Targeted gene correction: a new strategy for molecular medicine

OBJECTIVE: Microbiological testing evaluations of hygienic procedure properties using PALL BB22-15MS filters to prevent contamination of "single use" breathing circuits during anesthesia. DESIGN: Prospective study. SETTING: Two operating rooms in a University hospital. PATIENTS: One hundred and thirty eight patients underwent general anesthesia for urologic surgery procedures. Patients with positive tests for HIV, B and C hepatitis and those considered to be at risk for HIV infection were excluded. The study was divided into five phases on the basis of times of usage of the same circuit for an increasing number of patients: in phase I, microbiological tests were performed on 4 circuits used on 4 different patients; in phase II the same tests were performed on 2 circuits each used on two different groups of three patients; in phase III, a circuit was used on a group of 15 patients and another on a group of 16 patients and the results were analysed; in phase IV a circuit used in a group of 32 patients was evaluated and phase V involved the analysis of a circuit that was used in a group of 65 patients. INTERVENTIONS: A filter was left in place between the patient and the circuit's Y-piece during all phases of anesthesia. The level of microbial contamination of breathing circuits was analysed in order to evaluate the reliability of the procedure. RESULTS: All analysed circuits remained uncontaminated. Staphylococcus hominis was revealed in respiratory circuit no. 6 of phase II, probably as a consequence of secondary contamination. CONCLUSIONS: Our results confirm that this procedure with the routine placement of a PALL BB22-15MS filter at the circuit's Y-piece can provide an adequate level of protection against cross-infections during anesthesia. The hygienic protocol proposed may allow the change of the anesthetic breathing only once a month.     

Residual dipolar couplings as new conformational restraints in isotropically 13C-enriched oligosaccharides

Closed-circuit anesthesia (CCA) has certain advantages such as decreased cost, decreased anesthetic gas pollution, improved inhalational gas humidity and temperature in comparison to conventional inhalational anesthesia using a high fresh gas flow, i.e. more than 2 L x min(-1), with a semi-closed breathing circuit. The main disadvantage of CCA is the possibility of hypoxic anesthetic gas delivery. This potentially lethal situation is caused by an insufficient oxygen flow rate for the body metabolism or by the accumulation of inactive gas, usually nitrogen, within the breathing circuit in spite of a sufficient oxygen concentration in the fresh gas supply to the breathing circuit. In the latter case, the accumulation of inactive gas may also lead an increased risk of awareness because of its dilution effect on the concentrations of inhalational anesthetics. We herein present a case of air contamination of the breathing circuit through a sampling line of an anesthetic gas monitor. The air caused a decrease in the oxygen concentration during closed circuit anesthesia.     

Microbiological examination of bandage soft contact lenses used in laser refractive surgery

BACKGROUND: Disposable soft contact lenses are known to be colonized by bacteria and play a key role in bacterial keratitis pathogenesis. Such lenses, commonly used after laser refractive surgery procedures in which postoperative corneal infiltrations are sometimes observed, are potentially a substrate for bacterial inoculation. This study evaluates the extent of such a contamination. METHODS: Sixty disposable lenses collected from 60 eyes of patients who underwent photorefractive keratectomy (PRK), photoastigmatic refractive keratectomy (PARK), or laser in situ keratomileusis (LASIK) for the treatment of myopia or hyperopia were collected under sterile conditions over 4 months and cultured in various media. Results were statistically analyzed and the correlation with clinical and epidemiological data was examined. RESULTS: Eleven (18.3%) of the examined lenses were contaminated with Staphylococcus epidermidis. No other bacteria or fungi were found. Contamination was significantly more common among female patients (P = .036). Correlation with the other clinical or operative parameters examined was statistically insignificant. CONCLUSIONS: Contamination was independent of the surgical procedure and females who were frequent users of eyelid cosmetics displayed higher contamination frequencies, suggesting that bacteria possibly originate from eyelid flora. The isolation of Staphylococcus epidermidis requires close postoperative surveillance, since it is a known cause of keratitis. Prophylactic postoperative treatment with tobramycin, gentamycin, or sulphonamides could be indicated.     

