无菌性脑膜炎和脑炎病人脑脊液肠道病毒RNA的检测及其临床意义

目的 检测肠道病毒（EV）在中枢神经系统感染中的致病情况,探讨检测EV感染的方法。方法 就用逆转录－聚合酶链反应（RT－PCR0和病毒培技术检测46例无菌性脑膜炎及脑炎病人脑脊液（CSF）标本。结果 RT－PCR方法敏感特异;46例无菌性脑膜炎和脑炎急性期CSF标本中,31例EV阳性（67．4％）,14例病毒培养阳性（26．1％）。统计结果显示,RT－PCR敏感性明显高于病毒培养。结论 EV是引起无菌性脑膜炎和脑炎的重要病原;RT－PCR快速敏感特异,简单易行,易于推广,是诊断EV感染的有效方法。     

2001年徐州地区暴发性病毒性脑膜炎病原的研究

目的 确定引起2001年江苏省徐州地区无菌性脑膜炎流行的病原体。方法 组织培养法从患者脑脊液分离病毒，标准血清中和试验鉴定分离毒株；中和试验检测双份血清中和抗体效价；逆转录聚合酶链反应（RT-PCR）检测肠道病毒特异性基因片段。结果 22份脑脊液中分离出4株柯萨奇B5型、2株柯萨奇B3型、1株艾可7型肠道病毒，分离阳性率31．8％。RT-PCR检测脑脊液21份，肠道病毒阳性11份。阳性率52．4％。19例双份血清中11例中和效价呈4倍以上增长或转阳，阳性率57．9％。结论 此次江苏省徐州地区无菌性脑膜炎流行的病原体是以柯萨奇B5为主要血清型的肠道病毒。     

Rapid enterovirus genotyping in cerebrospinal fluids: a two-year prospective study in a virology laboratory setting

Enterovirus (EV - 68 serotypes) infections comprise a wide spectrum of clinical presentations including infections of the central nervous system. In severe clinical presentation or epidemics, the precise identification of the involved serotype is necessary. OBJECTIVES: To perform enterovirus genotyping directly in cerebrospinal fluid (CSF) samples, and to assess its feasibility in a laboratory setting. METHODS: Enterovirus genotyping was carried out directly with CSF specimens tested for the diagnostic procedure by amplifying the complete 1D gene encoding the VP1 protein of the HEV-B serotypes (the most frequent) - providing results in two days. Secondly, sequences 1A/1B encoding the VP4/VP2 capsid proteins, respectively, were analysed (results in five days). RESULTS: Direct enterovirus genotyping allowed the identification of enterovirus involved in 77 out of 81 (95%) meningitis cases between January 2006 and December 2007. In combination with the indirect genotyping of enterovirus isolates, identification of the type was achieved in 94 out of 97 (96.9%) patients included in the study. The most frequent serotypes were echovirus 6 (E6) and 13 in 2006, coxsackievirus B2 and E30 in 2007. Four children presented an EV71 associated meningitis. CONCLUSION: When prospectively applied in a laboratory setting, direct enterovirus genotyping in CSF samples allows the identification of the involved enterovirus in two to five days. This time frame is relevant for an optimal patient management, the rapid identification of a new enterovirus variant or in the context of an epidemic alert.     

Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens.

BACKGROUND: Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis. OBJECTIVE: To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens. STUDY DESIGN: After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay. RESULTS: Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80). CONCLUSIONS: The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.     

Inactivation of selected picornaviruses by high hydrostatic pressure

The potential of high hydrostatic pressure processing (HPP) to inactivate Aichi virus (AiV), human parechovirus-1 (HPeV-1), and the coxsackievirus strains A9 and B5 was investigated. For coxsackievirus A9 (CAV9), a 5-min HPP treatment in minimum essential growth medium (MEM) supplemented with 10% fetal bovine sera (FBS) resulted in 3.4-, 6.5-, and 7.6-log(10) tissue culture infectious dose (50%) (TCID(50)) reductions at 400, 500, and 600megaPascals (MPa), respectively. For HPeV-1, a 5-min treatment in MEM with 10% FBS resulted in reductions of 1.3-, 4.3-, and 4.6-log(10) TCID(50) at 400, 500, and 600MPa, respectively; however, AiV and coxsackievirus B5 (CBV5) in MEM supplemented with 2 and 10% FBS, respectively, remained fully infectious after a 5-min treatment at 600MPa. These data establish that different picornaviruses have widely variable pressure inactivation thresholds in response to HPP.     

