Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of <it>Brucella </it>DNA.</p> <p>Methods</p> <p>Five kits were evaluated using clinical specimens: QIAamp™ DNA Mini Kit (QIAGEN), peqGold™ Tissue DNA Mini Kit (PeqLab), UltraClean™ Tissue and Cells DNA Isolation Kit (MoBio), DNA Isolation Kit for Cells and Tissues (Roche), and NucleoSpin™ Tissue (Macherey-Nagel). DNA yield was determined using a quantitative real-time PCR assay targeting IS<it>711 </it>that included an internal amplification control.</p> <p>Results</p> <p>Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p < 0.05) and of diagnostic relevance. Sample volume, elution volume, and processing time were also compared.</p> <p>Conclusions</p> <p>We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.</p

Diagnosis of recent and relapsed cases of human brucellosis by PCR assay

BACKGROUND: Brucellosis affects human populations in many developing countries including the Middle East, and Latin America where it is still endemic. It has been prevalent in Jordan for years, where 7842 cases of human brucellosis were registered at the Ministry of Health during 10 year-period. This study was initiated by the recent increase in the number of human cases diagnosed in a rural area in the Northern Jordan to help assess the status of the disease in that area. For this purpose blood specimens from brucellosis suspected cases were tested by serology, culture and PCR. METHODS: Peripheral blood specimens from 50 healthy control subjects and 165 seropositive patients having compatible signs and symptoms that were clinically diagnosed to have brucellosis were tested by blood culture, and by PCR. The PCR assay used genus-specific primers from the conserved region of the 16S rRNA sequence, which showed high specificity for the Brucella spp. RESULTS: Diagnosis of Brucella was established by PCR in 120 cases (72.7%). All of them were seropositive and 20 were positive by culture. Forty-eight of 58 (82.8%) of the relapsed cases two months after completing the treatment with an increase in the previous serological titers were positive by PCR. The assay has 85.7% positive predicative value, 100% sensitivity and specificity since it correctly identified all cases that were positive by blood cultures, 95.8% by serology and none of the control group was positive. CONCLUSIONS: Results showed that PCR assay can be applied with serology for the diagnosis of brucellosis suspected cases and relapses regardless of the duration or type of the disease without relying on the blood cultures, especially in chronic cases

Spectrum of enteropathogens detected by the FilmArray GI Panel in a multicentre study of community-acquired gastroenteritis

The European, multicentre, quarterly point-prevalence study of community-acquired diarrhoea (EUCODI) analysed stool samples received at ten participating clinical microbiology laboratories (Austria, Finland, France, Germany, Greece, Ireland, Italy, Portugal, Romania, and the UK) in 2014. On four specified days, each local laboratory submitted samples from &lt;= 20 consecutive patients to the Austrian Study Centre for further testing with the FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT, USA). Of the 709 samples from as many patients received, 325 (45.8%) tested negative, 268 (37.8%) yielded only one organism, and 116 (16.4%) yielded multiple organisms. Positivity rates ranged from 41% (30 of 73 samples) in France to 74% (59 of 80 samples) in Romania. With the exception of Entamoeba histolytica and Vibrio cholerae, all of the 22 targeted pathogens were detected at least once. Enteropathogenic Escherichia coli, Cam pylobacter species, toxigenic Clostridium difficile, enteroaggregative E. coli, norovirus and enterotoxigenic E coli were the six most commonly detected pathogens. When tested according to local protocols, seven of 128 positive samples (5.5%) yielded multiple organisms. Overall, the FilmArray GI Panel detected at least one organism in 54.2% (384/709) of the samples, as compared with 18.1% (128/709) when testing was performed with conventional techniques locally. This underlines the considerable potential of multiplex PCR to improve routine stool diagnostics in community-acquired diarrhoea. Classic culture methods directed at the isolation of specific pathogens are increasingly becoming second-line tools, being deployed when rapid molecular tests give positive results. This optimizes the yield from stool examinations and dramatically improves the timeliness of diagnosis. Clinical Microbiology and Infection (C) 2015 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.</p

Further investigation of the role of HLA-DPB1 in adult Hodgkin's disease (HD) suggests an influence on susceptibility to different HD subtypes

