D-dimer levels and recurrence in patients with unprovoked VTE and a negative qualitative D-dimer test after treatment.

BACKGROUND: The rate of recurrent venous thromboembolism (VTE) in patients with a first unprovoked VTE who had a negative qualitative D-dimer test one month after stopping anticoagulant therapy was higher than expected in the D-dimer Optimal Duration Study (DODS). OBJECTIVES: To determine whether quantitative D-dimer levels using a low threshold, age- and sex-specific thresholds, or repeated measurements, would improve identification of patients at low risk of recurrent VTE. MATERIALS AND METHODS: D-dimer levels were quantified in banked samples from 307 patients in DODS who had a negative qualitative D-dimer test while on, and 1month after stopping, anticoagulant therapy and the rates of recurrent VTE were determined in patients with D-dimer levels below various predefined thresholds. RESULTS: The rate (per patient year) of recurrent VTE was: 5.9% with D-dimer levels<250µg/l at one month; 5.2% with D-dimer levels between 250 and 499µg/l at one month; 5.0% with D-dimer levels less than predefined age- and sex-specific thresholds at one month; and 6.3% when D-dimer levels were <500µg/l at both one and 7months after stopping anticoagulant therapy. These rates are similar to the overall event rate of 6.3% in patients who stopped treatment. CONCLUSIONS: Among unprovoked VTE patients who had a negative qualitative D-dimer test during and after anticoagulant therapy, low D-dimer thresholds, age and sex-adjusted thresholds or repeated measurements, did not identify subgroups with a very low rate of recurrence.

Exclusion of deep vein thrombosis using the Wells rule in clinically important subgroups: individual patient data meta-analysis.

OBJECTIVE: To assess the accuracy of the Wells rule for excluding deep vein thrombosis and whether this accuracy applies to different subgroups of patients. DESIGN: Meta-analysis of individual patient data. DATA SOURCES: Authors of 13 studies (n = 10,002) provided their datasets, and these individual patient data were merged into one dataset. ELIGIBILITY CRITERIA: Studies were eligible if they enrolled consecutive outpatients with suspected deep vein thrombosis, scored all variables of the Wells rule, and performed an appropriate reference standard. MAIN OUTCOME MEASURES: Multilevel logistic regression models, including an interaction term for each subgroup, were used to estimate differences in predicted probabilities of deep vein thrombosis by the Wells rule. In addition, D-dimer testing was added to assess differences in the ability to exclude deep vein thrombosis using an unlikely score on the Wells rule combined with a negative D-dimer test result. RESULTS: Overall, increasing scores on the Wells rule were associated with an increasing probability of having deep vein thrombosis. Estimated probabilities were almost twofold higher in patients with cancer, in patients with suspected recurrent events, and (to a lesser extent) in males. An unlikely score on the Wells rule (≤ 1) combined with a negative D-dimer test result was associated with an extremely low probability of deep vein thrombosis (1.2%, 95% confidence interval 0.7% to 1.8%). This combination occurred in 29% (95% confidence interval 20% to 40%) of patients. These findings were consistent in subgroups defined by type of D-dimer assay (quantitative or qualitative), sex, and care setting (primary or hospital care). For patients with cancer, the combination of an unlikely score on the Wells rule and a negative D-dimer test result occurred in only 9% of patients and was associated with a 2.2% probability of deep vein thrombosis being present. In patients with suspected recurrent events, only the modified Wells rule (adding one point for the previous event) is safe. CONCLUSION: Combined with a negative D-dimer test result (both quantitative and qualitative), deep vein thrombosis can be excluded in patients with an unlikely score on the Wells rule. This finding is true for both sexes, as well as for patients presenting in primary and hospital care. In patients with cancer, the combination is neither safe nor efficient. For patients with suspected recurrent disease, one extra point should be added to the rule to enable a safe exclusion.

Diagnosis of DVT: antithrombotic therapy and prevention of thrombosis: American College of Chest Physicians evidence-based clinical practice guidelines

BACKGROUND: Objective testing for DVT is crucial because clinical assessment alone is unreliable and the consequences of misdiagnosis are serious. This guideline focuses on the identification of optimal strategies for the diagnosis of DVT in ambulatory adults. METHODS: The methods of this guideline follow those described in Methodology for the Development of Antithrombotic Therapy and Prevention of Thrombosis Guidelines: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. RESULTS: We suggest that clinical assessment of pretest probability of DVT, rather than performing the same tests in all patients, should guide the diagnostic process for a first lower extremity DVT (Grade 2B). In patients with a low pretest probability of first lower extremity DVT, we recommend initial testing with D-dimer or ultrasound (US) of the proximal veins over no diagnostic testing (Grade 1B), venography (Grade 1B), or whole-leg US (Grade 2B). In patients with moderate pretest probability, we recommend initial testing with a highly sensitive D-dimer, proximal compression US, or whole-leg US rather than no testing (Grade 1B) or venography (Grade 1B). In patients with a high pretest probability, we recommend proximal compression or whole-leg US over no testing (Grade 1B) or venography (Grade 1B). CONCLUSIONS: Favored strategies for diagnosis of first DVT combine use of pretest probability assessment, D-dimer, and US. There is lower-quality evidence available to guide diagnosis of recurrent DVT, upper extremity DVT, and DVT during pregnancy.

Genetics informatics trial (GIFT) of warfarin to prevent deep vein thrombosis (DVT): rationale and study design.

The risk of venous thromboembolism (VTE) is higher after the total hip or knee replacement surgery than after almost any other surgical procedure; warfarin sodium is commonly prescribed to reduce this peri-operative risk. Warfarin has a narrow therapeutic window with high inter-individual dose variability and can cause hemorrhage. The genetics-informatics trial (GIFT) of warfarin to prevent deep vein thrombosis (DVT) is a 2 × 2 factorial-design, randomized controlled trial designed to compare the safety and effectiveness of warfarin-dosing strategies. GIFT will answer two questions: (1) does pharmacogenetic (PGx) dosing reduce the rate of adverse events in orthopedic patients; and (2) is a lower target international normalized ratio (INR) non-inferior to a higher target INR in orthopedic participants? The composite primary endpoint of the trial is symptomatic and asymptomatic VTE (identified on screening ultrasonography), major hemorrhage, INR ≥ 4, and death.

The effect of dietary protein level on performance characteristics of coccidiosis vaccinated and nonvaccinated broilers following mixed-species Eimeria challenge.

A series of experiments were conducted to investigate the effect of starter diet protein levels on the performance of broilers vaccinated with a commercially available live oocyst coccidiosis vaccine before subsequent challenge with a mixed-species Eimeria challenge. Data indicated that an increasing protein concentration in the starter diet improved broiler performance during coccidiosis vaccination. Prechallenge performance data indicated that vaccination could decrease BW and increase feed conversion ratio. The time period most important for the observed effects appeared to be between 13 and 17 d of age. This reduction in performance parameters of vaccinated broilers compared with nonvaccinated broilers was eliminated by the conclusion of the experiments (27 d) in the diet groups with higher protein. Vaccination was effective at generating protective immunity against Eimeria challenge, as evidenced by increased (P < 0.05) BW gain, improved feed conversion, reduced postchallenge mortality, and reduced lesion development in vaccinated broilers compared with nonvaccinated broilers. These observations support numerous other reports that confirm live oocyst vaccination can be used effectively as a preventive against avian coccidiosis in commercially reared broilers. More important, these findings suggest that reduced protein concentration of starter diets can lead to significant losses in broiler performance when using a vaccination program to prevent coccidiosis.

Molecular analysis of Salmonella serotypes at different stages of commercial turkey processing.

Salmonella isolates were collected from 2 commercial turkey processing plants (A and B) located in different US geographical locations. Isolates recovered at different stages of processing were subjected to 2 genotype techniques [PAGE and denatured gradient gel electrophoresis (DGGE)] to determine their usefulness for Salmonella serotyping. Primers used for PCR amplification were to a highly conserved spacer region located between the 16S and 23S rDNA genes. Sampling sites at plant A were 1) postscald, 2) pre-inside-outside bird wash, 3) post-IOBW, and 4) postchill with 30, 44, 36, and 12 Salmonella isolates recovered, respectively. Plant B had an additional site and these locations were 1) prescald, 2) postscald, 3) pre-inside-outside bird wash, 4) post-IOBW, and 5) postchill with 16, 54, 24, 35, and 24 Salmonella isolates recovered, respectively. In plant A, 4 different Salmonella serotypes were identified: Derby, Hadar, Montevideo, and Senftenberg. In plant B, 10 serotypes were identified: Agona, Anatum, Brandenburg, Derby, Hadar, Meleagridis, Montevideo, Reading, Senftenberg, and Typhimurium. Salmonella Derby was predominant in plant A (83%), whereas Salmonella Typhimurium was the most common serotype recovered in plant B (39%). Genotype analyses of the Salmonella serotypes were expressed in dendrograms with comparisons interpreted as percentage similarity coefficients. Both PAGE and DGGE were able to distinguish serotype band patterns. However, DGGE was more discriminating than PAGE. Isolates of the same serotypes were grouped together on the dendrogram of band patterns generated by DGGE. In contrast, PAGE failed to group all like serotypes together on the corresponding dendrogram. The results of the study suggest that genotyping techniques can be very useful in discriminating Salmonella serotypes collected from the processing plant environment of commercial poultry production. These molecular techniques may offer more cost-effective means to identify Salmonella serotypes from large numbers of isolates and with more immediate results than those currently achieved with conventional typing techniques.

