HFRS患者血清特异性抗体及特异性循环免疫复合物同步测…

为进一步研究ＨＦＲＳ免疫损伤机制，用ＥＬＩＳＡ法同步测定了１０８例不同临床型，不同病日，病期ＨＦＲＳ患者血清中特异性ＩｇＡ，ＩｇＧ，ＩｇＭ抗体以及ＨＦＲＳ病毒特异性ＩｇＡ，ＩｇＧＩｇＭ型循环免疫复合物（ＣＩＣ）的水平及检出率，发现ＨＦＲＳ－ＩｇＡ型抗体水平在轻型病例高于中，重型病例，其差异在病程早期（发热，低血压休克期，或是３～８病日）尤为突出，而ＩｇＡ型ＣＩＣ则未见到上述差异，ＨＦＲＳ－ＩｇＧ，     

血透患者检测抗HCV IgM的临床意义

为探讨丙型肝炎ＩｇＭ抗体测定在血液透析患者中的临床意义,采秀间接ＥＬＩＳＡ法检测抗ＨＣＶ ＩｇＭ。同时采用ＥＬＩＳＡ测抗ＨＣＶ ＩｇＧ,ＲＴ－ＰＣＲ法测ＨＣＶ ＲＮＡ,并进行比较。结果示６２例血液透析病人中,抗ＨＣＶ ＩｇＭ阳性２７例（４３．６％）,抗ＨＣＶ ＩｇＧ阳性２９例（４６．８％）,ＨＣＶ ＲＮＡ阳性３４例（５４．８％）,任一项阳性３７例（５９．７％）;抗ＨＣＶ ＩｇＭ与ＨＣＶ ＲＮＡ检测     

套式聚合酶链反应在母婴传播巨细胞病毒研究中的应用

本文建立了套式ＰＣＲ技术加限制酶分析方法，对１０５份母脐血配对进行套式ＰＣＲ，病毒分离，特异性ＩｇＭ及ＩｇＡ测定。结果母血ＨＣＭＶ ＤＮＡ阳性６份，脐血３份，母－脐传播率为３／６。三对被证实为母婴传播ＨＣＭＶ套式的标本中，二对套式ＰＣＲ、病毒分离、特异性ＩｇＭ及ＩｇＡ均阳性。一对套式ＰＣＲ病毒分离、特异性ＩｇＡ阳性，但特异性ＩｇＭ阴性，提示：套式ＰＣＲ是一种敏感、特异、简便快速，能早期诊断ＨＣＭＶ     

应用PCR和ELISA法检测新生儿不同标本中的巨细胞病毒

为了解新生儿感染巨细胞病毒检测方法的可靠性，本文用ＥＬＩＳＡ法筛１８１８例孕妇，查出ＣＭＶ－ＩｇＭ阳性４６例，并以５０例ＣＭＶ－ＩｇＭ阴性作为对照组，在其分娩时用ＰＣＲ检测脐血、新生儿血、新生儿尿中ＣＭＶ－ＤＮＡ，用ＥＬＩＳＡ法测血清ＤＣＭＶ－ＩｇＭ。结果显示对照组两种方法均为阴性，实验组ＥＬＩＳＡ法阳性率５％，ＰＣＲ法阳性率２７％有显著差异。两种方法比较ＰＣＲ法具有高度特异性和敏感性。并提出先天     

两种血清型HFRS患者病程中及病后4种特异性抗体测定分析

为评价汉滩及汉城型肾综合征出血热患者免疫状况，用ＥＬＩＳＡ法检测了５４例病程中，４７例病后１－６年ＨＦＲＳ患者（汉滩２４例，汉城２３例）血清中特异性ＩｇＡ、ＩｇＥ、ＩｇＧ、ＩｇＭ抗体。在病后血清中检出率及抗体滴度几何均数（ＧＭＴ）依次为ＨＦＲＳ－ＩｇＧ、ＩｇＡ、ＩｇＥ、ＩｇＭ，各本间差异显著（Ｐ〈０．００５），其中ＨＦＲＳ－ＩｇＧ抗体水平在病后第４年仍达１：１００以上（ＧＭＴ值）；ＨＦＲＳ－ＩｇＡ     

