Establishment And Application Of A Noninvasive Diagnostic Model For Liver Inflammation Activity And Liver Fibrosis In Chronic HBV Infected Patients

Background:Hepatitis B virus?HBV?is a global infectious disease,which is a great burden for national economy and health.According to the World Health Organization reports,approximately two billion people has serological evidence of past or present infection with HBV,among of which has an estimated of 240 million people are chronic HBV carriers.About 650 thousand people died of liver failure,cirrhosis and liver cancer caused by HBV infection each year.China has been a high prevalence area of HBV infection,where about 93 million people with chronic HBV infection.So far,there is hardly to eradicated hepatitis B virus from the nucleus of hepatocytes according to the current treatment schedule.Nowadays,antiviral therapy is the main method to prevent disease progression.However,the treatment effect is different from person to person.How to improve the antiviral effect for chronic HBV infection is a concerned problem for clinicians.Liver inflammation and fibrosis are the basic evidence and timing for antiviral treatment.Liver biopsy is still the golden standard for the assessment of liver necro-inflammation and fibrosis.Liver biopsy is an invasive examination methods,which has certain risks and complications that cannot be widely accepted by the patient.In addition,it cannot dynamic monitoring the disease progression,and the liver biopsy results interpretation will be affected by experience of pathologist.Besides that,there will be sampling error in liver biopsy for liver tissue always distributed unevenly in chronic liver disease.Therefore,liver biopsy is not fully representative of the entire liver disease.Alanine amiotransferase?ALT?is identified as the most direct and simple noninvasive serum marker for reflect the liver tissue inflammation to direct the antiviral treatment.Guidelines recommend that the people need to accept antiviral therapy if HBV-DNA positive and ALT is more than or equal to two times the upper limit of the normal range in chronic HBV infection.This argument is out of doubt.However,whether the ALT is in a low level?ALT<two times the upper limit?of chronic HBV infected patients need to be treated and the timing of antiviral therapy is still one of the difficult problems in clinics.Partly of chronic HBV infection with low levels of ALT are still in immune tolerance,which is no or only minor damage in liver tissue.These person always has a poor antiviral effect.So the Guidelines recommend these people do not need antiviral treatment temporarily.However,numerous studies have shown that some of chronic HBV infection with low levels of ALT,especially for those older than 30years old,is need antiviral therapy.Because the immune system of these people has broken the state of immune tolerance,and liver tissue began to have necrosis inflammation and/or fibrosis.Such people need antiviral therapy at once.Unfortunately,we have not yet found a good noninvasive serologic markers to assess the situation of liver inflammation and fibrosis,and still cannot distinguish real situation in the two subgroups in chronic HBV infection with low levels of ALT.Thus,for low levels of ALT in chronic HBV infection is often need for liver biopsy to assess liver inflammation and fibrosis to decide whether they should acccept antiviral therapy.Therefore,more and more research has focused on finding non-invasive diagnostic methods to instead of liver biopsy.To date,many studies have established a variety of non-invasive diagnostic techniques and models.Most of the non-invasive diagnostic models were established based on the patients with chronic hepatitis C infection.Furthermore,these non-invasive diagnostic models were always focused on the diagnosis of hepatic fibrosis,and the study of non-invasive diagnostic model of liver inflammation is limited,especially for the chronic HBV infection with ALT<2ULN.Liver inflammation is a pathological process accompanied by chronic liver tissue repair,which is mainly due to the immune damage caused by the immune system,rather than hepatitis virus itself.Studies have found that a variety of immune chemokines have significantly changed in the liver injury.Therefore,liver inflammation and immune response are inextricably linked.Previous studies have also found a variety of inflammatory cytokines and chemokines involved in inflammation of liver tissue.Monocyte chemoattractant protein?MCP-1?is a member of CC chemokines subfamily,which is the main factor to mediate macrophage and take part in