• 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.

• Biomedical Science Letters
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• v.19 no.2
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• pp.90-97
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• 2013

The tuberculin skin test (TST) and interferon gamma (IFN-${\gamma}$) release assay (IGRA) have been widely used for diagnosis of latent tuberculosis infection (LTBI). In order to overcome limitations of current LTBI diagnostic methods, the development of a novel molecular assay which is able to measure the IFN-${\gamma}$ messenger RNA (mRNA) expression level after stimulation with Mycobacterium tuberculosis (MTB) specific antigen was recently developed. The ability of a molecular assay to detect MTB infection was similar to commercial IGRA however, the optimal incubation time for stimulating IFN-${\gamma}$ was not yet established. Therefore, in this study the direct comparisons of MTB Ag stimulation times (4 and 24 hrs) were performed for diagnosis of MTB infection. Data showed that the coincident rate between QFT-GIT IFN-${\gamma}$ ELISA and IFN-${\gamma}$ RT-PCR (4 hrs) was 88.35% and that of QFT-GIT and IFN-${\gamma}$ RT-PCR (24 hrs) was 70.85%. Based on a receiver operating characteristic (ROC) curve, the 4 hrs-MTB specific Ag stimulation time for IFN-${\gamma}$ RT-PCR had the significant P value, 95% CI value, and AUC (P < 0.0001, 95% CI=0.82 to 1.02, and AUC=0.9214) in comparison with 24 hrs-MTB specific Ag stimulation time (P = 0.009, 95% CI=0.06 to 0.94, and AUC=0.7711). These results show that 4-hr was the most optimal MTB Ag stimulation time for performing IFN-${\gamma}$ RT-PCR. Although semi-quantitative RT-PCR had a few analytical limitations, it might be useful as an alternative molecular diagnostic method for detecting MTB infection.

• Tuberculosis and Respiratory Diseases
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• v.72 no.1
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• pp.44-49
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• 2012

Background: Multidrug-resistant tuberculosis (MDR-TB) is an increasing public health problem and poses a serious threat to global TB control. Fluoroquinolone (FQ) and aminoglycoside (AG) are essential anti-TB drugs for MDR-TB treatment. REBA MTB-FQ$^{(R)}$ and REBA MTB-KM$^{(R)}$ (M&D, Wonju, Korea) were evaluated for rapid detection of FQ and kanamycin (KM) resistance in MDR-TB clinical isolates. Methods: M. tuberculosis (n=67) were isolated and cultured from the sputum samples of MDR-TB patients for extracting DNA of the bacilli. Mutations in genes, gyrA and rrs, that have been known to be associated with resistance to FQ and KM were analyzed using both REBA MTB-FQ$^{(R)}$ and REBA MTB-KM$^{(R)}$, respectively. The isolates were also utilized for a conventional phenotypic drug susceptibility test (DST) as the gold standard of FQ and KM resistance. The molecular and phenotypic DST results were compared. Results: Sensitivity and specificity of REBA MTB-FQ$^{(R)}$ were 77 and 100%, respectively. Positive predictive value and negative predictive value of the assay were 100 and 95%, respectively, for FQ resistance. Sensitivity, specificity, positive predictive value and negative predictive value of REBA MTB-KM$^{(R)}$ for detecting KM resistance were 66%, 94%, 70%, and 95%, respectively. Conclusion: REBA MTB-FQ$^{(R)}$ and REBA MTB-KM$^{(R)}$ evaluated in this study showed excellent specificities as 100 and 94%, respectively. However, sensitivities of the assays were low. It is essential to increase sensitivity of the rapid drug resistance assays for appropriate MDR-TB treatment, suggesting further investigation to detect new or other mutation sites of the associated genes in M. tuberculosis is required.

• Biomedical Science Letters
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• v.26 no.3
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• pp.238-243
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• 2020

Tuberculosis is a global public health problem and manifests itself as a difference in the genetic susceptibility of the host, along with the properties of Mycobacterium tuberculosis (MTB). The single nucleotide polymorphisms (SNPs) and candidate genes proposed in the Genome-wide association study (GWAS) on tuberculosis in a recently published Chinese population were reported. In this study, we investigated whether the genetic polymorphism of candidate genes related to tuberculosis is reproduced when targeting Koreans. The CLCN6 (rs12404124, rs198391, rs535107), DOK7 (rs1203104, rs1203103) and HLA-DRA (rs1051336) gene polymorphisms showed statistically significant results. In addition, it was also found whether it acts as an expression quantitative trait loci (eQTL) that can influence gene expression. This study confirmed that the genetic polymorphism of the three genes (CLCN6, DOK7, HLA-DRA) affects the development of tuberculosis and will help to understand the genetic specificity of tuberculosis and the interaction between pathogens and hosts.

