A validation survey of 197 hospital steam sterilizers in The Netherlands in 2001 and 2002.

Steam sterilization is the most common method of sterilization used in hospitals and by companies sterilizing for hospitals. This study validated 197 steam sterilizers with respect to technical condition, various production processes and routine control tests, according to the European norms and standards for steam sterilization. Overall, only 40% of the validated steam sterilizers met the norms and standards. We recommend that adequate measures need to be taken, based on the comments in the validation reports, in order to guarantee the sterility of processed medical items.

Sequence variation in the hpd gene of nonencapsulated Haemophilus influenzae isolated from patients with chronic bronchitis.

The molecular diversity of protein D of nonencapsulated Haemophilus influenzae strains isolated from persistently infected patients with chronic bronchitis was studied by sequencing the hpd gene of four independently obtained isolates. The nucleotide (nt) sequences of the hpd genes of two strains were identical. The other two hpd sequences showed nt substitutions which were mostly synonymous. As a consequence the deduced amino acid (aa) sequences differed from the consensus sequence only by a few aa. No changes in the hpd genes were observed among the four variants of the four strains persisting in chronic bronchitis patients for 9, 11, 8 and 3 months, respectively, although variation in their major outer membrane proteins P2 and P5 occurred. We conclude that the hpd gene is conserved during chronic infections of nonencapsulated H. influenzae.

Absence of Lyme borreliosis among patients with presumed Bell's palsy.

In a prospective study, 69 patients with a presumed idiopathic (Bell's) peripheral facial palsy were clinically and serologically evaluated for the presence of Lyme borreliosis. In addition, their clinical spectrum was compared with clinical manifestations collected retrospectively in nine patients with symptomatic peripheral facial palsy due to Lyme borreliosis. The seroprevalence of Borrelia burgdorferi antibodies, determined by flagellum enzyme-linked immunosorbent assay, among 69 patients with idiopathic peripheral facial palsy (6%) and 153 healthy controls (4.5%) was not significantly different (odds ratio, 1.28; 95% confidence interval, 0.27 to 5.25). None of the patients with idiopathic peripheral facial palsy had or experienced the development of Lyme borreliosis. All patients with Lyme peripheral facial palsy had additional manifestations not present in patients with idiopathic peripheral facial palsy. These findings show that patients with a Lyme peripheral facial palsy can be differentiated from patients with idiopathic peripheral facial palsy by clinical examination. Therefore, screening of antibodies to B burgdorferi among patients with idiopathic peripheral facial palsy without additional manifestations is not recommended.

Isolation of the C9b fragment of human complement component C9 using urea in the absence of detergents.

The bactericidal activity of the C5b-9 complex of complement is dependent upon the terminal complement component C9. The precursor C5b-8 complex is not harmful to bacterial cells until C9 is added to complete the C5b-9 complex. The C9 molecule can be proteolytically cleaved by thrombin to yield an intact, nicked molecule that remains fully functional when added to either bacterial cells or erythrocytes bearing pre-formed C5b-8 complexes. In investigating the membranolytic function of C9 in the C5b-9 complex, the carboxyl-terminal portion of the nicked molecule (C9b) has been shown to be membranolytic when added to erythrocytes, liposomes, or bacterial inner membranes in the absence of any other complement components. The isolation of C9b from nicked C9 has been accomplished by preparative gel electrophoresis using detergents, however the study of the activity of C9b in membrane systems may be complicated by the possible presence of residual detergent. To address this concern, we have used 4 M urea in conjunction with hydroxyapatite chromatography and a phosphate elution procedure to separate the domains of nicked C9. The isolated C9b domain, free of detergents and in the absence of any other complement components, was found to be membranolytic. C9b isolated in this manner was capable of lysing erythrocytes and inhibiting the growth of bacterial spheroplasts.

Isolation of hepatitis B surface antigen (HBsAg) by affinity chromatography on antibody-coated immunoadsorbents.