liver inflammation.It also have been reported that MCP-1 could participate in acute or chronic liver injure and liver fibrosis.Recent research has found that serum MCP-1 could make change before the serum ALT,which means that MCP-1could be serving as a valuable serum marker to reflect liver injure.However,more research still needs to be done on the expression of MCP-1 in chronic HBV infection.Objective:1.The aim of the study was to detect the serum expression of MCP-1 in chronic HBV infection in different degree of liver inflammation activity and fibrosis,and to evaluate the diagnostic ability of MCP-1 in different degree of liver inflammation activity and fibrosis.2.To establish a noninvasive model to assess the liver inflammation activity and fibrosis in chronic HBV infection on this basis of routine clinical serum virological and biochemical marker and inflammatory chemokines.Methods:This study is divided into five parts.Part one:In the study,349 patients with chronic HBV infection and treat-naive were enrolled,among of them 122 chronic HBV-infected patients with ALT level?2 times of upper limit of normal?ULN?and 227chronic HBV-infected patients with normal or mildly elevated ALT level.277 chronic HBV-infected patients with biopsy-proven were meet the inclusion criteria.We further detected the routine clinical serum virological and biochemical marker with different liver different degree of liver inflammation activity and fibrosis,and assessed the diagnostic value of these clinical serum markers for liver inflammation activity and fibrosis.Part two:49 of 277 chronic HBV-infected patients with biopsy-proven were enrolled.We detected the expression of serum IL-17 and IL-29 in chronic HBV infection by enzyme-linked immunosorbent assay.We further explored the expression of serum IL-17 and IL-29 in different ALT level and liver inflammation activity.Part three:176chronic HBV infected patients with biopsy-proven and treat-na?ve was enrolled in this part.27 chronic HBV-infected patients with ALT level?2ULN and 149 chronic HBV-infected patients with normal or mildly elevated ALT level were included.We detected the serum MCP-1 expression in chronic HBV infection by enzyme-linked immunosorbent assay.We further explored the serum MCP-1 expression in different ALT level and liver inflammation activity.On this basis,we combined routine clinical serum virological and biochemical marker and inflammatory chemokines to establish a noninvasive model to assess the liver inflammation activity.Part four:176 chronic HBV infected patients with biopsy-proven and treat-na?ve were enrolled in this part.The serum MCP-1 expression in chronic HBV infection was detected by enzyme-linked immunosorbent assay.Similarly,we combined routine clinical serum virological and biochemical marker and inflammatory chemokines to establish a noninvasive model to assess the liver fibrosis.Part five:169 cases of chronic HBV infected patients who was positive of HBeAg and had ALT less than two times of upper limit were enrolled in this part.A non-invasive diagnostic model was established based on routine clinical biomarkers and immunological indicators.Then we evaluated the model's diagnostic capabilities in the validation queue.SPSS22.0 software was used for statistical analysis.For continuous variable,K-S test were conduct firstly.With normal distribution,the variables are expressed with mean value±standard deviation,and compared with T-tests.With abnormal distribution,the variables are expressed with median?25%-75%percentile?,and compared with rank sum tests.The diagnostic ability of noninvasive model were asses with receiver operator characteristic curve?ROC?,sensitivity,specificity,positive predictive value?PPV?and negative predictive value?NPV?.All the test were conduct with two-sided test,and P<0.05 means have significant differences.Results:1.In 277 biopsy-proven chronic HBV infected patients,significant liver inflammation rate was 47.7%,and significant liver fibrosis rate was 26%;In 72ALT?2ULN chronic HBV infected patients,significant liver inflammation rate was68.1%,and significant liver fibrosis rate was 37.5%;In 205 ALT<2ULN chronic HBV infected patients,significant liver inflammation rate was 40.5%,and significant liver fibrosis rate was 22.0%.2.The highest diagnostic ability of routine clinical serum virological and biochemical marker for significant liver inflammation and fibrosis were AST,ROC was 0.701 and 0.662,respectively.3.Serum levels of IL-17 in CHB patients with ALT?2ULN were 11.90±8.15.Serum IL-17 levels inchronic HBV-infected patients with ALT<2ULN were 14.06±18.93.There