聚合酶链反应技术在无菌性脑膜炎和脑炎病原诊断中的应用探讨

目的　提高无菌性脑膜炎和脑炎病原早期诊断的敏感性和特异性。方法　分别设计针对肠道病毒(EV) 5’UTR、Ⅰ型和Ⅱ型单纯疱疹病毒 (HSVⅠ ,Ⅱ )糖蛋白D基因、人巨细胞病毒 (CMV)立即早期蛋白基因、EBV核抗原 1基因和肺炎支原体 (MP)ATPase操纵子基因的巢式PCR引物 ,应用巢式 聚合酶链反应 (nested PCR)技术检测无菌性脑膜炎和脑炎患儿的脑脊液标本中EV、Ⅰ型和Ⅱ型HSV、CMV、EBV及MP。结果　所用不同病原体的引物之间无交叉反应 ,特异性高。收集到的 15 8例脑脊液标本中有 10 2例 (6 4 6 % )检测到病原 ,包括 4 2例EV (42 / 15 8,2 6 6 % ) ,2 7例HSVⅡ (2 7/ 15 8,17 1% ) ,16例EBV (16 / 15 8,10 1% ) ,8例MP(8/ 15 8,5 1% ) ,6例CMV(6 / 15 8,3 8% ) ,3例HSVⅠ (3/ 15 8,1 9% )。这些阳性标本中有 10例检测到 2种或 3种病原。结论　PCR技术在无菌性脑膜炎和脑炎病原的早期诊断中有潜在的应用价值 ,可在中枢神经系统感染的病原鉴别中作为一种辅助性实验室诊断为临床诊治提供参考依据。     

Relationship of interleukin-8 and colony-stimulating factors to neutrophil migration in aseptic meningitis

Polymorphonuclear neutrophilic leucocyte (PMNL) migration into the subarachnoid space in aseptic meningitis of probable enteroviral aetiology was evaluated in relation to cerebrospinal fluid and serum levels of interleukin-8 (IL-8), macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). IL-8 levels reached a plateau within 12 h of onset, while M-CSF and G-CSF levels reached a peak between 12 and 24 h after onset, corresponding to the peak increase in PMNL count. G-CSF levels had the closest correlation with PMNL count. M-CSF levels were weakly correlated with PMNL count. IL-8 levels were not correlated with PMNL count except within 12 h of onset. IL-8 and G-CSF were detected predominantly in cerebrospinal fluid, while the M-CSF levels in the two compartments were not different except between 12 and 24 h after onset. It is considered that IL-8 triggers rapid and transient migration of PMNL, and that G-CSF then promotes gradual and consistent infiltration of PMNL.     

儿童重症化脓性脑膜炎CD3＋ CD8＋ T细胞与炎症指标、体液免疫的变化

目的：通过观察儿童重症化脓性脑膜炎早期血CD3＋CD8＋T细胞的变化,以及与炎症指标、体液免疫指标之间的关系,探讨其在儿童重症化脓性脑膜炎发生发展中的临床意义。方法回顾性分析中国医科大学附属盛京医院PICU 2014年8月1日至2015年12月31日收治的39例1个月~14岁的重症化脓性脑膜炎患儿,血CD3＋CD8＋T细胞计数正常或升高(≥190个/mm3)为A组( n＝22),降低(<190个/mm3)为B组(n＝17),分析患儿的一般资料、血液炎症指标、体液免疫、脑脊液改变在两组患儿中的分布和差异。结果17例(43.6％)患儿CD3＋CD8＋T细胞明显下降;所有4例死亡均为B组患儿;虽然没有统计学差异,但 B 组 Glasgow 昏迷评分<8分者比例(58.8％)高于 A 组(31.8％)。B组C-反应蛋白、降钙素原中位数(最小值-最大值)分别为251.0(26.2-417.0)mg/L、32.7(0.9-100.0)ng/L,远远高于A组的106.5(12.0-458.0)mg/L、4.5(0.1-200.0)ng/L,差异有统计学意义(P<0.05);B组中6例(35.3％)外周血WBC<4×109/L,而 A组为1例(4.6％),中性粒细胞>80％的比例A组为7例(31.8％),而B组为12例(70.6％),两组比较差异有统计学意义(P<0.05)。 B组14例(82.3％)患儿脑脊液中糖含量<2.0 mmol/L,高于A组[11例(50.0％)],两组比较差异有统计学意义(P<0.05)。结论儿童重症化脓性脑膜炎CD3＋CD8＋T细胞可能受到抑制,其与患儿脑功能损伤程度、炎症反应以及预后相关。可能对指导临床免疫制剂的应用有一定帮助。     

