Tick-borne encephalitis: A comprehensive review of the epidemiology, virology, and clinical picture.

Tick-borne encephalitis virus (TBEV) is a flavivirus commonly found in at least 27 European and Asian countries. It is an emerging public health problem, with steadily increasing case numbers over recent decades. Tick-borne encephalitis virus affects between 10,000 and 15,000 patients annually. Infection occurs through the bite of an infected tick and, much less commonly, through infected milk consumption or aerosols. The TBEV genome comprises a positive-sense single-stranded RNA molecule of ∼11 kilobases. The open reading frame is > 10,000 bases long, flanked by untranslated regions (UTR), and encodes a polyprotein that is co- and post-transcriptionally processed into three structural and seven non-structural proteins. Tick-borne encephalitis virus infection results in encephalitis, often with a characteristic biphasic disease course. After a short incubation time, the viraemic phase is characterised by non-specific influenza-like symptoms. After an asymptomatic period of 2-7 days, more than half of patients show progression to a neurological phase, usually characterised by central and, rarely, peripheral nervous system symptoms. Mortality is low-around 1% of confirmed cases, depending on the viral subtype. After acute tick-borne encephalitis (TBE), a minority of patients experience long-term neurological deficits. Additionally, 40%-50% of patients develop a post-encephalitic syndrome, which significantly impairs daily activities and quality of life. Although TBEV has been described for several decades, no specific treatment exists. Much remains unknown regarding the objective assessment of long-lasting sequelae. Additional research is needed to better understand, prevent, and treat TBE. In this review, we aim to provide a comprehensive overview of the epidemiology, virology, and clinical picture of TBE.

Benefits of Aerosolized Phages for the Treatment of Pneumonia Due to Methicillin-Resistant Staphylococcus aureus: An Experimental Study in Rats.

BACKGROUND: The optimal method for delivering phages in the context of ventilator-associated pneumonia (VAP) is unknown. In the current study, we assessed the utility of aerosolized phages (aerophages) for experimental methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. METHODS: Rats were ventilated for 4 hours before induction of pneumonia. Animals received one of the following: (1) aerophages; (2) intravenous (IV) phages; (3) a combination of IV and aerophages; (4) IV linezolid; or (5) a combination of IV linezolid and aerophages. Phages were administered at 2, 12, 24, 48, and 72 hours, and linezolid was administered at 2, 12, 24, 36, 48, 60, and 72 hours. The primary outcome was survival at 96 hours. Secondary outcomes were bacterial and phage counts in tissues and histopathological scoring of the lungs. RESULTS: Aerophages and IV phages each rescued 50% of animals from severe MRSA pneumonia (P < .01 compared with placebo controls). The combination of aerophages and IV phages rescued 91% of animals, which was higher than either monotherapy (P < .05). Standard-of-care antibiotic linezolid rescued 38% of animals. However, linezolid and aerophages did not synergize in this setting (55% survival). CONCLUSIONS: Aerosolized phage therapy showed potential for the treatment of MRSA pneumonia in an experimental animal model and warrants further investigation for application in humans.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

Patterns of Neointima Formation After Coil or Stent Treatment in a Rat Saccular Sidewall Aneurysm Model.

BACKGROUND AND PURPOSE: Endovascular aneurysm treatment relies on a biological process, including cell migration for thrombus organization and growth of a neointima. To better understand aneurysm healing, our study explores the origin of neointima-forming and thrombus-organizing cells in a rat saccular sidewall aneurysm model. METHODS: Saccular aneurysms were transplanted onto the abdominal aorta of male Lewis rats and endovascularly treated with coils (n=28) or stents (n=26). In 34 cases, GFP+ (green fluorescent protein)-expressing vital aneurysms were sutured on wild-type rats, and in 23 cases, decellularized wild-type aneurysms were sutured on GFP+ rats. Follow-up at 3, 7, 14, 21, and 28 days evaluated aneurysms by fluorescence angiography, macroscopic inspection, and microscopy for healing and inflammation status. Furthermore, the origin of cells was tracked with fluorescence histology. RESULTS: In animals with successful functional healing, histological studies showed a gradually advancing thrombus organization over time characterized by progressively growing neointima from the periphery of the aneurysm toward the center. Cell counts revealed similar distributions of GFP+ cells for coil or stent treatment in the aneurysm wall (54.4% versus 48.7%) and inside the thrombus (20.5% versus 20.2%) but significantly more GFP+ cells in the neointima of coiled (27.2 %) than stented aneurysms (10.4%; P=0.008). CONCLUSIONS: Neointima formation and thrombus organization are concurrent processes during aneurysm healing. Thrombus-organizing cells originate predominantly in the parent artery. Neointima formation relies more on cell migration from the aneurysm wall in coiled aneurysms but receives greater contributions from cells originating in the parent artery in stent-treated aneurysms. Cell migration, which allows for a continuous endothelial lining along the parent artery's lumen, may be a prerequisite for complete aneurysm healing after endovascular therapy. In terms of translation into clinical practice, these findings may explain the variability in achieving complete aneurysm healing after coil treatment and the improved healing rate in stent-assisted coiling.

