Nucleic acid amplification-based diagnosis of respiratory virus infections

The appearance of eight new respiratory viruses in the human population in the past 9 years, including two new pandemics (SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009), has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid amplification tests (NATs) that first appeared two decades ago have been developed for both conventional and emerging viruses and now form the backbone of the clinical laboratory. NATs provide fast, accurate and sensitive detection of respiratory viruses and have significantly increased our understanding of the epidemiology of these viruses. Multiplex PCR assays have been introduced recently and several commercial tests are now available. The final chapter in the evolution of respiratory virus diagnostics will be the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance to multiplex assays. These resistance assays together with new viral load tests will enable clinical laboratories to provide physicians with important information for optimal treatment of patients.     

病毒核酸检测在献血者血液筛查中的应用

目的　建立献血者血液混合核酸检测方法 ,调查北京现有检测体系下血液的残余风险度 ,评估核酸检测 (NAT)的必要性和可行性。方法　用世界卫生组织标准品对国产丙型肝炎病毒(HCV)和人免疫缺陷病毒 (HIV)荧光 聚合酶链反应核酸扩增检测试剂进行灵敏度、重复性和精密度试验 ;对 2 0 0 2年 2～ 10月 34373份常规血清学检测 (ALT、HBsAg、抗 HCV、抗 HIV、梅毒抗体 )合格的献血者血样进行HCVRNA和HIV 1RNA核酸扩增分析。采取 2 4人份混合血样测定 ,超离心浓缩病毒 ,Roche核酸提取柱提取病毒核酸。结果　扩增系统能 10 0 %检出 5 0IU/mlHCV及 5 0IU/mlHIV 1标准品核酸 (n =16 ) ;常规血清学检测合格的献血者血液中 ,没有检出HCV或HIVNAT阳性。结论　该核酸检测体系适用于献血者血液病毒筛查 ;北京市血液的病毒安全性已有相当高的保障。为更准确地评估NAT检测项目的可行性和必要性 ,检测标本量尚待增加。     

Point-of-care diagnostics for respiratory viral infections

Introduction: Successful treatment outcomes for viral respiratory tract infections presenting from primary health care to quaternary hospitals will only be achieved with rapid, sensitive and specific identification of pathogens to allow effective pathogen-specific antiviral therapy and infection control measures.

Areas covered: This review aims to explore the different point-of-care tests currently available to diagnose viral respiratory tract infections, discuss the advantages and limitations of point-of-care testing, and provide insights into the future of point-of-care tests. The following databases were searched: Medline (January 1996 to 30 September 2017) and Embase (1988 to 30 September 2017), using the following keywords: ‘point of care’, ‘respiratory virus’, ‘influenza’, ‘RSV’, ‘diagnostics’, ‘nucleic acid test’ and ‘PCR’.

Expert commentary: Viral respiratory tract infections cause significant morbidity and mortality worldwide, and point-of-care tests are facilitating the rapid identification of the pathogen responsible given the similarities in clinical presentation.     

Nucleic acid analysis by sandwich hybridization

One of the most significant achievements of the biochemist during the past two decades is the use to which immunologically based assays have been put in clinical diagnosis (Hood et al.: Immunology, 1984). The problem faced and surmounted by immunologists in effecting the transition from research tool to routine clinical assay bears a remarkable similarity to that confronting the molecular biologist today; i.e., how can nucleic acid hybridization, a technique of obvious potential (Meinkoth and Wahl: Anal Biochem 138:267-284, 1984; Syvanen: Med Biol 64:313-324, 1986; Matthews and Kricka: Anal Biochem 169:1-25, 1988), be modified in order to fulfill all necessary parameters of a routine diagnostic assay? There are several such requirements, and the importance placed on each depends on the objectives of the assay: the technique must be sensitive, specific, and reproducible. Other advantages would be cost-effectiveness, ease of manipulation, and amenability to automation. Ideally, the signal detection should be based on a non-radioactive system, because of the instability of probes labelled with isotopes like 32p, and the potential hazards involved in their handling and disposal. The sandwich hybridization for the analysis of nucleic acid sequences was first used in 1977 (Dunn and Hassell: Cell 12:23-36, 1977), but its potential as a diagnostic assay was not realized until 1983, when it was applied to the detection of adenovirus DNA in nasopharyngeal aspirates from children with acute respiratory infection (Ranki et al: Gene 21:77-85, 1983). It has since been modified and used not only for the detection of microbial infection (Virtanen et al.: Lancet i:381-383, 1983; Ranki et al.: Cur Top Microbiol Immunol 104:307-318, 1983; Lehtomaki et al.: J Clin Microbiol 24:108-111, 1986; Virtanen et al.: J Clin Microbiol 20:1083-1088, 1984; Palva and Ranki: Clin Lab Med 5:475-490, 1985; Polsky-Cynkin et al.: Clin Chem 31:1438-1443, 1985; Parkkinen et al.: J Med Virol 20:279-288, 1986; Palva: FEMS Microbiol Lett 28:85-91, 1985; Palva et al: FEMS Microbiol Lett 23:83-89, 1984; Zolg et al.: Mol Biochem Parasitol 22:145-151, 1987; Palva: J Clin Microbiol 18:92-100, 1983), but also for the analysis of nucleotide sequence variations (Langdale and Malcolm: Gene 36:201-210, 1985). We will discuss the development of the sandwich technique and the advantages it conveys over the more conventional nucleic acid hybridization formats, together with new developments which will ensure that it earns a place alongside immunoassay in the diagnostic laboratory.     

