Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-02-16T18:56:46Z No. of bitstreams: 1 Tese Doutorado - Tatiana Kurtz -FINAL (1).pdf: 3145488 bytes, checksum: 8d9c7ca2d5630d7d9e3c1060cce48762 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-02-22T16:58:20Z (GMT) No. of bitstreams: 1 Tese Doutorado - Tatiana Kurtz -FINAL (1).pdf: 3145488 bytes, checksum: 8d9c7ca2d5630d7d9e3c1060cce48762 (MD5) / Made available in DSpace on 2018-02-22T17:05:19Z (GMT). No. of bitstreams: 1 Tese Doutorado - Tatiana Kurtz -FINAL (1).pdf: 3145488 bytes, checksum: 8d9c7ca2d5630d7d9e3c1060cce48762 (MD5) Previous issue date: 2017-08-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Introduction and Objectives: Tuberculosis (TB) is a complex infectious disease that can occur in any age group. When the host comes in contact with Mycobacterium tuberculosis (MTB) the immune response of the organism may be sufficient to prevent the disease, resulting in total destruction of the bacteria or establishment of latency, termed latent tuberculosis (TBL). Due to the difficulty in demonstrating MTB in the clinical specimens of the child, the diagnosis of TB disease is based on the clinical, epidemiological and radiological bases associated with the interpretation of the cutaneous tuberculin (TT) test. In this context, Mycobacterium tuberculosis infection, mostly latent, represents an important reservoir for reactivation of the disease. This contingent is sufficient to continue generating new cases for many decades, even if the chain of transmission is interrupted. Thus, the precise definition of the criteria for diagnosis of latent TB has great relevance and TT is an important tool. The objective of the study is to evaluate the effect of the neonatal BCG vaccine on the tuberculin skin test cutaneous reaction and to define cutoff points to detect tuberculosis in the first two years of life. Methods: A cross-sectional study was carried out in children from the municipality of Santa Cruz do Sul, who met the inclusion criteria of the study: infants up to 2 years of age who received BCG vaccine during the neonatal period. Exclusion criteria were birth weight <2,000 grams, being the mother of HIV positive mother, or mother with persistence of tuberculosis in the perinatal period, or cases where there was evidence of primary immunodeficiency, absence of BCG vaccine scar after 6 months of life, in addition to TB research and TB contact. The children were identified and included through the authorization of the person responsible, explaining the project and accepting the consent term. The project was approved by the Research Ethics Committee of the Santa Cruz Hospital, where patient data were collected and the University of Santa Cruz do Sul (UNISC). The variable under study was the cutaneous induration reaction of the tuberculin test, in the first two years post-vaccination, using different cutoff points. A descriptive analysis of the variables was performed. Numerical variables were represented by mean and standard deviation and categorical variables by means of absolute number and percentage. To describe the data, we used means and standard deviation, or median and interquartile range for the quantitative variables; percentage for qualitative variables. For analysis of the Tuberculin Test, the sample was submitted to the analysis of variance (ANOVA), with significance level of p?0.05. The data analyzed in the SPSS Program 17.0. Results: Potentially eligible participants totaled 808, of which 90 were selected from the inclusion / exclusion criteria. Data collected included demographic characteristics, nutritional indexes, vaccination status and previous exposure to TB. TTs were administered and induration measured after 48-72 hours. The selected ones were of both sexes, with ages varying between 3 and 24 months. Of these, eleven were excluded because they did not attend the reading of the tuberculin test (TT), resulting in a sample of 79 patients. The median age was 9.5 months for boys and 11 months for girls. It was divided into 3 groups according to the age range: between 3-9 months (group 1), 10-18 months (group 2) and 19-24 months of age (group 3). We found that, when comparing the 3 groups, we showed a decrease in the mean response to tuberculin as the age group progresses, presenting statistical significance (p = 0.041). Considering the probable absence of Mycobacterium tuberculosis infection in the sample of patients included in the study, we observed that the tuberculin test with the highest reaction occurs in group 1. From the age of 10 months, no patient shows a reaction to the tuberculin test above 5 mm. The finding shows the decline in the tuberculin reaction curve in the first year of life. A complementary analysis was performed excluding patients who did not present an induration reaction (TT = 0 mm), and 28 patients were excluded from the interpretation. The 51 patients with Test Tuberculin reactor were divided into the same 3 groups according to age group. Between 3-9 months (group 1), 10-18 months (group 2) and 19-24 months of age (group 3), we found that when comparing the 3 groups, again we showed a decrease in the reaction to tuberculin according to age progresses, presenting significance (p = 0.031). We found that there were no adverse effects, described in the literature, in patients who underwent the Tuberculin Test. Conclusions: Based on the data from the study, we demonstrated that the induration reaction occurs in the tuberculin test in the first 12 months of age in previously healthy and BCG-vaccinated patients in the neonatal period. Therefore our results suggest that the cutoff point could be modified from 10mm to 5mm of induration after 12 months of age, improving the specificity of the TT diagnostic test to identify cases of TB infection. This reevaluation of the lowest cutoff point in the first two years of life may prevent inappropriate management in patients with tuberculosis. / Introdu??o e Objetivos: A tuberculose (TB) ? uma doen?a infecciosa complexa, podendo ocorrer em qualquer faixa et?ria. Quando o hospedeiro entra em contato com o Mycobacterium tuberculosis (MTB) a resposta imunit?ria do organismo pode ser suficiente para evitar a doen?a, ocorrendo destrui??o total das bact?rias ou estabelecimento de um estado de lat?ncia, denominado de tuberculose latente (TBL). Devido ? dificuldade em demonstrar MTB nos esp?cimes cl?nicos da crian?a, o diagn?stico da TB doen?a ? fundamentado em bases cl?nicas, epidemiol?gicas e radiol?gicas associados ? interpreta??o do teste tubercul?nico (TT) cut?neo. Neste contexto, verifica-se que a infec??o pelo Mycobacterium tuberculosis, na sua maioria forma latente, representa um importante reservat?rio de reativa??o da doen?a. Este contingente ? suficiente para continuar gerando novos casos por muitas d?cadas, mesmo que a cadeia de transmiss?o seja interrompida. Dessa forma, a defini??o precisa dos crit?rios para diagn?stico de TB latente tem grande relev?ncia e o TT ? uma importante ferramenta. O objetivo do estudo ? avaliar o efeito da vacina BCG neonatal na rea??o de endura??o cut?nea do Teste Tubercul?nico e definir pontos de corte para detectar tuberculose nos dois primeiros anos de vida. M?todos: Estudo transversal, em crian?as do munic?pio de Santa Cruz do Sul, que se adequaram aos crit?rios de inclus?o do estudo: lactentes at? 2 anos de idade que receberam vacina BCG durante o per?odo neonatal. Crit?rios de exclus?o foram: peso ao nascimento <2,000 gramas, ser filho de m?e HIV positiva, ou m?e com vig?ncia de tuberculose no per?odo perinatal, ou ainda os casos em que houve evid?ncia de imunodefici?ncia prim?ria, aus?ncia de cicatriz vacinal de BCG ap?s 6 meses de vida, al?m de investiga??o de TB e contato de TB. As crian?as foram identificadas e inclu?das atrav?s de autoriza??o do respons?vel, mediante explica??o do projeto e aceita??o do termo de consentimento. O projeto foi aprovado pela Comiss?o de ?tica em Pesquisa do Hospital Santa Cruz, onde foi realizada a coleta de dados dos pacientes e Universidade de Santa Cruz do Sul (UNISC). A vari?vel em estudo foi a rea??o de endura??o cut?nea do teste tubercul?nico, nos dois primeiros anos p?s-vacina??o, utilizando diferentes pontos de corte. Realizada uma an?lise descritiva das vari?veis. As vari?veis num?ricas foram representadas por meio de m?dia e desvio padr?o e as categ?ricas por meio de n?mero absoluto e porcentagem. Para descri??o dos dados, foram utilizados m?dias e desvio padr?o, ou mediana e intervalo interquartil para as vari?veis quantitativas; porcentagem para as vari?veis qualitativas. Para an?lise do Teste Tubercul?nico a amostra foi submetida ao teste de an?lise de vari?ncia (ANOVA), com n?vel de signific?ncia de p?0,05. Os dados analisados no Programa SPSS 17.0. Resultados: Os participantes potencialmente eleg?veis totalizaram 808, desses 90 foram selecionados a partir dos crit?rios de inclus?o/exclus?o. Dados coletados inclu?ram caracter?sticas demogr?ficas, ?ndices nutricionais, estado de vacina??o e exposi??o pr?via ? TB. TTs foram administrados e a endura??o medida ap?s 48-72 horas. Os selecionados foram de ambos os sexos, com idade variando entre 3 e 24 meses. Destes, onze foram exclu?das, pois n?o compareceram a leitura do teste tubercul?nico (TT), resultando em amostra final de 79 pacientes. A mediana das idades foi de 9,5 meses, entre os meninos, e 11 meses entre as meninas. Realizada divis?o em 3 grupos conforme faixa et?ria: entre 3-9 meses (grupo 1), 10-18 meses (grupo 2) e 19-24 meses de idade (grupo 3). Constatamos que, quando comparados os 3 grupos, evidenciamos queda na m?dia de rea??o ? tuberculina conforme a faixa et?ria progride, apresentando signific?ncia estat?stica (p= 0.041). Considerando a prov?vel aus?ncia de infec??o por Mycobacterium tuberculosis na amostra de pacientes inclu?dos no estudo, observamos que o teste tubercul?nico com rea??o mais elevada ocorre no grupo 1. A partir dos 10 meses de idade nenhum paciente demonstra rea??o ao teste tubercul?nico acima de 5 mm. O achado evidencia o decl?nio na curva de rea??o ? tuberculina j? no primeiro ano de vida. Realizada an?lise complementar excluindo os pacientes que n?o apresentaram rea??o de endura??o (TT= 0 mm), sendo exclu?dos da interpreta??o 28 pacientes. Os 51 pacientes com Teste Tubercul?nico reator foram divididos nos mesmos 3 grupos conforme faixa et?ria. Entre 3-9 meses (grupo 1), 10-18 meses (grupo 2) e 19-24 meses de idade (grupo 3), onde constatamos que quando comparados os 3 grupos, novamente evidenciamos queda na rea??o ? tuberculina conforme a faixa et?ria progride, apresentando signific?ncia (p= 0.031). Constatamos que n?o ocorreram efeitos adversos, descritos em literatura, nos pacientes que se submeteram a aplica??o do Teste Tubercul?nico. Conclus?es: A partir dos dados do estudo demonstramos que ocorre queda da rea??o de endura??o no teste tubercul?nico nos primeiros 12 meses de idade em pacientes previamente h?gidos e vacinados com BCG no per?odo neonatal. Portanto nossos resultados sugerem que o ponto de corte poderia ser modificado de 10mm para 5mm de endura??o ap?s os 12 meses de idade, melhorando a especificidade do teste diagn?stico TT para identifica??o dos casos de TB infec??o. Esta reavalia??o do ponto de corte menor nos dois primeiros anos de vida pode evitar manejos inadequados nos pacientes com contato com tuberculose.

Recomendações para rastreamento de Tuberculoses Latente (TBL) em pacientes que receberão tratamento com antagonistas do TNF-alfa (anti- TNF) permanecem controversas para regiões endêmicas Objetivo: Esse estudo buscou demonstrar a eficácia em longo prazo do rastreamento e tratamento da TBL em pacientes portadores de Artrite Reumatoide (AR) recebendo anti-TNF. Métodos: 202 pacientes com AR, antes do início do anti-TNF, foram rastreados para TBL por meio do teste tuberculínico (TT), Radiografia de tórax (RX) e história de prévia de exposição à tuberculose (EXP). Todos os pacientes foram seguidos com intervalos de um a três meses. Resultados: 85 pacientes (42%) foram tratados com um único agente anti-TNF e 117 pacientes (58%) mudaram de anti-TNF uma ou duas vezes. O rastreamento para TBL foi positivo em 66 pacientes, 44 apresentaram TT positivo, 23 apresentavam história de exposição (EXP), e 14, alterações radiográficas (RX). EXP isoladamente foi responsável por 14 diagnósticos em pacientes TT negativos. Pacientes portadores de TBL receberam tratamento com Isoniazida (300 mg/dia por seis meses) e nenhum deles desenvolveu TB. Durante os seguimentos, o TT foi repetido em 51 pacientes. A conversão foi observada em cinco: três foram diagnosticados com TBL e dois com TB ativa (14 e 36 meses após receber terapia anti-TNF), sugerindo nova exposição a TB. Conclusão: O rastreamento e tratamento da TBL antes do início da terapia com anti-TNF é efetiva em regiões endêmicas, e reforça a relevância da história de contato com TB para o diagnóstico da TBL em pacientes com AR / Recommendations for screening of latent tuberculosis infection (LTBI) in patients eligible for anti-TNF agents remain unclear in endemic regions. Objective: This study aimed to evaluate the long-term efficacy of LTBI screening/treatment in patients with rheumatoid arthritis (RA) receiving TNF blockers. Design: 202 RA patients were screened for LTBI prior to receiving anti-TNF treatment, by means of tuberculin skin test (TST), chest radiography (X-Ray), and history of tuberculosis exposure (EXP). All subjects were regularly followed at 1- to 3-month intervals. Results: Eighty-five patients (42%) were treated with a single anti-TNF agent, and 117 patients (58%) switched anti-TNF agents once or twice. LTBI screening was positive in 66 patients, 44 presented positive TST, 23 had a history of EXP, and 14, abnormal X-Ray. Exposure alone accounted for LTBI diagnosis in 14 patients with negative TSTs. LTBI patients were treated with Isoniazid (300 mg/day during six months) and none developed TB. During follow up, TST was repeated in 51 patients. Conversion was observed in five: three were diagnosed with LTBI and two with active TB (14 and 36 months after receiving anti-TNF therapy, suggesting new TB exposure). Conclusion: LTBI screening and treatment prior to anti-TNF treatment is effective in endemic areas and reinforces the relevance of contact history for diagnosing LTBI in RA patients

