Comparison of the Standard E TB-Feron ELISA and QuantiFERON-TB Gold PLUS assays: the advantageous use of whole recombinant protein antigens for latent tuberculosis diagnosis.

The laboratory diagnosis of latent tuberculosis is often performed using interferon-gamma release assays. Here, we compared two enzyme-linked immunosorbent assay-based interferon-gamma release assays, namely, the newly developed Standard E TB-Feron enzyme-linked immunosorbent assay (STFE) and the QuantiFERON-TB Gold PLUS assay (QFT-GP), using samples from 155 participants. The STFE is based on using whole EAST6 and CFP10 recombinant antigens for latent tuberculosis diagnosis. The participants were classified into four groups and screened using both assays per the manufacturers' instructions. Thereafter, two statistical analyses were conducted to compare the obtained results. First, the STFE results were compared with the QTF-GP results (used as the gold standard) to calculate the total concordance, sensitivity, and specificity of STFE. Second, positivity and negativity concordances were calculated to differentiate healthy participants from participants with tuberculosis. The STFE showed 97% and 94% sensitivity and specificity, respectively. Furthermore, its positivity and negativity concordances were 91% and 98%, respectively. These results indicate the coordinated clinical performance of STFE in detecting latent tuberculosis and its improved performance in targeting tuberculosis-infected participants. Based on the comparison of the latent tuberculosis diagnostic abilities of STFE and QFT-GP, we establish the suitability and superior performance of STFE as a diagnostic tool.

Transrenal DNA detection of Mycobacterium tuberculosis in patients with pulmonary tuberculosis.

Background: Multiple attempts have been made to use biological samples other than sputum to diagnose tuberculosis (TB). Sputum acid-fast bacillus (AFB) microscopy is the fastest, most straightforward, and most inexpensive method for diagnosing pulmonary TB. However, urine can be used in place of sputum owing to its various advantages, such as a noninvasive method of collection, convenient handling and storage, and minimal risk of infection in health-care workers involved in sample collection. In this study, we aimed to assess the suitability of urine as a sample to obtain transrenal DNA (trDNA) to diagnose TB. This study involved several patients with TB undergoing inpatient treatment, whose AFB microscopy showed negative inversion. Methods: Here, 51 urine samples were collected from 40 patients with TB and examined to confirm the presence of trDNA. First, we compared the efficiency of two trDNA extraction methods.An automated magnetic bead-based method and a more efficient anchoring extraction method. Statistical analyses were performed using Excel software (Microsoft Office Professional Plus 2019). Results: Although molecular diagnosis using GeneXpert yielded negative results, a peculiarity was observed. There was no significant difference between GeneXpert findings and our results nor was there any difference in the sequential trDNA samples obtained. However, even when GeneXpert results were negative, trDNA was detected in seven out of ten samples using the anchor extraction method. Conclusions: Further studies are needed to establish biomarkers for the progression of TB treatment.

Label-free affinity screening, design and synthesis of inhibitors targeting the Mycobacterium tuberculosis L-alanine dehydrogenase.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.

The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe.

Background: Tuberculosis (TB) is a severe public health challenge in Korea. Of all Mycobacterium tuberculosis (M. tb) strains, the Beijing genotype strain reportedly correlates with hypervirulence and drug resistance. Hence, an early identification of the Beijing genotype strain of M. tb plays a significant role in initial TB treatment. Kogenebiotech® (KoRT-polymerase chain reaction [PCR]) has developed a real-time PCR 17 18 kit to determine the Beijing genotype strain classified as M. tb. To determine the feasibility of the commercially produced KoRT-PCR kit in identifying the M. tb strain. Methods: We used 100 clinical isolates of M. tb and 100 non-M. tb samples for the assessment. We evaluated the overall concordance between the KoRT-PCR kit and the mycobacterial interspersed repetitive unite variable number tandem repeat typing kit (GenoScreen, Lille, France). Moreover, we measured the detection limits based on the chromosomal DNA copies for the KoRT-PCR kit. In addition, we determined the reproducibility among individual technicians using the KoRT-PCR. Results: The KoRT-PCR kit successfully discriminated all M. tb (confidence interval [CI]: 96.38%-100.00% for both sensitivity and specificity) and Beijing genotype strain (CI: 95.70%-100.00% for sensitivity and 96.87%-100.00% for specificity). We confirmed no significant deviation in the reproducibility between the technicians. Conclusions: The KoRT-PCR kit displayed sufficient capability of discriminating the Beijing genotype strain, which enabled the rapid identification of the Beijing genotype strain from the M. tb clinical isolates.

