Escherichia coli SPFH Membrane Microdomain Proteins HflKC Contribute to Aminoglycoside and Oxidative Stress Tolerance.

Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs), also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the stomatin, prohibitin, flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. In contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K-12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner membrane FMMs and the regulation of cellular processes. Here, we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCE Eukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane microdomains. These domains are also called lipid rafts and contain proteins of the stomatin, prohibitin, flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but have been mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacterium E. coli is altered in the absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli.

Intermittent antibiotic treatment of bacterial biofilms favors the rapid evolution of resistance.

Bacterial antibiotic resistance is a global health concern of increasing importance and intensive study. Although biofilms are a common source of infections in clinical settings, little is known about the development of antibiotic resistance within biofilms. Here, we use experimental evolution to compare selection of resistance mutations in planktonic and biofilm Escherichia coli populations exposed to clinically relevant cycles of lethal treatment with the aminoglycoside amikacin. Consistently, mutations in sbmA, encoding an inner membrane peptide transporter, and fusA, encoding the essential elongation factor G, are rapidly selected in biofilms, but not in planktonic cells. This is due to a combination of enhanced mutation rate, increased adhesion capacity and protective biofilm-associated tolerance. These results show that the biofilm environment favors rapid evolution of resistance and provide new insights into the dynamic evolution of antibiotic resistance in biofilms.

Small-Molecule-Induced Activation of Cellular Respiration Inhibits Biofilm Formation and Triggers Metabolic Remodeling in Staphylococcus aureus.

Staphylococcus aureus, a major pathogen of community-acquired and nosocomial-associated infections, forms biofilms consisting of extracellular matrix-embedded cell aggregates. S. aureus biofilm formation on implanted medical devices can cause local and systemic infections due to the dispersion of cells from the biofilms. Usually, conventional antibiotic treatments are not effective against biofilm-related infections, and there is no effective treatment other than removing the contaminated devices. Therefore, the development of new therapeutic agents to combat biofilm-related infections is urgently needed. We conducted high-throughput screening of S. aureus biofilm inhibitors and obtained a small compound, JBD1. JBD1 strongly inhibits biofilm formation of S. aureus, including methicillin-resistant strains. In addition, JBD1 activated the respiratory activity of S. aureus cells and increased the sensitivity to aminoglycosides. Furthermore, it was shown that the metabolic profile of S. aureus was significantly altered in the presence of JBD1 and that metabolic remodeling was induced. Surprisingly, these JBD1-induced phenotypes were blocked by adding an excess amount of the electron carrier menaquinone to suppress respiratory activation. These results indicate that JBD1 induces biofilm inhibition and metabolic remodeling through respiratory activation. This study demonstrates that compounds that enhance the respiratory activity of S. aureus may be potential leads in the development of therapeutic agents for chronic S. aureus-biofilm-related infections. IMPORTANCE Chronic infections caused by Staphylococcus aureus are characterized by biofilm formation, suggesting that methods to control biofilm formation may be of therapeutic value. The small compound JBD1 showed biofilm inhibitory activity and increased sensitivity to aminoglycosides and respiratory activity of S. aureus. Additionally, transcriptomic and metabolomic analyses demonstrated that JBD1 induced metabolic remodeling. All JBD1-induced phenotypes were suppressed by the extracellular addition of an excess amount of menaquinone, indicating that JBD1-mediated respiratory stimulation inhibits biofilm formation and triggers metabolic remodeling in S. aureus. These findings suggest a strategy for developing new therapeutic agents for chronic S. aureus infections.

