The Study Related To Hepatitis C Virus Genotypes In The Patients With Hepatitis C In Guangzhou Region

BackgroundInfection with hepatitis C virus(HCV) can cause chronic liver disease in75–85%of the infected individuals， which is an important pathogen of chronic hepatitis、cirrhosis and hepatocellular carcinoma， presenting a major threat to global publichealth. The global prevalence of HCV infection is approximately3.0%，meaning thereare170million-2.0million people infected with HCV in the world. In China，the rateof HCV infection ranges from2.5%to4.9%with more than30million HCV-positivepeople.HCV can be spread by intravenous drug use(IDU)、 blood transfusion ortransfusion of blood products、sexual activities、vertical transmission、broken skin andmucous membranes such as healthcare exposure、tattoos、shared personal items and etal. The main transmission route is IDU in many developed countries. In the earlystage of China， blood transfusion or transfusion of blood products was a majortransmission route which decreased sharply after blood screening for HCV. However，along with the increase of intravenous drug users(IDUs)， the proportion of HCVinfection through IDU increases.HCV genotypes are multifarious. Currently， Simmonds nomenclature thatdivided HCV into six genotypes and more than80subtypes are often used nationallyand internationally. HCV genotypes vary distinctly in patterns of geographicaldistribution. Subtypes la、lb、2a、2b、3a are global pandemic and lb distributes most widely in the world. In China，1b is the most predominant followed by2a whichmainly distributes in the northern areas. Genotype3mainly distributes in southwestChina，such as Yunnan. Genotype4and5are rarely reported in China. Genotype6mainly distributes in Hong Kong，Macau and the southern border provinces of China.Genotype6is the most complex and its23variants have been found up to date. It wasreported that1b and6a are main genotypes and new variants of genotype6have beenfound in blood donors and IDUs in Guangdong province. The prevalence of HCVgenotypes among the patients with hepatitis C is still needed to investigate.The degree of liver damage is various with different genotype-HCV infection. Forexample，1b is more serious to the liver than the others. At present， the maintherapeutic regimen of hepatitis C is pegylated interferon (Peg-IFN)α withcombination of ribavirin (RBV). HCV genotypes are closely related to curativeefficacy of hepatitis C. The virological response rates of genotypes2、3are higherthan that of genotype1、4. Therefore，accurate genotyping is very important to thechoice of therapeutic regimen and prognosis.In order to genotyping accurately， the technologies for HCV-genotyping havebeen studied for a long-time in the world. At present， HCV genotyping methodsinclude gene sequencing， type-specific primers amplification， Line probehybridization assay，gene chip，restriction fragment length polymorphism assay，Heterouduplex mobility assay and et al. The most accurate method is the full-lengthgene sequencing undoubtedly. However，the full-length gen of HCV is so long that itis difficult to be amplified，also it is time-consuming and expensive. So it’s difficult tobe wildely used clinically. Partial gene-sequencing is relatively simple and feasible，and now sequencing on the NS5B region has been recognized as "gold standard" forHCV genotyping. But in China，domestic commercial reagent(probe-hybridization) isa routine used method for HCV genotyping in clinical，which can only distinguishgenotype1from Non-1with a lower accuracy. Considering the problems of genotyping methods and the complexity of genotypesin Guangdong area，the study related to hepatitis C virus gene in the patients withhepatitis C in Guangzhou region was performed to meet the needs of the diagnosisand treatment of hepatitis C in clinical，the study includes two parts: one is accuracycomparison of the two genotyping methods which are practical in clinical，and twois HCV molecular epidemiological study.Part Ⅰ Accuracy comparison of the two genotyping methods which are practicalin clinical（partial gene-sequencing and probe-hybridization）Content and ObjectivesAccuracy of partial gene-sequencing and probe-hybridization for HCV genotypingwas compared to provide a reliable basis for diagnosis and therapy of Hepatitis C andmolecular epidemiology of HCV.MethodsSerum samples were collected from191patients with hepatitis C hospitalized inthe8th People’s Hospital of Guangzhou during2012to2013, which HCV weregenotyped by domestic probe-hybridization. HCV genotyping results derived fromHCV full-length gene sequencing in international database were used as a "goldstandard". Designed amplification primers of C and NS5B regions were used toamplify and sequence C and NS5B regions of HCV in the sera. The sequencings weregenotyped by phylogenetic analysis with the "gold standard". Then genotyping resultswere compared with that of probe-hybridization.Results1. Total92HCV full-length sequences representing7genotypes、54subtypesand5recombinant genotypes were chosen from international database。Phylogenetictrees based on the whole genome，C and NS5B region sequences of92representativestrains showed that the genotyping results were consistent with each other. 