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PubMed1994

Determination of BRL 46470 in human plasma by high performance liquid chromatography with ultraviolet absorbance detection followed by post-column photochemical reaction and fluorescence detection.

A very sensitive and specific quantitative assay for BRL 46470, a selective 5-HT3 receptor antagonist, in human plasma was developed. The method uses HPLC with serial UV absorbance detection followed by post-column photochemical reaction and fluorescence detection to provide an ultra-sensitive and specific method with a wide quantitative range. The post-column photochemical reaction enhances the very weak native fluorescence of BRL 46470 by a factor of approximately 150. The quantification ranges were determined to be 0.1-1.5 ng ml-1 (fluorescence detection) and 1.5-200 ng ml-1 (UV absorbance detection) for BRL 46470. Results from a 3 d validation at nominal BRL 46470 concentrations of 0.1, 0.4, 1.0 and 1.5 ng ml-1, using post-column photochemical reaction and fluorescence detection, demonstrated precision ranges of 3.4-5.8% (average within-day) and 1.6-5.6% (between-day). The average accuracy ranged from 93.4 to 114.5%. Results from a 3 d validation at nominal BRL 46470 concentrations of 1.5, 4.0, 25 and 200 ng ml-1, using UV absorbance detection, demonstrated precision ranges of 2.0-8.2% (average within-day) and 1.0-3.4% (between-day). The average accuracy ranged from 86.3 to 103.7%. The recovery of BRL 46470 from human plasma was approximately 64%. Assay specificity was confirmed by HPLC-MS.

Bridged Bicyclo Compounds↗

PubMed2005

Does computer-aided detection assist in the early detection of breast cancer?

PURPOSE: To evaluate whether breast cancers detected at screening are visible in previous mammograms, and to assess the performance of a computer-aided detection (CAD) system in detecting lesions in preoperative and previous mammograms. MATERIAL AND METHODS: Initial screening detected 67 women with 69 surgically verified breast cancers (Group A). An experienced screening radiologist retrospectively analyzed previous mammograms for visible lesions (Group B), noting in particular their size and morphology. Preoperative and previous mammograms were analyzed with CAD; a relatively inexperienced resident also analyzed previous mammograms. The performances of CAD and resident were then compared. RESULTS: Of the 69 lesions identified, 36 were visible in previous mammograms. Of these 36 "missed" lesions, 14 were under 10 mm in diameter and 29 were mass lesions. The sensitivity of CAD was 81% in Group A and 64% in Group B. Small mass lesions were harder for CAD to detect. The specificity of CAD was 3% in Group A and 9% in Group B. Together, CAD and the resident found more "missed" lesions than separately. CONCLUSION: Of the 69 breast cancers, 36 were visible in previous mammograms. CAD's sensitivity in detecting cancer lesions ranged from 64% to 81%, while specificity ranged from 9% to as low as 3%. CAD may be helpful if the radiologist is less subspecialized in mammography.

Breast Neoplasms↗

PubMed1997

Trinucleotide repeats in the human genome: size distributions for all possible triplets and detection of expanded disease alleles in a group of Huntington disease individuals by the repeat expansion detection method.

Using a modified Repeat Expansion Detection (RED) assay, that was optimized for individual oligonucleotides, unrelated individuals were systematically screened for maximal repeat sizes of each of the ten possible trinucleotide repeats. Cloned trinucleotide repeats were generated and used as standards for the detectability of single copy trinucleotide repeat fragments. When the size distributions of trinucleotide repeats were compared to previously reported data, significant differences were found for the CTT repeat, which corresponds to the expanded GAA repeat in Friedreich ataxia, as well as for ATT, CCT and GTT repeats. Since 30-35% of normal individuals have CTG/CAG trinucleotide repeat sizes of 180 bp or more, we investigated the question whether small-scale CTG/CAG repeat expansions are detectable on a population basis by using the RED technique. We blindly screened 20 HD probands with CAG expansions of the HD gene, ranging in size between 120 and 174 bp, and found that a shift to larger CAG size ranges is clearly detectable when comparing the distribution of maximal repeat sizes in the disease group to a control group. Our study, therefore, demonstrates that the application of the RED assay to a population of probands and a population of controls allows the detection of small-scale CTG/CAG repeat expansions in the size range of the expanded HD gene and present in a single allele. We also provide standards and control data for the detection of other trinucleotide repeat expansions.

Adult↗

PubMed1998

Proof of hepatitis A virus negative-sense RNA by RNA/DNA-hybrid detection: a method for specific detection of both viral negative- and positive-strand RNA species.

