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PubMed2002

Osmotic characteristics of mouse spermatozoa in the presence of extenders and sugars.

Successful cryopreservation requires cells to tolerate volume excursions experienced during permeating cryoprotectant equilibration and during cooling and warming. However, prior studies have demonstrated that mouse spermatozoa are extremely sensitive to osmotically induced volume changes. A series of three experiments were conducted 1) to test the efficacy of two commonly used extender media components, egg yolk (EY) and skim milk (SM), in broadening the osmotic tolerance limits (OTL) of ICR and B6C3F1 murine spermatozoa; 2) to determine if the extender components affected sperm plasma membrane permeability coefficients for water and cryoprotective agent (CPA) characteristics; and 3) to test the effects of permeating and nonpermeating CPA on mouse sperm morphology. In experiment 1, sperm samples were added to 150, 225, 300, 450, or 600 mOsm NaCl, EY, SM, sucrose, or choline chloride at 22 degrees C and then returned to isosmotic conditions. In experiment 2, epididymal sperm were preequilibrated in 1 M glycerol (Gly) or 2 M ethylene glycol (EG) prepared in SM extender, abruptly exposed to isosmotic conditions at 22, 15, or 2 degrees C, and the corresponding volume excursions were measured and analyzed. In experiment 3, the effects of permeating CPA (0.3 M EG or dimethyl sulfoxide) or nonpermeating CPA (12% sucrose or 18% raffinose) on sperm morphology (i.e., principle midpiece folding and putative membrane fusion) were evaluated. Experiment 1 showed that spermatozoa from ICR and B6C3F1 mice have effectively broader OTL when exposed to EY or SM extenders. The results of experiment 2 indicated that, for ICR sperm, the activation energy (E(a)) for the hydraulic conductivity (L(p)) was unchanged in SM extender. However, for B6C3F1 sperm, there were significant differences in E(a) of L(p) in the presence of Gly and EG. The result of experiment 3 indicated that permeating CPAs damage sperm membrane integrity, causing a high frequency of head-to-tail or tail-to-tail membrane fusion, whereas this occurrence in the presence of nonpermeating CPA was less than 3%. Finally, the results of experiments 1 and 2 were combined in a mathematical model to predict Gly and EG addition and removal in the presence of SM extender, which would prevent mouse sperm membrane damage. These predictions indicated that, for ICR sperm, both Gly and EG may be added and removed in a single step. However, for B6C3F1 spermatozoa, Gly required a two-step addition while EG only required a single step. For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal.

Animals↗

PubMed2002

Extended hepatic resection for hepatocellular carcinoma in patients with cirrhosis: is it justified?

OBJECTIVE: To evaluate the perioperative outcomes and long-term survival of extended hepatic resection for hepatocellular carcinoma (HCC) in patients with cirrhosis. SUMMARY BACKGROUND DATA: Hepatic resection is a well-established treatment for HCC in cirrhotic patients with preserved liver function and limited disease. However, the role of extended hepatic resection (more than four segments) for HCC in cirrhotic patients has not been elucidated. METHODS: Between 1993 and 2000, 45 consecutive patients with histologically confirmed cirrhosis underwent right or left extended hepatectomy for HCC (group A). Perioperative outcomes and long-term survival of these patients were compared with 161 patients with HCC and cirrhosis who underwent hepatic resection of a lesser extent in the same period (group B). All clinicopathologic and follow-up data were collected prospectively. RESULTS: Group A patients had significantly higher intraoperative blood loss, longer operation time, and longer hospital stay than group B. However, the two groups were similar in overall morbidity and hospital mortality. There were no significant differences in the incidence of liver failure or other complications. The resection margin width was similar between the two groups. Despite significantly larger tumor size in group A compared with group B, long-term survival was comparable between the two groups. CONCLUSIONS: Extended hepatic resection for HCC can be performed in selected cirrhotic patients with acceptable morbidity, mortality, and long-term survival that are comparable to those of lesser hepatic resection. Extended hepatectomy for large HCC extending from one lobe to the other or central HCC critically related to the hepatic veins is justifiable in cirrhotic patients with preserved liver function and adequate liver remnant.

