Routine Laboratory Conditions Research Articles

First Experiences with the Measurement of Thrombin Generation (TGA) on the Ceveron® Alpha in the Routine Lab

Aims: Aim of the study was to show the suitability of the new coagulation analyzer Ceveron® alpha for determination of thrombin generation (TGA) in routine laboratories for discrimination of normal plasma samples from haemophilic and thrombophilic patient samples, from patients with a Lupus syndrome and patients with bleeding disorders.Methods: TGA was determined by a thrombin generation assay based on monitoring the formation of thrombin by means of a fluorigenic substrate upon activation of the coagulation cascade by tissue factor and phospholipids using 2 reagents with different phospholipid concentrations (RClow and RChigh) on Ceveron® alpha. Thrombin generation is measured with a specially adapted TGA fluorimetric module which is placed over the cuvette rotor. Readout parameters were lag time (tLag), peak thrombin (Peak), time to peak(tPeak), velocity index (VI) and area under the curve (AUC). In case of patients with Lupus Anticoagulants a ratio of peak thrombin values triggered with RClow and RChigh was formed. Four laboratory groups participated in the study. All patient samples used for thrombin generation measurement were characterized by routine parameters.Results: Group 1 analyzed samples with bleeding disorders. Peak thombin values showed the best discrimination between Hemophilia B patients (41.9, SD12.7nM), Hemophilia B patient after treatment (188.9nM) and normal controls (208.5nM, SD= 74.0). In Hemophilia A patients tLag was the best parameter to discriminate severe Hemophilia A (8.5min) from acquired hemophilia A and VWD (4.6 min, SD=2.1).Normal ranges for TGA established in group 1 (n=16) were tLag 2.7min (SD=0.8), tPeak 7.8min (SD=1.9), peak thrombin 208.5nM (SD= 74), AUC 1769.4 (SD=327.9).Group 2 analyzed samples of thrombophilic patients with pregnancy (n=30). Patient samples showed peak thrombin values (310.9nM, SD= 65.7) above normals (174.2nM, SD= 22.5). Even patients with heparin treatment had high peak thrombin values (340.5nM, SD=67.2). In heparin treated patients D-dimer values were pathologic as well.Group 3 determined thrombin generation in patients with Lupus Anticoagulants determined by dRVVT, APTT-LA, Lupus-Ceveron and anti-cardiolipin antibodies (ACA). Patients only with high ACA values showed normal peak thrombin ratios of RClow/RChigh 0.6 (SD= 0.07) whereas the ratio in patients with high lupus anticoagulants as measured in the coagulation tests was 0.98 (SD= 0.31) for high antibody titer and 0.83 (SD 0.01) for low antibody titer. Inter-assay CVs in the normal control determined by this laboratory were 2.67% for peak thrombin and 4.83% for AUC and in thrombophilic plasma 3.0% for peak thrombin and 1.41% for AUC.Group 4 determined TGA in orally anticoagulated patients. Normal range for peak thrombin established in this laboratory is 220nM (SD=52, n=12). Oral anticoagulant treatment lowered the AUC (from 1713.3nM, SD= 435.4 thrombin in samples with INR in the range 1 – 1.5 to 894.4nM, SD= 229.2 thrombin in samples with INR in the range 2 – 2.5) and peak thrombin formation (from 204.1nM, SD= 73.5 peak thrombin in samples with INR in the range 1 –1.5 to 79.0nM, SD= 35.2 peak thrombin in samples with INR in the range 2 – 2.5) and prolonged the tLag (from 5.5min, SD= 1.7 in samples with INR in the range 1 –1.5 to 7.6min, SD= 2.4 in samples with INR in the range 2 – 2.5.Conclusions: TGA can be measured reproducibly with low variation under routine laboratory conditions. Peak thrombin is the best parameter for discrimination of patient samples from normals in haemophiliacs, in thrombophilia, patients with a Lupus syndrome, pregnancy and bleeding disorders. With automated, standardised thrombin generation measurement on the Ceveron® alpha large prospective studies can be conducted to confirm these results.

