Thesis (MSc MedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Background The QuantiFERON In-Tube (QFT IT) assay is an Interferon-gamma release assay (IGRA) which is currently used to detect Mycobacterium tuberculosis (M. tb) infection. It however cannot differentiate between latent infection and active tuberculosis (TB) disease. In an attempt to improve this tool to accurately diagnose active TB, the release of a variety of markers should be assessed in combination with Interferon gamma (IFN- γ). Luminex analysis was previously done on QFT plasma and promising candidates were identified which could be of great value in treatment response studies. IFN-γ ELISpot, are not only used to detect M.tb infection, but is also implicated in vaccine trails to assess immunogenicity. The IFN-γ ELISpot and flow cytometry are the most common assays to assess these phenomena during clinical trials. Our aim therefore was to develop a multi platform immune analysis assay using the QFT IT system. Study design and method The first approach of this study was to optimize the QFT IT assay for flow cytometry applications. The following questions formed part of the optimization study: How does the QFT whole blood assay (QFT-WBA) compare to the currently used WBA? Is antigen re-stimulation required after the initial incubation time and for how long should cells be re-stimulated in the presence of Brefeldin A? The second approach was to use the optimized QFT-WBA for community controls (CTRL), household contacts (HHC) and TB cases, which were recruited from the high TB incidence areas Ravensmead, Uitsig and Elsies River. The infection status of each participant was determined by IFN-γ ELISA and Luminex analysis was performed to measured wide range of cytokine expression. In addition immune cell markers like CD14, CD4, CD8, CD19, and T cell receptor gamma delta (TCRγδ) were characterized; polyfunctional characteristics (IFN-γ, Tumor necrosis factor-alpha (TNF-α) and Interleukin-2 (IL-2)) and proliferation (Ki-67+) of T cells determined by flow cytometry. Results After stimulating the whole blood of the study participants for 22 hours with the M. tb specific antigens, early secreted antigenic target 6 kDa (ESAT-6), culture filtrate protein-10 kDa (CFP-10) and TB7.7 the levels of TNF-α producing CD4 T cells were elevated in TB cases compared to HHCs. After stimulating the whole blood for 6 days TNF-α producing T cells declined in TB cases and HHC showed a higher expression. CD40L+CD4+ (p=0.0225) was increased in HHC while IL-9+CD8+ (0.3230) was decreased in HHC compared to TB cases. Other markers such as IL-5(AG-NIL), IL-13(Ag- NIL), FGF basicAg, GM-CSFNIL, VEGFNIL/(Ag-NIL), MIP-1βAg and MCP-1Ag/(Ag-NIL) showed significant differences between HHC and TB cases. Conclusions The responses in the QFT-based assay were generally comparable to the WBA that is routinely used. The differences of TNF-α expression seen in QFT-WBA and QFTLPA could be explained by the fact that effector T cell responses were measured in the short term assay and the central memory T cell responses in the long term assay. Our study therefore shows that the QFT-based tests can be used to simultaneously assess a wide range of immunological markers and not only IFN-γ expression. / AFRIKAANSE OPSOMMING: Agtergrond Die QuantiFERON In Tube (QFT IT) toets is ‘n Interferon-gamma vrystellingstoets (IGRA) wat huidiglik dien as ‘n maatstaf van Mycobacterium tuberculosis (M. tb) infeksie. Hierdie toets kan egter nie onderskei tussen latente infeksie en aktiewe tuberkulose (TB) nie. ‘n Noemenswaardige verbetering in die vermoë van hierdie toets om aktiewe TB te diagnoseer, berus op die studie van ‘n verskeidenheid vrygestelde merkers, insluitend Interferon gamma (IFN-γ). In vorige Luminex studies op QFT plasma, is belowende kandidate geïdentifiseer wat van groot waarde kan wees vir studies wat fokus op die reaksie tot behandeling. Die IFN-γ ELISpot dien nie net as ‘n maatstaf van M.tb infeksie nie, maar word ook in vaksienproewe betrek om die aard van immuniteit te ondersoek. Die IFN-γ ELISpot toets sowel as vloeisitometriese toetse, is van die mees algemene toetse om hierdie verskynsels te meet, tydens kliniese proewe. Die doel van hierdie studie was dus om die QFT IT sisteem te ontwikkel as ‘n basis vir ‘n multiplatform immunologiese analiseringstoets. Studie ontwerp en metode Die inleidende benadering van hierdie studie was die optimisering van die QFT IT toets, vir vloeisitometrie doeleindes. Die volgende vrae het deel uitgemaak van die optimiseringstudie: Hoe vergelyk die QFT heelbloedtoets (QFT-WBA) met huidige WBAs wat in gebruik is? Word meermalige antigeenstimulasies benodig na die oorspronklike inkubasieperiode en hoe lank moet die tydperk wees vir sellulêre opvolgstimulasie, in die teenwoordigheid van Brefeldin A? As ‘n tweede benadering, was om die geoptimiseerde QFT-WBA te gebruik vir gemeenskapskontroles (CTRL), huishoudelike kontakte (HHC) en TB gevalle. Al drie hierdie groepe was opgeneem uit Ravensmead, Uitsig en Elsies Rivier, areas met betreklik hoë vlakke van TB infeksie. Elke persoon in die studie se vlak van infeksie is vasgestel met behulp van die IFN-γ ELISA en Luminex analiese was uitgevoer, om ‘n wye verskeidenheid uitdrukkingsvlakke van sitokiene te meet. Dies meer, was