Longitudinal study of meningococcal carrier rates in teenagers.

To gain actual information concerning the oropharyngeal carriage of Neisseria meningitidis among teenagers aged 15-18 years in Germany especially in a region with increased incidence of meningococal-related diseases prompted the study. Each teenager was swabbed three times with an interval of 2 months between the examinations. The 901 recovered N. meningitidis strains were characterized using serological (serogrouping, serotyping/serosubtyping) and molecular methods (PCR, PFGE) each. The results of the study demonstrate an overall average carrier rate of 18.8% for the three collection periods. There were, however, significant differences between the carrier rates within a given school and of different towns and counties. Of all isolates, 60.6% were not serogroupable. Serogroup B dominated (12.3%), followed by serogroup Y (9.0%) and serogroup C (3.6%). After PCR-based serogrouping of not serogroupable strains the percentages for serogroups enhanced to 18.8% for B, 10.8% for Y and 4.1% for C. Serotyping led to 305 different phenotypes with the most common being 29E:NT:P1.2,5 followed by Y:14:NST. In the 6 study towns the number of different N. meningitidis clones (PFGE types) isolated, varied between 30 and 87. In Wenden, where a prolonged outbreak had taken place, serogroup C (14.8%) was predominant. Only in this town C:2a isolates were found, all belonging to the ST-11/ET-37 complex and 12/13 matched identically to the ET-15 clone. Of the colonized teenagers, 26.7% were carriers over at least 23 weeks, 22.6% with the same strain, 36.0% were carrier for at least 15 weeks. Over all three collection periods 36.7% of the adolescents acquired a new strain. The highest acquisition rate was related to PFGE type 12.

The higher disinfectant resistance of nosocomial isolates of Klebsiella oxytoca: how reliable are indicator organisms in disinfectant testing?

The Children's Clinic in Giessen, Germany recently reported several severe infections with Klebsiella oxytoca resulting in deaths of two neonates. The putative source of the infections was a contaminated infusion solution. The resistance to disinfectant of the K. oxytoca isolates was investigated in three independent laboratories and was indeed found to be significantly increased. Comparative tests with standard strains of K. oxytoca and other recommended bacterial surrogates showed the disinfection procedures used were fully effective. The higher resistance of the nosocomial isolates may have developed due to improper handling and storage of the cleaning utensils. This report describes the events and draws conclusions concerning the use of disinfectants, the treatment of cleaning utensils, the reliability of procedures for testing disinfectants, and suggests additional measures.

Tissue and serum concentrations of levofloxacin in orthopaedic patients.

The level of levofloxacin was determined in serum, bone and several tissues after a single dose of 500 mg i.v. Twenty-one patients (mean age: 56.8 years) undergoing bone surgery (nine patients) or surgical debridement of a decubitus ulcer (12 patients) who received levofloxacin as perioperative prophylaxis were included in the study. During surgery, blood and tissue samples were obtained approximately 1.5 h (range 40-210 min) postdosing. Levofloxacin concentrations in 87 specimens including 21 serum samples were determined using high-performance liquid chromatography (HPLC). The mean serum concentration at 1.5 h was 8.6+/-2.3 microg/ml. Concentrations above the MIC of common pathogens were reached in all tissues during the collection period with a maximum in skin samples (19.9+/-9.9 microg/g) followed by wound tissue and granulation tissue with 17.3+/-6.5 and 13.7+/-6.4 microg/g respectively. In muscle and fatty tissue mean levofloxacin concentrations of 8.0+/-0.9 and 4.0+/-2.2 microg/g were attained. Mean levels in cancellous bone were 6.6+/-3.6 microg/g, lowest levels were measured in cortical bone (2.8+/-1.1 microg/g). Twenty-two different pathogens were cultivated from the lesions of 11 of 12 patients with pressure ulcers. MIC values for levofloxacin were determined and compared with the corresponding tissue concentrations. Levofloxacin may be useful for perioperative prophylaxis and treatment in orthopaedic patients due to its good tissue penetration.