Introduktion: Tuberkulos är en av världens mest spridda infektionssjukdomar. En tredjedel av jordens befolkning beräknas bära på bakterien utan att vara sjuka (latent tuberkulos, LTBI). Närmare tio procent av bärarna utvecklar aktiv tuberkulos någon gång under livet. Europa har jämförelsevis låg incidens av tuberkulos och i Sverige har antalet tuberkulosfall minskat sedan mitten av 40-talet. I Sverige ses de senaste årens ökade antal migranter som en förklaring till en tillfälligt ökad prevalens av tuberkulos. Tidig diagnostik och behandling av tuberkulos är avgörande faktorer för att förhindra smittspridning och ger den enskilde individen möjlighet att hantera sin hälsa, vilket är angeläget ur ett folkhälsoperspektiv. Landstingen/regionerna har valt olika strategier för vilka som ska inkluderas i screening för tuberkulos vid hälsoundersökningar av migranter. Syfte: Syftet med studien var att undersöka prevalens och uppföljning av latent tuberkulos bland migranter som genomgått hälsoundersökning vid vårdcentraler i Region Jönköpings län. Metod: Studien var en retrospektiv observationsstudie utifrån kvantitativ ansats. Data samlades in genom journalgranskning och 361 journaler inkluderades i analyserna. Förekomst av latent och aktiv tuberkulos korrelerades med kön, ålder och ursprungsland. Resultat: Resultaten från studien visade att prevalensen av LTBI i studiepopulationen var 9.4 %. Resultaten visade att LTBI var vanligare hos män och individer &gt; 35 år. Däremot sågs inga skillnader utifrån ursprungsland. Slutsats: Studien är av begränsad storlek och därmed generaliserbarhet. LTBI är relativt vanligt bland migranter. Ytterligare studier behövs för att identifiera optimala rutiner för screening och förebyggande insatser i migrantgruppen. / Introduction: Tuberculosis is one of the world's most widespread infectious diseases. One third of the world’s population is expected to carry the bacterium without being ill (latent tuberculosis, LTBI). Nearly ten percent of the carriers develop active tuberculosis sometime during their lifetime. Europe has a comparatively low incidence of tuberculosis and in Sweden the number of cases of tuberculosis has decreased since the mid-40s. In Sweden, the increased number of immigrants in recent years is seen as an explanation for a temporarily increased prevalence of tuberculosis. Early diagnosis and treatment of tuberculosis are key factors in preventing the spread of infection and giving the individual the opportunity to manage their health, which is important from a public health perspective. The county councils/regions have chosen different strategies for whom to be included in screening for tuberculosis in health surveys of immigrants. Aim: The aim of the study was to investigate the prevalence and follow-up of latent tuberculosis among immigrants who have undergone a health examination at health centers in the County of Jönköping. Method: The study was a retrospective observation study based on quantitative approach. Data was collected through journal review and 361 records were included in the analysis. The presence of latent and active tuberculosis was correlated with gender, age and country of origin. Results: The results of the study showed that the prevalence of LTBI in the study population was 9.4%. The results showed that LTBI was more common in men and individuals &gt; 35 years. However, no differences were seen by country of origin. Conclusion: The study is of limited size and thus generalisability. LTBI is relatively common among immigrants. Further studies are needed to identify optimal routines for screening and preventive measures in the immigrant group.

Recomendações para rastreamento de Tuberculoses Latente (TBL) em pacientes que receberão tratamento com antagonistas do TNF-alfa (anti- TNF) permanecem controversas para regiões endêmicas Objetivo: Esse estudo buscou demonstrar a eficácia em longo prazo do rastreamento e tratamento da TBL em pacientes portadores de Artrite Reumatoide (AR) recebendo anti-TNF. Métodos: 202 pacientes com AR, antes do início do anti-TNF, foram rastreados para TBL por meio do teste tuberculínico (TT), Radiografia de tórax (RX) e história de prévia de exposição à tuberculose (EXP). Todos os pacientes foram seguidos com intervalos de um a três meses. Resultados: 85 pacientes (42%) foram tratados com um único agente anti-TNF e 117 pacientes (58%) mudaram de anti-TNF uma ou duas vezes. O rastreamento para TBL foi positivo em 66 pacientes, 44 apresentaram TT positivo, 23 apresentavam história de exposição (EXP), e 14, alterações radiográficas (RX). EXP isoladamente foi responsável por 14 diagnósticos em pacientes TT negativos. Pacientes portadores de TBL receberam tratamento com Isoniazida (300 mg/dia por seis meses) e nenhum deles desenvolveu TB. Durante os seguimentos, o TT foi repetido em 51 pacientes. A conversão foi observada em cinco: três foram diagnosticados com TBL e dois com TB ativa (14 e 36 meses após receber terapia anti-TNF), sugerindo nova exposição a TB. Conclusão: O rastreamento e tratamento da TBL antes do início da terapia com anti-TNF é efetiva em regiões endêmicas, e reforça a relevância da história de contato com TB para o diagnóstico da TBL em pacientes com AR / Recommendations for screening of latent tuberculosis infection (LTBI) in patients eligible for anti-TNF agents remain unclear in endemic regions. Objective: This study aimed to evaluate the long-term efficacy of LTBI screening/treatment in patients with rheumatoid arthritis (RA) receiving TNF blockers. Design: 202 RA patients were screened for LTBI prior to receiving anti-TNF treatment, by means of tuberculin skin test (TST), chest radiography (X-Ray), and history of tuberculosis exposure (EXP). All subjects were regularly followed at 1- to 3-month intervals. Results: Eighty-five patients (42%) were treated with a single anti-TNF agent, and 117 patients (58%) switched anti-TNF agents once or twice. LTBI screening was positive in 66 patients, 44 presented positive TST, 23 had a history of EXP, and 14, abnormal X-Ray. Exposure alone accounted for LTBI diagnosis in 14 patients with negative TSTs. LTBI patients were treated with Isoniazid (300 mg/day during six months) and none developed TB. During follow up, TST was repeated in 51 patients. Conversion was observed in five: three were diagnosed with LTBI and two with active TB (14 and 36 months after receiving anti-TNF therapy, suggesting new TB exposure). Conclusion: LTBI screening and treatment prior to anti-TNF treatment is effective in endemic areas and reinforces the relevance of contact history for diagnosing LTBI in RA patients

Most of the chemical reactions of living cells are catalyzed by protein enzymes. These enzymes are very efficient and display a high degree of specificity with respect to the reaction catalyzed. Cellular activities depend critically on the precise three-dimensional structure and function of thousands of enzymes. Many enzymes require binding of metal ions or small organic molecules for their function. The organic molecules that are indispensible components of catalysis by proteins are called coenzymes. Pyridoxal 5ʹ-phosphate (PLP) is a versatile coenzyme found in all living cells. PLP-dependent enzymes play a key role in the function of most of the enzymes catalyzing reactions in the metabolic pathways of amino acid synthesis and degradation. The enzyme pyridoxal kinase serves to make available the co-enzyme PLP to apo-PLP dependent enzymes. Because of their key role in cellular function and their medical importance, the structure and function of PLP-dependent enzymes have been extensively investigated. In the past decade, detailed investigations on the structure and function of several PLP-dependent enzymes have been carried out in our laboratory. The enzymes studied are B. subtilis serinehydroxymethyl transferase (SHMT), S. typhimurium acetylornithine aminotransferase (AcOAT), S. typhimurium and E. coli diaminopropionate ammonia lyase (DAPAL), S. typhimurium D-serine dehydratase (DSD), S. typhimurium D-cysteine desulfhydrase (DCyD) and S. typhimurium arginine decarboxylase (ArgD). The extensive studies conducted on PLP-dependent enzymes in our laboratory during the past decade has not only resulted in deeper understanding of their structure and function but also raised several new questions regarding substrate recognition, reaction specificity, role of active site residues in the catalytic reaction, mechanism of catalysis and potential applications of these enzymes. This thesis is an attempt to answer some of these questions. The thesis also presents the structure and function of a new protein, Salmonella typhimurium pyridoxal kinase, the enzyme that provides PLP for PLP-dependent enzymes. Single crystal X-ray diffraction technique is the most powerful tool currently available for the elucidation of the three-dimensional structures of proteins and other biological macromolecules and for revealing the relationship between their structure and function. X-ray diffraction studies have provided in depth understanding of the topology of secondary structural elements in the three-dimensional structures of proteins, the hierarchical organization of protein domains, structural basis for the substrate specificity of enzymes, intricate details of mechanisms of enzyme catalyzed reactions, allosteric regulation of enzyme activity, mechanisms of feed-back inhibition, structural basis of protein stability, symmetry of oligomeric proteins and their possible biological implications and a myriad of other biochemical and biophysical properties of proteins. The work reported in this thesis is primarily based on X-ray diffraction studies. X-ray crystal structure investigations are complemented by spectral and biochemical studies on the catalyzed reactions. The thesis begins with an introduction to PLP-dependent enzymes and presentation of a brief summary of the earlier work carried out in our laboratory on PLP-dependent enzymes (Chapter 1). A brief description of earlier functional classification of PLP-dependent enzymes and the more recent classification of these enzymes into the four groups based on their three-dimensional structure is provided. Although enzymes belonging to these four structural classes have evolved from independent evolutionary lineages, they share some common features near their active sites and in the mode of PLP binding. Earlier work carried out elsewhere on pyridoxal kinase and its key role in maintaining PLP at a low concentration in the cytosol is presented. Different mechanisms that have been proposed for the transfer of PLP from pyridoxal kinase to other apo PLP-dependent enzymes are briefly described. The experimental procedures and computational methods used during the course of these investigations to obtain the results reported in chapters 3-6 are presented in Chapter 2. Most of these methods are applicable to the isolation of plasmids, cloning, over expression, protein purification, mutant construction, crystallization, X-ray diffraction data collection and processing, structure elucidation and refinement, validation and structural analysis presented in the next three chapters. Various programs and protocols used for data processing, structure determination, refinement, model building, structure validation and analysis are also briefly described. In chapter 3, the role of a number of active site residues in the reaction catalyzed by EcDAPAL, a fold type II PLP-dependent enzyme, the structure of which was determined earlier in the laboratory is explored by mutational, biochemical and structural analyses. Earlier studies had established the probable role of Asp120 and Lys77 in the reaction leading to the breakdown of D-DAP and L-DAP, respectively (Bisht et al., 2012). To further validate the earlier observations, a number of active site mutants were generated for Asp 120 (D120N, D120C, D120S and D120T), Asp 189 (D189N, D189C, D189S and D189T), Lys77 (K77T, K77H, K77R and K77A), His 123 (H123L) and Tyr 168 (Y168F). The structure of D120N mutant crystal obtained after soaking in crystallization cocktail containing D-DAP revealed the presence of an intact external aldimine complex at the active site supporting the earlier proposal that Asp120 is the base abstracting the Cα proton from the D-isomer of DAP. Biochemical and structural observations suggested that none of the Asp189 mutants may bind PLP and were catalytically inactive suggesting an essential role for Asp189 in catalysis. In contrast to type I PLP-dependent enzymes, none of the Lys 77 mutants of EcDAPAL could bind PLP either covalently or non-covalently and were inactive with both the isomers of DAP. Thus, Lys77 appears to be important for both PLP binding and catalysis. H123L mutant formed an external aldimine with D-DAP and a gem-diamine complex with L-DAP indicating that this residue is also crucial for catalysis. These studies have provided additional support to the catalytic mechanism of EcDAPAL proposed earlier. The next Chapter 4 explores the structure, function and catalytic mechanism of Salmonella typhimurium DAPAL (StDAPAL). The protein was purified from a construct carrying a hexa-histidine tag at the C-terminus by Ni-NTA chromatography. The purified protein was demonstrated to be homogeneous by SDS-PAGE and MALDI-TOF. Crystals of StDAPAL belonging to the C-centred monoclinic space group (C121) with four molecules in the asymmetric unit were obtained by the micro batch method and used for collecting X-ray diffracting data. The crystal structure was determined by molecular replacement using the homologous enzyme from E. coli (PDB code 4D9M, Bisht et al., 2012), which shares a sequence identity of 50% with the S. typhimurium enzyme as the phasing model in the program Phaser (McCoy et al., 2007) of the CCP4 suite. The