Evaluation of repetitive extragenic palindromic-polymerase chain reaction and denatured gradient gel electrophoresis in identifying Salmonella serotypes isolated from processed turkeys.

The current study was conducted to determine the usefulness of 2 molecular techniques, automated repetitive extragenic palindromic-PCR (REP-PCR) and denaturing gradient gel electrophoresis (DGGE), to identify Salmonella serotypes of poultry origin. Salmonella continues to be a foodborne pathogen of principal concern in the United States. The interspersed conserved repetitive sequence of the bacterial genome and the 16-23S rDNA intergenic spacer region were amplified for REP-PCR and DGGE, respectively. Fifty-four Salmonella isolates from 2 turkey processing plants (A and B) were used for this comparison. Serotypes consisted of Brandenburg, Derby, Hadar, and Typhimurium, with n=6, 21, 12, and 15, respectively. The REP-PCR was fully automated, whereas DGGE was run on an acrylamide gel and the image was captured digitally. Both dendrograms were created using the unweighted pair group method with arithmetic average. There were more variations in percentage similarity in DGGE when compared with REP-PCR. The banding patterns were more distinct and uniform in the REP-PCR group than with DGGE. The results from the REP-PCR were generated within 1 h, whereas the DGGE required approximately 1 d to run. These data suggest that DGGE and REP-PCR are useful tools for identifying Salmonella serotypes isolated from poultry production or processing environments. In addition, REP-PCR is more rapid, may have a higher discriminatory power, but may be less cost-effective than DGGE. However, more research may be needed to validate this argument. Both DGGE and REP-PCR displayed high sensitivity in discriminating among Salmonella serotypes and either method could be considered as an alternative to more expensive and time-consuming conventional antibody-based serotyping methodologies.

Measurement of Acetylcholine in Rat Brain Microdialysates by LC - Isotope Dilution Tandem MS.

An LC-MS/MS method was developed for measuring acetylcholine (ACh) in an aqueous medium using reversed-phase ion-pair chromatography, electrospray ionization on a quadrupole ion trap instrument and a tetradeuterated analogue (ACh-1,1,2,2-d(4)) as an internal standard. A rapid separation was achieved on a 5-cm long octadecylsilica column (2.1 mm i.d.) by employing heptafluorobutyric acid (0.1% v/v) as an ion-pairing agent and requiring 10% v/v acetonitrile in 20 mM ammonium formate buffer under isocratic elution at 200 µl/min flow rate. The instrument's response was calibrated with samples containing known mole ratios of ACh and ACh-1,1,2,2-d(4) in an artificial cerebrospinal fluid, which afforded the conclusion that analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with the known molar concentration of the ACh-1,1,2,2-d(4) added. The rapid and simple assay was tested by measuring the basal neurotransmitter concentration in rat brain microdialysates without the use of a cholinesterase inhibitor upon sample collection.

The flagellar filament of Rhodobacter sphaeroides: pH-induced polymorphic transitions and analysis of the fliC gene.

Flagellar motility in Rhodobacter sphaeroides is notably different from that in other bacteria. R. sphaeroides moves in a series of runs and stops produced by the intermittent rotation of the flagellar motor. R. sphaeroides has a single, plain filament whose conformation changes according to flagellar motor activity. Conformations adopted during swimming include coiled, helical, and apparently straight forms. This range of morphological transitions is larger than that in other bacteria, where filaments alternate between left- and right-handed helical forms. The polymorphic ability of isolated R. sphaeroides filaments was tested in vitro by varying pH and ionic strength. The isolated filaments could form open-coiled, straight, normal, or curly conformations. The range of transitions made by the R. sphaeroides filament differs from that reported for Salmonella enterica serovar Typhimurium. The sequence of the R. sphaeroides fliC gene, which encodes the flagellin protein, was determined. The gene appears to be controlled by a sigma(28)-dependent promoter. It encodes a predicted peptide of 493 amino acids. Serovar Typhimurium mutants with altered polymorphic ability usually have amino acid changes at the terminal portions of flagellin or a deletion in the central region. There are no obvious major differences in the central regions to explain the difference in polymorphic ability. In serovar Typhimurium filaments, the termini of flagellin monomers have a coiled-coil conformation. The termini of R. sphaeroides flagellin are predicted to have a lower probability of coiled coils than are those of serovar Typhimurium flagellin. This may be one reason for the differences in polymorphic ability between the two filaments.

