Evaluation of the IDI-MRSA assay on the SmartCycler real-time PCR platform for rapid detection of MRSA from screening specimens.

Rapid accurate detection is a prerequisite for the successful control of meticillin-resistant Staphylococcus aureus (MRSA). The IDI-MRSA real-time polymerase chain reaction (PCR) assay was designed to provide rapid results from nasal specimens collected in Stuart's liquid transport medium. This study has evaluated the IDI-MRSA kit for use in a clinical laboratory by investigating the following parameters: (1) limits of detection (LoD), (2) performance with Amies' gel-based transport medium, (3) ability to detect strains of MRSA in a collection representative of MRSA in Ireland since 1974 (n=113) and (4) performance in a clinical trial with swabs from nose, throat and groin/perineum sites from 202 patients. LoDs (colony-forming units per ml) of the IDI-MRSA kit, direct culture on MRSA-Select chromogenic agar (CA) and salt-enrichment culture (with subculture onto CA) were 10(3), 10(3) and 10(2), respectively. LoDs with Stuart's and Amies' transport media were comparable. All except one of the 113 MRSA isolates were detected by the kit but, when six control strains carrying staphylococcal cassette chromosome mec (SCCmec) type IV element subtypes IVa-d and SCCmec types V and V(T) were tested, the kit failed to detect MRSA carrying SCCmec V. The sensitivity and specificity for detection of MRSA from nose, throat and groin/perineum specimens were comparable with slightly lower sensitivities from throat and groin/perineum specimens compared with nasal swabs (90%, 97%; 89%, 99%; 88%, 99%, respectively). Overall sensitivity, specificity and positive and negative predictive values for specimens from all sites were 88%, 99%, 94% and 97%, respectively. Further developments to improve the sensitivity of this highly worthwhile assay are required.

Epidemiological typing of MRSA isolates from blood cultures taken in Irish hospitals participating in the European Antimicrobial Resistance Surveillance System (1999-2003).

Between 1999 and 2003, meticillin-resistant Staphylococcus aureus (MRSA) isolates recovered from blood cultures in Irish hospitals that participate in the European Antimicrobial Resistance Surveillance System were investigated by epidemiological typing using antibiogram-resistogram (AR) typing, biotyping, and DNA macrorestriction digestion using SmaI followed by pulsed-field gel electrophoresis (PFGE). PFGE patterns were assigned five-digit pulsed-field type (PFT) numbers, and PFTs of apparently related patterns were abbreviated to two-digit PFT groups (PFGs). AR and PFGE typing results were combined to produce AR-PFG types. Representative isolates of each AR-PFG type recovered in 2002 were typed by multilocus sequence typing and staphylococcal cassette chromosome (SCC) mec analysis. Isolates from 1999 and 2000 were also typed by phage typing. The extent to which epidemiological types of MRSA from blood cultures could be extrapolated to the total MRSA population was investigated by comparing results obtained with isolates from the total MRSA population versus those obtained with blood cultures during three study periods. Over the 5 years from 1999 to 2003, 1,580 blood culture isolates from 1,495 patients were analysed. Typeability and discriminatory indices were as follows: AR typing, 1 and 0.97; phage typing, 0.29 and 0.89; PFGE, 0.99 and 0.95; AR-PFG typing, 1 and 0.95. The most frequently occurring AR-PFG types were 06-01, 07-02, 13-00, and 14-00 and were exhibited by 57, 7, 14, and 12% of isolates, respectively. During the study period, the distribution of AR-PFG type changed markedly, with the prevalence of one type (AR-PFG 06-01) increasing by 880%, from 22% (39/181) in 1999 to 80% (343/430) in 2003. Investigation of whether epidemiological types among blood culture isolates of MRSA were representative of the total MRSA population showed that there was no significant difference in most instances. MLST and SCCmec typing showed that AR-PFG types 06-01, 07-02, 13-00, and 14-00 were ST22-MRSA-IV, ST36-MRSA-II, ST8-MRSA-IID, and ST8-MRSA-IIE, respectively.

Catalysis and binding in L-ribulose-5-phosphate 4-epimerase: a comparison with L-fuculose-1-phosphate aldolase.

