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.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 50-57).

Thesis (M.Med.Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 39-49).

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-116). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-116). Also available in print.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Mycobacterium tuberculosis (MTB) is a major infective agent causing human tuberculosis (TB) in the worldwide. Although tuberculosis can be treated by a six-month course of antibiotics, the prevalence of extensively drug-resistance TB (XDR-TB) made the disease becomes a global health problem. In addition to the conventional MTB detection methods, molecular methods become significant in drug resistant MTB detection which can enhance effective drug treatment. In this study, 200 MTB respiratory specimens were collected from patients with suspected tuberculosis in Tuen Mun Hospital in Hong Kong. Based on the culture method as a gold standard for MTB detection, the presence of MTB in clinical samples was determined by IS6110single tube nested real-time PCR. In addition, by using High Resolution Melting (HRM) analysis, the presence of mutant type KatG315 gene for detecting isoniazid resistant MTB was determined. Among 66 MTB culture positive samples, 10 samples had positive acid fast bacilli (AFB) smears giving the diagnostic sensitivity 15.1%. IS6110 single tube nested PCR was amplified in 51 specimens giving 77.2% MTB detection sensitivity and 97.8% specificity. Among 51 samples positive for IS6110 PCR, 66.7% showed successful amplification in subsequent KatG-HRM assay. Two samples were confirmed to be isoniazid (INH) resistance in Public Health Laboratory Centre (PHLC). However, there was only one sample showing detectable KatG315 mutation in clinical specimen by using HRM while the other was only detected in the corresponding culture isolate. From the result of this study, single tube nested real-time PCR demonstrated MTB detection in clinical samples and INH resistant strain with KatG315mutationcan be detected by HRM analysis. Early detection of mycobacteria allow earlier treatment of the patient, thus transmission of the disease can be controlled. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Background Tuberculosis (TB) infection is a contagious disease due to infection by Mycobacterium tuberculosis(MTB) causing global health burden. There is increasing effort to develop early case detection methods and also to address the issue of multidrug resistance TB (MDR-TB). Molecular methods have been applied to provide rapid and accurate diagnosis. In addition to commercial kits being available for the detection of MTB from clinical specimens, In-house PCR assays have also been developed for the detection of MTB, and can be adjusted according to the laboratories’ own demand. Several molecular techniques like TaqMan probes and Hybridization probes may be applied to target for markers of MTB, e.g. 16s rRNA and IS6110.Detection of the mutation genes, for example, katGfor isoniazid (INH), enables determination of susceptibility of the antibiotic more rapidly than traditional culture methods, and is especially useful due to the increasing emergence of MDR-TB. A wide range of genes have been reported to be related to the resistance of INH, katG315 mutation is the most common gene among them. Therefore, genotyping katG315 allows determination of the susceptibility of INH. Objective The first objective is to evaluate the diagnostic performance of IS6110 One-tube Nested Real-Time PCR for the detection of MTB. Clinical pulmonary specimens collected from Tuen Mun Hospital were retrieved for investigation. All the specimens have already been tested for COBAS TaqMan MTB test and culture results have been obtained for all the samples. During the first stage of the study, all the specimens were tested with IS6110 One-tube Nested Real-Time PCR. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were obtained from the comparison with the gold standard of MTB detection, i.e., culture. During the second stage of the study, samples were selected to undergo katG315 HybProbe Real-Time PCR assay to determine the genotype of katG. The performance of the assay was evaluated statistically. Result In the first stage of the study, 200 samples were tested with IS6110 One-tube Nested Real-Time PCR. The assay was found to have a sensitivity of 76.92%, specificity of 98.52%, positive predictive value of 96.15%, negative predictive value of 89.86% and the diagnostic odds ratio of 221.667. In the second stage of the study, 66 samples were selected and tested for katG315 HybProbe Real-Time PCR assay, 36 samples were successfully genotyped while 30 samples failed to be genotyped. The only culture proven INH resistance specimen was not amplified at first, and culture isolate was extracted for genotyping again. The repeated test confirmed the genotype of the resistance strain to be a mutant. Conclusion katG315 HybProbe Real-Time PCR assay is a valid approach for genotyping katG. However, the sensitivity and efficiency has to be improved before application for clinical use. From the statistics obtained, COBAS TaqMan PCR assay, which is routinely used in Tuen Mun Hospital, is statistically proven to have comparatively better performance than the IS6110 One-tube Nested Real-Time PCR. Improvement on the assay is required for IS6110 One-tube Nested Real-Time PCR. However, there is great potential of applying both IS6110 One-tube Nested Real-Time PCR and katG315 HybProbe Real-Time PCR assay in clinical use with the same platform available. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

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