Carcinoma, Hepatocellular↗

PubMed2000

Survivors of catastrophic illness: outcome after direct transfer from intensive care to extended care facilities.

OBJECTIVE: To describe outcomes of adult survivors of prolonged critical illness after direct transfer to extended care facilities. DESIGN: A retrospective cohort study. SETTING: All adult intensive care units (ICUs) in a tertiary care university hospital. PATIENTS: A consecutive series of 97 adult survivors with an ICU stay of > or =7 days transferred directly from intensive care to extended care facilities between 1990 and 1996. INTERVENTIONS: None. METHODS AND MAIN RESULTS: Hospital and extended care facility charts were reviewed for patient characteristics, resource utilization, and survival. Survivors were for a minimum of 1 yr and a maximum of 6 yrs, and were interviewed to assess quality of life and functionality. The mean age of the patients was 66+/-16 (range, 19-93) yrs. The median length of ICU stay for these patients was 39 (range, 7-276) days. Only 18 of the 71 ventilator-assisted patients were weaned from mechanical ventilation after transfer to the extended care facility. Survival for the study period, at 1 yr after discharge from the ICU, was 49.5%. One year after discharge from the ICU, 11.5% of all patients had returned home, were breathing spontaneously, had a fair or better quality of life, and had good physical functionality. Each successive year, an increasing proportion of patients underwent direct transfer to an extended care facility. This strategy decreased the patients' length of stay (p<.002) in the ICU from year to year, but was significantly associated with an increase in readmissions to acute care hospitals (p<.002). CONCLUSIONS: Survivors of catastrophic illness who are so debilitated that they require transfer to an extended care facility have a low likelihood of achieving both survival and functional independence 1 yr after discharge from the ICU. Aggressive cost-conscious strategies to accelerate the transfer of these patients successfully reduced the length of ICU stay and hospital costs, but were associated with a high rate of readmission to tertiary care facilities.

Adult↗

PubMed2000

Superior gluteal artery in the extended iliofemoral approach.

OBJECTIVE: To determine the incidence of superior gluteal artery injury following fracture of the acetabulum and to determine whether the combination of a superior gluteal artery injury and the use of an extended iliofemoral approach to the acetabulum creates abductor muscle necrosis. DESIGN: Prospective protocol, consecutive cases. SETTING: A consecutive series from the referral practice of the senior author plus seven cases from the practices of two other authors. PATIENTS: Two hundred twenty-seven patients with fractures of the acetabulum were treated operatively between November 1992 and January 1995. Forty-one were treated with the use of the extended iliofemoral approach. Preoperative angiograms were not performed for any of the patients. All fractures involved the posterior column, and all but two fractures had displacement of the greater sciatic notch. The average displacement of the notch was 2.5 centimeters (range 6 to 60 millimeters). INTERVENTION: All patients were treated with open reduction and internal fixation via the extended iliofemoral approach. Intraoperative Doppler examination of the superior gluteal artery was performed before and after reduction and fixation of the posterior column. MAIN OUTCOME MEASURE: Wound complications, abductor manual muscle testing, hip range of motion. RESULTS: Pulsatile flow was confirmed in forty of forty-one patients. All patients were followed for a minimum of six months with an average follow-up of 1.4 years. At most recent follow-up, no patients had evidence of complete loss of abductor function. Sixty-three percent of patients had achieved Grade 4 of 5 motor strength, and 25 percent of them had achieved normal motor strength. CONCLUSIONS: No instances of superior gluteal artery laceration and only one instance of superior gluteal artery thrombosis were encountered in these forty-one patients despite significant fracture displacement involving the sciatic notch. The incidence of superior gluteal artery injury was significantly less than would be expected from previous studies. Massive abductor necrosis resulting from superior gluteal artery injury combined with an extended approach has been described primarily in animal and cadaver studies. Although arteriograms are useful in the control of hemodynamic instability, we cannot support the recommendation of preoperative angiographic study of all patients undergoing acetabular fracture surgery via an extended approach. In one case, an extended iliofemoral approach was tolerated in a patient with absent superior gluteal artery flow.

