Mycobacteria and allograft heart valve banking: an international survey.

Since the 1970s many tissue banks have been testing allograft heart valves (HVs) for Mycobacterium tuberculosis (MTB). Donor selection for low risk of tuberculosis (TB) was introduced in the 1980s and appears to have reduced the risk of TB transmission. Regulatory guidance does not specify testing for TB, but does exclude donors with a recent history of TB. This survey of HV international bank practices revealed variations in donor selection, testing and processing of valves. Participant banks (from Europe and the USA) reported that over a period of 15 years, HV tissues from 38,413 donors were banked and 32,289 donors were tested for TB, none being positive. HV-associated tissue from 27,840 donors was stained and underwent microscopy; none of these were positive for acid-fast bacilli (AFB). Non-tuberculosis mycobacteria (NTBM) were detected by culture on 24 HVs. It is recommended that HV banks employ donor selection to exclude donors at risk of TB, to culture material for mycobacteria, and to investigate potential sources when clusters of NTBM are found to facilitate corrective and preventative actions.

Residual antibiotics in allograft heart valve tissue samples following antibiotic disinfection.

Antibiotics are routinely used for the decontamination of allograft heart valves. To monitor the efficacy of this process, samples of tissue are sent for microbiological analysis. This investigation was undertaken to determine residual antibiotic concentrations in decontaminated tissue and to assess the likely inhibitory effect on microbiological cultures. After a typical decontamination protocol, both gentamicin and vancomycin were present in all tissue samples and the majority of enrichment broths at concentrations sufficient to inhibit most bacteria. The data presented indicate that protocols used by heart valve banks and associated microbiology laboratories should be reviewed, and support the use of predecontamination cultures to identify particularly virulent micro-organisms.

Treatment of ocular infections with topical antibacterials.

Topically applied ophthalmic antibacterial preparations are widely used in the treatment of patients with superficial ocular infections. In addition, they are frequently used to augment treatment for intraocular infection administered systemically or via local instillation. Direct application delivers high concentrations of antimicrobial agents to the surface of the eye conveniently, quickly and with minimal systemic exposure to the agent. However, antibacterials are rapidly dissipated from the tear film and intraocular penetration of topical antibacterial agents is generally poor, necessitating intensive application for successful treatment of corneal infections. Therapeutic concentrations are rarely achieved at other sites in the eye. This article reviews what is known of the pharmacokinetics of topical ocular agents and how this information can be used to optimise ocular persistence and penetration and minimise systemic absorption of antibacterials. A review of the features of the most commonly employed topical antibacterials suggests that for the treatment of uncomplicated bacterial conjunctivitis there is little difference between the various agents in terms of clinical efficacy, although chloram-phenicol should be used with care because of its potential haematological toxicity. Carefully considered therapy is imperative for bacterial keratitis; fortified beta-lactam/aminoglycoside combinations are often used for these infections. The fluoroquinolones appear promising, but caution is necessary in treating keratitis of unknown aetiology with these agents alone because of inherent and emerging acquired resistance among Gram-positive bacteria.

The pathological and ecological significance of microorganisms colonizing acne vulgaris comedones.

A microbiological survey has been undertaken of comedones isolated by micro-dissection from skin biopsies. Of closed comedones 10.7% and of open comedones 7.1% did not contain Pityrosporum spp., Propionibacterium spp. or Staphylococcus spp., the organisms most frequently associated with the pathogenesis of acne. Mature comedones were more frequently colonised than were young comedones. These results support the argument that the presence of microorganisms is not a prerequisite for comedo formation. Other pathological and ecological implications of these results are discussed.

Wound disinfection with ultraviolet radiation.

