Comparison of the FilmArray RP, Verigene RV+, and Prodesse ProFLU+/FAST+ multiplex platforms for detection of influenza viruses in clinical samples from the 2011-2012 influenza season in Belgium.

Respiratory tract infections (RTIs) are caused by a plethora of viral and bacterial pathogens. In particular, lower RTIs are a leading cause of hospitalization and mortality. Timely detection of the infecting respiratory pathogens is crucial to optimize treatment and care. In this study, three U.S. Food and Drug Administration-approved molecular multiplex platforms (Prodesse ProFLU+/FAST+, FilmArray RP, and Verigene RV+) were evaluated for influenza virus detection in 171 clinical samples collected during the Belgian 2011-2012 influenza season. Sampling was done using mid-turbinate flocked swabs, and the collected samples were stored in universal transport medium. The amount of viral RNA present in the swab samples ranged between 3.07 and 8.82 log10 copies/ml. Sixty samples were concordant influenza A virus positive, and 8 samples were found to be concordant influenza B virus positive. Other respiratory viruses that were detected included human rhinovirus/enterovirus, respiratory syncytial virus, parainfluenza virus type 1, human metapneumovirus, and coronavirus NL63. Twenty-five samples yielded discordant results across the various assays which required further characterization by sequencing. The FilmArray RP and Prodesse ProFLU+/FAST+ assays were convenient to perform with regard to sensitivity, ease of use, and low percentages of invalid results. Although the limit of sensitivity is of utmost importance, many other factors should be taken into account in selecting the most convenient molecular diagnostic assay for the detection of respiratory pathogens in clinical samples.

Analysis of interactions of signaling proteins with phage-displayed ligands by fluorescence correlation spectroscopy.

Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed.

Sampling and detection of airborne influenza virus towards point-of-care applications.

Airborne transmission of the influenza virus contributes significantly to the spread of this infectious pathogen, particularly over large distances when carried by aerosol droplets with long survival times. Efficient sampling of virus-loaded aerosol in combination with a low limit of detection of the collected virus could enable rapid and early detection of airborne influenza virus at the point-of-care setting. Here, we demonstrate a successful sampling and detection of airborne influenza virus using a system specifically developed for such applications. Our system consists of a custom-made electrostatic precipitation (ESP)-based bioaerosol sampler that is coupled with downstream quantitative polymerase chain reaction (qPCR) analysis. Aerosolized viruses are sampled directly into a miniaturized collector with liquid volume of 150 µL, which constitutes a simple and direct interface with subsequent biological assays. This approach reduces sample dilution by at least one order of magnitude when compared to other liquid-based aerosol bio-samplers. Performance of our ESP-based sampler was evaluated using influenza virus-loaded sub-micron aerosols generated from both cultured and clinical samples. Despite the miniaturized collection volume, we demonstrate a collection efficiency of at least 10% and sensitive detection of a minimum of 3721 RNA copies. Furthermore, we show that an improved extraction protocol can allow viral recovery of down to 303 RNA copies and a maximum sampler collection efficiency of 47%. A device with such a performance would reduce sampling times dramatically, from a few hours with current sampling methods down to a couple of minutes with our ESP-based bioaerosol sampler.

DNA computing using single-molecule hybridization detection.

DNA computing aims at using nucleic acids for computing. Since micromolar DNA solutions can act as billions of parallel nanoprocessors, DNA computers can in theory solve optimization problems that require vast search spaces. However, the actual parallelism currently being achieved is at least a hundred million-fold lower than the number of DNA molecules used. This is due to the quantity of DNA molecules of one species that is required to produce a detectable output to the computations. In order to miniaturize the computation and considerably reduce the amount of DNA needed, we have combined DNA computing with single-molecule detection. Reliable hybridization detection was achieved at the level of single DNA molecules with fluorescence cross-correlation spectroscopy. To illustrate the use of this approach, we implemented a DNA-based computation and solved a 4-variable 4-clause instance of the computationally hard Satisfiability (SAT) problem.

The effect of exchange of bacteriopheophytin a with plant pheophytin a on charge separation in Y(M210)W mutant reaction centers of Rhodobacter sphaeroides at low temperature.

The bacteriopheophytin a molecules at the H(A) and H(B) binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H(A)(-), presumably by raising the free energy level of the P(+)B(A)(-) state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B(A). Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H(A), inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P(+)B(B)(-) state, where B(B) is the accessory bacteriochlorophyll in the "inactive" B-branch of the cofactors. The B(B) molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q(y) region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P(+)B(B)(-) state and absorption properties of the B(B) bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the B(B) binding pocket.

Magneto-capillary valve for integrated purification and enrichment of nucleic acids and proteins.

We describe the magneto-capillary valve (MCV) technology, a flexible approach for integrated biological sample preparation within the concept of stationary microfluidics. Rather than moving liquids in a microfluidic device, discrete units of liquid are present at fixed positions in the device and magnetic particles are actuated between the fluids. The MCV concept is characterized by the use of two planar surfaces at a capillary mutual distance, with specific features to confine the fluids by capillary forces, and the use of a gas or a phase-change material separating the stationary aqueous liquids. We have studied the physics of magneto-capillary valving by quantifying the magnetic force as a function of time and position, which reveals the balance of magnetic, capillary and frictional forces in the system. By purification experiments with a fluorescent tracer we have measured the amount of co-transported liquid, which is a key parameter for efficient purification. To demonstrate the versatility of the technology, several MCV device architectures were tested in a series of biological assays, showing the purification and enrichment of nucleic acids and proteins. Target recovery comparable to non-miniaturized commercial kits was observed for the extraction of DNA from human cells in buffer, using a device architecture with patterned air valves. Experiments using an enrichment module and patterned air valves demonstrate a 40-fold effective enrichment of DNA in buffer. DNA was also successfully purified from blood plasma using paraffin phase-change valves. Finally, the enrichment of a protein biomarker (prostate-specific antigen) using geometrical air valves resulted in a 7-fold increase of detection signal. The MCV technology is versatile, offers extensive freedom for the design of fully integrated systems, and is expected to be manufacturable in a cost-effective way. We conclude that the MCV technology can become an important enabling technology for point-of-care systems with sample in-result out performance.

