Horizontal Gene Transfer of blaNDM-5 Among Three Different Enterobacteriaceae Species Isolated from a Single Patient.

BACKGROUND: In this study, Escherichia coli, Klebsiella oxytoca, and Citrobacter amalonaticus carrying blaNDM-5 were isolated from a single patient. METHODS: The antibiotic susceptibility of the isolates was evaluated by using E-test and agar dilution methods, and blaNDM-5 was identified in genomic and plasmid DNA by using polymerase chain reaction and sequencing. Whole genome sequencing and de novo assembly were used for species characterization, resistance gene identification, and plasmid analysis. RESULTS: All three species had identical plasmids, which were similar to pEC463-NDM5, a plasmid harboring blaNDM-5. Transconjugation experiments confirmed the horizontal gene transfer of blaNDM-5, highlighting the need for a close monitoring of Enterobacteriaceae species harboring this gene. CONCLUSIONS: This study conclusively demonstrates the propensity for horizontal gene transfer of blaNDM-5 among Enterobacteriaceae species, underlining the importance of vigilant monitoring to combat antibiotic resistance.

Changes of frequency and expression level of CD161 in CD8+ T cells and natural killer T cells in peripheral blood of patients with systemic lupus erythematosus.

Immunologic abnormalities of natural killer (NK) cells and T cells play a role in the pathogenesis of systemic lupus erythematosus (SLE). CD161 is expressed on most of the NK cells and on some T cells. The quantities of CD161-expressing cells and expression levels of CD161 were analyzed in T cells and NK cells from patients with SLE compared with normal controls. The expression of CD161 on NK cells, NKT cells, CD4+ T cells, and CD8+ T cells in peripheral blood from patients with inactive SLE and active SLE, and from the normal controls group were determined using flow cytometry. The frequency and expression level of CD161 in the lymphocyte subsets and its relationship with the quantity of regulatory T cells, anti-double stranded DNA antibody, and the titer of antinuclear antibody were evaluated. Both the percentages of the CD161+ subpopulation and the mean fluorescence intensities (MFIs) of CD161 in CD8+ T cells and NKT cells decreased significantly in SLE patients compared with normal controls (P < .001). The CD161 expression in CD8+ T cells and NKT cells also decreased in the anti-dsDNA (+) group (P < .05). The counts of Treg cells were lower in SLE patients and were weakly correlated with the percentage of the CD161 subpopulation (r = 0.229, P = .016) and the MFIs of CD161 expression in CD8+ T cells (r = .232, P = .014). The frequencies and levels of CD161 expression on CD8+ T cells and NKT cells were reduced in SLE patients, suggesting that an abnormality of these cells was related to the pathogenesis of SLE.

Emergence and transmission of New Delhi metallo-beta-lactamase-5-producing Escherichia coli Sequence Type 361 in a Tertiary Hospital in South Korea.

BACKGROUND: The emergence of carbapenem-resistant Escherichia coli (E coli) is a serious global health threat, but little is known about carbapenemase-producing E coli in Daejeon, South Korea. The aim of this study was to investigate characteristics of thirteen carbapenem-resistant E coli isolates in a tertiary hospital. METHODS: Thirteen non-duplicate carbapenem-resistant E coli strains were collected from October 2017 to January 2018. Antimicrobial susceptibility was determined with the E test or disk diffusion method. The carbapenem minimum inhibitory concentrations (MICs) were determined by the agar dilution method. The colistin and tigecycline MICs were determined by broth microdilution. The resistance genes, including carbapenemase genes, were evaluated by polymerase chain reaction, and DNA sequencing was performed to characterize the genes. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) were performed to evaluate the clonal relatedness of isolates. The clinical data of patients were retrospectively reviewed. RESULTS: All the E coli isolates harbored blaNDM-5 gene and were resistant to most of the antimicrobial agents, such as carbapenem, cephalosporins, ciprofloxacin, and chloramphenicol, excluding amikacin and colistin. Other resistant genes, such as blaTEM-1 , blaCTX-M-15 , blaCMY-2 , aac(6')-Ib-cr, and qepA, were detected. The E coli isolates harboring blaNDM-5 belonged to ST361 (n = 11), ST12 (n = 1), ST410 (n = 1), and PFGE types A (n = 11), B (n = 1), and C (n = 1). CONCLUSIONS: This study reports on an outbreak of a predominant epidemic clone, the NDM-5 producing, multidrug-resistant E coli ST361 isolate. These findings suggest that we should pay attention to infection control measures to limit the spread of NDM-5-producing pathogens.