Low molecular weight chitooligosaccharide inhibits infection of SARS-CoV-2 in vitro.

AIMS: The discovery of antiviral substances to respond to COVID-19 is a global issue, including the field of drug development based on natural materials. Here, we showed that chitosan-based substances have natural antiviral properties against SARS-CoV-2 in vitro. METHODS AND RESULTS: The molecular weight of chitosan-based substances was measured by the gel permeation chromatography analysis. In MTT assay, the chitosan-based substances have low cytotoxicity to Vero cells. The antiviral effect of these substances was confirmed by quantitative viral RNA targeting the RdRp and E genes and plaque assay. Among the substances tested, low molecular weight chitooligosaccharide decreased the fluorescence intensity of SARS-CoV-2 nucleocapsid protein of the virus-infected cells in a dose-dependent manner. CONCLUSIONS: In conclusion, the chitooligosaccharide, a candidate for natural treatment, has antiviral effects against the SARS-CoV-2 virus in vitro. SIGNIFICANCE AND IMPACT OF STUDY: In this study, it was suggested for the first time that chitosan-based substances such as chitooligosaccharide can have an antiviral effect on SARS-CoV-2 in vitro.

Susceptibility of ß-Lactam Antibiotics and Genetic Mutation of Drug-Resistant Mycobacterium tuberculosis Isolates in Korea.

BACKGROUND: Mycobacterium tuberculosis (Mtb) is resistant to the ß-lactam antibiotics due to a non-classical transpeptidase in the cell wall with ß-lactamase activity. A recent study showed that meropenem combined with a ß-lactamase inhibitor clavulanate, was effective in MDR and XDR tuberculosis (TB). However, clavulanate can only be used in drugs containing amoxicillin in Korea. In this study, we investigated the susceptibility and genetic mutations of drug-resistant Mtb isolates to amoxicillin-clavulanate and meropenem-clavulanate to improve the diagnosis and treatment of drug-resistant TB patients. METHODS: The minimum inhibitory concentration (MIC) of amoxicillin-clavulanate and meropenem-clavulanate was examined by resazurin microtiter assay. We used 82 MDR and 40 XDR strains isolated in Korea and two reference laboratory strains. Mutations of drug targets blaC, blaI, ldtA, ldtB, dacB2, and crfA were analyzed by PCR and DNA sequencing. RESULTS: The MIC90 values of amoxicillin and meropenem with clavulanate in drug-resistant Mtb isolates were 64 and 16, respectively. Gene mutations related to amoxicillin/clavulanate and meropenem/clavulanate resistance could not be identified, but T448G mutation of was found in the blaC gene related to ß-lactam antibiotics high susceptibility. CONCLUSION: Our results provide clinical consideration of ß-lactams in treating drug-resistant TB and potential molecular markers of amoxicillin-clavulanate and meropenem-clavulanate susceptibility.

Instant inactivation of aerosolized SARS-CoV-2 by dielectric filter discharge.

This study aimed to evaluate the instant inactivation effect of dielectric filter discharge (DFD) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aerosols. The filter consisted of one layer of ZrO2 beads covered by aluminum mesh electrodes; this porous structure of DFD part generates filter-type surface discharge and reactive oxygen species. In a closed cylindrical chamber, DFD treated air flow containing SARS-CoV-2 aerosols, primarily composed of particle diameters of ≤ 1 µm. A polypropylene melt-blown filter collected the treated bioaerosols for inactivation analysis. Plaque and polymerase chain reaction assays showed that the aerosolized SARS-CoV-2 that passed through the filter were more than 99.84% inactivated with degradation of SARS-CoV-2 genes (ORF1ab and E). However, ozone exposure without DFD passage was not found to be effective for bioaerosol inactivation in plaque assay.

