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PubMed2004

Comparison of the Roche LightCycler vanA/vanB detection assay and culture for detection of vancomycin-resistant enterococci from perianal swabs.

We compared the performance characteristics of a real-time PCR method, the LightCycler vanA/vanB detection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 microg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 microg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 microg/ml (n = 25 versus n = 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 microg/ml (n = 31 versus n = 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 microg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 microg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (approximately 3.5 versus > or =72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.

Anal Canal↗

PubMed1996

Splicing mutations in DMD/BMD detected by RT-PCR/PTT: detection of a 19AA insertion in the cysteine rich domain of dystrophin compatible with BMD.

We have used an RNA based mutation detection method to screen the total coding region of the dystrophin gene of a Duchenne and a Becker muscular dystrophy patient in whom DNA based mutation detection methods have so far failed to detect mutations. By RT-PCR and the protein truncation test (PTT) we could identify point mutations in both cases. DMD patient DL184.3 has a T-->A mutation in intron 59 at position -9, creating a novel splice acceptor site for exon 60. As a result seven intronic bases are spliced into the mRNA, causing a frameshift and premature translation termination 20 codons downstream. Since this patient had died and only fibroblasts were available, we applied MyoD induced myodifferentiation of stored fibroblasts to enhance muscle specific gene expression. With the results of this mutation analysis, prenatal diagnosis could subsequently be performed in this family. BMD patient BL207.1 carries a G-->C mutation at position +5 of intron 64, disrupting the splice donor consensus sequence and activating a cryptic splice donor site 57bp downstream. The inclusion of these 57 intronic bases in the mRNA leaves the reading frame open and results in the insertion of 19 amino acids into the cysteine rich domain of dystrophin. Interestingly, this insertion in a part of the dystrophin considered to interact with the dystrophin binding complex of the sarcolemma is apparently compatible with mild BMD-like clinical features. Both mutations reported are missed by analysis of multiplex PCR products designed for deletion screening of the coding region. Extrapolation from existing point mutation detection efficiencies by DNA and RNA based methods emphasises that RNA based methods are more sensitive and that most of the remaining undetected mutations may affect splice or branch sites or create cryptic splice sites.

Adolescent↗

PubMed1995

Volume detection threshold: quantitative comparison of computed radiography and screen-film radiography in detection of pneumothoraces in an animal model that simulates the neonate.

PURPOSE: To quantitatively compare computed radiography (CR) and screen-film radiography (SFR) in the detection of pneumothorax in an animal model that simulates the neonate. MATERIALS AND METHODS: Three rabbits underwent placement of 5-F catheters in the right pleural space. Eight CR and eight SFR images were obtained. Volume-controlled pneumothorax was induced by incrementally injecting 4 cm3 of air (24 cm3 total) and four CR and four SFR images were obtained after each injection. Four radiologists blindly viewed the images twice (eight readings per image, 1,600 total observations). A dichotomous yes-no score for the presence of pneumothorax was used to calculate the volume detection threshold (VDT), defined as the air volume at which 50% of the pneumothoraces can be detected. The authors plotted the likelihood of a pneumothorax against the air volume by using the confidence indicator. RESULTS: Intraobserver VDT values for CR and SFR were not significantly different. CONCLUSION: CR is as accurate as SFR in the detection of pneumothorax in this quantitative animal model.

Animals↗

PubMed2001

Mammographic characteristics of 115 missed cancers later detected with screening mammography and the potential utility of computer-aided detection.

PURPOSE: To retrospectively determine the mammographic characteristics of cancers missed at screening mammography and assess the ability of computer-aided detection (CAD) to mark the missed cancers. MATERIALS AND METHODS: A multicenter retrospective study accrued 1,083 consecutive cases of breast cancer detected at screening mammography. Prior mammograms were available in 427 cases. Of these, 286 had lesions visible in retrospect. The 286 cases underwent blinded review by panels of radiologists; a majority recommended recall for 112 cases. Two experienced radiologists compared prior mammograms in 110 of these cases with the subsequent screening mammograms (when cancer was detected), noting mammographic characteristics of breast density, lesion type, size, morphology, and subjective reasons for possible miss. The prior mammograms were then analyzed with a CAD program. RESULTS: There were 110 patients with 115 cancers. On the prior mammograms with missed cancers, 35 (30%) of the 115 lesions were calcifications, with 17 of 35 (49%) clustered or pleomorphic. Eighty of the 115 (70%) were mass lesions, with 32 of 80 (40%) spiculated or irregular. For calcifications and masses, the most frequently suggested reasons for possible miss were dense breasts (12 of 35; 34%) and distracting lesions (35 of 80; 44%), respectively. CAD marked 30 (86%) of 35 missed calcifications and 58 (73%) of 80 missed masses. CONCLUSION: Detection errors affected cases with calcifications and masses. CAD marked most (77%; 88 of 115) cancers missed at screening mammography that radiologists retrospectively judged to merit recall.

