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PubMed2003

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR.

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.

Colony Count, Microbial↗
PubMed2000

Quantification of boundary segregation in the analytical electron microscope

When studying equilibrium grain-boundary segregation using the small ( approximately 1 nm) electron probe of the scanning transmission electron microscope and X-ray energy dispersive spectroscopy, the assumptions made about the size and shape of the beam-specimen interaction volume may introduce errors in quantification of up to a factor of five. Comparisons between experimental segregation profiles and different theoretical models have shown that a Gaussian model for the electron distribution will provide the best description for the interaction volume. Calculations of minimum detectable segregation levels have shown that optimum sensitivity is not achieved in the thinnest samples or even with the smallest probe sizes. In addition, operating at 300 keV (rather than 100 keV) will halve the minimum detectable segregation level, assuming all other experimental conditions are equal. Rastering the electron probe over a fixed area while acquiring spectra improves the accuracy of quantification but at the price of reducing sensitivity by at least a factor of three.

Journal Article↗
PubMed2002

Quantification of fibrosis progression in patients with chronic hepatitis C using a Markov model.

The knowledge of fibrosis progression in chronic hepatitis C and the impact of new treatments on progression is limited by the number of available liver biopsies per patient. Moreover, liver biopsies identify a patient's stage of fibrosis at a given point in time, but cannot quantify the time spent in that stage nor the date of transition to that stage. This paper assesses the potential of Markov modelling to overcome these difficulties. The data from interferon-treated (n=185) and untreated patients (n=102) are analysed to illustrate the power of this technique. The model accurately reproduced the distributions of patients in the different fibrosis stages at two subsequent biopsies. A quantification of the role of cofactors in the progression of the disease, and the impact of interferon treatment are given. In subjects who are 40 years old and have been infected for 10 years, the model predicted that 274 of 1000 untreated patients, but only 42 of 1000 treated patients, would progress from F0 or F1 to F3 or F4 fibrosis over the next 5 years. The model also confirms that as age and duration of infection increase, the risk of fibrosis progression increases, while the impact of treatment with interferon decreases. Hence Markov modelling is an accurate tool in the analysis of fibrosis progression in chronic hepatitis C. It will be valuable for the quantification of the effect of new treatments on fibrosis progression in hepatitis C.

Adolescent↗
PubMed1999

A human whole-blood assay for analysis of T-cell function by quantification of cytokine mRNA.

A whole blood assay was developed for T-lymphocyte analysis which allows the quantification of induced cytokine mRNA expression. We applied a novel kinetic reverse transcription polymerase chain reaction method which directly measures product accumulation using Taqman technology. Quantitative results were obtained by using beta-actin and cytokine standard curves generated from synthetic external standards. Since quantification relies on threshold cycles for fluorescence detection (Ct), this technique proved to be accurate over a dynamic range of at least five orders of magnitude. To evaluate the method a study was undertaken to find optimal conditions for whole-blood stimulation with soluble anti-CD3 monoclonal antibodies in the presence of a costimulatory signal mediated by anti-CD28 monoclonal antibody. Therefore, whole blood was taken from healthy individuals (n = 10) and aliquots for mRNA measurement were withdrawn after 0, 4, 8 and 24 h of stimulation. Optimal assay conditions were reached with 1 : 10 diluted heparinized whole blood and after stimulation with equimolar amounts of anti-CD3 and anti-CD28 monoclonal antibodies (1 microgram/ml). Interferon-gamma and tumour necrosis factor-alpha proved to be early response cytokines with peak expression at 4 h. In contrast, interleukin (IL)-2, IL-4 and IL-10 required 8 h of stimulation. This novel whole-blood assay is potentially useful for monitoring T-cell-specific immune functions in a variety of clinical settings. Using whole blood obviates the need for T-cell purification and may therefore closely approximate the state of responsiveness of circulating T cells in vivo.

Biological Assay↗
PubMed2001

Quantification of murine IFN-gamma mRNA and protein expression: impact of real-time kinetic RT-PCR using SYBR green I dye.

