[Management of the influenza pandemic on a local health authority level]. / Management der Influenzapandemie auf kommunaler Ebene.

In most cities and districts, the influenza pandemic of 2009 could be handled without any restrictions in providing medical care or any disturbance in public life. Despite its relatively mild course, the local public health services reached their limits of capacity. Based on nationwide regulations, the local management determines the success of the measures. Evaluating the experience on the community level offers the chance of facing future pandemics more efficiently. Press conferences, press releases, and the internet are the most reliable tools to inform the public even in terms of personnel expenses. Telephone conferences and internet platforms help to reduce time-consuming meetings. An electronic database and logbook provide up-to-date information for all parties involved and allow quick, rational, coordinated, and transparent decision-making. Local evaluation of registration data, reports on cases of illness, and the availability of hospital beds on a daily basis allow intervention at an early stage to cope with the pandemic efficiently and helps save resources. Recruitment of external personnel, e.g., for the call center and the vaccination campaign, relieves the public health employees in charge with respect to their main tasks of directing and management functions.

Multiplex real-time PCR and blood culture for identification of bloodstream pathogens in patients with suspected sepsis.

Severe sepsis is increasingly a cause of death. Rapid and correct initial antimicrobial treatment reduces mortality. The aetiological agent(s) cannot always be found in blood cultures (BCs). A novel multiplex PCR test (SeptiFast (alpha version)) that allows identification of 20 bacterial and fungal species directly from blood was used, comparatively with BC, in a multicentre trial of patients with suspected bacterial or fungal sepsis. Five hundred and fifty-eight paired samples from 359 patients were evaluated. The rate of positivity was 17% for BC and 26% for SeptiFast. Ninety-six microorganisms were isolated with BC, and 186 microorganisms were identified with SeptiFast; 231 microorganisms were found by combining the two tests. Of the 96 isolates identified with BC, 22 isolates were considered to be contaminants. Of the remaining 74 non-contaminant BC isolates available for comparison with SeptiFast, 50 were identified as a species identical to the species identified with SeptiFast in the paired sample. Of the remaining 24 BC isolates for which the species, identified in the BC, could not be detected in the paired SeptiFast sample, 18 BC isolates were identified as a species included in the SeptiFast master list, and six BC isolates were identified as a species not included in the SeptiFast master list. With SeptiFast, 186 microorganisms were identified, 12 of which were considered to be contaminants. Of the 174 clinically relevant microorganisms identified with SeptiFast, 50 (29%) were detected by BC. More than half of the remaining microorganisms identified with SeptiFast (but not isolated after BC) were also found in routine cultures of other relevant samples taken from the patients. Future clinical studies should assess whether the use of SeptiFast is of significant advantage in the detection of bloodstream pathogens.

Development and evaluation of a molecular assay for detection of nontuberculous mycobacteria by use of the cobas amplicor platform.

We have developed and evaluated a semiautomated assay for detection of nontuberculous mycobacteria (NTM) from clinical samples based on the Cobas Amplicor Mycobacterium tuberculosis test (Roche Diagnostics, Switzerland). A capture probe, specific for mycobacteria at the genus level, was linked to magnetic beads and used for the detection of amplification products obtained by the Cobas Amplicor M. tuberculosis assay. We demonstrate that the analytical sensitivity of the genus assay is similar to that of Cobas Amplicor M. tuberculosis detection. Four hundred sixteen clinical specimens were evaluated for the presence of NTM DNA. Sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. Specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. The genus assay is easy to perform, produces reliable results, and was found to be a valuable diagnostic tool for rapid diagnosis of infections with NTM. The genus assay has the potential to detect NTM not routinely recovered by culture and to discover new mycobacterial species.

Leprosy in a pregnant woman.

Herein, we describe a case of leprosy in a 29-year-old pregnant southeast-asian woman who presented with joint pain and multiple disseminated erythematous macules, papules and plaques. Histological examination and stains for acid-fast bacilli from skin biopsies substantiated the clinical suspicion of a cutaneous mycobacterial disease and both should be performed in all patients with unidentified skin lesions. The definitive laboratory diagnosis of leprosy was achieved by the application of a species-specific real-time polymerase chain reaction from infected tissue.