Hepatitis tb surface antigen (HBsAg) was isolated from human serum by two steps of affinity chromatography on antibody-coated gels. HBsAg-positive serum was passed through a column packed with guinea pig anti-HBsAg antibodies covalently bound to CNBr-activated beaded agarose gel. The majority of non-specifically bound proteins was removed by washing the gel with increased concentrations (0.5 M) of NaCl in Tris buffer. Elution of the specifically bound HBsAg was carried out with 3 M NaSCN. Residual normal human serum proteins present in the eluate were removed by passing the partially purified HBsAg through an immunoadsorbent coated with rabbit antibodies directed against human serum proteins. After this treatment normal human serum proteins could no longer be demonstrated by passive hemagglutination in the isolated HBsAg. Cross-reactions between HBsAg and normal human serum proteins could not be demonstrated. Both antibody-coated immunoadsorbents could be used over ten times without significant loss of their binding capacity.

Incidence of postextraction bacteremia under penicillin cover in children with cardiac disease.

To assess the efficacy of penicillin prophylaxis as recommended by the American Heart Association to prevent the onset of bacterial endocarditis, the incidence of postextraction bacteremia was determined in 82 children with cardiac disease who were receiving prophylactic penicillin. Aerobic and anaerobic blood cultures were taken five minutes after dental extraction, as was a blood sample to assay the serum penicillin concentration. The incidence of postextraction bacteremia was 21%. Streptococcal species accounted for half of the number of aerobes isolated. Of the isolated microorganisms, 16% were strict anaerobes. Susceptibility testing of the isolates showed that 24 penicillin-sensitive and eight penicillin-resistant microorganisms caused the bacteremia. There was neither a significant difference between the serum penicillin concentrations of children with and without bacteremia, nor between the serum penicillin concentrations of children with bacteremia due to penicillin-sensitive microorganisms and children with bacteremia due to penicillin-resistant microorganisms. It is concluded that the occurrence of postextraction bacteremia is not prevented by penicillin prophylaxis and that the serum penicillin concentration at extraction is not the discriminating factor in preventing this bacteremia. None of the children developed bacterial endocarditis. It is speculated that mechanisms not thoroughly studied are involved in the prevention of bacterial endocarditis after dental extraction.

Immunoglobulin A antibodies against Pseudomonas aeruginosa in the tear fluid of contact lens wearers.

PURPOSE: Pseudomonas aeruginosa is the most important cause of contact lens-associated ulcerative keratitis, especially for those who use extended-wear lenses. Until now, the presence of specific anti-P. aeruginosa immunoglobulin A (IgA) antibodies in the tears of contact lens wearers has not been investigated and is the purpose of the current study. METHODS: The levels of specific IgA antibodies against P. aeruginosa and total secretory IgA (s-IgA) concentrations were measured in tears of various groups of contact lens and non-contact lens wearers using enzyme-linked immunosorbent assays. Contact lens groups were divided into the following categories: daily-wear rigid gas-permeable lenses (n = 23), daily-wear soft lenses (n = 22), extended-wear soft lenses (n = 17), and non-contact lens wearers (n = 23). As a positive control group, we tested tears obtained from patients with cystic fibrosis (n = 5) because the respiratory tract of these persons often are colonized by P. aeruginosa. RESULTS: The percentage of nonresponders (< 15 U/ml) varied between 9% in daily-wear rigid gas-permeable contact lens users to 23% in daily-wear soft contact lens users. The percentage of nonresponders in controls was 13%. The frequency of nonresponders was not significantly different among the different groups tested. All patients with cystic fibrosis showed a very high anti-P. aeruginosa IgA response in their tears. When analyzing the mean anti-P. aeruginosa IgA response, a significantly lower level was found in extended-wear contact lens users (38 U/ml) compared to non-contact lens wearers (82 U/ml). Total s-IgA levels in the tears of the various groups tested were not significantly different. CONCLUSIONS: A substantial number of persons in the population of contact lens wearers tested lack detectable IgA antibodies against P. aeruginosa in their tears and may be susceptible to P. aeruginosa keratitis if the physiological condition of their cornea is compromised.