Reliable quantification of cytokine mRNA expression is an important technique for analyzing immune responses. Up until now, little to no information has been available as to whether different mRNA quantification techniques lead to similar results. Recently, real time quantitative reverse transcriptase (RT)-PCR using SYBR Green I as a double stranded DNA specific dye has been introduced. This novel method enables simple and rapid measurement of PCR product accumulation during the log-linear reaction phase and obviates the need for expensive hybridization probes. Here, we analyzed murine gamma interferon (IFN)-gamma mRNA expression in splenocytes by this technique in comparison to semiquantitative noncompetitive RT-PCR, Northern blot analysis, and ELISA after stimulation of the cells with interleukin (IL)-12, IL-18 and a combination of both cytokines. The results clearly show that all of the techniques detect differences in the IFN-gamma gene expression induced by these distinctive stimuli qualitatively exactly in the same order. However, real-time kinetic RT-PCR offers several advantages, notably its high sensitivity that allows the detection of basal IFN-gamma mRNA expression in unstimulated samples. In addition it provides the lowest interassay variability of all techniques investigated. Finally, the gene expression measured by this method eliminates any post-PCR manipulations because the PCR product identification can be easily performed by melting curve analysis.

Animals↗
PubMed2003

Use of a quantitative TaqMan-PCR for the fast quantification of mycobacteria in broth culture, eukaryotic cell culture and tissue.

The quantification of slow-growing mycobacteria such as Mycobacterium tuberculosis or M. bovis from in vitro and in vivo samples is complicated by their long generation time, their ability to form aggregates, and their capacity to persist in a state of dormancy. We compared different methods for the establishment of growth curves for broth cultures of M. bovis bacille Calmette-Guérin (BCG). A quantitative TaqMan-PCR yielded results comparable with those obtained by protein quantification and measurement of the ATP content of the cultures. The quantitative TaqMan-PCR furthermore turned out to be particularly suitable for the measurement of multiplication of BCG within eukaryotic cells. Furthermore, it is a fast method allowing an estimation of the mycobacterial load in tissue long before colony counts can be obtained.

Animals↗
PubMed2003

Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR.

We developed a real-time RT-PCR method for the quantification of dissimilatory (bi)sulphite reductase (DSR) mRNA in Desulfobacterium autotrophicum cells. The amount of DSR mRNA was determined relative to the amount of 16S rRNA at different growth conditions during transition from exponential to stationary phase: sulphate respiration with lactate, thiosulphate respiration with lactate, sulphate respiration with H2 and pyruvate fermentation. The dsr gene was expressed constitutively, although DSR mRNA content per-cell varied under different growth conditions. The maximum DSR mRNA per-cell content was 2.0 to 4.1-fold higher during sulphate or thiosulphate respiration than during pyruvate fermentation. After transfer of a pyruvate-fermenting culture into sulphate-rich medium, upregulation of the DSR mRNA content was observed. Irrespective of the mode of metabolism the per-cell DSR mRNA content changed significantly during growth (up to 310-fold from the early to the late exponential phase during respiration with thiosulphate). The maximum DSR mRNA per-cell contents correlated with cell-specific sulphate reduction rates for all experiments. Environmental applications for the quantification of DSR mRNA are discussed.

Culture Media↗
PubMed2001

Quantification of fibre type regionalisation: an analysis of lower hindlimb muscles in the rat.

Newly developed concepts and methods for the quantification of fibre type regionalisation were used for comparison between all muscles traversing the ankle of the rat lower hindlimb (n = 12). For each muscle, cross-sections from the proximodistal midlevel were stained for myofibrillar ATPase and classified as type I ('slow') or II ('fast'). For the 11 'fast' muscles (i.e. all except soleus), the muscle outline and the position of each type I fibre were digitised for further computer processing. Two potentially independent aspects of type I fibre regionalisation were evaluated quantitatively: (1) the degree to which type I fibres were restricted to a limited portion of the total cross-sectional area ('area-regionalisation'): (2) the extent and direction of the difference (if any) between the centre of the muscle cross-section and the calculated centre for the type I fibre cluster ('vector regionalisation'). Statistical analysis showed that type I fibres were vector regionalised in practically all investigated muscles and area regionalised within most of them, the only consistent exceptions being peroneus brevis and peroneus digitorum 4, 5. In muscles with a high degree of area regionalisation the population of type I fibres also had a markedly eccentric intramuscular position (i.e. high vector regionalisation). A significant relationship was observed between the relative position of a muscle within the hindlimb (transverse plane) and the direction and degree of its type I fibre eccentricity. On average, the degree of type I fibre eccentricity was greater for muscles remote from the limb centre than for those situated more centrally. In addition, the intramuscular concentration of type I fibres was typically greatest towards the centre of the limb, the most striking exception being tibialis posterior. For the slow soleus muscle, which is centrally placed within the limb, our analysis concerned the type II fibres, which were found to be weakly vector regionalised but not significantly area regionalised. It is concluded that, within muscles of the rat's lower hindlimb, fibre type regionalisation is a general and graded phenomenon which may reflect differentiating (embryological?) mechanisms of a transmuscular significance. Furthermore, the analysis demonstrated the usefulness of our new methods and concepts for the quantification of fibre type regionalisation.

