Detection of Specific SARS-CoV-2 IgM and IgG Antibodies in COVID-19 and Its Clinical Application

Xiao-mei LUO; Jing WANG; Ya ZHANG; Cheng-jun XUE; Kun YANG; Li-xiang XIANG; Hai-jun LI; Mei HAN; Mei LI; Ren-ni DENG; Fu-long LUO; Chang-hua HU; Yao-kai CHEN

doi:10.13718/j.cnki.xdzk.2020.03.005

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To investigate the diagnostic value of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to SARS-CoV-2 in the coronavirus disease 2019 (COVID-19), a retrospective study was made, in which blood samples were collected from patients of Chongqing Public Health Medical Center from February 4, 2020 to February 24, 2020, of whom 101 were cases of COVID-19, and 54 were non-COVID-19 patients with lung diseases or fever, who were used as the control. GICA (gold immunochromatography assay) was made to detect the specific IgM and IgG antibodies, with the results of RT-PCR detection as the gold standard. The sensitivity, specificity and diagnostic accuracy were calculated in different subpopulations and were used to evaluate the diagnostic value. The relationship between disease progress and disease classification was analyzed and χ2 test was made for statistical analysis. The sensitivity, specificity and diagnostic accuracy of IgM and IgG antibodies of SARS-CoV-2 were found to be 92.1%, 90.7% and 91.6%, respectively. Analysis of the relationship between the positivity of SARS-CoV-2 IgM and IgG antibodies and the course of the disease showed that there was a significant difference among different disease stages, and there was no statistically significant difference in positivity of SARS-CoV-2 IgM and IgG antibodies among different clinical types of disease. In conclusion, detection of blood SARS-CoV-2 IgM and IgG antibodies is an effective diagnostic tool for the diagnosis of COVID-19. However, there is a risk of false negativity in the early stage of disease.

Corresponding author: Yao-kai CHEN

Received Date: 04/03/2020

Available Online: 20/03/2020

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Abstract: To investigate the diagnostic value of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to SARS-CoV-2 in the coronavirus disease 2019 (COVID-19), a retrospective study was made, in which blood samples were collected from patients of Chongqing Public Health Medical Center from February 4, 2020 to February 24, 2020, of whom 101 were cases of COVID-19, and 54 were non-COVID-19 patients with lung diseases or fever, who were used as the control. GICA (gold immunochromatography assay) was made to detect the specific IgM and IgG antibodies, with the results of RT-PCR detection as the gold standard. The sensitivity, specificity and diagnostic accuracy were calculated in different subpopulations and were used to evaluate the diagnostic value. The relationship between disease progress and disease classification was analyzed and χ2 test was made for statistical analysis. The sensitivity, specificity and diagnostic accuracy of IgM and IgG antibodies of SARS-CoV-2 were found to be 92.1%, 90.7% and 91.6%, respectively. Analysis of the relationship between the positivity of SARS-CoV-2 IgM and IgG antibodies and the course of the disease showed that there was a significant difference among different disease stages, and there was no statistically significant difference in positivity of SARS-CoV-2 IgM and IgG antibodies among different clinical types of disease. In conclusion, detection of blood SARS-CoV-2 IgM and IgG antibodies is an effective diagnostic tool for the diagnosis of COVID-19. However, there is a risk of false negativity in the early stage of disease.

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目前正在暴发流行的新型冠状病毒肺炎(novel coronavirus pneumonia，NCP)又称为2019-冠状病毒病(corona virus disease 2019，COVID-19)，其病原体是严重急性呼吸综合征冠状病毒2(SARS-CoV-2)，系单股正链RNA病毒，传染性强，人群普遍对其缺乏免疫力^[1].目前，COVID-19尚缺乏特效疗法，对该疾病的预后与疫情防控，做到早发现、早诊断、早隔离、早治疗具有十分重要的意义.目前，主要使用SARS-CoV-2核酸检测作为COVID-19的病原学确诊方法^[1]，但由于样本采集与存放、检测灵敏度、病毒感染部位、RNA提取方法和核酸检测试剂盒质量问题等多方面因素，易出现假阴性结果，不易完全做到早期临床诊断^[2-4].血清学实验是临床病原学的主要诊断方法之一，其主要原理是利用病原体特异性抗原或特异性抗体对病毒特异性抗体和抗原进行检测.目前，COVID-19血清学试剂刚刚面世，如何有效地应用血清学检测方法，急需要进行评价和验证.本研究主要通过胶体金免疫层析法检测确诊COVID-19患者、非COVID-19呼吸疾病组和发热患者的SARS-CoV-2特异性IgG/IgM抗体，并分析抗体与疾病病程和疾病分型的关系，来评估SARS-CoV-2特异性IgM和IgG抗体检测在对COVID-19的临床应用价值.

