Diagnostic tools for Toscana virus infection

Toscana virus (TOSV; Phlebovirus, Bunyaviridae) is an important etiological agent of acute meningitis and meningoencephalitis in Mediterranean countries. Laboratory diagnosis has been carried out in serological studies using ELISA, immunofluorescence and/or neutralization tests that are not influenced by the virus viability; however, in the acute phase of the infection, nucleic acid amplification techniques are the methods of choice to diagnose viral meningitis from cerebrospinal fluid samples. Molecular methods are rapid and sensitive and, unlike traditional methods, such as virus isolation by cell culture, they are not influenced by the viability of the virus in the clinical specimen; however, the RNA integrity is crucial for the success of these methods. Real-time PCR is the most important molecular method used in laboratories worldwide, since it is less time-consuming and it reduces the risk of contamination. Therefore, a sensitive real-time PCR has been developed for diagnosis of suspected cases of TOSV infection either autochthonous and/or imported, since a new lineage of TOSV, divergent from the Italian prototype, has recently been reported in Spain.     

Transfusion-transmitted infectious diseases in Argentina, 1995 through 1997

BACKGROUND: Assessment of the safety of the blood supply, the quality of screening procedures, and the risk of transfusion transmission of infectious diseases in any country can be estimated by reviewing the records of blood donations and screening procedures and the prevalence of serologic markers of infectious diseases. STUDY DESIGN AND METHODS: Information on blood donors, particularly the number of screened donors, and on the prevalence of serologic markers of infectious diseases was available from Argentina for 1995 through 1997. This information permitted the estimation of the risks and costs of preventing transfusion transmission of infectious diseases within the country during this period. RESULTS: Screening coverage was higher in the private sector. The proportion of donors screened for HIV increased from 84.52 percent in 1995 to 97.97 percent in 1997; in the same period, serologic screening for HbsAg increased from 83.71 percent to 98.48 percent; that for HCV from 69. 92 percent to 97.83 percent; and that for syphilis from 87.94 percent to 98.71 percent. One hundred percent of donors were screened for Trypanosoma cruzi throughout the period. The overall prevalence of HIV per year varied from 2.42 to 3.36 per 1,000 donors; that of HBV, from 5.80 to 9.76 per 1,000; of HCV, from 7.39 to 16.61 per 1,000; and of syphilis, from 5.25 to 7.65 per 1,000. The overall prevalence of antibodies to T. cruzi ranged from 36.53 to 49.20 per 1,000 donors. The overall index of the spread of infectious viral disease through blood transfusion decreased from 47. 74 per 10,000 donations in 1995 to 4.75 per 10,000 in 1997. The ratio of acquired infections to donations improved from 1:209 to 1:2, 102 during the same period. The risk of T. cruzi infection from 1995 through 1997 was, in theory, nil, given the 100-percent screening. The greatest threat to the quality of the blood supply throughout the period studied was HCV. CONCLUSION: The status of the blood supply in Argentina improved steadily from 1995 to 1997, as shown by the increase in screening coverage.     

2513例抗核抗体与抗核抗体谱检测结果对比分析

目的分析本实验室抗核抗体(ANA)与抗核抗体谱(ANAs)15项联合检测的结果,并探讨两者之间的关系。方法分别用间接免疫荧光法(IIF)和免疫印迹法(IBT)检测患者ANA和ANAs,判断其ANA滴度、荧光模型和ANAs结果,并对ANA和ANAs检测结果进行对比分析。结果 471例ANA阳性患者标本中,颗粒型161例,均质型110例,胞质型96例、核仁型37例、着丝点型28例,其他型合计39例;滴度为1∶100者196例,占41.61%(196/471),其中ANAs阳性53例,占27.04%(53/196);滴度为1∶320者123例,占26.11%(123/471),其中ANAs阳性67例,占54.47%(67/123);滴度为1∶1 000者152例,占32.27%(152/471),其中ANAs阳性132例,占86.84%(132/152);2 042例ANA阴性标本中45例ANAs检测阳性,阳性率为2.2%(45/2042);高滴度ANA患者血清中ANAs的阳性率高于低滴度(χ2=123.132,P0.05)。结论不同滴度的ANA与ANAs之间有一定的关系,如果以ANA进行过筛,阳性再做ANAs会漏检部分患者,建议IIF和IBT联合应用,防止风湿病的漏诊。     

