Changing epidemiology and viral interplay of hepatitis B, C and D among injecting drug user-dominant prisoners in Taiwan

The spreading of viral hepatitis among injecting drug users (IDU) is an emerging public health concern. This study explored the prevalence and the risks of hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV) among IDU-dominant prisoners in Taiwan. HBV surface antigen (HBsAg), antibodies to HCV (anti-HCV) and HDV (anti-HDV), viral load and HCV genotypes were measured in 1137(67.0%) of 1697 prisoners. 89.2% of participants were IDUs and none had HIV infection. The prevalence of HBsAg, anti-HCV, dual HBsAg/anti-HCV, HBsAg/anti-HDV, and triple HBsAg/anti-HCV/anti-HDV was 13.6%, 34.8%, 4.9%, 3.4%, and 2.8%, respectively. HBV viremia rate was significantly lower in HBV/HCV-coinfected than HBV mono-infected subjects (66.1% versus 89.9%, adjusted odds ratio/95% confidence intervals [aOR/CI] = 0.27/0.10–0.73). 47.5% anti-HCV-seropositive subjects (n = 396) were non-viremic, including 23.2% subjects were antivirals-induced. The predominant HCV genotypes were genotype 6(40.9%), 1a(24.0%) and 3(11.1%). HBsAg seropositivity was negatively correlated with HCV viremia among the treatment naïve HCV subjects (44.7% versus 72.4%, aOR/CI = 0.27/0.13–0.58). Anti-HCV seropositivity significantly increased the risk of anti-HDV-seropositivity among HBsAg carriers (57.1% versus 7.1%, aOR/CI = 15.73/6.04–40.96). In conclusion, IUDs remain as reservoirs for multiple hepatitis viruses infection among HIV-uninfected prisoners in Taiwan. HCV infection increased the risk of HDV infection but suppressed HBV replication in HBsAg carriers. An effective strategy is mandatory to control the epidemic in this high-risk group.

Statistical analysis. The continuous variables were compared using Student's t test. The categorical variables were accessed using χ 2 or Fisher's exact test. Multivariate logistic regression test was used to analyze the risk factors of virus exposure and viremia. A two-tailed p-value < 0.05 was considered statistically significant. Anti-HCV-seropositive subjects with undetectable HCV RNA was defined as spontaneous HCV clearance if they never received anti-HCV therapy, and as treatment-induced HCV clearance if they had ever received anti-HCV therapy. Subjects who were HCV viremic or who were treatment-induced HCV clearance were further classified as supposed HCV viremia. All statistical analyses were performed using Statistical Product and Service Solutions (SPSS) software, version 17.0. (SPSS Inc., Chicago, IL).
In univariate analysis, age, levels of AST, ALT, and Fibrosis-4 (FIB-4) index, the proportion of IDU, anti-HBc, and anti-HDV-seropositivity in HBsAg carriers were significantly higher in anti-HCV-seropositive subjects than in anti-HCV-negative subjects. Among HBsAg-seropositive subjects, the proportion of HBV viremia was significantly lower in anti-HCV seropositive subjects than in anti-HCV-seronegative subjects (66.1% vs 89.9%, p = 2.6 × 10 -4 ). Of 396 anti-HCV-seropositive subjects, the levels of AST, ALT and FIB-4 were significantly higher, but body mass index (BMI) and HBsAg-seropositive rate were significantly lower in the HCV viremia subjects than in those of HCV non-viremic, whatever spontaneous loss of HCV RNA or eradicated by antiviral therapy (Table 4).

