A tuberculose (TB) embora passível de tratamento efetivo, ainda é um grave problema de saúde pública em diversos países, inclusive no Brasil. Nas últimas décadas houve progressos consistentes no controle da doença, porém o avanço da resistência bacilar ainda é um desafio a ser superado, já que os mecanismos da resistência são bastante complexos e não totalmente conhecidos, o que dificulta o desenvolvimento de testes de sensibilidade com elevada acurácia. O objetivo deste trabalho foi caracterizar as mutações gênicas de cepas de Mycobacterium tuberculosis de pacientes do Brasil e de Moçambique com doença resistente a drogas através do sequenciamento genômico total, além de descrever padrões de mutações obtidos por testes moleculares comerciais e comparar estes dados com resultados de testes fenotípicos. Estudo descritivo e transversal que incluiu 30 isolados (17 do Brasil e 13 de Moçambique), submetidos aos testes moleculares comerciais Genotype MTBDRplus®, Genotype MTBDRsl®, Xpert MTB/RIF® e teste fenotípico BACTEC MGIT 960 SIRE®. Todos os isolados também foram avaliados pelo sequenciamento genômico realizado pelo Illumina MiSeq Sequencing System® e submetidos a análise de mutações que conferem resistência às drogas contra TB utilizando o TB profiler online tool. A sensibilidade e especificidade do sequenciamento genômico para detecção de resistência a rifampicina foi de 87,5% e 92,3%, respectivamente. Além disso, o sequenciamento detectou a mutação (Val170Phe) no gene rpoB em dois isolados de M. tuberculosis de Moçambique. Esta mutação não é detectada pelos testes genotípicos comerciais. A sensibilidade do sequenciamento para a isoniazida foi de 95,6% e a especificidade de 100%. Para a estreptomicina, a sensibilidade foi de 85,7% e a especificidade de 93,3%. Para o etambutol, observamos sensibilidade de 100% e especificidade de 77,2%. As mutações mais frequentes associadas à resistência à rifampicina foram a Ser450Leu e a His445Tyr no gene rpoB. Em relação à isoniazida, predominou a mutação Ser315Thr no gene katG. O sequenciamento genômico, dado seu alto poder discriminatório, tem grande potencial de fornecer informações mais acuradas sobre mecanismo gênicos da resistência bacilar, possibilitando futuramente o aprimoramento de testes diagnósticos mais precisos. / Although there is an effective treatment for tuberculosis (TB), it is still a serious public health problem in several countries, including Brazil. In the last decades, there has been consistent progress in disease control, but the increasing number of disease caused by resistant strains is still a challenge to be overcome, since the mechanisms of resistance are quite complex and not fully known, which difficult the development of susceptibility tests with high accuracy. The aim of this work was to characterize gene mutations of Mycobacterium tuberculosis strains from Brazilian and Mozambican patients with drug-resistant disease through whole genome sequencing, as well as to describe patterns of mutations obtained by commercial molecular tests and to compare these data with results of phenotypic susceptibility tests. It was a cross-sectional study that included 30 isolates (17 from Brazil and 13 from Mozambique). Commercial molecular tests Genotype MTBDRplus(TM), Genotype MTBDRsl(TM), Xpert MTB / RIF(TM) and BACTEC MGIT 960 SIRE(TM) phenotypic test were performed for all isolates. All of them were also evaluated by whole genome sequencing performed by the Illumina MiSeq Sequencing System(TM) and submitted to analysis of mutations that confer drug resistance against TB using the TB profiler online tool. The sensitivity and specificity of whole genome sequencing for detection rifampicin resistance was 87.5% and 92.3%, respectively. Also, whole genome sequencing detected the mutation (Val170Phe) in the rpoB gene in two isolates of M. tuberculosis from Mozambique. This mutation is not detected by commercial genotypic tests. The sensitivity of the whole genome sequencing for isoniazid was 95.6%, and the specificity was 100%. For streptomycin, the sensitivity was 85.7%, and the specificity was 93.3%. For ethambutol, we observed a sensitivity of 100% and specificity of 77.2%. The most frequent mutations associated with rifampicin resistance were rpoB Ser450Leu and His445Tyr. About isoniazid, the katG Ser315Thr mutation was the most frequent. Whole genome sequencing, given its high discriminatory power, has great potential to provide more accurate information about the gene mechanisms of bacilli resistance, making possible the improvement of more accurate diagnostic tests in the future.

