Next generation sequencing improves detection of drug resistance mutations in infants after PMTCT failure

Abstract

Background Next generation sequencing (NGS) allows the detection of minor variant HIV drug resistance mutations (DRMs). However data from new NGS platforms after Prevention-of-Mother-to Child-Transmission (PMTCT) regimen failure are limited. Objective To compare major and minor variant HIV DRMs with Illumina MiSeq and Life Technologies Ion Personal Genome machine (PGM) in infants infected despite a PMTCT regimen. Study Design We conducted a cross-sectional study of NGS for detecting DRMs in infants infected despite a zidovudine (AZT) and Nevirapine (NVP) regimen, before initiation of combination antiretroviral therapy. Sequencing was performed on PCR products from plasma samples on PGM and MiSeq platforms. Bioinformatic analyses were undertaken using a codon-aware version of the Smith-Waterman mapping algorithm and a mixture multinomial error filtering statistical model. Results Of 15 infants, tested at a median age of 3.4 months after birth, 2 (13%) had non-nucleoside reverse transcriptase inhibitor (NNRTI) DRMs (K103N and Y181C) by bulk sequencing, whereas PGM detected 4 (26%) and MiSeq 5 (30%). NGS enabled the detection of additional minor variant DRMs in the infant with K103N. Coverage and instrument quality scores were higher with MiSeq, increasing the confidence of minor variant calls. Conclusions NGS followed by bioinformatic analyses detected multiple minor variant DRMs in HIV-1 RT among infants where PMTCT failed. The high coverage of MiSeq and high read quality improved the confidence of identified DRMs and may make this platform ideal for minor variant detection.

Antiretroviral Treatment and Resistance Patterns in HIV-Infected Children

“…However, these methods were limited by mismatches in primer binding [45] and the number of reactions that can be investigated. A newer alternative diagnostic method, known as nextgeneration sequencing (NGS) with potential for detection of various DRMs across the HIV-1 reverse transcriptase coding region, was used to compare major and minor variant HIV DRMs with both Illumina MiSeq and Life Technologies Ion Personal Genome Machine (Ion PGM) in South African infants [46]. These infants had already failed dual AZT and NVP PMTCT regimen.…”

“…Furthermore, NGS allowed detection of extra minor variant DRMs in the children with K103N. The instrument quality and coverage scores were higher with MiSeq, thereby increasing the confidence of minor variant detection [46].…”

Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure

“…Despite extensive studies on HIV-1 drug resistance in sub-Saharan Africa, few studies have screened for minority drug-resistant variants in treatment-naive or -experienced patients. However, minority NNRTI-resistant variants appeared to feature prominently in treatment failures for women in Africa who had received an NVP-based treatment regimen following single-dose NVP treatment to prevent mother-tochild transmission (47,48). Interestingly, NVP selective pressure, rather than the natural occurrence of K103N and Y181C at low frequencies (Ͼ1%) in the intrapatient HIV-1 population, was related to failure of NNRTI-based treatment regimens (49).…”

A Pan-HIV Strategy for Complete Genome Sequencing

“…The utility of next-generation sequencing (NGS) has been demonstrated for a variety of applications related to HIV patient monitoring, including identification of drug resistance mutations (6)(7)(8)(9)(10) and prediction of coreceptor tropism (11,12). Examples generally entail sequencing only of pol or env amplicons, respectively, to a depth necessary to identify low-level variants.…”