• Genomics & Informatics
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• 제21권3호
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• pp.42.1-42.23
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• 2023

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Tolllike receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

• 대한의생명과학회지
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• 제11권4호
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• pp.465-471
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• 2005

Ofloxacin has antimycobacterial activity that possibly contributes a pivotal role in the second-line drug regimens that are used for the treatment of multidrug-resistant tuberculosis. However, in some communities, the resistance rate of Mycobacterium tuberculosis to this agent is surging. Therefore, a rapid and accurate method that can be used to determine the resistance of M tuberculosis to the ofloxacin can be very useful for effective treatment of the patients. As an effort to develop such a method, this study was set up to reveal general types of mutations that are related to ofloxacin resistance of M tuberculosis. From previous studies, it has been well known that ofloxacin resistance is associated with mutations in a gene encoding the gyrase A subunit protein. In this study, we obtained 43 ofloxacin-resistant and 50 ofloxacin-susceptible M tuberculosis clinical isolates from Masan National TB Hospital, and sequences of DNA fragment of 320 bp, region of gyrA corresponding to the ofloxacin resistance-determining region were analyzed. In brief, the results showed that a total of seven mutation types were found at gyrA. Theses mutations were all clustered within nucleotides 2574 to 2586 of the gyrA gene (codons 88 to 94). Codon 94 was the most frequently substituted site. Twenty-four of the 43 isolates had mutations at this position resulting in a total of five different types of amino acid changes $(Asp{\to}Ala,\;Asp{\to}Gly,\;Asp{\to}His,\;Asp{\to}Tyr,\;and\;Asp{\to}Asn)$. Five isolates contained a mutation at codon 90 resulting $Ala{\to}Val$ change. Four isolates had mutations at codon 91 causing a $Ser{\to}Pro$ change at this site. Two isolates contained a mutation at codon 88 and each of them resulted in different types of amino acid changes $(Gly{\to}Cys,\;Gly{\to}Ala)$. On the other hand, polymorphic site at codon 95 was found in both ofloxacin-resistant and ofloxacin-susceptible isolates. From these results, we concluded that the rate of mutations present in gyrA among ofloxacin-resistant M. tuberculosis in Korea is similar to the general rates of mutations found throughout the world. Subsequently, an oligonucleotide probe was designed based on the results of sequence analysis and was used to develop a dot blot hybridization assay system to determine ofloxacin-resistance of M tuberculosis. To evaluate this probe, dot-blot hybridization was carried out using other 57 clinical isolates, and the results showed that the dot-blot hybridization assay is good for detecting sequence alterations atgyrA gene.

• Tuberculosis and Respiratory Diseases
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• 제82권2호
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• pp.151-157
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• 2019

Background: Tuberculosis (TB) is the most important disease screened for upon patient history review during preimmigration medical examinations as performed in South Korea in prospective immigrants to certain Western countries. In 2007, the U.S. Centers for Disease Control and Prevention (CDC) changed the TB screening protocol from a smear-based test to the complete Culture and Directly Observed Therapy Tuberculosis Technical Instructions (CDOT TB TI) for reducing the incidence of TB in foreign-born immigrants. Methods: This study evaluated the effect of the revised (as compared with the old) protocol in South Korea. Results: Of the 40,558 visa applicants, 365 exhibited chest radiographic results suggestive of active or inactive TB, and 351 underwent sputum tests (acid-fast bacilli smear and Mycobacterium tuberculosis culture). To this end, using the CDOT TB TI, 36 subjects (88.8 per $10^5$ of the population) were found to have TB, compared with only seven using the older U.S. CDC technical instruction (TI) (p<0.001). In addition, there were six drug-resistant cases which were identified (16.7 per $10^5$ of the population), two of whom had multidrug-resistance (5.6 per $10^5$ of the population). Conclusion: The culture-based 2007 TI identified a great deal of TB cases current to the individuals tested, as compared to older U.S. CDC TI.

