[Significance of the doctorate in scientific medical education]. / Stellenwert der Promotion in der wissenschaftlichen Medizinerausbildung.

According to European and German law, the medical education of physicians must take place in a scientific degree program at a university or under the supervision of a university. To keep up the ideal of a scientific degree program, various organizations and associations, such as the German Research Foundation, the German Council of Science and Humanities and the German Medical Faculty Association, see the need for an even stronger anchoring of academic learning content in the course of study. Traditionally, a scientific project, which is carried out during the studies, provides the basis for the Doctor of Medicine (Dr. med.) after graduation, although the research projects as a basis for medical degrees are currently not obligatory parts of the curricula. The number of medical students performing such research projects is significantly decreasing, thus they are missing major skills for working in science. To counteract these developments, faculties of medicine are currently developing model curricula including deepened scientific education. Despite these efforts, the German Association of Faculties of Medicine argues that the performance of research projects leading to the doctoral degree is most suitable for obtaining expertise in scientific work. According to recommendations by the German Council of Science on the requirements for quality assurance of graduation doctoral degree programs have been introduced. This and further measures, like MD/PhD programs or research-based additional study programs serving the scientific qualification of medical students, are the subject of this article.

Sterilizing immunity elicited by Neisseria meningitidis carriage shows broader protection than predicted by serum antibody cross-reactivity in CEACAM1-humanized mice.

Neisseria meningitidis asymptomatically colonizes the human upper respiratory tract but is also the cause of meningitis and severe septicemia. Carriage or disease evokes an immune response against the infecting strain. Hitherto, we have known little about the breadth of immunity induced by natural carriage of a single strain or its implications for subsequent infectious challenge. In this study, we establish that transgenic mice expressing human CEACAM1 support nasal colonization by a variety of strains of different capsular types. Next, we nasally challenged these mice with either of the N. meningitidis strains H44/76 (serogroup B, ST-32) and 90/18311 (serogroup C, ST-11), while following the induction of strain-specific immunoglobulin. When these antisera were tested for reactivity with a diverse panel of N. meningitidis strains, very low levels of antibody were detected against all meningococcal strains, yet a mutually exclusive "fingerprint" of high-level cross-reactivity toward certain strains became apparent. To test the efficacy of these responses for protection against subsequent challenge, CEACAM1-humanized mice exposed to strain 90/18311 were then rechallenged with different N. meningitidis strains. As expected, the mice were immune to challenge with the same strain and with a closely related ST-11 strain, 38VI, while H44/76 (ST-32) could still colonize these animals. Notably, however, despite the paucity of detectable humoral response against strain 196/87 (ST-32), this strain was unable to colonize the 90/18311-exposed mice. Combined, our data suggest that current approaches may underestimate the actual breadth of mucosal protection gained through natural exposure to N. meningitidis strains.

Ampicillin resistance of invasive Haemophilus influenzae isolates in Germany 2009-2012.

In this retrospective study covering a four-year observation period (2009-2012) the prevalence of aminopenicillin resistance of invasive Haemophilus influenzae (Hi) in Germany was analyzed. The main resistance mechanism against aminopenicillins is conferred by ß-lactamase production, which can be inhibited by clavulanate or sulbactam. Apart from that, ß-lactamase negative ampicillin resistance (BLNAR) has been reported due to mutations in the penicillin-binding protein PBP3. The prevalence of BLNAR varies considerably in different countries. Representative data from Germany have not been reported. We analyzed 704 culture positive cases with bacteraemia or detection of Hi in cerebrospinal fluid; 82 isolates (11.6%) were phenotypically resistant to ampicillin. Among these isolates, 65 (79.3%) showed ß-lactamase production, and 17 isolates (20.7%) were phenotypic BLNAR Hi. The proportion of ampicillin resistant isolates remained stable over the observation period. Analysis of the PBP3 sequences of 133 isolates with different susceptibility phenotypes including susceptible, BLNAR, and ß-lactamase positive isolates, revealed a high genetic diversity. Previously described PBP3 mutations were associated to elevated MIC values, albeit not exclusively, since few highly susceptible strains were found to be positive for the mutations. Furthermore, since ampicillin susceptible strains with elevated MIC values frequently harboured these mutations, prediction of the resistance phenotype using ftsI sequencing appears to be impossible.

Description and nomenclature of Neisseria meningitidis capsule locus.

Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference.

Prevalence dependent calibration of a predictive model for nasal carriage of methicillin-resistant Staphylococcus aureus.

