes containing amino acids and organic moieties designed for photo induced DNA cleavage” of this thesis presents a detailed presentation of the above mentioned work.

Avec plus d’un million et demi de morts chaque année, la tuberculose reste aujourd’hui la seconde cause de mortalité liée à un agent infectieux. De plus l’organisation mondiale de la santé (OMS) a estimé en 2011 qu’un tiers de la population mondiale était porteuse du bacille Mycobacterium tuberculosis responsable de la maladie. Depuis la fin des années 1980, une recrudescence du nombre de cas de tuberculose est observée à l’échelle mondiale. Cette recrudescence est due à la fois à l’apparition de souches résistantes, mais également à l’épidémie de VIH qui est un facteur de prédisposition au déclenchement de la maladie.En 2000, le répresseur transcriptionnel mycobactérien EthR a été identifié comme étant un régulateur clé dans la bioactivation de l’éthionamide (ETH), un antituberculeux utilisé pour le traitement de seconde intention. En 2009, l’inhibition de ce répresseur par le développement de molécules « drug-like » a permis de potentialiser l’activité de l’éthionamide d’un facteur 3 chez la souris infectée et a permis de valider cette cible pour une future approche thérapeutique.Ce travail repose sur la découverte et l’optimisation de nouveaux inhibiteurs de ce répresseur transcriptionnel mycobactérien, à partir d’une petite molécule appelée « fragment » qui a été cocristallisée avec la protéine. Par la combinaison d’un criblage in silico, d’un criblage in vitro des touches identifiées, de l’étude des structures radiocristallographiques des complexes ligands/protéines et de la chimie médicinale, le développement de trois approches complémentaires dites « fragmentgrowing », « fragment-merging » et « fragment-linking » a permis de développer des composés présentant de fortes activités. Ces résultats permettront très prochainement de sélectionner une nouvelle molécule issue de ce travail dans la perspective de nouveaux essais sur le modèle murin. / Tuberculosis (TB) remains the leading cause of death due to a single infective agent with more than 1.5 million people killed each year. In 2011, the world health organization (WHO) estimated that one third of the world’s population is infected with Mycobacterium tuberculosis, the pathogen responsible for the disease. This phenomenon may be due to an explosive escalation of TB incidence that occurred in the 1980s due to the emergence of both resistant strains and HIV epidemic.In 2000, EthR, a mycobacterial transcriptional repressor, was identified as a key modulator of ethionamide (ETH) bioactivation. ETH is one of the main second-line drugs used to treat drug resistant strains. In 2009, it was shown that co-administration of ETH and drug-like inhibitors of EthR was able to boost ETH activity threefold in a mouse-model of TB-infection, thus validating the target for a new therapeutic strategy.This work deals with the discovery and optimisation of new EthR inhibitors, based on a small molecule, called a “fragment”, co-crystallized with the protein. We combined in silico screening, in vitro evaluation of the hit compounds, study of co-crystal structures and medicinal chemistry to develop three complementary approaches called “fragment growing”, “fragment merging” and “fragment linking” that led to the discovery of very potent inhibitors. Based on these results, we are currently selecting a potential candidate for new in vivo experiments.