The detection of hepatitis A virus (HAV) negative-strand RNA, which is synthesized during replication of the positive-strand RNA genome, proved to be difficult. We developed a method for the specific detection of HAV negative-strand RNA by RNA-DNA hybridization and luminescence detection using an anti-RNA:DNA hybrid antibody. This method, which is also applicable for the specific detection of positive-strand RNA, offers a simple, yet relatively rapid and certain means of detecting low amounts of RNA such as HAV negative-strand RNA. By using appropriate hybridization DNA probes, the method should be applicable for the detection of single-stranded RNA species of different viruses in general.

Animals↗

PubMed1999

Novel non-isotopic detection of MutY enzyme-recognized mismatches in DNA via ultrasensitive detection of aldehydes.

A highly sensitive method to detect traces of aldehyde-containing apurinic/apyrimidinic (AP) sites in nucleic acids has been developed. Based on this method, a novel approach to detect DNA base mismatches recognized by the mismatch repair glycosylase MutY is demonstrated. Open chain aldehydes generated in nucleic acids due to spontaneous depurination, DNA damage or base excision of mismatched adenine by MutY are covalently trapped by a new linker molecule [fluorescent aldehyde-reactive probe (FARP), a fluorescein-conjugated hydroxylamine derivative]. DNA containing AP sites is FARP-trapped, biotinylated and immobilized onto neutravidin-coated microplates. The number of FARP-trapped aldehydes is then determined via chemiluminescence using a cooled ICCD camera. AP sites induced in plasmid or genomic calf thymus DNA via mild depurination or by simple incubation at physiological conditions (pH 7, 37 degreesC) presented a linear increase in chemiluminescence signal with time. The procedure developed, from a starting DNA material of approximately 100 ng, allows detection of attomole level (10(-18) mol) AP sites, or 1 AP site/2 x 10(7) bases, and extends by 1-2 orders of magnitude the current limit in AP site detection. In order to detect MutY-recognized mismatches, nucleic acids are first treated with 5 mM hydroxylamine to remove traces of spontaneous aldehydes. Following MutY treatment and FARP-labeling, oligonucleotides engineered to have a centrally located A/G mismatch demonstrate a strong chemiluminescence signal. Similarly, single-stranded M13 DNA that forms mismatches via self-complementation (average of 3 mismatches over 7429 bases) and treated with MutY yields a signal approximately 100-fold above background. No signal was detected when DNA without mismatches was used. The current development allows sensitive, non-isotopic, high throughput screening of diverse nucleic acids for AP sites and mismatches in a microplate-based format.

Aldehydes↗

PubMed2006

Automated microaneurysm detection using local contrast normalization and local vessel detection.

Screening programs using retinal photography for the detection of diabetic eye disease are being introduced in the UK and elsewhere. Automatic grading of the images is being considered by health boards so that the human grading task is reduced. Microaneurysms (MAs) are the earliest sign of this disease and so are very important for classifying whether images show signs of retinopathy. This paper describes automatic methods for MA detection and shows how image contrast normalization can improve the ability to distinguish between MAs and other dots that occur on the retina. Various methods for contrast normalization are compared. Best results were obtained with a method that uses the watershed transform to derive a region that contains no vessels or other lesions. Dots within vessels are handled successfully using a local vessel detection technique. Results are presented for detection of individual MAs and for detection of images containing MAs. Images containing MAs are detected with sensitivity 85.4% and specificity 83.1%.

Algorithms↗

PubMedDate not supplied

Computer-aided detection in full-field digital mammography: detection in dependence of the BI-RADS categories.

The object of this study was to determine the performance of a computer-aided detection system in full-field digital mammography (Senographe 2000D, General Electric, Buc, France) in detecting carcinomas in breasts in dependence of the initial Breast Imaging Reporting and Data System (BI-RADS) categories. A total of 226 mediolateral oblique (MLO) and 186 craniocaudal (CC) view mammograms of histologically proven cancers were retrospectively evaluated with a primary digital computer-aided detection system (Image Checker V2.3; R2 Technology, Los Altos, CA). According to BI-RADS of the American College of Radiology (ACR), the lesions were classified in MLO view as BI-RADS 1 in 2 cases, BI-RADS 2 in 11 cases, BI-RADS 3 in 37 cases, BI-RADS 4 in 56 cases, and BI-RADS 5 in 120 cases, and in CC view as BI-RADS 1 in 2 cases, BI-RADS 2 in 8 cases, BI-RADS 3 in 26 cases, BI-RADS 4 in 46 cases, and BI-RADS 5 in 104 cases. The computer-aided detection system shows markers also in mammograms classified as BI-RADS categories 1-3 by the radiologist. Furthermore, BI-RADS categories 4 and 5 in most cases demonstrate masses in mammography. With increasing BI-RADS category, the computer-aided detection system shows decreasing numbers of overlooked carcinomas. In MLO view, no markers were found in 100% (2/2), 81.8% (9/11), 59.5% (22/37), 46.4% (26/56), and 15% (18/120) for BI-RADS categories 1-5, respectively. False-positive markers, however, were seen in 0.5 per image (205/412). In conclusion, the high rate of false-positive markers shows that the specificity of the computer-aided detection system is limited and that improvements are necessary.

