PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples

A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.     

I-123 MIBG scintigraphy in adults. A report of clinical experience

Thirty-one adult patients with clinical findings suggestive of pheochromocytoma were studied with I-123 MIBG. All patients had images obtained at 24 and 48 hours. Five patients had abnormal uptake proved to be because of I-123 MIBG avid tumors. The remaining 26 patients had no proven tumors and showed physiologic uptake in various organs. The 24-hour images were of high quality. In all cases, the 48-hour images contributed no significant additional information. Only in 1 patient did the 48-hour image add some certainty. Physiologic uptake was seen in the salivary glands, liver, G.I. tract, and urinary bladder in all patients (100%). Uptake was also observed in the lung and heart (90%), normal adrenal glands (32%), thyroid (29%), spleen (23%) uterus (13%), and neck muscles (6%). The authors' experience indicates that I-123 MIBG gives superior images compared to the previously used I-131 MIBG, that the optimum imaging time for adults is 18-24 hours, and that normal distribution patterns including uterine and neck muscle uptake should be familiar to physicians interpreting the studies.     

Atrial and accessory pathway activation direction in patients with orthodromic supraventricular tachycardia: insights from vector mapping

OBJECTIVES: The purpose of this study was to utilize vector mapping to investigate atrial and accessory pathway activation direction during orthodromic supraventricular tachycardia. BACKGROUND: Although advances have been made in the electrophysiologic evaluation and management of accessory pathways, our understanding of accessory pathway anatomy and physiology remains incomplete. Vector mapping has been validated as a method of studying local myocardial activation. METHODS: In 28 patients with a left-sided or posteroseptal accessory atrioventricular (AV) pathway referred for ablation, atrial and accessory AV pathway activation direction was determined during ventricular pacing or orthodromic supraventricular tachycardia, or both, by summing three orthogonally oriented bipolar electrograms recorded from the coronary sinus to create three-dimensional vector loops. Atrial and accessory AV pathway activation direction was determined in all patients from the maximal amplitude vectors of the vector loops. Because of beat to beat variability in the directions of the vector loops, data from 8 of 28 patients could not be analyzed. RESULTS: At 81 of 83 sites, atrial activation direction along the long axis of the coronary sinus corresponded with the direction suggested by activation time mapping. Activation direction along the anteroposterior and inferosuperior axes was variable, potentially due to variations in the level of the atrial insertion of the accessory AV pathway and in the depth or angling of pathway fibers in the AV fat pad. In eight patients, at least one recording was obtained at the site of an accessory AV pathway potential. Accessory AV pathway activation proceeded superiorly and to the right in seven of eight patients; in one patient with a posteroseptal pathway, accessory AV pathway activation proceeded superiorly and to the left. CONCLUSIONS: 1) Vector mapping is a useful technique for localizing accessory AV pathways; 2) left-sided accessory AV pathways angle from left to right as they traverse the AV groove; and 3) variable activation directions of the atrial myocardium adjacent to the coronary sinus suggest that accessory AV pathway insertion into the atrium differs from patient to patient.     

Gelonin analogs with engineered cysteine residues form antibody immunoconjugates with unique properties

We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired cysteine residue. The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of Gel, or with the positions of endogenous cysteine residues. In most cases, enzymatic activity in vitro was unaltered by this modification. The rGel analogs were conjugated via their unpaired cysteine residue to the anti-CD5 antibody H65, or to H65 Fab and F(ab')2. Several rGel analogs formed immunoconjugates that were up to 6-fold more cytotoxic to antigen-bearing cells than those made with linker-modified rGel, whereas others were less potent. In the rat, the in vivo clearance rates of whole antibody conjugates correlated with their relative in vitro disulfide bond stability, and deconjugation to intact antibody and rGel was the predominant clearance mechanism. Fab conjugates to rGel analogs which differed in their in vitro disulfide bond stability had similar serum clearance rates, suggesting that clearance occurs mainly by removal of intact immunoconjugate from the serum, and is less dependent on deconjugation. Our results demonstrate that rGel analogs with a single cysteine at various positions on the solvent exposed surface are produced efficiently in Escherichia coli (>1 g/liter), and that the position of the cysteine greatly influences the potency and stability of the resulting immunoconjugates.     

A homozygous deletion in a small cell lung cancer cell line involving a 3p21 region with a marked instability in yeast artificial chromosomes

All types of lung carcinoma are characterized by a high frequency of loss of sequences from the short arm of chromosome 3, the smallest region of overlap containing D3F15S2 in band p21. Here we characterize a 440-kilobase segment from this region, which we found homozygously deleted in one of our small cell lung cancer-derived cell lines. The homozygous deletion maps between UBE1L and ZnF16, just centromeric to D3F15S2. Yeast artificial chromosomes with inserts originating from the deleted region are very unstable and readily lose parts of their insert.     

