Analysis of hepatitis B virus-mixed genotype infection by ultra deep pyrosequencing in Sudanese patients, 2015–2016
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The frequency of detection of HBV co-infection with multiple HBV genotypes is influenced by the detection method; usually co-infections are detected by multiplex PCR or hybridization assays, and are rarely confirmed by sequencing and conventional cloning. The objective of this study was to confirm by ultra-deep pyrosequencing (UDPS) mixed HBV infections, previously detected by multiplex-nested PCR.
Sixteen samples from HBV co-infected Sudanese patients detected by multiplex-nested PCR, were amplified targeting the P/S region and sequenced by UDPS.
The only genotypes identified using UDPS were D and E, while A, B, C and F genotypes, previously detected by multiplex-nested PCR, were not detected. Specifically, 10 samples were shown to be mono-infected (D or E); in 3 out of 10 mono-infected D patients, a subtype combination was observed: D1 + D7 in 2 cases and D2 + D6 in 1 case. The remaining 6 subjects were D + E co-infected (harboring different mixtures of D subtypes).
Overall, UDPS is more effective than multiplex-nested PCR for identifying multiple HBV genotypes and subtypes infections.
KeywordsHepatitis B virus High-throughput nucleotide sequencing Sudan Co-infection
This work was supported by Italian Ministry of Health (funds Ricerca Corrente, linea 3 hepatitis to National Institute for Infectious Diseases INMI IRCCS L. Spallanzani); the European Union’s Horizon 2020 research and innovation program European Virus Archive goes Global under Grant agreement no. 653316.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
- 7.Tabor E, Gerety RJ, Smallwood LA, Barker LF. Coincident hepatitis B surface antigen and antibodies of different subtypes in human serum. J Immunol Baltim Md. 1950;1977:369–70.Google Scholar
- 8.Ding X, Gu H, Zhong Z-H, Zilong X, Tran HT-T, Iwaki Y, et al. Molecular epidemiology of hepatitis viruses and genotypic distribution of hepatitis B and C viruses in Harbin, China. Jpn J Infect Dis. 2003;56:19–22.Google Scholar
- 11.Candotti D, Diarra B, Bisseye C, Tao I, Pham Quang K, Sanou M, et al. Molecular characterization of hepatitis B virus in blood donors from Burkina Faso: prevalence of quasi-subgenotype A3, genotype E, and mixed infections. J Med Virol. 2016;88:2145–56. https://doi.org/10.1002/jmv.24589.CrossRefGoogle Scholar
- 16.Osiowy C, Villeneuve J-P, Heathcote EJ, Giles E, Borlang J. Detection of rtN236T and rtA181 V/T mutations associated with resistance to adefovir dipivoxil in samples from patients with chronic hepatitis B virus infection by the INNO-LiPA HBV DR line probe assay (version 2). J Clin Microbiol. 2006;44:1994–7. https://doi.org/10.1128/JCM.02477-05.CrossRefGoogle Scholar
- 21.Vincenti D, Piselli P, Solmone M, D’Offizi G, Capobianchi MR, Menzo S. Evolutionary trends of resistance mutational patterns of HBV reverse transcriptase over years (2002–2012) of different treatment regimens: the legacy of lamivudine/adefovir combination treatment. Antiviral Res. 2017;143:62–8. https://doi.org/10.1016/j.antiviral.2017.03.008.CrossRefGoogle Scholar
- 24.Ramírez C, Gregori J, Buti M, Tabernero D, Camós S, Casillas R, et al. A comparative study of ultra-deep pyrosequencing and cloning to quantitatively analyze the viral quasispecies using hepatitis B virus infection as a model. Antiviral Res. 2013;98:273–83. https://doi.org/10.1016/j.antiviral.2013.03.007.CrossRefGoogle Scholar
- 31.Lim CK, Tan JTM, Ravichandran A, Chan YC, Ton SH. Comparison of PCR-based genotyping methods for hepatitis B virus. Malays J Pathol. 2007;29:79–90.Google Scholar
- 36.Repp R, Rhiel S, Heermann KH, Schaefer S, Keller C, Ndumbe P, et al. Genotyping by multiplex polymerase chain reaction for detection of endemic hepatitis B virus transmission. J Clin Microbiol. 1993;31:1095–102.Google Scholar
- 41.Ochwoto M, Chauhan R, Gopalakrishnan D, Chen C-Y, Nganga Z, Okoth F, et al. Genotyping and molecular characterization of hepatitis B virus in liver disease patients in Kenya. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2013;20:103–10. https://doi.org/10.1016/j.meegid.2013.08.013.Google Scholar