Comparative detection of enterovirus RNA in cerebrospinal fluid: GeneXpert system vs. real-time RT-PCR assay

Enteroviruses (EVs) constitute the most common cause of aseptic meningitis in both children and adults. Molecular techniques have now been recognized as the reference standard for the diagnosis of EV infections, and the rapidity of the molecular diagnosis of EV meningitis has been shown to be a determining factor in the management of patients. The rapid documentation of EV RNA in cerebrospinal fluid (CSF) is key to adapting patient management and the therapeutic regimen. To shorten the time needed for virological documentation, we implemented EV RNA detection in two point-of-care (POC) laboratories. Here, we present the results of the POC detection of EV RNA with the Xpert EV kit on the GeneXpert integrated system, and a comparison with the real-time RT-PCR (rtRT-PCR) assay routinely used in the core virology laboratory. From January to September 2009, a total of 310 CSF samples were tested. The rtRT-PCR gave 81 positive, 225 negative and four ‘indeterminate’ results. POC results were concordant in 81.6% (253/310). Most of the discrepancies consisted of ‘indeterminate’ results at the POC level (16%). Calculated performances (excluding the indeterminate results) of the Xpert EV kit on the GeneXpert system in POC settings were 100%, 98.9%, 97.6% and 100% for Sensibility, Specificity, positive predictive value and negative predictive value, respectively. Taken together, these results indicate that the implementation of POC detection of EV RNA can provide robust results in <4 h, and may have a significant impact on patient management, therapeutic attitude, and hospitalization costs.     

Rapid detection of enterovirus in cerebrospinal fluid by a fully-automated PCR assay is associated with improved management of aseptic meningitis in adult patients

BackgroundEnterovirus (EV) is the most frequent cause of aseptic meningitis (AM). Lack of microbiological documentation results in unnecessary antimicrobial therapy and hospitalization.ObjectivesTo assess the impact of rapid EV detection in cerebrospinal fluid (CSF) by a fully-automated PCR (GeneXpert EV assay, GXEA) on the management of AM.Study designObservational study in adult patients with AM. Three groups were analyzed according to EV documentation in CSF: group A = no PCR or negative PCR (n = 17), group B = positive real-time PCR (n = 20), and group C = positive GXEA (n = 22). Clinical, laboratory and health-care costs data were compared.ResultsClinical characteristics were similar in the 3 groups. Median turn-around time of EV PCR decreased from 60 h (IQR (interquartile range) 44–87) in group B to 5 h (IQR 4–11) in group C (p < 0.0001). Median duration of antibiotics was 1 (IQR 0–6), 1 (0–1.9), and 0.5 days (single dose) in groups A, B, and C, respectively (p < 0.001). Median length of hospitalization was 4 days (2.5–7.5), 2 (1–3.7), and 0.5 (0.3–0.7), respectively (p < 0.001). Median hospitalization costs were $5458 (2676–6274) in group A, $2796 (2062–5726) in group B, and $921 (765–1230) in group C (p < 0.0001).ConclusionsRapid EV detection in CSF by a fully-automated PCR improves management of AM by significantly reducing antibiotic use, hospitalization length and costs.     

Quantitation of C-reactive protein in cerebrospinal fluid and serum by zone immunoelectrophoresis assay (ZIA)

The zone immunoelectrophoresis assay (ZIA) for C-reactive protein (CRP) determinations is easy to perform and requires only small amount of antiserum, e.g., 25–100 and 0.5–1.0 μ1 anti-CRP antibody/20 serum and CSF samples, respectively. For quantitating CSF-CRP the immunoprecipitates formed were stained using lakaline phosphatase-conjugated secondary antibodies and the lowest standard concentration used was 30 μg/1. The immunoprecipitates formed when measuring CRP in serum were stained by Coomasie brilliant blue R250 with a detection limit of about 300 μg/l.

CRP was determined in cerebrospinal fluid in 27 patients with bacterial meningitis (range <0.03–0.23 mg/l) and in 25 patients with viral meningitis (range <0.03–0.23 mg/l).

CRP was quantitated in 52 sera by both the CRP ZIA method (y) and by electroimmunoassay (x). The correlation coefficient was r = 0.992 with the regression line y = 1.024 x + 0.855.     

