Diagnosis of Clostridium difficile-associated disease: examination of multiple algorithms using toxin EIA, glutamate dehydrogenase EIA and loop-mediated isothermal amplification.

The laboratory diagnosis of Clostridium difficile infection (CDI) needs to be accurate and timely to ensure optimal patient management, infection control and reliable surveillance. Three methods are evaluated using 810 consecutive stool samples against toxigenic culture: CDT TOX A/B Premier enzyme immunoassay (EIA) kit (Meridian Bioscience, Europe), Premier EIA for C. difficile glutamate dehydrogenase (GDH) (Meridian Bioscience, Europe) and the Illumigene kit (Meridian Bioscience, Europe), both individually and within combined testing algorithms. The study revealed that the CDT TOX A/B Premier EIA gave rise to false-positive and false-negative results and demonstrated poor sensitivity (56.47%), compared to Premier EIA for C. difficile GDH (97.65%), suggesting this GDH EIA can be a useful negative screening method. Results for the Illumigene assay alone showed sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of 91.57%, 98.07%, 99.03% and 84.44%, respectively. A two-stage algorithm using Premier EIA for C. difficile GDH/Illumigene assay yielded superior results compared with other testing algorithms (91.57%, 98.07%, 99.03% and 84.44%, respectively), mirroring the Illumigene performance. However, Illumigene is approximately half the cost of current polymerase chain reaction (PCR) methods, has a rapid turnaround time and requires no specialised skill base, making it an attractive alternative to assays such as the Xpert C. difficile assay (Cepheid, Sunnyvale, CA). A three-stage algorithm offered no improvement and would hamper workflow.

Guidelines on the facilities required for minor surgical procedures and minimal access interventions.

There have been many changes in healthcare provision in recent years, including the delivery of some surgical services in primary care or in day surgery centres, which were previously provided by acute hospitals. Developments in the fields of interventional radiology and cardiology have further expanded the range and complexity of procedures undertaken in these settings. In the face of these changes there is a need to define from an infection prevention and control perspective the basic physical requirements for facilities in which such surgical procedures may be carried out. Under the auspices of the Healthcare Infection Society, we have developed the following recommendations for those designing new facilities or upgrading existing facilities. These draw upon best practice, available evidence, other guidelines where appropriate, and expert consensus to provide sensible and feasible advice. An attempt is also made to define minimal access interventions and minor surgical procedures. For minimal access interventions, including interventional radiology, new facilities should be mechanically ventilated to achieve 15 air changes per hour but natural ventilation is satisfactory for minor procedures. All procedures should involve a checklist and operators should be appropriately trained. There is also a need for prospective surveillance to accurately determine the post-procedure infection rate. Finally, there is a requirement for appropriate applied research to develop the evidence base required to support subsequent iterations of this guidance.

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide.

The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.

Synthesis of six novel N,N-dialkyl derivatives of spermidine and effects on growth of the fungal plant pathogen Pyrenophora avenae.

Six novel N,N-dialkyl derivatives of spermidine were synthesised and examined for activity against the oat stripe pathogen Pyrenophora avenae. Two of these spermidine analogues, N,N-dimethyl-N1-(3-aminopropyl)-1,3-diaminopropane trihydrochloride (27) and N,N-dimethyl-N1-(3-aminopropyl)-1,4-diaminobutane trihydrochloride (28), reduced radial extension of P. avenae on plates when used at 2 mM, and caused more substantial reductions in fungal growth in liquid culture when used at 1 mM. Preliminary data suggest that neither compound affected polyamine biosynthesis, determined by following the incorporation of label from ornithine into polyamines and examining intracellular polyamine concentrations in fungal tissue.

Effect of polyamine depletion on caspase activation: a study with spermine synthase-deficient cells.

Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.

Effect of spermine synthase deficiency on polyamine biosynthesis and content in mice and embryonic fibroblasts, and the sensitivity of fibroblasts to 1,3-bis-(2-chloroethyl)-N-nitrosourea.