Acetabulum↗

PubMed2001

Safety, tolerability, and pharmacokinetics of an extended-release formulation of fluvastatin administered once daily to patients with primary hypercholesterolemia.

Fluvastatin sodium (Lescol, Novartis Pharmaceutical Corp., East Hanover, NJ, U.S.A.), a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A) reductase inhibitor that limits cholesterol biosynthesis, is available as a 40-mg immediate-release formulation capsule. An extended-release formulation for once-daily administration has been developed for patients with primary hypercholesterolemia who may benefit from doses higher than 40 mg/day. This phase I study evaluated the safety, tolerability, and pharmacokinetics of a new fluvastatin extended-release formulation at doses ranging from 80-640 mg/day in 40 hypercholesterolemic patients. After a 2-week dietary stabilization phase, patients (Fredrickson type IIa/IIb), 18-55 years of age, were randomly assigned to four groups to receive oral fluvastatin extended-release (80, 160, 320, or 640 mg) or matching placebo once daily for 13 days. Fluvastatin extended-release was generally safe and well tolerated at doses of 80-320 mg/day. Within this dose range, linear pharmacokinetics was observed after single and multiple dosing. At 640 mg, fluvastatin extended-release was not well tolerated. Six of the seven actively treated patients at this dose experienced adverse events, including diarrhea, headache, and clinically relevant elevations in serum transaminase concentrations. In addition, nonlinear pharmacokinetics, possibly due to saturation of first-pass metabolism, was observed at this dose, causing higher than expected serum drug concentrations. Once-daily administration of fluvastatin extended-release at doses of 80-320 mg/day was generally safe and well tolerated in patients with primary hypercholesterolemia over a 13-day dosing period.

Adolescent↗

PubMed1977

A systems analysis of the impact of physician extenders on medical cost and manpower requirements.

This paper presents a descriptive narrative of a mathematical manpower model and the results of an analysis of the effect physician extenders have on medical costs and manpower requirements. The model is extensively developed, through the use of a new medical classification system in the area of delegation of specific task areas and patient visits to physician extenders. Additionally the models incorporate a complete cost structure for a group practice. Field trials in seven HMOs indicate that the models accurately represent the actual system and can be used effectively as planning aids. Results are presented that analyze the use of physician extenders from the following viewpoints: minimum cost solution for adult medicine, pediatrics and obstetrics/gynecology (OB/GYN); maximum physician extender use; effect of physician extender salary on minimum cost utilization; level of independence exercised; size of clinic and regional manpower planning; and a case study of HMO planning. The type of results presented include cost analysis, manpower analysis, and the types of patient visits best delegated to physician extenders (PE).

Adult↗

PubMed2005

Extended use of transdermal norelgestromin/ethinyl estradiol: a randomized trial.

OBJECTIVE: To compare bleeding profiles and satisfaction among women using a norelgestromin/ethinyl estradiol (E2) transdermal contraceptive patch in an extended regimen to those among women using a traditional 28-day patch regimen. METHODS: Healthy, regularly menstruating women (N = 239) were randomly assigned (2:1 ratio) to receive the norelgestromin/ethinyl E2 transdermal patch in an extended regimen (weekly application for 12 consecutive weeks, 1 patch-free week, and 3 more consecutive weekly applications, n = 158) or a cyclic regimen (4 consecutive cycles of 3 weekly applications and 1 patch-free week, n = 81). Subjects recorded bleeding data daily and completed satisfaction questionnaires. Subjects and investigators provided overall assessments of the regimens. RESULTS: Extended use of the norelgestromin/ethinyl E2 transdermal patch resulted in fewer median bleeding days (6 compared with 14, P < .001), bleeding episodes (1 compared with 3, P < .001), and bleeding or spotting episodes (2 compared with 3, P < .001) compared with cyclic use during days 1-84; median numbers of bleeding or spotting days were similar between regimens (14 compared with 16, P = .407) during this time. Extended use delayed median time to first bleeding to 54 days compared with 25 days with cyclic (P < .001). Subjects were highly satisfied with both regimens. Although not statistically significant, slightly more adverse events were reported with the extended than with the 28-day regimen. CONCLUSION: Compared with cyclic use, extended use of the norelgestromin/ethinyl E2 transdermal patch delayed menses and resulted in fewer bleeding days. This regimen may represent a useful alternative for women who prefer fewer episodes of withdrawal bleeding.