Intravenous calcium chloride in the conversion of paroxysmal supraventricular tachycardia to normal sinus rhythm

OBJECTIVE: To determine whether a specially designed antisuction device can prevent the bacterial contamination of the drive air lines of the dental turbine that is caused by suction when the turbine is stopped. STUDY DESIGN: A dental unit with and without the antisuction device and three different types of sterilized handpieces were used in the tests. Each turbine was operated in air, then submerged into a bacterial suspension of E. coli and enterococci for 3 seconds, removed, and stopped. This procedure was repeated 10 times. Possible bacterial contamination of the drive air lines was examined by submersing the head of a sterilized handpiece with the turbine running into a nutrient broth for 30 seconds. The broth was incubated at 35 degrees C up to 2 days. RESULTS: After use of the conventional dental unit, bacterial growth of drive air lines was found in 10 of 150 broth samples. After the installation of the antisuction device no bacterial growth was found in any of the 138 samples. The difference in the contamination frequencies is statistically significant (p = 0.011, Fisher's two-sided exact test). CONCLUSIONS: The drive air lines of the turbine in the dental unit may become contaminated despite the sterilization of handpieces. The antisuction device installed into the dental unit was found to prevent the contamination. With the exception of possibly immunocompromised patients, the transmission of microbes by exhaust air may be too small to cause infections. However, transmission of oral material between patients should be prevented in dental practice.     

Differential diagnosis of Staphylococcus aureus from Staphylococcus epidermidis and Staphylococcus saprophyticus by alphazurine A dye

Staphylococcal bacterial suspensions were streaked on Trypticase soy agar with 5% sheep blood culture plates. Paper discs containing alphazurine A, a triphenylmethane dye, were placed on the inoculated plates which were incubated at 37 degrees C for 24 hours. A wide zone of inhibition of growth of Staphylococcus aureus was present around the paper discs. Growth of Staphylococcus epidermidis and Staphylococcus saprophyticus was not inhibited.     

Contamination of diagnostic ophthalmic solutions in primary eye care settings

Pharmaceutical agents and irrigating solutions are widely used in both optometric and ophthalmologic practices. Contamination of these containers or solutions could possibly pose some risk of infection to a patient. We set out to investigate the possible contamination of a representative sample of these containers in small office practices. Representative bottles of two diagnostic pharmaceutical agents and an irrigating solution were obtained from primary care optometric and ophthalmologic practices in the San Francisco-Oakland Bay area. These bottles were tested to investigate the rate of contamination and to identify the types of microorganisms in the contaminated solutions. Sixty total samples (proparacaine, tropicamide, and an irrigating solution) were randomly cultured, and 11.7% of the samples showed contamination. Pseudomonas cepacia, Staphylococcus epidermidis, Pseudomonas putida, and Streptococcus species were the predominant organisms isolated from the contaminated bottles. In addition, 17 of the original 60 containers were further cultured for investigation of the dried residue particles around the threads of the containers. Of these 17 containers, 13 (76.5%) tested positive for Staphylococcus and Micrococcus species.     

Bacteria inhibit biosynthesis of bone matrix proteins in human osteoblasts

The effect of extracts from Staphylococcus aureus and Staphylococcus epidermidis on bone matrix production were assessed by analyzing the biosynthesis of osteocalcin and Type I collagen in a human osteoblastic osteosarcoma cell line (MG-63). In MG-63 cells, extracts from Staphylococcus aureus and Staphylococcus epidermidis decreased 1,25(OH)2-vitamin D3 stimulated osteocalcin biosynthesis, and insulin-like growth factor I induced production of Type I collagen in a concentration dependent manner. The basal rate of osteocalcin and Type I collagen formation was unaffected by the bacterial extracts. The inhibitory effect of the bacteria on osteocalcin biosynthesis was seen after 24 hours of treatment and was maintained for at least 96 hours. The extracts of Staphylococcus aureus and Staphylococcus epidermidis enhanced prostaglandin E2 formation in the MG-63 cells. Abolition of the prostaglandin E2 response by treatment with indomethacin and flurbiprofen did not affect bacteria induced inhibition of osteocalcin production. Stimulation of osteocalcin biosynthesis by 1,25(OH)2-vitamin D3 was associated with a decreased rate of cell proliferation. The inhibitory action of the bacterial extracts was not linked to any inhibition of [3H]-thymidine incorporation into deoxyribonucleic acid. These data show that extracts of Staphylococcus aureus and Staphylococcus epidermidis have the ability to inhibit the biosynthesis of bone matrix proteins by a nonprostaglandin and noncytotoxic dependent mechanism and suggest that bone loss in inflammatory processes containing Staphylococcus aureus or Staphylococcus epidermidis may not be caused only by enhanced bone resorption but also by decreased bone formation.     