immuunselmerkers soos CD14, CD4, CD8, CD19 en T sel reseptor gamma delta (TCRγδ) gekarakteriseer. Meervuldige funskionele karakteristieke (IFN-γ, Tumor nekrose faktor-alpha (TNF-α) en Interleukin-2 (IL-2)) en vermenigvuldiging van T-selle, was vasgestel deur middel van vloeisitometrie. Resultate Nadat die heelbloed van studiedeelnemers gestimuleers was met M. tb spesifieke antigene, vroeë afskeidings antigeniese teiken 6kDa (ESAT-6), kultuurfiltraatproteïn 10kDa (CFP-10) en TB7.7, vir 22 uur, was gevind dat vlakke van TNF-α produserende CD4 T selle hoër was in TB pasïente, in vergelyking met HHCs. Nadat die heelbloed vir 6 dae gestimuleer was, het die vlak van TNF-α produserende T-selle afgeneem in TB pasïente, terwyl dit hoër was in HCC. CD40L+CD4+ (p=0.0225) het hoër vlakke bereik in HHC, terwyl IL-9+CD8+ (0.3230) vlakke afgeneem het, in vergelyking met TB pasïente. Ander merkers soos,onder andere, IL-5(AG-NIL), IL-13(Ag-NIL), FGF basicAg, GMCSFNIL, VEGFNIL/(Ag-NIL), MIP-1βAg and MCP-1Ag/(Ag-NIL), het noemenswaardige verskille geopenbaar tussen HHC en TB pasïente.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Setting Study conducted in Tygerberg, Cape Town in South Africa. Hypothesis Host biomarkers associated with the antimycobacterial immune response during active infection with M. tuberculosis and during anti-tuberculosis chemotherapy are indicative of bacterial killing in the host and can be used in models to predict eventual treatment outcome. Objectives 1. To investigate immune parameters that were selected in a biological context as biomarkers of the extent of disease and early response to anti-tuberculosis treatment. 2. To use selected immune parameters to characterise fast and slow responders to anti-tuberculosis therapy. Findings Evaluation of cytokine multiplex fluorescent bead-based immunoassays as a screening tool in the search for biomarkers The data showed that cytokine multiplex fluorescent bead-based immunoassays achieved acceptable recoveries to detect antigen-specific IFN- responses in whole blood supernatant making it attractive for biomarker screening. However, proper optimisation needs to be done and proper controls included when using these kits. Markers of extent of disease High levels of CRP at diagnosis were found to be associated with the presence of multiple cavities on chest X-rays. A high level of suPAR and sICAM-1 at diagnosis were associated with the extent of alveolar disease. Also significant were the associations between the level of granzyme B, LAG-3 at diagnosis and the size of the cavities. No significant associations were observed between sTNFRs or DR5 with the chest X-ray grading of tuberculosis disease. Early classification of fast and slow responders to anti-tuberculosis treatment After cross-validation classification, discriminant analysis (DA) and support vector machine (SVM) analysis of selected immune parameters (sICAM-1 CRP, granzyme B, suPAR, sTNFRs, LAG-3 and CD3dim/CD56+ (% of CD45+) resulted in a 75% to 100% correct classification of the fast responders and a 82% to 100% correct classification of the slow responders when using DA. For SVM, the correct classification of the fast responders ranged from 88% to 100%, and that for the slow responders ranged from 95% to 100%. Differential gene expression in fast and slow responders to treatment Direct comparison of fast and slow responders showed that IL-4 transcripts were significantly higher in the fast responders at week one after initiation of treatment when compared to slow responders. IL-42 was also differentially expressed. Although IL- was significantly up-regulated in both fast and slow responders after one week of treatment compared to diagnosis, IL- expression was more than two folds higher in slow responders than in fast responders. No significant differences between the fast and slow responders were observed in the expression of TGF-, TGF-RII, Foxp3 and GATA-3. Conclusion Predictive models for differential anti-tuberculous treatment responses combining host proteins are promising and should be included in larger prospective studies to find the optimal markers for inclusion into clinical trials of new drugs and for implementation into clinical practice. / AFRIKAANSE OPSOMMING: Ligging Studie onderneem in Tygerberg, Kaapstad, Suid-Afrika. Hipotese Gasheerbiomerkers wat verband hou met die antimikobakteriële immuunrespons tydens aktiewe infeksie deur M. tuberculosis en tydens teentuberkulose chemoterapie dui op bakteriële doding in die gasheer en kan in modelle gebruik word om die uiteindelike uitkoms van die behandeling te voorspel. Doelwitte 1. Om gekose immuunparameters in ’n biologiese konteks as biomerkers van die omvang van siekte en vroeë reaksie op behandeling te ondersoek. 