抗心磷脂抗体与反复自然流产关系的探讨

应用间接ＥＬＩＳＡ法检测１０５例反复自然流产患者血清中的抗心磷脂抗体，同时对ＡＮＡ、扩ｄｓ－ＤＮＡ、抗－Ｓｍ，抗－ＲＮＰ、ＲＦ进行了测定。结果表明：ＲＳＡ组ＡＣＡ阳性率为２８．６％，明显高于其他自身抗体及对照组（Ｐ＜０．０１），ＡＣＡ与ＲＳＡ患者的孕龄及流产次数无相关性（Ｐ＞０．０５），三种类别Ｉｇ中，以ＩｇＧ、ＩｇＧ、ＩｇＭ型ＡＣＡ检出率较高，中等或高水平的阳性结果占３３．３％（１０／３０），其     

逆转录套式ＰＣＲ检测丙型肝炎病毒感染者ＨＣＶ ＲＮＡ

目的　探讨人血清和外周血单个核细胞（ＰＢＭＣ）中丙型肝炎病毒（ＨＣＶ）ＲＮＡ与血清ＩｇＧ和ＩｇＭ抗体联合检测对丙型肝炎的诊断价值。方法对以ＥＬＩＳＡ方法检测所得的４６例ＨＣＶ—ＩｇＧ阳性的标本用逆转录套式聚合酶锭反应（ＲＴ—ＮｅｓｔｅｄＰＣＲ）检测血清和ＰＢＭＣ中ＨＣＶＲＮＡ，同时用ＥＬＩＳＡ方法检测血清中ＨＣＶ—ＩｇＭ。结果血清中有１７例ＨＣＶ　ＲＮＡ阳性和６例ＨＣＶ—ＩｇＭ阳性，而ＰＢＭＣ中有１９例ＨＣＶＲＮＡ阳性。结论血清中ＨＣＶ—ＩｇＭ可作为丙型肝炎的初筛诊断，ＨＣＶ—ＩｇＭ可作为ＨＣＶ近期感染的指标，但二者均不能准确反映病毒在体内的存在情况，而套式ＰＣＲ方法检测ＨＣＶＲＮＡ可直接反映ＨＣＶ在体内的活动情况，其中ＰＢＭＣ中检测ＨＣＶＲＮＡ的阳性率高于血清，提示ＨＣＶ可能在ＰＢＭＣ中潜伏乃至复制。     

临床肝病患者血清中抗庚型肝炎病毒抗体的检测及分析

由于特异性的引物进行的反转录聚合酶链反应（ＲＴ－ＰＣＲ）检测方法复杂,成本高,不利于进行临床的普遍检测。ＥＬＩＳＡ法操作简单,成本低,可作为临床病人的初检。我们用此方法检测了２０４份血清,其中有５１例抗ＨＧＶ－ＩｇＧ阳性,现报告如下。１资料与方法１?..     

幽门螺杆菌基因的检测与分型

我们用幽门螺杆菌(Ｈｐ)空泡毒素(ｖａｃＡ)和细胞毒相关蛋白(ｃａｇＡ)基因检测以及ｖａｃＡ基因分型的聚合酶链反应(ＰＣＲ)方法,并对幽门螺杆菌ｖａｃＡ基因型和ｃａｇＡ状态的关系作了调查,报告如下。一、材料和方法1标本:48株Ｈｐ临床分离株和124份用我院自制采样器所取Ｈｐ阳性胃粘液标本[1],经ＰＣＲ检测Ｈｐ16ＳｒＲＮＡ基因和尿素酶试验以及14Ｃ呼气试验确定为Ｈｐ阳性的标本。以上取样的172位Ｈｐ阳性患者中,经内窥镜取样病检诊断为萎缩性胃炎(ＡＧ ＣＡＧ)的患者为49例,浅表性胃炎(ＳＧ ＣＳＧ)的患者为64例。2.试剂:ＴａｑＤＮ…     

急性脑血管病脑脊液免疫球蛋白变化与CT

急性脑血管病脑脊液免疫球蛋白变化与ＣＴ侯景玉①李彤吉四辈脑脊液免疫球蛋白（ＣＳＦ－ＩｇＧ）、白蛋白（ＡＩｂ）、ＣＳＦ－ＩｇＧ／ＡＩｂ比值、ＣＳＦ－ＩｇＧ指数及补体Ｃ３等（下称ＣＳＦ－Ｉｇ系列指标）的测定，对于判定中枢神经系统（ＣＮＳ）疾病的血脑屏障（...     

Diagnostic value of PCR for detection of Borrelia burgdorferi DNA in clinical specimens from patients with erythema migrans and Lyme neuroborreliosis.

