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PubMed1998

Evaluation of an indirect ELISA for the diagnosis of bovine brucellosis in milk and serum samples in dairy cattle in Argentina.

An indirect enzyme-linked immunosorbent assay (ELISA) for Brucella abortus antibodies detection in bovine milk and serum samples was validated. The assay use B. abortus smooth lipopolysaccharide as antigen, immobilized on a polystyrene matrix; milk diluted 1:2 or serum diluted 1:50, in a buffer containing divalent cation chelating agents EDTA and EGTA (ethyleneglycol-bis-aminoether-N,N,N',N'-tetraacetic acid) to reduce non-specific reactions; and a mouse monoclonal antibody specific for an epitope of bovine IgG1, conjugated with horseradish peroxidase. A total of 2646 sera and 2119 milk samples from cows older than 24 months were obtained from 12 brucellosis-free herds for at least the previous 5 years. Milk samples were obtained in parallel with serum samples. The remaining 527 serum samples were from dry cows. All cattle were vaccinated with B. abortus strain 19 between 3-10 months of age. Five hundred and fifty-two milk samples and 562 serum samples were obtained from 6 infected herds with abortions where B. abortus was isolated at least once no more than 6 months before sampling. The complement-fixation test (CFT) on serum samples was considered the gold standard. Serum samples were also tested with the official screening test: the buffered plate antigen (BPA) test. The cut-off point was determined using receiver-operating characteristic (ROC) analysis. For milk samples, it was fixed at 36 percent positivity (PP) giving a sensitivity of 99.6% with a 95% confidence interval (CI) of 98.6-99.9%. The specificity was 99.1% (CI 98.9-99.4%). For serum samples, the cut-off was fixed at 53 PP giving a sensitivity of 99.6% (CI 98.6-99.9%) and a specificity 98.6% (CI 98-99%). The BPA test showed a relative sensitivity of 99.6% (CI 98.6-99.9%) and a relative specificity of 98.6% (CI 98.1-99%). Our results indicate that the indirect ELISA is a highly sensitive and specific test and can be adapted to process a large number of samples.

Animals↗
PubMed1977

Masticatory function and post-Pleistocene evolution in Nubia.

The present research focuses on craniofacial variation in Nubia over approximately 10,000 years. Samples were grouped according to their temporal location and subsistence pattern, and represent a transition from a hunting-gathering adaptation (Mesolithic) to a transitional hunting-gathering-agricultural adaptation (A-C Group) and finally to a fully agricultural adaptation (Meroitic/X-Group/Christian). The purposes were: (1) to compare the Mesolithic sample with the later Nubian populations; and (2) to evaluate further the hypothesis that change in Nubian craniofacial morphology was due to changing functional demands associated with the progressive change in subsistence adaptation and associated behavior. The results tend to support recent views that the Nubian Mesolithic population is probably ancestral to later Nubian groups, and that the masticatory-functional hypothesis can best account for craniofacial change among the Nubians since 12,000 B.P. According to this hypothesis systematic reduction in functional demand placed on the masticatory complex from the Mesolithic led, secondarily, to an alteration of the growth of the maxillomandibular complex such that the face became progressively less robust and more inferoposteriorly located relative to the cranial vault. Both the increase in the height of the vault relative to its length, producing a more "globular" appearance, and the reduction in dental size were tertiary, compensatory responses to altered facial size and position.

Biological Evolution↗
PubMed2001

Temperature-driven adaptation of the bacterial community in peat measured by using thymidine and leucine incorporation.

The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25 degrees C to 35, 45, or 55 degrees C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures. Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature. Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community. Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures. The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55 degrees C (thermophilic activity) and 25 degrees C (mesophilic activity). Shifting the peat incubation temperature from 55 to 25 degrees C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity. The availability of substrate for bacterial growth varied over time and among different peat samples. To avoid confounding effects of substrate availability, a temperature adaptation index was calculated. This index consisted of the log(10) ratio of TdR incorporation at 55 and 25 degrees C. The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift. There were no differences between the slopes of the temperature adaptation indices over time for peat samples incubated at 55 degrees C 3 or 11 days before incubation at 25 degrees C. Thus, different levels of bacterial activity did not affect the temperature-driven adaptation of the bacterial community.

Adaptation, Physiological↗
PubMed1990

A new approach to the analysis of nystagmus: an application for order-statistic filters.

