Computational protocol: Antiretroviral treatment outcome in HIV-1-infected patients routinely followed up in capital cities and remote areas of Senegal, Mali and Guinea-Conakry

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Protocol publication

[…] Total HIV-1 nucleic acids were extracted from two spots with NucliSENS miniMAG (bioMérieux, Craponne, France) with magnetic silica as previously described []. Briefly, two spots from each sample were punched and placed in a tube containing 2 mL of lysis buffer. After 30 min of gentle rocking at room temperature, the supernatant was clarified by centrifugation at 2500 rpm during two minutes and then transferred to a clean 15 mL conical tube. Extracted nucleic acids were eluted in 25 µL of elution buffer and stored at 4°C for immediate use (VL quantitation or PCR amplification) or at −80°C for longer storage.HIV-1 VL was quantified using the NucliSENS EasyQ HIV-1 v1.2 (bioMérieux, Marcy l'Etoile, France) according to manufacturers’ instructions. The VL cut-off was 800 copies/mL with this assay for DBS []. In the present study, we set the VF to 3log10 (1000) copies/mL as recently recommended by WHO [].HIV-1 drug resistance test was performed according to the ANRS AC11 protocol (http://www.hivfrenchresistance.org/) by amplifying separately the entire Protease (PR) gene and first 240 codons of Reverse Transcriptase (RT) using, respectively, 5′Prot1/3′Prot1 and MJ3/MJ4 as outer primers and 5′Prot2/3′Prot2 and A35/NE35 as inner primers. Second round PCR products were purified with QIAquick Gel Extraction Kit® (Qiagen, Courtaboeuf, France) according to the manufacturers’ instructions. Purified DNA was sequenced directly on ABI 3100 Avant Genetic Analyzer using Big Dye Terminator Technology®v3.1 (Applied Biosystems, Carlsbad, CA) and their respective inner primers. Sequences obtained were assembled and edited manually using SeqMan™ II 5.08 from DNAstar®software (Lasergene, Konstanz, Germany). Drug resistance analysis and interpretation were performed using the Stanford University HIV database version 6.0.8 (http://hivdb.stanford.edu/). HIV-1 subtypes were determined by phylogeny. Nucleotide sequences were aligned with a set of reference sequences of HIV-1 group M subtype and circulating recombinant forms (CRFs) downloaded from Los Alamos HIV database (http://www.hiv.lanl.gov/content/index). Each subtype was represented by at least three reference sequences. Sequences were aligned with MUSCLE (and gap positions removed by using Gblocks program on SEAVIEW v4.4.1). Maximum Likelihood phylogeny was inferred online using the PhyML software (http://www.atgc-montpellier.fr/phyml) with branch supports determined by the approximate likelihood ratio test method (aLRT) SH-like option, and the substitution model was GTR+I+G. The recombinant strains analysis (similarity and bootscanning) were performed on Simplot software v3.5.1 [].The new PR and RT generated sequences were deposited in EMBL with the following accession Numbers: HG380024 to HG380051, HG380054 to HG380063, HG380065 to HG380069, HG424394 to HG424413, HG424415, HG424417, HG424418 and HG424420 to HG424423. […]

Pipeline specifications

Software tools Gblocks, SeaView, PhyML, SimPlot
Databases ATGC
Application Phylogenetics
Organisms Human immunodeficiency virus 1, Homo sapiens
Diseases HIV Infections, Dyssomnias, Renal Insufficiency