Telomere length in paroxysmal nocturnal hemoglobinuria correlates with clone size

OBJECTIVE: To study if telomere length can be used as a surrogate marker for the mitotic history in normal and affected hematopoietic cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: The telomere length was measured by automated multicolor flow fluorescence in situ hybridization in glycosyl-phosphatidyl-inositol anchored protein (GPI)-negative and GPI-positive peripheral blood leukocytes. Eleven patients were studied, two with predominantly hemolytic PNH and nine with PNH associated with marrow failure. RESULTS: Telomere length in GPI-negative cells was significantly shorter than in GPI-positive cells of the same patient (p < 0.01, n = 11). The difference in telomere length (telomere length in GPI-positive minus telomere length in GPI-negative cells) correlated with the percentage of GPI-negative white blood cells. CONCLUSION: Our results support the hypothesis that telomere length is correlated to the replicative history of GPI-positive and GPI-negative cells and warrant further studies of telomere length in relation to disease progression in PNH.

Outcome after radical cystectomy with limited or extended pelvic lymph node dissection

PURPOSE: We compared recurrence patterns and survival of patients with urothelial bladder cancer undergoing radical cystectomy who either had limited or extended pelvic lymph node dissection at 2 institutions between 1987 and 2000. MATERIALS AND METHODS: Two consecutive series of patients treated with radical cystectomy and limited pelvic lymph node dissection (336; Cleveland Clinic) and extended pelvic lymph node dissection (322; University of Bern) were analyzed. All cases were staged N0M0 prior to radical cystectomy, and none were treated with neoadjuvant radiotherapy or chemotherapy. Patients with PTis/pT1 and pT4 disease were excluded from analysis. Pathological characteristics based on the 1997 TNM system and recurrence patterns were determined. RESULTS: The overall lymph node positive rate was 13% for patients with limited and 26% for those who had extended pelvic lymph node dissection. The 5-year recurrence-free survival of patients with lymph node positive disease was 7% for limited and 35% for extended pelvic lymph node dissection. The 5-year recurrence-free survival for pT2pN0 cases was 67% for limited and 77% for extended pelvic lymph node dissection, and the respective percentages for pT3pN0 cases were 23% and 57% (p <0.0001). The 5-year recurrence-free survival for pT2pN0-2 cases was 63% for limited and 71% for extended pelvic lymph node dissection, and for pT3pN0-2 cases the respective figures were 19% and 49% (p <0.0001). Incidence of local and systemic failure correlated closely with pathological stage for both series. CONCLUSIONS: Our data suggest that limited pelvic lymph node dissection is associated with suboptimal staging, poorer outcome for patients with node positive and node negative disease, and a higher rate of local progression. Extended pelvic lymph node dissection allows for more accurate staging and improved survival of patients with nonorgan confined and lymph node positive disease.

Nonacid reflux episodes reaching the pharynx are important factors associated with cough

BACKGROUND: Gastroesophageal reflux is implicated in the pathogenesis of asthma and chronic cough. To date most studies have focused on acid reflux measured by pH below the upper esophageal sphincter (UES). The aim of this study was to assess the relationship between cough and reflux through the UES into the pharynx. METHODS: Thirty-seven patients with asthma (19) and chronic cough (18) were recruited from the respiratory clinic. Reflux was monitored using a combined multichannel intraluminal impedance and pH probe by detecting (1) bolus reflux episodes within the esophagus and in the pharynx and (2) acidic reflux episodes within the esophagus and in the pharynx. All acid suppressive therapy was stopped for at least 7 days before the study. Demonstration of cough being linked to reflux was achieved using the symptom association probability (SAP). This was calculated using a 2-minute association window between symptoms and bolus entry into the esophagus. SAP was considered positive if >95%. RESULTS: A positive SAP for cough was noted in 7/26 patients reporting symptoms on the day of monitoring. Compared with SAP-negative patients, SAP-positive patients had both a greater number [median (interquartile range), 5(2 to 8) vs. 2(0 to 4), P<0.05] and a higher proportion of reflux episodes crossing the UES into the pharynx [25%(14% to 28%) vs. 7% (2% to 14%), P<0.02]. There was no difference in the number of reflux episodes or acid exposure time in the distal esophagus between SAP-positive and SAP-negative patients. Only 1% to 2% of episodes were detected by the pharyngeal pH sensor. CONCLUSIONS: Impedance detected pharyngeal reflux episodes are important factors in symptom production in cough patients.

