Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.

The Potential Role of Matrix Metalloproteinases 8 and 9 and Myeloperoxidase in Predicting Outcomes of Bacterial Meningitis of Childhood.

BACKGROUND: Matrix metalloproteinases (MMPs) and myeloperoxidase (MPO) contribute to the inflammatory cascade in the cerebrospinal fluid (CSF) during bacterial meningitis. We determined levels of MPO, MMP-8, MMP-9, and tissue inhibitor of metalloproteinase- (TIMP-) 1 in the CSF of children with bacterial meningitis and investigated how these inflammatory mediators relate to each other and to the disease outcomes. METHODS: Clinical data and the diagnostic CSF samples from 245 children (median age eight months) with bacterial meningitis were obtained from a clinical trial in Latin America in 1996-2003. MMP-9 levels in the CSF were assessed by zymography, while MMP-8, MPO, and TIMP-1 concentrations were determined with immunofluorometric and enzyme-linked immunosorbent assays. RESULTS: MPO correlated positively with MMP-8 (rho 0.496, P < 0.001) and MMP-9 (rho 0.153, P = 0.02) but negatively with TIMP-1 (rho -0.361, P < 0.001). MMP-8 emerged as the best predictor of disease outcomes: a CSF MMP-8 concentration above the median increased the odds of death 4.9-fold (95% confidence interval 1.8-12.9). CONCLUSIONS: CSF MMP-8 presented as an attractive prognostic marker in children with bacterial meningitis.

Raised adenosine deaminase in the cerebrospinal fluid: A tool for the diagnosis of tuberculous meningitis in developing countries.

CONTEXT: The rapid diagnosis followed by the early treatment of tuberculous meningitis (TBM) is important in preventing fatal outcomes. The mainstay of diagnosis lies in cerebrospinal fluid (CSF) analysis, radiological investigations, and clinical findings. AIM: The present study was conducted to determine the efficacy, sensitivity, and specificity of raised adenosine deaminase (ADA) level in CSF to differentiate TBM from non-TBM cases as a rapid, cost-effective, and noninvasive test. PATIENTS AND METHODS: This was a retrospective study conducted over a 1-year period in a tertiary teaching institute of Malwa region, India. A total of 143 patients presented with symptoms and signs of meningitis were included and divided into TBM and non-TBM groups on the basis of the diagnostic criteria. CSF ADA estimation was drafted and analyzed by using ≥10 U/L as a cutoff value. A statistical comparison of the ADA levels between the study groups was made by using unpaired Student's t-test. RESULTS: Out of the 143 cases, 40 were TBM, and 103 were non-TBM. The mean ADA level in TBM and non-TBM cases was 17.18 ± 9.59 and 6.33 ± 2.48, respectively, and the difference was statistically significant. Using a cutoff level ≥10 U/L, CSF ADA had a sensitivity of 92.5% and a specificity of 89.32%. Positive and negative likelihood ratios of the test were 8.66 and 0.08, respectively, and positive and negative predictive values, were 77.08 and 96.84%, respectively. CONCLUSION: The present study reflects the importance of a CSF ADA level ≥10 U/L in the diagnosis of TBM. Thus, it can be used as an adjunctive diagnostic tool to differentiate TBM from other non-TBM cases, when there is a diagnostic dilemma.

[Cerebrospinal fluid adenosine deaminase and its dynamic changes in tuberculous meningitis].

