Naive and Memory CD4+ T-cells in the Cerebrospinal Fluid of Children with Aseptic Meningitis Following Measles-Mumps-Rubella Vaccination and Enteroviral Meningitis

We investigated the distribution of memory (CD45RO⁺) and naive (CD45RA⁺CD62L⁺) CD4⁺ T-cells as well as CD8⁺ T-cells and total T-cells in the CSF of children with aseptic meningitis following measles-mumps-rubella (MMW) vaccination and those with enteroviral meningitis. Flow cytometric analysis of CSF cells was performed in 12 children with MMR vaccine-associated meningitis and 11 children with enteroviral meningitis. Percentages of total T-cells, CD4⁺ and CD8⁺ T-cells and monocytes in CSF of patients from the two groups were not significantly different. The majority of CD4⁺ T-cells in the CSF of both patient groups were of memory phenotype. Percentages of CSF naive CD4⁺ T-cells were increased in children with aseptic meningitis following MMR vaccination. Further studies focused on the more detailed immunophenotyping of CSF cells are needed to fully establish the usefulness of flow cytometry in the diagnostic workup of inflammatory CNS diseases in children.     

Production of Specific Antibodies by Cerebrospinal Fluid Lymphocytes in Patients with Herpes Zoster, Mumps Meningitis and Herpes Simplex Virus Encephalitis

We applied a new method consisting of short-term culture (18 h) of lymphocytes from cerebrospinal fluid (CSF-L) and peripheral blood (PBL) in viral antigen-coated ELISA plates and subsequent measurement of IgG and IgM antibodies bound to antigen. Utilizing mumps virus, herpes simplex virus (HSV), varicella zoster virus (VZV), and measles virus as antigens, we demonstrated production by CSF-L of antibodies against the aetiological agent only in all patients with mumps meningitis and HSV encephalitis and also in all patients with herpes zoster without central nervous system (CNS) symptoms. This might be considered as direct evidence that specific antibodies are produced within the CNS in inflammatory nervous system diseases. CSF-L usually produced higher amounts of antibodies than the corresponding number of PBL. In comparison with concentrations of free antibodies determined in parallel, our method had higher specificity and sensitivity and gave more precise information about the antibody response in infections of the nervous system.     

B and T Lymphocytes in Blood and Cerebrospinal Fluid in Acute Aseptic Meningitis

B and T cells in blood and cerebrospinal fluid (CSF) were calculated by using the surface membrane immunoglobulin marker for B cells and the capacity to bind sheep erythrocytes to form 'rosettes' as a marker to T cells. Patients with acute aseptic meningitis due to mumps virus or other etiologic agents and with various chronic neurological diseases were investigated. Significantly higher T-cell and lower B-cell values were observed in CSF than in blood in all three groups. No significant differences were found between the three patient groups. An elevation of B cells and a depression of T cells were observed in blood in patients with aseptic meningitis during the course of the disease.     

Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the “gold standard” were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.     

Broad-Range 16S rRNA PCR with Cerebrospinal Fluid May Be Unreliable for Management of Postoperative Aseptic Meningitis

