Dried Blood Spot: A Tool to Ensure Broad Access to Hepatitis D Screening, Diagnosis, and Treatment Monitoring

ABSTRACT

Background and Aims

Chronic hepatitis D is the most severe form of viral hepatitis. Despite recommendations for systematic screening of HBsAg‐positive individuals, hepatitis D infection remains underdiagnosed. Standard HDV virological markers are classically assessed in serum or plasma specimens obtained through venous whole blood sampling. This approach is costly and challenging in resource‐limited settings. Dried blood spot (DBS) sampling offers a practical alternative for large‐scale screening, diagnosis, and monitoring of viral hepatitis. The goal of the study is to evaluate the performance of commercially available HDV diagnostic assays using DBS.

Methods

Plasma and DBS‐collected whole blood specimens from individuals chronically infected with HBV or coinfected with HBV and HDV were analysed for HDV antibody detection, HDV RNA quantification, HBsAg levels, and HDV genotyping.

Results

HDV antibodies were reliably detected in DBS after threshold adjustment, with the LIAISON XL Murex Anti‐HDV assay showing superior sensitivity (99.3%) compared to HDV Ab DIA.PRO (90.3%), while both assays exhibited excellent specificity. HDV RNA was quantifiable in 86.8% of DBS specimens from patients with active infection, although levels were about 1.5 log₁₀ lower than plasma, showing a strong correlation (r = 0.786; p < 0.0001). HBsAg was detectable and quantifiable in all DBS samples, correlating closely with plasma values (r = 0.98). DBS‐based HDV genotyping was successful in 84.8% of samples and concordant with plasma results.

Conclusions

DBS collection of whole blood from DBS appears to be a promising and practical alternative to conventional venous blood sampling for HDV screening, diagnosis, and monitoring.

Key Points

• Universal HDV testing of all HBsAg‐positive is recommended.

• DBS sampling is recommended by liver societies and WHO for HBV and HCV testing.

• Serological and molecular assays and platforms should be developed.

Lay Summary

DBS sampling is a valuable tool for the large‐scale screening, diagnosis, and monitoring of viral hepatitis B and C. HDV virological markers can be easily detected in the whole blood. Collecting whole blood on filter paper offers a practical approach that could significantly contribute to the WHO's goal of eliminating viral hepatitis.

Abbreviations

95% CI

95% confidence interval

AASLD

American Association for the Study of Liver Diseases

ALT

alanine aminotransferase

anti‐HDV

HDV antibody

APASL

Asian Pacific Association for the Study of the Liver

AST

aspartate aminotransferase

AU/mL

arbitrary unit per millilitre

AUROC

area under the reciever operating characteristic

CASL

Canadian Association for the Study of the Liver

CLIA

chemiluminescence immunoassay

Co/S

cutoff‐to‐sample

CTM

COBAS TaqMan

DBS

dried blood spot

EASL

European Association for the Study of the Liver

EIA

enzyme immunoassay

EMA

European Medicines Agency

HBeAg

hepatitis B e antigen

HBsAg

hepatitis B surface antigen

HBV

hepatitis B virus

HCC

hepatocellular carcinoma

HDV

hepatitis D (or delta) virus

HIV‐1

human immunodeficiency virus type 1

IQR

interquartile range

IU/mL

international unit per millilitre

LIS

laboratory information system

SD

standard deviation

U/L

unit per litre

WHO

World Health Organization

1. Introduction

Hepatitis D virus (HDV) is a defective virus that requires the presence of hepatitis B virus (HBV) envelope proteins for virion assembly and transmission. Chronic hepatitis D is the most severe form of viral hepatitis and is associated with a 2‐ to 3‐fold increased risk of developing cirrhosis, hepatocellular carcinoma (HCC), and decompensated liver disease in patients with detectable HDV RNA compared to those with HBV monoinfection [1]. Persistent HDV viremia is the strongest predictor of poor outcomes, and emerging data suggest that HDV genotype may also influence disease progression [2].