16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory.

Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non-Pseudomonas aeruginosa) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.

[Pneumonia induced by nocardia -- a case report]. / Nokardien-Pneumonie - eine Kasuistik.

We describe a case of lung manifestation of nocardiosis with upper lobe shrinking of the right lung in a 45 year old patient without evident signs of an immuno-compromising illness. The patient came to the hospital in a reduced general state of health with severe cough, red and brown sputum and exertional dyspnoea. X-ray pictures of the thorax showed inflammatory infiltration and shrinking of the upper left lobe of the right lung. Gram-positive, branching rods were detected in the patient's bronchial secretion with the microscope and in cultures. Nocoardia transvalensis was identified via polymerase chain reaction (PCR). The antibiotic therapy was planned according to the bacterial resistance pattern. Imipenem was administered for 5 weeks and Amikacin was added for 3 weeks in the 3 (rd) week of therapy. The patient left the hospital in a good general state of health. There was no relapse.

Removal of PCR inhibitors by silica membranes: evaluating the Amplicor Mycobacterium tuberculosis kit.

The effectiveness of PCR inhibitor removal by silica membranes in combination with the Amplicor Mycobacterium tuberculosis kit was analyzed for 655 respiratory and nonrespiratory specimens. The overall inhibition rate was reduced from 12.5%, when applying the Amplicor kit alone, to 1.1% with the addition of silica membrane DNA purification.

Rapid molecular typing of methicillin-resistant Staphylococcus aureus by PCR-RFLP.

OBJECTIVE: To establish a new, rapid, and reliable genotypic fingerprinting technique for methicillin-resistant Staphylococcus aureus (MRSA) typing in routine epidemiological surveillance. DESIGN: The method is based on polymerase chain reaction (PCR) restriction fragment-length polymorphism (RFLP) following HaeII digestion of simultaneously amplified parts of the protein A gene, the coagulase gene, and the hypervariable region adjacent to mecA. A total of 46 MRSA initial isolates were analyzed, including 14 isolates from five countries; the six German epidemic strains; 16 isolates from the Frankfurt metropolitan area, which were known to be heterogeneous by pulsed-field gel electrophoresis (PFGE); and 10 isolates obtained during three epidemics, all of which displayed an identical genotype. RESULTS: Restriction analysis by PCR-RFLP permitted discrimination of 10 of 14 international isolates, all six German epidemic strains, and 15 of 16 national isolates. It also confirmed the homogeneous character of the 10 outbreak isolates. CONCLUSIONS: This new and rapid PCR-RFLP typing method is an attractive tool in routine epidemiological surveillance. Its impressive characteristics are ease of performance and interpretation, while at the same time guaranteeing good discriminatory power, reproducibility, and typeability.

Superoxide potently induces ceramide formation in glomerular endothelial cells.

Recent evidence suggests that the sphingolipid-derived second messenger ceramide and oxidative stress are intimately involved in apoptosis induction. Here we report that exposure of microcapillary glomerular endothelial cells to superoxide-generating substances, including hypoxanthine/xanthine oxidase and the redox cyclers DMNQ and menadione results in a dose-dependent and delayed increase in the lipid signaling molecule ceramide. Long-term incubation of endothelial cells for 2-30 h with either DMNQ or hypoxanthine/xanthine oxidase leads to a continuous increase in ceramide levels. In contrast, short-term stimulation for 1 min up to 1 h had no effect on ceramide formation. The DMNQ-induced delayed ceramide formation is dose-dependently inhibited by reduced glutathione, whereas oxidized glutathione was without effect. Furthermore, N-acetylcysteine completely blocks DMNQ-induced ceramide formation. All superoxide-generating substances were found to dose-dependently trigger endothelial cell apoptosis. In addition, glutathione and N-acetylcysteine also prevented superoxide-induced apoptosis and implied that ceramide represents an important mediator of superoxide-triggered cell responses like apoptosis.

