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.

An optimized in vitro model of the respiratory tract wall to study particle cell interactions

As a part of the respiratory tissue barrier, lung epithelial cells play an important role against the penetration of the body by inhaled particulate foreign materials. In most cell culture models, which are designed to study particle-cell interactions, the cells are immersed in medium. This does not reflect the physiological condition of lung epithelial cells which are exposed to air, separated from it only by a very thin liquid lining layer with a surfactant film at the air-liquid interface. In this study, A549 epithelial cells were grown on microporous membranes in a two chamber system. After the formation of a confluent monolayer the cells were exposed to air. The morphology of the cells and the expression of tight junction proteins were studied with confocal laser scanning and transmission electron microscopy. Air-exposed cells maintained monolayer structure for 2 days, expressed tight junctions and developed transepithelial electrical resistance. Surfactant was produced and released at the apical side of the air-exposed epithelial cells. In order to study particle-cell interactions fluorescent 1 microm polystyrene particles were sprayed over the epithelial surface. After 4 h, 8.8% of particles were found inside the epithelium. This fraction increased to 38% after 24 h. During all observations, particles were always found in the cells but never between them. In this study, we present an in vitro model of the respiratory tract wall consisting of air-exposed lung epithelial cells covered by a liquid lining layer with a surfactant film to study particle-cell interactions.

Oral purified bacterial extracts in acute respiratory tract infections in childhood: a systematic quantitative review

BACKGROUND: Recurrent acute respiratory tract infections (ARTI) are a common problem in childhood. Some evidence suggests a benefit regarding the prevention of ARTI in children treated with the immunomodulator OM-85 BV (Bronchovaxom). METHODS: We summarised the evidence on the effectiveness of the immunomodulator OM-85 BV in the prevention of ARTI in children. We searched randomised comparisons of oral purified bacterial extracts against inactive controls in children with respiratory tract diseases in nine electronic databases and reference lists of included studies. We extracted salient features of each study, calculated relative risks (RR) or weighted mean differences (WMD) and performed meta-analyses using random-effects models. RESULTS: Thirteen studies (2,721 patients) of low to moderate quality tested OM-85 BV. Patients and outcomes differed substantially, which impeded pooling results of more than two trials. Two studies (240 patients) reporting on the number of patients with less than three infections over 6 month of follow-up in children not in day care showed a trend for benefit RR 0.82 (95% CI, 0.65-1.02). One out of two studies examining the number of children not in day care without infections over 4-6 month reported a significant RR of 0.42 (95% CI, 0.21-0.82) whereas the smaller, second study did not [RR 0.92 (95% CI, 0.58-1.46)]. Two studies reporting the number of antibiotic courses indicated a benefit for the intervention arm [WMD 2.0 (95% CI, 1.7-2.3)]. Two out of the three studies showed a reduction of length of episodes of 4-6 days whereas a third study showed no difference between the two groups. CONCLUSION: Evidence in favour of OM-85 BV in the prevention of ARTI in children is weak. There is a trend for fewer and shorter infections and a reduction of antibiotic use.

Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy

ABSTRACT: Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions.This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

Susceptibility of Escherichia coli strains isolated from outpatient children with community-acquired urinary tract infection in southern Switzerland

BACKGROUND: Based on antimicrobial resistance patterns found in Swiss university hospitals, treatment with a third-generation cephalosporin is currently advised for Swiss children with urinary tract infection. OBJECTIVE: The aim of this study was to prospectively assess the susceptibility of Escherichia coli strains isolated from children with symptomatic community-acquired urinary tract infection. METHODS: The antimicrobial susceptibility of E coli strains causing symptomatic community-acquired urinary tract infections was assessed in outpatient children attending the emergency management unit at the Department of Pediatrics, Mendrisio and Bellinzona Hospitals, Switzerland. Strains from children receiving antimicrobial prophylaxis or prescribed antimicrobials in the previous 4 weeks were excluded. Clinical and Laboratory Standards Institute methods were used for culture and identification of pathogens. E coli susceptibility testing was performed using the disk diffusion technique. RESULTS: Strains from 100 consecutive outpatient children (73 girls, 27 boys; aged 5 weeks-17 years [median, 33 months]; 100% white) were assessed. High rates of ampicillin and cotrimoxazole resistance (39 and 21 strains, respectively) and low rates of nitrofurantoin resistance (4 strains) were identified. No resistance was identified for coamoxiclav or third-generation cephalosporins. CONCLUSIONS: In these Swiss outpatient children with symptomatic community-acquired urinary tract infection, without antimicrobial prophylaxis or recent prescription of antimicrobials, uropathogenic E coli strains resistant in vitro to ampicillin and cotrimoxazole were common. However, in vitro resistance to nitrofurantoin, coamoxiclav, and third-generation cephalosporins was uncommon.