Adenocarcinoma↗

PubMed2005

Optimized Scansystem platelet kit for bacterial detection with enhanced sensitivity: detection within 24 h after spiking.

BACKGROUND AND OBJECTIVES: The prevention and detection of bacterial contamination of platelet concentrates remains a major challenge for transfusion medicine. To be suitable for blood-transfusion services, the contamination detection method must be highly sensitive, easy to perform and preferably of low cost. In this spiking study, we evaluated the new optimized Scansystem Platelet Kit detection method for use on apheresis platelets. STUDY DESIGN AND METHODS: Apheresis platelet concentrates (APCs) were individually spiked with 10 colony-forming units (CFU)/ml of one of 10 different strains of bacteria. The spiked APCs were analysed at specific time-points during incubation by using the optimized Scansystem Platelet Kit. Bacterial enumeration was performed by plating onto blood agar. RESULTS: All the bacterial strains tested were detected by using the optimized Scansystem Platelet Kit when sampled 24 h after spiking. Compared to the Scansystem standard kit, sensitivity was increased to < 50 CFU/ml. The identity of the spiked bacteria was confirmed by Gram staining and DNA fingerprinting. CONCLUSION: The optimized Scansystem Platelet Kit was able to reliably detect, within 70 min, 10 transfusion-relevant bacterial species in APCs when a sample volume was taken 24 h after spiking. This is the first study carried out by using the optimized Scansystem bacterial detection that was found to have an enhanced sensitivity compared to the standard kit.

Bacteria↗

PubMedDate not supplied

Computer-aided detection of clustered microcalcifications: an improved method for grouping detected signals.

A computerized scheme for the automated detection of clustered microcalcifications from digital mammograms is being developed. This scheme is one part of an overall package for computer-aided diagnosis (CAD), the purpose of which is to assist radiologists in detecting and diagnosing breast cancer. One important step in the computer detection scheme is to group or cluster microcalcifications, since clustered microcalcifications are more clinically significant than are isolated microcalcifications. Previously a "growing" technique in which signals (possible microcalcifications) were clustered by grouping those that were within some predefined distance from the center of the growing cluster was used. In this paper, a new technique for grouping signals, which consists of two steps, is introduced. First, signals that may be several pixels in area are reduced to single pixels by means of a recursive transformation. Second, the number of signals (nonzero pixels) within a small region, typically 3.2 x 3.2 mm, are counted. Only if three or more signals are present within such a region are they preserved in the output image. In this way, isolated signals are eliminated. Furthermore, this method can eliminate falsely detected clusters, which were identified by a previous detection scheme, based on the spatial distribution of signals within the cluster. The differences in performance of the CAD scheme for detecting clustered microcalcifications using the old and new clustering techniques was measured using 78 mammograms, containing 41 clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Diseases↗

PubMed2001

Quantitative detection of hepatitis B virus DNA by real-time nucleic acid sequence-based amplification with molecular beacon detection.

We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 10(3) to 10(9) HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.

Centrifugation, Density Gradient↗

PubMed2006

Detection of chronic kidney disease in patients with or at increased risk of cardiovascular disease: a science advisory from the American Heart Association Kidney And Cardiovascular Disease Council; the Councils on High Blood Pressure Research, Cardiovascular Disease in the Young, and Epidemiology and Prevention; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: developed in collaboration with the National Kidney Foundation.

Chronic kidney disease (CKD) occurs commonly in patients with cardiovascular disease. In addition, CKD is a risk factor for the development and progression of cardiovascular disease. In this advisory, we present recommendations for the detection of CKD in patients with cardiovascular disease. CKD can be reliably detected with the combined use of the Modification of Diet in Renal Disease equation to estimate glomerular filtration rate and a sensitive test to detect microalbuminuria. All patients with cardiovascular disease should be screened for evidence of kidney disease with these two determinations.

American Hospital Association↗

PubMed2003

Detectability of onsets versus offsets in the change detection paradigm.

