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PubMed1978

Partial characterization of a beta-lactamase from Vibrio parahaemolyticus by a new automated microiodometric technique.

A number of characteristics were determined with a new automated method for a partially purified beta-lactamase from Vibrio parahaemolyticus. The enzyme had a molecular weight of 28,000 by gel filtration, a pH optimum between 6.5 and 7.0, and a temperature optimum at 36 degrees C. With penicillin G as the substrate, the K(m) value for the beta-lactamase was 54.4 muM. The beta-lactamase was inhibited by cloxacillin but not by p-chloromercuribenzoate. The enzyme was similar but not identical to beta-lactamases from gram-negative, "nonhalophilic" organisms described by other workers. The microiodometric assay to measure beta-lactamase activity was automated with the use of a centrifugal analyzer that permitted 14 simultaneous determinations. Within-run precision was tested by putting the same reaction mixture in each well, and the coefficient of variation was only about 3%. Four extracts from different strains of halophilic vibrios were used to demonstrate that reaction rates were linear with enzyme concentration. The correlation coefficient of activity by the automated method with activity by the spectrophotometric method was 0.9721, demonstrating that the methods compared favorably with each other. The automated method greatly facilitated the characterization of the beta-lactamase.

Autoanalysis↗
PubMed2002

Fully automated detection of hepatitis C virus RNA in serum and whole-blood samples.

In this study, we established a fully automated molecular assay for qualitative detection of hepatitis C virus (HCV) in serum and whole-blood samples and compared it with conventional molecular assays, including manual HCV RNA extraction protocols. Whole-blood samples were collected from patients with and without chronic HCV infection in EDTA tubes and nucleic acid stabilization tubes (NASTs). Prior to HCV RNA extraction, the HCV Internal Control (IC), derived from the COBAS AMPLICOR HCV test, version 2.0 (Roche Molecular Diagnostics), was added. The new assay was based on an automated extraction protocol on the MagNA Pure LC instrument (Roche Applied Science), followed by automated reverse transcription, amplification, hybridization, and detection on the Cobas Amplicor analyzer (Roche Molecular Diagnostics). The detection limit of the new assay was found to be similar to those of conventional molecular assays. In clinical samples, 100% agreement between the new assay and conventional methods was observed. The introduced amount of IC was detected in 45 of 45 serum samples, 41 of 45 EDTA tube whole-blood samples, and 43 of 45 NAST whole-blood samples. Retesting led to more frequent IC detection. The fully automated molecular assay was found to be suitable for detection of HCV RNA in different kinds of sample materials. It may be recommended for use in the high-throughput routine molecular diagnostic laboratory.

Hepacivirus↗
PubMed1982

Analysis of the disagreement between automated bioluminescence-based and culture methods for detecting significant bacteriuria, with proposals for standardizing evaluations of bacteriuria detection methods.

A fully automated method for detecting significant bacteriuria is described which uses firefly luciferin and luciferase to detect bacterial ATP in urine. The automated method was calibrated and evaluated, using 308 urine specimens, against two reference culture methods. We obtained a specificity of 0.79 and sensitivity of 0.75 using a quantitative pour plate reference test and a specificity of 0.79 and a sensitivity of 0.90 using a semiquantitative standard loop reference test. The majority of specimens negative by the automated test but positive by the pour plate reference test were specimens which grew several bacterial species. We suggest that such disagreement was most likely for urine containing around 10(5) colony-forming units per ml (the culture threshold of positivity) and that these specimens were ones contaminated by urethral or vaginal flora. We propose standard procedures for calibrating and evaluating rapid or automated methods for the detection of significant bacteriuria and have analyzed our results using these procedures. We recommend that identical analyses should be reported for other evaluations of bacteriuria detection methods.

Adenosine Triphosphate↗
PubMed2002

Automated ribotyping of vancomycin-resistant Enterococcus faecium isolates.

Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.

Alleles↗
PubMed2006

Accuracy of three automated systems (MicroScan WalkAway, VITEK, and VITEK 2) for susceptibility testing of Pseudomonas aeruginosa against five broad-spectrum beta-lactam agents.

