title: Cost-efficient HIV-1 drug resistance surveillance using multiplexed high-throughput amplicon sequencing: implications for use in low- and middle-income countries.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Halime
authors.initials: H
authors.lastname: Ekici
authors.affiliation: Hematology Research Unit, Division of Molecular Medicine, St John's Research Institute, Bangalore, India.
authors.forename: Shwetha D
authors.initials: SD
authors.lastname: Rao
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Unit of Infectious Diseases, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Sönnerborg
authors.affiliation: SciGenom Labs Pvt. Ltd, Cochin, Kerala, India.
authors.forename: Vedam L
authors.initials: VL
authors.lastname: Ramprasad
authors.affiliation: SciGenom Labs Pvt. Ltd, Cochin, Kerala, India.
authors.forename: Ravi
authors.initials: R
authors.lastname: Gupta
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Hematology Research Unit, Division of Molecular Medicine, St John's Research Institute, Bangalore, India ujjwal.neogi@ki.se.
authors.forename: Ujjwal
authors.initials: U
authors.lastname: Neogi
mesh_terms: D000328:Adult; D015331:Cohort Studies; D003906:Developing Countries; D024882:Drug Resistance, Viral; D062665:Epidemiological Monitoring; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007194:India; D008297:Male; D008875:Middle Aged; D010949:Plasma / Q000821:virology
keywords: genotypic resistance testing; next-generation sequencing; primary drug resistance surveillance; therapy naive