• 대한임상검사과학회지
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• 제41권1호
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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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• 제83권1호
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• pp.20-30
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• 2020

Tuberculosis (TB) remains a threat to public health and is the leading cause of death globally. Isoniazid (INH) is an important first-line agent for the treatment of TB considering its early bactericidal activity. Resistance to INH is now the most common type of resistance. Resistance to INH reduces the probability of treatment success and increases the risk of acquiring resistance to other first-line drugs such as rifampicin (RIF), thereby increasing the risk of multidrug-resistant-TB. Studies in the 1970s and 1980s showed high success rates for INH-resistant TB cases receiving regimens comprised of first-line drugs. However, recent data have indicated that INH-resistant TB patients treated with only firs-tline drugs have poor outcomes. Fortunately, based on recent systematic meta-analyses, the World Health Organization published consolidated guidelines on drug-resistant TB in 2019. Their key recommendations are treatment with RIF-ethambutol (EMB)-pyrazinamide (PZA)-levofloxacin (LFX) for 6 months and no addition of injectable agents to the treatment regimen. The guidelines also emphasize the importance of excluding resistance to RIF before starting RIF-EMB-PZA-LFX regimen. Additionally, when the diagnosis of INH-resistant TB is confirmed long after starting the first-line TB treatment, the clinician must decide whether to start a 6-month course of RIF-EMB-PZA-LFX based on the patient's condition. However, these recommendations are based on observational studies, not randomized controlled trials, and are thus conditional and based on low certainty of the effect estimates. Therefore, further work is needed to optimize the treatment of INH-resistant TB.

• Clinical and Experimental Pediatrics
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• 제52권5호
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• pp.529-537
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• 2009

Drug-resistant tuberculosis in children has important implications for both the patients and tuberculosis control programs. In Korea, among all new patients, the isoniazid resistance rate was 9.9% and multidrug-resistant tuberculosis rate was 2.7% in 2004 (in patients aged 10-19 yr, the multidrug-resistant tuberculosis rate reached 2.1%). Tuberculosis in pediatric patients is difficult to diagnose because many children have nonspecific clinical signs and the detection rates of acid-fast bacilli smears and cultures are low. Therefore, every effort should be made to identify adult sources and obtain information on drug susceptibility because symptomatic adult patients have a higher chance of culture positivity and drug-susceptibility patterns are the same in most adult-child pair patients. Korean children are at significant risk of drug-resistant tuberculosis. As the isoniazid resistance rate is greater than 4% among the new cases in Korea, a four-drug regimen should be considered for initial treatment of children with active tuberculosis, unless drug-susceptibility test results are available. Treatment of drug-resistant tuberculosis in children is challenging and there are only few available data. Tuberculosis control programs should be continuous with specific focus on pediatric populations because they can serve as reservoirs for future active cases. Further studies are needed regarding treatment of drug-resistant tuberculosis in children.

• Tuberculosis and Respiratory Diseases
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• 제82권2호
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• pp.143-150
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• 2019

Background: The purpose of this study was to analyze the relationship between the gene mutation patterns by the GenoType MTBDRplus (MTBDRplus) assay and the phenotypic drug susceptibility test (pDST) results of isoniazid (INH) and prothionamide (Pto). Methods: A total of 206 patients whose MTBDRplus assay results revealed katG or inhA mutations were enrolled in the study. The pDST results were compared to mutation patterns on the MTBDRplus assay. Results: The katG and inhA mutations were identified in 68.0% and 35.0% of patients, respectively. Among the 134 isolated katG mutations, three (2.2%), 127 (94.8%) and 11 (8.2%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Among the 66 isolated inhA mutations, 34 (51.5%), 18 (27.3%) and 21 (31.8%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Of the 34 phenotypic Pto resistant isolates, 21 (61.8%), 11 (32.4%), and two (5.9%) had inhA, katG, and both gene mutations. Conclusion: It is noted that Pto may still be selected as one of the appropriate multidrug-resistant tuberculosis regimen, although inhA mutation is detected by the MTBDRplus assay until pDST confirms a Pto resistance. The reporting of detailed mutation patterns of the MTBDRplus assay may be important for clinical practice, rather than simply presenting resistance or susceptibility test results.

