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.

[The first effect of the national vaccination campaign against meningococcal-C disease: a rapid and sharp decrease in the number of patients]. / Eerste effect van landelijke vaccinatiecampagne tegen meningokokken-C-ziekte: snelle en sterke afname van het aantal patiënten.

OBJECTIVE: To describe the initial effects of the large-scale vaccination campaign in June-July of 2002 (1-5- and 15-18-year-olds) and September-November of 2002 (6-14-year-olds) on the incidence of group-C meningococcal disease in the Netherlands. DESIGN: Descriptive. METHOD: The incidence of meningococcal disease and the serogroup distribution were determined on the basis of the patient data associated with isolates of Neisseria meningitidis that were sent to the Netherlands Reference Laboratory for Bacterial Meningitis during the period from 1 January 1999 to 31 January 2003. RESULTS: The highest monthly incidence of serogroup-C disease was reported in January-April 2002 (2.2-3.1/100,000), after which the incidence remained more or less unchanged after September at a level of 0.1-0.4/100,000. The incidence of meningococcal-C disease was 73% lower in August-October 2002 compared with the same period in 2001. In the same months, the incidence of meningococcal-C disease for the 0, 1-5, 6-14, 15-18 and > 18 year-olds was 49%, 80%, 89%, 89% and 42%, respectively, lower than in the same months in the previous year. The percentage of meningococcal disease caused by serogroup-C fluctuated from 35 to 49% in January-August 2002 and decreased to 9-19% in September-December 2002. There was also a decrease in the unvaccinated age groups. CONCLUSION: The vaccination campaign led almost immediately to a sharp decrease in the number of patients with meningococcal serogroup-C disease.

Functional activity of antibodies against the recombinant OpaJ protein from Neisseria meningitidis.

The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

Usefulness of Gram stain for diagnosis of lower respiratory tract infection or urinary tract infection and as an aid in guiding treatment.

During a prospective study of 8 months duration conducted in the Department of Internal Medicine and the Department of Pulmonary Diseases of the Academic Medical Centre, Amsterdam, Gram stainings of sputa and urine were performed for all patients whose clinical symptoms indicated an acute urinary tract infection or pulmonary infection. On the test request, the physician reported which antibiotic treatment he would prescribe if a microscopic examination was not available. The results of the Gram stain were discussed by the microbiologist with the physician, and the antibiotic therapy recommended by the microbiologist was recorded. This recommendation was compared with the antibiotic prescription noted in the patient record 1 day later. Two days after the results of final cultures and susceptibility tests became available, the patient record was again investigated for changes in the antibiotic regimen. Of 57 urine samples and 103 sputa, 27 and 85, respectively, were derived from patients with an infection of the urinary tract or respiratory tract. The results of the Gram stain confirmed the physician's suspicion 91% of the time for urinary tract infections and 81% of the time for pulmonary infections. In 67% of the patients with suspected lower respiratory tract infections and in 58% of patients with suspected urinary tract infections, the antibiotic treatment recommended on the basis of the results of the Gram stain differed significantly from the antibiotic treatment that the physician would have prescribed had a microscopic examination not been performed. The microbiologist's advice on antibiotic treatment was followed in 79% of the cases of respiratory tract infection and in 65% of the cases of urinary tract infection. The antibiotic treatment was adjusted to the final results of culture and antimicrobial susceptibility testing in 54% of the urinary tract infections and in 31% of the respiratory tract infections. The results indicate that the examination of sputa and urine in patients suspected to have an infection of the respiratory tract or urinary tract influences the antibiotic choice considerably.

Outbreak of meningococcal disease caused by PorA-deficient meningococci.

An outbreak of 7 cases of group C meningococcal disease occurred during the last week of July and the first week of August 2001 in the southwestern part of The Netherlands. Characterization of the 7 patients' isolates by various typing methods showed that the isolates were identical, except for the expression of PorA. Isolates from 5 patients were PorA deficient. These results show that transmission of PorA-deficient meningococci occurs and that PorA-deficient meningococci can cause invasive disease. PorA-based meningococcal vaccines may provide limited protection.

Listeria monocytogenes meningitis: serotype distribution and patient characteristics in The Netherlands, 1976-95.

Two hundred and seven cases of listeria meningitis that occurred in The Netherlands over 20 years were reviewed to study associations between Listeria monocytogenes serotype, age, underlying disease, and outcome. The mean annual incidence per 100,000 population was 0.12 in 1981-90, decreasing to 0.07 in 1991-5. Underlying disease was present in 50% of non-neonatal patients, most often haematological malignancy (15%) and the use of immunosuppressive therapy (14%). The meningitis-related case fatality rate was 16%; a significantly higher rate was associated with the presence of underlying disease (30%) or age > or = 70 years (29%). Serotype 4b was most frequent (65%) and L. monocytogenes types 1/2a, 1/2b, or 1/2c (30% of cases) were significantly more often isolated from non-neonatal patients with underlying disease, suggesting a higher virulence of listerial serotype 4b.