• Tuberculosis and Respiratory Diseases
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• 제80권2호
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• pp.159-168
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• 2017

Background: Streptomycin (SM) is recommended by the World Health Organization (WHO) as a part of standard regimens for retreating multidrug-resistant tuberculosis (MDR-TB) cases. The incidence of MDR-TB in retreatment cases was 19% in Thailand. To date, information on SM resistance (SMR) gene mutations correlated to the SMR of Mycobacterium tuberculosis Thai isolates is limited. In this study, the mutations in rpsL, rrs, gidB, and whiB7 were investigated and their association to SMR and the lineage of M. tuberculosis were explored. Methods: The lineages of 287 M. tuberculosis collected from 2007 to 2011 were identified by spoligotyping. Drug susceptibility profiles were evaluated by the absolute concentration method. Mutations in SMR genes of 46 SM-resistant and 55 SM-susceptible isolates were examined by DNA sequencing. Results: Three rpsL (Lys43Arg, Lys88Arg, and Lys88Thr) and two gidB (Trp45Ter and Gly69Asp) mutations were present exclusively in the SM resistant M. tuberculosis. Lys43Arg rpsL was the most predominant SMR mutations (69.6%) and prevailed among Beijing isolates (p<0.001). No SMR-related mutation in was found rrs. The combination of rpsL and gidB mutations provided 76.1% sensitivity for detecting SMR in M. tuberculosis Thai isolates. whiB7 was not responsible for SMR in SM resistant isolates lacking rpsL and rrs mutations. The significance of the three gidB mutations, 276A>C, 615A>G, and 330G>T, as lineage signatures for Beijing and EAI were underscored. This study identified 423G>A gidB as a novel sub-lineage marker for EAI6-BGD1. Conclusion: Our study suggested that the majority of SMR in M. tuberculosis Thai isolates were responsible by rpsL and gidB polymorphisms constantly providing the novel lineage specific makers.

• Tuberculosis and Respiratory Diseases
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• 제80권2호
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• pp.143-152
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• 2017

Background: Fluoroquinolones are considered important substitutes for the treatment of tuberculosis. This study investigates the current status of fluoroquinolone for the treatment of tuberculosis. Methods: In 2009, a retrospective analysis was performed at one tertiary referral center for 953 patients diagnosed with tuberculosis. Results: A total of 226 patients (23.6%), who received fluoroquinolone at any time during treatment for tuberculosis, were enrolled in this study. The most common reasons for fluoroquinolone use were adverse events due to other anti-tuberculosis drugs (52.7%), drug resistance (23.5%), and underlying diseases (16.8%). Moxifloxacin (54.0%, 122/226) was the most commonly administered fluoroquinolone, followed by levofloxacin (36.3%, 82/226) and ofloxacin (9.7%, 22/226). The frequency of total adverse events from fluoroquinolone-containing anti-tuberculosis medication was 22.6%, whereas fluoroquinolone-related adverse events were estimated to be 2.2% (5/226). The most common fluoroquinolone-related adverse events were gastrointestinal problems (3.5%, 8/226). There were no significant differences in the treatment success rate between the fluoroquinolone and fluoroquinolone-$na{\ddot{i}}ve$ groups (78.3% vs. 78.4%, respectively). Conclusion: At our institution, fluoroquinolones are commonly used for the treatment of both multidrug-resistant tuberculosis and susceptible tuberculosis, especially as a substitute for adverse event-related drugs. Considering the low adverse event rates and the comparable treatment success rates, fluoroquinolones seem to be an invaluable drug for the treatment of tuberculosis.

• Tuberculosis and Respiratory Diseases
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• 제70권3호
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• pp.251-256
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• 2011

The prevalence of multi-drug resistant tuberculosis (MDR-TB), which is resistant to isoniazid and rifampin, has been increasing in Korea. And the side effects of 2nd line anti-tuberculosis medications, including drug-induced hepatitis, are well known. Although prothionamide (PTH) is one of the most useful anti-TB medications and although TB medication-induced acute hepatitis is a severe complication, there are only a few published case reports about prothionamide induced hepatitis. In this case report, a 22 year old male was diagnosed with pulmonary MDR-TB and was administered 2nd line anti-TB mediations, including PTH. Afterwards, he had a spiking fever and his liver enzymes were more than 5 times greater than the upper limit of the normal range. He was then diagnosed with drug-induced hepatitis by liver biopsy. His symptoms and liver enzyme elevation were improved after stopping PTH. Accordingly, we report this case of an association between PTH and acute hepatitis.

