REFERENCES

01. Caviglia GP, Ciancio A, Rizzetto M. A review of HDV infection. Viruses. 2022; 14(8): 1749. doi: https://doi.org/10.3390/v14081749.

02. Botelho-Souza LF, Vasconcelos MPA, dos Santos AO, Salcedo JMV, Vieira DS. Hepatitis delta: virological and clinical aspects. Virol J. 2017; 14(1): 177. doi: https://doi.org/10.1186/ s12985-017-0845-y.

03. Le Gal F, Brichler S, Drugan T, Alloui C, Roulot D, Pawlotsky JM, et al. Genetic diversity and worldwide distribution of the Deltavirus genus: a study of 2,152 clinical strains. Hepatology. 2017; 66(6): 1826-41. doi: https://doi.org/10.1002/hep.29574.

04. Stockdale AJ, Kreuels B, Henrion MYR, Giorgi E, Kyomuhangi I, de Martel C, et al. The global prevalence of hepatitis D virus infection: systematic review and meta-analysis. J Hepatol. 2020; 73(3): 523-32. doi: https://doi.org/10.1016/j.jhep.2020.04.008.

05. Asselah T, Rizzetto M. Hepatitis D virus infection. N Engl J Med. 2023; 389(1): 58-70. doi: https://doi.org/10.1056/NEJMra2212151.

06. Kamal H, Aleman S, D-SOLVE Consortium. Natural history of untreated HDV patients: always a progressive disease? Liver Int. 2023; 43(Suppl. 1): 5-21.

07. Papatheodoridi M, Papatheodoridis GV. Is hepatitis delta underestimated? Liver Int. 2021; 41(Suppl. 1): 38-44. doi: https://doi. org/10.1111/liv.14833.

08. Ceesay MM, Bouherrou H, Tan J, Lemoine M, Ndow G, Testoni B, et al. Viral diagnosis of hepatitis B and delta: what we know and what is still required? Specific focus on low- and middle-income countries. Microorganisms. 2022; 10(11): 2096. doi: https://doi. org/10.3390/microorganisms10112096.

09. Cabezas C, Braga WSM. Hepatitis B virus and delta infection: special considerations in the indigenous and isolated riverside populations in the Amazon region. Clin Liver Dis (Hoboken). 2020; 16(3): 117-22. doi: https://doi.org/10.1002/cld.1009.

10. Lago BV, Mello FC, Barros LM, Mello FCA, Villar LM, Lewis- Ximenez LL, et al. Hepatitis D infection in Brazil: prevalence and geographical distribution of anti-delta antibody. J Med Virol. 2018; 90(8): 1358-63. doi: https://doi.org/10.1002/jmv.25196.

11. Roca TP, Queiroz MA, Passos-Silva DG, Araújo NM, Lago BV, Mello FCA, et al. Genetic spatio-temporal analysis of hepatitis D virus infection in Western Brazilian Amazon. Viruses. 2024; 16(11): 1690. doi: https://doi.org/10.3390/v16111690.

12. Ton T, Passos-Silva DG, Ton AP, De Castro Silva A, Santos AO, Araújo NM, et al. Clinical evaluation of the progression of liver disease in patients coinfected with HBV and HDV in the Western Amazon region of Brazil. Int J Hepatol. 2025; 2025: 2054487. doi: https://doi.org/10.1155/ijh/2054487.

13. Castro E Silva A, Roca TP, Passos-Silva DG, Borzacov LM, Silva Oliveira GC, Queiroz MA, et al. Clinical profile and parameters of patients monoinfected with HBV and infected with HDV in Western Amazon. Heliyon. 2024; 10(10): e31065. doi: https://doi. org/10.1016/j.heliyon.2024.e31065.

14. MS - Ministério da Saúde. Boletim epidemiológico: hepatites virais 2025. Brasília: Ministério da Saúde; 2025.

15. Ledsgaard L, Ljungars A, Rimbault C, Sørensen UB, Tulika T, Wade J, et al. Advances in antibody phage display technology. Drug Discov Today. 2022; 27(8): 2151-69. doi: https://doi. org/10.1016/j.drudis.2022.05.002.

16. Barbas CF, Burton DR, Silverman GJ. Phage display: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 2001.

17. Pande J, Szewczyk MM, Grover AK. Phage display: concept, innovations, applications and future. Biotechnol Adv. 2010; 28(6): 849-58. doi: https://doi.org/10.1016/j.biotechadv.2010.07.004.

18. Smith GP. Phage display: simple evolution in a petri dish (Nobel lecture). Angew Chem Int Ed Engl. 2019; 58(41): 14428-37. doi: https://doi.org/10.1002/anie.201908308.

19. De Souza JS, Carneiro MT, Narciso LG, Barros LM, Alves RC, Marson FA, et al. Eosinophilic esophagitis auxiliary diagnosis based on a peptide ligand to eosinophil cationic protein in esophageal mucus of pediatric patients. Sci Rep. 2022; 12(1): 12226. doi: https://doi.org/10.1038/s41598-022-16293-1.

20. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011; 7: 539. doi: https://doi.org/10.1038/msb.2011.75.

21. Dhanushkumar T, Kamaraj B, Vasudevan K, Gopikrishnan M, Dasegowda K, Rambabu M, et al. Structural immunoinformatics approach for rational design of a multi-epitope vaccine against triple negative breast cancer. Int J Biol Macromol. 2023; 243: 125209. doi: https://doi.org/10.1016/j.ijbiomac.2023.125209.

22. Jespersen MC, Peters B, Nielsen M, Marcatili P. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes. Nucleic Acids Res. 2017; 45(W1): W24-9. doi: https://doi.org/10.1093/nar/gkx346.

23. Du Z, Su H, Wang W, Ye L, Wei H, Peng Z, et al. The trRosetta server for fast and accurate protein structure prediction. Nat Protoc. 2021; 16(12): 5634-51. doi: https://doi.org/10.1038/ s41596-021-00628-9.

24. Zuccola HJ, Rozzelle JE, Lemon SM, Erickson BW, Hogle JM. Structural basis of the oligomerization of hepatitis delta antigen. Structure. 1998; 6(7): 821-30. doi: https://doi.org/10.1016/S0969- 2126(98)00084-7.

25. Queiroz MAS, Roca TP, Souza BF, De Souza AJ, Passos-Silva DG, Da Silva A, et al. Development of quantitative multiplex RT-qPCR one step assay for detection of hepatitis delta virus. Sci Rep. 2023; 13(1): 12073. doi: https://doi.org/10.1038/s41598-023-37756-z.

26. Zhang YY, Tsega E, Hansson BG. Phylogenetic analysis of hepatitis D viruses indicating a new genotype I subgroup among African isolates. J Clin Microbiol. 1996; 34(12): 3023-30. doi: https://doi. org/10.1128/jcm.34.12.3023-3030.1996.

27. Casey JL, Brown TL, Colan EJ, Wignall FS, Gerin JL. A genotype of hepatitis D virus that occurs in northern South America. Proc Natl Acad Sci USA. 1993; 90(19): 9016-20. doi: https://doi. org/10.1073/pnas.90.19.9016.

28. Tharwani A, Hamid S. Elimination of HDV: epidemiologic implications and public health perspectives. Liver Int. 2023; 43(Suppl. 1): 101-7. doi: https://doi.org/10.1111/liv.15579.

29. Chen HY, Pang XH, Goyal H, Yang L, Xu HG. Hepatitis D: challenges in the estimation of true prevalence and laboratory diagnosis. Gut Pathog. 2021; 13(1): 66. doi: https://doi.org/10.1186/ s13099-021-00462-0.

30. Salles BC, Costa LE, Alves PT, Dias AC, Vaz ER, Menezes-Souza D, et al. Leishmania infantum mimotopes and a phage-ELISA assay as tools for a sensitive and specific serodiagnosis of human visceral leishmaniasis. Diagn Microbiol Infect Dis. 2017; 87(3): 219-25. doi: https://doi.org/10.1016/j.diagmicrobio.2016.11.012.

31. Lempp FA, Roggenbach I, Nkongolo S, Sakin V, Schlund F, Schnitzler P, et al. A rapid point-of-care test for the serodiagnosis of hepatitis delta virus infection. Viruses. 2021; 13(12): 2371. doi: https://doi.org/10.3390/v13122371.

32. Coller KE, Butler EK, Luk KC, Rodgers MA, Cassidy TA, Gersch J, et al. Development and performance of prototype serologic and molecular tests for hepatitis delta infection. Sci Rep. 2018; 8(1): 2095. doi: https://doi.org/10.1038/s41598-018-20455-5.

33. Oberhardt V, Hofmann M, Thimme R, Neumann-Haefelin C. Adaptive immune responses, immune escape and immune-mediated pathogenesis during HDV infection. Viruses. 2022; 14(2): 298. doi: https://doi.org/10.3390/v14020298.

34. Villiers M-B, Cortay J-C, Cortès S, Bloquel B, Brichler S, Brakha C, et al. Protein-peptide arrays for detection of specific anti-hepatitis D Virus (HDV) genotype 1, 6, and 8 antibodies among HDV-infected patients by surface plasmon resonance imaging. J Clin Microbiol. 2015; 53(4): 1164-71. doi: https://doi.org/10.1128/JCM.03002-14.

35. Wang KS, Jansen RW, Brown EA, Lemon SM. Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies. J Virol. 1990; 64(3): 1108-16. doi: https://doi.org/10.1128/JVI.64.3.1108-1116.1990.

36. Lin HH, Wu WY, Wang CH, Ko YC, Chen DS, Chen PJ, et al. Performance of commercially available anti-HDV enzyme-linked immunosorbent assays in Taiwan. Virol J. 2020; 17(1): 76. doi: https://doi.org/10.1186/s12985-020-01355-5.