Complement activation and formation of the membrane attack complex on serogroup B Neisseria meningitidis in the presence or absence of serum bactericidal activity.

Encapsulated meningococci are complement sensitive only in the presence of bactericidal antibodies by yet-unexplored mechanisms. The objective of this study was to investigate the involvement of major bacterial surface constituents on complement activation and membrane attack complex (MAC) formation on serogroup B meningococci in the presence or absence of antibody-dependent serum bactericidal activity (SBA). The strains used were the encapsulated H44/76, five of its variants differing in capsulation and expression of the class 1 porin (PorA), and its lipopolysaccharide (LPS)-deficient isogenic mutant (LPS(-)) pLAK33. Two normal sera, one with high SBA (SBA(+)) and one with no bactericidal activity (SBA(-)) against H44/76 as well as an a-gamma-globulinemic serum were used for sensibilization of the bacteria. C3b and iC3b deposition on H44/76, its unencapsulated variant v24, and pLAK33 was similar in SBA(+) and SBA(-) serum, and no difference was present between the strains. MAC deposition on H44/76 was higher in SBA(+) serum than in SBA(-) serum and the a-gamma-globulinemic serum. The amounts of C3b on H44/76, v24, and pLAK33 in the a-gamma-globulinemic serum were also not different, indicating immunoglobulin G (IgG)- and LPS-independent complement activation. H44/76 PorA(+) and its PorA(-) variant and the v24 PorA(+) and its PorA(-) variant incubated in SBA(-) serum induced comparable amounts of MAC, despite their different serum sensitivities. Complement formation on the surface of the bacteria occurred almost exclusively via the classical pathway, but the considerable amounts of Bb measured in the serum indicated alternative pathway activation in the fluid phase. We conclude that complement deposition on meningococci is, for the most part, independent of classical pathway IgG and is not influenced by the presence of PorA or LPS on the meningococcal surface. Addition of an anti-PorA chimeric antibody to the nonbactericidal normal serum, while promoting a dose-related bacterial lysis, did not influence the amounts of C3b, iC3b, and MAC formed on the bacterial surface. These findings support the hypothesis that proper MAC insertion rather than the quantity of MAC formed on the bacterial surface is of importance for efficient lysis of meningococci.

Nosocomial outbreak of Clostridium difficile-associated diarrhoea due to a clindamycin-resistant enterotoxin A-negative strain.

A clindamycin-resistant toxin A-negative, toxin B-positive Clostridium difficile strain caused an outbreak among 24 hospitalized patients at the Department of Surgery, the Intensive Care unit, and the Department of Internal Medicine of an 800-bed academic hospital. Nineteen patients had undergone a surgical intervention and all 24 patients received at least one dose of antibiotics prior to the development of Clostridium difficile-associated diarrhoea. Twenty-seven episodes of Clostridium difficile-associated diarrhoea in 24 patients were categorized as mild (n=19), severe (n=7), or fatal (n=1). Relapses occurred in three patients. Nineteen of the 27 episodes required anti-Clostridium difficile treatment. Molecular typing performed by arbitrary primer polymerase chain reaction (PCR) and PCR amplification of rRNA intergenic spacer regions revealed that the outbreak strains recovered from culture were identical. The outbreak strain belonged to serogroup F and was resistant to erythromycin, clindamycin, and tetracycline, whereas susceptibility to chloramphenicol varied. No phenotypic activity of enterotoxin A was detected. A deletion of approximately 1.7 kb was found in the toxin A gene. Cytotoxin B had an unusual effect on cell culture assays that, at first, was not recognized as Clostridium difficile specific but could be neutralized with anti-Clostridium difficile B cytotoxin.

Decontamination of laryngoscopes in The Netherlands.

In this study the decontamination procedures of laryngoscopes in Dutch hospitals are described, based on a structured telephone questionnaire. There were substantial differences between decontamination procedures in Dutch hospitals and the standards of the APIC (Association of Professionals in Infection Control and Epidemiology), CDC (Centers of Disease Control) and ASA (American Society of Anesthesiology) were met in full in 19.4% of the hospitals. The standards of manual decontamination, used in 78% of the 139 hospitals, were particularly disappointing; manual cleaning was considered inadequate in 22.9% of these hospitals and manual disinfection did not meet the standards of the APIC, CDC or ASA in any of these hospitals. Decontamination by instrument cleaning machines as a standard procedure was used in 30 (22%) hospitals. In three of these hospitals the blades were subsequently sterilized. We suggest adherence to the infection control guidelines of the CDC, APIC and ASA, until the safety of less conservative infection control practices are demonstrated.