L-Ribulose-5-phosphate (L-Ru5P) 4-epimerase and L-fuculose-1-phosphate (L-Fuc1P) aldolase are evolutionarily related enzymes that display 26% sequence identity and a very high degree of structural similarity. They both employ a divalent cation in the formation and stabilization of an enolate during catalysis, and both are able to deprotonate the C-4 hydroxyl group of a phosphoketose substrate. Despite these many similarities, subtle distinctions must be present which allow the enzymes to catalyze two seemingly different reactions and to accommodate substrates differing greatly in the position of the phosphate (C-5 vs C-1). Asp76 of the epimerase corresponds to the key catalytic acid/base residue Glu73 of the aldolase. The D76N mutant of the epimerase retained considerable activity, indicating it is not a key catalytic residue in this enzyme. In addition, the D76E mutant did not show enhanced levels of background aldolase activity. Mutations of residues in the putative phosphate-binding pocket of the epimerase (N28A and K42M) showed dramatically higher values of K(M) for L-Ru5P. This indicates that both enzymes utilize the same phosphate recognition pocket, and since the phosphates are positioned at opposite ends of the respective substrates, the two enzymes must bind their substrates in a reversed or "flipped" orientation. The epimerase mutant D120N displays a 3000-fold decrease in the value of k(cat), suggesting that Asp120' provides a key catalytic acid/base residue in this enzyme. Analysis of the D120N mutant by X-ray crystallography shows that its structure is indistinguishable from that of the wild-type enzyme and that the decrease in activity was not simply due to a structural perturbation of the active site. Previous work [Lee, L. V., Poyner, R. R., Vu, M. V., and Cleland, W. W. (2000) Biochemistry 39, 4821-4830] has indicated that Tyr229' likely provides the other catalytic acid/base residue. Both of these residues are supplied by an adjacent subunit. Modeling of L-Ru5P into the active site of the epimerase structure suggests that Tyr229' is responsible for deprotonating L-Ru5P and Asp120' is responsible for deprotonating its epimer, D-Xu5P.

New xanthorin glycosides from a Dermocybe species.

A mixture of the 8-O-beta-D-glucopyranoside of omega-hydroxyxanthorin 1-O-methyl ether (3) and the 8-O-beta-D-gentiobioside of xanthorin 1-O-methyl ether (4) was isolated from the water-soluble extractives of the Australian toadstool Dermocybe sp. WAT 22963. Compounds 3 and 4 were identified by characterization of their respective peracetyl derivatives 5 and 6.

Peptide growth factors and preimplantation development.

This paper examines some of the problems and questions that must be considered in relation to research on the role of growth factors in preimplantation embryos. It reviews and summarizes the large body of work on gene expression of growth factor receptors and ligands in preimplantation embryos and in oviduct and uterine tissue. It also reviews the literature on the effects of gene knockout in preimplantation embryos and concludes with a review of work on the effects of growth factors on cultured embryos.

Immunohistochemical analysis of tissues regenerated from within periodontal defects treated with expanded polytetrafluoroethylene membranes.

Immunocytochemical analysis was carried out on samples of 5-, 6-, and 9-week old regenerated soft tissue taken from healing periodontal defects treated by guided tissue regeneration using expanded polytetrafluorethylene (ePTFE) membranes. A panel of monoclonal and polyconal antibodies to cytokeratins, vimentin, and collagen was used to label cells and collagen types I, III, and IV. Epithelium was identified in 7 of the 9 samples examined, in addition to mesenchymal cells staining positively for vimentin and co-distribution of collagen types I, III, and IV in all samples. Clinical observations indicated that exposure of the ePTFE membranes during healing was a frequent occurrence, and the presence and quantity of epithelium found within the healing defect beneath the membrane may be related to the extent to which this occurs.

Protein content of rabbit embryos: one cell to peri-implantation blastocyst.

The protein content of rabbit embryos during the first 7 days of development in vivo was determined. The protein content of intact embryos, embryonic cells (intact embryos without mucin coats for developmental stages up to 96 h post-coitum and free of blastocyst coverings for later stages) and blastocyst coverings were determined by the Pierce Micro BCA assay. The mean protein content of intact one-cell or two-cell embryos was 0.16 micrograms and increased at the four- to six-cell stage with no further increase until the late morula/early blastocyst stages (days 3 to 4). There was a 53-fold increase in protein from the early to late blastocyst stages. The protein content of embryonic cells was stable at a mean value of 0.16 micrograms until the late morula stage (day 3) and then increased to a mean of 6.85 micrograms on day 6 and 50.38 micrograms on day 7. The increase in protein content of intact embryos up to about 72 h appeared to be due solely to an increase in the protein content of the mucin coat. The protein content of the blastocyst coverings increased from a mean of 2 micrograms on day 5 to a mean of 35 micrograms on day 7. For blastocyst stages, the total protein content of intact blastocysts and of embryonic cells was correlated with the surface area of the embryos (r2 = 0.895 and 0.873, respectively) and, thus, an increase in blastocyst size is a true index of blastocyst development.

Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate.

A factor of low M(r) with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M(r) 10,000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M(r) of 192. The embryotrophic factor in the low M(r) extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.

In vitro maturation of ovarian oocytes from unstimulated rhesus monkeys: assessment of cytoplasmic maturity by embryonic development after in vitro fertilization.