抗核抗体核型与抗核抗体谱检测结果对比分析

目的：分析本实验室抗核抗体（ANA）与抗核抗体谱（ANAs）17项联合检测的情况,并探讨两者之间的相关性。方法分别用间接免疫荧光法和免疫印迹法检测患者 ANA 和 ANAs,判断其 ANA 核型和 ANAs 结果,并对各荧光核型和各种特异性抗核抗体结果进行统计分析。结果340例 ANA 阳性患者标本中,颗粒型214例,着丝点型51例,胞浆颗粒型28例,核仁型25例,均质型17例,周边型5例,颗粒型是检出的主要核型。ANAs 17项抗体阳性率为82．94％。214例荧光颗粒型标本中,常见的抗体为抗 SSA/Ro52kd（31．78％）、抗 SSA/Ro60k（d30．84％）和抗 SSB/La（19．16％）抗体;17例荧光均质型标本中,常见的抗体为抗核小体（47．06％）和抗组蛋白（41．18％）抗体;抗 CenpB 对着丝点核型的诊断具有较高的特异性,阳性率为92．16％（47/51）。结论不同荧光核型的 ANA 与 ANAs 抗体之间有一定的关系,但并无绝对的规律可寻,两种 ANA 的检测方法,间接免疫荧光法和免疫印迹法联合应用,有助于自身免疫性疾病（AID）的诊断和预后观察。     

TaqMan实时定量RT—PCR与常规RT—PCR检测登革病毒的比较

目的:比较TaqMan实时定量逆转录-聚合酶链反应(real-timeRT-PCR)法与常规逆转录-聚合酶链反应(RT-PCR)法检测登革病毒的敏感性、特异性和时效性。方法:建立TaqMan实时定量RT-PCR方法和常规RT-PCR方法,分别对登革病毒、乙型脑炎病毒及西尼罗病毒进行检测,并比较两法的特异性、敏感性和时效性。结果:两种方法都能检测出登革病毒,而对乙型脑炎病毒和西尼罗病毒的检测呈阴性;常规RT-PCR的敏感性为0.1TCID50/mL,TaqMan实时定量RT-PCR的敏感性提高了100倍,达到了0.001TCID50/mL,且至少节约了2h。结论:与常规RT-PCR比较,TaqMan实时定量RT-PCR更适用于登革病毒的实验室快速诊断。     

3种流感病毒检测方法的比较和应用

目的探讨比较荧光定量RT-PCR、普通RT-PCR和细胞培养3种方法在流感病毒检测中的应用,从而确定每种方法各自的实用领域。方法通过荧光定量RT-PCR、普通RT-PCR和狗肾传代细胞(MDCK)培养3种方法检测398件流感样病例(ILI)咽拭子样本。比较3种方法的灵敏度和特异性。结果实时荧光RT-PCR检测阳性样本129件(阳性率为32.4%);普通RT-PCR检测出阳性样本93件(阳性率为23.4%);MDCK细胞培养法分离出流感病毒64株(阳性率为16.1%)。荧光定量RT-PCR的灵敏度达到0.01TCID50,特异性强。结论无论从敏感性、特异性还是从时间上,荧光RT-PCR对于疫情的爆发更有应用价值。细胞培养法分离流感病毒对于核实疫情的实验室诊断和病原学监测具有重大意义。     

一起诺沃克病毒引起的急性胃肠炎爆发的分子流行病学研究

目的 查明2005年深圳市一起急性胃肠炎的病原体,为制定控制疾病流行策略及指导临床合理用药提供依据.方法 收集该次腹泻患者及相关人员的粪便标本15份,采用逆转录-聚合酶链反应(RT-PCR)和实时荧光PCR方法检测诺沃克病毒核糖核酸(RNA).结果 15份标本中,RT-PCR及实时荧光PCR方法均检测出10例诺沃克病毒阳性,阳性率为56.7%;2种方法的符合率为100%.同时对其中的10份RT-PCR阳性标本进行脱氧核糖核酸(DNA)序列测定,并进行同源性分析和进化分析.结论 该次群体性胃肠炎是由诺沃克病毒G2型感染引起;10份阳性标本中9株的核苷酸同源性为100%,另外1株与其他9株的同源性为99.8%;本次流行的毒株(05-53-61及05-63)与日本东京的SaitamaU201及SaitamaU18亲缘关系较近,核苷酸同源性分别为96%及95%,而与1997年深圳本地株97-11及97-30亲缘关系则较远,核苷酸同源性分别为76.4%及76.7%.     