The human visual system is particularly sensitive to abrupt onset of new objects that appear in the visual field. Onsets have been shown to capture attention even when other transients simultaneously occur. This has led some authors to argue for the special role that object onset plays in attentional capture. However, evidence from the change detection paradigm appears contradictory to such findings. Studies of change blindness demonstrate that the onset of new objects can often go unnoticed. Assessing the relative detectability of onsets compared with other visual transients in a change detection procedure may help resolve this contradiction. We report the results of four experiments investigating the efficacy with which onsets capture attention compared with offsets. In Experiment 1, we employed a standard flicker procedure and assessed whether participants were more likely to detect the change following a frame containing an onset or following a frame containing an offset. In Experiment 2, we employed the one-shot method and investigated whether participants detected more onsets or offsets. Experiment 3 used the same method but assessed whether onsets would be detected more rapidly than offsets. In Experiment 4, we investigated whether the effect obtained in Experiments 1-3 using simple shapes would replicate when images of real-world objects were used. Results showed that onsets were less susceptible to change blindness than were offsets. We argue that the preservation of information is greater in onsets than in offsets.

Attention↗

PubMed2003

Airborne rhinovirus detection and effect of ultraviolet irradiation on detection by a semi-nested RT-PCR assay.

BACKGROUND: Rhinovirus, the most common cause of upper respiratory tract infections, has been implicated in asthma exacerbations and possibly asthma deaths. Although the method of transmission of rhinoviruses is disputed, several studies have demonstrated that aerosol transmission is a likely method of transmission among adults. As a first step in studies of possible airborne rhinovirus transmission, we developed methods to detect aerosolized rhinovirus by extending existing technology for detecting infectious agents in nasal specimens. METHODS: We aerosolized rhinovirus in a small aerosol chamber. Experiments were conducted with decreasing concentrations of rhinovirus. To determine the effect of UV irradiation on detection of rhinoviral aerosols, we also conducted experiments in which we exposed aerosols to a UV dose of 684 mJ/m2. Aerosols were collected on Teflon filters and rhinovirus recovered in Qiagen AVL buffer using the Qiagen QIAamp Viral RNA Kit (Qiagen Corp., Valencia, California) followed by semi-nested RT-PCR and detection by gel electrophoresis. RESULTS: We obtained positive results from filter samples that had collected at least 1.3 TCID50 of aerosolized rhinovirus. Ultraviolet irradiation of airborne virus at doses much greater than those used in upper-room UV germicidal irradiation applications did not inhibit subsequent detection with the RT-PCR assay. CONCLUSION: The air sampling and extraction methodology developed in this study should be applicable to the detection of rhinovirus and other airborne viruses in the indoor air of offices and schools. This method, however, cannot distinguish UV inactivated virus from infectious viral particles.

Air Microbiology↗

PubMed2004

Early detection of Pseudomonas aeruginosa--comparison of conventional versus molecular (PCR) detection directly from adult patients with cystic fibrosis (CF).

BACKGROUND: Pseudomonas aeruginosa (PA) is the most important bacterial pathogen in patients with cystic fibrosis (CF) patients. Currently, routine bacteriological culture on selective/non- selective culture media is the cornerstone of microbiological detection. The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum. METHODS: Adult patients (n = 57) attending the regional adult CF centre in Northern Ireland, provided fresh sputum following airways clearance exercise. Following processing of the specimen with sputasol (1:1 vol), the specimen was examined for the presence of PA by plating onto a combination of culture media (Pseudomonas isolation agar, Blood agar & McConkey agar). In addition, from the same specimen, genomic bacterial DNA was extracted (1 ml) and was amplified employing two sequence-specific targets, namely (i) the outer membrane protein (oprL) gene locus and (ii) the exotoxin A (ETA) gene locus. RESULTS: By sputum culture, there were 30 patients positive for PA, whereas by molecular techniques, there were 35 positive patients. In 39 patients (22 PA +ve & 17 PA -ve), there was complete agreement between molecular and conventional detection and with both PCR gene loci. The oprL locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the oprL target negative. Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that 5 of these patients converted to being culture-positive at times ranging from 4-17 months later, with a mean lag time of 4.5 months. CONCLUSIONS: This study indicates that molecular detection of PA in sputum employing the oprL gene target, is a useful technique in the early detection of PA, gaining on average 4.5 months over conventional culture. It now remains to be established whether aggressive antibiotic intervention at this earlier stage, based on PCR detection, has any significant benefits on clinical outcome.

Journal Article↗

PubMed2000

Application of a bioluminescence method for the beer industry: sensitivity of MicroStar-RMDS for detecting beer-spoilage bacteria. Rapid Microbe Detection System.