Effects of three different types of management on the elimination kinetics of volatile anaesthetics. Implications for malignant hyperthermia treatment

The effectiveness of three types of management on the elimination kinetics of volatile anaesthetics was studied prospectively in 45 patients randomised to one of three groups. Patients were anaesthetised using isoflurane. Inspiratory and expiratory isoflurane concentrations were measured. After reaching a steady-state isoflurane concentration, the vaporizer was turned off. In group 1, only the fresh gas flow was increased from 40 to 120 ml.kg-1 x min-1. Patients in group 2, in addition to the increase in the fresh gas flow, had a charcoal filter connected in the inspiratory limb of the circuit. Patients in group 3 had the fresh gas flow increased and the anaesthetic machine and breathing system changed. There was a statistically significant difference in the isoflurane washout from the anaesthetic machines between group 1 (90% elimination time 39 [10] s) and group 2 (90% elimination time 25 [5] s) (p < 0.01). However, there was no significant difference in the isoflurane washout from the patients in any of the groups. Thus the use of a charcoal filter or a change of the anaesthetic machine and breathing system proved to be of no clinical advantage.     

Anaesthetic machine and breathing system contamination and the efficacy of bacterial/viral filters

Contamination of the anaesthetic machine and breathing system by the environment and by patient exposure has been shown to occur. Outside the intensive care setting, however, it is difficult to demonstrate that the anaesthetic machine and breathing system are a vector for patient cross-infection. Bacterial and viral filters for use within the breathing system have been shown to be very effective for filtration, yet their use has not been demonstrated to be of benefit in the prevention of cross-infection between patients. Several instances of patient morbidity are a direct consequence of filter use. The use of bacterial/viral filters may represent another step towards defensive medical practice.     

Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter

Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both Staphylococcus epidermidis and Staphylococcus aureus. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum to the 32-kDa S. epidermidis lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of S. aureus was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in S. epidermidis and S. aureus and of 40 kDa in Staphylococcus hominis. No reactivity was detected with Staphylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemolyticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis has been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type transporter. The operon contains three genes, designated sitA, -B, and -C, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively. SitC shows significant homology both with a number of bacterial adhesins, including FimA of Streptococcus parasanguis and ScaA of Streptococcus gordonii, and with lipoproteins of a recently described family of ABC transporters with proven or putative metal ion transport functions. Although the solute specificity of this novel transporter has not yet been determined, we speculate that it may be involved in either siderophore- or transferrin-mediated iron uptake in S. epidermidis.     

Characteristics of the staphylococci isolated from the udder of cows with mastitis

A total of 175 strains of Staphylococcus aureus and 67 strains of Staphylococcus epidermidis were studied, isolated from 486 samples of milk secretion taken aceptically from the individual quarters of the udder of cows affected with subclinical and purulent (clinical) mastitis. The staphylococci were referred to as the causative agent of mastitis in case they were the only microflora in the seedings of the investigated material. Tests were applied as given in Fig. 1 to characterize the strains. It was found that mastitis in cows could be due to both plasma coagulating staphylococci (Staphylococcus aureus) and coagulase-negative Staphylococcus epidermidis organisms. The two Staphylococcus species were isolated from cows with clinical and subclinical mastitis. The division between pathogenic and nonpathogenic Staphylococcus strains by the plasma-coagulating symptom proved impossible, and this made it necessary to use other tests for pathogenicity. It became evident that the thing Staph. aureus and Staph. epidermidis had in common when isolated from cows with mastitis was the production of a gold-like pigment and delta hemolysin. Similarly to Staph. aureus isolated animals, the bovine Staph. epidermidis organisms did not possess fibrinolysin and rarely produced hemolysin. The isolated organisms belonging to the coagulase-positive staphylococci corresponded by their basic properties to Staphylococcus aureus var. bovis as described in the literature. The cultures of Staphylococcus epidermidis isolated under similar conditions showed in a considerable per cent of the cases somewhat different behaviour.     

