Visceral fat obesity is the key risk factor for the development of reflux erosive esophagitis in 40-69-years subjects.

BACKGROUND: Visceral fat obesity can be defined quantitatively by abdominal computed tomography, however, the usefulness of measuring visceral fat area to assess the etiology of gastrointestinal reflux disease has not been fully elucidated. METHODS: A total of 433 healthy subjects aged 40-69 years (234 men, 199 women) were included in the study. The relationship between obesity-related factors (total fat area, visceral fat area, subcutaneous fat area, waist circumference, and body mass index) and the incidence of reflux erosive esophagitis was investigated. Lifestyle factors and stomach conditions relevant to the onset of erosive esophagitis were also analyzed. RESULTS: The prevalence of reflux erosive esophagitis was 27.2% (118/433; 106 men, 12 women). Visceral fat area was higher in subjects with erosive esophagitis than in those without (116.6 cm2 vs. 64.9 cm2, respectively). The incidence of erosive esophagitis was higher in subjects with visceral fat obesity (visceral fat area ≥ 100 cm2) than in those without (61.2% vs. 12.8%, respectively). Visceral fat obesity had the highest odds ratio (OR) among obesity-related factors. Multivariate analysis showed that visceral fat area was associated with the incidence of erosive esophagitis (OR = 2.18), indicating that it is an independent risk factor for erosive esophagitis. In addition, daily alcohol intake (OR = 1.54), gastric atrophy open type (OR = 0.29), and never-smoking history (OR = 0.49) were also independently associated with the development of erosive esophagitis. CONCLUSIONS: Visceral fat obesity is the key risk factor for the development of reflux erosive esophagitis in subjects aged 40-69 years.

EGF and BMPs Govern Differentiation and Patterning in Human Gastric Glands.

BACKGROUND & AIMS: The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organized inside glands and crypts. Regeneration depends on Wnt/ß-catenin pathway activation, but to understand homeostasis and its dysregulation in disease, we need to identify the signaling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen-, and gastric acid-producing cells. METHODS: We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief, and parietal cells. We localized the source of the growth factors in the tissue of origin. RESULTS: We show that epidermal growth factor is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with bone morphogenetic factor/Noggin signals, epidermal growth factor controls the differentiation of foveolar cells vs parietal or chief cells. We also show that epidermal growth factor is likely to underlie alteration of the gastric mucosa in the precancerous condition atrophic gastritis. CONCLUSIONS: Use of our recently established mucosoid cultures in combination with analysis of the tissue of origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signaling microenvironment in the human gastric glands.

RNAi-based small molecule repositioning reveals clinically approved urea-based kinase inhibitors as broadly active antivirals.

Influenza viruses (IVs) tend to rapidly develop resistance to virus-directed vaccines and common antivirals targeting pathogen determinants, but novel host-directed approaches might preclude resistance development. To identify the most promising cellular targets for a host-directed approach against influenza, we performed a comparative small interfering RNA (siRNA) loss-of-function screen of IV replication in A549 cells. Analysis of four different IV strains including a highly pathogenic avian H5N1 strain, an influenza B virus (IBV) and two human influenza A viruses (IAVs) revealed 133 genes required by all four IV strains. According to gene enrichment analyses, these strain-independent host genes were particularly enriched for nucleocytoplasmic trafficking. In addition, 360 strain-specific genes were identified with distinct patterns of usage for IAVs versus IBV and human versus avian IVs. The strain-independent host genes served to define 43 experimental and otherwise clinically approved drugs, targeting reportedly fourteen of the encoded host factors. Amongst the approved drugs, the urea-based kinase inhibitors (UBKIs) regorafenib and sorafenib exhibited a superior therapeutic window of high IV antiviral activity and low cytotoxicity. Both UBKIs appeared to block a cell signaling pathway involved in IV replication after internalization, yet prior to vRNP uncoating. Interestingly, both compounds were active also against unrelated viruses including cowpox virus (CPXV), hantavirus (HTV), herpes simplex virus 1 (HSV1) and vesicular stomatitis virus (VSV) and showed antiviral efficacy in human primary respiratory cells. An in vitro resistance development analysis for regorafenib failed to detect IV resistance development against this drug. Taken together, the otherwise clinically approved UBKIs regorafenib and sorafenib possess high and broad-spectrum antiviral activity along with substantial robustness against resistance development and thus constitute attractive host-directed drug candidates against a range of viral infections including influenza.

Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection.

OBJECTIVE: Helicobacter pylori causes life-long colonisation of the gastric mucosa, leading to chronic inflammation with increased risk of gastric cancer. Research on the pathogenesis of this infection would strongly benefit from an authentic human in vitro model. DESIGN: Antrum-derived gastric glands from surgery specimens served to establish polarised epithelial monolayers via a transient air-liquid interface culture stage to study cross-talk with H. pylori and the adjacent stroma. RESULTS: The resulting 'mucosoid cultures', so named because they recapitulate key characteristics of the gastric mucosa, represent normal stem cell-driven cultures that can be passaged for months. These highly polarised columnar epithelial layers encompass the various gastric antral cell types and secrete mucus at the apical surface. By default, they differentiate towards a foveolar, MUC5AC-producing phenotype, whereas Wnt signalling stimulates proliferation of MUC6-producing cells and preserves stemness-reminiscent of the gland base. Stromal cells from the lamina propria secrete Wnt inhibitors, antagonising stem-cell niche signalling and inducing differentiation. On infection with H. pylori, a strong inflammatory response is induced preferentially in the undifferentiated basal cell phenotype. Infection of cultures for several weeks produces foci of viable bacteria and a persistent inflammatory condition, while the secreted mucus establishes a barrier that only few bacteria manage to overcome. CONCLUSION: Gastric mucosoid cultures faithfully reproduce the features of normal human gastric epithelium, enabling new approaches for investigating the interaction of H. pylori with the epithelial surface and the cross-talk with the basolateral stromal compartment. Our observations provide striking insights in the regulatory circuits of inflammation and defence.

Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies.

As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease.

Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA.

Streptococcus pneumoniae (S.pn.) is the most common bacterial pathogen causing community acquired pneumonia. The pore-forming toxin pneumolysin (PLY) is the major virulence factor of S.pn. and supposed to affect alveolar epithelial cells thereby activating the immune system by liberation of danger-associated molecular patterns (DAMP). To test this hypothesis, we established a novel live-cell imaging based assay to analyse mitochondrial function and associated release of mitochondrial DNA (mtDNA) as DAMP in real-time. We first revealed that bacterially released PLY caused significant changes of the cellular ATP homeostasis and led to morphologic alterations of mitochondria in human alveolar epithelial cells in vitro and, by use of spectral live-tissue imaging, in human alveoli. This was accompanied by strong mitochondrial calcium influx and loss of mitochondrial membrane potential resulting in opening of the mitochondrial permeability transition pore and mtDNA release without activation of intrinsic apoptosis. Moreover, our data indicate cellular mtDNA liberation via microvesicles, which may contribute to S.pn. related pro-inflammatory immune activation in the human alveolar compartment.

Helicobacter pylori Depletes Cholesterol in Gastric Glands to Prevent Interferon Gamma Signaling and Escape the Inflammatory Response.

BACKGROUND & AIMS: Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosa for decades. H pylori expression of cholesterol-α-glucosyltransferase (encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response. METHODS: MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patients undergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-ß-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed by immunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Δcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1-/- mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Δcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy. RESULTS: In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human ß-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNG-response gene IRF1 was substantially higher in PMSS1Δcgt-infected mice than PMSS1-infected mice. Ifngr1-/- mice were colonized by PMSS1 to a greater extent than control mice. CONCLUSIONS: H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which H pylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell-based vaccines against H pylori.

InFusion: Advancing Discovery of Fusion Genes and Chimeric Transcripts from Deep RNA-Sequencing Data.

Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis.