2. Om gekose immuunparameters te gebruik om vinnige en stadige reageerders op teentuberkulosebehandeling te karakteriseer. Bevindings Evaluering van die sitokien veelvuldige fluoresseer-pêrelbaseerde immuuntoets (cytokine multiplex fluorescent bead-based immunoassays) as ’n siftingsinstrument in die soeke na biomerkers Die data het getoon dat die sitokien veelvuldige fluoresseer-pêrelgebaseerde immuuntoets in staat was om antigeenspesifieke IFN--respons te meet wat dit aanloklik maak vir biomerkersifting. Sorgvuldige optimering moet egter gedoen word en behoorlike beheer moet ingesluit word wanneer hierdie stelle gebruik word. Merkers van omvang van siekte Hoë vlakke van CRP by diagnose is getoon om verband te hou met die teenwoordigheid van veelvoudige holtes op die pasiënte se borskas x-strale. Hoë vlakke van suPAR en sICAM-1 by diagnose was assosieer met die omvang van alveolêre siekte. Die assosiasie tussen die vlakke van granzyme B, LAG-3 by diagnose en die grootte van die holtes was ook betekenisvol. Daar was geen betekenisvolle assosiasies toe sTNFRs of DR5 en die borskas x-straalgradering van tuberkulosesiekte nie. Vroeë klassifikasie van vinnige en stadige reageerders op teentuberkulosebehandeling Ná klassifikasie op grond van kruisstawing het diskriminant-analise (DA) en ondersteuningsvektormasjiene (SVM) van geselekteerde immuunparameters (sICAM-1 CRP, gransiem B, suPAR, sTNFRs, LAG-3 en CD3dim/CD56+ (% van CD45+)) gelei tot ’n 75% tot 100% korrekte klassifikasie van die vinnige reageerders met DA en ’n 82% tot 100% korrekte klassifikasie van stadige reageerders. Vir SVM het die korrekte klassifikasie van vinnige reageerders gewissel van 88% tot 100%, en vir stadige reageerders het dit gewissel van 95% tot 100%. Differensiële geenuitdrukking in vinnige en stadige reageerders op behandeling In vergelyking met die vlak by diagnose is die uitdrukkingsvlak van IL-4 in die vinnige reageerders betekenisvol opgereguleer met ’n faktor van 9.2 teen die eerste week ná die aanvang van behandeling, in kontras met die stadige reageerders. Daar was geen verskille tussen die vinnige en die stadige reageerders met betrekking tot die uitdrukking van TGF-, TGF-RII, Foxp3 en GATA-3 nie. Gevolgtrekking Voorspellende modelle vir differensiële tuberkulose behandelingsresponse wat gasheerproteïene kombineer, hou belofte in en behoort in groter prospektiewe studies ingesluit te word om die mees geskikte merkers te vind vir insluiting in kliniese proewe van nuwe middels en vir implementasie in kliniese praktyk.

Le séquençage intégral du génome de Mycobacterium tuberculosis a permis de mettre en évidence l’existence de onze Sérine/Thréonine Protéine-Kinases (STPKs) chez cette bactérie. Bien que la quasi-totalité des STPKs aient été biochimiquement caractérisées, très peu de substrats endogènes ont pu être identifiés. Par conséquent, le rôle physiologique de ces couples kinase/substrat reste à élucider. Tout d’abord, les études réalisées au cours de ce travail ont concerné la caractérisation biochimique de la protéine-kinase PknL, ainsi que l’identification de ses substrats potentiels, et notamment la protéine Rv2175c. En effet, l’analyse de l’environnement génétique du gène pknL de la kinase a révélé la présence du gène adjacent rv2175c, pouvant ainsi représenter un substrat éventuel de PknL. Les différentes approches mises en oeuvre ont permis d’identifier cinq sites de phosphorylation sur PknL, et de mettre en évidence le caractère essentiel des résidus K48, T173 et T175 dans les mécanismes d’autophosphorylation de PknL et de phosphorylation de Rv2175c, confirmant ainsi Rv2175c comme substrat spécifique de PknL. Par ailleurs, la caractérisation par RMN de la structure de Rv2175c a permis de déterminer la fonction de cette protéine. Rv2175c possède toutes les caractéristiques structurales d’une protéine capable de fixer l’ADN. Des études fonctionnelles ont permis de confirmer la capacité de Rv2175c de fixer l'ADN et ont mis en évidence le mécanisme de régulation via phosphorylation régissant son activité de fixation. Ensuite, nous avons mis en évidence la phosphorylation des protéines chaperonnes mycobactériennes et, plus particulièrement, caractérisé GroEL1. Nous avons démontré que GroEL1 était phosphorylée par PknF, et identifié les résidus T25 et T54 comme étant les sites de phosphorylation de GroEL1. L’ensemble de cette étude nous a donc permis de caractériser de nouveaux substrats de phosphorylation chez M. tuberculosis, de mieux appréhender les interactions kinase/substrat et d’impliquer la phosphorylation dans la régulation de l’activité de ces substrats / Analysis of the genome sequence of Mycobacterium tuberculosis predicted the presence of eleven Serine / Threonine Protein-Kinases (STPKs). Although most kinases have been investigated for their physiological roles, little information is available regarding how STPK-dependent phosphorylation regulates the activity of kinase substrates. As a result, the physiological role of these kinase / substrate couples remains to be clarified. During the course of this work, we first characterized a substrate/kinase pair, PknL/Rv2175c. Moreover, pknL (rv2176) is adjacent to rv2175c, a gene encoding a putative DNA-binding transcriptional regulator. We demonstrated that PknL can recruit and phosphorylate Rv2175c and that phosphorylation of Rv2175c was dependent on a specific phosphorylated residue located within the activation loop of PknL. However, although Rv2175c harbours a DNAbinding domain carrying a helix-turn-helix (HTH) motif, it shares only weak similarity to transcriptional regulatory proteins. Therefore, to provide further evidence for the function of Rv2175c, we have solved the soluble NMR structure of Rv2175c. In addition, we confirmed by gel shift mobility assays that Rv2175c was indeed able to bind DNA. More importantly, we identified Thr9 as the unique phosphorylation site in Rv2175c, and demonstrated that phosphorylation of Rv2175c strongly altered its DNA-binding activity. In addition, although mycobacterial GroEL1 proteins have been extensively studied, no data were available with respect to their potential post-translational modifications. We reported here, for the first time, phosphorylation of the M. tuberculosis GroEL1 chaperone. We demonstrated that M. tb GroEL1 is phosphorylated by PknF at two positions, Thr25 and Thr54. Unexpectedly, Mycobacterium smegmatis GroEL1 is not a substrate of its cognate PknF. This study showed that the phosphorylation profile of conserved proteins is species dependent and provides insights that may explain the numerous biological functions of these important proteins

