Search

Abstract Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.

IntroductionAn increasing number of studies demonstrate that viral meningitis and meningoencephalitis, even those with a mild course of meningitis, can result in residual sequelae.MethodsWe aimed to investigate the long-term outcome in both viral meningitis and meningoencephalitis/encephalitis patients and impact of long-term sequelae on patients’ social and professional daily lives in a prospective observational study with a follow-up period of 20 months.ResultsA total of 50 patients (12% encephalitis, 58% meningoencephalitis and 30% meningitis) and 21 control persons participated in the study. The most common cause was the tick-borne encephalitis (TBE) virus. The most important persistent signs and symptoms after 2 years were subjective cognitive impairment (36%), fatigue and/or excessive daytime sleepiness (31%), disturbed nighttime sleep (31%) and headaches (13%), as well as feeling more rapidly exhausted after cognitive effort (53%). Independent of disease severity in the acute phase, almost one third of patients still reported mildly impaired social and/or professional life due to the long-term sequelae, with scores in the health status assessment still significantly lower compared to healthy controls.DiscussionRegardless of the severity of the acute illness and despite constant improvement within 2 years, 67% of patients still had persistent signs and symptoms, but these were only relevant to everyday social or professional life in about 30% of these patients.

ABSTRACTDuring the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent—something that is unknown at the time of sequencing—and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses.

Abstract Drug repurposing is an important strategy in COVID-19 treatment, but many clinically approved compounds have not been extensively studied in the context of embryogenesis, thus limiting their administration during pregnancy. Here we used the zebrafish embryo model organism to test the effects of 162 marketed drugs on cardiovascular development. Among the compounds used in the clinic for COVD-19 treatment, we found that Remdesivir led to reduced body size and heart functionality at clinically relevant doses. Ritonavir and Baricitinib showed reduced heart functionality and Molnupiravir and Baricitinib showed effects on embryo activity. Sabizabulin was highly toxic at concentrations only 5 times higher than Cmax and led to a mean mortality of 20% at Cmax. Furthermore, we tested if zebrafish could be used as a model to study inflammatory response in response to spike protein treatment and found that Remdesivir, Ritonavir, Molnupiravir, Baricitinib as well as Sabizabulin counteracted the inflammatory response related gene expression upon SARS-CoV-2 spike protein treatment. Our results show that the zebrafish allows to study immune-modulating properties of COVID-19 compounds and highlights the need to rule out secondary defects of compound treatment on embryogenesis. All results are available on a user friendly web-interface https://share.streamlit.io/alernst/covasc_dataapp/main/CoVasc_DataApp.py that provides a comprehensive overview of all observed phenotypic effects and allows personalized search on specific compounds or group of compounds. Furthermore, the presented platform can be expanded for rapid detection of developmental side effects of new compounds for treatment of COVID-19 and further viral infectious diseases.

BackgroundMagnetic resonance imaging (MRI) findings in meningoencephalitis have mainly been described in terms of their diagnostic value rather than their prognostic potential, except for herpes simplex virus (HSV) encephalitis. The aims of our study were to describe frequency and anatomic locations of MRI abnormalities specific to limbic, circadian and motor systems in a cohort of meningoencephalitis patients, as well as to investigate the prognostic value of these MRI findings.MethodsA secondary, selective analysis of a retrospective database including all meningitis, meningoencephalitis and encephalitis cases treated between 2016 and 2018 in the University hospital of Bern, Switzerland was performed. Patients with meningitis of any cause, bacterial or autoimmune causes of encephalitis were excluded.ResultsMRI scans and clinical data from 129 meningoencephalitis cases found that the most frequent causes were tick-borne encephalitis (TBE, 42%), unknown pathogens (40%), VZV (7%), and HSV1 (5%). At discharge, median modified Rankin Score (mRS) was 3 (interquartile range, IQR, 1), 88% of patients had persisting signs and symptoms. After a median of 17 months, median Glasgow Outcome Score (GOS) was 5 (IQR 1), 39% of patients still had residual signs or symptoms. All patients with HSV, 27% with TBE and 31% of those with meningoencephalitis of unknown etiology had fluid-attenuated inversion recovery (FLAIR) and to a lesser extent diffusion-weighted imaging (DWI) lesions in their initial MRI, with highly overlapping anatomical distribution. In one fifth of TBE patients the limbic system was affected. Worse outcome was associated with presence of DWI and/or FLAIR lesions and lower normalized apparent diffusion coefficient (ADC) signal intensities.ConclusionPresence of FLAIR lesions, restricted diffusion as well as the extent of ADC hypointensity in initial MRI are parameters which might be of prognostic value regarding the longterm clinical outcome for patients with meningoencephalitis of viral and of unknown origin. Although not described before, affection of limbic structures by TBE is possible as shown by our results: A substantial proportion of our TBE patients had FLAIR signal abnormalities in these regions.

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach called NASCarD (Nanopore Adaptive Sampling with Carrier DNA), which allows a low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using COVID-19 positive samples available during the omicron wave, we demonstrate how the method may lead to >99% genome completeness of the SARS-CoV-2 genome sequences within 7 h of sequencing at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples.

Abstract Background/Aims Tick-borne encephalitis (TBE) is a disease affecting the central nervous system. Over the last decade, the incidence of TBE has steadily increased in Europe and Asia despite the availably of effective vaccines. Up to 50% of patients after TBE suffer from post-encephalitic syndrome that may develop into long-lasting morbidity. Altered sleep–wake functions have been reported by patients after TBE. The mechanisms causing these disorders in TBE are largely unknown to date. As a first step toward a better understanding of the pathology of TBEV-inducing sleep dysfunctions, we assessed parameters of sleep structure in an established infant rat model of TBE. Methods 13-day old Wistar rats were infected with 1 × 106 FFU Langat virus (LGTV). On day 4, 9, and 21 post infection, Rotarod (balance and motor coordination) and open field tests (general locomotor activity) were performed and brains from representative animals were collected in each subgroup. On day 28 the animals were implanted with a telemetric EEG/EMG system. Sleep recording was continuously performed for 24 consecutive hours starting at day 38 post infection and visually scored for Wake, NREM, and REM in 4 s epochs. Results As a novelty of this study, infected animals showed a significant larger percentage of time spend awake during the dark phase and less NREM and REM compared to the control animals (p

Abstract One-third of pneumococcal meningitis (PM) survivors suffer from neurological sequelae including learning disabilities and hearing loss due to excessive neuroinflammation. There is a lack of efficacious compounds for adjuvant therapy to control this long-term consequence of PM. One hallmark is the recruitment of leukocytes to the brain to combat the bacterial spread. However, this process induces excessive inflammation, causing neuronal injury. Maraviroc (MVC)—a CCR5 antagonist—was demonstrated to inhibit leukocyte recruitment and attenuate neuroinflammation in several inflammatory diseases. Here, we show that in vitro, MVC decreased nitric oxide production in astroglial cells upon pneumococcal stimulation. In vivo, infant Wistar rats were infected with 1 × 104 CFU/ml S. pneumoniae and randomized for treatment with ceftriaxone plus MVC (100 mg/kg) or ceftriaxone monotherapy. During the acute phase, neuroinflammation in the CSF was measured and histopathological analyses were performed to determine neuronal injury. Long-term neurofunctional outcome (learning/memory and hearing capacity) after PM was assessed. MVC treatment reduced hippocampal cell apoptosis but did not affect CSF neuroinflammation and the neurofunctional outcome after PM. We conclude that MVC treatment only exerted limited effect on the pathophysiology of PM and is, therefore, not sufficiently beneficial in this experimental paradigm of PM.