Adult↗

PubMed1998

Consensus on microembolus detection by TCD. International Consensus Group on Microembolus Detection.

Transcranial Doppler ultrasound is capable of detecting microembolic material, both gaseous and solid, within the intracranial cerebral arteries. To avoid discrediting this promising and exciting new technique, experts in this field met in January 1997 in Frankfurt, Germany, to discuss the limitations and problems of embolus detection and to determine guidelines for its proper use in clinical practice, as well as in scientific investigations. In particular, the authors suggest that studies report the following parameters: (1) ultrasound device, (2) transducer type and size, (3) insonated artery, (4) insonation depth, (5) algorithms for signal intensity measurement, (6) scale settings, (7) detection threshold, (8) axial extension of sample volume, (9) fast Fourier transform (FFT) size (number of points used), (10) FFT length (time), (11) FFT overlap, (12) transmitted ultrasound frequency, (13) high-pass filter settings, and (14) recording time. There was agreement that no current system of automatic embolus detection has the required sensitivity and specificity for clinical use.

Humans↗

PubMed2000

Stimulated acoustic emission detected by transcranial color doppler ultrasound : a contrast-specific phenomenon useful for the detection of cerebral tissue perfusion.

BACKGROUND AND PURPOSE: Experimental and clinical data suggest that insonation of echo-contrast agents with high acoustical power produces disintegration of microbubbles, resulting in a pseudo-Doppler phenomenon called stimulated acoustic emission (SAE). The purpose of this study was to investigate whether SAE might be detected by transcranial color Doppler imaging and whether these signals might be used for cerebral tissue perfusion measurements. METHODS: Nonmoving microbubbles (SHU 563 A) were insonated in vitro through the temporal parts of a human cadaver skull, and contrast signals were detected by velocity-coded color Doppler and power Doppler recordings. Transcranial color as well as power Doppler investigations were performed in 10 healthy volunteers with the echo-contrast agent Levovist (SHU 508 A). RESULTS: Color Doppler signals indicating SAE were observed in vitro and in transcranial human investigations. These signals were characterized by a mosaic of color Doppler pixels ranging over the full color scale. Apparent velocity information and spatial distribution of SAE signals changed from image frame to image frame. In the experimental model, the intensity of SAE signals decreased exponentially over time. With an increase of acoustic power, there was a significant increase of the maximum signal intensity (P<0.01) and a significantly shortened signal duration (P<0.01), consistent with stronger and more rapid disintegration. In humans, SAE signals were clearly detected in cerebral tissue regions. The intensity of SAE signals in those regions (eg, temporal cortex, 3.7+/-1.2 dB) was approximately 8 times lower than the signal enhancement in the major cerebral arteries (eg, in the MCA, 29.5+/-5.6). CONCLUSIONS: Echo-contrast specific color Doppler signals known as SAE are detectable by transcranial color and power Doppler sonography. Signals due to SAE might represent tissue perfusion, thereby providing a method for imaging flow with transcranial ultrasound.

Adult↗

PubMed1994

Survival of patients with breast cancer diagnosed in the United Kingdom trial of early detection of breast cancer. United Kingdom Trial of Early Detection of Breast Cancer Group.

OBJECTIVE: To examine the survival of patients with breast cancer diagnosed in different centres and by different methods in the United Kingdom trial of early detection of breast cancer, in order to investigate the contribution of different factors to the previously observed reductions in breast cancer mortality. SETTING: A non-randomised trial of the early detection of breast cancer, in which women aged 45-64 in two districts were offered annual screening for seven years, women in a further two districts were offered education about breast self examination (BSE), and those in four districts formed a comparison group. METHODS: Patients with breast cancer are classified according to the type of centre, method of detection, and attendance for BSE education. Univariate and multivariate survival analyses are carried out, including tumour size, dissemination status, and use of adjuvant treatment as additional variables. RESULTS: In the univariate analysis, patients with breast cancer who are non-attenders for screening have a significantly worse prognosis than those in the comparison centres. Patients whose cancer is detected by mammography have the best survival rate. The inclusion of size and dissemination status in the multivariate analysis explains only about one third of the improved prognosis in these cases. There is a significant difference between prognosis in the two BSE centres. CONCLUSIONS: The use of prognostic factors as recorded in this trial to predict breast cancer mortality may be inadequate.