Bacteria were counted concurrently in the air and wounds during the first 20 min of total joint arthroplasty procedures in two theatres: a conventional plenum ventilated theatre with ultraviolet C (UVC) tubes installed and a filtered vertical laminar flow theatre. Four theatre environments were tested: conventional theatre and clothing; conventional theatre with UVC protective clothing, with UVC set to produce 100 or 300 microW cm-2 s-1 irradiation; and filtered vertical laminar flow air with staff wearing cuffed cotton/polyester clothing. When used, the UVC was activated 10 min after starting an operation to assess the effect of UVC clothing alone, and of UVC radiation on bacteria already present in the wound. Compared with conventional theatres, UVC clothing reduced air counts by 38%, UVC at 100 microW cm-2 s-1 by 81%, at 300 microW cm-2 s-1 by 91%, and laminar flow by 92%. Wounds counts fell correspondingly by 66% with UVC clothing, 87% with UVC at 100 microW cm-2 s-1 and 92% both with UVC at 300 microW cm-2 s-1 and laminar flow. In conventional and laminar flow theatres air and wound counts correlated closely but in UVC theatres wound counts were lower than levels expected from prevailing air counts suggesting that UVC kills bacteria in wounds as well as in air.

Assessment of airborne bacterial contamination of clean wounds: results in a tissue model.

Ovine skeletal muscle was used as a model wound and inoculated with airborne bacteria collected from a busy communal room. A specialized counting technique involving agar overlay and post-incubation tetrazolium staining was developed to allow accurate counting of small numbers of bacteria on the surfaces of muscle and membrane filters coated with substantial quantities of muscle and fat debris. Two techniques of recovering the inoculated airborne bacteria from the model wound were compared. Pulsed jet lavage with membrane filtration of the recovered fluid showed substantially better recovery, less variability and correlated more closely with controls than a tetrazolium stained 5 microns membrane filter imprint technique. Pulsed jet lavage with membrane filtration is likely to be the more appropriate technique in the assessment of contamination of wounds created in ultraclean air.

The efficacy of filters used in respiratory function apparatus.

The ability of two low resistance barrier filters (Collins DC-1 and Pall Pf 305) to remove bacteria from expired air was assessed. A specially designed coupling device was used to hold each filter or a disposable plain cardboard mouthpiece a fixed distance (4.5 cm) from a blood agar plate. Volunteers performed maximal forced vital capacity manoeuvres through the assembled apparatus and bacteria impinged on to the agar plate were enumerated. Both filters allowed the transmission of approximately one-third of expired colony forming units. The efficacy of these filters for reducing the likelihood of cross-infection during spirometry is not supported by this study.

Topical 0.3% ciprofloxacin, norfloxacin, and ofloxacin in treatment of bacterial keratitis: a new method for comparative evaluation of ocular drug penetration.

AIMS: This study was designed to assess the relative corneal penetration of topical drops of three antibiotics and to relate those levels to minimum inhibitory concentrations for organisms associated with bacterial keratitis. METHODS: Four drops of each of ciprofloxacin, norfloxacin, and ofloxacin (0.3% topical ophthalmic preparations) were given to 12 patients undergoing corneal transplantation. After the recipient tissue was removed, corneal drug penetration was measured using high performance liquid chromatography. RESULTS: Intracorneal concentrations of ofloxacin (geometric mean 0.81 mg kg-1) were significantly higher than both ciprofloxacin (0.60 mg kg-1; p = 0.048) and norfloxacin (0.54 mg kg-1; p = 0.012). Ciprofloxacin and norfloxacin concentrations did not differ significantly (p = 0.33). CONCLUSIONS: Review of the minimum inhibitory concentrations of the fluoroquinolones against ocular pathogens reveals that ciprofloxacin is more potent than ofloxacin against many bacteria; ofloxacin is in turn more potent than norfloxacin. These data favour the selection of ciprofloxacin and ofloxacin rather than norfloxacin for the empirical treatment of corneal infection. The greater potency of ciprofloxacin offsets the superior penetration of ofloxacin. There is a need for improved clinical trial data concerning the use of fluoroquinolone eyedrops in ulcerative keratitis; some encouraging data are available for ciprofloxacin but not (in humans) for norfloxacin or ofloxacin.