IntroductionStreptococcus pneumoniae bacteria cause life-threatening invasive pneumococcal disease (IPD), including meningitis. Pneumococci are classified into serotypes, determined by differences in capsular polysaccharide and both serotype and pneumolysin toxin are associated with disease severity. Strains of serotype 8, ST 53, are increasing in prevalence in IPD in several countries.MethodsHere we tested the virulence of such an isolate in a rat model of meningitis in comparison with a serotype 15B and a serotype 14 isolate. All three were isolated from meningitis patients in South Africa in 2019, where serotype 8 is currently the most common serotype in IPD.Results and DiscussionOnly the serotype 8 isolate was hypervirulent causing brain injury and a high mortality rate. It induced a greater inflammatory cytokine response than either the serotype 15B or 14 strain in the rat model and from primary mixed-glia cells isolated from mouse brains. It had the thickest capsule of the three strains and produced non-haemolytic pneumolysin. Pneumolysin-sequestering liposomes reduced the neuroinflammatory cytokine response in vitro indicating that liposomes have the potential to be an effective adjuvant therapy even for hypervirulent pneumococcal strains with non-haemolytic pneumolysin.

Abstract Background Depending on geographic location, causes of encephalitis, meningoencephalitis and meningitis vary substantially. We aimed to identify the most frequent causes, clinical presentation and long-term outcome of encephalitis, meningoencephalitis and meningitis cases treated in the Inselspital University Hospital Bern, Switzerland. Methods In this monocentric, observational study, we performed a retrospective review of clinical patient records for all patients treated within a 3-year period. Patients were contacted for a telephone follow-up interview and to fill out questionnaires, especially related to disturbances of sleep and wakefulness. Results We included 258 patients with the following conditions: encephalitis (18%), nonbacterial meningoencephalitis (42%), nonbacterial meningitis (27%) and bacterial meningoencephalitis/meningitis (13%). Herpes simplex virus (HSV) was the most common cause of encephalitis (18%); tick-borne encephalitis virus (TBEV) was the most common cause of nonbacterial meningoencephalitis (46%), enterovirus was the most common cause of nonbacterial meningitis (21%) and Streptococcus pneumoniae was the most common cause of bacterial meningoencephalitis/meningitis (49%). Overall, 35% patients remained without a known cause. After a median time of 16 months, 162 patients participated in the follow-up interview; 56% reported suffering from neurological long-term sequelae such as fatigue and/or excessive daytime sleepiness (34%), cognitive impairment and memory deficits (22%), headache (14%) and epileptic seizures (11%). Conclusions In the Bern region, Switzerland, TBEV was the overall most frequently detected infectious cause, with a clinical manifestation of meningoencephalitis in the majority of cases. Long-term neurological sequelae, most importantly cognitive impairment, fatigue and headache, were frequently self-reported not only in encephalitis and meningoencephalitis survivors but also in viral meningitis survivors up to 40 months after acute infection.

Abstract Human adenoviruses (HAdVs) are highly contagious pathogens of clinical importance, especially among the pediatric population. Studies on comparative viral genomic analysis of cases associated with severe and mild infections due to HAdV are limited. Using whole-genome sequencing (WGS), we investigated whether there were any differences between circulating HAdV strains associated with severe infections (meningitis, sepsis, convulsion, sudden infant death syndrome, death, and hospitalization) and mild clinical presentations in pediatric patients hospitalized between the years 1998 and 2017 in a tertiary care hospital group in Bern, Switzerland covering a population base of approx. 2 million inhabitants. The HAdV species implicated in causing severe infections in this study included HAdV species C genotypes (HAdV1, HAdV2, and HAdV5). Clustering of the HAdV whole-genome sequences of the severe and mild cases did not show any differences except for one sample (isolated from a patient presenting with sepsis, meningitis, and hospitalization) that formed its own cluster with HAdV species C genotypes. This isolate showed intertypic recombination events involving four genotypes, had the highest homology to HAdV89 at complete genome level, but possessed the fiber gene of HAdV1, thereby representing a novel genotype of HAdV species C. The incidence of potential recombination events was higher in severe cases than in mild cases. Our findings confirm that recombination among HAdVs is important for molecular evolution and emergence of new strains. Therefore, further research on HAdVs, particularly among susceptible groups, is needed and continuous surveillance is required for public health preparedness including outbreak investigations.

Abstract Objective Bacteriophages (or phages) are viruses which infect and lyse bacteria. The therapeutic use of phages (phage therapy) has regained attention in the last decades as an alternative strategy to treat infections caused by antimicrobial-resistant bacteria. In clinical settings it is most likely that phages are administered adjunct to antibiotics. For successful phage therapy it is therefore crucial to investigate different phage-antibiotic combinations in vivo. This study aimed to elucidate the combinatorial effects of systemic daptomycin and nebulised bacteriophages for the treatment of experimental pneumonia due to methicillin-resistant Staphylococcus aureus (MRSA). Results Using a rat model of ventilator-associated pneumonia caused by MRSA, the simultaneous application of intravenous daptomycin and nebulised phages was not superior to aerophage therapy alone at improving animal survival (55% vs. 50%), or reducing bacterial burdens in the lungs, or spleen. Thus, this combination does not seem to be of benefit for use in patients with MRSA pneumonia.

The recommended empiric ceftriaxone dosing regimen for acute bacterial meningitis in adults is 2 g every 12 h. After penicillin-susceptible Streptococcus pneumoniae is isolated as a causative microorganism, the ceftriaxone dose may be continued or reduced to a single dose of 2 g every 24 h, per institutional preference. There is no clear guidance that indicates the superiority of one regimen over the other. The objective of this study was to evaluate the susceptibility of S. pneumoniae in the cerebral spinal fluid (CSF) of patients with meningitis and the relationship between ceftriaxone dose and clinical outcomes. We identified 52 patients with S. pneumoniae meningitis with positive CSF cultures who were treated at the University Hospital, Bern, Switzerland, over a 19-year period. We collected clinical and microbiological data for evaluation. Broth microdilution and Etest methods were performed to test penicillin and ceftriaxone susceptibility. All isolates were susceptible to ceftriaxone. Ceftriaxone was empirically used in 50 patients, with a starting dosing regimen of 2 g every 24 h in 15 patients and 2 g every 12 h in 35 patients. In 32 patients started on a twice-daily regimen (91%), doses were reduced to once daily after a median of 1.5 (95% CI 1–2) days. The overall in-hospital mortality was 15.4% (n = 8), and 45.7% of patients reported at least one sequela of meningitis at the last follow-up (median 375, 95% CI 189–1585 days). We found no statistical difference in outcome between the 2 g every 24 h and the 2 g every 12 h ceftriaxone dosing regimens. A ceftriaxone total daily dose of 2 g may be associated with similar outcomes to a 4 g total daily dose, provided that the causative organism is highly susceptible to ceftriaxone. The persistence of neurological and infection sequelae at the last follow-up underscores the need for optimal treatment of these complex infections.