环媒恒温扩增技术在病毒感染检测中的应用

环媒恒温扩增法(loop mediated isothermal amplification,LAMP)是一种新型的恒温核酸扩增技术,它利用了2对特异引物和一种有链取代活性的DNA聚合酶,通过使扩增产物形成单链环结构实现核酸的恒温扩增.LAMP自身的一系列特性使其可以达到对特定核酸片段高效、特异的扩增.由于反应结果可以通过测定反应副产物--焦磷酸镁沉淀引起的浊度来判断,因此使其成为一种十分有效的分子检测手段.     

核酸扩增检测在血液筛查的初步应用

目的为提高血站供血安全性,探讨核酸扩增检测在血站血液筛查中的可行性。方法在酶联免疫吸附试验(ELISA)常规筛查血液的基础上,采用荧光定量聚合酶链反应（PCR）方法,检测乙型肝炎表面抗原(HBsAg)、丙型肝炎抗体(抗-HCV)和人免疫缺陷病毒抗体(抗-HIV)1/2呈阴性的献血者微量血浆汇集池标本（20人份×50μl汇集）中的丙肝病毒（HCV）和乙肝病毒（HBV）核酸,再对阳性汇集池中的标本进行单份检测。结果8805份（分442个汇集池）血液被检测HCVRNA,结果有1例（0.01%）为阳性;1441份血液被检测HBVDNA,结果6例（0.4%）为阳性。从标本汇集到筛查出单份阳性献血者大约需要3d。结论ELISA结合荧光定量PCR检测微量血浆汇集池标本的方法筛查血液HBV和HCV感染是可行的,能够进一步提高输血的安全性。     

呼吸道病毒感染的实验室诊断

急性呼吸道疾病(acute respiratory disease,ARD)在发达国家急性病中发生率占75%,其中约80%是病毒性的,它们引起急性上呼吸道感染(upper respiratory tract infections,URTI),如鼻炎、咽炎和喉炎等.随着URTI病情进展,可导致哮喘急性发作、急性中耳炎和下呼吸道感染(lower respiratory tract infection,LRTI),如支气管炎和肺炎[1].     

Molecular diagnosis of respiratory virus infections

The appearance of eight new respiratory viruses, including the SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009, in the human population in the past nine years has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid based amplification tests (NATs) for respiratory viruses were first introduced two decades ago and today are utilized for the detection of both conventional and emerging viruses. These tests are more sensitive than other diagnostic approaches, including virus isolation in cell culture, shell vial culture (SVC), antigen detection by direct fluorescent antibody (DFA) staining, and rapid enzyme immunoassay (EIA), and now form the backbone of clinical virology laboratory testing around the world. NATs not only provide fast, accurate and sensitive detection of respiratory viruses in clinical specimens but also have increased our understanding of the epidemiology of both new emerging viruses such as the pandemic H1N1 influenza virus of 2009, and conventional viruses such as the common cold viruses, including rhinovirus and coronavirus. Multiplex polymerase chain reaction (PCR) assays introduced in the last five years detect up to 19 different viruses in a single test. Several multiplex PCR tests are now commercially available and tests are working their way into clinical laboratories. The final chapter in the evolution of respiratory virus diagnostics has been the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance. These assays are now being multiplexed with primary detection and subtyping assays, especially in the case of influenza virus. These resistance assays, together with viral load assays, will enable clinical laboratories to provide physicians with new and important information for optimal treatment of respiratory virus infections.     

