Strategic Retesting to Reduce False Positive SARS-CoV-2 PCR Test Results.

BACKGROUND: Nucleic acid amplification testing is the gold standard for SARS-CoV-2 diagnostics, although it may produce a certain number of false positive results. There has not been much published about the characteristics of false positive results. In this study, based on retesting, specimens that initially tested positive for SARS-CoV-2 were classified as true or false positive groups to characterize the distribution of cycle threshold (CT) values for N1 and N2 targets and number of targets detected for each group. METHODS: Specimens that were positive for N-gene on retesting and accompanied with S-gene were identified as true positives (true positive based on retesting, rTP), while specimens that retested negative were classified as false positives (false positive based on retesting, rFP). RESULTS: Of the specimens retested, 85/127 (66.9%) were rFP, 16/47 (34.0%) specimens with both N1 and N2 targets initially detected were rFP, and the CT values for each target was higher in rFP than in rTP. ROC curve analysis showed that optimal cutoff values of CT to differentiate between rTP and rFP were 34.8 for N1 and 33.0 for N2. With the optimal cutoff values of CT for each target, out of the 24 specimens that were positive for both N1 and N2 targets and classified as rTP, 23 (95.8%) were correctly identified as true positives. rFP specimens had a single N1 target in 52/61 (85.2%) and a single N2 target in 17/19 (89.5%). Notably, no true positive results were obtained from any specimens with only N2 target detected. CONCLUSIONS: These results suggest that retesting should be performed for positive results with a CT value greater than optimal cutoff value for each target or with a single N1 target amplified, considering the possibility of a false positive. This may provide guidance on indications to perform retesting to minimize the number of false positives.

Performance of the Assays for Hepatitis B Virus DNA using the µTASWako g1 in Comparison with the COBAS TaqMan Test.

BACKGROUND: The quantification of hepatitis B virus (HBV) DNA is used to monitor antiviral treatment for HBV infection. Recently, an HBV-DNA quantification reagent and assay protocol have been developed for the µTASWako g1 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), a fully automated genetic analyzer that uses PCR-capillary electrophoresis. We evaluated the performance of the newly developed µTASWako HBV-DNA assay using standard and clinical samples. METHODS: The performance of µTASWako HBV-DNA was evaluated using 3rd World Health Organization International Standard for HBV. Thereafter, we evaluated the correlation between the serum HBV DNA concentrations obtained using the µTASWako HBV-DNA and the Roche Cobas AmpliPrep/COBAS TaqMan HBV test, version 2.0 (CAP/CTM HBV test v.2.0) using 190 serum samples from possible HBV carriers. RESULTS: The limit of detection of the µTASWako HBV-DNA was 7.1 IU/mL and that of the CAP/CTM HBV test v.2.0 was 14.6 IU/mL. Seventy-six of the 190 samples yielded values between 1.3 - 7.8 log IU/mL from both assays. The correlation between the results of the assays for these samples was good, with a Deming regression equation of y = 0.929x + 0.041, 95% confidence intervals (CI) for the slope and intercept of 0.892 - 1.12 and -0.474 - 0.110, respectively, and a correlation coefficient r = 0.924. In the low concentration range of 1.3 - 4.0 log IU/mL (n = 64), the Deming regression equation was y = 0.893x + 0.126, and the 95% CIs for the slope and intercept were 0.915 - 1.342 and -0.930 - 0.025, respectively, and r = 0.809. There was also a close correlation for HBV DNA genotype C (n = 41), with a Deming regression equation of y = 0.975x - 0.048, 95% CIs of the slope and intercept of 0.872 - 1.183 and -0.591 - 0.188, respectively, and r = 0.950. The correlations of the four HBV DNA level categories (not detected, < LLOQ (< 1.3 log IU/mL), 1.3 - 3.2 log IU/mL, and ≥ 3.3 log IU/mL) between the two assays for the 190 samples was also compared, and the overall concordance rate was 81.6% (155/190), with a κ statistic of 0.73 (standard error, 0.040; 95% CI, 0.647 - 0.803). CONCLUSIONS: The µTASWako HBV-DNA has a performance comparable with that of the CAP/CTM HBV test, v.2.0. Thus, µTASWako HBV-DNA is useful for the monitoring of HBV infection.