Evaluation of the new ARCHITECT anti-HCV screening test under routine laboratory conditions

Background An improved test version of the Abbott ARCHITECT anti-hepatitis C virus (HCV) test became available at the end of 2005. Study design We compared the new test version with the Ortho Vitros anti-HCV test by evaluating 2034 serum samples in parallel on both systems under routine laboratory conditions. Discordant samples were tested in the Inno-LIA HCV Score assay as well as in the RIBA HCV 3.0. Results Of the 2034 samples 140 (6.9%) yielded positive and 1856 (91.2%) negative results in both assays. We observed discordant results in 38 samples (1.9%). All discrepant samples showed a low S/CO ratio of 1.0–6.9 (mean 2.8) in the Ortho assay and of 1.3–3.0 (mean 1.96) in the ARCHITECT assay. As expected, most of them could not be confirmed by immunoblot testing. Comparison of the results of the two immunoblots (Inno-LIA and RIBA) revealed a great variability in test results. Conclusions This study represents the first comparative evaluation of the modified version of the Abbott ARCHITECT anti-HCV assay in comparison with the Ortho Vitros anti-HCV test. Under routine laboratory testing, we observed good overall concordance between the two assays and no evidence that one assay shows more false-reactive or negative results than the other.

A HIGHLY EFFICIENT AND HIGHLY RELIABLE PROTOCOL FOR TRANSFORMATION OF ESCHERICHIA COLI BY ELECTROPORATION

ABSTRACT Electroporation is widely adopted for highly efficient transformation of Escherichia coli of different purposes, but its efficiency is highly varied in the range of several orders of magnitude. Here we describe a modified and highly reliable electroporation protocol which combines low temperature growth in the presence of sucrose with a postelectroporation heat pulse, which not only gives rise to an over 250‐fold enhancement in transformation compared with conventional protocols, but also alleviates the need for a strict low temperature control at all times of an electroporation‐competent cell preparation experiment and highly pure MilliQ grade water which is not readily available in many laboratories. Transformations for a 6‐kb E. coli and yeast shuttle vector were always higher than 107 transformants/µg, and more than 108 transformants/µg were sometimes recorded in routine experiments.PRACTICAL APPLICATIONSTransformation efficiency reached up to 8 × 109 transformants/µg DNA with ElectroMAXTM DH10BTMEscherichia coli cells and a 6‐kb pYES2/CT plasmid using our procedure, when combined with reduction of ampicillin concentration from 100 µg/mL to 20 µg/mL in the selection plates. It is highly efficient and highly reliable on DNA library construction of all kinds, especially from minute cell and tissue samples, under routine laboratory conditions. It will find widespread application in molecular manipulations in Escherichia coli, the workhorse of molecular biology.

Comparison of Chemical Methods of Assessing Potentially Available Organic Nitrogen from Organic Residues Applied to a Sandy Soil

More than 90% of the nitrogen (N) in soils is bond as organic N compounds. The available N can be estimated on the mineral N released during time‐consuming incubations of soil. Several chemical methods have been developed as substitutes for incubations. On the other hand, there has been an increase in waste production. Residues could potentially offset the need for mineral fertilizers, being both an economic and environmental benefit. Thus, the development of a routine method for prediction of N supply both from soil organic matter (SOM) and the application of organic residues is of great interest. An incubation experiment was performed in a Cambic Arenosol to evaluate different chemical methods. Air‐dried soil was mixed with increasing amounts of composted solid municipal waste, secondary pulp‐mill sludge, hornmeal, poultry manure, the solid phase from pig slurry, and composted pig manure. Samples were incubated for 244 days under a controlled environment. Among the chemical extractants studied, hot 2 M potassium chloride (KCl) and hot 0.01 M calcium chloride (CaCl2) showed promise in indicating values of N0 (potentially available nitrogen), and these simple methods are suitable for use in routine laboratory conditions.