Nucleic acid hybridization in the diagnosis of viral infections

Recombinant DNA technology, including molecular cloning and nucleic acid hybridization, is now being applied to problems in clinical virology. Although viral isolation in cell culture remains the most sensitive and specific diagnostic test for many viruses, for some viruses, isolation in cell culture is lengthy or difficult or has not yet been achieved. Utilization of hybridization techniques has already resulted in important new information concerning the pathogenesis of a number of viruses, such as Epstein-Barr virus, hepatitis B virus, and human papillomavirus. In addition, time to diagnosis for viruses such as cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus can be significantly shortened to 36 to 48 hours, a great improvement over standard isolation with obvious importance for patient management. Hybridization techniques have also been applied to screening of antiviral agents. Although results of studies to date have been encouraging, significant problems remain to be solved before these techniques can be applied in a routine diagnostic laboratory. First, more sensitive assays must be developed. One approach is the generation of probes with higher specific activities. Synthesis of single-stranded probes using recombinant M13 bacteriophage as a template results in probes of higher specific activities that also cannot re-anneal to themselves because they are not complementary. Thus, more probe is available to anneal to sample DNA. Synthesis of cRNA probes that form more stable hybrids with DNA is another approach that is receiving attention. A second problem is reagent safety and stability. The most sensitive and commonly used label in the studies reviewed in this article has been 32P. With its half-life of 2 weeks, potential hazards to personnel, and disposal problems, it is probably not suitable for clinical laboratories. A major step in the development of nonradioactive, stable probes has been synthesis of biotinylated nucleotide analogues that can be efficiently incorporated into DNA or RNA. Biotinylated probes are stable for 1 to 2 years at -20 degrees C, and their use obviates the need for autoradiography, thus shortening reaction times. In addition, very high concentrations of probes can be used without the background problems encountered with radiolabels. To date, biotinylated probes have been significantly less sensitive than those labeled with 32P, but continued efforts to improve sensitivity have yielded promising results.(ABSTRACT TRUNCATED AT 400 WORDS)     

新型冠状病毒核酸临床检测要点及经验

2019年12月以来,新型冠状病毒感染引起的肺炎病例数快速攀升。因患者体内病毒核酸的存在是目前唯一的确诊依据,而目前临床上普遍反映核酸检测阳性率不够理想,故对病毒核酸检测的实验室条件、检验人员及检测过程中各个环节都有较高要求。本文着重阐述了新型冠状病毒核酸检测各个环节中的检测要点、最新的行业规范及共识内容,同时探讨临床检测中的疑难问题,以期为新型冠状病毒所致疾病的准确诊断提供理论依据。     

纳米金探针液相杂交法检测核酸序列依赖扩增产物诊断侵袭性曲霉病

目的建立纳米金探针液相杂交法检测核酸序列依赖扩增(nucleic acid sequence-based amplification,NASBA)产物,用于快速诊断侵袭性曲霉病(IA)。方法采用NASBA扩增曲霉菌属特异性18S rRNA保守序列,扩增产物与5'端经巯基化俢饰的特异性纳米金探针进行液相杂交,用该法检测106例临床血液标本(14例确诊,32例拟诊,60例未感染IA),并与GM试验结果进行比较以评价该方法的准确性。结果纳米金探针只与曲霉菌属菌株的NASBA产物出现阳性杂交反应,而其他非曲霉菌均为阴性。NASBA-纳米金液相杂交法检测106例临床标本的敏感性和特异性分别为82.61%(38/46)、81.67%(49/60),ROC曲线下面积(AUC~(ROC))为0.890。GM试验检测的敏感性和特异性分别为56.52%(26/46)和83.33%(50/60),AUCROC为0.723。结论 NASBA-纳米金液相杂交技术检测曲霉菌感染具有敏感性高、特异性强、操作简单的特点,适合实验室常规开展。     

新型冠状病毒的研究现状与实验室诊断

新型冠状病毒(SARS-CoV-2)在全球范围内的传播对世界公共健康构成了巨大威胁。目前,预防和治疗SARS-CoV-2的选择仍然非常有限。解析病毒感染宿主的致病机制及治疗性疫苗的研制迫在眉睫。与此同时,研发快速、有效的SARS-CoV-2实验室检测技术,对于实现快速确诊感染病例、排除疑似病例、促进疫情的防控与临床救治至关重要。该文首先从SARS-CoV-2的结构特征、分子遗传变异特征、致病机制及潜在的药物靶点等方面介绍了“如何认识SARS-CoV-2”;同时立足于临床检验,重点介绍了现有的病毒实验室检测技术及问题解析、即时检验及高通量病毒筛查芯片等病毒筛查新技术,回答了“如何检测SARS-CoV-2”。最后对实验室从业人员如何安全防护进行了要点总结。     

