乙型肝炎病毒相关性肾小球肾炎患儿血清中HBV-cccDNA水平检测及其临床意义

Detection of HBV-cccDNA level in children with hepatitis B virus associated glomerulonephritis and its clinical significance

目的:分析血清中乙肝病毒共价闭合环状双链DNA (HBV-cccDNA)水平对乙型肝炎病毒相关性肾小球肾炎(HBV-GN)患儿肝肾功能、肾组织中HBV抗原的检出、肝组织病理分级和分期的影响,评价HBVcccDNA水平检测对HBV-GN患儿诊断的临床应用价值。方法:选取39例HBV-GN初治患儿作为研究对象(观察组),所有患儿均接受肝脏和肾脏穿刺;选取肝功能正常HBV携带患儿40例作为对照组。检测2组患儿丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平,采用PCR荧光分子信标技术检测患儿血清中HBV-cccDNA水平,HE染色检测患儿肝脏和肾脏组织的形态表现,免疫荧光法检测患儿肾组织中HBsAg、HBeAg和HBcAg检出率,采用受试者工作特征(ROC)曲线及曲线下面积(AUC)评价HBV-cccDNA水平检测在HBV-GN诊断中的价值。以HBV-cccDNA的Ax荧光值>21判定为阳性,反之判定为阴性,将HBV-GN患儿分为HBV-cccDNA阳性组(n=24)和HBV-cccDNA阴性组(n=15)。在抗病毒治疗后第2、4、8和12周时检测患儿血清中HBV DNA及HBV-cccDNA水平。结果:HBV-cccDNA阳性组患儿ALT和AST水平异常率、HBeAg和尿蛋白阳性率均高于HBV-cccDNA阴性组(χ~2=4.454,P=0.035;χ~2=5.912,P=0.022;χ~2=8.770,P=0.007)。HBV-cccDNA阳性和阴性组HBV-GN患儿肝组织炎症和纤维化程度比较差异无统计学意义(P>0.05),HBV-GN患儿肾脏活检病理类型以膜性肾病(MN)为主,HBV抗原成分以HBeAg及HBcAg为主,HBV-cccDNA阳性组患儿HBeAg及HBcAg检出率明显高于HBV-cccDNA阴性组(χ~2=5.652,P=0.027;χ~2=12.523,P=0.001)。ROC曲线评价,HBV-cccDNA水平检测能有效鉴别观察组HBV-GN患儿和对照组患儿,AUC=0.804 (95%CI:0.709~0.883)。10例规范治疗且有完整追踪治疗资料的HBV-GN患儿在治疗第2周时其HBV-cccDNA水平明显降低,治疗至第12周时其中7例患儿HBeAg转阴,无蛋白尿、血尿症状,ALT和AST水平均正常;治疗无效的3例患儿血清中HBV-cccDNA水平均高于其余7例标本。结论:HBV-cccDNA高表达与HBV-GN患儿肝功能、蛋白尿及肾脏HBV抗原检出有密切关联,检测HBVcccDNA水平对儿童HBV-GN患儿的辅助诊断及疗效评估具有潜在的临床应用价值。

Objective:To analyze the effects of serum hepatitis B virus covalently closed circular DNA(HBV-cccDNA)level on the liver and kidney functions,detection of HBV antigens in kidney tissue,pathological grading and staging of liver tissue in the children with hepatitis B virus associated glomerulonephritis(HBV-GN),and to evaluate the clinical value of HBV-cccDNA level detection in the diagnosis of the children with HBV-GN.Methods:A total of 39 HBV-GN children(observation group)were selected and all of them underwent the liver and kidney biopsy.A total of 40 HBV-carried children with normal liver function were selected as control group.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)of the children were detected.Molecular beacons PCR method was used to detect the serum HBV-cccDNA level of the children.The morphology of liver and kidney tissue s of the children was detected with HE staining.The detection rates of HBsAg,HBeAg and HBcAg in kidney tissue of the children were detected by immunofluorescence.Receiver operating characteristic(ROC)curve and area under the ROC curve(AUC)were used to evaluate the value of HBV-cccDNA level detection in the diagnosis of HBV-GN.The Ax fluorescence value of HBV-cccDNA>21 was determined as positive,and the Ax fluorescence value of HBV-cc cDNA<21 was determined as negative;the children with HBV-GN were divided into HBV-cccDNA positive group(n=24)and HBV-cccDNA negative group(n=15).The serum levels of HBV DNA and HBV-cccDNA were measured at the 2nd,4th,8th and 12th weeks after antiviral therapy.Results:The abnormal rates of ALT and AST levels,positive rates of HBeAg and urine protein of the children in HBV-cccDNA positive group were higher than those in HBV-cccDNA negative group(χ2=4.454,P=0.035;χ2=5.912,P=0.022;χ2=8.770,P=0.007).There were no significant differences in the degrees of liver inflammation and fibrosis in the children between HBV-cccDNA positive and negative groups(P>0.05).Membranous nephropathy(MN)was the main pathological type of kidney biopsy in the HBV-GN.HBeAg and HBcAg were the main components of HBV antigens.The detection rates of HBeAg and HBcAg of the children in HBV-cccDNA positive group were significantly higher than those in HBV-cccDNA negative group(χ2=5.652,P=0.027;χ2=12.523,P=0.001).The ROC curve analysis results showed that the HBV-cccDNA level detection could effectively distinguish the HBV-GN children in observation group from those in control group,AUC=0.804(95%CI 0.709-0.883).The levels of HBV-cccDNA of 10 cases of HBV-GN children underwent standardized treatment with complete follow-up treatment data were decreased significantly at the 2nd week after treatment.At the 12th week after treatment,in 7 cases the HBeAg turned negative,without proteinuria and hematuria symptoms,and the AST and ALT levels were normal.The HBV-cccDNA levels of 3 cases with ineffective treatment were higher than that those of the remaining 7 cases.Conclusion:The high expression of HBV-cccDNA is closely related to the liver function,proteinuria and HBV antigen detection in kindney tissue of the HBV-GN children.The detection of HBV-cccDNA level has potential clinical value for the auxiliaty diagnosis and evaluation on the curative effect of the HBV-GN.