BACKGROUND: Published models predicting nasal colonization with Methicillin-resistant Staphylococcus aureus among hospital admissions predominantly focus on separation of carriers from non-carriers and are frequently evaluated using measures of discrimination. In contrast, accurate estimation of carriage probability, which may inform decisions regarding treatment and infection control, is rarely assessed. Furthermore, no published models adjust for MRSA prevalence. METHODS: Using logistic regression, a scoring system (values from 0 to 200) predicting nasal carriage of MRSA was created using a derivation cohort of 3091 individuals admitted to a European tertiary referral center between July 2007 and March 2008. The expected positive predictive value of a rapid diagnostic test (GeneOhm, Becton & Dickinson Co.) was modeled using non-linear regression according to score. Models were validated on a second cohort from the same hospital consisting of 2043 patients admitted between August 2008 and January 2012. Our suggested correction score for prevalence was proportional to the log-transformed odds ratio between cohorts. Calibration before and after correction, i.e. accurate classification into arbitrary strata, was assessed with the Hosmer-Lemeshow-Test. RESULTS: Treating culture as reference, the rapid diagnostic test had positive predictive values of 64.8% and 54.0% in derivation and internal validation corhorts with prevalences of 2.3% and 1.7%, respectively. In addition to low prevalence, low positive predictive values were due to high proportion (> 66%) of mecA-negative Staphylococcus aureus among false positive results. Age, nursing home residence, admission through the medical emergency department, and ICD-10-GM admission diagnoses starting with "A" or "J" were associated with MRSA carriage and were thus included in the scoring system, which showed good calibration in predicting probability of carriage and the rapid diagnostic test's expected positive predictive value. Calibration for both probability of carriage and expected positive predictive value in the internal validation cohort was improved by applying the correction score. CONCLUSIONS: Given a set of patient parameters, the presented models accurately predict a) probability of nasal carriage of MRSA and b) a rapid diagnostic test's expected positive predictive value. While the former can inform decisions regarding empiric antibiotic treatment and infection control, the latter can influence choice of screening method.

Prospective single-center study of health-related quality of life after COVID-19 in ICU and non-ICU patients.

Long-term sequelae in hospitalized Coronavirus Disease 2019 (COVID-19) patients may result in limited quality of life. The current study aimed to determine health-related quality of life (HRQoL) after COVID-19 hospitalization in non-intensive care unit (ICU) and ICU patients. This is a single-center study at the University Hospital of Wuerzburg, Germany. Patients eligible were hospitalized with COVID-19 between March 2020 and December 2020. Patients were interviewed 3 and 12 months after hospital discharge. Questionnaires included the European Quality of Life 5 Dimensions 5 Level (EQ-5D-5L), patient health questionnaire-9 (PHQ-9), the generalized anxiety disorder 7 scale (GAD-7), FACIT fatigue scale, perceived stress scale (PSS-10) and posttraumatic symptom scale 10 (PTSS-10). 85 patients were included in the study. The EQ5D-5L-Index significantly differed between non-ICU (0.78 ± 0.33 and 0.84 ± 0.23) and ICU (0.71 ± 0.27; 0.74 ± 0.2) patients after 3- and 12-months. Of non-ICU 87% and 80% of ICU survivors lived at home without support after 12 months. One-third of ICU and half of the non-ICU patients returned to work. A higher percentage of ICU patients was limited in their activities of daily living compared to non-ICU patients. Depression and fatigue were present in one fifth of the ICU patients. Stress levels remained high with only 24% of non-ICU and 3% of ICU patients (p = 0.0186) having low perceived stress. Posttraumatic symptoms were present in 5% of non-ICU and 10% of ICU patients. HRQoL is limited in COVID-19 ICU patients 3- and 12-months post COVID-19 hospitalization, with significantly less improvement at 12-months compared to non-ICU patients. Mental disorders were common highlighting the complexity of post-COVID-19 symptoms as well as the necessity to educate patients and primary care providers about monitoring mental well-being post COVID-19.

Genome sequence of a Neisseria meningitidis capsule null locus strain from the clonal complex of sequence type 198.

Neisseria meningitidis is a commensal and accidental pathogen exclusively of humans. Although the production of polysaccharide capsules is considered to be essential for meningococcal virulence, there have been reports of constitutively unencapsulated strains causing invasive meningococcal disease (IMD). Here we report the genome sequence of a capsule null locus (cnl) strain of sequence type 198 (ST-198), which is found in half of the reported cases of IMD caused by cnl meningococcal strains.