ABSTRACT Despite best available therapy, cerebral aspergillosis is an often-lethal complication of disseminated aspergillosis. There is an urgent need to expand the currently limited therapeutic options. In this study, we assessed cerebral drug exposure and efficacy of caspofungin (CAS) using a lethal infant rat model of cerebral aspergillosis. Eleven-day-old Wistar rats were infected by intracisternal injection of Aspergillus fumigatus conidia. Treatment started after 22 h and was continued for 10 days. Regimens were CAS 1 mg/kg/day intraperitoneally (i.p.), liposomal amphotericin B (L-AmB) 5 mg/kg/day i.p., and both drugs combined at the same dose i.p. Infected controls were given NaCl 0.85% i.p. Primary endpoints assessed were survival, cerebral fungal burden, galactomannan index, and drug concentrations in brain homogenate at 2, 3, 5, and 11 days after infection. Compared to those of controls (4.4 ± 2.7 days), survival times were increased by treatment with CAS alone (10.3 ± 1.7 days; P

The SARS-CoV-2 Delta variant, corresponding to the Pangolin lineage B.1.617.2, was first detected in India in July 2020 and rapidly became dominant worldwide. The ARTIC v3 protocol for SARS-CoV-2 whole-genome sequencing, which relies on a large number of PCR primers, was among the first available early in the pandemic, but may be prone to coverage dropouts that result in incomplete genome sequences. A new set of primers (v4) was designed to circumvent this issue in June 2021. In this study, we investigated whether the sequencing community adopted the new sets of primers, especially in the context of the spread of the Delta lineage, in July 2021. Because information about protocols from individual laboratories is generally difficult to obtain, the aims of the study were to identify whether large under-sequenced regions were present in deposited Delta variant genome sequences (from April to August 2021), to investigate the extent of the coverage dropout among all the currently available Delta sequences in six countries, and to propose simple PCR primer modifications to sequence the missing region, especially for the first circulating Delta variants observed in 2021 in Switzerland. Candidate primers were tested on few clinical samples, highlighting the need to further pursue primer optimization and validation on a larger and diverse set of samples.

Abstract Background Pneumococcal meningitis (PM) remains a global public health concern and affects all age groups. If acquired during infancy or childhood, permanent neurofunctional deficits including cognitive impairment, cerebral palsy, and secondary epilepsy are typical sequelae of neuronal injury. Determination of patients at risk for the development of brain injury and subsequent neurofunctional sequelae could help to identify patients for focused management. Neurofilament light chain (NfL) is an axonal cytoskeletal protein released upon neuronal injury into the cerebrospinal fluid (CSF) and blood. As little is known about the course of neurofilament release in the course of PM, we measured CSF and serum NfL levels longitudinally in experimental PM (ePM). Methods Eleven-day-old infant Wistar rats were infected intracisternally with Streptococcus pneumoniae and treated with ceftriaxone. At 18 and 42 h post-infection (hpi), the blood and CSF were sampled for NfL measurements by a single molecule array technology. Inflammatory cytokines and MMP-9 in CSF were quantified by magnetic bead multiplex assay (Luminex®) and by gel zymography, respectively. Results In ePM, CSF and serum NfL levels started to increase at 18 hpi and were 26- and 3.5-fold increased, respectively, compared to mock-infected animals at 42 hpi (p < 0.0001). CSF and serum NfL correlated at 18 hpi (p < 0.05, r = 0.4716) and 42 hpi (p < 0.0001, r = 0.8179). Both CSF and serum NfL at 42 hpi strongly correlated with CSF levels of IL-1β, TNF-α, and IL-6 and of MMP-9 depending on their individual kinetics. Conclusion Current results demonstrate that during the peak inflammatory phase of ePM, NfL levels in CSF and serum are the highest among CNS disease models studied so far. Given the strong correlation of CSF versus serum NfL, and its CNS-specific signal character, longitudinal measurements to monitor the course of PM could be performed based on blood sample tests, i.e., without the need of repetitive spinal taps. We conclude that NfL in the serum should be evaluated as a biomarker in PM.

Abstract Background Meningococcal meningitis (MM) is a life-threatening disease associated with approximately 10% case fatality rates and neurological sequelae in 10–20% of the cases. Recently, we have shown that the matrix metalloproteinase (MMP) inhibitor BB-94 reduced brain injury in a mouse model of MM. The present study aimed to assess whether doxycycline (DOX), a tetracycline that showed a neuroprotective effect as adjuvant therapy in experimental pneumococcal meningitis (PM), would also exert a beneficial effect when given as adjunctive therapy to ceftriaxone (CRO) in experimental MM. Methods BALB/c mice were infected by the intracisternal route with a group C Neisseria meningitidis strain. Eighteen h post infection (hpi), animals were randomised for treatment with CRO [100 mg/kg subcutaneously (s.c.)], CRO plus DOX (30 mg/kg s.c.) or saline (control s.c.). Antibiotic treatment was repeated 24 and 40 hpi. Mouse survival and clinical signs, bacterial counts in cerebella, brain damage, MMP-9 and cyto/chemokine levels were assessed 48 hpi. Results Analysis of bacterial load in cerebella indicated that CRO and CRO + DOX were equally effective at controlling meningococcal replication. No differences in survival were observed between mice treated with CRO (94.4%) or CRO + DOX (95.5%), (p > 0.05). Treatment with CRO + DOX significantly diminished both the number of cerebral hemorrhages (p = 0.029) and the amount of MMP-9 in the brain (p = 0.046) compared to untreated controls, but not to CRO-treated animals (p > 0.05). Levels of inflammatory markers in the brain of mice that received CRO or CRO + DOX were not significantly different (p > 0.05). Overall, there were no significant differences in the parameters assessed between the groups treated with CRO alone or CRO + DOX. Conclusions Treatment with CRO + DOX showed similar bactericidal activity to CRO in vivo, suggesting no antagonist effect of DOX on CRO. Combined therapy significantly improved mouse survival and disease severity compared to untreated animals, but addition of DOX to CRO did not offer significant benefits over CRO monotherapy. In contrast to experimental PM, DOX has no adjunctive activity in experimental MM.

Background: In coronavirus disease-2019 (COVID-19) patients, there is increasing evidence of neuronal injury by the means of elevated serum neurofilament light chain (sNfL) levels. However, the role of systemic inflammation and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific immune response with regard to neuronal injury has not yet been investigated. Methods: In a prospective cohort study, we recruited patients with mild–moderate ( n = 39) and severe ( n = 14) COVID-19 and measured sNfL levels, cytokine concentrations, SARS-CoV-2-specific antibodies including neutralizing antibody titers, and cell-mediated immune responses at enrollment and at 28(±7) days. We explored the association of neuro-axonal injury as by the means of sNfL measurements with disease severity, cytokine levels, and virus-specific immune responses. Results: sNfL levels, as an indicator for neuronal injury, were higher at enrollment and increased during follow-up in severely ill patients, whereas during mild–moderate COVID-19, sNfL levels remained unchanged. Severe COVID-19 was associated with increased concentrations of cytokines assessed [interleukin (IL)-6, IL-8, interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α)], higher anti-spike IgG and anti-nucleocapsid IgG concentrations, and increased neutralizing antibody titers compared with mild–moderate disease. Patients with more severe disease had higher counts of defined SARS-CoV-2-specific T cells. Increases in sNfL concentrations from baseline to day 28(±7) positively correlated with anti-spike protein IgG antibody levels and with titers of neutralizing antibodies. Conclusion: Severe COVID-19 is associated with increased serum concentration of cytokines and subsequent neuronal injury as reflected by increased levels of sNfL. Patients with more severe disease developed higher neutralizing antibody titers and higher counts of SARS-CoV-2-specific T cells during the course of COVID-19 disease. Mounting a pronounced virus-specific humoral and cell-mediated immune response upon SARS-CoV-2 infection did not protect from neuro-axonal damage as by the means of sNfL levels.