病原菌明确的细菌性脑膜炎146例临床及病原学分析

目的 分析细菌性脑膜炎患儿临床表现和病原特点,为临床诊治提供帮助。方法 收集复旦大学附属儿科医院2001年1月至2012年12月病原菌诊断明确的细菌性脑膜炎住院患儿,采集临床表现、辅助检查、并发症、病原菌种类和耐药情况等资料,分为0~28 d（新生儿组）,~1岁、~3岁和>3岁组进行描述分析。结果 146例细菌性脑膜炎患儿进入分析,新生儿组58例,~1岁组36例,~3岁组20例,>3岁组32例。①新生儿组发热、呕吐和脑膜刺激征的发生率较低,>3岁组伴有颅面、脊柱解剖畸形50%（16/32）。②革兰阳性菌占54.8%（80株）,以凝固酶阴性葡萄球菌（33株）、肠球菌（19株）和肺炎链球菌（12株）为主;革兰阴性菌占45.2%（66株）,以大肠埃希菌（30株）和鲍氏不动杆菌（10株）为主。③革兰阳性菌对万古霉素和利奈唑胺均敏感,革兰阴性菌除3/10株鲍氏不动杆菌和1/3株铜绿假单胞菌对美罗培南耐药外,余均敏感;大肠埃希菌、克雷伯菌和阴沟肠杆菌产超广谱β内酰胺酶阳性率分别达到66.7%、60.0%和100%。④63例（43.2%）出现急性期并发症,以硬膜下积液（26例）、脑积水（20例）和局部神经损害（13例）最常见;出现并发症主要病原菌为大肠埃希菌(15/30)、凝固酶阴性葡萄球菌（14/33）、肺炎链球菌（6/12）和鲍氏不动杆菌(6/10)。⑤出院结局：死亡或放弃治疗31例（21.2%）,其中肺炎链球菌性脑膜炎为5/12例（41.7%）。结论 细菌性脑膜炎新生儿临床表现多不典型,>3岁患儿应关注神经系统相关解剖结构缺陷的可能。可根据病原菌分布特点和药敏结果选用敏感抗生素。肺炎链球菌性脑膜炎患儿预后可能不良。     

Cerebrospinal fluid concentrations of alpha-melanocyte-stimulating hormone in bacterial and aseptic meningitis

-Melanocyte-stimulating hormone (-MSH) has potent anti-inflammatory effects in several experimental models of inflammation. It inhibits both the actions and production of proinflammatory cytokines and neutrophil migration. We investigated whether -MSH in cerebrospinal fluid (CSF) increases during the acute stage in patients with bacterial and aseptic meningitis by measuring -MSH in CSF via radioimmunoassay. The -MSH concentrations in CSF from the children with bacterial meningitis who survived (n = 8), those with aseptic meningitis (n = 16), and the control subjects (n = 23) were all below the detection limit. However, CSF -MSH was elevated in four of the five children with bacterial meningitis who had neurological sequelae. We speculate that elevated -MSH levels in CSF during acute bacterial meningitis reflect negative feedback in response to severe inflammation associated with neurological sequelae induced by proinflammatory cytokines. Conclusion: CSF -MSH is elevated in children with severe bacterial meningitis who had neurological sequelae.     

Childhood bacterial meningitis in Pondicherry, South India

Objective: To identify causative bacteria from cerebrospinal fluid (CSF) of children with miningits and analyse various clinical and laboratory parameters.Methods: Over a 20 month period, September 1994 to April 1996, one hundred episodes of acute bacterial meningitis in children aged 1 month–12 years were studied in a tertiary urban hospital in South India. Organisms were isolated from the cerebrospinal fluid (CSF) in 35% of cases. Among infants and children, the two major pathogens wereH. influenzae (17%) andS.pneumoniae (12%).Results: The illness at presentation was mild in 13% and severe in 36% of cases. The association of subdural effusion in children with Salmonella Gp B meningitis merits attention. The overall case fatality rate was 25%.S.pneumoniae had a higher case fatality rate than Salmonella Gp B andH.influenzae (50% vs 17% vs 12%). All the three infants below 3 months of age withS.pneumoniae meningitis died. On analysis of selected clinical and laboratory features by discriminant analysis, CSF culture was the significant (P=0.02) variable in relation to outcome. In pneumococcal meningitis, CSF WBC count was a highly significant variable in relation to outcome (Wilk’s Lambda 0.15, F=24.64, P=0.0002).Conclusion: Prevention of infections due toH.influenzae andS.pneumoniae should be given higher priority.     

