Factors Influencing Hepatitis E Virus Antigen-capture EIA And Significance Of Antigen On Diagnosis Of Hepatitis E Virus Infection

Hepatitis E virus(Hepatitis E virus,HEV)is the causative agent of hepatitis E.HEV typically causes an acute and self-limiting infection.However,it has a higher mortality in pregnant women,up to 20%.Chronic HEV infection also has been rqported recently in transplant patients and other immunocompromised patients.Hepatitis E is considered to be endemic in many developing countries in Asia,Africa and Latin America.Hepatitis E virus infection manifests as epidemic and sporadic hepatitis in disease-endemic areas.In the recent few years,it has occurred in several high income countries,in an increasing number of sporadic cases.It has been estimated that two billion people,representing one third of the world's population,live in endemic areas for HEV.Therefore,HE is a major health concern worldwide.The clinical diagnosis of hepatitis E mainly depends on detection of anti-HEV IgM and IgG antibodies in serum.However,in the window period or HEV infection in immunocompromised patients whose antibody production is limited,nucleic acid and antigen detection become to be more necessary.Enzyme linked immunosorbent assay(EIA)antigen detection reagent according to the HEV ORF2 has been applied in China,India,Europe and the United States,and attracted more and more attention.To understand the influencing factors of HEV antigen reagent and the significance of detection of HEV antigen on diagnosis of HEV infection,it's very important for evaluating whether the ORF2 antigen can be used as early diagnostic markers and potential value of antigen detection reagent as a method for early diagnosis.In this study,a number of factors influencing the detection of antigen,such as the presence of antibodies,antigen detection limits,different types of matrix,proportion of antigen and nucleic acid in different type samples were studied.Another aims of this study is to understand the relationship between the antigen and other viral diagnostic markers in patients with HEV infection;to determinate the distribution of antigen in the cynomolgus monkeys and urine infectious capacity for HEV by cynomolgus monkeys animal model.Factors infuencing the detection of hepatitis E virus antigen To determine the effect of anti-HEV antibodies on the detection of HEV-Ag,serial dilutions of the two HEV samples were spiked into anti-HEV IgG positive or negative sera in vitro and tested by the EIA.By statistical analysis,the HEV-Ag EIA S/CO values of the serum sample and cell culture supernatant mixed with anti-HEV IgG positive sera were significantly reduced compared to the control.The detection limit of the HEV-Ag EIA was determined using 2-fold serial dilutions of the genotype 4 strain cell culture supernatant validated using the HEV WHO standard for NAT.All dilutions with an HEV load greater than 50 IU/mL were HEV-Ag positive and none of the samples with a concentration of 6.3 IU/mL were positive.By probit analysis,the minimum detection limit with 95%confidence was 54.2 IU/mL,and the 95%confidence interval(95%CI)ranged from 38.5 to 129.8 IU/mL.In comparison,the detection limit of real time RT-PCR based on the same serial dilutions was 24 IU/mL,and the 95%CI was 15.1 to 56.5 IU/mL.Thus,the antigen EIA is less sensitive than HEV RNA detection by real time RT=PCR.The supernatant of genotype 4 HEV strain was diluted serially half-log10 fold with serum,urine and fecal suspension.HEV-Ag EIA was performed on the serial dilutions.The results showed there was no significant impact of different matrix types on HEV antigen detection.The sensitivities of nucleic acid detection in serum,urine,feces matrix were in 30?300 times higher than the antigen.By comparing the ratio of antigen and RNA(Ag/RNA)in different types of samples,the results showed that the ratio of antigen and RNA in urine samples was significantly higher than those in serum,cell culture and feces.The minimum ratio was in fecal samples.The proportion of viral antigen was the highest in urine samples,the lowest proportion was in stool samples.The exist ratio of HEV antigen and RNA in the urine,serum,cell culture virus solution or feces were not same.The influence of temperature,storage time and repeated freezing and thawing and other factors on antigen and RNA detection were studied.It turned out that the stability of antigen and RNA in serum and stool samples stored at-20 ? was better than 4? and room temperature.However,in urine,the stability of HEV antigen and RNA at 4? was better than that of-20?and room temperature.There was less influence by repeated freezing and thawing on the antigen detection,but the impact on nucleic acid detection was significant,especially in urine.The results suggest that urine samples for RNA detection should avoid repeated freezing and thawing.Detection of HEV antigen in patients with clinical infection of HEV It was firstly found HEV antigen in the urine of chronic hepatitis E patient.After a series of trace detection of urine and serum of the patients before treatment,the dynamic changes of liver function markers results showed liver function was abnormal and the antigen S/CO value in urine was significantly higher than that in serum,the concentration of RNA in urine was higher than that of serum.After treatment,the antigen