Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing.

Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine microbiological testing. Direct PCR of such samples enables the identification of microbes that may be fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative brain abscess samples by molecular methods could enable detection and/or treatment of the source of infection.

Mutations in the ENG, ACVRL1, and SMAD4 genes and clinical manifestations of hereditary haemorrhagic telangiectasia: experience from the Center for Osler's Disease, Uppsala University Hospital.

AIM: The aim of this retrospective single-centre study was to evaluate whether mutations in the ENG, ACVRL1, and SMAD4 genes were associated with different phenotypes in hereditary haemorrhagic telangiectasia (HHT). METHODS: The case records of 21 HHT patients with verified mutations in ENG, ACVRL1, or SMAD4 genes were reviewed. The numbers of HHT diagnostic criteria fulfilled for the three genotypes were compared, as was the prevalence of complications such as iron deficiency anaemia, gastrointestinal haemorrhage, stroke, and cerebral abscess. RESULTS: Our results indicate that mutations in the ENG (HHT1), ACVRL1 (HHT2), and SMAD4 genes result in different HHT phenotypes. Epistaxis debuts earlier and may be more severe in HHT1 than in HHT2. The prevalence of pulmonary arteriovenous malformations (AVM) is higher in HHT type 1, whereas hepatic AVMs are more common in HHT2. One patient with mutations in both ENG and ACVRL1 genes was identified, as were two SMAD4-mutated patients suffering from the overlapping juvenile polyposis-HHT syndrome. Nearly one in five patients in our HHT population has been diagnosed with stroke or cerebral abscess, indicating a high prevalence of cerebral complications. CONCLUSION: Our results showing that ENG and ACVRL1 gene mutations result in different HHT phenotypes confirm the results from other HHT centres worldwide. Cerebral complications of HHT are common, underscoring the importance of regular screening for pulmonary AVMs and early intervention against such AVMs. We have identified an HHT patient with simultaneous mutations in the ENG and ACVRL1 genes. Surprisingly, this patient has had a mild course of the disease.

The proteome of pus from human brain abscesses: host-derived neurotoxic proteins and the cell-type diversity of CNS pus.

OBJECTIVE What determines the extent of tissue destruction during brain abscess formation is not known. Pyogenic brain infections cause destruction of brain tissue that greatly exceeds the area occupied by microbes, as seen in experimental studies, pointing to cytotoxic factors other than microbes in pus. This study examined whether brain abscess pus contains cytotoxic proteins that might explain the extent of tissue destruction. METHODS Pus proteins from 20 human brain abscesses and, for comparison, 7 subdural empyemas were analyzed by proteomics mass spectrometry. Tissue destruction was determined from brain abscess volumes as measured by MRI. RESULTS Brain abscess volume correlated with extracellular pus levels of antibacterial proteins from neutrophils and macrophages: myeloperoxidase (r = 0.64), azurocidin (r = 0.61), lactotransferrin (r = 0.57), and cathelicidin (r = 0.52) (p values 0.002-0.018), suggesting an association between leukocytic activity and tissue damage. In contrast, perfringolysin O, a cytotoxic protein from Streptococcus intermedius that was detected in 16 patients, did not correlate with abscess volume (r = 0.12, p = 0.66). The median number of proteins identified in each pus sample was 870 (range 643-1094). Antibiotic or steroid treatment prior to pus evacuation did not reduce the number or levels of pus proteins. Some of the identified proteins have well-known neurotoxic effects, e.g., eosinophil cationic protein and nonsecretory ribonuclease (also known as eosinophil-derived neurotoxin). The cellular response to brain infection was highly complex, as reflected by the presence of proteins that were specific for neutrophils, eosinophils, macrophages, platelets, fibroblasts, or mast cells in addition to plasma and erythrocytic proteins. Other proteins (neurofilaments, myelin basic protein, and glial fibrillary acidic protein) were specific for brain cells and reflected damage to neurons, oligodendrocytes, and astrocytes, respectively. Pus from subdural empyemas had significantly higher levels of plasma proteins and lower levels of leukocytic proteins than pus from intracerebral abscesses, suggesting greater turnover of the extracellular fluid of empyemas and washout of pus constituents. CONCLUSIONS Brain abscess pus contains leukocytic proteins that are neurotoxic and likely participate actively in the excessive tissue destruction inherent in brain abscess formation. These findings underscore the importance of rapid evacuation of brain abscess pus.

Association of ICAM-1 (K469E) and MCP-1 -2518 A>G gene polymorphism with brain abscess.