Neisseria meningitidis induces brain microvascular endothelial cell detachment from the matrix and cleavage of occludin: a role for MMP-8.

Disruption of the blood-brain barrier (BBB) is a hallmark event in the pathophysiology of bacterial meningitis. Several inflammatory mediators, such as tumor necrosis factor alpha (TNF-alpha), nitric oxide and matrix metalloproteinases (MMPs), contribute to this disruption. Here we show that infection of human brain microvascular endothelial cells (HBMEC) with Neisseria meningitidis induced an increase of permeability at prolonged time of infection. This was paralleled by an increase in MMP-8 activity in supernatants collected from infected cells. A detailed analysis revealed that MMP-8 was involved in the proteolytic cleavage of the tight junction protein occludin, resulting in its disappearance from the cell periphery and cleavage to a lower-sized 50-kDa protein in infected HBMEC. Abrogation of MMP-8 activity by specific inhibitors as well as transfection with MMP-8 siRNA abolished production of the cleavage fragment and occludin remained attached to the cell periphery. In addition, MMP-8 affected cell adherence to the underlying matrix. A similar temporal relationship was observed for MMP activity and cell detachment. Injury of the HBMEC monolayer suggested the requirement of direct cell contact because no detachment was observed when bacteria were placed above a transwell membrane or when bacterial supernatant was directly added to cells. Inhibition of MMP-8 partially prevented detachment of infected HBMEC and restored BBB permeability. Together, we established that MMP-8 activity plays a crucial role in disassembly of cell junction components and cell adhesion during meningococcal infection.

Immunogenicity of meningococcus C vaccination in a patient with atypical hemolytic uremic syndrome (aHUS) on eculizumab therapy.

We report successful kidney transplantation in a 10-yr-old boy with aHUS and heterozygous factor H mutation using the terminal complement inhibitor eculizumab to avoid recurrence of aHUS in the renal graft. Vaccination against meningococcus C (Men C) is essential in patients with dysfunction of the complement system, as induced by eculizumab. In our patient, we report waning SBA titers but maintenance of protective SBA titers (≥1:8) after kidney transplantation under immunosuppressive therapy with mycophenolate mofetil, tacrolimus, steroids, and eculizumab over a 27-month observational period. Our case illustrates that a humoral immune response to conjugate Men C vaccination may be mounted and maintained despite chronic renal disease, kidney transplantation, immunosuppressive drugs, and immunomodulatory therapy with eculizumab. However, it remains unclear whether serologically defined protective SBA titers mediate true protection from invasive meningococcal disease in an immunocompromised patient, particularly under treatment with a complement inhibitor. Thus, close monitoring of SBA titers seems mandatory in this patient.

Whole-genome sequence of the transformable Neisseria meningitidis serogroup A strain WUE2594.

Serogroup A meningococci are a leading cause of bacterial meningitis in children and young adults worldwide. However, the genetic basis of serogroup A strains' virulence and their epidemiological properties remain poorly understood. Therefore, we sequenced the complete genome of the transformable Neisseria meningitidis serogroup A strain WUE2594.

Transmission of Armillifer armillatus ova at snake farm, The Gambia, West Africa.

Visceral pentastomiasis caused by Armillifer armillatus larvae was diagnosed in 2 dogs in The Gambia. Parasites were subjected to PCR; phylogenetic analysis confirmed relatedness with branchiurans/crustaceans. Our investigation highlights transmission of infective A. armillatus ova to dogs and, by serologic evidence, also to 1 human, demonstrating a public health concern.

Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review.

Invasive disease caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae remains an important cause of morbidity and mortality worldwide, despite the introduction of successful conjugate polysaccharide vaccines that target disease-associated strains. In addition, resistance, or more accurately reduced susceptibility, to therapeutic antibiotics is spreading in populations of these organisms. There is therefore a continuing requirement for the surveillance of vaccine and non-vaccine antigens and antibiotic susceptibilities among isolates from invasive disease, which is only partially met by conventional methods. This need can be met with molecular and especially nucleotide sequence-based typing methods, which are fully developed in the case of N. meningitidis and which could be more widely deployed in clinical laboratories for S. pneumoniae and H. influenzae.

Pathogen-specific DNA enrichment does not increase sensitivity of PCR for diagnosis of invasive aspergillosis in neutropenic patients.

