Clinical prevalence and primary sources of OBI
Among 76428 admitted patients diagnosed with HBV infection, 977 were serum HBsAg negative and HBV DNA positive at the time of enrollment. After screening again by HBsAg titer and HBV DNA load, 279 patients with HBsAg titer < 0.05IU/mL and HBV DNA ≥ 10IU/mL were in OBI state. 279 patients with OBI were diagnosed with different disease types at admission, and the proportions were as follows: AHB accounted for 16.20%(58/279), CHB accounted for 69.27%(248/279), LC accounted for 5.31%(19/279), HCC accounted for 3.35%(12/279), others accounted for 15.92%(57/279). Therefore, it can be analyzed that the clinical OBI mainly comes from the cases of chronic hepatitis B and acute hepatitis B after antiviral treatment or spontaneous HBsAg clearance, but HBV DNA still exists in the liver and serum.
Basic clinical data and laboratory test results of OBI and HBsAg positive patients
According to the sex, age and disease type of 279 OBI patients, 279 HBsAg positive patients with chronic HBV infection were matched. The OBI patients were mainly male, accounting for 67.74%(189/279), and the median age was 49 years old. Most OBI patients and HBsAg positive patients have received antiviral therapy with nucleotide analogs and/or interferon in their clinical history, but the antiviral treatment time of OBI patients is generally longer than that of HBsAg positive patients. The positive rate of anti-HBs and anti-HBe in OBI patients were 45.88%(128/279) and 83.87%(234/279), which was significantly higher than that in HBsAg positive patients, while the positive rate of HBeAg in OBI patients was 2.51%(7/279), which was significantly lower than that in HBsAg positive patients, and the differences were statistically significant(P<0.05). The HBV DNA load of 94.98% of OBI patients was lower than that of 200IU/mL, and the median HBV DNA load was significantly lower than that of HBsAg positive patients (1.38 vs 2.52 IU/mL), and the difference was statistically significant (P<0.05). Compared with HBsAg positive patients, the median levels of ALT, AST, DBIL,γ-GT and ALP in OBI patients were lower (24 vs 29 U/L, 25 vs 29 U/L, 5.20 vs 5.70μmol/L, 25 vs 29 U/L, 77 vs 83 U/L), albumin and albumin/globulin had higher median levels (45.80 vs 44.20 g/L, 1.51 vs 1.45), and the differences were statistically significant(P<0.05)(Table 1).
Characteristics of HBV serologic markers in OBI patients
There are four main serological patterns in OBI patients: anti-HBs、anti-HBe and anti-HBc positive, anti-HBe and anti-HBc positive, anti-HBs and anti-HBc positive, and only anti-HBc positive. These four serological patterns were named pattern A, pattern B, pattern C, and pattern D, accounting for 35.84%(100/279), 47.67%(133/279), 7.89%(22/279), and 6.09%(17/279), respectively. Pattern B accounts for the highest proportion(47.67%), so it is the most important serological pattern of OBI patients. The above four serological models were all HBeAg negative, and there were also HBeAg positive cases in OBI patients, but only 7 cases were positive(2.51%, 7/279).
Clinical basic information of serum samples in OBI group, HBsAg positive group and healthy control group
According to the age, sex and type of disease diagnosed on admission of 30 OBI patients who collected clinical residual serum, 20 patients were matched from 279 HBsAg positive patients. Their clinical residual serum was collected as OBI group and HBsAg positive group, respectively. Information on gender, age distribution, serological markers and viral load of the OBI group and HBsAg positive group are described in Table 3. In the OBI group, 83.33%(24/30) had received antiviral therapy in the past, 66.67%(20/30) had received antiviral therapy for more than 3 months, and 70.00%(14/20) of them were treated with NA combined with peg-IFN-α2b for more than three months. In the HBsAg positive group, 70.00% had received antiviral therapy in the past, 45% had received antiviral therapy for more than three months, and 44.44% of them had been treated with NA combined with peg-IFN-α2b for more than three months. Previous studies have shown that NA combined with PEG-IFN treatment can improve the clearance rate of HBsAg. In this study, more patients in the OBI group were treated with NA combined with PEG-IFN-α2b, which proves that NA combined with PEG-IFN-α2b treatment can promote the development of HBV infected patients to OBI infection state.
