【摘要】 目的　建立人巨细胞病毒 (HCMV)短时培养快速诊断改良技术 ;检验临床样本 ,与病毒分离比较。方法　①用含细胞飞片培养板离心吸附AD16 9株 ,设时间梯度培养后用SABC法检测培养物中HCMV即刻早期抗原 (IEA) ;②用改良法检测 15 6例患儿尿样本 (7例留取脑脊液 ) ,其中 5 4例与病毒分离对照 ;14 7例检测血清特异性IgM ;③ 9例更昔洛韦 (GCV)治疗后随访尿排毒和特异性IgM变化。结果　①标准毒株培养 16～ 2 4h ,能 10 0 %检出培养物中HCMVIEA。②快速法检测尿样本 178份 ,阳性率 4 6 6 %。与病毒分离比较 ,敏感性和特异性分别达 10 0 %和 91 7%。③ 14 7例检测血清HCMVIgM ,5 0例阳性 (34 0 % ) ,其中 4 9例和另 2 5例特异性IgM阴性者尿快速培养阳性。后 2 5例中 ,年龄 <1岁 2 2例 ;2例免疫抑制者快速培养阳性。④ 2例先天感染儿脑脊液快速培养阳性伴蛋白增高。⑤ 9例GCV治疗后随访 2～ 4次 ,2例无效 ,1例尿排毒减少 ;6例尿排毒停止。结论　改良快速培养法敏感性高、特异性强 ,操作更为简便 ,是快速诊断活动性HCMV感染和评估抗HCMV疗效的可靠方法更多还原

【Abstract】 Objective To establish a modified rapid cell culture system (shell vial assay) and to use it to detect human cytomegalovirus (HCMV) in clinical specimens, comparing with the classical cell culture, a "golden" standard for diagnostic methods. Methods AD169 strains of HCMV were inoculated onto the monolayer of human embryonic lung fibroblast cells on cover slips within the wells of culture plate by low speed centrifugation enhanced way and incubated in 5% CO 2 at 37℃ for 4, 8, 16, 20 and 24 hours, respectively. Then, the inoculated tissue culture cells were stained with a streptavidin biotin enzyme complex conjugated monoclonal antibody to immediate early antigens (IEA) of HCMV by means of SABC immunohistochemical assay. There were three samples for each incubated time and tests were repeated for three times. Using the established modified method, 178 urine and 7 cerebrospinal fluid (CSF) specimens from 156 patients, aged from 4 days to 13 years, were examined, and 54 urine samples were confirmed by classical virus isolation. Serum anti HCMV IgM were also measured for 147 cases. Nine patients with HCMV hepatitis were followed up for 2 to 4 (mean: 2.6) times after the end of ganciclovir (GCV) treatment. The changes of their urine viral shedding and serum specific IgM were monitored. Results HCMV IEA antigens were found in all cell cultures inoculated with AD169 strain after incubating for 16 to 24 hours in three times of repeated tests. Out of 178 (46.6%) urine specimens, 83 were positive for HCMV in the rapid cell culture tests. Compared with conventional viral isolation, the sensitivity and specificity of this modified rapid culture system were 100% and 91.7%, respectively. Of 147 (34.0%) patients, 50 were also positive for anti HCMV IgM antibodies in their sera. Forty nine cases with positive specific IgM antibody and 25 cases (22 were under one year of age; two immunocompromised patients and one out patient without clinical data) with negative IgM were positive for urine rapid viral culture. Another infant aged 3 months was positive for serum anti HCMV IgM and urine viral isolation negative, who just received 2 units of blood platelet suspension and 2.5 grams of immunoglobulin infusion because of idiopathic thrombocytopenic purpura Wichin 3 days before collecting blood sample, which suggested that there was a higher possibility for him to get passive anti HCMV IgM antibodies. CSF rapid culture was positive in two congenitally infected patients who had urine viral shedding and elevated CSF protein levels as well (one had 529 mg/L of proteins in CSF with microcephaly and multiple cerebral calcification and the other had 495 mg/L of proteins in CSF without any nervous system abnormality), and the assay was negative in the remaining 5 patients who had normal results on CSF routine examination (two were urine viral isolation positive). The results of the follow up study showed that 2 patients were non responsive for GCV therapy, one had reduced urine viral shedding and in 6 cases the viral shedding stopped (2 without specific IgM seroconversion, 3 had prolonged IgM absence for up to 70 days and in only one IgM disappeared at the same time). Conclusion The modified cell culture system had higher sensitivity and specificity for detecting HCMV in clinical specimens and was easy to manipulate, rapid and reliable, indicating its potential applications for the evaluation in the efficacy of anti HCMV therapy and the rapid diagnosis of active HCMV infections, especially in younger infants and immunosuppressed patients whose serum specific IgM usually could not be detectable.更多还原