title: Evaluation of dried blood spots for human immunodeficiency virus type 1 drug resistance testing.
abstract: Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at -20 degrees C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at -20 degrees C or at -70 degrees C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that -20 degrees C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.
authors.affiliation: Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: McNulty
authors.forename: Cheryl
authors.initials: C
authors.lastname: Jennings
authors.forename: Diane
authors.initials: D
authors.lastname: Bennett
authors.forename: Joseph
authors.initials: J
authors.lastname: Fitzgibbon
authors.forename: James W
authors.initials: JW
authors.lastname: Bremer
authors.forename: Michael
authors.initials: M
authors.lastname: Ussery
authors.forename: Marcia L
authors.initials: ML
authors.lastname: Kalish
authors.forename: Walid
authors.initials: W
authors.lastname: Heneine
authors.forename: J Gerardo
authors.initials: JG
authors.lastname: García-Lerma
mesh_terms: D001800:Blood Specimen Collection / Q000379:methods*; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D016333:HIV Protease