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La tuberculosis es una enfermedad infecciosa que constituye un grave problema de salud pública a nivel mundial principalmente en países en vías de desarrollo, como el Perú. Las limitaciones de los métodos clásicos de diagnostico (baciloscopía y cultivo) así como la alta frecuencia de ésta enfermedad han creado la necesidad de implementar nuevas estrategias para incrementar la sensibilidad de las pruebas y a su vez reducir el tiempo necesario para establecer el diagnostico confirmatorio. Considerando que los estudios correspondientes a la región del Tracto Respiratorio Inferior (TRI) son escasos, el objetivo de esta tesis fue determinar el perfil molecular de Mycobacterium tuberculosis en muestras biológicas del TRI de pacientes limeños con sospecha de tuberculosis. En el presente trabajo se evaluaron 43 muestras de pacientes limeños con sospecha clínica de tuberculosis las cuales fueron obtenidas a través de lavado bronquial, aspirado bronquial, secreción bronquial, lavado bronco-alveolar, aspirado bronco-alveolar y broncofibroscopía. De estas muestras se extrajo DNA y se realizó la prueba del PCR - Nested, para ello se empleó como secuencia diana el gen de la proteína A (65KDa) de Mycobacterium tuberculosis, los productos amplificados fueron evidenciados mediante electroforesis en gel de agarosa y tinción con bromuro de etidio. Los resultados obtenidos muestran que el 77% de las muestras procesadas poseen DNA de Mycobacterium tuberculosis, demostrándose la alta sensibilidad y especificidad del método empleado, el 82% de estas muestras correspondieron a la región del árbol bronquial superior, debido a que esta región es el punto de partida para la diseminación de esta micobacteria hacia otras regiones del tracto respiratorio y además, a que la mayoría de muestras fueron tomadas directamente de esta región. Asimismo, la aplicación de la prueba PCR- Nested incrementó la sensibilidad de detección 5.5 veces con respecto a un único evento de amplificación, lo cual demuestra la utilidad de esta prueba en el análisis de material biológico con baja carga micobacteriana como las empleadas en este trabajo. Finalmente, se concluye que la prueba PCR- Nested es un método altamente sensible y específico para detectar DNA de Mycobacterium tuberculosis a partir de muestras procedentes del TRI. / Tuberculosis is an infectious disease that constitutes a serious public health problem worldwide, mainly in developing countries such as Peru. The limitations of the traditional methods of diagnosis (smear and culture) as well as the high incidence of this disease have created the need to implement new strategies to increase the sensitivity of tests to reduce time to establish a confirmed diagnosis. Since there are not many researches at the region of the Lower Respiratory Tract (LRT), the objective of this study was to determine the molecular profile of Mycobacterium tuberculosis in biological samples taken from the LRT of patients with suspicion tuberculosis. In the present study, 43 samples from patients with clinical suspicion of tuberculosis were evaluated. The samples were obtained through bronchial lavage, bronchial aspirate, bronchial secretions, bronco-alveolar lavage, and sucked bronco-alveolar broncofibroscopy. DNA extraction was prepared from each sample, and it was used in a PCR- Nested test targeting the gene encoding the protein A (65KDa) from M. tuberculosis. The results showed that DNA from M. tuberculosis was detected in 77% of the processed samples, revealing a high sensitivity and specificity of this method. From the positive samples, 82% corresponded to those obtained from the bronchial tree top region. This last result is explained because of the fact that most of the samples were taken directly from this region, which is the starting point for the mycobacteria dissemination toward other regions of the respiratory tract. In addition, the application of the PCR-Nested test increased the sensitivity of detection by 5.5 fold compared to a single event amplification, demonstrating the usefulness of this test in the analysis of biological material with low mycobacterial load as those used in this work. Finally, we conclude that the PCR-Nested test is a highly sensitive and specific method for detecting DNA from Mycobacterium tuberculosis in samples from the LRT.

Publicación a texto completo no autorizada por el autor / Produce un complejo de elongación ternario de la ARN polimerasa de Mycobacterium tuberculosis recombinante independiente del promotor transcripcionalmente activo. Para ello, prueba el efecto de las diferentes proporciones molares de las subunidades en la reconstitución in vitro de la ARN polimerasa de Mycobacterium tuberculosis, compara la coexpresión de la ARN polimerasa de Mycobacterium tuberculosis en Escherichia coli bajo dos temperaturas distintas: 25 y 37 °C y establece un método para la evaluación de la transcripción de dicha polimerasa independiente de promotor. / Tesis