Brain abscess develops in response to a parenchymal infection. Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play vital role in central nervous system (CNS) diseases. We studied ICAM-1 (K469E) and MCP-1 (-2518 A>G) polymorphisms among brain abscess patients. The genotypic distributions of ICAM-1 (K469E) and MCP-1 (-2518 A>G) were significantly different between patients and controls. Further, patient with predisposing factors, and also with culture result, we found significant association. The study revealed that the polymorphisms of these molecules lead to increased production, which appears to be a risk for the development of brain abscess.

Hereditary hemorrhagic telangiectasia patient presenting with brain abscess due to silent pulmonary arteriovenous malformation.

Hereditary hemorrhagic telangiectasia is a rare autosomal dominant inherited disease that is usually complicated by visceral vascular malformations. Patients harboring such malformations are at increased risk of brain abscess formation, which despite advances in diagnostic and surgical methods remains a life threatening medical emergency with high mortality and morbidity rates. In the present report we describe a case of cerebral abscess due to silent pulmonary arteriovenous malformation (AVM) in a young patient previously undiagnosed for hereditary hemorrhagic telangiectasia syndrome (HHT).

Tumor necrosis factor-α and interleukin-1ß gene polymorphisms and risk of brain abscess in North Indian population.

OBJECTIVE: Brain abscess develops in response to a parenchymal infection with pyogenic bacteria. Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are the most crucial pro-inflammatory cytokines with both beneficial and destructive properties for the central nervous system. The present study evaluated the association of specific alleles/genotypes of TNF-α and IL-1ß with brain abscess. MATERIAL AND METHODS: A total of 90 brain abscess patients and 100 healthy controls were included in the study. Predisposing factors were identified in 56 (62.2%) patients with brain abscess. TNF-α (-308 G>A) and IL-1ß (-511 CA) and C allele in IL-1ß (-511 CA) and IL-1ß (-511 C

Escherichia coli brain abscess in a twin pair associated with TLR4 gene mutation.

Brain abscesses are uncommon complications of bacterial meningitis or sepsis in neonates and infants. The causative pathogens of brain abscess in newborns are various. Of those, Escherichia coli is rarely seen as a pathogen in brain abscess at this age. Herein we reported brain abscesses in twin infants caused by E. coli sepsis. Interestingly, genetic analysis identified heterozygous Toll-like receptor 4 (TLR4) gene mutation in the twins. Because TLR plays an important role in the natural response to bacterial products and initiates specific immune response against these pathogens, this may explain the development of brain abscess in the present case.

Differential effects of interleukin-17 receptor signaling on innate and adaptive immunity during central nervous system bacterial infection.

Although IL-17A (commonly referred to as IL-17) has been implicated in the pathogenesis of central nervous system (CNS) autoimmune disease, its role during CNS bacterial infections remains unclear. To evaluate the broader impact of IL-17 family members in the context of CNS infection, we utilized IL-17 receptor (IL-17R) knockout (KO) mice that lack the ability to respond to IL-17, IL-17F and IL-17E (IL-25). In this article, we demonstrate that IL-17R signaling regulates bacterial clearance as well as natural killer T (NKT) cell and gamma-delta (γδ) T cell infiltrates during Staphylococcus aureus-induced brain abscess formation. Specifically, when compared with wild-type (WT) animals, IL-17R KO mice exhibited elevated bacterial burdens at days 7 and 14 following S. aureus infection. Additionally, IL-17R KO animals displayed elevated neutrophil chemokine production, revealing the ability to compensate for the lack of IL-17R activity. Despite these differences, innate immune cell recruitment into brain abscesses was similar in IL-17R KO and WT mice, whereas IL-17R signaling exerted a greater influence on adaptive immune cell recruitment. In particular, γδ T cell influx was increased in IL-17R KO mice at day 7 post-infection. In addition, NK1.1high infiltrates were absent in brain abscesses of IL-17R KO animals and, surprisingly, were rarely detected in the livers of uninfected IL-17R KO mice. Although IL-17 is a key regulator of neutrophils in other infection models, our data implicate an important role for IL-17R signaling in regulating adaptive immunity during CNS bacterial infection.

Immunodeficiency associated with FCN3 mutation and ficolin-3 deficiency.

Ficolin-3, encoded by the FCN3 gene and expressed in the lung and liver, is a recognition molecule in the lectin pathway of the complement system. Heterozygosity for an FCN3 frameshift mutation (rs28357092), leading to a distortion of the C-terminal end of the molecule, occurs in people without disease (allele frequency among whites, 0.01). We describe a patient with recurrent infections who was homozygous for this mutation, who had undetectable serum levels of ficolin-3, and who had a deficiency in ficolin-3-dependent complement activation.

[When genetics matters: an unusual case of left-sided hemiparesis]. / Wenn der Stammbaum zählt: ein ungewöhnlicher Fall einer linksseitigen Hemiparese.

