Anti‐HDV reflex testing in HBsAg‐positive subjects: An efficacious strategy to identify HDV infection

The prevalence of HDV infection in HBsAg carriers is about 9.9% in Italy. However, the real prevalence is underestimated because the anti‐HDV test is not performed routinely in all HBsAg carriers. The aim of this study was to compare the prevalence and the absolute number of HDV infection identified in HBsAg‐positive subjects tested at University Hospital Federico II before and after the introduction of anti‐HDV reflex testing.


| INTRODUC TI ON
Hepatitis delta virus (HDV) is a single-stranded circular RNA virus, firstly identified in Italy in the 1970s. 1 Because HDV is a defective virus, it requires the hepatitis B virus surface antigen (HBsAg) to propagate. 2HDV infection can occur either simultaneously with hepatitis B virus (HBV) infection (coinfection) or in chronic carriers of HBsAg (superinfection). 3Chronic HDV infection causes one of the most severe forms of chronic viral hepatitis with the development of cirrhosis in nearly 80% of patients within 2-10 years 4 and with a significantly increased risk of hepatocellular carcinoma (HCC) compared to HBV infection alone. 5plementation of vaccination programs against HBV was critical to the control of the infection and changed the HDV incidence and prevalence in the target population (young people). 6,7In highincome countries, due to changes in epidemiology, HDV infection can mainly be found in young immigrants from endemic areas and in patients with advanced chronic liver disease (ACLD) that acquired the infection decades ago. 8In Italy, in 2019, the estimated prevalence of anti-HDV in HBsAg carriers was 9.9% (6.4% in Italian natives and 26.4% in immigrants). 9wever, the prevalence of HDV infection seems to be underestimated due to the lack of screening programs in HBsAg-positive subjects. 10According to the European Association for the Study of the Liver (EASL) guidelines, anti-HDV testing is recommended in all HBsAg-positive patients. 11Conversely, the American Association for the Study of Liver Diseases (AASLD) guidelines recommend anti-HDV testing only in HBsAg-positive at risk, such as people who inject drugs, immigrants from high endemic areas and individuals at risk of acquiring sexually transmitted diseases. 12However, many HBV patients were not tested for HDV, even if recommended by major hepatology societies.Two recent papers showed that the percentage of HBsAg-positive subject, tested for anti-HDV, ranged between 2% and 40%, with a high probability to underestimate the prevalence and the absolute number of HDV-positive patients. 13,14rthermore, Italian National Surveillance System for Acute Viral Hepatitis (SEIEVA) data clearly indicated that, also in HBV acute infection, the HDV coinfection is poorly investigated. 7e knowledge of exact HDV prevalence and the real number of subjects with HDV infection is crucial to establish how many patients may be amenable to therapies, since a new antiviral treatment is now available.In this scenario, the main aim of this study was to compare the prevalence and the absolute number of patients with HDV infection identified before and after the introduction of anti-HDV reflex testing in all HBsAg-positive subjects analysed at Virological Laboratory of University Hospital Federico II of Naples.
Secondary aims were: (1) to describe the epidemiological and clinical characteristics of anti-HDV-positive subjects in this cohort; and (2)   to assess the potential feasibility, in a real world setting, of bulevirtide therapy according to EMA/AIFA recommendations. 15

| Patients and study design
This is a single-centre study conducted at University Hospital of Naples Federico II.The Liver and Virological Units were involved.
The study was conducted in accordance with the Declaration of Helsinki.The protocol was approved by the local ethic board of the promoting centre (Federico II University of Naples, n. 86/2020).All patients involved in the study provided written informed consent to participate.From January to December 2022, the reflex test for the detection of total HDV antibodies was performed in all consecutive HBsAg positive subjects revealed.None of them refused to participate.To compare the prevalence and the absolute number of HDV infection identified with anti-HDV reflex testing, we identified as control group the cohort of HBsAg-positive subjects tested at the same laboratory between January and December 2019, before the implementation of anti-HDV reflex testing, when the anti-HDV test was performed only if requested by clinicians.Baseline demographic, clinical, laboratory and virological characteristics were collected for HBsAg-positive subjects tested in 2022.
The severity of liver disease, at the time of the enrollment, was determined with both non-invasive tests, such as transient elastography (TE) by Fibroscan® and with the biomarker score Fibrosis-4 index (FIB-4), which was calculated from published formula. 16ver stiffness measurements (LSM) were performed by a single well-trained operator using a TE-FibroScan instrument (502Touch, EchosenseTM, Paris, France).The results were expressed in kilo-Pascals (kPa) with a range from 2.5 to 75 kPa.Interquartile range (IQR) was defined as an index of the intrinsic variability of LSM.
Only those measurements with more than ten successful acquisitions, with a success rate of at least 60% and an IQR lower than 30%, were classified as valid and taken into consideration for statistical anti-HDV reflex testing, epidemiology, HBV, HDV screening

