The prevention and diagnosis of central venous catheter-related infection

The potential source of CVC colonisation was assessed. Isolates of coagulase-negative staphylococci (CoNS) recovered from the skin and CVC components of 3 cardiothoracic surgery patients were characterised by pulsed-field gel electrophoresis (PFGE). The genetic heterogeneity of CoNS isolated from the skin was demonstrated and specific genotypes implicated in catheter colonisation. In addition, phenotypic and genotypic typing techniques were assessed for their ability to characterise strains of CoNS recovered from 33 patients who developed catheter-related bloodstream infection (CR-BSI) on a bone marrow transplant (BMT) unit and Siaphylococcus aureus recovered from 6 cardiothoracic surgery patients with surgical site infection (SSI) following median sternotomy. This epidemiological investigation revealed that common strains of CoNS and 51 aureus where not associated with infection in patients with CR-BSI or sternal SSI during the study period. Furthermore, there was no correlation between phenotypic and genotypic characterisation results. The variable expression of phenotypic traits within strains of staphylococci was evident whilst PFGE and randomly amplified polymorphic DNA (RAPD) were highly discriminatory for the molecular characterisation of S. aureus and CoNS. This was highlighted in 8 stem cell transplant (SCT) patients whereby it was demonstrated that routine identification and characterisation of CoNS by phenotypic techniques may not be adequate for the diagnosis of CR-BSI by current guidelines. The potential of the lipid S ELISA to facilitate the diagnosis of CR-BSI in 38 haematology/SCT patients and sternal SSI in 57 cardiothoracic surgery patients was also assessed. The ELISA proved to be a sensitive test for the rapid serodiagnosis of infection due to staphylococci in immunocompetent patients. The acridine orange leucocyte cytospin test (AOLC) was also evaluated for the rapid diagnosis of CR-BSI in 16 haematology/SCT patients with Hickman CVC in situ. Although the sensitivity of the test was low, it may provide a useful adjunct to conventional methods for the in situ sampling of catheters to predict and diagnose CR-BSI, preventing the unnecessary removal of CVC.

A rapid ELISA for the diagnosis of intravascular catheter related sepsis caused by coagulase negative staphylococci

Aim: To develop and evaluate a rapid enzyme linked immunosorbent assay (ELISA) for the diagnosis of intravascular catheter related sepsis caused by coagulase negative staphylococci. Methods: Forty patients with a clinical and microbiological diagnosis of intravascular catheter related sepsis and positive blood cultures, caused by coagulase negative staphylococci, and 40 control patients requiring a central venous catheter as part of their clinical management were recruited into the study. Serum IgG responses to a previously undetected exocellular antigen produced by coagulase negative staphylococci, termed lipid S, were determined in the patient groups by a rapid ELISA. Results: There was a significant difference (p = < 0.0001) in serum IgG to lipid S between patients with catheter related sepsis and controls. The mean antibody titre in patients with sepsis caused by coagulase negative staphylococci was 10 429 (range, no detectable serum IgG antibody to 99 939), whereas serum IgG was not detected in the control group of patients. Conclusions: The rapid ELISA offers a simple, economical, and rapid diagnostic test for suspected intravascular catheter related sepsis caused by coagulase negative staphylococci, which can be difficult to diagnose clinically. This may facilitate treatment with appropriate antimicrobials and may help prevent the unnecessary removal of intravascular catheters.

Evaluation of routine microbiological techniques for establishing the diagnosis of catheter-related bloodstream infection caused by coagulase-negative staphylococci

Microbiological diagnosis of catheter-related bloodstream infection (CR-BSI) is often based on isolation of indistinguishable micro-organisms from an explanted catheter tip and blood culture, confirmed by antibiograms. Whether phenotypic identification of coagulase-negative staphylococci (CoNS) allows an accurate diagnosis of CR-BSI to be established was evaluated. Eight patients with a diagnosis of CR-BSI had CoNS isolated from pure blood cultures and explanted catheter tips which were considered as indistinguishable strains by routine microbiological methods. For each patient, an additional three colonies of CoNS isolated from the blood and five from the catheter tip were subcultured and further characterized by antibiogram profiles, analytical profile index (API) biotyping and PFGE. PFGE distinguished more strains of CoNS compared to API biotyping or antibiograms (17, 10 and 11, respectively). By PFGE, indistinguishable micro-organisms were only isolated from pure blood and catheter tip cultures in four out of eight (50%) patients thus supporting the diagnosis of CR-BSI. In another patient, indistinguishable micro-organisms were identified in both cultures; however, other strains of CoNS were also present. The remaining three patients had multiple strains of CoNS, none of which were indistinguishable in the tip and blood cultures, thus questioning the diagnosis of CR-BSI. Phenotypic characterization of CoNS lacked discriminatory power. Current routine methods of characterizing a limited number of pooled colonies may generate misleading results as multiple strains may be present in the cultures. Multiple colonies should be studied using a rapid genotypic characterization method to confirm or refute the diagnosis of CR-BSI. © 2007 SGM.

