Ana Alberola, Adolfo de Salazar, A. Fuentes, Raquel Carracedo, Marta Illescas‐López, María del Carmen Rodríguez Baños, Carolina Freyre, Berta Cristina Soria Becerril, Isabel Viciana, Inmaculada López‐Rodríguez, Natalia Belén Montiel, Ana María Domínguez Castaño, María Pilar Luzón‐García, Encarnación Ramírez Arellano, Carmen Liébana‐Martos, Francisco Franco Álvarez de Luna, Maria Del Carmen Lozano, Begoña Caparrós Palop, Antonio Sampedro, Ana Belén Pérez, Sonia Algarate, Susana Rojo Alba, Pablo A. Fraile, Dolores Ocete, María Jesús Alcaráz, Juan Carlos Rodríguez, Francisco Javier Hernández‐Felices, Olalla Martínez, Victoria Domínguez, Nieves Orta, Dolores Gómez, Carmen Cortell Cortell, Jorge Camacho, Susana Sabater, Luis Ángel Iglesias Sánchez, Yasmina M. Martos Martín, Aldara Vallejo, Lourdes Molina, María Jesús Castaño Aroca, Beatriz Castro, Magdalena Lara, María José Pena, Ana Rodríguez‐Fernández, Alejandro González Praetorius, José Joaquín Blas, Alicia Beteta, César Pereiro Gómez, M. Carmen Soriano Rodríguez, Soledad Illescas, Melanie Abreu, Julio García Díez, Inocencio Beltrán, Marta Sandoval Torrientes, Ruth Saez, Teresa Martín, Pilar Tajada Alegre, Belén Huerta Lorenzo, María Teresa García Valero, Isabel Jiménez Sansegundo, Susana García-de Cruz, Sonsoles Garcinuño, Miguel Ángel Sáiz, Santiago J. Muñoz, Isabel Fernández, Helena Miriam Lorenzo Juanes, María Reyes Vidal-Acuña, Luz Moldes, Noelia Calvo, Matilde Trigo, María José Gude, Patricia Ordóñez, Sandra Cortizo, Miriam Blasco, Mayra Jeanneth Saldaña Sigcha, Iker Falces, Galán Jc, A Arribi, R. Muley Alonso, Asunción Iborra, María Jesús Del Amor Espín, Eva Cascales, Cayetano Pérez, Pablo Fernández, María Dolores Navarro, María Luz Núñez, Jorge Galán Ros, Luis Javier Gil‐Gallardo, Ana Miqueleiz, Gabriel Reina, Luis Elorduy, Mikele Macho, Carmen Gómez, Paloma Liendo, Yolanda Salicio, Antonio Aguilera, F Segovia Garcia
BACKGROUND: Hepatitis delta virus accelerates liver disease progression in chronic hepatitis B, yet remains underdiagnosed. Guidelines recommend double reflex diagnostic testing with antibody testing followed by viral load testing, but uptake remains inconsistent. AIMS: To evaluate double reflex diagnostic implementation in Spain during the retrospective phase of the Spain Double Reflex Diagnostic study. METHODS: Nationwide, multicenter retrospective analysis of individuals positive for hepatitis B surface antigen (2022-2024) from 80 centers. Laboratory data included hepatitis B surface antigen, hepatitis delta virus antibody and hepatitis delta virus viral load. Uptake rates, hepatitis delta virus antibody positivity, viral load confirmation and viral load-confirmed prevalence were calculated with 95% confidence intervals. RESULTS: Among 65,763 individuals positive for hepatitis B surface antigen, hepatitis delta virus antibody testing uptake increased from 55.1% (54.4-55.8) in 2022 (n = 18,986) to 72.5% (71.9-73.1) in 2023 (n = 22,463) and 85.8% (85.1-86.4) in 2024 (n = 24,314; p < 0.0001). Antibody positivity remained stable (5.6%, 4.9%, 5.3%). Viral load confirmation among antibody-positive patients improved from 72.6% in 2022 to 90.8% in 2023 and 91.0% in 2024. Viral load-confirmed prevalence among antibody-positive individuals was 40.3% in 2022, 31.6% in 2023, and 30.1% in 2024. Regional heterogeneity was marked, with several autonomous communities achieving near-universal uptake (> 95%). CONCLUSION: Between 2022 and 2024, double reflex diagnostic implementation expanded rapidly, demonstrating national feasibility and describing hepatitis D virus seroprevalence. However, regional inequities persist. Sustained uptake will require standardised reflex protocols, harmonization of viral load assays and continuous training to reduce underdiagnosis and improve care of coinfected patients.