title: A pragmatic approach to HIV-1 drug resistance determination in resource-limited settings by use of a novel genotyping assay targeting the reverse transcriptase-encoding region only.
abstract: In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples.
authors.affiliation: University Medical Centre, Utrecht, The Netherlands. s.c.aitken@umcutrecht.nl
authors.forename: Susan C
authors.initials: SC
authors.lastname: Aitken
authors.forename: Michelle
authors.initials: M
authors.lastname: Bronze
authors.forename: Carole L
authors.initials: CL
authors.lastname: Wallis
authors.forename: Lieven
authors.initials: L
authors.lastname: Stuyver
authors.forename: Kim
authors.initials: K
authors.lastname: Steegen
authors.forename: Sheila
authors.initials: S
authors.lastname: Balinda
authors.forename: Cissy
authors.initials: C
authors.lastname: Kityo
authors.forename: Wendy
authors.initials: W
authors.lastname: Stevens
authors.forename: Tobias F
authors.initials: TF
authors.lastname: Rinke de Wit
authors.forename: Rob
authors.initials: R
authors.lastname: Schuurman
mesh_terms: D000349:Africa; D002648:Child; D017931:DNA Primers / Q000235:genetics; D003906:Developing Countries; D024882:Drug Resistance, Viral; D005060:Europe; D005838:Genotype; D015658:HIV Infections / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010949:Plasma / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
references.citation: Lancet Infect Dis. 2011 Oct;11(10):750-9
references.pmid: 21802367
references.citation: AIDS Res Treat. 2011;2011:769627
references.pmid: 21490784
references.citation: AIDS. 2009 Jun 1;23(9):1127-34
references.pmid: 19417582
references.citation: Lancet Infect Dis. 2010 Mar;10(3):155-66
references.pmid: 20185094
references.citation: Top HIV Med. 2010 Dec;18(5):156-63
references.pmid: 21245516
references.citation: J Clin Virol. 2012 May;54(1):21-5
references.pmid: 22306272
references.citation: J Antimicrob Chemother. 2008 Dec;62(6):1191-8
references.pmid: 18927229
references.citation: J Clin Microbiol. 2007 Oct;45(10):3342-51
references.pmid: 17670924
references.citation: J Acquir Immune Defic Syndr. 2010 Apr 1;53(4):480-4
references.pmid: 19801944
references.citation: J Virol Methods. 2006 May;133(2):137-45
references.pmid: 16375980
references.citation: AIDS Res Ther. 2010 Oct 15;7:38
references.pmid: 20950432
references.citation: Int J Epidemiol. 2012 Feb;41(1):43-54
references.pmid: 21071386
references.citation: J Virol Methods. 2010 Feb;163(2):505-8
references.pmid: 19917318
references.citation: AIDS. 2007 Nov 30;21(18):2503-11
references.pmid: 18025887