Key points
Chronic hepatitis delta (HDV) is a rapidly progressive form of chronic hepatitis.The diagnosis of HDV infection is often late because only a minority of physicians tests HBV patients for searching HDV infection.The HDV reflex testing in all HBsAg subjects has proven to increase the diagnoses of HDV infection.evaluation. 17According to EASL guidelines, a FIB-4 index ≥1.3and/ or LSM ≥12 kPa were indicative of advanced liver fibrosis. 18However, these cut-offs did not show a good diagnostic performance in HDV patients, which have generally elevated liver serum enzymes.
Therefore, we used a FIB-4 index ≥3.25 and/or LSM ≥12.5 kPa to define advanced liver fibrosis, showing, respectively, an AUROC of .7 and .9 in HDV patients. 19,20

| Virological testing
The ADVIA Centaur HBsAgII assay (Siemens Healthcare) was used for the determination of qualitative HBsAg 21 and the Liaison Murex HBsAg (DiaSorin) for the quantitative determination. 22Fourhundred and eighty-four samples positive for HBsAg were screened for total HDV antibodies using a commercial chemiluminescence assay, the Liaison Murex Total Anti-HDV Qualitative (DiaSorin). 23V-DNA was quantified by real-time PCR with Abbott RealTime HBV Assay (Abbott Laboratories).HDV-RNA was quantified with RealStar HDV RT-PCR Kit 1.0 (Altona Diagnostics), with the limit of detection between 100 and 400.000.000IU/mL.All assays were performed as specified in the individual instructions for use.
The absolute number of patients tested and diagnosed anti-HDV positive prior and after the implementation of reflex test was compared.

| Statistical analysis
The Shapiro-Wilks test was used to assess the conformity of the parameters to the normal distribution.Categorical variables were described as absolute frequency and percentage.Quantitative data are depicted as median with IQR and compared by U Mann-Whitney test.Categorical variables were compared using the χ 2 test with Yates' correction or Fisher's exact test when appropriate.A pvalue < .05 was considered statistically significant.
Statistical analysis was performed using SPSS software, version 28 (Statistical Package for Social Sciences Inc.).

| Hepatitis D screening
At University Hospital Federico II of Naples, from January to December 2019, before the implementation of anti-HDV reflex testing, 32.649 patients were tested for HBsAg and 512 (1.5%) resulted positive.Anti-HDV testing was performed only in 84 (16.4%)HBsAg-positive patients and 14 (16.6%) of them were positive.HDV-RNA was detectable in 8 (57.1%) of the 14 HDV positive subjects.
Reflex anti-HDV testing was implemented in January 2022 and data were collected from January to December 2022.In this time, 33,788 patients were tested for HBsAg and 484 (1.4%) of them resulted positive.All HBsAg-positive patients were tested for anti-HDV and 52 (10.7%) subjects resulted positive.HDV-RNA was positive in 26 (53%) HDV positive subjects.The overall prevalence of anti-HDV was lower than before reflex testing implementation (16.6% in 2019 vs. 10.7% in 2022), but the absolute number of patients diagnosed with HDV increased from 14 cases in 2019 to 52 cases in 2022 (Figure 1).Assuming that demographics characteristics of the population before and after the introduction of the reflex testing are comparable and that the 10.7% of HDV prevalence obtained with reflex testing is the real one, we can estimate a 3.9-fold increase in the number of HBV patients diagnosed with HDV infection.