1. 研究对象与方法

1.1. 研究对象

选择重庆市公共卫生医疗救治中心2020年2月4-24日收治的COVID-19患者101例作为研究对象，诊断标准为《新型冠状病毒肺炎诊疗方案》第6版.研究对象与对照具体情况见表 1，对照组均经过新冠核酸检测确诊为阴性，病例组与对照组患者的年龄和性别比较，差异无统计学意义(p＞0.05)，具有可比性.本实验经重庆市公共卫生医疗救治中心医学伦理委员会批准(批准号：2020-019-01-KY).

1.2. 研究方法

SARS-CoV-2特异性IgM和IgG抗体检测试剂由江苏美克医学技术有限公司提供.实验所需血液标本的采集保存处理和检测程序以及结果判定按照试剂说明书进行：取全血样本15 μL加入检测孔，滴入样本稀释液3滴，室温下孵育15 min，进行结果判定.出现质控线证明结果有效，出现检测线判定为阳性.

1.3. 统计学分析

使用诊断特异性、敏感度和准确度等指标对诊断价值进行评价^[5].采用SPSS 17.0统计学软件进行数据分析，组间比较采用χ²检验，以p＜0.05表示差异有统计学意义.

2. 结果

2.1. SARS-CoV-2特异性IgM和IgG抗体检测结果

101名COVID-19患者中，IgM单独阳性91例(90.1%)，IgG单独阳性63例(62.4%)，IgM和IgG抗体联合测定(IgM和IgG抗体任一阳性即确定为阳性)阳性93例，阳性率92.1%. 54名非COVID-19患者中，4名患者出现IgM阳性，1名出现IgG阳性.

2.2. SARS-CoV-2特异性IgM和IgG抗体对COVID-19的诊断效能

以RT-PCR结果为金标准，计算SARS-CoV-2特异性IgM和IgG抗体对COVID-19的诊断效能.实验结果显示，敏感度92.1%，特异度90.7%，诊断准确度91.6%，阳性似然比9.90，阴性似然比0.087，尤登指数0.83.与RT-PCR方法相比较，SARS-CoV-2特异性IgM/IgG抗体二者联合诊断COVID-2019，经χ²检验，p=0.581，p大于0.05，说明2种指标诊断2019-nCoV感染的差异无统计学意义.

2.3. SARS-CoV-2特异性IgM和IgG抗体阳性率与病程的关系

SARS-CoV-2特异性IgM和IgG抗体阳性率与病程的关系见表 2，其中8例因属于无症状感染者，经过主动筛查发现，无法确定发病时间，未统计在内.经过卡方检验，不同发病天数各组间差别有统计学意义.从表中可以看出，发病天数少于或等于28 d，IgM与IgG的阳性率差异有统计学意义(p＜0.05).本研究显示，最早在发病4 d时可检测出IgM阳性.在发病后8 d，抗体IgM阳性率达到100%.随着病程的进展，发病28 d后，IgM开始消失，IgG抗体阳性率开始提高.

2.4. 不同临床类型COVID-19患者SARS-CoV-2特异性IgM和IgG抗体阳性率

不同临床类型COVID-19患者SARS-CoV-2特异性IgM和IgG抗体阳性率见表 3，从表 3中可以看出，不同疾病分型，IgM和IgG抗体阳性率差别无统计学意义.