Accumulating evidence from experimental animal models suggests that antibodies play a protective role against tuberculosis (TB). However, little is known about the antibodies generated upon Mycobacterium tuberculosis (MTB) exposure in humans. Here, we performed a molecular and functional characterization of the human B-cell response to MTB by generating recombinant monoclonal antibodies from single isolated B cells of untreated adult patients with acute pulmonary TB and from MTB-exposed healthcare workers. The data suggest that the acute plasmablast response to MTB originates from reactivated memory B cells and indicates a mucosal origin. Through functional analyses, we identified MTB inhibitory antibodies against mycobacterial antigens including virulence factors that play important roles in host cell infection. The inhibitory activity of anti-MTB antibodies was directly linked to their isotype. Monoclonal as well as purified serum IgA antibodies showed MTB blocking activity independently of Fc alpha receptor expression, whereas IgG antibodies promoted the host cell infection. Together, the data provide molecular insights into the human antibody response to MTB and may thereby facilitate the design of protective vaccination strategies.

Changes in Surgical Site Infections after Living Donor Liver Transplantation.

Surgical site infections (SSIs) are a major threat for liver transplant recipients. We prospectively studied SSIs after living donor liver transplantation (LDLT) at Kyoto University Hospital from April 2001 to March 2002 (1st period) and from January 2011 to June 2012 (2nd period). We investigated the epidemiology of SSIs after LDLT and determined the differences between the two periods. A total of 129 adult recipients (66 during the 1st period and 63 during the 2nd period) and 72 pediatric recipients (39 and 33) were included in this study. The SSI rates for each period were 30.3% (1st period) and 41.3% (2nd period) among the adult recipients and 25.6% and 30.3% among the pediatric recipients. The overall rates of 30-day mortality among adult transplant recipients with SSIs were 10.0% (1st period) and 3.9% (2nd period). No pediatric recipient died from SSIs after LDLT in either period. The incidence of Enterococcus faecium increased from 5.0% to 26.9% in the adults and from 10.0% to 40.0% in the pediatric patients. Extended-spectrum ß-lactamase-producing Enterobacteriaceae were emerging important isolates during the 2nd period. For this period, a univariate analysis showed that ABO incompatibility (P = 0.02), total operation duration (P = 0.01), graft-to-recipient body weight ratio (GRWR [P = 0.04]), and Roux-en-Y biliary reconstruction (P<0.01) in the adults and age (P = 0.01) and NHSN risk index (P = 0.02) in the children were associated with SSI development. In a multivariate analysis, lower GRWR (P = 0.02) and Roux-en-Y biliary reconstruction (P<0.01) in the adults and older age (P = 0.01) in the children were independent risk factors for SSIs during the 2nd period. In conclusion, SSIs caused by antibiotic resistant bacteria may become a major concern. Lower GRWR and Roux-en-Y biliary reconstruction among adult LDLT recipients and older age among pediatric LDLT recipients increased the risk of developing SSIs after LDLT.

Pneumocystis polymerase chain reaction and blood (1→3)-ß-D-glucan assays to predict survival with suspected Pneumocystis jirovecii pneumonia.

Pneumocystis polymerase chain reaction (PCR) and blood (1→3)-ß-D-glucan assays are known to be useful for the diagnosis of Pneumocystis pneumonia (PCP). However, their impact on the outcome of clinically suspected PCP patients has not yet been elucidated. Between January 2008 and July 2011, we prospectively observed 190 immunocompromised patients who had ground-glass opacity on chest computed tomography scans and were suspected to have PCP. The blood ß-D-glucan levels of these patients were measured, and PCR was used to detect Pneumocystis jirovecii in the respiratory samples. The 30-day mortality rates and related factors were assessed. The 30-day mortality rate of all included patients was 21.6%. Both ß-D-glucan-positive (10.1%) and PCR-positive patients (15.0%) had significantly lower mortality rates than ß-D-glucan-negative (28.1%) or PCR-negative patients (30.1%). All of the 13 definite PCP patients had positive PCR and ß-D-glucan results, received anti-PCP treatments, and survived. Among the 72 patients who were negative for microscopic detection of P. jirovecii but received anti-PCP treatments, positive PCR results (odds ratio [OR], 0.14; 95% confidence interval [CI], 0.02-0.74), a high Sequential Organ Failure Assessment score (OR, 1.42; CI, 1.08-1.88), and positive ß-D-glucan levels (OR 0.25, CI 0.06-1.02) were associated with mortality rates using stepwise logistic regression analyses. A positive Pneumocystis PCR or ß-D-glucan test was a candidate predictor of survival in patients who were suspected of having PCP, even though negative for visual detection by microscopy.