A computer program has been designed for the analysis of nystagmus. This program employs a class of nonlinear digital filters called order-statistic (OS) filters. Two OS filters and one linear filter are used. First, the eye-movement signal is smoothed using a predictive finite-impulse response (FIR), median hybrid filter. Then the smoothed signal is processed by a linear band-limited differentiating filter to calculate eye velocity. And finally, the slow-phase velocity (SPV) envelope is extracted from the eye-velocity signal using an adaptive asymmetrically trimmed-mean filter. This approach yields an evenly sampled SPV estimate without resorting to the various interpolation or extrapolation schemes generally used. The adaptive filter estimates SPV based on the local statistical properties of the eye-velocity signal. The adaptive strategy works under the assumption that, on the average, the eyes spend more time in slow-phase than in fast-phase. No assumptions are made about the direction of the nystagmus or the nature of the stimulus used to elicit the nystagmus. This method eliminates all the usual threshold tests and decision logic common to other nystagmus analysis programs. The robust performance of OS filters and the use of adaptive filter structures totally eliminates the need to custom "tune" the program parameters for atypical data sets.

Aircraft↗
PubMed1980

[Problems of maintaining hygienic and microbiological quality standards in the production of foodstuffs, particularly those of vegetable origin. Second report: Demands regarding hygiene and microbiology-theory is useless without practice (author's transl)].

Many legal regulations exist on the hygienic-microbiological properties of food products. The 'Länder-Speiseeisverordnungen' (County Ice Cream Regulations), the 'Diät-Verordnung' (Dietary Regulation) and the 'Trinkwasser-Verordnung' (Regulation on Potable Water) are examples for the frequent inadequacies inherent in these regulations. Caution is indicated against any new proposals for the establishment of additional hygienic-microbiological regulations without sufficient practice-related reasoning unless a revision and improvement of inadequate, already existing regulations has been carried out. Practicable procedures have to be prepared not only to maintain the hygiene in Catering facilities, but also for the evaluation of food products and raw materials according to hygienic-microbiological criteria: Although commonly assumed, the 'Microbe Counts' alone are not sufficient for the determination of the hygienic conditions. Therefore, inspite of all the weak points inherent in quality control procedures - e. g. possible sick, co-workers, only random sample control of raw material supplies - such adapted systems (good manufacturing practice) have to be enforced with special emphasis on the economy of the control expenditure, interdisciplinary aspects, governmental control and consumer information. Examples from literature and own experience demonstrate that the importance of the 'Fecal Indicators' for the judgement of 'hygienic conditions' in food production are rather limited and a correct evaluation only by 'Bacterial Plate Counts' is not possible due to the variation of germ density within one sample. Microbiological investigations of food products according to statistic rules require such an expenditure that they cannot generally be used as routine control procedures. A possible documentation is shown which allows - even without statistic investigations, at least retrospectively - the preparation of a survey on the microbiological properties of raw materials generally available on the market and which makes it possible to recognize and reject any unqualified microbiological limiting value requirements.

Bacteria↗
PubMed2000

A two-sample test with interval censored data via multiple imputation.

Interval censored data arise naturally in large scale panel studies where subjects can only be followed periodically and the event of interest can only be recorded as having occurred between two examination times. In this paper we consider the problem of comparing two interval-censored samples. We propose to impute exact failure times from interval-censored observations to obtain right censored data, then apply existing techniques, such as Harrington and Fleming's G(rho) tests to imputed right censored data. To appropriately account for variability, a multiple imputation algorithm based on the approximate Bayesian bootstrap (ABB) is discussed. Through simulation studies we find that it performs well. The advantage of our proposal is its simplicity to implement and adaptability to incorporate many existing two-sample comparison techniques for right censored data. The method is illustrated by reanalysing the Breast Cosmesis Study data set.

Algorithms↗
PubMed2004

Microirradiation of cells with energetic heavy ions.

The ion microprobe SNAKE at the Munich 14 MV tandem accelerator achieves beam focussing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. Because of these properties, SNAKE is particularly attractive for biological microbeam experiments. Here we describe the adaptation of SNAKE for microirradiation of cell samples. This includes enlarging of the focal distance in order to adjust the focal plane to the specimen stage of a microscope, construction of a beam exit window in a flexible nozzle and of a suitable cell containment, as well as development of procedures for on-line focussing of the beam, preparation of single ions and scanning by electrostatic deflection of the beam. When irradiating with single 100 MeV (16)O ions, the adapted set-up permits an irradiation accuracy of 0.91 microm (full width at half maximum) in the x-direction and 1.60 microm in the y-direction, as demonstrated by retrospective track etching of polycarbonate foils. Accumulation of the repair protein Rad51, as detected by immunofluorescence, was used as a biological track detector after irradiation of HeLa cells with geometric patterns of counted ions. Observed patterns of fluorescence foci agreed reasonably well with irradiation patterns, indicating successful adaptation of SNAKE. In spite of single ion irradiation, we frequently observed split fluorescence foci which might be explained by small-scale chromatin movements.