Roles of lytic transglycosylases in biofilm formation and ß-lactam resistance in methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is responsible for numerous community outbreaks and is one of the most frequent causes of nosocomial infections with significant morbidity and mortality. While the function of lytic transglycosylases (LTs) in relation to cell division, biofilm formation, and antibiotic resistance has been determined for several bacteria, their role in S. aureus remains largely unknown. The only known LTs in S. aureus are immunodominant staphylococcal antigen A (IsaA) and Staphylococcus epidermidis D protein (SceD). Our study demonstrates that, in a strain of methicillin-resistant S. aureus (MRSA), IsaA and SceD contribute differently to biofilm formation and ß-lactam resistance. Deletion of isaA, but not sceD, led to decreased biofilm formation. Additionally, in isaA-deleted strains, ß-lactam resistance was significantly decreased compared to that of wild-type strains. Plasmid-based expression of mecA, a major determinant of ß-lactam resistance in MRSA, in an isaA-deleted strain did not restore ß-lactam resistance, demonstrating that the ß-lactam susceptibility phenotype is exhibited by isaA mutant regardless of the production level of PBP2a. Overall, our results suggest that IsaA is a potential therapeutic target for MRSA infections.

Impact of renal function-based anti-tuberculosis drug dosage adjustment on efficacy and safety outcomes in pulmonary tuberculosis complicated with chronic kidney disease.

BACKGROUND: Dosages of anti-tuberculosis (TB) drugs are recommended to be adjusted according to renal function for patients complicated with chronic kidney disease (CKD). However, the efficacy and safety outcomes of such renal function-based dosage adjustments are not fully elucidated. METHODS: We retrospectively reviewed cases of pulmonary TB susceptible to first-line drugs that were treated at Jikei University Daisan Hospital between 2005 and 2014 with standard regimens based on dosage adjustments according to renal function recommended by international guidelines. Patients were divided into four groups, those with no, mild, moderate or severe CKD. In-hospital TB-related mortality, the rate of sputum culture conversion at 2 months, the frequency of adverse events (AEs), for which at least the temporal discontinuation of the suspect drug was required for patient improvement, and the rate of regimen change due to AEs were assessed. RESULTS: In the 241 enrolled patients (mean age, 64.1 years; 143 men), fourteen patients (5.8%) died due to TB during their hospitalization. The rate of sputum culture conversion at 2 months was 78.0%. The frequency of in-hospital TB-related death and the conversion rate in the groups did not vary significantly according to CKD severity including those in the non-CKD group (P = 0.310 and P = 0.864). Meanwhile, a total of 70 AEs were observed in 60 patients (24.9%) and the difference between the groups in the overall frequency of AEs was almost significant (P = 0.051). Moreover, for the 154 patients with CKD, severe CKD stage was a significant risk factor for regimen change (OR = 5.92, 95% CI = 1.08-32.5, P = 0.041), as were drug-induced hepatitis and cutaneous reaction (OR = 35.6, 95% CI = 8.70-145, P < 0.001; OR = 17.4, 95% CI = 3.16-95.5, P = 0.001; respectively). CONCLUSIONS: Adjusting the dosage of TB treatment for CKD patients according to the guidelines was efficient in terms of similar therapeutic outcome to that of the non-CKD group. However, AEs warrant attention to avoid regimen change in patients with severe CKD, even if the renal function-based dosage adjustment is performed.

Detection of bacterial DNA from central venous catheter removed from patients by next generation sequencing: a preliminary clinical study.

BACKGROUND: Catheter-related infection (CRI) is one of the serious challenges in clinical practice. This preliminary clinical study aimed to examine whether next-generation sequencing (NGS) targeting 16S rDNA, which was PCR-amplified directly from the tip of a central venous catheter (CVC), can be used to identify causative pathogens in CRI, compared to the culture method. METHODS: Hospitalized patients, from whom a CVC had just been removed, were prospectively enrolled and divided into the CRI-suspected and routine removal groups. DNA was extracted from the sonication fluid of CVC specimens derived from patients. For analysis of bacterial composition by NGS, the V3-V4 fragments of bacterial 16S rDNA were PCR-amplified, followed by index PCR and paired-end sequencing on an Illumina MiSeq device. Conventional culture methods were also performed in the CRI-suspected group. RESULTS: Of CVCs collected from the 156 enrolled patients (114 men; mean age 65.6 years), a total of 14 specimens [nine out of 31 patients suspected with CRI and five out of 125 patients without infection symptoms (routine removal group)] were PCR-positive. In five patients with definite CRI, Staphylococcus was the most frequently detected genus by NGS (4/5 specimens), although no pathogens were detected by NGS in the one remaining specimen. The genera identified by NGS were consistent with those from conventional culture tests. There was high agreement between NGS and the culture method in the CRI-suspected group, with sensitivity and specificity values of 80.0% and 76.9%, respectively; meanwhile, the false-positive rate of NGS was as low as 4.0% in the routine removal group. Moreover, several genera, besides the genus identified by culture test, were detected in each patient with definite CRI and surgical site infection (SSI). Additionally, in one patient with SSI, Enterococcaceae were detected not only by NGS but also by abdominal abscess drainage culture. CONCLUSIONS: NGS targeting 16S rDNA was able to analyze the bacterial composition of CVC specimens and detect causative pathogens in patients with CRI and was therefore suggested as a promising diagnostic tool for CRI.