Serum cytokines, chemokines and growth factors level
By comparing the median levels of cytokines between patients with OBI and HBsAg positive group, it was found that there were statistically significant differences in sCD40L, G-CSF, INF‐γ, MIP-1α, RANTES, Eotaxin, IL-4, IL-6, IL-8, IL-13, IL-17A, PDGF-AA, TGF-α, and TNF-β(P<0.05). We found that the higher median level of sCD40L(P=0.028), G-CSF(P=0.019), INF‐γ(P=0.024), MIP-1α(P=0.027), and RANTES(P=0.008) in OBI group, while the higher median level of Eotaxin(P=0.017), IL-4(P=0.025), IL-6(P=0.027), IL-8(P=0.014), IL-13(P=0.013), IL-17A(P=0.018), PDGF-AA(P=0.008), TGF-α(P=0.008), and TNF-β(P=0.030) in HBsAg positive group. There was no significant difference in other cytokines between the two groups. The median level of sCD40L, MIP-1α, PDGF-AA, and PDGF-AB/BB in OBI group and HBsAg positive group was lower than that in healthy control group, and the difference was statistically significant(P<0.05). The median level of EGF and IP-10 in HBsAg positive group was also lower than that in healthy control group, but the difference was not statistically significant(P>0.05). We found that the median levels of G-CSF, RANTES, and IFN-γ in OBI group were the highest, and there were significant differences among each group (P<0.05). However, when comparing the mean values of the measured cytokines, we found that unlike the median, the mean values of IP-10 and G-CSF were higher in HBsAg positive group, the difference was not statistically significant(P>0.05). The median(P25-P75) and mean±SD values of cytokines in these two groups are presented in Figure 2 and Table 4.
We found higher medians of sCD40L, IP-10, MIP-1α, PDGF-AA, and PDGF-AB/BB in the healthy control group; a significant difference was found for sCD40L(P<0.001), IP-10(P<0.001), MIP-1α(P=0.009), PDGF-AA(P<0.001), and PDGF-AB/BB(P<0.001) between the OBI group and healthy control group. The median of RANTES, IP-10(P<0.001), FGF-2, FLT-3L, Fractalkine, G-CSF, GRO-α, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, IL-10, IL-12(P40), IL-12(P70), IL-15, IL-17A, IL-17F, IL-18, MCP-1, M-CSF, TGF-α, and TNF-α were higher in healthy control group when compared to the OBI group, and the difference was statistically significant(P<0.05). There were no significant differences in the levels of other cytokines between the OBI and healthy control groups(Figure 3 and Table 4). IL-4, IL-13, MIP-1β,and TNF-β median values were similar between healthy control group and OBI group, but severalfold higher in HBsAg positive group(Table 4).
Correlation between serum cytokines/chemokines and ALT, AST, HBV DNA load in OBI patients
The correlation between serum cytokines/chemokines and ALT, AST, HBV DNA load, and the correlation between various cytokines and chemokines in OBI patients were analyzed. To explore possible relationships between them. We can observe a positive correlation between the levels of ALT and AST(P=0.001), IL-15(P=0.040), IL-18(P=0.031), and IL-17F(P=0.047). The levels of AST was positively correlated with IFN-γ(P=0.003), IL-6(P=0.032), IL-15(P=0.009), IL-18(P=0.003), IP-10(P=0.018), M-CSF(P=0.006), and TNF-α(P=0.019). HBV DNA load was positively correlated with GRO-α(P=0.039) and IL-6(P=0.019). It was possible to observe that there is a positive correlation between cytokines, Fractakine vs G-CSF(P=0.004), Fractakine vs IFN-α2(P=0.005), IFN-α2 vs IFN-γ(P=0.003), IFN-α2 vs IL-10(P<0.001), IFN-α2 vs IL-15(P<0.001), IFN-α2 vs MCP-1(P=0.003), IFN-γ vs IL-1RA(P<0.001), IFN-γ vs IL-15(P<0.001), IL-1β vs IL-17A(P=0.003), IL-1β vs TGF-α(P=0.002). They all have moderate or strong correlation. There is also a correlation between some other cytokines and chemokines, which is shown in Figure 4.