A multidrug efflux system is involved in colony growth in Streptomyces lividans

Multidrug resistance (MDR) genes are abundant in Streptomyces genomes, and yet these bacteria are generally drug sensitive under routine laboratory conditions, indicating low or no expression of these genes. Drug-resistant mutations have been isolated that lie in regulatory genes adjacent to the MDR genes, suggesting that resistance arises by derepression. This study identified a divergently oriented pair consisting of a TetR-family regulator (ebrS) and a major facilitator-family MDR pump (ebrC) gene in Streptomyces lividans, which is widely conserved in Streptomyces species. EbrS represses transcription of ebrC as well as its own transcription. Deletion of ebrS causes overexpression of ebrC, resulting in elevated resistance to many drugs. The ebrS and ebrC promoters were used in a reporter system to test inducibility by various chemicals. Among the 15 compounds (including five EbrC target drugs) tested, none induced ebrC transcription. On the other hand, the ebrS promoter was induced by rifampicin and high concentrations of calcium and magnesium. Deletion of ebrS-ebrC did not change rifampicin sensitivity, indicating that the EbrC pump is not involved in rifampicin efflux. Moreover, deletion of ebrC caused retardation of colony growth on selected media, and the defect could be suppressed by supplementation with high concentrations of Ca(2+), Mg(2+), Na(+) or K(+). Based on these results, it is proposed that the primary biological role of most MDR systems in Streptomyces species is not removal of extrinsic drugs, but rather export of specific toxic compounds endogenously synthesized during growth.

Ejaculate Volume Is Seriously Underestimated When Semen Is Pipetted or Decanted Into Cylinders From the Collection Vessel

The total sperm count (number of spermatozoa per ejaculate) rather than sperm concentration (number of spermatozoa per unit volume of semen) is the more important semen variable related to fertility. It reflects testicular volume (Handelsman et al, 1984; Andersen et al, 2000; Behre et al, 2000), and thus is a measure of total testicular sperm output (MacLeod and Wang, 1979), which is directly related to the chances of pregnancy after coitus. The concentration of spermatozoa in the ejaculate, however, depends on the extent of dilution of epididymal spermatozoa by secretions of the prostate and seminal vesicles occurring at ejaculation and is therefore influenced by the secretory capacity of the accessory sex glands. This is an important distinction, for when comparing semen quality from older and younger men, sperm concentrations do not differ, yet semen volume is reduced in the older men, and so the total number of spermatozoa per ejaculate is lower in the older men (Ng et al, 2004; Nieschlag et al, 1982). The total number of spermatozoa per ejaculate is obtained by multiplying the concentration of spermatozoa by the semen volume. The latter is best measured by weighing (Eliasson, 2003), assuming a density of 1.0 g/mL (Auger et al, 1995; Jorgensen et al, 1997, 2001; Brazil et al, 2004), but alternative methods, such as collection into graduated cylinders (Behre et al, 2000), pipetting from the collection vessel (Mortimer 1994; Jorgensen et al, 1997), and pouring from the collection vessel into a graduated tube (Jorgensen et al, 1997), are in current practice. Two recent studies have found that pipetting semen from the collection vessel leads to an underestimation of about 0.5 mL (range 0.3–0.8 mL; Brazil et al, 2004; Iwamoto et al, 2006) compared with weighing, but no data are available about losses incurred when pouring semen into graduated cylinders. Because the area of contact with the sides of the collection vessel while decanting semen into a graduated cylinder is likely to be far larger than that during pipetting, retention within the vessel could be much larger, leading to a larger underestimation of volume with this method. In this study, new data are obtained on the loss of semen volume during decanting to a cylinder and previously published results on losses because of pipetting, and the density of semen is reanalyzed together with additional data.

NT-proBNP as a tool to stratify disease severity in pulmonary arterial hypertension

The recent development of treatment modalities for patients with idiopathic pulmonary arterial hypertension has been based on the evaluation of many different markers such as functional capacity, addressed by NYHA classification, six-minute walk test (6MWT) and hemodynamic parameters. The aim of this study was to evaluate the correlation of N-terminal fragment (NT-proBNP) with other markers in IPAH and its potential for patient stratification. We studied 42 IPAH patients consecutively evaluated through right heart catheterization in the absence of any specific treatment for pulmonary hypertension. Blood samples, clinical evaluation and 6MWF distance were collected at baseline. The levels of NT-proBNP showed a high correlation with hemodynamic parameters, such as pulmonary vascular resistance (r=0.80, P<0.001). A significant difference was found among patients with different functional classes, addressed by NYHA classification (P< 0.02 for all groups comparison). The discriminant analysis reinforced the ability of NT-proBNP to stratify patients according to NYHA functional class. Compared to the other variables studied (hemodynamics and 6MWT), NT-proBNP had the lowest level of overlap in the stratification of IPAH patients. We conclude that NT-proBNP differs among the different functional classes and correlates with other measures of disease severity, although its role in predicting survival still needs to be addressed.

