Half-Year Longitudinal Seroprevalence of SARS-CoV-2-Antibodies and Rule Compliance in German Hospital Employees.

COVID-19, which is caused by SARS-CoV-2, is an occupational health risk, especially for healthcare employees due to their higher exposure and consequently higher risk of symptomatic and asymptomatic infections. This study was designed to determine the longitudinal seroprevalence of specific immunoglobulin-G (IgG) antibodies in employees in a hospital setting. All employees in a secondary care hospital, including healthcare and non-healthcare workers, were invited to participate in this single-center study. After an initial screening, a 6-month follow-up was carried out, which included serological examination for SARS-CoV-2 IgG antibodies and a questionnaire for self-reported symptoms, self-perception, and thoughts about local and national hygiene and pandemic plans. The seroprevalence of SARS-CoV-2 IgG antibodies was 0.74% among 406 hospital employees (0.75% in healthcare workers, 0.72% in non-healthcare workers), initially recruited in April 2020, in their follow-up blood specimens in October 2020. In this study, 30.54% of the participants reported using the official German coronavirus mobile application and the majority were content with the local and national rules in relation to coronavirus-related restrictions. At the 6-month follow-up, the 0.74% seroprevalence was below the reported seroprevalence of 1.35% in the general German population. The prevalence in healthcare workers in direct patient care compared with that in workers without direct patient contact did not differ significantly. Further follow-up to monitor the seroprevalence in the high-risk healthcare sector during the ongoing global pandemic is essential.

Prospective Sero-epidemiological Evaluation of SARS-CoV-2 among Health Care Workers in a German Secondary Care Hospital.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the pulmonary disease coronavirus disease 2019 (COVID-19, which has challenged health care facilities worldwide. The sustainability of health care systems is largely reliant on the health status of their health care workers (HCW). This study aimed to detect the SARS-CoV-2 virus and specific antibodies among HCWs in a German hospital as a model system for the potential spread of the pandemic. METHODS: Between March and June 2020, we used a combination of RT-PCR testing to detect SARS-CoV-2 RNA and an enzyme-linked immunosorbent assay to detect the presence of anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies among HCWs in a German hospital based on repetitive oropharyngeal swabs (OPSs) and blood samples. RESULTS: In total, 871/1081 employees participated in this prospective longitudinal study. During the study period of 9 weeks, 5329 OPSs and 2136 blood samples were analyzed. SARS-CoV-2 RNA was detected in three participants (0.34%). Anti-SARS-CoV-2 IgG antibodies were detected in 38 (4.36%) participants. CONCLUSION: Our study determined a low prevalence of COVID-19 in HCW, which may reflect the effectiveness of hygiene protocols. However, it could also indicate a low prevalence of SARS CoV-2 in hospital employees. Our study protocol may serve as an instructive example for future pandemic containment protocols in hospitals.

Combined antifungal therapy is superior to monotherapy in pulmonary scedosporiosis in cystic fibrosis.

Cystic fibrosis (CF) is characterised by chronic airway infection with bacteria and fungi. Infections caused by Scedosporium/Lomentospora species can occur and are difficult to treat. Moulds belonging to the genus Scedosporium/Lomentospora are detected most frequently in respiratory samples of patients with CF, next to Aspergillus spp. Our aim was to define pulmonary fungal infections due to Scedosporium/Lomentospora in CF and to study the antimycotic treatment. In this multicentre study (12 centres; duration January 2008 to December 2014) 31 patients with a lung infection caused by moulds of the genus Scedosporium/Lomentospora were included. 36 courses of antifungal treatment were documented. Scedosporium apiospermum sensu stricto accounted for 48.4% of cases. In 20/31 patients a therapeutic response under antimycotics (median duration 3.9 months) was achieved. Triple and double therapy was significantly more effective compared to monotherapy regarding FEV1, radiology, and symptoms. This data suggests that combined treatment is superior to monotherapy in patients with CF.

Ongoing epidemic of lymphogranuloma venereum in HIV-positive men who have sex with men: how symptoms should guide treatment.

