• Tuberculosis and Respiratory Diseases
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• v.49 no.5
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• pp.546-557
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• 2000

Background : Oligonucleotide chip technology has proven to be a very useful tool in the rapid diagnosis of infectious disease. Rifampin resistance is considered as a useful marker of multidrug-resistance in tuberculosis. Mutations in the rpoB gene coding $\beta$ subunit of RNA polymerase represent the main mechanism of rifampin resistance. The purpose of this study was to develop a diagnosis kit using oligonucleotide chip for the rapid and accurate detection of rifampin-resistance in Mycobacterium tuberculosis. Method : The sequence specific probes for mutations in the rpoB gene were designed and spotted onto the glass slide, oligonucleotide chip. 38 clinical isolates of Mycobacterium were tested. A part of rpoB was amplified, labelled, and hybridized on the oligonucleotide chip with probes. Results were analyzed with a laser scanner. Direct sequencing was done to verify the results. Result : The low-density oligonucleotide chip design어 to determine the specific mutations in the rpoB gene of M. tuberculosis accurately detected rifampin resistance associated with mutations in 28 clinical isolates. Mutations at codons 531, 526, and 513 were confirmed by direct sequencing analysis. Conclusion : Mutant detection using oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of multidrug-resistance in M. tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.47 no.6
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• pp.747-756
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• 1999

Background: Acid-fast stain and cultures for diagnosis of pulmonary tuberculosis are primary and essential method, but have their limitation : low sensitivity and time consuming. The objective of this study is comparison of amplified Mycobacterium tuberculosis direct test(MTD) by the conventional AFB smears and cultures in the detection of Mycobacterium tuberculosis in respiratory specimens. Methods: During the period between November, 1997 and May, 1998 a total of 267 respiratory specimens (sputum 173, bronchial washing 94) from 187 patients suspected pulmonary tuberculosis were subjected to AFB smears, cultures and MID test. MID is based on nucleic acid amplification. We compared the MID with 3% Ogawa culture method. In positive AFB smear and negative MID specimen, positive culture identification between nontuberculous mycobacterium and M.tuberculosis was assesed by using Accuprobe M.tuberculosis complex probe. In negative AFB smear and negative AFB culture, MTD results are assessed by clinical follow-up. Results : 1) Compared with culture in sputum and bronchial fluid specimens, sensitivity and specificity of MTD in positive AFB smear is 79.7% and 20.0%, sensitivity and specificity of MTD in negative AFB smear specimens is 75.0% and 79.7%. 2) Discrepant analysis is assessed by clinical follow-up and other specimen results beyond study. Culture negative but MTD positive specimens were proved to be true positive and gave MTD sensitivity 79.2%, specificity of 84.4%, positive predictive value 80.5% and negative predictive value 83.2%. 3) 14 out of 31 specimens in negative AFB smear, negative AFB culture and positive MTD showed pulmonary tuberculosis diagnosed on clinical follow-up and sensitivity is 45.2%. 4) 2 out of 13 specimens in positive AFB smear, positive AFB culture and negative MID diagnosed as non tuberculous mycobacterium by Accuprobe culture. Conclusion: This study suggested that MID in respiratory specimens is simple and rapid diagnostic method, but considered adjuvant method rather than replace the conventional AFB smear and culture.