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PubMed2001

A rapid screening procedure for cholesterol and dehydrocholesterol by electrospray ionization tandem mass spectrometry.

The mono-(dimethylaminoethyl) succinyl (MDMAES) ester is a new derivative for rapid, mild, and sensitive electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of cholesterol and dehydrocholesterol. It is an order of magnitude more sensitive than the previous most practical alternative, the N-methylpyridyl ether derivative. The MDMAES derivative was used to develop a rapid screening procedure for the biochemical diagnosis of Smith-Lemli-Opitz syndrome (SLOS) by measuring the dehydrocholesterol/cholesterol ratio in plasma (5 microl) and plasma spotted onto filter paper. Details of the synthesis of [25,26,26,26,27,27,27-(2)H7]-7-dehydrocholesterol, used as a standard for quantitation, are included. The measurement of total sterols as MDMAES esters, after base hydrolysis of plasma, afforded a dehydrocholesterol/cholesterol ratio of 0.05-2.95 for SLOS patient samples (n = 5) compared with 0.001-0.003 for normal adult controls (n = 20). Direct hexane extraction of plasma without base hydrolysis enabled the measurement of free sterols with a total sample analysis time of <1 h. The free dehydrocholesterol/cholesterol ratio was 0.10-4.47 for SLOS patient samples (n = 5) and 0.003-0.011 for normal adult controls (n = 20).

Adult↗
PubMed1986

Electroblot and immunoperoxidase staining for rapid screening of the abnormalities of the multimeric structure of von Willebrand factor in von Willebrand's disease.

A new electrophoretic method is described for rapid screening of abnormalities of the multimeric structure of von Willebrand factor in von Willebrand's disease. The method is based on the transfer of the separated proteins from agarose gels onto nitrocellulose foils followed by immunoperoxidase staining. It has the advantage of not requiring radio-iodinated antibodies and reduces the working time for the entire procedure from 5-6 days to 3 days. Electroblotting followed by immunoperoxidase staining differentiates patients with intact multimeric structure from those without intermediate and/or large multimers. The more subtle defects of the inner structure of the smallest multimers found in patients with type II von Willebrand's disease can also be identified. A potential disadvantage of electroblotting and immunoperoxidase staining is the lesser sensitivity of this technique, which results in the detection of a smaller number of multimers (11-12 bands) than by autoradiography without transfer onto nitrocellulose (16-17 bands).

Autoradiography↗
PubMed2003

The advantages of using a modified microbatch method for rapid screening of protein crystallization conditions.

In this study, characterization and optimization of a modified microbatch crystallization technique has been attempted in order to provide a rapid screening method. Using this method for screening has certain advantages over standard vapour-diffusion methods: no sealing of drops is required, no reservoir solutions are needed and the experiments can easily be performed over a range of temperatures.

Crystallization↗
PubMed1996

Rapid screening for acidic non-steroidal anti-inflammatory drugs in urine by gas chromatography-mass spectrometry in the selected-ion monitoring mode.

A rapid screening procedure is described for the simultaneous determination of various acidic non-steroidal anti-inflammatory drugs (NSAIDs) at sub-nanogram levels. The procedure involves solid-phase extraction (SPE) of NSAIDs using Chromosorb P as the adsorbent in partition mode, with subsequent single-step conversion to tert-butyldimethylsilyl (TBDMS) derivatives, followed by direct analysis by gas chromatography-mass spectrometry (GC-MS). The characteristic [M-57]+ high-mass ions constituting the base peaks in the electron-impact mass spectra of most TBDMS derivatives permitted sensitive detection of NSAIDs by GC-MS in selected-ion monitoring (SIM) mode, even in the presence of higher levels of coextracted urinary organic acids. The detection limit for SIM of each drug was in the range 0.03-0.9 pg. When applied to urine samples (250 microliters) spiked with NSAIDs, the present GC-SIM-MS method allowed simultaneous screening for various NSAIDs with good overall precision and accuracy in the range of 10-40 ng.