β Trace-protein as marker for cerebrospinal fluid fistula

Summary The detection of trace-protein with the help of an easy-to-perform Ouchterlony test is strongly indicative of liquorrhea. The protein has been purified from pooled cerebrospinal fluid and an antiserum has been produced in rabbits. An open CSF fistula was diagnosed in 24 cases and there was no false positive result.Abbreviations CSF Cerebrospinal fluid - SDS Sodium dodecyl sulfate     

Tau in cerebrospinal fluid: a sensitive sandwich enzyme-linked immunosorbent assay using tyramide signal amplification

Tau in cerebrospinal fluid (CSF) has been proposed as a diagnostic marker for Alzheimer's disease (AD). This paper presents a new sensitive sandwich ELISA allowing quantitation of tau from 8 microl CSF/well. A human specific monoclonal tau antibody HT7 was used as a capture antibody and a mixture of polyclonal tau antibodies, 92e and R134d was used as reporter antibodies. Tyramide signal amplification (TSA) technology was used in the last step to increase the sensitivity. With this TSA-ELISA, the lowest detection limit for tau was 14.3 pg/ml. Tau levels in CSF were found to be increased in AD patients (807+/-304 pg/ml, p<0.001) compared with controls (252+/-94 pg/ml). Thirty-five of 38 AD cases (92% sensitivity) yielded signals greater than cutoff, while only 1 of 38 control cases (97% specificity) was greater. A highly significant correlation was found between this assay and a commonly used kit, INNOTEST hTAU Antigen.     

Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients

ObjectivesTuberculous meningitis (TBM) is difficult to diagnose. Digital PCR (dPCR) is a novel method which can quantify trace nucleic acids. This study sought to evaluate the diagnostic accuracy of dPCR analysis of cerebrospinal fluid (CSF) for TBM.MethodsWe collected CSF specimens from hospitalized TBM and non-TBM patients. Total CSF DNA was purified and the concentrations of Mycobacterium tuberculosis insert sequence 6110 (IS6110) and gyrase subunit B (gyrB) were quantified using droplet dPCR. The receiver operating characteristic curves of dPCR were established and the diagnostic performances were obtained. We also compared the sensitivity of dPCR with routine diagnostic tests.ResultsA total of 101 patients were recruited, 68 of whom suffered from TBM (26 definite, 34 probable and eight possible TBM) and 33 from non-TBM. The sensitivity of IS6110-dPCR assay for total TBM was higher than that of gyrB-dPCR assay (57.4% (44.8–69.3%) vs. 22.1% (12.9–33.8%)), and there was no significant difference for specificity between them (97.0% (84.2–99.9%) vs. 100% (89.4–100.0%)). The sensitivity of IS6110-dPCR in definite TBM was higher than that in probable and possible TBM (73.1% vs. 52.9% and 25.0%, respectively). IS6110-dPCR assay showed a higher sensitivity than smear microscopy (53.3% vs. 6.7%), mycobacterial culture (50.0% vs. 12.5%), IS6110-quantitative PCR (53.1% vs. 21.9%) and Xpert MTB/RIF (70.4% vs. 29.6%). Long anti-tuberculosis treatment time was found to be significantly associated with negative dPCR results.ConclusionCSF IS6110-dPCR assay is a rapid and sensitive molecular test, which has the potential to be used to enhance the diagnosis of TBM.     

Should we reconsider cefazolin for treating staphylococcal meningitis? A retrospective analysis of cefazolin and cloxacillin cerebrospinal fluid levels in patients treated for staphylococcal meningitis

ObjectivesTo assess the meningeal penetration of cefazolin and cloxacillin in individuals treated for methicillin-susceptible staphylococcal meningitis.MethodsWe retrospectively identified individuals treated for Staphylococcus meningitis with measurements of cefazolin or cloxacillin concentrations in cerebrospinal fluid (CSF) using a validated assay of liquid chromatography coupled with mass spectrometry at the Nantes University Hospital between January 2009 and October 2019. Staphylococcus meningitis was defined by a compatible clinical presentation and a microbiological confirmation (positive CSF culture or positive specific PCR). Medical charts were retrospectively reviewed to collect microbiological and clinical data, and to assess therapeutic success.ResultsAmong the 17 included individuals, eight (47%) were treated with cefazolin and nine (53%) with cloxacillin. Median daily dosages of cefazolin and cloxacillin were 8 g (range 6–12 g) and 12 g (range 10–13 g), respectively. Cefazolin and cloxacillin were mainly administered by continuous infusion. Eleven individuals (65%) were men, median (interquartile range (IQR)) age was 54 years (50; 70), 14 (82%) had postoperative meningitis and 3 (18%) had haematogenous meningitis. Median (IQR) antibiotic CSF concentrations were 2.8 mg/L (2.1; 5.2) and 0.66 mg/L (0.5; 0.9) for cefazolin and cloxacillin groups, respectively. Cloxacillin was discontinued in two individuals for therapeutic failure.ConclusionsPatients with staphylococcal meningitis treated with high-dose continuous intravenous infusion of cefazolin achieved therapeutic concentrations in CSF. Cefazolin appears to be a therapeutic candidate that should be properly evaluated in this indication.     

