Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. MATERIAL AND METHODS: Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. RESULTS: Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. CONCLUSION: The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

Abdominal image segmentation using three-dimensional deformable models.

RATIONALE AND OBJECTIVES: The authors develop a three-dimensional (3-D) deformable surface model-based segmentation scheme for abdominal computed tomography (CT) image segmentation. METHODS: A parameterized 3-D surface model was developed to represent the human abdominal organs. An energy function defined on the direction of the image gradient and the surface normal of the deformable model was introduced to measure the match between the model and image data. A conjugate gradient algorithm was adapted to the minimization of the energy function. RESULTS: Test results for synthetic images showed that the incorporation of surface directional information improved the results over those using only the magnitude of the image gradient. The algorithm was tested on 21 CT datasets. Of the 21 cases tested, 11 were evaluated visually by a radiologist and the results were judged to be without noticeable error. The other 10 were evaluated over a distance function. The average distance was less than 1 voxel. CONCLUSIONS: The deformable model-based segmentation scheme produces robust and acceptable outputs on abdominal CT images.

Short- and long-term efficacy of single-dose subunit vaccines against Yersinia pestis in mice.

A single, subcutaneous, 30-microg dose of either a combination of the Yersinia pestis proteins F1+V or a F1-V fusion protein adsorbed to the adjuvant aluminum hydroxide, protected Hsd:ND4 mice for one year against pneumonic plague. The recombinant F1+V vaccine provided significant protection as early as day 14 postimmunization. The current Plague Vaccine USP in a single 0.2-ml dose did not provide significant protection in this mouse model. Antibody titers to F1 and V peaked at approximately 5-12 weeks postimmunization and were still detectable one year later. These F1 and V subunit vaccines may offer effective long-term immunity with a reduced dosage schedule when compared with the presently licensed, formalin-killed, whole-cell vaccine.

Cloning and expression of the gene for an immunoglobulin G Fc receptor protein from a group A streptococcus.

DNA containing the 5' end of the M-12 structural gene was used as a probe in colony hybridizations in an attempt to clone the M-76 gene from an M-type 76 group A streptococcal strain. A single positive colony was detected, and Southern hybridization analysis of plasmid DNA isolated from this colony indicated that the insert DNA had homology to the 5' end of the M-12 structural gene. Subclones were constructed to define the limits of the M-76 gene, and sonicates of these subclones were reacted with M-76-specific antiserum in immunodiffusion. A sonicate of one subclone, JM83(pDH56), reacted strongly with the M-76-specific antiserum but also reacted with preimmune rabbit serum. Protein expressed from this subclone bound immunoglobulin from horse and pig, as well as human myeloma immunoglobulin G (IgG) representing all four subclasses and purified human IgG Fc fragments. This indicated that JM83(pDH56) expressed a protein with characteristics previously attributed to the IgG Fc receptor protein from group A streptococci. Western blot analysis indicated that the cloned IgG Fc receptor protein had a molecular weight of approximately 29,000. Binding studies showed that the Fc receptor gene is expressed by the M-type 76 strain from which it was cloned and by an M- variant.

Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

The partial nucleotide sequence for an Fc-receptor gene from an M-type 76 group A streptococcus was determined. DNA sequence analysis revealed considerable sequence similarity between the Fc-receptor and M-protein genes in their proposed promoter regions, signal sequences, and 3' termini. Additional analysis indicated that the deduced Fc-receptor protein contains a proline-rich region and membrane anchor region highly similar to that of M protein. In view of these results, we postulated that Fc-receptor and M-protein genes of group A streptococci are the products of gene duplication from a common ancestral gene. It is proposed that DNA sequence similarity between these two genes may allow for extragenic homologous recombination as a means of generating antigenic diversity in these two surface proteins.

Construction of recombination-deficient strains of Streptococcus gordonii by disruption of the recA gene.

Degenerate oligonucleotide primers were used in a polymerase chain reaction (PCR) to amplify a region of the recA sequence of Streptococcus gordonii Challis. The resulting PCR fragment was cloned into the suicide vector pAM6199 and introduced into strain Challis, giving rise to recombination-deficient strains in which the recA gene was specifically inactivated.

Architecture of the vir regulons of group A streptococci parallels opacity factor phenotype and M protein class.