Administration, Cutaneous↗

PubMed2004

Once daily, extended release ciprofloxacin for complicated urinary tract infections and acute uncomplicated pyelonephritis.

PURPOSE: We assessed the efficacy and safety of 1,000 mg extended release ciprofloxacin orally once daily vs conventional 500 mg ciprofloxacin orally twice daily, each for 7 to 14 days, in patients with a complicated urinary tract infection (cUTI) or acute uncomplicated pyelonephritis (AUP). MATERIALS AND METHODS: In this prospective, randomized, double-blind, North American multicenter clinical trial adults were stratified based on clinical presentation of cUTI or AUP and randomized to extended release ciprofloxacin or ciprofloxacin twice daily. Efficacy valid patients had positive pretherapy urine cultures (105 or greater cFU/ml) and pyuria within 48 hours of study entry. Bacteriological and clinical outcomes were assessed at the test of cure visit (5 to 11 days after therapy) and the late followup visit (28 to 42 days after therapy). RESULTS: The intent to treat population comprised 1,035 patients (extended release ciprofloxacin in 517 and twice daily in 518), of whom 435 were efficacy valid (cUTI in 343 and AUP in 92). For efficacy valid patients (cUTI and AUP combined) bacteriological eradication rates at test of cure were 89% (183 of 206) vs 85% (195 of 229) (95% CI -2.4%, 10.3%) and clinical cure rates were 97% (198 of 205) vs 94% (211 of 225) (95% CI -1.2%, 6.9%) for extended release vs twice daily ciprofloxacin. Late followup outcomes were consistent with test of cure findings. Eradication rates for Escherichia coli, which accounted for 58% of pathogens, were 97% or greater per group. Drug related adverse event rates were similar for extended release and twice daily ciprofloxacin (13% and 14%, respectively). CONCLUSIONS: Extended release ciprofloxacin at a dose of 1,000 mg once daily was as safe and effective as conventional treatment with 500 mg ciprofloxacin twice daily, each given orally for 7 to 14 days in adults with cUTI or AUP. It provides a convenient, once daily, empirical treatment option.

Acute Disease↗

PubMed2004

Laparoscopic extended pelvic lymphadenectomy for bladder cancer: technique and initial outcomes.

PURPOSE: We describe the technique and our evolving outcomes of laparoscopic extended pelvic lymphadenectomy for bladder cancer. MATERIALS AND METHODS: Since 1999 laparoscopic radical cystectomy with pelvic lymphadenectomy and intracorporeal urinary diversion has been performed in 22 patients. The initial 11 patients underwent limited dissection (group 1) and the subsequent 11 consecutive patients underwent extended lymphadenectomy (group 2). Our split-and-roll technique of laparoscopic extended pelvic lymphadenectomy has evolved to achieve lymphatic tissue clearance by bilaterally skeletonizing the genitofemoral nerve, external iliac artery, external iliac vein, obturator nerve, hypogastric artery, common iliac artery and pubic bone. RESULTS: Extended lymphadenectomy added 1.5 hours of operative time. The median number of nodes retrieved was 3 and 21 in groups 1 and 2, respectively (p = 0.001). Three patients per group were found to have positive nodal disease. In 1 patient undergoing extended dissection injury to a deep pelvic vein was managed by intracorporeal suturing and resulted in 200 ml blood loss. Two other patients had deep venous thrombosis. At a mean followup of 11 months (range 2 to 43) there were no port site recurrences. CONCLUSIONS: Laparoscopic extended pelvic lymphadenectomy for bladder cancer can be performed with anatomical boundaries and nodal yields commensurate with those of current recommendations for open surgery.