False-positive PCR detection of cyclovirus Malawi strain VS5700009 in human cerebrospinal fluid

BackgroundCyclovirus (CyCV) Malawi strain VS5700009 has recently been discovered and reported in clinical cerebrospinal fluid (CSF) samples. Further epidemiological and case-control studies are warranted. The availability of a highly sensitive and specific detection assay for this new virus is thus crucial.ObjectivesTo evaluate the performance of the first and the only available PCR assay for CyCV-VS5700009.Study designA total of 100 CSF samples collected during January–December 2010 were selected for PCR detection of CyCV-VS5700009. Positive PCR amplicons were subjected to sequencing confirmation and BLAST analysis.ResultsInitial PCR screening for CyCV-VS5700009 identified one sample, showing a PCR band of expected size (380 bp). Sequencing and BLAST analysis, however, indicated that the band was 364 bp in length and showed >99% nucleotide homology to a human gene known as nuclear receptor coactivator 6 (NCOA6). Pairwise sequence alignment confirmed that both the forward and reverse PCR primers used had significant homology (>70%) to NCOA6. None of the CSF samples tested were positive for CyCV-VS5700009.ConclusionsThe original PCR assay for CyCV-VS5700009 detection may have potential cross-reactivity with contaminating human genomic DNA. The assay may be of little diagnostic use on clinical specimens that are rich in host DNA such as biopsy tissues.     

Age-related changes in the cerebrospinal fluid outflow glycosaminoglycans

The glycosaminoglycan distribution patterns of the cerebrospinal fluid (CSF) outflow pathway, dura mater and cerebral cortex of young New Zealand red rabbits and 1-, 3- and 12-week-old C-57 mice were identified by analyses of the glycosaminoglycan moieties and by the use of zone electrophoresis. The glycosaminoglycans were identified by specific degradation procedures, i.e., hyaluronate lyase, chondroitin ABC lyase, endo-gb-D-galactosidase and nitrous acid treatment. The CSF outflow pathway and dura mater glycosaminoglycan components were primarily hyaluronic acid and chondroitin sulfatedermatan sulfate, whereas the cerebral cortex glycosaminoglycan components were hyaluronic acid, chondroitin sulfatedermatan sulfate, keratan sulfate and heparan sulfate. The glycosaminoglycan components of the dura mater and cerebral cortex decreased and those of the CSF outflow pathway increased as a function of age. These results demonstrate the feasibility of analyses of the CSF outflow pathway glycosaminoglycan components and suggest that topographical changes in the glycosaminoglycan distribution profiles may contribute to the pattern of cerebrospinal fluid outflow.     

Evaluation of enterovirus serological tests IgM-EIA and complement fixation in patients with meningitis, confirmed by detection of enteroviral RNA by RT-PCR in cerebrospinal fluid

An enzyme immunoassay (EIA) for detection of anti-enterovirus IgM antibodies was compared with complement fixation test in 43 patients with confirmed enterovirus meningitis by RT-PCR of cerebrospinal fluids (CSF). In 34% of patients with enterovirus meningitis, IgM antibodies could be found, whereas complement fixation tests were positive in only 20%. The specificity was determined with sera of 105 patients with non-enterovirus meningitis. Specificity of IgM EIA and of complement fixation was 94% and 85%, respectively. In four patients with meningitis but without enterovirus detection in CSF, RT-PCR and virus isolation from stools were positive. In three of these patients, IgM antibodies were detected, giving a strong indication of an enterovirus-associated disease. Because of the high specificity of IgM EIA, diagnosis of enterovirus-associated diseases can be carried out in a single serum sample, whereas by complement fixation tests, only fourfold increases in antibody titres in paired sera indicate an acute infection. The application of IgM EIA is especially important in cases of meningitis when CSF samples are not available and for diagnosis of enterovirus diseases with other clinical symptoms such as fever, enteritis, and hand-foot-and-mouth disease.     