Base teórica: A introdução de agentes biológicos, especialmente os bloqueadores do fator de necrose tumoral (anti-TNF), para o tratamento de doenças reumáticas aumentou o risco de desenvolver tuberculose (TB). O rastreio para infecção tuberculosa latente (ILTB) é fortemente recomendado antes de iniciar a terapia com agentes anti-TNF. Os objetivos deste estudo foram identificar a prevalência de ILTB e TB entre pacientes com doenças reumáticas em uso dos medicamentos anti-TNF. Métodos: Estudo transversal. Foram revisados os registros médicos eletrónicos de todos os doentes adultos (≥ 18 anos) em uso da terapia anti-TNF. Todos os pacientes foram submetidos ao teste tuberculínico (TT) antes de iniciar o tratamento com os medicamentos anti-TNF. Resultados: No total, 176 pacientes foram incluídos no estudo. A idade média de todos os pacientes foi de 51,9 ± 12,4 anos, 34,7% eram do sexo masculino e 90,9% eram brancos. As doenças subjacentes mais comuns foram: Artite Reumatóide (AR) em 89 pacientes (50,6%), Espondilite Anquilosante (EA) em 49 (27,8%) e Artrite Psoriática (AP) em 31 (17,6%). A prevalência de TT positivo foi de 29,5%. O contato domiciliar com TB foi significativamente associado com TT positivo (p = 0,020). Os pacientes com AR apresentaram reações TT menores do que os pacientes com EA (p = 0,022). Houve seis casos de TB (3,4%) diagnosticados durante a terapia anti-TNF. Conclusões: Demonstrou-se alta prevalência de TT positivo (29,5%) em pacientes com doenças reumáticas em uma região com alta prevalência de TB. Nossos dados corroboram a recomendação do Colégio Americano de Reumatologia (ACR) de que os pacientes que vivem em configurações de alta incidência de TB devem ser testados anualmente para ILTB. / Background: The introduction of biological agents, especially the blockers of tumor necrosis factor (anti-TNF), for the treatment of rheumatic diseases increased the risk of developing tuberculosis (TB). Screening for latent TB infection (LTBI|) is strongly recommended before starting therapy with anti-TNF agents. The objectives of this study were to identify the prevalence of LTBI and TB among patients with rheumatic diseases on anti-TNF drugs. Methods: Cross-sectional study. The electronic medical records of all adult patients (≥ 18 years old) undergoing anti-TNF treatment were reviewed. Every patient underwent TST test before starting anti-TNF treatment. Results: In total, 176 patients were included in the study. The mean age of all patients was 51.9 ± 12.4 years, 34.7% were males, and 90.9% were white. The most common underlying diseases were: RA in 89 patients (50.6%), AS in 49 (27.8%), and PA in 31 (17.6%). The prevalence of positive TST was 29.5%. Household contact with TB was significantly associated with a positive TST (p=0.020). RA patients had lower TST reactions than AS patients (p=0.022). There were six cases of TB (3.4%) diagnosed during anti-TNF therapy. Conclusions: We demonstrated a high prevalence of positive TST (29.5%) among patients with rheumatic diseases in a region with high TB prevalence. Our data corroborates the ACR’s recommendation that patients who live in high TB incidence settings should be tested annually for LTBI.

Made available in DSpace on 2016-12-23T13:55:58Z (GMT). No. of bitstreams: 1 Solange Alves Vinhas.pdf: 4494197 bytes, checksum: c05b3a06ae983246a835977f36eb32e3 (MD5) Previous issue date: 2013-08-30 / Background: Based on the hypothesis that genetic variability of Mycobacterium tuberculosis (MTB) could influence virulence and immunopathology we analyzed genetic profiles of different MTB strains in order to detect relatedness between genetic diversity and presence of cavity (disease severity). Methods: We conducted a retrospective molecular study in Vitória ES, based on TB strains (2003 to 2006, n = 214) from patients with pulmonary cavitary and non-cavitary TB using IS6110-RFLP, Spoligotyping and MIRU-VNTR methodologies. RESULTS: Initially, we compared the association of the demographic and clinical characteristics of patients with the presence of cavities. After logistic regression the variables that most contributed to explain the model of the disease were smear positive (ORajust = 5.96; IC= 2.58-13.73) and sputum production (ORajust = 4.55; IC= 1.28-16.12), there was no statistically significant association with the remaining variables. The LAM family was the most frequent within the samples of