Tuberculosis (TB), a severe disease caused by Mycobacteria tuberculosis (MTb), remains a globally severe health problem. In modern days, emergence of drug resistant TB is a new threat to public health since it can lead to treatment failure, increased transmission of TB to other hosts and further development of drug resistant complications. The traditional diagnostic method for TB by Acid Fast Bacilli (AFB) smear and Löwenstein–Jensen medium (LJ) culture are poor in sensitivity and time consuming respectively. There is a need for rapid diagnosis and identification of MTb. Nucleic acid amplification like PCR and real-time PCR are options for rapid diagnosis. However, such techniques require sophisticated technique and complex equipment. The high cost would constitute a barrier for countries with a high demand but only limited resources. Loop-mediated isothermal amplification (LAMP) assay is a novel technique, proven by many studies as to its high sensitivity and highly specificity to MTb. Most importantly, LAMP assay is economical and affordable by developing countries. The first objective of this study was to evaluate the analytical sensitivity and specificity of LAMP assay. The specificity of LAMP assay was determined by performing LAMP assay on 19 clinical isolates, which had already been identified previously. The clinical isolates included 14 mycobacteria tuberculosis complex (MTb), and five mycobacteria other than tuberculosis (MOTT) strains that were positive for AFB smear and LJ culture but negative for IS6110 single-tube nested real time PCR. The specificity was 100%. The analytical sensitivity as well as the limit of detection (LOD) were determined by testing on a duplicate set of serial DNA dilution, where each duplicate consisted of dilution of 100,000, 10,000, 1000, 300, 100, 10 and 1 colony forming unit/milliliter (CFU/ml). The LOD of LAMP assay was about 3 CFU per reaction. The second objective of this study evaluated the diagnostic performance of LAMP assay against AFB culture and IS6110 single tube nested real time PCR for identification of MTb in 200 respiratory specimens from 123 patients. All the specimens have already been tested for IS6110 single tube nested real time PCR, and culture results and AFB smears results have been obtained for all the specimens. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval using LJ culture as gold standard. The LAMP assay had a sensitivity of 80%, specificity of 92.6%, PPV of 60% and NPV of 97.1%. However, MTb might fail to grow on the LJ culture medium for various reasons, for instance, MTb might already be dead after antibiotic treatment of the patient, or there might be poor laboratory practices during the processing of the specimens. Since the LJ culture method could produce false negatives in the situations described above, an alternative to the LJ culture method, ‘Hybrid Method’ was used as the gold standard. Under this method, a specimen was regarded as positive if the LJ culture result was positive. On the other hand, if a specimen generated a negative result using the LJ culture method, the results from the LAMP assay and IS6110 nested real time PCR would be considered, i.e., if both the LAMP assay and IS6110 nested real time PCR gave positive results while the results of LJ culture were negative, the specimen was referred to be positive in this case. In other words, a specimen would be regarded as negative if and only if the LJ culture result was negative and at least one of the LAMP assay or IS6110 was negative at the same time. Along the same line, the sensitivity, specificity, PPV and NPV of the three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval against the Hybrid Method. After resolution, the LAMP assay had a sensitivity of 87.0%, specificity of 100%, PPV of 100% and NPV of 97.1%. Our results showed that the LAMP assay has a great potential to be a new TB diagnostic test, especially in developing countries, with its lot of advantages like ease of use, cheap and fast. The LAMP assay in the study showed a high specificity, however, the sensitivity has to be improved before application in clinical use. For comparison of clinical performance, IS6110 single tube nested real time PCR had a higher sensitivity than that of LAMP assay (100% vs 80% using culture as gold standard; 100% vs 87% using ‘Hybrid Method’ as gold standard). However, LAMP assay had a higher specificity than that of IS6110 single tube nested real time PCR (92.6% vs 90.7% using culture as gold standard; 100% vs 98% using ‘Hybrid Method’ as gold standard). LAMP had been proven to be a potential and powerful tool in clinical diagnosis of MTb. Further improvement on its sensitivity is required to enable its extensive use in the clinic in the future. / published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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Made available in DSpace on 2016-01-26T12:51:28Z (GMT). No. of bitstreams: 1 maraluciamacedopaizan_dissert.pdf: 609664 bytes, checksum: dfade693fed390f4fb0a8e4705826a9f (MD5) Previous issue date: 2010-11-05 / Analyze the socio-demographic profile and the organization of health services as the primary gateway to the tuberculosis diagnosis. Methods: This is an exploratory descriptive study, conducted from a quantitative approach, with patients living in the city of Sao Jose do Rio Preto in 2009. A structured questionnaire that follows the reference of the Primary Care Assessment Tool for tuberculosis care was used, focusing on issues related to socio-demographic information and the gateway size. The data analysis was performed by means of frequency distribution and chi-square test for proportions and analysis of residuals. Results: The first health service sought after onset of symptoms was the Emergency Care, followed by Primary Health Care. Regarding the variables of gateway size there was a strong association between patients who sought the services closer to his home to the onset of signs and symptoms of tuberculosis and looked for the Primary Health Care and those who sought the services further away and went directly to the specialist services. Also was found between patients who were no preventive control of health and the search for Emergency Care. Conclusions: The main gateway to the respiratory symptoms is not Primary Health Care as recommended by the National Tuberculosis Control but the Emergency Care. / Analisar o perfil sócio-demográfico e a organização dos serviços de saúde da Atenção Básica como porta de entrada para o diagnóstico da Tuberculose. Método: Trata-se de um estudo descritivo exploratório, realizado a partir de uma abordagem quantitativa, com doentes residentes no município de São José do Rio Preto-SP em 2009. Utilizou-se o questionário estruturado que segue o referencial do Primary Care Assessment Tool, por meio do qual foram enfocadas questões relativas às informações sócio-demográficas e a dimensão porta de entrada. A análise dos dados foi realizada por meio da distribuição de frequência e do teste Qui-quadrado para proporções com análise de resíduo. Resultados: O primeiro serviço de saúde procurado após o início dos sintomas foi o Pronto Atendimento, seguido pela Atenção Básica. Em relação às variáveis da dimensão porta de entrada, verificou-se associação entre doentes que buscaram os serviços mais próximos de seu domicilio ao início dos sinais e sintomas da tuberculose, e procuraram pela Atenção Básica. Também houve associação estatística entre doentes que não faziam controle preventivo de saúde e a procura pelo Pronto Atendimento. Conclusões: A principal porta de entrada para os sintomáticos respiratórios não está sendo a Atenção Básica conforme preconizado pelo Programa Nacional de Controle da Tuberculose, e sim o Pronto Atendimento.