Early decortication for anaerobic empyema in children

Nine children underwent early decortication for empyema. Three had posttraumatic empyema and six had postpneumonic empyema. The decision for decortication was made 3-5 days after diagnosis of empyema, when it was judged that conventional treatment by antibiotics and intercostal catheter drainage was failing. Such failure was manifested by: loculated effusions (9), persistent fever (8), persistent respiratory distress (3), pulmonary air leak (3), and worsening parenchymal disease (1). The decortication procedure consisted of a standard posterolateral thoracotomy, removal of the fibrinous peel from the visceral and parietal pleurae, debridement or resection of necrotic lung tissue, irrigation of the pleural space, and drainage by intercostal catheters. In the 9 children who underwent this procedure, there were no deaths and a single complication, suppurative thrombophlebitis. Recovery was rapid in 6 out of 9 children, who were discharged within 10 days of operation. The remaining 3 out of 9 children had associated injuries or illnesses that necessitated a longer hospital stay. Bacteriologic studies confirmed anaerobic bacteria in the infected pleural contents of 5 out of 9 children. Bacteroides and anaerobic streptococci were the commonest isolates. Anaerobic infection of the pleural space cannot be cured without aggressive surgical drainage, in addition to antibiotics. Our experience suggests that early decortication is an efficacious procedure for selected children with empyema. The presence of anaerobic bacteria in the empyema may constitute an unequivocal indication for early decortication.     

Programmable open-loop micropump insulin delivery system

The application of "closed-loop" glucose-controlled insulin infusion in metabolic research and in the management of diabetic patients in acute emergency situations has stimulated the development of portable insulin infusion devices intended for the long-term stabilization of glucose levels. Since in vivo glucose sensors with long-term accuracy and reliability are not yet available, the currently projected portable insulin infusion systems are the "open-loop" type. Such systems should be reliable, safe, programmable, lightweight, and small in size. In addition, the power requirements should be as low as possible to avoid the need for heavy batteries and/or periodic recharging. In turn, these requirements govern the design criteria for the most essential component of such an instrument, the insulin delivery pump. A micropump has been developed in an attempt to meet all desirable design features. Evaluation data are presented.     

Effect of insulin resistance, apoE2 allele, and smoking on combined hyperlipidemia

Combined hyperlipidemia may result from the interaction of several metabolic and environmental factors. We explored to what extent fasting insulin concentration, apolipoprotein (apo) E2 frequency, and cigarette smoking explained the serum levels of triglyceride and high-density lipoprotein cholesterol (HDL-C) in patients with combined hyperlipidemia. Forty-nine untreated patients with combined hyperlipidemia were compared with 49 hypercholesterolemic patients who were matched for gender, age, and body mass index. All laboratory values were obtained after 9 weeks of standardized dietary intake and after an overnight fast. The patients with combined hyperlipidemia had a significantly higher (33 pmol/L, 50%) mean insulin concentration than matched hypercholesterolemic control subjects, indicating that the combined hyperlipidemic patients were more insulin resistant. However, the differences in the fasting insulin and triglyceride concentrations within the pairs were only slightly correlated (adjusted r = .29). The combined hyperlipidemic patients were also characterized by a higher frequency of apoE2 alleles (25% versus 6%) and smokers (41% versus 16%). In a matched multiple linear regression model, the differences in insulin concentration, apoE2 allele frequency, and smoking explained 12%, 8%, and 9%, respectively, of the mean paired difference in triglyceride concentration. The differences in insulin concentration or apoE2 allele frequency did not significantly explain the mean paired difference in HDL-C concentration, whereas smoking explained 17% of the difference. In conclusion, fasting insulin concentration, the presence of the apoE2 allele, and smoking may explain 30% of the hypertriglyceridemia and the low levels of HDL-C in nonobese patients with combined hyperlipidemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hospital inpatient costs resulting from road crashes in Western Australia

The purpose of this study was to estimate the inpatient costs of road crashes in Western Australia, and to investigate factors relating to casualties and their injuries that affect the hospital costs resulting from road crashes. All road crash casualties who were injured severely enough to be hospitalised in Western Australia in 1988 were included. A casemix classification system was used to classify patients into diagnostic related groups. Hospital costs were assigned to individual patients on the basis of their diagnostic related group and length of hospital stay. The annual cost of hospital treatment for road crash casualties was estimated as $13.9 million, and 33 per cent of this was incurred by those with lower extremity injuries and 27 per cent by those with head injuries. Hospital costs per casualty ranged from an average of $1388 for those sustaining minor (Abbreviated Injury Scale severity score of 1 or 2) spinal injuries to $16,580 and $33,424, respectively, for those sustaining severe (Abbreviated Injury Scale severity score of 4 or 5) head and spinal injuries. A multivariate analysis of variance revealed the following factors as having a significant independent effect on the hospital inpatient costs of road crash casualties: type of hospital (teaching or nonteaching), body region of injury, injury severity level and road user group. There were also significant interaction effects between different factors. Since hospital inpatient costs vary considerably across factors, using average cost data in the specific economic evaluation of road safety interventions for groups of road users is inappropriate.     

Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay

This study was performed to investigate the effects of clonidine on regional myocardial function in a canine model of regional myocardial ischemia. Myocardial systolic shortening (%SS) was used as an index of regional myocardial function. In eight dogs after thoracotomy, the left circumflex coronary artery (LCX) was occluded by screw clamp until regional myocardial function became impaired. After partial occlusion of the LCX, cumulative doses of clonidine (1.25, 2.5, and 5.0 micrograms/kg) were administered intravenously. After administration of clonidine, heart rate, mean arterial blood pressure (MAP), and norepinephrine concentration decreased in a dose-dependent manner. At a dose of 5.0 micrograms/kg of clonidine, the LCX flow and systolic shortening of the LCX area decreased to 76% and 81% of the poststenotic values (P < 0.05, respectively), whereas no significant changes were observed at a dose of 1.25 and 2.5 micrograms/kg. These results suggest that clonidine administration and an associated decrease in arterial blood pressure deteriorates regional myocardial function of the ischemic myocardium.