Re-evaluation of the etiology and clinical and radiological features of community-acquired lobar pneumonia in adults.

OBJECTIVE: The aims of this study were to elucidate the frequency and etiology of community-acquired lobar pneumonia (CALP) and the clinical and radiological differences between CALP and tuberculous lobar pneumonia (TLP). PATIENTS AND METHODS: We retrospectively reviewed medical records of patients with community-acquired pneumonia (CAP) (n = 1032) and tuberculosis (n = 1101) admitted to our hospital. RESULTS: Sixty-nine (6.7%) patients with CAP and 23 (2.1%) with pulmonary tuberculosis developed CALP. Legionella species were the most common pathogen (27 patients, 39.1%), followed by Streptococcus pneumoniae (19 patients, 27.5%) and Mycoplasma pneumoniae (18 patients, 26.1%). Symptom duration was longer in the patients with TLP than in those with CALP. On chest radiographs, cavitation in the area of lobar pneumonia and nodular shadows were radiological findings predictive of TLP. High-resolution computed tomography showed cavitation in the area of lobar pneumonia, well-defined centrilobular nodules, and tree-in-bud sign to be the radiological findings predictive of TLP by multivariate logistic regression models. CONCLUSION: Common causes of CALP are Legionella species, S. pneumoniae, and M. pneumoniae. TLP should be considered in patients with lobar pneumonia, particularly in patients with long symptom duration, cavitation, and nodular shadows on chest radiographs, and cavitation, well-defined centrilobular nodules, and tree-in-bud sign on CT.

Detection of pathogens by real-time PCR in adult patients with acute exacerbation of bronchial asthma.

BACKGROUND: Respiratory tract infection is a major cause of acute exacerbation of bronchial asthma (AEBA). Although recent findings suggest that common bacteria are causally associated with AEBA, a comprehensive epidemiologic analysis of infectious pathogens including common/atypical bacteria and viruses in AEBA has not been performed. Accordingly, we attempted to detect pathogens during AEBA by using real-time polymerase chain reaction (PCR) in comparison to conventional methods. METHODS: We prospectively enroled adult patients with AEBA from August 2012 to March 2014. Infectious pathogens collected in nasopharyngeal swab and sputum samples were examined in each patient by conventional methods and real-time PCR, which can detect 6 bacterial and 11 viral pathogens. The causal association of these pathogens with AEBA severity and their frequency of monthly distribution were also examined. RESULTS: Among the 64 enroled patients, infectious pathogens were detected in 49 patients (76.6%) using real-time PCR and in 14 patients (21.9%) using conventional methods (p < 0.001). Real-time PCR detected bacteria in 29 patients (45.3%) and respiratory viruses in 28 patients (43.8%). Haemophilus influenzae was the most frequently detected microorganism (26.6%), followed by rhinovirus (15.6%). Influenza virus was the significant pathogen associated with severe AEBA. Moreover, AEBA occurred most frequently during November to January. CONCLUSIONS: Real-time PCR was more useful than conventional methods to detect infectious pathogens in patients with AEBA. Accurate detection of pathogens with real-time PCR may enable the selection of appropriate anti-bacterial/viral agents as a part of the treatment for AEBA.

