Genotyping and phylogenetic analysis in HIV positive patients

Assessment of drug resistance is critical in clinical management of HIV infection. Although Sanger sequencing is considered gold standard in HIV genotyping, next-generation sequencing methods have emerged as a viable option for detecting low-frequency resistance mutations. Sanger and next generation sequencing methods were used in this study to genotype HIV-1 strains isolated from HIV/AIDS patients. Efficacy of methods in detecting resistance mutations was compared. Study included 47 samples routinely studied in Medical Virology Laboratory at Gazi University Medical Faculty. Following extraction, pol gene region encoding protease and reverse transcriptase were amplified. Sanger and next-generation sequencing were used to determine sequences. Sanger revealed 10.6%, 4.3% and 6.4% resistance to at least one drug, nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors, respectively. At 1% threshold and above, next generation sequencing revealed 46.8%, 19.1%, 27.7% and 19.2% resistance to at least one drug, protease inhibitors, nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors, respectively. At 5% and higher thresholds, 14.9% were found to be resistant to at least one drug. No resistance mutations observed for protease inhibitors above 1% threshold or with Sanger. All mutations detected by Sanger were also detected by next-generation sequencing. 66% of sequences were found to be subtype B followed by A (13%), G (9%), C (4%), CRF_02AG (4%), B/CRF02_AG (2%), CRF63_02A1 (2%). Prevalence of resistance mutations emphasizes significance of HIV-1 genotyping. Next-generation sequencing has improved sensitivity in detecting low-frequency resistance mutations below 5% threshold. Number of low-frequency resistance mutations below 5% threshold increased significantly. In Turkey, distribution of HIV-1 genotypes varies.