We previously demonstrated that discontinuing presumptive antibiotic treatment in cases of negative conventional cultures is safe and effective for patients with postoperative aseptic meningitis (PAM). Here, we prospectively investigated 32 patients with postoperative meningitis. All 26 patients with PAM diagnosed on the basis of conventional cultures demonstrated negative 16S rRNA PCR results. Our results suggest that the PCR technique does not change PAM management.Postoperative meningitis is a rare but life-threatening infection (6, 9, 13). Aseptic postoperative meningitis may account for up to 70% of all cases of postoperative meningitis (13, 15). Aseptic meningitis may be due to a local inflammatory reaction to blood breakdown products, sutures, tissue breakdown products, chemicals, etc. (7, 8), or to a small bacterial inoculum acquired perioperatively (5). Distinction between the two entities is clinically very difficult, and the results for direct bacteriological examination are often negative (1). Rapid diagnosis of postoperative bacterial meningitis is crucial, as the mortality rate can exceed 20%. The clinical outcome of aseptic postoperative meningitis is favorable, although recovery may be long. Whatever the mechanism of postoperative meningitis, the British Society for Antimicrobial Chemotherapy (2) recommends presumptive antibiotic therapy for all patients with signs of postoperative meningitis, based on local microbial ecology. Therapy is discontinued after a 48- or 72-h regimen in the case of negative cerebrospinal fluid (CSF) culture. We previously validated the safety and effectiveness of this approach (15).Several studies have examined the use of broad-range 16S rRNA PCR analysis of CSF in patients suffering from meningitis in different settings. This analysis has been found to be of little accuracy in the case of small-bacterial-inoculum meningitis (3, 12, 14). A single study specifically assessed the accuracy of this analysis for patients with aseptic postoperative meningitis. Surprisingly, the CSF PCR results were positive in all cases (5). The aim of the present study was therefore to prospectively investigate the accuracy of broad-range 16S rRNA PCR analysis for CSF samples drawn from patients with aseptic postoperative meningitis and to assess the help provided to clinicians in discriminating etiology and improving management.We studied all patients demonstrating postoperative meningitis diagnosed between October 2004 and October 2008 in our teaching hospital. Patients with external or internal CSF shunts were excluded. All consecutive patients who had undergone neurosurgery or ear, nose, and throat (ENT) surgery in the previous 3 months, who had a clinical indication for lumbar puncture, or who met the criteria for meningitis on the basis of the CSF analytical results (see below) were prospectively enrolled. Meningitis was considered to be of bacterial origin if (i) the results obtained from direct examination or culture of CSF were positive or (ii) a microbiological sample (blood, CSF leakage fluid, or surgical wound infection) obtained during the same episode was positive and the CSF contained more than 100 leukocytes/mm³ (8, 15). Meningitis was considered aseptic if the CSF contained more than 100 leukocytes/mm³ and showed negative results for direct examination and culture after 72 h (2, 11, 15). All patients diagnosed with postoperative meningitis received empirical antibiotic therapy combining vancomycin, ceftazidime, and ciprofloxacin on the basis of bacterial species and the resistance pattern encountered in our institution (15). During the first 3 days of treatment, the patient''s condition was reassessed. If the meningitis was proved to be of bacterial origin, the antimicrobial therapy was adapted to the bacterial isolate and was continued for 2 weeks (15). If the meningitis was found to be aseptic, the antimicrobial therapy was discontinued on the third day. PCR results were not available at the time the decision was made.CSF samples from all the patients with postoperative meningitis were sent for microscopic examination and bacterial culture. On arrival at the laboratory, 400 μl of each CSF sample was taken under sterile conditions and stored at −80°C until further processing. DNA extraction and PCR were carried out in separate areas. PCR was performed in a blinded manner, and cultures and PCR sequencing results were compared in retrospect. For each CSF sample, DNA of two aliquots (200 μl each) was extracted with a QIAamp DNA minikit in accordance with the manufacturer''s instructions (Qiagen, Courtaboeuf, France). For each batch of extraction, a negative control containing all reagents minus CSF was processed.PCR was done using universal primers (91E [5′-TCAAAKGAATTGACGGGGGC-3′] and 13BS [5′-GCCCGGGAACGTATTCAC-3′]) designed on the conserved sequences of the rrn gene coding for 16S rRNA and generating a 479-bp fragment as previously described (10). Concurrently, a 268-bp fragment of the human β-globin gene was amplified to ensure DNA extraction efficiency and the absence of inhibitors. Sequence analysis of the positive 16S rRNA amplicons was carried out with a 3130 XL genetic analyzer (Applied Biosystems, Courtaboeuf, France). The 16S rRNA sequences were compared with those available in the BIBI database and in the GenBank database with the BLASTN program (http://umr5558-sud-str1.univ-lyon1.fr/lebibi/lebibi.cgi and http://blast.ncbi.nlm.nih.gov/Blast.cgi). Identification at the genus or species level was defined according the recommendations of Drancourt et al. (4). The detection levels for the extraction method combined with 16S rRNA PCR for Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Haemophilus influenzae) bacteria in CSF were determined as follows. Strains were cultured and diluted 10-fold in 0.15 M NaCl and the viable counts determined. Bacterial suspensions were mixed with culture- and PCR-negative CSF samples in order to obtain inocula of 10⁶ to 10¹ CFU/ml. The detection levels were 10 to 10² CFU/ml for E. coli, P. aeruginosa, H. influenzae, and E. faecalis and 5 × 10³ CFU/ml for S. aureus and S. pneumoniae.For every patient, demographic and clinical data (indication for surgery, surgical approach, duration of surgery, antibiotic prophylaxis, clinical manifestations on the day of diagnosis, CSF leakage, delay between surgery and onset of meningitis, and surgical wound aspect) and biological data (CSF cell count and differential; protein and glucose levels; and results for bacteriological examination, blood culture, and CSF leakage fluid culture) involving the antimicrobial treatment and outcome were collected prospectively. PCR results were available after the clinical episode of meningitis and were compared with clinical and bacterial data. We used Student''s t test when comparing means and the chi-square test when comparing proportions. The sensitivity, specificity, positive predictive value, and negative predictive value of 16S rRNA PCR were determined with exact confidence intervals (CIs).Thirty-two patients were included. Twenty-six patients fulfilled the definition of aseptic postoperative meningitis and six of bacterial meningitis (Table ​(Table1).1). No patient was pretreated with antibiotics except for surgical prophylaxis, and none received selective decontamination of the digestive tract. As expected, no clinical or biological feature in CSF was significantly different between the two groups (Table ​(Table1).1). No patient had a positive blood culture or other focus of infection. The outcome was favorable in all cases, without any neurological sequelae. Among the 26 patients with aseptic meningitis, the results for all 16S rRNA PCRs were negative (Table ​(Table2).2). Among the six patients with bacterial meningitis, CSF samples had been drawn by lumbar puncture in 3 cases and from wound or CSF leakage in 3 other cases. In the first three cases (involving lumbar puncture), the CSF samples grew Streptococcus pneumoniae, Bacteroides thetaiotaomicron, and Staphylococcus capitis. The 16S rRNA PCR results were positive in the first two cases and negative in the case involving CSF growing S. capitis. In the 3 other cases (involving wound or leakage samples), the CSF samples grew a Corynebacterium sp., Propionibacterium avidum, and Enterobacter cloacae. The PCR results were negative for all three patients.