model was refined with Refmac5 of CCP4 suite to R and Rfree values of 25.5% and 30.9%, respectively. A superposition of the structure so obtained over EcDAPAL revealed that the two structures are very similar. A sulfate molecule bound to the active site of StDAPAL could be located. The position of the sulfate corresponds to that of the carboxyl group of aminoacrylate intermediate of EcDAPAL (4D9M). The PLP was bound to Lys78 as an internal aldimine. Since the active sites of the two protomers in fold type II PLP-dependent enzymes are independent, it might be possible to obtain functional monomers of EcDAPAL. With this view, mutation of a conserved Trp (Trp399) present in the dimeric interface resulted in the destabilization of the dimeric interface and partial conversion of the dimeric protein to a monomeric protein. However, the monomeric species of EcDAPALW399R was unable to bind PLP and hence did not possess any catalytic activity. This highlights the importance of dimeric organization for efficient binding of PLP as well as for the activity of the enzyme. A remarkable difference between EcDAPAL and StDAPAL is the absence of a disulfide bond between residues Cys271 and Cys299 in StDAPAL equivalent to the bond formed between Cys265 and Cys291 in EcDAPAL. Mutation of Cys265 and Cys291 of EcDAPAL to Ser did not affect the activity of the enzyme towards either of the isomers of the substrate indicating that the disulfide bond is not crucial for enzyme activity. The stability of the loop corresponding residues 261-295 of EcDAPAL was believed to be promoted by the disulfide bond. However, the equivalent loop was found to be ordered in StDAPAL even though the disulfide bond is absent. In contrast to StDAPAL, EcDAPAL did not show any metal dependent activity. The previous two chapters dealt with fold type II PLP-dependent enzymes. In contrast, Chapter 5 deals with revisiting the structure and function of a fold type I PLP-dependent enzyme, Salmonella typhimurium arginine decarboxylase (StADC). ADC is a very large polypeptide in comparison with other fold type I enzymes. It is induced when the bacterium is subjected to low pH and plays a major role in protecting the cells from acid stress. The structure of StADC was determined but not satisfactorily refined by Dr. S. R. Bharat earlier. The X-ray diffraction data collected by Bharat needed to be improved and the structure needed to be further refined and compared with the homologous E. coli enzyme. Therefore, the entire process of data processing, structure solution and refinement was repeated. The refined structure of StADC was found to correspond to the apo form of the enzyme with only a phosphate molecule occupying the position equivalent to that of 5’ phosphate of PLP observed in EcADC holo enzyme structure. This allowed examination of structural changes that accompany PLP binding and formation of an internal aldimine. The apo to holo transition in StADC involves the movement and ordering of two loops consisting of residues 151-164 and 191-196 which are in the linker and PLP binding domains of the protein, respectively. Phosphate binding by itself appears to be insufficient for these structural changes. These two loops are close to the PLP binding site of the other protomer of the dimer. Hence, these movements are probably important for the catalytic function of the enzyme. Holo ADC has been found as a decamer in other studies. The decameric form of the apo-StADC suggests that PLP binding may not be essential for the oligomeric state of the protein. ADC appears to reduce proton concentration inside the cell in two ways; (i) by surface charge neutralization and (ii) by arginine decarboxylation by extracting a proton from the cytoplasm. The resulting product agmatine is exchanged for extra cellular arginine by arginine-agmatine antiporter. The low sequence identity and lack of structural similarity of the inducible and constitutive forms of ADC from S. typhimurium shows that these are unlikely to be products of divergent evolution. The final chapter 6 of the thesis presents the work carried out on S. typhimurium pyridoxal kinase (PLK). In the salvage pathway of pyridoxal 5’phosphate (PLP), PLP is produced as the product of the reaction catalyzed by PLK using PL, PN and PM as substrates. Thus, PLK plays the critical role of ensuring availability of PLP to the large number of PLP-dependent enzymes. S. typhimurium PLK was purified to homogeneity, crystallized in its native as well as ligand bound forms. It was necessary to circumvent an unusual problem caused by spots arising from a contaminant crystal to obtain the structure of the native crystals of PLK that belonged to the P212121 space group with two protomers in the crystal asymmetric unit. It was then straight forward to determine the ligand bound structures of StPLK (space group P43212) obtained by co-crystallization with ATP, PL and Mg2+ by molecular replacement using the wild type structure as the phasing model. The structures obtained by co-crystallization revealed the presence of ADP, Mg2+ and a PL bound to the active site Lys233 via a Schiff base (internal aldimine). This is the first structure in which the presence of an internal aldimine in the active site of PLK has been observed. Formation of the internal aldimine might be one way to prevent the release of excess PLP and protecting the cell from PLP induced toxicity. The enzyme was shown to be inhibited by the product which will also help in maintaining PLP concentration at low levels. It was also demonstrated that PLK interacts with apo-PLP-dependent enzymes. This observation supports possible direct transfer of PLP from PLK to PLP-dependent enzymes. The thesis ends with an appendix where the work carried out during the course of the thesis work but not as part of the thesis is briefly described.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A tuberculose (TB) é uma doença infecto-contagiosa obtida a partir da inalação de aerossóis contendo seu agente etiológico, o Mycobacterium tuberculosis. A TB acomete principalmente os pulmões e é a patologia bacteriana líder em causar mortes no mundo. No Brasil, por ano, são notificados 69 mil casos de tuberculose, dos quais 4,6 mil evoluem para o óbito. Durante a infecção pelo M. tuberculosis, 90% dos indivíduos permanece na forma latente assintomática, e aproximadamente 10% evolui para doença. Este trabalho estudou parâmetros de resposta imune e inflamatória, em indivíduos de ambos os sexos, com idades de 18 a 65 anos, com diferentes graus de exposição ao M. tuberculosis (indivíduos não-expostos ao M. tuberculosis, TST < 5 mm, n= 30; indivíduos com tuberculose latente, TST &#8805; 5 mm, n=29; pacientes com tuberculose pulmonar n= 22). Nossos resultados mostraram que o TST isoladamente falhou em detectar todos os indivíduos expostos ao M. tuberculosis, e em 1/3 dos TST positivos não foi observada resposta in vitro a antígenos específicos de M. tuberculosis, avaliada com os biomarcadores IFN-&#947; e CXCL10. Houve uma alta correlação entre os biomarcadores IFN-&#947; e CXCL10 em culturas de sangue não fracionado estimuladas com antígenos específicos de M. tuberculosis. A utilização combinada destes 2 biomarcadores mostrou positividade para M. tuberculosis em 94,4% dos pacientes. Foram observadas diferenças marcantes de nível de expressão de RNA mensageiro específicos para CD64, GTPase associada a Ras, lactoferrina, PDL-1 e CXCL10, mas não para OASL em leucócitos sanguíneos, quando os pacientes com tuberculose pulmonar foram comparados com os dois outros grupos de voluntários. Da mesma forma, os níveis de expressão dos receptores CD64 e CD163 foram significativamente mais elevados em neutrófilos dos pacientes quando comparados com os grupos-controle. Tomadas em conjunto, nossas observações sugerem que o uso de mais de um biomarcador aumenta a sensibilidade e especificidade dos métodos para detecção de infecção latente por M. tuberculosis e tuberculose.