Adenocarcinoma↗

PubMed2004

Is extended hepatectomy for hepatobiliary malignancy justified?

BACKGROUND: Extended hepatectomy may be required to provide the best chance for cure of hepatobiliary malignancies. However, the procedure may be associated with significant morbidity and mortality. METHODS: We analyzed the outcome of 127 consecutive patients who underwent extended hepatectomy (resection of > or = 5 liver segments) for hepatobiliary malignancies. RESULTS: The patients underwent extended hepatectomy for colorectal metastases (n = 86; 67.7%), hepatocellular carcinoma (n =12; 9.4%), cholangiocarcinoma (n =14; 11.0%), and other malignant diseases (n =15; 11.5%). Thirty-two left and ninety-five right extended hepatectomies were performed. Eight patients also underwent caudate lobe resection, and 40 patients underwent a synchronous intraabdominal procedure. Twenty patients underwent radiofrequency ablation, and 31 underwent preoperative portal vein embolization. The median blood loss was 300 mL for right hepatectomy and 600 mL for left hepatectomy (P = 0.02). Thirty-six patients (28.3%) received a blood transfusion. The overall complication rate was 30.7% (n = 39), and the operative mortality rate was 0.8% (n = 1). Significant liver insufficiency (total bilirubin level > 10 mg/dL or international normalized ratio > 2) occurred in 6 patients (4.7%). Multivariate analysis showed that a synchronous intraabdominal procedure was the only factor associated with an increased risk of morbidity (hazard ratio [HR], 4.9; P = 0.02). The median survival was 41.9 months. The overall 5-year survival rate was 25.5%. CONCLUSIONS: Extended hepatectomy can be performed with a near-zero operative mortality rate and is associated with long-term survival in a subset of patients with malignant hepatobiliary disease. Combining extended hepatectomy with another intraabdominal procedure increases the risk of postoperative morbidity.

Adult↗

PubMed2005

Extended family studies for the identification of allogeneic stem cell transplant donors in Jewish and Arabic patients in Israel.

HLA-identified donors are the best source of allogeneic hematopoietic stem cell transplants, and are available in approximately 40% of cases. If no HLA-identical core family member is found, an extended family search may be performed. The aim of the study was to summarize the 10-year (1990-1999) experience of our tertiary care center with extended family donor search. During this period, 356 patients and 2659 of their family members were tissue-typed; 239 patients were Jewish (67%) and 117 were Arabic (33%). An HLA-identical core-family donor was identified for 168 patients (47%): 95 Jewish (40%) and 73 Arabic (62%) (p < 0.0001); 49 patients (14%) had more than one potential donor. An extended family search (grandmother/grandfather, aunts, uncles, etc.) was performed in 38 of the remaining families, which were found to be consanguineous: five Jewish and 33 Arabic. One HLA match was found in the Jewish families (20%) and 21 in the Arabic families (64%). The odds ratio for an Arabic patient to find a donor in the extended family search was 8.75, as opposed to a Jewish patient. Overall, HLA-matched donors were found by core and extended family search for 53% of the patients. The rate for Arabic patients was 80% and for Jewish patients, 40% (p < 0.001). This difference may be explained by the greater number of siblings and higher rate of consanguinity in the Arabic population. In conclusion, an extended family search for potential HLA-matched donors is worthwhile, especially in distinct ethnic populations with high consanguinity, such as Israeli Arabs.

Arabs↗

PubMedDate not supplied

Pharmacokinetic profile of a 300-mg extended phenytoin sodium capsule (Dilantin) formulation.

