Trend of nontuberculous mycobacteria species and minimal inhibitory concentration in a referral laboratory in Korea from 2013 to 2019.

BACKGROUND: This study investigated the trends of nontuberculous mycobacterial (NTM) isolates and the minimal inhibitory concentrations (MIC) of antimicrobial agents in Korea. METHODS: Data from 2013 to 2019 were collected from 69 medical institutions through 12 branches of the Korean Institute of Tuberculosis. NTM identification was conducted using the Advansure Mycobacteria Genoblot assay. The MIC of antibiotics against NTM species were measured using the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines. RESULTS: Over seven years, 86,194 NTM identifications were requested, with an annual increase from 8034 in 2013-17,229 in 2019. The most frequently identified NTM species were M. intracellulare (33,467; 47.3%) and M. avium (19,818; 27.2%), followed by M. abscessus (6858; 9.4%) and M. massiliense (3156; 4.3%). Regarding the antimicrobial agents, imipenem exhibited the greatest difference in MIC between M. intracellulare and M. avium, whereas clarithromycin showed the most significant difference between M. abscessus and M. massiliense. No notable changes were observed in the annual MIC distribution of most antibacterial agents, except for clarithromycin in M. abscessus. CONCLUSIONS: The prevalence of NTM in Korea is gradually increasing, and follow-up studies on NTM isolates identified as the causative agents of infection are needed.

A Study on the Factors Influencing Smoking in Multicultural Youths in Korea.

Based on the ecological integration model, this study examined the factors affecting smoking in adolescents from multicultural families by dividing them into two levels: microsystem and social network factors. The data were from the Korea Youth Risk Behavior Web-Based Survey (KYRBS) from 2016 to 2020. It included 4577 respondents whose fathers, mothers, or both, were not born in Korea. The factors affecting smoking among multicultural teenagers were determined by a composite-sample multiple logistic regression analysis. Male smoking rates among multicultural adolescents were 2.49 times higher than female rates in the microsystem. When the father was "Korean" rather than a "Foreigner", smoking was 0.55 times lower in family factors in terms of social network. In social factors of social networks, multicultural adolescents' smoking was 12.02 times greater when they were drinking than when they were not, and 3.62 times higher when the answer to the question of whether they had experienced violence was "yes" than "no." Based on the ecological model in this study, social factors such as drinking, and violence were highly related to smoking. Since multicultural adolescents were closely influenced by the surrounding environment, such as family, school, and social relationship, it was necessary to let parents and schoolteachers be involved in the intervention of smoking of multicultural adolescents so that they can help multicultural adolescents adjust better to school and perform better academically while decreasing risky behaviors for their health, such as drinking and, ultimately, smoking.

Performance Evaluation of the BACTEC MGIT 960 System for Rifampin Drug-Susceptibility Testing of Mycobacterium tuberculosis Using the Current WHO Critical Concentration.

The World Health Organization recently lowered the rifampin (RIF) critical concentration (CC) for drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) using the mycobacterial growth indicator tube (MGIT) 960 system. Here, we evaluated the diagnostic performance of the MGIT system with the revised CC for determining MTBC RIF resistance with 303 clinical MTBC isolates, including 122 isolates with rpoB mutations, of which 32 had single borderline-resistance mutations, and 181 wild-type rpoB isolates. The phenotypic RIF resistance was determined via the absolute concentration method (AC) and via MGIT using both previous (1 mg/L) and revised (0.5 mg/L) CCs for the latter method. The diagnostic accuracy of each phenotypic DST (pDST) was assessed based on rpoB genotyping as the reference standard. The overall sensitivity of the AC was 95.1% (95% confidence interval [CI], 89.6 to 98.2%), while the MGIT results with previous and revised CCs were 82.0% (95% CI 74.0 to 88.3%) and 83.6% (95% CI 75.8 to 89.7%), respectively. The 32 MTBC isolates with single borderline-resistance mutations showed a wide range of MICs, and sensitivity was not significantly increased by reducing the MGIT CC. All 181 wild-type rpoB isolates were RIF-susceptible in the AC and with MGIT using the previous CC, whereas 1 isolate was misclassified as RIF-resistant with the revised CC. Our results demonstrate that the overall diagnostic performances of the MGIT DST with the revised RIF CC and previous CC were comparable. A further large-scale study is required to demonstrate the optimal RIF CC for MGIT.

Wavelength-dependent label-free identification of isolated nontuberculous mycobacteria using surface-enhanced Raman spectroscopy.