Prolonged and secondary fever in childhood bacterial meningitis

In order to study the causes of prolonged and secondary fever in bacterial meningitis, a group of 102 infants and children with proven bacterial meningitis were studied. The causative agent wasHaemophilus influenzae in 58% of patients,Streptococcus pneumoniae in 25% andNeisseria meningitidis in 17%. Prolonged fever was observed in 12% of the patients. The established causes include, in order of frequency, subdural effusion, drug fever, otitis media, gastroenteritis and urinary tract infection. Secondary fever was noted in 18% of the patients. The causes, in order of frequency, were urinary tract infection, subdural effusion, otitis media, phlebitis, pneumonia and drug fever. Neither relapse of the meningitis nor inadequate response to antibiotic therapy was the cause for prolonged or secondary fever. Neurological sequalae were observed in 21 patients. There was no correlation between prolonged or secondary fever and neurological sequalae. We conclude that prolonged and secondary fever in patients with treated bacterial meningitis is rarely caused by the primary infection.     

Anti-interleukin-8 auto-antibodies in cerebrospinal fluid of children with purulent meningitis

BACKGROUND: Cerebrospinal fluid (CSF) of patients with purulent meningitis contains a high concentration of interleukin (IL)-8. Recently, the presence of anti-IL-8 auto-antibodies was noted in blood and alveolar fluid. Therefore, measurement of the concentration of anti-IL-8 auto-antibodies was attempted in CSF of children with and without meningitis. METHODS AND RESULTS: We measured the concentration of anti-IL-8 auto-antibodies in CSF of children with purulent or aseptic meningitis and those without meningitis. The CSF obtained on admission showed a significantly higher concentration of anti-IL-8 IgG and IgM auto-antibodies in children with purulent meningitis, compared with those with aseptic meningitis or without meningitis. Among the three groups of children, the concentration of IL-8 was also significantly higher in CSF of children with purulent meningitis. CONCLUSION: Because the anti-IL-8 IgG auto-antibody binds to IL-8 and inhibits IL-8 interaction with specific receptors on neutrophils, the presence of anti-IL-8 auto-antibodies seems to provide a mechanism that limits the bioavailability of free IL-8 in CSF.     

Aseptic meningitis: Diagnosis and management

The term aseptic meningitis encompasses all types of inflammations of the brain meninges other than that caused by pus producing organisms. It is usually a benign illness. Etiology of aseptic meningitis is very wide and includes many infections—both viral and non viral, drugs, malignancy and systemic illness. The most common cause is viral infection and enteroviruses—Coxsackie and ECHO viruses account for more than half of all cases. Clinical manifestations include headache, fever, malaise, photophobia and meningeal signs. Convulsions, neurological deficits and severe obtundation are rare except with certain non viral infectious meningitis. Diagnostic work up includes blood and cerebrospinal fluid (CSF) examination and serology for infectious meningitis. The polymerase chain reaction is a rapid and accurate method for detection of microbial DNA in CSF. Treatment is mainly supportive except for the nonviral infectious etiology.     

Serum cortisol levels in children with acute bacterial and aseptic meningitis.

OBJECTIVE: To study serum cortisol levels in acute childhood meningitis with respect to the severity of illness and the outcome. DESIGN: Prospective observational study. SETTING: Pediatric services of a tertiary care teaching and referral hospital. SUBJECTS: A total of 30 consecutive children, 2 months to 12 yrs of age, with suspected bacterial meningitis. METHODS: Serum cortisol levels (by enzyme-linked immunosorbent assay) obtained at admission were correlated with clinical characteristics (including Glasgow Coma Scale and Pediatric Risk of Mortality scores) recorded at admission and with neurologic and hearing status 2 months after discharge using SPSS 10.0. RESULTS: Mean +/- sd serum cortisol was 467 +/- 251 ng/dL in patients with bacterial (n = 16) and 319 +/- 159 ng/dL in aseptic meningitis (n = 14, p = .068). Glasgow coma scale score, systolic blood pressure, age, Pediatric Risk of Mortality, and cerebrospinal fluid protein were significant independent predictors of serum cortisol on stepwise multivariate regression analysis (each had an R change of >5%). Patients with neurologic or hearing sequelae had significantly higher median serum cortisol (450 ng/mL, n = 12) than those without sequelae (300 ng/mL, n = 17; p = .043 by Mann-Whitney U test). On multivariate logistic regression analysis, a serum cortisol of >/=420 ng/mL (odds ratio, 0.022; 95% confidence interval, 0.01-0.43) and systolic blood pressure (odds ratio, 1.35; 95% confidence interval, 1.04-1.74) were significant independent predictors of neurologic and hearing sequelae. CONCLUSION: Low serum cortisol is uncommon in acute bacterial meningitis of nonmeningococcal pathogenesis. Very high levels are likely to be associated with sequelae.     

