SC12 Characteristics and phylogenetic analysis of HIV-1 sub-subtype A6 circulating/epidemic in Italy

Background A6, originally identified as a sister clade of A1 circulating in former Soviet Union, was recently re-classified as a new HIV-1 sub-subtype and many European countries had experienced an increase in the frequency of A6. Sub-subtype A6 is a risk factor for virological failure of long-acting injectables cabotegravir-rilpivirine. This study aimed to assess the prevalence of the A6 sub-subtype in Italy over the years and to characterise these strains in term of HIV diversity, drug resistance patterns, and clustering. Material and Methods All sequences from ARCA database previously attributed to subtype A sub-subtypes and those classified as CRF01_AE (n=597) were collected for re-subtyping through phylogenetic analysis. The first available PR-RT (n=157) and integrase (n=105) sequences collected from 2002 to 2025 were analysed. MEGA12 with Neighbour Joining approach was used for subtyping analysis. Transmission clusters were identified by using maximum likelihood approach and IqTree (TVM+F+R5 model) and Cluster Picker software. HIV Stanford database was used to define resistance mutations. Results A total of 157 (26.3%) subjects carrying A6 subtype were retrieved. Most individuals were male (87/157, 55.4%), with a median age of 37 years (IQR: 32-49 years). Based on partially available data, caucasian ethnicity was the most represented (77/83, 92.8%), with most individuals originating from Italy (n=49) and Ukraine (n=23). Sexual transmission was the most reported route of infection (42/59, 71.2%), followed by intravenous drug use (12/59, 20.3%). The prevalence of subjects carrying A6 significantly increased over time (p<0.0001), both when stratified into four-year intervals (0.1% to 1%) and when comparing the period before (2002–2021) and after (2022–2025) the start of the Russian/Ukrainian war (0.3% to 1%). Transmitted drug resistance (TDR) was detected in 15.1% (n=8), 3.8% (n=2) and 5.6% (n=2) for NRTI, NNRTI and INI, respectively. A62V, which is polymorphic in sub-subtype A6, was the most represented NRTI-associated mutation (n=8), K103N was the most frequent NNRTI-associated (n=2). No differences were observed in the distribution of resistance between Italians and foreign-born. Thirteen significant transmission clusters were identified (bootstrap ≥ 70), the largest encompassing 33 sequences collected between 2014 and 2025. A significantly higher proportion of Italians were included in transmission clusters compared to foreign-born (89% vs. 60%, p<0.001) and individuals with sexual transmission compared to intravenous drug users (73.8% vs. 41.7%, p=0.037). No differences were observed in cluster distribution considering sampling period. Conclusions This study provides valuable insight on HIV-1 A6 molecular epidemiology in Italy, demonstrating significantly increasing prevalence in recent years. Despite incomplete epidemiological data, the higher proportion of Italians in transmission cluster suggests ongoing local transmission in our country.