We investigated the effect of Raman excitation wavelengths on the surface-enhanced Raman spectroscopy (SERS)-based identification of isolated nontuberculous mycobacteria (NTM). The SERS spectra with 3 commonly used excitation wavelengths, 532, 638, and 785 nm, were compared across 6 representative NTM species that primarily cause human NTM infections in Korea and the United States; these species were identified. The statistical differences among NTM SERS spectra at each Raman excitation wavelength were verified using 1-way analysis of variance, and the 6 NTM species were identified using principal components-linear discriminant analysis with leave-one-out cross validation. The identification accuracies with aromatic amino acid biomarkers were 99.3%, 91.3%, and 90.7% for 532, 638, and 785 nm, respectively. We believe that the proposed SERS protocol with aromatic amino acid biomarkers at the 532-nm Raman excitation wavelength will enable fast and accurate identification of NTM compared to previous identification methods.

Antimicrobial Resistance Profiles and Molecular Characteristics of Coagulase-Negative Staphylococci Isolated from Two Tertiary Hospitals Before and 15 Years After Implementation of the Separation of Drug Prescribing and Dispensing Policy of Korea.

This study compared changes in antimicrobial susceptibilities and molecular characteristics of coagulase-negative staphylococci (CNS) between the year 2000 and the year 2014-2015 to evaluate the policy of separating drug prescribing and dispensing in Korea. We obtained 68 CNS clinical isolates from two tertiary general hospitals before (the year 2000; n = 25) and after (the year 2014 - 2015; n = 43) implementation of the separation. Isolates were identified as Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, and Staphylococcus warneri. When minimal inhibitory concentrations of 14 antimicrobials were applied to isolates, resistance rates to gentamicin and oxacillin in 2000 were significantly higher than in 2014-2015 (p < 0.05). Fifty-seven isolates were methicillin-resistant CNS (MR-CNS), 42 of which were also multidrug resistant; overall, multidrug resistance decreased from 72% in the year 2000 to 55.8% in 2014-2015. Staphylococcal cassette chromosome mec (SCCmec) type III was the dominant type of MR-CNS in the year 2000, while SCCmec type IV was the dominant type in 2014-2015. Twenty-five sequence types (STs) were identified; ST2 appeared most frequently in both periods. After 15 years of implementation of this policy, multidrug resistance as well as methicillin and gentamicin resistance in CNS decreased, but not resistance to other antibiotics. Long-term surveillance at both genotypic and phenotypic levels of various species is necessary for further evaluation of this policy.

Serotype Distribution and Antimicrobial Resistance of Invasive and Noninvasive Streptococcus pneumoniae Isolates in Korea between 2014 and 2016.

BACKGROUND: Several factors contribute to differences in Streptococcus pneumoniae serotype distribution. We investigated the serotype distribution and antimicrobial resistance of S. pneumoniae isolated between 2014 and 2016 in Korea. METHODS: We collected a total of 1,855 S. pneumoniae isolates from 44 hospitals between May 2014 and May 2016, and analyzed the serotypes by sequential multiplex PCR. We investigated the distribution of each serotype by patient age, source of the clinical specimen, and antimicrobial resistance pattern. RESULTS: The most common serotypes were 11A (10.1%), followed by 19A (8.8%), 3 (8.5%), 34 (8.1%), 23A (7.3%), and 35B (6.2%). The major invasive serotypes were 3 (12.6%), 19A (7.8%), 34 (7.8%), 10A (6.8%), and 11A (6.8%). Serotypes 10A, 15B, 19A, and 12F were more common in patients ≤5 years old, while serotype 3 was more common in patients ≥65 years old compared with the other age groups. The coverage rates of pneumococcal conjugate vaccine (PCV)7, PCV10, PCV13, and pneumococcal polysaccharide vaccine 23 were 11.8%, 12.12%, 33.3%, and 53.6%, respectively. Of the 1,855 isolates, 857 (46.2%) were multi-drug resistant (MDR), with serotypes 11A and 19A predominant among the MDR strains. The resistance rates against penicillin, cefotaxime, and levofloxacin were 22.8%, 12.5%, and 9.4%, respectively. CONCLUSIONS: There were significant changes in the major S. pneumoniae serotypes in the community. Non-PCV13 serotypes increased in patients ≤5 years old following the introduction of national immunization programs with the 10- and 13-polyvalent vaccines.

Prevalence of ST131 and ST1193 Among Bloodstream Isolates of Escherichia coli not Susceptible to Ciprofloxacin in a Tertiary Care University Hospital in Korea, 2013 - 2014.