Pacientes com epilepsia apresentam comorbidades que podem afetar a sua qualidade de vida e a sua adequação social. Por esta razão há um interesse crescente pelo estudo das condições associadas à epilepsia. Pacientes com epilepsia apresentam maior risco de desenvolver transtornos de sono. Entretanto, a relação entre o sono e a epilepsia permanece negligenciada. Os objetivos deste estudo foram: (i) avaliar o cronotipo, a presença de sonolência diurna e a qualidade do sono, através de medidas paramétricas, comparando pacientes farmacorresistentes e farmacossensíveis, e (ii) verificar a associação entre estes fatores e a qualidade de vida e o funcionamento social. Para tal, nós entrevistamos 114 pacientes (58 pacientes com epilepsia farmacossensível e 56 pacientes farmacorresistentes) e 62 indivíduos saudáveis sem epilepsia. Os grupos foram pareados por idade e sexo Todos os indivíduos entrevistados foram submetidos a questionários de autopreenchimento que verificam o cronotipo (Questionário de Matutinidade e Vespertinidade), a qualidade do sono (Índice de Qualidade de Sono de Pittsburgh) e a sonolência diurna (Escala de Sonolência de Epworth), bem como a qualidade de vida (Inventário de Qualidade de Vida em Epilepsia, versão com 31 perguntas) e o funcionamento social (Escala de Adequação Social). Também foram analisadas outras variáveis relacionadas à epilepsia como tipo de epilepsia (focal ou generalizada), quantidade de fármacos utilizados (monoterapia ou politerapia) e frequência de crises epilépticas. A caracterização das variáveis que podem interferir na análise das variáveis de desfecho foi realizada através da mensuração dos efeitos adversos de fármacos antiepilépticos (Perfil de Eventos Adversos de Liverpool), dos sintomas depressivos (Inventário de Depressão de Beck 2ª versão) e dos sintomas de ansiedade (Inventário de Ansiedade Traço-Estado). Os grupos não diferiram quanto ao cronotipo e à sonolência diurna. Foi observada uma diferença significativa no resultado do PSQI do grupo farmacorresistente refletindo melhor qualidade de sono em relação aos outros dois grupos (diferença farmacossensível x farmacorresistente 1,90, p=0,003; diferença farmacorresistente x controle saudável -1,53, p=0,041). Em relação às variáveis clínicas, pacientes com epilepsia generalizada apresentaram maior vespertinidade do que os pacientes com epilepsia focal (diferença 11,99, p=0,0002). Pacientes em monoterapia, avaliados pelo PSQI, apresentaram pior qualidade de sono (diferença 1,75, p=0,01). A frequência de crises epilépticas não influenciou o resultado de cronotipo, qualidade de sono ou sonolência diurna. A qualidade de sono apresentou uma tendência à correlação com a qualidade de vida (p=0,070). Houve uma associação entre o cronotipo e a adequação social (p=0,027), porém apenas os farmacossensíveis se diferenciaram do grupo controle (p=0,011), apresentando um pior funcionamento social relacionado com a vespertinidade. Os nossos resultados sugerem que a relação entre o sono e a epilepsia está mais intimamente ligada ao tipo de epilepsia, independente da resposta terapêutica ou da frequência de crises epilépticas. Além do mais, esta relação está ligada ao cronotipo, não à qualidade do sono ou à sonolência diurna. A qualidade de vida e a adequação social também estão relacionadas aos fatores relacionados ao sono / Patients with epilepsy have comorbidities that may compromise their quality of life and social adjustment. For this reason, there is a growing interest in the study of conditions associated with epilepsy. Patients with epilepsy are at higher risk to develop sleep disorders. However, the relationship between sleep and epilepsy remains neglected. This study aimed to: (i) evaluate the chronotype, daytime sleepiness, and sleep quality by parametric measures, comparing patients with drug-responsive and drug-resistant epilepsy, and (ii) verify the association between these factors and quality of life and social functioning. For this purpose, we interviewed 114 patients (58 patients with drug-responsive epilepsy, and 56 patients with drug-resistant epilepsy) and 62 healthy controls without epilepsy. The groups were matched for age and gender. All individuals were interviewed with self-applied questionnaires that verified the chronotype (Morningness-Eveningness Questionnaire), sleep quality (Pittsburgh Sleep Quality Index) and daytime sleepiness (Epworth Sleepiness Scale), as well as quality of life (Quality of Life Inventory in Epilepsy, 31 questions version) and social functioning (Social Adjustment Scale). We also evaluated other epilepsy-related variables, such as the epilepsy type (focal or generalized), number of antiepileptic medications (mono or polytherapy), and seizure frequency. The characterization of variables that can influence the analysis of the outcome was measured by the adverse effects of antiepileptic drugs (Liverpool Adverse Effects Profile), depressive symptoms (Beck Depression Inventory 2ª version) and anxiety symptoms (State and Trait Anxiety Inventory). The groups did not differ in chronotype or daytime sleepiness. The drug-resistant group showed a better sleep quality compared to others (difference drug-responsive vs. drug-resistant 1.90, p=0.003; difference drug-resistant vs. healthy controls -1.53, p=0.041). Considering the clinical variables, patients with generalized epilepsy showed a more evening-oriented chronotype than patients with focal epilepsy (difference 11.99, p=0.0002). Patients under monotherapy, evaluated by PSQI, showed worse sleep quality (difference 1.75, p=0.01). Seizure frequency did not influence the results for chronotype, sleep quality or daytime sleepiness presented a correlation with quality of life (p=0,070). Sleep quality demonstrated a trend towards an association with quality of life (p=0,070). There was an association between chronotype and social functioning (p=0,027), however, only drug- responsive patients differed from controls without epilepsy (p=0,011) with a worse social functioning that was eveningness-related. Our results suggest that the relationship between sleep and epilepsy is closely related to epilepsy type, regardless of therapeutic or seizure frequency. Additionally, it is related to the chronotype, but not to the quality of sleep or daytime sleepiness. Sleep quality is also associated with sleep-related factors

Multidrug-resistant tuberculosis (MDR-TB) is a public health problem in several countries such as Angola, India, China, Kenya, and Nigeria. Due to the increasing high burden of MDR-TB, most of these countries do not have adequate capacities to manage MDR-TB patients effectively. This study investigated the effect of model of care; human immunodeficiency virus comorbidity; and demographic factors such as age, gender, and marital status on the treatment outcomes of MDR-TB patients in Nigeria. The study was based on the analysis of secondary data of 402 MDR-TB patients accessed from the data systems of the National Tuberculosis, Buruli Ulcer, and Leprosy Control Program. The theoretical framework for this study was the health belief model. The results of the study showed that treatment outcomes were similar for hospital and community-based models of care. Age was the only factor found to be significantly associated with treatment outcomes; age > than 40 years was a predictor of unsuccessful treatment outcomes among MDR-TB patients at a p-value of 0.026. In the multivariate logistics regression analysis, age and model of care were found to be significantly associated with treatment outcomes at p-values of 0.043 and 0.048, respectively. Marital status, gender, and HIV comorbidity were not significantly associated with treatment outcomes. Implications of the findings of this study for social change in a health care program include opportunities to help reduce the number of patients on waiting lists for MDR-TB treatment. These strategies may ultimately help to reduce the spread of MDR-TB infection as well as the mortality associated with late treatment.