Administration, Oral↗
PubMed1998

Rapid screening for psychologic distress in men with prostate carcinoma: a pilot study.

BACKGROUND: As more oncology care is moved to the outpatient setting, the need for a rapid means for oncologists to identify patients with significant distress has increased. Concurrent with this move has been the pressure to reduce time spent with each patient, adding to the likelihood that a distressed patient will not be recognized and will remain untreated in the current health care environment. METHODS: A pilot program was conducted in a prostate carcinoma oncology clinic to test the feasibility of a two-stage approach that identifies patients in significant distress and refers them for treatment. Two pencil and paper self-report measures were used to detect psychologic distress in patients over the previous week: 1) The Hospital Anxiety and Depression Scale (HADS) and 2) "The Distress Thermometer." Patients who scored above an agreed upon cutoff score on either measure (HADS = 15+; Thermometer = 5+) were referred to the psychiatric liaison in the clinic for evaluation. RESULTS: Compliance in filling out the measures was excellent; only 8 of 121 patients (6.6%) refused. Thirty-one percent of evaluable patients were referred based on elevated scores. Seventeen of 29 patients actually were evaluated. Eight of 17 patients met Diagnostic and Statistical Manual (of Mental Disorders)-IV criteria for a psychiatric disorder. CONCLUSIONS: This approach for rapid screening for distress was acceptable in prostate carcinoma patients, although these older men were reluctant to agree to evaluation and treatment. This simple screening method needs further testing and the identification of barriers on the part of the patient and oncologist that impede the identification of the most distressed patients.

Aged↗
PubMed2003

A rapid screen for functional mutants of TraM, an autoregulatory protein required for F conjugation.

TraM is an autoregulatory protein required for conjugative transfer of the F plasmid. A rapid screening procedure was developed to select for traM mutants constructed by random PCR mutagenesis. The mutated traM gene was cloned into pT7-5, without the traM promoters (collectively called P( traM)), such that these mutants were expressed from the downstream traJ promoter, resulting in constitutive, low-level, transcription of traM by polymerases that had circumnavigated the plasmid. P( traM) was cloned into pPR9tt as a translational fusion in which a DNA fragment containing P( traM), the ribosome binding site and first 24 codons of traM was fused to the 5' end of lacZ. To downregulate beta-galactosidase expression, a -1 frameshift mutation was introduced at the junction between traM and lacZ in the fusion. Selected TraM mutants were further characterized for their intracellular levels, electrophoretic mobility on nondenaturing gels, and activity in F conjugation. Point mutations throughout TraM were found to affect both autoregulation and conjugative function.

Bacterial Proteins↗
PubMed2006

Molecularly imprinted solid-phase extraction for rapid screening of mycophenolic acid in human plasma.

A molecularly imprinted solid-phase extraction coupled with high performance liquid chromatography (MISPE-HPLC) method was developed for rapid screening of mycophenolic acid (MPA) in human plasma. MPA imprinted polymers (MPA-MIP) were synthesized and then tested for their performance both in organic and in aqueous solution. MPA was selectively trapped and preconcentrated on the MPA-MIP sorbent using different loading and washing conditions. The good selectivity of MPA-MIP enabled further clean-up of the interfering components in human plasma. For the proposed MISPE-HPLC method, the linearity between responses (peak area) and concentration was found over the range of 1-100microg/ml with a linear regression coefficient (R(2)) of 0.9989. The limit of detection (LOD) and theoretical limit of quantification (LOQ) for MPA in plasma were 0.10 and 0.32microg/ml, respectively. The precisions were 7.3, 3.5 and 4.7% RSD for intra-day assay and 9.2, 4.1 and 5.5% RSD for inter-day reproducibility, respectively, at three concentration levels of MPA in spiked plasma (1, 10 and 100microg/ml). Both recoveries for the extraction (more than 74%) and for the analytical method (more than 87%) were acceptable for screening MPA in plasma samples. Twelve-hour pharmacokinetic profile of MPA for a renal transplant recipient receiving chronic oral dosing of 500mg mycophenolate mofetil (MMF) was investigated. Results indicated that this method could be applied for therapeutic drug monitoring of mycophenolic acid in patient plasma.