The serological diagnosis of staphylococcal infective endocarditis

Objectives: Establishing the diagnosis of infective endocarditis (IE) can be difficult when blood cultures remain sterile or echocardiography is inconclusive. Staphylococcus aureus is a common aetiological microorganism in IE and is associated with severe valvular destruction and increased mortality. Early diagnosis using culture and antibiotic independent tests would be preferable to allow prompt antibiotic administration. We have developed and evaluated 2 serological assays for the rapid identification of a staphylococcal aetiology in infective endocarditis. The assays measure IgG against whole cells of S. aureus and IgG against lipid S, a novel extracellular antigen released by Gram-positive microorganisms. Methods: Serum was collected from 130 patients with IE and 94 control patients. IgG against whole cells of S. aureus and against lipid S was measured by enzyme linked immunosorbent assay (ELISA). Results: Anti-lipid S IgG titres were higher in IE caused by Gram-positive microorganisms than in controls (p < 0.0001) and higher in staphylococcal IE than in both controls and IE caused by other microorganisms (p = 0.0003). Anti-whole cell staphylococcal IgG was significantly higher in serum from patients with staphylococcal IE than in IE caused by other microorganisms and control samples (p < 0.0001). Conclusion: High anti-whole cell IgG titres are predictive of a staphylococcal aetiology in IE. Elevated serum anti-lipid S IgG titres are predictive of Gram-positive infection compared to controls, very high titres being associated with staphylococcal IE. © 2005 The British Infection Society.

A study of potential serum markers for a diagnosis of gram-positive and gram-negative bacterial sepsis

Sepsis continues to be a major cause of morbidity and mortality as it can readily lead tosevere sepsis, septic shock, multiple organ failure and death. The onset can be rapid and difficult to define clinically. Despite the numerous candidate markers proposed in the literature, to date a serum marker for sepsis has not been found. The aim of this study was to assay the serum of clinically diagnosed patients with eithera Gram-negative or Gram- positive bacterial sepsis for elevated levels of nine potentialmarkers of sepsis, using commercially produced enzyme linked immunosorbent assays(ELISA). The purpose was to find a test marker for sepsis that would be helpful toclinicians in cases of uncertain sepsis and consequently expose false positive BC'scaused by skin or environmental contaminants. Nine test markers were assayed including IL-6, IL-I 0, ILI2, TNF-α, lipopolysaccharide binding protein, procalcitonin, sE-selectin, sICAM -1 and a potential differential marker for Gram-positive sepsis- anti-lipid S antibody. A total of 445 patients were enrolled into this study from the Queen Elizabeth Hospital and Selly Oak Hospital (Birmingham). The results showed that all the markers were elevated in patients with sepsis and that patients with a Gram-negative sepsis consistently produced higher median/range serum levels than those with a Gram-positive sepsis. No single marker was able to identify all the septic patients. Combining two markers caused the sensitivities and specificities for a diagnosis of sepsis to increase to within a 90% to 100% range. By a process of elimination the markers that survived into the last phase were IL-6 with sICAM -1, and anti-lipid S IgG assays Defining cut-off levels for a diagnosis of sepsis became problematic and a semi-blind trial was devised to test the markers in the absence of both clinical details and positive blood cultures. Patients with pyrexia of unknown origin and negative BC were included in this phase (4). The results showed that IL-6 with sICAM-l are authentic markers of sepsis. There was 82% agreement between the test marker diagnosis and the clinical diagnosis for sepsis in patients with a Gram-positive BC and 78% agreement in cases of Gram-negative Be. In the PUO group the test markers identified 12 cases of sepsis and the clinical diagnosis 15. The markers were shown to differentiate between early sepsis and sepsis, inflammatory responses and infection. Anti-lipid S with IL-6 proved be a sensitive marker for Gram-positive infections/sepsis.