Evaluation of the Analytical Performance of the Fully Automated Gene Analyzer µTASWako g1 for the Detection of SARS-CoV-2.

BACKGROUND: The worldwide spread of coronavirus disease 2019 (COVID-19) has led to an urgent need for nucleic acid amplification test (NAAT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because NAAT has many manual processes, results may vary depending on the operator. Therefore, it has been required to develop a fully automated testing device and reagent that detects genetic material from SARS-CoV-2. The µTASWako g1 system (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), a genetic analyzer, provides results in 75 minutes by performing a fully automated PCR process. METHODS: We evaluated the analytical and clinical performance of the µTASWako g1 system for the detection of SARS-CoV-2 RNA. RESULTS: The µTASWako g1 system had the limit of detection at 2,000 copies/mL using a known concentration of RNA. In clinical samples, the µTASWako g1 system had a sensitivity of 88.0% and 100% specificity compared to conventional RT-PCR. The µTAS Wako g1 system could detect three variants of concern carrying spike mutations including N501Y, E484K, and L452R. CONCLUSIONS: As the assay on the µTASWako g1 system is highly accurate for the detection of SARS-CoV-2 regardless of the experience of operator, it can be widely applicable in clinical laboratories.

First report of New Delhi metallo-ß-lactamase-1-producing Acinetobacter soli in Japan.

New Delhi metallo-ß-lactamase (NDM)-producing gram-negative rods, including Acinetobacter species, are a global problem but have rarely been isolated in Japan. To our knowledge, this is the first study to isolate an NDM-1-producing Acinetobacter soli strain, KUH106, in Japan. We analyzed this strain using next-generation sequencing to examine the plasmid carrying NDM-1. This plasmid, named pKUH106_NDM1, is 41,135 bp in length and contains genetic contexts with the structure ISAba14-aph(3')-VI-ISAba125-blaNDM-1ble-MBL. Comparative analysis of the plasmid revealed that it resembled the plasmids of Acinetobacter detected in various countries, such as the A. soli isolate from Taiwan and the Acinetobacter baumannii isolate from a healthcare facility in Osaka Prefecture, Japan. These results suggest that blaNDM-1 may spread via this plasmid in Acinetobacter species. This phenomenon needs to be confirmed through the genetic analysis of A. baumannii and other carbapenem-resistant Acinetobacter species. In particular, blaNDM-1 and other resistance genes must be investigated, and the spread of these genes in the community must be cautioned.

Study on the Mechanism of False Low Measurement of IgG-Binding (Affinity) IgM Type M Protein by Turbidimetric Immunoassay.

BACKGROUND: The purpose of this study was to investigate the immunological and physical characteristics of IgM-λ type M-protein from patients who were measured low in the turbidimetric immunoassay (TIA) IgM assay without error codes for high concentration to determine the cause of the false low levels and to clarify the mechanism of their occurrence. METHODS: Materials were IgM patient samples and 8 serum samples from other IgM M-protein patients as controls. Patient samples were assayed by the TIA method, in which five manufacturers and six models (two reagent manufacturers) share the principle, and the BN ProSpec method (nephelometric method), which has a different principle. Dilution linearity tests, IgG addition experiments, isoelectric point electrophoresis, and hydrophobic chromatography were performed on patients and subjects. In addition, the binding capacity of γ-globulin by BIACORE was also examined. RESULTS: The reaction curve of the patient IgM curved downward when the concentration of IgM exceeded 20 g/L, and no error code was obtained. In the measurement by the TIA method of five manufacturers and six models, patient IgM was measured at a false low level with no error code obtained in undiluted dilution by any of the instruments and reagents, but could be measured without any problem by the nephelometric method. In addition, in the patient IgG addition experiment, only patient IgM showed a false low level under high IgG concentration. Furthermore, the binding capacity of patient IgM to γ-globulin (IgG) by BIACORE was significantly higher than that of the control IgM-type M protein. CONCLUSIONS: Patient IgM has an affinity (binding capacity) for IgG and forms an IgM-IgG complex under conditions of high IgG concentration. It was speculated that this complex inhibited the reaction with the anti-IgM antibody and the absorbance of the second reaction did not increase, suggesting a false low.

Oltipraz ameliorates the progression of steatohepatitis in Nrf2-null mice fed a high-fat diet.

Oltipraz, a synthetic dithiolethione, has chemopreventive effect through nuclear factor erythroid 2-related factor 2 (Nrf2) activation. Nrf2 is known to be involved in the development of experimental steatohepatitis in rodents. In this study, to evaluate the effect of oltipraz on lipid and bile acid metabolism, wild-type and Nrf2-null mice were fed the standard diet (containing 4% soybean oil) with or without oltipraz. Based on these results, we examined the effect of oltipraz on the experimental steatohepatitis in high-fat diet (containing 4% soybean oil and 20% lard) fed Nrf2-null mice. Oltipraz induced hepatic mRNA expression of peroxisome proliferator-activated receptor α, carnitine palmityl transferase 1, and bile salt export pump by Nrf2 independent mechanisms. In Nrf2-null mice fed a high-fat diet for 12 weeks, moderate to severe inflammation and fibrosis were observed. Oral administration of oltipraz suppressed the degree of inflammation and fibrosis in Nrf2-null mouse liver fed a high-fat diet. These histopathological findings approximately corresponded to the data of mRNA expression of tumor necrosis factor α, monocyte chemoattractant protein-1, Timp-1, and collagen type 1α1. These results indicated that oltipraz administration ameliorated liver injury by Nrf2 independent manner in a model of steatohepatitis generated by Nrf2-null mice with high-fat diet.

Performance evaluation of leukocyte differential on the hematology analyzer Celltac G compared with two hematology analyzers, reference flow cytometry method, and two manual methods.

BACKGROUND: The automated hematology analyzer Celltac G (Nihon Kohden) was designed to improve leukocyte differential performance. Comparison with analyzers using different leukocyte detection principles and differential leukocyte count on wedge film (Wedge-Diff) shows its clinical utility, and comparison with immunophenotypic leukocyte differential reference method (FCM-Ref) shows its accuracy performance. METHODS: For method comparison, 598 clinical samples and 46 healthy volunteer samples were selected. The two comparative hematology analyzers (CAAs) used were XN-9000 (Sysmex) and CELL-DYN Sapphire (Abbott). The FCM-Ref provided by the Japanese Society for Laboratory Hematology was selected, and a flow cytometer Navios (Beckman-Coulter) was used. In manual differential, two kinds of automated slide makers were used: SP-10 (Sysmex) for wedge technique and SPINNER-2000 (Lion-Power) for spinner technique. The spinner technique avoids the issue of Wedge-Diff smudge cells by removing the risk of breaking cells and non-uniformity of blood cell distribution on films (Spinner-Diff). RESULTS: The Celltac G showed sufficient comparability (r = 0.67-1.00) with the CAAs for each leukocyte differential counting value at 0.00-40.87(109 /L), and sufficient comparability (r = 0.73-0.97) with FCM-Ref for each leukocyte differential percentage at 0.4-78.5. The identification ratio of the FCM-Ref in CD45-positive cells was 99.7% (99.4% to 99.8%). Differences were found between FCM-Ref/Celltac G/XN-9000/Spinner-Diff and Wedge-Diff for monocytes and neutrophils. The appearance ratio of smudge cells on wedge and spinner film was 12.5% and 0.5%. CONCLUSION: The Celltac G hematology analyzer's leukocyte differential showed adequate accuracy compared with the CAAs, FCM-Ref, and two manual methods and was considered suitable for clinical use.

Ezetimibe Markedly Reduces Hepatic Triglycerides and Cholesterol in Rats Fed on Fish Oil by Increasing the Expression of Cholesterol Efflux Transporters.

Besides diet therapy, hypolipidemic pharmacological therapy may be a crucial component of nonalcoholic fatty liver disease (NAFLD) treatment. Ezetimibe may be a promising drug for treatment of NAFLD. n-3 polyunsaturated fatty acids, which are abundant in fish oil, reduce serum and hepatic cholesterol and triglycerides in rodents. The aim of this study was to examine the combined effects of dietary fish oil and ezetimibe on lipid metabolism in rats. Seven-week-old male Sprague-Dawley rats were allocated to four different diets containing 1) 10% soybean oil (C), 2) 10% fish oil (F), 3) 10% soybean oil + 0.005% ezetimibe, and 4) 10% fish oil + 0.005% ezetimibe (F+E) for 4 weeks, when the liver, jejunum, blood, and fecal samples were collected. Compared with the C group, the F+E diet decreased hepatic triglycerides and cholesterol 84% and 86%, but it did not increase fecal cholesterol. In liver, the expression of lipogenic enzymes was decreased in the F+E diet, whereas ß-oxidation-related genes were not increased. Abcg5/g8 mRNA expression was increased 1380%/442% when ezetimibe was added to the F diet. These gene expression changes are related to the decrease in hepatic lipids. In jejunum, Abcg5/g8 mRNA was increased 244%/841% when ezetimibe was added to the F diet. Hepatic induction of Abcg5/8 rather than intestinal induction correlates with the marked decrease in liver cholesterol when ezetimibe was added to the F diet. These data suggest that fish oil diet and ezetimibe in combination may be a beneficial therapy for NAFLD by increasing hepatic Abcg5/g8 and decreasing lipogenic genes. SIGNIFICANCE STATEMENT: There is currently no single treatment for NAFLD. Thus, lifestyle modifications including dietary regulation and physical activity are also important options. In this study, ezetimibe, a cholesterol absorption inhibitor, was evaluated for the treatment of liver steatosis in rats fed on the different diets. We found that ezetimibe and fish oil in combination markedly improved fatty liver by increasing cholesterol efflux transporters. The combination therapy of fish oil agents and ezetimibe may be effective for NAFLD.

Glycosylated Bence Jones Protein with Poor Thermal Reactivity in Heat Coagulation Tests.

BACKGROUND: We experienced a patient with multiple myeloma whose urine contained a considerable amount of Bence Jones protein (BJP), which demonstrated poor thermal reactivity in heat coagulation test. The mechanism for this phenomenon was assessed. METHODS: Immunoelectrophoretic analyses reveal that a band corresponding to BJP in the urine had 2,600 Dalton by reduction after glycosidase treatment, but not after sialidase treatment. In addition, the glycosidase-treated urine tested positive in heat coagulation test. CONCLUSIONS: Glycosylation of the immunoglobulin light chain, which has rarely been seen, is the cause of the unexpected behavior of this patent's BJP in heat coagulation tests.

Epidemiological and molecular characterization of invasive Streptococcus pneumoniae isolated following introduction of 7-valent conjugate vaccine in Kinki region, Japan, 2008-2013.

Streptococcus pneumoniae is one of the most common bacteria causing community-acquired pneumonia and meningitis. The use of 7-valent pneumococcal conjugate vaccine (PCV7) has reduced the incidence of pneumococcal disease while changing pneumococcal population through herd immunity and non-vaccine pneumococci replacement. This study investigated molecular epidemiologic characteristics of pneumococcal strains in the Kinki region of Japan from 2008 to 2013. A total of 159 invasive pneumococcal isolates were characterized by serotyping, antibiotic susceptibility testing, PCR analysis of penicillin-binding protein genes, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). In adult populations, pediatric PCV7 introduction decreased isolates expressing PCV7 serotypes via herd immunity and increased isolates expressing non-PCV7 serotypes. The rate of penicillin resistance and isolates with alterations in all three pbp genes was higher in PCV7 type isolates than in non-PCV7 type isolates. In MLST analysis, all of serotype 19F isolates were of the same sequence type, ST236, which is the antimicrobial-resistant clone Taiwan19F-14, and the majority of serotypes 23F and 19A isolates were of ST1437 and ST3111 respectively, which are the predominant clones of antimicrobial-resistant pneumococci in Japan. In PFGE profiles, serotype 6B-ST2224, serotype 19F-ST236, serotype 19A-ST3111, and serotype 23F-ST1437 formed six separate clusters composed of genetically identical strains, and genetically identical serotype 22F-ST433 formed two different clusters between the pre- and post-PCV7 period. The results of molecular analysis suggest the spread and persistence of these identical antimicrobial resistant clones in the Kinki region and genetic changes of epidemic clone serotype 22F-ST433 before and after pediatric PCV7 introduction.

Laboratory surveillance of antimicrobial resistance and multidrug resistance among Streptococcus pneumoniae isolated in the Kinki region of Japan, 2001-2015.

The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced among children in Japan in 2010. There are no long-term multicenter surveillance studies of antimicrobial resistance in S. pneumoniae before and after the introduction of PCV7. Therefore, we examined chronological trends in antimicrobial resistance among 4534 strains of S. pneumoniae isolated from both children and adults in the Kinki region of Japan during 2001-2015. High-level penicillin and third-generation cephalosporin resistance in S. pneumoniae increased among both children and adults during the period before the introduction of PCV7 (2001-2010). Besides penicillin and cephalosporin, pneumococcal carbapenem and macrolide resistance increased among children. The rate of resistance to these antibiotics was higher among children than among adults. The introduction of PCV7 decreased the rate of non-susceptibility to ß-lactam antibiotics and the rate of multidrug resistant S. pneumoniae among children, but not among adults.

A Case of Waldenstrom Macroglobulinemia with Temporary Appearance of 7S IgM Half Molecule.

BACKGROUND: We encountered a rare case of Waldenstrom macroglobulinemia with temporary appearance of 7S IgM half molecule and with monoclonal proteins binding to agarose gel. METHODS: The patient's serum and urine were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The N-terminal amino acid sequences of the IgM with abnormal mass (68 kDa) were determined and compared with those of known immunoglobulin. RESULTS: The 68 kDa IgM consisted of a defective µ chain (36 kDa) and an intact κ chain. N-terminal amino acid sequence analysis demonstrated that the defective µ chain had the variable region of IgM. The agarose gel-binding ability of the IgM-κ M-protein was lost after reduction or alkaline treatment of serum. CONCLUSIONS: The 7S half molecule IgM in the present case may miss a large part of the constant region of the µ chain.

Role of Nrf2 in the alteration of cholesterol and bile acid metabolism-related gene expression by dietary cholesterol in high fat-fed mice.

Nuclear factor-E2-related factor 2 (Nrf2) is a regulator of lipid metabolism as well as various cytoprotective enzymes and may be involved in the pathogenesis of non-alcoholic fatty liver disease. Although, bile acids affect lipid metabolism, the role of Nrf2 in bile acid metabolism remains unclear. In this study, it was tested how Nrf2 modulates lipid and bile acid homeostasis in liver in response to changes of cholesterol absorption under high-fat diet using Nrf2-null mice. Eight-week-old male wild-type and Nrf2-null mice (n = 6/group) were divided into three groups fed the following diets: 1) control diet containing 4% soybean oil and 16% lard, 2) control diet plus ezetimibe, 3) control diet plus cholesterol. Blood and livers were removed after 4 weeks feeding. High cholesterol diet increased hepatic expression of liver X receptor α target genes related to fatty acid metabolism (FAS, ACC1, SREBP-1c, SCD-1c and CD36), cholesterol transport (Abcg5/abcg8) and bile acid synthesis (Cyp7a1) in wild type mice. However, these genes were not induced in Nrf2-null mice. These findings suggest that Nrf2 has a relation to liver X receptor α and controls the regulation of gene expressions related to lipid and bile acid metabolism.

[Relationship between serum hCG and urinary hCG in cases of pregnancy, abortion, and hydatidiform mole].

Human chorionic gonadotropin (hCG) is generally quantified in serum, but spot urine samples are also used to assess hCG levels in Japan. The purpose of the present study was to elucidate whether urinary hCG can be used clinically as a substitute for serum hCG. A total of 189 samples of serum and spot urine were collected from patients, including cases of normal pregnancy (NP) -13, abortion (AB) -21, extrauterine pregnancy (EP) 25, and hydatidiform mole (MOL) -7, during medical treatment and comparisons were made concerning serum and urinary hCG levels. The histogram of relative urinary/serum hCG(U-hCG.act/S-hCG) of the samples showed a wide distribution of values, but tended to converge to a narrow distribution by creatinine correction (U-hCG.cor/S-hCG). U-hCG.cor/S-hCG of the AB, EP, and MOL groups decreased 1 day to 14 days or was no earlier than 15 days postoperatively compared to preoperatively. The alteration of serum Intact/Total tended to be similar to that of U-hCG.cor/S-hCG in clinical course. The presented case indicated that U-hCG.act/S-hCG did not correspond to serum hCG levels. Because urinary hCG levels are inconsistent depending on whether spot urine is concentrated or diluted, the levels of hCG in spot urine do not always correlate with serum levels of hCG. Therefore, the data of urinary hCG should be interpreted after creatinine correction. Overall, it is recommended to determine serum hCG levels rather than creatinine corrected urinary hCG levels, considering that the relative urinary/serum hCG was not constant postoperatively.

Correction: Furugaito et al. Antimicrobial Susceptibility to 27 Drugs and the Molecular Mechanisms of Macrolide, Tetracycline, and Quinolone Resistance in Gemella sp. Antibiotics 2023, 12, 1538.

The authors wish to make the following corrections to this paper [...].

Ezetimibe increases hepatic iron levels in mice fed a high-fat diet.

Accumulating evidence suggests that ezetimibe may be a promising agent for treatment of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). Phlebotomy and dietary iron restriction reduce serum transaminase in NAFLD/NASH patients. Recent studies have shown that a mutual effect exists between lipid metabolism and iron metabolism. Accordingly, we examined the effect of ezetimibe on iron metabolism in mice fed a high-fat diet with or without iron. We fed C57BL/6 mice the following diets for 12 weeks. Experiment 1 comprised [1] a control diet (C), [2] C plus ezetimibe (0.3 mg/day; 4 weeks) (CE), [3] a high-fat diet (H), and [4] H plus ezetimibe (HE). Experiment 2 comprised [1] C containing carbonyl iron (average; 22.4 mg/day; 6 weeks) (CI), [2] CI plus ezetimibe (CIE), [3] H containing carbonyl iron (HI), and [4] HI plus ezetimibe (HIE). Blood, livers, and duodenum were removed after 12 weeks. In experiment 1, the hepatic iron levels were higher in HE than H, whereas there was no difference between C and CE. Hepatic mRNA expression of transferrin receptor 1 and 2, ferritins, and hepcidin were increased more in CE than C, and more in HE than H. In the duodenum, divalent metal transporter 1, ferritin H, and hephaestin mRNA levels were increased in CE compared with C. In experiment 2, hepatic iron concentrations were higher in HIE than HI. Hepatic mRNA expression of ferritin L and hepcidin were increased in HIE compared with HI. In duodenum, ferritin L mRNA was increased in HIE compared with CIE. Ezetimibe induced hepatic iron uptake transporter expression in mice fed a high-fat diet, causing increased hepatic iron concentrations.

Relationship between serum bilirubin and remnant lipoprotein cholesterol level.

BACKGROUND: Bilirubin has antioxidant properties and is known to have a role in the prevention of cardiovascular disease. Bilirubin is known to be negatively associated with serum parameters related to atherosclerosis. Triglyceride-rich lipoprotein is known to play an important role in the progression of atherosclerosis. The aim of the present study was to elucidate the relationship between serum bilirubin and serum remnant lipoprotein cholesterol (RLP-c). METHODS: We examined 270 Japanese males (mean age 64.8 years) without liver dysfunction and anemia from the Outpatient Clinic of Kinki University Hospital. Serum bilirubin, cholesterol, LDL cholesterol, HDL cholesterol, triglyceride and RLP-c levels were measured. RESULTS: Serum RLP-c level was significantly lower in patients in the high bilirubin group than those in the low bilirubin group. CONCLUSIONS: The findings of this study show that serum bilirubin within physiological levels may affect the serum RLP-c and suggest bilirubin may have a role in preventing atherosclerosis.

Antimicrobial Susceptibility to 27 Drugs and the Molecular Mechanisms of Macrolide, Tetracycline, and Quinolone Resistance in Gemella sp.

Gemella is a catalase-negative, facultative anaerobic, Gram-positive coccus that is commensal in humans but can become opportunistic and cause severe infectious diseases, such as infective endocarditis. Few studies have tested the antimicrobial susceptibility of Gemella. We tested its antimicrobial susceptibility to 27 drugs and defined the resistant genes using PCR in 58 Gemella strains, including 52 clinical isolates and six type strains. The type strains and clinical isolates included 22 G. morbillorum, 18 G. haemolysans (GH) group (genetically indistinguishable from G. haemolysans and G. parahaemolysans), 13 G. taiwanensis, three G. sanguinis, and two G. bergeri. No strain was resistant to beta-lactams and vancomycin. In total, 6/22 (27.3%) G. morbillorum strains were erythromycin- and clindamycin-resistant ermB-positive, whereas 4/18 (22.2%) in the GH group, 7/13 (53.8%) G. taiwanensis, and 1/3 (33.3%) of the G. sanguinis strains were erythromycin-non-susceptible mefE- or mefA-positive and clindamycin-susceptible. The MIC90 of minocycline and the ratios of tetM-positive strains varied across the different species-G. morbillorum: 2 µg/mL and 27.3% (6/22); GH group: 8 µg/mL and 27.8% (5/18); G. taiwanensis: 8 µg/mL and 46.2% (6/13), respectively. Levofloxacin resistance was significantly higher in G. taiwanensis (9/13 69.2%) than in G. morbillorum (2/22 9.1%). Levofloxacin resistance was associated with a substitution at serine 83 for leucine, phenylalanine, or tyrosine in GyrA. The mechanisms of resistance to erythromycin and clindamycin differed across Gemella species. In addition, the rate of susceptibility to levofloxacin differed across Gemella sp., and the quinolone resistance mechanism was caused by mutations in GyrA alone.

Dysregulated expression of fatty acid oxidation enzymes and iron-regulatory genes in livers of Nrf2-null mice.

BACKGROUND AND AIM: Hepatic excessive iron may play a role in the pathogenesis of non-alcoholic steatohepatitis (NASH). Nrf2 is a master regulator of antioxidative responses. However, the role of Nrf2 in lipid and iron homeostasis remains unclear. Accordingly, it was examined how Nrf2 regulates lipid-related and iron-regulatory genes after feeding a high-fat diet (HFD) with iron. METHODS: Wild-type and Nrf2-null mice were fed the following diets: (i) control diet (4% soybean oil) for 12 weeks, (ii) control diet for 8 weeks followed by control diet containing 0.5% carbonyl iron for 4 weeks, (iii) HFD (4% soybean oil and 16% lard) for 12 weeks, (iv) HFD for 8 weeks followed by HFD containing 0.5% carbonyl iron for 4 weeks. Blood and livers were removed after 12 weeks. RESULTS: Nrf2-null control mice exhibited a tendency towards higher hepatic triglycerides compared to wild-type control mice. Hepatic malondialdehyde was higher and hepatic iron levels tended to be higher in Nrf2-null mice than wild-type counterparts while on a HFD. The HFD with iron synergistically induced mRNA expression of Pparα targets, including Acox and Cpt1 in wild-type mice, yet the induction was diminished in Nrf2-null mice. Hepatic hepcidin and ferroportin 1 mRNA expression were increased in wild-type mice after feeding a HFD with iron, but were unchanged in any group of Nrf2-null mice. CONCLUSIONS: Nrf2 deletion dysregulates hepatic mRNA expression of ß-oxidation enzymes and iron-related genes, possibly causing a trend for increased hepatic triglyceride and iron concentrations. Nrf2 may have roles in the progression of NASH.

[Clinical microbiological investigation of vancomycin intermediate Staphylococcus aureus during glycopeptide therapy].

We isolated three strains of vancomycin intermediate Staphylococcus aureus (VISA) from a blood sample of a patient with infective endocarditis (VISA-1), postoperative pneumonia sputum (VISA-2), and pyogenic spondylitis blood sample (VISA-3). These VISA strains did not carry vanA, vanB, vanC1, or vanC2/C3 genes. Cell wall thickening was observed. VISA-1 and VISA-3 PFGE patterns showed the completely same pattern compared to the PFGE pattern of methicillin-resistant Staphylococcus aureus first isolated from patients 1 and 3. After 10 days on brain heart infusion agar, wall thickening in all three type of VISA was unchanged, but VISA-2 and VISA-3 reversed vancomycin susceptibility. The most suitable use of vancomycin in patients with MRSA infection thus appears to be in reducing the opportunity for cell wall thickening.