37. ANVISA - Agência Nacional de Vigilância Sanitária. Consultas - Agência Nacional de Vigilância Sanitária. Available from: https:// consultas.anvisa.gov.br/#/ (Accessed 4 Dec 2024).

38. Gomes-Gouvêa MS, Soares MC, Bensabath G, de Carvalho-Mello IM, Brito EMF, Souza OS, et al. Hepatitis B virus and hepatitis delta virus genotypes in outbreaks of fulminant hepatitis (Labrea black fever) in the western Brazilian Amazon region. J Gen Virol. 2009; 90(Pt 11): 2638-43. doi: https://doi.org/10.1099/vir.0.013615-0.

Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 121 | 2026

Research Articles

Novel epitope-based diagnostic probes selected by phage display for the serological detection of HDV

Mariana Araújo Costa^1,2,3,4,+, Rayany Cristina de Souza^1,2, Tárcio Peixoto Roca^5,6, Adrhyan Araújo⁶, Luiz Fellype Alves de Souza⁷, Rutilene Barbosa Souza^7,8, Rafaela Sabatini^3,4, Bruno Silva Andrade⁹, Iara Pereira Soares¹, Douglas Carvalho Caixeta^1,2, Marco Guevara-Vega^1,2, Ildercílio Mota de Souza Lima⁴, Ana Maísa Passos-Silva⁶, Mario Machado Martins¹, Abel Dib Rayashi¹, Daniel Archimedes da Matta⁷, Deusilene Vieira⁶, Luiz Ricardo Goulart^1†, Robinson Sabino-Silva^1,2, Fabiana de Almeida Araújo Santos¹

¹Universidade Federal de Uberlândia, Instituto de Biotecnologia, Laboratório de Nanobiotecnologia Prof Dr Luiz Ricardo Goulart Filho, Uberlândia, MG, Brasil
²Universidade Federal de Uberlândia, Centro de Inovação em Diagnóstico Salivar e Nanobiotecnologia, Instituto de Ciências Biomédicas, Departamento de Fisiologia, Uberlândia, MG, Brasil
³Secretaria de Saúde do Estado do Acre, Laboratório Central de Saúde Pública do Acre, Rio Branco, AC, Brasil
⁴Universidade Federal do Acre, Laboratório de Biologia Celular e Molecular Aplicada, Rio Branco, AC, Brasil
⁵Fundação Oswaldo Cruz-Fiocruz, Laboratório de Hepatites Virais, Rio de Janeiro, RJ, Brasil
⁶Fundação Oswaldo Cruz-Fiocruz, Laboratório de Virologia Molecular, Porto Velho, RO, Brasil
⁷Fundação Hospital Estadual do Acre, Centro de Infectologia Charles Merieux & Laboratório Rodolphe Merieux, Rio Branco, AC, Brasil
⁸Universidade Federal da Bahia, Programa de Pós-Graduação em Medicina e Saúde, Salvador, BA, Brasil
⁹Universidade Estadual do Sudoeste da Bahia, Departamento de Ciências Biológicas, Laboratório de Bioinformática e Química Computacional, Jequié, BA, Brasil

DOI: 10.1590/0074-02760250343

382 views 607 downloads

Print PDF Supplementary data

ABSTRACT

BACKGROUND Hepatitis delta virus (HDV) is associated with the worst prognosis among viral hepatitis infections; however, it remains largely underdiagnosed, particularly in endemic developing regions, underscoring the need for new, accessible serological methods for large-scale screening.
OBJECTIVES To develop HDV-mimetic molecules using phage display for application in immunodiagnostic platforms.
METHODS HDV-mimetic peptides were selected via phage display biopanning, sequenced and screened by phage-enzyme-linked immunosorbent assay (ELISA). Based on the sequences of these peptides, a recombinant protein (rHDV) was constructed and employed in an ELISA. The test was validated using 87 anti-HDV-positive samples and 93 hepatitis B virus (HBV) control samples collected from a public diagnostic laboratory in the Amazon region. Statistical analyses were performed to evaluate the diagnostic performance of the synthetic peptides and the recombinant protein.
FINDINGS Overall, rHDV exhibits a sensitivity of 74.71%, specificity of 97.85%, and area under the curve (AUC) of 0.8906. In HDV ribonucleic acid (HDV RNA)-positive patients, diagnostic performance improved, with a sensitivity of 88.0%, specificity of 98.92%, and AUC of 0.96.
MAIN CONCLUSIONS These findings highlight that the rHDV protein used in ELISA effectively discriminated HDV-infected individuals from patients monoinfected with hepatitis B. This demonstrates the potential of rHDV as an effective, rapid, and low-cost tool for HDV detection for broader HDV screening.

key words: hepatitis delta virus phage display diagnostic ELISA

REFERENCES