Objective: To explore the diagnostic value of cerebrospinal fluid (CSF) adenosine deaminase (ADA) level in tuberculous meningitis. Methods: We retrospectively analyzed 139 patients (73 males, 66 females) who visited Beijing Chest Hospital for suspected TBM from January 2010 to June 2015. Of them, 99 patients were diagnosed to have TBM, with 45 males and 54 females, and a mean age of (33±15) years. Forty patients were diagnosed as having Non-TBM, with 28 males and 12 females, and a mean age of (35±18) years. All patients underwent lumbar puncture, and CSF ADA, routine, biochemical and bacteriological tests were performed. Thirty-five TBM patients reviewed CSF ADA test after treatment for 4 weeks, 8 weeks and 6 months. Results: The level of CSF ADA in TBM group was higher than that in the non-TBM group, the difference being statistically significant (5.6 U/L vs 2.3 U/L, P=0.000). When the cut-off value of the CSF ADA was 3.8 U/L , the sensitivity and specificity for diagnosis of TBM were 60.6% (95%CI 50.3%-70.1%) and 87.5% (95%CI 72.4%-95.3%), respectively, and the area under the ROC curve was 0.734.The CSF ADA level was (6.7±4.2) U/L in the 35 cases of TBM before treatment. After 4 weeks, 8 weeks and 6 months of anti-tuberculosis treatment, the CSF ADA levels were (4.5±3.3) U/L, (3.7±2.7) U/L and (2.0±1.5) U/L, respectively; all significantly decreased as compared to that before treatment (P<0.001). There was no significant change in the ADA level between 8 weeks and 4 weeks (P=0.128). After 6 months of treatment, the level of CSF ADA was significantly lower than those after 4 and 8 weeks' treatment (P<0.001). Conclusions: CSF ADA in TBM patients was significantly higher than in non-TBM patients. The sensitivity of CSF ADA level in the diagnosis of TBM was poor, but the specificity was better. CSF ADA was significantly reduced and showed dynamic changes with effective anti-tuberculosis treatment and maybe helpful in evaluating the effect of treatment.

Association of kynurenine aminotransferase II gene C401T polymorphism with immune response in patients with meningitis.

BACKGROUND: The kynurenine (KYN) pathway has been shown to be altered in several diseases which compromise the central nervous system (CNS) including infectious diseases such as bacterial meningitis (BM). The aim of this study was to assess single nucleotide polymorphisms (SNPs) in four genes of KYN pathway in patients with meningitis and their correlation with markers of immune response in BM. METHODS: One hundred and one individuals were enrolled in this study to investigate SNPs in the following genes: indoleamine-2,3-dioxygenase (IDO1 gene), kynureninase (KYNU gene), kynurenine aminotransferase I (CCBL1 gene), and kynurenine aminotransferase II (AADAT gene). SNP analyses were performed by primer-introduced restriction analysis-PCR (PIRA-PCR) followed by RFLP. Cytokines were measured using multiplex bead assay while immunoglobulins (IG) by immunodiffusion plates and NF-kappaB and c-Jun by dot blot assay. RESULTS: The variant allele of SNP AADAT+401C/T showed prevalent frequency in patients with BM. A significant decrease (p < 0.05) in TNF-α, IL-1ß, IL-6, MIP-1αCCL3 and MIP-1ß/CCL4 levels was observed in BM patients homozygous (TT) to the SNP AADAT+401C/T. Furthermore, a significant (p < 0.05) decrease in cell count was observed in cerebrospinal fluid (CSF) from patients with TT genotype. In addition, an increase in the IgG level in adults (p < 0.05) was observed. The variant allele for KYNU+715G/A was found with low frequency in the groups, and the SNPs in IDO1+434T/G, KYNU+693G/A, CCBL1+164T/C, and AADAT+650C/T had no frequency in this population. CONCLUSIONS: This study is the first report of an association of SNP AADAT+401C/T with the host immune response to BM, suggesting that this SNP may affect the host ability in recruitment of leukocytes to the infection site. This finding may contribute to identifying potential targets for pharmacological intervention as adjuvant therapy for BM.

Pattern of cerebrospinal fluid lactic dehydrogenase during treatment of bacterial meningitis.