Anti-dsDNA antibody subtypes and anti-C1q antibodies: toward a more reliable diagnosis and monitoring of systemic lupus erythematosus and lupus nephritis

The putative distinct diagnostic and pathogenic potential of aDNA-Ab subtypes, differing in their affinity or epitope specificity, was subject of several studies with controversial results. Comparing five assays, characterized by different reaction conditions and nature/source of dsDNA, we investigated the abovementioned problem in a retrospective study on 100 systemic lupus erythematosus (SLE) patients and 100 controls (other CTD, autoimmune hepatopathies). As demonstrated, only assay 3 (Farrzyme, TBS, UK) and 5 (Farr-RIA, Trinity Biotech, Ireland) are really suitable to detect primarily high avidity aDNA-Ab. Both were significantly linked to lupus nephritis (specificity 84%) and highly specific for SLE (95 and 96%). Thereby, assay 3 was found to be the first solid phase ELISA probably suitable to replace the Farr-RIA. Classical ELISAs (assay 1, Orgentec, Germany, and 2, Bindazyme, TBS, UK), detecting aDNA-Ab more or less independent from their avidity, or tests with only intermediate specificity for high avidity Ab (assay 4, ELIAdn, Sweden Diagnostics, Germany), were less specific for SLE (83, 79, 91%, respectively) and not associated with renal involvement (specificity 54-57%). At least in the patients studied here, obvious antigen-related differences could not be observed. With slight differences, all assays were suitable to monitor disease activity and therapy in SLE, agreeing with the ECLAM score in about 70-80% of cases. For lupus nephritis, aC1q-Ab are as specific as high avidity aDNA-Ab and capable to close a diagnostic gap in some cases. Thus, to enhance the specificity (up to 98%) and to consider the distinct diagnostic/pathogenic potential of aDNA-Ab subtypes in SLE, under routine clinical laboratory conditions it should be recommended to combine a sensitive screening test with a more specific second assay.

Risk of culture-confirmed borrelial persistence in patients treated for erythema migrans and possible mechanisms of resistance

Erythema migrans (EM) develops at the site of the tick bite in 77–90% of Lyme borreliosis (LB) patients and is therefore a common manifestation of early disease. Clinical treatment failures have been reported in early LB cases for almost every suitable antimicrobial agent. The exact risk of resistance to antibiotic treatment in patients with EM, however, is not known and there are few published cases of culture-proven treatment failure. Moreover, currently available diagnostic techniques cannot reliably discriminate between possible reinfection, true endogenous relapse and co-infection with other tick-borne pathogens. These drawbacks together with the phenomenon of resistance to therapy in individual patients undoubtedly contribute to the inconsistencies surrounding the optimal treatment regimens for LB and are often misinterpreted and misused to support prolonged antibiotic treatment regimens. The question for the underlying mechanisms of possible antimicrobial resistance in Borrelia burgdorferi sensu lato remains unresolved but a better understanding of such genetic or phenotypic mechanisms would be helpful for the treatment of LB and other spirochetal diseases. Investigations on this issue, at best, should start with borrelial isolates cultured from patients before the start of antibiotic therapy and again after the conclusion of treatment. This task, however, remains challenging insofar, as culture is rarely successful under routine laboratory conditions after antimicrobial therapy. Here, we review recent clinical and experimental data on treatment resistance in EM patients suggesting that, although rare, borrelial persistence does occur at the site of the infectious lesion after antibiotic treatment. Borrelial persistence, however, is unlikely to result from acquired resistance against antimicrobial agents that were used for initial specific chemotherapy.