Analysis of Variance↗

PubMed1998

Bone metastases in patients with gastrinomas: a prospective study of bone scanning, somatostatin receptor scanning, and magnetic resonance image in their detection, frequency, location, and effect of their detection on management.

PURPOSE: To determine whether bone scan, magnetic resonance imaging (MRI), or somatostatin receptor scintigraphy (SRS) is best for identifying bone metastases in patients with gastrinomas, as well as their frequency and location, whether their detection affects management, and what patient subgroups should be examined. MATERIALS AND METHODS: One hundred fifteen patients with gastrinoma were prospectively studied. Patients were examined yearly and those with liver metastases were reexamined every 3 months. Based on clinical history, histology, growth pattern, and development of new bone lesions, possible bone metastases were classified as to whether they were or were not bone metastases. Imaging results were correlated at different times in the disease course and with disease extent. RESULTS: Bone scan was positive in 52 patients, MRI in seven, and SRS in six. Eight patients (7%) were determined to have bone metastases and MRI was correctly positive in seven, SRS in six, and bone scan in five. SRS or MRI was positive in all patients with bone metastases. Bone scan had significantly lower specificity and sensitivity, and a higher rate (P < .02) of false-negative results than MRI or SRS. Bone metastases occurred in 31% of patients with liver metastases and 0% with only lymph node metastases. The initial bone metastases were in the spine or sacrum (75%) followed in descending order by the pelvis or sacroiliac joints (38%), scapula or shoulder, and ribs. In all cases, detection of bone metastases changed the management. CONCLUSION: SRS and MRI, because of high sensitivity and specificity, are recommended over bone scanning to screen for bone metastases in patients with gastrinomas. However, because bone metastases can occur initially outside the axial skeleton, SRS is the recommended initial localization method of choice. Bone metastases occur in 7% of all patients and 31% of patients with liver metastases, only occur in patients with liver metastases, are usually in the axial skeleton initially, and their detection changes management in all cases. Patients with pancreatic endocrine tumors with liver metastases should undergo SRS every 6 months to 1 year to detect bone metastases.

Adolescent↗

PubMed1995

Rapid detection of mycoplasma contamination in cell cultures by enzymatic detection of polymerase chain reaction (PCR) products.

Enzymatic detection of polymerase chain reaction (ED-PCR) was applied for rapid and easy identification of mycoplasmas from contaminated cell culture. This method was based on the capture of amplified products via biotin-streptavidine affinity and the detection of an incorporated hapten in amplified products with enzyme-linked antibody. Primers corresponding to common sequence of Mollicutes in 16S ribosomal RNA dominated gene was used. Nineteen of twenty Mollicutes so far reported as cell contaminants appeared positive by ED-PCR, whereas remaining one, Acholeplasma axanthum, appeared negative. Samples from sixty-two cell culture were tested for contamination of mycoplasmas by means of ED-PCR, cultivation, and electronmicroscopy. The results of ED-PCR were the same as those of cultivating method. The time required for all the detection process in ED-PCR was about 5 hr for 20 samples. We suggest that ED-PCR can be used in the rapid detection of mycoplasms from cell culture.

Animals↗

PubMed1991

Detection uncertainty and the facilitation of chromatic detection by luminance contours.

A suprathreshold luminance flash (1 degree, 200 msec) on a large uniform yellow field facilitates detection of a coincident (1 degree, 200 msec) red or green equiluminant flash and approximately linearizes the psychometric function for detecting the chromatic flash. The facilitation is produced by the suprathreshold contour created by the luminance flash. We tested whether the contour facilitates detection by reducing spatiotemporal uncertainty in detecting the chromatic flash. Uncertainty increase false alarms, and this effect can be factored out by correcting yes-no psychometric functions for guessing. Uncertainty also alters the shape of the receiver operating characteristic. Measurements of yes-no psychometric functions and receiver operating characteristics do not support the uncertainty reduction hypothesis.