Multiplex PCR for the simultaneous detection of pseudorabies virus, porcine cytomegalovirus, and porcine circovirus in pigs

The use of porcine organs is being developed as a means to alleviate the shortage of human organs for transplantation. Recommendations have been published for the microbiological specifications of organ-source pigs to reduce the possibility of a microorganism from pigs being inadvertently transferred to the recipient of the xenograft. The pseudorabies virus (PRV), porcine cytomegalovirus (PCMV), and porcine circovirus (PCV) are infectious agents in pigs that are considered to be of significance for the microbiological safety of xenotransplantation. A multiplex polymerase chain reaction (mPCR) was developed to detect and differentiate among PRV, PCMV, and PCV. The sensitivities of the multiplex PCR were 10(2.5) TCID(50)/ml for PRV, 10(1.8) TCID(50)/ml for PCMV, and 10(1.8) TCID(50)/ml for PCV. The lowest viral concentrations detected by single PCR were 10(1.5) TCID(50)/ml for PRV, 10(1.0) TCID(50)/ml for PCMV, and 10(1.4) TCID(50)/ml for PCV2. Non-specific reactions were not observed when other viruses, bacteria, and Vero cells were used to assess the multiplex PCR. The multiplex PCR was effective in detecting various combinations of one or more of these viruses in pig specimens collected for xenotransplantation.     

Detection of enteroviral RNA by polymerase chain reaction in faecal samples from patients with aseptic meningitis.

An assay based on the polymerase chain reaction (PCR) for detection of enteroviral RNA in stool samples was carried out using specimens from 74 patients with aseptic meningitis. The primer pair and probe were derived from the highly conserved 5' non-coding enterovirus genomic region. Enteroviral RNA was detected in faeces of all 36 patients in whom an enterovirus was isolated from stool. The PCR assay yielded positive results in additionally 3/6 cases where enterovirus diagnoses were obtained by virus isolation from cerebrospinal fluid and/or serological tests. Thus, the positive outcome of the PCR assay was 39 (93%) among the 42 patients with enterovirus diagnoses. Furthermore, 7/19 (37%) cases with an etiology that was not established by other means were positive in the test indicating that the PCR assay may give considerable additional etiological information in patients with aseptic meningitis. The limit of RNA detectability in the PCR assay was about 100 TCID50 when highly cytopathogenic enterovirus types (coxsackievirus type B5 and echovirus type 11) were tested. The PCR was negative in all 13 patients with non-enterovirus diagnoses except in one case with a herpes simplex virus type 2 infection. Since enterovirus-specific IgM antibodies could be detected in this case a dual infection seemed probable. All the negative controls, included in the study, were PCR-negative and no contamination was encountered. This study proves the usefulness of the PCR assay for detection of enteroviral RNA in stool samples and suggests that the test may be an alternative to virus isolation for rapid enterovirus diagnosis in patients with aseptic meningitis.     

Evaluation of a rapid and completely automated real-time reverse transcriptase PCR assay for diagnosis of enteroviral meningitis

Nucleic acid amplification tests (NAATs) for enterovirus RNA in cerebrospinal fluid (CSF) have emerged as the new gold standard for diagnosis of enteroviral meningitis, and their use can improve the management and decrease the costs for caring for children with enteroviral meningitis. The Xpert EV assay (Cepheid, Sunnyvale, CA) is a rapid, fully automated real-time PCR test for the detection of enterovirus RNA that was approved by the U.S. Food and Drug Administration for in vitro diagnostic use in March 2007. In this multicenter trial we established the clinical performance characteristics of the Xpert EV assay in patients presenting with meningitis symptoms relative to clinical truth. Clinical truth for enteroviral meningitis was defined as clinical evidence of meningitis, the absence of another detectable pathogen in CSF, and detection of enterovirus in CSF either by two reference NAATs or by viral culture. A total of 199 prospectively and 235 retrospectively collected specimens were eligible for inclusion in this study. The overall prevalence of enteroviral meningitis was 26.04%. The Xpert EV assay had a sensitivity of 94.69% (90% confidence interval [CI] = 89.79 to 97.66%), specificity of 100% (90% CI = 99.07 to 100%), positive predictive value of 100%, negative predictive value of 98.17, and an accuracy of 98.62% relative to clinical truth. The Xpert EV assay demonstrated a high degree of accuracy for diagnosis of enteroviral meningitis. The simplicity and on-demand capability of the Xpert EV assay should prove to be a valuable adjunct to the evaluation of suspected meningitis cases.     

