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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

MMed, Haematology, Faculty of Health Sciences, University of the Witwatersrand / Background: Mycobacterium tuberculosis (MTB) infection remains a serious public health challenge in sub-Saharan Africa. Rapid and early diagnosis is critical in the successful control of this eminently treatable infection. This study compared the diagnostic usefulness of culture, bone marrow trephine biopsy granulomata, bone marrow trephine biopsy Ziehl-Neelsen (ZN) stain and bone marrow mycobacterial polymerase chain reaction (PCR) in establishing the diagnosis of mycobacterial infection in HIV infected patients. Materials and methods: The trephine biopsies of HIV positive patients done for the investigation of suspected tuberculosis were reviewed for granulomata and stained with ZN stain. The corresponding bone marrow aspirates were subjected to DNA real-time PCR analyses using LightCyler TB Kit® (Roche Diagnostic). Culture results were used as diagnostic gold standard. Results: Of the 60 patients studied, 24 were culture negative. Of the 34 culture positive, 62% were Mycobacterium tuberculosis and 38% were Mycobacterium avium intracellulare. Using the culture method as a gold standard, the sensitivities and specificities were 97% and 23% for bone marrow trephine biopsy granulomata, 65% and 58% for bone marrow trephine biopsy ZN staining and 50% and 73% for bone marrow aspirate PCR analysis respectively. Ninety-seven percent of all trephine biopsies with positive ZN stain had granulomata. Conclusion: The presence of granulomata in bone marrow trephine biopsies of HIV infected patients appear to have a high diagnostic yield whilst mycobacterial PCR has the lowest yield but highest specificity. These results should be confirmed in a prospective case controlled study because the sample size in this study was small, and the study was a retrospective one.

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 dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. 2017 / Mycobacterium tuberculosis assembles a complex cell wall consisting of mycolic acids, arabinogalactan and peptidoglycan layers. The peptidoglycan is important for structural maintenance and osmotic protection of the cell. Beta-lactam antibiotics, such as penicillin, perturb biogenesis of cross-linked peptidoglycan by inhibition of penicillin-binding proteins and cause cell death. As a result, penicillin-binding proteins have been extensively used in antimicrobial development. However, penicillin insensitive enzymes involved in peptidoglycan biogenesis such as amidases, transglycosylases and endopeptidases remain to be exploited for anti-TB drug development, a field that urgently requires new drugs in light of the rapid emergence of drug resistant strains. In this study, we functionally characterize a novel class of LytM domain containing peptidoglycan endopeptidases (also known as M23 peptidases) in mycobacteria. Bio-informatics tools were used to identify LytM domain-containing homologues in Mycobacterium smegmatis, designated MepB1-MepB4. These were deleted using standard allelic exchange mutagenesis and recombination techniques and the resulting mutants were assessed for cell wall related defects. We found that mycobacterial LytM endopeptidases have important roles in bacterial growth as demonstrated by delayed cell growth kinetics in a ΔmepB1 deletion mutant. We noted no growth defects in ΔmepB2 and ΔmepB3 single deletion mutants but observed defective cell division in a ΔmepB2 ΔmepB3 double deletion mutant. In this double mutant, spatial localization of new cell wall biosynthesis revealed the inability to degrade the septal bridge joining two daughter cells, pointing to a critical role for these enzymes in cell separation. MepB1 is sequestered from the peptidoglycan by cytosolic localization and its absence causes a septal and polar buldging phenotype. To further investigate the biological roles of these putative peptidoglycan endopeptidases, protein interaction studies were conducted using the bacterial two-hybrid mycobacterial protein fragment complementation assay. This analysis identified FtsX, a key cell division protein, as an interacting partner for both MepB2 and MepB3, thus identifying these proteins as novel components of the mycobacterial divisome. Collectively, these observations provide the first insight into a new group of potential drug targets for tuberculosis disease and notably enhance the overall understanding of peptidoglycan turnover, which is of general revelevance in bacterial pathogens. / MT2017

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.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) has infected one third of the world’s population and continues to claim more lives annually than any other infectious disease agent. A significant proportion of individuals carry latent TB infection (LBTI) which is characterized by the absence of clinical symptoms and it has been postulated that the tubercle bacilli are in a dormant-like state during this type of infection. Resuscitation promoting factors (Rpfs) are cell wall hydrolases which cleave glycosidic bonds within the peptidoglycan (PG), a mechanism thought to result in reactivation of bacteria from the state of dormancy. M. tuberculosis encodes five rpf-like homologues which are collectively dispensable for growth but are required for reactivation from dormancy in vitro, and for virulence in the mouse model of infection. LTBI thus poses a huge threat to the global burden of active disease. The purpose of this study was to further investigate the biological roles of Rpfs by assessing the effects of rpf gene deletion in M. smegmatis, a model organism used for TB research. M. smegmatis encodes four rpf-like genes designated rpfA, rpfB, rpfC and rpfE, and deletion mutants that lack one or more of these genes were constructed by allelic exchange mutagenesis. M. smegmatis mutant strains that lack either rpfA or rpfB display no significant differences in growth both on solid and in liquid medium when compared to wild type. However, loss of rpfA resulted in bacterial clumping during stationary-phase growth in broth culture and changes in cell morphology. Moreover; the rpfA rpfB double mutant and its derivative strain lacking rpfC, rpfA rpfB rpfC displayed a ca. 2-4 log increase in susceptibility to erythromycin and vancomycin. Furthermore, unusual colonial morphologies with reduced serpentine cording and smooth peripheries were observed for these multiple mutants. Cell surface defects and cell distortions were evidenced as wrinkle-textured, bent cells and polar tip bulges for the abovementioned mutants. The multiple deletion strains also displayed a defect in biofilm formation revealing an inability for the mutants to form complex cell-cell interactions. Collectively, the data are suggestive of a loss of bacterial cell wall integrity due to rpf gene deletion and it is proposed that rpfA, in combination with other genes, is largely responsible for cell wall integrity maintenance. Our data indicate that these factors play an important role in cell growth and division and therefore represent an untapped source of novel targets for anti-tubercular drug discovery.

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.