Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Radiographic features of pulmonary tuberculosis in patients infected by HIV: is there an objective indicator of co-infection?

This study aimed to compare the radiographic characteristics of patients with pulmonary tuberculosis (TB) and human immunodeficiency virus (HIV) infection with those of HIV-negative patients. In all, 275 TB patients attending the outpatients clinics at the University Hospital/UFPE, were studied from January 1997 to March 1999. Thirty nine (14.2%) of them were HIV+, with a higher frequency of males in this group (p=0.044). Seventy-five percent of the HIV+ patients and 19% of the HIV- had a negative tuberculin test (PPD) (p < 0.001). The proportion of positive sputum smears in the two groups was similar. The radiological finding most strongly associated with co-infection was absence of cavitation (p < 0.001). It may therefore be concluded that the lack of cavitation in patients with pulmonary TB may be considered a useful indicator of the need to investigate HIV infection. This approach could contribute to increasing the effectiveness of local health services, by offering appropriate treatment to co-infected patients.

Low sensitivity of polymerase chain reaction for diagnosis of tuberculous meningitis in southeastern Brazil

Two polymerase chain reaction (PCR) protocols showed low sensitivity (36% and 53% for TB AMPLICOR and MPB64 nested PCR, respectively), when compared with classic microbiological methods (73% and 54% for Ziehl-Neelsen staining and culture, respectively), in the diagnosis of tuberculous meningitis in 91 patients in southeastern Brazil. Only three PCR-positive, microbiologically negative patients were found. Analysis of sequential cerebrospinal fluid samples by nested PCR detected Mycobacterium tuberculosis DNA up to 29 days after the introduction of antituberculosis chemotherapy.

Intestinal nematodes and pulmonary tuberculosis

We report a significantly higher prevalence of intestinal nematodes in patients with pulmonary tuberculosis (TB) compared to a matched control group: 33/57 (57.8%) in patients with TB and 18/86 (20.9%) in the control group; OR=5.19; 95% CI= 2.33-11.69; p=0.000). When TB patients eosinophilia was also significantly higher among those with intestinal parasites (69.8%) compared to those without this condition (45.6%). We hypothesized that the immune modulation induced by nematodes is a factor that enhances TB infection/progression and that eosinophilia seen in TB patients is a consequence of helminth infection.

Characteristics of pulmonary tuberculosis in HIV seropositive and seronegative patients in a Northeastern region of Brazil

The aim of this study was to analyse the clinical, epidemiological and bacteriological features present in 60 pulmonary tuberculosis patients who were also infected with human immunodeficiency virus (HIV) and to compare these with 120 TB patients who were not infected with HIV. The patients with pulmonary tuberculosis and HIV coinfection were mostly male (p = 0.001), showed a higher frequency of weight loss >10 kilos (p <0.001), had a higher rate of non-reaction result to the tuberculin skin test (p <0.001), a higher frequency of negative sputum smear examination for acid-fast bacilli (p = 0.001) and negative sputum culture for Mycobacterium tuberculosis (p = 0.001). Treatment failure was more common in those who were HIV positive (p <0.000). No higher frequency of resistance to antituberculosis drugs was found to be associated with TB/HIV coinfection (p = 0.407). Association between extrapulmonary and pulmonary tuberculosis was more frequent in those seropositive to HIV than those without HIV virus, 30% and 1.6% respectively. These findings showed a predominance of atypical clinical laboratory features in co-infected patients, and suggest that health care personnel should consider the possibility this diagnosis.

Prevalence and incidence of Mycobacterium tuberculosis infection in nursing students in Vitória, Espírito Santo

This prospective study evaluated the incidence of Mycobacterium tuberculosis infection among nursing students at the Federal University of Espírito Santo, using the purified protein derivative test. In parallel, we evaluated whether knowledge on tuberculosis transmission mechanisms had any impact on nursing students' attitudes in relation to the risk of tuberculosis infection. The incidence of tuberculin conversion was 10.5% per year (p=0.035, 95% CI = 3.63-17.43), whereas the expected conversion rate in the overall population in Brazil is 0.5% per year. These results indicate nursing students as a group at risk for TB infection. Among the risk factors studied only the use of NIOSH95 masks was associated with protection against infection (RR=0.2). Furthermore there was no statistical difference among students that PPD converted and those who remained negative regarding disease knowledge and the existence of adequate facilities for patient hospitalization. Our data reinforces the need for implementing special biosafety programs targeting this professional group.