Mutant Gy male mice, which have previously been described as having disruption of the phosphate-regulating Phex gene and a spermine synthase gene [Meyer, Henley, Meyer, Morgan, McDonald, Mills and Price (1998) Genomics, 48, 289-295; Lorenz, Francis, Gempel, Böddrich, Josten, Schmahl and Schmidt (1998) Hum. Mol. Genet. 7, 541-547], as well as mutant Hyp male mice, which have disruption of the Phex gene only, were examined along with their respective normal male littermates. Biochemical analyses of extracts of brains, hearts and livers of 5-week-old mice showed that Gy males lacked any significant spermine synthase activity as well as spermine content. Organs of Gy males had a higher spermidine content. This was caused not only by the lack of conversion of spermidine into spermine, but also because of compensatory increases in the activities of other polyamine biosynthetic enzymes. Gy males were half the body weight of their normal male littermates at weaning age. Hyp males, however, were no different in size when compared with their controls. High mortality of Gy males occurs by weaning age and this mortality was shown to be largely post-natal. Embryonic fibroblasts were isolated from Gy males and their normal male littermates and were similarly shown to lack any significant spermine synthase activity as well as spermine content. The lack of spermine, however, had no significant effect on the growth of immortalized fibroblasts or of primary fibroblast cultures. Similarly, there was no difference in the time of senescence of primary fibroblast cultures from Gy males compared with cultures derived from normal male littermates. However, the lack of spermine did increase the sensitivity of immortalized fibroblasts to killing by the chloroethylating agent 1, 3-bis(2-chloroethyl)-N-nitrosourea. Therefore both the Gy male mice and derived embryonic fibroblasts provide valuable models to study the importance of spermine and spermine synthase, without the use of inhibitors which may have additional side effects.

Modulation of potassium channels in the hearts of transgenic and mutant mice with altered polyamine biosynthesis.

Inward rectification of cardiac I(K1)channels was modulated by genetic manipulation of the naturally occurring polyamines. Ornithine decarboxylase (ODC) was overexpressed in mouse heart under control of the cardiac alpha -myosin heavy chain promoter (alpha MHC). In ODC transgenic hearts, putrescine and cadaverine levels were highly elevated ( identical with 35-fold for putrescine), spermidine was increased 3.6-fold, but spermine was essentially unchanged. I(K1)density was reduced by identical with 38%, although the voltage-dependence of rectification was essentially unchanged. Interestingly, the fast component of transient outward (I(to,f)) current was increased, but the total outward current amplitude was unchanged. I(K1)and I(to)currents were also studied in myocytes from mutant Gyro (Gy) mice in which the spermine synthase gene is disrupted, leading to a complete loss of spermine. I(K1)current densities were not altered in Gy myocytes, but the steepness of rectification was reduced indicating a role for spermine in controlling rectification. Intracellular dialysis of myocytes with putrescine, spermidine and spermine caused reduction, no change and increase of the steepness of rectification, respectively. Taken together with kinetic analysis of I(K1)activation these results are consistent with spermine being a major rectifying factor at potentials positive to E(K), spermidine dominating at potentials around and negative to E(K), and putrescine playing no significant role in rectification in the mouse heart.

Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy.

Two lines of transgenic mice were produced with constitutive expression of antizyme-1 in the heart, driven from the cardiac alpha-myosin heavy chain promoter. The use of engineered antizyme cDNA in which nucleotide 205 had been deleted eliminated the need for polyamine-mediated frameshifting, normally necessary for translation of antizyme mRNA, and thus ensured the constitutive expression of antizyme. Antizyme-1 is thought to be a major factor in regulating cellular polyamine content, acting both to inhibit ornithine decarboxylase (ODC) activity and to target it for degradation, as well as preventing polyamine uptake. The two transgenic lines had substantial, but different, levels of antizyme in the heart, as detected by Western blotting and by the ability of heart extracts to inhibit exogenous purified ODC. Despite the high levels of antizyme, endogenous ODC activity was not completely abolished, with 10-39% remaining, depending on the transgenic line. Additionally, a relatively small decrease (30-32%) in cardiac spermidine content was observed, with levels of putrescine and spermine unaffected. Interestingly, although the two lines of transgenic mice had different antizyme expression levels, they had almost identical cardiac polyamine content. When treated with a single acute dose of isoprenaline (isoproterenol), cardiac ODC activity and putrescine content were substantially increased (by 14-fold and 4.7-fold respectively) in non-transgenic littermate mice, but these increases were completely prevented in the transgenic mice from both founder lines. Prolonged exposure to isoprenaline also caused increases in cardiac ODC activity and polyamine content, as well as an increase in cardiac growth, in non-transgenic mice. Although the increases in cardiac ODC activity and polyamine content were prevented in the transgenic mice from both founder lines, the increase in cardiac growth was unaffected. These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress.

Synthesis and antifungal activity of two novel spermidine analogues.