3,4–Methylenedioxymethamphetamine (MDMA, “Ecstasy”) is a serotonin- and noradrenaline-releasing substance, currently among the most widely used illicit substances worldwide. In animal studies, repeated exposure to MDMA has been associated with dendritic but also axonal degeneration in the brain. However, translation of the axonal findings, specifically, to humans has been repeatedly questioned and the few existing studies investigating white matter alterations in human chronic MDMA users have yielded conflicting findings. In this study, we combined whole-brain diffusion tensor imaging and neurofilament light chain (NfL) analysis in blood to reveal potential MDMA-induced axonal neuropathology. To this end, we assessed 39 chronic MDMA users and 39 matched MDMA-naïve healthy controls. MDMA users showed increased fractional anisotropy in several white matter tracts, most prominently in the corpus callosum as well as corticospinal tracts, with these findings partly related to MDMA use intensity. However, the NfL levels of MDMA users were not significantly different from those of controls. We conclude that MDMA use is not associated with significant white matter lesions due to the absence of reduced fractional anisotropy and increased NfL levels commonly observed in conditions associated with white matter lesions, including stimulant and ketamine use disorders. Hence, the MDMA-induced axonal degradation demonstrated in animal models was not observed in this human study of chronic MDMA users.

Background Treatment failure in pneumococcal meningitis due to antibiotic resistance is an increasing clinical challenge and alternatives to antibiotics warrant investigation. Phage-derived endolysins efficiently kill gram-positive bacteria including multi-drug resistant strains, making them attractive therapeutic candidates. The current study assessed the therapeutic potential of the novel endolysin PlyAZ3aT in an infant rat model of ceftriaxone-resistant pneumococcal meningitis. Methods Efficacy of PlyAZ3aT was assessed in a randomized, blinded and controlled experimental study in infant Wistar rats. Meningitis was induced by intracisternal infection with 5 x 107 CFU/ml of a ceftriaxone-resistant clinical strain of S. pneumoniae, serotype 19A. Seventeen hours post infection (hpi), animals were randomized into 3 treatment groups and received either (i) placebo (phosphate buffered saline [PBS], n = 8), (ii) 50 mg/kg vancomycin (n = 10) or (iii) 400 mg/kg PlyAZ3aT (n = 8) via intraperitoneal injection. Treatments were repeated after 12 h. Survival at 42 hpi was the primary outcome; bacterial loads in cerebrospinal fluid (CSF) and blood were secondary outcomes. Additionally, pharmacokinetics of PlyAZ3aT in serum and CSF was assessed. Results PlyAZ3aT did not improve survival compared to PBS, while survival for vancomycin treated animals was 70% which is a significant improvement when compared to PBS or PlyAZ3aT (pConclusion In experimental pneumococcal meningitis, PlyAZ3aT failed to cure the infection due to an inability to reach the CSF. Optimization of the galenic formulation e.g. using liposomes might enable crossing of the BBB and improve treatment efficacy.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

Incidence of invasive pneumococcal disease (IPD) has been low during the peak of the COVID-19 pandemic. In this study, we found that the IPD numbers again increased in Switzerland during the first six months of 2021 and that this coincides with the loosening of COVID-19 measures. Vaccine pneumococcal serotypes have continued to decrease and non-vaccine type serotype 23B has emerged (8% of the isolates in 2021). Worryingly, serotype 23B is associated with reduced susceptibility to penicillin.

Sensorineural hearing loss is prevalent within society affecting the quality of life of 460 million worldwide. In the majority of cases, this is due to insult or degeneration of mechanosensory hair cells in the cochlea. In adult mammals, hair cell loss is irreversible as sensory cells are not replaced spontaneously. Genetic inhibition of Notch signaling had been shown to induce hair cell formation by transdifferentiation of supporting cells in young postnatal rodents and provided an impetus for targeting Notch pathway with small molecule inhibitors for hearing restoration. Here, the oto-regenerative potential of different γ-secretase inhibitors (GSIs) was evaluated in complementary assay models, including cell lines, organotypic cultures of the organ of Corti and cochlear organoids to characterize two novel GSIs (CPD3 and CPD8). GSI-treatment induced hair cell gene expression in all these models and was effective in increasing hair cell numbers, in particular outer hair cells, both in baseline conditions and in response to ototoxic damage. Hair cells were generated from transdifferentiation of supporting cells. Similar findings were obtained in cochlear organoid cultures, used for the first time to probe regeneration following sisomicin-induced damage. Finally, effective absorption of a novel GSI through the round window membrane and hair cell induction was attained in a whole cochlea culture model and in vivo pharmacokinetic comparisons of transtympanic delivery of GSIs and different vehicle formulations were successfully conducted in guinea pigs. This preclinical evaluation of targeting Notch signaling with novel GSIs illustrates methods of characterization for hearing restoration molecules, enabling translation to more complex animal studies and clinical research.

Abstract Background Pneumococcal meningitis is associated with high risk of neurological sequelae such as cognitive impairment and hearing loss. These sequelae are due to parenchymal brain and inner ear damage primarily induced by the excessive inflammatory reaction in response to bacterial brain invasion. Metformin—a biguanide drug to treat diabetes mellitus type 2—was recently found to suppress neuroinflammation and induce neuroregeneration. This study evaluated the effect of metformin adjunctive to antibiotics on neuroinflammation, brain and inner ear damage, and neurofunctional outcome in experimental pediatric pneumococcal meningitis. Methods Eleven-day-old Wistar rats were infected intracisternally with 5.22 ± 1.27 × 103 CFU Streptococcus pneumoniae and randomized for treatment with metformin (50 mg/kg, i.p., once daily for 3 weeks) plus ceftriaxone (100 mg/kg, i.p., bid, n = 61) or ceftriaxone monotherapy (n = 79). Cortical damage and hippocampal apoptosis were evaluated histomorphometrically 42 h post infection. Cerebrospinal fluid cytokine levels were analyzed during acute infection. Five weeks post infection, auditory brainstem responses were measured to determine hearing thresholds. Spiral ganglion neuron density and abundance of recently proliferated and integrated hippocampal granule neurons were assessed histologically. Additionally, the anti-inflammatory effect of metformin was studied in primary rat astroglial cells in vitro. Results Upon pneumococcal infection, metformin treatment significantly reduced levels of inflammatory cytokines and nitric oxide production in cerebrospinal fluid and in astroglial cell cultures in vitro (p

Abstract Background Streptococcus pneumoniae is the cause of a highly lethal form of meningitis in humans. Microglial cells in the brain represent the first line of defense against pathogens, and they participate in the inflammatory response. The cholesterol-dependent cytolysin pneumolysin and the bacterial capsule are key pathogenic factors, known to exacerbate the course of pneumococcal meningitis. Methods We utilized live imaging and immunostaining of glial cells in dissociated and acute brain slice cultures to study the effect of pneumococcal factors, including the cholesterol-dependent cytolysin pneumolysin and the pneumococcal capsule, on microglial motility and taxis. Results In brain tissue, primary microglia cells showed an enhanced response towards lysates from bacteria lacking capsules and pneumolysin as they moved rapidly to areas with an abundance of bacterial factors. The presence of bacterial capsules and pneumolysin cumulatively inhibited microglial taxis. In mixed cultures of astrocytes and microglia, the motility of microglia was inhibited by capsular components within minutes after exposure. The reduced motility was partially reversed by mannan, a mannose receptor inhibitor. The effects on microglia were not mediated by astrocytes because pure microglial cells responded to various pneumococcal lysates similarly with distinct cell shape changes as seen in mixed cultures. Conclusions Our data indicate that microglia possess the capacity for a very agile response towards bacterial pathogens, but key pathogenic factors, such as pneumococcal capsules and pneumolysin, inhibited this response shortly after a bacterial challenge. Furthermore, we demonstrate for the first time that the bacterial capsule affects cellular behaviors such as motility and taxis.