One hundred recent clinical Pseudomonas aeruginosa isolates were used to assess the quantitative (MIC) and qualitative (susceptibility category) accuracies of the MicroScan WalkAway, VITEK, and VITEK 2 automated susceptibility test systems when five-broad spectrum beta-lactams, aztreonam, cefepime, ceftazidime, imipenem, and piperacillin-tazobactam, were tested. Isolates were selected so that the MICs for the isolates overrepresented the MICs near the breakpoints to assess precisely the agreement between the results obtained with the automated systems and the results obtained by the reference tests. The categorical and MIC results from the automated systems were compared to the consensus result of three reference methods: broth microdilution, agar dilution, and disk diffusion. The consensus categorical testing (susceptibility and resistance) rates were 47 and 27%, respectively, for aztreonam; 59 and 14%, respectively, for cefepime; 44 and 43%, respectively, for ceftazidime; 71 and 19%, respectively, for imipenem; and 50 and 50%, respectively, for piperacillin-tazobactam. All systems tested exhibited a high, unacceptable level of very major (false-susceptible) errors for piperacillin-tazobactam (19 to 27%). Major (false-resistant) error rates were generally acceptable (0 to 3%), but minor error rates were elevated (8 to 32%) for cefepime (VITEK 2 and VITEK) and for aztreonam (all three systems), leading to consistent trends toward false resistance. Manufacturer reevaluation of these automated systems for the testing of selected beta-lactams with current clinical isolates of P. aeruginosa that exhibit contemporary resistance mechanisms would be prudent to minimize the potential for serious reporting errors.

Bacteriological Techniques↗
PubMed1997

Mammographic findings after 14-gauge automated needle and 14-gauge directional, vacuum-assisted stereotactic breast biopsies.

PURPOSE: To compare findings from first imaging follow-up mammography for breast lesions shown to be benign at stereotactic biopsy with 14-gauge automated needles or 14-gauge directional, vacuum-assisted probes. MATERIALS AND METHODS: In 495 stereotactic breast biopsies, the mammographic appearance of the biopsy site or target lesion was evaluated at first imaging follow-up with a four-point scale (1 = progression of lesion or suspicious interval change [repeat biopsy], 2 = no clinically relevant change, 3 = interval decrease in size of lesion or number of microcalcifications, and 4 = no residual mammographic lesion). An automated needle was used in 363 biopsies and a directional, vacuum-assisted probe was used in 132 biopsies. Patient and lesion variables and time to first imaging follow-up were compared for the two techniques. RESULTS: No biopsy site or target lesion was assigned a score of 1 at first imaging follow-up with either technique. No lesion was referred for repeat biopsy because of the mammographic appearance at first imaging follow-up. First imaging follow-up was performed an average of 6.6 months for the directional, vacuum-assisted biopsies and 8.6 months for the automated needle biopsies (P < .0001). This difference reflected a difference in scheduling methods. CONCLUSION: Directional, vacuum-assisted and automated needle breast biopsies produced no distortion or suspicious interval change at the biopsy site at the first follow-up mammographic examination.

Aftercare↗
PubMed1997

Detection of malignant and benign breast lesions with an automated US system: results in 120 cases.

PURPOSE: To evaluate clinically an automated ultrasound (US) system for detecting benign and malignant breast lesions. MATERIALS AND METHODS: A prototype automated US system was used to examine 119 patients: 38 patients with 39 proved malignant breast lesions (7-50 mm), 41 patients with 41 proved benign breast lesions (8-40 mm), and 40 patients without breast lesions. The device yields a three-dimensional set of B-mode scans and reconstructed US images comparable to mammograms. All patients had undergone mammography. Four radiologists who had not performed the examinations independently assessed the mammograms and US images to detect benign and malignant breast lesions. RESULTS: Each of the four readers did not recognize one to three detectable malignant lesions on mammograms, one to two detectable malignant lesions on US images, two to four detectable benign lesions on mammograms, and five to seven detectable benign lesions on US images. All readers identified the 39 cancers with at least one of the modalities. The 40 cases without lesions were diagnosed correctly more frequently on the US images by three readers and on the mammograms by one reader. CONCLUSION: Depiction of breast lesions at automated US is reproducible. Automated US is complementary to mammography.

