A PCR detection method for testing Mycoplasma contamination of veterinary vaccines and biological products.

A rapid test method was developed for detecting mycoplasma contamination in veterinary biological products. The method reduces testing time by 2 weeks and shows comparable sensitivity to the current agar-based detection model. The primary goals for the development of the test were to reduce the testing time, incorporate a method that was easily adaptable across the veterinary biologics industry and reduce the subjective interpretation of results. We found that biological enrichment is necessary to maintain sensitivity of the detection method when compared to the standard culture-based test and that periodic sampling of enrichment cultures is essential to detect a wide variety of mycoplasma species that may be present as contaminants. The PCR detection method is comparable to the agar-based model and can reduce the overall testing time by up to 14 days.

Periosteal chondroma at birth.

Periosteal chondroma is a slow-growing, cartilaginous, surface tumor that usually occurs in the second and third decades of life. The youngest reported age at diagnosis is 5 years. Marginal excision is the treatment of choice. We report a case of a periosteal chondroma noted at birth and treated conservatively. This report expands the age range of periosteal chondroma to include neonates and suggests a role for observation in its management.

Aneurysmal bone cyst of the extremities. Factors related to local recurrence after curettage with a high-speed burr.

BACKGROUND: Aneurysmal bone cyst is a benign, locally destructive lesion of bone. The rates of local recurrence after curettage have varied widely. Therefore, we performed a retrospective study of patients who had had an aneurysmal bone cyst in order to identify the rate of local recurrence and the prognostic factors related to local recurrence after use of contemporary methods of curettage with a high-speed burr. METHODS: We reviewed the cases of forty patients who had been managed by the same surgeon for an aneurysmal bone cyst, as diagnosed on the basis of the latest pathological review, between January 1, 1976, and December 31, 1993. The patients were evaluated with regard to age, gender, the duration and type of symptoms, the presence or absence of pathological fracture, the status of the growth plate, the bone and part of the bone that were involved, the type of operative procedure, the outcome, the radiographic stage, the findings on magnetic resonance imaging and computerized tomography (when it became available) and on bone scintigraphy, and histological parameters. The median duration of follow-up was eighty-seven months (range, fifteen to 267 months). According to the criteria of Enneking, no patient had a stage-1 lesion (one with a surrounding rim of cortical bone), twenty-four had a stage-2 lesion (one with a clearly defined border but no cortical bone), and sixteen had a stage-3 lesion (one with no clearly defined border). RESULTS: Of the forty patients, thirty-four had curettage with use of a high-speed burr. Of these thirty-four, twenty-two had filling of the defect with a cancellous autogenous graft; four, with a cancellous allograft; and three, with polymethylmethacrylate. In five patients, no material was put into the defect. The remaining six patients had resection through the margin of the lesion. Four (12 percent) of the thirty-four patients who had curettage had a local recurrence. No patient who had an excision through the margin of the lesion had a local recurrence. All local recurrences were in skeletally immature girls who were three, four, ten, and eleven years old. Univariate analysis with use of the chi-square, Fisher exact, and Wilcoxon log-rank tests showed that local recurrence was associated only with a young age (p = 0.0036) and open growth plates (p = 0.039). All local recurrences occurred within two years postoperatively, at two, seven, nine, and twenty-four months, and all were treated successfully with a second operation. CONCLUSIONS: Rates of local control of almost 90 percent can be achieved with thorough curettage with use of a mechanical burr and without use of liquid nitrogen, phenol, or other adjuvants in patients who have an aneurysmal bone cyst of an extremity. A young age and open growth plates are associated with an increased risk of local recurrence.

Oral intake policies on labor and delivery: a national survey.

STUDY OBJECTIVE: To examine current policies on oral intake during labor among hospitals throughout the United States. DESIGN AND SETTING: Anonymous questionnaire survey distributed to the directors of anesthesia and obstetrics departments of 740 hospitals. Completed surveys were then grouped by number of deliveries performed each year. MEASUREMENTS AND MAIN RESULTS: A total of 2,265 surveys were distributed. Of that number, 902 (33% response rate) surveys, representing 740 U.S. hospitals, were returned. Of the surveys returned, 419 surveys were received from obstetricians and 401 surveys were received from anesthesiologists. Oral intake during labor is limited primarily to clear liquids, although hospitals with fewer deliveries allow significantly more oral intake during latent phase than do hospitals with larger services. Allowing nonclear liquids or solid foods is uncommon in either phase of labor, regardless of hospital size. CONCLUSIONS: The results give an indication of oral intake policies used by labor and delivery units in the United States, and they may be helpful for obstetric services that are in the process of developing their own policies.