Animals↗
PubMed2000

Anti-D quantification by flow cytometry:a comparison of five methods.

UNLABELLED: BACKGROUND Three flow cytometric methods for anti-D quantification have been published. All use different cell sensitization and antibody detection conditions that may lead to varied results. Therefore, a direct comparison of the three methods is timely. STUDY DESIGN AND METHODS: The published flow cytometric methods and two new in-house modifications were compared. Ten serum samples containing anti-D at levels between 11.6 and 915 IU per mL were selected for analysis, and each was tested a minimum of three times. Anti-D bound to cells was detected with fluorescence-labeled anti-human IgG reagents. RESULTS The interassay CV of the standard curves for each of the five methods was less than 10 percent. The intra-assay CV was consistently <10 percent with four out of the five methods, but, by the fifth method, it was >20 percent in more than one-third of the tests. In 72 percent of the sample and method combinations, the interassay CV was <25 percent. Plotting of the mean anti-D value for each sample as a percentage of the value determined by an automated technique (AutoAnalyzer) revealed wide variability between the methods. CONCLUSION: Anti-D quantification by flow cytometry is influenced by the serum antibody characteristics and the method used. The differences between the flow cytometric and AutoAnalyzer techniques indicate that further validation of the flow cytometric method is required before routine use.

Flow Cytometry↗
PubMed2003

Quantification of glycophorin A and glycophorin B on normal human RBCs by flow cytometry.

BACKGROUND: The quantification of antigens and proteins on RBCs has been achieved by different approaches. Flow cytometry allows the results of the earliest studies to be to reappraised because it offers the possibility of measuring the immunofluorescence intensity of single cells and integrating the individual data of a large number of cells within a very short time. STUDY DESIGN AND METHODS: Flow cytometry was used in this work to analyze the binding of four MoAbs to glycophorin A (GPA) and glycophorin B (GPB). RBCs in their native state (nonfixed) were utilized. To avoid the agglutination problem, cells were disaggregated before measurements, dates were taken on 20,000 events on the single-cell region, and the fluorescence intensity of the principal peak present in the fluorescence histograms was used for the analysis. The quantification of sites per RBC was estimated by applying the Langmuir adhesion model. RESULTS: The numbers of GPA and GPB sites obtained for samples from healthy donors were similar to those found in the literature (1.86-4.9) x 10(5) and (0.48-1.61) x 10(5) for GPA and (0.21-1.14) x 10(5) and (0.47-0.88) x 10(5) for GPB. Differences between antibodies were found that depend on the binding site of each one. CONCLUSION: A simple method to quantify antigen sites on RBCs was developed. It could be applied whenever one antibody is assumed to bind exactly one antigen.

Agglutination↗
PubMed2005

Emergence of quantification in clinical investigation and the quest for certainty in therapeutics: the road from Hammurabi to Kefauver.

Throughout most of history, medical knowledge was descriptive in nature and derived from the work of individual investigators of independent mind pursuing careful but often chance observations. Using deductive reasoning, these findings were then generalized, authoritatively presented, and dogmatically promulgated. This, coupled with firmly grounded principles of divine determinism, precluded any serious consideration of randomness, even when variations from recorded, but erroneous, statements were actually observed. Although probability remained an integral component of diagnosis and therapy, it was only as an attribute of opinion and not one supported by numbers. The gradual erosion of this edifice began during the scientific revolution of the seventeenth century that led to the burgeoning of the sciences basic to medicine. Although clinicians applauded these contributions, they failed to apply the inductive method of investigation to the study of disease or to therapy. The "numerical method" of Pierre Louis (1787-1872) first introduced systematic quantification into medicine during the first half of the nineteenth century. Analysis of quantifiable data found its principal application in epidemiology, which flourished during the second half of the nineteenth century. The subsequent adoption of probability calculus for the analysis of quantifiable data, during the first half of the twentieth century, refined the process further and led to the gradual emergence of medical statistics, with a distinct role in clinical research. The mathematical precision provided by quantification and statistical analysis established certainty in medicine and ultimately changed the conjectural art of clinical practice into a disciplined science founded on clinical investigation, the very basis of present-day, evidence-based medicine.

