Deleterious role of hepatitis B virus infection in therapeutic response among patients with rheumatoid arthritis in a clinical practice setting: a case-control study

Patients with RA who fulfilled the 1987 revised criteria of the ACR [15] or the 2010 ACR/European League Against Rheumatism (EULAR) criteria for the classification of RA [16] were retrospectively recruited between June 2012 and August 2016 at Sun Yat-Sen Memorial Hospital from a prospective cohort, as described in our previous study [17]. All patients were treated according to the “treat-to-target” strategy [18, 19] and completed at least one year of follow up. Consecutive patients with RA with HBsAg positivity persisting in serum for more than 6 months were recruited as the CHB group. Each patient with RA in the CHB group was matched by age (± 5 years), sex, and disease activity at baseline to four subjects without HBsAg positivity or HBV DNA in serum, who comprised the non-CHB group. The timing of baseline for the four matched patients in the non-CHB group was as close as possible to baseline for the corresponding patient in the CHB group. Other inclusion criteria were patients aged ≥ 18 years and patients with active disease defined as Disease Activity Score 28-joint assessment (DAS28) with four variables including C-reactive protein (DAS28-CRP) ≥2.6. The exclusion criteria were elevated aminotransferase at baseline; other overlapping autoimmune diseases (e.g. systemic lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, cryoglobulinaemia, nephritis); Wilson’s disease, steatohepatitis, hemochromatosis, Schistosomiasis japonica, or drug-induced hepatitis; concomitant infection with hepatitis C virus, hepatitis D virus, or human immunodeficiency virus; other serious infection, organ dysfunction, or malignancy; and lactating or pregnant women. This study was conducted in compliance with the Helsinki Declaration. Due to the retrospective nature of the study, informed consent was waived. The study was approved by the Medical Ethics Committee of Sun Yat-Sen Memorial Hospital (identifier SYSEC-KY-KS-011).

All patients were treated according to the 2008/2012 ACR [20, 21] and the 2010/2013 EULAR [18, 19] recommendations for the management of RA. The therapeutic target was defined as DAS28-CRP <2.6 in all patients or <3.2 in patients with long disease duration (>24 months) [18, 19]. Available clinical data on patients with RA were collected in this study at baseline and at visits at months 1, 3, 6, and 12, as described before [17], including 28-joint tender and swollen joint counts (TJC28 and SJC28), patient and provider global assessment of disease activity (PtGA and PrGA, respectively), pain visual analog scale (pain VAS), the Stanford Health Assessment Questionnaire (HAQ), erythrocyte sedimentation rate (ESR), CRP, rheumatoid factor (RF), and anti-cyclic citrullinated peptide antibody (ACPA). Besides DAS28-CRP, disease activity was also assessed using the Simplified Disease Activity Index (SDAI), the Clinical Disease Activity Index (CDAI), and the Routine Assessment of Patient Index Data 3 (RAPID3) [22]. Cumulative doses of oral glucocorticosteroids (GCs) and disease-modifying anti-rheumatic drugs (DMARDs) were recorded during one-year follow up. GC doses were converted to a prednisone-equivalent dose.

Serological markers of HBV infection, including HBsAg, HBV e antigen (HBeAg), the corresponding antibodies to these antigens (anti-HBs and anti-HBe, respectively), and antibodies to HBV core antigen (anti-HBc), were tested in all patients with RA using enzyme-linked immunosorbent assay (Zhongshan Biotechnology CO., LTD, Guangdong, China) or electrochemluminescence-immunoassay (Roche Diagnostics, Mannheim, Germany). HBV DNA was measured using a commercially available quantitative real-time polymerase chain reaction kit (Da An Gene Co., Ltd. of Sun Yat-Sen University, Guangdong, China), with the lowest detection threshold of 500 IU/mL. Liver function including alanine aminotransferase (ALT, U/L, normal range 5–40 U/L) and aspartate transaminase (AST, U/L, normal range 5–40 U/L) was tested at each visit during follow up. HBV serological markers and HBV DNA levels were evaluated in all patients with RA at baseline and every 1–3 months during follow up in the CHB group. These parameters in the non-CHB group were re-examined if aminotransferase was elevated during follow up. Antiviral prophylaxis by entecavir or tenofovir was suggested for all patients with RA with CHB before RA treatment. Moreover, serum levels of soluble matrix metalloproteinase (MMP-3) were tested as described before [23]. The normal ranges of serum MMP-3 concentrations were 18–60 ng/mL (women) and 24–120 ng/mL (men).