| Comparison of HBV mono-infection and HBV/HDV coinfection
Clinical, laboratory and virological data were available in 420 HBsAgpositive subjects of the 2022 cohort.Among these, 369 patients F I G U R E 1 Number of anti-HDV testing and positive cases before and after implementation of anti-HDV reflex testing.
had HBV mono-infection and 51 patients had HBV/HDV infection.
The main characteristics of the two groups of patients are shown in Table 1.There were no age, sex or nationality differences between the two groups.
Quantitative HBsAg (qHBsAg) and HBV-DNA determinations were available in 329 and 338 subjects, respectively.Patients with HBV/HDV coinfection had higher qHBsAg values and lower percentages of HBV-DNA detectable compared to HBV mono-infected subjects (p = .037and p < .001,respectively).Significantly more subjects of the HBV/HDV cohort showed HBV antiviral treatment experience in comparison with HBV mono-infected ones (p < .001).
The FIB-4 values and the presence of advanced liver fibrosis were higher in HBV/HDV subjects compared to HBV mono-infected ones (p < .001).

| Comparison of HDV-RNA positive and HDV-RNA negative subjects
HDV-RNA was determined in 49 of 52 (94.2%) anti-HDV positive subjects and 26 (53%) of them had detectable HDV-RNA.The main epidemiological, clinical, serological and virological characteristics of anti-HDV-positive individuals, stratified for HDV-RNA detectable or undetectable, are shown in Table 2.There were no differences in age and sex between the two groups.Patients with HDV-RNA positive were more likely non-Italian natives compared to HDV-RNA negative ones (p = .024).qHBsAg and HBV-DNA determinations were available in 41 and 48 subjects, respectively.Higher qHBsAg values were observed in HDV-RNA detectable group (p = .07),while there was no difference in percentage of HBV-DNA detectable between the two groups, although patients with HDV-RNA positive were more often under HBV antiviral therapy compared to HDV-RNA negative individuals (p = .035).

| Feasibility of bulevirtide therapy
We assessed, in a real world setting, the percentage of HDV-RNA positive patients amenable of HDV antiviral therapy with bulevirtide TA B L E 1 Comparison of demographic, clinical, virological and biochemical data in HBV monoinfected compared to HBV/HDV coinfected.