It has been suggested in a number of studies that susceptibility to adult Hodgkin's disease (HD) is influenced by the HLA class II region, and specifically by alleles at the HLA-DPB1 locus. Since HD is diagnostically complex, it is not clear whether different HLA-DPB1 alleles confer susceptibility to different HD subtypes. To clarify this we have extended a previous study to type DPB1 alleles in 147 adult HD patients from a single centre. We have analysed patients with nodular sclerosing (NS), mixed cellularity (MC) or lymphocyte predominant (LP) HD, and gender in relation to HLA-DPB1 type, in comparison with 183 adult controls. The results confirmed previously reported associations of DPB1*0301 with HD susceptibility (relative risk (RR) = 1.42; 95% confidence interval (CI) 0.86-2.36) and DPB1*0201 with resistance to HD (RR = 0.49; CI 0.27-0.90). However, analysis by HD subtype and gender showed that *0301-associated susceptibility was confined to females with HD (RR = 2.46; CI 1.02-5.92), and *0201-associated resistance to females with NS-HD (RR = 0.28; CI 0.10-0.79). Susceptibility to NS-HD was also associated in females with *1001 (RR = 11.73; CI 1.32-104.36), and resistance with *1101 (RR = 0.08; CI 0.01-0.65). In contrast, susceptibility to LP-HD was associated in males with *2001 (RR = 32.14; CI 3.17-326.17), and to MC-HD with *3401 (RR = 16.78; CI 2.84-99.17). Comparison of DPB1-encoded polymorphic amino-acid frequencies in patients and controls showed that susceptibility to MC-HD was associated with Leucine at position 35 of DPB1 (RR = 8.85; CI 3.04-25.77), Alanine-55 (RR = 15.17; CI 2.00-115.20) and Valine-84 (RR = 15.94; CI 3.55-71.49). In contrast, Glutamic acid 69 was significantly associated with resistance to MC-HD (RR = 0.14; CI 0.03-0.60). Certain DPB1 alleles and individual DPbeta1 polymorphic amino acid residues may thus affect susceptibility and resistance to specific HD subtypes. This may be through their influence on the binding of peptides derived from an HD-associated infectious agent, and the consequent effect on immune responses to the agent

Transfer vs. Breakup in the interaction of the 7Be Radioactive Ion Beam with a 58Ni target at Coulomb barrier energies

We measured for the first time 7Be elastically scattered nuclei as well as 3,4He reaction products from a 58Ni target at 22.3 MeV beam energy. The data were analyzed within the optical model formalism to extract the total reaction cross section. Extensive kinematical, Distorted Wave Born Approximation (DWBA)and Continuum Discretized Coupled Channel (CDCC) calculations were performed to investigate the 3,4He originating mechanisms and the interplay between different reaction channels

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Susceptibility breakpoints for amphotericin B and Aspergillus species in an in vitro pharmacokinetic-pharmacodynamic model simulating free-drug concentrations in human serum

Contains fulltext : 136894.pdf (publisher's version ) (Open Access)Although conventional amphotericin B was for many years the drug of choice and remains an important agent against invasive aspergillosis, reliable susceptibility breakpoints are lacking. Three clinical Aspergillus isolates (Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus) were tested in an in vitro pharmacokinetic-pharmacodynamic model simulating the biphasic 24-h time-concentration profile of free amphotericin B concentrations in human serum with free peak concentrations (fCmax) of 0.1, 0.3, 0.6, 1.2, and 2.4 mg/liter administered once daily. Drug concentrations were measured with a bioassay, and fungal growth was monitored for 72 h with galactomannan production. The fCmax/MIC corresponding to half-maximal activity (P50) was determined for each species, and the percentage of target attainment was calculated for different MICs for the standard (1 mg/kg of body weight) and a lower (0.6-mg/kg) dose of amphotericin B with Monte Carlo simulation analysis. The fCmax/MICs (95% confidence intervals) corresponding to P50 were 0.145 (0.133 to 0.158), 0.371 (0.283 to 0.486), and 0.41 (0.292 to 0.522) for A. fumigatus, A. flavus, and A. terreus, respectively. The median percentages of P50 attainment were >/=88%, 47%, and 0% for A. fumigatus isolates with MICs of /=2 mg/liter, respectively, and >/=81%, 24%, and 0% and >/=75%, 15%, and 0% for A. flavus and A. terreus isolates with MICs of /=1 mg/liter, respectively. The lower dose of 0.6 mg/kg would retain efficacy for A. fumigatus, A. flavus, and A. terreus isolates with MICs of /=2 mg/liter for A. fumigatus and /=1 mg/liter for A. flavus and A. terreus were determined for conventional amphotericin B with a pharmacokinetic-pharmacodynamic model simulating free-drug serum concentrations

Identification of Parametric Ground Motion Random Fields from Spatially Recorded Seismic Data

A methodology for the identification of random field models for the description of seismic ground motions recorded over extended areas is presented. The approach (i) embeds seismic ground motion parameter estimation in a probabilistic framework, and (ii) identifies simultaneously point and spatial models for the seismic ground motion random field from either smoothed or unsmoothed non-parametric spectral estimates. It is shown that parametric coherency estimates identified by means of this approach are stable and insensitive to the amount of smoothing performed on the empirical data. The approach and its findings have a significant impact on the evaluation of parametric ground motion models for the seismic response evaluation of lifelines