BACKGROUND: The aim of the study is to evaluate the diagnostic sensitivity of a 16S ribosomal RNA-based PCR on clinical specimens from patients with erythema migrans (EM) and neuroborreliosis and to compare the sensitivities with those obtained by in vitro culture and serological testing. A semiquantitative detection system, representing the input amount of specific DNA and thus the density of spirochetes in clinical specimens, indicated the preferred clinical sample to obtain for PCR testing. METHODS AND RESULTS: Skin biopsy and urine samples from 31 patients with EM and cerebrospinal fluid (CSF) and urine samples from 30 patients with neuroborreliosis were investigated. Borrelia burgdorferi DNA was detected in 71% of the skin biopsy specimens and 13% of the urine samples from patients with EM. Forty-one percent of the patients with EM were found to have B burgdorferi-specific antibodies in serum, and B burgdorferi was cultured in 29% of the EM specimens. For patients with neuroborreliosis, the diagnostic sensitivities in CSF and urine samples were 17% and 7%, respectively. Specific intrathecal antibody production was found in 90% of the patients, and 87% showed elevated B burgdorferi antibodies in serum. In general, PCR of skin biopsy samples yielded very high amounts of amplicons versus low amounts for CSF and urine samples. CONCLUSIONS: PCR of skin biopsy specimens is currently the most sensitive and specific test for the diagnosis of patients with EM, superior to culture and serological testing. For B burgdorferi-specific CSF disgnosis in patients with neuroborreliosis, the measurement of specific intrathecal antibody synthesis is superior to PCR. However, in patients with a short duration of disease (<14 days), PCR may be a useful diagnostic supplement. PCR of urine samples cannot be recommended at the present time for routine diagnosis of patients with EM or neuroborreliosis.     

小儿单纯疱疹病毒脑炎的临床分析

目的；通过分子生物学方法了解小儿中枢神经系统单纯疱疹病毒（HSv）感染情况，分析其临床特点。方法：收集150例住院的中枢神经系统病毒感染患儿的脑脊液标本，用套式PCR检测脑脊液中HSV DNA用酶联免疫吸附法检测脑脊液中特异性HSV IgM抗体。结果：150例中有6例脑脊液HSV1 DNA（＋），另1例HSV1 IgM（＋）；病例呈散发性起病，无明显季节、年龄、性别分布特点。与其他病毒感染相比，惊厥持续状态、精神症状发生率高（P〈0．01），意识障碍、病死率无显著差异。结论：单纯疱疹病毒感染占儿童中枢神经系统感染的4．67％。儿童中枢神经系统单纯疱疹病毒感染多为HSVl的原发感染，可导致脑炎、脑干脑炎、急性播散性脊髓膜炎；起病较危重，出现精神症状较多，及时有效的抗病毒治疗能明显改善病情，降低病死率。     

Detection of Toxoplasma gondii by polymerase chain reaction in cerebrospinal fluid from human immunodeficiency virus-1-infected Japanese patients with focal neurological signs

We aimed to determine the effectiveness of using the polymerase chain reaction (PCR) to detect Toxoplasma gondii in cerebrospinal fluid (CSF) specimens from Japanese patients infected with human immunodeficiency virus (HIV)-1. Twenty-six HIV-positive individuals presenting with focal neurological signs and a possible diagnosis of T. gondii encephalitis (TE) were enrolled in the study between April 1997 and March 2003. Eight patients were diagnosed as having TE using the accepted diagnostic criteria; PCR amplified the T. gondii B1 gene in CSF samples from five of these eight patients. CSF samples from the 18 patients without TE were negative for T. gondii DNA. The sensitivity, specificity and positive and negative predictive values for detecting T. gondii in CSF using PCR were 62.5%, 100%, 100% and 85.7%, respectively. These results suggest that PCR might be a clinically useful technique for detecting T. gondii DNA in patients infected with HIV showing focal neurological signs. Improvements in sensitivity are needed, however.     

Immuno-PCR for detection of antigen to Angiostrongylus cantonensis circulating fifth-stage worms