Differential effect of rpoB mutations on antibacterial activities of rifampicin and KRM-1648 against Staphylococcus aureus.

The in vitro antibacterial activities of the rifamycin derivatives rifampicin and KRM-1648 against 150 Staphylococcus aureus isolates were determined. The MICs of rifampicin and KRM-1648 for 90% of rifampicin-susceptible S. aureus isolates (n = 100) were 0.016 and 0.001 mg/L, respectively. In rifampicin-resistant S. aureus isolates (n = 50), different levels of resistance to rifamycins were associated with mutations at different sites in rpoB. Mutations at some sites were associated with high-level resistance to both rifamycins, while certain mutations were associated with the activity of KRM-1648 being < or = 100-fold better than that of rifampicin.

Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance.

Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.

Mycobacterium malmoense infections in immunocompetent patients.

While Mycobacterium malmoense infections were originally restricted to northern Europe, there has been an increasing number of reports of cases of infection in other countries. Two cases of infections due to Mycobacterium malmoense in immunocompetent patients in Germany are presented. In both cases a presumptive diagnosis of tuberculosis was established initially. Mycobacterium malmoense was cultured after a long incubation period (6-8 weeks). The patients were successfully treated with a triple regimen consisting of rifampicin, ethambutol and clarithromycin. The epidemiology and difficulties in diagnosis of Mycobacterium malmoense infection are discussed.

Molecular characterization of rpoB mutations conferring cross-resistance to rifamycins on methicillin-resistant Staphylococcus aureus.

Mutations of the rpoB gene conferring resistance to rifampin were analyzed in 40 methicillin-resistant Staphylococcus aureus isolates obtained from six countries. Interestingly, the majority of clinical isolates showed multiple mutations within rpoB. The amino acid substitution 481His-->Asn was the most prevalent one, capable of conferring low-level resistance on its own. Cross-resistance to rifampin, rifabutin, and rifapentine was demonstrated for all mutants identified. The level of resistance to rifamycins correlated with both the mutation position and type of amino acid substitution.

Detection and identification of mycobacteria by amplification of rRNA.

Oligonucleotides specific at a genus, group, or species level were defined by a systematic comparison of small-subunit rRNA sequences from Mycobacterium tuberculosis, M. bovis, M. africanum, M. bovis BCG, M. avium, M. kansasii, M. marinum, M. gastri, M. chelonae, M. smegmatis, M. terrae, M. nonchromogenicum, M. xenopi, M. malmoense, M. szulgai, M. scrofulaceum, M. fortuitum, M. gordonae, M. intracellulare, M. simiae, M. flavescens, M. paratuberculosis, M. sphagni, M. cookii, M. komossense, M. phlei, and M. farcinica. On the basis of the defined oligonucleotides, the polymerase chain reaction technique was explored to develop a sensitive taxon-specific detection system for mycobacteria. By using M. tuberculosis as a model system, fewer than 10 bacteria could be reliably detected by this kind of assay. These results suggest that amplification of rRNA sequences by the polymerase chain reaction may provide a highly sensitive and specific tool for the direct detection of microorganisms without the need for prior cultivation.

Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level.

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationship among different serovars of the Mycobacterium avium-M. intracellulare complex as well as to define signature nucleotides characteristic for different serovars. In general, the groups defined by rRNA sequencing reflect the classification obtained with sensitin tests and pathogenicity examinations in chickens. Unique 16S rRNA sequence patterns could be defined for (1) M. avium, (2) M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11, (3) M. intracellulare serovars 12, 13, 14, 15, 17, 19 and 20, (4) M. intracellulare serovar 7 and (5) M. intracellulare serovar 18. Phylogenetically, groups 1 and 2 on one hand and groups 3, 4 and 5 on the other hand each share a common ancestor. M. paratuberculosis was indistinguishable from M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11 by this kind of analysis.