High risk of false positive results in a widely used diagnostic test for detection of the porcine reproductive and respiratory syndrome virus (PRRSV)

During 2003 and 2004, increasing numbers of positive PRRSV RT-PCR results were reported from herds negative for PRRSV infection. Interestingly, three herds represent nucleus herds with no animal contacts from outside and without clinical symptoms of PRRS until now. Since these positive results that were obtained using a PCR protocol adapted to routine laboratory conditions could not be reproduced with other PRRSV specific RT-PCRs, controlled negative and positive samples were used to examine this phenomenon. A RT-PCR assay for detection and differential diagnosis of the European and North American genotypes of the porcine reproductive and respiratory syndrome virus (PRRSV) according to the method previously published by Oleksiewicz et al. [Oleksiewicz, M.B., Botner, A., Madsen, K.G., Storgaard, T., 1998. Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes. Vet. Microbiol. 64, 7–22] was investigated in parallel to another recently published method [Pesch, S., 2003. Etablierung einer Nachweismethode für die zwei Genotypen von dem porcine reproductive and respiratory syndrome virus (PRRSV) und ein Beitrag zu seiner molekularen Epidemiologie. Thesis, Institute of Virology, Faculty of Veterinary Medicine, University of Leipzig]. A panel of 228 clinical samples sent in for PRRSV routine diagnostics served as test panel. It was found that both methods have similar analytical sensitivity. However, the primers published by Oleksiewicz were shown to yield a very high proportion of false positive results under routine diagnostic laboratory conditions, i.e. they resulted in RT-PCR products with non-PRRSV sequences, that were indistinguishable from truly positive reagents in standard gel electrophoresis settings. The reason for and possible implications of this finding as well as the risk of modifying published methods without control are discussed.

Determination of adefovir in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to determine the concentrations of adefovir [9-(2-phosphonylmethoxyethyl)adenine, PMEA] in human plasma. After one-step protein precipitation of plasma samples by methanol, adefovir was analyzed by LC/MS/MS using positive electrospray ionization. Chromatography was performed on a C18 column. The extraction recoveries of adefovir were found to be 85.1-89.3%. Adefovir was stable under routine laboratory conditions. A minimal matrix effect resulting in a slight ionization enhancement of adefovir (<10.9%) was observed, which did not markedly affect the behavior of the calibrations curves and accuracy and precision data. The method had a chromatographic run time of 7.8 min and a linear calibration curve over the concentration range 1.5-90 ng/mL for adefovir. The lower limit of quantification of the method was 1.5 ng/mL. The intra- and inter-day precision was less than 8.4%. These results indicated that this LC/MS/MS method has high selectivity and efficiency, and acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method has been successfully used in a pharmacokinetic study in healthy volunteers treated with oral adefovir dipivoxil at 10 and 20 mg.

Effect of Short Time Morphine Addiction on the Kidney Tissue in Rat

Opium is an important substance that abuses and has very pharmacological and pathological effects. Morphine is the main substance of opium. In the recent study, it has been tried to identify the short effects of morphine on the kidney pathology of Rats. The randomized double blind study was designed. Twenty rats were selected and divided in two groups and maintained under routine laboratory conditions and similar intervention. One group had been addicted orally by morphine powder and control were not addict. After three weeks, all rats were anesthetized and underwent autopsy. Their kidneys were removed en-block and sent to pathology laboratory and reviewed by one pathologist. The major considered variables were tubulo-interstitial and glomerular changes, glomerular amount and size on light microscope view. Glomerular, tubular and vascular changes, glomerular counts, glomerular size, and cortical thickness showed no significant differences between groups of study. Only Lymphocytic infiltration was higher frequency in addict group, significantly P=0.007. It seems short course of morphine consumption is very little effect on kidney, only Lymphocytic infiltration observe in addicted renal tissue which, may be precursor of other changes in kidney. It is better long term studies are implemented in this course.

