Development of EUCAST zone diameter breakpoints and quality control range for Staphylococcus aureus with ceftaroline 5-µg disk.

This ceftaroline MIC/disk comparison study for Staphylococcus aureus was performed for the purpose of establishing EUCAST zone diameter breakpoints. Ceftaroline susceptibility for a challenge set of 70 methicillin resistant- and 30 methicillin susceptible-S. aureus was determined by 5-µg disk diffusion and broth microdilution methods. Seventeen isolates were retested by disk and MIC, and the remaining 83 isolates were retested by MIC. Molecular testing was performed on 19 isolates with borderline susceptible ceftaroline MIC results to assess any differences in mecA and epidemiological correlation. An additional set of 101 consecutive clinical S. aureus isolates were tested using the 5-µg disk. S. aureus ATCC 29213 was tested by multiple sites and media for QC range determination. Replicate MIC results were within ±1 doubling dilution, with tendency for slightly lower repeat MICs, and there was minimal variation in replicate zone results. Based on susceptible breakpoints for MIC of ≤1 mcg/mL and for disk of >20 mm, there was 100 % categorical agreement for 30 MSSA and 92 % categorical agreement for 70 MRSA. There were no common MLST or PBP changes for strains with MICs of 1 and 2 mcg/mL. All ceftaroline disk results for the consecutively collected isolates were >20 mm. EUCAST selected the ceftaroline 5-µg disk breakpoint of Susceptible ≥20, Resistant <20 mm because it correlated best with the MIC breakpoint of Susceptible ≤1, Resistant >1 mg/L. A ceftaroline 5-µg disk QC range for S. aureus ATCC 29213 of 24-30 mm was also established by EUCAST.

Design and synthesis of thrombin inhibitors: analogues of MD-805 with reduced stereogenicity and improved potency.

Mitsubishi's MD-805, a potent and selective inhibitor of thrombin which contains four stereogenic centers, has been the starting point for an optimization program. A systematic synthetic study resulted in thrombin inhibitors achiral at P2 and P3 but with a 10-fold increase in potency over the original lead. A number of 4-substituted piperidines were synthesized and examined as replacements for 2-carboxy-4-methylpiperidine at P2; 4-fluoroethylpiperidine (FEP) among others provided inhibitors (e.g. 45g) of increased potency. An enantioselective route was developed to 3(R)-methyl-1,2,3,4-tetrahydroquinolinesulfonyl chloride. Inhibitors containing this enantiomerically pure P3 (42d) had similar potency to the racemic material and provided support, with modeling studies, for the preparation of the gem 3,3-disubstituted compounds. A series of inhibitors containing the novel 3, 3-dimethyl-1,2,3,4-tetrahydroquinolinesulfonyl (DMTHQS) P3 (Table 5) were synthesized and showed a similar activity profile as the monomethyl series. The combination of P3-DMTHQS, P2-FEP, and P1-arginine (45g) had a K(i) of 6 nM (MD-805 K(i) = 85 nM). In animal models of both venous and arterial thrombosis, one inhibitor (42e) was shown to produce a dose-dependent inhibition of thrombus formation that in some situations was superior to that of MD-805.

Recombinant desulphatohirudin (CGP 39393) anticoagulant and antithrombotic properties in vivo.

The effects of the newly available biotechnology product, recombinant desulphatohirudin (CGP 39393) have been investigated in rats. This highly potent and selective thrombin inhibitor exhibited marked anticoagulant properties with controllable titration of anticoagulant effect, as measured by activated partial thromboplastin time (APTT), up to nearly four times control values. Furthermore, CGP 39393 exhibited impressive antithrombotic activity in vivo. In an arteriovenous shunt model of thrombus formation on a cotton-thread, the compound was capable of complete inhibition of thrombus development (ED50 = 0.3 mg/kg i.v. and 1.0 mg/kg s.c.). Venous stasis thrombosis was also highly susceptible to inhibition by CGP 39393 (ED50 = 0.01 mg/kg i.v. and 0.45 mg/kg s.c.). Comparison of the anticoagulant and antithrombotic activities of the compound shows that potent antithrombotic effects (83-97% inhibition in the rat shunt model) are achieved within the generally acceptable range of anticoagulation. These results suggest a clear potential for this new agent in the clinical treatment of thrombotic disease.

CGS 12970: a novel, long acting thromboxane synthetase inhibitor.