Group A streptococci have traditionally been categorized into two broad groups based on the presence or absence of serum opacity factor (OF). Recent studies show that these two groups vary in a number of properties in addition to the OF phenotype, including sequence variations in the constant region of the antiphagocytic M protein genes, the presence or absence of immunoglobulin G Fc receptor proteins, and the presence or absence of multiple M protein-like genes situated in a tandem array. The M protein genes (emm) in OF- streptococcal strains are known to be part of a regulon of virulence-related genes controlled by the trans-acting positive regulatory gene, virR, situated just upstream of emm. In OF+ strains, however, the region adjacent to virR is occupied by an M protein-related, type IIa immunoglobulin G Fc receptor gene (fcrA), and the relative position of emm has not been determined. To further define the vir regulon in OF+ streptococci, we used the polymerase chain reaction to show that fcrA49 is situated immediately upstream of emm49 in the OF+ type 49 strain CS101. This result shows for the first time the separate identity and genetic linkage of these two genes in the vir regulon of an OF+ group A streptococcal strain and confirms our previous hypothesis that emm49 exists as the central gene in a trio of emm-like genes. Additionally, using DNA hybridizations, we found considerable sequence divergence between OF- and OF+ group A streptococci in virR and in the noncoding sequences between virR and the emm or fcrA expression site. We found, however, a high degree of sequence conservation in this region within each of the two groups of strains.

Virtual angioscopy using spiral CT and real-time interactive volume-rendering techniques.

Our purpose was to describe a technique for visualizing the inner contours of the vasculature using contrast enhanced spiral CT and volume rendering techniques. Because the technique is similar to using a camera to look inside vessels, we call this technique "virtual angioscopy." Preliminary results suggest virtual angioscopy using volumetric 3D rendering techniques as a potentially useful technique for the noninvasive evaluation of vascular pathology.

Isolated DNA repeat region from fcrA76, the Fc-binding protein gene from an M-type 76 strain of group A streptococci, encodes a protein with Fc-binding activity.

The DNA repeat region of fcrA76, the gene encoding a group A streptococcal Fc-binding protein, was subcloned in-frame into an Escherichia coli plasmid expression vector. The expressed protein product displayed the same Fc-binding properties as the full-length Fc-binding protein expressed from fcrA76. The affinity-purified, full-length Fc-binding protein was found to compete with staphylococcal protein A or streptococcal protein G for binding to beads coated with human IgG. These results are consistent with earlier studies suggesting that the binding sites on human IgG for protein A, protein G and the type II Fc-binding protein from group A streptococci are located at the interface of the CH2 and CH3 domains of the Fc region.

Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region.

pAD1 (60 kb) is a conjugative, hemolysin/bacteriocin plasmid in Enterococcus faecalis. It confers a mating response to the peptide sex pheromone cAD1 produced by recipient (plasmid-free) cells, leading to highly efficient plasmid transfer in broth matings. Control of the physiological response to cAD1 can been overridden by a reversible phase variation event at frequencies on the order of 10(-4) to 10(-3) per cell per generation (L. T. Pontius and D. B. Clewell, Plasmid 26:172-185, 1991). The variant forms are designated Dryc and Dry+, which reflects the colony morphologies of cells whose conjugation functions are switched on and off, respectively. Here we show that Dryc variants exhibit a structural change in a region between repA and repB that contains two clusters of 8-bp iterons. The change involved a 31- or 32-bp increase in size of this region. In three or four independent variants examined, one of the iteron clusters increased in size from 13 to 17 iterons. When iteron DNA was placed on a multicopy plasmid and introduced into a wild-type pAD1 derivative, the Dryc phenotype was generated. Since traA, a key negative regulator of conjugation, bears several centrally located iteron-like sequences with the same orientation, we speculate that the protein(s) that normally binds iterons (possibly RepA and/or RepB) blocks traA transcription in Dryc variants.

Automatic liver segmentation technique for three-dimensional visualization of CT data.

PURPOSE: To develop a system for automatic segmentation of the liver from computed tomographic (CT) scans of the abdomen for three-dimensional volume-rendering displays. MATERIALS AND METHODS: An automated liver segmentation system was developed, which combined domain knowledge with analysis of a global histogram, morphologic operators, and the parametrically deformable contour model. Boundaries of the thresholded liver volume were modified section-by-section by exploiting information from adjacent sections. These boundaries were refined by optimization of the parametrically deformable contour model. Volume-rendered images were created by using the boundaries to exclude tissues outside the liver. The system was tested on CT data sets from 10 cases of potentially resectable hepatic neoplasm. RESULTS: Of the 401 sections in the 10 cases, 53 sections (13.2%) required user modifications during segmentation. The utility of the three-dimensional-rendered images with use of these liver boundaries was judged by a radiologist as being comparable to that of three-dimensional images created with manual editing. Twenty-eight of the sections were deemed imperfect by the radiologist and might need further modifications. CONCLUSION: An effective technique for automatic segmentation of the liver from CT images has been developed. This technique promises to save time and simplify the creation of three-dimensional liver images by minimizing operator intervention.

