Pérez-García, FelipeVirseda-Berdices, AnaPita-Martínez, CarlosMuñoz Monte, MarioSepulveda-Crespo, DanielCodina Márquez, HelenaAlonso, RobertoMesones, LaraRodrigo, SandraMacías, JuanReal, Luis MiguelCuadros-González, JuanMartinez, IsidoroResino, Salvador2026-06-262026-06-262026-02-11Pérez-García F, Virseda-Berdices A, Pita-Martínez C, Muñoz Monte M, Sepúlveda-Crespo D, Codina H, Alonso R, Mesones L, Rodrigo S, Macías J, Real LM, Cuadros-González J, Martínez I, Resino S. 2026. Challenges in accurate HDV RNA quantification: inter-assay variability and the impact of thermal shock. J Clin Microbiol 64:e01517-25. https://doi.org/10.1128/jcm.01517-25https://hdl.handle.net/20.500.12105/27567Quantitative RT-PCR (qRT-PCR) is essential for monitoring hepatitis delta virus (HDV) RNA, yet assays lack standardization. We aimed to evaluate the performance of three qRT-PCR assays and to assess the impact of a pre-analytical thermal shock procedure. We conducted a comparative study using 206 samples (106 with anti-HDV antibodies and 100 anti-HDV-negative as a control group), which were tested in parallel with three qRT-PCR assays: Vircell, Certest, and Altona. Performance was evaluated for inter-assay agreement (kappa index), quantitative correlation (R²), and bias (Bland-Altman). Altona detected 56 HDV-RNA positive samples, whereas Vircell and Certest detected 55 positive samples. Inter-assay agreement was perfect comparing Vircell vs Certest (agreement = 100%, к = 1.000) and almost perfect comparing Altona with both Vircell and Certest (agreement = 99.5%, к = 0.988). Quantitatively, Vircell and Certest assays showed relevant systematic biases compared to Altona, overestimating viral loads by approximately 0.24 log International Units (IU)/mL (Certest) and 0.33 log IU/mL (Vircell). The correlation with Altona was strong for Certest (R² = 0.864) and moderate for Vircell (R² = 0.793), while the correlation between Certest and Vircell was weaker (R² = 0.720). Thermal shock improved sensitivity in one case (Certest vs Vircell) but increased quantitative variability, worsening the inter-assay correlation (R² = 0.684). In conclusion, all three assays were highly concordant for the qualitative diagnosis of HDV infection, but their quantitative biases prevent their interchangeable use for treatment monitoring. Thermal shock is not recommended for routine monitoring, as a significant compromise in quantitative accuracy and precision outweighs any potential gains in sensitivity.IMPORTANCEThis study evaluates three hepatitis delta virus (HDV) RNA quantitative real-time PCR (qRT-PCR) assays, crucial for managing the HDV infection, particularly in the setting of new therapies like Bulevirtide, where assessing viral load reduction and accurate monitoring is paramount. We reveal significant quantitative biases among widely used assays, precluding their interchangeable use and risking misinterpretation of treatment response. Furthermore, our systematic assessment of the thermal shock pre-analytical procedure highlights its detrimental impact on quantitative precision, despite modest sensitivity gains. This work provides essential evidence for clinicians and laboratories, guiding assay selection and standardization efforts to optimize HDV diagnosis and patient monitoring.engVoRhttp://creativecommons.org/licenses/by/4.0/Hepatitis D virusInter-assay variabilityqRT-PCRThermal shockViral loadHepatitis DHepatitis Delta VirusHumansAttribution 4.0 International41474328642e015172510.1128/jcm.01517-25Journal of Clinical Microbiologyopen access