Background: Pulmonary nocardiosis (PN) is an uncommon but potentially life-threatening infection. Most of our knowledge on PN is derived from case reports and small case series. Increasing incidence rates of PN have been reported recently. The aim of this study was to describe the clinical course of and risk factors for PN in four Western European countries and to estimate population-based annual hospitalization rates. Methods: This was a retrospective evaluation (1995–2011) of the clinical course of and risk factors for PN in patients at 11 hospitals in four European countries (Germany, Austria, Switzerland, and the Netherlands). Population-based estimates of hospitalization rates for PN in Germany (2005 to 2011) were calculated using official German nationwide diagnosis-related groups (DRG) hospital statistics. Results: Forty-three patients fulfilled stringent criteria for proven (n = 8) and probable (n = 35) PN; seven had extrapulmonary dissemination. For these 43 patients, the major risk factors for PN were immunocompromising (83.7%) and/or pulmonary (58.1%; as only comorbidity in 27.9%) comorbidities. The median duration of PN targeted therapy was 12 weeks. Distinctive patterns of resistance were observed (imipenem susceptibility: Nocardia farcinica 33.3%; Nocardia asteroides 66.7%). The overall mortality rate was 18.9% (50% in disseminated PN). Over time, annual PN hospitalization rates remained unchanged at around 0.04/100 000, with the highest rate among men aged 75–84 years (0.24/100 000). Conclusions: PN is a rare, but potentially life-threatening disease, and mainly affects immunocompromised elderly males. Overall, annual hospitalization rates remained stable between 2005 and 2011. Keywords: Nocardiosis, Nocardia, Pulmonary nocardiosis

Objective: Anti-CD20-treated patients are at risk of a reduced humoral immune response during the SARS-CoV-2 pandemic. Our aim was to compare the antibody response after two vaccinations with the mRNA vaccines BNT162b2 or mRNA-1273 in patients with multiple sclerosis. Methods: Data from the University Hospital of Bern and Cantonal Hospital of Lucerne were retrospectively collected from medical records and then analyzed. Anti-spike IgG serum titers were collected from both centers and were considered to be protective from a value of ≥100 AU/mL. Continuous variables were given as the mean and 95% confidence interval (95% CI); categorical variables were given as frequencies. A Mann–Whitney test and Fisher’s exact test as well as a multivariable linear regression analysis with anti-spike IgG (AU/mL) as the dependent variable were run using SPSS Statistic 25 (IBM Corp., Amonk, NY, USA). Results: A total of 74 patients were included; 41/74 (63.51%) were female patients and the mean age was 46.6 years (95% CI 43.4–49.9). Of these patients, 36/74 were vaccinated with BNT162b2 and 38/74 with mRNA-1273, following the national vaccination recommendation. In both vaccine groups, protective anti-spike IgG titers (≥100 AU/mL) were infrequently achieved (5/74: mRNA-1273 3/38; BNT162b2 2/36). Conclusions: In addition to a low rate of protective anti-spike IgG titers in both vaccine groups, we identified a drop in anti-spike IgG serum titers over time. This observation bears therapeutic consequences, as initial positive titers should be checked in case of an infection with the SARS-CoV-2 virus to identify patients who would benefit from an intravenous anti-spike IgG treatment against acute COVID-19.

Translational research organizations (TROs) face specific challenges to secure resilience and longevity. This perspective article provides the rationale for six hands-on recommendations for the management of legitimacy building in TROs.

BackgroundMicroglia initiates and sustains the inflammatory reaction that drives the pathogenesis of pneumococcal meningitis. The expression of the G-protein cannabinoid receptor type 2 (CB2) in the brain is low, but is upregulated in glial cells during infection. Its activation down-regulates pro-inflammatory processes, driving microglia towards an anti-inflammatory phenotype. CB2 agonists are therefore therapeutic candidates in inflammatory conditions like pneumococcal meningitis. We evaluated the effects of JWH-133, a specific CB2 agonist on microglial cells, inflammation, and damage driven by S. pneumoniaein vitro and in experimental pneumococcal meningitis.Materials/methodsPrimary mixed glial cultures were stimulated with live or heat-inactivated S. pneumoniae, or lipopolysaccharide and treated with JWH-133 or vehicle. Nitric oxide and cytokines levels were measured in the supernatant. In vivo, pneumococcal meningitis was induced by intracisternal injection of live S. pneumoniae in 11 days old Wistar rats. Animals were treated with antibiotics (Ceftriaxone, 100 mg/kg, s.c. bid) and JWH-133 (1 mg/kg, i.p. daily) or vehicle (10% Ethanol in saline, 100 µl/25g body weight) at 18 h after infection. Brains were harvested at 24 and 42 h post infection (hpi) for histological assessment of hippocampal apoptosis and cortical damage and determination of cyto/chemokines in tissue homogenates. Microglia were characterized using Iba-1 immunostaining. Inflammation in brain homogenates was determined using membrane-based antibody arrays.ResultsIn vitro, nitric oxide and cytokines levels were significantly lowered by JWH-133 treatment. In vivo, clinical parameters were not affected by the treatment. JWH-133 significantly lowered microglia activation assessed by quantification of cell process length and endpoints per microglia. Animals treated with JWH-133 demonstrated significantly lower parenchymal levels of chemokines (CINC-1, CINC-2α/β, and MIP-3α), TIMP-1, and IL-6 at 24 hpi, and CINC-1, MIP-1α, and IL-1α at 42 hpi. Quantitative analysis of brain damage did not reveal an effect of JWH-133.ConclusionsJWH-133 attenuates microglial activation and downregulates the concentrations of pro-inflammatory mediators in pneumococcal infection in vitro and in vivo. However, we didn’t observe a reduction in cortical or hippocampal injury. This data provides evidence that inhibition of microglia by adjuvant CB2 agonists therapy effectively downmodulates neuroinflammation but does not reduce brain damage in experimental pneumococcal meningitis

Background:L. monocytogenes meningoencephalitis has a mortality rate of up to 50% and neurofunctional sequelae are common. Type I restriction-modification systems (RMS) are capable of adding methyl groups to the host genome. Some contain multiple sequence recognition (hsdS) genes that recombine, resulting in distinct DNA methylation patterns and patterns of gene expression. These phenotypic switches have been linked to virulence and have recently been discovered in multiple clonal complexes of L. monocytogenes. In the present study, we investigated the significant of RMS on L. monocytogenes virulence during the acute phase of experimental meningitis.Methods:L. monocytogenes strains containing RMS systems were identified, and purified clones enriched for single hsdS alleles were isolated. In vivo, 11-day old Wistar rats were infected with an inoculum containing (a) one of 4 single RMS allele variants (A, B, C, D) treated with amoxicillin (AMX 50 mg/kg/dosis, q8h), (b) a mixture of all 4 variants with or without AMX treatment, or (c) different mixtures of 2 RMS allele variants. At selected time points after infection, clinical and inflammatory parameters, bacterial titers and brain damage were determined. Changes in the relative frequency of the occurring RMS alleles in the inoculum and in CSF or cerebellum of infected animals were analyzed by capillary electrophoresis.Results: We have identified a phase variable RMS locus within L. monocytogenes CC4 and generated stocks that stably expressed each of the possible hsdS genes within that loci. Generation of these allele variants (A, B, C, D) allowed us to determine the methylation pattern associated with each hsdS through SMRT sequencing. In vivo infections with these single allele variants revealed differences in disease severity in that C induced the worst clinical outcome and more pronounced hippocampal apoptosis; D showed the most pronounced weight loss and the highest bacterial titer in the cerebellum. A caused the least severe disease.Conclusion: We identified that L. monocytogenes expressing hsdS (A) causes less damage than when other hsdS genes are expressed. While expression of hsdSC and D worsened the outcome in L. monocytogenes meningitis. We also demonstrate a competitive advantage of variants C and B over variant A in this model. Phenotypical switching may therefore represent a mechanism of virulence regulation during the acute phase of CNS infections with L. monocytogenes.

