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Analysis of hepatitis B virus-mixed genotype infection by ultra deep pyrosequencing in Sudanese patients, 2015–2016

  • Khalid Abdallah Enan
  • Claudia Minosse
  • Abdel Rahim Mohammed El Hussein
  • Marina Selleri
  • Emanuela Giombini
  • Maria Rosaria Capobianchi
  • Isam Mohamed Elkhidir
  • Mohamed Omer Mustafa
  • Osama Mohamed Khair
  • Dina Ahamed Hassan
  • Anna Rosa GarbugliaEmail author
Original Paper



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.


Hepatitis 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.

Supplementary material

15010_2019_1306_MOESM1_ESM.docx (41 kb)
Supplementary material 1 (DOCX 40 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Khalid Abdallah Enan
    • 1
  • Claudia Minosse
    • 2
  • Abdel Rahim Mohammed El Hussein
    • 1
  • Marina Selleri
    • 2
  • Emanuela Giombini
    • 2
  • Maria Rosaria Capobianchi
    • 2
  • Isam Mohamed Elkhidir
    • 3
  • Mohamed Omer Mustafa
    • 1
  • Osama Mohamed Khair
    • 3
  • Dina Ahamed Hassan
    • 1
  • Anna Rosa Garbuglia
    • 2
    Email author
  1. 1.Department of Virology, Central LaboratoryThe Ministry of Higher Education and Scientific ResearchKhartoumSudan
  2. 2.Laboratory of VirologyNational Institute for Infectious Diseases “Lazzaro Spallanzani”-IRCCSRomeItaly
  3. 3.Department of Microbiology and Parasitology, Faculty of MedicineUniversity of KhartoumKhartoumSudan

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