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A tuberculose (TB) é uma doença infecto-contagiosa obtida a partir da inalação de aerossóis contendo seu agente etiológico, o Mycobacterium tuberculosis. A TB acomete principalmente os pulmões e é a patologia bacteriana líder em causar mortes no mundo. No Brasil, por ano, são notificados 69 mil casos de tuberculose, dos quais 4,6 mil evoluem para o óbito. Durante a infecção pelo M. tuberculosis, 90% dos indivíduos permanece na forma latente assintomática, e aproximadamente 10% evolui para doença. Este trabalho estudou parâmetros de resposta imune e inflamatória, em indivíduos de ambos os sexos, com idades de 18 a 65 anos, com diferentes graus de exposição ao M. tuberculosis (indivíduos não-expostos ao M. tuberculosis, TST < 5 mm, n= 30; indivíduos com tuberculose latente, TST &#8805; 5 mm, n=29; pacientes com tuberculose pulmonar n= 22). Nossos resultados mostraram que o TST isoladamente falhou em detectar todos os indivíduos expostos ao M. tuberculosis, e em 1/3 dos TST positivos não foi observada resposta in vitro a antígenos específicos de M. tuberculosis, avaliada com os biomarcadores IFN-&#947; e CXCL10. Houve uma alta correlação entre os biomarcadores IFN-&#947; e CXCL10 em culturas de sangue não fracionado estimuladas com antígenos específicos de M. tuberculosis. A utilização combinada destes 2 biomarcadores mostrou positividade para M. tuberculosis em 94,4% dos pacientes. Foram observadas diferenças marcantes de nível de expressão de RNA mensageiro específicos para CD64, GTPase associada a Ras, lactoferrina, PDL-1 e CXCL10, mas não para OASL em leucócitos sanguíneos, quando os pacientes com tuberculose pulmonar foram comparados com os dois outros grupos de voluntários. Da mesma forma, os níveis de expressão dos receptores CD64 e CD163 foram significativamente mais elevados em neutrófilos dos pacientes quando comparados com os grupos-controle. Tomadas em conjunto, nossas observações sugerem que o uso de mais de um biomarcador aumenta a sensibilidade e especificidade dos métodos para detecção de infecção latente por M. tuberculosis e tuberculose.

Made available in DSpace on 2014-07-29T15:30:38Z (GMT). No. of bitstreams: 1 Dissertacao Daniela Graner Schuwartz Tannus Silva.pdf: 2699876 bytes, checksum: 9f1addca3b98dc8f124ef4cc53bfe67a (MD5) Previous issue date: 2010-12-09 / The diagnosis of latent tuberculosis (TBIL) in patients with rheumatoid arthritis (RA) has got special importance with the advent of anti tumor necrosis factor (anti-TNF-&#945;) and the arise of cases of active tuberculosis in these patients. The tuberculin skin test (TST) is a test used for more than a century on the diagnosis of TBIL but has limited value in patients with RA. New tests that are based on the production and release of interferon-gamma (IFN-&#947;) have been studied but its role has not yet been well established in this group of patients. This paper shows a review about the use of anti-TNF-&#945; and its role on the development of tuberculosis, the use of the TST for the diagnosis of TBIL in patients with RA and new tests for diagnosis of TBIL. The review is illustrated with a case of a patient who developed tuberculosis after starting the use of anti-TNF-&#945;. In the sequence, a study comparing the diagnostic TBIL in a group of RA patients by cellular immune response, compared to TST, T.SPOT-TB and measurement of IFN-&#947; by flow cytometry after stimulation with Hspx and through computed tomography changes consistent with TBIL. It was observed that the response to TST was lower in RA patients (13.5%) than the expected response to the general population. We also observed that the T.SPOT-TB identified a greater number of patients with TBIL compared to PT (36.8%). The IFN-&#947; in response to Hspx was not statistically different among the groups considered TBIL (TST and / or T.SPOT-TB positives) or NO TBIL (TST and T.SPOT-TB negatives). Finally, HRCT showed changes consistent with TBIL in 52.9% of patients, including eight of the eleven patients with TST and T.SPOT TB negatives. Conclusion: The TST alone is not appropriate for the diagnosis of TBIL. The T SPOT TB showed a higher number of positives results compared to TST but was negative in a large percentage of patients with CT changes highly suggestive of TBIL. HRCT is accessible in most major centers and should be incorporated in the diagnostic strategy of TBIL in patients with RA. / O diagnóstico de tuberculose latente (TBIL) em pacientes com artrite reumatoide (AR) ganhou especial importância com o advento do uso dos agentes anti necrose tumoral (anti-TNF-&#945;) e o surgimento de casos de tuberculose ativa nesses pacientes. A prova tuberculínica (PT), teste utilizado há mais de um século no diagnóstico de TBIL, apresenta valor limitado entre os pacientes com AR. Novos exames que se baseiam na produção e liberação de interferon-gamma (IFN-&#947;) vêm sendo estudados, mas ainda não têm seu papel bem estabelecido neste grupo de pacientes. No presente trabalho é realizada uma revisão sobre o uso dos anti-NF-&#945; e seu papel no desenvolvimento da tuberculose, a utilização da PT para o diagnóstico de TBIL nos pacientes com AR e os novos testes para o diagnóstico de TBIL ilustrado com um caso de uma paciente que desenvolveu tuberculose após o início do uso do anti-TNF-&#945;. Segue-se um estudo de comparação do diagnóstico de TBIL em um grupo de pacientes com AR através da resposta imune celular, frente à prova tuberculínica, T.SPOT-TB e medida da produção de IFN-&#947; pela citometria de fluxo após estímulo com Hspx e através de alterações tomográficas compatíveis com TBIL. Observou-se que a resposta à PT foi menor nos pacientes com AR (13,5%) do que a resposta esperada para população geral. Observou-se ainda que o T.SPOT-TB identificou um maior número de pacientes com TBIL em relação à PT (36,8%). A produção de IFN-&#947; em resposta ao Hspx não foi estatisticamente diferente entre os grupos considerados como TBIL (PT e/ou T.SPOT-TB positivos) ou NÃO TBIL (PT e T.SPOT-TB negativos). E, finalmente, a tomografia computadorizada de alta resolução apresentou alterações compatíveis com TBIL em 52,9% dos pacientes estudados, inclusive entre oito dos onze pacientes com PT e T.SPOT-TB negativos. Conclusão: A PT isoladamente é insuficiente para o diagnóstico de TBIL. O T.SPOT TB apresentou maior número de resultados positivos em relação à PT mas foi negativo em grande porcentagem de pacientes com alterações tomográficas bastante sugestivas de TBIL. A TCAR é um exame acessível na maior parte dos grandes centros e deve ser incorporada na estratégia diagnóstica de TBIL em pacientes com AR.