• Korean Journal of Clinical Laboratory Science
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• v.41 no.1
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• pp.24-30
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• 2009

Rifampicin (RIF) and isoniazid (INH) are the most important drug for the treatment of Mycobacterium tuberculosis. Mutations correlated to rifampicin and isoniazid-resistance have been detected in rpoB gene and katG gene, respectively. Of the rifampicin-resistant isolates, 90% showed mutations in rpoB gene at codon 507 to 533. Isoniazid-resistant isolates analysed had a mutation in katG at codon 315. The aim of this study is to develop a pyrosequencing-based approach for rapid detection of ripampin or isoniazid resistant M. tuberculosis based on characterization of all possible mutation in the target region. For this study, the DNA selected from 35 cases of MTB PCR positive clinical sample such as bronchial washing, sputum, and pleural fluid. RIF or INH resistant was analyzed by pyrosequencing data of rpoB and katG gene. 28 (80%) and 7 (20%) of 35 MTB PCR positive DNAs were occured rifampicin-sensitivity and resistant, respectively. For INH, 30 (85.7%) and 5 (14.5%) cases were detected isoniazid-sensitivity and resistant, respectively. When pyrosequencing analysis was compared with ABI sequencing analysis, both analysis were presented same result, but pyrosequencing analysis was more rapid than ABI sequencing analysis. In conclusion, we found that pyrosequencing technology offers high accuracy, specificity, short turn around time and a high throughput in detection of rifampicin or isoniazid resistance in M. tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.45 no.4
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• pp.714-722
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• 1998

Background: Rifampicin (RFP) is a key component of the antituberculous short-course chemotherapy and the RFP resistance is a marker of multi-drug resistant (MDR) tuberculosis. RPoB gene encodes the $\beta$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. And the mutations of rpoB gene have been found in about 96% of rifampicin resistant clinical isolates of M. tuberculosis. So in order to find a rapid and clinically useful diagnostic method in identifying the RFP resistance, we compared the PCR -line probe method with PCR-SSCP for the detection of the rpoB gene mutation in cultured M. tuberculosis. Methods: 45 clinical isolates were collected from patients who visited Sung Kyun Kwan University Hospital. The RFP susceptibility test was referred to the referral laboratory of the Korean Tuberculosis Institute. 33 were rifampicin resistant and 12 were rifampicin susceptible. The susceptibility results were compared with the results of the PCR-BSCP and PCR-line probe method. Results: We could find rpoB mutations in 27/33(81.8%) RFP-resistant strains by PCR-line probe method, and in 23/33 (69.7%) by PCR-SSCP and there was no significant difference between two methods. There was no mutation in rifampicinn susceptible strains by both methods. Conclusion: PCR-line probe method would be a rapid, sensitive and specific method for the detection of rifampicin resistant Mycobacterium tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.59 no.3
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• pp.257-265
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• 2005

Background : Rifabutin (ansamycin) is a spiro-piperidyl rifamycin, which is highly active against Mycobacterium tuberculosis. It has been found that some clinical isolates of tubercle bacilli that are resistant to rifampicin are susceptible to rifabutin, with some patients with multi-drug resistant pulmonary tuberculosis having shown favorable clinical and bacteriological responses to the rifabutin. This study was conducted to find the proportion of rifabutin-susceptible strains among rifampicin-resistant isolates from Korean MDR-TB patients, and investigate the presence of specific rpoB mutations, which may confer resistance to rifampicin, but not to rifabutin. Methods : 201 rifampicin-resistant and 50 pan-susceptible M. tuberculosis isolates were randomly selected for this study. The isolates were retested at rifampicin and rifabutin concentrations of 0, 20, 40 and $80{\mu}g/ml$, respectively. The isolates that grew at and/or over a rifabutin concentration of $20{\mu}g/ml$ were judged rifabutin-resistant. The rpoB gene was extracted from the isolates, and then amplified for direct sequencing to investigate specific rpoB mutations that conferred rifabutin- susceptibility but rifampicin-resistance. Results : Out of the 201 rifampicin-resistant M. tuberculosis, 41 strains (20.4%) were susceptible to rifabutin using the absolute concentration method on Lowenstein-Jensen media. The rpoB mutation types that showed susceptibility to rifabutin were Leu511Pro, Ser512Arg, Gln513Glu, Asp516Ala, Asp516Gly, Asp516Val, Asp516Tyr, Ser522Leu, His526Asn, His526Leu, His526Cys, Arg529Pro and Leu533Pro. A reverse hybridization technique was able to detect 92.5% of the rifabutin-susceptible isolates, with a specificity of 96.1% among 195 M. tuberculosis isolates with the rpoB mutation. Conclusions : Around 20% of the rifampicin-resistant isolates in Korea showed susceptibility to rifabutin, which was associated with some specific mutations of rpoB. Rifabutin could be used for the treatment of MDR-TB patients, especially when drug susceptibility testing reveals susceptibility to rifabutin.

