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PLoS One. 2017 May 4;12(5):e0172101. doi: 10.1371/journal.pone.0172101. eCollection 2017.

Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population.

Author information

Roche Diagnostics International Ltd, Rotkreuz, Switzerland.
Bioscientia Institute for Medical Diagnostics, Ingelheim, Germany.
Department of Molecular Genetics and Microbiology, MVZ Labor Dr. Limbach & Kollegen GbR, Heidelberg, Germany.
Roche Diagnostics GmbH, Penzberg, Germany.
Department of Laboratory Medicine, Yonsei University College of Medicine, Severance Hospital, Seoul, South Korea.
Roche Diagnostics GmbH, Mannheim, Germany.
Hepatology Department, Medic Medical Center, Ho Chi Minh City, Vietnam.
Gastroenterology Department, Ho Chi Minh City University Medical Center, Ho Chi Minh City, Vietnam.
Division of Hepatology and Department of Medicine, University of Cape Town and Groote Schuur Hospital, Cape Town, South Africa.
AIT Austrian Institute of Technology, Health and Environment Department, Molecular Diagnostics, Vienna, Austria.
Cerba Spécimen Services, Saint-Ouen l'Aumône, France.
Infectious Diseases and Viral Hepatitis Unit, Second University of Naples, Naples, Italy.
Infectious Diseases Unit, Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy.


The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the "a" determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of "a" determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

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