Most-probable-number rapid viability PCR method to detect viable spores of Bacillus anthracis in swab samples.

A comparison of Most-Probable-Number Rapid Viability (MPN RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs from a multi-center validation study was performed. The purpose of the study was to compare environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were typically within 1-log of the values from a plate count method for all three levels of spores tested (3.1x10(4), 400, and 40 spores sampled from surfaces with swabs) even in the presence of debris. The MPN method tended to overestimate the expected result, especially at lower spore levels. Blind negative samples were correctly identified using both methods showing a lack of cross contamination. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols, enhancing its utility for characterization and clearance following a biothreat agent release.

Rapid, high-throughput, culture-based PCR methods to analyze samples for viable spores of Bacillus anthracis and its surrogates.

To rapidly remediate facilities after a biothreat agent release, improved turnaround times are needed for sample analysis. Current methods to confirm the presence of a viable biothreat agent are limited by low sample throughput. We have developed a rapid-viability-polymerase chain reaction (RV-PCR) method to determine the presence of viable spores. The method combines high-throughput sample processing with 96-well PCR analysis, which measures a change in real-time, quantitative PCR response arising from increased target-cell populations during culturing. The method accurately detects 1 to 10 live spores in a high-dead spore background (10(6)). Field tests using approximately 1000 biological indicators, each containing 10(6) spores of the B. anthracis surrogate, Bacillus atrophaeus, exposed to seven lethal and sub-lethal chlorine dioxide levels showed no significant difference (p>0.05) between RV-PCR and standard culturing methods for detecting the percent survival of spores. RV-PCR results were obtained in <17 h compared to 7 days for the standard culturing method. High-throughput sample processing and RV-PCR protocols were also developed and tested for synthetic wipe samples containing reference dirt material. RV-PCR protocols allowed processing and accurate analysis of approximately100 dirty wipe samples (2''x2'' synthetic) containing approximately10 viable B. atrophaeus spores in <24 h. Quantitative RV-PCR protocols based on a Most-Probable-Number (MPN) statistical approach developed for B. anthracis Sterne resulted in more rapid turnaround times than those for traditional culturing and no significant difference in log colony-forming units compared to traditional viability analysis. Integration of RV-PCR assays with high-throughput protocols will allow the processing of 200 wipe samples per day per robot using commercially available automation.

An application of spatial deconvolution to a capillary-based high-pressure chamber for fluorescence microscopy imaging.

Capillary-based high-pressure chambers for which the wall serves as both the optical window and mechanical support have been reported for fluorescence microscopy imaging. Although capillary chambers are straightforward and economical to construct, the curved capillary wall introduces image aberrations. The significance of these aberrations in imaging sub-cellular-dimension objects has yet to be assessed. Using a capillary chamber that is routinely pressurized to between 20 and 30 MPa, a pressure range suitable for studying a wide variety of cellular processes, we demonstrate sub-cellular-dimension spatial resolution in the imaging of fluorescent micro-spheres. Objectives with a range of numerical apertures (0.5-1.3) and working distances (0.1-7.4 mm) are considered. We show that spatial (or point-spread function, PSF) deconvolution improves image contrast in capillary-based images by comparing deconvolution results with those obtained from slide-mounted controls. Furthermore, similar deconvolution results between a measured PSF and a calculated, flat-geometry PSF indicate that the capillary wall is optically flat on cellular length scales. Results here facilitate the application of contemporary techniques in fluorescence microscopy to high-pressure imaging fields.

Decontamination issues for chemical and biological warfare agents: how clean is clean enough?

The objective of this assessment is to determine what level of cleanup will be required to meet regulatory and stakeholder needs in the case of a chemical and/or biological incident at a civilian facility. A literature review for selected, potential chemical and biological warfare agents shows that dose information is often lacking or controversial. Environmental regulatory limits or other industrial health guidelines that could be used to help establish cleanup concentration levels for such agents are generally unavailable or not applicable for a public setting. Although dose information, cleanup criteria, and decontamination protocols all present challenges to effective planning, several decontamination approaches are available. Such approaches should be combined with risk-informed decision making to establish reasonable cleanup goals for protecting health, property, and resources. Key issues during a risk assessment are to determine exactly what constitutes a safety hazard and whether decontamination is necessary or not for a particular scenario. An important conclusion is that cleanup criteria are site dependent and stakeholder specific. The results of a modeling exercise for two outdoor scenarios are presented to reinforce this conclusion. Public perception of risk to health, public acceptance of recommendations based on scientific criteria, political support, time constraints, and economic concerns must all be addressed in the context of a specific scenario to yield effective and acceptable decontamination.

Prostatic cryosurgery: use of MR imaging in evaluation of success and technical modifications.