the two groups analyzed, representing 65 (62%) of the isolates in the cavitary group and 40 isolates (38%) of the non-cavitary. After comparing the proportions of LAM and other spoligotyping families there was no statistically significant difference between the groups (p=0.17). In relation to deletions RDRio (p=0.65) and RD174 (p=0.65) there were no statistically significant difference between the groups. Amongst the 205 isolates analyzed, 25 (12%) belonging to the non-cavitary group and 43 (21%) belonging to the cavitary group, were grouped in clusters. The statistical analysis of the association of the occurence of clusters with the presence of cavity showed no statistically significant difference between the quantity of clusters and the groups that were analyzed, (p= 0.4). Conclusion: The genotipic profile for the isolates from patients with cavitary and non-cavitary disease was determined. Our data showed that LAM9 was the most frequent among the strains between cavitary and noncavitary groups, corroborating findings that this family is the most frequent in Brasil. There were no statistical differences that could show association among the variables analyzed related to presence of cavity or disease severity / Introdução: Baseado na hipótese de que a variabilidade genética de Mycobacterium tuberculosis (MTB) pode influenciar a virulência e a gravidade da doença os perfis genéticos de isolados clínicos de MTB foram avaliados para detectar associação entre diversidade genética e gravidade da doença. Objetivos: Analisar características genéticas de isolados de MTB e verificar sua possível associação com a gravidade da TB pulmonar. Métodos: Estudo retrospectivo, caso controle, conduzido em Vitória-ES, utilizando isolados de MTB (2003 a 2006, n=214) de pacientes com TB pulmonar, cavitária (127) e não cavitária (87). Realizou-se genotipagem por meio de RFLP-IS6110, Spoligotyping, MIRU-VNTR 24 loci, e a análise de deleções e inserções, como RDRio, RD174 utilizando PCR multiplex, bem como a detecção do Ag85C103. Realizou-se análise estatística, para verificação dos padrões de distribuição das variáveis, seguida de análises bivariadas para verificação de associações entre elas, empregando-se os teste exato de Fisher ou Chi-quadrado, ambos com 95% de intervalo de confiança e nível de significância (&#61554;) < 0,05. Resultados: Após a regressão logística, as variáveis que contribuíram no modelo explicativo da doença foram baciloscopia (ORajust = 5,96; IC= 2,58-13,73) e produção de escarro (ORajust = 4,55; IC= 1,28- 16,12). Não houve associação estatisticamente significativa com o restante das variáveis.A família LAM foi a mais frequente entre os dois grupos analisados, representando 65 (62%) dos isolados no grupo cavitário e 40 isolados (38%) do grupo não cavitário. Não houve diferença estatisticamente significativa entre os grupos em relação à deleção RDRio (p=0,65) e com relação à deleção RD174 (p=0,65). Dentre os 205 isolados analisados, 25 (12%) isolados do grupo não cavitário e 43 (21%) do grupo cavitário, estavam em cluster. não houve diferença estatisticamente significativa entre a quantidade de clusters e os grupos analisados (p= 0,4). Conclusões: Foi determinado o perfil genotípico dos isolados de pacientes com doença pulmonar, cavitária e não cavitária. Não houve associação entre a presença de cavidade e os genótipos encontrados. Não houve associação do genótipo com nenhum dos marcadores moleculares avaliados

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The Mycobacterium genus includes M. tuberculosis complex (CMTB) species and others called nontuberculous mycobacteria (MNT). The CMTB bacilli cause tuberculosis (TB), a bacterial infectious disease, which commonly affects the lungs. Since the MNT cause other mycobacterial infections. The correct diagnosis of diseases caused by mycobacteria is essential for determining treatment. The tuberculosis treatment regimen in Brazil is recommended by the National Tuberculosis Control/Ministry of Health (PNCT/MS) and was recently modified. The main changes proposed by the Technical Advisory Committee of PNCT/MS were: to introduce a fourth drug, ethambutol, during the attack and take the combination of drugs in tablet form, with fixed-dose combinations (FDC) 4 in 1, for intensive treatment phase, and 2 in 1 for the maintenance phase. Due to the high incidence of tuberculosis in Brazil and worldwide, the