Two spermidine analogues were synthesised and examined for antifungal activity. Both compounds used as 1 mM post-inoculation sprays reduced infection of barley seedlings by the powdery mildew fungus, Erysiphe graminis f.sp. hordei, infection of broad bean seedlings by the rust fungus, Uromyces viciae-fabae, and infection of apple seedlings by the powdery mildew fungus, Podosphaera leucotricha. Since these fungal pathogens cannot be cultured axenically, the effects of the two spermidine analogues on mycelial growth in vitro, as well as preliminary investigations on polyamine biosynthesis, were undertaken using the oat stripe pathogen, Pyrenophora avenae. Although neither compound affected radial growth of the fungus on plates, both analogues reduced fungal biomass in liquid culture substantially. The two spermidine analogues, used at a concentration of 1 mM, had no significant effect on the conversion of labelled ornithine into polyamines in P. avenae.

An evaluation of surveillance methods for detecting infections in hospital inpatients.

Eight selective surveillance methods were compared with a reference method for their ability to detect hospital infections in patients was also assessed. In the reference method, case records were reviewed three times a week, and during the 11-month period of study, 668 infections were identified amongst 3326 patients. Three hundred and thirty-eight were community acquired infections (CAI) and 330 were hospital acquired infections (HAI). The time for data collection was 18.1 h per 100 beds per week. Of the selective surveillance methods, those based on the review of treatment and temperature charts detected the highest proportion (70%) of CAI; and the review of microbiology reports with regular ward liaison identified the highest proportion (71%) of HAI. The time for data collection in the eight methods ranged from 1.2 h per 100 beds per week to 6.5 h per 100 beds per week. After considering the sensitivity for identifying patients with HAI and time for data collection, the review of microbiology reports with regular ward liaison was judged to be an effective and efficient method of surveillance.

An 11-month incidence study of infections in wards of a district general hospital.

Between March 1988 and January 1989, an incidence study of infections in patients occupying 122 beds in a district general hospital was undertaken. Nursing notes, medical notes, temperature charts, drug prescription charts and laboratory information were reviewed three times a week to determine if patients had infection which met strict case definitions. In addition, the surveyor consulted with ward nursing and medical staff for clarification of symptoms and signs indicative of infection. During the study, 668 infections were identified amongst 3326 patients. Three hundred and thirty-eight (51%) were community-acquired infections (CAI) and 330 hospital-acquired infections (HAI). Excluding 24 HAI acquired in other hospitals, the incidence rates were 9.2 HAI per 100 discharges, and 1.1 HAI per 100 patient days. The common types of CAI were pneumonia, abdominal infection and urinary tract infection. The main types of HAI were urinary tract infection, surgical wound infection and pneumonia. The microorganisms most frequently associated with CAI and HAI were Gram-negative bacilli.

Surveillance of methicillin-resistant Staphylococcus aureus in England and Wales, 1986-1990.

The incidence of methicillin-resistant Staphylococcus aureus in England and Wales was monitored by a weekly reporting scheme from early 1986 to March 1990. Potential coverage was approximately two-thirds of hospital beds. Reporting centres fell from a peak of 210 in 1986 to a low of 101 centres early in 1989 with later recovery. There were 2367 positive reports in 1986, 2174 in 1987, 1700 in 1988, 1701 in 1989 and 632 in the first quarter of 1990. Colonizations outnumbered infections by 2:1. There were marked regional differences: North-East Thames was dominant in 1986 and 1987, and then declined; South-East Thames showed a dramatic increase in 1988 which continued. Other regions showed less significant changes but there were continuing problems in the South-Western Region and in the West Midlands. Some of these changes were related to the decline of EMRSA-1, possibly due to the introduction of effective control measures, and to the emergence of EMRSA-3 in South-East Thames and its spread to Wessex.

A survey of methicillin-resistant Staphylococcus aureus affecting patients in England and Wales.

For a six-month period between October 1987 and March 1988, 660 isolates of methicillin-resistant Staphylococcus aureus (MRSA) from 570 patients were sent to the Staphylococcus Reference Laboratory at Colindale to supplement the National reporting survey of MRSA in England and Wales. The isolates were characterized by phage typing, antibiotic susceptibility and by selected biochemical tests. Patient details were also surveyed. Fourteen strains affected more than one hospital and were called multi-hospital epidemic strains. One strain, EMRSA-1, accounted for more than 40% of isolates and of patients. Other epidemic strains were defined. Ten additional strains were restricted to single hospitals. Only 25 primary isolates were non-typable but 67 sporadic typable strains occurred. The patients affected were approximately equally either infected or colonized. The sexes were represented equally. Orthopaedic and geriatric wards were over-represented. Epidemic strains were clumping factor positive while some sporadic strains were weak producers. Urea alkalinization and protein A production could supplement phage typing and antibiotic resistance in strain recognition.

The dispersal of bacteria and skin scales from the body after showering and after application of a skin lotion.