An HPLC assay and a microbiological assay to determine levofloxacin in soft tissue, bone, bile and serum.

A simple, specific and sensitive HPLC assay for levofloxacin in serum, bile, soft tissue and bone was evaluated and validated. The samples were prepared by protein precipitation with acids and methanol, which yielded high recoveries (for serum and bile>98% and for bone and soft tissue>90%). The compounds were separated on a reversed phase column with an acidic mobile phase containing triethylamine. The eluate was monitored by fluorescence detection. The HPLC assay is linear over the usable concentration range (0.1-40 microg/ml) and it provides good validation data for accuracy and precision. Although comparison of HPLC results to the results of a microbiological assay showed congruent results (regression coefficients>0.967). HPLC should be the method of choice for determination of levofloxacin in biological matrices.

Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis.

A rapid and sensitive PCR assay for the detection of Candida albicans DNA in serum was established. DNA from human serum samples was purified using the QIAamp blood kit, which proved to be a fast and simple method for isolating minute amounts of Candida DNA from clinical specimens for diagnosis of invasive candidiasis. Universal primer sequences used in the PCR assay are derived from the internal transcribed spacer rRNA gene of fungi, whereas the biotinylated hybridization probe used in a DNA enzyme immunoassay (DEIA) binds specifically to C. albicans DNA. The sensitivity of this PCR-DEIA method is very high; the detection limit for genomic Candida DNA is one C. albicans genome per assay. Blood from uninfected and infected persons, ranging from healthy volunteers, patients with mucocutaneous infections, and patients at risk to develop a systemic Candida infection to patients with an established systemic candidiasis, was analyzed for the presence of C. albicans to diagnose fungal infection. Candida DNA could not be detected in sera of 16 culture-negative controls and from 11 nonsystemic candidal infections by PCR or DEIA. Blood cultures from patients at risk were all negative for Candida, whereas all blood cultures from systemic candidiasis patients were positive. However, Candida DNA could be detected by PCR and DEIA in the serum from three out of nine patients who were at risk for a systemic infection and in the serum of all seven patients who had already developed an invasive Candida infection. PCR is more sensitive than blood culture, since some of the patients at risk for invasive yeast infection, whose blood cultures were all negative for Candida, tested positive in the PCR amplification. These results indicate the potential value of PCR for detecting C. albicans in serum samples and for identifying patients at risk for invasive candidiasis.

[Meningococcal infections: aspects of microbiology, epidemiology and prevention]. / Meningokokkeninfektionen: Aspekte zur Mikrobiologie, Epidemiologie und Prävention.

Neisseria meningitidis (meningococcus) is responsible for an average of 40% of all cases of bacterial meningitis in Germany. Cerebrospinal fluids, blood cultures, throat swabs and scratches, aspirations and biopsies of the skin rash are appropriate materials for the diagnosis of meningococcal disease. The materials should reach the laboratory without delay. Since 1993, the incidence of meningococcal disease in Germany is less than 1 case per 100,000 inhabitants. The case fatality rate is about 10%. Most cases of meningococcal disease occur in the first quarter of the year. Almost half of all invasive N. meningitidis isolates are from children under five years of age. In the period 1990-1998, in Germany an average of 74% of cases were caused by serogroup B and 21% by serogroup C. In serogroup B disease, isolates of serotype 15, in group C disease strains of serotype 2a are predominating. Chemoprophylaxis should be given to all household members and all contacts living in institutions with household-like character, contacts in institutions for children under six years of age and all persons who had contact with the oropharyngeal secretions of the patient. At present, only capsular polysaccharide vaccines against serogroups A, C, Y and W135 are available for immunoprophylaxis in Germany.

[Quality assurance (general) at the medical school of Heidelberg as a model]. / Qualitätsförderung (allgemein-) medizinischen Hochschulunterrichts im Rahmen des Heidelberger Modells.

