A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis

Advances in techniques of molecular biology have made possible the amplification of specific genes from single cells. This has a major clinical application in preimplantation diagnosis of monogenic disorders. However, the incidence of allele specific amplification failure (allele drop out) in heterozygous single cells can lead to misdiagnosis and the transfer of affected embryos. Few studies have been done to investigate the actual cause of allele drop out, although some investigators have succeeded in reducing but not eliminating it. Here we report the efficiency of amplifying both alleles in heterozygous cells lysed according to two different protocols. A total of 177 heterozygous cells from carriers of cystic fibrosis (CF) and haemoglobin C (HbC) were lysed using two different lysis buffers. Interestingly none of the cells that were lysed with sodium dodecyl sulphate/proteinase K showed any example of allele specific amplification failure whereas in those lysed by KOH/dithiothreitol it was present in 17.6 and 4.7% of the CF and HbC cells respectively. Our results suggest that the phenomenon of allele specific amplification failure is at least in part dependent on the lysis buffer used.      

FIELD TRIAL OF Bacillus thuringiensis var israelensis PELLET FORMULATION IN THE CONTROL OF MOSQUITOES

In a simulated field trial Bacillus thuringiensis var israelensis (BTI) pellet formulation exhibited an enhanced efficacy with increasing dose. A dosage of 1.0 and 1.5 ppm was most effective under simulated field conditions. In field trials persistence of BTI pellet (1.0 ppm) was observed for 35 days in moderately polluted water collection as compared to 21 days in highly polluted water bodies.KEY WORDS: Bacillus thuringiensis, Malaria, Mosquito control     

Gender and Ethnic Differences in Survival in a Cohort of HIV Positive Clients

Objectives: The purpose of this study was to examine gender and ethnic differences in survival of persons receiving treatment for HIV infection to determine if differences existed, and if they did, to assess the possibility of explaining these differences by examining other factors, such as age, disease severity when beginning treatment, alcohol, illicit drugs, tobacco, educational level, living arrangements, antiretroviral treatment, PCP prophylaxis, sexually transmitted diseases, mode of transmission and opportunistic infections.

Design: A retrospective cohort study of all clients receiving treatment at an HIV only clinic from its opening in early 1988 until the end of May 1993. Statistical methods used to examine the data included incidence density ratios, Kaplan‐Meier survival curves, Breslow (generalized Wilcoxon) tests of equality of survival curves and Cox proportional hazards models both with and without time dependent covariates.

Results: In the cohort (37% African American, 7% Hispanic American and 25% female), 220 deaths occurred during 1223 person years of follow‐up. Compared to European American males, the following incidence density ratios were observed: European American females: 0.50, Hispanic American females: 0.70, Hispanic American males: 0.96, African American females: 1.28 and African American males: 2.38. The differences were noted above for gender/ethnicity groups were significant at the p < 0.0001 level. After adjusting for disease stage (as measured by laboratory testing of CD4 positive T‐lymphocytes), educational level, and age, no differences in survival by gender or ethnicity remained. Disease stage and educational level had the greatest prognostic significance.

Conclusions: European Americans entered treatment at a much earlier disease stage (as measured by CD4 positive T‐lymphocyte counts) and had higher educational levels (a surrogate for socioeconomic status) than African Americans. These factors may explain the longer survival in European Americans as compared to African Americans in this cohort.     

No evidence of endothelial dysfunction in patients with Alzheimer's disease

In view of accumulating evidence of vascular pathology in Alzheimer's disease (AD), we tested the hypothesis that AD patients have impaired endothelial function. This was assessed using the technique of strain-gauge venous occlusion plethysmography, which measures forearm blood flow (FBF). Intra-arterial (brachial) infusion of acetylcholine (ACh) and sodium nitroprusside (SNP) was used to assess local endothelial dependent and independent responses, respectively. There was no difference in the basal FBF of patients and controls. ACh and SNP caused dose-related increases in FBF from baseline, but no difference was recorded between the AD and control group. This study provides no evidence of endothelial dysfunction in the systemic circulation of patients with AD.     