Molecular epidemiology and virulence factors of methicillin-resistant Staphylococcus aureus isolated from patients with bacteremia.

BACKGROUND: The various virulence factors of methicillin-resistant Staphylococcus aureus bacteremia (MRSAB) are associated with a high mortality rate worldwide. Further studies are warranted to confirm the significant relationship between the strains and virulence genes. Here, we prospectively investigated the molecular characteristics underlying the genotypes and virulence factors of MRSA isolated from patients with bacteremia. METHODS: We collected 59 MRSA isolates from adult patients with bacteremia. Antimicrobial susceptibility results were obtained with the Vitek2 automated system. Genotypes were identified with multi-locus sequence typing (MLST) and pulse-field gel electrophoresis (PFGE), and 21 virulence genes were detected with polymerase chain reaction (PCR). RESULTS: The 59 MRSA isolates mainly comprised ST5 (n = 31, 52.5%) and ST72 (n = 22, 37.2%). Most ST5 isolates and all ST72 isolates were clustered into one and two PFGE groups, respectively. The mean number of virulence genes was higher in ST5 than in ST72. Sel was more frequently detected in ST5 than in ST72, whereas sec and sed were found only in ST5. ST5 had significantly higher resistance against many antibiotics than ST72. CONCLUSION: Most MRSA isolates causing bacteremia were ST5 (CC5) and ST72 (CC8), and those belonging to the same STs were divided into only a few PFGE groups. ST5 was associated with higher antibiotic resistance and staphylococcal superantigen toxin genes, than ST72, which may be related to its higher virulence.

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates.

BACKGROUND: The emergence and transmission of tigecycline- and carbapenem-resistant Klebsiella pneumoniae (TCRKP) have become a major concern to public health globally. Here, we investigated the molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant K pneumoniae (CRKP) isolates. METHODS: Forty-five non-duplicate CRKP isolates were collected from January 2017 to June 2019. We performed antimicrobial susceptibility tests, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). PCR and DNA sequencing were performed for the detection and mutation analysis of acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes, which are related to tigecycline resistance. The expression levels of efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed by quantitative real-time PCR. RESULTS: The resistance rate to tigecycline in CRKP isolates was 37.8% (17/45). K pneumoniae ST307 was a predominant clone type (70.6%, 12/17) among the TCRKP isolates. The expression levels of acrB (P < .001) and marA (P = .009) were significantly higher in the tigecycline-resistant group than in the tigecycline-intermediate and tigecycline-susceptible groups. Increased expression of acrB was associated with marA expression (r = 0.59, P = .013). CONCLUSIONS: We found that the activated MarA-induced overexpression of AcrAB efflux pump plays an important role in the emergence of tigecycline resistance in CRKP isolates.

Comparison of the Genedia MTB/NTM Detection Kit and Anyplex plus MTB/NTM Detection Kit for detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria in clinical specimens.