Clinical data in children with meningococcal meningitis in a Spanish hospital

Neisseria meningitidis is the main cause of bacterial meningitis in Spain. Of the 213 children included in this study with meningococcal meningitis, 7 died. Mortality was linked to a shorter time from the first symptom to diagnosis (mean time for fatal cases was 9.5 h, mean time for survivors was 19h, p = 0.034), to deteriorated consciousness (DC) (mortality rate (MR) with DC = 6/87, MR without DC = 1/124, p = 0.02) and to shock (MR with shock = 5/7, MR without shock = 2/206, p < 0.0001). Previous treatment reduced the yield from blood culture (36/54 versus 45/137, p < 0.0001). Positivity in both Gram stain (GS) and cerebrospinal fluid (CSF) culture increased with longer duration of symptoms (mean GS+= 25h, GS^-= 16h, p = 0.004; CSF+= 20h, CSF^-= 12h, p = 0.001), and blood culture (BC) gave more positive results when carried out earlier (mean BC+= 14 h, BC = 24 h, p < 0.001). Reduced susceptibility to penicillin was seen in 34% of the strains, and rapidly evolving forms were responsible for most of the deaths; reduced susceptibility was more frequent among strains responsible for death or sequelae (9/15 = 60%) as compared with the more harmless strains (69/ 215 = 32%) ( p = 0.04). The progressive reduction of susceptibility to penicillin indicates that it should be replaced by a third-generation cephalosporine.     

Aseptic meningitis in children: Correlation between fever and interferon-gamma level

We studied the correlation between interleukin-1 beta (IL-1), tumour necrosis factor alpha (TNF-) and interferon gamma (IFN-) in cerebrospinal fluid (CSF) and clinical/laboratory findings in children with aseptic meningitis. In 19/27 patients (70%), the CSF IFN level was high at initial diagnosis, and reduced to a low or undetectable level during the convalescent phase (5–14 days later) of the disease. There were no differences in IL-1 and TNF- levels between the acute and convalescent phase of the patients. The serum IFN- levels in the patients, which were simultaneously examined were undetectable in the acute phase. When we compared the clinical/laboratory findings between the 29 patients with detectable CSF IFN- level and the 21 patients with an undetectable CSF IFN- level in the acute phase, the former demonstrated higher body temperature (P<0.01), and higher cell number and protein level in the CSF (P<0.05) than the latter. On the other hand, there were no significant differences in the duration of meningeal signs, the titre of C-reactive protein, and the peripheral leucocyte count between the two groups. By the Spearman's rank sum test, the CSF IFN- levels correlated more definitively with the severity of febrile episode (maximal body temperature, duration of fever and body temperature at the first lumbar tap), and the cell number and protein level in the CSF. These results suggest that IFN- produced in the inflamed intrathecal space may be associated with the pathogenesis of aseptic meningitis, especially the production of fever.     

Lack of effectiveness of dexamethasone in neonatal bacterial meningitis

A clinical trial was conducted to determine whether dexamethasone as adjunctive therapy alters the outcome of bacterial meningitis in neonates. Fifty-two full-term neonates with bacterial meningitis were enrolled in a prospective study. Infants were alternately assigned to receive either dexamethasone or not. Twenty-seven received dexamethasone in addition to standard antibiotic treatment and 25 received antibiotics alone. Dexamethasone therapy was started 10–15 min before the first dose of antibiotics in a dose of 0.15 mg/kg per 6 h for 4 days. Baseline characteristics, clinical and laboratory features in the two groups were virtually similar. Both groups showed a similar clinical response and similar frequency of mortality and sequelae. Six (22%) babies in the treatment group died compared to 7 (28%) in the control group (P = 0.87). At follow up examinations up to the age of 2 years, 6 (30%) of dexamethasone recipients and 7 (39%) of the control group had mild or moderate/severe neurological sequelae. Audiological sequelae were seen in two neonates in the dexamethasone group compared to one in the control group. Conclusion Adjunctive dexamethasone therapy does not improve the outcome of neonatal bacterial meningitis. Received: 22 December 1997 / Accepted: 14 March 1998     

