医院实验室样本的生物安全管理问题及对策

2019年底暴发的新冠肺炎疫情给医院实验室样本的生物安全管理带来了全新考验.2021年正式实施的《中华人民共和国生物安全法》也对医院实验室样本的管理提出了更高的要求.为适应新形势、新要求,不断做好相关样本的生物安全管理工作.本文从医院实验室样本的生物安全管理的特点、医院实验室样本的生物安全管理的问题、规范医院实验室样本的生物安全管理三方面进行了研究,以期为医院实验室样本管理做出贡献.     

影响SARS-CoV-2疑似感染者检出率的多因素分析

目的 由于轻症和无症状携带者的病毒载量较低以及试剂敏感性的影响,新型冠状病毒(severe acute respiratory syndrome coronavirus-2,SARS-CoV-2)的检测常会出现假阴性.因此分析试剂性能和该群体的病毒分布特征,选择性能较优试剂并优化采样部位十分必要.方法 我们收集了新诊断的SARS-CoV-2感染轻症患者和无症状携带者的咽拭子、血浆、尿液和肛拭子样本.分析不同类型样本的病毒载量差异,同时分析试剂性能和样本灭活对检出率的影响.结果 早期批号检测试剂的检出率低,重复性差.SS2试剂检测结果重复性较好(CV= 1.01％).随着批号的增加,SS试剂(ORF1ab)和ZJ试剂(ORF1ab)检测结果的检出率和重复性有提高的趋势.与未灭活相比,56℃灭活1h未显著降低SARS-CoV-2病毒完整RNA含量(34.31± 3.80 vs 35.43±3.99,t = 0.61,P = 0.55).不同部位标本分析显示,咽拭子的阳性检出率高于肛拭子(77.78％vs 44.44％,x2 =4.208,P = 0.040),血浆和尿液中未检出病毒核酸.结论 在使用新试剂或改变试剂批号前,实验室应进行性能验证,以满足检测要求.对病毒保存液中的样本进行加热灭活处理,不会影响检测结果.建议在轻症患者和与确诊患者有密切接触的疑似人群中,同时采集咽拭子和肛拭子来提高检出率.     

依据CNAS-GL039在Bio-rad CFX96荧光定量PCR仪进行SARS-CoV-2核酸检测的性能验证

目的 对本实验室实时荧光聚合酶链反应(RT-PCR)方法检测SARS-CoV-2进行方法学评价,建立本实验室分子诊断定性检验程序性能验证的标准化方法,为日后性能验证工作提供参考.方法 依据CNAS-GL039和厂家说明对SARS-CoV-2核酸检测进行符合率、精密度、检测限、交叉反应和抗干扰能力的验证,实验结果在厂家声称的范围内或是满足本实验室的执行标准为性能验证通过.结果 国家临检中心室间质评10个质控样本的定性检测结果均符合,符合率验证通过;批内精密度变异系数(CV)为ORF1ab基因:1.11％、N基因:0.95％;批间精密度变异系数(CV)为ORF1ab基因:0.89％、N基因:1.99％、阴性内参:0.54％;变异系数满足厂家声明的≤5％;精密度验证通过;定值质控品S1(2.01×103 copies/mL)稀释至厂家声明的检测限500 copies/mL;靶基因检出率100％,检测限符合要求;病原体交叉反应检测结果均为阴性,干扰物质检测结果均为弱阳性,分析特异性符合要求.结论 SARS-CoV-2核酸检测的符合率、精密度、检测限、交叉反应和抗干扰能力的验证,结果均符合检测要求.     

医学实验室生物安全管理的规范化

实验室生物安全是社会安全的一部分,实验室安全管理必须警钟长鸣。本文根据我国医学实验室生物安全的现状和特点,结合国家实验室安全管理的法律法规,提出加强实验室生物安全管理应采取的一些安全防范措施。     

一起新型冠状病毒暴发疫情的基因组特征与溯源分析

目的:了解引起河南省2021年11月COVID-19暴发疫情的新型冠状病毒(SARS-CoV-2)基因组特征,分析核苷酸和氨基酸变异情况并进行溯源。方法:收集病例急性期咽拭子标本,应用实时荧光RT-PCR方法对SARS-CoV-2核酸进行检测。选取SARS-CoV-2核酸阳性样本进行高通量基因序列测定和全基因组序列比对...     