Dual-phase spiral CT angiography with volumetric 3D rendering for preoperative liver transplant evaluation: preliminary observations.

PURPOSE: The goal of our study was to determine whether dual-phase spiral CT angiography with 3D volume rendering could be used for preoperative evaluation and patient selection for orthotopic liver transplantation candidates. METHOD: Fifty consecutive potential candidates for liver transplantation were evaluated with dual-phase spiral CT with 3D volume rendering. Intravenous contrast medium was administered as bolus peripheral injection at 3 ml/s. The protocol consisted of a contrast-enhanced dual-phase spiral CT (arterial phase acquisition at 30 s after initiation of contrast medium injection followed by portal venous phase beginning at 60 s) with scan parameters of 0.75 s gantry rotation speed, 3 mm collimation, 5 to 6 mm/s table speed, and reconstruction at 1 mm intervals for arterial-phase images and 3 mm collimation for portal venous-phase studies (Siemens Plus 4 scanner; Siemens Medical Systems, Iselin, NJ, U.S.A.). All scan information was sent to a free-standing workstation (Silicon Graphics Onyx or Infinite Reality, Mountain View, CA, U.S.A.) for interactive real-time 3D volume rendering using a customized version of the Volren volume renderer (Silicon Graphics; Advanced Imaging Laboratory, Johns Hopkins Medical Institutions, Baltimore, MD, U.S.A.). The arterial phase was used to create vascular maps of the celiac axis including the origin(s) of the hepatic artery and origin of the superior mesenteric artery. The portal phase was used to define portal venous patency as well as the hepatic venous anatomy. All images were analyzed for vascular patency, shunting, or collateralization as well as the status of the underlying liver (i.e., liver size, cirrhosis, tumor, etc.). RESULTS: All 50 studies were successfully completed without complication. The 3D CT angiograms defined key arterial and venous structures including origin(s) of the hepatic artery, portal vein and/or superior mesenteric vein thrombosis, cavernous transformation of the portal vein, and/or other collateral vasculature. Ten patients (20%) demonstrated anomalous anatomy at the origin(s) of the hepatic artery. Portal vein thrombosis with cavernous transformation of the portal vein was shown in six patients, and there were three cases of partial venous thrombosis. Underlying liver tumors as well as parenchymal liver disease were well defined. Hepatic masses were found in five patients. Masses were pathologically proven as hepatocellular carcinoma (n = 1), giant cavernous hemangioma (n = 1), hepatic adenoma (n = 1), and focal nodular hyperplasia (n = 2). CONCLUSION: Preliminary results suggest that dual-phase spiral CT with CT angiography can provide a comprehensive preoperative liver transplant evaluation, supplying the necessary information for patient selection and surgical planning. As a single, minimally invasive examination, this should significantly impact patient care by minimizing procedures and avoiding potential complications.

CT angiography with volume rendering for quantifying vascular stenoses: in vitro validation of accuracy.

OBJECTIVE: The purpose of this study was to evaluate the accuracy of CT angiography with volume rendering for quantifying vascular stenoses in vitro. MATERIALS AND METHODS: Vascular models with three degrees of stenosis (33%, 67%, and 83%) were imaged at three orientations to the axial plane (parallel, perpendicular, or 45 degrees ) using helical CT with 2-mm collimation and two pitches (1 or 2), two reconstruction intervals (1 or 2 mm), and two scan times (.75 or 1 sec). Diameter and percentage of stenosis were measured from volume renderings using full width at half maximum. Images were measured in two planes whenever resolution varied with direction. Statistical analysis was performed using analysis of variance. RESULTS: Mean absolute error of the measured percentage of stenosis was 7% (range, 0-27%). The actual percentage of stenosis and vessel orientation had the most significant effects on accuracy (p < .001). The measured percentage of stenosis was significantly less accurate with phantoms parallel to the axial plane than with other orientations (p < .001). Mean absolute error in the measured percentage of stenosis was 4% when the parallel-to-the-axial-plane orientation was excluded. Overlapping (1-mm) reconstructions were significantly more accurate than 2-mm reconstructions (p < .05) and direction of measurement significantly affected accuracy (p < .05), but these effects were secondary. CONCLUSION: CT angiography with volume rendering can accurately quantify vascular stenoses, but it is less accurate for vessels in the axial plane. With 2-mm collimation, vessel characteristics have greater effects on accuracy than do acquisition parameters.