Soil Lime Requirement by Direct Titration with a Single Addition of Calcium Hydroxide

Direct titration of acid surface soils is often used to measure soil acidity. However, titration has not been used for routine determination of the lime requirement (LR) in soil testing laboratories, because it is too time‐consuming, requiring multiple additions of base and long equilibration times between additions. Since soil pH as a function of added base can be described well by a linear equation, titration may be adopted by soil testing laboratories using a minimum number of base additions. Our objective was to evaluate the accuracy of a simplified titration procedure, based on an initial pH reading and a second reading following the addition of one dose of Ca(OH)2, followed by extrapolation to the target pH. Seventeen soils were titrated with Ca(OH)2 in both water and 0.01 M CaCl2 with a 30‐min interval between additions. The slopes from the first two data points of the titration in water were frequently in error for estimation of the slopes regressed by all data points to pH 6.5. However, the first two data points in the 0.01 M CaCl2 titration were found to be more reliable for estimating the slopes. Both the initial pH in water and in 0.01 M CaCl2 were used to calculate the LR with the two‐point slope in 0.01 M CaCl2. The LR predicted using the initial pH in 0.01 M CaCl2 gave better estimates of the LR than the initial water pH when compared with the standard 3‐d Ca(OH)2 incubation. A linear regression of LR by the single addition method in 0.01 M CaCl2 (Y) on LR determined with the 3‐d Ca(OH)2 incubation (X) gave the linear equation Y = 0.88 X, r2 = 0.93. It is concluded that the LR predictions with the single addition method in 0.01 M CaCl2 are sufficiently accurate to justify further evaluation under routine laboratory conditions.

Performance of the New VITEK 2 GP Card for Identification of Medically Relevant Gram-Positive Cocci in a Routine Clinical Laboratory

The VITEK 2 gram-positive (GP) identification card (bioMerieux, Marcy l'Etoile, France) has been redesigned to achieve greater accuracy in the identification of gram-positive cocci. A total of 43 biochemical tests, including 17 enzymatic tests, are present in the card and interpreted in a kinetic mode, for up to 8 h. The VITEK 2 database, used in conjunction with the GP identification card, allows the identification of 115 different taxa. A total of 364 strains of GP cocci (217 Streptococcaceae strains and 147 Micrococcaceae strains) belonging to 31 taxa were tested with the new VITEK 2 GP identification card. Of the 364 strains, 105 were taken from routine primary plating media. A total of 344 strains (94.5%) were correctly identified to the species level and 17 strains (4.7%) were identified with low discrimination, requiring additional tests, whereas 1 strain (0.3%) was incorrectly identified and 2 strains (0.5%) remained unidentified. Within 7 h of the start of incubation, more than 90% of all strains were identified. Of the 105 primary cultures, 97% were correctly identified to the species level, 2% were identified with low discrimination, and 1% remained unidentified. Identification performance data were independent of each of the three plating media used. It is concluded that the new VITEK 2 GP identification card provides reliable results for the identification of GP cocci under routine laboratory conditions.

Evaluation of the automated coagulation analyzer Sysmex® CA-7000

Introduction Centralization of laboratory diagnostics and an increasing number of urgent requests and nonstandard samples raise the need for short turn-around times and high-throughput analyzers for coagulation tests. The aim of the present study was to evaluate the analytical and technical performance of the Sysmex® CA-7000 coagulation analyzer under routine laboratory conditions. Materials and methods We evaluated the Sysmex® CA-7000 in comparison to the Sysmex® CA-6000 analyzer for PT, INR, aPTT, Clauss, and derived fibrinogen. We also compared antithrombin (AT) measured on the Sysmex® CA-7000 and Dimension® RxL. Imprecision studies were performed, and throughput, online, and STAT functions and the handling of routine samples were evaluated. Results The Sysmex® CA-7000 showed very low intra-assay and interassay variability for all parameters. The method comparison study showed good comparability to the Sysmex® CA-6000 and Dimension® RxL. The throughput of the Sysmex® CA-7000 was 2.5–3 times faster than that of Sysmex® CA-6000. No interference was seen for total bilirubin up to 710 μmol/L. Triglyceride levels >11.4 mmol/L resulted in invalid PT measurements and levels >3.42 mmol/L gave invalid results for Clauss fibrinogen on the CA-7000 analyzer. Conclusions This is the first published evaluation of the analytical and technical performance of the coagulation analyzer Sysmex® CA-7000. We conclude that the CA-7000 is well suited for coagulation laboratories with high sample throughput and a high number of nonstandard samples.