INTRODUCTION: Lymphogranuloma venereum (LGV) is a sexually transmitted infection (STI) caused by chlamydia trachomatis (CT) genotype L (L1, L2 and L3). Recent outbreaks of LGV in Europe and North America affected mainly men who have sex with men (MSM). Infections with CT serotypes D-K are mostly associated with mild symptoms or may be clinically silent. However, infections with L genotypes are more invasive and induce anogenital ulcer or inguinal lymphadenopathy. MATERIALS AND METHODS: Between 2003 and 2013, anal or genital CT infections were diagnosed in 471 symptomatic patients attending the Infectious Diseases Center Hamburg (ICH). CT DNA was detected by PCR. CT genotyping of positive samples was performed by sequence analyses of omp1 PCR products (samples between 2003 and 2010). Samples between 2012 and 2013 were analyzed by genotype L specific real-time PCR. RESULTS: In total, 221 LGV patients were identified (3 in 2003; 11 in 2004; 26 in 2005; 33 in 2006; 24 in 2007; 16 in 2008; 18 in 2009; 15 in 2010; 17 in 2011; 26 in 2012 and 32 in 2013). One hundred ninety-eight of 221 (89.6%) patients with LGV were HIV-infected; 10 of 221 (4.5%) were HIV-negative, and the HIV-status unknown in 13 (5.9%). The majority of LGV positive patients (204/221; 92%) had anorectal symptoms (bloody proctitis, rectal pain, purulent or mucous discharge, tenesmus, constipation), compared to 17/221 (8%) who presented with genital symptoms as urethritis, genital ulcer or inguinal lymphadenopathy. Of 283 CT-positive patients with anorectal symptoms, genotype L was detected in 205 (72%), while non-L genotypes (D-K) were found in 78 (28%). In contrast, genotype L was found in only 6% of patients with genital symptoms (11/177), whereas 94% (166/177) were infected with non-L genotypes only. CONCLUSIONS: The epidemic of LGV among predominantly HIV+ MSM is ongoing. LGV might be underdiagnosed, especially in patients with anorectal symptoms. Infections with CT serotypes D-K are more often associated with urogenital symptoms or asymptomatic infection, whereas LGV genotypes are found most frequently in patients with rectal/intestinal symptoms. Anorectal CT infections in MSM should be further characterized by genotyping for LGV, as for LGV three weeks of doxycycline treatment are recommended. CT genotypes D-K generally require shorter antibiotic treatment. If CT genotyping is not available, the duration of treatment should be prolonged to three weeks empirically for CT-positive patients with anorectal or intestinal symptoms.

S100A7/psoriasin expression in the human lung: unchanged in patients with COPD, but upregulated upon positive S. aureus detection.

BACKGROUND: Progressive airway inflammation and susceptibility to the airway colonisation and infection are characteristic for the pathophysiology of chronic obstructive pulmonary disease (COPD). Antimicrobial peptides (AMPs) are central to the function of the innate host immune response against microbial pathogens and are regulators of inflammation and immunity. S100A7/psoriasin, a recently described AMP, is an essential component of the human epithelia against invading pathogens and acts as an effector molecule of the host innate defence in the skin. We hypothesized that S100A7/psoriasin is involved in the airway mucosal immunity and differently regulated and expressed in the lung during progression of COPD. METHODS: S100A7/psoriasin gene expression was assessed in bronchial biopsies and bronchoalveolar lavage (BAL) fluid cells of healthy controls and COPD patients. Using confocal microscopy and immunohistochemistry, the protein expression of S100A7/psoriasin was investigated. RESULTS: Here, we report that S100A7/psoriasin, the major antimicrobial peptide of the human skin, is constitutively expressed in perinuclear granules of human bronchial epithelial cells and alveolar macrophages. Whereas typical activators of the innate immune response like TLR ligands and cytokines induced the upregulation of CXCL-8 mRNA and release of CXCL-8 by epithelial cells, S100A7/psoriasin mRNA expression was not modulated. To investigate a potential association of S100A7/psoriasin with COPD, S100A7/psoriasin mRNA expression was assessed in bronchial biopsies and BAL fluid cells of patients at different stages of COPD and controls. Overall, 10 healthy individuals and 34 COPD patients were enrolled in this study. We found an association of S100A7/psoriasin mRNA expression with bacterial detection in the tracheobronchial system (p = 0.0304), which was the strongest in individuals positive for with S. aureus (p = 0.0005). However, S100A7/psoriasin mRNA expression was not altered during the progression of COPD. CONCLUSIONS: S100A7/psoriasin gene expression is unchanged in the airways during COPD. The newly identified association of S100A7/psoriasin with S. aureus may provide new insights into the antimicrobial defence response of the human airways, leading to the induction of S100A7/psoriasin upon microbial challenge.