Lipoprotein alterations in children with bacterial meningitis

Abnormalities in serum lipids, including hypertriglycerideinia, are common during infectious disorders. However, the lipoprotein pattern during infections, particularly in children, has been investigated to only a limited extent. We have monitored alterations in serum lipoproteins in eight children with a severe baclcrial infcction (meningitis) by a quantitating method measuring cholesterol and triglyccrides in each major class. The levels of triglyccrides in serum and in low-density lipoproteins were markedly elevated during the infection, whereas the amount of cholesterol in high-density lipoprotcins was decreased. The cholesterol to triglyceride ratio was decreased in low-, as well as in high-density lipoproteins. These lipoprotein abnormalities may, at least in part, be explained by a depressed lipolytic activity of lipoprotein lipasc, the key enzyme for removal of triglycerides in man. Serum triglycerides and the levels of cholesterol in high-density lipoproteins, as well as the ratio between these parameters, may be used as indicators of inflammatory activity.     

新生儿细菌性脑膜炎的诊治及观点更新

新生儿细菌性脑膜炎起病隐匿,临床症状不典型,病死率高,存活者可能会遗留神经系统并发症,因此及早诊断,规范治疗非常重要.2016年欧洲临床微生物与感染疾病协会(European Society for Clinical Microbiology and Infectious Diseases)发布了急性细菌性脑膜炎的诊治指南.本文结合该指南及最新文献,对新生儿细菌性脑膜炎的诊治进行了详述.     

Lipoprotein alterations in children with bacterial meningitis

Abnormalities in serum lipids, including hypertriglycerideinia, are common during infectious disorders. However, the lipoprotein pattern during infections, particularly in children, has been investigated to only a limited extent. We have monitored alterations in serum lipoproteins in eight children with a severe baclcrial infcction (meningitis) by a quantitating method measuring cholesterol and triglyccrides in each major class. The levels of triglyccrides in serum and in low-density lipoproteins were markedly elevated during the infection, whereas the amount of cholesterol in high-density lipoprotcins was decreased. The cholesterol to triglyceride ratio was decreased in low-, as well as in high-density lipoproteins. These lipoprotein abnormalities may, at least in part, be explained by a depressed lipolytic activity of lipoprotein lipasc, the key enzyme for removal of triglycerides in man. Serum triglycerides and the levels of cholesterol in high-density lipoproteins, as well as the ratio between these parameters, may be used as indicators of inflammatory activity.     

Controversies in the management of bacterial meningitis

Bacterial meningitis is an important infection of childhood with significant morbidity and mortality, and clinicians are faced with controversies over steroid use and fluid restriction in its initial management because the standard of practice is not clear. A 1999 survey of paediatric infectious diseases specialists demonstrated that only 56% of respondents recommended dexamethasone for Haemophilus influenzae type b meningitis and only 34% recommended dexamethasone for Streptococcus pneumoniae meningitis, despite recommendations for dexamethasone in the 1997 Red Book. The present article illustrates a typical case presentation of bacterial meningitis, and discusses dexamethasone use and fluid restriction. The use of intravenous fluid therapy is also reviewed, based on results from the single prospective randomized clinical trial in this area.     

Clinical decision rules to distinguish between bacterial and aseptic meningitis

Background

Clinical decision rules have been derived to distinguish between bacterial and aseptic meningitis in the emergency room to avoid unnecessary antibiotic treatments and hospitalisations.

Aims

To evaluate the reproducibility and to compare the diagnostic performance of five clinical decision rules.

Methods

All children hospitalised for bacterial meningitis between 1995 and 2004 or aseptic meningitis between 2000 and 2004 have been included in a retrospective cohort study. Sensitivity and specificity were calculated by applying each rule to the patients. The best rule was a priori defined as the one yielding 100% sensitivity for bacterial meningitis, the highest specificity, and the greatest simplicity for a bedside application.

Results

Among the 166 patients included, 20 had bacterial meningitis and 146 had aseptic meningitis. Although three rules achieved 100% sensitivity (95% CI 84–100), one had a significantly lower specificity (13%, 95% CI 8–19) than those of the other two rules (57%, 95% CI 48–65; and 66%, 95% CI 57–73), which were not statistically different. The ease of manual computation of the rule developed by Nigrovic et al (a simple list of five items: seizure, blood neutrophil count, cerebrospinal fluid (CSF) Gram stain, CSF protein, CSF neutrophil count) was higher than the one developed by Bonsu and Harper.

Conclusion

On our population, the rule derived by Nigrovic et al had the best balance between accuracy and simplicity of manual computation and could help to avoid two thirds of unnecessary antibiotic treatments and hospitalisations.