emergence of resistant strains and the deployment of new therapies by the Ministry of Health, this study aimed to evaluate the susceptibility to antituberculosis or individually associated clinical isolates of Mycobacterium tuberculosis from the University Hospital Mary. The antimicrobial susceptibility alone or associated (rifampicin, isoniazid, pyrazinamide and ethambutol) was evaluated using the microdilution method (MMC) and compared to the proportion method (MP), gold standard method for susceptibility to mycobacteria. The MMC has proven to be a rapid, easily performed and well correlated with the MP. We have found various clinical isolates of M. tuberculosis resistant to one, two or three drug when tested against four drugs alone. However, when they were tested against four drugs associated with the FDC no strain was considered resistant. This fact is against the concept of FDC which aims to unite the four anti-TB drugs to combat the resistance of the bacilli. / O gênero Mycobacterium é constituído por espécies do Complexo M. tuberculosis (CMTB) e outras denominadas de micobactérias não tuberculosas (MNT). Os bacilos do CMTB causam a tuberculose (TB), uma doença infecciosa bacteriana, a qual comumente afeta os pulmões. Já as MNT causam outras micobacterioses. O diagnostico correto das doenças causadas pelas micobactérias é essencial para a definição do tratamento. O esquema de tratamento para a tuberculose no Brasil é preconizado pelo Programa Nacional de Controle da Tuberculose/Ministério da Saúde (PNCT/MS) e foi recentemente modificado. As principais mudanças propostas pelo Comitê Técnico Assessor do PNCT/MS foram: introduzir um quarto fármaco, o etambutol, na fase de ataque e adotar a associação dos fármacos em forma de comprimidos, com doses fixas combinadas (DFC) 4 em 1, para a fase de tratamento intensivo, e, 2 em 1, para fase de manutenção. Devido à grande incidência da tuberculose no Brasil e no mundo, à emergência de cepas resistentes e à implantação de novas terapias pelo Ministério da Saúde, este trabalho objetivou avaliar a suscetibilidade aos tuberculostáticos associados ou individualmente dos isolados clínicos de Mycobacterium tuberculosis do Hospital Universitário de Santa Maria. A suscetibilidade aos antimicrobianos isolados ou associados (rifampicina, isoniazida, pirazinamida e etambutol) foi avaliada através do método de microdiluição em caldo (MMC) e comparados ao método das proporções (MP), método padrão ouro para suscetibilidade de micobactérias. O MMC mostrou-se ser um método rápido, de fácil realização e boa correlação com o MP. Foram encontrados vários isolados clínicos de M. tuberculosis resistentes a um, dois ou três fármacos quando testados frente aos quatro fármacos isoladamente. Porem, quando estes foram, testados contra os quatro fármacos da DFC associados nenhuma cepa foi considerada resistente. Este fato vem de encontro ao conceito da DFC a qual pretende unir os quatro medicamentos anti-TB para combater a resistência dos bacilos.

A tuberculose é uma doença crônica granulomatosa caracterizada por um déficit de imunidade antígeno específica do hospedeiro, cuja resposta imune é ativamente regulada por citocinas. No Brasil há mais de 50 milhões de habitantes infectados pelo Mycobacterium tuberculosis. O objetivo foi avaliar a linfoproliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) estimuladas por quatro diferentes antígenos do M. tuberculosis, um complexo, o antígeno sonicado, e três purificados, ESAT-6, antígeno 85B e antígeno HBHA, eventuais candidatos à vacina anti-tuberculose. Para avaliação da produção de IFN-g e IL-10 foram utilizados dois métodos: Elispot e Elisa à partir de sobrenadante de cultura de PBMC. Para essas avaliações, os pacientes com tuberculose ativa (TB-A) foram comparados a dois subgrupos de indivíduos controles. O primeiro subgrupo foi constituído por indivíduos saudáveis PPD+ e o segundo por indivíduos curados de um episódio de tuberculose (TB-C). Nossos resultados de linfoproliferação e de Elisa revelaram diminuição da resposta linfoproliferativa e da produção de IFN-g dos pacientes em comparação com os indivíduos PPD+, enquanto os indivíduos TB-C apresentaram em geral resultados intermediários. Observou-se também que as respostas à PHA não diferiam significativamente entre os grupos, ressaltando a natureza antígeno específica da hiporreatividade na tuberculose. Adicionalmente, verificamos