Monitoring of tienopyridines or glycoprotein IIb/IIIa-receptor antagonists: simple platelet function tests

Platelet function analyses were known as mystified, complicated, and time consuming laboratory investigations with a high coefficient of variation. Only in rare cases they led to precise interpretations under routine clinical laboratory conditions. In the first line they were focussed on bleeding tendencies. Only few were suited to evaluate the state of hyperaggregability of platelets. This short presentation of the GTH-Symposium 2004 gives a survey on the most simple and practicable tests for checking platelet function which may be used for the monitoring of therapy with tienopyridines and glycoprotein IIb/IIIa-receptor antagonists.

Characterization of Pseudomonas putida genes responsive to nutrient limitation.

The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation and biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high availability of nutrients and oxygen. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 were examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7 and NRM17, were selected for identification of the tagged genes. In strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased 4.9-26.4-fold under various low-nutrient conditions. In NRM7, expression of the novel NADPH : quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low-nutrient and anoxic conditions. The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low-nutrient conditions but its absolute expression level was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that in Escherichia coli.

In-house validation of a real-time PCR method for rapid detection of Salmonella ssp. in food products

A real-time polymerase chain reaction (PCR) method for Salmonella ssp. detection in food samples has been developed and validated in-house. The specificity of the assay was confirmed by tests with 295 different Salmonella strains, including four strains of Salmonella bongori. When tested with extracted Salmonella DNA the lowest detected amount was found to be 5 fg, which is equivalent to approximately one genome copy. The detection limit was further determined by artificial contamination of minced meat with S. Typhimurium cells and of pastry with S. enteritidis using the most probable number approach for cell dose dilutions. It was calculated that even one colony forming unit of Salmonella was still detectable in 25 g food after enrichment culture for 18 h. An additional PCR system for internal positive control, which was included in each reaction and detected in parallel via another reporter fluorescence dye, has no negative impact on the sensitivity of the assay. The method was evaluated with 1,293 naturally contaminated food samples and compared to the conventional cultural method. Of 55 positive PCR samples, 45 were confirmed by the cultural method. The statistical comparison revealed a correlation of 99.2% for specificity, of 100% for sensitivity and of 99.2% for trueness. The results of the comparative analysis and the advantages of the real-time PCR method for detection of Salmonella ssp. under routine laboratory conditions are discussed.

Next Day Detection of Salmonella spp. In Naturally Contaminated Meat Products Using Molecular Approach

Salmonella species are a well known food borne pathogen in developed and developing countries with a wide variety of illnesses conditions. The aim of this study was to compare a traditional isolation and identification method for Salmonella spp from meat products with a newer DNA/rRNA gene hybridization method [LUMIprobe 24 Salmonella] under the routine food laboratory conditions. A total of 133 samples [30 pork sausage, 40 chicken meat coated with oil and spices, 40 kebab and 23 beef burger] were collected from February to May 2004, samples were processed according to the guideline of the British Standard Cultural Method; BS EN 12824:1998, as well as, the DNA/rRNA method as guided by the manufacture. Results showed that the two methods gave equivalent sensitivity and selectivity for Salmonella spp detection. Significant time saving was achieved using the LUMIprobe 24 test allowing earlier release of results on the next day and the new molecular method experienced a reasonable level of flexibility and labor saving.

Monitoring von Tienopyridinen und GPIIb/IIIa-Antagonisten

ZusammenfassungThrombozytenfunktionstests galten in der Vergangenheit als mystifizierte, komplizierte und zeitaufwändige Untersuchungen mit hohem Variationskoeffizient bezüglich Präzision. Sie ermöglichten zudem oft keine schlüssige Interpretation unter Routinebedingungen im klinischen Labor. Diese Tests wurden primär zur Evaluierung von Störungen der primären Hämostase eingesetzt. Nur wenige erfassten auch die Plättchenhyperreaktivität. Diese Zusammenfassung der Präsentation im Rahmen des GTH-Symposiums 2004 gibt einen Überblick über eine Auswahl von einfachen, leicht handhabbaren Plättchenfunktionstests, die sich zum Monitoring der Therapie mit Tienopyridinen und Glykoprotein-IIb/IIIa-Rezeptor-Antagonisten eignen könnten.