Bacterial and aseptic meningitis are characterized by distinctive lactic dehydrogenase isoenzyme patterns. No studies have quantified the dynamics of lactic dehydrogenase isoenzyme distribution during treated bacterial meningitis. We used a retrospective case-series design, and reviewed files of all neonates with bacterial meningitis who attended our pediatric tertiary medical center for 8 years period. We identified neonates in whom a repeated lumbar puncture was indicated. Findings of cerebrospinal fluid analysis, including levels of lactic dehydrogenase isoenzymes, were compared with an age-matched reference group. In two patients with meningitis, lumbar puncture with cerebrospinal fluid analysis was repeated because of inadequate response to treatment or initially obscure etiologic pathogens. Both patients had initially low levels of lactic dehydrogenase-1 and lactic dehydrogenase-2 and high levels of lactic dehydrogenase-4 and lactic dehydrogenase-5, similar to other patients with bacterial meningitis. The distribution pattern of lactic dehydrogenase isoenzyme normalized after adequate antibiotic treatment. In light of the encouraging results in these two patients, further studies are warranted regarding the value of lactic dehydrogenase isoenzyme measurements for follow-up purposes and for evaluations of response to treatment.

Comparative analysis of cerebrospinal fluid adenosine deaminase in tuberculous and non-tuberculous meningitis.

OBJECTIVE: To calculate cut-off point for the adenosine deaminase (ADA) activity in the CSF of patients with tuberculous meningitis (TBM). PATIENTS AND METHODS: The ADA assay was based on the automatic indirect method in which ADA catalyzes adenosine to inosine. ADA activity in the CSF was calculated based on ammonia liberated from adenosine and quantified spectrophotometrically. Arithmetic mean values and standard deviation of each variable were measured. Mann-Whitney U and Fisher exact tests were applied to compare continuous and dichotomous variables between tuberculous and non-tuberculous groups. A receiver operating characteristic curve was plotted to identify various cut-off points to determine the best level for ADA activity. RESULTS: Totally 42 patients were enrolled into the study. The median of ADA activity in the TBM group was 22 and in the non-TBM group was 8.0. The mean CSF-ADA activity was found to be significantly higher in TBM group (23.05+/-13.1IU/L) than in the CSF from non-TBM patients (9.39+/-5.18IU/L). The highest accuracy is at the cut-off value of 10.5IU/L. The sensitivity and specificity of the test at this cut-off to differentiate TBM from non-tuberculous meningitis is 81% and 86% respectively. CONCLUSION: Considering that a high positive value of ADA activity cannot confirm TBM, however, in suspected patients it may lead the physician to treat patient earlier before the confirmatory diagnostic reports will be received. The suggested cut-off value in this pilot study is 10.5IU/L with high sensitivity and specificity.

von Willebrand factor activation, granzyme-B and thrombocytopenia in meningococcal disease.

SUMMARY BACKGROUND: During invasive meningococcal disease, severe thrombocytopenia is strongly associated with a poor outcome. OBJECTIVES: In order to elucidate the pathophysiological mechanism behind the development of thrombocytopenia, we studied the role of von Willebrand factor (VWF) in meningococcal disease. PATIENTS/METHODS: Thirty-two children with severe meningococcal disease admitted to our university hospital were included in this study. VWF and related parameters were measured and results were correlated with the development of shock and thrombocytopenia. RESULTS: At admission, all patients had increased levels of (active) VWF and VWF propeptide. The highest VWF propeptide levels were observed in patients with shock, indicating acute endothelial activation. Although VWF propeptide levels in patients with shock, with or without thrombocytopenia, were similar, increased active VWF was significantly lower in patients with thrombocytopenia as compared with patients without thrombocytopenia. ADAMTS13 was moderately decreased. However, the VWF multimeric pattern was minimally increased. We assume that these findings are explained by VWF consumption and perhaps by granzyme B (GrB). In vitro experiments showed that GrB is able to cleave VWF multimers in plasma, whereas GrB was high in patients with shock, who developed thrombocytopenia. CONCLUSIONS: Our results demonstrate that consumption of VWF, derived from endothelial cells, could be a key feature of meningococcal disease and primary to the development of thrombocytopenia during shock.

Cerebrospinal fluid lactate dehydrogenase isoenzymes in children with bacterial and aseptic meningitis.