Nasal carriage of Staphylococcus aureus and endonasal activity in Wegener s granulomatosis as compared to rheumatoid arthritis and chronic Rhinosinusitis with nasal polyps.

OBJECTIVES: Nasal colonisation with Staphylococcus aureus (S. aureus) has been implicated in Wegener's granulomatosis (WG) disease activity. In this study, the frequency of nasal colonisation with S. aureus in WG was compared to healthy and disease control groups for the first time. Moreover, endonasal activity was correlated to colonisation. PATIENTS AND METHODS: Nasal carriage of S. aureus of a well-defined group of 89 patients with WG was compared to 40 patients with chronic rhinosinusitis with nasal polyps (CRS), 35 patients with rheumatoid arthritis (RA), 50 hospital staff members and 25 subjects without regular hospital contact and correlation analysis of nasal carriage and endonasal activity of WG was performed. RESULTS: WG patients showed significantly higher rates (72%) of nasal colonisation with S. aureus compared to CRS patients (28%) and healthy subjects without regular hospital contact (25%, 95%-CI), but not to RA patients (46%) and hospital staff members (58%). WG patients with nasal carriage of S. aureus had significantly higher endoscopically proven endonasal activity (p=0.01), significantly more often first manifestation of WG in the upper respiratory tract (p=0.02) and higher relapse-rates (p=0.052) than WG patients without such carriage. CONCLUSIONS: Endonasal activity in WG is associated with higher nasal S. aureus colonisation rates and subsequent higher relapse rates. The higher frequency of S. aureus colonisation could be a consequence of a recently shown mucosal barrier defect in WG and facilitate chronic inflammation and granuloma formation in the upper respiratory tract.

Monoclonal antibody S60-4-14 reveals diagnostic potential in the identification of Pseudomonas aeruginosa in lung tissues of cystic fibrosis patients.

The lipopolysaccharide (LPS) of Pseudomonas aeruginosa has been identified to contain an inner-core structure expressing a Pseudomonas-specific epitope. This target structure is characterized by a highly phosphorylated and 7-O-carbamoyl-l-glycero-alpha-d-manno-heptopyranose (CmHep) and was found to be present in all human-pathogenic Pseudomonas species of the Palleroni (RNA)-classification I scheme. We raised and selected the monoclonal antibody S60-4-14 (mAb S60-4-14, subtype IgG1) from mice immunized with heat-killed Pseudomonas bacteria. The epitope of this mAb was found to reside in the inner-core structure of P. aeruginosa and, hence, successfully evaluated for the immunohistochemical detection of P. aeruginosa in formalin- or HOPE-fixed (Hepes-glutamic acid buffer-mediated organic solvent protection effect) and paraffin-embedded human lung tissue slices. Lung specimens, mainly from explanted lungs of cystic fibrosis (CF) patients, as well as P. aeruginosa isolates from patients suffering from CF and patients with extrapulmonar Pseudomonas infections were investigated by PCR, immunohistochemistry, and Western blot analysis with mAb S60-4-14. The results revealed an unequivocal coincidence of PCR and immunohistochemistry. Together with the Western blot results mAb S60-4-14 displays a potential diagnostic tool for the specific identification of P. aeruginosa in infected lungs of CF.

Manno(rhamno)biose-containing capsular polysaccharides of Klebsiella pneumoniae enhance opsono-stimulation of human polymorphonuclear leukocytes.