Discussion
This study revealed the prevalence of hepatitis B, C, and D in Penghu Prison was 13.6%, 34.8%, 3.4%, respectively. IDU was significantly associated with HCV and HDV, but not HBV infection. The prevalence rates of HBV/ HCV, HBV/HDV dual infections and HBV/HCV/HDV triple infections was 5%, 3.4% and 2.8%, respectively. The predominant HCV genotypes in IDUs were GT6 (40.9%), GT1a (24.0%) and GT3 (11.1%). The risk of HBV viremia was significantly reduced in the anti-HCV seropositive subjects. HBsAg seropositive and body mass index were negatively correlated with HCV viremia among the treatment naïve HCV subjects. Positive for anti-HCV antibody significantly increased the risk of HDV infection. In Taiwan, the implementation of universal hepatitis B vaccination since 1986 has substantially decreased the HBsAg carrier rate from-15% to < 1% in young adult 6 . This study showed the prevalence of hepatitis B among IDUs was similar to that of the general population. An outbreak of HIV infection among IDUs occurred in  www.nature.com/scientificreports/ Taiwan between 2004 and 2006. In this outbreak, the HCV infection rate was up to 98% in HIV-infected IDUs, and some unprecedented HCV genotypes (e.g., 6 g, 6 k, 6w…) had been identified in Taiwan 16 . The prevalence of anti-HCV seropositive was not only extremely high in HIV-infected IDUs (98.7%) but also in HIV-uninfected IDUs (83%) 10 . Elevated HDV seroprevalence among HBV carriers was simultaneously observed in both HIVinfected IDUs (74.9-84.2%) and HIV-uninfected IDUs (40.0-66.7%) 11,12,17 To avoid the spread of this epidemic, the Taiwan Centers for Disease Control (CDC) has implemented harm reduction project for IDUs since 2005. The core interventions include the disposable syringe practices, methadone maintenance treatment, HIV counseling, antiretroviral therapy, and public health education programs. Our study revealed the prevalence of HCV and HBV/HDV dual infection among the inmates in Penghu Prison had steadily declined to 34.8% and 25.2% in 2019 (Supplementary Fig. S4 and Table S1). The superinfection of viral hepatitis among IDUs is common because of the same routes of transmission. Subjects with dual infection have a greater risk of advanced liver disease, cirrhosis and HCC compared with mono-infected subjects 18,19 . Our survey revealed the presence of HCV RNA significantly reduced the risk of HBsAg seropositive. Positive for anti-HCV antibody significantly decreased the risk of HBV viremia. In addition, HBV/HDV dual infected subjects had a less HBV DNA level compared with HBV mono-infected subjects. The previous studies revealed HCV exhibited stronger inhibitory action among the subjects with HBV/HCV dual infection [20][21][22] . HBV reactivation occurs frequently in HBV/HCV coinfected patients receiving DAA therapy but is rare among patients with resolved HBV infection 20,21 . HDV appeared to be the predominant virus in either HBV/HDV dual infection or in HBV/HCV/HDV triple infection 23,24 . The complex interplay among these viruses remains poorly understood. Some possible mechanism had been elucidated. (1) Direct inhibition HBV replication by HCV core protein; (2) Eradication of one virus provides available replication space for another; (3) Loss of host immune responses to one virus may improve replication of the other 25 . Murai et al. found HCV infection suppresses HBV replication via the RIG-1 (retinoic acid-inducible gene-1) like helicase pathway 26 . MicroRNAs play a role in facilitation of HCV dominance as well. miR-122 can restrain HBV replication through cyclin G1-mediated P53 activity 27 . In contrast, miR-122 stabilizes HCV genome by protecting the 5′ terminus of the HCV RNA from degradation by the host exonuclease 28 . In addition, HDV can suppress HBV replication via inhibition the host DNA-dependent RNA polymerase involving HBV transcription 29 . Small hepatitis delta antigen exerts a strong inhibition of HBV mRNAs synthesis or stability 30,31 . HDV proteins inhibit HBV replication by repressing HBV enhancers and by activating the IFN-α-inducible MxA (myxovirus resistance A) gene 32 .