Calibration↗
PubMed1984

Evaluation of two bioluminescence-measuring instruments, the Turner Design and Lumac systems, for the rapid screening of urine specimens.

Two bioluminescence-measuring instruments, the Turner Design and Lumac systems, were compared with a standard plate culture method for their ability to rapidly screen 400 urine specimens. For cultures with less than 1,000 CFU/ml the Turner Design, with old and new evaluation formulas, gave 6.5 and 50.6% false-positive results, respectively, versus 17.6% at greater than or equal to 500 relative light units with the Lumac. For cultures which had greater than 10(5) CFU/ml the Turner Design gave 39% (old formula) and 14% (new formula) false-negative results compared with 4% at less than 200 relative units with the Lumac. The microorganisms most frequently isolated in the false-negative cultures from either system were gram-positive cocci. Predictive values for a positive test at greater than 10(5) CFU/ml were 77.4% (old formula) and 35.7% (new formula) for the Turner Design versus only 50% for the Lumac at greater than or equal to 500 relative light units. Predictive values for a negative test for both instruments were greater than 88% at greater than 10(5) CFU/ml. The Turner Design and Lumac systems were 4.0 and 3.7 times as expensive, respectively, as the plate culture method. Although both systems greatly reduce the time required to process urine specimens, their high costs as compared with that of plate culture, their failure to detect many specimens having greater than 10(5) CFU of gram-positive cocci per ml, and the numerous false-positives reported by both instruments suggest that additional improvements in the systems are warranted.

Bacteria↗
PubMed1999

Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library.

A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5-1-mm layer of agarose containing 130 micron diameter TentaGel beads at a density of 2-20 beads/mm2. Employing this technique a 9-mer, 442,368-member peptide library (designed around the 13 amino acid alpha-MSH peptide sequence) made up of 12 separate pools of 36,864 peptides/pool was assayed. Initially, a fraction (approximately 10%) of each pool was scanned (approximately 3700 beads from each pool) in 60-mm petri dishes to identify the most active pools. Upon direct photocleavage of the beads with UV light (365 nm), each petri dish was photographed over a 60-min period with a CCD camera to record changes in light intensity as an index of melanosome dispersion. Active beads were those that were surrounded by a localized decrease in light transmittance indicating melanosome dispersed cells. Upon examination with a dissecting microscope, single beads centrally located to a circular array of dispersed cells were identified and removed from the agarose and sequenced by Edman degradation to determine the peptide sequence. Re-synthesized peptides were re-examined against alpha-MSH receptor to confirm and quantify the activity. Several 9-mer peptides were identified with potencies similar to the natural 13-mer peptide. This method allows for the rapid screening of large bead-based photo-cleavable peptide libraries with the advantage that each compound is screened as a discrete molecule in a well-less format.

Animals↗
PubMed2002

[Rapid screening of katG gene mutation in isoniazid-resistant Mycobacterium tuberculosis]

OBJECTIVE: To evaluate the relationship between katG gene mutation and isoniazid (INH) resistance and to develop a rapid screening method of point mutation in the katG gene associated with MTB resistance. METHODS: Twenty-four clinical isolates of MTB with 8 INH resigtance isolates and 16 INH-sensitive isolates were analyzed by PCR-RFLP, with the H(37)Rv reference strain as the control. RESULTS: G-->C point mutations were detected in 7 of 8 isoniazid-resistant strains and no gene mutation was shown in 16 isoniazid-sensitive isolates. The sensitivity and specificity were 87.5 % and 100 % respectively. No katG gene sequence deletion was observed in any specimen. CONCLUSION: Our results suggest katG gene mutation is one of the most important mechanisms of INH-resistant TB. PCR-RFLP may be useful in detection of katG gene mutation.