流感病毒亚型鉴定的两种方法比较

目的 比较直接免疫荧光法（DFA）与间接免疫荧光法（IFA）检测流感病毒亚型的特异性和敏感性,评价2种方法在流感病毒亚型检测中的应用效果.方法 采用DFA法和IFA法对90份流行性感冒患者及120份健康者的鼻炎分泌物、血清进行检测.结果 DFA法检测腺病毒的阳性率为17%（36/210）,IFA法检测的阳性率为26%（56/210）,二者差异无统计学意义（P〉0.05）.DFA法检测的敏感性为54.6%,特异性为97.2%;IFA法检测的敏感性为87.5%,特异性为48.2%.二者敏感性和特异性比较,差异有统计学意义（P〈0.05）.结论 DFA与IFA法检测流感病毒具有较好的特异性和敏感性.     

Molecular diagnostic testing for primary biliary cholangitis

Introduction: A reliable liver autoimmune serology for the diagnosis of primary biliary cholangitis (PBC) is of particular importance. Recognition of patients at early stages and prompt treatment initiation may alter the outcome, slow progression, delays liver failure, and improves survival.

Areas covered: In this review, we summarize and discuss the published data obtained from literature searches from PubMed and The National Library of Medicine (USA) and our own experience on the current and potential molecular based approaches to the diagnosis of PBC.

Expert commentary: Standardization of liver diagnostic serology and clinical governance are two major points as antimitochondrial antibodies are the diagnostic hallmark of the disease and PBC-specific antinuclear antibodies could assist in the diagnosis and estimation of prognosis. New biomarkers such as novel autoantibodies, genetic polymorphisms, metabolomic profiling, micro-RNA and epigenetics may assist to the understanding, diagnosis and management of the disease.     

间接免疫荧光法及免疫印迹法检测抗核抗体在SLE中的临床价值

目的研究间接免疫荧光法(IIF)筛查的抗核抗体(ANA)和免疫印迹法(IBT)检测的特异性ANA谱在系统性红斑狼疮(SLE)患者的相关性和临床意义。方法对80例SLE患者(SLE组)、80例其他风湿病患者(疾病对照组)及80例健康体检者(健康对照组)分别采用IIF、IBT检测血清中的ANA。结果 SLE组ANA阳性率(96.25%)显著高于疾病对照组(32.50%)和健康对照组(5.00%),差异有统计学意义(P0.05);SLE组荧光模式主要是颗粒型(43例,53.75%)、均质型(16例,20.00%)、胞浆颗粒型(14例,17.50%)。SLE组ANA的阳性率为97.5%,其中抗nRNP/Sm抗体、抗Sm抗体、抗dsDNA抗体、抗核小体抗体、抗组蛋白抗体、抗Rib-P抗体阳性率均显著高于疾病对照组和健康对照组,差异有统计学意义(P0.05)。结论 ANA和ANA谱联合检测对SLE的诊断及治疗具有重要意义。     

浙江省台州地区未成年人呼吸道合胞病毒流行特点分析

目的对台州地区未成年人呼吸道合胞病毒(RSV)感染进行监测,了解其流行特点。方法对2005年1月至2008年12月来浙江省台州医院就诊的呼吸道感染的患者3556名采集呼吸道分泌物,利用直接免疫荧光法进行RSV抗原检测。结果 2005-2008年的RSV阳性率分别为42.23%、20.24%、46.40%、22.16%。不同年度的RSV阳性率比较差异有统计学意义。RSV阳性率总体上从10-11月开始明显增高,持续到次年的3-4月,全年均有散发存在,但每年的流行趋势存在差别。3556例标本中,共有RSV阳性1281例,RSV阳性率为36.02%,RSV1岁组1081例(30.40%),1～3岁组174例(4.89%),3岁组26例(0.73%),各年龄组间检出率差异有统计学意义(P0.01)。男性908例(35.75%),女性373例(36.71%),男女性差异无统计学意义(P0.05)。结论 RSV是台州地区冬春季节引起小儿呼吸道感染的主要病毒,但RSV感染率存在年度差异。年龄越小RSV感染率越高,各年龄阶段的男性与女性对RSV同等易感。     

A combined indirect elisa and immunoblotting for the detection of intrathecal herpes simplex virus igg antibody synthesis in patients with herpes simplex virus encephalitis