We investigated the ST distribution of bloodstream isolates of E. coli that were not susceptible to ciprofloxacin (CIP-NS isolates), collected from 2013 to 2014 in a tertiary care hospital. Fifty-nine CIP-NS isolates were collected. ST131 was the most frequent ST (37.3%) isolate, followed by ST1193 (23.7%). ST131 and ST1193 showed significant differences in ESBL production and lactose fermentation. Further study should be continued on the monitoring of resistant strains.

Fluoroquinolone Resistance Mechanisms by Molecular Epidemiologic Study of Ciprofloxacin-Nonsusceptible Escherichia coli Sequence Types Isolated from Clinical Specimens in a Tertiary Care University Hospital in Korea: Emergence of Clone ST131, 2006-2008.

To investigate the sequence types (STs) and fluoroquinolone resistance related mutations among ciprofloxacin (CIP)-non-susceptible extended-spectrum ß-lactamase (ESBL)-producing E. coli isolated from Korean patients from 2006-2008. The prevalence of fluoroquinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, and parE and plasmid-mediated quinolone resistance (PMQR) genes were also studied. Multilocus sequence typing (MLST) was performed to identify STs. The most common ST was ST131 (33/51, 64.7%). All isolates, except one isolate, showed three mutations at codons 83 (S83L) and 87 (S87N) in gyrA and 80 (S80I) in parC The prevalence of ST131 in our hospital was much higher than reported in other Asian studies during a similar time period. The mutations found in ST131 were concordant with other studies.

Prevalence of Clostridium perfringens toxin in patients suspected of having antibiotic-associated diarrhea.

BACKGROUND: Although Clostridium perfringens has been reported as a cause of antibiotic-associated diarrhea (AAD), it is uncommon to detect this pathogen in clinical microbiology laboratories in Korea. The aim of this study was to investigate the prevalence of C. perfringens toxin in patients suspected of having AAD. METHODS: A total of 135 stool specimens submitted to a clinical microbiology laboratory for C. difficile toxin assay were tested. We tried to detect both C. difficile and C. perfringens toxins using the Seeplex Diarrhea ACE Detection kit (Seegene, Seoul, Korea). We evaluated the prevalence of 10 bacteria and 5 viruses. RESULTS: A total of 40 Clostridium spp. were detected in 34 specimens (29.6%). The C. perfringens toxin was detected in 14 of 135 specimens (10.4%), while C. difficile toxin was detected in 26 specimens (19.3%). Other bacteria and viruses, including 8 Aeromonas spp., were detected in 15 specimens. All tests were negative in 92 of the 135 specimens (68.1%). CONCLUSION: Clostridium perfringens toxin is relatively common, and we should consider the possibility of its presence in patients suspected of having AAD, especially if C. difficile tests are negative.

A bloodstream infection by Ruminococcus gnavus in a patient with a gall bladder perforation.

Ruminococcus gnavus is frequently found among human gut microbiome. However, human bloodstream infections by R. gnavus have been reported only three times. Clinical details were lacking for one case; the other two cases with concurrent bacteremia in patients with diverticulitis. We report a case of R. gnavus bloodstream infection in a patient with a gall bladder perforation suggesting its association with damage to the gastrointestinal tract. R. gnavus was misidentified using biochemical test but 16S rRNA sequencing and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were useful for correct identification. With the advancement of identification method in clinical laboratory, more frequent identification of R. gnavus from clinical specimens is expected.

Rapid label-free identification of Klebsiella pneumoniae antibiotic resistant strains by the drop-coating deposition surface-enhanced Raman scattering method.

Although many methodologies have been developed to identify unknown bacteria, bacterial identification in clinical microbiology remains a complex and time-consuming procedure. To address this problem, we developed a label-free method for rapidly identifying clinically relevant multilocus sequencing typing-verified quinolone-resistant Klebsiella pneumoniae strains. We also applied the method to identify three strains from colony samples, ATCC70063 (control), ST11 and ST15; these are the prevalent quinolone-resistant K. pneumoniae strains in East Asia. The colonies were identified using a drop-coating deposition surface-enhanced Raman scattering (DCD-SERS) procedure coupled with a multivariate statistical method. Our workflow exhibited an enhancement factor of 11.3×106 to Raman intensities, high reproducibility (relative standard deviation of 7.4%), and a sensitive limit of detection (100 pM rhodamine 6G), with a correlation coefficient of 0.98. All quinolone-resistant K. pneumoniae strains showed similar spectral Raman shifts (high correlations) regardless of bacterial type, as well as different Raman vibrational modes compared to Escherichia coli strains. Our proposed DCD-SERS procedure coupled with the multivariate statistics-based identification method achieved excellent performance in discriminating similar microbes from one another and also in subtyping of K. pneumoniae strains. Therefore, our label-free DCD-SERS procedure coupled with the computational decision supporting method is a potentially useful method for the rapid identification of clinically relevant K. pneumoniae strains.