The European Association for the Study of the Liver (EASL), the Asian‐Pacific Association for the Study of the Liver (APASL), the Canadian Association for the Study of the Liver (CASL), and the 2024 World Health Organization (WHO) guidelines recommend HDV antibody (anti‐HDV) screening for all HBsAg‐positive individuals at least once [3, 4, 5, 6, 7]. In contrast, the American Association for the Study of Liver Diseases (AASLD) guidelines advocate HDV testing only in HBsAg‐positive individuals who are at risk of infection, including people who have ever injected drugs, men who have sex with men, individuals infected with HIV and/or HCV infection, persons with multiple sexual partners, persons born in regions with reported high HDV endemicity, or individuals with HBV DNA levels below 2000 IU/mL but elevated ALT (alanine aminotransferase) or AST (aspartate aminotransferase) levels [8]. In the US, only an estimated 12.9% of chronic HBsAg carriers have received an HDV antibody test [9]. In Europe, where universal HDV testing is recommended, many cases remain undiagnosed. Between 2018 and 2022, 35% of patients newly diagnosed as HBsAg‐positive in our experience had not been screened for HDV infection [10].

The HDV virological markers are classically assessed in serum or plasma specimens obtained through venous whole blood sampling. However, this approach is costly and may not be suitable for populations or settings with limited access to healthcare or laboratory facilities. Dried blood spot (DBS) sampling offers a practical alternative and has been successfully used for screening and diagnosis of viral infections such as human immunodeficiency virus type 1 (HIV‐1), hepatitis B, and hepatitis C [11, 12, 13]. The performance of DBS for HDV testing remains limited [14]. In the present study, we assess the performance of DBS specimens for HDV antibody detection, HDV RNA quantification, HBsAg measurements, and HDV genotyping, compared to conventional biological matrices in individuals chronically infected with HBV or coinfected with HBV and HDV.

2. Materials and Methods

2.1. Study Design and Participants

All individuals diagnosed with HBV infection at Henri Mondor Hospital between January 2018 and December 2022 were tested for the presence of HDV antibodies, and all HDV antibody‐positive patients (n = 38) were included in the study. Demographic (age, sex, country of birth), biochemical (ALT levels), virological (HCV and HIV status), and histological (stage of liver disease) data were extracted from the laboratory information system (LIS). In parallel, all blood donors identified with an HBV infection between 2000 and 2022 through national blood donor surveillance were tested for HDV antibodies and all blood donors who tested positive (n = 31) were included. Demographic (age, sex, country of origin) and virological (HBV, HCV, and HIV status) data were retrieved from the National Epidemiological Donor Database. HDV infection was defined by the presence of HDV antibodies, with or without detectable HDV RNA. Controls consisted of 36 individuals followed at the Henri Mondor Hospital and 43 blood donors with chronic HBsAg carriage and no HDV infection, matched for age and sex (see Table S1).

Whole blood was reconstituted by mixing plasma or serum with a globular concentrate of human group O red blood cells adjusted to a 45% haematocrit as previously described [15]. Fifty microliters of the reconstituted whole blood were spotted onto Whatman 903 filter paper cards (GE Healthcare Europe, Freiburg, Germany), dried overnight, and stored individually at −20°C with desiccant until analysis. Dried blood spots for HDV antibody‐positive individuals, including 10 patients and 10 blood donors, were stored at room temperature (24°C) or −20°C for 4 weeks to assess the stability of HDV markers (HDV antibodies and HDV RNA) on this matrix.

This retrospective study was approved by the Ethics Committee of “Sud Ouest et Outre Mer I”, France (ID n°APHP230425) and conducted in accordance with the Declaration of Helsinki. Informed consent for the use of blood samples for epidemiological and scientific purposes was obtained from all blood donors during the pre‐donation interview.

2.2. Laboratory Measurements

Plasma HBV DNA levels were measured using a real‐time PCR assay (COBAS TaqMan [CTM] HBV Test or the cobas HBV Test on the cobas 4800 System, Roche Diagnostics, Basel, Switzerland). The limit of detection was 6 IU/mL for the CTM HBV test and 10 IU/mL for the cobas HBV Test. HBsAg levels were quantified using the Alinity i HBsAg Quant assay on an Alinity i System (Abbott, Des Plaines, IL, USA). The hepatitis B e antigen (HBeAg) levels were determined using a quantitative chemiluminescent immunoassay (CLIA; LIAISON HBeAg, DiaSorin, Saluggia, Italy). HBV genotype was determined by phylogenetic analysis of the 630‐bp fragment of the S/P overlapping genes [16].

Total HDV antibodies were detected in plasma and whole blood using two immunoassays: a chemiluminescence assay (CLIA; LIAISON XL Murex Anti‐HDV; DiaSorin, Saluggia, Italy) and an enzyme immunoassay (EIA; HDV Ab DIA.PRO; Diagnostic Bioprobes Srl, Milan, Italy). Nucleic acids were extracted from plasma and whole blood using the EMAG automated extractor (bioMérieux, Marcy‐l'Étoile, France). HDV RNA levels were quantified using the EurobioPlex HDV EBX‐004 qRT‐PCR assay (Eurobio Scientific, Les Ulis, France) on the cobas z 480 analyser (Roche Diagnostics, Switzerland). Quantification was calibrated using the 1st WHO International Standard HDV RNA Standard for Hepatitis D Virus RNA (PEI code 7657/12). The limit of detection (LoD) was 100 IU/mL and the lower limit of quantification (LLOQ) was 1000 IU/mL. The hepatitis D virus genotype was determined by phylogenetic analysis of a portion of the so‐called R0 region of the viral genome (positions 920–1289), as previously described [17, 18]. Viral RNA was extracted from 400 μL of plasma or whole blood using the EMAG automated extractor (bioMérieux, Marcy‐l'Étoile, France). A 397‐base pair (bp) fragment of the R0 region was amplified using the QIAGEN OneStep RT‐PCR kit (Qiagen, Hilden, Germany) with primers 900 s (5′‐CAT GCC GAC CCG AAG AGG AAA G‐3′) and 1280as (5′‐GAA GGA AGG CCC YCG AGA ACA AGA‐3′) [17]. PCR products were purified and sequenced using the BigDye Terminator Cycle 3.1 Sequencing kit (Thermo Fisher Scientific, Courtaboeuf, France) on a SeqStudio Genetic Analyser (Applied Biosystems, Waltham, MA, USA). Phylogenetic analyses were conducted using PHYLIP, version 3.695 with reference sequences for HDV genotypes 1 to 8 available in GenBank.

For DBS specimens, a punched disk was eluted in 1 mL of PBS for 60 min at 4°C with gentle agitation, followed by centrifugation at 36200 × g for 2 min. Supernatant was used as follows: 170 μL for the LIAISON XL murex Anti‐HDV assay, 100 μL for the HDV Ab DIA.PRO assay, and 125 μL for HBsAg quantification using the Alinity i HBsAg Quant assay. Alternatively, a punched disk was eluted into 1.1 mL of NUCLISENS easyMAG Lysis Buffer (bioMérieux, Marcy‐l'Étoile, France) for 30 min at room temperature, then centrifuged as above. HDV RNA was extracted from 400 μL of pre‐extraction supernatant using the EMAG. A volume of 10 or 5 μL of the nucleic acid extract was used for HDV RNA quantification using the EurobioPlex HDV EBX‐004 qRT‐PCR assay or for hepatitis D virus genotyping, respectively.

2.3. Statistical Analysis

The performance of immunoassays for HDV antibody detection in whole blood DBS samples was evaluated using the area under the receiver operating characteristic (AUROC) with 95% confidence interval (95% CI). Assay thresholds were optimized using the Youden Index to maximize sensitivity and specificity compared to plasma. Categorical variables are reported as numbers and percentages, and continuous variables as mean with standard deviation (SD) or median with interquartile range (IQR). Relationships between quantitative variables were assessed using regression analysis, and differences between quantitative assays were visualized by means of the Bland–Altman plot method. Comparisons were made using Fisher's exact test or the Mann–Whitney U test for categorical or continuous variables, respectively. Statistical analyses were performed using Stata 13.0 (StataCorp LP, College Station, TX, USA). Values of p < 0.05 were considered statistically significant.