Improving diagnosis and oral vaccination strategies against bovine tuberculosis

In this work peptide antigens [ESAT-6,p45 in water (1ml, 1mg/ml)] have been adsorbed onto 10mg inorganic substrates (hydroxyapatite (MHA P201;P120, CHA), polystyrene, calcium carbonate and glass microspheres) and in vitro release characteristics were determined. The aim of formulation was to enhance the interaction of peptides with antigen presenting cells and to achieve rapid peptide release from the carrier compartment system in a mildly acidic environment. Hydroxyapatite microparticle P201 has a greater surface area and thus has the largest peptide adsorption compared to the P120. CHA gave a further higher adsorption due to larger surface area than that available on microparticles. These particles were incorporated into the BOVIGAMTM assay to determine if they improve the sensitivity. After overnight incubation the blood plasma was removed and the amount of IFN-g in each plasma sample was estimated. CHA and MHA P201 gave a significantly higher immune response at low peptide concentration compared to the free peptide, thus indicating that these systems can be used to evaluate Tuberculosis (TB) amongst cattle using the BOVIGAMTM assay. Badgers are a source of TB and pass infection to cattle. At the moment vaccination against TB in badgers is via the parenteral route and requires a trained veterinary surgeon as well as catching the badgers. This process is expensive and time consuming; consequently an oral delivery system for delivery of BCG vaccines is easier and cheaper. The initial stage involved addition of various surfactants and suspending agents to disperse BCG and the second stage involved testing for BCG viability. Various copolymers of Eudragit were used as enteric coating systems to protect BCG against the acidic environment of the stomach (SGF, 0.1M HCl pH 1.2 at 37oC) while dissolving completely in the alkaline environment of the small intestine (SIF, IM PBS solution pH 7.4 at 37oC). Eudragit L100 dispersed in 2ml PBS solution and 0.9ml Tween 80 (0.1%w/v) gave the best results remaining intact in SGF loosing only approximately 10-15% of the initial weight and dissolving completely within 3 hours. BCG was incorporated within the matrix formulation adjusted to pH 7 at the initial formulation stage containing PBS solution and Tween 80. It gave viability of x106 cfu/ml at initial formulation stage, freezing and freeze-drying stages. After this stage the matrix was compressed at 4 tons for 3 mins and placed in SGF for 2 hours and then in SIF until dissolved. The BCG viability dropped to x106 cfu/ml. There is potential to develop it further for oral delivery of BCG vaccine.

Rapid access wet AmD referrals using teleophthalmology and a central retinal grading centre

Introduction: We have adapted the existing , optometry diabetic retinopathy screening pathway and software , so that it can be used for wet AMD fast track referral. Purpose: To compare the conventional, optometry wet AMD fast track referral service using FAX transmission, with a teleophthalmology service using colour fundus photography transmitted to a central retinal grading centre. Method: 40 optometry practices involved in diabetic retinopathy screening were enrolled and had modified computer software installed. Referrals were made by conventional fast track FAX to the macular clinic, and patients were photographed by the optometrist and images transmitted to a central grading centre Results of the two pathways were compared in terms of 1)speed of diagnosis and 2)sensitivity and specificity of diagnosis of wet AMD. Results: Over a ten month period, 62 consecutive patients were referred. The mean time for conventional pathway was 20.8 days (range 3-34),and for new teleophthalmology pathway was 6.9 days (range 1-13). Sensitivity of technician grading of images was 96%, Specificity 53%, and consultant ophthalmologist was sensitivity 96%, specificiity 87%. The technician showed a learning effect with specificity increasing from 30.7% for first 31 patient cohort, to 70.6% for the second cohort. One patient had images that could not be graded. Conclusion: Rapid referral of wet AMD cases by optometrists using modified diabetic retinopathy screening software, allows fast and accurate diagnosis, and may reduce unnecessary referrals. Retinal grading technicians can be trained to grade wet AMD images.

Rapid serodiagnosis of Staphylococcus aureus surgical site infection following median sternotomy

Objectives: To determine the sensitivity and specificity of a novel ELISA for the serodiagnosis of surgical site infection (SSI) due to staphylococci following median sternotomy. Methods: Twelve patients with a superficial sternal SSI and 19 with a deep sternal SSI due to Staphylococcus aureus were compared with 37 control patients who also underwent median sternotomy for cardiac surgery but exhibited no microbiological or clinical symptoms of infection. A further five patients with sternal SSI due to coagulase-negative (CoNS) staphylococci were studied. An ELISA incorporating a recently recognised exocellular short chain form of lipoteichoic acid (lipid S) recovered from CoNS, was used to determine serum levels of anti-lipid S IgG in all patient groups. Results: Serum anti-lipid S IgG titres of patients with sternal SSI due to S. aureus were significantly higher than the control patients (P<0.0001). In addition, patients with deep sternal SSI had significantly higher serum anti-lipid S IgG titres than patients with superficial sternal SSI (P=0.03). Serum anti-lipid S IgG titres of patients with sternal SSI due to CoNS were significantly higher than the control patients (P=0.001). Conclusion: The lipid S ELISA may facilitate the diagnosis of sternal SSI due to S. aureus and could also be of value with infection due to CoNS. © 2005 Published by Elsevier Ltd. on behalf of The Bristish Infection Society.