Accuracy of serological testing for SARS-CoV-2 antibodies: First results of a large mixed-method evaluation study.

BACKGROUND: Serological immunoassays that can identify protective immunity against SARS-CoV-2 are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. To date, however, the utility of such immunoassays remains unclear. In a mixed-design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various SARS-CoV-2 proteins and assessed the neutralizing activity of antibodies in patient sera. METHODS: Consecutive patients admitted with confirmed SARS-CoV-2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. An in-house enzyme-linked immunosorbent assay utilizing recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was developed and compared to three commercially available enzyme-linked immunosorbent assays (ELISAs) targeting the nucleoprotein (N), the S1 domain of the spike protein (S1), and a lateral flow immunoassay (LFI) based on full-length spike protein. Neutralization assays with live SARS-CoV-2 were performed. RESULTS: One thousand four hundred and seventy-seven individuals were included comprising 112 SARS-CoV-2 positives (defined as a positive real-time PCR result; prevalence 7.6%). IgG seroconversion occurred between day 0 and day 21. While the ELISAs showed sensitivities of 88.4% for RBD, 89.3% for S1, and 72.9% for N protein, the specificity was above 94% for all tests. Out of 54 SARS-CoV-2 positive individuals, 96.3% showed full neutralization of live SARS-CoV-2 at serum dilutions ≥ 1:16, while none of the 6 SARS-CoV-2-negative sera revealed neutralizing activity. CONCLUSIONS: ELISAs targeting RBD and S1 protein of SARS-CoV-2 are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against SARS-CoV-2.

Innate and adaptive immune responses following PD-L1 blockade in treating chronic murine alveolar echinococcosis.

BACKGROUND: Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) immune checkpoint blockade are efficacious in certain cancer therapies. OBJECTIVES: The present study aimed to provide a picture about the development of innate and adaptive immune responses upon PD-L1 blockade in treating chronic murine AE. METHODS: Immune treatment started at 6 weeks post-E. multilocularis infection, and was maintained for 8 weeks with twice per week anti-PD-L1 administration (intraperitoneal). The study included an outgroup-control with mice perorally medicated with albendazole 5 d/wk, and another one with both treatments combined. Assessment of treatment efficacy was based on determining parasite weight, innate and adaptive immune cell profiles, histopathology and liver tissue cytokine levels. RESULTS/CONCLUSIONS: Findings showed that the parasite load was significantly reduced in response to PD-L1 blockade, and this blockade (a) contributed to T-cell activity by increasing CD4+ /CD8+ effector T cells, and decreasing Tregs; (b) had the capacity to restore DCs and Kupffer cells/Macrophages; (c) suppressed NKT and NK cells; and thus (d) lead to an improved control of E. multilocularis infection in mice. This study suggests that the PD-L1 pathway plays an important role by regulating adaptive and innate immune cells against E. multilocularis infection, with significant modulation of tissue inflammation.

Meningitis, meningoencephalitis and encephalitis in Bern: an observational study of 258 patients.

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.

Digest the Sugar, Kill the Parasite: A New Experimental Concept in Treating Alveolar Echinococcosis.