Radiographs of the bilateral hands and wrists (anteroposterior view) of all patients were performed at baseline and month 12. The Sharp/van der Heijde-modified total Sharp score (mTSS), joint erosion (JE), and joint space narrowing (JSN) were scored by two experienced observers (MJD from the Department of Rheumatology and YZH from the Department of Radiology), who were blinded to the patients’ clinical data. As described previously [17], reliability and agreement were assessed based on the intra-class correlation coefficient (ICC): the mean ICC for inter-observer agreement was 0.950. Bony erosion was defined as a cortical break identified on radiography [24]. Radiographic progression was defined as a change in mTSS (ΔmTSS) ≥0.5 units [25], and rapid radiographic progression (RRP) was defined as ΔmTSS ≥5 units from baseline to month 12 [26].

The primary outcome was the percentage of patients with one-year radiographic progression. The secondary outcomes were determined at each visit. These were the percentages of patients achieving therapeutic target and remission, rates of EULAR responses and ACR20/50/70 responses [27], and changes in disease activity indicators.

Side effects were recorded and evaluated at each visit. Neutropenia was defined as neutrophil count <2.0 × 10⁹/L. HBV reactivation was defined as the reappearance of HBsAg in a patient with resolved HBV infection, the detection of previously undetectable HBV DNA or > 1 log10 (10-fold) IU/mL increase in serum HBV DNA, and rise in HBV DNA level above an arbitrary cutoff (for example, 20,000 IU) in patients with biochemical worsening [28‐30]. A hepatitis flare was determined to be present when ALT was greater than two times the upper limit of the normal range (ULN) [30].

Statistical analyses were performed using IBM SPSS Statistics version 20.0 software (IBM, Armonk, NY, USA). Descriptive statistics (median, interquartile range (IQR) or 5th/95th percentile ranges) were calculated for continuous variables and categorical variables were presented as frequencies and percentages. Conditional logistic regression analysis was used to compare continuous and categorical variables between the CHB group and the non-CHB group, and odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to identify risk factors for one-year radiological progression and failure to achieve therapeutic target within 6 months. Variables were included in the equation when the p value was < 0.05 or removed when the p value was > 0.10, following the step-forward selection rule. A two tailed p value <0.05 was considered statistically significant.

A total of 32 patients with RA with CHB were included in the study, of whom 27 (84%) were female. There were 72% of patients who were both RF and ACPA positive, and 78% of patients had bony erosion at baseline. Eleven (34%) patients were treatment-naïve, without previous GC or DMARD therapy for 6 months before enrollment. Fourteen (44%) patients had a level of HBV DNA above 500 IU/mL, of whom 11 (34%) had levels above 10³ IU/mL and 8 (25%) had levels above 10⁴ IU/mL, all with normal liver function. A total of 128 age-matched, sex-matched, and baseline disease activity-matched RA controls in the non-CHB group were compared with patients with RA with CHB. There was no significant difference in baseline demographic and clinical characteristics between groups, except for significantly higher levels of JE subscore and mTSS, and significantly greater proportions of patients using sulfasalazine (SSZ) and hydroxychloroquine (HCQ) in the previous 6 months before enrollment in the CHB group (both p < 0.05, Table 1).