BACKGROUND: Real-time (RT) PCR is a rapid and accurate method that is widely used for the detection of Mycobacterium tuberculosis complex (MTB). The aim of this study was to evaluate and compare the performance of the Genedia MTB/NTM Detection Kit and the Anyplex plus MTB/NTM Detection kit in the detection of MTB and nontuberculous mycobacteria (NTM) in clinical specimens. METHODS: From October 2017 to February 2018, 236 respiratory specimens and 137 non-respiratory specimens, from patients with suspected tuberculosis, were examined. AFB smear, culture, and RT-PCR using the Genedia MTB/NTM Detection kit (Green Cross Medical Science Corp.) and the Anyplex plus MTB/NTM Detection kit (Seegene) were applied. PCR performance in the detection of MTB and NTM was evaluated in relation to culture results and between the two assays. RESULTS: Culture was positive for MTB in 30 (8.0%) of the 373 specimens and for NTM in 23 (6.2%). The sensitivity and specificity of MTB detection with the Genedia kit were 76.7% and 99.7%, respectively, whereas the Anyplex kit sensitivity and specificity for MTB detection were 86.7% and 97.5%, respectively. Both kits exhibited the same sensitivity (73.9%) for NTM detection, and the specificity was 100% and 99.4% for the Genedia and Anyplex kits, respectively. CONCLUSIONS: The Genedia and Anyplex kits demonstrated high sensitivity and specificity for the detection of MTB and NTM. Both kits have a high concordance rate and can be used more widely in clinical laboratories for the early detection of tuberculosis.

Cytogenetic evolution in myeloproliferative neoplasms with different molecular abnormalities.

We investigated the changes in chromosomal abnormalities in myeloproliferative neoplasm (MPN) patients during long-term follow-up. In total, 28 MPN patients (22 with primary myelofibrosis and 6 with polycythemia vera) were included. Among them, 25 patients underwent serial bone marrow (BM) biopsies during disease progression, and 3 patients had cytogenetic abnormalities at initial diagnosis but lacked follow-up BM biopsies. JAK2, CALR, and MPL mutation analyses were performed. Targeted sequencing analysis was conducted in 11 patients. Among the 28 patients, 21 (75.0%) had cytogenetic abnormalities either at diagnosis (8/26) or during follow-up. The median time from the initial analysis to the appearance of additional cytogenetic abnormalities was 8.4 years. Among the chromosomal abnormalities at initial diagnosis, trisomy 8 (3/26, 11.5%) was the most frequent, followed by gain of 1q, del(20q), and del(9q) (each in 2/26). Among all chromosomal abnormalities, including those that occurred during follow-up, the most frequent was del(20q) and +1q (8/28, 28.6%), followed by del(6p) (14.3%) and trisomy 8 (10.7%). Del(20q) was more frequent in CALR-mutated patients (4/6, 66.7%) than in JAK2-mutated patients (3/19, 15.8%, P = 0.016). The presence of cytogenetic abnormalities at initial diagnosis was associated with poor prognosis. Cytogenetic evolution may provide interesting insights into the disease course.

Rapid Screening of Urinary Tract Infection and Discrimination of Gram-Positive and Gram-Negative Bacteria by Automated Flow Cytometric Analysis Using Sysmex UF-5000.

Rapid screening of urinary tract infection is important to determine antibiotic treatment and reduce unnecessary urine culture. We evaluated the performance of the new flow cytometry-based UF-5000 automated urine analyzer (Sysmex, Kobe, Japan). A total of 1,430 urine samples from 1,226 patients were analyzed and compared to urine cultures to which a Previ Isola (bioMérieux, Marcy l'Etoile, France) system was applied. In total, 878 of 1,430 urine cultures (61.4%) produced ≥103 CFU/ml bacterial growth (309 with Gram-negative [GN] bacteria, 517 with Gram-positive [GP] bacteria, and 52 mixed cultures), with 336 samples (23.5%) presenting ≥105 CFU/ml bacterial growth. The ≥105 CFU/ml bacterial growth was detected by a ≥71 bacteria/µl UF-5000 bacterial count with 95% sensitivity and 84% specificity. Using a cutoff of <15 bacteria/µl to determine whether or not to culture, 50.9% of samples were below the cutoff, 94.8 and 99.5% of which presented <104 and <105 CFU/ml of bacterial growth, respectively. The bacterial discrimination performance of the UF-5000 for GN bacteria was superior to that for GP bacteria, and in ≥105 CFU/ml monobacterial samples, the sensitivity and specificity for reporting GN bacteria were 91.7 and 90.0%, respectively. In summary, UF-5000 demonstrated potential utility for the rapid screening of negative bacterial cultures. However, this utility is dependent on the patient population; cutoff optimizations must be performed for specific populations. In addition, UF-5000 presented improved performance in characterizing GP and GN bacteria, although the concurrence rates were not high enough to replace routine cultures.