A combined indirect ELISA and immuno-blotting assay was used for the detection of intrathecal synthesis of IgG antibodies to herpes simplex virus (HSV) in patients with HSV encephalitis (HSVE). By using these two assays as well as three markers for blood-brain barrier, leakage can be easily excluded. A total of 21 sera and 24 cerebrospinal fluid (CSF) samples from 11 patients with HSVE were examined. For seven patients more than one pair of serum and CSF were available. For one patient IgG antibodies began to be detectable in CSF after the sixth day from the onset of the disease. In the other 10 patients the intrathecal synthesis of HSV IgG antibodies was detected later than the sixth day and reached high optical density (OD) values after the 10th day from the onset of disease, at the earliest. In contrast, intrathecal HSV antibody synthesis was not found in specimens taken from 20 patients with acute meningitis who composed our negative control group. The use of a combined indirect ELISA and of an immunoblotting assay on a single dilution of serum and CSF for HSV IgG synthesis in the central nervous system (CNS) allowed the diagnosis of HSVE after the first week of disease.     

五重荧光定量RT-PCR法检测呼吸道病毒

目的 建立一种五重荧光定量RT-PCR检测并鉴别流感病毒A( Flu A)、流感病毒B( Flu B)、呼吸道合胞病毒(RSV)以及新甲型H1N1流感病毒(nH1N1)的方法.方法 以人细胞RNA酶P基因为内参,用Primer Express 3.0设计PCR引物和探针.用一系列不同滴度的病毒培养物和不同来源的呼吸道...     

Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients

The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009–10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples.     

A contemporary perspective on the molecular characteristics of mitochondrial autoantigens and diagnosis in primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune hepatobiliary disease characterized by immune mediated destruction of the intrahepatic small bile ducts and the presence of antimitochondrial antibodies (AMAs). The mitochondrial autoantigens have been identified as the E2 subunits of the 2-oxo-acid dehydrogenase complex, including the E2 subunits of pyruvate dehydrogenase, branched-chain 2-oxo acid dehydrogenase complex, oxoglutarate dehydrogenase complex, E3 binding protein and PDC E1 alpha subunit. The AMA epitope is mapped within the E2 lipoic acid binding domain, which is particularly important for oxidative phosphorylation. In addition, lipoic acid, which serves as a swinging arm to capture electrons, is particularly susceptible to an electrophilic attack and may provide clues to the etiology of PBC. This review emphasizes the molecular characteristics of AMAs, including detection, immunochemistry and the putative role in disease. These data have significance not only specifically for PBC, but generically for autoimmunity.     

茂名地区儿童非典型肺炎病原体流行病学调查

目的了解茂名地区儿童9种呼吸道病原体的IgM抗体检测结果及流行情况。方法取6 241例初步诊断为吸道感染患者的血清,采用间接免疫荧光法同时检测9种病原体IgM抗体。9种非典型病原体分别是嗜肺军团菌(LP)、肺炎支原体(MP)、Q热立克次体(COX)、肺炎衣原体(CP)、腺病毒(ADV)、呼吸道合胞病毒(RSV)、甲、乙型流感病毒(INFA/B)和副流感病毒(PIVS)。结果共检测6 241份血清,其中1 320份阳性,非典型病原体感染阳性率为21%,其中MP(15.12%)感染率最高,其次为INFB、LP、ADV和PIVS,它们的感染率分别为3.03%、1.92%、0.54%和0.22%。感染率最小的病原群体为RSV、COX、INFA和CP,它们的感染率分别为0.14%、0.11%、0.048%和0.016%。结论茂名地区儿童急性呼吸道感染的病原体主要是MP,病原体检出率与季节有关。     

9项呼吸道病原体血清IgM抗体检测在呼吸道感染中的临床价值

目的通过检测695例呼吸道感染者病原体IgM抗体表达情况,为临床更好地选择治疗方案提供依据。方法采用间接免疫荧光法检测2015年9月至2016年3月来兰州军区总医院安宁分院和兰州军区总医院呼吸科、小儿科、胸外科门诊就诊和住院的呼吸道感染患者695例血液中的9项病原体IgM抗体,并分析检出情况。结果 695例患者中,9项病原体进行检测,单项病原体检测阳性率33.9%,排在前3位的是肺炎支原体(MP)、乙型流感病毒(IFB)、呼吸道合胞病毒(RSV),检出率依次为MP 14.1%,IFB 9.6%,RSV 4.8%,2项病原体混合感染检出率7.79%;MP、IFB在不同季节检出率有明显区别,Q热立克次体检出率无明显季节变化,男、女比例有明显差异。结论本地区呼吸道感染病原体检出率以MP、IFB、RSV感染为前3位。