INTRODUCTION: The E. multilocularis laminated layer (LL) is a heavily glycosylated parasitic structure that plays an important role in protecting the larval stage (metacestode) of this parasite from physiological and immunological host reactions. We elaborated an experimental design with the idea to modify the (glycan) surface of the LL by a targeted digestion. This should allow the host defense to more easily recognize and attack (or kill) the parasite by immune-mediated effects. METHODS: Experimentally, E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or ß1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry. RESULTS: Our experiments demonstrated that the parasite vesicular structure can be directly destroyed by adding galactosidases into the in vitro culture system, resulting in the fact that the parasite metacestode vesicles could not anymore infect and develop in mice after this glycan digestion. Moreover, when compared to the mice inoculated with E. multilocularis metacestode without galactosidases, PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with ß1-3-galactosidase, with a lower IL-10 secretion from CD4+ Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teff from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galac-tosidase. DISCUSSION: We raised our hypothesis that this "aborting" effect may be linked to an altered PD1 and IL-10 response fine-tuning between immunopathology and immune protection. These findings justify a continuation of these experiments upon therapeutical in vivo administration of the enzymes.

Evaluation of neurofilament light chain in the cerebrospinal fluid and blood as a biomarker for neuronal damage in experimental pneumococcal meningitis.

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-1ß, 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.

A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing.

Amplicon sequencing of the 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations, with the most significant being the inability to resolve mixed amplicons when closely related species are coamplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read next-generation sequencing (NGS) technologies could solve the mixed amplicon issue but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling a flexible number of samples per run and an adjustable sequencing time. Here, we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (long read consensus analysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN's behavior when dealing with samples with known cross-contamination levels. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single-base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability, and availability of amplicon sequencing in diagnostic settings.

Nebulized Bacteriophages for Prophylaxis of Experimental Ventilator-Associated Pneumonia Due to Methicillin-Resistant Staphylococcus aureus.

OBJECTIVES: There is a need for alternative strategies to combat and prevent antibiotic-resistant bacterial infections. Here, we assessed the potential for bacteriophage prophylaxis in the context of experimental ventilator-associated pneumonia due to methicillin-resistant Staphylococcus aureus in rats. DESIGN: Nebulized phages (aerophages) were delivered to the lungs of rats using a modified vibrating mesh aerosol drug delivery system. Animals were intubated and ventilated for 4 hours, at which point they were infected with methicillin-resistant S. aureus strain AW7 via the endotracheal tube, extubated, and then monitored for 96 hours. SETTING: Ventilator-associated pneumonia. SUBJECTS: Male Wistar rats. INTERVENTIONS: A single application of aerophages prior to ventilation at one of two concentrations (~1010 plaque forming units/mL or ~1011 plaque forming units/mL). MEASUREMENTS AND MAIN RESULTS: 1) Animal survival at 96 hours, 2) enumeration of bacteria and phages in the lungs and spleen, and 3) lung tissue histopathology. Animals that received aerophages prior to ventilation and methicillin-resistant S. aureus challenge showed a higher survival rate compared with untreated controls (60% for animals that received 3 × 10 plaque forming units; 70% for animals that received 3 × 10 plaque forming units; 0% for controls; p < 0.01 for each treatment versus untreated). Surviving animals that received aerophage prophylaxis had fewer methicillin-resistant S. aureus in the lungs compared with untreated control animals that succumbed to pneumonia (1.6 × 10 colony forming units/g vs 8.0 × 10; p < 0.01). CONCLUSIONS: Prophylactically administered nebulized bacteriophages reduced lung bacterial burdens and improved survival of methicillin-resistant S. aureus infected rats, underscoring its potential in the context of ventilator-associated pneumonia.

Combined therapy with ceftriaxone and doxycycline does not improve the outcome of meningococcal meningitis in mice compared to ceftriaxone monotherapy.

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.

MMPs and ADAMs in neurological infectious diseases and multiple sclerosis.

Metalloproteinases-such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs)-are involved in various diseases of the nervous system but also contribute to nervous system development, synaptic plasticity and neuroregeneration upon injury. MMPs and ADAMs proteolytically cleave many substrates including extracellular matrix components but also signaling molecules and receptors. During neuroinfectious disease with associated neuroinflammation, MMPs and ADAMs regulate blood-brain barrier breakdown, bacterial invasion, neutrophil infiltration and cytokine signaling. Specific and broad-spectrum inhibitors for MMPs and ADAMs have experimentally been shown to decrease neuroinflammation and brain damage in diseases with excessive neuroinflammation as a common denominator, such as pneumococcal meningitis and multiple sclerosis, thereby improving the disease outcome. Timing of metalloproteinase inhibition appears to be critical to effectively target the cascade of pathophysiological processes leading to brain damage without inhibiting the neuroregenerative effects of metalloproteinases. As the critical role of metalloproteinases in neuronal repair mechanisms and regeneration was only lately recognized, the original idea of chronic MMP inhibition needs to be conceptually revised. Recently accumulated research urges for a second chance of metalloproteinase inhibitors, which-when correctly applied and dosed-harbor the potential to improve the outcome of different neuroinflammatory diseases.