Obstetric anesthesia work force survey, 1981 versus 1992.

BACKGROUND: In 1981, with support from the American Society of Anesthesiologists and the American College of Obstetricians and Gynecologists, anesthesia and obstetric providers were surveyed to identify the personnel and methods used to provide obstetric anesthesia in the United States. The survey was expanded and repeated in 1992 with support from the same organizations. METHODS: Comments and questions from the American Society of Anesthesiologists Committee on Obstetrical Anesthesia and the American College of Obstetricians and Gynecologists Committee on Obstetric Practice were added to the original survey instrument to include newer issues while allowing comparison with data from 1981. Using the American Hospital Association registry of hospitals, hospitals were differentiated by number of births per year (stratum I, > or = 1,500 births; stratum II, 500-1,499 births; stratum III, < 500 births) and by U.S. census region. A stratified random sample of hospitals was selected. Two copies of the survey were sent to the administrator of each hospital, one for the chief of obstetrics and one for the chief of anesthesiology. RESULTS: Compared with 1981 data, there was an overall reduction in the number of hospitals providing obstetric care (from 4,163 to 3,545), with the decrease occurring in the smallest units (56% of stratum III hospitals in 1981 compared with 45% in 1992). More women received some type of labor analgesia and there was a 100% increase in the use of epidural analgesia. However, regional analgesia was unavailable in 20% of the smallest hospitals. Spinal analgesia for labor was used in 4% of parturients. In 1981, obstetricians provided 30% of epidural analgesia for labor; they provided only 2% in 1992. Regional anesthesia was used for 78-85% (depending on strata) of patients undergoing cesarean section, resulting in a marked decrease in the use of general anesthesia. Anesthesia for cesarean section was provided by nurse anesthetists without the medical direction of an anesthesiologist in only 4% of stratum I hospitals but in 59% of stratum III hospitals. Anesthesia personnel provided neonatal resuscitation in 10% of cesarean deliveries compared with 23% in 1981. CONCLUSIONS: Compared with 1981, analgesia is more often used by parturients during labor, and general anesthesia is used less often in patients having cesarean section deliveries. In the smallest hospitals, regional analgesia for labor is still unavailable to many parturients, and more than one half of anesthetics for cesarean section are provided by nurse anesthetists without medical direction by an anesthesiologist. Obstetricians are less likely to personally provide epidural analgesia for their patients. Anesthesia personnel are less involved in newborn resuscitation.

Oncological outcomes of operative treatment of subcutaneous soft-tissue sarcomas of the extremities.

We reviewed the cases of sixty-two patients who had had a subcutaneous sarcoma to determine the effect of tumor and treatment-related variables on the rates of survival and local recurrence. Fifty-nine (95 per cent) of the patients had had an operation at another hospital before being referred to us. Twenty-nine (47 per cent) of the sixty-two tumors were high-grade, forty-two (68 per cent) were small (five centimeters or less), and thirty (48 per cent) were malignant fibrous histiocytomas. We followed a treatment strategy that consisted of repeat excision with the goal of obtaining wide margins. Excluding thirteen patients who had had a palpable local recurrence at the time of presentation, twenty (49 per cent) of forty-one patients who had had a marginal excision at another hospital had microscopic residual tumor on repeat excision. At a median of fifty-six months after the repeat excision, fifty (81 per cent) of the sixty-two patients had been continuously disease-free, one had no evidence of disease, eight had died of the disease, and three had died of other causes. The five-year rate of disease-free survival was 85 per cent (fifty-three of sixty-two patients). There were three local recurrences, all in patients who had had a marginal resection. No recurrences were noted in patients who had had a wide local excision of the tumor or of the previous operative field. Multivariate analysis revealed that a large tumor (greater than five centimeters), a marginal excision, and adjuvant radiation therapy were associated with a worse prognosis. Excellent rates of survival for patients who have a subcutaneous sarcoma, including those who have a large or high-grade tumor and those who have residual tumor following a previous operation, can be obtained with carefully planned operative treatment alone. We recommend operative excision or repeat excision with wide margins because of the high prevalence of residual tumor. Size is the most important tumor-related factor, and the operative margin is the most important treatment-related factor. The additional value of adjuvant radiation therapy remains unproved.