The American Cancer Society predicts there will be 110,070 new cases and 32,120 deaths due to gynecological malignancies in 2018. A major contributing factor to the high mortality associated with gynecological cancers is the recurrence of treatment-resistant tumors. Ovarian cancer (OC) remains the most lethal gynecological malignancy, yet the mechanisms responsible for regulating tumor resistance and vulnerability are largely unknown or undruggable. Therefore, the goal of this research is to identify mechanisms responsible for therapeutic resistance in gynecological cancers and discover innovative approaches to circumvent these molecular alterations. Our efforts began in OC where secondary analysis of gene expression data from OC studies revealed that Sab, an outer mitochondrial membrane (OMM) scaffold protein, was down-regulated in OC tumors compared to normal tissue controls. Our previous studies demonstrate that Sab-mediated OMM signaling induces cell death in cervical cancer. In the current study, we found that Sab concentrations corresponded to chemoresponsiveness in a panel of OC cells; wherein, OC cells with low Sab levels were chemo resistant. Dynamic BH3 profiling revealed that cells with high Sab expression were primed for apoptosis. Furthermore, over-expression of Sab in chemo resistant cells enhanced apoptotic priming and restored cellular vulnerability to cisplatin/paclitaxel treatment. Additionally, an examination of treatment-resistant metastatic uterine cancer (UC) cells were found to have low Sab concentrations compared to vulnerable primary site-derived UC cells. Ectopic expression of Sab in chemo resistant UC cells enhanced the susceptibility towards megestrol acetate and BH3-mimetic ABT-737. To exploit the relationship between Sab concentrations and chemo-responsiveness in gynecological cancer cells, we developed a high-throughput screening assay to detect Sab levels in chemo-resistant OC cells. In collaboration with the Torrey Pines Institute for molecular studies, we have identified compounds that can increase Sab levels in resistant OC cells. The identified compounds improved the effectiveness of cisplatin/paclitaxel therapy. We propose that Sab may be a prognostic marker to discern personalized treatments for gynecological cancer patients. Furthermore, pharmacologically enhancing Sab-mediated signaling may increase the efficacy of chemotherapeutic agents, which would mean lower doses that would limit toxic side-effects.

On the verge of entering the post-antibiotic era, numerous efforts are in place to regain the waning charm of antibiotics which are proving ineffective against most “Superbugs”. Engineered nanomaterials, especially gold nanoparticles (GNPs) capped with antibacterial agents, are proving to be an effective and novel strategy against multidrug resistant (MDR) bacteria. In this study, we report a one-step synthesis of antibioticcapped GNPs (25 ± 5 nm) utilizing the combined reducing and capping ability of a cephalosporin antibiotic, ceftazidime. No signs of aggregation or leaching of ceftazidime from GNP surface was observed upon its storage. Antibacterial testing showed dosedependent broad spectrum activity of Cef-GNPs against both Gram-positive (S. bovis and E. durans) and Gram-negative (P. aeruginosa and E. aerogenes) bacteria. A significant reduction in the minimum inhibition concentration (MIC) of Cef-GNPs was observed as compared to the ceftazidime by itself against Gram-negative bacteria. The MIC of Cef- GNPs were 0.1 mg mL-1 (P. aeruginosa and E. aerogenes) and 1.2 mg mL-1 (E. durans and S. bovis). Cef-GNPs exerted bactericidal action on both P. aeruginosa and E. durans by disrupting the cellular membrane resulting in leakage of cytoplasmic content and death of bacterial cell. Our investigation and results provides an additional step in the development of antibiotic capped GNP as potent next generation antibacterial agents.