Le génome de Mycobacterium tuberculosis, l’agent causal de la tuberculose, code pour une protéine ayant la capacité de se fixer sur la cellulose (Rv1987), une cellulase potentielle (Rv1090), et une cellulase pleinement active (Rv0062). Cette observation est surprenante, car la cellulose est un composant majeur des parois des cellules végétales, tandis que M. tuberculosis est un pathogène humain sans contact connu avec des plantes. Nous avons émis l’hypothèse que ces protéines pourraient jouer un rôle dans les interactions entre les mycobactéries du complexe M. tuberculosis avec les kystes d’amibes libres, dont la paroi contient également de la cellulose. Dans notre travail de thèse, nous avons cherché par une analyse in silico la présence de ces trois gènes chez toutes les bactéries ayant un génome complètement séquencé présentes dans la base de données CAZy (accessible en ligne à l’adresse www.cazy.org). Cette étude a montré que seulement 2,5% des bactéries codent pour les trois gènes simultanément. Parmi ces bacteries, nous avons ensuite confirmé expérimentalement par PCR et séquençage la présence des gènes Rv0062, Rv1090 et Rv1987 chez les mycobactéries du complexe M. tuberculosis. Nous avons ensuite vérifié la transcription de ces trois gènes chez la souche de référence M. tuberculosis H37Rv, puis produit dans Escherichia coli des protéines de fusion Rv1090 et Rv1987 et montré qu'elles étaient capables d'hydrolyser la cellulose (Rv1090) et de s’y fixer (Rv1987). De plus, nous avons mis en place un model expérimental d’interaction entre les mycobactéries du complexe M. tuberculosis et les amibes libres dans le but de comprendre le rôle des gènes Rv0062, Rv1090 et Rv1987. Dans un premier temps nous avons montré que M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii ainsi que Mycobacterium avium utilisé ici comme un controle positif étaient capables de survivre dans le cytoplasme des amibes libres telles que Acanthamoeba polyphaga. Ensuite, nous avons montré que M. tuberculosis et M. bovis mais pas M. canettii étaient capables de survivre à l’intérieur des kystes d’amibes. Enfin nous avons montré que M. tuberculosis, M. bovis et M. canettii étaient capables de survivre dans le sol pendant au moins 6 mois. Les données établies dans cette thèse soutiennent le rôle des cellulases dans la survie environnementale des mycobactéries du complexe M. tuberculosis, et ouvrent la voie à l’étude de cette phase méconnue dans le cycle de ces organismes / The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes a protein with the ability to bind to cellulose (Rv1987), one potential cellulase (Rv1090), and one fully active cellulase (Rv0062). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. We hypothesized that these genes could play a role in the interactions between M. tuberculosis complex organisms and amoebal cysts, whose wall contains cellulose.In our thesis work, we have searched by in silico analysis for the presence of these three genes in all bacteria with complete sequenced genomes present in the CAZy database (available online at www.cazy. org). This study showed that only 2.5% of bacteria encode the three genes simultaneously. Among these bacteria we have confirmed experimentally by PCR and sequencing the presence of Rv0062, Rv1090 and Rv1987 in the M. tuberculosis complex organisms. We have checked the transcript of the three genes in the reference strain M. tuberculosis H37Rv and we subsequently produced Rv1090 and Rv1987 fusion proteins in Escherichia coli and demonstrated that they were indeed able to hydrolyze (Rv1090) and to bind (Rv1987) cellulose. In addition, we have developed an experimental model of interaction between M. tuberculosis organisms and the free-living amoebae in order to understand the role of Rv0062, Rv1090 and Rv1987 genes. Initially we have shown that M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii and Mycobacterium avium used here as a positive control were able to survive in the cytoplasm of the free-living amoeba such as Acanthamoeba polyphaga. We have further shown that M. tuberculosis and M. bovis but not M. canettii were able to survive within the amoebal cysts. Finally we have shown that M. tuberculosis, M. bovis and M. canettii were able to survive in soil for at least 6 months. The data obtained in this thesis support the role of cellulase in the survival of M. tuberculosis complex organisms in the environment and pave the way for the study of this unknown phase in the cycle of these organisms.