一种快速核酸提取试剂在流感病毒检测中的应用评价

目的 探讨一种快速核酸提取试剂在流感病毒检测中的适用性及有效性。方法 选取流感病毒H3亚型、乙型Yamagata系细胞株和甲型H1N1型鸡胚株各1株,对其倍比稀释液及2015年北京市流感病原学监测阳性的67份咽拭子样本,同时采用快速提取法及离心柱提取法提取病毒核酸,经实时荧光PCR法进行定性、定量检测,检测结果采用SPSS 19.0软件进行统计分析。结果 快速提取法提取细胞培养液、鸡胚培养液中流感病毒核酸的最低检测限高于离心柱法10倍,咽拭子样本则高100倍;快速提取法提取核酸后检测到的病毒载量均低于离心柱法,其中对含有病毒量较高的毒株检测到的病毒载量低于离心柱法10倍,而对病毒量较少的咽拭子样本,则低100倍;快速提取法稳定性优于离心柱法;提取后的核酸-80℃保存10 d后冻融复测,快速提取法核酸损失量大于离心柱法。67份离心柱提取法检测阳性咽拭子样本使用快速提取法提取核酸后检测,两种方法阳性符合率总计为92.54%,其中10～102拷贝/ml组,阳性符合率为76.92%,102～103拷贝/ml组,阳性符合率为92.59%,103拷贝/ml组,阳性符合率为100%。结论 快速提取法具有稳定性高、易于操作、不易污染的优势,适用于细胞培养液、鸡胚培养液等病毒含量较高的大量样本中流感病毒核酸提取,适于检测样本量大、人手不足、设备欠缺的基层实验室使用;但-80℃冻融后核酸损失量较大,不宜反复冻融后使用;传统的离心柱法对于病毒含量较少的咽拭子样本及核酸需被反复冻融使用时具有优势。     

Enzyme immunoassay for respiratory syncytial virus: Rapid detection in nasopharyngeal secretions and evaluation of isolates representing different RSV subgroups

The presence of respiratory syncytial virus (RSV) was investigated by immunofluorescent antibody (IFA) technique and by an enzyme immunoassay (EIA) in 169 samples of nasopharyngeal secretions of infants and children with acute respiratory infections. Of 31 samples positive by EIA, 25 were positive by IFA. In 24 samples from a retrospective study, RSV positive by IFA and/or tissue culture isolation (TCI), 22 were also positive by EIA. The EIA was also evaluated with 111 RSV isolates in Hep2 cell cultures representing different RSV subgroups. All were positive by EIA. © 1993 Wiley-Liss, Inc.     

Management of respiratory infections in the elderly

Respiratory infections are common at all ages but are particularly sinister among the elderly because of the fragility and chronic comorbidity associated with this age group. The three types of respiratory infection in the elderly are community-acquired pneumonia, acute exacerbation of chronic obstructive pulmonary disease and nonpneumonic respiratory tract infection. The etiology of these three types of infection includes classic bacteria, atypical pathogens and respiratory viruses. The relative frequency of each of the etiological groups as the causative agent of the infection varies significantly among these types of infection, but in all three types a significant proportion of infections involves more than one pathogen. The causative agent of respiratory infection in the elderly cannot be determined on the basis of clinical manifestation or the results of routine imaging procedures or laboratory tests. Thus, initial antibiotic therapy in these patients should be empiric, based on accepted guidelines. In recent years, the antipneumococcal fluoroquinolones have gained in stature as one of the best options to treat these infections. Pneumococcal and influenza vaccinations can reduce morbidity and mortality from respiratory infections in the elderly, so it is important that all elderly individuals are vaccinated through a structured program in the framework of primary care. The economic impact of respiratory infections in the elderly is primarily associated with the requirement for hospitalization in many of the cases. Any action that can reduce hospitalization rates has important economic ramifications. In light of the difficulty in reaching an early etiologic diagnosis in respiratory infections, it is essential to invest in the development of a compact diagnostic kit for the early stages of the disease, which could change reality in this important area of medicine.     

Profile of the Alere i Influenza A & B assay: a pioneering molecular point-of-care test

Introduction: The Alere i Influenza A & B assay incorporates the Nicking Enzyme Amplification Reaction technique on the Alere i instrument to detect and differentiate influenza virus (Flu) A and B nucleic acids in specific specimens.

