During the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a ...modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples.
Comparison of our results with those of a conventional culture-based method revealed a sensitivity of 96% (initial sensitivity of 82%) and specificity of 98%. Furthermore, only one cross-reaction was observed upon investigating 102 culture isolates from 70 untargeted bacteria. The total assay time was only three hours, including the time required for the DNA extraction, PCR and microarray steps in sequence.
The assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.
High incidence of septic patients increases the pressure of faster and more reliable bacterial identification methods to adapt patient management towards focused and effective treatment options. The ...aim of this study was to assess two automated DNA extraction solutions with the PCR and microarray-based assay to enable rapid and reliable detection and speciation of causative agents in the diagnosis of sepsis.
We evaluated two automated DNA instruments NucliSENS® easyMAG® and NorDiag Arrow for the preparation of blood culture samples. A set of 91 samples flagged as positive during incubation was analyzed prospectively with the high-throughput generation of Prove-it™ Sepsis assay designed to identify over 60 gram-negative and gram-positive bacterial species as well as methicillin resistance marker from a blood culture. Bacterial findings were accurately reported from 77 blood culture samples, whereas 14 samples were reported as negative, containing bacteria not belonging to the pathogen panel of the assay. No difference was observed between the performance of NorDiag Arrow or NucliSENS® easyMAG® with regard to the result reporting of Prove-it™ Sepsis. In addition, we also assessed the quality and quantity of DNA extracted from the clinical Escherichia coli isolate with DNA extraction instruments. We observed only minor differences between the two instruments.
Use of automated and standardized sample preparation methods together with rapid, multiplex pathogen detection offers a strategy to speed up reliably the diagnostics of septic patients. Both tested DNA extraction devices were shown to be feasible for blood culture samples and the Prove-it™ Sepsis assay, providing an accurate identification of pathogen within 4.5 hours when the detected pathogen was in the repertoire of the test.
Hypervirulent Clostridium difficile clade has been shown to include several lineages of ribotype 027 and also other ribotypes. We present data on additional non-027 strains, identified as presumptive ...027 by two commercial molecular C. difficile assays.
The tested clinical isolates were selected from the national reference laboratory collection on the basis of toxin gene profile similarities with ribotype 027 and tested with XpertC. difficile/Epi and Amplidiag C. difficile+027 assay.
Xpert misclassified five ribotypes (016, 019, 080, 176 and variant of type 046) as presumptive 027 and Amplidiag two ribotypes (016, 176). The misclassified strains were rare, covering 1.6% of reference laboratory strain collection.
Our findings confirm the concept that there are closely related outliers to hypervirulent 027 clones that can be misclassified as 027, and that these comprise numerous ribotypes, including previously reported four ribotypes (198, 176, 244, 019), and additional three (016, v046, 080) identified in the present study.
Low levels of oxygen (O2) in the hypolimnion layer of lakes are harmful to benthic animals and fish; they may also adversely affect nutrient cycles. Artificial aeration is often used in lake ...management to counteract these problems, but the effects of aeration on nitrogen (N) cycling are not known. We studied the effects of hypolimnetic aeration on N dynamics and temperature in a eutrophic lake by comparing continuous and pulsed aeration with a nonaerated station. Aeration decreased the accumulation of NH4+–N deep in the lake (20–33 m) by supplying O2 for nitrification, which in turn provided substrate for denitrification and promoted N removal. Aeration also increased the temperature in the hypolimnion. Denitrification rate was highest in the nonaerated deep areas (average, 7.62 mg N m−2 d−1) due to very high rates during spring turnover of the water column, demonstrating that natural turnover provides O2 for nitrification. During stratification, denitrification was highest at the continuously aerated station (4.06 mg N m−2 d−1) and lowest at the nonaerated station (3.02 mg N m−2 d−1). At the periodically aerated station, aeration pauses did not restrict the increase in temperature but resulted in accumulation of NH4+–N and decreased the contribution of denitrification as a nitrate reduction process. Our findings demonstrate that hypolimnetic aeration can substantially affect N cycling in lakes and that the effect depends on the aeration strategy. Because N is one of the main nutrients controlling eutrophication, the effects of aeration methods on N removal should be considered as part of strategies to manage water quality in lakes.