We tested the relationship between the capsular and the O-antigen structures and the ability of bacteria to trigger respiratory burst in human polymorphonuclear leukocytes (PMNL). Capsulated and non-capsulated variants as well as capsule-switched derivatives of Klebsiella serotypes bearing or lacking manno(rhamno)biose repeats in their capsular polysaccharides and expressing either mannose-rich or mannose-poor O antigens were tested for their ability to induce respiratory burst and survive in human PMNL. Luminol-enhanced chemiluminescence (CL) was measured to quantify respiratory burst. Intracellular survival was quantified by determining the viable counts of intracellular bacteria. K serotypes and the capsule-switched derivative lacking manno(rhamno)biose induced significantly lower CL than those expressing manno(rhamno)biose. Manno(rhamno)biose-lacking serotypes survived in the cells significantly better than serotypes expressing these repeats. C1q depletion did not affect CL induced by the manno(rhamno)biose-containing serotype, whereas factor B depletion revealed a significantly reduced CL. Likewise, EGTA in the presence of Mg(2+) significantly decreased CL, but the values were higher than those induced by the bacterium opsonized with factor B-depleted serum. In the presence of EGTA, Mg(2+)-treated factor B-depleted serum revealed a significant reduction in the CL response compared with the responses induced by opsonization with factor B-depleted serum alone. These results indicate, in addition to the alternative pathway, a manno(rhamno)biose pattern recognition of Klebsiella by PMNL probably by the complement lectin pathway.

Klebsiella oxytoca: opportunistic infections in laboratory rodents.

Opportunistic pathogens have become increasingly relevant as the causative agents of clinical disease and pathological lesions in laboratory animals. This study was conducted to evaluate the role of Klebsiella oxytoca as an opportunistic pathogen in laboratory rodents. Therefore, K. oxytoca-induced lesions were studied from 2004 to early 2006 in naturally infected rodent colonies maintained at The Jackson Laboratory (TJL), Bar Harbor, USA, the Animal Research Centre (Tierforschungszentrum, TFZ) of the University of Ulm, Germany and the Central Animal Facility (ZTM) of the Hannover Medical School, Germany. K. oxytoca infections were observed in substrains of C3H/HeJ mice, which carry the Tlr4(Lps-d) allele; in LEW.1AR1-iddm rats, the latter being prone to diabetes mellitus; in immunodeficient NMRI-Foxn1(nu) mice; and in mole voles, Ellobius lutescens. The main lesions observed were severe suppurative otitis media, urogenital tract infections and pneumonia. Bacteriological examination revealed K. oxytoca as monocultures in all cases. Clonality analysis performed on strains isolated at the ZTM and TFZ (serotyping, pulse field gel electrophoresis [PFGE], enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction, sequencing of 16S rRNA and rpoB genes) revealed that the majority of bacteria belonged to two clones, one in each facility, expressing the capsule type K55 (ZTM) or K72 (TFZ). Two strains, one isolated at the ZTM and one at the TFZ, showed different PFGE and ERIC pattern than all other isolates and both expressed capsule type K35. In conclusion, K. oxytoca is an opportunistic pathogen capable of inducing pathological lesions in different rodent species.

Rapid emergence of resistance to linezolid during linezolid therapy of an Enterococcus faecium infection.

We report the emergence of linezolid resistance (MICs of 16 to 32 mg/liter) in clonally related vancomycin-susceptible and -resistant Enterococcus faecium isolates from an intensive care unit patient after 12 days of linezolid therapy. Only linezolid-susceptible isolates of the same clone were detected at 28 days after termination of linezolid therapy.

Identification of RNase 8 as a novel human antimicrobial protein.

The antimicrobial activity of the human RNase A superfamily member RNase 8 was evaluated. Recombinant RNase 8 exhibited broad-spectrum microbicidal activity against potential pathogenic microorganisms (including multidrug-resistant strains) at micro- to nanomolar concentrations. Thus, RNase 8 was identified as a novel antimicrobial protein and may contribute to host defense.

Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection.

