Introduced in the 1980s and 1990s, competitive and non-competitive RIAs were employed to detect anti-HDV antibodies using radioisotopes as markers to label antigens (Rizzetto et al., 1979; Gowans et al., 1990). The Abbott Laboratories anti-HDV RIA kit gained wide use (Bezeaud et al., 1989; Govindarajan et al., 1991; Shattock and Morris, 1991). This method relies on radioactivity intensity for detection, making it highly sensitive. However, it necessitates specialized facilities, involves a time-consuming protocol, and uses hazardous isotopes. RIA was subsequently replaced by an enzyme-linked immunosorbent assay (ELISA) with similar specificity and comparable sensitivity (Kuo et al., 2012).

ELISA stands as the most commonly used method for antibody detection (Lin et al., 2020). These kits can detect specific anti-HDV IgG, specific anti-HDV IgM, and total anti-HDV. Anti-HDV IgG constitutes the majority of total antibodies and is present in all patients with a normal immune response to HDV infection (Smedile et al., 1994; Negro and Rizzetto, 1995; Roingeard et al., 1992). The competitive ELISA (e.g., Dia. Pro Diagnostic Bioprobes Srl HDV-Ab ELISA) can detect anti-HDV IgG by binding it to HDAg adsorbed onto the plate. After washing, a peroxidase-conjugated anti-HDV antibody is added, binding to free HDAg. The amount of bound enzyme becomes inversely proportional to the anti-HDV IgG concentration in the tested sample. It’s important to note that the anti-HDV IgM level is low in the late phase of acute HDV infection, and its non-specific binding to HDAg has minimal interference with anti-HDV IgG detection. Total anti-HDV are a crucial marker and can be detected using a competitive ELISA (e.g., DiaSorin ETI-AB-DELTAK-2).

Anti-HDV IgM typically becomes detectable 2–3 weeks after the onset of HDV infection symptoms and disappears within 2 months after acute HDV infection (Koh et al., 2019; Wranke et al., 2014; Mederacke et al., 2012). The principle behind the anti-HDV IgM ELISA involves a capture method that specifically detects anti-HDV IgM, excluding IgG detection (e.g., Dia. Pro Diagnostic Bioprobes Srl HDV-IgM ELISA; DiaSorin ETI-DELTA-IGMK-2). A microplate is coated with an anti-human IgM μ-chain antibody, capturing all IgM antibodies in the sample. Specific HDAg and peroxidase-conjugated anti-HDV Fab antibody are subsequently added for signal development, enabling more accurate detection of anti-HDV IgM compared to other methods (Mederacke et al., 2012).

Previous studies have demonstrated that RIA and ELISA exhibit similar sensitivity for detecting low-titer IgG antibodies. However, in the case of high-titer IgG samples, ELISA reaches the endpoint faster than RIA (Govindarajan et al., 1991; Kuo et al., 2012). ELISA is a straightforward and safer method compared to RIA, with practical advantages. Nevertheless, it can only be utilized for serum/plasma samples, and the assay is qualitative since most commercial kits do not provide anti-HDV standard samples.

CLIA technology utilizes magnetic particles coated with recombinant HDAg to capture anti-HDV IgM and IgG from the samples. A monoclonal antibody targeting human IgM/IgG, labeled with luminol, isoluminol, or other luminescent substrates, is employed for signal development. The emitted photons are then detected by chemiluminescence detectors (e.g., Dia. Pro Diagnostic Bioprobes Srl Sara/CLIA; DiaSorin LIAISON XL Murex). CLIA offers several advantages, including higher sensitivity compared to ELISA and a shorter assay time. Notably, DiaSorin S. p.A. developed an automated anti-HDV CLIA, LIAISON XL Murex, which provides random access to samples and delivers results in as little as 32 min. Rocco et al. conducted a comparison between LIAISON and the ETI-AB-DELTAK-2 ELISA for total anti-HDV (Rocco et al., 2019), revealing that CLIA had a lower detection limit and reasonable concordance with ELISA. However, it remains a qualitative method and requires specialized laboratory equipment.