• BMB Reports
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• 제42권2호
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• pp.81-85
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• 2009

Early and accurate detection of drug resistant Mycobacterium tuberculosis can improve both the treatment outcome and public health control of tuberculosis. A number of molecular-based techniques have been developed including ones using probe molecules that target drug resistance-related mutations. Although these techniques are highly specific and sensitive, mixed signals can be obtained when the drug resistant isolates are mixed with drug susceptible isolates. In order to overcome this problem, we developed a new drug susceptibility test (DST) for one of the most effective anti-tuberculosis drug, isoniazid. This technique employed a microplate hybridization assay that quantified signals from each probe molecule, and was evaluated using clinical isolates. The evaluation analysis clearly showed that the microplate hybridization assay was an accurate and rapid method that overcame the limitations of DST based on conventional molecular techniques.

• Annals of Laboratory Medicine
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• 제38권6호
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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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• 제55권1호
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• pp.41-58
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• 2003

연구배경 : 약제내성 결핵권의 조기 진단을 위해서, 최근 돌연변이 검출 및 질병의 진단 등에 새로운 기술로 대두되고 있는 올리고뉴콜레오티드 칩 기술을 이용하여 결핵균의 리팜핀, 아이소나아지드와 스트렙토마이신 내성과 관련된 rpoB, katG와 rpsL 유전자의 주요 돌연변이를 신속하고 정확하게 검출하고자 하였다. 방 법 : 리팜핀 내성 검출의 야생형 7개와 돌연변이형 13개, 아이소니아지드 내성 검출의 야생형 2개와 돌연변이형 3개, 그리고 스트렙토마이신 내성 검출을 위한 야생형과 돌연변이형 프로브 각 2종류를 고안한 후, 유리 슬라이드에 고정시켜 올리고뉴클레오티드 칩을 제작하였고, 약제내성을 가지고 있는 배양균주 55균주를 선택하여 PCR 증폭반응과 혼성화 반응을 실시한 후 비공초점 레이저 스케너를 이용하여 돌연변이를 검출하였다. 이를 염기서열방법으로 확인하여 돌연변이 다형성을 분석하였다. 결 과 : 리팜핀 내성은 코돈 531과 코돈 526에서 65%의 돌연변이를 검출하였고, 현재까지 보고되어 있지 않은 D516F의 새로운 돌연변이도 검출하였다. 아이소니아지드 내성은 S315T와 R463L 돌연변이가 45.2%로 검출되었고, 스트렙토마이신 내성은 K43R과 K88R 돌연변이가 78%로 검출되었다. 리팜핀 내성의 88%(35/40), 아이소니아지드 내성의 50%(20/42), 그리고 스트렙토마이신 내성의 78%(7/9)를 검출함으로써 현재까지 보고되어 있는 세가지 약제내성과 관련된 rpoB, katG와 rpsL 유전자의 주요 돌연변이는 대부분 검출할 수 있음을 확인할 수 있었고, 염기서열분석 결과와 비교하였을 때 모두 일치하는 결과를 얻음으로써 올리고뉴콜레오티드 칩의 유용성을 확인할 수 있었다. 결 론 : 따라서 본 연구에서 개발한 올라고뉴클레오티드 칩은 약재내성 결핵균의 조기 진단에 유용한 도구가 될 것으로 사료된다.