This quantitative, descriptive study investigated factors that contributed to TB patients registered in four Tembisa clinics in 2001, defaulting treatment. An interview schedule with closed and open-ended questions was used for 30 patients who could be traced who had interrupted treatment. The reasons for treatment interruption were related to socio-economic, TB policy-related and health care worker-related factors. The findings illustrate that TB management requires a multi-sectoral approach and joint efforts to tackle the disease that continues to kill people even though it is curable. / Health Studies / M.A. (Health Studies)

The study investigated the knowledge levels of clients on long-term tuberculosis (TB) treatment at Kwekwe General Hospital, Zimbabwe. A quantitative, descriptive research design was chosen and data was collected using a structured questionnaire with a convenience sample of 60 clients on TB treatment and 10 professional nurses. The major findings of the study were that although clients had some knowledge about their condition, there was a lack of knowledge regarding critical aspects such as information on drug-resistant TB and the Directly Observed Therapy Short Course. The professional nurses experienced constraints such as insufficient time for appropriate health education and home visits. Based on the study findings and conclusions, several recommendations were made. / Health Studies / Thesis(M.A(Health Studies))

A tuberculose (TB) embora passível de tratamento efetivo, ainda é um grave problema de saúde pública em diversos países, inclusive no Brasil. Nas últimas décadas houve progressos consistentes no controle da doença, porém o avanço da resistência bacilar ainda é um desafio a ser superado, já que os mecanismos da resistência são bastante complexos e não totalmente conhecidos, o que dificulta o desenvolvimento de testes de sensibilidade com elevada acurácia. O objetivo deste trabalho foi caracterizar as mutações gênicas de cepas de Mycobacterium tuberculosis de pacientes do Brasil e de Moçambique com doença resistente a drogas através do sequenciamento genômico total, além de descrever padrões de mutações obtidos por testes moleculares comerciais e comparar estes dados com resultados de testes fenotípicos. Estudo descritivo e transversal que incluiu 30 isolados (17 do Brasil e 13 de Moçambique), submetidos aos testes moleculares comerciais Genotype MTBDRplus®, Genotype MTBDRsl®, Xpert MTB/RIF® e teste fenotípico BACTEC MGIT 960 SIRE®. Todos os isolados também foram avaliados pelo sequenciamento genômico realizado pelo Illumina MiSeq Sequencing System® e submetidos a análise de mutações que conferem resistência às drogas contra TB utilizando o TB profiler online tool. A sensibilidade e especificidade do sequenciamento genômico para detecção de resistência a rifampicina foi de 87,5% e 92,3%, respectivamente. Além disso, o sequenciamento detectou a mutação (Val170Phe) no gene rpoB em dois isolados de M. tuberculosis de Moçambique. Esta mutação não é detectada pelos testes genotípicos comerciais. A sensibilidade do sequenciamento para a isoniazida foi de 95,6% e a especificidade de 100%. Para a estreptomicina, a sensibilidade foi de 85,7% e a especificidade de 93,3%. Para o etambutol, observamos sensibilidade de 100% e especificidade de 77,2%. As mutações mais frequentes associadas à resistência à rifampicina foram a Ser450Leu e a His445Tyr no gene rpoB. Em relação à isoniazida, predominou a mutação Ser315Thr no gene katG. O sequenciamento genômico, dado seu alto poder discriminatório, tem grande potencial de fornecer informações mais acuradas sobre mecanismo gênicos da resistência bacilar, possibilitando futuramente o aprimoramento de testes diagnósticos mais precisos. / Although there is an effective treatment for tuberculosis (TB), it is still a serious public health problem in several countries, including Brazil. In the last decades, there has been consistent progress in disease control, but the increasing number of disease caused by resistant strains is still a challenge to be overcome, since the mechanisms of resistance are quite complex and not fully known, which difficult the development of susceptibility tests with high accuracy. The aim of this work was to characterize gene mutations of Mycobacterium tuberculosis strains from Brazilian and Mozambican patients with drug-resistant disease through whole genome sequencing, as well as to describe patterns of mutations obtained by commercial molecular tests and to compare these data with results of phenotypic susceptibility tests. It was a cross-sectional study that included 30 isolates (17 from Brazil and 13 from Mozambique). Commercial molecular tests Genotype MTBDRplus(TM), Genotype MTBDRsl(TM), Xpert MTB / RIF(TM) and BACTEC MGIT 960 SIRE(TM) phenotypic test were performed for all isolates. All of them were also evaluated by whole genome sequencing performed by the Illumina MiSeq Sequencing System(TM) and submitted to analysis of mutations that confer drug resistance against TB using the TB profiler online tool. The sensitivity and specificity of whole genome sequencing for detection rifampicin resistance was 87.5% and 92.3%, respectively. Also, whole genome sequencing detected the mutation (Val170Phe) in the rpoB gene in two isolates of M. tuberculosis from Mozambique. This mutation is not detected by commercial genotypic tests. The sensitivity of the whole genome sequencing for isoniazid was 95.6%, and the specificity was 100%. For streptomycin, the sensitivity was 85.7%, and the specificity was 93.3%. For ethambutol, we observed a sensitivity of 100% and specificity of 77.2%. The most frequent mutations associated with rifampicin resistance were rpoB Ser450Leu and His445Tyr. About isoniazid, the katG Ser315Thr mutation was the most frequent. Whole genome sequencing, given its high discriminatory power, has great potential to provide more accurate information about the gene mechanisms of bacilli resistance, making possible the improvement of more accurate diagnostic tests in the future.