Inhibin-α gene mutations and mRNA levels in human lymphoid and myeloid leukemia cells.

The inhibin-α gene was identified as a tumor suppressor gene in the gonads and adrenal glands by functional studies using knockout mice. Methylation of CpG sites within the regulatory regions of tumor suppressor gene is frequently associated with their transcriptional silencing. We investigated epigenetic modifications, changes in loss of heterozygosity (LOH), and mutation of the inhibin-α gene, and regulation of transcriptional expression in response to inhibitors of DNA methylation (5-aza-2'-deoxycytidine, 5-AzaC) in human lymphoid (Jurkat, Molt-4, Raji, and IM-9) and myeloid (HL-60, Kasumi-1, and K562) leukemia cells. The inhibin-α promoter was hypermethylated in lymphoid (Molt-4 and Raji) and myeloid (HL-60 and Kasumi-1) leukemia cells. Inhibin-α gene mutations differed significantly between lymphoid (heterozygote) and myeloid (homozygote) leukemia cells. LOH in the inhibin-α gene was detected in lymphoid and myeloid leukemia cells, with the exception of Jurkat cells. Treatment with 5-AzaC, a demethylating agent, resulted in increased inhibin-α mRNA and protein levels in most of the cell lines. Also, 5-AzaC treatment inhibited cell proliferation and induced apoptosis. Taken together, our results reveal that the inhibin-α gene is transcriptionally silenced in human leukemia cells and that reactivation is suppressed by a demethylating agent. In addition, mutations in, and expression levels of, the inhibin-α gene differed between human lymphoid and myeloid leukemia cells.

Mutations in DNA Gyrase and Topoisomerase IV in Ciprofloxacin-Nonsusceptible Extended-Spectrum -Lactamase-Producing Escherichia coli and Klebsiella pneumoniae.

BACKGROUND: We investigated mutations in the quinolone resistance-determining region (QRDR) of ciprofloxacinnonsusceptible extended-spectrum -lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae by a statistical analysis. METHODS: We collected 97 clinical isolates of ciprofloxacin-nonsusceptible ESBL-producing E. coli (55 strains) and K. pneumoniae (42 strains) from a tertiary-care university hospital in Seoul, Republic of Korea, between 2006 and 2008. The QRDR of the gyrA, gyrB, parC, and parE genes were amplified by PCR and sequenced. RESULTS: Most E. coli isolates (53/55; 96.4%) with a minimum inhibitory concentration of ≥ 64 mg/L against ciprofloxacin had double mutations in gyrA (Ser-83Leu and Asp-87Asn) and at least one mutation in parC (Ser-80 Ile or Glu-84Val), with or without one in parE. Fifty E. coli (90.9%) isolates had a mutation in parE, of which Ile-529Leu (70.9%) was the most frequent. However, we could not find statistically significant variables in increasing ciprofloxacin resistance in E. coli isolates. Thirty-six K. pneumoniae isolates (36/42; 85.7%) had at least one mutation in gyrA, gyrB, or parC, and the mutation in gyrA might have been associated with plasmid-mediated quinolone-resistance (PMQR). Ser-80Ile in parC and aac(6')-Ib-cr in the K. pneumoniae isolates were significantly associated with an increased MIC of ciprofloxacin by ordinal logistic regression analysis. CONCLUSIONS: The Ser-80Ile in parC and aac(6')-Ib-cr in K. pneumoniae are supposed to play an important role in increased ciprofloxacin resistance, but statistically significant variables could not be found in E. coli isolates in the present study.

RNA expression analysis of efflux pump genes in clinical isolates of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis in South Korea.