BACKGROUND: Definitive diagnosis of infestation with Angiostrongylus cantonensis is difficult because the parasitic nematode is undetectable in the cerebrospinal fluid (CSF) of one-half of afflicted patients and the diagnostic sensitivity of ELISA for circulating worm antigens in patient sera is low. We studied immuno-PCR as a diagnostic tool. METHODS: We studied 30 controls and 60 afflicted patients (30 confirmed by parasitologic analysis of CSF). We used a monoclonal antibody to capture circulating A. cantonensis antigens in serum samples. A DNA label generated by PCR amplification with biotinylated primer was bound by use of streptavidin to a biotinylated third antibody. Circulating antigens sandwiched by monoclonal antibody were detected by PCR amplification of the DNA label. RESULTS: The detection limit of the ELISA was 100-1000 times higher than that of the immuno-PCR. The concentrations of circulating antigens in patients were markedly higher than those in controls (Wilcoxon rank-sum test, P <0.001). At a cutoff of 0.1 ng/L, sensitivity and specificity for immunodiagnosis of patients with angiostrongyliasis by immuno-PCR were 98% (95% confidence interval, 91-99%) and 100% (93-100%), respectively. The test was positive in all parasitologically confirmed cases. CONCLUSIONS: Immuno-PCR is a promising technique for diagnosis of A. cantonensis infestation.     

Analysis of cross-reactivity between flaviviruses with sera of patients with Japanese encephalitis showed the importance of neutralization tests for the diagnosis of Japanese encephalitis

Japanese encephalitis (JE) is one of the most important viral encephalitis in Asia. JE is caused by the Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus, family Flaviviridae. The diagnosis of JE is usually based on serological assays, and it has been reported that cross-reactivity between flaviviruses has complicated the interpretations of results from serological assays. Therefore, analysis of the cross-reactivity is an important subject for serological diagnosis of JE and other diseases caused by flaviviruses. In the present study, the cross-reactivity of the sera of patients with JE to other flaviviruses was analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization tests. Sixteen serum samples were collected from patients with JE and were tested for: i) IgM antibody against West Nile virus (WNV), dengue virus (DENV), zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) using IgM-ELISA, ii) IgG antibody against DENV and TBEV using IgG-ELISA, and iii) neutralization tests with DENV 1–4, ZIKV, TBEV, and WNV. Out of the 16 samples tested using ELISA, 11 and 14 samples were positive for IgM and IgG, respectively, against at least one of the other flaviviruses. In neutralization tests, neutralizing potency against DENV, ZIKV, or TBEV was not detected in any samples. Although 13 samples showed neutralizing potency against WNV, their neutralizing antibody titers were equal to or less than one-eighth of those against JEV. These results show that neutralization tests are more specific than ELISA, indicating the importance of the neutralization tests in the diagnosis of JE.     

流行性脑脊髓膜炎病例实验室不同方法监测结果分析

目的应用实验室免疫学、病原学方法监测合肥市流行性脑脊髓膜炎(流脑)病例感染状况和菌群分布,比较不同方法监测结果。方法采集流脑疑似病例急性期和恢复期血液、急性期脑脊液,实验室诊断方法包括免疫学血清抗体检测、脑脊液抗原检测,病原学血液脑膜炎奈瑟菌(INm/I)培养、脑脊液INm/I培养,病原学血清INm/I的DNA特异片段检测。结果2005-2008年流脑疑似病例421份,实验室监测病例311份,免疫学血清抗体检测方法病例阳性率最高72.4％,其中C群流脑病例阳性率70.9％;临床诊断病例391份,临床诊断病例率92.9％,实验室诊断病例181份,实验室诊断病例率43.0％。结论合肥市流脑监测病例中流脑感染以C群INm/I为主;流脑病例实验室诊断与临床诊断之间存在差距,实验室诊断需要结合多种方法,加强质量管理。     

Prospective case‐control study of enterovirus detection differences in children’s cerebrospinal fluid between multiplex PCR and real‐time RT‐PCR assay

BackgroundViral encephalitis is common in childhood. It is an acute brain parenchymal inflammation caused by a variety of viral infection, and enterovirus accounts for the majority. Due to atypical clinical manifestations, pathogenic testing is important for assisting clinical diagnosis. The purpose of this study was to evaluate the performance of the multiplex PCR assay compared with quantitative real‐time PCR for enterovirus detection.MethodsA prospective case‐control study was performed involving 103 pediatric patients suspected for viral encephalitis and cerebrospinal fluid (CSF) samples were collected and tested for 9 pathogens using multiplex PCR assay during April to November in 2018. In parallel, an aliquot of samples was tested for enterovirus infection by real‐time PCR assay.ResultsThere were 85.4% children were confirmed as viral encephalitis on discharge, the remaining ones were diagnosed as other CNS diseases, such as epilepsy. The specificity of the two methods was the same as that of the clinical diagnosis, but the sensitivity and consistency with clinical diagnosis of multiplex PCR were both higher than the real‐time PCR. Besides of enterovirus, multiplex PCR could also detect coinfection of enterovirus with Epstein‐Barr virus and mumps virus.ConclusionResults of multiplex PCR method are more consistent with the clinical diagnosis and are superior to real‐time PCR for detecting enterovirus in CSF.     