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-06-29T12:16:23Z No. of bitstreams: 2 Dissertação - Daniel Batista Gomes - 2016.pdf: 3477482 bytes, checksum: 39d73011b1245110ab18fca9b832b249 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-07-10T14:14:40Z (GMT) No. of bitstreams: 2 Dissertação - Daniel Batista Gomes - 2016.pdf: 3477482 bytes, checksum: 39d73011b1245110ab18fca9b832b249 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-10T14:14:40Z (GMT). No. of bitstreams: 2 Dissertação - Daniel Batista Gomes - 2016.pdf: 3477482 bytes, checksum: 39d73011b1245110ab18fca9b832b249 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-12-20 / Tuberculosis is still a major global health problem. One of the strategies recommended for the control of tuberculosis is the identification and early treatment of individuals with latent M. tuberculosis infection (ILTB). In Brazil, ILTB is not part of the compulsory notification aggravations, but there is a recommendation for States to create instruments for notification and follow-up of cases. In Goiás, a notification form for ILTB was developed in 2012 by the State Department of Health. The objective of the research is to analyze the epidemiological profile of ILTB cases and to characterize the surveillance processes related to the diagnosis and treatment of this disease in this state. The reports of ILTB cases treated between 2013 and 2015 were analyzed. A database linking was carried out considering the cases of tuberculosis reported in the SINAN NET Notification System and the ILTB records. To evaluate the technical and structure aspects of ILTB control services, a structured questionnaire was applied to the supervisors of health surveillance in the 18 health regions of the State. A descriptive and exploratory data analysis was carried out using software SPSS 13.0 and TABWIN 1.6 EPI INFO. 345 cases of ILTB were reported in the study period. The patients' ages ranged from 0 to 92 years (median age 38 years); 65.2% were adults and 10.1% were up to 10 years. Five municipalities (Goiânia, Aparecida de Goiânia, Jataí, Anápolis and Formosa) reported 77.7% of the cases. In 24.6% of the cases, the criterion for treatment of ILTB was the result of Tuberculin Test (TT)> 10mm. In this group all cases were asymptomatic and 78.8% had contact with active tuberculosis. It was identified that 39 cases were HIV positive, corresponding to 12.7% of indications for ILTB treatment. According to health surveillance supervisors, all 246 municipalities had a Tuberculosis Control Program. Concerning the specific training on ILTB, 74 municipalities (30.1%) received this training, reaching 141 health professionals. In relation to the specific training for the application of TT, only three (16.7%) health regions were trained, of which two managed to decentralize this training to some of their jurisdictions. Eleven regional health (61.1%) reported that the number of TT provided by the State Department of Health was inadequate to meet the demands of municipalities. According to supervisors, 88.2% of the municipalities in Goiás do not have the tools to monitor cases of co-infection with HIV. The present study contributed to the knowledge of the epidemiological profile of the reported cases of ILTB, as well as to the process of control of this aggravation in the State. Failures were identified in the ILTB control process in the different regions of the State of Goiás. This study is expected to support effective actions to control tuberculosis in the State. / A tuberculose ainda é um grande problema de saúde global. Uma das estratégias preconizada para controle da tuberculose consiste na identificação e tratamento precoce dos indivíduos com infecção latente pelo M. tuberculosis (ILTB). No Brasil, a ILTB não faz parte dos agravos de notificação compulsória, porém existe recomendação para que os Estados criem instrumentos para notificação e acompanhamento dos casos. Em Goiás, uma ficha de notificação para ILTB foi desenvolvida em 2012 pela Secretaria de Estadual da Saúde. O objetivo da pesquisa consiste em analisar o perfil epidemiológico dos casos de ILTB e caracterizar os processos de vigilância relacionados ao diagnóstico e tratamento dessa doença neste estado. Foram analisadas as notificações de casos de ILTB tratados entre 2013 e 2015. Foi realizada a vinculação de base de dados considerando os casos de tuberculose notificados no Sistema de Informação de Agravos de Notificação (SINAN NET) e os registros de ILTB. Para avaliar aspectos técnicos e de estrutura de serviços de controle de ILTB foi aplicado questionário estruturado para os supervisores de vigilância em saúde das 18 regiões de saúde do Estado. Foi realizada análise descritiva e exploratória de dados por meio dos softwares SPSS 13.0 e TABWIN 1.6 EPI INFO. 345 casos de ILTB foram notificados, no período de estudo. A idade dos pacientes variou de 0 a 92 anos (mediana de 38 anos); 65,2% eram adultos e 10,1% tinham até 10 anos. 05 municípios (Goiânia, Aparecida de Goiânia, Jataí, Anápolis e Formosa) notificaram 77,7% dos casos. Em 24,6% dos casos, o critério para tratamento da ILTB foi o resultado do Teste Tuberculínico (TT) >10mm. Nesse grupo todos os casos eram assintomáticos e 78,8% tinham contato com caso de tuberculose ativa. Identificou-se que 39 casos eram HIV positivos, correspondendo a 12,7% das indicações para tratamento ILTB. De acordo com os supervisores de vigilância em saúde, todos os 246 municípios contavam com Programa de Controle da Tuberculose. 74 municípios (30,1%), receberam treinamento sobre ILTB, alcançando 141 profissionais de saúde. Em relação ao treinamento especifico para aplicação do TT apenas 03 (16,7%) regiões de saúde foram capacitadas, das quais duas conseguiram descentralizar esta capacitação para alguns de seus municípios jurisdicionados. 11 regionais de saúde (61,1%) informaram que o número de TT disponibilizado pela Secretaria de Estado da Saúde foi inadequado para atender as demandas dos municípios. Ainda segundo os supervisores, 88,2% dos municípios goianos não dispõem de ferramentas para acompanhamento dos casos de co-infecção com HIV. O presente estudo contribuiu para o conhecimento do perfil epidemiológico dos casos notificados de ILTB, bem como para o processo de controle desse agravo no Estado. Foram identificadas falhas no processo de controle da ILTB, nas diferentes regiões de Saúde do Estado de Goiás. Espera-se que esse estudo possa subsidiar ações efetivas para o controle da tuberculose no Estado.