CT angiography with volume rendering: advantages and applications in splanchnic vascular imaging.

The authors compared volume rendering with maximum intensity projection (MIP) and shaded surface display as a technique for generating three-dimensional (3D) images of the vasculature from spiral computed tomography (CT) data sets. In four patients with pathologic splanchnic vasculature, the advantages of volume-rendered display are illustrated for depiction of 3D vascular anatomy, vascular and visceral interrelationships, variant vasculature, tumor encasement, and hepatic tumor localization for presurgical planning.

Skeletal 3-D CT: advantages of volume rendering over surface rendering.

Both surface rendering and volume rendering have been extensively applied to CT data for 3-D visualization of skeletal pathology. The review illustrates potential limitations of each technique by directly comparing 3-D images of bone pathology created using volume rendering and surface rendering. Surface rendering show gross 3-D relationships most effectively, but suffer from more stairstep artifacts and fail to effectively display lesions hidden behind overlying bone or located beneath the bone cortex. Volume-rendering algorithms effectively show subcortical lesions, minimally displaced fractures, and hidden areas of interest with few artifacts. Volume algorithms show 3-D relationships with varying degrees of success depending on the degree of surface shading and opacity. While surface rendering creates more three-dimensionally realistic images of the bone surface, it may be of limited clinical utility due to numerous artifacts and the inability to show subcortical pathology. Volume rendering is a flexible 3-D technique that effectively displays a variety of skeletal pathology with few artifacts.

Fraction 1 capsular antigen (F1) purification from Yersinia pestis CO92 and from an Escherichia coli recombinant strain and efficacy against lethal plague challenge.

As a first step in formulating an improved plague vaccine, we developed a simple purification strategy that produced high yields of pure cell-associated and culture supernatant-derived fraction 1 capsular antigen (F1) from both avirulent Yersinia pestis C092 (Pgm- Lcr-) and an Escherichia coli F1-producing recombinant strain. Cell-associated F1 was partially purified by sequential ammonium sulfate precipitations of a sodium chloride extract of acetone-dried bacteria harvested from broth cultures. Cell-free F1 was precipitated directly from culture supernatants with a single application of 30% ammonium sulfate. By exploiting the aggregative property of F1, large quantities of purified high-molecular-weight F1 species from both cell extracts and supernatants were isolated in the void volume of a preparative gel filtration column. Highly purified, endotoxin-free F1, combined with two different adjuvants, induced very high F1 titers in mice and protected them against either subcutaneous (70 to 100% survival) or aerosol (65 to 84% survival) challenge with virulent organisms. This protection was independent of the source of the antigen and the adjuvant used. F1-induced protection against both subcutaneous and aerosol challenge was also significantly better than that conferred by immunization with the licensed killed whole-cell vaccine. Our results indicate that F1 antigen represents a major protective component of previously studied crude capsule preparations, and immunity to F1 antigen provides a primary means for the host to overcome plague infection by either the subcutaneous or respiratory route.

Identification of two type IIa IgG-binding proteins expressed by a single group A streptococcus.

Functional heterogeneity associated with Ig-binding proteins expressed by group A streptococci is well documented. In this study we have demonstrated that treatment of group A streptococcal isolate 64/14 with CNBr resulted in the solubilization of two different sized proteins that displayed identical functional reactivity with human IgG1, IgG2, and IgG4 (characteristics of a type IIa binding protein). Monospecific polyclonal antibodies to each form of type IIa molecule were prepared and no antigenic cross-reactivity between the two m.w. forms of type IIa binding protein could be detected. The smaller m.w. protein was shown to be identical or closely related to the recombinant type IIa protein cloned from strain CS110. These studies provide further evidence for the heterogeneity of type II Ig-binding proteins expressed by pathogenic group A streptococci.

Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas.

RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulmonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms.

Three-dimensional CT stereoscopic visualization of renal masses: impact on diagnosis and patient treatment.

OBJECTIVE: The objective of this study was to determine whether three-dimensional reconstruction with stereoscopic display of helical CT data sets and CT angiography are useful in the examination of patients with known or suspected renal masses. CONCLUSION: Volume-rendering techniques applied to helical CT data sets coupled with three-dimensional stereoscopic imaging provide a complete examination of patients with known or suspected renal masses. Such information can help guide patient treatment and provide a single preoperative study when nephron-sparing surgery or total nephrectomy is considered.