Because of new social and professional challenges, especially in the developed countries, there is a trend towards change and quality assurance is taking place in medical education over the past 20 years. In Heidelberg, the new way of teaching the students by general practitioners includes quality assurance by questionnaires, reports of practice visits, quality conferences of the teachers, and co-operation of the students. 90% of the students recommended the practice-based structured program as very useful, especially the work with the patients in the general practices, 87% of the teaching general practitioners accepted special criteria for teaching; by this way an academic general practice can be established to meet the future expectations of primary medical care.

Detection of endotoxins and other pyrogens using human whole blood.

When cells of the immune system, i.e. primarily blood monocytes and macrophages, come into contact with pyrogens (fever-inducing contaminations) they release mediators transmitting the fever reaction through the organism to the thermoregulatory centres of the brain. The new test discussed here exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample. If there is pyrogen contamination, the endogenous pyrogen interleukin-1 is released, which is then determined by ELISA. According to the pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample. In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species. Compared to the well established in vitro alternative, i.e. the limulus amebocyte lysate assay (LAL), the new blood assay is not restricted to endotoxins of gram-negative bacteria, it is not affected by endotoxin-binding blood proteins and it reflects the potency of different endotoxin preparations in mammals. Here, interim results of the ongoing optimization and pre-validation are reported and the present state of the evaluation for biological and pharmaceutical drugs are presented.

Ceftazidime in combination with glycopeptide antibiotic is an effective first-line therapy for patients undergoing high-dose therapy with autologous peripheral blood stem cell support.

It was the objective of this study to evaluate the efficacy and toxicity of an empirical antibiotic therapy consisting in ceftazidime and a glycopeptide antibiotic. All patients enrolled in the study had hematological malignancies and underwent high-dose therapy with peripheral blood stem cell (PBSC) support. In this retrospective study, 183 of 207 patients who had received a PBSC-supported high-dose therapy were evaluable. Any patients who had fever higher than 38.5 degrees C received ceftazidime in combination with vancomycin (105 patients) or teicoplanin (69 patients). In 80 of 174 patients with fever (45%) the fever resolved within 72 h as a result of the treatment with ceftazidime and the glycopeptide antibiotic. In nonresponding patients, the changes included the replacement of ceftazidime by imipenem/cilastin (94 patients) and the addition of erythromycin (12 patients) or metronidazole (3 patients). Amphotericin B was administered in 29 patients. Following hematological reconstitution, the fever and clinical signs, including radiographic findings, resolved in 20 primarily nonresponding patients. In blood cultures, a significantly higher incidence of gram-positive than of gram-negative bacteria was observed (26 vs 7). The toxicity of the first-line antibiotic therapy was limited to allergic skin reactions in 12 patients. Ceftazidime in combination with a glycopeptide antibiotic provides an effective and safe first-line therapy for patients with neutropenic fever following PBSC-supported high-dose therapy.

Chlorate as an inorganic disinfection by product in swimming pools.

Chlorate and chlorite concentrations were determined in water samples taken from 33 swimming pools. In the pools under investigation, disinfection of the water is carried out either by gaseous chlorine (n = 14) or hypochlorite solution in conjunction with flocculation and sand filtration. A number of the pools also use ozone treatment to augment the disinfection process. Chlorite was not detectable in any of the samples (detection limit 1 mg/l). High concentrations of chlorate were detected in samples from a number of the pools; in one case as high as 40 mg/l. Higher chlorate concentrations were found to be associated with those pools using hypochlorite solution as a disinfecting agent. In contrast, relatively low chlorate concentrations were found in pools treated with gaseous chlorine. In order to elucidate any relationship between the chlorate content of pool water and that of the respective hypochlorite stock solution, chlorate and bromate concentrations were determined in the hypochlorite stock solutions of nine pools. Bromate concentration in the stock solutions were not found to exceed 1.2 g/l, chlorate was measured in concentrations of up to 44.5 g/l. The additional use of ozone as part of the water purification process appears to have no significant influence on chlorate concentration. Chlorate has no bactericidal properties and does not interfere with the measurement of certain parameters relevant to hygiene in swimming pools such as free and combined chlorine, pH or redox potential. At present, the effects of high chlorate concentrations in swimming pool water are unclear. Our initial investigations indicate that chlorate has no cytotoxic (Neutral-Red assay) or irritating properties (HET-CAM assay). However, both chlorate and chlorite are known to interfere with the haematopoetic system. In Germany, the MCL for chlorite in drinking water is 0.2 mg/l. It is therefore strongly recommended that measures should be taken to reduce chlorate concentrations in swimming pool water.