History, 15th Century↗
PubMed2000

Standardization of hepatitis C virus RNA quantification.

It was recently recommended that hepatitis C virus (HCV) RNA quantification be used to tailor the duration of combined interferon alfa (IFN-alpha)/ribavirin therapy in patients infected by HCV genotypes 1, 4, and 5. This recommendation has been difficult to implement in the absence of standardized quantitative units for HCV RNA. The aim of this work was to define clinically relevant HCV RNA loads in standardized international units (IU), for use in routine clinical and research applications based on standardized quantitative assays. Two hepatitis C virus RNA quantitative assays were used: (1) the Superquant assay (National Genetics Institute, Los Angeles, CA), for which possibly relevant thresholds were established; and (2) the semi-automated Cobas Amplicor HCV Monitor assay version 2.0 (Cobas v2.0, Roche Molecular Systems, Pleasanton, CA) that measures HCV RNA loads in IU/mL. Quantification in the Cobas v2.0 assay was linear over the entire range of values tested, including viral loads higher than 850,000 IU/mL after 100-fold dilution. The accuracy and precision of the measures in IU/mL were satisfactory with Cobas v2.0. The results obtained with Superquant and Cobas v2.0 correlated (r =.932; P <.0001). A value of 2,000,000 copies/mL (6.3 log(10) copies/mL) with Superquant was converted to nearly 800,000 IU/mL (5.9 log(10) IU/mL). In conclusion, all HCV RNA quantitative assays should give HCV RNA loads in international units and be validated with appropriate calibrated panels; 800,000 IU/mL in any of these assays should be used as the decision threshold to tailor the IFN-alpha/ribavirin treatment duration in patients infected by HCV genotypes 1, 4, and 5.

Antiviral Agents↗
PubMed2002

Critical flicker frequency for quantification of low-grade hepatic encephalopathy.

Subclinical hepatic encephalopathy (SHE) is currently diagnosed by psychometric tests or neurophysiologic techniques. In view of its sociomedical relevance, simple and reproducible tests for routine diagnosis are required. This study evaluates critical flicker-frequency thresholds for quantification of low-grade hepatic encephalopathy. A total of 115 patients (92 with cirrhosis, 23 controls) were analyzed for HE severity (mental state, computerized psychometric tests), and the threshold frequencies at which light pulses are perceived as fused (fusion frequency) or flickering light (critical flicker frequency [CFF]). CFF was a highly reproducible parameter with little age, day-time, and training dependency. CFFs in cirrhotic patients without HE (HE 0) were not different from those found in noncirrhotic controls. Significantly lower CFFs were found in cirrhotic patients with subclinical or manifest HE, and the various HE groups separated from each other at a high level of significance (P <.01). By using a CFF cut-off value of 39 Hz, a 100% separation of patients with manifest HE from noncirrhotic controls and HE 0 cirrhotic patients was obtained. SHE patients separated from HE 0 cirrhotic patients with high sensitivity (55%) and specificity (100%). The HE severity-dependent differences were found in both, alcoholic and posthepatitic cirrhosis. Statistically significant correlations (P <.01) were found between CFFs and individual psychometric tests. Aggravation of preexisting HE after transjugular intrahepatic portosystemic stent shunt (TIPS) implantation was accompanied by a corresponding decrease of CFF, whereas improvement of HE increased CFF. In conclusion, CFF is a sensitive, simple, and reliable parameter for quantification of low-grade HE severity in cirrhotic patients and may be useful for the detection and monitoring of SHE.

Aged↗
PubMedDate not supplied

Detection and quantification of coronary stenosis severity with myocardial contrast echocardiography.

The development of microbubble contrast agents and new imaging modalities now allows the assessment of myocardial perfusion during echocardiography. These microbubbles are excellent tracers of red blood cell kinetics. Apart from providing a spatial assessment of myocardial perfusion, myocardial contrast echocardiography (MCE) can also be used to quantify the 2 specific components of myocardial blood flow-flow velocity and myocardial blood volume. The method to quantify myocardial blood flow velocity is based on rapid destruction of microbubbles by ultrasound, and subsequent assessment of the rate of replenishment of microbubbles into the myocardial microcirculation within the ultrasound beam elevation. Assessment of steady state myocardial video intensity (VI) provides a measure of myocardial or capillary blood volume. Perfusion defects that develop distal to a stenosis during hyperemia are therefore due to capillary derecruitment. We have shown that the degree of derecruitment (and therefore the severity of a perfusion defect) is proportional to stenosis severity. Because the capillary bed also provides the greatest resistance to hyperemic flow, decreases in capillary blood volume distal to a stenosis during hyperemia result in increases in microvascular resistance, which is the mechanism underlying the progressive decrease in flow reserve in the presence of a stenosis. Consequently, both the severity of a perfusion defect and quantification of abnormal myocardial blood flow reserve on MCE can be used to determine stenosis severity. As imaging methods with MCE continue to be refined, the optimal imaging algorithms for clinical practice still need to be determined. MCE, however, holds promise as a noninvasive, instantaneous, on-line method for the detection and quantification of coronary artery disease.