Erratum: Author Correction: Norgestimate inhibits staphylococcal biofilm formation and resensitizes methicillin-resistant Staphylococcus aureus to ß-lactam antibiotics.

[This corrects the article DOI: 10.1038/s41522-017-0026-1.].

Norgestimate inhibits staphylococcal biofilm formation and resensitizes methicillin-resistant Staphylococcus aureus to ß-lactam antibiotics.

Formation of bacterial biofilms on medical devices can cause severe or fatal infectious diseases. In particular, biofilm-associated infections caused by methicillin-resistant Staphylococcus aureus are difficult to eradicate because the biofilm is strongly resistant to antibiotics and the host immune response. There is no effective treatment for biofilm-associated infectionss, except for surgical removal of contaminated medical devices followed by antibiotic therapy. Here we show that norgestimate, an acetylated progestin, effectively inhibits biofilm formation by staphylococcal strains, including methicillin-resistant S. aureus, without inhibiting their growth, decreasing the selective pressure for emergence of resistance. 17-Deacetyl norgestimate, a metabolite of norgestimate, shows much weaker inhibitory activity against staphylococcal biofilm formation, indicating that the acetyl group of norgestimate is important for its activity. Norgestimate inhibits staphylococcal biofilm formation by inhibiting production of polysaccharide intercellular adhesin and proteins in the extracellular matrix. Proteome analysis of S. aureus indicated that norgestimate represses the expression of the cell wall-anchored protein SasG, which promotes intercellular adhesion, and of the glycolytic enzyme enolase, which plays a secondary role in biofilm formation. Notably, norgestimate induces remarkable changes in cell wall morphology, characterized by increased thickness and abnormal rippled septa. Furthermore, norgestimate increases the expression level of penicillin binding protein 2 and resensitizes methicillin-resistant S. aureus to ß-lactam antibiotics. These results suggest that norgestimate is a promising lead compound for the development of drugs to treat biofilm-associated infections, as well as for its ability to resensitize methicillin-resistant S. aureus to ß-lactam antibiotics.

Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4.

Accumulation of profibrotic myofibroblasts is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF) pathogenesis. TGFB (transforming growth factor ß) is one of the major profibrotic cytokines for myofibroblast differentiation and NOX4 (NADPH oxidase 4) has an essential role in TGFB-mediated cell signaling. Azithromycin (AZM), a second-generation antibacterial macrolide, has a pleiotropic effect on cellular processes including proteostasis. Hence, we hypothesized that AZM may regulate NOX4 levels by modulating proteostasis machineries, resulting in inhibition of TGFB-associated lung fibrosis development. Human lung fibroblasts (LF) were used to evaluate TGFB-induced myofibroblast differentiation. With respect to NOX4 regulation via proteostasis, assays for macroautophagy/autophagy, the unfolded protein response (UPR), and proteasome activity were performed. The potential anti-fibrotic property of AZM was examined by using bleomycin (BLM)-induced lung fibrosis mouse models. TGFB-induced NOX4 and myofibroblast differentiation were clearly inhibited by AZM treatment in LF. AZM-mediated NOX4 reduction was restored by treatment with MG132, a proteasome inhibitor. AZM inhibited autophagy and enhanced the UPR. Autophagy inhibition by AZM was linked to ubiquitination of NOX4 via increased protein levels of STUB1 (STIP1 homology and U-box containing protein 1), an E3 ubiquitin ligase. An increased UPR by AZM was associated with enhanced proteasome activity. AZM suppressed lung fibrosis development induced by BLM with concomitantly reduced NOX4 protein levels and enhanced proteasome activation. These results suggest that AZM suppresses NOX4 by promoting proteasomal degradation, resulting in inhibition of TGFB-induced myofibroblast differentiation and lung fibrosis development. AZM may be a candidate for the treatment of the fibrotic lung disease IPF.

Pirfenidone inhibits myofibroblast differentiation and lung fibrosis development during insufficient mitophagy.