登革病毒TaqManMGB实时荧光定量PCR检测方法的建立及初步应用

目的建立登革病毒TaqMan实时荧光定量PCR快速检测方法及应用于临床。方法根据1～4型登革病毒3’端非编码区的一段高度保守序列,设计一套型通用的引物和TaqManMGB探针,以4个血清型登革病毒标准株为标准,以日本乙脑病毒和丙肝病毒作阴性对照,以包含登革病毒2型标准株（DENV-2NGC株）3’非编码区349bD片段的质粒DNA作标准品,对引物和TaqManMGB探针的特异性、灵敏度进行分析,从而建立登革病毒实时荧光定量PCR检测方法。用该法对登革病毒野毒株和10份登革病毒患者血清进行检测。结果TaqMan实时荧光定量PCR检测的1～4型登革病毒标准株及野毒株均为阳性,日本乙脑病毒和丙肝病毒均为阴性;检测灵敏度可达到每反应2个基因拷贝;检测的10份登革患者血清样本中,8份检测结果为阳性。结论TaqManMGB实时荧光定量PCR方法是一个快速、特异性强、敏感性高的检测登革病毒的方法,适用于登革病毒的临床早期诊断。     

A prospective study of the polymerase chain reaction for detection of herpes simplex virus in cerebrospinal fluid submitted to the clinical virology laboratory.

Polymerase chain reaction (PCR) was prospectively performed with cerebrospinal fluid (CSF) from 51 patients whose CSF was available for analysis and was submitted for viral culture and/or herpes simplex virus (HSV) serology and 20 patients whose CSF was submitted exclusively to the Clinical Biochemistry Laboratory. Primers were used that flanked a 92 bp segment of the HSV DNA polymerase gene (35 cycles). Amplified products were electrophoresed on agarose gel, blotted onto nylon membrane, and probed with a 32P-labelled sequence internal to the primers. For nested PCR, 1 microliter of PCR product was amplified for an additional 35 cycles before electrophoresis and Southern blot analysis. Review of the clinical records revealed that 15 patients had central nervous system (CNS) infections. Specific HSV DNA sequences were detected in CSF specimens of three of the individuals [PCR(2), nested PCR(1)]. Two of these patients had disseminated HSV infection including encephalitis and one patient had aseptic meningitis. The diagnoses of the 12 patients with CNS infection who did not have HSV DNA detected in CSF included encephalitis [varicella-zoster virus (1), cytomegalovirus (1), Mycoplasma pneumoniae (1)], meningitis [Neisseria meningitidis (1), Coccidioides immitis (1), Enterovirus (1), aseptic meningitis (1)], varicella-zoster radiculitis (2), human immunodeficiency virus dementia (2), and transverse myelitis due to Epstein-Barr virus (1). Importantly, HSV DNA was also not detected in the CSF of the 36 patients who did not have CNS infection and 20 samples submitted exclusively to the Clinical Biochemistry Laboratory. Our findings demonstrate the utility of PCR as a rapid, non-invasive method for the routine laboratory diagnosis of CNS infection due to HSV.     

Rapid detection of Neisseria meningitidis in cerebrospinal fluid by one-step polymerase chain reaction of the nspA gene

A polymerase chain reaction (PCR) protocol for the rapid detection of meningococcal DNA in cerebrospinal fluid (CSF) was developed and optimized. A set of primers based on Neisseria surface protein A (nspA) gene sequence was designed to amplify a 481-bp product specific for N. meningitidis. We tested 85 N. meningitidis strains obtained from patients with meningococcal meningitis and 112 CSF samples from patients with suspected meningococcal meningitis. No amplification of the nspA gene was observed from other Neisseriaceae species (except from N. gonorrhoeae) and from other bacteria frequently associated with meningitis. N. meningitidis belonging to different serogroups yielded the same product after PCR amplification. The sensitivity and specificity of our protocol was determined by comparing the results of specific amplification of nspA gene by PCR reaction (nspA-PCR) with those obtained by conventional methods. All positive samples by conventional methods were confirmed by nspA-PCR, whereas 48% of negative samples after culture and latex agglutination tested positive by nspA-PCR. The use of nspA-PCR proved to be a rapid diagnostic method, in which sensitivity and specificity may not be affected by prior antibiotic treatment.