Comparative evaluation of subtyping tools for the surveillance of newly emerging HIV-1 strains

HIV-1 non-B subtypes/circulating recombinant forms (CRFs) are increasing worldwide. Since subtype identification can be clinically relevant, we assessed the added value in HIV-1 subtyping using updated molecular phylogeny (Mphy), and the performance of routinely used automated tools. Updated Mphy (2015 updated reference sequences), used as gold standard, was performed to subtyping of 13,116 HIV-1 protease/reverse transcriptase sequences, and then compared with previously Mphy (reference sequences until 2014), and with COMET, REGA, SCUEAL and Stanford subtyping tools. Updated Mphy classified subtype B as the most prevalent (73.4%), followed by CRF02_AG (7.9%), C (4.6%), and F1 (3.4%), A1 (2.2%), G (1.6%), CRF12_BF (1.2%), and other subtypes (5.7%). 2.3% of sequences were reassigned as different subtypes or CRFs because misclassified by previous Mphy. Overall, the best concordant tool with updated Mphy was Stanford-v8.1 (95.4%), followed by COMET (93.8%), REGA-v3 (92.5%), Stanford-old (91.1%), and SCUEAL (85.9%). All the tools had a high sensitivity (≥98.0%) and specificity (≥95.7%) for subtype B. Regarding non-B subtypes, Stanford-v8.1 was the best tool for C, D, F subtypes, and for 01, 02, 06, 11, and 36 CRFs (sensitivity ≥92.6%, and specificity ≥99.1%). A1 and G subtypes were better classified by COMET (92.3%) and REGA-v3 (98.6%), respectively.Our findings confirm Mphy as the gold standard for an accurate HIV-1 subtyping, though Stanford-v8.1, occasionally combined with COMET or REGA-v3, represents an effective subtyping approach in clinical settings. Periodical updating of HIV-1 reference sequences is fundamental to improve subtype characterization in the context of an effective epidemiological surveillance of non-B strains.