Nesse trabalho foi desenvolvido um Teste Colorimétrico de Hibridização Reversa (TCHR-TB) que verifica a presença de mutações em uma região específica do gene rpoB, que confere resistência à rifampicina. Foram analisados 156 DNAs de M. tuberculosis obtidos de cultura. Quando comparado com teste convencional de susceptibilidade às drogas, a sensibilidade e a especificidade do TCHR-TB foi 92.3% e 98.0% respectivamente. Comparando com o seqüenciamento, o qual é considerado padrão ouro, a sensibilidade e a especificidade do TCHR-TB foram 90% e 100%, respectivamente. Mutações no códon 531 seguido de mutações no códon 526 são as mutações mais comuns mundialmente, sendo responsáveis por aproximadamente 75% das amostras resistentes à RIF. O TCHR-TB detectou corretamente 100% destas mutações. O método desenvolvido pôde também detectar mutações no gene rpoB de DNA de M. tuberculosis extraídos diretamente de amostras clínicas. Trinta e três amostras clinicas foram testadas e os resultados do TCHR-TB foram 100% concordantes quando comparados com método das proporções. Os resultados do nosso estudo demonstraram uma alta taxa de concordância do TCHR-TB quando comparado com os testes de sensibilidade convencionais e com o seqüenciamento. O teste pode ser aplicado com sucesso quando se faz necessária uma rápida e sensível detecção de resistência à rifampicina e conseqüentemente um correto gerenciamento dos pacientes. / In this work a Reverse-Line Blot Hybridization (RLBH) assay was developed which allows the detection of mutation in a specific region of rpoB gene, that confer rifampicin resistance, in a panel of 156 DNAs of M. tuberculosis obtained from cultures. When compared to the conventional drug susceptibility testing (DST), sensitivity and specificity of the RLBH were 92.3% and 98.0%, respectively. Comparing to sequencing, which is considered a “gold standard” reference, sensitivity and specificity of the RLBH were 90% and 100%, respectively. Mutations at codon 531, followed by mutation at codon 526 are the most common mutations worldwide accounting for approximately 75% of RIF resistance. The RLBH assay correctly detected 100% of these mutations. The method developed could also detect rpoB mutations of M. tuberculosis in clinical specimens. Thirty-three clinical specimens were tested and the results of RLBH were 100% concordant when compared to DST. The results of our study demonstrate a high concordance rate when the RLBH results were compared to conventional methods and sequencing data. The test can be successfully applied when a rapid sensitivity testing is required for the correct management of patients.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-01-05T12:31:55Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_InvestigacaoPolimorfimosGenes.PDF: 1141933 bytes, checksum: 471a75c00025493bea9afec0bcc8cf90 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-01-09T17:53:54Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_InvestigacaoPolimorfimosGenes.PDF: 1141933 bytes, checksum: 471a75c00025493bea9afec0bcc8cf90 (MD5) / Made available in DSpace on 2017-01-09T17:53:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_InvestigacaoPolimorfimosGenes.PDF: 1141933 bytes, checksum: 471a75c00025493bea9afec0bcc8cf90 (MD5) Previous issue date: 2015-11-06 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Tuberculose pulmonar é uma doença infectocontagiosa de transmissão por via aérea, que segundo a Organização Mundial de Saúde (OMS), infecta cerca dois bilhões de pessoas ao redor do mundo. É a principal causa de morte por doença infecciosa em adultos nos países em desenvolvimento, representando um grave problema de saúde pública, devido principalmente, a não aderência ao tratamento, ao diagnóstico tardio e subdiagnóstico e ao não controle de contatos, o que faz com que a população continue susceptível à infecção. No presente estudo se objetivou investigar associações de três polimorfismos genéticos nos genes IFNɣ e INFGR1, responsáveis pela suscetibilidade à tuberculose em pacientes acometidos pela doença; avaliar se ocorrem diferenças nas frequências alélicas e genotípicas dos polimorfismos nos genes IFNɣ871A>T, INFGR1611(C>T) e INFGR1 -56(A>G) entre indivíduos com tuberculose e indivíduos sem tuberculose da população de Belém. O controle de efeito de subestruturação foi realizado pelo emprego de um painel de 48 Marcadores Informativos de Ancestralidade Genética, tanto na amostra de pacientes como na amostra controle. Para realização deste estudo nos utilizamos amostras de sangue periférico de 148 pacientes diagnosticados com tuberculose,e 125 indivíduos sem tuberculose (controles) residentes no Estado do Pará, Brasil, atendidos no Hospital Universitário João de Barros Barreto (HUJBB) durante o período de 2006 a 2012.De cada indivíduo foram obtidos 5mL de sangue venoso colhido de veia periférica.A extração de DNA foi realizada segundo método descrito por Sambrook et al., (1989).A genotipagem dos polimorfismos para os genes IFNɣ(rs1130562) e INFGR1 (rs1327474, rs2234711) foi realizada por técnica de PCR em tempo real (Rtq-PCR) utilizando-se o sistema TaqMan.As análises estatísticas foram realizadas nos softwares SPSS 17.0, usando o teste de Mann-Whitney, considerando como significantes valores de p<0,05. Os resultados obtidos não demonstraram significância dos polimorfismos investigados em relação a suscetibilidade para tuberculose. / Pulmonary tuberculosis is an infectious disease transmission by air, which according to the World Health Organization (WHO), infects about two billion people around the world. It is the leading cause of death from infectious disease in adults in developing countries, representing a serious public health problem, mainly due to non-adherence to treatment, late diagnosis and underdiagnosis and no control contacts, which makes our population susceptible to infection. The present study aimed to investigate associations three genetic polymorphisms in IFNɣ and INFGR1 genes responsible for susceptibility to tuberculosis in patients affected by the disease; evaluate if there are differences in allelic and genotypic frequencies of polymorphisms in genes IFNɣ 871A> T, INFGR1 611 (C> T) and INFGR1 -56 (A> G) among individuals with TB and those without TB population of Bethlehem. The control substructures effect was carried out by the use ofa 48 markers Informational Genetic Ancestry panel in both patient sample and the control sample. For this study we used peripheral blood samples from 148 patients diagnosed with tuberculosis, and 125 individuals without tuberculosis (controls) resident in the State of Pará, Brazil, attended at University Hospital João de Barros Barreto (HUJBB) during the period from 2006 to 2012. each specimen was obtained 5 ml of venous blood collected from a peripheral vein. DNA extraction was performed according to the method described by Sambrook et al., (1989). Genotyping for polymorphisms IFNɣ gene (rs1130562) and INFGR1 (rs1327474, rs2234711) was performed by PCR in real time (RTQ-PCR) using the TaqMan system. Statistical analyzes were performed in SPSS 17.0 software, using the Mann- Whitney test, with significance set at p <0.05. The results did not show significance of the polymorphisms investigated in relation to susceptibility to tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic Mtb:ΔRv2745c mutant is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb, implicating a role for ClgR in the management of intraphagosomal redox stress. Our data indicates that ClgR plays a role in multiple regulatory networks in response to different stress conditions. Thus, redox stress leads to dysregulation of the σH/σE regulon in Mtb:ΔRv2745c. Induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions. Disruption of genes involved in sulfate assimilation also occurred in the knock out, implicating clgR as a possible regulator of downstream signaling cascades that facilitate Mtb survival. On the other hand, the expression of clgR during hypoxia is known to result in Clp protease induction. As such, the isogenic mutant has a significantly different growth profile upon hypoxia and reaeration. Transcriptomics reveal disruption of the dosR regulon, σH/σE regulon, and mycolic acid synthesis genes. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in vivo. Our in vivo findings in a low dose aerosolized model reveal deficiencies of the isogenic mutant when establishing an infection, leading to skewed immune responses throughout the course of infection. Thus, clgR plays a critical role in both establishing an infection that influence the immunogenic outcome. Additional studies investigating the role of clgR in a nonhuman primate model will further elucidate the contributions of clgR to the pathogenesis of Mtb in an animal model that is more representative of human TB disease. / acase@tulane.edu

In paleopathology, the assessment of disease occurs through macroscopic observation, which is dependent on the preservation of the sample and the experience of the observer. Many disease events do not leave any visible signatures and therefore go undetected. The relatively new field of paleomicrobiology incorporates molecular techniques where microbial DNA, if present, is amplified from an archaeological sample. The identification of genetic material from micro-organisms, including bacteria and viruses, can confirm a diagnosis that was originally based on visible osteological or mummified tissue changes. Even more promising is the capability of molecular technology to detect microbial DNA evidence of disease processes that were not visibly evident.