We report the case of a 35-year-old woman who has been admitted to our emergency room because of the sudden onset of a left-sided hemiparesis. The physical examination showed disseminated teleangiectases on the upper and lower lip, on the mucosal surface of the tongue and on the skin. The cerebral CT scan presented a right-sided fronto-parietal lesion, more likely an abscess, confirmed on surgical removal. A meticulous family history showed the presence of similar clinical features among several family members, raising the certainty about the presence of the Rendu-Osler-Weber disease. This case is highly instructive in emphasizing the value of taking a careful medical history and how doing it is extremely important in our daily practice.

Brain abscess and hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant disorder that can lead to serious central nervous system complications including hemorrhage, ischemia, and infection. Symptoms can be mild, making diagnosis problematic. Fifty-three prior cases of HHT and brain abscess are described, in addition to two new cases. The clinical manifestations and current methods for diagnosis and management of patients with HHT are reviewed. Early recognition of HHT is important because screening in these patients and affected family members may help prevent complications. In addition, advancements in imaging, surgical techniques, antibiotics, and genetic testing may improve outcomes.

Microbial genome dynamics in CNS pathogenesis.

The balancing act between microbes and their host in commensal and disease states needs to be deciphered in order to fully treat and combat infectious diseases. The elucidation of microbial genome dynamics in each instance is therefore required. In this context, the major bacterial meningitis pathogens are Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae. In prokaryotic CNS pathogenesis both the intact organism as well as its released components can elicit disease, often resulting in neurological sequelae, neurodegeneration or fatal outcome. The study of microbial virulence in CNS disease is expected to generate findings that yield new information on the general mechanisms of brain edema and excitatory neuronal disturbances due to meningitis, with significant potential for discoveries that can directly influence and inspire new strategies for prevention and treatment of this serious disease.

MyD88-dependent signals are essential for the host immune response in experimental brain abscess.

Brain abscesses form in response to a parenchymal infection by pyogenic bacteria, with Staphylococcus aureus representing a common etiologic agent of human disease. Numerous receptors that participate in immune responses to bacteria, including the majority of TLRs, the IL-1R, and the IL-18R, use a common adaptor molecule, MyD88, for transducing activation signals leading to proinflammatory mediator expression and immune effector functions. To delineate the importance of MyD88-dependent signals in brain abscesses, we compared disease pathogenesis using MyD88 knockout (KO) and wild-type (WT) mice. Mortality rates were significantly higher in MyD88 KO mice, which correlated with a significant reduction in the expression of several proinflammatory mediators, including but not limited to IL-1beta, TNF-alpha, and MIP-2/CXCL2. These changes were associated with a significant reduction in neutrophil and macrophage recruitment into brain abscesses of MyD88 KO animals. In addition, microglia, macrophages, and neutrophils isolated from the brain abscesses of MyD88 KO mice produced significantly less TNF-alpha, IL-6, MIP-1alpha/CCL3, and IFN-gamma-induced protein 10/CXCL10 compared with WT cells. The lack of MyD88-dependent signals had a dramatic effect on the extent of tissue injury, with significantly larger brain abscesses typified by exaggerated edema and necrosis in MyD88 KO animals. Interestingly, despite these striking changes in MyD88 KO mice, bacterial burdens did not significantly differ between the two strains at the early time points examined. Collectively, these findings indicate that MyD88 plays an essential role in establishing a protective CNS host response during the early stages of brain abscess development, whereas MyD88-independent pathway(s) are responsible for pathogen containment.

An essential role for tumor necrosis factor in the formation of experimental murine Staphylococcus aureus-induced brain abscess and clearance.

Tumor necrosis factor-alpha (TNF-alpha) is a central mediator of the immune response to pathogens, but may also exert neurotoxic effects, thereby contributing to immunopathology. To define the role of TNF during the course of brain abscess, TNF-deficient (TNF(0/0) mice were stereotaxically infected with Staphylococcus (S.) aureus-laden agarose beads. In comparison to 100% survival of wild type (WT) mice, TNF(0/0) mice displayed high mortality rates (54%) in the initial phase of abscess development as well as significantly increased morbidity in the course of the disease. The worse clinical outcome was due to an increased intracerebral (i.c.) bacterial load in TNF(0/0) mice as compared to WT mice. The impaired control of S. aureus was associated with reduced inductible nitric oxide synthase (iNOS) mRNA and protein expression in TNF(0/0)mice. Similarly, numbers of inflammatory leukocytes, cytokine expression of IL-6, IL-12p40, IFNgamma IL-beta mRNA, and brain edema were significantly increased in TNF(0/0)mice as compared to WT animals. In addition, resolution of i.c. infiltrates was delayed in TNF(0/0)mice correlating with reduced apoptosis of inflammatory leukocytes and formation of a fibrous abscess capsule. Collectively, these data demonstrate that TNF is of key importance for the control of S. aureus-induced brain abscess and regulates the ensuing host immune response.