Differentiation of bacterial from aseptic meningitis may be difficult. Our aim was to determine the pattern of distribution of lactate dehydrogenase (LDH) isoenzymes in the cerebrospinal fluid (CSF) of patients with bacterial and aseptic meningitis. One hundred and fifty-seven patients with suspected meningitis were enrolled in the study. They were divided into 3 groups according to the culture- or bacterial antigen assay-proven diagnosis and CSF findings: bacterial meningitis (n = 31), aseptic meningitis (n = 65), and non-meningitis (n = 61). Total LDH level and percentages of LDH isoenzymes in the CSF were measured in each patient. Each group showed a distinct LDH isoenzyme distribution pattern, with a statistically significant difference among the groups in the percentages of the various isoenzymes. Compared with the non-meningitis group, total LDH activity in the CSF was high in the aseptic meningitis group (49.82+/-35.59 U/L, P < 0.001) and exaggerated in the bacterial meningitis group (944.53+/-112.3 U/L, P < 0.001). Low LDH-2 levels were unique to bacterial meningitis (P < 0.01), whereas high LDH-3 levels were characteristic of aseptic meningitis (P < 0.05). Both groups had low levels of LDH-1 and high levels of LDH-4 and LDH-5. In conclusion, the LDH isoenzyme pattern may be of clinical diagnostic value in meningitis, particularly when culture results are pending.

[Metalloproteinases in meningoencephalitis]. / Metaloproteinazy w zapaleniach opon mózgowo-rdzeniowych i mózgu.

Meningoencephalitis remains a devastating disease with high morbidity and mortality. Despite advances in antibiotic treatment and critical care, mortality rate in bacterial meningoencephalitis is close to 25%. Moreover, neurological and neuropsychological sequelae emerge in up to 50% of survivors. Adverse outcome is significantly associated with events secondary to meningitis and damage of the blood-brain barrier. Several studies conducted on animals confirmed that matrix-metalloproteinases (MMP), a family of enzymes with major actions in the remodeling of exracellural matrix components facilitate this process which results in acute neurological complications. Gelatinases (MMP-2, MMP-9), the most complex family member, through degradation of gelatine and collagen IV play an important role in the pathogenesis of brain's inflamatory diseases (e.g. Guillian-Barre syndrom) and contribute to spreading the disease beyond the central nervous system. Infection (bacterial, viral or fungal) can lead to increased concentration and activity of metalloproteinases due to excessive enzyme's secretion or decrease in level of its natural inhibitors. A detailed analysis of those enzymes could help in developing new diagnostic and prognostic markers for meningoencephalitis and could facilitate new treatment approaches.

Dynamic changes of matrix metalloproteinase-9 in patients with Klebsiella pneumoniae meningitis.

To quantitate cerebrospinal fluid (CSF) concentrations of matrix metalloproteinase 9 (MMP-9) in adult patients with Klebsiella pneumoniae meningitis and to correlate levels of MMP-9 with parameters of intrathecal inflammation and analyze the kinetic changes of MMP-9. In a prospective cohort study, levels of MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP-1) concentrations were measured in the CSF of six adult patients with meningitis and 11 controls. MMP-9 and TIMP-1 were detected in all of the six patients at presentation and follow up lumbar puncture. CSF levels of MMP-9 (6.71+/-7.29 ng/ml) and TIMP-1(454.3+/-242.9 ng/ml) were higher in patients than in the control group (0.07+/-0.11 ng/ml and 27.14+/-39.34 ng/ml, respectively). Levels of MMP-9 correlated with CSF concentrations of protein, cell count and lactate. Repeated lumbar punctures showed that levels of MMP-9 decrease during clinical recovery, although the levels of MMP-9 in the CSF are variable because of the small number of cases. The relative change in gelatin zymography is comparable to the changes of MMP-9 levels found in ELISA. MMP-9 levels in CSF may be a useful tool in follow-up in patients with K. pneumoniae meningitis.

Serum matrix metalloproteinase-2 levels indicate blood-CSF barrier damage in patients with infectious meningitis.