Bacterial strains isolated from neutropenic patients and their resistance to antibiotics

BACKGROUND: Although Gram negative as well as Gram positive bacteria participate in febrile episodes of neutropenic patients, in particular recently the ratio of Gram positive bacteria is increasing. The objective of the present work was to investigate the incidence and antibiotic resistance of pathogenic bacterial agents in neutropenic patients. METHODS AND RESULTS: The presence of bacteria was investigated in 446 neutropenic patients hospitalized at the Haematological Clinic in 1995. Haemocultures (apparatus Bact/Alert 120, cultivation media Organon-Teknika) and urine were examined. The sensitivity for antibiotics was tested by the standard dilution micromethod. In blood most frequently Staphylococcus epidermidis was isolated (45.4%), coagulase-negative strains of Staphylococcus haemolyticus, Staphylococcus hominis and Staphylococcus saprophyticus (14.4%), Acinetobacter calcoaceticus-baumannii (complex 6.3%) and Pseudomonas aeruginosa (6.3%). In urine the following were detected: Staphylococcus epidermidis (36.5%), Enterococcus sp. (14.5%), Escherichia coli (13.1%), Enterococcus faecalis (11.6%) and Enterococcus solitarius (6.5%). In all strains resistance to antibiotics and chemotherapeutic drugs was assessed. CONCLUSIONS: Investigation of the frequency of different bacterial species, along with monitoring of the resistance is an essential prerequisite of initial antibiotic therapy of febrile episodes in neutropenic patients.     

Zoonoses as occupational diseases in the DDR

To determine the incidence of bacteremia in healthy adults, blood cultures were obtained from 240 patients who had no demonstrable foci of infection. Five patients (2.1%) had positive blood cultures. Staphylococcus epidermidis was isolated from four patients and Alcaligenes faecalis from one. In each of these patients, the organism isolated probably represented contamination rather than bacteremia.     

Upregulation of urokinase-type plasminogen activator by endogenous and exogenous HIV-1 Tat protein in tumour cell lines derived from BK virus/tat-transgenic mice

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods was developed. The protocol used a universal culture medium and the same PCR conditions with 13 sets of specific primers. The 13 species of foodborne pathogens examined were Escherichia coli, E. coli-ETEC, E. coli-O157:H7, Shigella spp., Salmonella spp., Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. No interference was observed using the PCR assay when food sample was artificially inoculated with each individual bacterial species. Twelve different seafood samples and two soft cheese samples without artificial inoculation were examined by this protocol. Vibrio vulnificus, Salmonella spp., E. coli, Listeria monocytogenes and Bacillus cereus were detected in some foods. Internal probe hybridization and nested PCR procedures were used to confirm the above findings.     

Comparing two heat and moisture exchangers, one hydrophobic and one hygroscopic, on humidifying efficacy and the rate of nosocomial pneumonia