Chen et al. devised a high-throughput Q-MAC assay, involving the microarray printing of recombinant S-HDAg on slides coated with a plasmonic gold film. Diluted serum samples were applied to each well, followed by the addition of IRDye800-labeled anti-human IgG secondary antibodies. The fluorescent signal was then detected using a Licor Odyssey scanner. Purified anti-HDV IgG from patient sera served as an internal standard (Chen et al., 2017). An important aspect of this assay is its ability to provide quantitative and defined anti-HDV titers, which can be used to predict HDV RNA positivity prospectively (Mahale et al., 2018; Mahale et al., 2019).

LFA is a point-of-care test that does not require a laboratory instrument. It involves immobilizing an engineered pan-genotypic L-HDAg and anti-goat antibody (for detection and control bands, respectively) on a membrane. A filter paper strip with colloidal gold-conjugated anti-human IgG is placed in front of the membrane. In comparison to the DiaSorin ELISA, LFA exhibited a 94.6% sensitivity for ELISA-positive samples and 100% specificity for ELISA-negative samples (Lempp et al., 2021). Although the developer did not test only genotype 4, it showed high specificity and concentration dependence when performing the other genotypes. Expanded access is warranted in regions such as Africa, Asia, and South America, where almost all HDV genotypes are prevalent. And Roggenbach et al. conducted a HDV seroprevalence study in China, utilizing LFA technology on over 4,000 HBsAg-positive sera (Roggenbach et al., 2021).

Lopes et al. (2025) developed a new method for hepatitis D detection based on the recombinant antigen DTH10.1. The method consists of a DTH10.1 ELISA, an ICT for anti-HDV IgG and a multiplex ICT. The DTH10.1 antigen is based on the bioinformatics analysis of eight HDV genotypes designed to contain a common sequence of HDV antigen. The core of the working principle of the multiplex ICT is the use of colloidal gold nanoparticle labeling technology: DTH10.1 recombinant antigen (covering the conserved sequences of the 8 HDV genotypes) is conjugated to colloidal gold in a 1:1 ratio with a hepatitis B monoclonal antibody (J), which serves as the detection reagent; two lines of detection are set up on a nitrocellulose membrane to immobilize an anti-HBs monoclonal antibody (C, capturing HBsAg) and an anti-human IgG antibody (to capture anti-HDV IgG). When 50 μL of serum sample is added to the assay, HBsAg, if present, binds to colloidal gold-labeled antibody J and migrates to line 1 where it is captured by antibody C to show a red band; anti-HDV IgG binds to colloidal gold-labeled DTH10.1 and is captured by the anti-human IgG antibody in line 2, and the result can be read by the naked eye within 20 min.

These methods are highly sensitive for the detection of different genotypes of hepatitis D virus, ELISA is stable, and the multiplex ICT is suitable for use in remote endemic areas, with analytical sensitivities of up to 5 IU/mL for HBsAg and 95.2% sensitivity and 98.0% specificity for anti-HDV IgG. The ICT is easy to transport and manipulate and provides rapid results. Given the global distribution and genetic variability of HDV, the test should demonstrate high sensitivity to detect infection with all eight HDV genotypes. Moreover, compared to other HBsAg tests, the multiplex ICT detects both markers simultaneously, which is an advantage in endemic areas and provides a more efficient and practical tool for hepatitis D diagnosis.

Histology remains the gold standard for the most accurate characterization of liver disease and also allows for the classification grading and staging of necroinflammation and fibrosis, respectively (European Association for Study of LiverAsociacion Latinoamericana para el Estudio del Higado, 2015; European Association for the Study of the Liver, 2021). IHC/IF is employed to detect intrahepatic HDAg in liver biopsies. This technique is typically used after serological and other diagnostics have been performed before surgery, allowing IHC/IF to confirm the initial diagnosis. The HDV genotype can be characterized through an IHC assay when genotype-specific anti-HDV are applied (Hsu et al., 2000). IHC/IF is helpful in estimating the prevalence of HDV infection,but unfortunately these additional tests are not available in most pathology laboratories (Olivero and Smedile, 2012).