Hyperthermia is usually used at a sub-lethal level in cancer treatment to potentiate the effects of chemotherapy. The purpose of this study is to investigate the role of heating rate in achieving synergistic cell killing by chemotherapy and hyperthermia. For this purpose, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted. The cytotoxicitiy, mode of cell death, induction of thermal tolerance and P-gp mediated MDR following the two different modes of heating were studied. Doxorubicin (DOX) was used as the chemotherapy drug. Indocyanine green (ICG), which absorbs near infrared light at 808nm (ideal for tissue penetration), was chosen for achieving rapid rate hyperthermia. A slow rate hyperthermia was provided by a cell culture incubator. The results show that the potentiating effect of hyperthermia to chemotherapy can be maximized by increasing the rate of heating as evident by the results from the cytotoxicity assay. When delivered at the same thermal dose, a rapid increase in temperature from 37 °C to 43 °C caused more cell membrane damage than gradually heating the cells from 37 °C to 43 °C and thus allowed for more intracellular accumulation of the chemotherapeutic agents. Different modes of cell death are observed by the two hyperthermia delivery methods. The rapid rate laser-ICG hyperthermia @ 43 °C caused cell necrosis whereas the slow rate incubator hyperthermia @ 43 °C induced very mild apoptosis. At 43 °C a positive correlation between thermal tolerance and the length of hyperthermia exposure is identified. This study shows that by increasing the rate of heating, less thermal dose is needed in order to overcome P-gp mediated MDR.

PURPOSE: The purpose of this study was to assess factors associated with the development of drug resistant tuberculosis in Ethiopia. DESIGN: A quantitative case-control study was conducted to determine if there were any significant differences in prevalence of pre-defined factors between cases and controls. METHODS: Cases were patients with drug resistant tuberculosis who had a confirmed diagnosis by culture drug-susceptibility or gene expert tests. Successfully treated, tuberculosis symptom free patients who had been on first-line tuberculosis treatment and who were registered as cured or treatment completed were taken as controls. An equal number of cases (N=181) and controls (N=181) was selected using a systematic random sampling method and was used in the study. A structured questionnaire developed by the researcher was used to collect data. Odds ratio and multiple logistic regression were used to quantify the strength of association between dependent and independent variables. RESULTS: The development of drug resistant tuberculosis was significantly associated with two or more previous episodes of tuberculosis illness (adjusted odds ratio (AOR): 14.84; 95% CI 8.90 –24.75), previous first-line tuberculosis treatment not directly observed by a health worker for 7 to 8 weeks (AOR: 13.41; 95% CI 8.06 – 22.29) and previous first-line tuberculosis treatment outcome of failure (AOR: 39.19; 95% CI 12.05 -127.46). Interruption of first-line tuberculosis treatment for one day or more (AOR = 4.28; 95% CI 2.76 – 6.64) and history of treatment in the first-line tuberculosis treatment category for previously treated patients (AOR: 3.70; 95% CI 2.40 – 5.72) were also significantly associated with the development of drug resistant tuberculosis in the current study. CONCLUSION: Patients with a history of previous first-line tuberculosis treatment, patients who interrupted previous first-line tuberculosis treatment and patients with previous first-line tuberculosis treatment outcome of failure were at high risk of developing drug resistant tuberculosis. Therefore, the full course of first-line tuberculosis treatment should be given, following the Directly Observed Treatment (DOT) guide. Patients with recurrent tuberculosis and unfavourable first-line tuberculosis treatment outcome should be tested for drug susceptibility. / Health Studies / D. Litt. et Phil. (Health Studies)