Ocular penetration of topical ciprofloxacin and norfloxacin drops and their effect upon eyelid flora.

A double blind, prospective study was undertaken to compare aqueous humour penetration of topical 0.3% norfloxacin and 0.3% ciprofloxacin and their effect upon normal eyelid flora in 39 patients undergoing cataract surgery. Lid swabs were taken before and after six 1 hourly applications of single drops of ciprofloxacin or norfloxacin given before surgery. Aqueous humour was aspirated at surgery and antibiotic concentration assayed using high performance liquid chromatography. The mean aqueous humour concentrations were: ciprofloxacin 220 ng ml-1, norfloxacin 140 ng ml-1. Although this difference was not statistically significant (p = 0.112) the trend demonstrated may be relevant clinically, especially considering the greater activity of ciprofloxacin. Both coagulase negative staphylococcal (p = 0.004) and total bacterial (p = 0.019) lid counts dropped sixfold after ciprofloxacin treatment but the smaller reductions noted after norfloxacin application did not achieve statistical significance (p > 0.1). The reduction of external eye flora experienced with ciprofloxacin suggests that this may be a useful presurgical prophylactic agent.

An antimicrobial effect associated with rabbit primary aqueous humour.

Viability curves were recorded for a variety of bacteria suspended in rabbit primary aqueous humour. Controls included neat rabbit serum, dilute rabbit serum (1/50), dilute heat treated fetal bovine serum (1/50), and balanced salt solution. Rabbit aqueous humour was bactericidal in vitro for Staphylococcus aureus and Pseudomonas aeruginosa. Micrococcus spp, Streptococcus pneumoniae and Escherichia coli were not affected. The factor mediating the bactericidal effect survives filtration (0.2 micron porosity) and freezing in liquid nitrogen (-196 degrees C) but is labile when stored in air at temperatures of 4 degrees C, 25 degrees C, and 37 degrees C for 1 hour. The bactericidal effect was stable when aqueous humour was stored in an atmosphere of 5% CO2 at 37 degrees C for 3 hours. The antibacterial component in rabbit aqueous humour has yet to be identified.

Infection control of lung function equipment: a practical approach.

The degree of risk of cross-infection of patients via lung function testing equipment has yet to be quantified. Based on current evidence, elaborate precautions are not justified for the majority of patients attending the laboratory, but attention to appropriate routine cleaning and disinfection protocols is important. Disinfection and sterilization can be achieved by a variety of methods, although chemical methods should be used with caution. Identification of factors increasing the susceptibility or infectivity of particular patients is important in determining appropriate precautions. Where patients are known to be infectious or are immunocompromized, additional precautions such as using a barrier filter may be appropriate. However, because of cost constraints, the routine use of barrier filters is difficult to justify based on current evidence of minimal cross-infection associated with lung function equipment. Until further studies have been conducted to quantify the degree of risk of cross-infection that lung function test equipment poses, the recommendations given in this review provide a practical approach to dealing with this problem.

Reservoirs of coagulase negative staphylococci in preterm infants.

This investigation was undertaken to determine the magnitude of, and interrelations between, reservoirs of coagulase negative staphylococci on infants' skin at various sites (including sites used for insertion of intravascular catheters) and in faeces during the first six months of life. Sites with large numbers of coagulase negative staphylococci were identified by sampling 16 skin sites and stools from 20 preterm neonates at 8-30 days of life. A more detailed survey of numbers and types of coagulase negative staphylococci in stool and at six skin sites of 10 preterm infants was then performed over the first six months of life. Isolates of coagulase negative staphylococci were collected and characterised by speciation, antibiotic susceptibility profiling, and plasmid restriction fragment length polymorphism analysis. Large, relatively stable reservoirs were identified in the faeces, around the ear, and in the axilla and nares. Skin on the forearm and leg, sites at which peripheral catheters are frequently sited, carried small unstable numbers of coagulase negative staphylococci, which were usually indistinguishable from coagulase negative staphylococci isolated from other body sites on the same baby. Contamination of catheter insertion sites with coagulase negative staphylococci from reservoir sites on the same baby could explain these observations. These data suggest that interventions reducing cross-contamination between sites on the same baby might be as important in preventing coagulase negative staphylococcal bacteraemias as measures taken to prevent cross infection between babies. Procedures which are likely to result in heavy coagulase negative staphylococcal contamination of the hands of healthcare staff, such as changing soiled nappies, should receive particular attention.