CGS 12970 is a potent selective inhibitor of human platelet thromboxane synthetase in vitro (IC50 = 12 nM). It is four orders of magnitude less potent as an inhibitor of sheep seminal vesicle cyclooxygenase, bovine aorta prostacyclin synthetase and human leucocyte 15-lipoxygenase. The compound inhibited collagen-induced thromboxane B2 production by human platelets in vitro without an effect on the accompanying platelet aggregation induced by collagen, ADP, platelet activating factor, thrombin, arachidonic acid or the prostaglandin mimetic, U 46619. Administration of CGS 12970 to rats inhibited collagen-induced thromboxane B2 production but had no effect on platelet aggregation ex vivo. It also had no effect on platelet aggregation induced by thrombin or on plasma clotting times. A single oral dose of 1 or 3 mg kg-1 to rabbits inhibited thromboxane B2 production in clotting blood ex vivo for 12 or 24 h respectively. CGS 12970 inhibited thromboxane B2 production in vivo induced by intravenous administration of collagen to rats or calcium ionophore to guinea-pigs. In both cases there was a concomitant elevation of immunoreactive 6-keto-prostaglandin F1 alpha but no effect on the induced thrombocytopenia. As with other thromboxane synthetase inhibitors, CGS 12970 prolonged tail bleeding time in the rat. However, CGS 12970 was not as potent as other thromboxane synthetase inhibitors in this test. In addition to these usual effects of thromboxane synthetase inhibitors, CGS 12970 inhibited the thrombocytopenia induced by the Forssman reaction or ADP administration. In these tests the effect of the compound was of short duration. 8 CGS 12970 had no effect on the thrombocytopenia associated with the Arthus reaction which distinguishes it from cyclo-oxygenase inhibitors. It also had no effect on thrombus formation on a cotton thread in an arteriovenous shunt in the rat.

Prolongation of platelet survival in hypercholesterolaemic rabbits by CGS 12970 (3-methyl-2-(3-pyridyl)-1 indoleoctanoic acid) and dazoxiben.

In rabbits receiving a normal laboratory diet the platelet half-life was 40.4 +/- 2.5h (mean +/- S.D., n = 35). In animals fed the cholesterol-enriched diet for 12 weeks the platelet half-life was reduced to 31.6 +/- 3.6h (mean +/- S.D., n = 35). Treatment of cholesterol-fed animals with a single daily dose of CGS 12970 (a long acting inhibitor of thromboxane synthase) normalised the platelet half-life. Single daily doses of the relatively shorter acting thromboxane synthase inhibitors (CGS 13080 and dazoxiben) failed to correct the reduced platelet survival. However, twice daily dosing with dazoxiben was effective. The cyclooxygenase inhibitors, aspirin and sulphinpyrazone, failed to correct the reduced platelet survival.

Multicentre study of the in vitro evaluation of moxifloxacin and other quinolones against community acquired respiratory pathogens.

The in vitro activity of moxifloxacin was compared with that of ciprofloxacin, levofloxacin, ofloxacin and trovafloxacin against 710 strains (180 Streptococcus pneumoniae, 180 Haemophilus influenzae, 160 Moraxella catarrhalis and 190 Streptococcus pyogenes) isolated from patients with community-acquired respiratory tract infections. MIC values for moxifloxacin, trovafloxacin were 0.25/0.25, 0.03/0.03, 0.06/0.03 and 0.125/0.0125 mg/l for S. pneumoniae, H. influenzae, M. catharralis and S. pyogenes. Based upon the MIC(90) values and the MIC distributions, moxifloxacin and trovafloxacin were the most active of the quinolones tested. They showed enhanced activity against Gram-positive organisms including penicillin non susceptible S. pneumoniae strains. Moxifloxacin was also highly active against ciprofloxacin-resistant S. pneumoniae strains.

The management of retinal detachment complicating degenerative retinoschisis.

We repaired six retinal detachments complicating degenerative retinoschisis by using simultaneous external subretinal fluid drainage and intraocular gas injection without a scleral buckle or vitrectomy. The outer wall breaks were 30 to 135 degrees in size, and in three cases, extended close to the arcade vessels. We achieved retinal reattachment and collapse of the schisis cavity at surgery in all six cases. In one case, the retina redetached postoperatively, but it was repaired with a scleral buckle and gas injection. This technique simplified the management of retinal detachments complicating degenerative retinoschisis, particularly those with large or posterior outer-layer breaks.