Anesthesia-related deaths during obstetric delivery in the United States, 1979-1990.

BACKGROUND: Anesthesia-related complications are the sixth leading cause of pregnancy-related death in the United States. This study reports characteristics of anesthesia-related deaths during obstetric delivery in the United States from 1979-1990. METHODS: Each state reports deaths that occur within 1 yr of delivery to the Centers for Disease Control and Prevention as part of the ongoing Pregnancy Mortality Surveillance. Maternal death certificates (with identifiers removed) matched with live birth or fetal death certificates when available from 1979-1990 were reviewed to identify deaths due to anesthesia, the cause of death, the procedure for delivery, and the type of anesthesia provided. Maternal mortality rates per million live births were calculated. Case fatality rates and risk ratios were computed to compare general to regional anesthesia for cesarean section deliveries. RESULTS: The anesthesia-related maternal mortality rate decreased from 4.3 per million live births in the first triennium (1979-1981) to 1.7 per million in the last (1988-1990). The number of deaths involving general anesthesia have remained stable, but the number of regional anesthesia-related deaths have decreased since 1984. The case-fatality risk ratio for general anesthesia was 2.3 (95% confidence interval [CI], 1.9-2.9) times that for regional anesthesia before 1985, increasing to 16.7 (95% CI, 12.9-21.8) times that after 1985. CONCLUSIONS: Most maternal deaths due to complications of anesthesia occurred during general anesthesia for cesarean section. Regional anesthesia is not without risk, primarily because of the toxicity of local anesthetics and excessively high regional blocks. The incidence of these deaths is decreasing, however, and deaths due to general anesthesia remain stable in number and hence account for an increased proportion of total deaths. Heightened awareness of the toxicity of local anesthetics and related improvements in technique may have contributed to a reduction in complications of regional anesthesia.

Genome plasticity in Neisseria gonorrhoeae.

The pathogenic Neisseria have exploited the processes of horizontal DNA transfer and genetic recombination as mechanisms for the generation of extensive protein variation and modulation of gene expression. Localized recombinations have been well documented in members of multigene families as have alterations in short repetitive sequences. Here we report an analysis of the chromosomal structure of a defined lineage of Neisseria gonorrhoeae strain MSl1 pilin variants. This study reveals the occurrence of large rearrangements, including the amplification of a 26 kb region and an inversion involving more than a third of the chromosome. Additionally, a restriction site polymorphism that correlates with pilin expression has been observed. These findings highlight the flexibility of the gonococcal genome.

Construction of Hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformable Neisseria mutants.

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformable Neisseria mutants. By random insertion of a selectable marker into the conjugative Neisseria plasmid ptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned in Escherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of an E. coli replicon that does not support autonomous replication in Neisseria, e.g. ColE1, p15A, or ori(fd), fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformable Neisseria strain involves a three-step process: (i) insertion of the desired gene into a +Hermes vector; (ii) transformation of Hermes into a Neisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the final Neisseria recipient. Several applications for the genetic manipulation of pathogenic Neisseriae are described.

Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia.

The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).

Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae to human epithelial cells.

Pili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra-strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA- strain were selected that had lost the ability to bind to human cornea epithelial cells (A-) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence-negative mutants failed to produce detectable levels of PilC1 or PilC2 proteins, representing piIC phase variants generated in the absence of RecA. The A- pilC phase variants were indistinguishable from their A+ parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus-mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PilC1 nor PilC2 is obligatory for the assembly of gonococcal pili.

Physical and genetic map of the Neisseria gonorrhoeae strain MS11-N198 chromosome.

A macro-restriction map of the Neisseria gonorrhoeae chromosome was constructed using the enzymes Nhel and Spel. Combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA were performed to align the restriction fragments. The chromosome is circular, with an estimated size of 2.33 Mb +/- 35 kb. A genetic map was derived from the physical map; positions of over 60 defined loci were determined by Southern hybridization.

The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes.

Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.

Training anesthesia personnel to perform bleeding times.

Template bleeding time results generated by residents in anesthesiology correlate well with those obtained by trained laboratory personnel (p less than 0.002). Anesthesia personnel can accurately perform bleeding time tests when laboratory support is not available. The results of these tests can be used to guide clinical decisions.