Application of a skin lotion to the body after showering greatly reduced the number of bacteria and skin scales dispersed from 10 men and 10 women. This effect lasted for at least 4 h when surgical clothing was worn. The use of a skin lotion to reduce bacterial dispersal could provide a simple and inexpensive alternative to an ultraclean air system or uncomfortable operating clothing during surgery requiring these procedures.

Evaluation of three serological tests for detection of antibody to Pseudomonas aeruginosa in human sera.

Four hundred and ninety-five sera from 325 patients from whom Pseudomonas aeruginosa had been isolated and 86 control sera were tested for antibody by indirect haemagglutination tests (HAT) and complement fixation tests (CFT) using a polyvalent pseudomonas serotype-specific vaccine antigen, PEV-02. Sera were also tested by countercurrent immunoelectrophoresis (CIE) for precipitins to a species-specific protein antigen. Control sera gave titres of 160 or less by HAT and 20 or less by CFT. 2-Mercaptoethanol resistant antibody titres (immunoglobulin G) were below 40 for all control sera and none of the latter contained precipitins to common antigen. Of 325 patients, 156 (48%) gave titres of 320 or greater by HAT and of these, 114 (73%) showed elevated immunoglobulin G titres. Less patients with positive blood cultures than expected were positive by HAT and more patients with bone infections gave raised immunoglobulin G titres than expected. Cystic fibrosis patients were invariably seropositive by all tests. There was a correlation between positive CIE and CFT tests, especially in patients who were positive by HAT. Approximately half of 83 patients tested gave a serotype-specific antibody response. The tests were of little value in confirming clinically evident acute infections, but in cases of doubtful infection they did provide confirmatory evidence of an antibody response in approximately one-third of patients culture-positive for Pseudomonas aeruginosa.

Microbiological aspects of the 1980 national prevalence survey of infections in hospitals.

The records of the 1980 national prevalence survey of infection in hospitals were re-assessed from a microbiological point of view. Of 407 records of Escherichia coli, 71 per cent came from the urinary tract while the commonest source of Staphylococcus aureus was from skin infections. These yielded only 41 per cent of the 303 records. Proteus spp. were recorded 166 times, Pseudomonas spp. 115 times and Klebsiella spp. 101 times. These came mainly from the urinary tract but other sources were important. Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis and the viruses were associated with community infections while E. coli, Proteus spp., Pseudomonas spp., Klebsiella spp., Str. faecalis and non-aureus staphylococci were associated with hospital-acquired infections. The prevalence of bacteraemia was re-assessed.

An extended model for transfer of micro-organisms via the hands: differences between organisms and the effect of alcohol disinfection.

A model for contact transfer of micro-organisms by hand has been extended to include representatives of bacterial species responsible for a majority of hospital-acquired infections. The ability of the organisms to transfer from contaminated fabrics to hands and from hands to sterile fabrics was measured, as was their ability to survive on the skin of the hands. There were differences between the species. Staphylococcus saprophyticus transferred well to the hand but not as well from hand to fabric as the other species; it survived well on skin. Pseudomonas aeruginosa, Klebsiella aerogenes and Serratia marcescens transferred moderately well overall and also survived on the skin. These results were in contrast to those obtained with a strain of Escherichia coli and one of Streptococcus pyogenes. The contact transfer model was used to investigate the use of small volumes of alcohol in preventing transfer via the hands. An alcohol handrub of either 0.3 ml 80% ethanol or 0.3 ml 70% isopropanol gave reductions in transfer slightly less than that of a soap and water wash. Raising the volume, and consequently the contact time, to 0.5 ml 70% isopropanol gave a 14000-fold reduction in transfer, statistically indistinguishable from that of a thorough soap and water wash (9800-fold reduction).

The evaluation of fabrics in relation to their use as protective garments in nursing and surgery. III. Wet penetration and contact transfer of particles through clothing.

A method is described for comparing the resistance to penetration by aqueous fluids, under rubbing contact, of a representative series of fabrics. Untreated woven fabrics are rapidly penetrated, but some non-woven synthetic materials resist penetration for much longer and tightly woven proofed cotton fabrics for prolonged periods, even after repeated washing and sterilizing. If a wetting agent is added to water, penetration occurs more quickly, but fabrics containing natural cotton are penetrated more slowly by serum. The same fabrics were examined by a test designed to simulate transfer of dry particulate material, e.g. skin scales, through them during nursing contact. The proportionate differences observed were much greater than for air dispersal during exercise and closely resembled those obtained by a laboratory rubbing test. In particular, one of the non-woven fabrics showed much greater relative penetration when examined by these methods than the relative dispersal of skin scales through it during exercise.