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.

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

doi:10.1371/journal.pone.0140809

Abstract

BackgroundPregnant 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).MethodsPlasma 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.ResultsDrug-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.ConclusionsBoth 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.

Highlights

Antiretroviral regimens for the prevention of mother-to-child transmission (PMTCT) of HIV have a proven efficacy in resource-limited countries
allele-specific real-time PCR (ASPCR) has a higher sensitivity than ultradeep sequencing (UDS), but is restricted to single resistance mutations
Improvements to the UDS limit of detection are in progress, and UDS could facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies

Summary

Introduction

Antiretroviral regimens for the prevention of mother-to-child transmission (PMTCT) of HIV have a proven efficacy in resource-limited countries. A major drawback of such temporary regimens is the emergence of resistant HIV-1 strains. This was extensively shown for the nevirapine single-dose (NVP-SD) regimen [1]. The implementation of the 2006 WHO-recommended triple antiretroviral regimen consisting of antenatal mono-administration of zivoduvine (AZT), NVP-SD at labor onset, and AZT plus lamivudine (3TC) for one week postpartum was assumed to reduce the development of drug resistance [1,2,3]. Drug resistant variants in HIV-1 protease and reverse transcriptase (RT) are routinely detected by Sanger population sequencing [4]. The study offered the opportunity to compare amplicon-based 454 ultradeep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT)

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