Journal Article

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

Bannert, Norbert, Kunz, Andrea, Hauser, Andrea, Theuring, Stefanie, Radonić, Aleksandar, Nitsche, Andreas, Dugange, Festo, Dabrowski, Piotr Wojtek, Sewangi, Julius, Kuecherer, Claudia, Mbezi, Paulina, Harms, Gundel, Meixenberger, Karolin

2015

Overview

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). 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. 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. 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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Alleles / Anti-HIV Agents - therapeutic use / Antiretroviral agents / Antiretroviral drugs / Comparative analysis / Development and progression / Disease transmission / DNA sequencing / Drug resistance / Drug Resistance, Viral - genetics / Drug therapy / Female / Genetic aspects / GLP-1 receptor agonists / High-Throughput Nucleotide Sequencing - methods / HIV / HIV Infections - diagnosis / HIV Infections - drug therapy / HIV Infections - virology / HIV-1 - genetics / Human immunodeficiency virus / Humans / Infectious Disease Transmission, Vertical - prevention & control / Mutation / Physiological aspects / Post-Exposure Prophylaxis - methods / Pregnancy / Pregnancy Complications, Infectious - classification / Pregnancy Complications, Infectious - diagnosis / Pregnancy Complications, Infectious - drug therapy / Pregnancy Complications, Infectious - virology / Prognosis / Prophylaxis / Real time / Real-Time Polymerase Chain Reaction - methods / RNA-directed DNA polymerase / Sampling methods / Tanzania / Treatment Failure / Viruses / Women / Zidovudine