Pneumococcal meningitis (PM) causes damage to the hippocampus, a brain structure critically involved in learning and memory. Hippocampal injury–which compromises neurofunctional outcome–occurs as apoptosis of progenitor cells and immature neurons of the hippocampal dentate granule cell layer thereby impairing the regenerative capacity of the hippocampal stem cell niche. Repetitive transcranial magnetic stimulation (rTMS) harbours the potential to modulate the proliferative activity of this neuronal stem cell niche. In this study, specific rTMS protocols–namely continuous and intermittent theta burst stimulation (cTBS and iTBS)–were applied on infant rats microbiologically cured from PM by five days of antibiotic treatment. Following two days of exposure to TBS, differential gene expression was analysed by whole transcriptome analysis using RNAseq. cTBS provoked a prominent effect in inducing differential gene expression in the cortex and the hippocampus, whereas iTBS only affect gene expression in the cortex. TBS induced polarisation of microglia and astrocytes towards an inflammatory phenotype, while reducing neurogenesis, neuroplasticity and regeneration. cTBS was further found to induce the release of pro-inflammatory cytokines in vitro. We conclude that cTBS intensified neuroinflammation after PM, which translated into increased release of pro-inflammatory mediators thereby inhibiting neuroregeneration.

Tick-borne encephalitis (TBE) is an infectious disease affecting the central nervous system. Recently, the occurrence of TBEV infections has steadily increased, reaching all-time high incidence rates in European countries. Up to 50% of patients with TBE present neurological sequelae, among them sleep–wake and circadian disorders (SWCD), which are poorly characterized. The aim of this review is to investigate the prevalence, clinical characteristics, and prognosis of SWCD after TBE. The literature review was performed in accordance with PRISMA guidelines. The quality of the paper was assessed using a standardized quality assessment. The analysis of SWCD was categorized into four different time intervals and two age groups. The literature search identified 15 studies, five including children and 10 including adults. In children, fatigue was most frequently observed with a prevalence of 73.9%, followed by somnolence/sleepiness, restlessness, and sleep-wake inversion. In adults, tiredness/fatigue was the most reported sequela with a prevalence of 27.4%, followed by extensive daytime sleepiness/somnolence, and insomnia (3.3%). Two studies showed impaired social outcomes in patients after TBE infections. SWCD after TBE in children and adults is a newly recognized sequela. Additional clinical and experimental research is needed to gain more precise insight into the clinical burden of SWCD after TBE and the underlying mechanisms.

Abstract Background Pneumococcal meningitis is associated with high mortality and morbidity rates. Up to 50% of survivors show neurologic sequelae including hearing loss, cognitive impairments and learning disabilities, being particularly detrimental in affected infants and children where adjuvant therapy with dexamethasone has no proven beneficial effect. We evaluated the effect of concomitantly targeting specific pathophysiological mechanisms responsible for brain damage—i.e. matrix-metalloproteinase (MMP) activity and the exacerbated cerebral inflammation provoked through antibiotic-induced bacterial lysis. Here, we combined adjunctive therapies previously shown to be neuroprotective when used as single adjuvant therapies. Methods Eleven-day-old Wistar rats were infected intracisternally with 6.44 ± 2.17 × 103 CFU Streptococcus pneumoniae and randomised for treatment with ceftriaxone combined with (a) single adjuvant therapy with daptomycin (n = 24), (b) single adjuvant therapy with Trocade (n = 24), (c) combined adjuvant therapy (n = 66) consisting of daptomycin and Trocade, or (d) ceftriaxone monotherapy (n = 42). Clinical parameters and inflammatory CSF cytokine levels were determined during acute meningitis. Cortical damage and hippocampal apoptosis were assessed 42 h after infection. Morris water maze and auditory brainstem responses were used to assess neurofunctional outcome 3 weeks after infection. Results We found significantly reduced apoptosis in the hippocampal subgranular zone in infant rats receiving adjuvant Trocade (p

Abstract Background Nonencapsulated Streptococcus pneumoniae bacteria are successful colonizers of the human nasopharynx and often possess genes aliB-like ORF 1 and 2 in place of capsule genes. AliB-like ORF 2 binds peptide FPPQSV, found in Prevotella species, resulting in enhanced colonization. How this response is mediated is so far unknown. Results Here we show that the peptide increases expression of genes involved in release of host carbohydrates, carbohydrate uptake and carbohydrate metabolism. In particular, the peptide increased expression of 1,5-anhydro-D-fructose reductase, a metabolic enzyme of an alternative starch and glycogen degrading pathway found in many organisms, in both transcriptomic and proteomic data. The peptide enhanced pneumococcal growth giving a competitive advantage to a strain with aliB-like ORF 2, over its mutant lacking the gene. Possession of aliB-like ORF 2 did not affect release of inflammatory cytokine CXCL8 from epithelial cells in culture and the nonencapsulated wild type strain was not able to establish disease or inflammation in an infant rat model of meningitis. Conclusions We propose that AliB-like ORF 2 confers an advantage in colonization by enhancing carbohydrate metabolism resulting in a boost in growth. This may explain the widespread presence of aliB-like ORF 2 in the nonencapsulated pneumococcal population in the human nasopharynx.

Abstract Background The production of reactive oxygen species (ROS) during pneumococcal meningitis (PM) leads to severe DNA damage in the neurons and is the major cause of cell death during infection. Hence, the use of antioxidants as adjuvant therapy has been investigated. Previous studies have demonstrated the possible participation of apurinic/apyrimidinic endonuclease (APE1) during PM. The aims of this study were to investigate the APE1 expression in the cortical and hippocampal tissues of infant Wistar rats infected with Streptococcus pneumoniae and its association with cell death and understand the role of vitamin B6 (vitB6) as a protective factor against cell death. Methods APE1 expression and oxidative stress markers were analyzed at two-time points, 20 and 24 h post infection (p.i.), in the cortex (CX) and hippocampus (HC) of rats supplemented with vitB6. Statistical analyses were performed by the nonparametric Kruskal–Wallis test using Dunn’s post test. Results Our results showed high protein levels of APE1 in CX and HC of infected rats. In the CX, at 20 h p.i., vitB6 supplementation led to the reduction of expression of APE1 and apoptosis-inducing factor, while no significant changes in the transcript levels of caspase-3 were observed. Furthermore, levels of carbonyl content and glutamate in the CX were reduced by vitB6 supplementation at the same time point of 20 h p.i.. Since our data showed a significant effect of vitB6 on the CX at 20 h p.i. rather than that at 24 h p.i., we evaluated the effect of administering a second dose of vitB6 at 18 h p.i. and sacrifice at 24 h p.i.. Reduction in the oxidative stress and APE1 levels were observed, although the latter was not significant. Although the levels of APE1 was not significantly changed in the HC with vitB6 adjuvant therapy, vitB6 supplementation prevented the formation of the truncated form of APE1 (34 kDa) that is associated with apoptosis. Conclusions Our data suggest that PM affects APE1 expression, which can be modulated by vitB6. Additionally, vitB6 contributes to the reduction of glutamate and ROS levels. Besides the potential to reduce cell death and oxidative stress during neuroinflammation, vitB6 showed enhanced effect on the CX than on the HC during PM.

