Viral load distribution in SARS outbreak

An unprecedented community outbreak of severe acute respiratory syndrome (SARS) occurred in the Amoy Gardens, a high-rise residential complex in Hong Kong. Droplet, air, contaminated fomites, and rodent pests have been proposed to be mechanisms for transmitting SARS in a short period. We studied nasopharyngeal viral load of SARS patients on admission and their geographic distribution. Higher nasopharyngeal viral load was found in patients living in adjacent units of the same block inhabited by the index patient, while a lower but detectable nasopharyngeal viral load was found in patients living further away from the index patient. This pattern of nasopharyngeal viral load suggested that airborne transmission played an important part in this outbreak in Hong Kong. Contaminated fomites and rodent pests may have also played a role.     

用逆转录聚合酶链反应方法检测SARS冠状病毒

目的 应用双位点逆转录聚合酶链反应(RT-PCR)和改良巢式实时RT-PCR方法检测临床标本中SAILS冠状病毒(SAILS-CoV)核酸,以提高检测的可靠性和灵敏度。方法 对2003年杭州市3例严重急性呼吸综合征(SARS)临床确诊患者、4例疑似和27名医学观察者的咽拭子标本用巢式实时RT-PCR检测SAILS-CoV核酸,对阳性扩增产物进行核酸序列测定,同时对3例患者的标本应用半巢式RT-PCR检测另一位点SARS-CoV核酸片段,并应用常规实时RT-PCR技术及改良实时RT-PCR技术进行检测。结果 3例患者的巢式RT-PCR检测结果2例阳性,4例疑似和27名医学观察者均为阴性;阳性扩增产物的核酸序列与SAILS-CoV基因组相应部分序列完全一致。3例患者标本的另一位点的半巢式RT-PCR及实时RT-PCR结果均相同。在检测低拷贝数标本时,常规实时RT-PCR技术在约35个循环后出现弱阳性信号,但改良的巢式实时RT-PCR技术可使强阳性信号在10个扩增循环后出现。结论 双位点RT-PCR方法是提高检测准确性的有效方法,改良巢式实时RT-PCR技术较常规实时RT-PCR技术具有更高的灵敏度。     

检测SARS冠状病毒的套式RT-PCR方法的建立与初步应用

[目的]建立套式RT-PCR检测SARS冠状病毒的方法。[方法]从广东地区SARS患者尸解肺组织、咽拭子、嗽口水及本实验室分离的SARS冠状病毒（SAPS—CoV）组织培养上清中提取RNA，利用针对SARS冠状病毒多聚酶基因的特异性引物，进行逆转录及套式PCR扩增。扩增出的阳性片段连接入pGEM—T载体中，测序后比较其与其他已知SARS冠状病毒的同源性。[结果]从SARS患者尸解肺组织、咽拭子、嗽口水及SARS冠状病毒组织培养上清中均可扩增出阳性片段，经测序后证实是SARS冠状病毒特有序列。[结论]针对SARS冠状病毒多泵酶基因所建立的套式RT—PCR方法适用于临床标本及组织培养标本中SARS冠状病毒的检测。     

SARS恢复期患者血液与排泄物中SARS-CoV RNA的检测

目的应用巢式逆转录聚合酶链反应(RT—PCR)检测严重急性呼吸综合征(SAILS)恢复期患者血液与排泄物中的病毒RNA。方法对连续3次收集的23例确诊SARS恢复期(病程≥21天)患者的血、尿、痰、粪便标本进行核酸提取，设计特异性内外引物，使用巢式RT—PcR的方法进行病毒RNA检测。结果通过巢式RT—PCR，共检测到6份阳性标本，其中粪便标本中检测到4份，检出率为5．8％；痰标本中检测到2份，检出率为2．9％。在尿液和血液标本中未检测到病毒．RNA。结论个别SARS恢复期患者排泄物中有SARS病毒RNA存在，因此对恢复期患者的排泄物应慎重处理，对其引起SARS的再次传播的危险性需要进一步评估。     

SARS患者某定点收治医院空气样本中SARS-CoV及其RNA的检测

目的 了解SARS某定点收治医院空气中的SARS病毒污染情况及SARS病毒在空气中的分布和传播特性。方法 采用FA-2型空气微生物采样器在病房区及病房阳台连续采样。将采集到的空气样本洗脱后分别采用细胞分离和逆转录-聚合酶链反应分析,并对细胞分离阳性者作进一步的系列分析鉴定。结果 SARS患者某定点收治医院病房区及阳台空气样本中均有部分PCR结果阳性,SARS阳性率病房区为29％,阳台为20％;其中一份样品中分离出活性病原体,并稳定传代;经免疫荧光染色鉴定阳性,RT-PCR扩增产物的测序结果显示该病原体与已知SARS-CoV的同源性在98％以上。结论 SARS急性期后期与恢复期早期的患者仍然从呼吸道排毒,病毒在距传染源周围1m之内的空气中具有感染活性,在此范围之内存在气溶胶传播的潜在威胁。     

医院污水中SARS冠状病毒核酸的检验

目的　了解收治SARS病人的定点医院污水中是否含有病毒和病毒核酸。方法　采用载阳电荷滤材吸附浓集污水中的SARS冠状病毒 ,消毒前浓集污水 2 5L ,消毒后 2 5～ 5 0L ;采用VERO细胞培养及RT -PCR技术检测SARS冠状病毒。结果　建立的阳电荷浓集方法对污水中加标f2 噬菌体回收率平均 12 7% ,而SARS冠状病毒回收率只有 1 0 2 %。消毒前在 30 9医院和小汤山医院污水的6d监测中均检测出SARS冠状病毒核酸 ;消毒后 ,在 30 9医院只有 3d监测出病毒核酸 ,其余样品均未检出病毒核酸 ;两医院所有的污水回收液中均未检出活病毒或具有感染性的病毒。结论收治SARS病人医院污水中含有SARS冠状病毒核酸 ,但没有检出活病毒或具有感染性的病毒 ,因此 ,在当时条件下 ,医院污水 ,尤其是消毒后污水传播SARS冠状病毒可能性较小。     

Merkel Cell Polyomavirus in Respiratory Tract Secretions

Merkel cell polyomavirus (MCPyV), associated with Merkel cell carcinoma, was detected in 27 of 635 nasopharyngeal aspirate samples by real-time PCR. MCPyV was more commonly found in adults than in children. Presence in the upper respiratory tract may be a general property of human PyVs.     

Encephalitis-Associated Human Metapneumovirus Pneumonia in Adult,Australia

Human metapneumovirus pneumonia, most commonly found in children, was diagnosed in an adult with encephalitis. This case suggests that testing for human metapneumovirus RNA in nasopharyngeal aspirate and cerebrospinal fluid samples should be considered in adults with encephalitis who have a preceding respiratory infection,     

实时荧光定量RT-PCR对新型冠状病毒3种不同生物学样本检出率的比较

目的用实时荧光定量RT-PCR(Real-time RT-PCR)方法对新型冠状病毒肺炎患者的鼻咽拭子样本、粪便样本、痰液标本进行病毒核酸检测,并比较其检出率。方法采用成都市公共卫生临床医疗中心确诊的处于病程前期的新型冠状病毒患者的鼻咽试子样本(242份)、粪便样本(78份)、痰液标本(230份),其中包括同一时间采集患者的两种不同生物学样本(55份)和同一时间采集患者三种不同生物学样本(44份),均应用Real-time RT-PCR方法对病毒核酸进行检测,比较3种标本单独和联检的阳性检出率。结果鼻咽拭子样本、粪便样本、痰液样本中SARS-CoV-2核酸阳性检出率分别为32.64%、39.74%和68.26%,差异有统计学意义(P<0.05)。三种样本类型联合检测的总阳性率(95.45%)大于两种样本联合。结论本研究中三种生物学样本单独检测阳性率以痰液检出率最高,粪便样本次之,鼻咽拭子样本最低,三种样本联合检测将大大提高COVID-19的检出率,为COVID-19实验室检测样本的选择和提高核酸检出率提供参考。     

Detection of SARS coronavirus in patients with suspected SARS

Cases of severe acute respiratory syndrome (SARS) were investigated for SARS coronavirus (SARS-CoV) through RNA tests, serologic response, and viral culture. Of 537 specimens from patients in whom SARS was clinically diagnosed, 332 (60%) had SARS-CoV RNA in one or more clinical specimens, compared with 1 (0.3%) of 332 samples from controls. Of 417 patients with clinical SARS from whom paired serum samples were available, 92% had an antibody response. Rates of viral RNA positivity increased progressively and peaked at day 11 after onset of illness. Although viral RNA remained detectable in respiratory secretions and stool and urine specimens for >30 days in some patients, virus could not be cultured after week 3 of illness. Nasopharyngeal aspirates, throat swabs, or sputum samples were the most useful clinical specimens in the first 5 days of illness, but later in the illness viral RNA could be detected more readily in stool specimens.     