PURPOSE: To evaluate the usefulness of contrast material-enhanced magnetic resonance (MR) imaging in objective assessment of prostatic cryosurgery and the role of MR imaging in the modification of prostatic cryosurgical technique. MATERIALS AND METHODS: Thirty-eight consecutive patients with localized (T1-3, N0, M0) prostatic adenocarcinoma treated with prostatic cryosurgery underwent MR imaging without contrast enhancement before cryosurgery and unenhanced and gadolinium-enhanced MR imaging within 1-3 weeks after cryosurgery. The first 20 patients also underwent MR imaging at 3 months after cryosurgery. MR imaging findings were correlated with those from transrectal ultrasound-directed prostatic staging biopsy. RESULTS: Cryonecrotic prostate was identified as avascular regions characterized by absolute signal void on contrast-enhanced images. With progressive modification of cryosurgical technique, complete cryoablation of the prostate was achieved in the latter nine of the 38 patients. When cryoablation was considered complete according to MR imaging criteria, findings invariably correlated with those at biopsy, with no residual prostate tissue or tumor. CONCLUSION: Gadolinium-enhanced MR imaging of the prostate after cryosurgery provides a highly accurate means of monitoring success. Objective MR imaging findings allow modifications to the technology and technique, resulting in optimal therapeutic results with prostatic cryosurgery.

Asymmetric hearing loss: toward cost-effective diagnosis.

OBJECTIVE: Unilateral audiovestibular symptoms are commonly seen in clinical practice, and the treating physician frequently considers retrocochlear disease in the differential diagnosis. These considerations often precipitate a lengthy and expensive battery of tests. This study was conducted to evaluate the diagnostic yield of the various tests available in the investigation of these symptoms and to recommend changes to practice patterns, if appropriate. METHODS: We examined a cohort of 310 patients with unilateral audiovestibular symptoms who were referred to our radiology department for investigation. All patients had complete auditory assessments, and 144 underwent auditory evoked potential testing (ABR). There were 258 contrast-enhanced CT (CECT) scans and 86 gadolinium-enhanced MRI (Gd-MRI) scans performed. RESULTS: A total of 12 cerebellopontine angle (CPA) tumours were diagnosed. CONCLUSIONS: MRI is the diagnostic tool of choice in patients with asymmetric sensory neural hearing loss (ASHL), but it is expensive and not always easily available. Analysis of diagnostic accuracy of CECT and ABR, both used as screening tests, demonstrate their limitations and reveals that CECT is no more accurate than ABR, although it is three times more costly. Based on these data, we have changed our diagnostic approach to patients with unilateral audiovestibular symptoms. Patients are either evaluated with MRI or are screened initially with ABR based on clinical suspicion. CECT is no longer routinely included in the evaluation of patients with ASHL. Finally, newer emerging MRI techniques promise faster and less expensive examinations with increased diagnostic accuracy.

Patellar tendinitis: MR imaging features, with suggested pathogenesis and proposed classification.

PURPOSE: To characterize the magnetic resonance (MR) imaging features of patellar tendinitis. MATERIALS AND METHODS: Fifteen patients with a clinical diagnosis of patellar tendinitis underwent gadolinium-enhanced MR imaging of the knee. RESULTS: Grades of patellar abnormality, based on findings in the enthesial region at MR imaging, correlated with signs of increasing fibrovascular repair: grade 1 (n = 4), enhancing area adjacent to patellar apex, with marginal zone of intermediate signal intensity, and a patellar apical chondral-bone avulsion; grade 2 (n = 5), same signs as grade 1 damage but without avulsion; grade 3 (n = 6), homogeneous, nonenhancing area of intermediate signal intensity adjacent to the patellar apex seen on all images. Changes were most obvious posteriorly and involved the central and medial thirds of the tendon. Chronic injury to the medial retinaculum was a common associated finding. CONCLUSION: Patellar tendinitis demonstrates a consistent spectrum of changes at MR imaging that can aid understanding of the origin and treatment of damage.

Severity of disease score as a predictor of mortality after caval filter insertion.

PURPOSE: To estimate 30-day mortality after vena caval filter insertion and assess the usefulness of a severity of disease score in predicting postprocedure 30-day survival. PATIENTS AND METHODS: Records of 40 consecutive patients undergoing inferior vena caval filter insertion over a 2 1/2-year period were retrospectively reviewed. A severity of illness score for each patient was calculated based on the weighting system described for the APACHE (Acute Physiology and Chronic Health Evaluation) II system. In addition, 30-day postprocedure survival was determined. RESULTS: Seven patients died within 30 days of the procedure (18%). The use of a severity of disease score of greater than 15 as a predictor of postprocedure 30-day mortality had a sensitivity of 50%, specificity of 97%, positive predictive value of 75%, a negative predictive value of 91.4%, and accuracy of 90%. CONCLUSIONS: The 30-day mortality after caval filter insertion is significant. A severity of disease score is a useful predictor of patients likely to survive following caval filter insertion. On this basis it may be possible to establish criteria for more beneficial use of vena caval filters.