Abstract People who inject drugs (PWID) are commonly exposed to hepatitis B virus (HBV) and hepatitis D virus (HDV). We evaluated the prevalence of HDV viremia among hepatitis B surface antigen (HBsAg)-positive PWID (n = 73) using a new quantitative microarray antibody capture (Q-MAC) assay, HDV western blot, and HDV RNA. HDV Q-MAC performed well in this cohort: anti-HDV, 100% sensitivity and specificity; HDV viremia, 61.5% sensitivity and 100% specificity. Hepatitis D viremia was present in 35.6% of HBsAg-positive participants and was more common in those with resolved compared to chronic hepatitis C (5.1% vs 0.6%; adjusted odds ratio, 9.80; P < .0001). epidemiology, hepatitis B virus, hepatitis C virus, hepatitis D virus, HDV RNA, people who injected drugs, treatment, viremia Hepatitis D virus (HDV) requires hepatitis B virus (HBV) for its life cycle; people who inject drugs (PWID) are commonly exposed to both viruses . Compared to those with HBV monoinfection, HBV-HDV coinfected individuals experience a more rapid progression to cirrhosis, higher rates of hepatocellular carcinoma, and mortality [1–3]. In the United States, PWID are the group at highest risk of acquiring and transmitting HDV, yet epidemiological data among such individuals are limited and largely based on anti-HDV measurements among individuals with chronic hepatitis B [4–6]. For clinical and public health purposes, the presence of HDV RNA is more relevant, as viremic individuals can transmit the virus and might benefit from antiviral treatment. Furthermore, the prevalence of hepatitis C virus (HCV) infection among PWID is high, therefore triple infection with chronic hepatitis viruses occurs [7, 8] and the relationship between HDV, HBV, and HCV appears to be complex . Assessment of HDV prevalence has been limited by the lack of reliable and convenient antibody assays, and varying sensitivity of reverse transcriptase polymerase chain reaction (RT-PCR) assays for HDV RNA . A recently developed HDV quantitative microarray antibody capture (Q-MAC) assay showed excellent performance characteristics in a Mongolian population [10, 11], but this assay must be evaluated in other populations. We evaluated HDV Q-MAC among PWID from San Francisco and examined the prevalence of HDV viremia in this cohort. METHODS As previously described , the Urban Health Study (UHS) was a serial cross-sectional epidemiological study that recruited PWID from street settings in the San Francisco Bay area from 1985 through 2002; individuals included in the current analysis were evaluated between 1998 and 2000. Participants were ≥18 years of age and had injected illicit drugs within the past 30 days. Blood samples were collected at study sites and stored at −80°C. Further details about UHS are provided in Supplementary Methods. The study was approved by the Committee on Human Subjects Research at the University of California, San Francisco and the Institutional Review Board of the National Cancer Institute. Methods used to determine status for HBV, HCV, and human immunodeficiency virus (HIV) infections have been described previously  (Supplementary Methods). Participants were tested for anti-HBc and HBsAg; anti-HBc–positive individuals were considered to have been infected with HBV and HBsAg-positive individuals were considered to have “active infection.” Subjects who were negative for anti-HBc and HBsAg, but positive for anti-HBsAg were considered to have serological evidence for HBV vaccination. Subjects who were anti-HCV–positive were considered to have been infected with HCV and tested for HCV RNA. Subjects with a positive HCV RNA result were considered “chronically infected with HCV,” while those with a negative result were considered to have “resolved HCV infection.” Participants were considered HIV-infected if they tested positive for antibodies to HIV-1. In the present study, specimens from HBsAg-positive participants were tested by HDV Q-MAC. Detailed methods for the assay are provided in Supplementary Methods. As previously described , the assay was constructed on noncontinuous, nanostructured plasmonic gold slides with enhanced near-infrared fluorescence detection. A microarray printing robot was used to place recombinant full-length HDV small delta antigen on the slides. Slides were blocked with fetal bovine serum (FBS), washed with phosphate-buffered saline, and 1 μL of sample (diluted to 50 μL with FBS) was applied to each well. Slides were washed with phosphate-buffered saline and IRDye800-labeled donkey antihuman IgG (diluted 1:1000 in FBS solution) was applied for 1 hour followed by further washing and drying. Slides were then scanned using a Licor Odyssey instrument and the fluorescent intensity measured. All HBsAg-positive specimens were also tested with an HDV western blot assay and specimens that were positive by western blot were assayed for HDV RNA levels by one-step RT-PCR as described elsewhere . We examined previously defined Q-MAC assay cutoffs for predicting anti-HDV positivity (0.164 fluorescence intensity units) and for predicting HDV RNA positivity (1.659 units) . Samples from participants with active infection who had sufficient remaining serum amount (n = 70) were also tested for HBV DNA levels (Qiagen, Hilden, Germany). HDV replication requires HBsAg, therefore, we assumed that individuals who tested negative for HBsAg were negative for hepatitis D viremia. On that basis, we calculated the prevalence of viremia among UHS participants overall and in the subset of participants who were anti-HBc–positive. We fitted a logistic regression model to determine the association between risk factors and HDV infection as measured by the adjusted odds ratio (aOR), as well as the corresponding 95% confidence interval (CI) and P value. Finally, we evaluated the relationship between HDV RNA, HBV DNA, and HCV RNA levels among people with active HBV infection. RESULTS Study Population The characteristics of the UHS participants are shown in Supplementary Table 1. Median age at study visit was 45 years, median age at first drug use was 19 years, and median duration of injection drug use at study visit was 24 years. Most participants were men (71%) and 49.5% were African American. Regarding HBV infection, 1764 (76.8%) were anti-HBc–positive, among whom 73 (3.2% of total) had active HBV infection. Chronic and resolved HCV infection was present in 1717 (74.8%) and 375 (16.3%) of participants, respectively. HIV prevalence was 11.9%. HDV Q-MAC Testing HDV Q-MAC testing was conducted among the 73 HBsAg-positive participants, with replicate testing performed on samples from 8 subjects. Based on the Q-MAC fluorescence intensity cutoff for anti-HDV positivity (≥ 0.164 units) , all replicates yielded concordant results (4 positive, 4 negative). Results for western blot testing were fully consistent with the Q-MAC results; therefore, HDV Q-MAC yielded a sensitivity and specificity of 100% compared to western blot (Figure 1). Figure 1. View largeDownload slide Performance of the HDV Q-MAC assay compared to the western blot and HDV RNA assays: fluorescence intensity derived from Q-MAC assay against the findings for the same samples from western blot and HDV RNA assays. Of the 73 samples tested, 26 were positive for both western blot and HDV RNA and 47 were both western blot and HDV RNA negative. There was 100% concordance between the western blot and Q-MAC results using the proposed Q-MAC assay cutoff of 0.164 units for anti-HDV positivity . Of the 26 HDV RNA-positive samples, however, only 16 exceeded 1.659 fluorescence intensity units in the Q-MAC assay, the proposed cutoff for predicting HDV RNA positivity. The sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) for predicting anti-HDV and HDV RNA are provided at the bottom of the figures. Abbreviations: CI, confidence intervals; HDV, hepatitis D virus; Q-MAC, quantitative microarray antibody capture. Figure 1. View largeDownload slide Performance of the HDV Q-MAC assay compared to the western blot and HDV RNA assays: fluorescence intensity derived from Q-MAC assay against the findings for the same samples from western blot and HDV RNA assays. Of the 73 samples tested, 26 were positive for both western blot and HDV RNA and 47 were both western blot and HDV RNA negative. There was 100% concordance between the western blot and Q-MAC results using the proposed Q-MAC assay cutoff of 0.164 units for anti-HDV positivity . Of the 26 HDV RNA-positive samples, however, only 16 exceeded 1.659 fluorescence intensity units in the Q-MAC assay, the proposed cutoff for predicting HDV RNA positivity. The sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) for predicting anti-HDV and HDV RNA are provided at the bottom of the figures. Abbreviations: CI, confidence intervals; HDV, hepatitis D virus; Q-MAC, quantitative microarray antibody capture. All 16 specimens that met the previously defined Q-MAC threshold for predicting HDV RNA positivity (≥1.659 units) , were positive for HDV RNA, as were 10 samples with Q-MAC values between 0.164 and 1.659 units (Figure 1, Supplementary Figure 1). Therefore, in UHS, the Q-MAC threshold of 1.659 units yielded 61.5% sensitivity and 100% specificity for HDV