• YAKHAK HOEJI
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• v.43 no.1
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• pp.124-127
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• 1999

Bifidobacterium infantis K-525 isolated from healthy Korean was susceptible to rifampicin and fluoroquinolones and resistant to other antituberculosis agents. When the preparation of this strain is taken as a therapeutics for human intestinal disorders with rifampicin or fluoroquinolones, its therapeutic effect can not be expected. So, B, infantis RFR-525 resistant to rifampicin was obtained by treating the parent B. infantis 525 with N-methyl-N'-nitro-N-nitrosoguanidine. B. infantis OFR-525 was produced by serial passage of B. infantis RFR-525 on agar with 2-fold minimal inhibitory concentration of ofloxacin. B. infantis OFR-525 was resistant to antituberculosis agents and fluoroquinolones up to 4∼128 fold higher than that for the original strain. The resistance of B. infantis OFR-525 against rifampicin and ofloxacin was maintained in vivo and in vitro. Conclusively, B. infantis OFR-525 can be regarded as a promising strain which can be developed as the preparation for the treatment of the intestinal disorders of the tuberculosis patients under rifampicin and ofloxacin therapy.

• Genomics & Informatics
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• v.12 no.4
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• pp.276-282
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• 2014

The disease tuberculosis, caused by Mycobacterium tuberculosis (MTB), remains a major cause of morbidity and mortality in developing countries. The evolution of drug-resistant tuberculosis causes a foremost threat to global health. Most drug-resistant MTB clinical strains are showing resistance to isoniazid and rifampicin (RIF), the frontline anti-tuberculosis drugs. Mutation in rpoB, the beta subunit of DNA-directed RNA polymerase of MTB, is reported to be a major cause of RIF resistance. Amongst mutations in the well-defined 81-base-pair central region of the rpoB gene, mutation at codon 450 (S450L) and 445 (H445Y) is mainly associated with RIF resistance. In this study, we modeled two resistant mutants of rpoB (S450L and H445Y) using Modeller9v10 and performed a docking analysis with RIF using AutoDock4.2 and compared the docking results of these mutants with the wild-type rpoB. The docking results revealed that RIF more effectively inhibited the wild-type rpoB with low binding energy than rpoB mutants. The rpoB mutants interacted with RIF with positive binding energy, revealing the incapableness of RIF inhibition and thus showing resistance. Subsequently, this was verified by molecular dynamics simulations. This in silico evidence may help us understand RIF resistance in rpoB mutant strains.

• Tuberculosis and Respiratory Diseases
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• v.46 no.5
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• pp.618-627
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• 1999