Clinical characteristics and risk factors of non-Candida fungaemia.

BACKGROUND: The incidence of fungaemia has been increasing worldwide. It is important to distinguish non-Candida fungaemia from candidaemia because of their different antifungal susceptibilities. The aims of this study were to investigate the clinical characteristics of non-Candida fungaemia and identify the clinical factors that differentiate it from candidaemia. METHODS: We investigated the clinical manifestations and mortality of non-Candida fungaemia in Kyoto University Hospital from 2004 to 2009. RESULTS: There were 110 episodes of fungaemia during the study period. There were 11 renal replacement therapy episodes of fungaemia due to non-Candida yeasts (10.0%), including 6 episodes with Cryptococcus neoformans, 4 with Trichosporon asahii, and 1 with Kodamaea ohmeri, in addition to 99 episodes of candidaemia (90.0%). The presence of collagen disease [odds ratio (OR) 9.00; 95% confidence interval (CI) 1.58-51.4; P=0.01] or renal replacement therapy (OR 15.0; 95% CI 3.06-73.4; P<0.01) was significantly more common in non-Candida fungaemia patients than in candidaemia patients. Prior colonisation by the species may be a predictor of non-Candida fungaemia. Non-Candida fungaemia had a higher mortality than candidaemia (54.5% versus 21.2%, P=0.03). CONCLUSIONS: Although Candida species frequently cause fungaemia, we should also be aware of non-Candida yeasts because of their high mortality, particularly among high-risk patients, such as those with collagen disease and those under renal replacement therapy. Prior colonisation by the causative organisms may be an important predictor of non-Candida fungaemia.

Risk factors associated with elevated blood cytomegalovirus pp65 antigen levels in patients with autoimmune diseases.

OBJECTIVES: To further assess the relationship between elevated levels of cytomegalovirus (CMV) pp65 antigen in blood, as indicative of viral load, during treatment-free follow-up and CMV diseases in patients with autoimmune diseases and to identify any risk factors associated with elevated viral loads. METHODS: This was a retrospective review of the electronic medical charts of 148 patients with autoimmune diseases who tested positive for CMV pp65 antigen in the blood. RESULTS: A total of 106 patients were analyzed. During follow-up, elevated viral loads were detected in 35 patients who were not on antiviral therapy, of whom five developed CMV diseases. Elevated viral load was significantly associated with CMV diseases [5/35 vs. 0/71 (no elevated viral load); P = 0.001). Multivariate analysis revealed that lymphopenia [lymphocyte numbers <700/mm(3), odds ratio (OR) 34.44, 95 % confidence interval (CI), 7.82-151.66; P < 0.001], systemic lupus erythematosus (SLE) (OR 6.71, 95 % CI, 1.23-36.49; P = 0.028), and polymyositis/dermatomyositis (PM/DM) (OR 10.62, 95 % CI 1.41-79.77; P = 0.022) were significantly associated with elevated viral load. CONCLUSIONS: Elevated viral load was significantly associated with CMV diseases. Patients with SLE or PM/DM and lymphopenia would therefore benefit from a detailed viral load follow-up and careful physical examination.

Emergence and spread of B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups among extended-spectrum-ß-lactamase-producing Escherichia coli in Japan.