Detecting legionellosis by unselected culture of respiratory tract secretions and developing links to hospital water strains.

For a 13-month period, all respiratory tract secretions submitted for routine bacteriology from a large hospital complex were cultured for legionella, irrespective of clinical diagnosis and laboratory requests. Ten cases of legionellosis were detected in this manner, three of which met a strict epidemiological definition of hospital-acquired. Therefore, the 16 warm-water systems of the hospitals, spread out over two locations, were examined for the presence of legionella. Legionella pneumophila was found in 15 warm water systems, with a distinct pattern of serogroups between the two locations. Legionella of the same serogroups as those isolated from patients were present in each hospital water supply. The isolates were further typed by monoclonal antibodies and by genomic macrorestriction analysis. Similarity between clinical and environmental isolates was found in seven cases. In these cases, acquisition from the hospital water supply appears very likely. The strains of the remaining three patients did not match those in hospital water, suggesting that community-acquired legionellosis was occurring as well. This study suggests that routinely culturing respiratory tract secretions of pneumonia patients for legionella can help diagnose unsuspected cases of legionellosis. Typing legionella strains beyond the serogroup level with tools such as macrorestriction analysis is useful to define sources of infection, which can then be targeted for control measures.

Molecular probes for the detection of pathogenic fungi in the presence of human tissue.

Four primer systems, amplifying fragments of the gene coding for the small ribosomal subunit (18S rRNA) were characterised with pure cultures of 65 medically relevant fungal species plus two mushrooms. A primer cocktail (TR1/CA1-TR2/AF2) amplified 59 of 67 fungal species; the universal fungal primer 1 (UF1) in combination with the eukaryotic primers S3 or EU1 amplified 64 and 65 of 67 fungal species, respectively. The design of an additional primer (RZY1) enabled the amplification of the missing members of the zygomycetes. The primer systems amplified all the medically relevant fungi tested. These included eight Candida spp. and seven other yeast species, 13 dermatophytes, 32 moulds (including six zygomycetes and five dimorphic fungi) and two mushrooms. Eleven controls including DNA from Schistosoma mansoni, Escherichia coli, Mycobacterium tuberculosis and man were not amplified. The oligonucleotide CA hybridised with C. albicans, C. tropicalis and C. parapsilosis; the oligonucleotide TR hybridised with the 13 dermatophytes; the oligonucleotide AF hybridised with Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, A. versicolor, A. tamarii, A. clavatus, A. fischeri, but not with A. niger or A. versicolor; and the oligonucleotide HC hybridised with three varieties of Histoplasma capsulatum. These oligonucleotides did not hybridise with the other fungi nor the controls. The specificity of the newly designed primer systems was confirmed by selective amplification of fungal DNA from human lung tissue spiked with fungal biomass and from vitrectomy fluid of a patient with candida endophthalmitis.

Identification and analysis of a new vacA genotype variant of Helicobacter pylori in different patient groups in Germany.

