How to evaluate the HbeAg-positive during the natural course of Chronic Hepatitis B in clinic? A cross-sectional study

Background The previous studies showed the correlation between HBsAg and serum HBV DNA levels was weak or missing. Objective The study aims to investigate the correlation between HBeAg and HBV DNA levels, and to find an alternative tool to evaluate the HBV DNA level for clinicians. A total of 1020 patients with CHB were enrolled in this cross-sectional study. We divided the patients into four groups as: HBeAg positivity and negativity groups, and high and low HBV DNA levels groups. Further, as per the levels of serum HBV DNA, we performed subgroups’ analyses for the HBeAg-positive and HBeAg-negative groups. Results Results showed that the ALT, ALB and HBeAg are independent factors to estimate the serum HBV DNA in CHB patients. But diagnosing the high levels of HBV DNA is not credible (the AUC=0.622, Fig1-A). In HBeAg-positive group, when the level of HBeAg is higher than 16.15 S/CO, we can predict the patient with high levels of HBV DNA (> 2000 IU/ml, AUC=0.787, Fig1-C) and the patients were 4 folds to have the high levels of HBV DNA than the HBeAg-negativ (table3). The levels of ALT and TB are the independent risk factors for the patients in HBeAg-negative group. When the levels of ALT and TB are higher 36.5 IU/L and 11.15 umol/L,respectively, the patient would have a high levels of HBV DNA (> 2000 IU/ml, AUC=0.609, Fig2-B).

positive. On the other hand, the patients with HBeAg-negative are not mean having a low levels of HBV DNA, which can be evaluated by the levels of ALT and TB.
Testing the HBV DNA of the serum is a common method of evaluating the effect of treatment decisions on patients and assessing the response to antiviral therapy [9,10].
Previous studies have reported a correlation between the HBsAg and HBV DNA levels and have suggested [6,[11][12][13][14] serum HBsAg quantitation can be a marker to predict HBV DNA levels. However, similar studies have shown no correlations of HBsAg with HBV DNA [15][16]. Research trials have tested the quantification of serum HBsAg accurately, which is difficult in clinic to test the quantification of serum HBeAg in our center, especially, Gupta E, Kumar A, et al. [11] reported that the best cut-off point of serum HBsAg quantification to predict the high HBV DNA levels is 3.36×10 3 IU/ml. However, when the HBsAg level is >3000 IU/mL in our center, the results were shown to be >3000 IU/mL. Thus, HBsAg quantification is difficult to apply in the clinical setting, and the correlation with HBV DNA is weak or absent. In contrast, the HBV DNA levels are not exactly similar with the natural course of CHB. Generally, the patients with HBeAg positivity (IT phase or IC phase) have high levels of serum HBV DNA. However, in the clinical setting, the levels of serum HBV DNA remain undetected in some patients. Patients with HBeAg-negativity (LR phase and HBeAg-negative hepatitis phase) are believed to have low HBV DNA levels. However, a survey of HBeAg-negative patients showed that 47.5% of the patients in LR phase and 63.4% in the HBeAg-negative hepatitis phase had high serum HBV DNA levels (> 10 4 copies/mL) [17]. Thus, HBV DNA levels are high or undetected in both, HBeAg-positive and HBeAg-negative, patients. This phenomenon poses a challenge to clinicians, regarding whether and when we should recommend a HBV DNA test for patients with CHB, because HBV DNA testing is expensive, and there is insufficient evidence to convince the patient.
Therefore, there is a need for an alternative tool to evaluate the levels of HBV DNA easily in CHB.
HBeAg plays a crucial role in HBV infection, meaning the high replication and high infectivity of CHB [18], but it has not been reported the correlation with HBV DNA in previous studies. This study aimed to determine the correlation between HBeAg and HBV DNA, to evaluate the difference between HBeAg-positive and HBeAg-negative patients, and to find an alternative tool to serum HBV DNA levels in CHB patients to establish whether to have a serum HBV DNA test.

Patients
We retrospectively evaluated HBV patients who underwent serum HBV and HBV-DNA tests at our center, in the Department of Liver Surgery, Liver Transplantation Center, West China Hospital of Sichuan University, from 2011 to 2013. The criteria for patient inclusion were as follows: i) Age > 18 years, ii) first visit to our center, and iii) positivity in HBsAg and HBeAg, or positive with HBsAg on serum HBV test. Patients were excluded when they had i) co-infection with other hepatitis viruses, such as hepatitis C, A, and D; ii) had acute hepatitis, especially acute liver failure; or iii) had received antivirus treatment at other hospitals. According to previous studies [7,8,11], high HBV DNA level is defined to be >2000 IU/mL.

Methods
We divided the patients into four groups as per HBeAg positivity and negativity, high and low HBV DNA levels, respectively. Further, as per the levels of serum HBV DNA, we performed subgroup analyses for the HBeAg-positive and HBeAg-negative groups.

Statistical analysis
All the data were analyzed using SPSS 22.0 data statistical software (SPSS Inc., Chicago, IL, USA). Continuous variables are expressed as mean ± standard deviation (± sd) values.
Between-group comparisons of the continuous variables were made using T-test, and the optimal predictive cut-off value was determined by Receiver Operating Characteristics (ROC) curves. Using logistic regression multivariate analysis to identify the independent risk and getting a model predict. The categorical variables were analyzed using chi-square test (c 2 ). For all the analyses, P value < 0.05 was considered statistically significant.

Comparison between HBeAg (+) and HBeAg (−) patients
We enrolled 1020 patients in this study from January Table 1 shows the feature of high HBV DNA levels and low HBV DNA levels of the whole course of natural CHB. The continuous variables of PLT, AST, ALT, and ALB were significantly different between the high HBV DNA group and the low HBV DNA group, with the ROC shown in figure 1-B. The AUC of AST, ALT, ALB, and PLT was 0.635, 0.642, 0.432, and 0.473, respectively, and the optimal cutoff points were 46.5 IU/L, 42.5 IU/L, 25.5 g/L, and 74.5 × 10 9 /L, respectively.The levels of AST and ALT were higher in high HBV DNA group, and the levels of PLT and ALB were lower than low HBV DNA group. However, the logistic regression multivariate analyses showed no significance in the PLT and AST.

Different HBV DNA levels in CHB
Through the logistic regression multivariate analysis and univariate analysis, the independent factors to evaluate the levels of HBV DNA are ALT, ALB and HBeAg (table 3).
Based on the results, we drew the following predictive model:  Table 2  they experience a prolonged IC phase. In contrast, infected after early children, the patients generally do not experience the IT phase, they will enter the LR phage quickly.

HBV DNA levels in HBeAg (−) patients
And the levels of serum HBV DNA did not mean low [19]. So the age is not a reasonable factor to predict the serum HBV DNA of patients with HBeAg-positive .
The PLT count is different between the HBeAg-positive and HBeAg-negative patients as well as those with high and low levels of serum HBV DNA (p 1 =0.001 and p 2 =0.011, table 1). However, in subgroups' analyses showed a non-significant difference (p 1 =0.739 and p 2 =0.086 , table 2). An animal model has suggested a link between the PLT count and immune control of HBV infection [20,21]. The model analyzed the whole natural course of the CHB, so when we divided the situation into two groups: i) the PLT count in HBeAg-positive and HBeAg-negative group; ii) the PLT count in high or low HBV DNA levels group.
We can find that the PLT is significant difference between HBeAg-positive versus HBeAgnegative groups ( 121.6 vs 140.81, p=0.001, table 1), and the high serum HBV DNA levels versus the low serum HBV DNA groups ( 129.84 vs 142.87, p=0.011, table 1), but the levels of PLT count is in the normal range, which can't convey a useful information to identify the levels of serum HBV DNA. Further, the multivariate analyses also eliminates the PLT count to predict the serum HBV DNA. Therefore, the correlation between PLT and HBV DNA level needs further research.
The level of ALT is very important in CHB patients because it is a marker of liver function damage. The natural course of CHB is based on biochemical, serological, and virological characteristics, including serum ALT levels, HBeAg serostatus, and HBV DNA levels [4][5][6][7].
Some studies have pointed out that although the level of AST is normal, the levels of serum HBV DNA need to be tested [22,23]. On the other hand, the high AST levels may be associated with HBV replication throughout the course of chronic HBV infection that do harm to the liver [24]. Combining with the multivariate analysis, the levels of ALT is an independent factor to predict the levels of serum HBV DNA, but the odds ratio (OR) are 1.004 and 1.005 in natural course of HBV and the patient with HBeAg-negative, respectively (table 3). The correlation is weak, especially for the patient in HBeAg-positive group, because the HBeAg-positive has a strong correlation of HBV DNA ( OR is 4.104, table 3). However, the levels of ALT as a factor to predict the levels of serum HBV DNA in HBeAg-negative group is credible, as to AUC is 0.655 ( figure 1-D) and there is no a strong factor in this group. There are also some questions that several studies have reported that the AST levels may vary with body mass index, abnormal lipid and carbohydrate metabolism, and the time of the day [25,26]. We need to pay attention to these factors while evaluating the HBV DNA levels of HBeAg-negative patient with CHB.
Previous studies have reported a weak or absent correlation between HBsAg levels and HBV DNA levels [11][12][13][14][15][16]. The serum HBsAg levels were higher in the HBeAg-positive patients than in the HBeAg-negative patients [6,24]. HBeAg can be as a sign of the high replication and infectivity of CHB [17]. According to HBeAg-positive to diagnose the high levels of serum HBV DNA is not accurate, because the AUC of ROC is just 0.622 ( figure 1-A), which is less than 0.7 ( an AUC < 0.7 indicates poor diagnostic ability ). However, combining with logistic regression multivariate analysis, HBeAg is an independent factor for the natural course of HBV to predict the levels of serum HBV DNA with an OR of 4. HBeAg-negativity with CHB is usually correlated with lower intrahepatic cccDNA levels [27][28][29]. Therefore, the serum HBV DNA levels are different between HBeAg-positive and HBeAg-negative patients. Lai CL, Ratziu V, et al. [19] reported that HBeAg-negative patients didn't mean the levels of serum HBV DNA is low. With the analysis of logistic regression multivariate analyses, the independent factors to predict the serum HBV DNA in HBeAg-negative are the levels of TB and ALT, and the cut-off values are 11.15 umol/L and 36.5 IU/L, respectively (table 2). Following the predict model ( Y 2 ), both of TB and ALT are higher than 11.15 umol/L and 36.5 IU/L, respectively, the levels of serum HBV DNA should undergo a test, though the levels of TB and ALT are in normal range, which is contradict with phages of low-replicate and HBeAg-negative hepatitis [5][6][7][8]. On the other hand, the HBV infection is throughout the all phages, we can not judge the effectivity of the levels of HBV DNA just by HBeAg-negative.
The present study has the following limitations: i) cross-sectional retrospective studies need a long-term follow-up to identify the parameters that reflect the response of patients who had received the antivirus treatment, especially those who had high levels of serum HBV DNA and were HBeAg-positive. In the current study, we did not perform follow-up to study the effect of antiviral treatment ii) We did not divide the patients into different phases as per the natural course of CHB. We studied the entire cohort of CHB patients; this might not reflect the real levels of serum HBV DNA in HBeAg-negative patients iii) The levels of serum HBV DNA may be different between HBV-genotype A and D; we did not assess the HBV-genotype in the patients.

In conclusion
HBeAg is an independent factor that reflects the levels of serum HBV DNA with a strong