Color Perception↗

PubMed1990

Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system).

A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.

Animals↗

PubMed1987

A new, very sensitive, bioluminescence-enhanced detection system for protein blotting. Ultrasensitive detection systems for protein blotting and DNA hybridization, I.

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blots is described. The method utilizes antibodies conjugated with alkaline phosphatase. The alkaline phosphatase then takes part in a reaction by releasing D-luciferin (Photinus pyralis) from D-luciferin-O-phosphate. Liberated D-luciferin reacts with luciferase, ATP and oxygen with light emission. The light is detected by a sensitive photographic film, thereby permitting the visualization of the alkaline phosphatase-conjugated antibodies. Under non-optimized conditions the limit of detection is at present 5 to 50 pg of protein, corresponding e.g. to 30 to 300 x 10(-18) mol of rabbit immunoglobulin G. The detection system is therefore 100 times more sensitive than other systems used at present.

Alkaline Phosphatase↗

PubMed1998

Comparison of the Difco EZ Coil rapid detection system and Petrifilm test kit-HEC for detection of Escherichia coli O157:H7 in fresh and frozen ground beef.

The Difco EZ Coli Rapid Detection System was compared to the 3M Petrifilm method for detection of Escherichia coli O157:H7 in raw ground beef. Raw meatballs (25 g) were inoculated with 10 to 15 cells of Escherichia coli O157:H7, stored for various times and at different temperatures, and then stomached for 2 min in 225 ml of EZ Coli enrichment broth, which was then incubated at 42 degrees C for 18 to 24 h. A 1-ml sample of the enrichment broth was loaded into the top of the detector tips and the remaining EZ Coli broth held at 35 degrees C before streaking onto MacConkey sorbitol agar and tryptic soy agar with yeast extract. A duplicate set of meatballs were tested using the 3M Petrifilm Test Kit-HEC for hemorrhagic Escherichia coli O157:H7. In this method raw meatballs (25 g) were enriched for 6 h in modified EC broth containing novobiocin at 37 degrees C prior to inoculation of the Petrifilm E. coli Count Plates, which were incubated at 42 degrees C for 18 h. The immunoblot ELISA was performed following this incubation. Presumptive positive isolates from both methods were confirmed using Oxoid E. coli Latex Agglutination and Difco Pasco ID Tripanels. Both methods permitted detection of 10 to 15 cells of E. coli O157:H7 per ml (i) immediately following inoculation, (ii) after 3 days of refrigerated storage at 8 degrees C, and (iii) after 30 days in frozen storage at -20 degrees C. The Difco EZ Coli Detection System proved to be a simpler and faster screening method with identification of negative and presumptive positive samples within 15 to 18 h.

Animals↗

PubMedDate not supplied

[Evaluation of the reincubation of positive blood cultures for detection in the BACT-ALERT system for the detection of polymicrobial bacteremia].

Between February and September 1997, 6588 blood cultures at the Instituto de Cardiología y Cirugía Cardiovascular and Hospital de Niños Ricardo Gutiérrez were studied by using the Bact-Alert system (Organon Teknika) 341 contaminants and 294 episodes of bacteremia (600 samples) were analyzed. From these samples, 280 (95.3%) were monomicrobial episodes and 14 (4.7%) polymicrobial episodes. Positive blood cultures detected by the Bact-Alert system were processed and then reincubated during 7 days, when they were Gram stained and subcultured in blood agar, chocolate agar (both in 5-10% CO2), laked blood agar supplemented with hemin and vitamin K in anaerobic atmosphere (only anaerobic bottles) and CLDE (aerobic conditions). Following reincubation, 3 out of 14 polymicrobial bacteremias were detected, rising the level of detection from 3.7% to 4.7%. Taking into account the total number of bacteremias, only in 3 out of 294 (1%), a second microorganism was detected. Otherwise, in blood cultures where a contaminating microorganism was initially isolated, no further isolates representing a true bacteremia were recovered. Reincubation and terminal subculture of initially positive blood cultures did not provide relevant data in order to change therapeutic measures in the studied population. Due to the increase in costs and labor we consider that this methodology is not routinely advised.

Bacteremia↗

PubMedDate not supplied

Minimum detectable level of Salmonellae using a binomial-based bacterial ice nucleation detection assay (BIND).