OBJECTIVE: Many heat and moisture exchangers with filter (HMEF) have been developed. In-house data from companies provide some information about their performances; unfortunately, to our knowledge, no comparative evaluation in clinical conditions has been undertaken of these newer products. The aim of this study was to compare the efficiency of two HMEFs, one hydrophobic and one hygroscopic, on humidifying capacity and the rate of bronchial colonization and ventilator-associated pneumonia in ICU patients. DESIGN: Prospective, randomized study. SETTING: ICU of a university hospital. PATIENTS: All patients who required mechanical ventilation for > or = 24 h during the study period. INTERVENTIONS: On admission to the ICU, patients were randomly assigned to one of two groups. In one group, the patients were ventilated with a hygroscopic device (Humid-Vent Filter Light HMEF; Gibeck; Upplands Vaesby, Sweden). The condensation surface was made of paper (Microwell) impregnated with CaCl2. The filter membrane was made of polypropylene. In the other group, the patients were ventilated with a hydrophobic device (Pall BB100 HMEF). The condensation surface was made of a hydrophobic resin with a hydrophylic layer. The filter membrane was made of ceramic fibers. In both groups, HMEFs were changed daily. MEASUREMENTS AND RESULTS: Both groups of patients were similar for the tested characteristics, including parameters of mechanical ventilation. Sixty-six patients were ventilated for 11.7+/-11 days with the Humid-Vent Filter Light HMEF and 70 patients for 12.2+/-12 days with the Pall BB 100. Patients ventilated with the Humid-Vent Filter Light underwent 6.0+/-3.0 tracheal aspirations and 1.7+/-2.0 instillations per day, and those with the Pall BB 100, 6.0+/-3.0 and 1.6+/-2.0 per day, respectively (not significant [NS]). Abundance of tracheal secretions, presence of blood, and viscosity, evaluated by semiquantitative scales, were similar in both groups. No difference in the rate of atelectasis was observed between the two groups (7.5% and 7.1%, NS). One episode of tracheal tube occlusion was observed with the Humid-Vent Filter Light HMEF, and one with the other HMEF (NS). One patient in each group (NS) was switched to an active heated humidifier because of very tenacious bronchial secretions despite repeated instillations. Tracheal colonization was observed at a rate of 67% with the Humid-Vent Filter Light and 58% with the Pall BB 100 (NS). A small, but NS difference was observed in the rate of ventilator-associated pneumonia: Humid-Vent Filter Light, 32% (27.1 per 1000 ventilator days); and Pall BB 100, 37% (30.4 per 1000 ventilator days). Bacteria responsible for tracheal colonization and pneumonia were similar in both groups. Three patients in each group died from their nosocomial pneumonia. CONCLUSION: Despite differences in their components, the two HMEFs tested achieved similar performances in terms of humidification and heating of inspired gases. Only one episode of endotracheal tube occlusion was detected and very few patients (one in each group) had to be switched to an active heated humidifier. No difference was observed either in the rate of tracheal colonization or of ventilator-associated pneumonia. These data show that the hygroscopic HME (Humid-Vent Filter Light) and the hydrophobic HME (Pall BB 100) are suited for use in ICU patients.     

Bacterial growth in human vitreous humor

The aim of this study was to investigate the capacity of human vitreous to support bacterial growth and to show differences in the growth kinetics of gram-positive and gram-negative bacteria. Vitreous gel of 70 keratoplasty donor eyes was sampled under sterile conditions, screened microscopically for cellular components and tested for sterility and levels of antibiotic drugs by bio-assay. The samples were inoculated with clinical isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans and Streptococcus pyogenes. As control each strain was added both to 0.9% sodium chloride solution and to Mueller-Hinton broth. In order to determine bacterial growth the number of colony forming units was determined 4, 6, 24, 48 and 72 hr after inoculation by viable count. Vitreous gel did not support bacterial growth; the tested strains could not be recovered after 48 hr. Similar results could be obtained with sodium chloride; whereas in Mueller Hinton broth the strains showed normal pattern of growth. It seems that vitreous humor has inherent antibacterial capacity in vitro, although the responsible factors remain unknown.     

Differentiation of staphylococci from sheep and goat milk samples

A total of 447 micrococcaceae strains isolated from 88 ewe and 359 goat milk samples from cases of chronic mastitis were differentiated by means of the ATB 32 STAPH-test. Of these strains 389 (= 87%) could be identified. Fourteen strains were sensitive in the bacitracin-resistance-test and therefore classified as Micrococcus spp. In ewe milk following Staphylococcus spp. were found: S. epidermidis, S. aureus, S. lentus, S. xylosus, S. warneri, S. equorum, S. haemolyticus, S. simulans, S. hominis and S. saprophyticus. Staphylococcus spp. identified in goat milk samples were: S. epidermidis, S. aureus, S. caprae, S. lentus, S. simulans, S. capitis, S. lugdunensis, S. xylosus, S. chromogenes, S. hominis, S. arlettae, S. warneri, S. sciuri, and S. saprophyticus. Highest cell counts in the milk of both animals species, and the highest incidence of clinical udder alterations were caused by S. aureus. Increases in milk cell counts as well as pathological udder findings were observed in coagulase-negative staphylococcal infections for novobiocin-sensitive Staphylococcus spp. (S. epidermidis, S. warneri, S. simulans, S. lugdunensis, and S. chromogenes) and several S. lentus strains.     