Areas covered: The Alere i Influenza A & B assay was cleared by the US Food and Drug Administration for use with nasal swabs (NS) and nasopharyngeal swabs, either directly or in viral transport medium. Notably, direct use on NS was the first ever CLIA-waived nucleic acid-based test. Previously published evaluations have reported sensitivities and specificities of 55.2–100% and 62.5–100% for Flu A and 45.2–100% and 53.6–100% for Flu B, respectively.

Expert commentary: The Alere i Influenza A & B assay provides a rapid and simple platform for detection and differentiation of Flu A and B. Efforts are expected to further improve sensitivity and user-friendliness for effective and widespread use in the true point-of-care setting.     

粪便Gene Xpert MTB/RIF检测对HIV阴性肠结核的早期快速诊断价值

目的:评价Gene Xpert结核分枝杆菌/利福平(MTB/RIF,简称"Xpert")在人类免疫缺陷病毒(HIV)阴性肠结核患者早期快速诊断中的价值。方法:选取2018年1月至2019年6月在山东省胸科医院、北京胸科医院、苏州市第五人民医院3家医院就诊的254例HIV阴性疑似肠结核患者。采集所有患者的粪便标本,每份标本分别进行Xpert检测、涂片查抗酸杆菌、BACTEC MGIT 960(简称"MGIT 960")分枝杆菌培养。以临床诊断为标准,评价Xpert检测粪便标本的敏感度和特异度,若培养结果为阳性,则对菌株进行利福平药物敏感性检测,并对Xpert检测结果与MGIT 960药敏检测结果一致性进行分析。结果:254例患者中139例(54.7%)临床诊断为肠结核,115例(45.3%)临床诊断为非肠结核。139例肠结核患者中,60例(43.2%)MGIT 960分枝杆菌培养阳性;79例(56.8%)按临床表现、肠镜检查结果、抗结核治疗效果等临床诊断为肠结核。以临床诊断为标准,Xpert检测粪便标本的敏感度[60.4%(84/139)]优于抗酸杆菌涂片敏感度[13.2%(17/139)],两者敏感度比较有统计学差异(P0.001),Xpert检测特异度为100.0%(84/84);MGIT 960分枝杆菌培养敏感度为43.2%(60/139),与Xpert检测比较有统计学差异(P=0.004)。结核分枝杆菌(MTB)培养阳性患者中,Xpert检测的敏感度为98.3%(59/60)。在涂片和培养均为阴性的肠结核患者中,Xpert检测的阳性率为31.6%(25/79)。结论:Xpert检测粪便标本中MTB较抗酸杆菌涂片和MGIT 960分枝杆菌培养敏感度更高,并且可以同时检测利福平耐药性,为早期快速诊断肠结核提供细菌学依据。     

Quality assessment in general practice: diagnosis and antibiotic treatment of acute respiratory tract infections

Abstract

Objective: To investigate areas in need of quality improvement within the diagnostic process and antibiotic treatment of acute respiratory tract infections (RTIs) in Danish general practice by using quality indicators (QIs).

Design and setting: During a 4-week period in winter 2017, a prospective registration of patients diagnosed with RTIs was conducted in general practice in two regions of Denmark.

Subjects: Throughout the registration period each patient with symptoms of an RTI was registered. Information about age, symptoms and findings, duration of symptoms, the use and result of clinical tests, allergy towards penicillin, referral to secondary care and the antibiotic given were recorded.

Main outcome measures: Values and acceptable ranges for QIs focusing on the diagnostic process, the decision to prescribe antibiotics and the choice of antibiotics for patients with RTIs.

Results: Regarding the diagnostic process nearly all QIs for patients diagnosed with acute pharyngotonsillitis and pneumonia fell within the acceptable range. Contrarily, the diagnostic QIs for patients with acute otitis media and acute rhinosinusitis were outside the acceptable range. All indicators designed to measure overuse of antibiotics were outside the acceptable range and nearly all indicators assessing if patients were sufficiently treated fell within the acceptable range. QIs assessing use of the recommended type of antibiotic were only within the acceptable range for patients diagnosed with acute pharyngotonsillitis.

Conclusion: The findings indicate an overuse of antibiotics for RTIs in Danish general practice. Especially management of acute rhinosinusitis and acute bronchitis should be targeted in future quality improvement projects.

• KEY POINTS:

• To improve antibiotic prescribing in general practice it is important to focus on both the diagnostic process and the prescribing patterns.

• The findings indicate an overuse of antibiotics for acute respiratory tract infections in Danish general practice.

• Especially the diagnostic process and antibiotic prescribing patterns for acute rhinosinusitis and acute bronchitis could benefit from future quality improvement interventions.