viremia. Lowering the HDV RNA cutoff value to 0.164 would have yielded 100% sensitivity for predicting HDV RNA positivity. Prevalence of HDV Viremia The prevalence of hepatitis D viremia (as reflected by HDV RNA) was 1.1% (26/2296) in all participants, 1.5% (26/1764) in those who had been infected with HBV (anti-HBc–positive), and 35.6% (26/73) among those with active HBV infection (Supplementary Table 2). Prevalence did not differ by age at study visit, gender, or race, either overall or in subgroups defined by HBV infection status. However, among those with active HBV infection, higher HDV prevalence was observed with increasing duration of drug use (Ptrend = 0.02). There was no association between HIV infection status and HDV prevalence. Among anti-HBc–positive PWID, those with resolved HCV infection were approximately 8-fold more likely to have HDV infection compared to chronic HCV infection (5.1% vs 0.6%, respectively; P < .0001); among actively infected individuals that difference was approximately 2-fold (45.7% vs 25.0%; P = .08). Relationship Between HDV, HBV, and HCV Using multivariable logistic regression to explore the relationship between chronic hepatitis C and hepatitis D viremia among the individuals who were anti-HBc–positive, individuals with resolved HCV infection were approximately 10-times more likely to be infected with HDV (aOR, 9.80; 95% CI, 4.13–23.19; P < .0001) than those with chronic HCV infection (Supplementary Table 3). No other predictors were associated with HDV infection; however, statistical comparisons were limited by sparse data. We compared the characteristics of the 73 participants with active HBV infection by whether they tested positive or negative for HDV RNA (Supplementary Table 4). Individuals with HDV viremia had longer duration of drug use (median, 27.5 vs 22 years; P = .03) and tended to be older (median, 45.7 vs 41.6 years; P = .13), but the groups did not differ by gender or race. We examined relationships between HDV RNA, HBV DNA, and HCV RNA among participants with active HBV infection. HDV RNA levels were higher in individuals who were positive for HBV DNA; however, that difference did not approach statistical significance (P = .29) (Figure 2A). HDV RNA levels were higher in those with resolved compared to chronic HCV infection (median [log10IU/mL], 4.79 vs 3.00, respectively; P = .03) (Figure 2B). HCV RNA levels tended to be lower among HDV-infected compared with HDV-uninfected individuals (median [log10IU/mL], 5.44 vs 6.60; P = .21). Only 9 subjects were infected with both HDV and HCV, which limited the statistical power for analyzing the correlation between HDV RNA and HCV RNA levels (Pearson’s correlation coefficient = −0.23; P = .55). The relationship between absolute values of HBV DNA and HDV RNA by presence or absence of HCV RNA is depicted in Supplementary Figure 2. Figure 2. View largeDownload slide Interaction between hepatitis viruses among people who injected drugs who tested positive for HBsAg. A, Distribution of HDV RNA according to HBV DNA status. B, Distribution of HDV RNA levels according to HCV RNA status. Median HDV RNA levels are specified besides the boxes. The P values for comparison were derived from Wilcoxon rank-sum test. C, Distribution of HBV DNA levels in the context of HCV RNA and HDV RNA levels. Median values for the HBV DNA are specified besides the boxes. The P values for comparison were derived from Wilcoxon rank-sum test. Abbreviations: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus Figure 2. View largeDownload slide Interaction between hepatitis viruses among people who injected drugs who tested positive for HBsAg. A, Distribution of HDV RNA according to HBV DNA status. B, Distribution of HDV RNA levels according to HCV RNA status. Median HDV RNA levels are specified besides the boxes. The P values for comparison were derived from Wilcoxon rank-sum test. C, Distribution of HBV DNA levels in the context of HCV RNA and HDV RNA levels. Median values for the HBV DNA are specified besides the boxes. The P values for comparison were derived from Wilcoxon rank-sum test. Abbreviations: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus We also examined HBV DNA levels in the context of HCV and HDV infections (Figure 2C). Markedly higher HBV DNA levels were found among individuals who were negative for both HCV RNA and HDV RNA compared to those with chronic HCV alone (P = .0003), chronic HDV alone (P < .0001), or both chronic HCV and chronic HDV (P = .001). HBV DNA levels did not differ among those last 3 groups (P > .50, all comparisons). Discussion In this