Manual versus automated streaking system in clinical microbiology laboratory: Performance evaluation of Previ Isola for blood culture and body fluid samples.

BACKGROUND: The process of plate streaking has been automated to improve routine workflow of clinical microbiology laboratories. Although there were many evaluation reports about the inoculation of various body fluid samples, few evaluations have been reported for blood. In this study, we evaluated the performance of automated inoculating system, Previ Isola for various routine clinical samples including blood. METHODS: Blood culture, body fluid, and urine samples were collected. All samples were inoculated on both sheep blood agar plate (BAP) and MacConkey agar plate (MCK) using Previ Isola and manual method. We compared two methods in aspect of quality and quantity of cultures, and sample processing time. To ensure objective colony counting, an enumeration reading reference was made through a preliminary experiment. RESULTS: A total of 377 nonduplicate samples (102 blood culture, 203 urine, 72 body fluid) were collected and inoculated. The concordance rate of quality was 100%, 97.0%, and 98.6% in blood, urine, and other body fluids, respectively. In quantitative aspect, it was 98.0%, 97.0%, and 95.8%, respectively. The Previ Isola took a little longer to inoculate the specimen than manual method, but the hands-on time decreased dramatically. The shortened hands-on time using Previ Isola was about 6 minutes per 10 samples. CONCLUSION: We demonstrated that the Previ Isola showed high concordance with the manual method in the inoculation of various body fluids, especially in blood culture sample. The use of Previ Isola in clinical microbiology laboratories is expected to save considerable time and human resources.

Incidence, puberty, and fertility in 45,X/47,XXX mosaicism: Report of a patient and a literature review.

Turner syndrome (TS), characterized by short stature and premature ovarian failure, is caused by chromosomal aberrations with total or partial loss of one of the two X chromosomes. Spontaneous puberty, menarche, and pregnancy occur in some patients depending on the abnormality of the X. Moreover, spontaneous pregnancy is uncommon (<0.5%) for TS with 45,X monosomy. Among TS patients, 45,X/47,XXX karyotype is extremely rare. Previous reports have demonstrated that TS with 45,X/47,XXX is less severe than common TS due to higher occurrence of puberty (83%), menarche (57-67%), and fertility (14%) and lower occurrence of congenital anomalies (<5%). However, TS mosaicism may not reduce the frequency of short stature. We diagnosed a 10-year-girl with TS with 45,X/47,XXX mosaicism who presented with short stature. She showed mild TS phenotype including short stature but had spontaneous puberty. Based on our case and previous reports, we expect that girls with 45,X/47,XXX mosaicism may progress through puberty normally, without estrogen therapy. Therefore, it is necessary to consider specific guidelines for clinical decisions surrounding pubertal development and fertility in TS with 45,X/47,XXX karyotype.

Evaluation of the LABGEO PT10 Point-Of-Care Testing Device: Comparison of Analyte Measurements in Capillary Whole Blood and Lithium Heparin Whole Blood Samples With Those in Central Laboratory.