The population structure of Neisseria meningitidis serogroup A fits the predictions for clonality.

The population structure of Neisseria meningitidis is supposedly epidemic according to. The model predicts that linkage disequilibrium in N. meningitidis populations is only temporary and arises due to the outgrowth of highly successful clonal genotypes from an essentially sexual population. These clones should disappear after a few years because of frequent recombination. In contrast, multilocus enzyme electrophoresis (MLEE) data had previously been interpreted as showing that serogroup A meningococci are truly clonal and possess only limited genetic variability (Wang et al., 1992). The two interpretations are contradictory. In order to elucidate the true population structure of serogroup A meningococci, we analyzed data for a representative group of 84 serogroup A isolates obtained by MLEE, random amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). Analysis of linkage disequilibrium and bootstrap analyses of cluster analysis showed a strongly structured population with highly significant linkage disequilibrium. This was not due to the overrepresentation of certain genotypes, in contrast to the expectations for an epidemic population. The analyses identify two main clades, within each of which linkage disequilibrium was also highly significant, thus, excluding a cryptic speciation model. These observations support a population structure based on clonal evolution, in which clones are much more stable than expected for epidemic clonality. We propose that serogroup A meningococci may possess a different population structure from other serogroups of Neisseria meningitidis.

Two distinct ospA genes among Borrelia valaisiana strains.

Borrelia valaisiana is a recently described bacterial species in the Borrelia burgdorferi sensu lato complex. To further characterize this bacterium, the plasmid-encoded ospA genes from eight B. valaisiana isolates were amplified by PCR, cloned and sequenced. All B. valaisiana isolates studied possessed an ospA gene with a size of 822-825 bp. The identity of the predicted amino acid sequences of the OspA proteins among B. valaisiana isolates was 69.1-100%, and ranged from 68.2 to 79.1% between B. valaisiana and other B. burgdorferi sensu lato species. Based on the OspA protein sequences, the eight B. valaisiana isolates could be distinguished into two subgroups. Subgroup I contained six B. valaisiana isolates of which OspA sequences were almost identical, but clearly differed from other LB spirochetes. Subgroup II consisted of two isolates with identical OspA sequences which were only 70% identical to subgroup I B. valaisiana isolates and similarly distant from the OspA sequences of other B. burgdorferi sensu lato genospecies. Phylogenetic analysis indicates that B. valaisiana isolates belonging to subgroups I and II possibly evolved from two distinct ancestors. Our data showed for the first time a major difference in OspA proteins within a well-defined B. burgdorferi sensu lato species at the evolutionary level, suggesting that it is not always reliable to assign Borrelia isolates to a definite species solely based on data from ospA gene sequence analysis.

Reinfection versus recrudescence in Helicobacter pylori infection.

Antimicrobial treatment of Helicobacter pylori is the proper management strategy in patients with ulcers. A high rate of H. pylori reinfection after successful eradication therapy however, may give rise to ulcer recurrence. The risk of reinfection, depending on the prevalence and the rate of acquisition of H. pylori infection, varies with socioeconomic status, age and geographical location. The rate of reinfection may vary in a similar way. The available data in the literature reveal that reinfection by H. pylori is low or absent in developed countries and may be lower than the initial rate of acquisition. In addition, reported cases of H. pylori reinfection are often cases of recrudescent H. pylori infection. Acquisition rate in developing countries is high, so the reinfection rate is expected to be higher than in developed countries. However, studies discriminating reinfection from recrudescence are lacking and therefore more data from developing regions are needed to settle if 'cured once, cured forever' holds true.

Treatment of Helicobacter pylori infection with low or high dose omeprazole combined with amoxycillin and the effect of early retreatment.