Molecular typing is increasingly essential to tuberculosis (TB) control programmes, providing public health practitioners with a tool to characterize transmission patterns, track the emergence and spread of strains of M. tuberculosis complex (MTC) in populations. While molecular typing is already used extensively as a tool for TB control in many developed settings across the globe, its use in resource-poor settings is still limited. Moreover, information on the role, contribution and burden of nontuberculous mycobacteria (NTM) and other pathogens in aetiology of TB-like syndromes is also lacking in such settings. The broad objective of this dissertation was to determine the genetic diversity of MTC and their drug resistance profiles as well as the prevalence of NTM and other potentially pathogenic bacteria among TB suspects in Northeastern, Tanzania in order to generate insights that may inform the design of a rational TB control programmes. A total of 18 distinct spoligotypes were identified in this study area, with CAS1-KILI and EAI8 being the most predominant families. Major lineages prediction by conformal Bayesian network (CBN) revealed that 70% of TB infections in this area is due to modern lineages, whereas 30% of TB infections is due to the ancestral lineages mainly of Indo-oceanic lineage. The study also revealed that the overall proportions of any drug resistance and MDR-TB were 12.7% and 6.3% respectively. With the prevalence of any drug resistance and MDR-TB among new cases being 11.4% and 4.3% respectively, among previously, treated cases were 22.2%. The prevalence of NTM was found to be 9.7 %, with HIV being a significant predictor of NTM detection (P < 0.001). Four out of 30 patients with NTM diagnosed by culture received 1st line anti-TB treatment suggesting that a proportion of patients diagnosed by smear microscopy (4/65, 6.2%) were mistreated as TB patients. Our findings further showed that 17 (4.6%) out of 372 TB suspects were due to pulmonary nocardiosis. Overall this dissertation has revealed that TB is still a major problem in Tanga and is characterized by a diverse array of MTB strains. Additionally, modern MTB strains contribute significantly to TB infections in this area. High proportions of anti-TB drug resistance among new treated cases observed suggest that more efforts need to be done to identify individual cases at facility level for improved TB control programmes. Inefficient screening of TB patients and a prevalent increase of NTM may contribute to both unrealistic and mismanagement of TB cases. A diverse array of pathogenic Nocardia species among TB suspects further indicates that they are likely cause of human disease in this population. Therefore, need to integrate NTM and pathogens causing TB-like syndromes in diagnosis and management of TB is urgent. Results of these investigations contribute to the understanding of the dynamics of TB transmission in resource poor settings of Tanzania and highlight key factors that should be considered in the development of rational approaches to design effective TB prevention and control programmes in the country.

Apesar das recomendações da Organização Mundial da Saúde, na reunião ministerial realizada em Amsterdam, Holanda em 2000, a tuberculose continua em ritmo crescente, atingindo principalmente os países em desenvolvimento e pessoas com o sistema imunológico comprometido, principalmente as infectadas pelo vírus da imunodeficiência adquirida. Os métodos utilizados no diagnóstico continuam os mesmos utilizados por muitos anos, cujas limitações impedem a ação rápida dos programas de saúde que buscam interromper a cadeia de transmissão da doença. Continuando uma linha de pesquisa do Laboratório de Soroepidemiologia e Imunobiologia do IMTSP, utilizando o método do Western Blotting, procuramos neste trabalho ampliar os conhecimentos da resposta imunológica de anticorpos em pacientes com tuberculose pulmonar, clinica e laboratorialmente definida, avaliando a participação das imunoglobulinas IgG, IgA e IgM, subclasses de IgG (IgG1 e IgG3) e a avidez da imunoglobulina IgG em amostras de soros colhidas no início e no final do tratamento. Nossos resultados mostraram que o melhor marcador imunológico foi a imunoglobulina IgG por apresentar melhor desempenho diagnóstico quando comparada com os resultados dos métodos microbiológicos e de dosagem de interferon gama, Quantiferon TB Gold. As frações protéicas que apresentaram melhor desempenho diagnóstico foram as de 38 e 30 KDa / Despite the recommendations of the World Health Organization, during the Ministerial meeting held in Amsterdam, Holland in 2000, tuberculosis continues at an important increasing rate, affecting mostly developing countries and people with severe compromised immune systems, especially those infected with human acquired immunodeficiency virus. The methods used for diagnosis are the same used for many years, whose limitations prevent the rapid action of countries health programs that seek to stop the chain of disease transmission. Continuing a line of research of the Laboratory of Seroepidemiology and Immunobiology of IMTSP using the method of Western blotting, in this study we tried to broaden the knowledge of the immune response of antibodies in patients with pulmonary tuberculosis, clinical and laboratory defined by evaluating the participation of IgG, IgA and IgM, IgG subclasses (IgG1 and IgG3) and immunoglobulin IgG avidity in serum samples collected in the beginning and end of treatment. Our results showed that the immunoglobulin IgG was best immunological marker when compared with the results of microbiological methods and determination of interferon gamma, QuantiFERON TB Gold. The proteins fractions that showed better diagnosis performance were 38 and 30 KDa