Translocation and cellular entering mechanisms of nanoparticles in the respiratory tract

Anthropogenic nano-sized particles (NSP), ie, particles with a diameter of less than 100 nm, are generated with or without purpose as chemically and physically well-defined materials or as a consequence of combustion processes respectively. Inhalation of NSP occurs on a regular basis due to air pollution and is associated with an increase in respiratory and cardiovascular morbidity and mortality. Manufactured NSP may intentionally be inhaled as pharmaceuticals or unintentionally during production at the workplace. Hence the interactions of NSP with the respiratory tract are currently under intensive investigation. Due to special physicochemical features of NSP, its biological behaviour may differ from that of larger sized particles. Here we review two important themes of current research into the effects of NSP on the lungs: 1) The potential of NSP to cross the blood-air barrier of the lungs, thus gaining access to the circulation and extrapulmonary organs. It is currently accepted that a small fraction of inhaled NSP may translocate to the circulation. The significance of this translocation requires further research. 2) The entering mechanisms of NSP into different cell types. There is evidence that NSP are taken up by cells via well-known pathways of endocytosis but also via different mechanisms not well understood so far. Knowledge of the quantitative relationship between the different entering mechanisms and cellular responses is not yet available but is urgently needed in order to understand the effects of intentionally or unintentionally inhaled NSP on the respiratory tract.

Physiologic cold shock increases adherence of Moraxella catarrhalis to and secretion of interleukin 8 in human upper respiratory tract epithelial cells

Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid and prolonged downshifts of environmental temperature when humans breathe cold air. In the present study, we show that a 26 degrees C cold shock up-regulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, by prolonging messenger RNA half-life. Cold shock promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin, an extracellular matrix component that mediates bacterial attachment. Exposure of M. catarrhalis to 26 degrees C increases the outer membrane protein-mediated release of the proinflammatory cytokine interleukin 8 in pharyngeal epithelial cells. Furthermore, cold shock at 26 degrees C enhances the binding of salivary immunoglobulin A on the surface of M. catarrhalis. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C affects the nasopharyngeal host-pathogen interaction and may contribute to M. catarrhalis virulence.

Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization

BACKGROUND: Cystic fibrosis (CF) is associated with the appearance of serum autoantibodies directed against bactericidal/permeability-increasing protein (BPI). OBJECTIVES: To determine the age-specific seroprevalence rates of anti-BPI-IgG and IgA in a population of patients with CF and to correlate anti-BPI antibody concentrations with microbial respiratory tract colonization and pulmonary function variables at the time of serum sampling and 6 years thereafter. METHODS: Determination of BPI antibodies of the IgG and IgA isotypes using a commercial enzyme-linked immunosorbent assay in sera of a CF serum bank of 1992; correlation of anti-BPI antibody concentrations with age, clinical score, pulmonary function variables in 1992 and 1998, total serum immunoglobulin isotype concentrations and respiratory tract colonization with Pseudomonas aeruginosa and Aspergillus spp. RESULTS: Seventy-one patients (age in 1992, 14.1 +/- 7.5 years) were studied. Reactivities for anti-BPI-IgG and IgA were found in 28 (39%) and 26 (37%) patients, respectively. The seroprevalence of anti-BPI-IgA, but not IgG, increased significantly with age. P. aeruginosa colonization was associated with elevated concentrations of anti-BPI-IgG (P = 0.003) and IgA (P = 0.037). There were significant negative correlations between pulmonary function variables (vital capacity, forced expiratory volume in 1 s) in 1992 and 1998, respectively, and concentrations of anti-BPI-IgG or IgA in a multiple regression analysis. Anti-BPI-IgG, but not IgA, remained significantly associated with P. aeruginosa colonization (P = 0.006) and with reduced vital capacity (P = 0.01) in 1998 after correction for total serum isotype concentration. CONCLUSIONS: Anti-BPI-IgG are strongly associated with concurrent P. aeruginosa colonization and with long term restrictive pulmonary function abnormalities.

Human coronavirus NL63 associated with lower respiratory tract symptoms in early life

Coronavirus NL63 has been identified as a new member of the coronavirus genus, but its role as a cause of respiratory disease needs to be established. We studied the first episode of lower respiratory tract symptoms in a cohort of healthy neonates. NL63 was identified in 6 (7%) of 82 cases and was as frequent as other coronaviruses (9%). NL63 was recovered at the onset of symptoms and was cleared within 3 weeks in half of the cases. Our data suggests that coronavirus NL63 causes lower respiratory tract symptoms and is acquired in early life.