An investigation into the suitability of a commercial real-time PCR assay to screen for Taylorella equigenitalis in routine prebreeding equine genital swabs.

REASONS FOR PERFORMING STUDY: Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real-time PCR assay was evaluated for its suitability in screening swabs. OBJECTIVE: To compare the results of a commercially available real-time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under 'field trial' conditions. MATERIALS AND METHODS: Routine prebreeding genital swabs (n=2072) collected from Thoroughbred mares and stallions during 2009 were examined together with stored T. equigenitalis positive material. Swabs were cultured for T. equigenitalis using standard microbiological techniques. Bacterial lysates were isolated from the swabs and examined for the presence of a 16S DNA fragment of T. equigenitalis, using a commercial multiplex real-time PCR assay system. RESULTS: There was complete concordance between positive and negative results obtained by the 2 methods. Real-time PCR also detected T. equigenitalis DNA from swabs that were negative using standard microbiological culture after 6 months' storage at +4 degrees C but from which T. equigenitalis had been isolated following collection. The sensitivities of real-time PCR and bacterial culture were both 10(-3) (equivalent to 3 colony-forming units). CONCLUSION AND CLINICAL RELEVANCE: Routine bacterial culture of T. equigenitalis requires an incubation period of not less than 7 days before a conclusive negative result can be obtained, whereas bacterial extraction and real-time PCR assay can be completed in less than 6 h. The commercially-available PCR assay tested provided a rapid and reliable method for the identification of T. equigenitalis from equine genital swabs and could be usefully employed for the screening of mares and stallions for preseason Horserace Betting Levy Board (HBLB) Code of Practice and in other situations such as for bloodstock sales screening requirements, overcoming the current delays imposed by bacterial culture requirements. Its use could be quality assured by the existing HBLB biannual testing scheme for designated laboratories.

Effect of urokinase antiserum on plasminogen activators: demonstration of immunologic dissimilarity between plasma plasminogen activator and urokinase.

Antiserum against purified human urokinase was produced by immunization of Hartley strain guinea pigs. The antiserum was capable of neutralizing the plasminogen activator activity of the antigen and of native urokinase in human urine. The antiserum did not inhibit plasminogen activators of bacterial origin, i.e., streptokinase and staphylokinase; neither did it inhibit urokinase from nonprimate mammals, i.e., dog, pig, rabbit, guinea pig, nor tissue activator or tissue culture supernatants from porcine sources. Partial cross-reactivity against urokinase from primates, i.e., rhesus monkey and baboon, was noted as well as with supernatant from rhesus kidney tissue culture. In vitro studies showed lack of immunologic identity between human urokinase and human milk activator or human tissue activator from adrenal sources but demonstrated immunologic identity between human urokinase and the supernatant from human kidney tissue culture. In vivo studies in man failed to show detectable levels of urokinase activity in peripheral venous or renal venous blood under a variety of clinical states and when stimuli such as exercise, electroshock therapy, or nicotinic acid were used to enhance plasminogen activator activity in the plasma. The results establish that human plasma activator, milk activator, and tissue activator from the adrenals are immunologically distinct from human urokinase.

Reduction of coagulation factor XIII concentration in patients with myocardial infarction, cerebral infarction, and other thromboembolic disorders.

Coagulation factor XIII and plasma fibrinogen chromatographic assays have been performed serially in patients suffering from acute myocardial and cerebral infarction, and in others with disseminated intravascular coagulation. The findings were compared with 2 groups of "controls"; normal clinically-well subjects and hospitalized patients with cerebral infarction who exhibited minimal, stable, or improving neurological deficits. Substantial depression of factor XIII concentrations developed in the 3 patient groups, together with concomitant significant increases in the proportion and concentration of plasma high molecular weight fibrin(ogen) complexes (HMWFC). An inverse correlation (p less than 0.05) between coagulation factor XIII concentration and percentage of HMWFC was demonstrated in the early stages of the illness. These findings suggest that depression of coagulation factor XIII concentration in these states, is secondary to extravascular or intravascular coagulation and may reflect its degree.

Profile of a large-scale cohort study.

An event-monitoring, observational cohort study was conducted in 10,800 patients treated with the nonsteroidal anti-inflammatory agent (NSAID) nabumetone and in 2005 patients treated with a selection of other NSAIDs. A total of 2986 doctors in general practice in the UK participated in the study, which involved the provision of a registration report at the beginning of the study and follow-up reports at 1, 3, 6, and 12 months. The methodology which was developed was successful in collecting demographic, diagnostic, drug use, clinical event and adverse reaction data in everyday medical practice.

Spontaneous adverse drug reaction reporting vs event monitoring: a comparison.

Spontaneous adverse drug reaction (ADR) reporting is the mainstay of national and international drug safety evaluation in the post-approval phase. A major criticism of the method has been a high, but essentially unquantifiable, level of under-reporting by doctors. A direct comparison has been made between spontaneous ADR reporting and an observational event monitoring system for a group of more than 44,000 patients receiving one or other of a group of seven new drugs. The data suggests that under-reporting by the spontaneous system may be as high as 98% for several clinical events believed to be associated with drug treatment.