<p> Based on phylogenetic analyses of modern isolates, scientists have concluded that micro-organisms such as <i>Mycobacterium tuberculosis</i> and <i>Helicobacter pylori</i> have been associated with humans for thousands of years. <i>M. tuberculosis</i> is the causative agent of the disease tuberculosis, and <i>H. pylori</i> is known for its role in gastritis and peptic ulcers. Both are pathogenic bacteria that still impact the health of modern populations. Through the analysis of microbial DNA from these two bacteria in skeletal and mummified tissue, data can be accumulated regarding the spatial and temporal impact of these infections. Interestingly, due to the lengthy association between these bacteria and humans, phylogenetic studies on modern strains have shown that strain characterizations of both <i>M. tuberculosis</i> and <i>H. pylori</i> bacteria reveal connections with past human migrations.<p> In 1999, human remains were discovered eroding out of a glacier in northern British Columbia, Canada on the traditional territory of the Champagne and Aishihik First Nations. The Aboriginal elders named the site Kwäday Dän Tsìnchi, which means long ago person found. Radiocarbon testing of bone collagen and artifacts from the site suggested a time-frame of approximately AD 1670 to 1850, which is either pre-European contact or early post-contact for that area. I analyzed the tissues of the ancient individual specifically for genetic evidence of <i>M. tuberculosis</i> and <i>H. pylori</i> to identify partial health status and determine if a connection could be made to strains associated with European populations to clarify whether the site was pre or post-European contact.<p> Through polymerase chain reaction (PCR) testing of the individuals tissues with primers specific for the IS<i>6100</i> insertion sequence, <i>TbD1</i>, and <i>Rv3479</i>, <i>katG</i> and <i>gyrB</i> genes, I identified evidence of a possible latent tuberculosis infection. Genetic characterization of the <i>katG</i> gene associated with the ancient <i>M. tuberculosis</i> strain revealed a potential connection with European strains. Amplification and sequencing of the <i>gyrB</i> gene fragment indicated the presence of two alleles that may have been the result of a selective pressure.<p> PCR testing of the individuals stomach tissue with specific primers for regions with the <i>vacA</i> gene resulted in a positive identification of <i>H. pylori</i> DNA. Genetic characterization of this virulence-associated gene indicated that the strain contained a <i>vacA</i> signal (s) region s2 allele. This allele is more commonly identified in Western strains that do not cause disease, which suggests that the individual had no gastric symptoms and that European strains were present in northwestern Canada at that time. The vacA middle (m) region contained a hybrid m2a/m1d sequence. Modern hybrids are rare but they have been identified in Asian strains. Studies have shown that the m2a allele is more common in Western strains. A phylogenetic analysis identified that the m1d region clusters with previously published novel strains associated with Aboriginal individuals that are closely related to Asian strains. This indicates a past connection between the ancient individual and his ancestors who arrived in the New World from Asia thousands of years ago.

In paleopathology, the assessment of disease occurs through macroscopic observation, which is dependent on the preservation of the sample and the experience of the observer. Many disease events do not leave any visible signatures and therefore go undetected. The relatively new field of paleomicrobiology incorporates molecular techniques where microbial DNA, if present, is amplified from an archaeological sample. The identification of genetic material from micro-organisms, including bacteria and viruses, can confirm a diagnosis that was originally based on visible osteological or mummified tissue changes. Even more promising is the capability of molecular technology to detect microbial DNA evidence of disease processes that were not visibly evident.<p> Based on phylogenetic analyses of modern isolates, scientists have concluded that micro-organisms such as <i>Mycobacterium tuberculosis</i> and <i>Helicobacter pylori</i> have been associated with humans for thousands of years. <i>M. tuberculosis</i> is the causative agent of the disease tuberculosis, and <i>H. pylori</i> is known for its role in gastritis and peptic ulcers. Both are pathogenic bacteria that still impact the health of modern populations. Through the analysis of microbial DNA from these two bacteria in skeletal and mummified tissue, data can be accumulated regarding the spatial and temporal impact of these infections. Interestingly, due to the lengthy association between these bacteria and humans, phylogenetic studies on modern strains have shown that strain characterizations of both <i>M. tuberculosis</i> and <i>H. pylori</i> bacteria reveal connections with past human migrations.<p> In 1999, human remains were discovered eroding out of a glacier in northern British Columbia, Canada on the traditional territory of the Champagne and Aishihik First Nations. The Aboriginal elders named the site Kwäday Dän Tsìnchi, which means long ago person found. Radiocarbon testing of bone collagen and artifacts from the site suggested a time-frame of approximately AD 1670 to 1850, which is either pre-European contact or early post-contact for that area. I analyzed the tissues of the ancient individual specifically for genetic evidence of <i>M. tuberculosis</i> and <i>H. pylori</i> to identify partial health status and determine if a connection could be made to strains associated with European populations to clarify whether the site was pre or post-European contact.