中山市医疗机构实验室生物安全管理现状调查

目的了解中山市医疗机构实验室生物安全管理现状,为进一步加强和规范医疗机构实验室生物安全管理工作提供依据。方法设计《实验室生物安全自查表》,对中山市各类医疗卫生机构共29家单位进行问卷调查,使用Excel软件对数据进行统计分析。结果中山市医疗机构生物安全实验室在安全管理制度的制定、防护设备使用方面与生物安全管理要求尚有一定差距．尤其在菌毒种管理和运送方面存在安全隐患。结论中山市生物安全实验室的管理工作需要加强,应尽快建立健全的实验室生物安全管理体制。     

病理科生物安全问题的思考与对策

病理科的质量管理是质量保证的基本前提.它不仅包括严格遵循科学的工作流程,遵守各项操作的规章制度,实验室的环境和生物安全也是其中不可忽略的一个部分.生物安全是一个广义的概念,病理科的生物安全是指在病理制片过程中,大量使用的甲醛、二甲苯、丙酮、氨水、冰醋酸、DAB显色剂等有毒、致癌的化学物品及其废弃物对实验室环境造成的不安全的防范,以及工作人员接触到的患者的组织标本、血液标本、尿液标本、痰液标本、体液标本、尸体等对工作人员健康造成的不安全的防范 .这些不安全因素轻者造成对工作人员的伤害,重者殃及社会,造成环境的污染和大范围传染病的流行,都直接和间接的影响了病理科的质量安全体系.因此病理科生物安全也是值得我们认真分析和?屯     

新型冠状病毒肺炎病例粪便及咽拭子核酸平行检测结果比对分析

目的:通过平行比对确诊患者咽拭子和粪便样本中SARS-CoV-2核酸检测的阳性率,为实验室诊断提供参考。方法:收集解放军总医院41例确诊住院患者的粪便和咽拭子样本,采用荧光定量PCR方法进行平行检测,比较两种不同类型样本的阳性率和一致性。结果:41例患者中,咽拭子样本共检出9例阳性,占30%,粪便检出5例阳性,占12....     

茂名市疾病预防控制机构实验室生物安全现状分析

目的全面掌握广东省茂名市疾病预防控制机构实验室的生物安全现状,为卫生部门进行生物安全管理提供决策依据。方法采用发放调查表和现场核实相结合的方法对全市疾病预防控制机构实验室生物安全现状进行调查分析。结果全市7个疾病预防控制机构中有6个建立了微生物实验室,其中4个为2级生物安全实验室;6家实验室虽然都配备了一定的生物安全设备,但与国家的标准相比还有比较大的差距;6家实验室均制定了一定的实验室生物安全管理制度,但制度落实不到位,实验室的生物安全日常管理工作做得也较差;6家实验室都能正确处理医疗废弃物,但生物安全培训工作开展得不够,人员健康监护做得也不合要求。结论应加强对茂名市疾病预防控制机构实验室相关人员的培训,提高他们的生物安全意识,加大实验室生物安全硬件投入,制定完善的生物安全管理规范和加大监管力度。     

医学神经生物学实验室安全管理探究

高校实验室安全问题逐渐得到了各大高校的重视。以人为本的实验室生物安全管理体系是一个实验室正常运转的前题。顺应国家和学校的实验室安全管理的响应,系统梳理了神经生物学实验室中存在的隐患,并提出了可行的解决方案。通过开展安全教育、制定安全管理制度、建立规范操作流程和建立广泛的群众监督制度从思想上、制度上以及管理规范上对神经生物学实验室的医学生进行约束,旨在提高学生的实验室安全意识,培养良好的实验操作习惯,确保医学生在神经生物学实验室中的安全。     

Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation

Virus Genes - Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in...     

Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories

Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is <1%; however, fatality rates in those with severe clinical disease have been reported as high as 29%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. An existing one-step real-time RT-PCR (rRT-PCR) assay was modified for quick virus inactivation for use in BSL-2 laboratories, evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 10^6.5 plaque forming units/ml, or up to 10¹⁰ viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.     

Value of anal swabs for SARS-COV-2 detection: a literature review

Facing the unprecedented global public health crisis caused by coronavirus disease 2019 (COVID-19), nucleic acid tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the gold standard for diagnosing COVID-19. The asymptomatic carriers were not suspected of playing a significant role in the ongoing pandemic, and universal nucleic acid screening in close contacts of confirmed cases and asymptomatic carriers has been carried out in many medium- and high-risk areas for the spread of the virus. Recently, anal swabs for key population screening have been shown to not only reduce missed diagnoses but also facilitate the traceability of infectious sources. As a specimen for the detection of viruses, the goal of this paper is to briefly review the transmission route of SARS-CoV-2 and the necessity of using anal swabs for SARS-CoV-2 screening to minimize transmission and a threat to other people with COVID-19.     