Discontinuation of and changes in treatment after start of new courses of antihypertensive drugs: a study of a United Kingdom population.

OBJECTIVE: To evaluate the incidence of discontinuation of and changes in treatment after newly prescribed courses of antihypertensive drugs of the four primary therapeutic classes: beta blocker, calcium channel blocker, and angiotensin converting enzyme inhibitor. DESIGN: A retrospective analysis of patients on an automated database of 1.2 million patients was conducted on visits between 1 October 1992 and 30 September 1993. SETTING: General practices in the United Kingdom. SUBJECTS: 37,643 patients with hypertension receiving a relevant drug in the time period were identified. A new course of treatment in at least one of the four therapeutic classes, defined as a drug not prescribed in the previous four months, was observed in 10,222 patients aged > or = 40 years. MAIN OUTCOME MEASURES: Patients changing to other treatment or discontinuing after initiating a new course of treatment, defined as the absence of a refill prescription for the new drug or another in its category within a six month observation period. RESULTS: Changes in or discontinuation of treatment were frequently observed, and by month six continuation rates ranged between 40% to 50% for all four classes of drugs. CONCLUSION: Low rates of continuation with a newly prescribed antihypertensive drug exist regardless of which drug is prescribed.

Building a regional medical center. Interview by Donald E.L. Johnson.

Allen P. Fletcher is president of a 372-bed community hospital that is between Atlanta and Birmingham and loses patients to hospitals in those cities more frequently than he likes. Northeast Alabama Regional Medical Center, Anniston, Alabama, runs an average daily census of about 165 patients and many of its 100 active physicians are so busy that they have waiting lists. Fletcher has been president of Northeast for six years. He holds an MHA from Georgia State University. In an interview with Donald E.L. Johnson, editor and publisher of Health Care Strategic Management, Fletcher talks about the small window of opportunity he sees for secondary hospitals.

An appraisal of spontaneous adverse event monitoring.

Awareness of the need to continue the safety assessment of new drugs into the post-marketing period has continued to increase over the past few years. The stage has been reached where there is, for all practical purposes, unanimous agreement that all new drugs, particularly those intended for widespread use and/or long-term administration, be subjected to some form of post-marketing monitoring. In many cases nationally sponsored spontaneous reporting systems are claimed to be sufficient to meet the needs without any critical appraisal being made of the suitability or reliability of this method. The adoption of 'pharmacovigilance' (spontaneous reporting) as the system for the whole European Community (EC) emphasizes the faith that has been placed in the system. At a recent symposium (Management Forum, 9-10 March, 1992) on post-marketing safety evaluation, more than half of the 16 papers presented were entirely or predominantly concerned with spontaneous reporting systems, which is a pattern common to other meetings on this topic. The Committee on Safety of Medicines (CSM) has referred to spontaneous reporting as the cornerstone of post-marketing surveillance which has further endorsed a general feeling of confidence that the system will meet our societal needs even though its scientific capability may be deficient. It is the purpose of this paper to challenge these assumptions and to make an objective appraisal of the various methods available.

Formation of soluble fibrin oligomers in purified systems and in plasma.

The kinetic parameters for release of fibrinopeptide A (FPA) from human fibrinogen by thrombin are: Km = 2.3 X 10(-6)M and Vmax. = 1.1 X 10(-10)mol of FPA/s per unit of thrombin; for fibrin formation, Km is similar to that for FPA release, but, the conditions of the present study, Vmax. was approximately half of that for FPA release. The formation of fibrin polymer before the sol-gel transition was studied by gel-permeation chromatography combined with effluent analysis for fibrinogen antigen and residual FPA. Polymer formation in purified fibrinogen incubated with thrombin proceeded as a bimolecular association of exposed sites in a manner predicted by probability calculations and assuming random FPA cleavage. Each oligomer consisted of n molecules of fibrin monomer and two fibrinogen molecules, each of the latter lacking one FPA molecule, i.e. each oligomer, regardless of molecular size, retains two FPA molecules. The addition of 5 mM-CaCl2 to the reaction mixture changed the rate of polymer formation, so that dimer was no longer the prevalent oligomer; in the presence of Ca2+, the trimer was the oligomer in highest concentration. The polymers formed in the presence of calcium were similar in composition to those without, i.e. 2 mol of FPA/mol of oligomer. EDTA-treated plasma samples incubated for short periods of time, 30s or less, with thrombin ranging in concentration up to 1 N.I.H. unit/ml did not form clots during the 10-15 min period of observation until they were applied to the column, though a large proportion of the available FPA was cleaved (maximum 45%). The soluble polymers in plasma were mostly of the high-Mr variety (tetramer and greater); these high-Mr polymers contained less than 2 mol of FPA/mol of polymer, whereas dimer and trimer in plasma were similar to those in the purified systems, i.e. 2 mol of FPA/mol.