Ultrasonic enhancement of antibiotic action on gram-negative bacteria

The effect of gentamicin upon planktonic cultures of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Staphylococcus aureus was measured with and without application of 67-kHz ultrasonic stimulation. The ultrasound was applied at levels that had no inhibitory or bactericidal activity against the bacteria. Measurements of the MIC and bactericidal activity of gentamicin against planktonic cultures of P. aeruginosa and E. coli demonstrated that simultaneous application of 67-kHz ultrasound enhanced the effectiveness of the antibiotic. A synergistic effect was observed and bacterial viability was reduced several orders of magnitude when gentamicin concentrations and ultrasonic levels which by themselves did not reduce viability were combined. As the age of the culture increased, the bacteria became more resistant to the effect of the antibiotic alone. Application of ultrasound appeared to reverse this resistance. The ultrasonic treatment-enhanced activity was evident with cultures of P. aeruginosa and E. coli but was not observed with cultures of gram-positive S. epidermidis and S. aureus. These results may have application in the treatment of bacterial biofilm infections on implant devices, which infections are usually more resistant to antibiotic therapy.     

Extracellular and bacterial factors influencing staphylococcal phagocytosis and killing by human polymorphonuclear leukocytes

Extracellular and bacterial factors that influence the phagocytosis and killing of staphylococci by human polymorphonuclear leukocytes have been studied. Staphylococcus epidermidis strains were, in general, more rapidly phagocytized than were S. aureus strains. However, two strains of S. epidermidis had a very slow rate of ingestion. Although the rate of phagocytosis of S. aureus Wood 46 was greater than that of S. aureus 502A, the Wood 46 strain was more difficult to kill. Serum was essential for phagocytosis of both S. aureus and S. epidermidis. The opsonic titer of pooled serum was similar for S. aureus and S. epidermidis. In normal pooled serum, heat-labile factors were more important for effective phagocytosis than they were in immune serum. Although a saturation point for ingestion was reached, the percentage of ingested bacteria that remained alive within the leukocyte remained relatively fixed. Heat-killed and live staphylococci were igested in a similar fashion. The rate of phagocytosis was greatly reduced at 41 degrees C.     

Response time studies of a new, portable mass spectrometer

Needleless connectors have been widely introduced into clinical practice to allow the connection of syringes and luers to peripheral and central vascular catheters. The potential for microbial contamination of catheters via these devices is currently unclear. A recently introduced connector, the 'Connecta Clave', was assessed by various in-vitro methods. The 'Connecta Clave' is specifically devised to separate external components from the fluid pathway. The compression seals of 50 devices were contaminated with 1 x 10(4) cfu Staphylococcus epidermidis, disinfected with isopropanol, and fluid passed through. Only one device allowed organisms to pass through, despite this challenge, representing a contamination rate of 2%. In comparison, when 50 connectors were challenged with 20 cfu of S. epidermidis, no organisms passed through the device during use. In the clinical situation, after manipulation, < 16 cfu of skin organisms were found associated with the compression seal of the devices. It is, therefore, likely that the contamination rates in clinical practice will be extremely low. Three methods of disinfecting the compression seals and associated rims were also evaluated. A combination of alcohol chlorhexidine spray, followed by a 70% isopropanol swab, resulted in the most efficacious disinfection. The isopropanol swabs produced an adequate disinfection rate. The overall results suggest that by use of specially designed connectors, not only are needlestick injuries reduced, but the likelihood of microbial contamination of catheters via the internal route may also be diminished.