Sungweon Ryoo

Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.

First– and Second–Line Drugs and Drug Resistance

Tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis, which is transmitted through inhalation of aerosolized droplets. TB mainly attacks the lungs, but can also affect other parts of the body. TB is highly contagious during the active stage of the disease and can infect an individual through inhalation of as few as 10 Mycobacterium tuberculosis (MTB) bacteria. After inhalation, these bacteria are mainly captured by the alveolar macrophages, but they can evade the host immune system and remain in the dormant stage for a long period of time, at which point they can reactivate to a virulent form under immune-compromised conditions of the host. This is possible because M. tuberculosis can persist in slow growing as well as in fast growing stages which makes treatment challenging. Almost all of the antibiotics that can be used to treat TB work when the bacteria are actively dividing. In the intensive phase of TB treatment, the antibiotics mainly kill rapidly growing bacteria, which causes rapid sputum conversion, and the eradication of clinical symptoms. However, in order to kill the persistent or slow growing strains of MTB, the continuation phase of the treatment is essential. TB can be treated effectively by using first line drugs (FLD) isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), ethambutol (EMB) and streptomycin (SM). However, this first line therapy often fails to cure TB for several reasons. Relapse and the spread of the disease contribute to the emergence of drug resistant bacteria. The emergence of multidrug resistant TB (MDR-TB), i.e. which is resistant to at least isoniazid (INH) and rifampicin (RIF), is of great concern, because it requires the use of second-line drugs that are difficult to procure and are much more toxic and expensive than FLDs [1]. Therefore, the detection and treatment of drug susceptible or single drug resistant TB is an important strategy for preventing the emergence of MDR-TB [2]. M. tuberculosis strains with extensively drug resistant-TB (XDR-TB), that is resistant to either isoniazid or rifampicin (like MDR tuberculosis), any fluoroquinolone, and at least one of three second-line antituberculosis injectable drugs—i.e., capreomycin, kanamy‐ cin, and amikacin have also been reported [3].

Evaluation of MGIT 960 System for the Second-Line Drugs Susceptibility Testing of Mycobacterium tuberculosis.

Many laboratories validate DST of the second-line drugs by BACTEC MGIT 960 system. The objective of this study is to evaluate the critical concentration and perform DST for the 2nd line drugs. We evaluated 193 clinical strains of M. tuberculosis isolated from patients in South Korea. Testing the critical concentration of six second-line drugs was performed by MGIT 960 and compared with L-J proportion method. The critical concentration was determined to establish the most one that gave the difference between drug resistance and susceptibility in MGIT960 system. Good agreement of the following concentrations was found: Concordance was 95% for 0.5 μg/mL of moxifloxacin; 93.6%, 1.0 μg/mL of levofloxacin; 97.5%, 2.5 μg/mL of kanamycin; 90.6%, 2.5 μg/mL of capreomycin; 86.2%, 5.0 μg/mL of ethionamide; and 90.8%, 2.0 μg/mL of ρ-aminosalicylic acid. The critical concentrations of the four drugs, moxifloxacin, levofloxacin, kanamycin, and capreomycin, were concordant and reliable for testing 2nd line drug resistance. Further study of ethionamide and ρ-aminosalicylic acid is required.

Evaluation of the GenoType® MTBDRsl assay in Korean patients with MDR or XDR tuberculosis

Abstract Background This study used the GenoType® MTBDRsl assay (MTBDRsl, Hain Lifescience, Nehren, Germany), an assay for anti-tuberculosis drug susceptibility testing, for the detection of resistance of 40 tuberculosis strains to fluroquinolones (FLQ), injectable drugs (amikacin or capreomycin) and ethambutol to identify multi-drug resistant and extensively drug-resistant tuberculosis patients previously identified by solid drug susceptibility test (DST). Methods These tuberculosis strains were analysed by both DNA sequencing and conventional drug susceptibility testing on solid medium. Results The overall agreement rates of the MTBDRsl assay and phenotypic drug susceptibility testing for the detection of ofloxacin (OXF), moxifloxacin (MXF), amikacin (AMK), capreomycin (CPM) and ethambutol (EMB) susceptibility in clinical strains were 87.5% (35/40), 87.5% (35/40), 97.5% (39/40), 60.0% (24/40) and 65.0% (26/40), respectively. Conclusions This study reconfirmed 37 extensively drug-resistant tuberculosis strains using the MTBDRsl assay and these strains were verified by phenotypic drug susceptibility testing. After identification with the MTBDRsl assay, the results were analysed by DNA sequencing with specific primers. From the various mutations identified in the DNA sequencing results, this study found new mutations that characterised EMB-resistant strains, namely, R507K and G406T mutations, which had not been previously reported.