One-hundred and sixty-six cumulus-enclosed oocytes, obtained from ovaries of unstimulated rhesus monkeys, were subjected to six different treatments in vitro--two types of media (simple = TALP; complex = CMRL) x three levels of gonadotropins (none, FSH, FSH + hCG)--to assess their ability to undergo maturation, fertilization, and embryo development. A summary of development in culture for all experimental treatments is as follows: 58% of oocytes underwent germinal vesicle breakdown; 37% extruded a first polar body; 17% had more than one pronucleus and/or two polar bodies after insemination (i.e., were activated/fertilized); and 12% cleaved (i.e., developed) to at least the 2-cell stage in vitro. Of 45 oocytes incubated only in medium (either simple or complex) without gonadotropins, only 3 were activated/fertilized (6.7%), and only one embryo developed to at least the 2-4-cell stage (2.2%). There were no differences between oocytes incubated with only FSH and oocytes incubated with FSH + hCG. Activation/fertilization (20.7% vs. 6.7%) and embryo development (greater than or equal to 2 cells; 15.7% vs. 2.2%) were significantly higher in treatments with than without gonadotropin supplementation. There were no statistically significant differences attributable to incubation in different media during oocyte maturation. Cumulus-enclosed oocytes recovered from unstimulated ovaries of rhesus monkeys can resume maturation during culture in vitro, as shown by their ability to be fertilized and by the cleavage in vitro of the resultant zygotes.

Relationships between follicular fluid steroid hormone concentrations, oocyte maturity, in vitro fertilization and embryonic development in the rhesus monkey.

Oocytes and matched samples of follicular fluid (FF) were obtained from 70 follicles of five rhesus monkeys stimulated with either pregnant mare serum gonadotropin or human menopausal gonadotropin. Follicular aspiration was performed 30-32 h after human chorionic gonadotropin administration. The concentrations of estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) in FF were measured. Twenty-six percent of oocytes were classified as mature (M), 41% matured in vitro (Miv), 13% were dysmature, and 20% atretic. M oocytes were associated with significantly higher levels of P and a higher P:E2 ratio. There were no differences in hormone levels associated with fertilized and nonfertilized oocytes. Thirty-five embryos developed to the six- to eight-cell stage in vitro, of which 13 exhibited optimal cleavage rates. Significantly lower levels of E2 and higher P:E2 ratios were associated with the more rapidly cleaving embryos. Proportionally more embryos showing optimal cleavage rates developed from M compared to Miv oocytes, and only embryos derived from M oocytes developed to blastocysts in culture. Optimal cleavage rates to the six- to eight-cell stage in vitro, rather than fertilization rates, are a better indicator of (subsequent) developmental capacity, and, in this study, embryonic development was closely associated with the maturity of the oocyte at recovery.

Evaluation of ultrasonography for monitoring follicular growth in rhesus monkeys.

The purpose of this study was to determine if the ovaries and uterus of rhesus monkeys could be visualized by ultrasonography and to detect changes associated with follicular growth and ovulation. Animals were examined during 15 menstrual cycles, for an average of nine consecutive days. Ultrasonic recordings were correlated with hormonal parameters (estradiol 17beta, E(2); luteinizing hormone, LH; and progesterone, P) and laparoscopic findings. The uterus and both ovaries were observed in more than 90% of the examinations. A dominant follicle (DF) was identified during all ovulatory cycles, on average 1 d preceding the E(2) peak. The maximal diameter of the DF ranged from 3 to 7 mm. Laparoscopic examinations to determine the site of the DF confirmed ultrasonic findings in 10 of 14 cycles (P < 0.1). There was no significant difference in the size of the dominant and contralateral ovaries; however, more follicles with a diameter of 2 to 7 mm were found on the dominant ovary (P < 0.05). Two animals stimulated with exogenous gonadotropins showed a linear increase in ovarian size for 6 d prior to oocyte recovery (P < 0.05), reflecting an increase in the number of developing follicles. Ultrasonography can be used to identify the DF during spontaneous cycles in rhesus monkeys and to monitor the response of monkeys to exogenous gonadotropins.

Ultrasonographic assessment of the endometrium in rhesus monkeys during the normal menstrual cycle.