Evaluate severe acute respiratory syndrome coronavirus 2 infectivity by pseudoviral particles

Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans in late 2019, it has rapidly spread worldwide. To identify the biological characteristics of SARS-CoV-2 in a normal laboratory environment (biosafety level 2 [BSL-2]), a lentiviral-based nucleocapsid was used to carry the spike protein of SARS-CoV-2 onto the surface of pseudoviral particles as a surrogate model to evaluate the infective characterization of SARS-CoV-2. This study indicated that SARS-CoV-2 has extensive tissue tropism for humans and may infect monkeys and tree shrews but not rodents. More importantly, the use of pseudoviral particles in this study allows rapid assessment of neutralizing antibodies in serum in a BSL-2 laboratory. This study will provide a quick and easy tool for evaluating neutralizing antibodies in the serum of recovering patients and assessing the potency of candidate vaccines.     

Automated nucleic acid isolation in viral molecular diagnostics: evaluation of the QIAsymphony SP

The Qiagen QIAsymphony SP is a high-throughput (up to 96 samples per run), fully-automated nucleic acid isolation system. It was implemented in the authors' laboratory to cope with the high demand for pandemic H1N1 influenza testing in 2009. This study evaluated the QIAsymphony SP for viral nucleic acid isolation from quality control materials, pure cultures and various clinical specimens. The effect of varying sample volume on detection sensitivity was investigated using serial 10-fold dilutions of pure viral specimens and target nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Little variability in detection sensitivity was observed for all the viral targets tested, although variation in cycle threshold values was apparent in some cases. Importantly, pathogens were detectable over a broad concentration range and from diverse clinical specimens. Removal of PCR inhibitors was generally effective, as demonstrated by detection of viral nucleic acids and/or internal controls. The results demonstrate that the QIAsymphony SP is suitable for use in routine virology molecular diagnostics, and provides a high-throughput capacity, which is needed in peak seasons of infection or in centralised laboratories.     

Development, technical performance, and clinical evaluation of a NucliSens basic kit application for detection of enterovirus RNA in cerebrospinal fluid

The combination of nucleic acid sequence-based amplification and electrochemiluminescence detection was used to develop an internally controlled, highly sensitive and specific assay for the detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF). The analytical performance of the assay was determined using both in vitro-transcribed EV RNAs and viral culture isolates. The sensitivity of the assay was 10 EV RNA copies per amplification reaction. The assay detected all enteroviral isolates tested with no cross-reactivity to 21 nonenteroviral species, including rhinovirus and parechovirus. The clinical performance of the assay was evaluated by testing 992 CSF specimens collected from adult and pediatric patients. NucliSens EV results from a subset of 327 CSF samples were compared to viral culture of nasopharyngeal specimens and rectal swabs (n = 195) and/or CSF (n = 212). Of the 212 CSF samples, 96 samples were positive by either the NucliSens EV assay (94/96; 97.9%) or culture (63/96; 65.6%), and 61/96 (63.5%) were positive by both methods. The inclusion of an EV-specific internal control monitored the entire process, including the efficiency of nucleic acid extraction, amplification, and detection. In total, only five blood-clotted CSF samples (0.5%) were inhibited. The NucliSens EV assay demonstrated superior sensitivity over viral culture (P < 0.001), excellent specificity, clear delineation of positive samples, and minimal amplification inhibition.     

Homogenization with heat treatment: A cost effective alternative to nucleic acid extraction for herpes simplex virus real-time PCR from viral swabs

Most laboratories use expensive commercial kits to purify nucleic acids and remove PCR inhibitors that may be present in clinical specimens. In this study a simple homogenization with heat treatment of herpes simplex virus types 1 and 2 (HSV-1/2) was shown to be equivalent to commercial kit-based nucleic acid extraction methods. With a cost of less than $1 USD per extraction, this method provides an economical, rapid, and effective method to recover HSV-1/2 DNA from swabs suitable for real-time HSV PCR.     

High-speed large-scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled to high-content screening

ObjectivesA novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the current coronavirus disease 2019 global pandemic. Only a few laboratories routinely isolate the virus, which is because the current co-culture strategy is highly time-consuming and requires a biosafety level 3 laboratory. This work aimed to develop a new high-throughput isolation strategy using novel technologies for rapid and automated isolation of SARS-CoV-2.MethodsWe used an automated microscope based on high-content screening (HCS), and we applied specific image analysis algorithms targeting cytopathic effects of SARS-CoV-2 on Vero E6 cells. A randomized panel of 104 samples, including 72 that tested positive by RT-PCR and 32 that tested negative, were processed with our HCS strategy and were compared with the classical isolation procedure.ResultsThe isolation rate was 43% (31/72) with both strategies on RT-PCR-positive samples and was correlated with the initial RNA viral load in the samples, in which we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with the HCS strategy, where 80% (25/31) of the positive samples were recovered by the third day of co-culture, compared with only 26% (8/30) with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive samples (31 with HCS versus 27 with classic strategy) after 1 week of co-culture.ConclusionsThis system allows the rapid and automated screening of clinical samples with minimal operator workload, which reduces the risk of contamination and paves the way for future applications in clinical microbiology, such as large-scale drug susceptibility testing.