3. Results

3.1. Characteristics of the Study Population

Table 1 summarizes the characteristics of the 148 individuals with chronic HBsAg carriage, including 69 subjects (46.6%) with serological evidence of HDV exposure. The cohort was predominantly male (69.6%), with a median age of 39 years (range: 18–85 years). More than 90% of subjects were born in a foreign country, including 63.2% born in regions with high HDV endemicity [19]. Of the 148 subjects, 124 (83.8%) were HBeAg‐negative. Compared to HBeAg‐positive individuals, HBeAg‐negative subjects were significantly older (40.0 vs. 31.5 years) and exhibited significantly lower HBV DNA levels (2.5 vs. 3.5 log₁₀ IU/mL; p = 0.016) and HBsAg levels (3.5 vs. 4.1 log₁₀ IU/mL; p = 0.0013). When detectable, the median HBV DNA level was 2.9 log₁₀ IU/mL. HBV DNA was undetectable in 45 subjects, whereas 31 exhibited levels exceeding 2000 IU/mL. The median HBsAg level was 3.6 log₁₀ IU/mL. The median ALT level was 31 U/L, and 29 patients (39.2%) had ALT levels above 40 U/L. Liver stiffness assessment was performed in 72 subjects, of whom 44 (61.1%) demonstrated significant fibrosis (≥F2), including 20 (27.8%) with cirrhosis. The proportion of patients with ALT > 40 U/L and significant fibrosis (≥F2) was numerically higher in HBeAg‐positive individuals compared to HBeAg‐negative individuals (44.4% versus 27.8%; p = 0.245), though this difference was not statistically significant. The most prevalent HBV genotypes were E (50.0%), D (31.2%), and A (15.0%). Coinfections were infrequent: eight patients (5.1%) were HIV antibody‐positive and seven (4.4%) were HCV antibody‐positive. Among these, only two had detectable and quantifiable HIV RNA, and one had detectable HCV RNA. According to the 2025 EASL Clinical Practice Guidelines, 52 patients met the eligibility criteria for antiviral therapy. These patients received entecavir (n = 24), tenofovir‐based therapy (n = 27), or a combination of both (n = 1).

TABLE 1.

Characteristics of the study population, including patients followed in a tertiary centre (n = 74) and blood donors (n = 74).

	Overall (n = 148)	Patients followed in a tertiary centre (n = 74)	Blood donors (n = 74)
Age (years), median [IQR] (range)	39.5 [28.75–50] (18–85)	46.5 [36.25–56] (20–85)	31 [21–42] (18–66)
Gender, male [n (%)]	102 (68.9)	47 (635)	55 (74.3)
Country of birth [n (%)] a
France	20 (14.5)	7 (9.5)	13 (20.3)
Europe, excluding France	12 (8.7)	7 (9.5)	5 (7.8)
North America	1 (0.7)	0 (0.0)	1 (1.6)
South America	1 (0.7)	0 (0.0)	1 (1.6)
North Africa	11 (8.0)	3 (4.0)	8 (12.5)
Middle East	6 (4.3)	1 (1.4)	5 (7.8)
Asia	8 (5.8)	6 (8.1)	2 (3.1)
Pacific	1 (0.7)	1 (1.3)	0 (0.0)
Sub‐Saharan Africa	78 (56.6)	49 (66.2)	29 (45.3)
Coinfections [n (%)]
HIV	8 (5.4)	8 (10.8)	0 (0.0)
Hepatitis C	7 (4.7)	6 (8.1)	1 (1.3)
HBeAg negative [n (%)]	124 (83.8)	56 (75.7)	68 (91.9)
Detectable HBV DNA [n (%)]	103 (69.6)	48 (64.9)	55 (74.3)
HBV DNA level (log10 IU/mL), median [IQR] (range)	2.9 [2.2–3.5] (0.8–6.1)	2.8 [1.7–3.6] (1.1–6.1)	2.9 [2.3–3.4] (0.8–5.8)
HBsAg level (log10 IU/mL), median [IQR] (range)	3.6 [3.0–4.0] (−1.2–5.2)	3.6 [2.8–4.1] (1.2–4.6)	3.6 [3.1–4.0] (−0.3–5.2)
HBV genotype [n (%)]
A	12 (14.8)	2 (7.4)	10 (18.5)
B	1 (1.2)		1 (1.8)
C	2 (2.5)	1 (3.7)	1 (1.8)
D	25 (30.9)	4 (14.8)	21 (38.9)
E	41 (50.6)	20 (74.1)	21 (38.9)
Anti‐HDV positive [n (%)]	69 (46.6)	38 (51.3)	31 (41.9)
Anti‐HDV titre (AU/mL), mean ± SD b	12.6 ± 4.1	13.9 ± 3.8	11.9 ± 3.8
Anti‐HDV cutoff‐to‐sample value (Co/S), mean ± SD c	15.7 ± 9.8	14.0 ± 9.1	16.2 ± 9.8
Detectable HDV RNA [n (%)]	38 (55.7)	18 (47.4)	20 (64.5)
HDV RNA level (log10 IU/mL), median [IQR] (range)	6.4 [5.2–7.3] (2.6–8.4)	6.4 [5.4–7.4] (2.6–8.4)	6.1 [5.0–7.0] (3.1–7.5)
HDV genotype [n (%)]
HDV‐1	31 (94.0)	15 (93.8)	16 (94.1)
HDV‐5	1 (3.0)	1 (6.2)
HDV‐8	1 (3.0)		1 (5.9)
Stage of liver disease
Liver stiffness (kPa), median [IQR] (range)		7.9 [5.5–10.9] (3.1–50)
Significant fibrosis (≥F2) [n (%)] d		44 (61.1)
Cirrhosis [n (%)] c		21 (29.2)
HCC (n)		3
ALT level (U/L), median [IQR] (range)		31 [22–50] (8–3564)
ALT > 40 U/L [n (%)]		27 (36.5)
Antiviral treatment
Previous IFN treatment (n)		9
Nucleos(t)ide analogue treatment (at any time) (n)		52
Tenofovir‐based regimen (n)		28
Entecavir (n)		25

^a Country of birth is missing in 10 blood donors.

^b Total antibodies to HDV were measured using the fully automated chemiluminescent immunoassay, the LIAISON XL murex Anti‐HDV (DiaSorin S.p.A., Italy).

^c Total antibodies to HDV were detected using the competitive enzyme immunoassay, the HDV Ab DIA.PRO (Diagnostic Bioprobes Srl, Italy).

^d Fibrosis stage was missing in two patients followed in Henri Mondor hospital.

Of the 148 HBsAg‐positive subjects, including 74 patients followed at a tertiary centre and 74 blood donors, 69 (46.6%) tested positive for HDV antibodies. Total HDV antibodies were measured using the LIAISON XL murex Anti‐HDV and HDV Ab DIA.PRO assays, yielding a mean HDV antibody titre of 12.6 ± 4.1 AU/mL (range: 1.5–18.4) and a cutoff‐to‐sample (Co/S) value of 15.7 ± 9.8 (range: 1.2–44.0), respectively. HDV RNA was assessed in all subjects, and 36 of 69 (52.2%) had detectable viremia, with a median HDV RNA level of 6.4 log₁₀ IU/mL. HDV genotype was determined in all subjects with detectable HDV RNA. HDV genotype 1 was predominant (n = 31) followed by genotypes HDV‐5 (n = 1) and HDV‐8 (n = 1). Subjects with HDV infection had a significantly lower proportion of detectable HBV DNA compared to those without HDV infection (46.4% vs. 83.5%; p < 0.001), and their median HBV DNA level was correspondingly lower (2.6 vs. 2.9 log₁₀ IU/mL; p = 0.14). In contrast, HBsAg levels did not differ significantly between the two groups (3.6 vs. 3.6 log₁₀ IU/mL; p = 1).

3.2. Detection of Total HDV Antibodies in DBS

All dried blood spot (DBS) samples were tested for total HDV antibodies using two methods: a chemiluminescent immunoassay (CLIA; LIAISON XL murex Anti‐HDV) and an enzyme immunoassay (EIA; HDV Ab DIA.PR0). The AUROC of CLIA for HDV antibody detection was 0.9965 (95% CI: 0.9886–1.000; p < 0.0001). An optimal threshold value was established at 0.16 AU/mL. This threshold had a sensitivity of 99.3% (95% CI: 96.25%–100%), and a specificity of 100% (95% CI: 95.5%–100%). The distribution of the AU/mL values among HDV‐negative (n = 79), HDV RNA‐negative (n = 33), and HDV RNA‐positive (n = 36) individuals is shown in Figure 1A. In whole blood, mean HDV antibody values were 0.1 ± 0.0 AU/mL in HDV‐negative, 8.0 ± 6.4 AU/mL in HDV RNA‐negative, and 13.0 ± 2.8 AU/mL in HDV RNA‐positive subjects. In plasma, corresponding values were 0.3 ± 0.4 AU/mL, 12.7 ± 4.6 AU/mL, and 14.1 ± 2.8 AU/mL, respectively. The AUROC of EIA for HDV antibody detection was 0.9642 (95% CI: 0.9414–0.9869; p < 0.0001). An optimal cutoff‐sample value (Co/S) was established at 0.42. This optimized cutoff value had a sensitivity of 90.3% (95% CI: 84.3%–94.1%), and a specificity of 100% (95% CI: 95.5%–100%). Figure 1B depicts the distribution of the Co/S values among HDV‐negative (n = 79), HDV RNA‐negative (n = 33), and HDV RNA‐positive (n = 36) individuals. In whole blood, mean Co/S values were 0.18 ± 0.05, 3.9 ± 3.7, and 6.6 ± 2.4, respectively, while in plasma the corresponding values were 0.3 ± 0.4, 12.0 ± 7.4, and 16.6 ± 10.2.

FIGURE 1.