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Haematology. Johannesburg, 2015. / Introduction: The Gene Xpert MTB/Rif assay (Xpert) is a nucleic acid amplification technique that has been studied in the diagnosis of both pulmonary and, to a lesser extent, extrapulmonary tuberculosis (TB). This study was performed in the National Health Laboratory Services (NHLS) laboratory at Charlotte Maxeke Hospital which services a population with a high prevalence of Mycobacterium Tuberculosis Complex (MTBC) infection. The study aimed to develop a protocol for the processing of pleural and ascitic fluid samples to be run on Xpert for MTBC diagnosis, to evaluate the sensitivity and specificity of the Xpert assay as compared to the gold standard MTBC culture assays and to assess the utility of the Xpert assay as part of the diagnostic algorithm for fluid samples received in high prevalence MTBC laboratories. Materials and methods: A total of 392 pleural and ascitic fluid specimens were received for routine immunophenotypic analysis between August 2012 and February 2013 at the NHLS flow cytometry laboratory in Charlotte Maxeke hospital. Of these specimens, 229 had sufficient residual volume (>0.5ml) after routine immunophenotypic analysis to be tested on Xpert. Specimens were processed as per the manufacturer’s guidelines for pulmonary specimens and results were compared to the gold standard culture for Mycobacterium tuberculosis. Results: Xpert positivity was detected in 8.7% (20/229) of the total specimens. Only 43% (99/229) of these specimens were submitted for concurrent MTBC liquid culture (Mycobacterium Growth Indicator tube, MGIT) testing based on the laboratory information system history. Positivity on Xpert was shown in 9% (9/99) of specimens compared to 17% (17/99) on MGIT. One false positive was detected on Xpert. More than half of the specimens, 57% (130/229) were not referred for concurrent MTBC culture. The Xpert detected MTBC in 8.5% (11/130) of these specimens, with 1 Rifampicin resistant case identified. Xpert sensitivity and specificity in this study were 50% (CI:26-75%) and 99% (CI:91-100%) respectively Conclusion: The sensitivity and specificity of Xpert in this study was comparable to that found in other studies performed on fluid samples. Importantly, this study demonstrates that in a high burden HIV/TB setting like South Africa, more than 50% of fluid specimens referred for immunophenotypic analysis to exclude lymphoma are not referred for concurrent MTBC culture testing. Incorporation of Xpert into the laboratory diagnostic algorithm (LDA) in the immunophenotypic laboratory would, therefore, have a number of benefits, improving overall patient work-up and care. Implementation and policy uptake, however, would require a full costing analysis as Xpert testing would be performed in addition to, and not instead of, routine testing.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree, Master of Medicine in Haematopathology. Johannesburg, 2014 / Mycobacterium Tuberculosis is the leading cause of morbidity and mortality due to infectious diseases worldwide. South Africa has ~20% of the world’s HIV associated Tuberculosis and has the second largest reported numbers of multidrug resistant (MDR) Tuberculosis in the world. Given the complexity of the mycobacterium and its ability to evade the immune system, there is a need for dissecting the immunological response to Tuberculosis including innate like lymphocytes such as gamma-delta T cells. Gamma-delta T cells are of particular relevance as they react to phospho-proteins of mycobacteria. Gamma-delta T cells can be divided into two subsets. Gamma-delta T cells using the Vdelta2 (VD2) segment as the variable segment in their T cell receptor and gamma-delta T cells using an alternative variable segment (non VD2 T cells). We aimed to enumerate both subsets of gamma-delta T cells in the immunological response to Tuberculosis. We collected samples from three patient populations at the Charlotte Maxeke Johannesburg Academic Hospital for comparison: HIV positive patients with no evidence of Tuberculosis disease, HIV positive patients with active pulmonary Tuberculosis and a healthy control group. We used a nine colour flow cytometric panel to enumerate the frequency of gamma-delta T cells in these participant groups. We found that the VD2 T cell subset was reduced in the HIV positive group and the dual HIV positive TB positive group compared with healthy controls, which mirrored the loss of CD4 T cells in these patients. Conversely, the non VD2 subset of gamma-delta T cells showed a statistically significant increased frequency in HIV positive patients and dual HIV positive TB positive patients compared to healthy controls. The frequency of gamma-delta T cells, expressed as a percentage of total T cells, was significantly increased in HIV positive patients and not non- significantly increased in the HIV positive TB positive groups compared to healthy controls. This skewing of the gamma-delta T cell repertoire in HIV positive patients and HIV positive patients with active Tuberculosis may have specific immune implications. The mechanism of the loss of VD2 T cells in HIV and HIV associated Tuberculosis has not been elucidated. The loss of VD2 gamma-delta T cells in HIV and HIV associated Tuberculosis may underlie susceptibility to Tuberculosis disease.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. / The prevalence of tuberculosis in South African miners is substantially higher than that of in the general population. Through exposure to dust which leads to different degrees of silicosis, and by working in enclosed spaces where coughed out bacilli can survive in droplet nuclei and be inhaled by other workers, miners are especially prone to to become infected with M. tuberculosis and develop the disease. It is not only the working conditions which promote transmission of M. tuberculosis, but the living conditions as well. Most miners live and sleep in rooms shared by up to eight other men, which increases the opportunity for transmission, leading to both primary and reinfection tuberculosis. / IT2018

Degree of Master of Science in Medicine by Research Only Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine by Research. Johannesburg, 2012 / Genetic heterogeneity of Mycobacterium tuberculosis demonstrating mixed infections or affecting single strains has been previously described. A single sputum culture from five patients with drug-resistant tuberculosis treated at Sizwe Hospital was analysed in-depth for genotypic and phenotypic heterogeneity. IS6110-based restriction fragment length polymorphism (RFLP) was performed on 20 colonies from each sputum for detection of mixed infections and clonal heterogeneity. No mixed infections were found, but IS6110-RFLP-linked clonal heterogeneity was observed in one patient. Drug susceptibility testing (DST) and sequencing of nine drug-resistance-associated genes performed on a total of 99 colonies from the five patients failed to show genotypic hetero-resistance. On DST, however, discordant rifampicin resistance findings were encountered in one patient. Minimal inhibitory concentrations performed on these colonies were close to the rifampicin critical concentration used for resistance determination, suggesting failure of the BACTEC MGIT 960 assay to reliably determine rifampicin susceptibility in strains with borderline resistance.

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