Detection of Bovine Antibodies against a Conserved Capsid Epitope as the Basis of a Novel Universal Serological Test for Foot-and-Mouth Disease.

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral nonstructural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus (FMDV) serotype and their sensitivity may be affected by antigenic variability within each serotype and mismatching between test reagents. As a consequence, FMD reference laboratories are required to maintain multiple type-specific SP assays and reagents. A universal SP test would simplify frontline diagnostics and facilitate large-scale serological surveillance and postvaccination monitoring. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterized using a panel of intertype-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide enzyme-linked immunosorbent assay (VP2-ELISA) was optimized using experimental and reference antisera from immunized, convalescent, and naïve animals (n = 172). The VP2-ELISA is universal and simple and provided sensitive (99%) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for serosurveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in countries where the disease is endemic.

Inhibition of biofilms associated with dentures and toothbrushes by tetrasodium EDTA.

AIMS: We examined the efficacy of tetrasodium EDTA in eradicating biofilms derived from salivary inocula or pure cultures of Candida albicans on discs of polymethyl methacrylate (PMMA) denture base or on toothbrushes that had been used normally for 4-8 weeks. Its efficiency in virus neutralization was also determined. METHODS AND RESULTS: Overnight (16 h) treatment with 4% (w/v) tetrasodium EDTA solution reduced salivary and C. albicans biofilm viable counts by > or =99%. Biofilm removal was confirmed using confocal laser scanning microscopy. Presence/absence of sucrose during biofilm formation had no effect on killing efficacy. Prolonged treatment of PMMA with tetrasodium EDTA did not influence subsequent formation of C. albicans biofilms or affect surface roughness of the PMMA, but it reduced subsequent biofilm formation from a salivary inoculum. Infectivities of herpes simplex virus and polio virus suspensions were reduced by >99.99% by treatment for 1 and 2 h, respectively. CONCLUSIONS: Tetrasodium EDTA solution efficiently disinfected toothbrushes and PMMA discs, with the detachment of biofilms, and rapidly neutralized both nonenveloped and enveloped viruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Dentures and toothbrushes become contaminated by bacterial biofilms and by viruses. There is a need for disinfection methods that are rapidly effective, cost-effective, nontoxic and easily implemented. These studies indicate that tetrasodium EDTA solution has disinfection applications in the oral care field.

Volume flow measurement using Doppler and grey-scale decorrelation.

A technique for volumetric blood flow measurement was developed by combining standard Doppler measurements with grey-scale decorrelation. Steered Doppler is used to determine the in-plane velocities, which are then used to extract the out-of-plane velocities from the temporal A-line decorrelation. As a result, a three-dimensional (3-D) vector flow field can be computed over the imaging plane using a single clinical transducer without knowledge of the vessel orientation. Volume flow is computed by integrating the out-of-plane flow over the vessel cross-section. The algorithm was tested using a scattering-enhanced fluid in a 6.4-mm diameter dialysis tubing. For a wide range of transducer angles, the volume flow was accurately measured to within 28% in these preliminary tests.

US artifacts: effects on out-of-plane us images reconstructed from three-dimensional data sets.

Three-dimensional volumetric data sets of stacked ultrasonographic (US) scans were obtained in vitro and in vivo to investigate the effect of common US artifacts on cross-sectional images reconstructed out-of-plane to the plane of acquisition of these data sets. The appearance of the artifacts on the reconstructed images was different from that on the source images. Such artifacts have the potential to simulate pathologic abnormalities.

Sequence, expression and reconstitution of an HCV genome from a British isolate derived from a single blood donation.

Morphological analysis of hepatitis C virus and development of antiviral drugs to eradicate this agent have been seriously hampered by the low viraemias observed during natural infection and the unavailability of a cell culture system for virus propagation. Recently a low-grade hepatitis has been reported in chimpanzees after intrahepatic transfection of full-length synthetic HCV RNA and successful infections shown to be critically dependent on the integrity and genetic homogeneity of the reconstituted clone. In this study we describe and characterize a full HCV RNA sequence derived from a case of chronic sporadic hepatitis. The genotype was shown to be 1a with a low level of intraclonal sequence heterogeneity, and processing of both structural and nonstructural proteins has been documented. The assembly of the full genome has also been achieved. The low level of intraclonal variation observed may reflect infection with a single isolate and the fact that cloning was performed on virus obtained from a single blood donation makes this clone a good candidate for future in vivo and in vitro transfection studies.

Effect of spectacular reflection on out-of-plane ultrasonographic images reconstructed from three-dimensional data sets.

The effect of specular reflection on ultrasonographic images reconstructed out of plane to the plane of acquisition of a three-dimensional volumetric data set was studied using two in vitro phantoms that incorporated structures exhibiting specular reflection. The phantoms were scanned transversely (axially) to form three-dimensional data sets, with coronal cross-sectional images reconstructed perpendicular to the plane of acquisition of the data sets. Directly scanned, nonreconstructed coronal images of the phantoms also were obtained in the same planes and from the same areas as the reconstructed coronal images. The direct and reconstructed coronal images were compared. Owing to the inherent directionality of specular reflectors, the reconstructed coronal images differed from the directly scanned images in two ways, containing some hyperechoic regions that were not present at direct coronal scanning and failing to contain other hyperechoic areas that were present at direct coronal imaging. We conclude that sonographic images reformatted from volumetric data sets may have a different appearance than images scanned directly in the same plane, independent of other factors such as resolution. This should be taken into account when such reformatted images are interpreted.

Speckle decorrelation flow measurement with B-mode US of contrast agent flow in a phantom and in rabbit kidney.

PURPOSE: To use speckle decorrelation in the presence of ultrasonographic (US) contrast agent as an alternative flow measurement technique to Doppler US. MATERIALS AND METHODS: In vivo and in vitro studies were performed. A tube with flowing saline solution containing contrast agent was positioned horizontally across a US image. The amount of decorrelation between a series of images was recorded. The flow profile across the tube was generated by averaging the decorrelation values and was compared with a Doppler frequency shift image. In addition, B-mode images of six rabbit kidneys were obtained during and after intravenous injection of contrast agent. Images were analyzed to compute the correlation between successive points in time. RESULTS: The velocity profiles across the tube were parabolic, with the fastest flow rates measured in the center of the tube. In the rabbit kidneys, measurements indicated the largest decorrelation rates occurred in the larger vessels. The cortical decorrelation rates were significantly slower than those for the hilar vessels (P < .05) and were relatively angle independent. CONCLUSIONS: Decorrelation flow measurements can be used to estimate flow in vitro and in vivo similar to measurements obtained with Doppler US but with less angle dependence. These measurements could lead to a US perfusion technique.

Frequency analysis of echo texture in tendon.

A texture discriminant based on spatial frequencies is proposed for characterizing B-scans of Achilles' tendon. The anisotropic echo texture of normal tendon has an ellipsoidal spatial spectrum that can be quantified by the ratio of the major-to-minor axis and by the direction of the major axis. Applying a moving window to the B-scan, a corresponding tissue elliptical axis ratio (TEAR) image is derived that segments out tendon. The algorithm was applied to B-scan images taken from 13 volunteers, 6 of whom had tendon abnormalities: tendon rupture (n = 3) or cholesterol deposits (xanthomas) in patients with heterozygous familial hypercholesterolemia (n = 3). The average TEAR value was 1.75 +/- 0.17 for normal tendon, 1.04 +/- 0.06 for torn tendon, and 1.31 +/- 0.16 for tendons with xanthomas. The dispersion of the directionality vectors was used to further differentiate tendons with xanthomas from normal tendons. This technique appears to be useful for characterizing both diffuse and focal tendon abnormalities.

Automated three-dimensional US frame positioning computed from elevational speckle decorrelation.

For ultrasonographic B-scan images collected by means of a handheld transducer moving in the elevational direction, frame spacings are computed with a speckle-decorrelation algorithm, without additional positioning hardware. Fully developed speckle volumes are automatically segmented and spacing computed from the decorrelation curves. Position accuracy is within 10% for phantoms and 15% for breast studies. The algorithm provides image-based registration, which allows accurate three-dimensional volume rendering.

Ultrasound properties of liver with and without particulate contrast agents.

Basic acoustic parameters are examined in rabbit liver with and without a solid contrast agent used for tumor detection. In normal liver, backscatter, attenuation, and sound speed are found to decrease with increasing water content. The addition of micron-sized particles made from iodipamide ethyl ester (IDE) can increase backscatter and attenuation depending on size and concentration. A discrepancy of the increased backscatter from theoretical predictions based on random scatterers is attributed to the particle's biodistribution in the liver.

Ultrasound contrast for hepatic tumors using IDE particles.

Iodipimide ethyl ester (IDE) can be formulated as dense spherical particles with narrow diameter distribution. When IDE particles are injected intravenously, the Kupffer cells of the hepatic sinusoids accumulate particles within 10 to 20 minutes, after which the clearance and excretion of IDE takes place. During the uptake phase, the dense particles act as scattering sites, increasing the echogenicity of normal liver tissue. In comparison, tumors and other lesions remain at pre-injection echogenicity, as they lack Kupffer cells and therefore do not retain particles. This report provides initial studies of contrast enhancement in rabbit livers with implanted VX2 tumors, scanned in vivo and evaluated ex vivo using pulse-echo techniques. The distribution of particles within hepatic lobules may explain why the observed echogenicity is greater than that predicted by single-particle backscatter theory. Directions for future improvements are discussed.

Liver glycogen and water storage: effect on ultrasound attenuation.

Glycogen has been shown in vitro to have a high specific absorption coefficient (ultrasound absorption in water per concentration) compared to other proteins. Depending on the amount of water which may accompany glycogen flux into and out of liver cells, the ultrasound attenuation coefficient of the liver may rise and fall with stored hepatic glycogen. This paper reports ex vivo studies on rats and in vivo studies on normal human volunteers before and after fasting. The results show a statistically significant difference in liver attenuation between well fed and fasted individuals. Generally, the attenuation difference is greater than 10%, and indicates that liver wet weight may not be strictly constant over glycogen storage cycles, as suggested in classic works. In contrast, no significant change in ultrasound backscatter is noted. The results point to the possible role of ultrasound attenuation measurements as a sensitive indicator of tissue physiology, and suggest that glycogen (feeding or fasting) must be controlled in tissue characterization experiments which compare liver attenuation coefficients of individuals and groups.

Deviations from Rayleigh statistics in ultrasonic speckle.

The statistics of speckle patterns in ultrasound images have potential for tissue characterization. In "fully developed speckle" from many random scatterers, the amplitude is widely recognized as possessing a Rayleigh distribution. This study examines how scattering populations and signal processing can produce non-Rayleigh distributions. The first order speckle statistics are shown to depend on random scatterer density and the amplitude and spacing of added periodic scatterers. Envelope detection, amplifier compression, and signal bandwidth are also shown to cause distinct changes in the signal distribution.

The role of glycogen and phosphate in ultrasonic attenuation of liver.

The excess ultrasonic attenuation caused by adding glycogen and inorganic phosphate to liver homogenates has been studied to determine the underlying differences between attenuation coefficients of normal and diffusely diseased livers. Results show that glycogen has a higher than average specific absorption coefficient compared to other large molecular weight biomolecules. Since the glycogen content of liver can vary from 1%-10% of wet weight, this compound may have a major time-varying effect on the liver ultrasonic attenuation coefficient, even in normal subjects. In contrast, the excess attenuation of liver homogenate resulting from addition of inorganic phosphate was not significant at presumed physiological levels. The implications of these findings are discussed relative to tissue characterization efforts based on in vivo measurements of ultrasonic attenuation coefficients of liver.

A particulate contrast agent with potential for ultrasound imaging of liver.

Ultrasonic backscatter and attenuation coefficients of a medium can be increased by the addition of solid, micron sized inhomogeneities. A potentially useful agent for ultrasonic contrast of liver images has been identified. Iodipamide ethyl ester (IDE) particles can be produced in the form of dense, relatively incompressible solids with high impedance mismatch to water. The chemical, biomechanical, and pharmacological properties of the small, uniform diameter IDE particles permit safe intravenous injection followed by rapid accumulation by reticuloendothelial (RE) cells of the liver and spleen, and later elimination from these organs. Since the particles are phagocytized by RE cells, present in normal liver but not in tumors and many lesions, the selective enhancement of ultrasonic backscatter should improve detectability of lesions which are hypo- or iso-echoic compared to surrounding tissue. The mechanisms of particle-ultrasound interaction may be described by relative motion attenuation, and scattering from a cloud of dense, incompressible spheres for the case of IDE particles in agar. Thus, values of attenuation and backscatter can be controlled by choice of ultrasound frequency and particle concentration and size. When the particles are accumulated in rat livers, additional mechanisms induce attenuation and backscatter in excess of that predicted by IDE in agar. This preliminary work demonstrates that solid, biocompatible particles may be useful as an ultrasonic contrast agent.