A manutenção da infecção latente pelo M. tuberculosis (TBIL) pode ser atribuída à sua capacidade de sobreviver durante anos no organismo humano em um estado não replicativo (dormente). A proteína HspX do M. tuberculosis, induzida sob condições de hipóxia, está fortemente associada com a manutenção da viabilidade do bacilo na TBIL. O presente estudo tem como objetivo, verificar se a superexpressão da proteína HspX altera a expressão de genes envolvidos com a síntese de componentes da parede celular, replicação do DNA e divisão celular de bacilos, assim como, na expressão de genes envolvidos com a resposta imune inata em macrófagos infectados com esses bacilos. O gene hspX foi amplificado pela PCR a partir do DNA do M. tuberculosis H37Rv, clonado no vetor de expressão pFPCA1GFP, e a proteína HspX expressa em M. smegmatis mc2155. As bactérias, nas quais, a presença da proteína recombinante foi confirmada por Western Blot, foram utilizadas, para a análise de expressão gênica tanto em bactérias quanto em macrófagos infectados. O estudo de expressão gênica foi realizado utilizando a RT-qPCR. Quando comparado aos controles, as bactérias que expressavam a proteína HspX apresentaram uma redução na expressão de genes de replicação do DNA e divisão celular, que foi acompanhado por uma tendência a filamentação das células e uma redução no tamanho das colônias. Além disso, nos macrófagos infectados com a bactéria expressando a proteína HspX, houve um aumento tanto da expressão do mRNA quanto da secreção de IL-1b, citocina importante para estabilização do granuloma, e uma redução na expressão de IRGM, gene relacionado com o processo autofágico, importante mecanismo de defesa do hospedeiro contra bactérias intracelulares. Portanto, em conjunto, essas alterações de expressão gênica, em consequência da presença da proteína HspX sugerem uma contribuição, direta ou indireta, dessa proteína para a adaptação morfológica e metabólica da bactéria dormente durante a TBIL, e consequentemente, para a resposta imune inata dos macrófagos infectados favorecendo a viabilidade intracelular dessas bactérias. / The maintenance of Mycobacterium tuberculosis infection latent (TBIL) may be attributed to its ability to persist for years in the host in a non-replicative state (dormant). The HspX protein from M. tuberculosis, induced under hypoxic, is strongly associated with maintaining the bacillus viability in TBIL. This study aims to determine if HspX overexpression chances the expression of genes involved in the synthesis of cell wall components, DNA replication and cell division of bacilli, as well as, the expression of genes involved in innate immune response of macrophages infected. The gene hspX was amplified by PCR from DNA of M. tuberculosis H37Rv, and cloned into the expression vector pFPCA1GFP. The HspX was expressed in M. smegmatis mc2155 and the recombinant protein was confirmed by Western blot. The bacterias expressing HspX were used for gene expression analysis both in bacteria and in infected macrophages by RT-PCRq. In bacterias expressing HspX, it was observed a reduction in expression of genes involved in DNA replication and cell division, and with cells more filamentous and smaller colonies, compared with controls. In addition, in macrophages infected with bacillus expressing HspX, there was an increase in both mRNA expression and secretion of IL-1b, an important cytokine for granuloma stability, and a reduction in expression of IRGM, an autophagic gene, important for host defense mechanism against intracellular bacteria. Together, these results suggest a direct or indirect contribution of HspX protein for metabolic and morphological adaptation of dormant bacteria in TBIL, and for the innate immune response in infected macrophages, improving the bacteria intracellular viability.

Avaliou-se a aplicação rotineira de um método automatizado TSMA em comparação ao convencional TSMP na determinação da sensibilidade a drogas anti-micobacterianas de 126 isolados clínicos de bactérias do complexo M. tuberculosis. Os isolados clínicos foram divididos em: sem tratamento (NT), tratado anteriormente (RT) e multirresistentes (MDR). A concordância entre TSMP e TSMA, para Rifampicina no NT, foi de 98.3%, p=1.000 e Kappa=0.659. No RT, foi de 94.3%,p=0.625 e Kappa=0.639, no MR, foi de 96.6%, p=0.625 e Kappa=0.651. Para Hidrazida, no NT, foi de 88.5%, p=0.125 e Kappa=0.408 no RT foram de 88.6%, p=0.625 e Kappa=0.645 no de pacientes MR, foi de 86,7%, p=0.625 e Kappa=0.053. Para Estreptomicina no NT, foi de 93.5%, p=0.125 e Kappa=0.635. No RT, foi de 79.4%, p=0.016 e KAPPA=0.296 no MR, foi de 76,6%, p=0.453 e Kappa=0.533. Para Etambutol, no NT, foi de 93.4%, p=0.125 e Kappa=0.315, no RT foi de 94.3%, p=0.500 e Kappa=0.478. No MR, foi de 72.4%, p=0.70ª e Kappa=0.455. A metodologia automatizada mostrou-se mais rápida. / We evaluated the routinely application of an automated system(STAM) and the conventional method (STPM), used to determine the profile of sensitivity to antimycobacterial drugs of 126 clinical isolates of the complex M. tuberculosis. Clinical isolates was group divided into: without treatment (WT), previously treated (PT) and multidrug resistant (MDR).Concordant result between STPM and STAM, for Rifampin, in WT, was 98.3%, p=1.000 and Kappa=0.659. PT, was 94.3%, p=0.625 and Kappa=0.639; in MDR, was 96.6%, p=0.625 and Kappa=0.651. For Hidrazin, WT, was 88.5%, p=0.125 and Kappa=0.408; in PT, 88.6%, p=0.625 and Kappa=0.645; for MDR, 86.7%, p=0.625 and Kappa=0.053. For Streptomycin, PT was 93.5%, p=0.125 and Kappa=0.635. In PT, was 79,5%, p=0.016 and Kappa=0.296; in MDR, 76.6%, p=0.453 and Kappa=0.533. For Ethambutol, WT was 93.4%, p=0.125; in PT 94.3%, p=0.500 and Kappa=0.478. MDR was 72.4%, p=0.70 and kappa=0.455. Evaluation of mycobacterial sensitivity was faster in the automated method when compared with the conventional method.