was no significant difference between them and the normal human.4.The IL-29 level in the serum of patients with CHB with ALT?2ULN was 670.02±39.56,and the IL-29 level in the chronic HBV-infected patients with ALT<2ULN was 689.25±71.26.There was no difference between the two groups,but they were all significant higher than normal serum levels.5.The serum expression of MCP-1 in chronic HBV infection was99.95?76.17,137.05?pg/ml,significant higher than normal person42.56?36.32,55.18?pg/ml?P<0.05?.The serum expression of MCP-1 in chronic HBV infection with different ALT level was significant difference.The serum expression of MCP-1 in chronic HBV infection with ALT?2ULN was 119.16?99.88,170.63?pg/ml,significant higher than those patients with ALT<2ULN 95.1?72.05,127.91??P<0.05?.The serum expression of MCP-1 in chronic HBV infection with ALT?2ULN or ALT<2ULN group was significant higher than normal person.6.The serum expression of MCP-1 in chronic HBV infection with different liver inflammation activity had significant difference.MCP-1_G0:40.06?38.79-40.06?pg/ml;MCP-1_G1:83.2?63.86-102.25?pg/ml;MCP-1_G2:116.04?91.61-174.79?pg/ml;MCP-1_G3:144.42?114.96-212.69?pg/ml.There were significant differences in groups.7.The serum expression of MCP-1 in chronic HBV infection with different liver fibrosis had significant difference.MCP-1_S0:89.62?62.82-105.81?pg/ml;MCP-1_S1:95.92?76.18-126.98?pg/ml;MCP-1_S2:101.88?80.4-147.73?pg/ml;MCP-1_S3:130.26?114.96-212.16?pg/ml;MCP-1_S4:139.40?110.56-203.25?pg/ml.There was significant difference in groups.8.The non-invasive model to assess the significant liver inflammation activity was:G_inflammation=-7.569+0.048*AST?U/L?+0.034*MCP-1?pg/ml?-0.009*PLT?*10⁹/L?.AUC was 0.878,95%confidence interval?CI?was 0.8291⁰.9269.The best cutoff point was-3.52,sensitivity was 67.42%,specificity was 90.80%,PPV was 88.23%and NPV was73.15%.The AUC of non-invasive model to assess the significant liver inflammation activity in chronic HBV infection with ALT<2ULN was 0.875,95%CI was0.8213⁰.9293.The best cutoff point was-4.5,sensitivity was 83.58%specificity was73.17%,PPV was 71.80%and NPV was 84.50%.9.The non-invasive model to assess significant liver fibrosis was:S_fibrosis=-6.711+0.007*MCP-1?pg/ml?-0.018*ALT?U/L?+0.042*AST?U/L?+0.016*ALP?U/L?+0.0181*APT?S?-0.014*PLT?*10⁹/L?.AUC was0.7951,95%CI was 0.7267⁰.8635.The best cutoff point was-0.12,sensitivity was55.56%,specificity 90.27%,PPV was 76.10%and NPV was 78.46%.10.Established and validated a noninvasive diagnostic model for assessing significant inflammation of liver tissue in HBeAg-positive chronic HBV-infected individuals with ALT<2ULN.G=0.047*MCP-1?Pg/ml?+0.083*AST?U/1?-0.002*HBeAg?S/CO?-6.09.In the training group,the AUC for moderate or severe necrotizing inflammation was 0.897,and the 95%CI was 0.840-0.955.In the validation group,the model predicted a moderate or severe necroinflammatory inflammation with an AUC of 0.844 and a 95%CI of0.740-0.948.-1.70 was consider as the low cutoff point of the model and 0.15 was consider as the high cutoff point of the model.This model allowed 83?73.5%?patients in the training group to avoid liver biopsy;40?71.4%?patients in the validation group avoided liver biopsy and a total of 123?72.8%?patients avoided liver biopsy.Conclusion:1.In chronic HBV infections,significant liver inflammation rate was higher than significant liver fibrosis rate.2.In chronic HBV infections with ALT<2ULN,AST showed asuitable diagnostic ability for significant liver inflammation and fibrosis.3.Serum IL-17 was not significantly elevated in patients with chronic HBV infection.Serum IL-29 was significantly elevated in the chronic HBV infection.However,neither of them can distinguish the grade of liver inflammation.4.The serum expression of MCP-1 was up-regulated in chronic HBV infection.The serum expression of MCP-1 in chronic HBV infection with different ALT level was significant difference.5.The serum expression of MCP-1 was positive relationship with liver inflammation activity and fibrosis,and can serve as the noninvasive marker for liver inflammation activity and fibrosis.6.The combination of PLT,AST and MCP-1 for the noninvasive inflammation model can be used to assess the liver inflammation activity in chronic HBV infection,especially for patients with ALT<2ULN.7.The combination of PLT,AST,ALT,APTT,ALP and MCP-1 for the noninvasive fibrosis model can be used to assess the liver fibrosis.8.A non-invasive diagnostic model for assessing hepatic inflammation in HBeAg-positive chronic HBV-infected patients with ALT<2ULN was constructed with high diagnostic value and reproducibility...