Delta hepatitis: Toward improved diagnostics
Authors: Saleem Kamili, Jan Drobeniuc, Tonya Mixson‐Hayden, Maja Kodani
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article on Page 1739 Hepatitis D virus (HDV), the etiologic agent of hepatitis D or delta hepatitis, is a unique human virus that requires the hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV) for its replication and to establish infection. HDV, the only member of its own separate genus, Deltavirus, is the smallest known infectious viral agent of humans.1 The HDV virion, which measures 35‐37 nm in diameter, contains a single‐stranded circular ∼1,700‐nucleotide RNA genome of negative polarity, which encloses only a single functional open reading frame encoding the sole viral protein, the hepatitis delta antigen (HDAg). The HDAg is associated with the genomic RNA to form a ribonucleoprotein complex which is encapsulated by a lipid envelope containing the small, middle, and large HBsAg proteins.2 HDV exhibits a high degree of genetic heterogeneity, with estimated mutation rates between 3 × 10–2 and 3 × 10–3 base substitutions per genomic site per year.1 The virus has a wide geographic distribution with eight distinct genotypes. HDV genotype 1 is the most common genotype, being prevalent in North America, Europe, the Middle East, and North Africa; genotypes 2 and 4 are found predominantly in East Asia; genotype 3 is found exclusively in the Amazon basin in South America; and genotypes 5‐8, also known as African genotypes, are predominantly found in West and central Africa.3 HDV is a highly pathogenic virus, and clinical presentation of hepatitis D ranges from fulminant hepatitis, exacerbation of the course of underlying HBV infection, and acceleration of progression to cirrhosis, leading to early decompensation of liver function and hepatocellular carcinoma in the majority of patients.4 However, a benign course of HDV infection has also been observed in Greece, Samoa, and the Far East; whether this is related to various viral characteristics such as the infecting genotype or host genetics remains to be determined.4 Outbreaks of severe and fulminant hepatitis D have been reported from Brazil, Russia, Greenland, and Mongolia.3 The laboratory diagnosis of coinfection or superinfection with HDV is based on simultaneous detection of various serologic markers of HBV and HDV infection. The markers of acute HDV infection include, along with the markers of HBV infection, HDAg, HDV RNA, and immunoglobulins M and G (IgM and IgG, respectively) antibodies to HDV (anti‐HDV). These markers of HDV infection are present only transiently and disappear during early convalescence; IgM anti‐HDV and even IgG anti‐HDV also disappear with time in acute resolving HDV infection.3 Superinfection of HBsAg carriers, which almost always leads to chronic hepatitis D, is marked by absence of IgM antibody to hepatitis B core antigen and presence of all the other markers of HBV and HDV infections (Fig. 1). However, markers of HBV replication, especially HBV DNA, may be suppressed during the acute phase of HDV infection and remain undetectable.1 Chronicity of HDV infection is associated with persistence of HDAg, HDV RNA, and IgM and IgG anti‐HDV. HDV RNA is the gold standard for diagnosis of current HDV infection because assays for detection of HDAg are fraught with sensitivity and specificity issues. Quantitative assays of HDV RNA are useful for monitoring response to antiviral therapy, and the recent availability of a World Health Organization standard has further helped in optimizing assays across various centers and laboratories. Sequencing of HDV RNA–positive samples is a reliable way to determine the HDV genotype.5Figure 1: Serologic course of acute resolving (A) and chronic (B) HDV infection. Abbreviations: anti‐HBc, antibody to hepatitis B core antigen; anti‐HBs, antibody to HBsAg.The prevalence of HDV is a measure of anti‐HDV positivity among HBsAg positive carriers. It is estimated that 10 million to 20 million individuals (∼5% of chronic HBV patients) worldwide are coinfected with HDV.6 Vaccination against hepatitis B, which is also the default vaccination against HDV, resulted in a decrease of HDV prevalence in industrialized countries with the implementation of routine hepatitis B immunization of children and other populations.7 However, in several European countries (e.g., Germany, Italy, and England), which have large and increasing numbers of migrants from HDV‐endemic areas (e.g., eastern Europe and Turkey), HDV prevalence