Análise espacial da co-infecção tuberculose/HIV: relação com níveis socioeconômicos em município do sudeste do Brasil

INTRODUÇÃO: Analisar espacialmente a co-infecção tuberculose/vírus da imunodeficiência humana e associá-la com variáveis socioeconômicos, São José do Rio Preto, SP, 1998-2006. MÉTODOS: Foram geocodificados casos novos de TB/HIV e calculados coeficientes de incidência segundo unidades espaciais. Utilizou-se o índice de Moran para avaliar a dependência espacial das incidências. Regressões múltiplas foram realizadas para selecionar variáveis com maior poder de explicação da dependência espacial. O indicador local de associação espacial foi utilizado para identificação de agrupamentos espaciais significantes. RESULTADOS: O índice de Moran foi de 0,0635 (p = 0,0000), indicando ocorrência de dependência espacial. A variável que apresentou maior poder de explicação da dependência espacial da incidência foi a porcentagem de chefes de família com até três anos de instrução. O LISA cluster map para os coeficientes de incidência de co-infecção TB/HIV evidenciou aglomerados de alta incidência na região norte e baixa incidência na sul e oeste do município. CONCLUSÕES: O estudo possibilitou a compreensão da distribuição geográfica espacial da co-infecção TB/HIV no município e apontou a sua associação com variáveis socioeconômicas dando subsídios para o planejamento orientado para a priorização das regiões com maior carência social e consequentemente maiores incidências da doença.

Estudo clínico-radiológico de crianças e adolescentes indígenas Suruí, Região Amazônica

INTRODUÇÃO: Entre os Suruí de Rondônia foram registradas incidências médias de TB > 2.500/100.000 habitantes, entre 1991-2002. Aproximadamente 50% desses casos foram notificados em < 15 anos. MÉTODOS: Trata-se de um estudo clínico-epidemiológico que teve como objetivo descrever as características clínico-radiológicas em crianças e adolescentes identificados como contatos de doentes de TB. Além disto, aplicar o sistema de pontuação para o diagnóstico de TB na infância e verificar se as condutas adotadas no nível local foram concordantes com as diretrizes nacionais. RESULTADOS: Foram analisados 52 Rx de 37 indígenas. Deste conjunto, 48,1% foram normais e 51,9% anormais. Alguns dos Rx apresentaram duas ou mais alterações, totalizando 36 eventos independentes. Observou-se infiltrados (38,9%), calcificações (38,9%), cavitações (11,1%) e atelectasias/derrame pleural (11,1%). Nas imagens anormais, 22,2% eram TB provavelmente ativa e 33,3% sequelas. A confrontação com as diretrizes constatou 52,6% de condutas discordantes. CONCLUSÕES: A presença da infecção tuberculosa latente (ITBL) e TB ativa, entre crianças e adolescentes, são indicadores de transmissão ativa e continuada do Mycobacterium tuberculosis. Os Rx mostrando alta frequência de infiltrados e calcificações é compatível com primo-infecção em idade precoce. Entretanto, essas alterações não são diferentes daquelas observadas entre outros grupos, não sugerindo comprometimento imunológico. As discordâncias apontadas indicam que o momento ideal para o tratamento da ITBL passou despercebido. Conclui-se que é fundamental a utilização do sistema de pontuação para o correto diagnóstico de TB na infância, assim como a realização de baciloscopia e cultura de escarro em adolescentes capazes de expectorar.