<p> Through polymerase chain reaction (PCR) testing of the individuals tissues with primers specific for the IS<i>6100</i> insertion sequence, <i>TbD1</i>, and <i>Rv3479</i>, <i>katG</i> and <i>gyrB</i> genes, I identified evidence of a possible latent tuberculosis infection. Genetic characterization of the <i>katG</i> gene associated with the ancient <i>M. tuberculosis</i> strain revealed a potential connection with European strains. Amplification and sequencing of the <i>gyrB</i> gene fragment indicated the presence of two alleles that may have been the result of a selective pressure.<p> PCR testing of the individuals stomach tissue with specific primers for regions with the <i>vacA</i> gene resulted in a positive identification of <i>H. pylori</i> DNA. Genetic characterization of this virulence-associated gene indicated that the strain contained a <i>vacA</i> signal (s) region s2 allele. This allele is more commonly identified in Western strains that do not cause disease, which suggests that the individual had no gastric symptoms and that European strains were present in northwestern Canada at that time. The vacA middle (m) region contained a hybrid m2a/m1d sequence. Modern hybrids are rare but they have been identified in Asian strains. Studies have shown that the m2a allele is more common in Western strains. A phylogenetic analysis identified that the m1d region clusters with previously published novel strains associated with Aboriginal individuals that are closely related to Asian strains. This indicates a past connection between the ancient individual and his ancestors who arrived in the New World from Asia thousands of years ago.

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. The high incidence is still found in Taiwan. There is strong evidence that host genes influence individual susceptibility to tuberculosis. Young children, like immunocompromised patients, once infected are at increased risk for TB disease and progression to extrapulmonary disease. Thus far, to identify the genes responsible for the variation in the human susceptibility/resistance to TB has remained elusive. Mannose-binding lectin (MBL) activates the complement system in an antibody-independent manner, enhances complement-mediated phagocytosis, and plays an important role in innate immunity in the regulation of inflammatory cytokine release by monocytes. It is one of the molecules that have been suggested to have a link to human susceptibility or protection against infection. According to some studies (mostly conducted in adult populations) , low levels of MBL associated with variant alleles at the promoter and exon 1 regions of MBL protect against tuberculosis. Other investigators instead claim that protection against the disease is associated with high levels of MBL. In this study we aimed to investigate the relationships between the susceptibility to TB and MBL gene polymorphisms in children with cervical mycobacterial lymphadenitis infected by M. tuberculosis.139 case patients with cervical mycobacterial lymphadenitis and 102 unrelated healthy control subjects were tested by real-time PCR for polymorphisms at the promoter and the exon 1 regions of the MBL gene. Diagnosis of mycobacterial lymphadenitis infected by M. tuberculosis, based on findings of pathological examination of the lymph nodes, was confirmed by acid-fast stain and TB PCR.The frequency of A allele was significantly higher in TB+ patients compared with TB- controls (82.7% vs 72.6%; odds ratio 1.813; p=0.007). The frequency of high-producer MBL2 genotypes (A/A) was higher in TB+ patients than in TB- subjects (70.5% vs 45.1%, odds ratio 2.91, p<0.001), while patients carried the B alleles (A/B and B/B) that have decreased levels of MBL was inversely associated with mycobacterial infectivity (29.5% vs 54.9%; odds ratio 2.910; p<0.001). The frequencies of MBL promoter -550 genotypes also revealed a significant difference between TB+ and TB- groups (p = 0.046), but in contrast, with significantly higher frequency of L/L genotype (of low MBL level) in TB+ patients (34.5% vs 21.6%; odds ratio 1.918; p=0.029). The frequencies of MBL promoter -221 genotypes (X and Y) was similar in TB+ and TB- groups.This study supports the conclusion that MBL can protect or predispose the host to tuberculosis, depending on the host¡¦s haplotype pair.

Thesis (Ph. D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on Nov. 25, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Chemistry, University of Alberta." Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 41-49).

Thesis (PhD (Veterinary Tropical Diseases, Veterinary Science))--University of Pretoria, 2009.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences