Validation of Vitek version 7.01 software for testing staphylococci against vancomycin.

We tested 143 isolates of staphylococci with vancomycin by the National Committee for Clinical Laboratory Standards broth microdilution (BMD) reference method and compared the results to those generated using the Vitek automated system (GPS-105 and GPS-107 cards and version 7.01 software). For ten isolates, the vancomycin MICs by BMD were 8 microg/ml. By Vitek, the vancomycin MICs ranged from 2 to 16 microg/ml. Vancomycin MICs of > or =32 microg/ml were reported for two additional isolates by Vitek; however, the MICs decreased to < or =0.5 microg/ml on retesting. By BMD, the vancomycin MICs for both isolates were 1 microg/ml. While the modal vancomycin MIC results by BMD for S. aureus and coagulase-negative staphylococci (CoNS) were both 1 microg/ml, Vitek results showed a mode of < or =0.5 microg/ml for S. aureus, and a mode of 2 microg/ml for CoNS. Vitek did not report vancomycin MICs of 1 or 4 microg/ml for any of the isolates tested. While the sensitivity of detecting staphylococci with reduced susceptibility to vancomycin appears to be improved with Vitek version 7.01 software, when compared to earlier software versions, laboratories may notice an overall shift in MIC data toward higher vancomycin MICs, although for the most part, this does not affect the categorical interpretations of the results.

Performance of eight methods, including two new rapid methods, for detection of oxacillin resistance in a challenge set of Staphylococcus aureus organisms.

Using a set of 55 Staphylococcus aureus challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSA-Screen, that require less than a day to determine the susceptibility of S. aureus to oxacillin. The methods were evaluated by using the presence of the mecA gene, as detected by PCR, as the "gold standard." The strains included 19 mecA-positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 microg/ml) and 36 mecA-negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 microg/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the mecA-negative strains increased to 4 to 8 microg/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.

Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods.

Extended-spectrum beta-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were > or =2 microg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of > or =5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by > or =3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were > or =32 microg/ml, suggesting either the presence of an AmpC-type beta-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a beta-lactamase with a pI of > or =8.3 suggestive of an AmpC-type beta-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and bla(OXA) genes. The IEF profiles of the remaining 16 isolates showed a variety of beta-lactamase bands, all of which had pIs of < or =7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla(OXA), and bla(CTX-M) genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained beta-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained beta-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.

Use of urokinase in pregnancy. Two success stories.

Use of urokinase in treatment of deep venous thrombosis in pregnancy has been limited because of concerns about teratogenic effects and potential hemorrhage before and after delivery. However, reports to date have been encouraging and our experience is supportive. In the cases described here, urokinase thrombolytic therapy was effectively used to treat deep venous thrombosis in two pregnant patients without any apparent complications.

Study of immune function in inbred miniature pigs vaccinated and challenged with suid herpesvirus 1.

Specific immune responses of inbred miniature pigs following vaccination and challenge with suid herpesvirus 1 (SHV-1) were determined. Vaccination of swine with SHV-1 elicited both specific neutralizing antibody and lymphoproliferative responses. Moreover, pigs vaccinated with SHV-1 were fully protected against a lethal virus challenge. Pigs vaccinated with a recombinant (r) SHV-1 virus, followed by challenge with a virulent SHV-1, had lower percentages of circulating T- and B-lymphocytes, and showed a significant (P < or = 0.05) reduction in peripheral blood mononuclear cell (PBMC) antibody-dependent cell-cytotoxicity than control (noninfected, SHV-1 sero-negative) animals. From the 5th through the 8th week of postchallenge, rSHV-1 was isolated from 2 of 4 pigs. Presence of r-virus was indicative that PBMC were infectious in vivo. The rSHV-1, with beta-galactosidase activity, was only recovered from ConA- and IL-2-stimulated primary PBMC cocultivated with porcine kidney cells. Control pigs exposed to challenge SHV-1 elicited both specific neutralizing antibody and lympho-proliferative responses followed by subsequent infection. These infected pigs, compared to control pigs, had significantly (P < or = 0.05) lowered percentages of T- and B-lymphocytes, lowered T-cell mitogenic responses, variable PBMC counts, and lowered blood phagocytic cell function. When PBMC from control pigs were cultured and infected with SHV-1, the virus caused a significant (P < or = 0.05) suppression of T-cell proliferation and PBMC mitochondrial dehydrogenase and macrophage activities.

Gliotoxin inhibits transformation and its cytotoxic to turkey peripheral blood lymphocytes.

Gliotoxin, an epipolythiodioxopiperizine mycotoxin, has been shown to be produced by, among other fungi, Aspergillus fumigatus Fresenius. This organism is the major causative agent of the respiratory disease aspergillosis in avian species, especially turkeys. Because gliotoxin has been shown to be immunosuppressive and has the potential for being involved in the pathogenesis of aspergillosis, the in vitro activity of this compound with avian lymphocytes was investigated. Immunosuppression was investigated using peripheral blood lymphocytes from turkeys in a lymphoblastogenesis assay and a cytotoxicity assay using conversion of the tetrazolium salt MTT to MTT formazan by the mitochondrial succinate dehydrogenase enzyme elaborated only by living cells. Gliotoxin appeared to have a threshold level in both tests because little or no response or stimulation was evident when cells were exposed to concentrations of the toxin below 100 ng/ml, but at 100 ng/ml, all cells appeared to be dead. Using T-2 mycotoxin as a known cytotoxic agent, the response in the MTT bioassay using turkey peripheral lymphocytes was linear with increasing concentrations of toxin. Gliotoxin may potentially cause immunosuppression in turkey poults through action on the lymphocytes or if this toxin were present in low concentrations stimulation could possibly occur.

Immunomodulating effects of intestinal absorbed maternal colostral leukocytes by neonatal pigs.

Intestinal absorption of fluorescein isothiocyanate-labelled maternal colostral leukocytes (FITC-CL) was studied in 49 neonatal colostrum-deprived (CD) pigs from nine Minnesota miniature sows. Within 2 h postfeeding (pf), maternal FITC-CL were absorbed from the sibling's digestive tract and migrated into blood. The peak appearance of FITC-CL in blood occurred in samples at 5 and 7 h pf. By 24 h pf, cells were detected in liver, lung, lymph nodes, spleen and gastrointestinal tissues. To confirm intercellular migration of FITC-CL, gastrointestinal explant cultures from neonatal CD pigs were used. Maternal FITC-CL were observed to intercellularly migrate in 24 to 48 h pf between duodenal- and jejunal-epithelial cells to lamina propria cells and submucosal spaces. Fluorescein isothiocyanate-labelled maternal colostral leukocytes were not absorbed via ileal explant cultures. Unlike FITC-CL, maternal FITC-peripheral blood mononuclear leukocytes (FITC-PBL) were not absorbed either in vivo or in vitro by gastrointestinal tissues. When maternal FITC-PBL were intravenously administered to siblings they were distributed in blood and organs similar to FITC-CL. Following exposure to FITC-labelled cells, treated- and mock (untreated)-pigs were compared on the basis of PBL proliferative responses to phytomitogens. Sibling CD-pigs fed maternal FITC-CL showed higher PBL T-cell responses to phytohemagglutinin (PHA) and concanavalin A (ConA), and a significant stimulation (p < or = 0.01) of B-cell responses to pokeweed mitogen (PWM). Pigs fed FITC-PBL showed little PBL responses to PHA, ConA and PWM over PBL from mock pigs. Similarly, the influence of noncellular constituents of colostrum were also assessed by proliferative studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Disseminated Mycobacterium avium complex infection: clinical identification and epidemiologic trends.

To evaluate the incidence of disseminated Mycobacterium avium complex infection (DMAC) and to define the association between signs and symptoms and development of DMAC in patients with human immunodeficiency virus (HIV) infection, all cases of DMAC at Grady Memorial Hospital Infectious Disease Clinic (Atlanta) between 1985 and 1990 were reviewed, and a prospective study of the association of symptoms with DMAC was done. Between 1985 and 1990, DMAC occurred in 16% of patients with AIDS. Incidence increased from 5.7% in 1985-1988 to 23.3% in 1989-1990 (P less than .001). Median time from AIDS diagnosis to diagnosis of DMAC increased from 4.5 months in 1985-1988 to 8 months in 1989-1990 (P less than .02). In the prospective study, DMAC was seen only in persons with a CD4+ count less than 100 cells/mm3 and was associated with fever (P less than .03), anemia (P less than .001), weight loss (P less than .01), diarrhea (P less than .01), and elevated alkaline phosphatase (P less than .01). It is recommended that all such HIV-infected persons have mycobacterial blood cultures done.