New non-cocaine-containing topical anesthetics compared with tetracaine-adrenaline-cocaine during repair of lacerations

OBJECTIVE: To compare the effectiveness of three new topical anesthetics that do not contain cocaine (prilocaine-phenylephrine, tetracaine-phenylephrine [tetraphen], and tetracaine-lidocaine-phenylephrine) to that of tetracaine-adrenaline-cocaine (TAC) during laceration repair in children. DESIGN: Prospective, randomized, double-blind clinical trial. SETTING: The emergency department of an urban children's hospital. PARTICIPANTS: Children 1 year of age or older with a laceration /= 5 years of age using a visual analogue scale (VAS). Suture technicians, research assistants, and parents also scored pain using a seven-point Likert scale. In addition, suture technicians completed an anesthetic effectiveness scale. RESULTS: There was consistently no difference demonstrated between the effectiveness of tetraphen and that of TAC for each outcome measure of each observer group. A statistically significant difference was seen among anesthetics when comparing VAS and Likert scale scores of suture technicians and Likert scale scores of research assistants. Based on post hoc analyses, these statistically significant differences were between TAC and prilocaine-phenylephrine (suture technician VAS and Likert scale) and between TAC and tetracaine-lidocaine-phenyl-ephrine (suture technician Likert scale), but not between TAC and tetraphen. When power analyses were performed using alpha = 0.05 and beta = 0.20, it was possible to detect a difference of 1.2 VAS units for each of the observer groups. Based on anesthetic effectiveness scale scores, the three new topical preparations collectively performed significantly better on the face and scalp than on the extremities (relative risk = 1.83; 95% confidence interval 1.20 < relative risk < 2.79). CONCLUSION: This study demonstrated the effectiveness and safety of three new non-cocaine-containing topical anesthetics. Consistently, there was no statistical difference demonstrated between the effectiveness of tetraphen and that of TAC for each outcome measure of each observer group. Tetraphen offers an effective alternative to TAC during laceration repair in children.     

Hypospadias trends in two US surveillance systems

OBJECTIVE: Hypospadias is a common congenital anomaly, the cause of which is unknown. Unexplained increases in the rates of hypospadias occurred in five European countries in the 1970s and 1980s. We examined data from two birth defects surveillance systems in the United States for evidence of similar trends. METHODOLOGY: The Metropolitan Atlanta Congenital Defects Program (MACDP) provided birth prevalence rates from 1968 to 1993. The nationwide Birth Defects Monitoring Program (BDMP) provided rates from 1970 to 1993. MACDP data are population-based and could be categorized by the severity of the hypospadias. BDMP data allowed analysis of rate trends for the four census regions of the United States. RESULTS: Data from both surveillance systems showed an approximate doubling of hypospadias rates in the 1970s and 1980s. MACDP data showed that the rate of severe cases increased while the ratio of mild to severe cases decreased. BDMP data showed that hypospadias rates increased markedly in all four regions of the United States. CONCLUSIONS: The observed increases are unlikely to be attributable to increased sensitivity of the surveillance systems or the identification of more mild cases by physicians over time, because either trend would have increased rather than decreased the ratio of mild to severe cases. If real, these trends represent the largest number of cases and the first report of an increase in hypospadias rates outside of Europe. Additional investigation of a possible increase in hypospadias rates is warranted.     

Acetabular revision with use of a bilobed component inserted without cement in patients who have acetabular bone-stock deficiency

