title: Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group.
abstract: OBJECTIVE
  We have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification.
  
  
  RESULTS
  The overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/10(6) PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /10(6) PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification.
  
  
  CONCLUSIONS
  Our results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.
authors.affiliation: Department of Experimental Medicine, University of Rome "Tor Vergata", Via Montpellier 1, 00133, Rome, Italy.
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mesh_terms: D000328:Adult; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011533:Proviruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D015728:Receptors, HIV / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D056189:Viral Tropism; D053586:Virus Internalization
chemical_list: D004279:DNA, Viral; D015728:Receptors, HIV