Breast Diseases↗
PubMed1997

Signal amplification of FISH for automated detection using image cytometry.

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Chromosomes, Human, Pair 3↗
PubMed1998

Fast-FISH detection and semi-automated image analysis of numerical chromosome aberrations in hematological malignancies.

A new fluorescence in situ hybridization (FISH) technique called Fast-FISH in combination with semi-automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL). Commercially available alpha-satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of the Fast-FISH protocol, and microscopic images of the fluorescence-labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with a standard FISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling and Fast-FISH was found. Automated spot counting after Fast-FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility of Fast-FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since the Fast-FISH technique requires a hybridization time as low as 1/20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours.

Aneuploidy↗
PubMed2004

Automated extraction and description of dark areas in surface microscopy melanocytic lesion images.

BACKGROUND: Identification of dark areas inside a melanocytic lesion (ML) is of great importance for melanoma diagnosis, both during clinical examination and employing programs for automated image analysis. OBJECTIVE: The aim of our study was to compare two different methods for the automated identification and description of dark areas in epiluminescence microscopy images of MLs and to evaluate their diagnostic capability. METHODS: Two methods for the automated extraction of 'absolute' (ADAs) and 'relative' dark areas (RDAs) and a set of parameters for their description were developed and tested on 339 images of MLs acquired by means of a polarized-light videomicroscope. RESULTS: Significant differences in dark area distribution between melanomas and nevi were observed employing both methods, permitting a good discrimination of MLs (diagnostic accuracy = 74.6 and 71.2% for ADAs and RDAs, respectively). CONCLUSIONS: Both methods for the automated identification of dark areas are useful for melanoma diagnosis and can be implemented in programs for image analysis.

Diagnosis, Differential↗
PubMed1996

Automated reasoning system in histopathologic diagnosis and prognosis of prostate cancer and its precursors.

OBJECTIVE: This article presents the rationale and options offered to diagnostic and prognostic decision support systems for prostate pathology by automated reasoning capabilities. METHODS: The symbolic information used in diagnostic decision-making is systematically ordered, compared, numerically assessed in its probability, and combined such that a conclusion can be drawn. The framework for the processing of such symbolic information may be an expert system, an inference network or a case-based reasoning system. Automated reasoning is implemented by the use of a rule base and information flow control modules. RESULTS: Automated reasoning allows decision support systems to follow highly adaptive decision sequences, capable of handling contradictory evidence, exceptions in diagnostic clue expression, and nonmonotonic decision-making. CONCLUSIONS: Automated reasoning capability in diagnostic and prognostic decision support systems allows highly flexible decision development, very close to human decision procedures.

Artificial Intelligence↗
PubMed2006

MannDB - a microbial database of automated protein sequence analyses and evidence integration for protein characterization.

BACKGROUND: MannDB was created to meet a need for rapid, comprehensive automated protein sequence analyses to support selection of proteins suitable as targets for driving the development of reagents for pathogen or protein toxin detection. Because a large number of open-source tools were needed, it was necessary to produce a software system to scale the computations for whole-proteome analysis. Thus, we built a fully automated system for executing software tools and for storage, integration, and display of automated protein sequence analysis and annotation data. DESCRIPTION: MannDB is a relational database that organizes data resulting from fully automated, high-throughput protein-sequence analyses using open-source tools. Types of analyses provided include predictions of cleavage, chemical properties, classification, features, functional assignment, post-translational modifications, motifs, antigenicity, and secondary structure. Proteomes (lists of hypothetical and known proteins) are downloaded and parsed from Genbank and then inserted into MannDB, and annotations from SwissProt are downloaded when identifiers are found in the Genbank entry or when identical sequences are identified. Currently 36 open-source tools are run against MannDB protein sequences either on local systems or by means of batch submission to external servers. In addition, BLAST against protein entries in MvirDB, our database of microbial virulence factors, is performed. A web client browser enables viewing of computational results and downloaded annotations, and a query tool enables structured and free-text search capabilities. When available, links to external databases, including MvirDB, are provided. MannDB contains whole-proteome analyses for at least one representative organism from each category of biological threat organism listed by APHIS, CDC, HHS, NIAID, USDA, USFDA, and WHO. CONCLUSION: MannDB comprises a large number of genomes and comprehensive protein sequence analyses representing organisms listed as high-priority agents on the websites of several governmental organizations concerned with bio-terrorism. MannDB provides the user with a BLAST interface for comparison of native and non-native sequences and a query tool for conveniently selecting proteins of interest. In addition, the user has access to a web-based browser that compiles comprehensive and extensive reports. Access to MannDB is freely available at http://manndb.llnl.gov/.