Long noncoding RNAs (lncRNAs) are key regulators of lineage specification during development. Here, the authors investigate remodeling of enhancers and regulation of the lncRNA transcriptome during mesendoderm specification, and identify a pluripotent stage-specific transcribed enhancer controlling adoption of the mesendodermal cell fate.

Abstract Background Throughout Europe, Ixodes ricinus transmits numerous pathogens. Its widespread distribution is not limited to rural but also includes urbanized areas. To date, comprehensive data on pathogen carrier rates of I. ricinus ticks in urban areas of Switzerland is lacking. Results Ixodes ricinus ticks sampled at 18 (sub-) urban collection sites throughout Switzerland showed carrier rates of 0% for tick-borne encephalitis virus, 18.0% for Borrelia burgdorferi (sensu lato), 2.5% for Borrelia miyamotoi, 13.5% for Rickettsia spp., 1.4% for Anaplasma phagocytophilum, 6.2% for "Candidatus Neoehrlichia mikurensis", and 0.8% for Babesia venatorum (Babesia sp., EU1). Site-specific prevalence at collection sites with n > 45 ticks (n = 9) significantly differed for B. burgdorferi (s.l.), Rickettsia spp., and "Ca. N. mikurensis", but were not related to the habitat type. Three hundred fifty eight out of 1078 I. ricinus ticks (33.2%) tested positive for at least one pathogen. Thereof, about 20% (71/358) were carrying two or three different potentially disease-causing agents. Using next generation sequencing, we could detect true pathogens, tick symbionts and organisms of environmental or human origin in ten selected samples. Conclusions Our data document the presence of pathogens in the (sub-) urban I. ricinus tick population in Switzerland, with carrier rates as high as those in rural regions. Carriage of multiple pathogens was repeatedly observed, demonstrating the risk of acquiring multiple infections as a consequence of a tick bite.

Sensorineural hearing loss is the most common long-term deficit after pneumococcal meningitis (PM), occurring in up to 30% of surviving patients. The infection and the following overshooting inflammatory host response damage the vulnerable sensory cells of the inner ear, resulting in loss of hair cells and spiral ganglion neurons, ultimately leading to elevated hearing thresholds. Here, we tested the oto-protective properties of the small heat shock protein alpha B-crystallin (HspB5) with previously reported anti-inflammatory, anti-apoptotic and neuroprotective functions, in an experimental model of PM-induced hearing loss. We analyzed the effect of local and systemic delivery of HspB5 in an infant rat model of PM, as well as ex vivo, using whole mount cultures. Cytokine secretion profile, hearing thresholds and inner ear damage were assessed at predefined stages of the disease up to 1 month after infection. PM was accompanied by elevated pro-inflammatory cytokine concentrations in the cerebrospinal fluid (CSF), leukocyte and neutrophil infiltration in the perilymphatic spaces of the cochlea with neutrophils extracellular trap formation during the acute phase of the disease. Elevated hearing thresholds were measured after recovery from meningitis. Intracisternal but not intraperitoneal administration of HspB5 significantly reduced the levels of TNF-α, IL-6 IFN-γ and IL-10 in the acute phase of the disease. This resulted in a greater outer hair cell survival, as well as improved hearing thresholds at later stages. These results suggest that high local concentrations of HspB5 are needed to prevent inner ear damage in acute PM. HspB5 represents a promising therapeutic option to improve the auditory outcome and counteract hearing loss after PM.

Pneumococcal conjugate vaccines (PCVs) have lowered the incidence of invasive pneumococcal disease (IPD) worldwide. However, the influence of regional vaccine uptake differences on the changing epidemiology of IPD remains unclear. We aimed to examine the overall impact of both seven- and 13-valent PCVs (PCV7 and PCV13) on IPD in Switzerland. Three-year periods from 2005–2010 and 2011–2019 were considered, respectively, as (early and late) PCV7 eras and (early, mid and late) PCV13 eras. Vaccine coverage was estimated from a nationwide survey according to east (German-speaking) and west (French/Italian-speaking) regions for each period. Reported incidence rate ratios (IRRs) were compared between successive periods and regions using nationwide IPD surveillance data. Overall IPD incidence across all ages was only 16% lower in the late PCV13 era compared to the early PCV7 era (IRR 0.83, 95% CI 0.79–0.88), due to increasing incidence of non-PCV-type IPD (2.59, 2.37–2.83) in all age groups, except children p < 0.001), and were accompanied by lower IPD incidences across all age groups in the former region. Post-PCV13, non-PCV serotypes 8, 22F and 9N were the major cause of IPD in adults ≥65 years. Increased PCV coverage in both areas of Switzerland resulted in a decrease in vaccine-type and overall IPD incidence across all age groups, in a regionally dependent manner. However, the rising incidence of non-vaccine-type IPD, exclusive to older adults, may undermine indirect beneficial effects.

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological and microbiological definitions. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 13,387 VoCs have been identified since the detection of the first Swiss case in October 2020, with 4194 being B.1.1.7, 172 B.1.351, and 7 P.1. The remaining 9014 cases of VoCs have been described without further lineage specification. Overall, all diagnostic centers reported a rapid increase of the percentage of detected VOCs, with a range of 6 to 46% between 25 to 31 of January 2021 increasing towards 41 to 82% between 22 to 28 of February. A total of 739 N501Y positive genomes were analysed and show a broad range of introduction events to Switzerland. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs.

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

Identification and characterization of viral genomes in vectors including ticks and mosquitoes positive for pathogens of great public health concern using metagenomic next generation sequencing (mNGS) has challenges. One such challenge is the ability to efficiently recover viral RNA which is typically dependent on sample processing. We evaluated the quantitative effect of six different extraction methods in recovering viral RNA in vectors using negative tick homogenates spiked with serial dilutions of tick-borne encephalitis virus (TBEV) and surrogate Langat virus (LGTV). Evaluation was performed using qPCR and mNGS. Sensitivity and proof of concept of optimal method was tested using naturally positive TBEV tick homogenates and positive dengue, chikungunya, and Zika virus mosquito homogenates. The amount of observed viral genome copies, percentage of mapped reads, and genome coverage varied among different extractions methods. The developed Method 5 gave a 120.8-, 46-, 2.5-, 22.4-, and 9.9-fold increase in the number of viral reads mapping to the expected pathogen in comparison to Method 1, 2, 3, 4, and 6, respectively. Our developed Method 5 termed ROVIV (Recovery of Viruses in Vectors) greatly improved viral RNA recovery and identification in vectors using mNGS. Therefore, it may be a more sensitive method for use in arbovirus surveillance.