TABLE 1.

Clinical and biological features of patients with postoperative meningitis according to their etiology

Diagnosis of Meningococcal Meningitis by Broad-Range Bacterial PCR with Cerebrospinal Fluid

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.     

Immunodetection of a Hepatitis C Virus (HCV) Antigen and Th1/Th2 Cytokines in Cerebrospinal Fluid of Meningitis Patients

Abstract

Infection with hepatitis C virus (HCV) has become the most important public health problem in Egypt. HCV infection has been implicated in diseases of the central nervous system. Cerebrospinal fluid (CSF) and serum samples from 91 patients with meningitis (62 males and 29 females, mean age of 37 years) were investigated. Anti‐HCV antibodies and HCV antigen were evaluated in patients CSF and serum using enzyme linked immunosorbent assay. The levels (mean?±?SD?pg/ml) of Th1 cytokines (IFN‐γ and TNF‐α) and Th2 interleukines (IL‐10 and IL‐4) were also determined. The anti‐HCV antibodies were detected in high percentages both in CSF samples (71%) and in sera (90%). Also, the HCV antigen was detected in about 60% of tested CSF and serum samples. The levels of IFN‐γ and IL‐10 cytokines were significantly higher (P?<?0.05) in both serum and CSF of patients positive for HCV antigen than those negative. HCV antigen was detected in the CSF of meningitis patients with a significant upregulation of Th1 and Th2 responses. The high incidence of HCV infection may draw light on the etiological role of HCV in the pathogensis of meningitis diseases in our study group.     