Immunoblotting for the detection of serum HDAg is an effective, sensitive, and noninvasive method for diagnosing and monitoring chronic HDV infection (Buti et al., 1989; Jardi et al., 1994). Bergmann, Negro, and Gerin were the first to develop an immunoblot assay for detecting HDAg in the serum and liver of patients as well as acutely infected chimpanzees and woodchucks (Bergmann and Gerin, 1986; Negro et al., 1988). An HDAg-specific antibody is used to detect both S-HDAg and L-HDAg, and a secondary antibody amplifies the signal, enabling its visualization.

The early characterization of monoclonal anti-HDV paved the way for the development of HDAg ELISA (Pohl et al., 1987). In this assay, samples are initially incubated with a detergent to dissolve HDAg from HDV particles. The freed HDAg is then captured by pre-coated anti-HDV and detected using a peroxidase-conjugated anti-HDV antibody (e.g., Dia. Pro Diagnostic Bioprobes Srl HDV-Ag ELISA). Despite its sensitivity, HDAg levels decline and become undetectable over years in chronic infections compared to HDV RNA (Negro et al., 1988).

Serum HDV RNA was probed using Northern blot hybridization, employing a radiolabeled cloned cDNA fragment or its RNA transcript (Denniston et al., 1986; Smedile et al., 1986). The Northern blot involves serum RNA extraction, denaturation, gel electrophoresis separation, transfer to cellulose nitrate or nylon membrane, hybridization with radiolabeled HDV DNA or transcripts, and signal development. The use of a riboprobe is more sensitive than a homologous DNA probe (Smedile et al., 1990). This assay is semi-quantitative, non-invasive, and the only way to visualize HDV RNA dimers and trimers. Slot blot simplifies the process by bypassing RNA fractionation and transfer steps, making it more straightforward than Northern blot (Taylor et al., 1987; Saldanha et al., 1989). Both blots have low throughput and lower sensitivity compared to PCR, and as a result, they have been largely replaced by PCR technology.

FISH utilizing a nonradioisotopic strand-specific HDV probe is a rapid and sensitive method for detecting HDV RNA genomes in paraffin-embedded liver biopsies. In certain cases, hepatic HDV RNA may be the only positive marker, while HDAg and serum HDV RNA are undetectable (Lopez-Talavera et al., 1993; Negro, 2004). When combined with confocal microscopy, this method (e.g., Advanced Cell Diagnostics RNAscope) enables the visualization of HDV RNAs in hepatocyte nuclei and quantification of genomic/antigenomic copy numbers (Gowans et al., 1988; Cunha et al., 1998).

In comparison to hybridization methods, PCR amplification of reverse-transcribed HDV RNA offers several advantages in terms of sensitivity, simplicity, and clinical feasibility (Madejón et al., 1990; Zignego et al., 1990). Madejón et al. demonstrated that RT-PCR is 10,000 times more sensitive than slot blot (Madejón et al., 1990). Serum HDV RNA is reverse transcribed, and the resulting complementary (c)DNA product serves as the template for exponential amplification using a primer pair that selectively binds to conserved sequences, such as the HDV ribozyme. RT-PCR products not only reveal HDV RNA levels but can also be utilized for HDV genotyping and cloning (Roggenbach et al., 2021). Moreover, the sensitivity and specificity of RT-PCR have been enhanced. Nested RT-PCR employs an external primer pair along with an internal pair that amplifies the first-round PCR product, making it suitable for detecting RNA with lower copy numbers (Roggenbach et al., 2021; Salichos et al., 2023). Thus, while RT-PCR has a detection limit of 1,000 genome copies/mL, nested RT-PCR can detect as few as 10 genome copies/mL (Smedile et al., 2004). Currently, the use of reverse transcription-nested PCR (RT-nested PCR) for genotyping HDV has become the gold standard in the diagnosis of occult hepatitis B infection (Ceesay et al., 2022). A 2016 study employed a semi-nested RT-nested PCR method to detect HDV isolates from blood donors in Sudan who were coinfected with HBV/HDV. This approach has been demonstrated to be both sensitive and specific for HDV/HBV co-infection in this population (Mohmed et al., 2016).