Symptomatic retinoschisis-detachment involving the macula.

We treated three patients (four eyes) in whom posteriorly situated retinoschisis-detachments became symptomatic because of elevation of a limited area of full-thickness retina at the macula adjacent to these lesions. Laser photocoagulation alone was successful in achieving long-term macular reattachment in one eye but failed in both eyes of a bilaterally affected patient. In this patient, retinal cryopexy, external drainage of subretinal and retinoschisis cavity fluid, and intravitreal air injection attained long-term macular reattachment and retinoschisis cavity collapse in both eyes. In the third patient, cryopexy, drainage of subretinal and retinoschisis cavity fluid, and scleral buckling failed to reattach the retina. Subsequent laser photocoagulation induced reabsorption of subretinal fluid but without retinoschisis cavity collapse. Alternative management strategies for these unusual cases include retinal cryopexy alone and vitrectomy techniques.

Isoelectric focussing of proteins on thick (500 micron) modified cellulose acetate gel membranes.

A method is described for isoelectric focussing proteins on thick (500 micron) cellulose acetate gel membrane modified by boron trigluoride in methanol. Longer pH gradients could be formed and larger sample volumes could be applied than on the normal thickness product.

Further observations on isoelectric focussing of serum proteins using modified cellulose acetate gel membranes, and direct isoenzyme staining.

1. Treatment of cellulose acetate gel strips by boron trifluoride in methanol has been shown previously to produce a material suitable for isoelectric focusing. Further observations are reported here on the changes in the strips induced by the reagent. It has shown by conventional protein electrophoresis, for example, that not only is the degree of electroendosmosis reduced, but also that other properties are altered. 2. Minor modifications are introduced for the general isoelectric focussing method including improved electrode solutions. 3. Isoenzyme detection following isoelectric focussing of serum proteins is discussed in the light of experience gained using alkaline phosphatase and lactate dehydrogenase as examples.

Isoelectric focussing of proteins on cellulose acetate gel membranes.

A method is described for analytical isoelectric focussing of proteins on cellulose acetate gel strips pretreated with boron trifluoride in methanol. It is satisfactory for separations requiring narrow pH gradients as well as for those requiring wide gradients. As well as conventional protein staining methods, the use of immunological reagents is described to identify directly specific proteins in a complex separation. The method is extremely quick and easy to perform and requires only low voltage electrophoresis equipment.

The Nambour study of ocular disease. I. Design, study population and methodology.

In association with a study of actinic skin disease, we undertook a comprehensive survey of ocular disease in a population sample of the town of Nambour, Queensland, Australia. Particular emphasis was placed on those diseases with a putative relationship to exposure to solar radiation. In addition to collecting prevalence data, a randomized controlled trial was commenced to determine, among other things, if daily ingestion of 30 mg of beta-carotene supplements reduces the incidence or progression of ocular diseases possibly related to solar radiation exposure. The study design, population and methodology of the study are described in detail as a background to the future reporting of the results. The study should provide unique epidemiological information about eye disease in an Australian community setting due to the representative nature of the subjects and the comprehensive examination performed.

A non-occlusive model of arterial thrombus formation in the rat and its modification by inhibitors of platelet function, or thrombin activity.

A technically simple model of arterial thrombosis in the rat, induced by a crush injury to the dorsal aorta is described. The mechanical injury to the artery caused deep medial injury and the formation of a platelet-rich thrombus with associated fibrin formation which was assessed both radiometrically and morphometrically. No significant inclusion of erythrocytes was noted in the thrombus. Administration of the platelet inhibitors aspirin, BM 13505 (a thromboxane receptor antagonist) or CGS 12970 (a thromboxane synthase inhibitor) reduced the extent of platelet deposition on the injured vessel, but no decrease in fibrin(ogen) was observed. In contrast, infusion of prostacyclin resulted in reductions in both these components of the thrombus. In studies involving inhibition of thrombin activity, the direct thrombin inhibitor CGP 39393 (recombinant desulphatohirudin) inhibited both the platelet and fibrin(ogen) deposition. The indirect thrombin inhibitors were less effective; unfractionated heparin and low-molecular-weight heparin inhibited both platelet and fibrin(ogen) deposition but only at doses which rendered the blood uncoagulable, as evaluated by the activated partial thromboplastin time. Dermatan sulphate only inhibited platelet deposition. The results suggest that thrombin plays a key role in the initiation of thrombus formation in this experimental model. The agonist prostaglandins (PGG2, PGH2, and TXA2) would appear to have a supporting role in the platelet deposition onto the thrombotic surface but do not have a role to play with respect to fibrin(ogen) deposition.