• Tuberculosis and Respiratory Diseases
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• 제49권4호
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• pp.412-420
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• 2000

연구배경 : 전세계적으로 약제 내성 결핵에 대한 관심이 높ㅇ지고 있다. 일차 내성 결핵의 유병률은 수년 간의 국가 결핵 관리 체계를 평가하는 역학적 지표로 사용된다. 저자들은 내성 결핵에서의 사회경제적 인자를 포함한 임상적 특정에 대해 평가하고자 하였다. 방법 : 1995년 3월부터 2000년 2월까지 이대목동병원에서 내성 결핵으로 진단 받은 환자 중 조사가 가능했던 68명을 대상으로 하였다. 결과 : 일차 내성 결핵 환자는 획득 내성 결핵환자보다 평균 연령이 더 젊고(39.6$\pm$16.3yr vs. 48.2$\pm$16.5yr ; p<0.05), 40대 미만 연령층의 구성비가 더 높았으며(62.9% vs. 36.4% ; p<0.05), 고학력자가 더 많았다 (38.9% vs. 11.1% ; p<0.05). 획득 내성 환자는 일차 내성 환자보다 통계학적 의의는 없었으나 가족력의 빈도가 더 높고 자가 주택이 없는 경우가 더 많았다. 획득 내성 환자는 일차 내성 환자보다 침범된 폐엽의 수가 더 많았고(2.0$\pm$0.8 vs. 1.4$\pm$0.7; p<0.01), 총 치료 기간이 더 길었다(18.3$\pm$7.2 months vs. 10.6$\pm$6.3 months ; p<0.05). 획득 내성 환자가 일차 내성 환자보다 통계학적 의의는 없었으나 내성 약제의 수가 더 많고 입원률이 더 낮았으며 임의로 투약을 중단하는 경우가 더 많았다. 일차 내성 환자가 획득 내성 환자보다 단일 약제 내성률이 더 높은 반면, 복수 약제 내성률과 다제 내성률은 더 낮았다. 약제별 내성률에서 isoniazid에 대한 내성률이 가장 높았고, 획득 내성 환자에서 일차 내성 환자보다 isoniazid에 대한 내성률이 유의하게 높았다(90.9% vs. 71.4% ; p<0.05). 결론 : 획득 내성 환자가 낮은 사회경제적 계층의 비율이 높은 반면, 일차 내성 환자는 젊고 활동적인 집단인 것으로 보인다. 약제 내성 결핵의 전파와 재발을 막기 위해서는 내성 환자의 적절한 격리와 적극적인 추적 관찰을 통한 치료의 종결이 중요하다.

• 대한의생명과학회지
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• 제11권4호
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• pp.455-463
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• 2005

Resistance to isoniazid (INH), which is one of the most important drugs in Mycobacterium tuberculosis chemotherapy, has been associated with mutations in genes encoding the mycobacterial catalse-peroxidase (katG), the enoyl acyl carrier protein (ACP) reductase (inhA), alkyl hydroperoxide reductase (ahpC), beta-ketoacyl acyl carrier protein synthase (kasA), and NADH dehydrogenase (ndh). In this study, we examined INH-resistance related genes in 50 INH-resistant and 24 INH-susceptible isolates by PCR-sequence analysis. In brief, mutations at the katG gene were found at codon 315 alone (2/50), at codon 463 alone (19/50), and both at 315 and 463 (29/50). However, while mutations at codon 315 were only detected in INH-resistant isolates, mutations at codon 463 were also detected in INH-susceptible isolates indicating mutations at 463 alone do not seem to confer resistance to INH. Similar to the case of katG 463, some of inhA mutations were also found among INH-susceptible isolates. For example, whereas mutations at 8 upstream of the start codon (UPS) and 15 UPS of the inhA gene were detected only in INH-resistant isolates, mutations at 101, 115, and 125 UPS were detected only in INH-susceptible isolates. Many different kinds of mutations were detected in INH­resistant isolates at ahpC, oxyR gene, and intergenic region of the oxyR-ahpC genes. Howerver, the mutations were not found oxyR and the intergenic regions in INH-susceptible isolates. No mutations were found at either kasA or at ndh gene among INH-resistant isolates. In conclusion, some of mutations such as katG 315, inhA promotor region, and oxyR-ahpC seem to be strongly related to INH-resistance. Currently we are developing a molecular diagnostic method based on these results.

• Tuberculosis and Respiratory Diseases
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• 제85권3호
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• pp.264-272
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• 2022

Background: The current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed. Methods: To evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results. Results: The susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST. Conclusion: In conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.