BACKGROUND: Cure rates of H. pylori infection, using dual therapy with omeprazole and amoxycillin, vary considerably and the efficacy of retreatment with this regimen in the case of initial failure is controversial. Therefore, we conducted a large prospective double-blind randomized trial, studying the efficacy of low vs. high dose omeprazole in dual therapy and of early retreatment with the same regimens. METHODS: One hundred and sixty-eight consecutive H. pylori-positive patients, suffering from either peptic ulcer disease or functional dyspepsia, were enrolled. Group I (n = 84) received omeprazole 20 mg b.d. plus amoxycillin 750 mg t.d.s., for 2 weeks. Group II (n = 84) received omeprazole 40 mg t.d.s. plus amoxicillin 750 mg t.d.s., for 2 weeks. RESULTS: The H. pylori eradication rate was 60.2% in group I and 64.3% in group II (P = 0.59). Cure of H. pylori infection was significantly better in patients with peptic ulcer disease, compared to non-ulcer dyspeptics (P = 0.016). Retreatment, given in 54 patients, was successful in 21.4% patients in group I and in 28% patients in group II (P = 0.58). CONCLUSIONS: High dose of omeprazole has no advantage compared to low dose in terms of eradication efficacy. Early retreatment with the same regimen offers limited improvement in cure rate. Presence of peptic ulcer disease influences cure rates significantly.

Adhesion of Escherichia coli on to a series of poly(methacrylates) differing in charge and hydrophobicity.

The adhesion of three Escherichia coli strains on to six poly(methacrylates) differing in hydrophobicity and surface charge was measured as a function of time under laminar flow conditions. Polymers used were poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA) and copolymers of MMA or HEMA with either 15% methacrylic acid (MAA) or 15% trimethylaminoethyl methacrylate-HCl salt (TMAEMA-Cl). Bacterial and polymer surfaces were characterized by means of water contact angles and zeta potentials. Both the sessile drop contact angles and the zeta potentials of the bacterial surfaces were significantly different. No significant differences in the sessile drop contact angles of the polymer surfaces were observed. Using the Wilhelmy plate technique large contact angle hysteresis was observed for the different polymer surfaces. Surfaces of copolymers with MAA had more negative zeta potentials than those of the corresponding homopolymers. Surfaces of copolymers with TMAEMA-Cl had positive zeta potentials. The highest numbers of adherent bacteria were found on materials with positive zeta potentials, irrespective of the bacterial strain used. Bacterial adhesion on to copolymers with MAA was less than on to the corresponding homopolymers. Bacterial equilibrium adhesion values correlate with the zeta potentials of the polymer surfaces (r greater than 0.85). On substrates with less negative zeta potentials high numbers of adhered bacteria were observed. Additionally, the equilibrium bacterial adhesion values could be related with receding contact angles of polymer surfaces with negative zeta potentials (r greater than 0.86). High equilibrium adhesion values were obtained for polymers with high contact angles. No correlation between the zeta potentials and contact angles of the bacteria with the adhesion values was found.

In vitro and in vivo evaluation of gelatin-chondroitin sulphate hydrogels for controlled release of antibacterial proteins.

Chemically cross-linked gelatin-chondroitin sulphate (ChS) hydrogels, impregnated in Dacron, were evaluated as drug delivery systems for antibacterial proteins. The gelatin-chondroitin sulphate gels, plain or impregnated in Dacron, were cross-linked with a water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The release of lysozyme and recombinant thrombocidin (rTC-1), an antibacterial protein derived from human blood platelets, from the gelatin-ChS gels in Dacron in phosphate-buffered saline at 37 degrees C was determined, and compared to the release from gelatin gels in Dacron and plain gelatin-ChS gels. The incorporation of chondroitin sulphate into gelatin gels, caused a marked increase in lysozyme loading capacity, and a slower release rate. The relative release profiles for rTC-1 and lysozyme were equal for cross-linked gelatin as well as for cross-linked gelatin-ChS gels. Furthermore, rTC-1 showed no loss of antibacterial activity after 1 week of release. The lysozyme concentration profiles in the samples and in the surrounding medium as a function of time were calculated using mathematical solutions for Ficks second law of diffusion for a semi-infinite composite medium, which is a schematic representation of a slab in a surrounding medium. The biocompatibility and degradation of the Dacron matrices impregnated with gelatin-ChS gels was studied after implantation in subcutaneous pockets in rats. Chemically cross-linked gelatin-Ch5 gels showed a mild tissue reaction, and almost complete degradation within 18 weeks of implantation.