The intermediate filament GFAP is important for the control of experimental murine Staphylococcus aureus-induced brain abscess and Toxoplasma encephalitis.

The functional role of astrocytes exerted via their intermediate protein glial fibrillary acidic protein (GFAP) in CNS infections was studied in Staphylococcus aureus-induced brain abscess. Compared to wild type (WT) mice, GFAP(0/0) mice developed larger and more poorly demarcated inflammatory lesions paralleled by a significantly increased intracerebral bacterial load, a diffuse leukocytic infiltration of the contralateral hemisphere, purulent ventriculitis, vasculitis, and severe brain edema. These observations were correlated with the lack of a bordering function of activated astrocytes that strongly upregulated their GFAP expression in the abscess surrounding of WT mice. Clinically important, this lack of restriction of inflammation markedly aggravated the course of disease with manifestation of seizures and a severe weight loss in GFAP(0/0) mice. These data were paralleled by observations in the model of Toxoplasma encephalitis (TE) during which the intracerebral parasitic load was significantly increased. Moreover, tachyzoite-induced tissue necrosis was exclusively found in the brains of GFAP(0/0) mice in chronic TE. Collectively, these findings delineate a host defense function of astrocytes via restricting pathogenic spread and multiplication within the CNS, thereby contributing to the protection of the highly vulnerable brain parenchyma.

[Familial brain abscess as a complication of hereditary hemorrhagic telangiectasia]. / Hereditaer haemorrhagiás teleangiectasia szövódményeként kialakult agytályog családi halmozódása.

The hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber disease) is an inherited autosomal dominant disease with angiodysplasia of the skin, mucosa, parenchymal organs, and it can affect the central nervous system. In 40% of the cases neurological complications, most frequently intracerebral abscesses occur. In this study, the case history of a patient with central nervous system manifestation of hereditary hemorrhagic telangiectasia showing familiar aggregation of brain abscess will be presented. A young male patient was admitted to Neurological Department because of his first epileptic seizure and progressive right hemispheric symptoms. His examinations showed frontal abscess, which was surgically removed. The frequent nose-bleeding of the patient and recurrent brain abscess in his brother's history provided the possibility of hereditary hemorrhagic telangiectasia. The background of brain abscess were multiple pulmonary arteriovenous malformation, which were embolized by repeated angiography. Familiar brain abscess is very rare. However, in the case of brain abscess especially with familiarity diagnosis of the Rendu-Osler-Weber disease should be considered.

Proinflammatory cytokine, chemokine, and cellular adhesion molecule expression during the acute phase of experimental brain abscess development.

Brain abscess represents the infectious disease sequelae associated with the influx of inflammatory cells and activation of resident parenchymal cells in the central nervous system. However, the immune response leading to the establishment of a brain abscess remains poorly defined. In this study, we have characterized cytokine and chemokine expression in an experimental brain abscess model in the rat during the acute stage of abscess development. RNase protection assay revealed the induction of the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, and tumor necrosis factor-alpha as early as 1 to 6 hours after Staphylococcus aureus exposure. Evaluation of chemokine expression by reverse transcription-polymerase chain reaction demonstrated enhanced levels of the CXC chemokine KC 24 hours after bacterial exposure, which correlated with the appearance of neutrophils in the abscess. In addition, two CC chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1alpha were induced within 24 hours after S. aureus exposure and preceded the influx of macrophages and lymphocytes into the brain. Analysis of abscess lesions by in situ hybridization identified CD11b+ cells as the source of IL-1beta in response to S. aureus. Both intercellular adhesion molecule-1 and platelet endothelial cell adhesion molecule expression were enhanced on microvessels in S. aureus but not sterile bead-implanted tissues at 24 and 48 hours after treatment. These results characterize proinflammatory cytokine and chemokine expression during the early response to S. aureus in the brain and provide the foundation to assess the functional significance of these mediators in brain abscess pathogenesis.

Identification of the cause of a brain abscess by direct 16S ribosomal DNA sequencing.

We report the case of a young man who apparently suffered successive episodes of meningitis and cerebral abscess over a 1-month period, both of which were diagnosed by two different molecular approaches; PCR for Neisseria meningitidis IS1106 from CSF and 16S rRNA gene sequencing on a specimen of brain pus. In each case, cultures were negative due to prior antibiotic therapy.