Investigation of Clofazimine Resistance and Genetic Mutations in Drug-Resistant Mycobacterium tuberculosis Isolates.

Recently, as clofazimine (CFZ) showed a good therapeutic effect in treating multi-drug-resistant tuberculosis (MDR-TB), the anti-tuberculosis activity and resistance were re-focused. Here, we investigated the CFZ resistance and genetic mutations of drug-resistant Mycobacterium tuberculosis (DR-Mtb) isolates to improve the diagnosis and treatment of drug-resistant TB patients. The minimal inhibitory concentration (MIC) of CFZ was examined by resazurin microtiter assay (REMA) with two reference strains and 122 clinical isolates from Korea. The cause of CFZ resistance was investigated in relation to the therapeutic history of patients. Mutations of Rv0678, Rv1979c and pepQ of CFZ resistant isolates were analyzed by PCR and DNA sequencing. The rate of CFZ resistance with MIC > 1 mg/L was 4.1% in drug-resistant Mtb isolates. The cause of CFZ resistance was not related to treatment with CFZ or bedaquiline. A CFZ susceptibility test should be conducted regardless of dugs use history. The four novel mutation sites were identified in the Rv0678 and pepQ genes related to CFZ resistance in this study.

Sustainable Antibacterial and Antiviral High-Performance Copper-Coated Filter Produced via Ion Beam Treatment.

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), disease prevention has become incredibly important. Consequently, mask and air-purifier use has increased. The filter is the core component of these items. However, most filter materials lack antimicrobial properties. Copper is a sustainable antimicrobial material. When copper is deposited onto the filter's surface, the microorganisms that come into contact with it can be effectively inactivated. In this study, we used an oxygen ion beam with a controlled process temperature to treat filter surfaces with copper. This enabled a strong adhesion of at least 4 N/cm between the copper and the filter fibers without damaging them. Upon exposing the filter to bacteria (Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 4352, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853) for one hour, a >99.99% removal rate was attained; when the filter was exposed to SARS-CoV-2 virus for one hour, it inactivated more than 99%. These beneficial properties minimize the risk of secondary infections, which are significantly more likely to occur when a conventional filter is replaced or removed.

Validation and comparative analysis of kogene mycobacterial interspersed repetitive unit-variable number of tandem repeat typing kit and its application on clinically isolated mycobacterium tuberculosis samples from national tuberculosis hospital, Republic of Korea.

Background: Tuberculosis (TB) remains a serious public health burden in Korea. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) is preferred for epidemiological TB investigation. Until recently, the difficulty lies in epidemiological TB investigation due to the absence of commercialized MIRU-VNTR in Korea. Here, we have evaluated the newly designed MIRU-VNTR kit by Kogenebiotech, Korea. Materials and Methods: A total of 200 samples, where 100 are Mycobacrerium tuberculosis (M. tuberculosis), and the other 100 are non-M. tuberculosis, were used. Initially, the Kogenebiotech MIRU-VNTR typing kit (KoMIRU) was compared with Multilocus Variable Number Tandem Repeat Genotyping of M. tuberculosis typing kit (MVNTR) by Philip Supply for validation purpose. Then, Limit of Detection for DNA copies was optimized. Finally, KoMIRU and Genoscreen MIRU-VNTR typing kit (GeMIRU) were tested and comparatively analyzed for its specificity and sensitivity. Results: The study showed that the KoMIRU has slightly higher discriminatory power over MVNTR, 100% versus 97.5%. In comparative analysis, the KoMIRU has shown comparable capability as GeMIRU, showing 100% for sensitivity and specificity with a 95% CI value of 96.38 to 100.00%. Also, no discrepancies were observed on discriminated lineage strains between KoMIRU and GeMIRU. Out of 100, 84 were identified as Beijing strains, and remains were identified as NEW-1 (n = 8), Uganda (n = 6), East African Indian (EAI) (n = 6), Turkey (n = 2), and Haarlem (n = 1). Conclusion: In this study, KoMIRU has shown a comparable capability to GeMIRU. Furthermore, previous researches had suggested an association between lineage strains and drug resistance; hence, the implementation of KoMIRU can help in TB control and prevention.