Effect of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on European and North American PRRSV shedding in semen from infected boars

The objective of the present study was to compare the effects of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health, St. Joseph, MO) on European and North American PRRSV shedding in the semen of experimentally infected boars. The boars were randomly divided into six groups. Vaccinated boars shed the North American PRRSV at the rate of 10(0.1) to 10(1.0) viral genome copies per ml and 3.63 to 10(1.1) 50% tissue culture infective doses (TCID(50))/ml, respectively, in semen, whereas nonvaccinated boars shed the North American PRRSV at the rate of 10(0.2) to 10(4.7) viral genome copies per ml and 1.14 to 10(3.07) TCID(50)/ml, respectively, in semen. Vaccinated boars shed the European PRRSV at the rate of 10(0.1) to 10(4.57) viral genome copies per ml and 1.66 to 10(3.10) TCID(50)/ml, respectively, in semen, whereas nonvaccinated boars shed the European PRRSV at the rate of 10(0.3) to 10(5.14) viral genome copies per ml and 1.69 to 10(3.17) TCID(50)/ml, respectively, in semen. The number of genomic copies of the European PRRSV in semen samples was not significantly different between vaccinated and nonvaccinated challenged European PRRSV boars. The present study demonstrated that boar vaccination using commercial modified live PRRSV vaccine was able to decrease subsequent shedding of North American PRRSV in semen after challenge but was unable to decrease shedding of European PRRSV in semen after challenge.     

肠道病毒71抗原ELISA检测方法构建及其应用

目的 建立肠道病毒71型(EV71)抗原的ELISA检测方法.用于EV71的抗原定量、TCID50试验及EV71免疫原性与免疫保护性的关系分析.方法 以高亲和力的EV71中和单抗K8G2和Y8H2-HRP构建检测EV71抗原的双抗体夹心ELISA方法.比较本方法与显微镜观察法检测的EV71 TCID50.分析EV71抗原的特异比活与免疫血清中和抗体水平的关系.结果 本试剂定量检测抗原的线性范围为0.125 ～4.0 U/ml,R2 =0.9911,试剂不与EV71之外的受试物反应,试剂的回收率为0.89 ～ 1.16,变异系数小于15％,37℃9 d的热回收率大于85％.本法与显微镜观察法检测的EV71 TCID50的相关系数r=0.990.21株EV71抗原的特异比活与免疫血清中和抗体水平的相关系数r=0.930.结论 构建了EV71抗原ELISA检测试剂,可用于EV71的抗原含量检测、TCID50分析,为特异比活与免疫血清中和性关系研究提供了一些有价值的信息.     

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.     

Coxsackievirus B3-associated aseptic meningitis: an emerging infection in Hong Kong

Enterovirus (EV) infection is a common disease of childhood and associated not uncommonly with aseptic meningitis. In the summer of 2008, laboratory surveillance has detected increased number of coxsackievirus B3 (CVB3) associated aseptic meningitis in Hong Kong, constituting 11.6% of those infected. This study analyzed the epidemiology, circulating pattern, and clinical presentations of CVB3 in Hong Kong over the last 10 years with reference to the circulation of EV in the locality. Enteroviruses (EV) were isolated from respiratory, cerebrospinal fluid (CSF), stool, and vesicular samples using human rhabdomyosarcoma, human laryngeal carcinoma (HEp2-C), human lung fibroblast (MRC-5), and African green monkey kidney (Vero) cell lines. Virus isolates were identified and characterized by indirect immunofluorescence (IF) using monoclonal antibodies (mAB), neutralization test as well as partial VP1 sequencing. Different from previous years, IF test result showed that majority of the isolates from 2008 were untypeable by the mAB suggesting antigenic change. Sequence analysis revealed that these isolates were clustered with recent isolate from Fuyang, China. Review of data from 1999 to 2008 showed increased activity of CVB3 in the years 2005 and 2008, and isolates in these 2 years displayed an amino acid change from threonine to alanine at codon 277 of the VP1 gene, which may be associated with central nervous system (CNS) disease.     