OBJECTIVES: The increasing prevalence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli has been associated with the emergence of the CTX-M-producing sequence type 131 (ST131) pandemic clonal group, a member of the O25b serogroup and the B2 phylogenetic group. To assess the clonal spread of ESBL-producing E. coli in Japan, a regional surveillance programme was conducted. METHODS: A total of 581 ESBL-producing clinical specimen E. coli isolates were collected between 2001 and 2010. Clonal groups, including ST131, D-ST405, D-ST393 and D-ST69, were determined using the PCR O type, phylogenetic grouping by triplex PCR, allele-specific PCR and multilocus sequence typing (MLST). A subset of clonal groups underwent PFGE. RESULTS: Among clonal strains, 215 isolates (37%) were identified as belonging to the ST131 group, 185 as B2-ST131-O25b (32%), 26 as B2-ST131-O16 (4%), 3 as B1-ST131-O25b (0.5%) and 1 as B2-ST131-O-non-typeable (0.1%). Forty-one isolates (7%) were identified as belonging to the D-ST405 clonal group, seven (1%) as D-ST69 and two (0.3%) as D-ST393. The B2-ST131-O16 clonal group was characterized by CTX-M-14 and a significantly lower ciprofloxacin resistance rate than the B2-ST131-O25b clonal group. The B2-ST131-O16 and B2-ST131-O25b clonal groups each made up a single PFGE cluster, with 65% similarity. The rate of ESBL-producing E. coli increased over the years (0.2% in 2001 to 9.7% in 2010) and corresponded to increases in the numbers of the B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups. CONCLUSIONS: The B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups have contributed to the spread of ESBL-producing E. coli in Japan.

Prevalence of plasmid-mediated AmpC ß-lactamase-producing Escherichia coli and spread of the ST131 clone among extended-spectrum ß-lactamase-producing E. coli in Japan.

In 2010, a total of 1327 clinical Escherichia coli isolates from five hospitals in the Kyoto and Shiga regions of Japan were analysed by PCR. The prevalences of plasmid-mediated AmpC ß-lactamase (pAmpC)-producers, extended-spectrum ß-lactamase (ESBL)-producers and co-producers of pAmpC and ESBL were 1.7%, 9.7% and 0.3%, respectively. Less than one-half of the pAmpC-producers were reported to be resistant to third-generation cephalosporins, cephamycins and ß-lactam/ß-lactam inhibitors using the old 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints. CMY-2 was the most prevalent pAmpC type (95%), and CTX-M-14 (38%), CTX-M-15 (26%) and CTX-M-27 (19%) were the most prevalent ESBL types. The worldwide O25b-ST131-B2 clone accounted for 11% of pAmpC-producers and 41% of ESBL-producers. The O25b-ST131-B2 clone was characterised by a CTX-M-27- or CTX-M-15-type ESBL and ciprofloxacin-non-susceptibility with quadruple mutations in the quinolone resistance-determining regions (S83L and D87N in GyrA and S80I and E84V in ParC). A significant proportion of pAmpC-producers and the O25b-ST131-B2 clone were found in Japan by a recent regional surveillance programme.

Molecular characterization of IMP-type metallo-ß-lactamases among multidrug-resistant Achromobacter xylosoxidans.

OBJECTIVES: To analyse the metallo-ß-lactamase (MBL) genes and their genetic environments in clinical isolates of Achromobacter xylosoxidans. METHODS: From January 2004 to December 2010, four MBL-producing, multidrug-resistant (MDR) A. xylosoxidans strains were isolated and analysed at a tertiary care university hospital in Japan. Species-level identification was confirmed by 16S ribosomal RNA gene sequencing. Molecular typing was performed using PFGE, the presence of MBL genes was detected using PCR, and integron gene cassettes were examined by cloning and sequence analysis of integron PCR products. The plasmids obtained from individual isolates were analysed based on EcoRI restriction patterns, Southern hybridizations using digoxigenin-labelled probes for bla(IMP-1) and bla(IMP-19) as well as conjugation and transformation experiments. RESULTS: No clonal relationship was found among the four A. xylosoxidans isolates. Three isolates harboured bla(IMP-1) and one isolate harboured bla(IMP-19). These MBL genes were carried on class 1 integrons. Four different class 1 integron gene cassette arrays were found, including orf1-bla(IMP-1)-aacA4, bla(IMP-1)-bla(IMP-1)-nit1/nit2-catB3-bla(IMP-1)-bla(IMP-1), aacA4-bla(IMP-1) and bla(IMP-19). The restriction pattern of the plasmid DNA obtained from each isolate was unique and the hybridization analyses revealed the presence of each MBL gene within a plasmid. Moreover, all of the plasmids carrying an MBL gene could be transformed into Escherichia coli HST08. CONCLUSIONS: This study provides genetic evidence for the existence of IMP-type MBL genes in MDR A. xylosoxidans isolates. Moreover, the present findings provide evidence that A. xylosoxidans can receive IMP-type MBL genes via plasmid-mediated transfer, which contributes to their carbapenem resistance.