BACKGROUND: Pirfenidone (PFD) is an anti-fibrotic agent used to treat idiopathic pulmonary fibrosis (IPF), but its precise mechanism of action remains elusive. Accumulation of profibrotic myofibroblasts is a crucial process for fibrotic remodeling in IPF. Recent findings show participation of autophagy/mitophagy, part of the lysosomal degradation machinery, in IPF pathogenesis. Mitophagy has been implicated in myofibroblast differentiation through regulating mitochondrial reactive oxygen species (ROS)-mediated platelet-derived growth factor receptor (PDGFR) activation. In this study, the effect of PFD on autophagy/mitophagy activation in lung fibroblasts (LF) was evaluated, specifically the anti-fibrotic property of PFD for modulation of myofibroblast differentiation during insufficient mitophagy. METHODS: Transforming growth factor-ß (TGF-ß)-induced or ATG5, ATG7, and PARK2 knockdown-mediated myofibroblast differentiation in LF were used for in vitro models. The anti-fibrotic role of PFD was examined in a bleomycin (BLM)-induced lung fibrosis model using PARK2 knockout (KO) mice. RESULTS: We found that PFD induced autophagy/mitophagy activation via enhanced PARK2 expression, which was partly involved in the inhibition of myofibroblast differentiation in the presence of TGF-ß. PFD inhibited the myofibroblast differentiation induced by PARK2 knockdown by reducing mitochondrial ROS and PDGFR-PI3K-Akt activation. BLM-treated PARK2 KO mice demonstrated augmentation of lung fibrosis and oxidative modifications compared to those of BLM-treated wild type mice, which were efficiently attenuated by PFD. CONCLUSIONS: These results suggest that PFD induces PARK2-mediated mitophagy and also inhibits lung fibrosis development in the setting of insufficient mitophagy, which may at least partly explain the anti-fibrotic mechanisms of PFD for IPF treatment.

Increased levels of prostaglandin E-major urinary metabolite (PGE-MUM) in chronic fibrosing interstitial pneumonia.

BACKGROUND: Dysregulation of the prostaglandin E2 (PGE2) signaling pathway has been implicated in interstitial pneumonia (IP) pathogenesis. Due to the unstable nature of PGE2, available detection methods may not precisely reflect PGE2 levels. We explored the clinical usefulness of measuring stable prostaglandin E-major urinary metabolite (PGE-MUM) with respect to pathogenesis and extent of chronic fibrosing IP (CFIP), including idiopathic pulmonary fibrosis (IPF), as PGE-MUM is reflective of systemic PGE2 production. METHODS: PGE-MUM was measured by radioimmunoassay in controls (n = 124) and patients with lung diseases (bronchial asthma (BA): n = 78, chronic obstructive pulmonary disease (COPD): n = 33, CFIP: n = 44). Extent of lung fibrosis was assessed by fibrosing score (FS) of computed tomography (CT) (FS1-4). Immunohistochemical evaluation of COX-2 was performed to find PGE2 producing cells in IPF. Human bronchial epithelial cells (HBEC) and lung fibroblasts (LFB) were used in in vitro experiments. RESULTS: Compared to control, PGE-MUM levels were significantly elevated in CFIP. PGE-MUM levels were positively correlated with FS, and inversely correlated with %DLCO in IP (FS 1-3). COX-2 was highly expressed in metaplastic epithelial cells in IPF, but lower expression of EP2 receptor was demonstrated in LFB derived from IPF. TGF-ß induced COX-2 expression in HBEC. CONCLUSIONS: PGE-MUM, elevated in CFIP, is a promising biomarker reflecting disease activity. Metaplastic epithelial cells can be a source of elevated PGE-MUM in IPF.

Effectiveness of hepatoprotective drugs for anti-tuberculosis drug-induced hepatotoxicity: a retrospective analysis.