Bacteriophages Improve Outcomes in Experimental Staphylococcus aureus Ventilator-associated Pneumonia.

Rationale: Infections caused by multidrug-resistant bacteria are a major clinical challenge. Phage therapy is a promising alternative antibacterial strategy.Objectives: To evaluate the efficacy of intravenous phage therapy for the treatment of ventilator-associated pneumonia due to methicillin-resistant Staphylococcus aureus in rats.Methods: In a randomized, blinded, controlled experimental study, we compared intravenous teicoplanin (3 mg/kg, n = 12), a cocktail of four phages (2-3 × 109 plaque-forming units/ml of 2003, 2002, 3A, and K; n = 12), and a combination of both (n = 11) given 2, 12, and 24 hours after induction of pneumonia, and then once daily for 4 days. The primary outcome was survival at Day 4. Secondary outcomes were bacterial and phage densities in lungs and spleen, histopathological scoring of infection within the lungs, and inflammatory biomarkers in blood.Measurements and Main Results: Treatment with either phages or teicoplanin increased survival from 0% to 58% and 50%, respectively (P < 0.005). The combination of phages and antibiotics did not further improve outcomes (45% survival). Animal survival correlated with reduced bacterial burdens in the lung (1.2 × 106 cfu/g of tissue for survivors vs. 1.2 × 109 cfu/g for nonsurviving animals; P < 0.0001), as well as improved histopathological outcomes. Phage multiplication within the lung occurred during treatment. IL-1ß increased in all treatment groups over the course of therapy.Conclusions: Phage therapy was as effective as teicoplanin in improving survival and decreasing bacterial load within the lungs of rats infected with methicillin-resistant S. aureus. Combining antibiotics with phage therapy did not further improve outcomes.

Virulence Traits of a Serogroup C Meningococcus and Isogenic cssA Mutant, Defective in Surface-Exposed Sialic Acid, in a Murine Model of Meningitis.

In serogroup C Neisseria meningitidis, the cssA (siaA) gene codes for an UDP-N-acetylglucosamine 2-epimerase that catalyzes the conversion of UDP-N-acetyl-α-d-glucosamine into N-acetyl-d-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (α2→9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogenic cssA knockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD50) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of the cssA mutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in the corpus callosum, indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of the corpus callosum in the mouse model of meningococcal meningitis.

Combining Ceftriaxone with Doxycycline and Daptomycin Reduces Mortality, Neuroinflammation, Brain Damage, and Hearing Loss in Infant Rat Pneumococcal Meningitis.

Despite appropriate antibiotic therapy, pneumococcal meningitis (PM) is associated with a case fatality rate of up to 30% in high-income countries. Survivors often suffer from severe lifelong disabilities. An excessive inflammatory reaction drives the pathophysiology, leading to brain damage and neurologic sequelae. We aimed to improve the outcome of experimental PM by simultaneously targeting different pathophysiological mechanisms with combined adjunctive therapies previously shown to be neuroprotective. In vitro, the anti-inflammatory effects of doxycycline and daptomycin were evaluated on primary rat astroglial cells stimulated with Streptococcus pneumoniae Eleven-day-old infant Wistar rats were infected intracisternally with S. pneumoniae and randomized for treatment with ceftriaxone or combination adjuvant therapy consisting of ceftriaxone, daptomycin, and doxycycline. During acute PM, combined-adjuvant therapy with ceftriaxone, daptomycin, and doxycycline increased the survival rate from 64.1% to 85.8% (P < 0.01) and alleviated weight loss compared to ceftriaxone monotherapy (P < 0.01). Levels of inflammatory cytokines were significantly reduced by combined-adjuvant therapy in vitro (P < 0.0001) and in cerebrospinal fluid in vivo (P < 0.05). In infected animals treated with combined adjunctive therapy, cortical damage was significantly reduced (P < 0.05), and animals showed a trend toward better hearing capacity 3 weeks after the infection (P = 0.089), an effect which was significant in mildly infected animals (48 decibels [dB] versus 67.22 dB; P < 0.05). These mildly infected animals showed significantly reduced cochlear fibrous occlusion (P < 0.01). By combining nonbacteriolytic daptomycin and anti-inflammatory doxycycline with ceftriaxone, the previously reported beneficial effects of the drugs were cumulated and identified the triple-antibiotic therapy as a promising therapeutic option for pediatric PM.