Algorithms↗
PubMed2006

Automated extraction protocol for quantification of SARS-coronavirus RNA in serum: an evaluation study.

BACKGROUND: We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay. METHODS: An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics) was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen) for quantitative performance, analytical sensitivity and precision. RESULTS: The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method. CONCLUSION: As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

Humans↗
PubMed2006

Automated surveillance for adverse drug events at a community hospital and an academic medical center.

OBJECTIVES: To compare the rates and nature of ADEs at an academic medical center and a community hospital using a single computerized ADE surveillance system. DESIGN: Prospective cohort study of patients admitted to two tertiary care hospitals. Outcome Measure Adverse drug events identified by automated surveillance and voluntary reporting. METHODS: We implemented an automated surveillance system across an academic medical center and a community hospital. Potential events identified by the computer were reviewed in detail by medication safety pharmacists and scored for causality and severity. Findings were compared between the two hospitals, and with voluntary reports from nurses and pharmacists. RESULTS: Over the 8 month study period, 25,177 patients were admitted to the university hospital and 8,029 to the community hospital. There were 1,116 ADEs in 900 patients at the university hospital for an overall rate of 4.4 ADEs per 100 admissions. At the community hospital, 399 patients experienced 501 ADEs for a rate of 6.2 events per 100 admissions. Rates of antibiotic-associated colitis, drug-induced hypoglycemia, and anticoagulation-related ADEs were significantly higher at the community hospital compared with the university hospital. Computerized surveillance detected ADEs at a rate 3.6 times that of voluntary reporting at the university hospital and 12.3 times that at the community hospital. CONCLUSIONS: Operation of a common automated ADE surveillance system across hospitals permits meaningful comparison of ADE rates in different inpatient settings. Automated surveillance detects ADEs at rates far higher than voluntary reporting, and the difference may be greater in the community hospital setting. Community hospitals may experience higher rates of certain types of ADEs compared with academic medical centers.

Academic Medical Centers↗
PubMed2006

Total laboratory automation can help eliminate the laboratory as a factor in emergency department length of stay.

We obtained data on laboratory turnaround time (TAT) and emergency department (ED) length of stay (LOS). We correlated potassium test TAT outlier percentage (TAT-OP) with ED LOS and found that for each outlier percentage (potassium result > 40 minutes), a projected impact on ED LOS was approximately 2.8 additional minutes (ED LOS = 2.79 TAT-OP + 78.77). To address this issue, we began implementation of a totally automated chemistry system to decrease TAT-OPs. Our TAT means did not change substantially with automation (potassium, 28 to 27 minutes); however, TAT-OPs decreased substantially (potassium, 18% to 5%). Preautomation average ED LOS correlated best with the TAT-OP (r(2) = 0.98; P = .01), but this relationship weakened substantially after automation (r(2) = 0.29; P > .05), suggesting the laboratory was no longer a factor in ED LOS. The postautomation ED LOS correlated best with ED patient volume (r(2) = 0.88; P = .06). Although laboratories have focused on TAT means for performance assessment, our study suggests TAT-OPs are more clinically relevant benchmarks. Furthermore, our findings suggest that total laboratory automation can effectively improve overall laboratory service reliability and help eliminate the laboratory as a factor in ED LOS.

Autoanalysis↗
PubMed2003

The efficacy of an automated feedback system for general practitioners.

