Computational protocol: HIV-1 Drug-Resistance Surveillance among Treatment-Experienced and -Naïve Patients after the Implementation of Antiretroviral Therapy in Ghana

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Protocol publication

[…] HIV-1 drug-resistance genotyping was performed as previously reported with some modifications . In brief, viral RNA was extracted from 200 µL of plasma samples using QIAamp viral RNA mini kit (Qiagen, Hilden, Germany). RT-PCR was performed with QIAGEN one-step RT-PCR kit (Qiagen), and nested PCR was subsequently performed using AmpliTaq DNA polymerase (Applied Biosystems, Foster City, USA). Specific primers known as DRPRO5, DRPRO2L, DRPRO1M, and DRPRO6 were used for the protease (PR) region (424 bp, positions 2,168 to 2,591 in the reference HXB2 sequence), and DRRT1L, DRRT4L, DRRT7L, and DRRT6L primers for the reverse transcriptase (RT) region (838 bp, positions 2,510 to 3,347) . Details of the primers used in the study are shown in . Nucleotide sequencing was performed using ABI 3730 auto-sequencer followed by editing with SeqScape software v2.5 (Applied Biosystems). HIV-1 drug-resistance mutations were detected according to the latest definition of the International AIDS Society-USA panel . In addition, transmitted HIV-1 drug-resistance mutations were defined using the mutation list proposed by Bennett et al. . [...] HIV-1 subtyping was performed using the pol gene sequences (1,095 bp, positions 2253 to 3347). Phylogenetic tree was constructed with the references of subtypes A-D, F-H, J, K, and all circulating recombinant forms (CRFs) 01 to 51, except 30, 41, and 50, obtained from the HIV Sequence Database at the Los Alamos National Laboratory. In addition, HIV-1 sub-subtype A3 (DDI579, DDJ360, and DDJ369) and A4 (97CD_KCC2, 97CD_KTB13, and 02CD_KTB035) isolates were added to the phylogenetic tree analysis, as these sub-subtypes have been reported as circulating in several African countries , . Multiple sequences were aligned using the MUSCLE program, and genetic distances were calculated based on the maximum composite likelihood model using MEGA software v5.05 . Phylogenetic trees were constructed using the neighbor-joining method with 1,000 bootstrap replicates. In similarity plotting and boot-scanning analyses, nine HIV-1 subtypes, A-D, F-H, J, and K, and three CRFs, CRF02_AG, CRF06_cpx, and CRF09_cpx, were used as references. Similarity plotting and boot-scanning were performed using SimPlot software v3.5.1 with window and step sizes of 250 and 20 nucleotides, respectively . One HIV-1 isolate identified with an unknown mosaic pattern both in similarity plotting and boot-scanning analyses was considered as a unique recombinant form (URF). […]

Pipeline specifications

Software tools SeqScape, MEGA, SimPlot
Applications Phylogenetics, Sanger sequencing
Organisms Human immunodeficiency virus 1, Homo sapiens
Diseases HIV Infections
Chemicals Zidovudine, Stavudine, Lamivudine, Nevirapine, Nelfinavir