title: Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical management.
abstract: BACKGROUND
  HIV-1 near full-length genome (HIV-NFLG) sequencing from plasma is an attractive multidimensional tool to apply in large-scale population-based molecular epidemiological studies. It also enables genotypic resistance testing (GRT) for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups. Thus, the main objective of this study was to develop a simplified subtype-independent, cost- and labour-efficient HIV-NFLG protocol that can be used in clinical management as well as in molecular epidemiological studies.
  
  
  METHODS
  Plasma samples (n=30) were obtained from HIV-1B (n=10), HIV-1C (n=10), CRF01_AE (n=5) and CRF01_AG (n=5) infected individuals with minimum viral load >1120 copies/ml. The amplification was performed with two large amplicons of 5.5 kb and 3.7 kb, sequenced with 17 primers to obtain HIV-NFLG. GRT was validated against ViroSeq™ HIV-1 Genotyping System.
  
  
  RESULTS
  After excluding four plasma samples with low-quality RNA, a total of 26 samples were attempted. Among them, NFLG was obtained from 24 (92%) samples with the lowest viral load being 3000 copies/ml. High (>99%) concordance was observed between HIV-NFLG and ViroSeq™ when determining the drug resistance mutations (DRMs). The N384I connection mutation was additionally detected by NFLG in two samples.
  
  
  CONCLUSIONS
  Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies. It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies. Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.|Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Grossmann
authors.affiliation: Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Piotr
authors.initials: P
authors.lastname: Nowak
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; ujjwal.neogi@ki.se.
authors.forename: Ujjwal
authors.initials: U
authors.lastname: Neogi
mesh_terms: D000328:Adult; D024882:Drug Resistance, Viral / Q000235:genetics; D005260:Female; D016679:Genome, Viral; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D017720:Molecular Epidemiology; D009154:Mutation; D019065:Virus Assembly
keywords: HIV-1 NFLG sequencing; molecular epidemiology; subtype independent