Novel digital droplet inverse PCR assay shows that natural clearance of hepatitis B infection is associated with fewer viral integrations
Hepatitis B virus (HBV) DNA integration into the host cell genome is reportedly a major cause of liver cancer, and a source of hepatitis B surface antigen (HBsAg). High HBsAg levels can alter immune responses which therefore contributes to the progression of HBV-related disease. However, to what ext...
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| Language: | English |
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Taylor & Francis Group
2025-12-01
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| Series: | Emerging Microbes and Infections |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/22221751.2025.2450025 |
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| author | Dong Li Vikki Ho Chiao-Fang Teng Hung-Wen Tsai Yuanyuan Liu Sarah Bae Harout Ajoyan Jochen M. Wettengel Ulrike Protzer Brian S. Gloss Rebecca J. Rockett Rafid Al Asady Jane Li Simon So Jacob George Mark W. Douglas Thomas Tu |
| author_facet | Dong Li Vikki Ho Chiao-Fang Teng Hung-Wen Tsai Yuanyuan Liu Sarah Bae Harout Ajoyan Jochen M. Wettengel Ulrike Protzer Brian S. Gloss Rebecca J. Rockett Rafid Al Asady Jane Li Simon So Jacob George Mark W. Douglas Thomas Tu |
| author_sort | Dong Li |
| collection | DOAJ |
| description | Hepatitis B virus (HBV) DNA integration into the host cell genome is reportedly a major cause of liver cancer, and a source of hepatitis B surface antigen (HBsAg). High HBsAg levels can alter immune responses which therefore contributes to the progression of HBV-related disease. However, to what extent integration leads to the persistent circulating HBsAg is unclear. Here, we aimed to determine if the extent of HBV DNA integration is associated with the persistence of circulating HBsAg in people exposed to HBV. We established a digital droplet quantitative inverse PCR (dd-qinvPCR) method to quantify integrated HBV DNA in patients who had been exposed to HBV (anti-HBc positive and HBeAg-negative). Total DNA extracts from both liver resections (n = 32; 14 HBsAg-negative and 18 HBsAg-positive) and fine-needle aspirates (FNA, n = 10; 2 HBsAg-negative and 8 HBsAg-positive) were analysed. Using defined in vitro samples for assay establishment, we showed that dd-qinvPCR could detect integrations within an input of <80 cells. The frequency of integrated HBV DNA in those who had undergone HBsAg loss (n = 14, mean ± SD of 1.514 × 10−3 ± 1.839 × 10−3 integrations per cell) was on average 9-fold lower than those with active HBV infection (n = 18, 1.16 × 10−2 ± 1.76 × 10−2 integrations per cell; p = 0.0179). In conclusion, we have developed and validated a highly precise, sensitive and quantitative PCR-based method for the quantification of HBV integrations in clinical samples. Natural clearance of HBV is associated with fewer viral integrations. Future studies are needed to determine if dynamics of integrated HBV DNA can inform the development of curative therapies. |
| format | Article |
| id | doaj-art-6fbc532eb5e9499e9bd9f26af2a1423b |
| institution | Kabale University |
| issn | 2222-1751 |
| language | English |
| publishDate | 2025-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Emerging Microbes and Infections |
| spelling | doaj-art-6fbc532eb5e9499e9bd9f26af2a1423b2025-01-13T18:29:13ZengTaylor & Francis GroupEmerging Microbes and Infections2222-17512025-12-0114110.1080/22221751.2025.2450025Novel digital droplet inverse PCR assay shows that natural clearance of hepatitis B infection is associated with fewer viral integrationsDong Li0Vikki Ho1Chiao-Fang Teng2Hung-Wen Tsai3Yuanyuan Liu4Sarah Bae5Harout Ajoyan6Jochen M. Wettengel7Ulrike Protzer8Brian S. Gloss9Rebecca J. Rockett10Rafid Al Asady11Jane Li12Simon So13Jacob George14Mark W. Douglas15Thomas Tu16Storr Liver Centre, The Westmead Institute for Medical Research, Westmead Hospital and The University of Sydney, Westmead, NSW, AustraliaStorr Liver Centre, The Westmead Institute for Medical Research, Westmead Hospital and The University of Sydney, Westmead, NSW, AustraliaGraduate Institute of Biomedical Sciences, China Medical University, Taichung, TaiwanDepartment of Pathology, National Cheng Kung University Hospital, Tainan, TaiwanStorr Liver Centre, The Westmead Institute for Medical Research, Westmead Hospital and The University of Sydney, Westmead, NSW, AustraliaStorr Liver Centre, The Westmead Institute for Medical Research, Westmead Hospital and The University of Sydney, Westmead, NSW, AustraliaStorr