title: Evaluation of different RNA extraction methods and storage conditions of dried plasma or blood spots for human immunodeficiency virus type 1 RNA quantification and PCR amplification for drug resistance testing.
abstract: The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays--a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)--were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n = 47; median VL, 4.13 log(10) copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log(10) copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log(10) copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log(10) copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20 degrees C but for only 1 month at 37 degrees C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37 degrees C and for 1 month at 20 degrees C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.
authors.affiliation: UMR 145, Institut de Recherche pour le Développement, and University of Montpellier 1, Montpellier, France.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Monleau
authors.forename: Céline
authors.initials: C
authors.lastname: Montavon
authors.forename: Christian
authors.initials: C
authors.lastname: Laurent
authors.forename: Michel
authors.initials: M
authors.lastname: Segondy
authors.forename: Brigitte
authors.initials: B
authors.lastname: Montes
authors.forename: Eric
authors.initials: E
authors.lastname: Delaporte
authors.forename: François
authors.initials: F
authors.lastname: Boillot
authors.forename: Martine
authors.initials: M
authors.lastname: Peeters
mesh_terms: D001769:Blood / Q000821:virology*; D003890:Desiccation; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques; D010949:Plasma / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D019562:Viral Load
chemical_list: D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic