Characterization of the enrolled patients

WE enrolled a total of 82 chronic HBV infected patients who were treatment- naïve. These were categorized into HBeAg-positive chronic HBV infection (n = 53) and HBeAg-negative chronic HBV infection (n = 29). In addition, according to the clinical diagnosis, these patients were also divided into CHB group (n = 62) and hepatitis B cirrhosis (LC) group (n = 20). The detailed baseline summary characteristics of the included patients are listed in Table 1. The median level of HBV DNA was exhibited to be higher than HBV RNA by 1–2 log10 in both HBeAg-positive and HBeAg-negative phases. The ratio of serum HBV RNA to HBV DNA was calculated to assess the reverse transcriptional efficiency of pgRNA [14]. As shown in Fig. 1, The median ratio of HBV RNA to HBV DNA was 0.59 (IQR0.55–0.63), 0.66 (IQR0.61–1.00), 0.59 (IQR0.55–0.68) and 0.62 (IQR0.60–0.66) for HBeAg-positive chronic HBV infection, HBeAg-negative chronic HBV infection, CHB and LC. The RNA to DNA ratio was higher in patients who were HBeAg-negative than HBeAg-positive chronic HBV infection (0.66 vs. 0.59; P < 0.05). The ratio was not significantly different for CHB and LC subgroup ((0.59 vs. 0.62; P = 0.14). To investigate the utility of HBV RNA in the clinic, we performed an observational, non-interventive, real clinical cohort study. Among the 82 patients who received first-line oral NAs, 59, 46, and 30 patients were followed up for 12, 24, and 48 weeks receiving first-line NAs, respectively. After 48 weeks of follow-up, five patients developed HBeAg seroconversion. No patient had HBsAg seroconversion.

Table1 Baseline characteristics of treatment-naïve patientsFig. 1

Serum HBV RNA to HBV DNA ration in chronic HBV infection

Correlation between HBV serological markers in treatment-naïve patients

We first analyzed the correlation coefficients between HBV serological markers in treatment-naïve patients. The summary of correlation coefficients between HBV serological markers in treatment-naïve patients are shown in Table 2. Among the total 82 patients at baseline, the serum levels of HBV RNA were positively associated with HBV DNA (r = 0.602, P < 0.05), HBsAg (r = 0.502, P < 0.05). In addition, we found a positive correlation between HBV DNA and HBsAg (r = 0.496, P < 0.05). Furthermore, we also analyzed the correlations of HBV serological markers in HBeAg-positive and HBeAg-negative chronic HBV infection subgroups. a moderate positive correlation of HBV RNA level and HBV DNA was also observed in either HBeAg-positive patients (r = 0.445, P < 0.05) or HBeAg-negative patients (r = 0.478, P < 0.05) at baseline. The levels of HBV RNA and HBsAg remained correlated in HBeAg-positive patients (r = 0.413, P < 0.05), but in HBeAg-negative patients there was no correlation (r = 0.352, P = 0.061). Moreover, there was a linear correlation with HBsAg and HBV DNA in HBeAg-positive patients (r = 0.442, P < 0.05) but not in HBeAg-negative patients (r = 0.265, P = 0.165). Further subgroup analysis was performed based on clinical diagnosis. In the CHB group, HBV RNA showed a positive linear correlation with HBV DNA and HBsAg (r = 0.598 and 0.515, P < 0.05). HBV DNA and HBsAg remained were positively correlated (r = 0.551, P < 0.05). Furthermore, in the LC group, HBV RNA only showed a strong linear correlation with HBV DNA (r = 0.700, P < 0.05), but not with HBsAg (r = 0.367, P = 0.112). There is almost no correlation between HBV DNA and HBsAg (r = 0.278, P = 0.235). The correlation plots for HBV serological markers are shown in Additional file 1: Fig. S1, Additional file 2: Fig. S2, Additional file 3: Fig. S3.

Table 2 Correlation between HBV serological markers in treatment-naïve patientsThe proportion of patients with detectable HBV RNA and HBV DNA during 48 weeks of antiviral therapy

At therapy baseline, HBV DNA was detected in 99% (n = 81) of enrolled patients, while HBV RNA was only found in 71 (87%) patients. At week 12, 59 patients completed the follow-up. Of those, 41 (69%) patients had detectable HBV DNA, and 41 (69%) patients had detectable HBV RNA. After 24 weeks of therapy, 46 patients completed the follow-up. Among those people, 54% (n = 25) of patients had detectable HBV DNA and 70% (n = 32) had detectable HBV RNA. After 48 weeks of therapy, only 30 patients had completed the follow-up, 37% (n = 11) patients still had detectable HBV DNA, and 67% (n = 20) had RNA (Fig. 2a). Subgroup analysis was performed for HBeAg-positive and HBeAg-negative patients separately (Fig. 2b–c). At baseline, almost all patients (n = 53, 100% for HBeAg-positive; n = 28, 98% for HBeAg-negative) had detectable HBV DNA in serum, while 52 (98%) HBeAg-positive patients and 19 (66%) HBeAg-negative had detectable HBV RNA in serum. At week 12, there were 36 HBeAg-positive patients, and 23 HBeAg-negative patients completed the follow-up. In the HBeAg-positive subgroup, 32 (89%) patients were positive for HBV DNA, and 31(86%) patients were positive for HBV RNA. In the HBeAg-negative subgroup, 9 (39%) patients were positive for HBV DNA, and 10 (43%) patients were positive for HBV RNA. After 24 weeks of NA therapy, there were 34 HBeAg-positive patients, and 12 HBeAg-negative patients completed the follow-up. In the HBeAg-positive subgroup, 21 (62%) patients had detectable HBV DNA, and 31 (91%) patients had detectable HBV RNA. However, in the HBeAg-negative subgroup, HBV DNA was detectable in 4 (33%) patients, and serum HBV RNA was detectable only in one (8%) patient. At week 48, there were 21 HBeAg-positive patients, and 9 HBeAg-negative patients completed the follow-up. In the HBeAg-positive subgroup, 9 (43%) patients were positive for HBV DNA, and 17 (81%) patients were positive for HBV RNA. In the HBeAg-negative subgroup, only 2 (22%) patients were positive for HBV DNA, and 3 (33%) patients were positive for HBV RNA. At the same time, we also analyzed CHB and LC subgroups (Fig. 2d, e). In the CHB subgroup, at baseline, 61 patients (98%) had detectable HBV DNA, and 52 (84%) patients had detectable HBV RNA. At week 12, 46 patients had completed the follow-up. Among them, both of 31(67%) patients had detectable HBV DNA and HBV RNA. At week 24, 38 patients had completed the follow-up. Among them, 21 (55%) and 28 (74%) patients had detectable HBV DNA and HBV RNA, respectively. At week 48, 21 patients completed the follow-up. Among them, 7 (33%) and 15 (71%) patients had detectable HBV DNA and HBV RNA, respectively. In the LC subgroup, at baseline, all patients (n = 20, 100%) had detectable HBV DNA and 19 (95%) patients had detectable HBV RNA. At week 12, 13 patients had completed the follow-up. Among them, both of 10 (77%) patients had detectable HBV DNA and HBV RNA, respectively. At week 24, only eight patients had completed the follow-up. Among them, both of four (50%) patients had detectable HBV DNA and HBV RNA. At week 48, nine patients had completed the follow-up. Among them, 4 (44%) and 5 (56%) patients had detectable HBV DNA and HBV RNA, respectively.

Fig. 2

Proportions of patients with detected HBV DNA and HBV RNA during NA therapy in cohort. a proportions of total patients. b proportions of HBeAg-positive patients. c proportions of HBeAg-negative patients. d proportions of CHB patients. e proportions of LC patients

Dynamic changes of HBV serological markers and biochemical indicators during 48 weeks of NA treatment therapy

The total HBV serological markers and biochemical indicators presented a decreasing trend from baseline to 48 weeks. As shown in Fig. 3a, the mean level of serum HBV RNA was 4.62 (IQR: 3.05–5.82) log10 copies/mL at baseline; and the mean levels of serum HBV RNA at weeks 12, 24, and 48 were 2.88 (IQR: 0–4.67), 2.71 (IQR: 0–4.22) and 2.96 (IQR: 0–4.32) log10 copies/ mL, respectively (P < 0.05). The mean level of serum HBV DNA was 6.26 (IQR: 5.20–7.21) log10 IU/mL at baseline, and the mean levels of serum HBV DNA at weeks 12, 24, and 48 was 1.99 (IQR: 1.30–2.79), 1.41 (IQR: 1.30–2.07) and0.00 (IQR: 0.00–1.50) log10 IU/ mL (P < 0.05) (Fig. 3b). The mean level of serum HBsAg was 3.62 log10 IU/mL at baseline, and the mean level of serum HBsAg at weeks 12, 24, and 48 was 3.37, 3.43, and 3.46 log10 IU/ mL (Fig. 3c). Furthermore, during 48 weeks of first-line NAs therapy, biochemical indicators such as ALT and AST were gradually normal in Fig. 3d, e. In summary, the current long-time NA therapy can induce viral suppression and normalization of liver function.

Fig. 3

Diagrammatic presentation of changes in individual serological and virological markers in different follow-up time point. a HBV RNA viral load [log10 copies/ml]. b HBV DNA viral load [log10 IU/ml]. c HBsAg levels [log10 IU/mL]. d ALT levels [log10 IU/L]. e AST levels [log10 IU/L]

Correlation between HBV serological markers during 48 weeks of NA treatment therapy

The correlation coefficients between the viral biomarkers upon 12, 24, and 48 weeks of NA therapy were analyzed (Table 3). In general, HBV RNA showed a strong linear correlation with HBV DNA upon 12, 24, and 48 weeks of NA treatment (r = 0.640, 0.715, and 0.656, respectively, P < 0.05), and a weakened correlation between HBV RNA and HBsAg was still preserved at 12 and 24 weeks of NA treatment (r = 0.440 and 0.438, respectively, P < 0.05). However, at 48 weeks of NA treatment, there was no correlation between HBV RNA and HBsAg (r = 0.216, P = 0.252). In addition, the correlation between HBV DNA and HBsAg was still preserved upon 12 and 24 weeks (r = 0.519 and 0.398, respectively, P < 0.05), but not upon 48 weeks of NA treatment (r = 0.094, P = 0.622).

Table 3 Correlation between HBV serological markers in NA-treated patientsSubgroup analysis of correlation between HBV serological markers during 48 weeks of NA treatment therapy

When subgroup analysis is performed for HBeAg-positive and HBeAg-negative patients separately, the linear correlation between serum HBV RNA and HBV DNA & HBsAg was stronger in HBeAg-positive than in HBeAg-negative patients. The specific results are shown in Table 4. In HBeAg-positive patients, HBV RNA correlated with HBV DNA (at week 12: r = 0.594, week 24: r = 0.792, and week 48: r = 0.770; all P < 0.05) and with HBsAg (at week 12: r = 0.540, P < 0.05; week 24: r = 0.377, P < 0.05; and week 48: r = 0.201, P = 0.383), but not in HBeAg-negative patients. Furthermore, we also analyzed the correlation coefficients between the viral biomarkers in CHB and LC patients. In CHB patients, HBV RNA correlated with HBV DNA (at week 12: r = 0.686 week 24: r = 0.703, and week 48: r = 0.609; all P < 0.05) and with HBsAg (at week 12: r = 0.423 P < 0.05; week 24: r = 0.440, P < 0.05; and week 48: r = 0.031, P = 0.894). In LC patients, HBV RNA correlated with HBV DNA (at week 12: r = 0.600, P < 0.05; week 24: r = 0.736, P < 0.05 and week 48: r = 0.934, P < 0.05) and HBsAg (at week 12: r = 0.608, P < 0.05; week 24: r = 0.482, P = 0.226; and week 48: r = 0.627, P = 0.071) (Additional file 4: Table S1).

Table 4 Correlation between HBV serological markers in NA-treated patients according to HBeAg Status