Reliability of Rapid Subtyping Tools Compared to That of Phylogenetic Analysis for Characterization of Human Immunodeficiency Virus Type 1 Non-B Subtypes and Recombinant Forms

Human immunodeficiency virus type 1 (HIV-1) subtyping is often estimated on the basis of pol sequences by using online websites instead of phylogenetic analysis (phy). We evaluated the reliability of distinct rapid subtyping tools versus phy with a large panel of HIV-1 non-B subtypes and circulating recombinant forms (CRF). pol sequences (277 protease [PR] and 171 reverse transcriptase [RT] sequences) previously assigned by phy to eight distinct HIV-1 non-B subtypes were obtained from 277 HIV-infected patients. Phy was run again to identify CRF. Subtyping was then performed using three rapid tools (the Stanford, NCBI, and REGA online tools). Thirty-three additional clade B sequences were tested as controls. New phylogenetic analyses reclassified two-thirds of pol sequences previously assigned to HIV-1 non-B clades as CRF. CRF02_AG variants were correctly assigned by the Stanford and NCBI tools for 92 to 97% and 96 to 99% of PR-RT sequences, respectively, while they were correctly assigned by the REGA tool for only 18 to 32% of PR-RT sequences. The Stanford, NCBI, and REGA tools failed to assign pure non-B clades correctly for 24 to 33%, 35%, and 57 to 64% of PR-RT sequences, respectively. For PR-RT sequences from CRF other than CRF02_AG, discrepancies occurred in 98 to 100%, 18 to 43%, and 80 to 87% of sequences, respectively. The concordance between those tools and phy was almost complete for subtype B assignment. Rapid subtyping tools show relatively low agreement with phy in identifying HIV-1 non-B clades and CRF other than CRF02_AG. The Stanford tool shows the best concordance with phy for the assignment of pure non-B clades, while the NCBI tool performs better at identifying CRF. Before entering routine clinical use, rapid subtyping tools should be optimized and updated periodically. Larger numbers of different non-B subtypes and CRF sequences should be included.