Identification of viridans streptococci on the Minitek Miniaturised Differentiation System.

A total of 227 strains of viridans streptococci were simultaneously identified on the Minitek Miniaturised System (BBL) and by a conventional method according to Colman and Williams. The Minitek discs were each overlaid with a drop of sterile liquid paraffin, and the trays were incubated in GaSPak jars (BBL) with CO2 generator envelopes. Identification was possible three to four days earlier than with the conventional method. The results were found to be in agreement with the conventional method. Compared to the identification schemes of Cowan and Steel and of Facklam, the results were also in good agreement. Minor differences were found in the number of positive and negative results in those reactions that are variable in all three schemes.

Purification of specific precipitinogen and extraction of endotoxin from Haemophilus influenzae.

After purifying a Haemophilus influenzae precipitinogen from endotoxic activity by means of ultracentrifugation, column chromatography (Sepharose 6B) and ion exchange chromatography (DEAE Sephadex A25) a fraction was obtained which still contained a specific precipitinogen that was virtually free of endotoxin. Furthermore, during the chromatographic procedures fractions with a high and a low molecular weight endotoxic activity were found. The limulus lysate test was more sensitive in the high molecular weight fractions and the LD50 in mice in the low molecular weight fractions with endotoxic activity.

Frequency of pathogenic and enteroadherent Escherichia coli in patients with inflammatory bowel disease and controls.

AIMS: To determine whether inflammatory bowel disease (IBD) is associated with pathogenic or enteroadherent Escherichia coli. METHODS: A least two stool specimens and one rectal biopsy were taken from 30 patients with IBD and from 20 controls. A large number of E coli-like colonies cultured from each stool sample and biopsy was tested, using DNA probes, for the presence of genes encoding shiga-like toxins, invasiveness, attachment-effacement and the ability to adhere to HEp-2 cells. Similarity among isolates from stool samples and rectal biopsies was determined by random amplified polymorphic DNA (RAPD) analysis. RESULTS: Enterohaemorrhagic and enteroinvasive E coli were not found in samples from either patients or controls. No significant difference in the detection rate of enteroadherent E coli between patients and controls was found. Rectal biopsies from 11 of 28 patients with IBD and 4 of 18 controls contained E coli, which hybridised with probes for detection of genes encoding diffuse adherence to HEp-2 cells, or encoding P-pili (p = 0.2). Enteroadherent E coli isolated from two or three stool specimens from the same patient or control appeared to be identical by RAPD analysis, and are considered to be residents in the colon. Probe positive isolates obtained from stool specimens and corresponding rectal biopsies were always identical on RAPD analysis. CONCLUSIONS: E coli strains possessing adherence factors reside in the large intestine and adhere to the rectal mucosa, irrespective of the presence of colitis.

Diagnosis of intestinal and disseminated microsporidial infections in patients with HIV by a new rapid fluorescence technique.