新型冠状病毒RNA核酸的检测方法研究及应用

[目的 ]　研究和建立引起严重的急性呼吸道综合征 (SARS)的新型冠状病毒RNA核酸的检测方法。　 [方法 ]　使用一步法巢式逆转录聚合酶链扩增法 (RT -PCR)。　 [结果 ]　采自临床留观、疑似和临床诊断病人的 10 46份咽漱液 /咽拭子样品中 ,3份样品SARS冠状病毒RNA核酸为阳性 ;在 482份血清样品中 ,有 5份为阳性。　 [结论 ]　一步法巢式RT -PCR能检测患者急性期咽漱液 /咽拭子和血清样品中的新型冠状病毒RNA核酸 ,但样本采集的时间和咽漱液 /咽拭子采集的质量直接影响检测结果     

Laboratory diagnosis of Mycoplasma pneumoniae infection. 2. Comparison of methods for the direct detection of specific antigen or nucleic acid sequences in respiratory exudates

The efficiency of the direct detection of Mycoplasma pneumoniae in respiratory exudates by an antigen capture, indirect enzyme immunoassay (Ag-EIA), has been compared with its detection with a cDNA probe ('Gen-Probe assay') directed against the specific ribosomal RNA sequences of the organism ('Mycoplasma pneumoniae Rapid Diagnostic System', Gen-Probe, San Diego, California). Both assays showed excellent specificity against a range of mycoplasma species suspended in negative nasopharyngeal aspirates; only M. pneumoniae and M. genitalium reacted. In experiments with graded doses of viable M. pneumoniae cells suspended in negative nasopharyngeal aspirate, the Gen-Probe assay was more sensitive than Ag-EIA; detection limits were respectively 2 X 10(3) c.f.u./ml (3.2 X 10(5) genomes) and 2.5 X 10(4) c.f.u./ml (4 X 10(6) genomes); detection levels 10-100 times less sensitive than culture. The two assays were also tested on nasopharyngeal aspirates or sputum specimens from 90 patients with respiratory infection; 67 of these were culture- or seronegative for M. pneumoniae and 23 were culture- or seropositive. Ag-EIA detected 21 (91%) of the latter but the Gen-Probe assay detected only 5 (22%). Both assays were negative with the 67 culture-/sero-negatives; there were no Gen-Probe assay positive/Ag-EIA negatives. Overall, it is concluded that although Ag-EIA and the Gen-Probe assay are effective substitutes for culture as a diagnostic procedure, there is a significant problem with samples which are culture-negative and from patients who have good serological evidence of current infection. Possible reasons for the disparity between the two assays are advanced.     

Hep-2细胞在严重急性呼吸综合征病原学检测中的应用研究

目的 应用人咽喉上皮癌细胞(Hep-2)对严重急性呼吸综合征(SARS)病例标本进行病原体检测与研究，为该病的预防与控制提供依据。方法 采用Hep-2细胞培养法，对2003年l～2月广东省收集的SARS病例的咽拭子等标本分离致病病原体，并应用电镜形态学、逆转录．多聚酶链反应(RT-PCR)扩增部分基因进行序列分析，并对分离的病原体进行研究。结果 132例SARS病例的3l份咽拭子、100份痰液和l份尸检肺组织标本中有73份标本致单层细胞出现“蚀斑病变”，细胞病变阳性率为55．3％，其中48h阳性率为36．4％。采用巢式PCR(Nested-PCR)对其中25份出现“蚀斑病变”细胞培养物进行检测，能扩增出冠状病毒的特异片段；对中2-18、中2-19等6份Nested-PCR产物进行基因序列分析，结果 显示其核苷酸序列与国内外公布的结果一致，氨基酸序列与已知的人或动物冠状病毒的同源性在58％～76％之间。通过电镜在SARS病例标本(中2-10)病变细胞及l份尸检肺组织标本中观察到衣原体样颗粒和病毒样颗粒。结论 从接种SARS病例标本的Hep-2细胞病变培养物中证实有冠状病毒。     

SARS in hospital emergency room

Thirty-one cases of severe acute respiratory syndrome (SARS) occurred after exposure in the emergency room at the National Taiwan University Hospital. The index patient was linked to an outbreak at a nearby municipal hospital. Three clusters were identified over a 3-week period. The first cluster (5 patients) and the second cluster (14 patients) occurred among patients, family members, and nursing aids. The third cluster (12 patients) occurred exclusively among healthcare workers. Six healthcare workers had close contact with SARS patients. Six others, with different working patterns, indicated that they did not have contact with a SARS patient. Environmental surveys found 9 of 119 samples of inanimate objects to be positive for SARS coronavirus RNA. These observations indicate that although transmission by direct contact with known SARS patients was responsible for most cases, environmental contamination with the SARS coronavirus may have lead to infection among healthcare workers without documented contact with known hospitalized SARS patients.     