BACKGROUND: The effectiveness of hepatoprotective drugs for DIH (drug induced hepatotoxicity) during tuberculosis treatment is not clear. We evaluated the effectiveness of hepatoprotective drugs by comparing the period until the normalization of hepatic enzymes between patients who were prescribed with the hepatoprotective drugs after DIH was occurred and patients who were not prescribed with the hepatoprotective drugs. METHODS: During 2006-2010, 389 patients with active tuberculosis were included in this study. DIH was defined as elevation of peak serum aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) of more than twice the upper limit of normal (ULN). We divided the patients into the severe (peak serum AST and/or ALT elevation of >5 times the ULN), moderate (peak serum AST and/or ALT elevation of >3 to ≤5 times the ULN), and mild DIH groups (peak serum AST and/or ALT elevation of >2 to ≤3 times the ULN). We compared the average period until the normalization of hepatic enzymes between patient subgroups with and without hepatoprotective drugs (ursodeoxycholic acid: UDCA, stronger neo-minophagen C: SNMC, and glycyrrhizin). RESULTS: In the severe group, there was no significant difference in the average period until the normalization between subgroups with and without hepatoprotective drugs (21.4 ± 10.8 vs 21.5 ± 11.1 days, P = 0.97). In the mild group, the period was longer in the subgroup with hepatoprotective drugs than that without hepatoprotective drugs (15.7 ± 6.2 vs 12.4 ± 7.9 days, P = 0.046). CONCLUSION: Regardless of the severity, hepatoprotective drugs did not shorten the period until the normalization of hepatic enzymes.

Identification of pathogens by comprehensive real-time PCR versus conventional methods in community-acquired pneumonia in Japanese adults.

BACKGROUND: Community-acquired pneumonia (CAP) has high morbidity and mortality. Unfortunately, the pathogen detection rate using conventional culture methods is relatively low. We compared comprehensive real-time polymerase chain reaction (real-time PCR) analysis of nasopharyngeal swab specimens (NPS) and sputum samples against conventional methods for ability to detect causative pathogens of CAP. METHODS: We prospectively enrolled adult CAP patients, including those with prior antibiotic use, from December 2012 to May 2014. For each patient, causative pathogens were investigated conventionally and by real-time PCR that can identify 6 bacterial and 11 viral pathogens. RESULTS: Patients numbered 92 (mean age, 63 years; 59 male), including 30 (33%) with prior antibiotic use. Considering all patients, identification of causative pathogens by real-time PCR was significantly more frequent than by conventional methods in all patients (72% vs. 57%, p = 0.018). In patients with prior antibiotic use, identification rates also differed significantly (PCR, 77%; conventional, 50%; p = 0.027). Mixed infections were more frequent according to real-time PCR than conventional methods (26% vs. 4%, p < 0.001). By the real-time PCR, Streptococcus pneumoniae was most frequently identified (38%) as a causative pathogen, followed by Haemophilus influenzae (37%) and Mycoplasma pneumoniae (5%). PCR also identified viral pathogens (21%), with sensitivity enhanced by simultaneous examination of both NPS and sputum samples rather than only NPS samples. CONCLUSIONS: Real-time PCR of NPS and sputum samples could better identify bacterial and viral pathogens in CAP than conventional methods, both overall and in patients with prior antibiotic treatment.

The Optimal Cut-off Value of Plasma BNP to Differentiate Heart Failure in the Emergency Department in Japanese Patients with Dyspnea.

OBJECTIVE: In the emergency department, it is sometimes difficult to differentiate heart failure (HF) from other diseases (e.g., respiratory diseases) in patients who develop dyspnea. The plasma B-type natriuretic peptide (BNP) levels increase in patients with HF, and various levels are associated with specific New York Heart Association classes. Although the diagnosis of HF should not be made based only on the plasma BNP levels, the identification of a cut-off value for BNP to diagnose HF would be helpful. METHODS: Patients admitted to the emergency department of our hospital with dyspnea between January 2010 and December 2011 were retrospectively reviewed. The patients whose estimated glomerular filtration rate was less than 30 mL/min/1.73 m(2) were excluded. Patients were divided into two groups: those with HF (n=131) and those without HF (n=138). The cut-off value for BNP was determined by the receiver-operating characteristic curve. RESULTS: The area under the curve of this curve was 0.934. The optimal cut-off point for detection of HF was 234 pg/mL. The sensitivity and specificity were 87.0% and 85.5%, respectively. The fifth and 95th percentiles of the HF group were 132.2 and 2,420.8 pg/mL, respectively. Those of the non-HF group were 9.7 and 430.2 pg/mL, respectively. CONCLUSION: Our study suggests that a plasma BNP level cut-off value of 234 pg/mL can be used to detect HF in the emergency department.