Dot plot of AU/mL (A) and signal‐to‐cutoff values (B) assessed by means of CLIA‐ (LIAISON XL murex Anti‐HDV) and EIA‐ (HDV Ab DIA.PR0) based assays according to HDV infection: HDV‐negative (n = 79), HDV‐positive RNA‐negative (n = 33), and HDV‐positive RNA‐positive (n = 36).

Four subjects, including one patient followed at a tertiary centre and three blood donors, exhibited AU/mL values below the optimized CLIA threshold of 0.16 AU/mL when tested using DBS and were considered as false negatives. All four were HDV RNA negative in plasma and had plasma HDV antibody levels of 3.0, 3.1, 5.3, and 7.5 AU/mL. Similarly, nine subjects, including three patients followed at a tertiary centre and six blood donors, demonstrated Co/S values below the optimized EIA threshold of 0.42 when tested using DBS and were also considered false negatives. All nine were HDV RNA negative in plasma, with corresponding plasma Co/S values of 5.3, 10.7, 3.7, 1.2, 3.0, 2.4, 4.6, 2.0, and 5.5.

No clinical difference in HDV antibody levels was observed between DBS specimens stored at −20°C and those stored at room temperature for 4 weeks, respectively. The mean differences (±SD) in the HDV antibody values were 0.32 ± 1.5 AU/mL and −1.23 ± 0.85 using the CLIA and EIA assays, respectively.

3.3. Detection and Quantification of HDV RNA in DBS

All DBS samples were tested for the presence of HDV RNA using a real‐time RT‐qPCR assay (EurobioPlex HDV EBX‐004 qRT‐PCR assay). HDV‐seronegative subjects showed no detectable HDV RNA in whole blood specimens from DBS, corresponding to 100% specificity (95% CI: 95.4%–100%). Of the 38 patients with active HDV infection (HDV RNA > 0), 33 (86.8%) also had quantifiable HDV RNA in paired DBS specimens, with an average level of 4.9 ± 0.9 log₁₀ IU/disk. The remaining five individuals, including two patients followed at a tertiary centre and three blood donors, had undetectable HDV RNA in DBS specimens, despite having detectable HDV RNA in plasma ranging from 2.0 to 3.4 log₁₀ IU/mL (Table 2). The sensitivity for HDV RNA detection from DBS was 86.8% (95% CI: 72.7%–94.2%). HDV RNA levels in whole blood specimens were strongly correlated with plasma levels (r = 0.786; p < 0.0001). Bland–Altman analysis showed a mean bias of 1.4 ± 0.7 log₁₀ IU. Higher values were consistently measured in plasma specimens (Figure 2).

TABLE 2.

Characteristics of the individuals with active HDV infection but missed by DBS testing.

Patient ID	Age (years)	ALT level (U/L)	Liver stiffness (kPa)	Cirrhosis	Antiviral treatment	HBeAg status	qHBsAg (log10 IU/mL)	Plasma viral load (log10 IU/mL)		Viral genotype
								HBV DNA	HDV RNA	HBV	HDV
#310363	61	15	4.4	Y	ETV	Negative	3.1	< 1	2.6	E	1
#050054	57	38	—	Y	ETV	Negative	1.2	< 1	3.1	ND	1
#1518 a	38	—	—	—	—	Negative	4.1	< 0.8	2.0	ND	1
#5525 a	42	—	—	—	—	Negative	2.7	2.2	3.1	E	8
#8363 a	31	—	—	—	—	Positive	3.8	3.8	3.4	E	1

Note: ND: not determined due to the absence of specimens with an HBV DNA level > 2.5 log IU/mL.

Abbreviations: ETV, entecavir; Y, yes.

^a Blood donors: limited data are available.

FIGURE 2.

Scatter plots showing correlation between HDV RNA levels measured by the EurobioPlex HDV EBX‐004 qRT‐PCR assay (Eurobio Scientific, Les Ulis, France) in plasma specimens and in whole blood from DBS. (A) Linear regression of HDV RNA levels measured in plasma specimens and in whole blood from DBS (slope = 0.7524; intercept = 0.1189). (B) Bland–Altman plot of the difference between plasma and whole blood from DBS. The bias was 1.40 log₁₀ IU, with 95% limits of agreement ranging from 0.1521 to 2.708.

No clinical difference in HDV RNA levels was observed between DBS specimens stored at −20°C and those stored at room temperature for 4 weeks, respectively. The mean difference (±SD) in HDV RNA levels was 0.08 ± 0.14 log₁₀ IU/mL, using the EurobioPlex HDV EBX‐004 qRT‐PCR assay.

3.4. Determination of HDV Genotype in DBS

Of the 36 individuals with detectable HDV RNA in plasma, the HDV genotype was identified in 33 cases, including HDV‐1 in 31 individuals and HDV‐5 and HDV‐8 in one individual each. Among the corresponding 33 DBS samples, amplification of the R0 region was achieved in 28 (84.8%) DBS samples. The five DBS specimens (15.2%) that failed to amplify were exclusively from individuals who were HDV RNA negative in whole blood specimens, indicating that amplification failure reflected a true absence of detectable RNA rather than assay limitations. In all 28 cases with successful DBS amplification, the HDV genotype matched concordantly with that obtained from paired plasma specimens, demonstrating high analytical concordance between plasma and DBS‐based genotyping.

3.5. Detection and Quantification of Hepatitis B Surface Antigen in DBS

All DBS samples were analysed for hepatitis B surface antigen of hepatitis B (HBsAg) using a CMIA‐based quantitative assay (Alinity i HBsAg Quant Assay). Among the 144 subjects with detectable and quantifiable HBsAg in plasma, all corresponding DBS samples also had detectable and quantifiable HBsAg, yielding a sensitivity of 100%. HBsAg concentrations measured in DBS samples strongly correlated with plasma values (r = 0.98; p < 0.0001), independent of HBV genotype. Bland–Altman analysis demonstrated a mean bias of 1.6 ± 0.3 log₁₀ IU, with smaller discrepancies observed at HBsAg concentrations below 1 IU/mL (Figure 3).

FIGURE 3.

Scatter plots showing correlation between HBsAg levels measured by Alinity i HBsAg Quant assay on an Alinity i system (Abbott, Des Plaines, IL, USA) in plasma specimens and in whole blood from DBS. (A) Linear regression of HBsAg levels measured in plasma specimens and in whole blood from DBS (slope = 0.8167; intercept = −1.028). (B) Bland–Altman plot of the difference between plasma and whole blood from DBS. The bias was 1.60 log₁₀ IU, with 95% limits of agreement ranging from 1.028 to 2.220.

4. Discussion

Screening for hepatitis D infection requires a combination of laboratory tests, beginning with HBsAg and HDV antibody screening, followed by quantitative HDV RNA testing for all anti‐HDV‐positive individuals to confirm active infection. Dried blood spot cards have long been used as standard tools for whole blood sampling. DBS technology supports a broad range of clinical applications, including the diagnosis of blood‐borne viral infections through the detection of antibodies, antigens, or nucleic acids.

Until now, no data have been available on the performance of commercially available HDV virological assays when applied to DBS specimens. This gap in knowledge is particularly important given the recent European Medicines Agency (EMA) approval of the novel entry inhibitor bulevirtide and several additional drugs through phase 2 or 3 clinical trials.

The present study demonstrates that DBS specimens can be confidently used for HDV antibody detection as well as for HDV RNA detection using standard commercial HDV assays, even though these methods are not formally validated for DBS. In the context of a reflex testing strategy, DBS sampling could help to improve the cascade of care by avoiding unnecessary medical visits to diagnose HDV infection and potential eligibility for antiviral therapy for HDV infection. However, DBS testing is not proposed as a full replacement for plasma or serum‐based HDV RNA testing. Because DBS has a lower sensitivity compared with conventional matrices. Accurate HDV RNA measurement is challenging using DBS. HDV RNA assays require better standardization using the WHO international standard with the expression of results in IU/mL, ideally on standard platforms, covering all eight HDV genotypes with similar analytical sensitivity and quantitative accuracy [20]. In this study, we used reconstituted whole blood, which closely mimics fresh capillary whole blood, enabling an extensive evaluation of DBS performance, as previously suggested [15].