医院SARS重症监护病区空气传播途径的研究

目的对SARS重症监护(ICU)病区患者及周围空气等进行研究,为SARS的传播途径提供帮助和证据支持.方法用日本产安德森空气采样器对SARSICU病区重复2次,对5个病房的空气和病区空调的过滤网、冷凝水取样,分别用RT-PCR和病毒分离培养方法检测.结果SARSICU病房空气样本中查出SARS病毒,并发现病毒既存在大的生物颗粒上,也存在较小的生物颗粒和病房外空调机过滤网上,表明在ICU病区SARS病毒可能存在近距离飞沫,也存在小的气溶胶的生物颗粒上,随空气在一定范围、一定的时间内传播.结论在SARSICU病房、病区空气中可能存在SARS病毒,所以加强病区的空气通风与ICU医务人员的呼吸道、黏膜等个人防护是防止SARS传播的关键.     

小汤山医院SARS病房内外空气中SARS病毒及其RNA的检测

目的：了解小汤山医院病区空气中的SARS病毒污染情况。方法：2003年6月4日-8日采用FA-Ⅱ型空气微生物采样器在病房、内走廊、护士站和病房排气口下风向5m处4个地点连续采样4d。之后进行空气样本的洗脱、Vero-E6细胞培养、RT-PCR及序列测定。结果：小汤山医院每天采集19个空气样品，4d共采集76个空气样品。病房、内走廊、护士站和病房排气口下风向5m处4个地点均有病毒核酸检出，其中以病房排气口下风向5m处的阳性率最高(58．3％)，护士站相对较低(25．0％)；病房(52．1％)和内走廊(50．0％)的阳性率相当。结论：小汤山医院空气中SARS病毒的污染比较严重，急性后期的病患仍然从呼吸道大量排毒；但在病房室内外的空气中没有检测到活的SARS病毒，初步评估认为，SARS病人病房采取通风和消毒的措施对降低室内污染程度是非常有益的；而空气消毒和环境因素对SARS病毒的存活有较大的影响，故SARS病人病房排出的空气对周边环境造成的危害很小。     

SARS患者粪便、痰标本病毒RNA阳性检出率与病程变化的关系

目的 探讨严重急性呼吸综合征(SARS)患者粪便、痰标本中病毒RNA阳性检出率与病程变化的关系。方法 采取一步法逆转录聚合酶链反应(RT-PCR)对不同病程的临床标本进行SARS冠状病毒(SARS-associated coronavirus,SARS-CoV)RNA的扩增和序列测定,并采用趋势X~2检验探讨标本阳性检出率与不同发病时间的关系。结果 被检样品中扩增出的特异性片段,经测序验证为SARS-CoV序列(同源性100％)。在检测的临床标本中,粪便标本阳性检出率最高(21.55％)。趋势X~2检验显示SARS患者粪便和痰标本中病毒阳性检出率均随着发病时间的增长而下降,X~2=12 55和16.408,P=0.0004和P=0.000 05。结论 一步法RT-PCR可以有效检测SARS患者临床标本存在的SARS-CoV;SARS患者粪便、痰标本RT-PCR阳性率随着发病时间的增长而下降。     

广州市严重急性呼吸综合征患者血清IgG抗体滴度变化规律和隐性感染情况调查

目的　了解严重呼吸道综合征 (SARS)患者血清中特异性IgG抗体滴度变化规律和健康人群感染SARS冠状病毒的状况 ,并为临床诊断和血清流行病学调查提供可靠依据。方法　 2 0 0 3年 5～ 6月份选择发病 1、 2、 3、 4、 5月的SARS患者 ,及无临床症状的健康人群作为调查对象 ,用间接ELISA法检测SARS冠状病毒IgG抗体。 结果　对 5 33份发病后 14～ 15 3d的SARS患者的血清进行IgG抗体检测 ,其中 2 6 5例阳性。发病 1个月IgG抗体GMT为 5 4 96 ,发病 2个月为 12 2 5 6 ,发病3个月为 135 2 5 ,发病 4个月为 15 2 17,发病 5个月为 183 79。SARS患者血清IgG抗体随着发病时间的延长而呈上升趋势。发病 1个月与 2、 3、 4、 5个月抗体水平相比差异有统计学意义 (P <0 0 1) ,发病 2、 3、 4、 5个月抗体水平相比差异无统计学意义。对照人群中共 2 16人份 ,仅发现 1例IgG抗体阳性。 结论　SARS发病 1个月IgG抗体水平比较低 ,1个月后迅速上升 ,第 4、 5个月仍持续在较高抗体水平。正常人群隐性感染水平低