Clinical characteristics and risk factors of ocular candidiasis.

Ocular candidiasis is a major complication of Candida bloodstream infection (BSI). This study was performed to reveal the clinical characteristics of ocular candidiasis. Of the 220 patients with Candida BSI, 204 cases received ophthalmology consultations between January 2005 and December 2011 at 2 teaching hospitals. Fifty-four (26.5%) cases had findings consistent with the diagnosis of ocular candidiasis. Of these 54 cases, 43 (79.6%) were diagnosed within 7 days after a positive blood culture. Among ocular candidiasis cases, more cases were due to Candida albicans (P =0.034 odds ratio [OR]; 3.68 95% confidence interval [CI] 1.11-12.2) and had higher ß-d-glucan values (P = 0.001 OR; 9.99 95% CI 2.60-21.3). We need to consider fundoscopic examination to be performed within the first 7 days of therapy, especially for those patients who have C. albicans BSIs and higher ß-d-glucan values. Additionally, follow-up fundoscopic examination should be considered before stopping therapy for high-risk patients.

Disseminated Nocardia farcinica infection in a patient with myasthenia gravis successfully treated by linezolid: a case report and literature review.

Nocardiosis is increasingly being diagnosed because of a growing population of immunocompromised hosts and improvements in the detection of Nocardia species in clinical laboratories. Historically, sulphonamides have been the first-line therapy for the treatment of nocardiosis, but sulphonamides tend to have a high rate of drug allergy in clinical settings. In this report, we described a disseminated Nocardia farcinica infection that occurred in a patient with myasthenia gravis who suffered from multiple drug allergies and was successfully treated using linezolid. We undertook a review of the literature of previously reported cases of nocardiosis treated with linezolid. To date, only 15 cases of nocardiosis treated with linezolid have been published. All cases exhibited long-term tolerance of linezolid, and 14 of 15 cases showed either an improvement in or complete clearance of the infection. According to the literature review, linezolid is an attractive alternative to trimethoprim-sulfamethoxazole for the treatment of disseminated nocardiosis, despite limited clinical evidence to support this claim.

[Evaluation of a UF-1000i screening method to identify the bacteriuria for cultures and susceptibility testing].

Urine culture remains the gold standard for diagnosing urinary tract infections, but most clinical samples yield negative results. Fast screening methods will improve the turnaround time and also reduce the associated costs. We evaluated the detection of bacteria using the Sysmex UF-1000i urine analyzer to identify negative samples which do not need further culture testings. The bacterial counts of the UF-1000i method and of the conventional culture of 197 samples, including 117 samples of midstream urine (MU) and 80 from catheter ports (CU), were prospectively compared, and the patient backgrounds were reviewed. The cut-off values to determine the necessity for culture were 2.7 bacteria/microL for MU and 11.0 bacteria/microL for CU samples, and 16.2% of the MU and 30.0% of the CU samples did not require cultures. These cut-off values are similar to those described in previous studies, however, our findings suggest that it would therefore be possible to reduce the need for unnecessary samples by the use of our cut-off values, which utilize the CU and MU samples separately. The cost reduction was calculated to be $239-306 per 100 samples. This UF-1000i screening method is an acceptable modality which improves the turnaround time, workload and cost to perform urine cultures.