Cells, Cultured↗
PubMed2002

Assessment of strawberry aroma through SPME/GC and ANN methods. Classification and discrimination of varieties.

To provide an efficient and running analytical tool to strawberry plant breeders who have to characterize and compare the aromatic properties of new cultivars to those already known, a HS-SPME/GC-MS analysis method has been coupled with a statistical treatment method issued from the current development of artificial neuron networks (ANN), and more specifically, the unsupervised learning systems called Kohonen self-organizing maps (SOMs). So, 70 strawberry samples harvested at CIREF from 17 known varieties have been extracted by using a DVB/Carboxen/PDMS SPME fiber according to the headspace procedure, and then chromatographed. A panel of 23 characteristic aromatic constituents has been selected according to published results relative to strawberry aroma. The complex resulting matrix, collecting the relative abundance of the 23 selected constituents for each sample, has been input into the SOM software adapted and optimized from the Kohonen approach described by one of the authors. After a period of training, the self-organized system affords a map of virtual strawberries to which real samples are compared and plotted in the best matching unit (BMU) of the map. The efficiency for discriminating the real samples according to their variety is dependent on the number of units selected to define the map. In this case, a 24-unit map allowed the complete discrimination of the 17 selected varieties. Moreover, to test the validity of this approach, two additional samples were blind-analyzed and the results were computed according to the same procedure. At the end of this treatment, both samples were plotted into the same unit as those of the same variety used for training the map.

Breeding↗
PubMed2007

Human papillomavirus quantification in urine and cervical samples by using the Mx4000 and LightCycler general real-time PCR systems.

During the last decade, growing efforts have focused on human papillomavirus (HPV) detection using liquid hybridization, conventional PCR, and real-time PCR-based methods to increase the overall proportion of patients participating in cervical cancer screening procedures. We proposed a new general HPV DNA real-time PCR on the Mx4000 (Stratagene) and LightCycler (Roche Diagnostics) systems usable for both cervical scrape specimens and urine samples. A linear range was obtained from 5 DNA copies to 8 log(10) DNA copies/ml, and intra- and interassay variations were between 1.8 and 4%. Cervical carcinoma and HPV DNA screening was performed in 333 individual women referred for gynecological examination at the university hospitals of Angers and Brest and enrolled in the PapU study. Among cervical specimens (n = 333), 45% were positive for HPV DNA, with a mean viral load at 5.00 log/ml (+/- 1.73). Among urine samples (n = 177), 37% were positive with a significant 50-fold-lower mean viral load (3.77 +/- 1.32 log/ml; P < 0.0001). Kappa agreement for HPV DNA between cervical and urine specimens was excellent (93%). Thus, we developed a highly sensitive and quantitative general HPV DNA real-time PCR method that allows mass screening of patients with HPV infection. The ongoing longitudinal and prospective multicenter PapU study should give us the opportunity to validate this method adapted to HPV DNA screening in urine samples in a larger population.

Cervix Uteri↗
PubMed1988

Detection and determination of antimalarial drugs and their metabolites in body fluids.

This review of methods for determining antimalarial drugs in biological fluids has focused on the various analytical techniques for the assay of chloroquine, quinine, amodiaquine, mefloquine, proguanil, pyrimethamine, sulphadoxine, primaquine and some of their metabolites. The methods for determining antimalarials and their metabolites in biological samples have changed rapidly during the last eight to ten years with the increased use of chromatographic techniques. Chloroquine is still the most used antimalarial drug, and various methods of different complexity exist for the determination of chloroquine and its metabolites in biological fluids. The pharmacokinetics of chloroquine and other antimalarials have been updated using these new methods. The various analytical techniques have been discussed, from simple colorimetric methods of intermediate selectivity and sensitivity to highly sophisticated, selective and sensitive chromatographic methods applied in a modern analytical laboratory. Knowledge concerning the method for a particular study is determined by the type of application and the facilities, equipment and personnel available. Often is it useful to apply various methods when conducting a clinical study in malaria-endemic areas. Field-adapted methods for the analysis of urine samples can be applied at the study site for screening, and corresponding blood samples can be preserved for subsequent analysis in the laboratory. Selecting samples for laboratory analysis is based on clinical, parasitological and field-assay data. The wide array of methods available for chloroquine permit carefully tailored approaches to acquire the necessary analytical information in clinical field studies concerning the use of this drug. The development of additional field-adapted and field-interfaced methods for other commonly used antimalarials will provide similar flexibility in field studies of these drugs.