Bedaquiline susceptibility test for totally drug-resistant tuberculosis Mycobacterium tuberculosis

This study aimed to provide information that bedaquilline is significantly effective for treatment of totally drug resistant (TDR) Mycobacterium tuberculosis that shows resistant to all first- and second-line drugs-using an innovative disc agarose channel (DAC) system. Time-lapse images of single bacterial cells under culture conditions with different concentrations of bedaquiline were analysed by image processing software to determine minimum inhibitory concentrations (MICs). Bedaquiline inhibited the growth of TDR M. tuberculosis strains, with MIC values ranging from 0.125 to 0.5 mg/L. The results of the present study demonstrate that bedaquiline, newly approved by the United States Food and Drug Administration (FDA), may offer therapeutic solutions for TDR-TB.

Systematic review and meta-analysis of the nitrate reductase assay for drug susceptibility testing of Mycobacterium tuberculosis and the detection limits in liquid medium

Recently, the need for rapid, reliable, and low-cost drug susceptibility testing (DST) methods has increased due to the emergence of multidrug-resistant Mycobacterium tuberculosis. Colorimetric methods of DST provide results more quickly than standard culture methods and are inexpensive than molecular methods. Thus, colorimetric methods, such as the nitrate reductase assay (NRA), are being recommended. We searched Medline PubMed for reports on the NRA for DST of M. tuberculosis written in English and published within the last five years. We selected 20 reports on six major anti-TB drugs and conducted a meta-analysis using Meta-Disc software. The pooled sensitivities for isoniazid, rifampicin, streptomycin, ethambutol, ofloxacin, and kanamycin were 95.4%, 96.4%, 91.5%, 93.1%, 99.3%, and 88.4%, and the pooled specificities were 98.5%, 99.2%, 92.9%, 97.8%, 97.4%, and 99.4%, respectively. The area under the summary receiver operator curve for all drugs was 0.9723-0.9952. The time to results (TTR) for the direct and indirect NRAs was 7-28days and 6-15days, respectively. Quality assessments were conducted using the quality of diagnostic accuracy studies tool (QUADAS-2) items, and most reports showed good performance. However, ethambutol, streptomycin, and kanamycin showed relatively low sensitivity. We performed a quantitative NRA in liquid media at various inoculum concentrations. The TTR at 4.94×106, 1.67×104, and 2.27×102CFU/mL was 4, 14, and 14days, respectively. The minimum absorbance and nitrite concentration for positive samples were 0.8 and 168μM, respectively. We propose a quantitative standard to determine sample positivity to address the problems with the current standard NRA which is much less expensive than the conventional assay conducted on solid medium.

Role of confirmatory interferon-gamma release assays in school outbreaks of tuberculosis in South Korea.