Tuberculosis (TB) remains the main cause of death in people living with HIV (PLHIV). Indeed, PLHIV have a 20-30% greater risk of developing TB compared to HIV-uninfected subjects and have lower TB treatment success rates. In 2014, among the 9.6 million incident cases of TB reported worldwide, 12% occurred in PLHIV and 0.4 million deaths from HIV-associated TB were recorded.Mycobacterium tuberculosis is the main etiological agent for TB. For a majority of individuals, the immune response upon infection by M. tuberculosis is sufficient to prevent the development of disease, but insufficient to clear the bacteria. This leads to the persistence of viable M. tuberculosis in diverse cells with no resulting clinical manifestations, an entity known as latent tuberculosis infection (LTBI). The resulting reservoir of M. tuberculosis is vast, and an estimated one third of the world population is concerned. For subjects with LTBI, the life-time risk of reactivation and progression to TB lies between 5 and 10%. However, if co-infected with HIV, the risk is much greater and reaches 10% per year. According to a Cochrane review in 2010, the screening and treatment of LTBI in PLHIV reduces this risk by 30-60%. This prevention strategy is therefore widely recommended. However, the implementation of LTBI screening and treatment into standard HIV-care has been limited. In this work, three different approaches have been used to understand and address this issue, focusing on a low TB-incidence and high-income setting.The first approach was to assess the implementation of LTBI screening in HIV-care across Belgium and identify its barriers as perceived by the caregivers on the field. Raising awareness to this issue was an indirect objective of the study. A multi-choice questionnaire was sent to 55 physicians working in a Belgian AIDS reference center or satellite clinic. A response rate of 62% was obtained. Only 20% of participants performed LTBI screening on all their patients and notable variations in the screening methods used were observed. A large majority of participants were in favor of targeting LTBI screening to HIV-infected patients at highest risk of TB rather than a systematic screening of all PLHIV. These results have been communicated to the Belgian LTBI working group, currently updating the national LTBI screening guidelines. Indeed, targeting screening to those at highest risk of TB is an attractive strategy in low-TB incidence countries and is already recommended in the United Kingdom. However, to date, no score assessing the risk of TB in PLHIV has been validated. Among the barriers to LTBI screening identified by the participants of this first study, the most frequently reported were lack of sensitivity of screening tools, risk associated to polypharmacy and toxicity of treatment. Improving the sensitivity of LTBI screening was the cornerstone of the second approach. The available screening tools for LTBI are the tuberculin skin test (TST) and two Interferon-gamma release assays (IGRAs): the QuantiFERON-TB Gold-IT (QFT-GIT) and the T-SPOT.TB®. All three lack sensitivity in PLHIV. Various strategies to discover superior LTBI screening tools are therefore being explored, including the development of IGRAs in response to alternative M. tuberculosis antigens to those used in the QFT-GIT or T-SPOT.TB®. A potential candidate is the native Heparin-Binding Haemagglutin (nHBHA), a methylated M. tuberculosis protein regarded as a latency-associated antigen. An in-house IGRA based on nHBHA (nHBHA-IGRA) has been shown to be a promising LTBI screening tool both in immunocompetent adults and in hemodialysed patients. The contribution of this nHBHA-IGRA to the detection of M. tuberculosis in PLHIV was therefore investigated. Treatment-naïve HIV-infected subjects were recruited from 4 Brussels-based hospitals. Subjects underwent screening for latent TB using the nHBHA-IGRA in parallel to the classical method consisting of medical history, chest X-ray, TST and QFT-GIT. Prospective clinical and biological follow-up ensued, with repeated testing with nHBHA-IGRA. Among 48 candidates enrolled for screening, 9 were diagnosed with LTBI by combining the TST and QFT-GIT results (3 TST+/QFT-GIT+, 1 TST+/QFT-GIT- and 5 TST-/QFT-GIT+). All 3 TST+/QFT-GIT+ patients, the TST+/QFT-GIT- patient as well an additional 3 subjects screened positive with the nHBHA-IGRA. These 3 additional patients had known M. tuberculosis exposure risks compatible with LTBI. During follow-up (median 14 months) no case of TB was reported and nHBHA-IGRA results remained globally constant. Multiplex analysis confirmed IFN- as the best read-out for the assay. From this study, we concluded that the nHBHA-IGRA appears complementary to the QFT-GIT for the screening of LTBI in PLHIV and the combination of the two tests may increase the sensitivity of screening. A large-scale study is however necessary to determine whether combining nHBHA-IGRA and QFT-GIT offers sufficient sensitivity to dismiss TST, as suggested by our results. In the same study, a group of HIV-infected adults with clinical suspicion of active TB were also recruited and tested with nHBHA-IGRA. Contrary to results in HIV-uninfected subjects, the nHBHA-IGRA could not discriminate between LTBI and active TB in PLHIV. This is an important caveat as HIV-infected subjects may present subclinical TB.A different angle was used for the third approach to the problem of LTBI in PLHIV. Systemic immune activation (SIA) is one of the principal driving forces in the natural course of HIV-infection. Despite long-term viral suppression by combination antiretroviral treatment (cART), a low-level SIA persists and is associated with an early-onset of age-associated disorders such as cardiovascular disease, dementia and osteoporosis. Causes of SIA in PLHIV are multiple and certain chronic infections appear to be implicated. A recent study in South Africa found that LTBI in PLHIV was associated with an increase in circulating activated CD8+ T-cells. If LTBI should contribute to the persistence of SIA, its screening and treatment could have an additional benefit on the clinical outcome of PLHIV. To investigate this theory, the expression of T-cell activation markers (CD38 and HLADR) as well as the level of plasmatic markers of immune activation (IL-6, sCD14, D-Dimers) were compared between subjects presenting active TB, subjects with LTBI and M. tuberculosis-free persons, with and without HIV-infection. In accordance with previous studies, active TB was associated with higher levels of SIA biomarkers in both HIV-infected and -uninfected groups. Among the HIV-uninfected subjects, no significant difference in biomarker level was found between those presenting LTBI and those with no evidence of M. tuberculosis. The effect of LTBI on activation biomarkers in the HIV-infected groups remained inconclusive because of the small number of individuals in the HIV+/LTBI group. Further investigation is therefore warranted. Interestingly, it was found that plasmatic markers may have a greater sensitivity for the detection of M. tuberculosis-associated SIA than the T-cell activation markers, an important result for future studies.Overall, LTBI in PLHIV is a challenging topic, in particular because of the lack of a gold-standard for the diagnosis of LTBI. Despite suboptimal tools, the evident clinical impact of LTBI screening and treatment in PLHIV on TB incidence justifies its implementation in standard HIV-care. In low TB-incidence countries, who, when and how to screen for LTBI in PLHIV remains unclear. This work offers an overview on the subject with particular focus on possible measures for improvement in the field. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished