Detection of extended-spectrum ß-lactamases producing Enterobacteriaceae using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based MBT STAR-BL software module with ß-lactamase inhibition assay depends on the bacterial strains.

Rapid and sensitive detection of extended-spectrum ß-lactamases (ESBLs) is essential for infection control and antimicrobial treatment. Recently, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based MBT STAR-BL software module has been used for detecting ß-lactamase activity; however, this system cannot differentiate ESBL producing bacteria from other third-generation cephalosporin-resistant strains. In this study, we utilized a MALDI-TOF MS-based MBT STAR-BL method to identify ESBL activity with ß-lactamase inhibitors. A cefotaxime (CTX) hydrolysis assay, ß-lactamase inhibition, clavulanic acid (CVA), and sulbactam (SBT) were used for detecting ESBL producers with the MBT STAR-BL software module. This software module automatically calculated the logRQ values in each assay. logRQ is the logarithm of the ratio of the summed hydrolyzed peak intensities to the summed non-hydrolyzed peak intensities and measured the efficiency of antibiotic hydrolysis. We divided the logRQ level of the ß-lactamase inhibition assay by the logRQ value in the CTX hydrolysis assay, and we used this logRQ ratio as a measure of ß-lactamase inhibition efficiency. We assessed the logRQ ratio calculated by this novel method for detecting ESBL producers in 132 Enterobacteriaceae. We performed the MALDI-TOF MS-based MBT STAR-BL approach with ß-lactamase inhibitors for detecting ESBL producers and showed that the results of the inhibition assay with ß-lactamase inhibitors depended on types of bacterial species. Furthermore, we improved elapsed times and accuracy in MBT STAR-BL methods by using proper ß-lactamase inhibitors against specific bacterial strains to compare with the conventional standard lab method. The results suggest that the target bacterial species and ß-lactamase inhibitors used were important for the utility of the MALDI-TOF MS-based MBT STAR-BL software module.

A rapid and simple detection method for phenotypic antimicrobial resistance in Escherichia coli by loop-mediated isothermal amplification.

PURPOSE: In infectious disease therapy, administration of adequate antimicrobial agents is essential for preventing the emergence and spread of resistant bacteria. However, conventional antimicrobial susceptibility testing (AST), based on bacterial growth, is time consuming; therefore, a rapid, simple assay is needed for the timely selection of appropriate antibiotics in clinical laboratories. Here, we established a simple, cost-effective, time-saving and highly sensitive AST assay based on loop-mediated isothermal amplification (LAMP). METHODOLOGY: The targeted bacteria were cultivated for a short period with or without antibiotic before the LAMP reaction. The time to detect a positive reaction with LAMP was used to generate a threshold time (Tt) value, and subtraction of the Tt value for an antibiotic-free sample from the Tt value in an antibiotic-exposed sample generated the ΔTt value, which was used as a marker of antimicrobial susceptibility. The ΔTt value generated using the LAMP-based assay simply and quickly detected antimicrobial resistance in clinical Escherichia coli isolates. RESULTS: Detection of susceptibility to levofloxacin using the ΔTt value perfectly matched with the results of the conventional assay. In addition, the sensitivity and specificity for the detection of ampicillin, trimethoprim-sulfamethoxazole and fosfomycin resistance were 100 %, 93.8 %, 100 % and 80.0 %, 93.3 %, 97.6 %, respectively. CONCLUSION: These results showed that this LAMP-based AST has high sensitivity and specificity for detecting resistant strains and a significant time advantage compared with the conventional method.

IL-13 regulates IL-17C expression by suppressing NF-κB-mediated transcriptional activation in airway epithelial cells.

The cytokine interleukin (IL)-17C is highly expressed in epithelial tissues and involved in innate immune responses; however, the regulation of IL-17C expression in the airways remains poorly understood. Here, we show that IL-1ß strongly induces both IL-17C mRNA and protein expression in primary normal human bronchial epithelial cells. Conversely, IL-13 significantly reduced the IL-1ß-induced IL-17C expression. Attenuation of the nuclear factor (NF)-κB-signaling pathway using an NF-κB-subunit p65-specific small-interfering RNA (siRNA), reduced IL-1ß-induced IL-17C expression, demonstrating the importance of NF-κB signaling in IL-17C regulation. The inhibitory effects of IL-13 on IL-17C expression were abolished when the Janus kinase (JAK)/signal transducer and activator of transcription 6 (STAT6)-signaling pathway was impaired, using either the JAK inhibitor ruxolitinib or a STAT6-specific siRNA. Western blot analysis demonstrated that IL-1ß promoted both IκB-α phosphorylation and degradation, and p65 nuclear translocation. Although IL-13 induced STAT6 phosphorylation and nuclear translocation, it did not affect the activation of the IL-1ß-mediated NF-κB-pathway. Using chromatin immunoprecipitation, we confirmed that IL-1ß enhanced p65 binding to regions within the IL-17C promoter that flank putative NF-κB-binding sites (-130/-120 and -157/-147). Interestingly, IL-13 treatment reduced the IL-1ß-mediated p65 binding to these regions. These findings demonstrate that NF-κB-mediated transcriptional mechanisms are critically involved in the IL-1ß-mediated IL-17C induction, and that IL-13 negatively regulates this induction by suppressing NF-κB-based transcriptional activation.

Nonspecific interstitial pneumonia preceding diagnosis of collagen vascular disease.

BACKGROUND: The aim of this study was to evaluate the incidence and clinical features of patients who developed collagen vascular disease (CVD) after an initial diagnosis of idiopathic nonspecific interstitial pneumonia (NSIP). METHODS: We conducted a retrospective review of 72 consecutive patients with NSIP who were diagnosed by surgical lung biopsy in our institution (idiopathic NSIP, n = 35; CVD-NSIP, n = 37 at initial diagnosis). No patients fulfilled the American College of Rheumatology criteria for a diagnosis with CVD within six months after the diagnosis of idiopathic NSIP. RESULTS: Of 35 patients initially diagnosed with idiopathic NSIP, six patients (17.1%) developed CVD during the follow-up period (5.5 ± 5.0 years); three patients were diagnosed with dermatomyositis (DM), two patients with overlap syndrome (DM and Sjogren's syndrome), and one patient with rheumatoid arthritis. The mean time until CVD diagnosis was 2.0 years (six months - 3.5 years), and the one-, two- and three-year incidences of CVD development were 3.6%, 15.2% and 20.0%, respectively. There was no significant difference in clinical characteristics and survival among patients with NSIP preceding CVD diagnosis, those with idiopathic NSIP, or those with CVD-NSIP. In addition, at the time of initial diagnosis, there was no significant difference for the fulfillment of previous criteria such as interstitial pneumonia with autoimmune feature (IPAF) between patients with NSIP preceding CVD diagnosis and those with idiopathic NSIP. CONCLUSIONS: It is difficult to predict CVD occurrence and careful attention is needed to detect the development of CVD in patients with idiopathic NSIP.

Evaluation of serum bone alkaline phosphatase activity in patients with liver disease: Comparison between electrophoresis and chemiluminescent enzyme immunoassay.

BACKGROUND: Serum bone alkaline phosphatase (ALP) is a marker of bone formation and metabolism. However, existing methods for measuring it have their limitations and their accuracy has not been determined. METHODS: We measured serum bone ALP activity in 127 patients with liver disease using 2 methods: electrophoresis and chemiluminescent enzyme immunoassay (CLEIA). The results of these 2 methods were compared and analyzed according to gender, age and several serum biochemical markers. RESULTS: When ALP3 (%; bone-type isozyme activity as a percentage of total ALP activity) values were high, the 2 methods showed good correlation. However, with a decrease in ALP3 (%) levels, the correlation coefficient (R) also decreased. Starting with ALP3 (%)<23, R values markedly decreased to <0.50 (p>0.05). Five outliers displayed low ALP3 (%) activity levels. Furthermore, in regard to genders, there were significant differences in total cholesterol (TC), γ-glutamyltransferase (γ-GTP), ALP and ALP3 (%) levels (p<0.05). CONCLUSIONS: When serum ALP3 (%) levels were high in patients with liver disease, the accuracy of electrophoresis was comparable to that of CLEIA. However, the accuracy of electrophoresis needs to be evaluated with further when patient samples under certain conditions.

Increased levels of serum Wisteria floribunda agglutinin-positive Mac-2 binding protein in idiopathic pulmonary fibrosis.

BACKGROUND: Mac-2 binding protein (M2BP) is a cell-adhesive glycoprotein of the extracellular matrix secreted as a ligand of galectin-3 (Mac-2). Recently, a Wisteria floribunda agglutinin positive-M2BP (WFA(+)-M2BP) assay developed using a lectin-antibody sandwich immunoassay has shown promise as a new fibrotic marker in liver fibrosis to detect unique fibrosis-related glycoalteration. The aim of this study is to evaluate the utility of serum WFA(+)-M2BP levels in patients with idiopathic pulmonary fibrosis (IPF). METHODS: We measured serum WFA(+)-M2BP levels in 116 patients with IPF and 42 healthy volunteers. We examined the relationship between serum WFA(+)-M2BP levels and clinical parameters and further investigated the prognostic significance of serum WFA(+)-M2BP levels in patients with IPF. RESULTS: Serum WFA(+)-M2BP levels were significantly higher in patients with IPF than in healthy controls (1.09 ± 0.89 cutoff index [COI], 0.57 ± 0.24 COI, respectively; P < 0.001). In patients with IPF, a significant positive correlation was found between serum WFA(+)-M2BP levels and age, KL-6, neutrophils in BAL, reticulation and honeycombing scores in HRCT, and fibrotic foci scores in pathological findings, and a significant negative correlation was found between serum WFA(+)-M2BP levels and FVC, %DLco and macrophages in BAL. Furthermore, patients with high serum WFA(+)-M2BP levels had a significantly worse prognosis than those with low levels (log-rank test, P = 0.0209), and a high serum WFA(+)-M2BP level was a significant prognostic factor in Cox proportional hazards regression analysis. CONCLUSIONS: Our results suggest that the serum WFA(+)-M2BP level is a potential biomarker in patients with IPF.

A case of treatment with voriconazole for chronic progressive pulmonary aspergillosis in a patient receiving tacrolimus for dermatomyositis-associated interstitial lung disease.

We report a successful treatment with voriconazole (VRCZ) for chronic progressive pulmonary aspergillosis (CPPA) in a patient with dermatomyositis-associated interstitial lung disease (DM-ILD) treated with tacrolimus. A 73-year-old man with DM-ILD, treated with tacrolimus and prednisolone, complained of productive cough and his chest X-ray showed infiltration in the left upper lung field. We diagnosed CPPA and added VRCZ. Although we reduced the dose of tacrolimus for drug interaction, serum VRCZ level increased after the treatment. The patient was found to have cytochrome P450 (CYP) 2C19 *2/*2, a genetic polymorphism in poor metabolizers of VRCZ. We adjusted the doses of both drugs and treated him successfully. We recommend performing individual therapeutic drug monitoring (TDM) in CYP-mediated drug interactions and considering the effect of CYP polymorphisms.

Plasma connective tissue growth factor levels as potential biomarkers of airway obstruction in patients with asthma.

BACKGROUND: Bronchial asthma is a chronic inflammatory disorder characterized by airway hyperresponsiveness and airflow limitation. Connective tissue growth factor (CTGF), one of the key profibrotic factors associated with transforming growth factor ß, may be related to airway remodeling in asthma. However, no data are available on the association between plasma CTGF levels and clinical and physiologic parameters in patients with asthma. Recently, we developed a novel subtraction method for determination of plasma CTGF levels. OBJECTIVE: To investigate the utility of plasma CTGF level as a surrogate biomarker in asthma. METHODS: Plasma CTGF levels were measured in 67 patients with stable asthma and 81 healthy volunteers, using the subtraction method. We evaluated correlations between plasma CTGF levels and clinical and physiologic parameters in patients with asthma. RESULTS: Plasma CTGF levels were higher in patients with asthma than in healthy volunteers. Asthmatic patients with a percentage of predicted forced expiratory volume in 1 second (FEV1) less than 80% had significantly higher levels of plasma CTGF than those with a percentage of predicted FEV1 of 80% or more. In patients with asthma, plasma CTGF levels had significantly negative correlations with forced vital capacity (FVC), FEV1, percentage of predicted FEV1, FEV1/FVC ratio, forced expiratory flow at 50% of the FVC (FEF50%), percentage of predicted FEF50%, forced expiratory flow at 75% of the FVC (FEF75%), and percentage of predicted FEF75%, parameters that reflect the degree of airway obstruction. Plasma CTGF levels were negatively correlated with Asthma Control Test scores, a patient-based index of clinical control of asthma. CONCLUSION: Plasma CTGF may be a potential biomarker for stable asthma when evaluating the degree of persistent airway obstruction. TRIAL REGISTRATION: umin.ac.jp/ctr Identifier: UMIN000013081.

Investigation of unexpected serum CA19-9 elevation in Lewis-negative cancer patients.

BACKGROUND: Cancer patients with a Lewis (a-b-) phenotype have no carbohydrate antigen 19-9 (CA19-9) in their serum. However, we found a small but distinct elevation in the serum CA19-9 level in three cancer patients with the Lewis-negative phenotype. Here, we investigated the reason of such phenomena. METHODS: Six cancer patients with a Lewis-negative phenotype were selected by very low CA19-9 concentrations: three showed a small elevation (Group A) and the other three showed no elevation (Group B) in the serum CA19-9. We investigated the difference by analyzing the Lewis/Secretor genotypes. RESULTS: All of the six patients with a Le (a-b-) phenotype were genuine Le-negative genotypes: four individuals were homozygous for le1 (le(59,508)), one patient was compound heterozygous for le1 (le(59,508)) and le2 (le(59,1067)) and one patient was compound heterozygous for le1 and le(202,314). As for the Secretor gene, the three patients in Group B were homozygous for Se2 (one patient) or compound heterozygous for Se2 and sej (two patients), while the patients in Group A were all homozygous for sej genotypes. CONCLUSIONS: Even genuinely Le-negative patients, who genetically lack the Le enzyme and theoretically never produce CA19-9, occasionally show a slight increase in serum CA19-9 level when they are homozygous for Se-negative genotypes and suffer from advanced cancer with overproduction of glycans as precursors of CA19-9. Although such cases are not frequent, we should be acquainted with the correlation between serum CA19-9 values and genotypes of Lewis and Secretor genes.

Plasma CCN2 (connective tissue growth factor; CTGF) is a potential biomarker in idiopathic pulmonary fibrosis (IPF).

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal pulmonary fibrotic disease and useful biomarkers are required to diagnose and predict disease activity. CCN2 (connective tissue growth factor; CTGF) has been reported as one of the key profibrotic factors associated with transforming growth factor-ß (TGF-ß), and its assay has potential as a non-invasive measure in various fibrotic diseases. Recently, we developed a new subtraction method for determination of plasma CCN2 levels. We examined the utility of plasma CCN2 levels as a surrogate marker in IPF. METHODS: Plasma CCN2 levels were calculated in 33 patients with IPF, 14 patients with non-IPF idiopathic interstitial pneumonias (IIPs) and 101 healthy volunteers by sandwich enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies for two distinct epitopes of human CCN2. We evaluated the utility of plasma CCN2 levels by comparison with clinical parameters. RESULTS: Plasma CCN2 levels were significantly higher in patients with IPF than in those with non-IPF IIPs and healthy volunteers. Importantly, plasma CCN2 levels showed significantly negative correlation with 6-month change of forced vital capacity (FVC) in patients with IPF. CONCLUSIONS: Plasma CCN2 is a potential biomarker for IPF.