We set up the original operating conditions of the MicroStar-Rapid Microbe Detection System (RMDS) to suppress false positives, which have kept this system from practical. The detection limit of our system was between 6.3 x 10(-16) mol and 3.1 x 10(-16) mol in terms of the amount of ATP, which is approximately equal to the ATP content of one yeast cell or 50 lactic acid bacteria cells. The detection time and the detection count were compared between the RMD method and the conventional plate count method (C.P.C. method) using 23 test samples of beer-spoilage Lactobacillus brevis. Judging from the detection time and detection count, 16-24 hours of cultivation for the RMD method corresponded to 40-96 hours of cultivation for the C.P.C. method. The RMD method reached a useful level for our practical use at the point of sensitivity.

Adenosine Triphosphate↗

PubMed1995

Molecular detection of hepatitis C virus: impact of detection methodology on clinical and laboratory correlations.

The clinical manifestations of hepatitis C virus (HCV) infection are generally indistinguishable from other causes of viral hepatitis. HCV infections are usually anicteric, asymptomatic, and rarely cause acute fulminant liver failure. Serological testing for HCV in conjunction with epidemiological studies have verified that HCV was the major cause of parenterally transmitted non-A, non-B hepatitis (NANBH). With the widespread introduction of serological screening of blood products for HCV antibody, the risk of transfusion-associated HCV infection has been dramatically reduced (to < 3 cases per 10,000 units transfused). Despite the virtual elimination of transfusion-associated infections, the diagnosis of HCV remains important because > 50% of infections are sporadic in origin, 50 to 70% of infected individuals develop chronic hepatitis, and these individuals are at risk of developing cirrhosis (> 20%) as well as hepatocellular carcinoma. Although currently available anti-HCV immunoassays function well as blood-donor screening assays, they are poor at detecting acute infection because of the prolonged lag time between infection and detection of seroconversion (approximately 10 to 26 weeks for second-generation immunoassays). In contrast, polymerase chain reaction (PCR)-based detection of HCV RNA in serum can detect infection in as little as 1 to 2 weeks after exposure. This review focuses on the impact of modern serologic and nucleic acid-based HCV detection methodology on the clinical understanding of HCV infection, its associated illnesses, and its transmissability. Quantitative and reproducible nucleic acid-based detection assays will be required to provide additional insights into the clinical spectrum of HCV infections as well as to assess the efficacy of antiviral agents.

Hepacivirus↗

PubMed2002

[A detection method of recombinant DNA from genetically modified potato (NewLeaf Plus potato) and detection of NewLeaf Plus potato in snack].

A detection method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) potato (NewLeaf Plus potato; NL-P), which has not been authorized as safe in foods in Japan. The potato sucrose synthase gene was used as an internal control. The DNA from NL-P specifically provided an amplified band using PCR with a primer pair recognizing PLRV-rep gene. In addition, to prevent false-positive results in processed potato foods infected with PLRV, we designed a primer pair recognizing sequences derived from two organisms to detect specifically NL-P in processed potato. The PCR product obtained using the designed primer pair was specific for NL-P. The DNA introduced into NL-P could be detected from potato powder samples containing 0.05% NL-P. The proposed method was applied to the detection of NL-P in 25 processed potato foods. NL-P was detected in 3 snack products.

DNA, Plant↗

PubMed1995

[Early detection of prostate cancer-results of a prostate specific antigen-based detection program at a "multiphasic screening"].

Determination was made of serum PSA in the early detection of prostate cancer in a population of 1,227 men aged 55 years or older who visited a "human dock". "Human dock" is a unique health examination facility open to public in Japan. Recommendation for biopsy was made based solely on this parameter when its value exceeded 2.0 ng/ml in IMx immunoenzymetric assay (Dinabot Co.). Of these, 162 (13.2%) had elevated values. The proportion of males with serum PSA greater than 4.0 ng/ml was only 3.6%. Of the 109 males who underwent ultrasound guided biopsies, 17 cancers were detected, the cancer detection rate thus being 1.4%. Most of these cases (82.4%) were clinically localized cancers. Eight patients with cancer had serum PSA levels below 4.0 ng/ml including 6 less than 3.0 ng/ml. Radical prostatectomy was conducted on 14 patients. All had histologic features of clinically significant cancer and 64.3% were pathologically confined. Oriental elderly males appear to have lower serum PSA than western counterparts. The optimal cutoff of serum PSA for early detection should be examined further in oriental male populations. Differences in the incidence of prostate cancer between ethnic groups may have been overestimated in previous studies owing in part to unawareness by the physician and general public. Though the application of PSA for early detection will likely disclose greater numbers of prostate cancers in Japan, whether early detection reduces mortality by minimizing risk of death from cancer remains a point to be clarified.

Aged↗