The vacuolating cytotoxin of Helicobacter pylori (VacA) is known to cause cell damage to mammalian cells and is suspected to give rise to gastric epithelial lesions that might lead to peptic ulcer disease. As shown recently, the gene encoding VacA exhibits genetic variation, with three different families of signal sequences (s1a, s1b, and s2) and two families of midregion sequences (m1 and m2). In order to investigate the relationship between the presence of specific vacA genotypes and peptic ulceration, the vacA genotypes of 158 clinical isolates of H. pylori were determined. The study group consisted of 106 patients with duodenal ulceration; 52 patients with nonulcer dyspepsia (NUD) were used as controls. H. pylori of genotype s1 was isolated from 96% of the patients with ulcerations, whereas genotype s2 was only present in 4%, indicating a strong correlation between the vacA genotype and peptic ulceration (P < 0.001). In contrast, 31% of the patients from the NUD control group were infected with strains of vacA genotype s2. Particular midregion genotypes (m1 and m2) were not associated with clinical manifestations. The midregions from 18% of the isolates could not be classified by the proposed scheme. DNA sequencing revealed high homology between the untypeable midregions and that of genotype m1, with multiple base pair exchanges, some affecting the primer annealing site. Compared to those of m1 and m2 alleles, the divergent midregions from untypeable strains showed clustering, indicating the presence of a further subfamily of sequences in the midregion of vacA in German isolates, for which we propose the term "m1a." A new specific primer that we designed for typing m1a isolates might be useful in other studies.

[Irritating effects of disinfection by-products in swimming pools]. / Irritierende Wirkung von Nebenprodukten der Schwimmbadwasserdesinfektion.

Compounds which can occur as disinfection by-products (DBP's) in swimming pool water were examined for their mucous membrane irritating potential. Previous studies using the rabbit eye test (Draizé test) have shown that the irritating potential of typical concentrations of free and combined chlorine are insufficient to explain the degree of eye irritation that can result from exposure to swimming pool water. Other DBP's which may be responsible for eye irritation include halogenated carboxyl compounds (HCC's) which act as precursors during the formation of chloroform. In this study, a modified HET-CAM Test (Hens Egg Test at the Chorion Allantois Membrane) has been used to investigate the mucous membrane irritating effects of HCC's. Some of the compounds tested were found to have a significantly increased irritating effect when compared to a chlorine/chloramine mixture of the same concentration, several mixtures of HCC's where even more active at lower concentrations than single compounds. However, the irritating effects of individual compounds as well as of mixtures of HCC's were not sufficiently intense to allow the identification of those compounds specifically responsible for the overall observed increase in irritation. HCC's were therefore tested in the presence of aqueous chlorine solution. When combined with aqueous chlorine, a number of compounds exhibited significantly enhanced effects. Our results show that the eye irritating effects of low concentrations of DBP's can be investigated using a modified HET-CAM assay. Moreover, results obtained using this assay suggest that the mucous membrane irritating potential of swimming pool water is a consequence of the effects and synergistic action of a number of DBP's in the presence of chlorine. Further work should be carried out in order to establish an indicator for eye irritating effects of swimming pool water.

[Fungal nucleic acid detection for invasive aspergillosis]. / Pilz-Nukleinsäure-Nachweis bei invasiven Aspergillosen.

A universal PCR-assay for the detection of fungal DNA was compared with microscopy and culture for the diagnosis of invasive aspergillosis using 78 samples from 42 patients. Eighteen patients were suffering from invasive aspergillosis, 5 patients were colonized with Aspergillus in the respiratory tract, 19 patients did not show any sign of aspergillosis. Samples from 6 of the 18 patients with invasive aspergillosis were microscopically positive with true mycelia, 15 of 18 grew Aspergillus in culture, 16 of 18 were PCR-positive. The combination of microscopy and culture led to the diagnosis in 17 of 18 patients, the combination of microscopy and PCR in 16 of 18 and the combination of culture and PCR in all the 18 patients. For 3 of 18 patients, PCR was the diagnostic key: in 2 biopsies the histologically detected fungal elements were identified as Aspergillus, in 3 bronchial lavages from 1 patients nothing but PCR was positive for Aspergillus. Four out of 5 culture positive patients with Aspergillus colonization were also PCR positive; one out of 19 patients without aspergillosis was culture positive, 3 out of 19 were falsely PCR positive. Candida colonization in the upper respiratory tract or Pneumocystis carinii pneumonia did not lead to false positive Aspergillus-PCR results. In conclusion, the evaluated fungal PCR-assay can supplement conventional methods for the diagnosis of invasive aspergillosis.