Host immune response influences the clinical outcome of Helicobacter pylori infection leading to ulcer disease, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A genetic risk profile for gastric cancer has been identified, but genetic susceptibility to develop MALT lymphoma is still unclear. We investigated the role of NOD1 and NOD2 as intracellular recognition molecules for pathogen-associated molecules in H. pylori infection in vitro and analysed the influence of single nucleotide polymorphisms on susceptibility to ulcer disease and MALT lymphoma. Expression of NOD1 and NOD2 significantly sensitized HEK293 cells to H. pylori-induced NF-kappaB activation in a cag pathogenicity island (cagPAI)-dependent manner. In cells carrying the Crohn-associated NOD2 variant R702W the NF-kappaB response was significantly diminished. NOD1/NOD2 expression levels were induced in the gastric epithelium in H. pylori-positive patients. No mutations were found to be associated with gastritis or gastric ulcer development. However, the R702W mutation in the NOD2/CARD15 gene was significantly associated with gastric lymphoma. Carrier of the rare allele T had a more than doubled risk to develop lymphoma than controls [odds ratio (OR): 2.4, 95% confidence interval (CI): 1.2-4.6; P < 0.044]. H. pylori-induced upregulation of NOD1 and NOD2 in vivo may play a critical role in the recognition of this common pathogen. A missense mutation in the leucine-rich region of CARD15 is associated with gastric lymphoma.

Lipid-specific membrane activity of human beta-defensin-3.

Defensins represent a major component of innate host defense against bacteria, fungi, and enveloped viruses. One potent defensin found, e.g., in epithelia, is the polycationic human beta-defensin-3 (hBD3). We investigated the role of the lipid matrix composition, and in particular the presence of negatively charged lipopolysaccharides (LPS) from sensitive (Escherichia coli, Salmonella enterica serovar Minnesota) or resistant (Proteus mirabilis) Gram-negative bacteria or of the zwitterionic phospholipids of human cells, in determining the action of polycationic hBD3 on the different membranes, and related to their biological activity. The main focus was directed on data derived from electrical measurements on a reconstitution system of the OM as a planar asymmetric bilayer composed on one side of LPS and on the other of a phospholipid mixture. Our results demonstrate that the antimicrobial activity and the absence of cytotoxicity can be explained by the lipid-specificity of the peptide. A clear correlation between these aspects of the biological activity of hBD3 and its interaction with lipid matrices could be found. In particular, hBD3 could only induce lesions in those membranes resembling the lipid composition of the OM of sensitive bacterial strains. The permeation through the membrane is a decisive first step for the biological activity of many antimicrobial peptides. Therefore, we propose that the lipid-specificity of hBD3 as well as some other membrane-active antimicrobial peptides is important for their activity against bacteria or mammalian cells.

Detection of diverse bacterial signatures in atherosclerotic lesions of patients with coronary heart disease.

BACKGROUND: Bacterial infection has been discussed as a potential etiologic factor in the pathophysiology of coronary heart disease (CHD). This study analyzes molecular phylogenies to systematically explore the presence, frequency, and diversity of bacteria in atherosclerotic lesions in patients with CHD. METHODS AND RESULTS: We investigated 16S rDNA signatures in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients with CHD, control material from postmortem patients (n=15), and heart-beating organ donors (n=11) using clone libraries, denaturating gradient gel analysis, and fluorescence in situ hybridization. Bacterial DNA was found in all CHD patients by conserved PCR but not in control material or in any of the normal/unaffected coronary arteries. Presence of bacteria in atherosclerotic lesions was confirmed by fluorescence in situ hybridization. A high overall bacterial diversity of >50 different species, among them Staphylococcus species, Proteus vulgaris, Klebsiella pneumoniae, and Streptococcus species, was demonstrated in >1500 clones from a combined library and confirmed by denaturating gradient gel analysis. Mean bacterial diversity in atheromas was high, with a score of 12.33+/-3.81 (range, 5 to 22). A specific PCR detected Chlamydia species in 51.5% of CHD patients. CONCLUSIONS: Detection of a broad variety of molecular signatures in all CHD specimens suggests that diverse bacterial colonization may be more important than a single pathogen. Our observation does not allow us to conclude that bacteria are the causative agent in the etiopathogenesis of CHD. However, bacterial agents could have secondarily colonized atheromatous lesions and could act as an additional factor accelerating disease progression.

Activity of human beta-defensins 2 and 3 against ESBL-producing Klebsiella strains.