and RNA in serum gradually decrease,but the antigen and nucleic acid of urine were still positive,and the duration of positive antigen was longer than that of RNA,IgM and IgG antibodies of serum were positive,but antibody could not be detected out in urine.It suggested that urine antigen had potential applications as a diagnostic marker.In order to observe whether these results also present in acute hepatitis E patients,the urine samples from five acute HE patients were collected from infectious hospital,three of them(60%)were both antigen and RNA positive,and two of them(40%)were only antigen-positive.Meanwhile,urine samples were collected from five chronic hepatitis B patients and five normal blood donors.The RNA,antigen,IgM and IgG antibodies were negative in urine.Our results showed that positive rate of antigen was 100%in urine during viremia of hepatitis E patients.Through the detection of HEV antigen and other markers in serum samples from acute hepatitis E patients or suspected HEV patients,the relationship between HEV antigen and other markers was analyzed.The results showed that positive rates of RNA and antigen were 60.7%was 55.6%respectively.The co-incidence rate was 77.8%among 117 samples.The 117 serum samples were divided into 4 groups as IgM-/IgG-,IgM+/IgG-,IgM+/IgG+ ? IgM-/IgG+.The positive rates of antigen were 100%(3/3),94.1%(16/17),59.2%(45/76)and 4.5%(1/22)respectively,showing a gradual decrease in these four groups.The positivity rates of HEV-Ag in the HEV RNA-positive samples were 100%(3/3),93.8%(15/16)and 71.2%(37/52)in the IgM-/IgG-,IgM+/IgG-and IgM+/IgG+ samples,respectively,also showing a gradual decrease in these three groups.The concordance rate of these two markers in each group varied and was inversely proportional to the titre of the antibodies.The correlation between HEV-Ag EIA activities(S/CO)and the corresponding HEV RNA concentrations(Log10IU/mL)was determined.The Pearson correlation coefficient r was 0.55(P ? 0.01),and thus,the correlation was significant.Antigen and other markers of fecal samples in 73 patients with acute hepatitis were detected.The results showed that the positive rate of antigen was 76.7%and that of nucleic acid was 84.9%.The coincidence rate was 80.8%.The antigen S/CO values and nucleic acid concentration(Log10IU/mL)in positive fecal samples showed a significant correlation using Pearson correlation analysis.The correlation coefficient r was 0.93.HEV sequences isolated from serum and urine samples of acute and chronic hepatitis E patients showed that all the sequences of HEV were belong to genotype 4a and 4d.Detection of HEV antigen in monkeys infected with HEV Three cynomolgus monkeys were infected by HEV genotype 1 and genotype 4 strains.ALT,AST and other liver index became abnormal within two weeks of infection.The average values of ALT and AST were 3 to 6 times higher than that of normal level.The HEV antigen and RNA were not only detected in serum and feces,but also in urine.The levels of ALT or AST in cynomolgus monkeys were increased along with the antibodies appearing,but with the antibody reaching peak,the levels of them began to decline.When the liver function tended to be normal,antigen concentration in serum decreased to the cutoff value,but the antigen levels in urine and stool were positive.This indicated that the dection of antigen levels in urine was an effective method for monitoring of HEV infection process.The ratio of antigen and RNA in serum,urine and faeces of different stages were analyzed.The results showed that the ratio of antigen and RNA in urine samples was significantly higher than those in serum and fecal samples at the same period whilst the lowest ratio was in stool samples.With the extension of infection,the concentration of antigen in urine increased,which resulted in the ratio of antigen and RNA decreasing in urine.The results indicated that complete virus in the urine was increasing.Healthy cynomolgus monkeys were challenged by urine from monkeys infected HEV,the antigen markers were first detected in the serum two weeks after challenging.On two weeks later,the antigens in urine and feces became to be positive.The ALT and AST turned to abnormal about five weeks later and the antibodies become positive gradually.The antigen in urine peaked at fifth week and tended to be negative at ninth week.The antigen in fecal peaked at sixth week and tended to be negative at eighth week.The dynamic change of HEV markers in monkeys were in accordance with infected patients.It was fully illustrated that there was infectious HEV in the urine.Pathological section of liver in viremia displayed cells degeneration,the cells of periportal infiltration turned inflammatory and Kupffer cells showed prominent sinus wall.The kidney also showed tubular protein like substances accumulation,interstitial infiltration of inflammatory cells and other changes.Renal tubule protein-like substances could be seen gathering,interstitial inflammatory cell occured.The expression of antigen could be seen in liver,kidney,bladder,ureter,duoderum,brain,ovary,pancreas,salivary gland and other tissues.The virus particles were observed by transmission electron microscopy in acute hepatitis E of cynomolgus monkey in liver,kidney,ureter,brain and colon tissue.The above results provided the evidence for hepatitis E virus replication out of liver.