Os casos de tuberculose vêm crescendo, coincidindo com o aumento dos casos de AIDS, sendo uma das doenças que causa maior número de mortes no mundo. Entretanto, a busca por novos fármacos cresce de forma lenta, principalmente pelo baixo interesse da indústria farmacêutica, uma vez que a tuberculose ocorre predominantemente em países em desenvolvimento. Tiossemicabazonas têm atraído a atenção de muitos pesquisadores por suas propriedades biológicas e farmacológicas. Esta classe de ligantes é biologicamente ativa contra Mycobacterium tuberculosis, agente causador da tuberculose. Este trabalho apresenta a síntese e caracterização de uma série de complexos de metais da primeira série de transição com ligantes tridentados do tipo 2-acetilpiridina-N(4)-R-tiossemicarbazona (Hatc-R). A caracterização dos compostos envolveu diversas técnicas, como análise elementar, espectroscopia na região do infravermelho e do UV-Vis, condutimetria, ressonância magnética nuclear (1H RMN), voltametria cíclica, voltametria de pulso diferencial, medidas de susceptibilidade magnética e difração de raios X em monocristais. Baseando-se nos resultados de caracterização, verificou-se a formação de complexos de geometria octaédrica do tipo [M(atc-R)2], onde M = Mn(II) (R = hidrogênio, metil, etil, fenil, ciclohexil e morfolinil), Ni(II) (R = etil) e Zn(II) (R = etil e fenil); [M(atc-R)2]X, onde M = Fe(III) (R = etil e fenil, X = HSO4-) e Co(III) (R = etil e fenil, X = Cl-), e complexos de cobre (II) com geometrias quadrática-plana [CuCl(atc-Me)] e pirâmide de base quadrada [Cu2(&micro;-atc-Me)2&micro;-SO4]. Com isto, foi possível avaliar a influência dos metais, da geometria formada e dos grupos periféricos (R) dos ligantes na atividade anti-Mycobacterium tuberculosis, bem como na citotoxicidade dos complexos obtidos, de modo a potencializar a atividade desta classe de ligantes e obter um complexo com alto índice de seletividade, com potencial para ser futuramente utilizado na terapia da tuberculose. Dentre os 15 complexos obtidos neste trabalho, destacaram-se os compostos [Mn(atc-Ph)2], [Co(atc-Et)2]Cl e [Co(atc-Ph)2]Cl com valores de IS igual à 50. Estes resultados são comparáveis ou melhores que alguns fármacos de primeira e segunda linha usados atualmente no tratamento de tuberculose, tornando estes complexos fortes candidatos à futuros fármacos. / Tuberculosis cases have been rising, coinciding with the increase of AIDS cases, becoming one of the diseases with highest mortality worldwide. However, the research for new drugs grows slowly, mainly due to the low interest of the pharmaceutical industry, once tuberculosis usually occurs in developing countries. Thiosemicarbazones have attracted the attention of many researchers due to their biological and pharmacological properties. This class of ligands is biologically active against Mycobacterium tuberculosis, the pathogenic agent of tuberculosis. This work presents the synthesis and characterization of a set of complexes of the first transition metals series with tridentate ligands of the type 2-acetylpyridine-N(4)-R-thiosemicarbazone (Hatc-R). The characterization involved many techniques such as elemental analyses, infrared and UV-Vis spectroscopies, conductometry, nuclear magnetic resonance (1H NMR), cyclic voltammetry, differential pulse voltammetry, magnetic susceptibility measurement and X-ray diffraction on single crystals. Based on the results of the characterization it was observed the formation of complexes in octahedral geometry of the type [M(atc-R)2], where M = Mn(II) (R = hydrogen, methyl, ethyl, phenyl, ciclohexyl and morpholinyl), Ni(II) (R = ethyl) and Zn(II) (R = ethyl and phenyl); [M(atc-R)2]X, where M = Fe(III) (R = ethyl and phenyl, X = HSO4-) and Co(III) (R = ethyl and phenyl, X = Cl-), and copper (II) complexes with square-planar [CuCl(atc-Me)] and square pyramidal [Cu2(&micro;-atc-Me)2&micro;-SO4] geometries. So, it was possible to evaluate the influence of the metals, of the generated geometry and of the peripheral groups (R) of the ligands on the anti-Mycobacterium tuberculosis activity, as well as the citotoxicity of the obtained complexes, in order to enhance the activity of the class of ligands and to obtain a complex with high selectivity index, with potential to be futurely used in tuberculosis therapy. Among the 15 complexes obtained in these work, the most selective compounds were [Mn(atc-Ph)2], [Co(atc-Et)2]Cl and [Co(atc-Ph)2]Cl with values of SI equal to 50. These results are comparable or better than some drugs of first and second line for the currently treatment of tuberculosis, making these complexes strong candidates for future drugs.