Liver Centre, The Westmead Institute for Medical Research, Westmead Hospital and The University of Sydney, Westmead, NSW, AustraliaInstitute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, GermanyInstitute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, GermanyScientific Platforms, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, AustraliaCentre for Infectious Diseases and Microbiology–Public Health, Westmead Hospital, Westmead, NSW, AustraliaDepartment of Radiology, Westmead Hospital, Westmead, NSW, AustraliaDepartment of Radiology, Westmead Hospital, Westmead, NSW, AustraliaDepartment of Radiology, Westmead Hospital, Westmead, NSW, AustraliaStorr Liver Centre, The Westmead Institute for Medical Research, Westmead Hospital and The University of Sydney, Westmead, NSW, AustraliaStorr Liver Centre, The Westmead Institute for Medical Research, Westmead Hospital and The University of Sydney, Westmead, NSW, AustraliaStorr Liver Centre, The Westmead Institute for Medical Research, Westmead Hospital and The University of Sydney, Westmead, NSW, AustraliaHepatitis B virus (HBV) DNA integration into the host cell genome is reportedly a major cause of liver cancer, and a source of hepatitis B surface antigen (HBsAg). High HBsAg levels can alter immune responses which therefore contributes to the progression of HBV-related disease. However, to what extent integration leads to the persistent circulating HBsAg is unclear. Here, we aimed to determine if the extent of HBV DNA integration is associated with the persistence of circulating HBsAg in people exposed to HBV. We established a digital droplet quantitative inverse PCR (dd-qinvPCR) method to quantify integrated HBV DNA in patients who had been exposed to HBV (anti-HBc positive and HBeAg-negative). Total DNA extracts from both liver resections (n = 32; 14 HBsAg-negative and 18 HBsAg-positive) and fine-needle aspirates (FNA, n = 10; 2 HBsAg-negative and 8 HBsAg-positive) were analysed. Using defined in vitro samples for assay establishment, we showed that dd-qinvPCR could detect integrations within an input of <80 cells. The frequency of integrated HBV DNA in those who had undergone HBsAg loss (n = 14, mean ± SD of 1.514 × 10−3 ± 1.839 × 10−3 integrations per cell) was on average 9-fold lower than those with active HBV infection (n = 18, 1.16 × 10−2 ± 1.76 × 10−2 integrations per cell; p = 0.0179). In conclusion, we have developed and validated a highly precise, sensitive and quantitative PCR-based method for the quantification of HBV integrations in clinical samples. Natural clearance of HBV is associated with fewer viral integrations. Future studies are needed to determine if dynamics of integrated HBV DNA can inform the development of curative therapies.https://www.tandfonline.com/doi/10.1080/22221751.2025.2450025HBV DNA integrationfunctional curefine needle aspirationdigital droplet PCRHBsAg |
| spellingShingle | Dong Li Vikki Ho Chiao-Fang Teng Hung-Wen Tsai Yuanyuan Liu Sarah Bae Harout Ajoyan Jochen M. Wettengel Ulrike Protzer Brian S. Gloss Rebecca J. Rockett Rafid Al Asady Jane Li Simon So Jacob George Mark W. Douglas Thomas Tu Novel digital droplet inverse PCR assay shows that natural clearance of hepatitis B infection is associated with fewer viral integrations Emerging Microbes and Infections HBV DNA integration functional cure fine needle aspiration digital droplet PCR HBsAg |
| title | Novel digital droplet inverse PCR assay shows that natural clearance of hepatitis B infection is associated with fewer viral integrations |
| title_full | Novel digital droplet inverse PCR assay shows that natural clearance of hepatitis B infection is associated with fewer viral integrations |
| title_fullStr | Novel digital droplet inverse PCR assay shows that natural clearance of hepatitis B infection is associated with fewer viral integrations |
| title_full_unstemmed | Novel digital droplet inverse PCR assay shows that natural clearance of hepatitis B infection is associated with fewer viral integrations |
| title_short | Novel digital droplet inverse PCR assay shows that natural clearance of hepatitis B infection is associated with fewer viral integrations |
| title_sort | novel digital droplet inverse pcr assay shows that natural clearance of hepatitis b infection is associated with fewer viral integrations |
| topic | HBV DNA integration functional cure fine needle aspiration digital droplet PCR HBsAg |
| url | https://www.tandfonline.com/doi/10.1080/22221751.2025.2450025 |
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