In contrast to RT-PCR, qRT-PCR is quantitative and real-time, relying on the detection of DNA-bound fluorescent reporters (e.g., SYBR-Green dye, Taqman probe, FRET probe, and molecular beacon). Yamashiro et al. were the first to use qRT-PCR to detect serum HDV RNA in 48 patients with positive HBsAg and anti-HDV, establishing a correlation between HDV RNA loads and disease stages (Yamashiro et al., 2004). Le Gal et al. further demonstrated that viral loads reflect profiles of virological responses during interferon therapy (Le Gal et al., 2005). Combining results from anti-HDV and HDV RNA via qRT-PCR (e.g., RoboGene HDV RNA kit) can differentiate between chronic and resolved infections (Castelnau et al., 2006). However, the vast inter-laboratory variability in diagnostic qRT-PCR assays, which employ different standards, primers, and protocols, necessitates international standardization for this widely-used method (Le Gal et al., 2016).

The third-generation PCR technology includes chip and droplet dPCR, which are highly sensitive, extremely accurate, and not dependent on standard samples. ddPCR employs microfluidic technology to divide one sample into 20,000 water-in-oil emulsified micro-droplets, with most containing a few nucleic acid templates for independent qPCR amplification and fluorescence detection. Absolute quantification of copy numbers is based on the events of positive and negative fluorescent droplets conforming to a Poisson distribution, eliminating the need for an external standard curve. This method operates within a quantitative dynamic range of 10¹–10⁶ IU/mL, with a lower limit of quantification at 8.76 IU/mL (Olivero et al., 2022; Xu et al., 2022). ddPCR is highly specific and capable of detecting exceptionally low levels of HDV, surpassing the accuracy of qRT-PCR (Xu et al., 2022; Hindson et al., 2013). Furthermore, a duplex ddPCR allows for the quantification of both non-edited and ADAR1-edited HDV genomes in a single sample following FTI-277 treatment (Verrier et al., 2022). Serological assays were validated to quantify HDV RNA editing rates, enabling comprehensive investigation of their correlation with disease progression patterns and therapeutic outcomes in chronic hepatitis D patients. It’s worth noting that this method is complex, expensive, and not widely adopted. Despite its advantages, ddPCR is currently limited by its complexity and high cost, restricting its routine clinical application. It remains primarily a research tool and has not yet been widely adopted in clinical settings due to these practical constraints.

RT-LAMP is a diagnostic assay that involves continuous cycling of DNA synthesis using inner and outer primers and Bst polymerase at temperatures ranging from 60°C–65°C (Notomi et al., 2000). Wang et al. developed an RT-LAMP system for HDV detection, requiring only a 50-min reaction at 65°C, with a lower detection limit of 75 fg/μL (Wang et al., 2013). This method is rapid, straightforward, 1,000 times more sensitive than PCR, and does not necessitate large instruments. However, it has thus far been applied to only a limited number of genotype 1 samples, requiring further optimization for broader patient sample and genotype detection. While RT-LAMP offers a potential solution for point-of-care testing with its simplicity and speed, it requires further optimization to cover a broader range of HDV genotypes. As of now, its application has been limited to specific genotypes, and it is not yet a fully validated clinical diagnostic method.

The HDV quasispecies that accumulate during prolonged HDV replication can be characterized using NGS (Beeharry et al., 2014; Homs et al., 2016). In comparison to Sanger sequencing, NGS offers more precise quantification of edited and unedited HDV genomes, as well as other mutations in patient serum samples (Sopena et al., 2018). Additionally, NGS enables rapid and accurate HDV genotyping (Sopena et al., 2018; Wu et al., 2020). Depending on the HDV genotype and strain, variable RNA editing rates in patients have been observed (Dziri et al., 2021). NGS can also analyze the evolution of ribozyme quasispecies (QS) and identify highly conserved regions as potential gene - silencing targets (Pacin-Ruiz et al., 2022). Owing to the varying replication capacities of HDV genotypes and the distinct impacts of the HBV envelope on viral particle assembly and infectivity, coupled with the clinical symptoms induced by different HDV genotypes differing, the employment of genetic sequencing to precisely delineate genotypes is particularly crucial. Furthermore, this is of significant importance for the compilation of regional prevalence rates of HDV. NGS, though powerful in providing detailed insights into HDV quasispecies and genotyping, is currently hindered by high costs and technical complexity. These factors limit its routine use in clinical laboratories, confining it mainly to research settings where resources and expertise are more readily available.