A tuberculose ainda é pontuada como uma doença de impacto mundial, sendo considerada desde 1993 um problema de saúde pública global. Uma das grandes preocupações é a contínua prevalência de cepas da Mycobacterium tuberculosis multidroga resistentes, especialmente o genótipo hipervirulento W-Beijing. Acredita-se que este genótipo apresenta alguma vantagem seletiva em relação a outros genótipos da M. tuberculosis, além de estar associado à falha terapêutica, tuberculose extrapulmonar, resistência à vacinação pela BCG e acentuada capacidade de disseminação. Estas cepas apresentam variável capacidade de sobrevivência dentro de macrófagos e do granuloma, modulando vias metabólicas específicas que culminam no escape do sistema imunológico e sucesso na infecção. Buscando entender esta vantagem seletiva e capacidade de persistência na infecção, este estudo teve como objetivo analisar e comparar o perfil transcriptômico de macrófagos infectados com as cepas da M. tuberculosis, W-Beijing 1471 e H37Rv. Os RNAs mensageiros dos macrófagos infectados foram sequenciados em plataforma HiScan Genome Analyzer Illumina. Foram gerados aproximadamente 30 milhões de sequências por amostra, em leituras single reads, com mais de 70% de sequências com valores de score Q de qualidade superior ou igual a 30. Foram mapeados e analisados 35.581 transcritos. Em média, 63% dos genes não apresentaram diferenças nos valores de expressões, 19% tiveram suas expressões reduzidas e 18% dos genes foram classificados como mais expressos, para todas as amostras de macrófagos sequenciadas. Após as análises terciárias e validação por PCR em tempo real, as amostras infectadas com a cepa W-Beijing 1471 apresentaram um aumento nas expressões de IFNs da classe I (p<0,001) e aumento exacerbado de TNF-alfa (p<0,001), comparativamente ao controle e as amostras infectadas com a cepa padrão H37Rv. Aditivamente foi observado um aumento nas expressões de duas quinases, RIPK1 e RIPK3 e de moléculas envolvidas na indução e controle de espécies reativas de oxigênio (ROS), que em infecções por bactérias intracelulares, estão correlacionadas com a morte de macrófagos por necroptose. A cepa hipervirulenta da M. tuberculosis, W-Beijing 1471, apresentou reduzida persistência intramacrofágica e induziu morte precoce dos macrófagos ao quinto dia de infecção. A morte observada nos macrófagos foi associada a ativação de IFNs da classe I/TNF-&#945;/RIPK1/RIPK3 e ROS, indicando necroptose. Ainda, foi observado um aumento na expressão do receptor TLR3 nas amostras infectadas com a cepa W-Beijing, comparativamente as amostras controles e infectadas com a cepa H37Rv. É provável que a ativação inicial dos IFNs da classe I tenha ocorrido via TLR3 através do reconhecimento de dsRNAs da M. tuberculosis. / Since 1993 the tuberculosis is considered as a disease of worldwide impact and a problem of public health. A major concern is the continuing prevalence of multidrug resistant Mycobacterium tuberculosis strains, especially the hypervirulent W - Beijing genotype. It is believed that this genotype has a selective advantage over other genotypes of M. tuberculosis and has being associated with treatment failure, extrapulmonary tuberculosis, resistance to BCG vaccination and marked ability to spread. These strains have varying ability to survive within macrophages and granuloma, modulating specific metabolic pathways that culminate in the escape of the immune system response. To understand this selective advantage and ability to persist in infection, this study aimed to analyze and compare the transcriptomic profile of macrophages infected with strains of M. tuberculosis W - Beijing 1471 and H37Rv. The mRNAs of infected macrophages were sequenced in HiScan Illumina Genome Analyzer platform. Were generated approximately 30 million sequences per sample, in single-reads readings. More than 70 % of sequences had values of Q score superior or equal to 30. Were mapped and analyzed 35,581 transcripts. On average, 63% of the genes showed no differences in the expressions, 19% were downregulated and 18% were upregulated, for all samples sequenced macrophages. After tertiary analysis and validation by real-time PCR, samples infected with the strain W -Beijing in 1471 showed an increase in expression of IFN class I (p <0.001) and exacerbated increase of TNF- alpha (p < 0.001) when compared to the control samples and those infected with standard strain H37Rv. Additively was observed an increase in expressions of the two kinases RIPK1 and RIPK3 and molecules involved in the induction and control of reactive oxygen species (ROS). In infections by intracellular bacteria, activation of RIPK1, RIPK3, ROS and TNF-&#945;, are correlated with death of macrophages by necroptosis. The hypervirulent M. tuberculosis W - Beijing 1471 strain showed reduced persistence inside macrophage and induced early death of macrophages in the fifth day of infection. The death observed in macrophages was associated with activation of IFNs class I/TNF-&#945;/RIPK1/RIPK3 and ROS, indicating necroptosis. Also was observed an increase in the expression of TLR3 receptor in infected samples with W-Beijing 1471 strain compared to controls and those infected with H37Rv strain. Probably the initial activation of IFNs class I occurred by TLR3 through the recognition of M. tuberculosis dsRNAs.