Initial treatment after enrollment and therapy adjustment were according to the “treat-to-target” strategy and patient’s willingness. There was no significant difference between groups in the initial therapy using GCs, methotrexate (MTX), iguratimod, or biologic agents after enrollment (all p > 0.05, Table 2). Compared with the non-CHB group, significantly higher percentages of patients in the CHB group took SSZ and HCQ (44% vs. 2% and 75% vs. 11%, respectively; both p < 0.001), while a significantly smaller proportion of patients with CHB took leflunomide (LEF) (16% vs. 84%, p < 0.001). Accordingly, patients in the CHB group received significantly higher cumulative doses of SSZ, HCQ, and cyclosporin A (CysA), while taking significantly lower cumulative doses of methotrexate (MTX) and LEF both within the initial 6 months after enrollment and during one-year follow up (all p < 0.05). Combined DMARDs were used in 31 (97%) patients in the CHB group and 125 (98%) in the non-CHB group. Compared with the non-CHB group, a greater proportion of CHB patients used the regimen of MTX combined with SSZ and HCQ (34% vs. 1%, p < 0.001), while a smaller percentage of patients in the CHB group used the regimen of MTX combined with LEF (9% vs. 72%, p < 0.001). Six (19%) patients in the CHB group and 38 (30%) in the non-CHB group were treated with a combination of conventional synthetic DMARDs (csDMARDs) and biologic agents (tocilizumab, infliximab, or recombinant human tumor necrosis factor-α receptor-II (Yi Sai Pu, biosimilar)).

Antiviral prophylaxis by entecavir or tenofovir was suggested for all patients with RA with CHB. However, due to economic reasons, only 14 (44%) patients finally accepted, of whom 5 (36%) chose lamivudine, 6 (43%) adefovir, and only 3 (21%) entecavir. Notably, there were no significant differences in GC therapy, DMARD therapy, or cumulative doses of the medications after enrollment between patients with and without antiviral prophylaxis. Antiviral therapy was commenced or switched to agents with a high barrier to resistance, such as entecavir or tenofovir, when HBV reactivation occurred. In addition, liver function was closely monitored as frequently as 2 to 4 weeks and GC, MTX, or LEF therapies were tapered or withdrawn, especially if hepatitis flare occurred.

Compared with the non-CHB group, there was a significantly higher percentage of patients in the CHB group experiencing one-year radiographic progression (53% vs. 17%, p < 0.001), with a similar trend in RRP (16% vs. 5%, p = 0.059). Significantly higher levels of JE subscore, JSN subscore, and mTSS at month 12 were observed and there were more increases in JE subscore and mTSS in the CHB group than those in the non-CHB group (all p < 0.05). The cumulative probability distribution of radiographic change from baseline to month 12 in patients with RA in both groups and the space between the curves indicated there was a significantly higher percentage of patients with CHB who had one-year radiographic progression (Fig. 1a-c).

At month 6, the proportions of patients achieving therapeutic target (53% vs. 82%, p = 0.003) and remission (50% vs. 72%, p = 0.039) were significantly smaller in the CHB group than those in the non-CHB group, with a similar trend of achieving therapeutic target and remission at month 12 (53% vs. 75%, p = 0.034 and 44% vs. 63%, p = 0.077, respectively) (Fig. 2a, b). The percentages of ACR20 and ACR50 responders were also significantly lower in the CHB group versus the non-CHB group at month 6 (56% vs. 81%, p = 0.013 and 47% vs. 70%, p = 0.029, respectively) (Fig. 2c, d). Additionally, compared with the non-CHB group, the percentage of patients achieving good or moderate EULAR response was significantly lower in the CHB group at month 3 (75% vs. 91%, p = 0.038) and month 12 (69% vs. 91%, p = 0.004); a significantly smaller proportion of patients achieving a good EULAR response was observed in the CHB group at month 6 (56% vs. 79%, p = 0.022) and month 12 (53% and 74%, p = 0.041) (Fig. 2e, f).

The results of dynamic disease activity indicators during one-year follow up are shown in Fig. 2g-n. Disease activity indicators, except for ESR, TJC28, HAQ, were significantly higher in the CHB group than those in the control group mainly at month 6 and month 12 (all p < 0.05). An additional file shows the other clinical responses that were not demonstrated in Fig. 2 (Additional file 1). Moreover, a significantly higher serum MMP-3 level was observed in female patients from the CHB group compared with that in the non-CHB group at month 12 (p = 0.020, Fig. 2o). Regardless of gender, a similar trend in the serum MMP-3 level was also seen at month 6 (p = 0.086, Additional file 1).