• Biomedical Science Letters
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• v.18 no.3
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• pp.201-209
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• 2012

Apoptosis is a physiological programmed cell death process. Tubercle bacilli inhibit apoptosis of alveolar macrophages and phagolysosome fusion. We investigated whether the Bcl-2 family anti-apoptotic member, Bfl-1/A1, plays an important role in the anti-apoptotic process during mycobacterial infection. PMA-treated human monocytoid THP-1 cells were infected with mycobacteria (H37Rv, BCG, and K-strain) at a multiplicity of infection (MOI) of 10 for 0, 1.5, 3, 6, 9, 12, 18, 24, 48, or 72 h. In addition, PMA-treated THP-1 cells were pretreated with specific inhibitors for 45 min before stimulation with mycobacteria at an MOI of 10 for 4 h. After the indicated time, the cells were subject to reverse transcription-polymerase chain reaction (RT-PCR) analysis, and a Bfl-1/A1-specific Western blot was performed. In PMA-differentiated THP-1 cells, the expression level of Bfl-1/A1 mRNA was increased by Mycobacterium tuberculosis (MTB) H37Rv infection. The mRNA level of Bfl-1/A1 peaked 3 h after MTB infection, then declined gradually until 9 h. However, Bfl-1/A1 mRNA induction gradually re-increased from 24 h to 72 h after MTB infection. No difference in Bfl-1/A1 expression was detected following infection with MTB H37Rv, K-strain, or M. bovis BCG. These results were not dependent on mycobacterial virulence. Moreover, mRNA levels of other anti-apoptotic molecules (Mcl-1, Bcl-2, and Bcl-xL) were not increased after MTB H37Rv or K-strain infection. These results suggest that mycobacteria induce the innate immune host defense mechanisms that utilize Bfl-1/A1 molecules at early time points, regardless of virulence.

• Tuberculosis and Respiratory Diseases
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• v.51 no.4
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• pp.315-324
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• 2001

Background : IL-8 is a potent chemotactic cytokine that plays an important role in the host defense mechanism against M. tuberculosis by recruiting inflammatory cells to the site of the infection. Lung epithelial cells, as well as alveolar macrophages are known to produce IL-8 in response to M. tuberculosis. IL-8 gene expression is mainly regulated on the level of transcription by NF-${\kappa}B$. This study investigated whether or not A549 cells produce IL-8 in NF-${\kappa}B$ dependent mechanism in response to macrophages phagocytosing M. tuberculosis. Methods : Peripheral blood monocytes that were obtained from healthy donors were cultured for 24 h with M. tuberculosis and a conditioned medium(CoMTB) was obtained. As a negative control, the conditioned medium without M. tuberculosis (CoMCont) was used. A549 cells were stimulated with M. tuberculosis, CoMCont and CoMTB and the IL-8 concentration in the culture media was measured by ELISA. The CoMTB induced IL-8 mRNA expression in the A549 cells was evaluated using RT-PCR, and CoMTB induced $I{\kappa}B{\alpha}$ degradation was measured using western blot analysis. CoMTB induced nuclear translocation and DNA binding of NF-${\kappa}B$ was also examined using an electrophoretic mobility shift assay(EMSA), and the CoMTB induced NF-${\kappa}B$ dependent IL-8 transcriptional activity was measured using a luciferase reporter gene assay. Results : CoMTB induced IL-8 production by A549 cells($46.8{\pm}4.8\;ng/ml$) was higher than with direct stimulation with M. tuberculosis ($6.8{\pm}2.9\;ng/ml$). CoMTB induced IL-8 mRNA expression increased after 2 h of stimulation and was sustained for 24 h. $I{\kappa}B{\alpha}$ was degraded after 10 min of CoMTB stimulation and reappeared by 60 min. CoMTB stimulated the nuclear translocation and DNA binding of NF-${\kappa}B$. The CoMTB induced NF-${\kappa}B$ dependent IL-8 transcriptional activity($13.6{\pm}4.3$ times control) was higher than either CoMCont($2.0{\pm}0.6$ times control) or M. tuberculosis ($1.4{\pm}0.6$ times control). Conclusion : A conditioned medium of peripheral blood monocytes phagocytosing M. tuberculosis stimulates NF-${\kappa}B$ dependent IL-8 production by the lung epithelial cells.

• Tuberculosis and Respiratory Diseases
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• v.74 no.6
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• pp.251-255
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• 2013

Tuberculosis (TB) is one of the largest health problems in the world today. And the incidence of nontuberculous mycobacteria (NTM) lung disease appears to be increasing worldwide. Recently, an automated, nucleic acid amplification assay for the rapid detection of both Mycobacterium tuberculosis and rifampin resistance was developed (Xpert MTB/RIF). And fixed-dose combinations of anti-TB drugs and linezolid have been introduced in the treatment of TB. And new NTM species, named Mycobacterium massiliense, which is very closely related to Mycobacterium abscessus was reported. In this review, these recent advances in the diagnosis and treatment of TB and clinical characteristics of M. massiliense lung disease are discussed.