Co-infection and risk factors of tuberculosis in a Mexican HIV+ population

INTRODUCTION: The situation of tuberculosis (TB) is being modified by the human immunodeficiency virus (HIV), which is increasing the occurrence of new cases and the generation of drug resistant strains, affecting not only the people infected with HIV, but also their close contacts and the general population, conforming a serious public health concern. However, the magnitudes of the factors associated to this co-infection differ considerably in relation to the population groups and geographical areas. METHODS: In order to evaluate the prevalence and risk factors for the co-infection of tuberculosis (TB) in a population with human immunodeficiency virus (HIV+) in the Southeast of Mexico, we made the analysis of clinical and epidemiological variables and the diagnosis of tuberculosis by isolation of mycobacteria from respiratory samples. RESULTS: From the 147 HIV+ individuals analyzed, 12 were culture positive; this shows a prevalence of 8% for the co-infection. The only variable found with statistical significance for the co-infection was the number of CD4-T < 200 cells/mm³, OR 13 (95%, CI 2-106 vs 12-109). CONCLUSIONS: To our knowledge this is the first report describing the factors associated with tuberculosis co -infection with HIV in a population from Southern Mexico. The low number of CD4 T-cells was the only variable associated with the TB co-infection and the rest of the variables provide scenarios that require specific and particular interventions for this population group.

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

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

Caracterização da tuberculose multidroga resistente durante a gravidez, em Campinas, Estado de São Paulo, Brasil, no período de 1995 a 2007

INTRODUÇÃO: Foi feito um acompanhamento das gestantes tratadas para tuberculose multidroga resistente (TB-MDR) durante a gravidez, em Campinas, Estado de São Paulo, no período de 1995 a 2007. MÉTODOS: No estudo retrospectivo, foram incluídas pacientes com cepa resistente, pelo menos à isoniazida e à rifampicina e gravidez em qualquer momento, durante o tratamento. Os casos receberam esquema individualizado baseado nos padrões de resistência medicamentosa e nos antecedentes de tratamento de cada paciente. RESULTADOS: Sete gestantes apresentaram resistência a duas ou mais drogas. Três já estavam grávidas, antes do início do tratamento e quatro engravidaram depois. Duas estavam com Aids, uma foi a óbito e em outra ocorreu falência de tratamento. Todas apresentaram tuberculose avançada com achados radiológicos graves e para cinco pacientes o tempo médio da conversão do escarro foi de quatro meses. Duas gestantes foram curadas. Dois recém-nascidos foram infectados com o HIV por suas progenitoras. CONCLUSÕES: O resultado do tratamento foi insatisfatório e a supervisão direta é imperativa na TB-MDR durante a gestação. O planejamento familiar deve ser fortemente recomendado.

Synthesis of Polymeric Nanoparticles for biomedical delivery applications

Polymeric nanoparticles (PNPs) have attracted considerable interest over the last few years due to their unique properties and behaviors provided by their small size. Such materials could be used in a wide range of applications such as diagnostics and drug delivery. Advantages of PNPs include controlled release, protection of drug molecules and its specific targeting, with concomitant increasing of the therapeutic index. In this work, novel sucrose and cholic acid based PNPs were prepared from different polymers, namely polyethylene glycol (PEG), poly(D,L-lactic-co-glycolic acid) (PLGA) and PLGA-co-PEG copolymer. In these PNP carriers, cholic acid will act as a drug incorporation site and the carbohydrate as targeting moiety. The uptake of nanoparticles into cells usually involves endocytotic processes, which depend primarily on their size and surface characteristics. These properties can be tuned by the nanoparticle preparation method. Therefore, the nanoprecipitation and the emulsion-solvent evaporation method were applied to prepare the PNPs. The influence of various parameters, such as concentration of the starting solution, evaporation method and solvent properties on the nanoparticle size, size distribution and morphology were studied. The PNPs were characterized by using atomic force microscopy (AFM), scanning electron microscopy (SEM) and dynamic light scattering (DLS) to assess their size distribution and morphology. The PNPs obtained by nanoprecipitation ranged in size between 90 nm and 130 nm with a very low polydispersity index (PDI < 0.3). On the other hand, the PNPs produced by the emulsion-solvent evaporation method revealed particle sizes around 300 nm with a high PDI value. More detailed information was found in AFM and SEM images, which demonstrated that all these PNPs were regularly spherical. ζ-potential measurements were satisfactory and evidenced the importance of sucrose moiety on the polymeric system, which was responsible for the obtained negative surface charge, providing colloidal stability. The results of this study show that sucrose and cholic acid based polymeric conjugates can be successfully used to prepare PNPs with tunable physicochemical characteristics. In addition, it provides novel information about the materials used and the methods applied. It is hoped that this work will be useful for the development of novel carbohydrate based nanoparticles for biomedical applications, specifically for targeted drug delivery.