Background : Rifampicin (RFP) is a key component of the antituberculous short-course chemotherapy. Usually the RFP resistant M.tuberculosis is also resistant to isoniazid (INH), so the RFP resistance is the marker of multi-drug resistant (MDR) tuberculosis. But unusual cases of mono-RFP-resistant tuberculosis have been recently reported with increasing frequency, especially associated with HIV infection in western countries. Therefore, we conducted a retrospective study to investigate the frequency, causes, and the clinical characteristics of mono-RFP-resistant tuberculosis in Korea. Methods : Of the bacteriologically confirmed and susceptibility-proven 699 pulmonary tuberculosis patients (921 isolates) who visited Asan Medical Center from January 1990 to August 1997, eighteen patients with INH-susceptible and RFP-resistant tuberculosis were evaluated. Previous history of tuberculosis, antituberculous drug compliances, associated systemic illness, drug susceptibility patterns, and clinical outcomes were analysed. And rpoB gene sequencing was done in 6 clinical isolates of M. tuberculosis. Results : The mean age of 18 patients was $43{\pm}14$ years, and the sex ratio is 12:6 (M : F). Sixteen (89%) patients had previous history of tuberculosis. None had diagnosed gastrointestinal disorders, and 2 HIV tests that were performed came out negative. Susceptibility tests were done repeatedly in eleven patients, and six (55%) were mono-RFP resistant repeatedly while five (45%) evolved to MDR tuberculosis. Eight (44%) patients were cured, six (33%) failed, three (17%) were lost to follow-up, and the other one is now on treatment. rpoB gene sequencing showed 5 mutations, codon 531 TCG to TIG mutation in 4 isolates and 526 CAC to TAC in 1 isolate. Conclusion : The clinical characteristics of mono-RFP resistant tuberculosis were similar to that of MDR tuberculosis in Korea where the HIV infection rate is lower than western countries. But some patients with mono-RFP-resistant tuberculous could be cured by primary drug regimens including RFP, suggesting that mono-RFP-resistant tuberculous is a different entity from MDR tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.84 no.3
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• pp.237-244
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• 2021

Background: The emergence of drug-resistant tuberculosis (TB), is a major menace to cast off TB worldwide. Line probe assay (LPA; GenoType MTBDRplus ver. 2) and Xpert MTB/RIF assays are two rapid molecular TB detection/diagnostic tests. To compare the performance of LPA and Xpert MTB/RIF assay for early diagnosis of rifampicin-resistant (RR) TB in acid-fast bacillus (AFB) smear-positive and negative sputum samples. Methods: A total 576 presumptive AFB patients were selected and subjected to AFB microscopy, Xpert MTB/RIF assay and recent version of LPA (GenoType MTBDRplus assay version 2) tests directly on sputum samples. Results were compared with phenotypic culture and drug susceptibility testing (DST). DNA sequencing was performed with rpoB gene for samples with discordant rifampicin susceptibility results. Results: Among culture-positive samples, Xpert MTB/RIF assay detected Mycobacterium tuberculosis (Mtb) in 97.3% (364/374) of AFB smear-positive samples and 76.5% (13/17) among smear-negative samples, and the corresponding values for LPA test (valid results with Mtb control band) were 97.9% (366/374) and 58.8% (10/17), respectively. For detection of RR among Mtb positive molecular results, the sensitivity of Xpert MTB/RIF assay and LPA (after resolving discordant phenotypic DST results with DNA sequencing) were found to be 96% and 99%, respectively. Whereas, specificity of both test for detecting RR were found to be 99%. Conclusion: We conclude that although Xpert MTB/RIF assay is comparatively superior to LPA in detecting Mtb among AFB smear-negative pulmonary TB. However, both tests are equally efficient in early diagnosis of AFB smear-positive presumptive RR-TB patients.

• Annals of Laboratory Medicine
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• v.38 no.6
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• pp.569-577
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• 2018

Background: The increasing prevalence of drug-resistant tuberculosis (TB) infection represents a global public health emergency. We evaluated the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform [QMAP], QuantaMatrix, Seoul, Korea) to rapidly identify the Mycobacterium tuberculosis complex (MTBC) and detect rifampicin (RIF) and isoniazid (INH) resistance-associated mutations. Methods: A total of 200 clinical isolates from respiratory samples were used. Phenotypic anti-TB drug susceptibility testing (DST) results were compared with those of the QMAP system, reverse blot hybridization (REBA) MTB-MDR assay, and gene sequencing analysis. Results: Compared with the phenotypic DST results, the sensitivity and specificity of the QMAP system were 96.4% (106/110; 95% confidence interval [CI] 0.9072-0.9888) and 80.0% (72/90; 95% CI 0.7052-0.8705), respectively, for RIF resistance and 75.0% (108/144; 95% CI 0.6731-0.8139) and 96.4% (54/56; 95% CI 0.8718-0.9972), respectively, for INH resistance. The agreement rates between the QMAP system and REBA MTB-MDR assay for RIF and INH resistance detection were 97.6% (121/124; 95% CI 0.9282-0.9949) and 99.1% (109/110; 95% CI 0.9453-1.0000), respectively. Comparison between the QMAP system and gene sequencing analysis showed an overall agreement of 100% for RIF resistance (110/110; 95% CI 0.9711-1.0000) and INH resistance (124/124; 95% CI 0.9743-1.0000). Conclusions: The QMAP system may serve as a useful screening method for identifying and accurately discriminating MTBC from non-tuberculous mycobacteria, as well as determining RIF- and INH-resistant MTB strains.