Quantifying the composition of human skin for glucose sensor development.

BACKGROUND: Glucose is heterogeneously distributed within human skin. In order to develop a glucose measurement method for human skin, both a good quantification of the different compartments of human skin and an understanding of glucose transport processes are essential. This study focused on the composition of human skin. In addition, the extent to which intersubject variability in skin composition alters glucose dynamics in human skin was investigated. METHODS: To quantify the composition of the three layers of human skin-epidermis, dermis, and adipose tissue-cell and blood vessel volumes were calculated from skin biopsies. These results were combined with data from the literature. The composition was applied as input for a previously developed computational model that calculates spatiotemporal glucose dynamics in human skin. The model was used to predict the physiological effects of intersubject variability in skin composition on glucose profiles in human skin. RESULTS: According to the model, the lag time of glucose dynamics in the epidermis was sensitive to variation in the volumes of interstitial fluid, cells, and blood of all layers. Data showed most variation/uncertainty in the volume composition of the adipose tissue. This variability mainly influences the dynamics in the adipose tissue. CONCLUSIONS: This study identified the intersubject variability in human skin composition. The study shows that this variability has significant influence on the glucose dynamics in human skin. In addition, it was determined which volumes are most critical for the quantification and interpretation of measurements in the different layers.

Effects of systemic administration of 0.5% tropicamide on intraocular pressure, pupillary diameter, blood pressure, and heart rate in normal cats.

OBJECTIVE: The objective of the study was to determine the effects of systemic 0.5% tropicamide on intraocular pressure (IOP), pupillary diameter (PD), blood pressure, and heart rate (HR) in normal felines with normotensive eyes. PROCEDURES: Intraocular pressure, PD, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and HR were measured in 18 clinically healthy cats. Each of the previously mentioned parameters was measured every 30 min during the trial period. At T(60), each cat was treated with one to two drops of 0.5% tropicamide ophthalmic solution placed on the dorsal aspect of the tongue. Changes in SBP, DBP, MBP, and HR were evaluated using one-way repeated measures analysis of variance, with time as the repeated factor. IOP and PD were evaluated using two-way repeated measures analysis of variance, with time and side (OD vs. OS) as the repeated factors. P values less than or equal to 0.05 were considered statistically significant. RESULTS: After lingual tropicamide administration, the mean PD at T(60) was 3.53 mm OD and 3.53 mm OS. The mean PD at T(90) was 6.36 mm OD and 6.31 mm OS. The mean PD at T(120) was 8.25 mm OD and 8.19 mm OS. This change in PD from T(60), T(90), and T(120) was statistically significant, demonstrating a linear increase in PD over time after tropicamide application on the tongue (P<0.0001). There was no statistically significant difference in PD when comparing the right to the left pupils (P=0.10). The mean IOP at T(60) was 14 mmHg OD and 12.94 mmHg OS. The mean IOP at T(90) was 14.5 mmHg OD and 14.23 mmHg OS. The mean IOP at T(120) was 14.94 mmHg OD and 14.89 mmHg OS. This change in IOP from T(60), T(90), and T(120) was statistically significant, demonstrating a linear increase in IOP over time after tropicamide application on the tongue (P=0.034). There was no statistically significant difference in IOP when comparing the right eye to the left eye (P=0.28). There were no statistically significant differences in SBP, DBP, MBP, and HR values over time for the duration of the study. CONCLUSIONS: We conclude that although lingual application of tropicamide appears to result in systemic absorption, causing significant pupillary dilation and elevations in IOP, systemic effects on SBP, DBP, MBP, and HR were not observed.

Canine ocular onchocerciasis in the United States: two new cases and a review of the literature.

Since 1991, 53 cases of canine ocular onchocerciasis have been reported in the literature worldwide, 43 of these were from Greece, five from Hungary, and five from the western United States. Information on the histopathologic features of canine ocular onchocerciasis is limited. We describe the histopathologic features of canine ocular onchocerciasis in two dogs from California that presented clinically with firm episcleral nodules and uveitis unilaterally. Pertinent literature and pathogenesis are reviewed; recognizable clinical features and treatment are discussed. The cases presented were diagnosed via histopathology of the enucleated globes and episcleral granulomas at the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW). Positive identification of adult Onchocerca within episcleral granulomas was made based on light-microscopy features. Histopathologic examination of both globes revealed episcleral parasites surrounded by granulomas containing few to moderate numbers of eosinophils. Other sequelae, in both cases, included lymphoplasmacytic uveitis, preiridal fibrovascular membranes, peripheral anterior synechiae, retinal degeneration, and optic nerve head cupping. Both male and female worms were present, as were in utero microfilariae in both cases. Worms in both cases were tentatively identified as Onchocerca lienalis. Ocular onchocerciasis should be a differential consideration in cases of canine conjunctival nodules or periorbital swelling, particularly in dogs from the western United States.