• Tuberculosis and Respiratory Diseases
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• 제59권6호
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• pp.613-618
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• 2005

배 경 : 최근 들어 다제내성 폐결핵의 증가로 항결핵제 치료로만으로는 만족스러운 균음전율을 보여주지 못하고 있다. 이에 저자들은 수술을 시행한 환자를 분석함으로써 향후 다제내성 폐결핵의 치료의 한 방법을 제시하고자 한다. 방 법 : 국립마산병원에서 1994년 1월부터 2003년 12월까지 10년간 다제내성 폐결핵으로 페절제술을 시행한 138례를 술전 환자의 특성, 술전 범위, 수술 방법, 합병증, 술후 추적관찰을 후향적으로 분석하였다. 결 과 : 대상 환자의 남녀비는 5.1:1으로 남자에서 많았고 평균연령은 42.6세이고 내성약제는 5.3개였다. 술전 객담에서 94례(68.1%)에서 균양성 소견이였고 128례(92.8%)에서 공동성 병소를 보였다. 수술술식은 24례에서 전폐적출술, 83례에서 폐엽절제술, 10례에서 쌍엽절제술, 19례에서 폐엽절제술과 구역절제술, 설상절제술, 2례에서 설상절제술과 공동성형술을 시행하였다. 술 후 균 음성율은 91.3%였다. 균 음전에 실패한 2례는 모두 공동성 병변이였고 균 음전 실패 후 항결핵제 변경과 재수술로 6례에서 균음전되었다. 재발한 10례 중 6례에서 항결핵제 변경과 재수술로 균음전되었다. 이상에서 술후 지속적인 약물 복용과 필요시 항결핵제 변경과 재수술로 높은 장기 균음전율(92.8%)을 보였다. 수술 후 치료 실패 및 재발한 22례는 전체적으로 술전 유병기간이 길었고 술전 치료 횟수 및 술전 내성약제가 많았다. 수술에 관련된 사망자는 없었고 술 후 합병증으로는 1주일 이상 지속되는 공기 유출이 6례, 출혈로 인한 재수술이 6례, 기관지 늑막루와 농흉이 8례, 사강이 16례, 무기폐가 1례, 상처 감염이 1례, 수술 부위에 낭종형성이 1례 등이 나타났다. 결 론 : 다제내성 폐결핵에 대한 수술의 적응증, 수술 후 처방, 수술 후 치료기간 등에 대한 많은 의견이 있지만 수술은 환자의 상태가 가능한 빨리, 가능한 적극적으로 시행하여 내과적인 치료와 병행함으로써 높은 치료 효과를 얻을 수 있을 것임을 확인할 수 있었다.

• Bulletin of the Korean Chemical Society
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• 제28권6호
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• pp.947-952
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• 2007

Mycobacterium tuberculosis is a pathogen responsible for 2-3 million deaths every year worldwide. The emergence of drug-resistant and multidrug-resistant tuberculosis has increased the need to identify new antituberculosis targets. Acetohydroxy acid synthase, (AHAS, EC 2.2.1.6), an enzyme involved in branched-chain amino acid synthesis, has recently been identified as a potential anti-tuberculosis target. To assist in the search for new inhibitors and “receptor-based” design of effective inhibitors of tubercular AHAS (TbAHAS), we constructed four different structural models of TbAHAS and used one of the models as a target for virtual screening of potential inhibitors. The quality of each model was assessed stereochemically by PROCHECK and found to be reliable. Up to 89% of the amino acid residues in the structural models were located in the most favored regions of the Ramachandran plot, which indicates that the conformation of each residue in the models is good. In the models, residues at the herbicide-binding site were highly conserved across 39 AHAS sequences. The binding mode of TbAHAS with a sulfonylurea herbicide was characterized by 32 hydrophobic interactions, the majority of which were contributed by residue Trp516. The model based on the highest resolution X-ray structure of yeast AHAS was used as the target for virtual screening of a chemical database containing 8300 molecules with a heterocyclic ring. We developed a short list of molecules that were predicted to bind with high scores to TbAHAS in a conformation similar to that of sulfonylurea derivatives. Five sulfonylurea herbicides that were calculated to efficiently bind TbAHAS were experimentally verified and found to inhibit enzyme activity at micromolar concentrations. The data suggest that this time-saving and costeffective computational approach can be used to discover new TbAHAS inhibitors. The list of chemicals studied in this work is supplied to facilitate independent experimental verification of the computational approach.