Colonization with resistant microorganisms in patients transferred from abroad: who needs to be screened?

BACKGROUND While multi-drug resistant organisms (MDRO) are a global phenomenon, there are significant regional differences in terms of prevalence. Traveling to countries with a high MDRO prevalence increases the risk of acquiring such an organism. In this study we determined risk factors for MDRO colonization among patients who returned from a healthcare system in a high-prevalence area (so-called transfer patients). Factors predicting colonization could serve as screening criteria to better target those at highest risk. METHODS This screening study included adult patients who had been exposed to a healthcare system abroad or in a high-prevalence region in Switzerland over the past six months and presented to our 950-bed tertiary care hospital between January 1, 2012 and December 31, 2013, a 24-month period. Laboratory screening tests focused on Gram-negative MDROs and methicillin-resistant Staphylococcus aureus (MRSA). RESULTS A total of 235 transfer patients were screened and analyzed, of which 43 (18 %) were positive for an MDRO. Most of them yielded Gram-negative bacteria (42; 98 %), with only a single screening revealing MRSA (2 %); three screenings showed a combination of Gram-negative bacteria and MRSA. For the risk factor analysis we focused on the 42 Gram-negative MDROs. Most of them were ESBL-producing Escherichia coli and Klebsiella pneumoniae while only two were carbapenemase producers. In univariate analysis, factors associated with screening positivity were hospitalization outside of Europe (p < 0.001), surgical procedure in a hospital abroad (p = 0.007), and - on admission to our hospital - active infection (p = 0.002), antibiotic treatment (p = 0.014) and presence of skin lesions (p = 0.001). Only hospitalization outside of Europe (Odds Ratio, OR 3.2 (95 % CI 1.5- 6.8)) and active infection on admission (OR 2.7 (95 % CI 1.07- 6.6)) remained as independent predictors of Gram-negative MDRO colonization. CONCLUSION Our data suggest that a large proportion of patients (i.e., 82 %) transferred to Switzerland from hospitals in high MDRO prevalence areas are unnecessarily screened for MDRO colonization. Basing our screening strategy on certain criteria (such as presence of skin lesions, active infection, antibiotic treatment, history of a surgical procedure abroad and hospitalization outside of Europe) promises to be a better targeted and more cost-effective strategy.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Bacterial decontamination of surgical wounds treated with Lavasept

In a prospective randomized controlled double-blind study in 50 acutely injured patients, bacterially contaminated type 2-4 soft tissue wounds were treated with moist dressings of 0.2% Lavasept (fractionated polyhexamethylenbiguanide and macrogolum 4000) solution (n=28) in comparison with Ringer solution (n=22). Standardized swabs were taken on days 0, 2, 8 and 15 and investigated for microorganisms. For a quantitative evaluation, the number of colony forming units (CFU) was determined by a serial dilution technique. The tissue compatibility and anti-inflammatory effect were rated on a scale of 0 (=bad) to 3 (=very good). The most frequently found microorganism was Staphylococcus aureus, which was isolated from 13 wounds. Use of Lavasept led to a faster and significant reduction in microorganisms on the wound surfaces. The number of CFU per wound remained constant or decreased, in contrast to the wounds treated with Ringer solution. This was true for both Gram-positive and Gram-negative bacteria. There was no evidence of impaired wound healing in either group. The anti-inflammatory effect and the tissue compatibility of Lavasept were rated significantly better than that of Ringer solution. It is concluded that Lavasept combines antiseptic action with good tissue compatibility.

Potent and broad-spectrum antimicrobial activity of CXCL14 suggests an immediate role in skin infections

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous gram-positive bacteria and Candida albicans as well as the gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human beta-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.

Bacteria causing bacteremia in pediatric cancer patients presenting with febrile neutropenia – species distribution and susceptibility patterns