A modified bacterial ice nucleation detection (BIND) assay was used for rapid and sensitive detection of several Salmonella species. For the BIND assay, Salmonella cells are infected with bacteriophage genetically modified to contain DNA encoding an ice nucleation protein (INP). After infection, de novo protein synthesis occurs and INPs are incorporated into the outer membrane of the organism. After supercooling (-9.3 degrees C), only buffer solutions containing transfected salmonellae freeze, causing a phase-sensitive dye to change color. This technique, and a probability-based protocol modification, provided quantitative detection with a minimum detectable level (MDL) of 2.0 +/- 0.3 S. enteritidis cells/mL in buffer (about 3 h). The MDLs for S. typhimurium DT104 and S. abaetetuba were 4.2 +/- 0.2 and 11.1 +/- 0.4 cells/mL, respectively. Using salmonellae-specific immunomagnetic bead separation technology in conjunction with the modified BIND protocol, we achieved an MDL of about 4.5 S. enteritidis cells/mL with an apparent capture efficiency of 56%.

Algorithms↗

PubMed2001

On-column amperometric detection of ofloxacin and pasiniazid in urine by capillary electrophoresis with an improved fractured joint and small detection cell.

An improved high-voltage electric field isolating joint and small detection cell have been carefully designed and fabricated. The joint possesses short steady time, high electric conductance efficiency and high performance. The cell is convenient to install and remove the capillaries with and without the joint, as well as to fix, adjust and insert the microelectrode into the detection capillary. Using the joint and the cell, an analytical method for determination of ofloxacin (Oflx) and pasiniazid (Ipa) in urine by capillary electrophoresis with on-column amperometric detection was developed. The calibration lines were linear in the range of 10-100 mg l-1 of Oflx and 1.0-50 mg l-1 of Ipa, respectively. The detection limits were 8.5 mg l-1 of Oflx and 0.80 mg l-1 of Ipa. Their recovery ranged from 101 to 104%. The accuracy and intra-day and inter-day reproducibility of Oflx and Ipa were determined with satisfactory results. This method was successfully used for determining Oflx and Ipa in human urine.

Aminosalicylic Acid↗

PubMed2001

Detection of mutations in the RB1 gene by single strand conformation polymorphism (SSCP) analysis, amplification mismatch detection (AMD) analysis and polymerase chain reaction sequencing.

Mutations of the retinoblastoma gene are known to cause both nonhereditary and hereditary forms of retinoblastoma. Most patients with hereditary retinoblastoma have bilateral disease. Hereditary predisposition to retinoblastoma is caused by a germline mutation at the retinoblastoma gene locus (RB1) and transmitted as an autosomal dominant trait with 90% penetrance. Three quarters of these alterations represent de novo mutations. Since 75% of these cases are new mutations, there is a need for methods which can be used to identify carriers, so that informed genetic counselling will be available to patients and close relatives. In the present study, leukocyte DNA and RNA from 5 patients with sporadic bilateral retinoblastoma. were subjected to single stranded conformation analysis (SSCP) and amplification and mismatch detection (AMD) analysis. SSCP band shifts were found in 3 of the 5 patients. AMD was applied to reverse-transcriptase PCR and exons of the RB1 gene in the patients with bilateral retinoblastoma. Cleavage was found in 2 patients. Neither of these patients corresponded to the 3 with SSCP band shifts. Thus in total, 5 patients with retinoblastoma had mutations detected by a combination of SSCP and AMD analysis, and proof was sought by means of sequencing. This approach has proved to be a useful method for the rapid detection of mutations in the RB1 gene. The five mutations detected in this study were all novel and emphasise the heterogeneity of the molecular pathology in this gene.

Amino Acid Substitution↗

PubMed2001

[Comparison of the clinical detection and the biological detection of dental caries].

Different methods can be used to detect dental caries. The comparison of the clinical detection by the use of explorer and mirror to the biological detection by the measurement of alkaline phosphatase activity (ALP) in 107 men of 19 to 25 years old was performed. Biological detection reveals more active caries(APL activity > 116 Ul/L) than the clinical one. Absence of caries activity is expressed by an ALP activity between 86 and 116 Ul/L. The Chi 2 test shows that alkaline phosphatase is a statistically significant marker of dental caries and more it reveals its carious activity.

Adult↗