Antimalarials↗
PubMed1995

An improved method for clenbuterol screening using high resolution selected ion recording.

A method has been developed using reliable GC derivatization techniques interfaced with a high resolution mass spectrometer. The method has proved successful for the detection of low levels (less than 1 ppm) of clenbuterol in complex biological matrices. Selected ion recording of two characteristic isotopic fragment ions provides a specific mode of detection by verifying the GC retention time of these ions and also by comparing their relative abundance. Analysis of urine samples demands higher mass spectrometric resolution, and 40 000 (10% valley) was found to be a prerequisite for accurate integration of the drug-related chromatographic peaks. The method developed is suitable for adaptation to a completely unattended automated routine incorporating sample injection, storage and retrieval of source tuning parameters, and data processing.

Adrenergic beta-Agonists↗
PubMed1981

Light-induced changes in energy metabolites, guanine nucleotides, and guanylate cyclase within frog retinal layers.

Freeze-dried sections were prepared from retinas of frogs which were dark-adapted or exposed to varying periods of light. Samples of the discrete layers were dissected, weighed, and analyzed for energy metabolites, guanylate compounds, and the enzyme guanylate cyclase. ATP and P-creatine were measured in both dark- and light-adapted retinas. There was a gradient in ATP and P-creatine levels in dark-adapted retinas, with the lower concentrations in the photoreceptors, and increasing concentrations in the inner retina. After light adaptation, concentrations increased, an observation which supports the concept that transmitter release occurs in the dark and ceases in the light. The sum of GTP plus GDP, GDP, and cyclic GMP were analyzed in dark-adapted retinas and after exposure to 2 min or 2 h of room light. GDP was rather uniformly distributed in the retinal layers, was increased by 2 min of light in all layers but the outer nuclear, and remained elevated at 2 h in the inner retina. GTP values showed a marked localization in the outer nuclear layer, which increased after 2 min or 2 h of illumination; in all other layers GTP was decreased by light. Cyclic GMP in the dark was highest in the photoreceptor cells, decreasing to one-third after 2 min of light; there were significant increases in the outer plexiform and inner nuclear layers at this time. Cyclic GMP remained low in the photoreceptor cells even after 2 h of light, while the inner layers returned to dark values. Guanylate cyclase, like cyclic GMP, was largely confined to the photoreceptor cells and showed a maximal increase after 2 min of light exposure.

Adenosine Triphosphate↗
PubMed1994

[Celioscopic cholecystectomy. 2 cases of infectious complications].

Despite the low morbidity and mortality of laparoscopic cholecystectomy, trauma and infection have been reported. Such complications can produce a misleading clinical picture, as in two cases we observed. Case 1. A symptomatic 56-year-old female patient underwent laparoscopic cholecystectomy. During the operation, the gall bladder ruptured and the contents had to be aspirated from the abdominal cavity. The patient complained of hepatalgia 2 weeks after the operation, then was not seen again for more than 1 year when fever and hepatalgia did not respond to symptomatic treatment. An inter-hepato-renal collection (6 cm in diameter) was punctured under echography. Aspirate culture yielded Pseudomonas aeruginosa. Adapted antibiotic therapy was unsuccessful and surgery was required to empty the abscess then remove a fibrous conjunctive tissue formation. Case 2. A 55-year-old female patient with a history of complete remission after mammectomy for breast cancer underwent laparoscopic cholecystectomy in 1991. Two days after the operation, fever (39 degrees C) was accompanied by abdominal defence. Biliary peritonitis due to imperfect suture of the bile duct was repaired followed by peritoneal lavage-drainage. Per-operative blood samples revealed type 6 Pseudomonas aeruginosa. Despite adapted parenteral antibiotics, fever persisted at 39 degrees C and intense jaundice was observed. A second laparoscopy 14 days later showed inflammatory narrowing of the main bile duct which was drained into a small bowel loop. Eight days later computed tomography revealed multiple abscess in the liver. Transparietal cholangiography was performed and showed that the contrast medium entered the abscesses via the biliary canals. The state of sepsis persisted, jaundice worsened and hepatic encephalopathy developed with obnubilation and flapping tremor. After 1 month of general antibiotherapy, no improvement was seen on computed tomography images and needle biopsy of an abscess led to the identification of resistant type 6 P. aeruginosa. Antibiotics were adapted and administered iv with no clinical improvement. Selective catheterism of the hepatic artery via the femoral access was performed to allow intra-hepatic antibiotic delivery. Three weeks later clinical situation remained unchanged when acute respiratory distress highly suggestive of pulmonary embolism led to death. Autopsy was not performed. In both of these rare cases of infectious complications due to P. aeruginosa after laparoscopic cholecystectomy, the source of contamination remained unknown. Nosocomial infection was suspected.