BACKGROUND: Point-of-care (POC) testing device has been widely used because of its rapid availability of results making diagnosis and management as early as possible. Capillary blood can reduce the difficulty of obtaining samples compared to venous blood and allows the prompt testing results. In this study, we evaluated the usefulness of capillary blood in Samsung LABGEO PT10. METHODS: Fifty-one patients and 18 healthy adults aged between 20 and 65 were enrolled and their capillary and venous blood samples were collected. Venous blood samples were split into lithium heparin (LiHep) tube and serum-separating tube. Measurements using capillary blood and LiHep whole blood were performed in LABGEO PT10. Serum was used for measurement by Toshiba 2000FR NEO in central laboratory. RESULTS: In comparison between measurements in LABGEO PT10 using capillary and LiHep whole blood, the slope ranged between 0.9289 and 1.0471, correlation coefficients (R2 ) were over 0.95 except albumin, high-density lipoprotein, and total protein. Comparison of measurements in capillary and LiHep whole blood using LABGEO PT10 with those in the central laboratory revealed that the slope ranged between 0.6433 and 1.1364 for capillary whole blood and 0.6255 and 1.1602 for LiHep whole blood except alkaline phosphatase. For most of analytes, R2 were over 0.95. CONCLUSION: Measurements in LABGEO PT10 using capillary blood was well correlated with those in LABGEO PT10 using LiHep whole blood and also with in the central laboratory. In conclusion, capillary blood provides reliable measurements and can be trustfully used in LABGEO PT10.

Epidemiology, Molecular Characteristics, and Virulence Factors of Carbapenem-Resistant Pseudomonas aeruginosa Isolated from Patients with Urinary Tract Infections.

PURPOSE: Pseudomonas aeruginosa is an important pathogen that causes urinary tract infections. Carbapenems are the drugs of choice for the treatment of P. aeruginosa infections. However, the emergence and spread of carbapenem-resistant P. aeruginosa (CRPA) is a serious global health threat. In this study, we investigated the epidemiology, molecular characteristics, drug resistance, and virulence factors of CRPA isolated from urine samples. METHODS: A total of 124 P. aeruginosa isolates were obtained from urine samples collected between March 2020 and February 2021. Clonal relatedness was evaluated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). We performed antimicrobial susceptibility tests and investigated the presence of carbapenemase genes and virulence factors in CRPA isolates. RESULTS: The carbapenem resistance rate of the isolated P. aeruginosa was 46.7% (59/124). A total of 54 (91.5%) out of the 59 CRPA isolates were identified as multidrug-resistant. The majority of the CRPA isolates (81.4%, 48/59) harbored carbapenemase genes, such as bla IMP-6 or bla NDM-1. In an epidemiological analysis using MLST, 88.1% of CRPA isolates were confirmed to be ST773 (50.8%, 30/59) or ST235 (37.3%, 22/59). The CRPA isolates harboring bla IMP-6 and bla NDM-1 belonged to ST235 (PFGE pulsotypes E1-E18, F) and ST773 (PFGE pulsotypes H1-H2, I1-I16) subtypes, respectively. The studied CRPA isolates simultaneously harbored 10 to 14 virulence factors of the 16 virulence factors examined. Nine virulence factor genes (toxA, exoT, plcH, plcN, phzM, phzS, lasB, aprA, and algD) were identified in all CRPA isolates. CONCLUSION: Our study shows that P. aeruginosa ST235 harboring bla IMP-6 and ST773 harboring bla NDM-1-known internationally as high-risk clones with multiple virulence factors-are widely spread in the study area. These findings suggest that continuous monitoring is necessary to prevent the further spread of carbapenemase-producing CRPA.

Identification of novel candidate target genes, including EPHB3, MASP1 and SST at 3q26.2-q29 in squamous cell carcinoma of the lung.

BACKGROUND: The underlying genetic alterations for squamous cell carcinoma (SCC) and adenocarcinoma (AC) carcinogenesis are largely unknown. METHODS: High-resolution array- CGH was performed to identify the differences in the patterns of genomic imbalances between SCC and AC of non-small cell lung cancer (NSCLC). RESULTS: On a genome-wide profile, SCCs showed higher frequency of gains than ACs (p = 0.067). More specifically, statistically significant differences were observed across the histologic subtypes for gains at 2q14.2, 3q26.2-q29, 12p13.2-p13.33, and 19p13.3, as well as losses at 3p26.2-p26.3, 16p13.11, and 17p11.2 in SCC, and gains at 7q22.1 and losses at 15q22.2-q25.2 occurred in AC (P < 0.05). The most striking difference between SCC and AC was gains at the 3q26.2-q29, occurring in 86% (19/22) of SCCs, but in only 21% (3/14) of ACs. Many significant genes at the 3q26.2-q29 regions previously linked to a specific histology, such as EVI1,MDS1, PIK3CA and TP73L, were observed in SCC (P < 0.05). In addition, we identified the following possible target genes (> 30% of patients) at 3q26.2-q29: LOC389174 (3q26.2),KCNMB3 (3q26.32),EPHB3 (3q27.1), MASP1 and SST (3q27.3), LPP and FGF12 (3q28), and OPA1,KIAA022,LOC220729, LOC440996,LOC440997, and LOC440998 (3q29), all of which were significantly targeted in SCC (P < 0.05). Among these same genes, high-level amplifications were detected for the gene, EPHB3, at 3q27.1, and MASP1 and SST, at 3q27.3 (18, 18, and 14%, respectively). Quantitative real time PCR demonstrated array CGH detected potential candidate genes that were over expressed in SCCs. CONCLUSION: Using whole-genome array CGH, we have successfully identified significant differences and unique information of chromosomal signatures prevalent between the SCC and AC subtypes of NSCLC. The newly identified candidate target genes may prove to be highly attractive candidate molecular markers for the classification of NSCLC histologic subtypes, and could potentially contribute to the pathogenesis of the squamous cell carcinoma of the lung.

Comparison of triglyceride glucose index, and related parameters to predict insulin resistance in Korean adults: An analysis of the 2007-2010 Korean National Health and Nutrition Examination Survey.

The triglyceride glucose (TyG) index, a product of triglyceride and fasting glucose, is a reliable marker for insulin resistance (IR). Obesity is also known to be closely related with IR. Recently, the efficiency of TyG-related markers that combine obesity markers with TyG index has been studied; however, earlier studies were limited in number and the results were inconsistent. Therefore, in this study, we investigated the efficiency of several combinations of TyG index and obesity indices, namely, body mass index (BMI), waist circumference (WC), and waist-to-height ratio (WHtR), in reflecting IR. Data were obtained from the Korean National Health and Nutrition Examination Survey from 2007-2010. A total of 11,149 subjects (4,777 men and 6,372 women) were included. IR was defined as the homeostasis model assessment for IR (HOMA-IR) of above the 75th percentile for each gender. Logistic regression analysis was performed after adjusting for confounding factors, to compare and identify the associations of the 4 parameters (TyG index, TyG-BMI, TyG-WC, and TyG-WHtR) with IR. For each parameter, odds ratios (OR) and 95% confidence intervals (CIs) of quartiles 2-4 were calculated and compared with quartile 1 as a reference. A receiver operating characteristic (ROC) curve analysis was conducted to evaluate the ability of each parameter to predict IR. The adjusted ORs of quartile 4 in comparison with quartile 1 (95% CIs) for IR were 7.60 (6.52-8.87) for TyG index, 12.82 (10.89-15.10) for TyG-BMI, 16.29 (13.70-19.38) for TyG-WC, and 14.86 (12.53-17.62) for TyG-WHtR. The areas under the ROC curve for each parameter were 0.690 for TyG index, 0.748 for TyG-BMI, 0.731 for TyG-WC, and 0.733 for TyG-WHtR. In conclusion, TyG-BMI was found to be superior to other parameters for IR prediction. We propose TyG-BMI as an alternative marker for assessing IR in clinical settings.

Automated Versus Conventional Microscopic Interpretation of Antinuclear Antibody Indirect Immunofluorescence Test.