• Tuberculosis and Respiratory Diseases
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• 제51권5호
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• pp.409-415
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• 2001

배 경 : 다제내성 폐결핵은 대부분은 획득내성에 의해 발생한다. 그러나 일부분에서 초회내성으로 발생하는데 이러한 환자들은 획득내성으로 인한 환자들과 차이가 있을 것으로 생각된다. 본 연구는 초회내성으로 진단된 다제내성 폐결핵 환자들의 임상적 특징을 조사하여 향후 이들에 대한 효과적인 치료에 지표로 삼고자 한다. 대상 및 방법 : 1995년 1월부터 1998년 12월까지 입원치료를 시행한 초회내성으로 진단된 다제내성 폐결핵 환자 30명을 대상으로 하였다. 임상적 특징을 조사하기 위하여 성별, 연령, 가족력, 균음전화 기간, 내성약제수, 치료약제와 흉부방사선상 NTA 분류를 이용한 병변의 정도 및 공동 유무와 치료기간 등을 조사하였다. 통계적 분석은 흉부방사선상 병변의 정도와 공동의 호전 여부를 윌콕슨 부호 순위 검정법을 이용하였고, Kaplan-Meier 방법으로 1년 및 4년 무병율을 조사하였다. 결 과 : 환자들의 평균나이는 평균 46.6세였고, 남녀비는 1:1이었다. 폐결핵 가족력이 있었던 경우는 6(20%)명이었다. 객담에서 균이 음전된 기간은 평균 2.6개월이었으며, 내성약제의 개수는 평균 7.6개였다. 환자들 중 23(67%)명에서 12개월 이하로 치료하였다. 그리고 초치료 처방으로 치료한 경우는 28(93%)명이었다. 흉부방사선상 병변의 정도와 공동은 치료 후 호전되었다(p<0.05). 총 13명의 환자들 평균 22.6개월간 외래 추적조사 결과 2(15%)명에서 재발을 관찰할 수 있었고, 1년 및 4년 무병율은 85%였다. 결 론 : 초회내성으로 진단된 다제내성 폐결핵의 경우에 있어서 초치료 처방으로 하여 흉부방사선상 병변의 정도와 공동의 호전 여부를 주의 깊게 관찰하면서 9-12개월을 치료한다면 성공적인 결과를 얻을 수 있을 것으로 생각된다.

• Tuberculosis and Respiratory Diseases
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• 제43권6호
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• pp.842-851
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• 1996

연구배경: Rifampicin(RFP)은 항결핵 단기지료의 근간이 되는 약제로서 RFP에 대한 내성을 다제약제내성의 지표로 보기도 한다. rpoB유전자는 RFP이 결합하여 약리작용을 나타내는 RNA poly-merase의 ${\beta}$-subunit을 coding하는 유전자이다. 다른 보고들에 의하면 RFP내성균주에서 rpoB유전자의 돌연변이가 관찰되고 특히 점돌연변이가 중요한 기전임이 알려지고 있다. 이에 저자는 rpoB유전자의 점돌연변이를 쉰게 관찰할 수 있는 PCR-SSCP법 을 이용하여 신속하게 RFP에 대한 내성여부를 확인할 수 있는지에 대하여 알아보았다. 방법: 전통적인 약제내성검사상 RFP에 내성을 보이는 25 배양균주와 RFP에 감수성인 27 배양균주 그리고 표준균주인 H37Rv를 대상으로 하였다. TR8, TR9 primer를 이용하여 rpoB 유전자의 511 codon에서 533 codon 부위를 포함하는 157bp의 DNA를 증폭한 후 폴리 아크릴아마이드젤 전기영동으로 PCR-SSCP를 시행하여 band의 이동양상을 비교하였다. 그리고 H37Rv 와 다른 band의 양상을 보인 균주에서 direct sequencing을 시행하여 H37Rv의 염기서열과 비교하였다. 또한 임상결과를 추적할 수 있었던 19예에서 임상결과와 전통적인 감수성검사 결과, 그리고 PCR-SSCP결과를 비교하였다. 결과: 1) PCR-SSCP결과로 RFP감수성 27균주 모두에서 H37Rv와 동일한 band의 양상을 보였고, RFP내성 25균주 모두에서 H37Rv와 다른 band의 양상을 보여서 전통적인 RFP 감수성검사와 rpoB유전자의 PCR-SSCP 사이에 100% 일치하는 결과를 보여주었다. 2) rpoB 유전자 돌연변이의 주된 기전은 점돌연변이였다. 3) rpoB 유전자의 PCR-SSCP 결과는 전통적인 RFP감수성검사나 항결핵치료의 임상경과와 잘 일치하였다. 결론: 결핵균 rpoB 유전자의 PCR-SSCP에 의한 돌연변이의 확인은 RFP내성 결핵균의 신속한 진단에 아주 유용한 검사로 기대된다. 향후 직접임상검체를 대상으로 한 연구가 필요하리라 사료된다.