• Journal of Yeungnam Medical Science
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• v.29 no.2
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• pp.83-88
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• 2012

Background: This study was conducted to evaluate the usefulness of the BACTEC MGIT (Mycobacterium Growth Indicator Tube) 960 system for mycobacteria culture and immunochromatographic assay to identify Mycobacterium tuberculosis (MTB) in positive MGIT culture. Methods: Mycobacteria-culture-positive cases were retrospectively analyzed from December 2010 to July 2011. The detection rates and the recovery times of the mycobacteria between the Ogawa media and the MGIT were compared. An immunochromatographic assay (ICA) (SD BIO-LINE) was also performed in the positive MGIT culture for identification, and the results were compared with those of the Ogawa media in the Korea National Tuberculosis Association. Results: Among the 261 patients (M:F, 168:93; mean age, $61.6{\pm}17.16$ yrs), 450 specimens (sputa, 365; bronchial washing, 61; and pleural effusion, 24) were found positive with mycobacteria. Mycobacteria were grown both on the MGIT and Ogawa media in 310 cases (68.9%); only on the MGIT in 115 cases (22.6%); and only on the Ogawa media in 25 cases (5.5%) (p<0.05). The recovery time was $28.2{\pm}8.9$ days in the Ogawa media and $11.1{\pm}5.8$ days in the MGIT (p<0.05). Among the 127 cases from the positive MGIT culture, all 92 cases that were confirmed as MTB cases bythe Korea National Tuberculosis Association were identified as MTB by ICA, with 100% sensitivity. Conclusion: MGIT increases the detection rate and shortens the recovery time of mycobacteria in clinical respiratory specimens, and the TB Ag MPT64 kit using ICA is useful in identifying MTB in a positive MGIT culture.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.3
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• pp.323-328
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• 2019

Tuberculosis, a chronic bacterial infection caused by Mycobacterium tuberculosis (MTB), differs in its status latency and activity because of the characteristics of MTB, immune status of the host, and genetic susceptibility. The host defense mechanism against MTB is caused mainly by interactions between macrophages, T cells, and dendritic cells. CD44 is expressed in activated T cells when infected with MTB and regulates lymphocyte migration. In addition, CD44 mediates leukocyte adhesion to the ECM and plays a role in attracting macrophages and $CD4^+$ T cells to the lungs. Therefore, genetic polymorphism of the CD44 gene will inhibit the host cell immune mechanisms against MTB. This study examined whether the genetic polymorphism of the CD44 gene affects the susceptibility of tuberculosis. A total of 237 SNPs corresponding to the CD44 genes were analyzed using the genotype data of 443 tuberculosis cases and 3,228 healthy controls from the Korean Association Resource (KARE). Of these, 17 SNPs showed a significant association with the tuberculosis case. The most significant SNP was rs75137824 (OR=0.231, CI: 1.51~3.56, $P=1.3{\times}10^{-4}$). In addition, rs10488809, one of the 17 significant SNPs, is important for the tuberculosis outbreak can bind to the JUND and FOS transcription factors and can affect CD44 gene expression. This study suggests that polymorphism of the CD44 gene modulates the host susceptibility to tuberculosis in a variety of ways, resulting in differences in the status of tuberculosis.

• Biomedical Science Letters
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• v.20 no.4
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• pp.250-255
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• 2014

Mycobacterium tuberculosis (MTB) resides and replicates inside macrophages. In our previous report, we reported that CD8+ T cell-mediated immune responses specific for the peptide derived from MTB RNA polymerase beta-subunit ($RpoB_{127-135}$) could be induced in TB patients expressing HLA-$A^*0201$ subtype. In order to examine whether $RpoB_{127-135}$ specific CD8+ T cells can recognize MTB infected macrophages in vitro, CD8+ T cell lines specific for $RpoB_{127-135}$ peptide were generated from peripheral blood mononuclear cells (PBMCs) of healthy HLA-$A^*0201$ subjects by in vitro immunization technique. In this study, we observed $RpoB_{127-135}$ specific CD8+ T cells could recognize and destroy macrophages infected with MTB for 2 to 4 days. $RpoB_{127-135}$ specific CD8+ T cell immune response was inducible from PBMC of healthy subjects expressing HLA-$A^*0206$ subtype, one of HLA-A2 supertype members. Next, we investigated the HLA-I processing mechanism of $RpoB_{127-135}$ peptide in MTB infected macrophages. As a result, the presentation of the MTB derived epitope peptide, $RpoB_{127-135}$, to CD8+ T cells was not inhibited by the treatment with brefeldin-A (ER-Golgi transport inhibitor) or lactacystin (proteasome inhibitor), which blocks the classical HLA-I processing pathway. However, $RpoB_{127-135}$ specific CD8+ T cell activity was blocked either by the blocking agent for the endocytosis (cytochalasin D) or by the blocking antibody (W6/32) for HLA-I molecules. Therefore, the $RpoB_{127-135}$ peptide may be processed by accessing the alternate HLA-I processing pathway. Understanding the processing and presentation mechanisms of the MTB derived proteins will help to improve the efficacy of vaccines and the efficiency of therapeutic agents for TB.