PCR assays designed for the diagnosis of invasive aspergillosis (IA) in high-risk patients have to detect minute amounts of target DNA to reach sufficient analytical sensitivity to be of clinical use. This prospective study assessed the use of a novel strategy for selective pathogen DNA enrichment for enhancing the performance of diagnostic PCR in a direct comparison with a highly sensitive in-house quantitative PCR (qPCR) assay and the galactomannan enzyme-linked immunosorbent assay (ELISA). Surprisingly, and in contrast to experience with other patient groups, the novel protocol for selective pathogen DNA enrichment did not enhance but instead significantly impaired sensitivity. This could be explained by the small amounts of host DNA in the specimens, which were derived mostly from severely neutropenic patients. In the qPCR assay, positive samples required an average of 43.5 amplification cycles (range, 39.2 to 50) for detection in the in-house PCR. Repetitive testing of selected samples showed test positivity to be variable, most likely due to the small amounts of target DNA. Despite this, the in-house protocol proved helpful in the diagnosis of IA, detecting 2 out of 3 patients with probable IA and 10 out of 19 patients with possible IA. Our results underline the necessity for diagnostic PCR protocols that help diagnose IA to be highly sensitive and show that selective pathogen DNA enrichment using affinity purification may not be useful in severely neutropenic patients.

New diagnostic PCR for Haemophilus influenzae serotype e based on the cap locus of strain ATCC 8142.

A new PCR protocol for molecular typing of Haemophilus influenzae serotype e (Hie) was developed. To this end, the sequence of the cap region II of Hie strain ATCC8142 was identified, which was >99% identical to the recently published sequence of Hie isolate 274. The PCR using primer pair TTL63/TTL64 amplifies an internal 592-bp fragment of ecs4, an e-specific capsule synthesis gene, in 40 of 40 Hie strains. Of all non-Hie strains, there were no false positives. False-negative results of the PCR proposed by Falla et al. (1994) are explained by single nucleotide insertions in the primer sequences.

Characterization of FarR as a highly specialized, growth phase-dependent transcriptional regulator in Neisseria meningitidis.

Transcriptional regulators play an important role for the survival of Neisseria meningitidis within its human host. We have recently shown that FarR acts as transcriptional repressor of the adhesin nadA in N. meningitidis. Here, we examined the FarR regulon by microarray analyses, qRT-PCR, and electrophoretic mobility shift assays, revealing that FarR is a highly specific repressor of nadA. We demonstrate by reporter gene fusion assays that alterations of the FarR binding site within the nadA promoter are sufficient to induce transcription of nadA. Furthermore, farR expression is growth phase-dependent. The highest transcription rate was observed in the late-exponential growth phase of meningococci. Upon contact with active components of the complement system in normal human serum, expression of farR is slightly downregulated. Concluding, we present FarR as an exquisitely specialized, growth phase-dependent, possibly complement-responsive transcriptional regulator in N. meningitidis.

Resistance to ampicillin, third-generation cephalosporins, ciprofloxacin, co-trimoxazole and azithromycin in clinical isolates of Salmonella enterica subsp. enterica from Germany: real problem or sporadic circumstance?

The objective of this study was to determine the in vitro activity of ampicillin, third-generation cephalosporins, ciprofloxacin, co-trimoxazole and azithromycin against Salmonella enterica isolates. None of the isolates tested showed resistance to third-generation cephalosporins or azithromycin. The rates of resistance to ampicillin, co-trimoxazole and ciprofloxacin were 16.8%, 3.2% and 0.8%, respectively. Moreover, 7.2% of the isolates showed reduced ciprofloxacin susceptibility, but none of them harboured qnr genes. To conclude, our data show that resistance to fluoroquinolones and third-generation cephalosporins in clinical isolates found in Germany still represents a rare circumstance.

Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis.

Neisseria meningitidis is a leading cause of infectious childhood mortality worldwide. Most research efforts have hitherto focused on disease isolates belonging to only a few hypervirulent clonal lineages. However, up to 10% of the healthy human population is temporarily colonized by genetically diverse strains mostly with little or no pathogenic potential. Currently, little is known about the biology of carriage strains and their evolutionary relationship with disease isolates. The expression of a polysaccharide capsule is the only trait that has been convincingly linked to the pathogenic potential of N. meningitidis. To gain insight into the evolution of virulence traits in this species, whole-genome sequences of three meningococcal carriage isolates were obtained. Gene content comparisons with the available genome sequences from three disease isolates indicate that there is no core pathogenome in N. meningitidis. A comparison of the chromosome structure suggests that a filamentous prophage has mediated large chromosomal rearrangements and the translocation of some candidate virulence genes. Interspecific comparison of the available Neisseria genome sequences and dot blot hybridizations further indicate that the insertion sequence IS1655 is restricted only to N. meningitidis; its low sequence diversity is an indicator of an evolutionarily recent population bottleneck. A genome-based phylogenetic reconstruction provides evidence that N. meningitidis has emerged as an unencapsulated human commensal from a common ancestor with Neisseria gonorrhoeae and Neisseria lactamica and consecutively acquired the genes responsible for capsule synthesis via horizontal gene transfer.