Purification of an antimicrobial peptide from rabbit aqueous humour.

PURPOSE: To identify an antimicrobial factor previously demonstrated in rabbit aqueous humour. METHODS: Rabbit aqueous humour was fractionated by a multi-stage process involving anion-exchange and size-exclusion liquid chromatography. The antimicrobial effect of aqueous humour fractions upon Staphylococcus aureus were evaluated in an in vitro model. The components of aqueous humour fractions mediating an antimicrobial effect were investigated by SDS-PAGE. RESULTS: A single peptide of molecular weight approximately 8 kDa was identified which mediated an antimicrobial effect upon Staphylococcus aureus. Attempts to identify the peptide have been unsuccessful. CONCLUSIONS: Rabbit aqueous humour contains an unidentified peptide that mediates an antimicrobial effect upon Staphylococcus aureus. If such a peptide is present in human aqueous humour it may contribute to the apparent resistance to bacterial infection manifest in the anterior chamber.

Effect of antiseptics, ultraviolet light and lavage on airborne bacteria in a model wound.

We modelled a 'clean' surgical wound lightly contaminated with airborne bacteria, using agar, ovine muscle and ovine adipose tissue. This was used to assess the effect on bacteria of ultraviolet C light (UVC) 1200 mu W/cm2, hydrogen peroxide 3%, povidone-iodine 1% and 10%, chlorhexidine 0.05%, pulsed jet lavage with UVC and syringe and needle lavage with chlorhexidine 0.05%. All the agents were effective on agar, but mixing with blood or plasma neutralised hydrogen peroxide and povidone-iodine 1%. All the agents were less effective on tissue specimens than on agar, but were more effective on adipose tissue than on muscle. All the antiseptics except chlorhexidine were less effective when blood or plasma was added to muscle specimens before disinfection. UVC after pulsed jet lavage had an additive effect. Syringe and needle lavage with chlorhexidine 0.05% was the most effective method tested; it reduced colony counts by 99.8% and warrants clinical investigation.

Molecular approaches for the diagnosis and epidemiological investigation of Aspergillus infection.

Molecular techniques have been applied to the diagnosis of invasive aspergillosis and to investigate the ecology and epidemiology of Aspergillus. Recent advances in diagnosis include the development of PCRs targeting either panfungal or Aspergillus-specific sequences, using whole blood or serum samples. When a sensitive PCR is used, invasive aspergillosis in bone marrow transplant patients can be detected several weeks before antigen tests become positive, and a positive PCR often pre-dates the institution of antifungal therapy. The role of PCR in monitoring response to therapy in immunocompromised patients is unclear. No prospective studies have yet demonstrated that management incorporating PCR alters the poor outcome of invasive aspergillosis in immunocompromised patients. Molecular typing of Aspergillus fumigatus has shown wide geographical dispersal of indistinguishable strains. This, combined with the observation that multiple strains may be isolated from individual colonised patients with cystic fibrosis and from immunocompromised patients with disseminated disease, makes the elucidation of the epidemiology of aspergillosis relatively complex.

Topical ciprofloxacin in the treatment of blepharitis and blepharoconjunctivitis.