OBJECTIVE: An automated feedback system that produces comments about the non-adherence of general practitioners (GPs) to accepted practice guidelines for ordering diagnostic tests was developed. Before implementing the automated feedback system in daily practice, we assessed the potential effect of the system on the test ordering behaviour of GPs. DESIGN: We used a randomised controlled trial with balanced block design. SETTING: Five times six participant groups of GPs in a computer laboratory setting. INTERVENTION: The GPs reviewed a random sample of 30 request forms they filled in earlier that year. If deemed necessary, they could make changes in the tests requested. Next, the system displayed critical comments about their non-adherence to the guidelines as apparent from the (updated) request forms. SUBJECTS: Twenty-four randomly selected GPs participated. MAIN OUTCOME MEASURES: The number of requested diagnostic tests (17% with 95% confidence interval [CI]: 12-22%) and the fraction of tests ordered that were not in accordance with the practice guidelines (39% with 95% CI: 28-51%) decreased due to the comments of the automated feedback system. The GPs accepted 362 (50%) of the 729 reminders. IMPLICATIONS: Although our experiment cannot predict the size of the actual effect of the automated feedback system in daily practice, the observed effect may be seen as the maximum achievable.

Decision Support Systems, Clinical↗
PubMed2004

Automated EEG preprocessing during anaesthesia: new aspects using artificial neural networks.

The computer-aided detection of artefacts became an essential task with increasing automation of quantitative electroencephalogram (EEG) analysis during anaesthesiological applications. The different algorithms published so far required individual manual adjustment or have been based on limited decision criteria. In this study, we developed an artificial neural networks-(ANN-)aided method for automated detection of artefacts and EEG suppression periods. 72 hr EEG recorded before, during and after anaesthesia with propofol have been evaluated. Selected parameterized patterns of 0.25 s length were used to train the ANN (22 input, 8 hidden and 4 output neurons) with error back propagation. The detection performance of the ANN-aided method was tested with processing epochs between 1 to10 s. Related to examiner EEG evaluation, the average detection performance of the method was 72% sensitivity and 80% specificity for artefacts and 90% sensitivity and 92% specificity for EEG suppression. The improvement in signal-to-noise ratio with automated artefact processing was 1.39 times for the spectral edge frequency 95 (SEF95) and 1.89 times for the approximate entropy (ApEn). We conclude that ANN-aided preprocessing provide an useful tool for automated EEG evaluation in anaesthesiological applications.

Algorithms↗
PubMed2000

Evaluation of two automated enzyme immunoassays for detection of antinuclear antibodies.

We evaluated two enzyme immunoassays (EIA) for detection of antinuclear antibodies (ANA) which became recently available and which were designed for application on a fully automated system; (i) the EIA-ANA screen kit from Sigma Diagnostics (St. Louis, MO, USA) applied on APTUS, an automated EIA analyser from Sigma Diagnostics and (ii) the Cobas Core Hep-2 EIA-ANA assay applied on the fully automated Cobas Core immunochemistry analyzer from Roche Diagnostics (Basel, Switzerland). The evaluation was done by an analytic comparison of the automated systems to an established indirect immunofluorescence (IF) method performed on SSA transfected human epitheloid cell substrate slides. Three hundred and thirty six samples were tested with the Sigma EIA-ANA assay and 603 samples with the Cobas Core Hep-2 EIA-ANA assay. For both EIA systems, there was a trend of generally increasing signal from the assay system with increasing IF-ANA titers. With an IF-ANA reference range of < 1:160, concordance between Sigma EIA-ANA and IF-ANA was 86% and concordance between Roche EIA-ANA and IF-ANA was 85%. When compared to IF-ANA with a reference range of < 1:160, sensitivity and specificity of the EIA-ANA screen was 0.65 and 0.92, respectively, for Sigma EIA-ANA and 0.61 and 0.91, respectively, for the Cobas Core assay. The low sensitivity observed with both methods is a major concern. Sigma EIA-ANA screen revealed the presence of autoantibodies in 23 of the 29 samples containing antibodies to extractable nuclear antigens (ENA) and/or double stranded DNA and the Cobas Core Hep-2 EIA-ANA assay revealed the presence of autoantibodies in 40 of the 43 samples containing antibodies to ENA and/or double stranded DNA.

Antibodies, Antinuclear↗