Core Ideas
Continuous hypolimnetic aeration prevented NH4+ accumulation in a lake at 20–33 m depth.
Continuous aeration promoted denitrification by enabling nitrification.
Periodical aeration did not restrict temperature changes, but NH4+ was accumulated.
Denitrification rate decreased during aeration pauses.
Benthic N removal in a lake can be enhanced via hypolimnetic aeration.
Diurnal variation in the density, biomass, and individual size of the predatory cladoceran Leptodora kindtii in Lake Vesijärvi (southern Finland) was studied. Day and night sampling was conducted in ...July and August at 16 different study stations with net hauls from the bottom to the surface. Independently on the month, estimated biomasses were more than two times higher at night than at day. This was due to both higher density and larger individual length at night. The day‐night differences could not be explained by vertical or horizontal migrations or by bottom‐dwelling behavior by Leptodora, but were attributed to net avoidance. When light was present, more Leptodora were able to avoid the net haul than in the darkness. The avoidance was more effective by large adult individuals (>5 mm) than by juveniles, which amplified the day‐night difference in the population biomass estimate. In order to evaluate the role of large pelagic invertebrate predators in aquatic food webs, nighttime sampling should be used.
Summary Background New DNA-based microarray platforms enable rapid detection and species identification of many pathogens, including bacteria. We assessed the sensitivity, specificity, and turnaround ...time of a new molecular sepsis assay. Methods 2107 positive blood-culture samples of 3318 blood samples from patients with clinically suspected sepsis were investigated for bacterial species by both conventional culture and Prove-it sepsis assay (Mobidiag, Helsinki, Finland) in two centres (UK and Finland). The assay is a novel PCR and microarray method that is based on amplification and detection of gyrB, parE , and mecA genes of 50 bacterial species. Operators of the test assay were not aware of culture results. We calculated sensitivity, specificity, and turnaround time according to Clinical and Laboratory Standards Institute recommendations. Findings 1807 of 2107 (86%) positive blood-culture samples included a pathogen covered by the assay. The assay had a clinical sensitivity of 94·7% (95% CI 93·6–95·7) and a specificity of 98·8% (98·1–99·2), and 100% for both measures for meticillin-resistant Staphylococcus aureus bacteraemia. The assay was a mean 18 h faster than was the conventional culture-based method, which takes an additional 1–2 working days. 34 of 3284 (1·0%) samples were excluded because of technical and operator errors. Interpretation Definitive identification of bacterial species with this microarray platform was highly sensitive, specific, and faster than was the gold-standard culture-based method. This assay could enable fast and earlier evidence-based management for clinical sepsis. Funding None.
We present a method for efficient air bubble removal in microfluidic applications. Air bubbles are extracted from a liquid chamber into a vacuum chamber through a semipermeable membrane, consisting ...of PDMS coated with amorphous Teflon(®) AF 1600. Whereas air is efficiently extracted through the membrane, water loss is greatly reduced by the Teflon even at elevated temperatures. We present the water loss and permeability change with the amount of added Teflon AF to the membrane. Also, we demonstrate bubble-free, multiplex DNA amplification using PCR in a PDMS microfluidic device.
Abstract Objective Probiotics may have potency in reducing upper respiratory infections, in particular in children. We studied findings from middle ear effusion (MEE) samples after randomized, ...placebo-controlled 3-week oral administration of probiotic Lactobacillus rhamnosus GG (L. GG). Methods 40 children referred to tympanostomy were randomized to receive either L. GG or placebo (1:1) for 3 weeks before surgery. MEE samples were collected from 13 children (in total, 25 samples, 19 from the L. GG group and 6 from the placebo group) and analyzed for L. GG and pathogenic bacterial and viral findings. Results L. GG was present in 5 of the 25 MEE samples (4 from the L. GG group). Haemophilus infuenzae was the most prominent pathogen in 12 samples (10 from the L. GG group). Rhinovirus was present in 12 samples (10 from the L. GG group) and enterovirus in 1 sample (L. GG group). Conclusions L. GG was present in the middle ear of children suffering from otitis media with effusion, but did not reduce the presence of pathogenic bacteria or viruses.
PDF