Safe, stable, whole blood samples for quality assessment of glucose measurement by non-laboratory staff.

A whole blood control material has been used to assess the analytical performance of non-laboratory staff who use glucose meters in clinical areas. It is prepared from sterile horse blood which is readily available from a commercial source. There are no known infection or disease transmission risks to users. When the material is stabilized by the addition of sodium fluoride less than 3% loss of glucose over 48 h is observed from an initial value of 10 mmol/L. However, we prefer to stipulate that the glucose is measured on the day of receipt. The material has been used successfully with Reflolux IIM meters and B-M sticks (Boehringer Mannheim, UK) for over a year in our hospital.

Post-UV survival of Escherichia coli strains carrying more than one UV-sensitising plasmid.

The post-UV phenotypes conferred by wild-type plasmids R391 and pYD1, which increase UV-induced mutagenesis but sensitise Escherichia coli AB1157 umuC+ uvrB+ to UV, were compared, alone and in combination with that of plasmid pGW16, which sensitises AB1157 to low, but protects against high UV doses. All three plasmids increased UV resistance when present in Shigella sonnei. No plasmid significantly affected the UV sensitivity of E. coli TK501 umuC uvrB, in which pKM101, the parent of pGW16 increases UV resistance up to 1000-fold. Both pYD1 and R391 reduced the UV protective effect of pKM101, and increased UV-sensitisation conferred by pGW16. UV-sensitisation conferred by pYD1 and R391 was additive when the plasmids were together in strain AB1157, and both pKM101 and pGW16 reduced this additive sensitisation.

Vascular leakage stimulates phenotype alteration in ocular cells, contributing to the pathology of proliferative vitreoretinopathy.

Plasma leaking from damaged retinal blood vessels can have a significant impact on the pathologies of the posterior segment of the eye. Inflammation in the eye and metabolic change resulting from diabetes mellitus causes vascular leakage with alteration of the phenotype of retinal pigment epithelial (RPE) cells and fibrocytes, resulting in changes in cell function. Phenotypically altered cells then significantly contribute to the pathogenesis of retinopathies by being incorporated into tractional membranes in the vitreous, where they secrete matrix molecules, such as fibronectin, and express altered cell surface antigens. We hypothesize that there is a direct relationship between the leaking of plasma and the proliferation and phenotypic change of RPE cells and fibroblasts, thus exacerbating the pathology of retinal disease. If the hypothesis is correct, control of vascular leakage becomes an important target of therapy in proliferative vitreoretinopathy.

GPs and eye skills. A brave new world?

OBJECTIVE: To evaluate the efficacy of a brief general practitioner eye upskilling intervention to enhance GP eye skills and increase their use in clinical practice. METHOD: Seventeen GPs from a Brisbane Division of General Practice were recruited to participate in a 7 hour clinical upskilling intervention, delivered over an 8 week period. RESULTS: The proportion of GPs able to accurately recognise glaucomatous fundal damage on clinical assessments rose from 24.0 to 54.2% (standard error [SE] 3.96). The proportion able to accurately recognise diabetic fundal disease on clinical assessment rose from 30.8 to 54.1% (SE 9.2). The ability to recognise fundal disease on slide presentations rose on average from 27.1 to 67.5% (SE 10.8). Although none of the practitioners were able to perform oculokinetic perimetry at the beginning of the intervention, all were able to accurately perform it at the conclusion of the intervention. CONCLUSION: GPs are capable of rapidly upskilling in important areas of ocular assessment involving preventable blindness.

When is the right time? complex issues around withdrawing life-sustaining treatment in children.

When should one withdraw treatment in children? The challenge is to recognise when a decision needs to be made. Parents may be in denial, and deciding which questions to ask may be difficult. Ethically, the guiding principle should be the child's best interests. May the parents or primary caregiver decide what the child's best interests are? Legislation in South Africa prevents a parent or caregiver from refusing treatment that professionals deem to be in the child's best interests. This article discusses the ethical and legal aspects around the decision to palliate in children.