OBJECTIVE: Protein components in cerebrospinal fluid (CSF) are maintained at a specific concentration by a dynamic gradient between the capillary and intrathecal spaces via the blood-cerebrospinal fluid barrier (BCB) in the brain and spinal cord. Permeability to proteins increases when there is structural damage to the BCB. Matrix metalloproteinase-2 (MMP-2; gelatinase A) has been shown to degrade type IV collagen, a major component of the cellular basement membrane. We analyzed alpha2 macroglobulin (alpha2M) indices and evaluated the relationship between alpha2M, as an indicator of BCB permeability, and MMP-2, which degrades the extra-cellular matrix in patients with infectious meningitis. MATERIALS AND METHODS: Albumin levels in CSF or serum were determined by turbidimetric immunoassay, or bromcresol green assay, respectively. alpha2M levels in CSF or serum were measured with enzyme-linked immunosorbent assay, or laser-nephelometry, respectively. Serum MMP-2 levels were determined by enzyme immuno assay. We calculated the alpha2M index, i.e. the ratio of alpha2M (CSF / serum) to albumin (CSF / serum; alpha2M in CSF / alpha2M in serum x albumin in serum / albumin in CSF). RESULTS: alpha2M indices were significantly increased in infectious meningitis compared to healthy controls (p < 0.05). They were highest in bacterial meningitis, and there was a significant difference between viral or mycotic and bacterial meningitis (p < 0.05). Serum MMP-2 levels were increased in infectious meningitis, being highest in bacterial meningitis, where they were significantly different from healthy controls (p < 0.05). There was a significant positive correlation between serum MMP-2 levels and alpha2M indices (r = 0.64, p < 0.0001). CONCLUSION: Markedly increased levels of serum MMP-2 in infectious, especially bacterial, meningitis may reflect the degree of damage to the BCB.

Immunohistochemical analysis of MMP-9, MMP-2 and TIMP-1, TIMP-2 expression in the central nervous system following infection with viral and bacterial meningitis.

Matrix metalloproteinases (MMPs) are capable of degrading components of the basal lamina of cerebral vessels, thereby disrupting the blood-brain barrier and inducing leukocyte recruitment. This study provides comprehensive information regarding the cell specificity of matrix metalloproteinases (MMP-2, MMP-9) and their binding tissue inhibitors (TIMP-1, TIMP-2) in the central nervous system during viral and bacterial meningitis. Specifically, we evaluated the immunoreactivity of MMPs and TIMPs in various cell types in brain parenchyma and meninges obtained from autopsy tissues. We found that a higher proportion of endothelial cells were positive for MMP-9 during meningitis when compared to controls. In addition, the immunoreactivity of MMP-9 decreased and the immunoreactivity of TIMP-1 increased in astrocytes upon infection. Furthermore, the results of this study revealed that mononuclear cells were highly immunoreactive for TIMP-1, TIMP-2 and MMP-9 during viral meningitis and that the expression of TIMPs in polymorphonuclear cells was even higher during bacterial meningitis. Taken together the results of this study indicated that the central nervous system resident cells and inflammatory infiltrates contribute to MMPs activity and that the expression patterns vary between cell types and in response to viral and bacterial meningitis.

Comparative study of cerebrospinal fluid adenosine deaminase activity in patients with meningitis.

Cerebrospinal fluid (CSF) adenosine deaminase (ADA) activity in tubercular meningitis (TBM) patients (n=20), non-tubercular meningitis (NTBM) patients (n=10) and non-tubercular non-meningitis (NTBNM) cases (n=15) were measured by the method based on Berthlot's reaction. The mean CSF ADA activity in TBM (13.62 +/- 8.45 IU/L) was found to be significantly higher as compared to NTBM (6.51 +/- 2.41 IU/ L, p<0.001) and NTBNM (2.35 +/- 1.16 IU/L, p<0.0001) respectively. The sensitivity and specificity of CSF ADA activity was 85.0% and 88.0% respectively at cut-off value of 6.97 IU/L to diagnose tubercular meningitis. The specificity and sensitivity of CSF ADA for TBM was found to be 85.0% and 70.0% as compared to NTBM and 85.0% and 100.0% as compared to NTBNM. We propose that estimation of that ADA activity in CSF of TBM patients, using a cut off value 6.97 IU/L can diagnose differentially tubercular meningitis. Since, most developing countries have the dubious distinction of having higher prevalence and incidence of tubeculosis and lack of well equipped laboratory services for proper diagnosis of tubercular meningitis, measurement of CSF ADA activity can be a better and reliable approach for the rapid diagnosis and management of tubercular meningitis vis a vis other types of meningitis.