Pathogens in COPD exacerbations identified by comprehensive real-time PCR plus older methods.

Respiratory infection is a major cause of exacerbation in chronic obstructive pulmonary disease (COPD). Infectious contributions to exacerbations remain incompletely described. We therefore analyzed respiratory tract samples by comprehensive real-time polymerase chain reaction (PCR) in combination with conventional methods. We evaluated multiple risk factors for prolonged hospitalization to manage COPD exacerbations, including infectious agents. Over 19 months, we prospectively studied 46 patients with 50 COPD exacerbations, collecting nasopharyngeal swab and sputum samples from each. We carried out real-time PCR designed to detect six bacterial species and eleven viruses, together with conventional procedures, including sputum culture. Infectious etiologies of COPD exacerbations were identified in 44 of 50 exacerbations (88%). Infections were viral in 17 of 50 exacerbations (34%). COPD exacerbations caused by Gram-negative bacilli, including enteric and nonfermenting organisms, were significantly associated with prolonged hospitalization for COPD exacerbations. Our results support the use of a combination of real-time PCR and conventional methods for determining both infectious etiologies and risk of extended hospitalization.

Clinical efficacy of anti-glycopeptidolipid-core IgA test for diagnosing Mycobacterium avium complex infection in lung.

BACKGROUND AND OBJECTIVE: It is difficult to verify the bacteriological diagnosis of Mycobacterium avium complex (MAC) infection. The anti-glycopeptidolipid (GPL)-core IgA antibody test was recently developed as a diagnostic method for MAC pulmonary disease. Only a few studies evaluate its clinical efficacy. We conducted retrospective evaluations of clinical characteristics of patients suspected of MAC infection to explore the usefulness of the anti-GPL-core IgA antibody test. METHODS: We retrospectively evaluated 296 patients who were suspected to have MAC infection and underwent anti-GPL-core IgA antibody test between March 2013 and July 2014 in Jikei University hospital. RESULTS: A total of 29 patients were diagnosed with 'definite MAC' based on the American Thoracic Society (ATS) criteria with multiple identifications of MAC. On the other hand, 106 patients were diagnosed with other pulmonary diseases than MAC. The sensitivity and specificity of anti-GPL-core IgA antibody test for MAC diagnosis were 58.6% and 98.1%, respectively. The definite MAC group showed no significant differences in strains, treatment history or number of segments involved. The duration of MAC disease in the positive-antibody group was significantly longer than in the negative-antibody group (P = 0.046). A significant increase in the false-negative rate was observed in patients with malignant disease (P = 0.029). CONCLUSIONS: The anti-GPL-core IgA antibody test demonstrated high sensitivity and specificity for the diagnosis of MAC infection especially in patients without malignant diseases.

Progressive diffuse pulmonary interstitial opacities due to complications of pulmonary tumor emboli: an autopsy case report.

A 76-year-old man complaining of exertional dyspnea was admitted to our hospital. Chest computed tomography revealed bilateral diffuse ground-glass opacities and small nodules. A transbronchial lung biopsy revealed tumor cell emboli in the pulmonary arteries. The patient was diagnosed with gastric adenocarcinoma using an endoscopic stomach biopsy; however, the interstitial opacities progressively worsened and he died of acute respiratory failure. An autopsy revealed extensive pulmonary tumor embolisms (PTE) with associated ischemic damages, e.g., infarctions, alveolar wall thickening with cuboidal metaplasia, hemorrhage, and diffuse alveolar damage. The ground-glass opacities in the chest computed tomography findings appear to correlate with the pathological ischemic changes associated with PTE.