术前腰大池外引流对经鼻蝶垂体腺瘤切除术中脑脊液漏的防治作用

目的：探讨术前腰大池外引流对经鼻蝶垂体腺瘤患者术中脑脊液漏的防治作用。方法回顾性分析2012年1月—2013年11月郑州大学第一附属医院神经外科行经鼻蝶垂体腺瘤切除术的194例患者的临床资料,其中54例患者术前行腰大池外引流（引流组),140例患者术前未行腰大池外引流（对照组)。两组患者性别、年龄、肿瘤类型、Wilson分级等一般资料差异均无统计学意义（P值均>0．05),具有可比性。对比2组术中脑脊液漏的发生情况。结果194例患者均顺利完成手术。引流组54例,除1例无脑脊液流出者,其余患者术中引流脑脊液40~80 mL;术中发生脑脊液漏3例（5．6%),常规给予修补鞍底+腰大池外引流,术后均未再发生脑脊液漏;术后发生脑脊液漏2例（3．7%),1例行腰大池外引流、1例予修补鞍底+腰大池外引流,均治愈。对照组140例,术中发生脑脊液漏54例（38．6%),常规给予修补鞍底+术后腰大池外引流治疗,49例痊愈,5例术后再次发生脑脊液漏;术后共发生脑脊液漏7例（5．0%),1例行腰大池持续外引流、6例再次手术行脑脊液漏修补+腰大池外引流,均痊愈。两组患者术中脑脊液漏发生率比较,引流组术中脑脊液漏发生率明显低于对照组,差异有统计学意义（χ2=20．473,P<0．01)。结论术中鞍膈及蛛网膜的直接或间接损伤可能是经鼻蝶垂体腺瘤患者术中发生脑脊液漏的原因。经鼻蝶垂体腺瘤切除术术前行腰大池外引流,术中腰大池外引流释放脑脊液可降低蛛网膜张力,减少鞍膈和蛛网膜的直接或间接损伤,进而降低术中脑脊液漏的发生率。     

The influence of cerebrospinal fluid turnover on age-related changes in cerebrospinal fluid protein concentrations

Studies have shown that ageing and several neurological diseases of the central nervous system are often accompanied with increase in concentrations of many cerebrospinal fluid (CSF) proteins. However, few studies have actually looked into the mechanisms behind the increase in CSF protein concentrations. In this study, CSF secretion rates and turnovers were measured using the in situ perfused choroid plexus (CP) technique in a group of sheep between 1 and 10 years of age. CSF protein concentrations were determined using quantitative proteomic techniques. CSF turnover in hours correlated significantly with age, changing from 10.5 ± 2.7 h in the young to 17.1 ± 2.4 h in the old. The amount of CSF replaced per hour decreased from 2.46 ± 0.42 mL in the young to 1.17 ± 0.16 mL in the old. The age-related reduction in CSF turnover was calculated to have a concentrating effect of approximately 1.32 times in middle-aged and 2.10 times in old CSF proteins. After CSF turnover normalization, CSF albumin (a plasma-derived protein) concentration still increased significantly with age; however, both brain-derived and partially brain-derived protein concentrations in the CSF decreased with age after normalization. Regression analysis between turnovers and albumin concentrations has shown that reduced CSF turnover was the cause of increased CSF albumin concentrations with age. Therefore, CSF protein concentrations should be normalized according to their age-specific turnovers first before their concentrations can be compared logically between different ages.     

Differentiation of lymphocytes in cerebrospinal fluid in disorders of the central nervous system

Summary In 73 patients with variable CNS-disorders Slg-lymphocytes and RF-lymphocytes originating from CF were characterized by demonstration of surface markers. In the CF the Slg-lymphocytes were increased in comparison to the blood whereas the RF-lymphocytes generelly were decreased. Patients suffering from acute or chronic inflammatory diseases had significantly more Slg-lymphocytes while RF-lymphocytes were significantly decreased in those patients as well as in patients with malignancies. A correlation between the number of Slg-lymphocytes and immunoglobulin-levels was not seen. The respective data in blood did not follow the changes in CF. The results suggest a prevailing stimulation of the humoral immune system of the CNS during inflammatory diseases.     

Detection of enterovirus RNA in cerebrospinal fluid (CSF) using NucliSens EasyQ Enterovirus assay.