OBJECTIVE To investigate the usefulness of confirmatory QuantiFERON(®) (QFT) testing among tuberculin skin test (TST) positive contacts to diagnose latent tuberculous infection (LTBI) in tuberculosis (TB) outbreaks among adolescents. DESIGN We used the Korean national claims database to identify the development of active TB disease in relation to initial TST (cut-off 10 mm induration) and subsequent QFT results. RESULTS A total of 7475 contacts in 89 schools were divided into four groups: TST- (n = 5714), TST+/QFT+ (n = 534), TST+/QFT- (n = 697) and TST+ only (n = 530). The mean duration of follow-up was 3.9 ± 0.9 years. For contacts with no LTBI treatment (n = 6868), TB incidence rates per 1000 person-years (py) and the adjusted hazard ratio (HR) compared with TST- individuals were as follows: TST+/QFT+, 66.2/1000 py (HR 35.59, 95%CI 14.03-90.31, P < 0.001); TST+ only, 10.1/1000 py (HR 5.16, 95%CI 2.91-9.17, P < 0.001); TST+/QFT-, 4.0/1000 py (HR 2.05, 95%CI 1.05-4.01, P = 0.035); and TST- 2.0/1000 py. The TB progression rate was significantly higher in TST+/QFT+ than in TST+/QFT- individuals (HR 16.82, 95 CI 5.84-48.46, P < 0.001). CONCLUSION A confirmatory QFT for TST+ contacts could reduce the number of candidates for LTBI treatment after school TB outbreaks.

Whole-Genome Sequence of Mycobacterium tuberculosis Korean Strain KIT87190.

ABSTRACT Mycobacterium tuberculosis is a contagious agent that causes tuberculosis. A specific type (called the K cluster) of M. tuberculosis with 10 copies of IS6110 in restriction fragment length polymorphism (RFLP) has been found in about 4% of M. tuberculosis isolates in Korea. Here, we report the complete genome sequence of M. tuberculosis Korean strain KIT87190 belonging to the K cluster.

Comparing Two Mycobacterium tuberculosis Genomes from Chinese Immigrants with Native Genomes Using Mauve Alignments

Background The number of immigrants with tuberculosis (TB) increases each year in South Korea. Determining the transmission dynamics based on whole genome sequencing (WGS) to cluster the strains has been challenging. Methods WGS, annotation refinement, and orthology assignment for the GenBank accession number acquisition were performed on two clinical isolates from Chinese immigrants. In addition, the genomes of the two isolates were compared with the genomes of Mycobacterium tuberculosis isolates, from two native Korean and five native Chinese individuals using a phylogenetic topology tree based on the Multiple Alignment of Conserved Genomic Sequence with Rearrangements (Mauve) package. Results The newly assigned accession numbers for two clinical isolates were CP020381.2 (a Korean-Chinese from Yanbian Province) and CP022014.1 (a Chinese from Shandong Province), respectively. Mauve alignment classified all nine TB isolates into a discriminative collinear set with matched regions. The phylogenetic analysis revealed a rooted phylogenetic tree grouping the nine strains into two lineages: strains from Chinese individuals and strains from Korean individuals. Conclusion Phylogenetic trees based on the Mauve alignments were supposed to be useful in revealing the dynamics of TB transmission from immigrants in South Korea, which can provide valuable information for scaling up the TB screening policy for immigrants.

A rapid culture system uninfluenced by an inoculum effect increases reliability and convenience for drug susceptibility testing of Mycobacterium tuberculosis

The Disc Agarose Channel (DAC) system utilizes microfluidics and imaging technologies and is fully automated and capable of tracking single cell growth to produce Mycobacterium tuberculosis (MTB) drug susceptibility testing (DST) results within 3~7 days. In particular, this system can be easily used to perform DSTs without the fastidious preparation of the inoculum of MTB cells. Inoculum effect is one of the major problems that causes DST errors. The DAC system was not influenced by the inoculum effect and produced reliable DST results. In this system, the minimum inhibitory concentration (MIC) values of the first-line drugs were consistent regardless of inoculum sizes ranging from ~103 to ~108 CFU/mL. The consistent MIC results enabled us to determine the critical concentrations for 12 anti-tuberculosis drugs. Based on the determined critical concentrations, further DSTs were performed with 254 MTB clinical isolates without measuring an inoculum size. There were high agreement rates (96.3%) between the DAC system and the absolute concentration method using Löwenstein-Jensen medium. According to these results, the DAC system is the first DST system that is not affected by the inoculum effect. It can thus increase reliability and convenience for DST of MTB. We expect that this system will be a potential substitute for conventional DST systems.