Metformin mediates neuroprotection and attenuates hearing loss in experimental pneumococcal meningitis.

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 < 0.05). Compared to animals receiving ceftriaxone monotherapy, adjunctive metformin significantly reduced cortical necrosis (p < 0.02) during acute infection and improved median click-induced hearing thresholds (60 dB vs. 100 dB, p < 0.002) 5 weeks after infection. Adjuvant metformin significantly improved pure tone hearing thresholds at all assessed frequencies compared to ceftriaxone monotherapy (p < 0.05) and protected from PM-induced spiral ganglion neuron loss in the inner ear (p < 0.05). CONCLUSION: Adjuvant metformin reduces brain injury during pneumococcal meningitis by decreasing the excessive neuroinflammatory response. Furthermore, it protects spiral ganglion neurons in the inner ear and improves hearing impairments after experimental pneumococcal meningitis. These results identify adjuvant metformin as a promising therapeutic option to improve the outcome after pediatric pneumococcal meningitis.

Pneumolysin and the bacterial capsule of Streptococcus pneumoniae cooperatively inhibit taxis and motility of microglia.

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.

Combined effect of non-bacteriolytic antibiotic and inhibition of matrix metalloproteinases prevents brain injury and preserves learning, memory and hearing function in experimental paediatric pneumococcal meningitis.

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 < 0.01) or combined adjuvant therapy (p < 0.001). Cortical necrosis was significantly reduced in rats treated with adjuvant daptomycin (p < 0.05) or combined adjuvant therapy (p < 0.05) compared to ceftriaxone monotherapy. Six hours after treatment initiation, CSF cytokine levels were significantly reduced for TNF-α (p < 0.01), IL-1ß (p < 0.01), IL-6 (p < 0.001) and IL-10 (p < 0.01) in animals receiving combined adjuvant intervention compared to ceftriaxone monotherapy. Importantly, combined adjuvant therapy significantly improved learning and memory performance in infected animals and reduced hearing loss (77.14 dB vs 60.92 dB, p < 0.05) by preserving low frequency hearing capacity, compared to ceftriaxone monotherapy. CONCLUSION: Combined adjuvant therapy with the non-bacteriolytic antibiotic daptomycin and the MMP inhibitor Trocade integrates the neuroprotective effects of both single adjuvants in experimental paediatric pneumococcal meningitis by reducing neuroinflammation and brain damage, thereby improving neurofunctional outcome. This strategy represents a promising therapeutic option to improve the outcome of paediatric patients suffering from pneumococcal meningitis.

Novel and preclinical treatment strategies in pneumococcal meningitis.

PURPOSE OF REVIEW: Pneumococcal meningitis is the most frequent form of bacterial meningitis in Europe and the United States. Although early antimicrobial and adjuvant therapy with dexamethasone have helped to improve disease outcome in adults, mortality and morbidity rates remain unsatisfactorily high, emphasizing the need for additional treatment options. Promising targets for adjuvant therapy have been identified recently and will be the focus of this review. RECENT FINDINGS: Brain disease in pneumococcal meningitis is caused by direct bacterial toxicity and excessive meningeal inflammation. Accordingly, promising targets for adjuvant therapy comprise limiting the release of toxic bacterial products and suppressing inflammation in a way that maximally protects against tissue injury without hampering pathogen eradication by antibiotics. Among the agents tested so far in experimental models, complement inhibitors, matrix-metalloproteinase inhibitors, and nonbacteriolytic antibiotics or a combination of the above have the potential to more efficiently protect the brain either alone (e.g., in children and outside the high-income settings) or in addition to adjuvant dexamethasone. Additionally, new protein-based pneumococcal vaccines are being developed that promise to improve disease prevention, namely by addressing the increasing problem of serotype replacement seen with pneumococcal conjugate vaccines. SUMMARY: Pneumococcal meningitis remains a life-threatening disease requiring early antibiotic and targeted anti-inflammatory therapy. New adjuvant therapies showed promising results in animal models but need systematic clinical testing.