Journal Article↗
PubMed1993

[Evaluation of rapid screening tests in the diagnosis of urinary infections].

Bacterial urine examination is the main bacterial examination used in microbiology laboratories. The comparative interest of urine aspect associated with rapid screening tests (catalase, leucocyte esterase, nitrite) and different urine examination methods (Gram stain of sediment, direct examination on Malassez cell and on centrifuged urine pellet) are evaluated for negative predictive value of urinary tract infection. In out-patient, the association urine aspect-leucocyte esterase has the best negative predictive value for urinary tract infection (99.4%). Either in outpatients or in hospitalized patients, the association Gram stain of sediment-leukocyturia evaluation has the best negative predictive value for urinary tract infection (100%). This data allows in our population to give back negative results without culture.

Bacteriological Techniques↗
PubMed1997

An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.

We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible.

Bacteriological Techniques↗
PubMed2005

Evaluation of a rapid screening method for detection of antimicrobial resistance in the commensal microbiota of the gut.

The assessment of antimicrobial resistance among commensal bacteria is an indicator of the spread of antimicrobial resistance. Rapid screening methods for detection of antimicrobial-resistant faecal Escherichia coli directly on MacConkey plates have been successfully adopted but suffer from lack of standardisation. The purpose of this study was to evaluate a direct plating method (DPM) for detection of antimicrobial-resistant faecal E. coli and to compare it with a conventional method. Faecal samples were collected from 71 healthy children from Peru and Bolivia. In the DPM, a faecal swab was directly plated onto a MacConkey agar plate and antimicrobial disks were applied onto the seeded plate. Raw data were obtained by direct reading of the plate and were subjected to confirmatory analysis. Good concordance between the DPM and a conventional method was observed in detecting carriage of resistant E. coli, with a higher sensitivity for the DPM. Analysis of the results allowed interpretive criteria to be defined for DPM raw data. The DPM showed good sensitivity and specificity at very low cost (ten times cheaper than the conventional method) to investigate the faecal carriage of drug-resistant E. coli. It may represent a useful tool to conduct large-scale resistance surveillance studies and to monitor resistance control programmes cost effectively, particularly in low-resource countries.

Bacteriological Techniques↗
PubMed1998

Duplex PCR for Autosomal Dominant Spinocerebellar Ataxia Testing: A Nonradioactive Rapid Screening Method.

Background: Current DNA diagnostic testing for spinocerebellar ataxias (SCAs) 1, 2, 3, and 6 involves four separate tests based on radioactively-labeled poly merase chain reaction (PCR). Although this approach allows accurate allele sizing, it also is time-consuming and requires manipulation with hazardous radioactive isotopes. Methods and Results: A rapid screening approach was developed based on a nonradioactive and duplexed PCR assay coupling SCA 1 and SCA 3 together and SCA 2 and SCA 6 together. This was achieved by designing new primers that amplify all normal and mutant alleles in nonoverlapping size ranges. A series of negative and positive samples previously tested with the single radioactively labeled PCRs was tested using the new duplex PCRs; 100% concordancy was achieved. Conclusion: This novel approach of nonradioactive, duplex PCRs for autosomal dominant spinocerebellar ataxias is a rapid, reliable, and cost-effective diagnostic screening approach that is suitable for laboratories with high sample load and/or laboratories seeking to reduce their use of radioisotopes.

Journal Article↗
PubMed1996

Young women with symptoms of urinary tract infection. Prevalence and diagnosis of chlamydial infection and evaluation of rapid screening of bacteriuria.