• Tuberculosis and Respiratory Diseases
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• v.44 no.6
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• pp.1245-1255
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• 1997

Background : Rifampicin(RFP) is a key component of the antituberculous short-course chemotherapy and the RFP resistance is a marker of multi-drug resistant(MDR) tuberculosis. RPoB gene encodes the $\beta$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. And rpoB gene mutations are the cause of RFP resistance of M. tuberculosis. Although several reports showed that PCR-SSCP would be a rapid diagnostic method for identifying the RFP resistance, there were few reports Performed using direct, clinical specimens. So we Performed PCR-SSCP analysis of rpoB gene of M. tuberculosis in direct, clinical specimens. Methods : 75 clinical specimens were collected from patients at Asan Medical Center from June to August 1996. After PCR of IS 6110 fragments, 43 both AFB smear-positive and IS6110 fragment PCR-positive specimens were evaluated. The RFP susceptibility test was referred to the referral laboratory of the Korean Tuberculosis Institute. DNA was extracted by bead beater method. And heminested PCR was done using 0.1ul(1uCi) [$\alpha-^{32}P$]-dCTP. SSCP analysis was done using non-denaturating MDE gel electrophoresis. Results : The results of PCR of IS6110 fragments of M. tuberculosis were positive in 55(73%) cases of 75 AFB smear-positive clinical specimens. Of the 55 specimens, RFP susceptibility was confirmed in only 43 specimens. Of the 43 AFB smear-positive and IS6110 fragment-positive specimens, 29 were RFP susceptible and 14 were RFP resistant. All the RFP susceptible 29 strains showed the same mobility compared with that of RFP sensitive H37Rv in SSCP analysis of ropB gene. And all the other RFP resistant 13 strains showed the different mobility. In other words they showed 100% identical results between PCR-SSCP analysis and traditional susceptibility test. Conclusion : The PCR-sseP analysis of rpoB gene in direct clinical specimens could be used as a rapid diagnostic method for detecting RFP resistant M. tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.43 no.6
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• pp.842-851
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• 1996

Background : Rifampicin(RFP) is a key component of the antituberculous shon-course chemotherapy and the RFP-resistance is a marker of multi-drug resistant(MDR) M. tuberculosis. rpoB gene encodes the ${\beta}$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. Recent reports show that rpoB gene mutations are the cause of RFP resistance of M. tuberculosis and the main mechanism of rpoB gene mutation is point mutation. And PCR-SSCP is a rapid and easy method for detecting point mutations. So we performed PCR-SSCP of rpoB gene of M. tuberculosis and compared the result with traditional RFP sensitivity test. Method : The 27 RFP sensitive M. tuberculosis culture isolates and 25 RFP resistant isolates were evaluated. The RFP sensitivity test was done at the Korean Tuberculosis istitute. The DNA was extracted by bead beater method and was amplified with primers TR-8 and TR-9 in a 20ul PCR reaction containing 0.1ul(luCi) [${\alpha}-^{32}P$] - dCTP. After amplification, SSCP was done using non-denaturaring polyacrylamide gel electrophoresis. Then direct sequencing was done in cases of different eletrophoretic mobility compared with that of H37Rv. In 19 cases, we compared PCR-SSCP results with patient's clinical course and the results of traditional RFP sensitivity test. Results : 1) All 27 RFP sensitive M. tuberculosis isolates showed the same electrophoretic mobility compared with that of H37Rv. And all 25 RFP resistant M. tuberculosis isolates showed different electrophoretic mobility. 2) The mechanism of rpoB gene mutation of M. tuberculosis is mainly point mutation. 3) The PCR-SSCP results correlate well with traditional RFP sensitivity and patient's clinical response to antituberculous treatment. Conclusion: The PCR-SSCP of rpoB gene is a very sensitive and rapid mehod in detecting RFP- resistant M. tuberculosis.