Medical savings accounts (MSAs)--big benefits for small business.

The Health Insurance Portability and Accountability Act, passed by Congress last year, implemented a four-year pilot project that allows employees of small businesses to set up tax-free interest-bearing medical savings accounts. These accounts offer advantages for both employees and employers

Estimating neonatal mortality risk: an analysis of clinicians' judgments.

BACKGROUND: Clinicians' estimates of mortality risk in the neonatal intensive care unit (NICU) have implications for patient triage, transfer, initiation and termination of life support, and allocation of medical resources. The accuracy of these judgments has not been studied, nor the differences between nurses and attending physicians. OBJECTIVES: 1) evaluate the accuracy of subjective judgments of NICU unit mortality risk, 2) identify the key components of clinician judgments, 3) compare accuracy between attending physicians and nurses, and 4) examine the utility of combining an objectively computed risk and clinician judgments to improve predictions. METHODS: We obtained estimates of mortality risk on 544 admissions to two NICUs on the day of admission from the attending physician and primary nurse. These were compared with an objective computed mortality risk based on birth weight and the Score for Neonatal Acute Physiology (SNAP) using a linear judgment analysis model, as well as with actual outcomes. RESULTS: Physicians and nurses had good discriminating power with actual mortality rates ranging from 0% among low risk patients to 67% among those with the highest mortality estimates. Physicians had a tendency to overestimate mortality risk. Clinicians base their estimates on the same factors and similar judgment weights as the empiric mortality risk model (22% birth weight, 62% illness severity (SNAP), 13% low Apgar, and 3% for intrauterine growth restriction). Clinicians place additional emphasis on therapeutic as well as physiologic factors. When the computed risk and physician judgment were combined, both made significant contributions in a logistic mortality risk model. CONCLUSIONS: Clinician judgments of mortality risk are fairly accurate and similar to an objective illness severity index. This simple method provides insight into clinical decision making and has important applications in improving direct patient care, appropriate allocation of medical resources, and medical training.

Rectal spirochaetosis in homosexual men: the association with sexual practices, HIV infection and enteric flora.

OBJECTIVE: To determine the prevalence of rectal spirochaetosis in homosexual men attending a sexually transmissible diseases clinic and investigate the association between their presence and sexual practices, HIV infection and enteric flora. DESIGN: The study included 144 male homosexual subjects who each completed a questionnaire, underwent physical examination, proctoscopy and investigations for STD and HIV screening, rectal biopsies and collection of faecal samples. SETTING: The Sexual Health Centre, Sydney Hospital, Sydney, Australia. RESULTS: Spirochaetes were detected in 39% of the rectal biopsies, using histological criteria. Logistic regression analysis showed that rectal spirochaetosis was significantly associated with: oral-anal contact. (P < 0.05, OR 3.45, 95% CI 1.48-8.05); detection of 3-5 different non-pathogenic protozoa in faeces (P < 0.01, OR 11.68, 95% C.I. 2.33-58) and a positive HIV antibody test (P < 0.01) OR 4.48, 95% C.I. 1.28-15.72). CONCLUSIONS: These findings indicate that rectal spirochaetosis is relatively common in homosexual men. The association with non-pathogenic protozoa is most likely attributable to the common mode of transmission viz oral-anal contact. However it is difficult to determine whether the association with HIV infection is cause or effect because of the limitations in the study design. Further information is required to determine the clinical significance of infection with these organisms.

Subclinical ischaemic episodes during the steady state of sickle cell anaemia.

AIMS: To determine the clinical, haematological, biochemical and rheological changes that occur in the asymptomatic steady state of sickle cell anaemia. METHODS: Patient self-assessment visual analogue scores (for wellbeing and tiredness), the blood concentration of acute phase proteins (C-reactive protein, orosomucoid, and fibrinogen), and blood rheology (percentage of dense cells and the number of sickled cells that occluded pores 5 microns in diameter) were studied longitudinally on 10 occasions in each of 20 outpatients with sickle cell anaemia. RESULTS: Patients in the steady state showed fluctuation in visual analogue scores, in concentration of acute phase proteins, and in rheological parameters consistent with minor episodes of tissue injury. Significantly more variation in acute phase proteins occurred in the steady state of 14 of the 20 patients who developed one or more vaso-occlusive crises during the 16 month study period. Rheological fluctuation in the steady state simulated rheological change during crisis, namely a transient rise and then fall in the number of dense and poorly filterable cells. CONCLUSIONS: The term "steady state" is a misnomer, being characterised by biochemical and rheological fluctuation consistent with minor episodes of microvascular occlusion that are insufficient to cause the overt tissue infarction of painful crisis.

Rheological changes in the prodromal and established phases of sickle cell vaso-occlusive crisis.

A rheological study has been made in 20 patients with sickle cell anaemia in the steady state and in the prodromal and established phases of 12 vaso-occlusive crises. Rheology of sickle cells was studied by discontinuous density gradient fractionation and by filtration through pores of 5 microns diameter. The prodromal phase of crisis (day 1), when compared with mean steady state values, was associated with the development of a sub-population of poorly deformable dense cells. This sub-population appeared 1 or more days before the acute-phase rise in C-reactive protein, orosomucoid, fibrinogen, plasma viscosity and leucocytes, and before the rise in serum lactate dehydrogenase. As crisis evolved, the sub-population decreased to steady-state values, or below, by days 6-7. Identification of the prodromal phase of sickle cell crisis has allowed the detection of rheological changes of potential aetiological significance.

Concurrent zidovudine-induced myopathy and hepatoxicity in patients treated for human immunodeficiency virus (HIV) infection.

Myopathy and hepatic toxicity are important complications of zidovudine (3'-azido-3'-deoxythymidine therapy) in patients infected with the human immunodeficiency virus (HIV) both may also occur in HIV infection in the absence of zidovudine therapy. We report 2 cases of myopathy caused by zidovudine, occurring within 16 wks of initiation of therapy, and a case of concurrent hepatic and muscle toxicity. In one case, electron microscopy demonstrated characteristic enlarged mitochondria with paracrystalline inclusions. This technique can distinguish the myopathies caused by either HIV or zidovudine. Both zidovudine-induced myopathy and hepatoxicity require discontinuation of the drug if severe.

The effects of anthranilic acid on gene expression.

1. A Drosophila pseudoobscura amylase gene cloned in Escherichia coli is expressed at high levels. The expression of this gene is repressed when glucose (0.5% final concentration) is added to a starch minimal medium culture of E. coli cells containing the amylase plasmid. 2. Addition of anthranilic acid (5 and 7 mM final exogenous concentration) to catabolite repressed cells mimics the action of adenosine 3'5' cyclic monophosphate (cAMP) by depressing the expression of the amylase. 3. The results suggest that anthranilic acid acts either indirectly, possibly through the glucose transport system, or directly, by way of an intercalative model of initiation, to alter the levels of transcription.

Effect of polymerization tendency on haematological, rheological and clinical parameters in sickle cell anaemia.

The polymerization tendency of sickle haemoglobin was estimated as a function of oxygen saturation in 30 patients with homozygous sickle cell anaemia. The deformability of their erythrocytes was also measured, by initial-flow-rate filtration at 37 degrees C through pores of 5 microns diameter, and clinical severity was assessed using a visual analogue scale. By means of partial correlation analysis, it was found that correlations between haematological, rheological, and clinical parameters in sickle cell anaemia could be explained on the basis of an association of each variable with polymerization tendency. Patients with the greatest tendency to form polymer had the least deformable erythrocytes and perceived their disease to be more severe as judged by the visual analogue scale. Polymer formation also appeared to be a determinant of the number of dense cells which, in turn, determine haemolytic rate and erythrocyte deformability.

An amylase gene from Drosophila pseudoobscura is expressed in Escherichia coli. Functional selection and biochemical comparisons of the fly- and clone-produced amylases.

An amylase gene from Drosophila pseudoobscura was isolated from a genomic library constructed in pBR322 and cloned in Escherichia coli by selecting for the ability of its product to hydrolyze starch, a carbon source not normally utilized by E. coli. Hybridization of pAMY17F to D. pseudoobscura polytene chromosomes shows a positive signal at the amylase pseudogene locus (bank 78, chromosome 3). The chimeric plasmid pAMY17F, has been altered in such a way as to increase amylase expression. Southern and Northern hybridizations to the cloned amylase DNA indicate that the source of the gene is from D. pseudoobscura. Biochemical properties such as pH optima, substrate specificities, electrophoretic analyses, inhibitor sensitivities, heat stabilities, temperature responsiveness and molecular weights indicate that the amylases produced by the fly and bacterial clone are similar and have similar properties. It appears that E. coli/pAMY17F is producing an amylase like that found in D. pseudoobscura.