Treatment of advanced epithelial ovarian carcinoma in pregnancy with cisplatin-based chemotherapy.

Malignant ovarian neoplasms associated with pregnancy are uncommon, and the majority of these are Stage I. While adjuvant chemotherapy for germ cell tumors during pregnancy has been reported, only one previous report exists on the use of cisplatin-based chemotherapy for epithelial ovarian carcinomas during pregnancy. A patient with Stage III cystadenocarcinoma of the ovary treated during pregnancy with five courses of dose-intensive cisplatin and cyclophosphamide is presented. No adverse fetal effects secondary to cisplatin-based chemotherapy were noted. A review of the literature is presented.

Host response to Pasteurella multocida turbinate atrophy toxin in swine.

A porcine strain of Pasteurella multocida (serotype D:3) produced a toxin causing turbinate atrophy (TA) in pigs. The toxin (TAT), processed on a high performance liquid chromatography size exclusion column, eluted as a single peak (molecular weight of about 160,000) containing trace amounts of endotoxin (lipopolysaccharide, LPS; protein:LPS, 85:1). The eluted fraction migrated on sodium dodecyl sulfate polyacrylamide gels as a single band. It could be prevented from dissociating into two prominent polypeptides by addition of a protease inhibitor. A single dose (2.0 to 79.0 micrograms/kg) of TAT given to pigs intravenously was lethal. Doses from 0.02 to 1.0 microgram/kg caused transient clinical signs of porcine systemic toxicosis with reduced appetite, generalized weakness, depression, lethargy, weight loss, and in some instances, death. Intradermal doses of TAT (greater than or equal to 0.1 microgram/site) produced hemorrhagic areas within four hours. Systemically, TAT causes bilateral TA, lymphopenia, liver dysfunctions, and possible renal impairment. Affinity of TAT for cells of epithelial origin was demonstrated in mice given 125I-TAT. In vitro, TAT stimulated DNA and protein syntheses of peripheral blood lymphocytes and suppressed syntheses in turbinate and kidney cell cultures without being cytolytic. Biological effects of TAT were eliminated by exposure to either heat, trypsin or anti-TAT antibody.

Immunological responses of cross-bred and in-bred miniature pigs to swine poxvirus.

Swine poxvirus (SPV), topically and subdermally applied to skin of the inguinal region of cross-bred and in-bred miniature pigs, caused typical pox lesions to occur with a pustular stage at 4 to 5 days p.i., and healing by 10 to 14 days p.i. Following inoculation, peripheral blood lymphocytes (PBLs) of the pigs showed lower transformation responses to SPV and mitogens (Concanavalin A, phytohemagglutinin and 12-0-tetradecanoyl-phorbol 13-acetate) than PBLs from uninoculated controls. The PBLs generally responded to SPV from 7 to 9 days p.i. to 23 to 30 days p.i. with a maximum transformation response at the 12 to 13 days p.i. interval. Sera from the animals generally showed presence of SPV-neutralizing antibody as early as 7 days p.i. and a peak titer at 20 days p.i. of 1:512. No detectable SPV-antibody was observed at 50 days p.i. By 51Cr release assays, PBLs displayed the ability to lyse target cells in the presence of SPV-antibody with peak lysis from the 11th through the 21st day p.i. An antibody-histocompatibility restricted cell lysis was observed at 11 and 14 days p.i. When PBLs were depleted of adherent cells, there was a reduction in lysis of target cells indicating the adherent cells were instrumental as effector cells in the presence of SPV-antibody. Control pigs not exposed to SPV showed no PBL response to SPV-antigen. Partially histocompatible and non-histocompatible porcine kidney cells were found useful as cell models for evaluating SPV-infected pigs in their effort to immunologically respond to SPV.

Effects of T-2 mycotoxin on gastrointestinal tissues: a review of in vivo and in vitro models.

