title: Stability of unfrozen whole blood DNA for remote genotypic analysis of HIV-1 coreceptor tropism.
abstract: BACKGROUND
  Maraviroc is an HIV-1 coreceptor antagonist that has shown good efficacy and tolerability in treatment-naive and treatment-experienced patients harboring CCR5-tropic virus. The use of Maraviroc in treatment simplification in patients with suppressed plasma HIV-1 RNA requires analysis of HIV-1 DNA. Coreceptor tropism testing is often performed remotely at reference laboratories. In this study paired whole blood stored at + 4 °C and at-20°C were compared as a source for genotypic coreceptor tropism testing.
  
  
  METHODS
  Two hundred paired whole blood samples from different patients were analysed. Each sample was stored in two different conditions: one aliquot was stored at-20 °C until spin column DNA extraction (WB20) and one aliquot was stored at +4°C for two weeks and then placed at room temperature (22-24 °C) for two days before DNA extraction (WB4). Subsequently, a fragment encompassing the HIV-1 gp120 V3 domain was amplified by a singlicate nested PCR followed by triplicate nested PCR in the negative samples. A randomly selected panel of 20 paired WB4 and WB20 duplicate amplification products were sequenced and coreceptor tropism was inferred by geno2pheno [coreceptor].
  
  
  RESULTS
  WB20 yielded a higher amount of DNA than WB4 (median [IQR] values 332.5 ng/μl [117.5-401] and 107 ng/μl [56.6-318], respectively; P < 0.001). However, the DNA purity was higher for WB4 than for WB20 (median distance from the optimal OD260/280 ratio, 0.14 [0.07-0.79] and 0.96 [0.36-1.10], respectively; P < 0.0001). The number of samples successfully amplified was 152 (76.0%) for WB20 and 155 (77.5%) for WB4 with the first PCR and 179 (89.5%) for WB20 and 181 (90.5%) for WB4 (P = ns) following subsequent triplicate analysis. The inferred coreceptor tropism was concordant in 18 out of 20 paired WB4 and WB20 samples. Two samples yielded discordant results, consistent with the discordance rate within duplicates from the same sample source (2/20 with WB4 and 1/20 with WB20) due to the inherent gp120 V3 variability.
  
  
  CONCLUSIONS
  Storing whole blood at +4°C for up to two weeks and shipping at room temperature is a convenient method for obtaining HIV-1 gp120 V3 sequence information via testing at a remote laboratory in patients with suppressed viremia.
authors.affiliation: Department of Medical Biotechnologies, University of Siena, Siena, Italy. meini4@unisi.it.
authors.forename: Genny
authors.initials: G
authors.lastname: Meini
authors.forename: Angelo
authors.initials: A
authors.lastname: Materazzi
authors.forename: Francesco
authors.initials: F
authors.lastname: Saladini
authors.forename: Andrea
authors.initials: A
authors.lastname: Rosi
authors.forename: Ilaria
authors.initials: I
authors.lastname: Vicenti
authors.forename: Michele
authors.initials: M
authors.lastname: Mancini
authors.forename: Antonella
authors.initials: A
authors.lastname: Pirazzoli
authors.forename: Cinzia
authors.initials: C
authors.lastname: Caudai
authors.forename: Maurizio
authors.initials: M
authors.lastname: Zazzi
mesh_terms: D004279:DNA, Viral / Q000097:blood* / Q000235:genetics* / Q000302:isolation & purification; D005838:Genotype; D015699:HIV Envelope Protein gp120 / Q000235:genetics*; D015658:HIV Infections / Q000097:blood* / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016133:Polymerase Chain Reaction; D015728:Receptors, HIV / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D056189:Viral Tropism
chemical_list: D004279:DNA, Viral; D015699:HIV Envelope Protein gp120; D015728:Receptors, HIV