Comparative evaluation of a commercial system for identification of gram-positive cocci.

The performance of a new commercial system for the identification of different groups of gram-positive cocci [BBL Crystal Gram-Positive (GP) Identification System; Becton Dickinson Microbiology Systems, Germany] was evaluated in comparison with two currently used commercial systems, the API Staph and the API Strep (bioMérieux Diagnostic, Germany). A total of 191 strains from seven different gram-positive genera comprising 32 different species were tested. For the BBL Crystal GP system, the correct identification rate without additional tests was 89.5% at the species level and 97.9% at the genus level. The findings suggest that the newly introduced BBL Crystal GP ID system provides an accurate method for the identification of gram-positive cocci, with an overall rate of correct species identification of about 90%, similar to that of the established API systems. Its major advantage is the extended spectrum of taxa included in a single test panel in contrast to the two different API test kits. Furthermore, the simplicity of use and the safe and rapid handling in a closed system conveniently accommodate existing laboratory workflow.

Ribosomal genes of Histoplasma capsulatum var. duboisii and var. farciminosum.

A total of 1704 basepairs of the 18S rDNA of Histoplasma capsulatum var. duboisii (HCD, strain CBS175.57) and H. capsulatum var. farciminosum (HCF, strain CBS478.64) were sequenced (EMBL accession no. Z75306 and no. Z75307). The 18S rDNA of HCD was 100% identical to a published sequence of H. capsulatum var. capsulatum (HCC). The 18S rDNA of HCF showed one transversional point mutation at the nucleotide position 114 (ref. Saccharomyces cerevisiae). Hybridization confirmed that, in the 18S rDNA of two out of five strains of HCF, guanine was substituted for cytosine at the nucleotide position 114. Furthermore, identical group 1C1 introns (403 bp) were found to be inserted after position 1165 in four out of five strains of HCF, including the two strains with point mutations in the 18S rDNA, and a slightly different group 1C1 intron (408 bp) was detected in one strain of HCC without this point mutation. Intraspecific sequence variability in the highly conserved 18S rDNA because of occurrence of introns and mutations as a possible source of error in molecular diagnostics is discussed. In addition, internal transcribed spacer regions between the 18S rDNA and the 5.8S rDNA (ITS1) of three strains of HCF, and one strain each of HCC and HCD showed significant sequence variability between varieties and strains of H. capsulatum.

Systematic analysis and controlling of health care organisations lead to numerical health care improvements.

The EFQM model for organisational excellence is used in the health care sector as a tool to diagnose and assess the starting position for the effective QM programme. Feedback reports cover the fields of acute medical care, rehabilitation and ambulant care and contain strengths areas for improvement. Building on the EFQM feedback reports, the Modular Concept for Quality in Health Care ("Heidelberg Model") improves QM both holistically and specifically by implementing so-called "Modules for Excellence". The implementation process follows principles of project management covering medical, nursing and managing issues and the performance is periodically evaluated against targets. QM projects that are designed in the dichotomic way follow three goals. Organisational diagnosis and therapy lead to numerical health care improvements in "Prevention of nosocomial infections" and "Optimising out-patient treatment". Different assessment approaches lead to a diagnosing feedback report for QM in health care. The Modular Concept for Quality in Health Care ("Heidelberg Model") clusters, priorities, implements and evaluates the organisation's key areas for improvement.