AIMS: To assess the value of a new rapid fluorescence method for the diagnosis of microsporidiosis in HIV seropositive patients. METHODS: Microsporidian spores in stools were demonstrated by using the fluorochrome stain Uvitex 2B. The new technique was evaluated in three groups of HIV seropositive patients with diarrhoea. Group 1: 19 patients with biopsy confirmed E bieneusi infection (186 stool samples); group 2: 143 consecutive patients from whom faeces were submitted for routine investigation of diarrhoea (318 samples); group 3: 16 patients with small intestinal biopsy specimens negative for microsporidia (55 samples). The new method was used to monitor spore shedding during experimental treatment with paromomycin and albendazole in four patients. RESULTS: Brightly fluorescent spores were detected in all stool samples of patients in group 1. In group 2 16 (11%) patients had spores in their stool samples. E bieneusi was found in 11 patients; in the other five another genus of microsporidia, Encephalitozoon, was recognised. Encephalitozoon spores were also found in the urine of three of these patients and in the maxillary sinus aspirate of two of them, suggesting disseminated infection. The results were confirmed by electron microscopic examination. In group 3 negative biopsy specimens were confirmed by negative stool samples in all cases. Treatment with albendazole and paromomycin did not affect the spore shedding in three patients with E bieneusi infection. By contrast, in a patient with Encephalitozoon sp infection albendazole treatment resulted in clinical improvement together with complete cessation of spore excretion in the stool. CONCLUSION: The Uvitex 2B fluorescence method combines speed, sensitivity, and specificity for the diagnosis and treatment evaluation of intestinal and disseminated microsporidiosis.

Ultrastructural distribution of the M form of creatine phosphokinase in human muscle by immunogold labeling.

Creatine phosphokinase regenerates ATP from ADP using creatine phosphate. Isoenzymes of creatine phosphokinase are bound to certain cellular structures or are compartmentalized in areas of the cell, and this has been used as a basis for defining the role of these isoenzymes in energy metabolism. The M isoenzyme of creatine phosphokinase has been morphologically associated with the M-line of striated muscle in many species. In this present study the ultrastructural distribution and the relative concentration of the M form of creatine phosphokinase in human muscle tissue was determined using immunogold and electron microscopy. The M-line of the sarcomere, comprising only 3-4% of the sarcomere area, was found to contain over 20% of the total M isoenzyme signal of the entire sarcomere. This technique represents a quantitative, ultrastructural method to study the subcellular distribution of this isoenzyme. These data suggest that localized concentrations of M-CPK may be important for normal energy metabolism, and may also serve as a foundation for a better understanding of the relationship between abnormal creatine metabolism and the pathogenesis of neuromuscular disease.

Evidence for frequent OspC gene transfer between Borrelia valaisiana sp. nov. and other Lyme disease spirochetes.

Molecular polymorphism of the ospC gene has been reported in Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii, the spirochetes causing human Lyme borreliosis. To assess the genetic relationship between ospC genes from the recently described Borrelia valaisiana sp. nov. and other B. burgdorferi sensu lato species, the ospC genes from eight B. valaisiana isolates were amplified by PCR, cloned and sequenced. The ospC genes of three B. valaisiana isolates were identical, but clearly distinct from ospC genes from other Borrelia species. Four B. valaisiana isolates possessed ospC genes more related to those of B. garinii, and fell into a cluster representing B. garinii species in the phylogenetic tree. One isolate had an ospC gene encoding a protein identical to that of B. afzelii strain. Since five of the eight (62.5%) B. valaisiana isolates contained a gene highly homologous or even identical to ospC genes found among B. garinii and B. afzelii strains, our findings indicate that ospC gene transfer occurs between B. valaisiana and other Lyme disease spirochetes.

Identification and characterization of a cross-reactive and a unique B-cell epitope on the hsp60 homologue from Neisseria gonorrhoeae.

Two monoclonal antibodies (G6 and 7B), generated against a 63-kDa stress protein (GSP63) from Neisseria gonorrhoeae strain VP1, were used to investigate the antigenic heterogeneity of GSP63 among the Neisseriaceae and its antigenic relationship with the Hsp60 heat-shock protein family. Immunoblotting experiments demonstrated antibody reactivity with all pathogenic Neisseria tested and with some of the commensal strains. One of the antibodies (7B) cross-reacted with the 65-kDa M. bovis BCG heat-shock protein and with 14 out of the 21 similarly sized proteins in other bacterial species. The other antibody (G6) specifically recognized neisserial GSP63 homologues. These results demonstrate that GSP63 is a conserved neisserial antigen bearing both a unique neisserial B-cell epitope and a more widely distributed Hsp60 epitope.