rates have remained unchanged.2 High‐risk populations like injection drug users continue to get impacted by superinfection with HDV, as has been observed in Europe and recently in the United States.7 Based on cross‐sectional studies, high rates of HDV infection, ranging from 10% to 70%, in HBsAg carriers have been reported from Nigeria, Gabon, India, Pakistan, Iran, the western Brazilian Amazon, Tajikistan, and Mongolia.6 A number of developing countries have reported a high endemicity of HDV, and prevalence rates of >20% have been reported; these include central Africa, mountainous regions of Venezuela and Colombia, Romania, Pakistan, Iran, the Amazon basin in South America, and Mongolia. However, reliable data on the accurate prevalence of HDV are not available from all regions due to either lack of testing of HBsAg carriers for HDV infection or lack of availability of anti‐HDV antibody assays with proven performance characteristics. In this context, the article by Chen et al. published in this issue of hepatology9 is of major significance. The authors found a substantially higher prevalence (∼60%) of HDV infections among HBV‐infected individuals identified in a national survey sampling of the Mongolian population. Of a total of 1,158 individuals chosen based on a three‐stage cluster sampling method to reflect the gender, age, and geographical origin representative of the entire country, 123 tested positive for HBsAg, of whom 75 (60%) tested anti‐HDV‐positive by a newly developed high‐throughput quantitative microarray antibody capture (Q‐MAC) assay that the authors describe.9 Modern diagnostic approaches, such as protein microarray–based Q‐MAC assays, allow for improvement in the sensitivities of various diagnostic assays. Protein microarrays have become an important tool in studies of protein–protein interactions, protein detection, and other applications in quantitative and functional proteomics and beyond. Protein microarrays are arrays of protein targets, frequently antibodies and/or antigens, immobilized on a solid support such as a glass slide. Plasmonic substrates, such as nanostructured plasmonic gold film, have been developed to improve the sensitivity and dynamic range of protein detection on microarrays and have been shown to detect cancer biomarkers and integrated human antibodies and antigens down to femtomolar ranges.10 Chen et al. have used this protein microarray technology to develop a Q‐MAC assay for sensitive quantitative fluorescent detection of anti‐HDV IgG from patient sera. The authors not only convincingly demonstrated the excellent performance characteristics of their Q‐MAC assay but also established quantitative thresholds of captured HDV antibody predictive of HDV RNA positivity. However, as the authors have righty stated, despite a good correlation between fluorescence intensity of the Q‐MAC assay and HDV RNA levels, determination of HDV RNA remains the gold standard for monitoring of treatment against hepatitis D. The Q‐MAC assay is rapid, requires a small volume of patient serum, and can easily be scaled to high‐throughput screening of large cohorts of HBsAg carriers for HDV infection. A true seroepidemiologic gauge of the prevalence of hepatitis D is to test HBsAg carriers only and estimate the prevalence rates using the HBsAg positives as the denominator. With this approach, Chen et al.9 found an alarming proportion of their HBsAg‐positive population in Mongolia superinfected with HDV. Compared to other HBsAg carriers, persons superinfected with HDV are at the highest risk for hepatocellular carcinoma, and HDV superinfection undoubtedly contributes to Mongolia's high rate of hepatocellular carcinoma. Assessments of HDV as a cause of excess morbidity and mortality among persons with HBV infection globally are limited by the lack of serologic studies. The data from Chen et al.9 highlight the importance of conducting such seroprevalence studies among HBsAg carriers in HDV‐endemic countries and high‐risk populations in developed countries. Given that an estimated 248 million are chronically infected with HBV worldwide and thus susceptible to superinfection with HDV, concerted efforts, like the one undertaken by the Hepatitis Delta International Network (http://hepatitis‐delta.org/), are needed for the determination of accurate estimates of hepatitis D disease burden, evidence‐based policies for HDV testing, and research to understand the biological mechanisms of HDV infection and find efficacious therapies for treatment of hepatitis D.