BACKGROUND: Massive deficiency of acetabular bone stock is a challenging problem in the increasing number of patients who need a revision of a failed hip arthroplasty. The bilobed cup has been presented as one alternative reconstruction technique for hips with extensive acetabular bone loss. The purpose of this study was to assess the results with use of a bilobed acetabular component inserted without cement for revision reconstruction in hips with acetabular bone deficiency in order to clarify the indications for its use and to identify the factors that influence the clinical and radiographic outcome. METHODS: Forty-one hips in thirty-eight patients had an acetabular revision with a bilobed acetabular component inserted without cement between December 1991 and December 1995. These hips were a subset of the 414 hips treated with an acetabular revision during the same period of time. One patient was lost to follow-up, and one died during the study period. Two patients who could not return for radiographic evaluation completed questionnaires. The remaining thirty-four patients (thirty-seven hips) were evaluated radiographically and clinically and were followed for an average of forty-one months (range, twenty-four to sixty-six months). RESULTS: Radiographic analysis demonstrated an improvement in the average vertical displacement of the hip center. At the time of the latest follow-up examination, 76 percent (twenty-eight) of the thirty-seven cups were stable, 8 percent (three) were probably unstable with a change in the screw position but no definite migration of the cup, and 16 percent (six) were unstable. Eight of the nine loose or probably loose components were in patients who had more than two centimeters of superior migration of the component and disruption of Kohler's line on preoperative radiographs. Additionally, implants were more likely to become unstable (demonstrating more than 4 degrees of change in the abduction angle or more than four millimeters of radiographic migration) when the inferior aspect of the component did not extend to or distal to the interteardrop line, which indicated that the component was undersized. CONCLUSIONS: On the basis of our early rate of probable or definite loosening of 24 percent (nine of thirty-seven cups) and the technical difficulties involved, we do not recommend the routine use of this component. We believe that this device is indicated when a patient has an oblong-shaped acetabular defect and the surgeon wants to correct an elevated hip center. However, the medial wall of the acetabulum (Kohler's line) should be intact if the failed component has migrated more than two centimeters. An alternative reconstruction technique, such as use of a structural allograft with or without an acetabular cage, is also an option in this situation.     

The accuracy and reproducibility of radiographic assessment of stress-shielding. A postmortem analysis

BACKGROUND: Although periprosthetic bone loss remains a major concern in total hip arthroplasty, radiographic assessment of such loss is both difficult and subjective. In the present study, we assessed the ability of orthopaedic surgeons to reproducibly recognize changes in periprosthetic bone density on radiographs. We hypothesized that assessment of periprosthetic bone loss on plain radiographs is not reliable enough to justify its use in outcomes research. METHODS: Twenty-nine unilateral total hip replacements and the surrounding bone were retrieved at autopsy, and radiographs were made; radiographs of the contralateral, normal femur were also made after implantation of an identical prosthesis and used as a control. Three orthopaedic surgeons independently examined the specimen radiographs and classified bone loss in each of sixteen femoral zones. Bone loss was recorded as present if the bone of the femur that had had in vivo implantation showed evidence of cortical thinning, increased porosity, or decreased density (either cortical or trabecular) when compared with the control femur. The kappa coefficient was used to quantify interobserver and intraobserver reproducibility in determining bone loss for the 464 zones examined and in determining the Engh and Bobyn stress-shielding classification of each femur. In fourteen femoral pairs, bone loss was also quantified with dual-energy x-ray absorptiometry, and the resulting value was then compared with the bone-loss classification that had been determined radiographically. RESULTS: First, the surgeons agreed on the presence or absence of bone loss in 73 percent (337) of the 464 zones. The interobserver kappa value of 0.58 denoted only good reproducibility. The intraobserver reproducibility was better; the surgeon's initial evaluation of bone loss agreed with his second evaluation for 90 percent of the zones (kappa = 0.74). Second, the three surgeons agreed on the degree of stress-shielding, according to the Engh and Bobyn classification, in 66 percent (nineteen) of the twenty-nine femora. The kappa value for this comparison was only 0.27, indicating marginal reproducibility. Third, although there was some agreement among reviewers when there was 20 to 60 percent reduction in bone-mineral content as determined with dual-energy x-ray absorptiometry, excellent agreement among the examiners (kappa = 0.85) was not achieved until bone loss averaged 70 percent. CONCLUSIONS: On the basis of these results, we suggest caution in interpreting results from studies of femoral bone loss that have used plain radiographic analysis if the authors have not provided interobserver reliability data. We question the utility of evaluating periprosthetic bone loss on radiographs, since the loss is not reproducibly recognized until 70 percent of the bone is gone.