| DISCUSS ION
This study shows that the anti-HDV reflex testing could be used as effective strategy to identify HBV/HDV infection.In contrast to EASL recommendations, 11 in real world practice, only a minority of HBsAg-positive individuals are screened for anti-HDV and the prevalence and the real number of subjects with HDV infection are consequently underestimated.El Bouzidi et al. 14 showed that, when HDV testing is performed after the request of clinicians, only the 40% of HBsAg-positive subjects were screened for anti-HDV, while when reflex-laboratory led HDV screening, it was performed in the 99.4% of HBsAg positive-subjects.Another recent study by Palom et al. 13 observed that, in Barcelona, before reflex testing, anti-HDV was tested in 7.6% of HBsAg-positive individuals: 23% in academic hospital and only 2% in primary care centers.After reflex testing was established, 93% of HBsAg-positive cases were evaluated for anti-HDV.Furthermore, a recent Italian study 7 showed that, also in HBV acute setting, anti-HDV was tested only in the 34.9% of HBsAgpositive patients.Interestingly, the rate of HBsAg-positive tested was significantly lower to the proportion of patients tested in the previous decades (34.1% in 2010-2019 vs. 37.5% in 2000-2009 vs. 50.1% in 1990-1999; p < .001).Therefore, it emerged that, over the last 30 years, there has been a progressive reduction of HDV testing in HBsAg-positive subjects.This data is probably due to the lack, in the past years, of an available efficacious antiviral therapy for HDV infection.Our study is the first one evaluating the use of anti-HDV reflex testing in an Italian cohort and, in line with European results, shows that, before the implementation of reflex testing, only the 16.4% of HBsAg-positive subjects was tested for anti-HDV, while, after the introduction of the reflex test, 100% of HBsAg-positive individuals was screened.The anti-HDV screening of HBsAg-positive subjects is crucial to the early diagnosis of HDV infection, characterized by high morbidity and to the identification of cirrhotic patients with dual infection at higher risk of HCC. 4,5 consequence of the lack of anti-HDV screening in HBsAgpositive subjects, the exact prevalence of HDV infection remain controversial.In a recent systematic review and meta-analysis, Chen et al. 24 suggested that more than 60 million individuals worldwide  could be infected with HDV and that the prevalence of HDV in HBsAg carriers reached 10.6%, which is twice as high as previously estimated.In Italy, the most recent overall estimated HDV prevalence is 9.9%. 9Particularly, anti-HDV prevalence decreased from 7.4% to 6.4% among Italians and increased from 12.2% to 26.4% among non-natives during 2001-2019 period. 9In our study, the real prevalence of HDV infection in a tertiary care centre was 10.7%, similar to other Italian studies, and the 53% of anti-HDV positive subjects had detectable HDV-RNA.
Moreover, in our cohort, there was a significant diagnostic yield after the introduction of the reflex HDV testing.The cost effectiveness of reflex test was already demonstrated in HCV setting, particularly for viremia detection.With this approach, additional blood drawn for HCV-RNA testing and an appointment for the results can be avoided in anti-HCV-positive cases.Moreover, a Markov disease progression model showed that the HCV reflex testing was the most cost-effective test compared to others to reach the World Health Organization target on HCV elimination. 25In the HDV setting, a recent paper of Polaris Observatory Group showed that the use of HDV double reflex testing (anti-HDV and HDV-RNA) in HBsAg positive could be cost-effective, because the prevalence of HBV patients to test is low in Europe and because the implementation of this test will reduce healthcare costs due to end-stage HBV/HDV liver disease. 268][29] Our results demonstrated that the infection is outliving in ageing patients with ACLD, as proved by higher prevalence of advanced fibrosis in HDV subjects compared to HBVmonoinfected ones (46.9% vs. 10.2% respectively; p < .001).Similarly, Caviglia et al., 27 in a recent survey on natural history of HDV infection in Italy, showed that HDV infection was still present in a cohort of ageing cirrhotic patients and maintained a discreet impact on liver transplantation programs.Also Ricco et al. 30  Moreover, in the present study, upon stratification by RNA positivity, we showed that, in non-Italian natives, the prevalence of HDV-RNA-positive subjects was higher.This result was in line with other studies demonstrating the higher HDV active infection prevalence in immigrants compared to Italian natives. 9,30Our cohort of HDV viremic patients shows higher transaminases values and higher prevalence of advanced liver fibrosis compared to HDV-RNA negative cohort.The significant association between HDV viraemia and increased ALT levels has also been described in both univariate and multivariate analyses in a cross-sectional study of over 1.500 patients from 15 countries. 31Also, in a recent retrospective analysis on prevalence and clinical characteristics of HDV in Canada, Osiowy et al. 32 showed that patients infected with HDV were also more likely to suffer severe liver disease and its complications, compared to HDV non-viremic subjects.
Finally, in our population, we identified the exact number of HDV infections amenable of antiviral drug treatments.Also in real world practice, the majority (80.7%) of patients with active HDV infection were eligible for antiviral therapy.This information is relevant for health policy programs and because the elimination of etiological agent in chronic liver disease (HDV-RNA suppression) is a cornerstone to avoid liver fibrosis progression. 33

| CON CLUS IONS
In summary, before the implementation of HDV-reflex testing, in clinical practice, anti-HDV testing rates were inadequate with a consequent high number of missed diagnosis.Our data showed that the introduction of anti-HDV reflex testing increased the diagnoses of HDV infection and favoured the right estimation of HDV prevalence.
Our results indicate that HDV infection is associated with significant liver fibrosis and the majority (80.7%) of HDV patients are candidate to antiviral treatment.

TA B L E 2
Demographic, clinical, virological and biochemical data of all anti-HDV positive subjects with HDV-RNA determination.
demonstrated that the prevalence of cirrhosis in HDV-positive patients increased in the decade 2010-2019 compared to 2000-2009 years, as a consequence of old infections.
Categorical variables were described as absolute frequency and percentage.Quantitative data are depicted as median with interquartile ranges (IQR).Categorical variables were compared using the χ 2 test with Yates' correction or Fisher's exact test when appropriate.The bold values are the statistically significant ones.