To determine risk factors for one-year radiographic progression, univariate logistic regression analysis was performed on variables including baseline characteristics, CHB status, and RA medications after enrollment (including categories of medications, one-year cumulative doses of medications, and different regimens of combined therapies) (Table 3). The results revealed that CHB status (OR 3.129, 95% CI 1.661–5.895; p < 0.001), higher baseline mTSS (OR 1.016, 95% CI 1.006–1.026; p = 0.001), and higher one-year cumulative dose of GCs (OR 1.000282, 95% CI 1.000067–1.000497, p = 0.010) were significant factors for one-year radiographic progression. In bivariate analyses that adjusted for baseline mTSS and one-year cumulative dose of GCs, one factor at a time, CHB status was still positively associated with one-year radiographic progression (OR 2.610 and OR 2.881, respectively; both p < 0.01). Moreover, multivariate logistic regression analysis that adjusted for all significant factors from univariate analyses showed that CHB status was independently associated with one-year radiographic progression (OR 2.403, 95% CI 1.218–4.743; p = 0.011).

Both ACR and EULAR recommendations for the management of RA emphasize tight control of disease activity and attaining therapeutic target within 6 months [19, 21]. Therefore, logistic regression analysis was also performed to determine risk factors for failure to achieve therapeutic target within 6 months (Table 4). The results of univariate logistic regression analysis revealed that CHB status (OR 3.077, 95% CI 1.349–7.017; p = 0.008), being medication-naïve (OR 0.300, 95% CI 0.102–0.881; p = 0.029), MTX therapy (OR 0.266, 95% CI 0.099–0.716; p = 0.009), a regimen of MTX combined with LEF (OR 0.365, 95% CI 0.155–0.861; p = 0.021), 6-month cumulative dose of MTX (OR 0.997, 95% CI 0.993–1.000; p = 0.049), and 6-month cumulative dose of CysA (OR 1.000077, 95% CI 1.000011–1.000143; p = 0.023) were recognized as significant factors for failure to achieve therapeutic target within 6 months. Bivariate analyses that adjusted for the significant factors in the univariate analyses, one factor at a time, demonstrated that CHB status was always an independent risk factor (OR 2.722–3.077, p = 0.019–0.008). Further multivariate logistic regression analysis was performed by adjusting for all significant factors in the univariate analyses. Due to the multicollinearity between MTX therapy and 6-month cumulative dose of MTX, two multivariate models were set up respectively and the results of both models showed that CHB status remained significantly associated with failure to achieve therapeutic target within 6 months (MTX therapy model, OR 2.617, 95% CI 1.140–6.007, p = 0.023; 6-month cumulative dose of MTX model, OR 2.844, 95% CI 1.245–6.498, p = 0.013).

Table 5 presents an overview of side effects during one-year follow up. Infections occurred in 16% of patients with CHB and 13% of patients in the non-CHB group. The most common infections were respiratory tract infection, with one patient (3%) in the CHB group and six paitents (5%) in the control group diagnosed as pneumonia respectively. One patient in the non-CHB group suffered from herpes zoster 7 months after initiation with tocilizumab. There was no significant difference between groups in the incidence of infections, trichomadesis, neutropenia, or gastrointestinal discomfort (all p > 0.05). No deaths occurred due to these side effects in this study.

The main side effects of RA treatment on HBV infection were HBV reactivation and aminotransferase elevation. No patient in the non-CHB group developed HBV reactivation. However, 34% of CHB patients developed virus reactivation, of whom 8 (72%) refused to accept antiviral prophylaxis, with hepatitis flare occurring in 2 patients (6%) during follow up. There was no significant difference in the incidence of aminotransferase elevation between groups (19% vs. 31%, p > 0.05). A flowchart of HBV reactivation and hepatitis flare occurring in patients with CHB is shown in Additional file 2. Fortunately, serum HBV DNA level and aminotransferase in all patients with RA returned to undetectable or normal after commencing antiviral therapy or adjusting GC, MTX, or LEF therapy. No liver cirrhosis, liver failure, or HBV-related deaths occurred during follow up in this study.