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.

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.

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.

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.

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.

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.

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.

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.

The restoration of the vasculature of skin autografts in the rabbit.

The restoration of the vasculature of full-thickness skin autografts was investigated on the rabbit ear, the grafted discs of skin being replaced with their original orientation, or rotated 90 or 180 degrees. Circulation in the graft was first detected 1 day after grafting, increasing on the 2nd and successive days. The larger arteries in the grafts survived and became permanently joined at the periphery of the grafts to adjacent severed arteries in the host by well-defined and often tortuous segments termed junction vessels. The pre-operative vascular pattern was re-established when grafts were replaced without rotation but comparable survival of arteries also occurred when the grafted discs of skin were rotated. In most grafts, the segment of artery became stabilized by 4-9 weeks; more than 75% of the length of the arterial segments survived in 83% of grafts, with minimal accompanying histological changes in their walls. There was some increase in number and aborization of small vessels in the established grafts, although the vascular pattern was essentially similar to that of unoperated skin. The factors possibly involved in the survival of the large arteries in the grafts include the relative rigidity of the arterial wall, the early establishment of circulation, and the favourable haemodynamic position of the grafts.

Necrotizing and giant cell granulomatous phlebitis of caecum and ascending colon.

A distinctive form of necrotizing and granulomatous phlebitis of a segment of large intestine is described in a previously healthy 36-year-old woman who presented with sudden severe abdominal pain and diarrhoea. At operation the caecum and ascending colon were oedematous and inflamed and right hemicolectomy was performed. Microscopically there was striking involvement of veins in all coats of the bowel ranging from recent fibrinoid necrosis of the whole vessel wall in the case of the caecum, to more chronic giant cell granulomas in parts of the vessel wall with partial or complete occlusion of the lumen in ascending colon. Arteries and lymphatics were entirely spared of these changes. The aitiology of this condition has not been elucidated but the histological appearances and site of involvement suggest an immunological reaction to material absorbed from the bowel. No evidence of food or other allergies or of infection has been obtained. The patient remains symptom free after 18 months. This form of phlebitis does not appear to have been previously described.

Atypical polypoid adenomyoma: a case report with ultrastructural examination.

A case of atypical polypoid adenomyoma occurring in a 40 yr-old female is reported. The lesion was composed of irregular glands lined by atypical epithelium lying in a spindle cell stroma containing smooth muscle cells. The mitotic count of the stroma was less than 1 per 10 high power fields. Squamous metaplasia was present in the glandular elements. Ultrastructural examination of the stroma confirmed the presence of smooth muscle cells. The lesion is compared with similar cases previously reported. The differential diagnosis is considered, including endometrial adenocarcinoma, adenomyosis, endometrial polyps and mixed Mullerian tumours. Atypical polypoid adenomyoma is a benign lesion, and conservative management is recommended.

Round-headed spermatozoa: a case report.

A case is presented of a patient in whom over 90% of spermatozoa had distinctive round heads as seen by light microscopy. Other features of his semen were only slightly abnormal. Electron microscopy revealed spermatozoa with no acrosome confirming the sterility of the patient. With such men other means of achieving a family must be considered.

Primary cerebral neuroblastomas. A clinicopathological study of one adolescent and five adult patients.

Neuronal differentiation was demonstrated by light microscopy, immunohistochemistry and electron microscopy in the cerebral neoplasms of one adolescent and five adult patients. The tumours exhibited a wide spectrum of cellular differentiation from low to high grade malignancy which seems to correlate well with their biological behaviour. The natural history of these 6 cerebral neuroblastomas is rather long compared to that of other malignant primary cerebral neoplasms of adulthood; however, 2 patients died, one survived about 5 yr after initial symptoms whilst an untreated patient survived more than 12 yr. It is suggested that morphological grading along the lines of Kernohan's system may be useful in determining the prognosis and postoperative management of patients with cerebral neuroblastomas.

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.

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.

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