PURPOSE Infections are a major cause of morbidity and mortality in pediatric cancer patients. The aim of this study was to establish the microbiological spectrum and the susceptibility patterns of bacteremia-causing bacteria in pediatric cancer patients with febrile neutropenia in relation to the use of prophylactic and empirical antibiotics. METHODS We analyzed positive blood cultures of pediatric cancer patients presenting with febrile neutropenia between 2004 and 2011 in Groningen and Amsterdam (the Netherlands) and in Bern (Switzerland), using different antibiotic prophylactic and empirical regimens. RESULTS A total of 156 patients with 202 bacteremias, due to 248 bacteria species, were enrolled. The majority (73%) of bacteremias were caused by Gram-positive bacteria. Gram-negative bacteria, especially Pseudomonas aeruginosa, were observed significantly more often in Bern, where no fluoroquinolone prophylaxis was used. Ciprofloxacin-resistant bacteria were cultured more often from patients who did receive ciprofloxacin prophylaxis, compared to the patients who did not (57 versus 11%, p = 0.044). CONCLUSIONS Gram-positive bacteria predominated in this study. We showed that the use of prophylactic antibiotics in pediatric cancer patients was associated with increased resistance rates, which needs further study. The strategy for empiric antimicrobial therapy for febrile neutropenia should be adapted to local antibiotic resistance patterns.

Seasonal controls on grassland microbial biogeography: Are they governed by plants, abiotic properties or both?

Temporal dynamics create unique and often ephemeral conditions that can influence soil microbial biogeography at different spatial scales. This study investigated the relation between decimeter to meter spatial variability of soil microbial community structure, plant diversity, and soil properties at six dates from April through November. We also explored the robustness of these interactions over time. An historically unfertilized, unplowed grassland in southwest Germany was selected to characterize how seasonal variability in the composition of plant communities and substrate quality changed the biogeography of soil microorganisms at the plot scale (10 m x 10 m). Microbial community spatial structure was positively correlated with the local environment, i.e. physical and chemical soil properties, in spring and autumn, while the density and diversity of plants had an additional effect in the summer period. Spatial relationships among plant and microbial communities were detected only in the early summer and autumn periods when aboveground biomass increase was most rapid and its influence on soil microbial communities was greatest due to increased demand by plants for nutrients. Individual properties exhibited varying degrees of spatial structure over the season. Differential responses of Gram positive and Gram negative bacterial communities to seasonal shifts in soil nutrients were detected. We concluded that spatial distribution patterns of soil microorganisms change over a season and that chemical soil properties are more important controlling factors than plant density and diversity. Finer spatial resolution, such as the mm to cm scale, as well as taxonomic resolution of microbial groups, could help determine the importance of plant species density, composition, and growth stage in shaping microbial community composition and spatial patterns. (C) 2014 The Authors. Published by Elsevier Ltd. All rights reserved.

CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection

CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.

Subtyping schizophrenia: A comparison of positive/negative and system-specific approaches

BACKGROUND Schizophrenia is a heterogeneous disorder. Over the years, different approaches have been proposed to approach this heterogeneity by categorizing symptom patterns. The study aimed to compare positive/negative and system-specific approaches to subtyping. METHODS We used the Positive and Negative Syndrome Scale (PANSS) and Bern Psychopathology Scale (BPS), which consists of subscales for three domains (language, affect and motor behavior) that are hypothesized to be related to specific brain circuits, to assess cross-sectional psychopathological characteristics in a sample of 100 inpatients with schizophrenia spectrum disorders. We then categorized participants into positive/negative and system-specific subgroups to allow comparisons of the two approaches. RESULTS The analyses revealed correlations between the PANSS positive subscore and the BPS affective subscore (r=.446, p<.001) and between the PANSS negative subscore and the BPS motor behavior subscore (r=.227, p=.023). As regards the positive and negative subtype, more participants were classified as positive in the language-dominant subtype (30.3%) and affect-dominant subtype (30.3%), whereas more were classified as negative in the motor behavior-dominant subtype (44.4%). However, most patients met the criteria for the mixed subtype. CONCLUSIONS The results suggest that the positive/negative and system-specific approaches can be regarded as complementary. Future studies should examine both approaches in a longitudinal assessment of psychopathological symptoms and link them with qualitative-phenomenological approaches.

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

Rapid qualitative urinary tract infection pathogen identification by SeptiFast real-time PCR

Background Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical. Aim To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast®) and to compare the results with dipslide and microbiological culture. Design of study Pilot study with prospectively collected urine samples. Setting University hospital. Methods 82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast®) was performed in all samples. Results 61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results. Conclusion The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods.

Role of Toll interleukin-1 receptor (IL-1R) 8, a negative regulator of IL-1R/Toll-like receptor signaling, in resistance to acute Pseudomonas aeruginosa lung infection

Toll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulator in vivo under different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused by Pseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acute P. aeruginosa infection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense against P. aeruginosa acute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8-/- IL-1RI-/- double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused by P. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.