人巨细胞病毒pp65特异性抗体测定在原发感染诊断中的应用

The clinical application of the measurement of HCMV pp65 specific antibodies for diagnosis of HCMV primary infection

目的通过测定患者单份血清人巨细胞病毒(HCMV)pp65特异性IgM抗体和IgG亲和指数(AI),建立HCMV原发感染的临床判断标准。方法从临床收集40份患儿血清和尿标本,以本室自制的pp65为抗原,运用间接酶联免疫吸附试验(ELISA)检测血清标本中HCMVpp65特异性IgM抗体;同时通过尿素变性实验,以6M尿素作为温和蛋白变性剂,测定HCMVpp65IgGAI。尿标本常规处理后接种人胚成纤维(HF)细胞进行病毒分离,观察HCMV特异性细胞病变效应(CPE),聚合酶链反应(PCR)试验检测细胞培养物UL83基因,间接免疫荧光试验检测细胞玻片HCMV抗原。并将病毒分离结果同血清学方法进行比较。结果40例标本中,IgM阳性13例,IgG阳性30例,其中,仅IgM阳性4例,病毒分离结果亦为阳性,可诊断为原发感染;30例IgG阳性标本中,2种抗体均为阳性9例(A组),其中,5例AI<50%,病毒分离结果均为阳性,判断为原发感染;1例AI在50%与60%之间,为可疑原发感染,需要进一步鉴定;3例AI>60%,判断为继发感染。IgG阳性21例(B组),其中仅3例(14.29%)AI<50%,但病毒分离结果为阴性,提示患者不久前曾发生HCMV原发感染,特异性IgM抗体已经转阴,病毒进入潜伏状态;余18例患者AI均>60%,判断为继发感染;2种抗体均为阴性的标本有6例,病毒分离结果亦为阴性,说明患者未被感染。统计学分析,A组与B组之间差异有统计学意义(P<0.05)。血清学方法与病毒分离比较,一致率为75.49%,该方法灵敏度为100%,特异度为87.10%,准确度为90.00%。结论以HCMVpp65重组蛋白为抗原检测特异性抗体以及相应IgGAI的ELISA,可快速诊断HCMV原发感染和继发感染;该方法具有高度特异性与敏感性,操作简便,重复性好,具有良好的临床应用前景。

ObjeCtive To establish a standard method for clinical diagnosis of HCMV primary infection through the measurement of HCMV pp65 specific immunoglobulin M （IgM） and immunoglobulin G （IgG） by measuring an avidity index（AI）.Methods An IgM and IgG antibody avidity assay was performed. This assay which uses protein-denaturing agents along with a modification of an enzyme-linked immunosorbent assay （ELISA） using recombinant protein pp65 as an antigen, was used to determine its usefulness in distinguishing primary HCMV infections from nonprimary infections. Urine samples were inoculated in human embryo fibroblasts （HF） after routine treatment for viral isolation.HCMV specific cytopathic effects （CPE） were observed daily, HCMV UL83 DNA from the cultures was tested by PCR.The HCMV antigen was detected on cell slides by indirect immunofluorescence. Results of both methods were compared. Results Of the total 40 samples taken from children,4 patients had only positive IgM and were classified as primary infection,whereas 9 patients had positive IgG and IgM and were classified as Group A and the remaining 21 patients, who had only positive IgG, were classified as Group B. A total of 30 samples, which were classified as HCMV pp65 IgG positive, were measured using an avidity index （AI） of HCMV-pp65-IgG,, An HCMV-IgG AI less than 50% was defined as primary infection, 50%-60% was defined as suspicious primary infection, and greater than 60% was considered a nonprimary infection. There were only 3 cases in Group B （ 14.29% ） whose AI was under 50% ,while all other cases in this group were above 60%. While in Group A,5 cases （55.56%） had an AI under 50% and therefore were considered to have a primary infection, 1 case whose AI was between 50% and 60% and classified as a doubtful primary infection,and 3 cases （62.50%） whose AI was above 60% were determined as nonprimary infection. The statistical analyses indicated that there were differences between Group A and Group B （ P 〈 0.05 ）. The concordance rate between the results of the new ELISA and viral isolation was 75.49% .These results indicate that the sensitivity of the new ELISA assay is 100%, the specificity is 87.10% and the accuracy rating is 90%. Conclusion Determination of HCMV pp65 IgM and HCMV pp65-IgG AI by ELISA is helpful for distinguishing primary infections from nonprimary infections.This method has a high degree of specificity and sensibility and can be generalized for clinical diagnosis.