Cost-efficient HIV-1 drug resistance surveillance using multiplexed high-throughput amplicon sequencing: implications for use in low- and middle-income countries

Abstract

Objectives: Increased trends of primary drug resistance mutations (DRMs) among treatment-naive HIV-1-infected patients in low- and middle-income countries (LMICs) and the non-availability of pre-antiretroviral therapy (ART) genotypic resistance testing (GRT) may severely affect future therapeutic outcomes. The main objective of this study was therefore to develop a simplified, cost- and labour-efficient but high-throughput GRT protocol to be applied in the large-scale surveillance of DRMs in LMICs.

Patients and methods: Ninety-six therapy-naive HIV-1-infected patients belonging to three cohorts were included: Indian patients followed at St John's Medical College Hospital, Bangalore, India (n = 49); East Africans (n = 21), who had migrated to Sweden; and Caucasians (n = 26) living in Sweden. GRT by population sequencing (GRT-PS) on individual plasma samples and GRT by next-generation sequencing (GRT-NGS) on equimolar multiplexed samples (n = 24) using Illumina MiSeq were performed.

Results: The multiplexing procedure was shown to be technically feasible and gave high-quality reads independent of whether HIV-1 subtype C or B was analysed. GRT-NGS detected all the DRMs found by GRT-PS. Additional clinically important low-abundance (<20% of the viral population) major DRMs (e.g. K101E, K103N, Y181C and M184V) were detected by GRT-NGS but not by GRT-PS. The frequency of low-abundance DRMs was higher among East African compared with Indian and Caucasian individuals.

Conclusions: Our high-throughput next-generation sequencing with a multiplexed amplicon is a cost-efficient and promising approach for the large-scale surveillance of primary DRMs in LMICs where routine pre-ART GRT is not the standard of care. This strategy may be useful in optimizing future therapeutic regimens in those settings.

Keywords: genotypic resistance testing; next-generation sequencing; primary drug resistance surveillance; therapy naive.