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is an important communicable disease. Various mechanisms of resistance to antituberculosis drugs have been reported; these are principally mutations in target genes. However, not all M. tuberculosis resistance can be explained by mutations in such genes. Other resistance mechanisms associated with drug transport, such as efflux pumps, have also been reported. In this study, we investigated the expression levels of three putative efflux pumps and mutations in target genes associated with injectable agents and fluoroquinolones with clinical MDR and XDR-TB isolates. Thirty clinical isolates of M. tuberculosis that had been phenotypically characterized were obtained from the Korean Institute of Tuberculosis. Of these, 14 were MDR-TB isolates resistant to at least one injectable aminoglycoside (amikacin; AMK, kanamycin; KAN, and/or capreomycin; CPM) and 16 were XDR-TB isolates. M. tuberculosis H37Rv (ATCC 27249) was used as a reference strain. Five putative genes (Rv1258c, Rv2686c, Rv2687c, Rv2688c and pstB) were selected for analysis in this study. Sequencing was performed to detect mutations in rrs and eis genes. qRT-PCR was performed to investigate expression levels of five efflux pump genes. Of the 30 isolates, 25 strains had mutations in rrs associated with resistance to KAN, CPM and AMK and two strains had eis mutations, as well as mutations in rrs. pstB (Rv0933) exhibited increased expression and Rv2687c and Rv2688c exhibited decreased expression compared to the reference strain. Increased expression of pstB in clinical drug-resistant tuberculosis isolates may contribute to drug resistance in M. tuberculosis. In our case, overexpression of Rv1258c may have been associated with resistance to kanamycin. No correlation was evident between Rv2686c, Rv2687c or Rv2688c expression and fluoroquinolone resistance. To explore the details of efflux pump drug-resistance mechanisms, further studies on efflux pump inhibitors, transcriptional regulators, such as whiB7, and additional efflux pump genes are needed.

Opportunities in low-level radiocarbon microtracing: applications and new technology.

14C-radiolabeled (radiocarbon) drug studies are central to defining the disposition of therapeutics in clinical development. Concerns over radiation, however, have dissuaded investigators from conducting these studies as often as their utility may merit. Accelerator mass spectrometry (AMS), originally designed for carbon dating and geochronology, has changed the outlook for in-human radiolabeled testing. The high sensitivity of AMS affords human clinical testing with vastly reduced radiative (microtracing) and chemical exposures (microdosing). Early iterations of AMS were unsuitable for routine biomedical use due to the instruments' large size and associated per sample costs. The situation is changing with advances in the core and peripheral instrumentation. We review the important milestones in applied AMS research and recent advances in the core technology platform. We also look ahead to an entirely new class of 14C detection systems that use lasers to measure carbon dioxide in small gas cells.

Invasive Fungal Sinusitis by Lasiodiplodia theobromae in an Patient with Aplastic Anemia: An Extremely Rare Case Report and Literature Review.

Human Lasiodiplodia theobromae infection has not been reported frequently. We report the first case of invasive L. theobromae nasal and neck infection. A 66-year-old male visited our hospital with anemia and general weakness. He showed pancytopenia, and his bone marrow examination revealed markedly decreased hematopoietic cells. The patient was presumed to have iatrogenic aplastic anemia due to mushroom toxicity. He began treatment for multiple organ infections with broad-spectrum antibiotics and antifungal agents. During hospitalization, he complained of nasal obstruction and left neck lymph node enlargement. A mass-like lesion was observed, and a nasal mass biopsy was performed. The mass was identified as a fungal ball. He underwent surgical excision for the nasal mass and the neck lymph node. The pathologic examination indicated an invasive fungal infection, and the lymph node revealed chronic granulomatous inflammation with fungal infection. 18s rRNA sequencing revealed that the sequence shared 99 % identity with L. theobromae. The nasal mass fungus was identified by internal transcribed spacer region sequencing from pathologic paraffin sections. The obtained sequence corresponded to Lasiodiplodia or Macrophoma. The sequence corresponded to the neck discharge sequence results. Hence, the patient was diagnosed with invasive fungal sinusitis with neck lymph node involvement caused by L. theobromae. To our knowledge, this is the first report of L. theobromae infection in Korea and the first report of invasive L. theobromae fungal sinusitis in the literature. We should include more precise evaluations of additional novel fungal species as possible candidates.

Mean Platelet Volume in Mycobacterium tuberculosis Infection.

Introduction. Mean platelet volume (MPV) has been thought as a useful index of platelet activation. It is supposed that MPV is also associated with several inflammatory and infectious diseases. Korea still has a high incidence of tuberculosis (TB). The aim of this study was to investigate MPV as an inflammatory marker in TB patients. Materials and Methods. MPV were determined in 221 patients with TB and 143 individuals for control group. MPV was estimated by an Advia 2120 (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Results. In the TB patient group, a positive correlation was found between CRP and MPV. Age and MPV had a positive correlation in TB patient group. Conclusions. We conclude that there is a significant relation between MPV and inflammatory conditions. MPV can be an inflammatory marker to determine the disease activity in TB patients.

MALDI-TOF MS is more accurate than VITEK II ANC card and API Rapid ID 32 A system for the identification of Clostridium species.

All 50 Clostridium difficile strains were definitely identified by Vitek2 system, Rapid ID 32A system, and MALDI-TOF. For 18 non-difficile Clostridium strains, the identification results were correct in 2, 0, and 17 strains by Vitek2, Rapid ID 32A, and MALDI-TOF, respectively [corrected].