study of PWID, over a third of participants who were positive for HBsAg also had chronic hepatitis D. Methodologic differences limit comparison of our results to findings from previous studies that examined anti-HDV prevalence [5, 6, 13, 14]; however, our study provides additional evidence that HDV infection is common among HBV-infected PWID in the United States. The novel HDV Q-MAC assay performed well in this population. Regarding anti-HDV status, both the sensitivity and the specificity were 100% compared to HDV western blot. Furthermore, although Q-MAC measures HDV antibody response, the assay also yielded 100% specificity for predicting hepatitis D viremia (based on previously proposed higher threshold of fluorescence intensity). The proposed cutoffs for this promising assay  must be evaluated in a wide range of populations. However, results to date suggest an algorithm might be developed whereby values <0.164 units are considered antibody negative, values of 0.165–1.658 are considered antibody positive/RNA indeterminate, and values ≥1.659 units are considered positive for both anti-HDV and RNA. That approach could allow efficient determination of HDV antibody and RNA status with limited testing for HDV RNA (and no western blot testing). Viral interaction patterns in the setting of triple hepatitis virus infections are complex. We found that individuals with chronic hepatitis C were much less likely to have HDV viremia and, if HDV RNA was present, the level tended to be lower than among individuals with resolved HCV infection. We also observed lower HBV DNA levels among people with either chronic hepatitis C or chronic hepatitis D. Our study was cross-sectional; therefore, we could not examine the timing of viral acquisition. However, these relationships could have implications for antiviral treatment of chronic hepatitis C. “Interferon-free” HCV regimens based on direct-acting antiviral agents (DAAs) lack the suppressive effect of interferon-α on HBV and HDV. HBV reactivation with fulminant hepatitis has been reported in the context of DAA treatment of HCV and it is possible that poorer control of HDV infection has contributed to some of these cases . Specimens for this study were collected between 1998 and 2000. Injection practices, prevalence of hepatitis virus infections, and HBV vaccination rates may change over time; however, our results are broadly consistent with results based on anti-HDV testing in the ALIVE cohort of PWID during 2 periods (1988–1989 and 2005–2006) . Given the growing problem of injection drug use in the United States, contemporary data on HDV prevalence among PWID from a wide range of geographic areas are needed. In conclusion, hepatitis D viremia was common in PWID with active HBV infection, but uncommon overall due to a low prevalence of active HBV infection. The HDV Q-MAC assay demonstrated excellent performance characteristics in this US cohort and could form the backbone of an efficient algorithm for determining HDV antibody and viremia status. We observed lower HDV RNA levels in individuals with chronic HCV infection, consistent with suppression of viral replication in those with triple hepatitis infections. Future research should examine the impact of therapeutic clearance of HCV infection among individuals who are also chronically infected with HBV and HDV. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Disclaimer. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. Financial support. This work was supported by the Intramural Research Program of the National Institutes of Health (National Cancer Institute, Division of Cancer Epidemiology and Genetics). The Urban Health Study was supported by NIH (grant numbers R01-DA09532, R01-DA12109, R01-DA13245, and R01-DA16159); National Cancer Institute (contracts numbers NO1-CO-12400 and N02-CP-91027); Substance Abuse and Mental Health Services Administration (grant number H79-TI12103); and the City and County of San Francisco Department of Public Health. Potential conflicts of interest. J. S. G. reports personal fees from Eiger BioPharmaceuticals Inc., outside the submitted work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: The Liver Meeting 2017, American Association for the Study of Liver Diseases, 20–24 October 2017, Washington, DC. References 1. Wedemeyer H , Manns MP . Epidemiology, pathogenesis and management of hepatitis D: update and challenges ahead . 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The Journal of Infectious Diseases – Oxford University Press
Published: Mar 21, 2018
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