The distribution of HCV genotypes varies by risk groups and geographically. This study showed the predominant HCV genotypes among the inmates of Penghu Prison were GT6 (40.9%), followed by GT1a (24.0%), and GT3 (11.1%). The previous studies reported the proportion of HCV genotype 6 (28.0-43.4%), 1a (14.9-29.2%), and 3a (7.8-20.2%) was apparently higher among IDUs compared with the general population (GT6: 0-0.49%; GT1a: 0-2.7%; GT3a: 0-0.98%) in Taiwan 3, 10, 16, 33, 34 . Phylogenetic analysis revealed that these distinct subtypes were derived from the spread of HCV along the drug trafficking routes via Vietnam and Thailand 35,36 . Genotype   www.nature.com/scientificreports/ 1a was common among IDUs in Europe and America as well 37,38 . In general, the distribution of HCV genotypes among IDUs in Taiwan did not alter tremendously in the past fifteen years. Dual and/or triple infections of HBV, HCV and/or HDV have been associated with poor long-term outcome 39,40 . The estimated prevalence rate of anti-HDV was 4.5% among HBsAg-positive carriers and around 0.16% in general population with a total of 12 million people seropositive for anti-HDV worldwide 39 . However, there is no global estimate for HBV/HCV/HDV triple infections available. HCV infection has been associated with risk of HDV infection among HBV carriers in previous study as well as in the current study 11,39 . Similar to previous study in general population and high-risk groups (PWID and/or HIV-positive patients) in Taiwan 11 , around 80% of HBV/HDV-infected subjects were seropositive for anti-HCV in the current study, with a 2.8% prevalence rate of HBV/HCV/HDV triple infections in the prisoners (Fig. 2).
In this study, 31.7% (95/300) chronic hepatitis C patient concomitant with HCV RNA seropositive had ever received antiviral therapy. The sustained virologic response rate was 87.8% (36/41) in interferon group and 98.2% (56/57) in DAA group, respectively. Asians have a higher likelihood of achieving an interferon-induced viral clearance than their Caucasians counterparts due to carriage of favorable interleukin-28 genotype 41 . With the recommended "standard interferon dose and duration regimens", SVR is achieved in Asia for around 70% of HCV genotype 1 (GT1) infected cases, approximately 90% of GT2/3, 65% of GT4, and 80% of GT6 patients 42 . The treatment efficacy in HCV infected prisoners was comparable with that in the community cohort.
Although treatment efficacy of the DAA is excellent, there is still a wide gap between the awareness of hepatitis C and access to antiviral therapy among this high-risk population. Irrespective of coinfection with HIV or not, the carrier rate of viral hepatitis is still high among IDUs. In addition to HIV, we recommend the viral hepatitis markers should be examined routinely for all the incarcerated IDUs in Taiwan prisons. The incarcerated inmates may face numerous barriers to health care services. It is necessary to adopt integrated strategies to prevent intravenous drug use, and to assist subjects searching for medical care aggressively. Apart from medical service, social-economic support is important to prevent the incarcerated IDUs from reuse intravenous drugs after being released.
There are three major pillars for controlling the epidemic of viral hepatitis in this high-risk groups. Firstly, universal vaccination with HBV vaccine, which could provide protection from HBV and HDV infection for HBV-unexposed subjects 43 ; secondly, education for avoiding percutaneous risk behaviors, such as abstinence of drugs, harm reduction with safe needles, and safe sex; thirdly, treatment as prevention. It is critical in particular  www.nature.com/scientificreports/ for HCV control since there are no vaccine available. The concept of HCV micro-elimination could help prevent the spreading of HCV in the high-risk groups 44 . We recently approved the concept that an outreach screening program with onsite DAA treatment is the key toward HCV micro-elimination in uremic HCV patients under maintenance hemodialysis 45,46 . Therefore, an outreach onsite HCV treatment program is proposed for HCV micro-elimination in the high risk setting.
There are limitations in our study. This was a cross-sectional study. It failed to find out the change of molecular epidemiology of hepatitis B, C and D over time. However, it is very difficult to follow the inmates when they are released or transferred. Since there is no HIV-infected subject in Penghu Prison, the results may not fully reflect the actual status of viral hepatitis among IDUs in Taiwan. Because of the highly coexistence between the viral hepatitis and HIV, the infection rate of viral hepatitis among IDUs may be underestimated.

Conclusion
This study revealed a marked decline in the prevalence of HBV, HCV, and HDV among HIV-uninfected IDUs over the past fifteen years in Taiwan. Although the universal HBV vaccination program and disposable syringe practices indeed prevent the spread of viral hepatitis, IDUs still emerge as a reservoir for viral hepatitis in Taiwan. Despite high efficacy DAAs been on the market for several years, there is a wide gap between awareness of viral hepatitis and antiviral therapy among IDUs. More effective public health policy is required to eliminate the epidemic in these high-risk groups.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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