The etiologic cause of encephalitis, meningitis or meningo-encephalitis is unknown in up to 70% of cases. Clinical shotgun metagenomics combined with host depletion is a promising technique to identify infectious etiologies of central nervous system (CNS) infections. We developed a straightforward eukaryotic host nucleic acid depletion method that preserves intact viruses and bacteria for subsequent shotgun metagenomics screening of clinical samples, focusing on cerebrospinal fluid (CSF). A surrogate CSF sample for a CNS infection paradigm was used to evaluate the proposed depletion method consisting of selective host cell lysis, followed by enzymatic degradation of the liberated genomic DNA for final depletion with paramagnetic beads. Extractives were subjected to reverse transcription, followed by whole genome amplification and next generation sequencing. The effectiveness of the host depletion method was demonstrated in surrogate CSF samples spiked with three 1:100 dilutions of Influenza A H3N2 virus (qPCR Ct-values 20.7, 28.8, >42/negative). Compared to the native samples, host depletion increased the amount of the virus subtype reads by factor 7127 and 132, respectively, while in the qPCR negative sample zero vs. 31 (1.4E-4 %) virus subtype reads were detected (native vs. depleted). The workflow was applied to thirteen CSF samples of patients with meningo-/encephalitis (two bacterial, eleven viral etiologies), a serum of an Andes virus infection and a nose swab of a common cold patient. Unlike surrogate samples, host depletion of the thirteen human CSF samples and the nose swab did not result in more reads indicating presence of damaged pathogens due to, e.g., host immune response. Nevertheless, previously diagnosed pathogens in the human CSF samples (six viruses, two bacteria), the serum, and the nose swab (Human rhinovirus A31) were detected in the depleted and/or the native samples. Unbiased evaluation of the taxonomic profiles supported the diagnosed pathogen in two native CSF samples and the native and depleted serum and nose swab, while detecting various contaminations that interfered with pathogen identification at low concentration levels. In summary, damaged pathogens and contaminations complicated analysis and interpretation of clinical shotgun metagenomics data. Still, proper consideration of these issues may enable future application of metagenomics for clinical diagnostics.

The rate of invasive group B Streptococcus (GBS) infections is steadily increasing, particularly in older persons and in adults with diabetes and other comorbidities. This population includes persons with a foreign body (e.g., who have undergone arthroplasty). In a rat tissue cage model, we evaluated the efficacy of adjunctive gentamicin (GEN) administered systemically (5 mg/kg body weight) every 24 h, or locally (12.5 mg/L tissue cage concentration) every 24 or 72 h, in combination with penicillin (PEN) administered systemically (250,000 IU/kg body weight three times per day). The efficacy was evaluated on two different sessile forms of GBS: transition (i.e., in between planktonic and biofilm) and biofilm. After 3 days of treatment, the mean bacterial load reduction of transition-form GBS was greater in all PEN–GEN combination groups than in the PEN monotherapy group (P ≤ 0.03). The 6-day regimen decreased the bacterial load significantly in comparison to the 3-day regimen, irrespective of growth form and adjunctive GEN (P < 0.01). After 6 days of treatment, the mean reduction in transition-form GBS was greater with PEN plus GEN administered locally every 24 h than with PEN monotherapy (P = 0.03). These results were not confirmed with biofilm GBS. The difference in mean bacterial load reduction between all PEN–GEN and PEN monotherapy groups was

Vertical transmission from an infected cow to its fetus accounts for the vast majority of new Neospora caninum infections in cattle. A vaccine composed of a chimeric antigen named recNcMIC3-1-R, based on predicted immunogenic domains of the two microneme proteins NcMIC1 and NcMIC3, the rhoptry protein NcROP2, and emulsified in saponin adjuvants, significantly reduced the cerebral infection in non-pregnant BALB/c mice. Protection was associated with a mixed Th1/Th2-type cytokine response. However, the same vaccine formulation elicited a Th2-type immune response in pregnant mice and did not prevent vertical transmission or disease, neither in dams nor in offspring mice. In this study, an alternative vaccine formulation containing recNcMIC3-1-R emulsified in Freund’s incomplete adjuvant, a stimulator of the cellular immunity, was investigated. No protection against vertical transmission and cerebral infection in the pregnant mice and a very limited protective effect in the non-pregnant mice were observed. The vaccine induced a Th1-type immune response characterized by high IgG2a titres and strong IFN-γ expression, which appeared detrimental to pregnancy.

Streptococcus pneumoniae bacteria can be characterized into over 90 serotypes according to the composition of their polysaccharide capsules. Some serotypes are common in nasopharyngeal carriage whereas others are associated with invasive disease, but when carriage serotypes do invade disease is often particularly severe. It is unknown whether disease severity is due directly to the capsule type or to other virulence factors. Here, we used a clinical pneumococcal isolate and its capsule-switch mutants to determine the effect of capsule, in isolation from the genetic background, on severity of meningitis in an infant rat model. We found that possession of a capsule was essential for causing meningitis. Serotype 6B caused significantly more mortality than 7F and this correlated with increased capsule thickness in the cerebrospinal fluid (CSF), a stronger inflammatory cytokine response in the CSF and ultimately more cortical brain damage. We conclude that capsule type has a direct effect on meningitis severity. This is an important consideration in the current era of vaccination targeting a subset of capsule types that causes serotype replacement.

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP3 and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

Pneumococcal meningitis is associated with caspase 3-dependent apoptosis of recently post-mitotic immature neurons in the dentate gyrus of the hippocampus. The death of these cells is implicated in the learning and memory deficits in patients surviving the disease. The stress-activated protein kinase c-Jun N-terminal kinase (JNK) has been shown to be an important mediator of caspase 3-dependent neuronal apoptosis. However, whether JNK is involved in hippocampal apoptosis caused by pneumococcal meningitis has so far not been investigated. Here we show in a neonatal rat model of pneumococcal meningitis that JNK3 but not JNK1 or JNK2 is activated in the hippocampus during the acute phase of infection. At the cellular level, JNK3 activation was accompanied in the dentate gyrus by markedly increased phosphorylation of its major downstream target c-Jun in early immature (Hu-positive) neurons, but not in migrating (doublecortin-positive) neurons, the cells that do undergo apoptosis. These findings suggested that JNK may not be involved in pneumococcal meningitis-induced hippocampal apoptosis. Indeed, although intracerebroventricular administration of D-JNKI-1 or AS601245 (two highly specific JNK inhibitors) inhibited c-Jun phosphorylation and protein expression in the hippocampus, hippocampal apoptosis was unaffected. Collectively, these results demonstrate that JNK does not mediate hippocampal apoptosis in pneumococcal meningitis, and that JNK may be involved in processes unrelated to apoptosis in this disease.

Inflammation of the subarachnoid and ventricular space contributes to the development of brain damage i.e. cortical necrosis and hippocampal apoptosis in pneumococcal meningitis (PM). Galectin-3 and -9 are known pro-inflammatory mediators and regulators of apoptosis. Here, the gene and protein expression profile for both galectins was assessed in the disease progression of PM. The mRNA of Lgals3 and Lgals9 increased continuously in the cortex and in the hippocampus from 22 h to 44 h after infection. At 44 h after infection, mRNA levels of Lgals9 in the hippocampus were 7-fold and those of Lgals3 were 30-fold higher than in uninfected controls (P

Pneumococcal meningitis (PM) is characterized by an intense inflammatory host reaction that contributes to the development of cortical necrosis and hippocampal apoptosis. Inflammatory conditions in the brain are known to induce tryptophan degradation along the kynurenine pathway, resulting in accumulation of neurotoxic metabolites. In the present study, we investigated the contribution of the kynurenine pathway to brain injury in experimental PM by measuring the concentration of its metabolites and the enzymatic activities and mRNA levels of its major enzymes in the vulnerable brain regions. In the late phase of acute PM, we found a significant transcriptional upregulation of kynurenine-3-hydroxylase and an accumulation of the neurotoxic metabolites 3-hydroxykynurenine (3-HKYN) and 3-hydroxyanthranilic acid in cortex and hippocampus. The positive correlation between the concentration of 3-HKYN and the extent of hippocampal apoptosis adds support to the concept that 3-HKYN contributes to brain injury in PM.