This study was undertaken to determine whether cyclical changes in the endometrium of the rhesus monkey could be observed by using ultrasound. Three indices of endometrial size were examined: the antero-posterior (or ventro-dorsal), longitudinal, and transverse diameters. Changes in the ultrasonic reflectivity of the endometrium were also assessed. We have attempted to correlate these endometrial parameters with the hormonal status of the animal. Ultrasonography was performed for an average of 12 consecutive days during 19 menstrual cycles. All ultrasonic recordings were normalized to the day of the estradiol (E2) peak (Day 0). We found that the reflectivity of the endometrium was dependent on the stage of the cycle: during the follicular phase, the endometrium appeared less echogenic (darker) compared to the myometrium; in the luteal phase, the endometrium was more echogenic (lighter). During the follicular phase (Days -9 to 0), there was a linear increase in the antero-posterior (p less than 0.001), longitudinal (p less than 0.05), and transverse (p less than 0.001) diameters. In the luteal phase (Days 1-15), no significant changes were observed in these diameters. An estimated endometrial volume (EEV) was obtained by the product of the antero-posterior, longitudinal, and transverse diameters. Each animal observed during the follicular phase (n = 14) exhibited a peak in the EEV, which correlated with the day of the E2 peak (p less than 0.01). From this study, we conclude that the sonographic appearance of the endometrium of the rhesus monkey reflects the cyclical changes that occur during the menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

The inaccuracy of total uterine volume measurements: sources of error and a proposed solution.

Inaccuracies in total intrauterine volumes calculated using the prolate ellipse equation have been reported. No previous study has examined all the sources of error. In this study, a comprehensive approach was undertaken. Measurements were obtained from scans of the pregnant uterus in the prone position using an automated water-path scanner (Octoson) and in the supine position using standard static B-mode scanners. Several conclusions could be drawn: 1) From the Octoson prone scans, uterine volumes obtained using the prolate ellipse formula were markedly different from the true uterine volumes obtained by the summation of stepped areas. This showed that the prolate ellipse formula was inaccurate. 2) From the static supine scans, many observer inconsistencies were found in uterine volumes obtained from the prolate ellipse formula. This made the prolate ellipse formula unreliable. 3) Previously published graphs calculated from the prolate ellipse equation, comparing fetal age with total intrauterine volume, were found to vary accuracy, presumably as a result of 1 and 2. A more accurate approach is proposed. Using the outer uterine wall as the boundary, the stepped area-to-volume values of transverse scans taken at 3-cm intervals were found to closely approximate true volumes, with an average error of only 3.5 per cent. Since these measurements encompass the intrauterine contents and the myometrium, it is suggested that the term "total uterine volume" be used instead of "total intrauterine volume."

Some parasitic and infectious diseases in waterfowl in Oklahoma.

Blood films and serum samples from free-ranging waterfowl wintering in and migrating through Oklahoma were examined for hematozoa and tested for antibody responses to Newcastle disease virus (NDV) and type-A influenza. One-hundred-eleven of 728 birds (15.24%) were positive for 1 or more hematozoa. Serologic testing revealed 11 of 280 (3.93%) positive for antibody to NDV and 5 of 171 (2.95%) positive for antibody to type-A influenza.

Seroepidemiological investigation of Brucella canis antibodies in different human population groups.

A study was conducted to determine the prevalence of Brucella canis antibodies in specified groups based on their exposure to dogs. The method used was a microtiter technique, and the presence of antibodies at a 1:12 or greater dilution of serum was considered a positive test. Eleven (5.7%) of the newborn infants had evidence of maternal antibodies, and 67.8% of the persons with an average exposure to dogs had B. canis antibodies, with a 62.1% prevalence in males and a 72.4% prevalence in females (P less than or equal to 0.001). Veterinarians had a much higher rate of infection (72.6%) than male blood donors (56.9%) (P less than or equal to 0.01). Patients with fevers of undetermined origin had significantly higher antibody titers to B. canis than all other patients (P less than or equal to 0.001). This study presents evidence that the prevalence of B. canis antibodies in humans is high, and that the incidence of brucellosis may increase when physicians consider B. canis as a possible etiological agent in febrile illnesses.

Dental health of Louisiana residents based on the ten-state nutrition survey.

The dental health status of 4,006 residents of Louisiana was analyzed, based on data in the 1968-70 Ten-State Nutrition Survey funded by the U.S. Government. These data were based on examinations of census districts in which the average per capita income was in the lowest quartile for the nation. A considerable variation in the prevalence of dental diseases was found among the Louisiana residents according to age. The females examined had a slightly higher DMF (decayed, missing, and filled permanent teeth) score, a lower OHI (oral hygiene index) score, and a slightly lower PI (periodontal index) score than did the males. The dental caries attack rate did not vary much by race, but the whites examined had received a much greater amount of dental care than had their black counterparts. The OHI scores of the blacks were higher than those for the whites in both the debris and calculus components. The PI scores were higher for the blacks than for the whites. More white persons than blacks were edentulous; this result, however, tends to confirm the observation of increased dental care in white persons. The percentages of persons with periodontal disease and periodontal pockets were considerably higher among persons with incomes below the poverty level, and a greater percentage of blacks had incomes below that level. The data thus apparently indicate that the major determinants of dental health status in Lousiana are age and level of income; race appears to be the major determinant of the amount of dental care received.