Comparative genome biology of a serogroup B carriage and disease strain supports a polygenic nature of meningococcal virulence.

Neisseria meningitidis serogroup B strains are responsible for most meningococcal cases in the industrialized countries, and strains belonging to the clonal complex ST-41/44 are among the most prevalent serogroup B strains in carriage and disease. Here, we report the first genome and transcriptome comparison of a serogroup B carriage strain from the clonal complex ST-41/44 to the serogroup B disease strain MC58 from the clonal complex ST-32. Both genomes are highly colinear, with only three major genome rearrangements that are associated with the integration of mobile genetic elements. They further differ in about 10% of their gene content, with the highest variability in gene presence as well as gene sequence found for proteins involved in host cell interactions, including Opc, NadA, TonB-dependent receptors, RTX toxin, and two-partner secretion system proteins. Whereas housekeeping genes coding for metabolic functions were highly conserved, there were considerable differences in their expression pattern upon adhesion to human nasopharyngeal cells between both strains, including differences in energy metabolism and stress response. In line with these genomic and transcriptomic differences, both strains also showed marked differences in their in vitro infectivity and in serum resistance. Taken together, these data support the concept of a polygenic nature of meningococcal virulence comprising differences in the repertoire of adhesins as well as in the regulation of metabolic genes and suggest a prominent role for immune selection and genetic drift in shaping the meningococcal genome.

Entry of Neisseria meningitidis into mammalian cells requires the Src family protein tyrosine kinases.

Neisseria meningitidis, the causative agent of meningitis and septicemia, is able to attach to and invade a variety of cell types. In a previous study we showed that entry of N. meningitidis into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin bound to the outer membrane protein Opc, which forms a molecular bridge to alpha 5 beta 1-integrins. This interaction results in cytoskeletal remodeling and uptake of the bacteria. In this study we identified and characterized the intracellular signals involved in integrin-initiated uptake of N. meningitidis. We determined that the Src protein tyrosine kinases (PTKs) are activated in response to contact with N. meningitidis. Inhibition of Src PTK activity by the general tyrosine kinase inhibitor genistein and the specific Src inhibitor PP2 reduced Opc-mediated invasion of HBMEC and human embryonic kidney (HEK) 293T cells up to 90%. Moreover, overexpression of the cellular Src antagonist C-terminal Src kinase (CSK) also significantly reduced N. meningitidis invasion. Src PTK-deficient fibroblasts were impaired in the ability to internalize N. meningitidis and showed reduced phosphorylation of the cytoskeleton and decreased development of stress fibers. These data indicate that the Src family PTKs, particularly the Src protein, along with other proteins, are important signal proteins that are responsible for the transfer of signals from activated integrins to the cytoskeleton and thus mediate the endocytosis of N. meningitidis into brain endothelial cells.

Vaccine preventability of meningococcal clone, Greater Aachen Region, Germany.

Emergence of serogroup B meningococci of clonal complex sequence type (ST) 41/44 can cause high levels of disease, as exemplified by a recent epidemic in New Zealand. Multiplication of annual incidence rates (3.1 cases/100,000 population) of meningococcal disease in a defined German region, the city of Aachen and 3 neighboring countries (Greater Aachen) prompted us to investigate and determine the source and nature of this outbreak. Using molecular typing and geographic mapping, we analyzed 1,143 strains belonging to ST41/44 complex, isolated from persons with invasive meningococcal disease over 6 years (2001-2006) from 2 German federal states (total population 26 million) and the Netherlands. A spatially slowly moving clone with multiple-locus variable-number tandem repeat analysis type 19, ST42, and antigenic profile B:P1.7-2,4:F1-5 was responsible for the outbreak. Bactericidal activity in serum samples from the New Zealand MeNZB vaccination campaign confirmed vaccine preventability. Because this globally distributed epidemic strain spreads slowly, vaccination efforts could possibly eliminate meningococcal disease in this area.