3. 讨论与结论

病原特异性抗体检测是协助临床诊断的重要手段之一，《中国成人社区获得性肺炎诊断和治疗指南(2016年版)》明确推荐血清特异性抗体检测作为病毒学检测方法之一^[6].国家卫生健康委发布的《新型冠状病毒肺炎防控方案(第5版)》附件4《新型冠状病毒肺炎实验室检测技术指南》^[7]指出，抗体检测可作为新型冠状病毒肺炎(COVID-19)的辅助诊断方法.在《军队支援湖北医疗队新型冠状病毒疾病诊疗方案(试行第1版)》中，已经将新冠病毒的抗体检测纳入诊断标准^[8]；在2020年3月3日最新公布的《新型冠状病毒肺炎诊疗方案(试行第7版)》中也明确提出了血清学用于疑似病例的排除和确诊病例的诊断^[9].本研究结果证实，全血SARS-CoV-2特异性IgM/IgG临床诊断敏感度、特异度和诊断准确度分别为92.1%、90.7%和91.6%，与RT-PCR方法相比，诊断COVID-19的差异无统计学意义，与临床确诊的结果具有非常好的一致性.

本研究显示，SARS-CoV-2特异性IgM和IgG抗体阳性率与发病天数相关，而与疾病的分型无关，与体液免疫的规律相符.本研究结果与徐万洲等^[10]的研究结果在联合诊断效率上一致，但在特异性抗体的分布上存在差异.本研究结果中IgM抗体的阳性率高于IgG抗体，而徐万洲等的研究中结果相反.分析其原因在于患者的群体存在差异，徐万洲等的研究中患者处于中期或者恢复期，而本研究标本采集时间离发病时间4~37 d，中位数在14 d左右，本研究的群体处于发病早期和中期的人数较多，而IgM存在于患者发病的早期和中期.因此，结合本研究结果与徐万洲等研究结果可以说明IgM和IgG联合检测对新型冠状病毒病的诊断、疗效及预后均具有价值，IgM阳性提示疾病的存在；IgM浓度的下降乃至消失，IgG浓度的升高，则预示患者逐渐痊愈，并产生了对SARS-CoV-2的免疫力.

本研究使用的是全血与胶体金免疫层析法，而徐万洲等^[10]研究使用的是化学发光法检测血清，二者各有千秋.胶体金方法操作简便快速，无需特殊设备，室温下即可进行，可以使用末梢血，15 min内判定结果，对人员环境仪器的要求低，可以有效辅助确认或排查疑似人群及密切接触人群中的COVID-19感染者，对患者的管理及疫情防控尤为重要.

在本研究中，有2例患者经过多次核酸检测才得以确诊，1例在入院当天的标本经过检测出现了IgM阳性，在徐万洲等的研究中也有19例核酸检测阴性，但基于临床症状确诊的COVID-19患者中，有16例患者2019-nCoV IgM阳性，说明抗体的检测可以在一定程度上弥补核酸检测漏检的风险.抗原抗体血清学检测使用外周血、血清或血浆标本，其采集和保存简单易行，抗体在血液标本中的稳定性高，且抗体的检测对于病原体存在机体的组织器官没有选择性，避免了因取样而导致的假阴性结果.

COVID-19为新发疾病，SARS-CoV-2感染后IgM和IgG抗体产生的规律还没有任何报道.根据以往经验，抗体检查通常适用于筛查，例如HIV、丙肝等的筛查策略^[10-12]. COVID-19潜伏期为1~14 d，多为3~7 d，但是本研究显示，最早在发病4 d时可检测出现IgM阳性.在发病后8 d，抗体IgM阳性率达到100%，其原因在于血清学抗体检测存在窗口期，个体免疫应答差异等影响因素，因此SARS-CoV-2特异性IgM和IgG抗体对早期COVID-19的检测仍然存在假阴性的可能，同时由于交叉反应，也可能产生假阳性，单独使用SARS-CoV-2特异性IgM和IgG抗体在早期诊断与筛查上存在较大的漏诊和一定的误诊风险.

综上所述，SARS-CoV-2特异性IgM和IgG抗体的重要意义在于可以与核酸检查联合检查，提高COVID-19诊断的敏感度.目前，有必要通过进一步研究确定血清学实验的窗口期，以及进行前瞻性研究，确定COVID-19的诊断和筛查策略，以达到更好更快地确定感染者的目的.

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Detection of Specific SARS-CoV-2 IgM and IgG Antibodies in COVID-19 and Its Clinical Application

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