2. HCV genes of C and NS5B regions were both successfully amplified in171of191(89.53%) patients with the designed amplification primers. Genotyping resultsof phylogenetic analysis were consistent between C and NS5B regions in167of171(97.66%) patients.3. The coincidence of167genotyping results compared betweengene-sequencing and probe-hybridization was only84.43%（141/167）. Twenty-sixincorrect results produced by probe-hybridization including:6a of23patients wasmistaken as genotype1,1b of2patients and1a of1patient was mistaken as genotypeNon-1. Four of171(2.34%) genotyping results were inconsistent between C regionand NS5B region. Genetyping6a of three patients in C region was typed as1b inNS5B region. Genetyping1b of one patient in C region was typed as6a in NS5Bregion. Only two results in C region and two results in NS5B region were consistentwith that produced by probe-hybridization.Conclusions1. Currently， the false detection rate of domestic commercial reagents(probe-hybridi zation)widely used for HCV genotyping in clinical was high and apt toconfuse genotype1and genotype6.2. Established a more precise method of HCV genotyping：combination sequenceanalysis with both C and NSSB regions of HCV could improve the accuracy ofgenotyping，which could replace HCV full-length sequencing when a consistent resultbetween C and NS5B regions was gotten. HCV infection with mixed or recombinantisolates should be considered when the genotyping results were inconsistent betweenC and NS5B regions and should be further analyzed. Part Ⅱ Molecular epidemiology of HCV gen in the patients with hepatitis C inGuangzhou regionContent and ObjectivesThe study of characteristics of HCV genotypes and the correlation betweengenotypes and ages，gender，transmission routes，viral titers among the patients withhepatitis C in Guangzhou area was performed to understand the changes of molecularepidemiology trend of HCV in clinical and its impact on clinical prognosis.MethodsSerum samples were collected from207hepatitis C patients with HCV RNApositive hospitalized in the8th People’s Hospital of Guangzhou during2012to2013.Reverse transcription nested PCR (RT-nPCR) was performed to amplify genes ofHCV C and NS5B regions. DNA products successfully amplified in both C and NS5Bregions were then sequenced. The sequences of C/NS5B regions were aligned andcompared with that of related references. Phylogenetic trees were constructed todetermine HCV genotypes when both genotyping results from C and NS5B regionswere consistent. Then， the relationship of HCV genotypes and transmission routeswere analyzed.The correlation between genotypes and ages，gender，viral titers wasanalyzed by SPSS13.0.Results1.179results included in analysisHCV C and NS5B regions were amplified by RT-nPCR respectively among207samples. The expected PCR products were401bp and375bp respectively.Electrophoretic DNA was single band and clear without non-specific bands.185weredouble-positive in gene amplification of the C and NS5B regions. Both genotypingresults from C and NS5B regions were consistent in179samples.2. Characteristics of HCV genotypes 1) HCV genetic characteristicsFour genotypes and eight subtypes were identified in total，in which1b and6awere the main genotypes. The proportions of each subtype were as follows:1baccounting for40.78﹪(73/179)，6a for27.37﹪(49/179)，3b for11.73﹪(21/179)，3a for8.94﹪(16/179)，2a for6.15﹪(11/179)，1a for3.91﹪(7/179)，2b for0.56﹪(1/179)，6n for0.56﹪(1/179).No new variant of genotype6was found.2) Characteristics of1b and6a phylogenyHCV1b isolates formed three clusters，designated groups A、B and C. Group Ahad high homology with1b isolates that prevalent throughout China，while group Bwas close to that predominant in the central and southern China，6a isolates alsoformed two clusters，designated groupsⅠand Ⅱ. Most6a isolates were close to thatfrom blood donors and intravenous drug users.3. Characteristics of molecular epidemiology1) Transmission routesThe patients infected with HCV mainly by IDU and blood transfusion ortransfusion of blood products. Genotype6a was mainly found in patients with IDU，accounting for57.45%（27/47）， followed by3a which accounted for21.28%（10/47）.While，1b was the main subtype found in patients with blood transfusionor transfusion of blood products，accounting for78.95%（30/38）.2) GenderThere were statistical differences on gender ratios between different subtypes（P=0.002）.3) AgesThere were statistical differences on average ages between different subtypes（P=0.007）. The average ages of patients with subtype2a were highest than theothers’（53.45±11.02y）. Pairwise comparisons were applied with the others’. Theresults show there were statistical differences(（all P<0.05）).While there were nostatistical differences among the other’. 4) Viral loadsThere were statistical differences on the average viral loads between differentsubtypes （P=0.000） The average viral loads of patients with subtype1b were6.51±0.90log10IU/ml， as the highest. Pairwise comparisons were applied with theothers’.The results show there were statistical differences(（all P<0.05）).While therewere no statistical differences among the other’.5) Characteristics of HCV/HIV-infected individualsOf207Hepatitis C patients，33were co-infected with HIV，especially amongpatients with subtype6a， which up to51.51%（17/33）. The average viral loadsbetween HCV and HIV co-infection had no significant differences by usingindependent samples t-test(P=0.691).Conclusions1. Many genotypes of HCV were found in the patients with hepatitis C inGuangzhou area，of which，1b、6a、3were the main genotypes/subtypes. The subtype1b covered most strains distributed in all of China. Subtypes6a、3replaced2abecoming the second and third prevalent subtypes respectively. No genotype6variants previously reported were found.2. There were statistical differences on patients’ gender ratios，ages，viral loadsbetween different subtypes. The average ages of patients with subtype2a were highest.While， the virus loads of patients with subtype1b were highest.This is consistentwith the report that the pathogenicity of subtype1b was stronger than the others’.1bwas the main subtype found in patients with blood transfusion or transfusion of bloodproducts，Subtype6a was mainly found in patients with IDU.3.6a was the main subtype among patients with HCV/HIV co-infected.Theaverage viral loads between HCV and HIV co-infection had no significantdifferences.