OBJECTIVES: To test the bactericidal activity of human beta-defensins (hBDs) 2 and 3 against extended-spectrum beta-lactamase (ESBL)-producing Klebsiella strains. METHODS: Thirty-six Klebsiella pneumoniae and seventeen Klebsiella oxytoca ESBL-producing isolates from nosocomial infections were tested. The bactericidal activity of recombinantly synthesized hBD-2 and -3 was tested and the results were given either as lethal doses killing > or = 90% of bacteria (LD90s) or as MBCs (> or = 99.9% killing). RESULTS: Except for one intermediately susceptible strain (MBC = 25 mg/L), all other ESBL-producing strains were highly susceptible to both defensins (LD90s and MBCs < or = 12.5 mg/L). CONCLUSIONS: The results underline the high efficacy of hBD-2 and -3 against ESBL-producing Klebsiella, making both defensins attractive candidates as antimicrobial agents to combat these increasingly troublesome bacteria.

Noncapsulated Klebsiella pneumoniae bearing mannose-containing O antigens is rapidly eradicated from mouse lung and triggers cytokine production by macrophages following opsonization with surfactant protein D.

To better understand the relationship between the surface polysaccharides of pulmonary pathogens and components of the lung innate immune system, we employed selected serotypes of Klebsiella pneumoniae expressing distinct capsular polysaccharides and/or O antigen in a murine model of K. pneumoniae infection. In addition, we examined the effect of surfactant protein D (SP-D) on the cytokine response of human monocyte-derived macrophages to these serotypes in vitro. Noncapsulated mannose-containing O3 serotypes (K50/n and K55/n), which react efficiently with SP-D in vitro, triggered high levels of interleukin-1beta (IL-1beta) and IL-6 production. In vivo, they were more efficiently cleared from the lungs of mice but not from macrophage-depleted mice. They also were more efficiently internalized by alveolar macrophages in vivo. In contrast, galactose-containing O1 serotypes (K2/n and K21a/n), which interact poorly with SP-D, exhibited significantly lower cytokine production and less efficient pulmonary clearance and were ineffectively internalized by alveolar macrophages. These findings are consistent with in vitro results showing that production of IL-1beta and IL-6 mRNA and IL-6 protein by human macrophages exposed to mannose-bearing Klebsiella O serotypes is significantly increased by SP-D. Thus, survival of inhaled bacteria in the lung depends partially on the lipopolysaccharide structure of the bacteria and their interactions with innate immunity components. We speculate that an imbalance of host SP-D and therefore cytokine levels may result in high susceptibility of the host to the pathogen.

Burkholderia is highly resistant to human Beta-defensin 3.

The bactericidal activity of the novel beta-defensin hBD-3 against 28 species and 55 strains of gram-positive cocci and gram-negative fermentative and nonfermentative rods was tested. All strains proved to be highly or intermediately susceptible to hBD-3 (minimal bactericidal concentration [MBC], 100 micro g/ml.

Surfactant protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing mannose-rich O-antigens.

Surfactant protein D (SP-D) plays important roles in the regulation of innate immune responses in the lung. We have previously shown that SP-D can agglutinate and enhance the macrophage-dependent killing of specific unencapsulated phase variants of Klebsiella pneumoniae. In the present studies, we used 16 clinical isolates of Klebsiella representing four O-serotypes and examined the interaction of SP-D with their isolated LPSs. Although SP-D bound to the core oligosaccharide of rough LPS from all isolates, it selectively bound to smooth forms of LPS expressed by O-serotypes with mannose-rich repeating units in their O-polysaccharides. SP-D was more potent in agglutinating unencapsulated phase variants of O-serotypes expressing these SP-D "reactive" O-polysaccharides, and more effectively inhibited the adhesion of these serotypes to lung epithelial cells. This novel anti-adhesion activity required the multimerization of trimeric SP-D subunits (dodecamers). Klebsiella serotypes expressing "nonreactive" LPS O-Ags were isolated at a significantly higher frequency from patients with K. pneumoniae. Our findings suggest that SP-D plays important roles in the clearance of opportunistic Gram-negative bacteria and contributes to known serotypic differences in the pathogenicity of Klebsiella through specific interactions with O-polysaccharides.