Mycoplasma pneumoniae P1 type 1- and type 2-specific sequences within the P1 cytadhesin gene of individual strains.

Mycoplasma pneumoniae strains traditionally are divided into two types, based on sequence variation in the P1 gene. Recently, however, we have identified 8 P1 subtypes by restriction fragment length polymorphism analysis. In the present study the P1 gene sequences of three P1 type 1 and two P1 type 2 M. pneumoniae strains were analyzed. A new P1 gene sequence in a type 1 strain with partial similarity to a recently reported variable region in the P1 gene of an M. pneumoniae type 2 strain (T. Kenri, R. Taniguchi, Y. Sasaki, N. Okazaki, M. Narita, K. Izumikawa, M. Umetsu, and T.Sasaki, Infect. Immun. 67:4557-4562, 1999) was identified. In addition, the P1 gene of the type 1 strain contained another region with nucleotide polymorphisms identical to a stretch in the P1 gene of one of our type 2 strains. These findings indicate that recombination between sequences specific for P1 type 1 and type 2 had occurred and that P1 type 1 and type 2 hybrid sequences can be present within the P1 gene of an individual strain. Identical or nearly identical variable P1 gene sequences were present in several repetitive regions outside the P1 gene locus in the genome of M. pneumoniae strain M129, implying recombination as a mechanism for generation of the P1 gene variation. Additionally, in the P1 gene sequences of four of the five strains studied, single-nucleotide polymorphisms different from the previously reported P1 type 1 and 2 characteristic sequences were identified. The polymorphic sites are candidate targets for genotyping of M. pneumoniae by direct sequencing of amplicons from clinical specimens.

Platelet microbicidal activity is an important defense factor against viridans streptococcal endocarditis.

To study the role of platelet microbicidal activity in host defense against infective endocarditis (IE) due to viridans streptococci (VS), the susceptibility to platelet releasate of blood and oral VS isolates from patients with and without IE was compared. The influence of neutralization of platelet microbicidal activity was studied in 2 experimental IE models. Resistance to platelet releasate was more prevalent among VS from blood of patients with IE than from blood of bacteremic patients without IE and among oral VS isolates. Serum from rabbits vaccinated with human platelet sonicate supernatants neutralized human and rabbit platelet-released microbicidal activity and had antibodies recognizing microbicidal proteins thrombocidin-1 and -2 and other human platelet proteins. In the 2 rabbit IE models, vaccination increased the susceptibility to experimental IE due to platelet releasate-susceptible VS. Thus, platelet-released microbicidal activity is an important host defense factor against IE due to VS.

[Sterilization of dental instruments]. / Het steriliseren van tandheelkundige instrumenten.

Instruments used by dentists are often sterilized with steam sterilization. At this moment three types of processes are described in the European norms: Type N, S and B. According to the norms type N can be used for non-wrapped non-hollow instruments and non-porous instruments, type S can be used for instruments specified by the manufacturer and type B can be used for wrapped, hollow and porous instruments. The principles on which the sterilizors are based are described. It is concluded that before purchasing the sterilizor the method of working and instruments to be sterilized must specified. In most cases a type B process is preferable in order to gaurantee to be certain to have an effective and reproducible process.

Clarithromycin-susceptible and -resistant Helicobacter pylori isolates with identical randomly amplified polymorphic DNA-PCR genotypes cultured from single gastric biopsy specimens prior to antibiotic therapy.

Of the Helicobacter pylori populations from 976 patients, six contained clarithromycin-resistant as well as -susceptible colonies. In each heterogeneous H. pylori population, resistant H. pylori colonies harbored identical 23S ribosomal DNA (rDNA) mutations associated with clarithromycin resistance, while the susceptible H. pylori colonies all had wild-type 23S rDNA. The resistant and susceptible colonies of each of the heterogeneous H. pylori populations had identical randomly amplified polymorphic DNA-PCR genotypes. In conclusion, evaluation of antimicrobial susceptibility can be misinterpreted if only a single colony from the primary H. pylori population is used to test for clarithromycin susceptibility.

Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation.

To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype.

Analysis of a VMP-like sequence (vls) locus in Borrelia garinii and Vls homologues among four Borrelia burgdorferi sensu lato species.