Contrast Media↗
PubMed2002

Simultaneous quantification of prostaglandins in human synovial cell-cultured medium using liquid chromatography/tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric (LC/MS-MS) method was developed for the simultaneous quantification of prostaglandin (PG) E(2), PGF(2alpha), 6-keto-PGF(lalpha) and thromboxane (TX) B(2). These eicosanoids and their deuterium derivatives, using as internal standards, were extracted by solid-phase extraction and analyzed using LC/MS-MS in the selected reaction-monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng for each eicosanoid was demonstrated. The accuracy of added eicosanoids ranged from 94.1 to 106.6% and coefficients of variation ranged from 0.62 to 7.8%. Furthermore, we applied this method for the determination of eicosanoids in the human synovial cell-cultured medium, stimulated by lipopolysaccharide (LPS). LPS produced each eicosanoid and they increased in a time-dependent manner. The production levels after 24 h stimulation were 6-keto-PGF(1alpha) > PGE(2) > TXB(2) >> PGF(2alpha). This simultaneous quantification method is so useful to clarify the function of synovial cells in rheumatoid arthritis (RA).

Calibration↗
PubMed1977

[Quantification and statistical analysis of ordinal scale type data in psychiatry (author's transl)].

Observational data from psychiatric patients are usually of the ordinal scale type. Therefore, in the traditional one-rating-per-symptom-and-patient-situation, the resulting score values can be analysed only by applying nonparametric statistical methods. These methods are rather inefficient when--as is usual--there are only four-point-scales for evaluating the symptoms. Moreover, non-parametric methods are not available as of yet to take into consideration the often complex structure of data from psychiatric studies. This leads to the conclusion that the quantification of ordinal scale type data would be very desirable in order that parametric statistical methods could be applied. Such a method of quantification is proposed on the basis of the audiovisual psychiatric rating technique. It is shown that the data resulting from the many-ratings-per-symptom-and-patient-situation can be subjected to parametric statistical methods. In applying the proposed method, therefore, much better use could be made of and a maximum of information could be extracted from the data in psychiatric studies.

Humans↗
PubMed1979

[A new method of pericardiocentesis and quantification of pericardial effusions by echocardiography (author's transl)].

With a new method for safe and complete evacuation of pericardial effusions it was possible to check the quantification of pericardial fluid is was possible to check the quantification of pericardial fluid by echocardiography. Using a formula based on the assumption that in systole the pericardial fluid is distributed equally around the contracting heart (like a coat enveloping a sphere), it was demonstrated that pericardial effusions ranging from 250 to 600 ml could be fairly accurately measured. Smaller effusions were not so readily determined. Effusions over 600 ml could be only roughly measured, usually being underestimated.

Echocardiography↗
PubMed2006

Quantification of the cephalosporin antibiotic cefditoren in human plasma by high-performance liquid chromatography.

A simple, sensitive and selective high-performance liquid chromatography (HPLC) method with ultraviolet detection (305 nm) was developed and validated for quantification of cefditoren (CAS 104145-95-1), a broad-spectrum orally administered cephalosporin in human plasma. Following solid-phase extraction using Waters Oasis SPE cartridges, the analyte and internal standard (hydrochlorothiazide, CAS 58-93-5) were separated using an isocratic mobile phase of 0.03 % trifluoro acetic acid buffer / acetonitrile (81/19, v/ v) on reverse phase Waters symmetry C18 column. The lower limit of quantification was 50 ng/mL, with a relative standard deviation of less than 4%. A linear range of 50 to 5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 0.5 to 3.7 % and 0.5 to 2.5%, respectively. The between-batch and within-batch accuracy was 96.9 to 103.8% and 97.5 to 102.3%, respectively. Stability of cefditoren in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.

Calibration↗