10-DEBC Hydrochloride as a Promising New Agent against Infection of Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections. Its resistance to current antimicrobial drugs makes it the most difficult non-tuberculous mycobacteria (NTM) to treat with a treatment success rate of 45.6%. Therefore, there is a need for new therapeutic agents against M. abscessus. We identified 10-DEBC hydrochloride (10-DEBC), a selective AKT inhibitor that exhibits inhibitory activity against M. abscessus. To evaluate the potential of 10-DEBC as a treatment for lung disease caused by M. abscessus, we measured its effectiveness in vitro. We established the intracellular activity of 10-DEBC against M. abscessus in human macrophages and human embryonic cell-derived macrophages (iMACs). 10-DEBC significantly inhibited the growth of wild-type M. abscessus and clinical isolates and clarithromycin (CLR)-resistant M. abscessus strains. 10-DEBC's drug efficacy did not have cytotoxicity in the infected macrophages. In addition, 10-DEBC operates under anaerobic conditions without replication as well as in the presence of biofilms. The alternative caseum binding assay is a unique tool for evaluating drug efficacy against slow and nonreplicating bacilli in their native caseum media. In the surrogate caseum, the mean undiluted fraction unbound (fu) for 10-DEBC is 5.696. The results of an in vitro study on the activity of M. abscessus suggest that 10-DEBC is a potential new drug for treating M. abscessus infections.

Polyimide Surface Dielectric Barrier Discharge for Inactivation of SARS-CoV-2 Trapped in a Polypropylene Melt-Blown Filter.

Surface dielectric barrier discharge (SDBD) was used to inactivate the infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) trapped in a polypropylene (PP) melt-blown filter. We used a dielectric barrier made of polyimide films with hexagonal holes through which air flowed. In a cylindrical wind tunnel, the SDBD device supplied reactive oxygen species such as ozone to the SARS-CoV-2 trapped in the PP filter. A plaque assay showed that SDBD at an ozone concentration of approximately 51.6 ppm and exposure time of 30 min induced more than 99.78% reduction for filter-adhered SARS-CoV-2. A carbon catalyst after SDBD effectively reduced ozone exhaust below 0.05 ppm. The combination of SDBD, PP filter, and catalyst could be a promising way to decrease the risk of secondary infection due to indoor air purifiers.

Clomiphene Citrate Shows Effective and Sustained Antimicrobial Activity against Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections and is the most difficult non-tuberculous mycobacteria (NTM) to treat due to its resistance to current antimicrobial drugs, with a treatment success rate of 45.6%. Thus, novel treatment drugs are needed, of which we identified the drug clomiphene citrate (CC), known to treat infertility in women, to exhibit inhibitory activity against M. abscessus. To assess the potential of CC as a treatment for M. abscessus pulmonary diseases, we measured its efficacy in vitro and established the intracellular activity of CC against M. abscessus in human macrophages. CC significantly inhibited the growth of not only wild-type M. abscessus strains but also clinical isolate strains and clarithromycin (CLR)-resistant strains of M. abscessus. CC's drug efficacy did not have cytotoxicity in the infected macrophages. Furthermore, CC worked in anaerobic non-replicating conditions as well as in the presence of biofilm. The results of this in vitro study on M. abscessus activity suggest the possibility of using CC to develop new drug hypotheses for the treatment of M. abscessus infections.

Copper-Coated Polypropylene Filter Face Mask with SARS-CoV-2 Antiviral Ability.