Rapid detection of enterovirus (EV) infections is essential in the management of aseptic meningitis. Molecular approaches have opened the way to such rapid, but also specific and sensitive, diagnostic tests. The aim of this study was to compare the performance of the CE marked NucliSens EasyQ Enterovirus assay with an in-house two-step RT-PCR assay using cerebrospinal fluid (CSF) and throat swab samples. In addition, specificity was tested with clinical isolates positive for viruses with clinical importance in CSF samples. For nucleic acid extraction, the NucliSens miniMAG and NucliSens magnetic extraction reagents were used. Subsequently real-time nucleic acid sequence-based amplification (NASBA) RNA amplification was performed using NucliSens EasyQ basic kit reagents and NucliSens EasyQ Enterovirus reagents. An EV-specific internal homologous control (IC) RNA was used to monitor the entire NucliSens EasyQ procedure at the individual sample level. No IC but an external inhibition control was available for the RT-PCR method. For the NucliSens EasyQ procedure, amplification and real-time detection reactions were carried out in the NucliSens EasyQ analyzer. The real-time NASBA enterovirus detection was based on NASBA amplification and real-time molecular beacon technology. Data were analyzed using the manufacturer's software on the NucliSens EasyQ analyzer. For the in-house assay, RT-PCR amplicons were detected using agarose gel analysis. The analysis of clinical samples positive for HSV-1, HSV-2, adenovirus, CMV, VZV, mumps and rhinovirus were all negative by NucliSens EasyQ Enterovirus assay. Three rhinovirus samples were, however, strongly positive in RT-PCR. A total of 141 clinical samples were retrospectively tested, including 126 cerebrospinal fluid (CSF) samples and 15 throat swabs. The 91 CSF samples were negative by both methods, 31 CSF samples and 14 throat swab samples were positive by both methods. The four CSF samples were positive by RT-PCR only. One throat swab sample was negative in NucliSens EasyQ but positive in RT-PCR. The sensitivity and specificity of both methods seem to be more or less comparable. However, the in-house RT-PCR assay appears to amplify some rhinovirus strains and should therefore not be used for throat swab samples. NucliSens EasyQ Enterovirus assay gave more invalid results than the in-house RT-PCR, which is obvious taken into account the difference in quality control between the CE marked NucliSens EasyQ Enterovirus assay and the in-house enterovirus assay. The NucliSens EasyQ procedure can be completed within 5h versus 9.5h for the RT-PCR. NucliSens EasyQ Enterovirus assay showed to be a standardized, rapid, specific, sensitive and reliable procedure for the detection of enterovirus RNA.     

Evaluation of real-time PCR versus PCR with liquid-phase hybridization for detection of enterovirus RNA in cerebrospinal fluid

A LightCycler and two TaqMan real-time PCR assays were evaluated against an older PCR with liquid-phase hybridization method for the detection of enterovirus RNA in 74 patient samples. The two-step LightCycler and the two-step TaqMan formats correlated well with each other (r(2) = 0.90) and were equally sensitive compared to the liquid-phase hybridization method, whereas the one-step recombinant Tth DNA polymerase format was rather insensitive, detecting enterovirus RNA in only about one-half of those patient samples previously positive by liquid-phase hybridization. The two-step TaqMan method was optimized utilizing 10 micro l of cDNA and demonstrated the highest degree of analytical sensitivity among the methods evaluated in our study, being able to reproducibly quantify down to 510 copies of enteroviral RNA/ml of cerebrospinal fluid. This new assay can be performed in 4 h, is much less labor intensive, and showed less cross-reactivity with rhinovirus than the liquid-phase hybridization assay. Thus, the two-step TaqMan assay should prove useful in the diagnosis of enteroviral meningitis versus bacterial meningitis, thereby resulting in timely and appropriate clinical management that can amount to significant cost savings to the patient and health care system.     

癫痫患者血液和脑脊液免疫异常在其发病机制中的作用

对７２例癫痫患者血液中的ＩｇＧ，ＩｇＡ，ＩｇＭ，Ｃ３，ＣＩＣ，白蛋白，ＣＤ４＾＋细胞，ＣＤ８＾＋细胞和脑脊液中的ＩｇＧ进行检测，并计算ＣＤ４＾＋／ＣＤ８＾＋比值，ＣＳＦ－ＩｇＧ／Ｓ－ＩｇＧ比率，ＣＳＦ－ＡＬＢ／Ｓ－ＡＬＢ比率和ＩｇＧ指数。结果表明癫痫组血液中的５项参数对比照组低，尤其是原发性和末治疗亚组。而ＣＳＦ呈现病理性的免疫增强反应，表现为原发性亚组ＩｇＧ合成，而继发性亚组患者存在血脑屏障功能