BACKGROUND: The detection of antinuclear antibodies (ANAs) is crucial for the diagnosis of systemic autoimmune rheumatic diseases. Here, we evaluated the performance of an automated ANA image analyzer, EUROPattern Suite. METHODS: We compared the automated and manually revised interpretations of EUROPattern Suite with conventional microscopic interpretations of indirect immunofluorescence (IIF) by calculating the analytical sensitivity and specificity for ANA detection and evaluating the concordance between these approaches for ANA detection, pattern recognition, and titer determination. RESULTS: The analytical sensitivity and specificity of EUROPattern Suite for ANA detection were 97.7% and 88.2%, respectively, relative to those of conventional IIF, with a concordance of 94.4% 3κ=0.874). After manual revision, the analytical sensitivity, specificity, and concordance improved to 98.5%, 94.1%, and 97.0% (κ=0.932), respectively. The concordance rates for pattern recognition with simple and mixed patterns were 72.1% and 56.0%, respectively, and varied depending on the type of pattern. After manual revision, the concordance rates with simple and mixed patterns increased to 97.1% and 84.0%, respectively. The concordance rate for titer determination with a simple pattern was 84.6%. CONCLUSION: Although automated pattern recognition has some limitations, EUROPattern Suite facilitates automated ANA detection with a high sensitivity and concordance with conventional IIF.

Microarray-Based Nucleic Acid Assay and MALDI-TOF MS Analysis for the Detection of Gram-Negative Bacteria in Direct Blood Cultures.

Objectives: To compare a microarray-based identification and resistance determination system (blood culture gram-negative [BC-GN]; Nanosphere, Northbrook, IL) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct blood cultures (BCs). Methods: BC-GN and MALDI-TOF MS assay results from direct BCs were compared with conventional test results after pure culture. Results: Among 124 BCs, 130 gram-negative rods (GNRs), including six cultures with mixed GNRs (117 bacteria were covered by the BC-GN panel), were detected. The BC-GN test presented 116/117 (99.1%) concordance for the identification of targeted GNRs. Among the six polymicrobial BCs, 10 targeted GNRs were correctly identified. Among the 100 BCs tested by MALDI-TOF MS, 88/106 (86.7%) GNRs were correctly identified, and 18 GNRs were not identified. Among the six polymicrobial samples, seven of 12 GNRs (58.3%) were correctly identified. Conclusions: The BC-GN assay exhibited superior performance compared with MALDI-TOF MS for the identification of targeted GNRs in direct BCs, particularly in polymicrobial samples.

Gain at chromosomal region 5p15.33, containing TERT, is the most frequent genetic event in early stages of non-small cell lung cancer.

Chromosomal imbalances resulting in altered gene dosage play a role in the molecular pathogenesis of non-small cell lung cancer (NSCLC), but the target genes remain to be identified. To identify early-stage genetic events that drive progression of NSCLC, we conducted a high-resolution array comparative genomic hybridization (CGH) study, using an array of 4,046 bacterial artificial chromosome clones to screen for DNA copy number changes associated with individual genes in 36 tumors obtained from patients in early stages of NSCLC. Multiple early genetic events occurring on chromosome 5p were identified, with a minimal detection region at 5p15.33 approximately 12. The most frequent finding involved gain of 5p15.33, observed in 15 of 19 stage I (A+B) cancers (79%) and in 28 of the total 36 NSCLC cases (78%). This locus harbors the genes TERT, SLC6A19, and SLC6A18 and is a telomeric boundary at bacterial artificial chromosome (BAC) clone 91_J20. Other potential candidate genes evidencing high numbers of genomic copy number changes (> or =40% of patients) included the following genes, encountered in >50% of 19 stage I (A+B) cancers: CEP72 and TPPP (14 of 19; 74%); AHRR, EXOC3 (previously SEC6L1), SLC9A3, LOC442126, ZDHHC11, BRD9, and TRIP13 (13/19; 68%); and CLPTM1L (alias CRR9), SLC6A3 (previously DAT1), and LOC401169 (10/19; 53%). Fluorescence in situ hybridization validated the array CGH findings. The gain of 5p15.33 is thus one of the most consistent alterations in the early stages of lung cancer, and a series of genes in the critical 5p15.33 region may be used as novel biomarkers for the early detection and classification of lung cancer.

Association between Biofilm Formation and Antimicrobial Resistance in Carbapenem-Resistant Pseudomonas Aeruginosa.