Drug resistance of Mycobacterium tuberculosis strains in southern Brazil

INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT) strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB) cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA) from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2% (11/120); 5% (6/120) were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7) of the eleven (11) samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01%), which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.

Spatio-temporal patterns of tuberculosis incidence in Ribeirão Preto, State of São Paulo, southeast Brazil, and their relationship with social vulnerability: a Bayesian analysis

INTRODUCTION: The purpose of this ecological study was to evaluate the urban spatial and temporal distribution of tuberculosis (TB) in Ribeirão Preto, State of São Paulo, southeast Brazil, between 2006 and 2009 and to evaluate its relationship with factors of social vulnerability such as income and education level. METHODS: We evaluated data from TBWeb, an electronic notification system for TB cases. Measures of social vulnerability were obtained from the SEADE Foundation, and information about the number of inhabitants, education and income of the households were obtained from Brazilian Institute of Geography and Statistics. Statistical analyses were conducted by a Bayesian regression model assuming a Poisson distribution for the observed new cases of TB in each area. A conditional autoregressive structure was used for the spatial covariance structure. RESULTS: The Bayesian model confirmed the spatial heterogeneity of TB distribution in Ribeirão Preto, identifying areas with elevated risk and the effects of social vulnerability on the disease. We demonstrated that the rate of TB was correlated with the measures of income, education and social vulnerability. However, we observed areas with low vulnerability and high education and income, but with high estimated TB rates. CONCLUSIONS: The study identified areas with different risks for TB, given that the public health system deals with the characteristics of each region individually and prioritizes those that present a higher propensity to risk of TB. Complex relationships may exist between TB incidence and a wide range of environmental and intrinsic factors, which need to be studied in future research.

Contributions of culture and antimicrobial susceptibility tests to the retreatment of patients with pulmonary tuberculosis

Introduction This study evaluated the efficacy of retreatment of pulmonary tuberculosis (TB) with regard to treatment outcomes and antimicrobial susceptibility testing (ST) profiles. Methods This retrospective cohort study analyzed 144 patients treated at a referral hospital in Brazil. All of them had undergone prior treatment, were smear-positive for TB and received a standardized retreatment regimen. Fisher's 2-tailed exact test and the χ2 test were used; RRs and 95% CIs were calculated using univariate and multivariate binary logistic regression. Results The patients were cured in 84 (58.3%) cases. Failure was associated with relapsed treatment and abandonment (n=34). Culture tests were obtained for 103 (71.5%) cases; 70 (48.6%) had positive results. ST results were available for 67 (46.5%) cases; the prevalence of acquired resistance was 53.7%. There were no significant differences between those who achieved or not therapeutic success (p=0.988), despite being sensitive or resistant to 1 or more drugs. Rifampicin resistance was independently associated with therapeutic failure (OR: 4.4, 95% CI:1.12-17.37, p=0.034). For those cases in which cultures were unavailable, a 2nd model without this information was built. In this, return after abandonment was significantly associated with retreatment failure (OR: 3.59, 95% CI:1.17-11.06, p=0.026). Conclusions In this cohort, the general resistance profile appeared to have no influence on treatment outcome, except in cases of rifampicin resistance. The form of reentry was another independent predictor of failure. The use of bacterial culture identification and ST in TB management must be re-evaluated. The recommendations for different susceptibility profiles must also be improved.