The VMP-like sequence (vls) locus that consists of one expressed vlsE gene and 15 silent vls cassettes has been described in Borrelia burgdorferi sensu stricto B31. In the present study, the vls locus from a Borrelia garinii isolate A87SA was analyzed. DNA fragments that contained three complete and five partial vls cassettes were cloned and sequenced. Pulsed-field gel electrophoresis (PFGE) analysis and Southern hybridization of the PFGE blot indicated that the vls locus of B. garinii A87SA, consisting of at least eight vls cassettes, was located on a 21-kb linear plasmid. The size of the three complete vls cassettes varied from 573 to 612 bp. They had 93.8-94.3% identity at the nucleotide level and 84.9-87.3% amino acid identity. The amino acid sequences of the three vls cassettes of B. garinii A87SA exhibited 45.9-50.8% identity to the VlsE sequence of B. burgdorferi B31, and 30.0-33.8% identity to the VMP17 sequence of B. hermsii HS1. Homologues of the vls locus of B. garinii were detected by dot blot hybridization among 24 of the 30 (80.0%) isolates representing four B. burgdorferi sensu lato species distributed widely in Europe. Our findings indicate that B. garinii might possess a similar vls structure to that described in B. burgdorferi sensu stricto. The highly conserved nature of the vls locus among various B. burgdorferi sensu lato species suggests that it may be important in the physiology and pathogenesis of Lyme disease spirochetes.

Induction of complement sensitivity in Escherichia coli by citric acid and low pH.

AIMS: The lytic functions of the complement system play an important role in the control of Gram-negative infections. Complement-resistant Escherichia coli LP1395 (O18) grown under normal conditions can survive the bactericidal action of complement present in human serum. Towards elucidating the mechanisms of complement resistance, the resistance of E. coli LP1395 grown under conditions of low pH and in the presence of citric acid was tested. METHODS AND RESULTS: E. coli LP1395 becomes sensitive to complement after growth in the presence of citric acid at pH 5. Complement resistance could be restored when the cells were transferred to pH 7 media. However, this recovery was greatly impaired when the cells were transferred to pH 7 media with chloramphenicol. This implies that protein synthesis may be involved in complement resistance. The cells exposed to citric acid at pH 5 showed no indication of a generalized outer membrane (OM) permeability when compared with those grown under normal conditions in terms of sensitivity to lysozyme, uptake of lipophilic dye, or sensitivity to a number of antibiotics. CONCLUSION: Complement-resistant LP1395 may acquire a sensitivity to complement due not to a generalized disruption of the OM barrier, but possibly to the alteration of the activity of one or more normal complement resistance factors. SIGNIFICANCE AND IMPACT OF THE STUDY: The elucidation of the mechanisms of complement resistance of Gram-negative pathogens would bring important information about bacterial infections. Complement resistance factors could also be potential targets in antimicrobial therapies.

The susceptibility of Mycobacterium tuberculosis to isoniazid and the Arg-->Leu mutation at codon 463 of katG are not associated.

A mutation (CCG-->CTG [Arg-->Leu]) in codon 463 of katG (catalase peroxidase) of Mycobacterium tuberculosis has been found in isoniazid (INH)-resistant strains. A PCR restriction endonuclease analysis to detect this mutation was applied to 395 M. tuberculosis isolates from patients in The Netherlands. The proportion of isolates with a detectable mutation was 32% (32 out of 100) and 29% (85 out of 295) among INH-susceptible isolates and INH-resistant or -intermediate isolates, respectively. Sequencing of five INH-susceptible isolates with such mutations showed that all five had the Arg463Leu mutation. We conclude that the Arg463Leu mutation of katG of M. tuberculosis is not a reliable indicator of INH resistance.

NmeSI restriction-modification system identified by representational difference analysis of a hypervirulent Neisseria meningitidis strain.

Neisseria meningitidis is a gram-negative bacterium that may cause meningitis, sepsis, or both. The increase in the incidence of meningococcal disease in various countries in the past 2 decades is mainly due the genotypically related lineage III meningococci. The chromosomal DNA differences between lineage III strains and non-lineage III strains were identified using representational difference analysis. Thus, a 1.8-kb locus that is specific for lineage III meningococci was identified. The locus contains three open reading frames encoding the NmeSI restriction-modification system. The methyltransferase gene was cloned and expressed in Escherichia coli. Site AGTACT was found to be modified by the enzyme. In conclusion, lineage III strains differ from endemic strains by the presence of a specific restriction-modification system. This restriction-modification system may contribute to the clonal and hypervirulent character of lineage III strains by influencing horizontal gene transfer and transcription.