Anti-Bacterial Agents↗
PubMed2004

Study of spectral analytical data using fingerprints and scaled similarity measurements.

A new chemoinformatic model has been developed for enlarging the differences between spectra and applied to differentiation of wines according to the criteria grape origin and variety and ageing process. The model is based on generation of fingerprints from normalised spectra, using empirical parameters and a set of 120 samples. After generation of the fingerprints, similarity matrixes were built on the basis of the Tanimoto similarity index between the fingerprints of the samples. Calculation of the Tanimoto index was modified to adapt the index to the characteristics of the analytical measurements. Thus, scaling factors taking into account pattern fingerprints generated from a group of samples with common characteristics were used. In addition, a modified expression for calculating the Tanimoto index was employed. Principal-components analysis (PCA) and soft independent modelling of class analogy (SIMCA) were applied to the similarity matrixes. The results obtained are discussed as a function of the normalisation method employed, the empirical factor used in generation of the fingerprints, and selection of samples for building the pattern fingerprint, etc. Finally, results from differentiation of wines are compared with those obtained by applying PCA to the unprocessed spectra as stated by the proposed model.

Journal Article↗
PubMed2001

Detection of Cryptosporidium parvum oocysts in environmental water in Hokkaido, Japan.

Control of cryptosporidiosis is important in public health. Rivers that are polluted with Cryptosporidium and drinking water that is treated for drinking water production from polluted rivers could result in the waterborne disease of cryptosporidiosis. We carried out an epidemiological study of natural water supplies in Hokkaido, one of the largest dairy prefectures in Japan. To detect Cryptosporidium oocysts in environmental water, the filtration method was used for 28 samples, which were collected from 10 rivers. A method adapted from the United States Environmental Protection Agency (U.S. EPA) filtration method using a cartridge filter has been used for the collection of samples. Oocysts were separated from a pellet by discontinuous sucrose gradient method. Twelve samples were collected from 10 rivers and parasites were purified by iron (III) flocculation method. Cryptosporidium parvum oocysts were identified with the immunofluorescence antibody technique using DIF kit (Cellabs Pty. Ltd., Sydney/Australia). We detected Cryptosporidium oocysts in 6 out of 10 rivers sampled. Fifty percentage (14/28) of the samples were Cryptosporidium-positive. The average number of Cryptosporidium oocysts was 16.73/100 L (max. 80/100 L).

Animals↗
PubMedDate not supplied

Evaluation of the Syva ETS-PLUS Ethyl Alcohol Assay with application to the analysis of antemortem whole blood, routine postmortem specimens, and synovial fluid.

A critical evaluation of the ETS-PLUS Ethyl Alcohol Assay indicates that it is simple to perform, sensitive enough to reliably detect ethanol above a concentration of 0.010 g/dL, and dynamic enough to reliably quantitate ethanol concentrations between 0.010 and 0.400 g/dL, correlating well with headspace gas chromatography. The technique is suitable for the analysis of relatively fresh (less than 1 month old) serum, plasma, urine, bile, vitreous humor, and gastric samples. With the aid of dilution, it is adaptable to the analysis of whole blood, relatively old specimens (more than 3 months old), viscous and highly pigmented plasma, bile, and the occasional extremely viscous vitreous humor specimens. Our evaluation further indicates that samples can remain in the system's covered sample wheel for up to 2 h without significant (less than 10%) diminution in ethanol concentration. This technique was further utilized to evaluate the suitability of synovial fluid as an alternative for vitreous humor.

Ethanol↗