maior reatividade ao antígeno complexo, sonicado, que aos antígenos purificados, e entre estes, a reatividade foi maior para ESAT-6 e 85B que para HBHA, A resposta ao HBHA pode ter sido eventualmente subestimada por razões técnicas, como utilização de dose sub-ótima ou perda da atividade biológica. Em relação ao Elispot para IFN-g, não pudemos observar diferenças entre os grupos, tanto quando se considerou o número total de spots, como quando se contou apenas spots com diâmetro > 65 mm, apresentando portanto uma sensibilidade aparentemente menor comparado aos outros 2 métodos. A comparação entre os métodos revelou pouca correlação entre seus resultados, que pode ser eventualmente explicado pela diferente contribuição das populações celulares (T CD4+ e T CD8+) para cada uma das provas munológicas. Finalmente, a análise da produção de IL-10 medida por Elisa no sobrenadante de cultura e por spots de IL10, também não revelou diferenças entre os grupos. Convém notar que o Elisa detectou baixas concentrações de IL-10 nos sobrenadantes, porém o Elispot demonstrou número elevado de spots e boa correlação entre as resposta aos antígenos. Em conclusão, nossos resultados sugerem que métodos \'clássicos\', e já estabelecidos, como linfoproliferação e Elisa, persistem válidos para se avaliar a imunidade celular, e que em nossas condições laboratoriais, a técnica de Elispot não representou, até o momento, uma melhora na qualidade da avaliação imunológica. / Tuberculosis is a chronic granulomatous disease characterized by a deficit of the antigen-specific immunity of the host, whose immune response is actively regulated by cytokines. In Brazil there are 50 million people infected with Mycobacterium tuberculosis. The objective of the present work was to evaluate the lymphoproliferative response e the IFN-g response by peripheral blood mononuclear cells (PBMC) indiced with 4 different antigens isolated from Mycobacterium tuberculosis: a complex, crude, the sonicate antigen, and 3 other, purified ones, Esat-6, 85B, and HBHA, the last 3 eventual candidates to the design of a vaccine against tuberculosis. We used 2 methods to evaluate the IFN-g and IL-10 productions, namely Elispot and Elisa of supernatant of PBMC cultures. We studied a group of active tuberculosis patients (TB-A), and compared them with controls individuals comprising 2 groups, one made of healthy PPD+ individuals and the second one of individuals who have been cured from an episode of tuberculosis in the past (TB-C). Our results of lymphoproliferation and Elisa revealed decrease in the lymphoproliferative and IFN-g responses by patients\' PBMC as compared to the PPD+ group, with the TB-C group in general presenting intermediate results. We also observed that the responses to the mitogen PHA were not statisically different among the groups, denoting the antigen-specific nature of the immune deficit in tuberculosis. In addition, we verified that stronger reactivity to the complex antigen than with the purified antigens, and, among the latter, the reactivity was stronger with Esat-6 and 85B as compared to HBHA, Reactivity to HBHA may have been understimated due to technical reasons, such as loss of .the biological activity of the molecule or use of a sub-optimal dose. By using the Elispot for IFN-g we were not able to detect differences among the groups, even when we counted all spots formed or spots with more than > 65 mm in diameter. Thus our Elispot for IFN-g apparently showed lower sensitivity than the other 2 methods. Furthermore, comparisons between the methods revealed low correlation between their results, a finding that may be explained by the differning contribution of different subpopulations (T CD4+ and T CD8+) to each of the results. Finally, analysis of the production of IL-10 as measured by Elisa in the culture supernatants as well as by Elispot revealed no differences among the groups. It is noteworthy that the levels of IL-10 detected by Elisa were low, but the Elispot revealed high number of spots and a good correlation between the antigen responses. In conclusion, we may say that our well standardized \'classical\' methods Elisa and lymphoproliferation persist useful to evaluate cellular immunity responses, and that the Elispot technique, up to now and in our laboratorial conditions, did not represent an improvement in the quality of the immunological evaluation.