Face masks will be used to prevent pandemic recurrence and outbreaks of mutant SARS-CoV-2 strains until mass immunity is confirmed. The polypropylene (PP) filter is a representative disposable mask material that traps virus-containing bioaerosols, preventing secondary transmission. In this study, a copper thin film (20 nm) was deposited via vacuum coating on a spunbond PP filter surrounding a KF94 face mask to provide additional protection and lower the risk of secondary transmission. Film adhesion was improved using oxygen ion beam pretreatment, resulting in cuprous oxide formation on the PP fiber without structural deformation. The copper-coated mask exhibited filtration efficiencies of 95.1 ± 1.32% and 91.6 ± 0.83% for NaCl and paraffin oil particles, respectively. SARS-CoV-2 inactivation was evaluated by transferring virus-containing media onto the copper-coated PP filters and subsequently adding Vero cells. Infection was verified using real-time polymerase chain reaction and immunochemical staining. Vero cells added after contact with the copper-coated mask did not express the RNA-dependent RNA polymerase and envelope genes of SARS-CoV-2. The SARS-CoV-2 nucleocapsid immunofluorescence results indicated a reduction in the amount of virus of more than 75%. Therefore, copper-coated antiviral PP filters could be key materials in personal protective equipment, as well as in air-conditioning systems.

Biobank for multidrug-resistant tuberculosis research: importance of sequential samples.

Since 2013, Masan National Tuberculosis Hospital has collected standardized specimens from its tuberculosis patients, which include a large number of multidrug-resistant strains. The repository collects matched participants and their bacilli samples, compiling sequential samples from the beginning of treatment. The repository aims to provide resources for in-depth international research.

Detection of airborne bacteria from patient spaces in tuberculosis hospital.

Background: The spread of nosocomial bacterial infection greatly threatens public health and the impact of nosocomial infection worsens if highly pathogenic bacteria, Mycobacterium tuberculosis as an instance, involves. In this study, we have investigated the presence of airborne M. tuberculosis in a specialized tuberculosis hospital. Methods: The study sites selected were waiting room I, II, and ward VI patient lounge, Masan National Tuberculosis Hospital, where the modern ventilation system is on the operation for opportunistic infection prevention. The air samples were collected from the different sites three times for 1 day, and after air collection, air sampled disposable filter membrane was incubated for 4 weeks on nine Middlebrook 7H11 agar plates. Results: Our data showed that out of nine incubated 7H11 plate agars, four plates showed bacterial growth and these grown bacterial colonies were isolated and identified. Among bacterial species identified, there was a colony of Mycobacterium mageritense, one of nontuberculous Mycobacteria. Although there was no M. tuberculosis, the cause of tuberculous disease and transmitted through the nosocomial infection, all pathogens detected were known to be associated with nosocomial infection. Conclusions: Hospitals dealing with infectious diseases should always be wary that ventilation system does not guarantee safety from airborne pathogen exposure hence should continuously monitor the presence of other hospital-associated infection causing pathogenic microorganisms.

Thiostrepton: A Novel Therapeutic Drug Candidate for Mycobacterium abscessus Infection.

Mycobacterium abscessus is a rapid-growing, multidrug-resistant, non-tuberculous mycobacterial species responsible for a variety of human infections, such as cutaneous and pulmonary infections. M. abscessus infections are very difficult to eradicate due to the natural and acquired multidrug resistance profiles of M. abscessus. Thus, there is an urgent need for the development of effective drugs or regimens against M. abscessus infections. Here, we report the activity of a US Food and Drug Administration approved drug, thiostrepton, against M. abscessus. We found that thiostrepton significantly inhibited the growth of M. abscessus wild-type strains, subspecies, clinical isolates, and drug-resistant mutants in vitro and in macrophages. In addition, treatment of macrophages with thiostrepton significantly decreased proinflammatory cytokine production in a dose-dependent manner, suggesting an inhibitory effect of thiostrepton on inflammation induced during M. abscessus infection. We further showed that thiostrepton exhibits antimicrobial effects in vivo using a zebrafish model of M. abscessus infection.

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.

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.