title: Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.
abstract: BACKGROUND
  Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT).
  
  
  METHODS
  Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.
  
  
  RESULTS
  Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage.
  
  
  CONCLUSIONS
  Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.
authors.affiliation: HIV and other Retroviruses, Robert Koch-Institute, Berlin, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Hauser
authors.affiliation: HIV and other Retroviruses, Robert Koch-Institute, Berlin, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Kuecherer
authors.affiliation: Institute of Tropical Medicine and International Health, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Kunz
authors.affiliation: Centre for Biological Threats and Special Pathogens 1, Highly Pathogenic Viruses, Robert Koch-Institute, Berlin, Germany.
authors.forename: Piotr Wojtek
authors.initials: PW
authors.lastname: Dabrowski
authors.affiliation: Centre for Biological Threats and Special Pathogens 1, Highly Pathogenic Viruses, Robert Koch-Institute, Berlin, Germany.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Radonić
authors.affiliation: Centre for Biological Threats and Special Pathogens 1, Highly Pathogenic Viruses, Robert Koch-Institute, Berlin, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Nitsche
authors.affiliation: Institute of Tropical Medicine and International Health, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Theuring
authors.affiliation: HIV and other Retroviruses, Robert Koch-Institute, Berlin, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Bannert
authors.affiliation: Regional AIDS Control Program Mbeya Region, Ministry of Health and Social Welfare, Dar es Salaam, Tanzania.
authors.forename: Julius
authors.initials: J
authors.lastname: Sewangi
authors.affiliation: PMTCT Service Mbeya Region, Ministry of Health and Social Welfare, Dar es Salaam, Tanzania.
authors.forename: Paulina
authors.initials: P
authors.lastname: Mbezi
authors.affiliation: Kyela District Hospital, Ministry of Health and Social Welfare, Dar es Salaam, Tanzania.
authors.forename: Festo
authors.initials: F
authors.lastname: Dugange
authors.affiliation: Institute of Tropical Medicine and International Health, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Gundel
authors.initials: G
authors.lastname: Harms
authors.affiliation: HIV and other Retroviruses, Robert Koch-Institute, Berlin, Germany.
authors.forename: Karolin
authors.initials: K
authors.lastname: Meixenberger
mesh_terms: D000483:Alleles; D019380:Anti-HIV Agents / Q000627:therapeutic use*; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D015658:HIV Infections / Q000175:diagnosis / Q000188:drug therapy* / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D018445:Infectious Disease Transmission, Vertical / Q000517:prevention & control*; D009154:Mutation; D056990:Post-Exposure Prophylaxis / Q000379:methods; D011247:Pregnancy; D011251:Pregnancy Complications, Infectious / Q000145:classification / Q000175:diagnosis / Q000188:drug therapy* / Q000821:virology; D011379:Prognosis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013636:Tanzania; D017211:Treatment Failure
chemical_list: D019380:Anti-HIV Agents