| ObjectiveThis study will determine the HBV genotypes of 60 familial grouped patients with chronic HBV infection by Real-time quantitative PCR combined with fluorescent probe technology and explore the genotype distribution and its relationship with viral load and liver pathology.Materials and methods1.PatientsA total of 60 HBV DNA-positive sera were collected from the familial grouped patients with chronic HBV infection in Shenyang,who were treated in the department of infecious diseases in Shengjing Hospital of China Medical University during the period of December 2006 to December 2008.All sera were both HBsAg and HBV DNA-positive(HBV DNA load≥10~3 copies/ml were positive).In 60 patients,16 were chronic HBV surface antigen carriers(ASC),32 were chronic hepatitis B(CHB) and 12 were liver cirrhosis(LC).The age of these patients was from 10 to 50 years old(average 32 years old).For all these patients,HBV genotyes were determined,HBV DNA load were detected and liver pathology were analyzed.Patients were selected according to the following criteria.1) the diagnostic criteria of familial grouped patients with chronic HBV infection:patients with chronic HBV infection whose mother or(and) father and at least one of his(her) brothers and sisters were HBsAg-positive.2) clinical diagnosis:patients were diagnosed according to the programme of prevention and cure for viral hepatitis in 2000.2.Determination of HBV genotype The HBV genotypes were detected by Real-time quantitative PCR combined with fluorescent probe technology.The test kit was purchased from Hangzhou Biocore Bio-Tech Company.This kit only can detect two HBV genotypes including genotype B and C,ABI 7500 machine and related software were used for the detection and data analyse.3.Quantitative detection of HBV DNAHBV DNA was determined by fluorescent quantitative PCR,The kit was purchased from Shenzhen-based Biological Engineering Company,the detection sensitivity was 10~3 copies/ml,Lanegene FQ-PCR machine was used.4.Detection of liver biopsyLiver biopsy was done by ultrasonic guidance,about 2 centimeters liver tissue of every patient was got,and then fixed in 4%formaldehyde,dyed by HE,MASSON and reticular fiber staining respectively.The diagnostic criteria of the inflammatory grade (G)/fibrosis stage(S) was decided according to the programme of prevention and cure for viral hepatitis in 2000.5.Statistical analysisStatistical analysis was performed with the aid of the software SPSS 15.0.The analysis method was X~2 analysis.P values＜0.05 were considered statistical significant.Results1.Genotype distributionHBV genotype classification showed that:In 60 patients,14 patients were of genotype B(23.3%),40 patients were of genotype C(66.7%),6 patients were unclassified(10%).In 16 ASC,3 patients were of genotype B(18.7%),12 patients were of genotype C(75%);In 32 patients with CHB,11 patients were of genotype B (34.4%),17 patients were of genotype C(53.1%);In 12 patients with LC,11 patients were of genotype C(91.7%).2.The relationship between HBV genotype and HBV DNA loadHBV DNA load were divided into two groups,low HBV DNA load(＜10~6 copies/ml) group and high HBV DNA load(＞10~6 copies/ml) group.All 16 ASC had a high HBV DNA load;In 32 patients with CHB,9 patients had a low HBV DNA load, 23 patients had a high HBV DNA load;In 12 patients with LC,6 patients had a high HBV DNA load,6 patients had a low HBV DNA load.Although most of the patients had a high HBV DNA load,there was no significant difference between genotype B and genotype C in HBV DNA load(P＞0.05).3.The relationship between HBV genotype and liver pathologyThe liver pathological injury of patients for the genotype B was in lighter extent, 13(92.9%) with a low stage of hepatic inflammation(≤G1) and 12(85.7%) with a low stage of hepatic fibrosis(≤S1).The liver pathological injury of patients for the genotype C was severer,21(52.5%) with a higher stage of hepatic inflammation(≥G2) and 18(45%) with a higher stage of hepatic fibrosis(≥S2).In 12 patients with LC,11 patients(91.7%) were of genotype C,suggesting that genotype C was related to the severe liver disease.In the 11 patients with CHB of genotype B,10 patients(90.9%) were with a low stage of hepatic inflammation(≤G1) and 9 patients(81.8%) were with a low stage of hepatic fibrosis(≤S1).However,in the 17 patients with CHB of genotype C,7 patients(41.2%) were with a low stage of hepatic inflammation(≤G1),8 patients(47.1%) were with a low stage of hepatic fibrosis(≤S1);10 patients(58.8%) were with a higher stage of hepatic inflammation(≥G2),9 patients(52.9%) were with a higher stage of hepatic fibrosis(≥S2),there was statistical significance by X~2 test (X~2=76.6,P＜0.05),these suggested that genotype C was related to the severe liver injury,too.There were 13 patients(81%)with a different degrees of liver injury in 16 ASC.Conclusions1.The majority of familial grouped patients with infection of HBV in Shenyang is genotype C.2.There is not significant difference in HBV DNA load between genotype B and genotype C. 3.Genotype C is related to the severe liver disease.
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