Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay

A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.     

Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. We studied the quantitative adsorption of fibronectin onto polymethylmethacrylate (PMMA) cover slips by using a 125I-labeled preparation of the purified plasma glycoprotein. Fibronectin in buffer solutions showed a high affinity to PMMA coverslips. Adherence of S. aureus Wood 46 was studied on PMMA pre-exposed to fibronectin, using an assay specifically adapted to the cover slip model. Whereas S. aureus adherence in an albumin-containing buffer was less than or equal to 10(3) CFU on control uncoated cover slips, adherence in the same medium increased up to maximum of 7.7 X 10(4) CFU on cover slips preincubated in a solution of fibronectin (125-micrograms/ml). At intermediate fibronectin concentrations, bacterial adherence was a linear function of both the quantity in solution and of the quantity adsorbed on the PMMA cover slips. The presence of human serum proteins, as represented by a fibronectin-depleted pool, essentially prevented adsorption of radiolabeled fibronectin on PMMA and subsequent bacterial adherence on the cover slips. Precoating of PMMA with denatured collagen resulted in increased fibronectin adsorption on PMMA, even in the presence of serum proteins, and S. aureus adherence was optimal on such surfaces. Collagen may therefore play a role as a cofactor contributing to S. aureus adherence onto fibronectin-coated substrata or foreign bodies.     

Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection

The world's first deliveries of normal babies after use of flow cytometric separated human sperm cells (MicroSort) for preconception gender selection are reported. Offspring were of the desired female gender in 92.9% of the pregnancies. Most of these pregnancies and births were achieved after simple intrauterine insemination.      

A 3 year retrospective review of intrauterine insemination, using cryopreserved donor spermatozoa and cycle monitoring by urinary or serum luteinizing hormone measurements

Insemination with donor spermatozoa is an integral part of infertility treatment. For the last 3 years in our unit, intrauterine insemination with donor spermatozoa (IUID) has been used in preference to vaginal insemination. In this retrospective study, patients were offered an initial course of five single intrauterine inseminations with cryopreserved donor spermatozoa and treatment was then reviewed. A total of 389 patients received 1465 inseminations. In all, 1119 cycles were monitored using luteinizing hormone serum analyses and 346 cycles using the urine home test kits. The clinical pregnancy rate per insemination for the cycles monitored by the serum assay was 18.0% (202/1119) compared with the urine cycles (13.7%, 46/346) (P <05). The pregnancy loss rate was not significantly different (14.4%, 29/202 and 21.7%, 10/46) (serum and urine cycles respectively). The viable clinical pregnancy rate was significantly higher (P <03) for the serum cycles than for the cycles using the urinary monitoring (15.5%, 173/1119 and 10.4%, 36/346 respectively). The cycles monitored by serum assay had a significantly higher cumulative viable clinical pregnancy rate (P <0001) of 70.2% after nine inseminations compared with the urine monitored cycles of 54.8%. The majority of patients opted for the serum cycles, with a minority self-selecting the urine cycles mainly for travelling convenience. The explanation for the significant differences between the viable clinical pregnancy rates per insemination and the cumulative viable clinical pregnancy rates may be due to the sensitivity of the urine home test kit or the patients' interpretation of the result.      

Striated intramural gallbladder lucencies on US studies: predictors of acute cholecystitis

Ultrasound scans of 51 consecutive patients with gallbladder wall thickening were reviewed, and specific sonographic features were correlated with surgical and clinical follow-up. Two patterns of thickening were identified as specific indicators of the presence or absence of acute cholecystitis. "Striated" wall thickening, consisting of several alternating, irregular, discontinuous, lucent and echogenic bands, was seen in eight of 13 patients (62%) with acute cholecystitis. This pattern was not encountered in any of the patients who did not have acute cholecystitis. Conversely, "three-layer" thickening, consisting of a single circumferential lucent zone between two relatively uniform echogenic layers, was seen in only one of 13 patients (8%) with acute cholecystitis but in 11 of 38 patients (29%) with other diagnoses. Other abnormalities, including the presence of intramural echogenic foci and wall irregularities, were more frequently seen in patients with acute cholecystitis but were not as helpful. Use of these features may suggest or help exclude a diagnosis of acute cholecystitis in those patients in whom the cause of gallbladder wall thickening is otherwise not apparent.     

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.