Recently, carbapenem resistance in P. aeruginosa is an increasingly important problem globally. Biofilm formation is a well-known pathogenic mechanism of P. aeruginosa, and the gene, pslA, plays an important role in its primary stages. We studied the association between biofilm formation and pslA in carbapenem-resistant P. aeruginosa isolates, along with antimicrobial resistance and the prevalence of metallo-ß-lactamase (MBL) genes, based on the presence of pslA 82 carbapenem-resistant P. aeruginosa isolates were collected from a tertiary hospital in Daejeon, Korea, between March 2008 and June 2014. Minimum inhibitory concentrations (MICs) of nine antimicrobial agents were determined using the agar dilution method. Biofilm formation was measured by microtiter plate assay. PCR and sequencing were used to identify pslA and the MBL gene. 76 (92.7%) carbapenem-resistant isolates were biofilm producers. These biofilm producers showed higher levels of amikacin, ceftazidime, and cefepime resistance than non-producers. pslA was detected in 71 (93.4%) biofilm-producing isolates and these results were statically significant (p<0.01). 11 isolates carrying pslA and blaIMP-6 were extremely resistant to all antimicrobials tested. In this study, biofilm formation was significantly associated with pslA Furthermore, the coexistence of pslA and the MBL gene in carbapenem-resistant isolates likely contributed to the increase in antimicrobial resistance.

Pyrosequencing-based quantitative measurement of CALR mutation allele burdens and their clinical implications in patients with myeloproliferative neoplasms.

BACKGROUND: We developed a pyrosequencing-based method for the quantification of CALR mutations and compared the results using Sanger sequencing, fragment length analysis (FLA), digital-droplet PCR (ddPCR), and next-generation sequencing (NGS). METHODS: Method validation studies were performed using cloned plasmid controls. Samples from 24 patients with myeloproliferative neoplasms were evaluated. RESULTS: Among the 24 patients, 15 had CALR mutations (7 type 1, 2 type 2, and 6 other mutations). The type 1 or type 2 mutation-positive results from pyrosequencing exhibited 100% concordance with the Sanger sequencing results. One novel CALR mutation was not detected by pyrosequencing. The CALR mutation allele burdens measured by pyrosequencing were slightly lower than those measured by FLA but slightly higher than the results obtained using ddPCR. Pyrosequencing exhibited high correlations with both methods. The mutation allele burdens estimated by NGS were significantly lower than those measured by pyrosequencing. An increased CALR mutation allele burden was associated with overt primary myelofibrosis. Patients with >70% mutation allele burdens in myeloid cells had a significantly longer time from diagnosis (P = 0.007), more bone marrow fibrosis (P = 0.010), and lower hemoglobin (P = 0.007). CONCLUSIONS: Pyrosequencing was a useful rapid sequencing method to determine the burden of CALR mutations.

Discriminating myelodysplastic syndrome and other myeloid malignancies from non-clonal disorders by multiparametric analysis of automated cell data.

BACKGROUND: We investigated the usefulness of novel complete blood count (CBC) data for discriminating myeloid malignancies from non-clonal CBC abnormalities. METHODS: Data were obtained during routine CBC tests of 119 samples from 37 myelodysplastic syndrome (MDS) patients, 92 samples from 45 myeloproliferative neoplasm (MPN) patients, and 15 samples from 11 chronic myelogenous leukemia (CML) patients using a DxH800 (Beckman Coulter). Data obtained from patients with hypocellular bone marrow and from those with other non-clonal diseases with CBC abnormalities were included in the comparisons. RESULTS: For cell population data of neutrophils, the means of median, upper median, lower median, and low angle light scatters were significantly lower in MDS patients than in patients without hematological malignancies. Low hemoglobin density (LHD) did not significantly differ between the MDS and non-clonal cytopenia patients, but it was significantly higher in the MPN and CML patients. We selected 13 parameters and scored the MDS diagnosis using cut-off values obtained from receiver operating characteristic (ROC) curve analysis. Using a score > 9, MDS was distinguished from non-clonal cytopenia with a sensitivity of 92.4% and a specificity of 85.4%. CONCLUSIONS: Multiparametric analyses of new automated parameters are useful for discriminating MDS from non-clonal cytopenia.