T-2 mycotoxin, a trichothecene, is the principal toxic component of Fusarium sp. Agricultural products and food are frequently contaminated with this toxin. Various animal models have been used to determine its metabolic fate, rate of excretion, and distribution. A modulation effect on cell-mediated immunity and alterations in gastrointestinal propulsion have been demonstrated. The toxin has been shown to produce some similar pathologic alterations in various animal species studied. The consistent alteration appears to mainly affect mitotic cells of the gastrointestinal tract and the lymphoid system. A host of bioassay systems are now being used as alternative methods to the use of animals for testing of the mycotoxin. These tests may accurately assess and define the role of the subject-toxin interactions following consumption of T-2 mycotoxin contaminated food sources. T-2 mycotoxin, as observed above with in vivo and in vitro models, promotes a chemically-induced change in structure and function of affected gastrointestinal cells from a transient and reversible aberration in a single enzymatic reaction to cell death. Regardless of the end point measured, the toxic response brought about in cells appears to involve the interactions of virtually all subcellular processes--membrane transport and permeability, chemical metabolism, DNA function, and energy production/expenditure--as cells attempt to maintain their functional integrity while disposing of the toxicant. The variation in the quality of the toxic response with dose suggests that more cellular processes are perturbed as the chemical dose is increased.

Immunological surveillance in a pseudorabies quarantined herd using gilts and their progeny as sentinels.

Specific pathogen free gilts and their progeny were evaluated to use as sentinels in a pseudorabies virus (PRV) infected herd by immunologically monitoring for PRV seroconversions. Time intervals targeted were pre- and post-PRV vaccinations, herd exposure, and farrowing to finishing. Post-PRV vaccinations, gilts showed low PRV lymphocyte stimulation and humoral responses. Following herd exposure, control gilts PRV seroconverted and PRV vaccinated gilts increased (2 to 4 times) in virus neutralization (VN) titers. Sixty-seven percent (4/6) of the progeny from a control gilt were PRV seropositive at finishing. Progeny from PRV vaccinated gilts were depleted of passive immunity by week 7, and were seronegative until week 9. At finishing 47% (14/30) of them were PRV seropositive indicating exposure to PRV. The VN test was not sensitive enough to detect weak positive serums, noted as positives by latex agglutination (LA) test, ELISA, and Western blots. The gilts and progeny detected PRV, respectively, in the herd housing quarters and in the farrow to finish facilities. A strategy for future sentinel experimental surveillances using primarily the LA test is proposed.

Identification of carriers of Duchenne muscular dystrophy: value of molecular analysis.

Currently, molecular methods are the most accurate diagnostic tools for carrier detection and prenatal diagnosis of Duchenne muscular dystrophy (DMD). This report illustrates the value of molecular diagnosis as opposed to previous diagnostic methods, the need for frequent re-evaluations as new methodologies develop, and the necessity for in-depth genetic counseling. In Family 1, the proposita was predicted to be a carrier by an indirect assay (abnormal in vitro muscle ribosomal protein synthesis). DNA analysis using restriction fragment length polymorphisms (RFLPs) indicated that she was not a carrier. She gave birth to a predicted non-affected male, who inherited the gene in question. In Family 2 the proposita, an obligate carrier, was initially evaluated by RFLP analysis. Two pregnancies were monitored by first trimester chorionic villous sampling. Re-evaluation indicated that all affected individuals, including one of the embryos, carried a deletion of the dystrophin gene. The identification of an RFLP within the region containing the deletion allowed unambiguous determination of the carrier status of all individuals.

Perinatal medical negligence closed claims from the St. Paul Company, 1980-1982.

One hundred fifty-three closed claims involving perinatal injury or death filed from 1980 through 1982 with the St. Paul Fire and Marine Insurance Company were studied. The claims included were those in which an indemnity was paid or $1,000 or more was expended on the legal defense. Five obstetricians reviewed these cases to identify obstetric and neonatal risk factors. In addition, cases were classified as to the presence or absence of medical negligence. Most of the complications leading to claims arose during labor and delivery. Many claims resulted from the failure to evaluate or treat in a manner consistent with accepted standards of care. Many lacked documentation of the physician's recognition of the risk factors involved. Low Apgar scores at both one and five minutes were the newborn risk factors seen most commonly. In the opinion of the reviewers, medical negligence occurred in 47% of the cases. Indemnity payment occurred with most of the claims judged to be associated with medical negligence. Payment to the claimant was made in a number of cases in which the reviewer thought no malpractice occurred. These results suggest that improvements may be needed in prenatal and perinatal health care as well as in the legal system used to address the problem of perinatal medical negligence.

Assay of disaturated phosphatidylcholine in amniotic fluid as a test of fetal lung maturity: experience with 2000 analyses.