Relationship between Intracranial Granulomas and Cerebrospinal Fluid Levels of Gamma Interferon and Interleukin-10 in Patients with Tuberculous Meningitis

Cerebrospinal fluid gamma interferon (IFN-γ) and interleukin-10 levels in 39 patients with tuberculous meningitis were serially measured. Cytokine levels did not predict intracranial granuloma (IG) development, but IFN-γ levels in the top quartile after 1 month of therapy were highly associated (odds ratio = 18) with detection of an IG by computed tomography scanning.     

YKL-40 Is Elevated in Cerebrospinal Fluid from Patients with Purulent Meningitis

YKL-40, a member of the family 18 glycosyl hydrolases, is secreted by activated neutrophils and macrophages. It is a growth factor for connective tissue cells and a potent migration factor for endothelial cells and may function in inflammation and tissue remodeling. YKL-40 was determined in 134 cerebrospinal fluid (CSF) samples taken on admission from patients suspected of having meningitis (48 with purulent meningitis, 49 with lymphocytic meningitis, 5 with encephalitis, and 32 without evidence of meningitis). YKL-40 levels in CSF were significantly higher in patients with purulent meningitis (median, 663 μg/liter [range, 20 to 8,960]) and encephalitis (5,430 μg/liter [620 to 11,600]) than in patients with lymphocytic meningitis (137 μg/liter [41 to 1,865]) or patients without meningitis (167 μg/liter [24 to 630]) (Kruskal-Wallis and Dunn multiple comparison tests, P < 0.001). CSF YKL-40 levels were also determined for 26 patients with purulent meningitis having a repuncture, and patients who died (n = 5) had significantly higher YKL-40 levels than patients who survived (n = 21) (2,100 μg/liter [1,160 to 7,050] versus 885 μg/liter [192 to 15,400], respectively; Mann-Whitney test, P = 0.018). YKL-40 was most likely locally produced, since patients with infections of the central nervous system had CSF YKL-40 levels that were at least 10-fold higher than the corresponding levels in serum (2,033 μg/liter [470 to 11,600] versus 80 μg/liter [19 to 195]). The CSF neopterin level was the biochemical parameter in CSF and blood that correlated best with CSF YKL-40 levels, indicating that YKL-40 may be produced by activated macrophages within the central nervous system. In conclusion, high levels of YKL-40 in CSF are found in patients with purulent meningitis.     

Microscopic Examination and Broth Culture of Cerebrospinal Fluid in Diagnosis of Meningitis

We reviewed the results of microscopic Gram stain examination and routine culture for 2,635 cerebrospinal fluid (CSF) samples processed in an adult hospital microbiology laboratory during 55 months. There were 56 instances of bacterial or fungal meningitis (16 associated with central nervous system [CNS] shunt infection), four infections adjacent to the subarachnoid space, four cases of sepsis without meningitis, and an additional 220 CSF specimens with positive cultures in which the organism isolated was judged to be a contaminant. Because 121 of these contaminants were isolated in broth only, elimination of the broth culture would decrease unnecessary work. However, 25% of the meningitis associated with CNS shunts would have been missed by this practice. The most common cause of meningitis was Cryptococcus neoformans, followed by Streptococcus pneumoniae and Neisseria meningitidis. In 48 of 56 (88%) of cases, examination of the Gram-stained specimen revealed the causative organism. If patients who had received effective antimicrobial therapy prior to lumbar puncture are excluded, the CSF Gram stain is 92% sensitive. Microscopic examination incorrectly suggested the presence of organisms in only 3 of 2,635 (0.1%) CSF examinations. Thus, microscopic examination of Gram-stained, concentrated CSF is highly sensitive and specific in early diagnosis of bacterial or fungal meningitis.     

Serum and Cerebrospinal Fluid Neuron-Specific Enolase for Diagnosis of Tuberculous Meningitis

Purpose

Late diagnosis and treatment lead to high mortality and poor prognosis in tuberculous meningitis (TbM). A rapid and accurate diagnosis is necessary for a good prognosis. Neuron-specific enolase (NSE) has been investigated as a biochemical marker of nervous tissue damage. In the present study, the usefulness of NSE was evaluated, and a cut-off value for the differential diagnosis of TbM was proposed.