OBJECTIVE: To estimate the prevalence of chlamydial infection among young women with UTI symptoms. To evaluate chlamydia diagnostics with the aid of enzyme immuno assay (EIA) on first-void urine. To evaluate rapid screening of bacteriuria, including low concentrations of common pathogens. DESIGN: EIA for detection of Chlamydia trachomatis antigen and confirmation with immunofluorescence test (DFA) in urine, cervical and urethral chlamydia culture, nitrite and granulocyte esterase test, urine sediment, chamber count, dipslide and conventional urine culture were performed. SETTING: Primary health care (PHC). PATIENTS: 217 women aged 15-35 years attending PHC for dysuria or urgency-frequency. MAIN OUTCOME MEASURES: Frequency of chlamydial infections. Sensitivity, specificity, predictive values of EIA and bacteriuria screening tests, respectively. RESULTS: The frequency of chlamydial infection was 3.7%. In spite of a high specificity of the EIA test (0.94 without DFA) the number of false positives exceeded the number of true positives. No single bacteriuria test showed sufficient diagnostic efficiency. CONCLUSIONS: Routine chlamydia testing in young women with UTI symptoms is recommended. EIA test on urine is of little use. Assessing diagnosis of UTI symptoms requires insight into the use of several rapid tests and a dialogue with the patient.

Adolescent↗
PubMed1998

Rapid screening of destruxins by liquid chromatography/mass spectrometry.

A liquid chromatographic/mass spectrometric (LC/MS) method utilizing an atmospheric pressure chemical ionization (APCI) interface and in-source collisionally induced dissociation (CID) was developed for the rapid screening of the cyclic hexadepsipeptides destruxins, produced by the fungi Metarhizium anisopliae and Trichothecium roseum. The APCI mass spectra are fully consistent with the high-energy CID data but the sequence ions are abundant over the whole mass range. In addition to 22 known destruxins, two new representatives of this family were detected by LC/MS analysis: destruxin Ed1 and roseotoxin A were isolated and their structures were inferred from MS/MS and NMR data.

Chromatography, High Pressure Liquid↗
PubMed2001

The potential of 19F NMR spectroscopy for rapid screening of cell cultures for models of mammalian drug metabolism.

The use of microbial cultures as a complementary model for mammalian drug metabolism has been well established previously. Here is a preliminary investigation into the potential of 19F NMR spectroscopy as a rapid screening tool to quantify the biotransformations of fluorine-containing model drugs. Biotransformations of three model drugs in 48 taxonomically diverse organisms were measured by acquiring 19F NMR spectra at 376 MHz. The presence of fluorine in the molecules allowed rapid, simultaneous detection of over 20 biotransformation products without sample pretreatment, chromatography, mass spectrometric techniques or the use of radiolabelled substrates. The detection limit at 376 MHz using 5 mm NMR tubes was ca. 0.3 microg ml(-1) using a typical analysis time of 20 min per sample. With the recent advent of flow injection NMR technology, analysis time of 5 min could be achieved with less sample. This approach may be used to develop fast small-scale microbial screens for the biosynthesis of metabolite standards and production of novel drug analogues, whilst also having a role in reducing animal experiments needed to identify animal and human metabolites of fluorinated xenobiotics.

Animals↗
PubMed2001

Combination of matrix solid-phase dispersion extraction and direct on-line liquid chromatography-nuclear magnetic resonance spectroscopy-tandem mass spectrometry as a new efficient approach for the rapid screening of natural products: application to the total asterosaponin fraction of the starfish Asterias rubens.

A combination of matrix solid-phase dispersion extraction (MSPD) and LC-NMR-MS hyphenation is proposed as a rapid screening method of natural products for unknown compounds. In this report, this new analytical approach is applied for the first time. MSPD represents a significant simplification compared to classical extraction procedures and is thus an excellent complement to the fast and powerful LC-NMR-MS: MSPD yields extracts suitable for LC-NMR-MS in one simple preparation step, while LC-NMR-MS yields a wealth of information in one single chromatographic run. The suitability of this technique to characterise glycosidic compounds in the molecular mass range of 1200 to 1400 a.m.u. is demonstrated. The information on the number of exchangeable protons provided by an additional back-exchange experiment proved to be particularly valuable for structural elucidation. The possibility of semi-quantitative LC-NMR measurements through methyl signals H(3)-18 and 19 of the steroidal skeleton is demonstrated and is ensuingly used to provide relative quantitative data of the steroid oligosaccharide fraction.

Animals↗