We determined concentrations of disaturated phosphatidylcholine (DSPC) in nearly 2000 amniotic fluid samples obtained either transabdominally or as vaginal pools. Here we report our comparison of these DSPC values with the lecithin/sphingomyelin (L/S) ratios for amniotic fluid samples obtained from diabetic and nondiabetic pregnancies and also between transabdominally or vaginally collected samples uncontaminated by blood or meconium. DSPC measurement is at least as good as the L/S ratio in predicting the absence of respiratory distress syndrome. DSPC concentrations were, however, lower in diabetic than in nondiabetic pregnancies, supporting the hypothesis that DSPC synthesis may be impaired in fetuses of diabetic mothers. Visually uncontaminated samples collected transabdominally or vaginally, when grouped according to length of gestation, have similar DSPC values but different L/S ratios. Thus, even in the absence of blood or meconium, DSPC may be a more useful test than the L/S ratio for vaginally pooled samples.

Pregnancy following renal transplantation in class T diabetes mellitus.

Nine cases of pregnancy complicated by diabetes and prior renal transplantation are reviewed. Maternal and fetal death occurred in a patient with foot and leg ulcers associated with preexisting peripheral vascular disease. Pregnancy-induced hypertension occurred in six cases. Spontaneous weight-bearing fractures occurred in two patients. No episodes of renal allograft rejection occurred. Evidence of fetal compromise was present in six cases. All fetuses were delivered by cesarean section prior to term, with live births occurring from 31 1/2 to 36 weeks' gestation. A single case of hypospadias was the only congenital defect. Prepregnancy screening for complications of diabetes and renal transplantation is advised and euglycemia should be achieved before and during pregnancy. Advanced diabetic vascular disease puts these gestations at significant risk.

Essential fatty acid patterns in preeclampsia.

Fatty acid patterns were determined for maternal and cord sera from normal and preeclamptic pregnancies using gas liquid chromatography. Ten normal and 11 preeclamptic maternal sera were used; 6 preeclamptic and 6 normal cord sera were measured. Fatty acid patterns were measured in the nonesterified fatty acids (NEFA), the triglycerides (TG), the cholesterol esters (CE), and the phospholipids (PL). Preeclampsia showed significant differences in the essential fatty acid patterns in the maternal and cord sera compared to normal pregnancy. Oleic acid, an early indicator of essential fatty acid deficiency, made up a significantly greater proportion of preeclamptic cord blood NEFA, TG, and PL than in normal cord blood. Abnormalities in essential fatty acid patterns may be associated with decreased prostacyclin production and dysmaturity which may be seen with preeclampsia.

The use of magnesium sulfate for tocolysis in preterm labor complicated by twin gestation and betamimetic-induced pulmonary edema.

Maternal cardiorespiratory compromise has been reported to occur during the treatment of preterm labor with tocolytic agents. The risk for these complications is increased by the following factors: multiple gestation, the combination of magnesium sulfate and beta-adrenergic agonist, and the use of adrenocortico-steroids to hasten fetal pulmonary maturity. A case is presented of labor at 28 weeks' gestation complicated by pulmonary edema associated with the three risk factors listed above. The beta-agonist was discontinued with resolution of pulmonary edema. Intravenous magnesium sulfate was continued for 11 days. When uterine contractions finally overcame the tocolysis, delivery occurred. It appeared that uterine quiescence was achieved through the continued use of magnesium sulfate despite pulmonary edema in this case. The risk of continued tocolysis seemed to be counterbalanced by the benefits of prolonged intra-uterine existence for the fetuses.

Investigation of obstetric malpractice closed claims: profile of event.

The files of 220 obstetric closed-claim cases were reviewed by five obstetricians to determine whether information could be collected an analyzed to identify common predisposing factors to claims and to suggest preventative measures. The data suggests these cases contain common easily identified obstetric risk factors, most of which occurred in labor and delivery (66%). Fifty-four percent of the risks were recognized, 32% correctly managed, and a high percentage of risks were considered by the reviewers to be directly related to the obstetric outcome leading to the claim (66%). The authors feel obstetric closed claims can be studied and suggestions made to aid obstetricians in providing care. Identification of common obstetric risks and correct management of these risks is poor in these cases. Recognition and management guidelines are imperative in ensuring good obstetric outcome. These two physician-controlled factors played important parts in the majority of cases reviewed. It would appear from this study that obstetric malpractice closed claims are amenable to study; physicians and their patients would benefit from better data collection systems to identify risks in individual pregnancies; physicians need readily available resources to aid their management of patients; only through modification of physician behavior can suits be avoided.