The pharmacokinetic profile of a newly developed Dilantin 300-mg Kapseal formulation was compared with that of currently marketed Dilantin 100-mg Kapseals in both a 300-mg single-dose bioequivalence study in nine healthy volunteers and a once-daily 300-mg multiple-dose study in 18 patients with seizures. Results of these studies indicate the rate and extent of absorption of the 300-mg extended phenytoin (PHT) sodium capsule formulation are similar to that of 100-mg extended PHT sodium capsules based on PHT plasma maximum concentrations and time to achieve them (Cmax, tmax), and area under the curve (AUC) values and the urinary excretion of total hydroxy phenylhydantoin (HPPH) in the single-dose study and steady-state PHT plasma Cmax, tmax, minimum plasma concentrations (Cmin), and AUC values and urinary excretion of total HPPH in the multiple-dose study. Control of seizures in patients was equally maintained on a once-daily 300-mg multiple-dosing regimen administered as either one 300-mg extended PHT sodium capsule daily or three 100-mg extended PHT sodium capsules daily. Therefore, 300-mg extended PHT sodium capsules can be safely and effectively interchanged with three 100-mg extended PHT sodium capsules in patients requiring a once-daily 300-mg PHT sodium dosing regimen.

Adult↗

PubMed1977

Time utilization, productivity and costs of solo and extended duty auxiliary dental practice.

A study was conducted to compare the time utilization of the dentist, and productivity and costs for solo (one dentist, one chairside assistant and one treatment room) and extended duty settings (one dentist, two extended duty dental hygienists, one chairside assistant and two treatment rooms). Only amalgam and composite restorations done in a general group practice were included. In the extended duty setting the dentist spent more time in managerial activities and less time in treatment than in the solo setting. Nevertheless, the dentist in the extended duty setting produced 53% more restorations as compared with solo practice. The cost ratio of solo to extended duty practice was computed to 1:1.52. From the point of view of microeconomics, the extended duty setting was found no worse than the solo setting.

Dental Assistants↗

PubMed1999

Cortical afferents to the extended amygdala.

The projections of the cerebral cortex to the extended amygdala were studied in the rat using anterograde and retrograde tract-tracing techniques. Most cortical areas with strong projections to the extended amygdala preferentially targeted either the medial extended amygdala (including the medial amygdalar nucleus, ventromedial substantia innominata, and the medial part of the bed nucleus the stria terminalis) or the central extended amygdala (including the central amygdalar nucleus, dorsolateral substantia innominata, and the lateral part of the bed nucleus of the stria terminalis). Some cortical areas, however, had equal projections to both medial and central portions. The main areas projecting preferentially to the medial extended amygdala were the ventral subiculum, infralimbic cortex, ventral agranular insular area, and the rostral part of the ventrolateral entorhinal area. The main areas projecting preferentially to the central extended amygdala were the prefrontal cortex, viscerosensory and somatosensory portions of the insular cortex, and the amygdalopiriform transitional area. It is suggested that these cortical inputs may be important for cognitive, mnemonic, and affective aspects of emotional and motivated behavior.

Afferent Pathways↗

PubMed1994

Comparison of the stride kinematics of the collected, working, medium and extended trot in horses.

Highly-trained dressage horses were studied to test the hypothesis that stride length is altered independently of stride duration in the transitions between the collected, working, medium and extended trot. Six well-trained dressage horses were filmed at a frame rate of 150 frames/s performing the collected, working, medium and extended trots in a sand arena. Temporal, linear and angular data were extracted from the films, with 4 strides being analysed for each horse and gait type. There were no significant asymmetries between the left and rights limbs or diagonals when data from the whole group were pooled, but 3 horses showed asymmetries in one or more variables (P < 0.01). Analysis of variance and post-hoc tests indicated that the speed increased significantly (P < 0.01) from the collected (3.20 m/s) to the working (3.61 m/s) to the medium (4.47 m/s) to the extended (4.93 m/s) trot. The increases in speed were associated with a significant increase in stride length from 250 cm in the collected trot, to 273 cm in the working trot, 326 cm in the medium trot and 355 cm in the extended trot (P < 0.01). The lengthening of the stride was a result of increases between each gait type in the over-reach distance, whereas the diagonal distance was significantly longer in the extended than the collected trot only (P < 0.01). The stride duration tended to decrease as speed increased, and the difference became significant between the collected and extended trots (P < 0.01).

Analysis of Variance↗

PubMed2000

Efficient correction for CT image artifacts caused by objects extending outside the scan field of view.

The purpose of this paper is to develop a method of eliminating CT image artifacts generated by objects extending outside the scan field of view, such as obese or inadequately positioned patients. CT projection data are measured only within the scan field of view and thus are abruptly discontinuous at the projection boundaries if the scanned object extends outside the scan field of view. This data discontinuity causes an artifact that consists of a bright peripheral band that obscures objects near the boundary of the scan field of view. An adaptive mathematical extrapolation scheme with low computational expense was applied to reduce the data discontinuity prior to convolution in a filtered backprojection reconstruction. Despite extended projection length, the convolution length was not increased and thus the reconstruction time was not affected. Raw projection data from ten patients whose bodies extended beyond the scan field of view were reconstructed using a conventional method and our extended reconstruction method. Limitations of the algorithm are investigated and extensions for further improvement are discussed. The images reconstructed by conventional filtered backprojection demonstrated peripheral bright-band artifacts near the boundary of the scan field of view. Images reconstructed with our technique were free of such artifacts and clearly showed the anatomy at the periphery of the scan field of view with correct attenuation values. We conclude that bright-band artifacts generated by obese patients whose bodies extend beyond the scan field of view were eliminated with our reconstruction method, which reduces boundary data discontinuity. The algorithm can be generalized to objects with inhomogeneous peripheral density and to true "Region of Interest Reconstruction" from truncated projections.

Algorithms↗

PubMed1999

Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene.

A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum beta-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a beta-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. Vmax values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. blaVEB-1 showed significant homology at the DNA level with only blaPER-1 and blaPER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known beta-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of blaVEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with blaVEB-1; (ii) a second 59-base element just upstream of blaVEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of blaVEB-1; and (iv) a second antibiotic resistance gene 3' of blaVEB-1, aadB. blaVEB-1 may therefore be the first class A extended-spectrum beta-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore, blaVEB-1 is encoded on a large (> 100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer.

Amino Acid Sequence↗

PubMed1999

Detection and reporting of organisms producing extended-spectrum beta-lactamases: survey of laboratories in Connecticut.

Extended-spectrum beta-lactamases (ESBLs) are enzymes produced in some gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. They are most common in Klebsiella spp. and Escherichia coli but are present in a variety of Enterobacteriaceae. Resistance mediated by these enzymes can be difficult to detect depending on the antimicrobial agents tested. AmpC beta-lactamases are related to the chromosomal enzymes of Enterobacter and Citrobacter spp. and also mediate resistance to extended-spectrum cephalosporins and aztreonam in addition to cephamycins, such as cefoxitin. Unlike ESBLs, however, AmpC beta-lactamases are not inhibited by clavulanic acid or other similar compounds. To assess the abilities of various antimicrobial susceptibility testing methods to detect ESBLs, we sent three ESBL-producing organisms, one AmpC-producing organism, and a control strain that was susceptible to extended-spectrum cephalosporins to 38 laboratories in Connecticut for testing. Eight (21.0%) of 38 labs failed to detect extended-spectrum cephalosporin or aztreonam resistance in any of the ESBL- or AmpC-producing isolates. Errors were encountered with both automated and disk diffusion methods. Conversely, seven (18.4%) labs categorized at least some of the four resistant isolates as potential ESBL producers and reported the results with the extended-spectrum cephalosporins and aztreonam as resistant as suggested by current National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The percentage of laboratories that failed to detect resistance in the ESBL or AmpC isolates ranged from 23.7 to 31.6% depending on the type of enzyme present in the test organism. This survey suggests that many laboratories have difficulty detecting resistance in ESBL and AmpC-producing organisms and may be unaware of the NCCLS guidelines on modifying susceptibility testing reports for ESBL-producing strains.

Aztreonam↗