Laboratory investigation and phylogenetic analysis of enteroviruses involved in an aseptic meningitis outbreak in Greece during the summer of 2007

BackgroundAseptic meningitis is the most commonly observed CNS infection and is mainly attributed to Non-Polio Enteroviruses (EV).ObjectiveIdentification and genetic analysis of the EV involved in the recent aseptic meningitis outbreak which occurred in Greece, during the summer of 2007.Study designIn total, 213 CSF and faecal samples were examined for EV presence by culture, while enteroviral RNA detection was performed by nucleic acid sequence-based amplification assay (NASBA). EV strains were typed by seroneutralization, as well as nested RT-PCR followed by VP1-2A gene partial sequencing. Phylogenetic analysis was carried out for the identification of the genetic relatedness among the isolated EV strains.ResultsEV detection rate in CSF and faecal samples was 43.9% and 70.8%, respectively. EV serotyping and VP1 region analysis revealed the predominance of echovirus 4 (ECV4) serotype and the circulation of ECV6, 9, 14, 25, Coxsackie A6, A15, A24 and Coxsackie B1 serotypes. All ECV4 isolates presented a 98.7% similarity in nucleotide sequence, with a Spanish ECV4 strain, isolated during a meningitis outbreak in 2006.ConclusionsIt is the first time that ECV4 is associated with an aseptic meningitis outbreak in Greece, during which 9 different EV serotypes were co-circulating. All Greek ECV4 isolates were closely related to the Spanish ECV4 strain. Genetic analysis of the VP1 gene can significantly contribute to the revelation of the endemic EV strains circulation pattern and their phylogenetic relationship with enteroviruses involved in epidemics of distant geographical areas at different time periods.     

Impact of rapid enterovirus molecular diagnosis on the management of infants,children, and adults with aseptic meningitis

Enteroviruses (EV) are the main etiological agents of aseptic meningitis. Diagnosis is made by detecting the genome using RT‐PCR. The aim of the study was to evaluate the impact of a positive diagnosis on the management of infants, children, and adults. During 2005, 442 patients were admitted to hospital with suspected meningitis. Clinical and laboratory data and initial treatment were recorded for all patients with enteroviral meningitis. The turnaround time of tests and the length of hospital stay were analyzed. The results showed that EV‐PCR detected EV in 69 patients (16%), 23% (16/69) were adults. About 18% of CSF samples had no pleocytosis. After positive PCR results, 63% of children were discharged immediately (mean 2 hr 30 min) and 95% within 24 hr. Infants and adults were discharged later (after 1.8 and 2 days, respectively). The use of antibiotics was significantly lower in children than in infants and adults. The PCR results allowed discontinuation of antibiotics in 50–60% of all patients treated. Patients received acyclovir in 16% of cases (7% children vs. 50% adults) and 23% (11% vs. 69%) underwent a CT scan. Clinical data were compared between patients whose positive EV‐PCR results were available within 24 hr (n = 32) and those whose results were available > 24 hr after collection of CSF (n = 14). Duration of antibiotic treatment (difference: 2.3 days; P = 0.05) was reduced between the two groups. No statistical difference in the length of stay was observed. The EV‐PCR assay should be performed daily in hospital laboratory practice and considered as part of the initial management of meningitis. J. Med. Virol. 81:42–48, 2009. © 2008 Wiley‐Liss, Inc.     

Improved diagnosis on a daily basis of enterovirus meningitis using a one-step real-time RT-PCR assay

The detection of the enterovirus genome in cerebrospinal fluid (CSF) by PCR techniques has proved to be more sensitive than traditional cell culture for the diagnosis of enterovirus meningitis. However, PCR assays are time consuming and labor intensive, particularly if separate hybridization steps are used to confirm the specificity of positive findings. The aim of this study was to develop a one-step real-time RT-PCR assay with LightCycler (LC) technology that was sensitive, rapid, and easy to perform in routine practice. The enterovirus detection limit was determined by testing 10-fold limiting dilution series of cell culture stocks with the echovirus 25 (E-25) prototype strain and with the third European Union Quality Control Concerted Action (EU-QCCA) enterovirus proficiency panel. A total of 100 CSF specimens were investigated in a comparative study. With the E-25 strain, the detection limit of the real-time assay was 286 TCID50/ml (50% tissue culture infective dose). When samples of the EU-QCCA panel were tested, our assay gave identical results (detection limit down to 3.6 TCID50/ml) to those of the reference laboratory, which used one-step RT-PCR assay. When CSF specimens were tested, there was a correlation between the real-time assay and the conventional in-house assay in 96 of 100 CSFs tested. This one-step real-time assay allows rapid enterovirus detection in CSF since results are obtained in 3 hr as against 36 hr with the "in-house" RT-PCR assay. This new assay is now being used in routine practice, and allows diagnosis on a daily basis.