An international multicentre study assessed the clinical and antibacterial efficacy of a new topical ophthalmic formulation of the quinolone antimicrobial agent ciprofloxacin and compared it with that of tobramycin ophthalmic solution in patients with blepharitis and blepharoconjunctivitis. The study consisted of a randomised double-masked between-group evaluation of 464 patients, 230 of whom were treated with ciprofloxacin and 234 with tobramycin. There was qualitative and quantitative bacteriology, and clinical assessment of ocular symptoms and signs before and after a seven-day course of treatment. Bacteriological cultures demonstrated eradication or reduction of potentially pathogenic bacteria in 93.7% of eyes (ciprofloxacin) versus 88.9% of eyes (tobramycin), seven days after starting treatment. Clinically more than 80% of patients in both treatment groups were cured or improved after seven days. No statistically significant differences were observed between the two treatment groups. No serious side-effects were observed after use of either antimicrobial agent. Ciprofloxacin ophthalmic solution appears safe and effective. The spectrum of activity and clinical efficacy of this new formulation are discussed in comparison with currently used antimicrobial agents.

Clinical relevance of a positive molecular test in the diagnosis of Clostridium difficile infection.

BACKGROUND: In 2011, the Department of Health advised that a two-stage test approach should be used to improve accuracy of Clostridium difficile infection (CDI) diagnosis. No specific test protocol was established at that time. AIM: To compare clinical features of inpatient CDI cases identified by toxin enzyme immunoassay (EIA) with those identified as polymerase chain reaction (PCR) positive but toxin EIA negative. METHODS: During a six-month period (2011-2012), 2181 liquid faeces samples submitted to North Bristol NHS Trust were tested by EIA for both toxin and glutamate dehydrogenase (GDH). A total of 215 toxin or GDH EIA-positive samples were tested by Cepheid Xpert PCR assay; 128 clinically evaluable inpatients were grouped by test result, and their duration of diarrhoea and 14-day mortality compared. FINDINGS: Inpatients with a positive PCR but negative toxin EIA had a significantly lower 14-day all-cause mortality [11%; 95% confidence interval (CI): 4-23%] than patients with a positive PCR and positive toxin EIA test (37%; 95% CI: 19-59%; P = 0.01), and a smaller proportion of patients had prolonged diarrhoea (>5 days or unresolved at death: 19%; CI: 9-32%, vs 67%; CI: 45-84%; P < 0.001). A positive toxin EIA test was a significant independent predictor of death [odds ratio (OR): 4.7, 95% CI: 1.4-15.4; P = 0.01] and prolonged diarrhoea (OR: 8.6; CI: 2.9-25.6; P < 0.001), but a positive PCR (given positive GDH EIA) was not. CONCLUSION: The clinical significance of a positive PCR result without a positive toxin EIA is questionable; such a result is associated with a significantly lower mortality and shorter duration of symptoms than patients with a positive toxin EIA.

A method for multiple synchronous collection of airborne organisms and the effects on colony counts of various processing procedures.

A method is described for synchronous collection on agar of 10 similar specimens of airborne bacterial colony-forming units (cfu) for comparative experiments. The system delivers 50 to 100 cfu per specimen with a coefficient of variance of 13 among the 10 specimens. After collection, different methods for removing colonies from the agar surface and counting them were employed. A progressive increase in colony counts was noted when increasingly destructive procedures were used. The increases noted were 4% by wetting, 30% by jet lavage, 58% by pulsed jet lavage, 82% by blending, 130% by spreading and 340% by grinding. As airborne cfu consist mainly of skin squames with multiple organisms attached, disruption of cfu is proposed as the cause of the increases. Membrane filtration of wash fluid containing cfu from the air resulted in a 47% decrease in colony counts when compared with pour-plating. Destructive processing techniques also resulted in increased variability in colony counts. The break up of occasional exceptionally large cfu is a probable explanation. The procedure described is suitable for investigating the behaviour of airborne micro-organisms and can be modified to model surgical wound contamination by replacing the agar with tissue.