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
Creators: name: Hauser, Andrea
name: Kücherer, Claudia
name: Kunz, Andrea
name: Dabrowski, Piotr Wojtek
name: Radonić, Aleksandar
name: Nitsche, Andreas
name: Theuring, Stefanie
name: Bannert, Norbert
name: Sewangi, Julius
name: Mbezi, Paulina
name: Dugange, Festo
name: Harms, Gundel
name: Meixenberger, Karolin
Publisher: name: Robert Koch-Institut, Infektionskrankheiten / Erreger
Year: 2015
Subjects: subject: Humans
subject: Female
subject: Anti-HIV Agents/therapeutic use
subject: HIV Infections/virology
subject: HIV-1/genetics
subject: Mutation
subject: HIV Infections/drug therapy
subject: Alleles
subject: Treatment Failure
subject: HIV Infections/diagnosis
subject: Drug Resistance Viral/genetics
subject: Pregnancy
subject: Pregnancy Complications Infectious/drug therapy
subject: Pregnancy Complications Infectious/virology
subject: High-Throughput Nucleotide Sequencing/methods
subject: Real-Time Polymerase Chain Reaction/methods
subject: Infectious Disease Transmission
subject: Vertical/prevention &amp; control
subject: Post-Exposure Prophylaxis/methods
subject: Pregnancy Complications Infectious/classification
subject: Pregnancy Complications Infectious/diagnosis
subject: Prognosis
subject: Tanzania
subject: 610 Medizin

description: 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.