Consistent with previous recommendation, detection of HDV antibodies in whole blood via DBS using CLIA or EIA assays required optimization and the definition of new threshold values to minimize false‐negative results [21, 22, 23, 24, 25]. Adjusting assay thresholds for DBS specimens has been shown to enhance the overall performance of testing for sexually transmitted and blood‐borne infections [26]. With the new threshold values, only 12 subjects were misclassified, all of whom were HDV RNA negative. As a first‐line screening tool, serological assays must demonstrate excellent concordance with reflex HDV RNA testing to cost‐effectively identify patients with active HDV infection eligible for treatment. Our results indicate that the LIAISON XL Murex Anti‐HDV assay shows superior sensitivity (99.3% versus 90.3%) compared to the manual HDV Ab DIA.PR0 while specificity was excellent and comparable between the two assays. Diagnosis of active HDV infection and monitoring of antiviral treatment response rely on accurate and reliable RNA assays. In this study, HDV RNA was quantified in 33 of 38 subjects with active hepatitis D infection using the NUCLISENS lysis buffer for pre‐extraction from DBS. HDV RNA levels in whole blood were, on average, approximately 1.5 log₁₀ lower than those in plasma. Although this reduction is commonly observed with other hepatitis viruses when using DBS, considerable variability in HDV RNA levels between plasma and whole blood samples remained. Variability between platforms and laboratories continues to challenge for HDV RNA assay standardization [20]. Although a WHO international standard for HDV RNA exists, it is limited to genotype 1. Differences in real‐time molecular techniques, including nucleic acid extraction methods and amplification platforms, contribute to inconsistencies not only across laboratories but also across and within assays [27]. Despite improvements in recent years, recent studies assessing the analytical and clinical performance of different assays and platforms have revealed significant sensitivity differences, mainly due to nucleic acid extraction methods [28, 29]. Several recent studies suggest that HDV genotype contributes to the severity of liver involvement [2, 30]. In our study, all subjects were correctly genotyped relative to plasma samples, demonstrating high concordance between DBS‐ and plasma‐based genotyping. Given that the hepatitis D virus is a replication‐defective virus requiring HBV surface proteins as its envelope proteins, we also evaluated the performance of DBS for the detection and quantification of HBsAg in individuals chronically infected with HBV or co‐infected with HBV and HDV. Our results further support the good performance and robustness of CLIA‐based assay for HBsAg detection and quantification using DBS [23, 31].

Importantly, we showed that HDV serological and molecular markers remain stable in DBS stored at room temperature, thereby facilitating sustainable transportation and storage, particularly in resource‐limited settings where maintaining a strict cold chain may not be feasible. However, subfreezing temperatures are recommended for DBS samples intended for long‐term storage (e.g., 3 years or longer) to ensure analyte stability and adequate recovery concentrations.

The study has several limitations. First, it was conducted in a reference laboratory by highly qualified personnel, which may not fully reflect routine clinical settings. Second, DBS performance was assessed using reconstituted whole blood rather than capillary whole blood, which may better reflect real‐world DBS collection, particularly in self‐collection settings. Therefore, further studies using capillary whole blood in real‐life clinical settings are warranted. Third, two spots due to constraints in the pre‐extraction step for whole blood using two different buffers are required for antibody detection and nucleic acid testing. Finally, the HDV genotype distribution in this study was consistent with that reported in France, dominated by HDV‐1 (93.9%). However, only a limited numbers of HDV‐5 and HDV‐8 were included, and other HDV genotypes (HDV‐2, HDV‐3, HDV‐4, HDV‐6, and HDV‐7) were not presented.

In conclusion, this study is the first to assess the clinical performance of DBS specimens for screening and diagnosing HDV infection. Collection of whole blood using DBS sampling appears to be a promising alternative to conventional blood sampling methods. Capillary whole blood collection on filter paper provides a practical and accessible method for screening, diagnosing, and monitoring within an expanding population, particularly among individuals at high risk. The implementation of comprehensive viral diagnostics, including hepatitis viruses and HIV, from a single blood draw has the potential to improve early detection, shorten the time to treatment initiation, and reduce patient loss to follow‐up. These advances could make meaningful contributions toward achieving the WHO's viral hepatitis elimination goals.

Author Contributions

Conception/design: S.C. and F.R.‐T. Patient selection: P.I. and V.L. Data collection and/or processing: R.M., V.O., O.G., and A.S. Analysis and interpretation: R.M. and S.C. Statistical analysis: F.R.‐T. Manuscript draft: R.M. and S.C. Manuscript editing and review: all authors.

Funding

This work was supported by the French National Reference Center (NRC) for Hepatitis B, C, and delta viruses, grant funded by Santé publique France (The French Public Health Agency).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Characteristics of the study population including patients followed in a tertiary centre and blood donors according to the HDV status.

Matar R., Roudot‐Thoraval F., Ortonne V., et al., “Dried Blood Spot: A Tool to Ensure Broad Access to Hepatitis D Screening, Diagnosis, and Treatment Monitoring,” Liver International 46, no. 7 (2026): e70759, 10.1111/liv.70759.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.