Fibrinolysis and host response in bacterial infections.

The plasminogen activation system is part of the fibrinolysis which is tightly regulated and protected against dysfunction by various activators and inhibitors. However, microorganisms including bacteria, fungi and also parasites have been proven to interact in a specific manner with components of the fibrinolytic pathways. Pathogenic bacteria are capable to subvert the function of proteases, activators or inhibitors for their own benefits including dissemination within the host and evasion of host inflammatory immune response. Here, we provide a state of the art overview of the divers strategies employed by bacteria to interact with components of the fibrinolytic system and to exploit the system for invasion. Moreover, the role of factors of the fibrinolytic cascade in inflammatory host response due to different bacterial infections will be presented.

beta-Glucuronidase activity in cerebrospinal fluid pleocytosis due to urinary tract infection.

AIM: This study examines the beta-glucuronidase activity in the cerebrospinal fluid (CSF) of patients with urinary tract infection (UTI) and sterile CSF pleocytosis and the feasibility of using these measurements for diagnostic purposes. METHODS: beta-Glucuronidase activity was measured in the CSF from 22 in each group neonates and infants with UTI and sterile CSF pleocytosis, bacterial meningitis, aseptic meningitis of apparently viral etiology and controls without CSF pleocytosis. RESULTS: The median (range) beta-glucuronidase activity in UTI with sterile CSF pleocytosis was 44.1 (33.2-57.1), whereas in the controls without CSF pleocytosis it was 19.1 (7.0-22.7), in aseptic meningitis of apparently viral etiology it was 26.5 (21.0-30.0) and in bacterial meningitis it was 168 (70.0-1152). The difference between the enzyme activity in the CSF of the patients with UTI and those in the other groups of neonates and infants is significant (p < 0.0001), with no overlapping between UTI and the other groups of children studied. Both the sensitivity and specificity of the activity was 100%. Conversely, there was a broad overlapping of the classic CSF laboratory parameters among the groups of subjects studied. CONCLUSION: beta-Glucuronidase activity in cell-free CSF discerns, with much greater accuracy than the classic CSF laboratory parameters, sterile CSF pleocytosis due to UTI from that of bacterial and viral meningitis and from control subjects without CSF pleocytosis.

[Evaluation of cerebrospinal fluid and plasma creatine kinase (CK) activity in patients with purulent, bacterial meningoencephalitis]. / Ocena aktywnósci kinazy kreatynowej (CK) w plynie mózgowo-rdzeniowym i w surowicy chorych z ropnymi, bakteryjnymi zapaleniami opon i mózgu.

The aim of the study was evaluation of usefulness of cerebrospinal fluid (CSF) creatine kinase (CK) activity assessment in diagnostics of purulent, bacterial meningoencephalitis in adults. The investigations were performed in 18 subjects. In all individuals CSF and plasma CK activity was estimated during the first 24 hours of hospitalization. Mean CSF CK activity in patients in very severe clinical state (group I) was 27,41 IU/L compared to 16,73 IU/L in subjects of group II with moderate and mild course of disease. The difference between mean CSF activities of this enzyme was statistically significant (p < 0,01). The obtained results indicate the usefulness of CSF CK activity assessment in estimation of severity of the patient's clinical state. The magnitude of this activity seems to be also helpful as prognostic marker in purulent, bacterial meningoencephalitis.

[Changes in erythrocyte superoxide dismutase and catalase activity in premature newborns with intraventricular haemorrhages].

Superoxide dismutase and catalase activities have been studied in peripherical blood erythrocytes of 49 premature newborns at 28-37 weeks gestation. Superoxide dismutase and catalase activities were lower in patients with bacterial infection of meninges. The standard therapy did not restore superoxide dismutase and catalase activities by the end of the neonatal period.