Materials and Methods

Patient charts were reviewed for levels of serum and cerebrospinal fluid (CSF) NSE, obtained from a diagnostic CSF study of samples in age- and gender-matched TbM (n=15), aseptic meningitis (n=28) and control (n=37) patients.

Results

CSF/serum NSE ratio was higher in the TbM group than those of the control and aseptic groups (p=0.001). In binary logistic regression, CSF white blood cell count and CSF/serum NSE ratio were significant factors for diagnosis of TbM. When the cut-off value of the CSF/serum NSE ratio was 1.21, the sensitivity was 86.7% and the specificity was 75.4%.

Conclusion

The CSF/serum NSE ratio could be a useful parameter for the early diagnosis of TbM. In addition, the authors of the present study suggest a cut-off value of 1.21 for CSF/serum NSE ratio.     

Appearance of Cytotoxic T Cells in Cerebrospinal Fluid of Mice with Ectromelia Virus-Induced Meningitis

In mice with meningitis induced by ectromelia virus, the influx of inflammatory cells into cerebrospinal fluid (CSF) was shown to be triggered by an immunologically specific, T cell-dependent mechanism. These cells were counted, and their cytotoxicity assayed in vitro. Cell numbers reached a peak 6 days after infection and showed specific cytotoxicity for ectromelia-infected target cells, an effect that reached a peak at day 6 and was retained for at least a further 4 days. Treatment with anti-thymocyte serum 2 to 3 days before harvest of CSF cells depressed the numbers found and their cytotoxicity, suggesting that they reached the exudate from the blood.     

结核性脑膜炎患者脑脊液基质金属蛋白酶-9的检测意义

在脑膜炎的发展过程中,基质金属蛋白酶-9(MMP-9)对降解基底膜和损伤血脑屏障发挥重要作用,结核分枝杆菌能够刺激单核-巨噬细胞过度表达MMP-9.为检测结核性脑膜炎患者脑脊液MMP-9水平,评价其对结核性脑膜炎诊断及治疗的意义,应用ELISA方法检测12例结核性脑膜炎和9例病毒性脑膜炎患者脑脊液MMP-9水平,并选取5例非肿瘤非感染性头痛患者脑脊液作对照组.结果显示,结核性脑膜炎脑脊液MMP-9的水平显著增高,中枢神经系统并发症发生率与MMP-9的水平相关.结论为脑脊液MMP-9水平对结核性脑膜炎的诊断和预后有参考价值.     

Detection of Leptospira DNA in Patients with Aseptic Meningitis by PCR

Samples of cerebrospinal fluid from 103 patients with aseptic meningitis were tested by PCR for detection of leptospires, and the results were compared with those of the microscopic agglutination test (MAT) and an enzyme-linked immunosorbent assay for detection of immunoglobulin M (ELISA-IgM). Of these samples, 39.80% were positive by PCR and 8.74 and 3.88% were positive by MAT and ELISA-IgM, respectively.     

牙周炎患者龈沟液中细胞因子水平的变化

研究牙周炎患者龈沟液 (GCF)中细胞因子水平的变化及其临床意义。用RIA检测了成人牙周炎组 (AP组 ) 16例 2 6个牙 ,快速进展型牙周炎组 (RPP组 ) 19例 2 9个牙和牙周健康者对照组 (H组 ) 2 4名 31个牙的GCF中白介素 1β(IL - 1β)、肿瘤坏死因子α(TNFα)、白介素 6 (IL - 6 )和转化生长因子α(TGFα)含量的变化并记录受检牙的牙周破坏指数PD、AL。结果表明 :AP组、RPP组患者GCF中IL - 1β、TNFα、IL - 6水平明显高于对照组(P <0 .0 5和P <0 .0 1)且与PD、AL呈显著正相关 (P <0 .0 1) ;TGFα 水平明显低于对照组 (P <0 .0 1) ,与PD、AL呈显著负相关 (P <0 .0 1)。提示IL - 1β、TNFα、IL - 6、TGFα 是参与牙周炎牙槽骨吸收破坏的最重要的细胞因子