Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture.

AIMS: We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials and assay methods on false-negative rate (FNR) and limit of detection (LOD95 ) for recovering Bacillus spores using a macrofoam-swab sampling procedure. METHODS AND RESULTS: Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2-500 per coupon) onto glass, stainless steel, vinyl tile and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD95 results. CONCLUSIONS: Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD95 was lowest for glass and highest for vinyl tile. LOD95 values overall were lower for mRV-PCR than for the culture method. SIGNIFICANCE AND IMPACT OF STUDY: This study adds to the limited data on FNR and LOD95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis.

False-negative rate, limit of detection and recovery efficiency performance of a validated macrofoam-swab sampling method for low surface concentrations of Bacillus anthracis Sterne and Bacillus atrophaeus spores.

AIMS: We sought to evaluate the effects of Bacillus species, low surface concentrations, and surface material on recovery efficiency (RE), false-negative rate (FNR) and limit of detection for recovering Bacillus spores using a validated macrofoam-swab sampling procedure. METHODS AND RESULTS: The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target surface concentrations (2 to 500 CFU per plate or coupon) to positive-control plates and test coupons (25·8064 cm(2) ) of four surface materials (glass, stainless steel, vinyl tile and plastic). The Bacillus species and surface material had statistically significant effects on RE, but surface concentration did not. Mean REs were the lowest for vinyl tile (50·8% with BAS and 40·2% with BG) and the highest for glass (92·8% with BAS and 71·4% with BG). FNR values (which ranged from 0 to 0·833 for BAS and from 0 to 0·806 for BG) increased as surface concentration decreased in the range tested. Surface material also had a statistically significant effect on FNR, with FNR the lowest for glass and highest for vinyl tile. Finally, FNR tended to be higher for BG than for BAS at lower surface concentrations, especially for glass. CONCLUSIONS: Concentration and surface material had significant effects on FNR, with Bacillus species having a small effect. Species and surface material had significant effects on RE, with surface concentration having a nonsignificant effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide valuable information on the performance of the macrofoam-swab method for low surface concentrations of Bacillus spores, which can be adapted to assess the likelihood that there is no contamination when all macrofoam-swab samples fail to detect B. anthracis.

Remote-sensing technique for determination of the volume absorption coefficient of turbid water.

We use remote-sensing reflectance from particulate R(rs) to determine the volume absorption coefficient a of turbid water in the 400 < lambda < 700-nm spectral region. The calculated and measured values of a(lambda) show good agreement for 0.5 < a < 10 (m(-1)). To determine R(rs) from a particulate, we needed to make corrections for remote-sensing reflectance owing to surface roughness S(rs). We determined the average spectral distribution of S(rs) from the difference in total remote-sensing reflectance measured with and without polarization. The spectral shape of S(rs) showed an excellent fit to theoretical formulas for glare based on Rayleigh and aerosol scattering from the atmosphere.

Effect of suspended particulate and dissolved organic matter on remote sensing of coastal and riverine waters.

We use remote sensing reflectance (RSR) together with the inherent optical properties of suspended particulates to determine the backscattering ratio b(b)/b for coastal waters. We examine the wavelength dependence of b(b)(lambda) and f(lambda)/Q(lambda) and establish the conditions when C(lambda) in RSR(lambda) approximately or = C(lambda)b(b)(lambda)/a(lambda) can be treated as a constant. We found that for case 2 waters, RSR was insensitive to the natural fluctuations in particle-size distributions. The cross-sectional area of the suspended particulate per unit volume, x(g), showed an excellent correlation with the volume scattering coefficient.

Structural and functional analysis of the S1 subunit of pertussis toxin using synthetic peptides.

Pertussis toxin (PT) is a major virulence factor of Bordetella pertussis, and also an important protective antigen. PT is an oligomeric A-B type toxin in which the S1 subunit has the ADP-ribosyltransferase activity whereas the B-oligomer mediates its binding to target cell receptors. To analyze the immunological properties of S1 and to generate probes to localize and characterize S1 functional domains, we synthesized four sets of peptides and peptide analogs corresponding to potentially critical regions of the S1 subunit. Two peptide-KLH conjugates were found to be capable of inducing PT-neutralizing antibodies in rabbits as judged by the CHO cell clustering assay. These peptides comprise residues 1-18 (N18-S1) and 121-138 (NAD-S1), respectively. Immunization with the unconjugated C-terminal peptide C35-S1 (residues 201-235) in the presence of Freund's adjuvant also elicited PT-neutralizing antibodies, indicating that the C-terminal region of S1 contains a potent functional T-helper cell epitope. Using truncated peptide analogs of N18-S1, we have demonstrated that the first three N-terminal residues are essential for inducing neutralizing antibodies. The NAD-S1 peptide elicited a neutralizing antibody response when coupled to KLH via its N-terminal end but not via its C-terminal residue. Identification of these B-cell neutralization epitopes represents a first step towards the rational design of a synthetic vaccine against whooping cough.

Identification of a potent synthetic HIV1 immunogen compromising gag-P24 tandem T- and B-cell epitopes.

Recent studies indicate that the gag gene products may play a crucial role in the immune response against HIV infection since clinical progression to AIDS is associated with a reduction in the level of circulating antibodies to gag p24 and antibodies raised against p17 peptide can inhibit HIV1 infection in vitro. Using conventional structure prediction algorithms for T-cell and B-cell epitopes, we have selected and chemically synthesized several gag peptides. In particular, an unconjugated HIV1-p24 peptide containing both B- and T-cell epitopes in tandem plus Freund's adjuvant induced a strong antibody response in both mice and rabbits against p24 and its precursor p55 as judged by immunoblotting. In addition, the peptide presented in the appropriate MHC context was shown to be highly stimulatory for p24 specific murine T-cell clones.

Computerized 3-D reconstruction of small blood vessels from high voltage electron-micrographs of thick serial cross sections.

Morphological analysis of compartments in terms of volumes and the orientation of the cells and their nuclei within very small mesenteric arteries and arterioles is being carried out. Samples were from simultaneously sham operated and instrumented control and one-clip two kidney Goldblatt sustained (30 days) hypertensive dogs. Collection of specimens was standardized, i.e. sampling location, perfusion fixation, embedding, staining, and sectioning by methods proven to preserve in vivo dimensions. Serial thick cross sections (0.5 mu) were subjected to high voltage electromicrography (1.0 MeV) at 2500x and then linearly enlarged 3x photographically. The glossy micrographs (7500x) were made into montages and the contours of the smooth muscle layer (vascular smooth muscle), the endothelium (E), nuclei (N), and the internal lamina (IL) were digitized on a large GTCO digi-pad with input to a microcomputer (PC-Limited, Turbo) equipped with a 20 MB hard disk and coprocessor. Solid and transparent image reconstructions and computerized dissection of cell compartments were performed. Compartment volumes of vascular smooth muscle, IL, E and N, and individual cell and nuclear volumes, as well as nuclear/cytoplasmic ratios were calculated. Image reconstructions are presented. The emphasis is on methods and these are discussed in terms of useability for determining accurate microvessel morphology in health and disease, with particular emphasis on assessment of hypertrophy and hyperplasia in experimental hypertension, and architectural angiogenesis in general.

Remote sensing of particulate concentrations in water.

Determination of residual radiance is essential in remote sensing measurements of suspended solids in the absence of real-time ground-truth data. We present some typical background corrections for Lake Superior and determine the spectral distribution of the residual radiance from three major categories of turbidity in the lake. The results tested with Landsat 2 digital tape data indicate that for large bodies of water general information on atmospheric scattering, water clarity, and optical properties of suspended solids enables one to estimate the concentrations of particulates to within +/-0.5 mg/liter in the absence of real-time ground-truth data.

Analysis of suspended solids by single-particle scattering.

Light scattering by individual particulates is used in a multiple-detector system to categorize the composition of suspended solids in terms of broad particulate categories. The scattering signatures of red clay and taconite tailings, the two primary particulate contaminants in western Lake Superior, along with two types of asbestiform fibers, amphibole and chrysotile, were studied in detail. A method was developed to predict the concentration of asbestiform fibers in filtration plant samples for which electron microscope analysis was done concurrently. Fiber levels as low as 5 x 10(4) fibers/liter were optically detectable. The method has application in optical categorization of samples for remote sensing purposes and offers a fast, inexpensive means for analyzing water samples from filtration plants for specific particulate contaminants.

Remote sensing of snow albedo for determination of dustfall.

Changes in snow reflectivity as a function of dustfall were used to measure dust deposition rates in terms of satellite reflectivity data. The results show that for dry snow conditions, snow reflectivity can be approximated by Kubelka-Munk theory. Dustfall above threshold values of 10 microg/cm(2) can be detected using Landsat computer tape data. Determination of dust deposition over ice-covered lakes and harbors appears in good agreement with sampling measurements.