Next-Generation Sequencing Analysis for HIV-1 Genotyping and Drug Resistance Mutations Mapping in Sicily, Italy

Abstract

Background: The advent and continuous improvement in antiretroviral therapy (ART) have profoundly altered the clinical course of HIV infection, shifting the focus from AIDS-related complications to the management of age-related comorbidities and non-AIDS-related hospitalizations. In this evolving context, optimizing ART is essential, with genotypic resistance testing (GRT), particularly through next-generation sequencing (NGS), playing a pivotal role.

Methods: This multicenter, retrospective cross-sectional study investigated HIV-1 subtypes, resistance mutations, and drug resistance profiles among 367 people living with HIV (PLWH) in Sicily, based on 384 GRTs performed at the Microbiology Laboratory of the University Hospital of Palermo.

Results: Subtype B was the most prevalent (50%), followed by circulating recombinant forms (30%). Among treatment-naïve individuals, resistance-associated mutations were infrequent, with prevalence rates of 0.4% for NRTIs, 5.5% for NNRTIs, 1.3% for PIs, and 0.8% for INIs. Conversely, treatment-experienced individuals showed significantly higher resistance rates, especially to NRTIs (16.3%), NNRTIs (10.6%), and INIs (9.6%). No significant differences in resistance patterns were observed between B and non-B subtypes.

Conclusions: This study provides the first regional overview of HIV drug resistance across Sicily. Despite the detection of resistance-associated mutations, the overall prevalence of clinically relevant resistance, particularly to currently recommended therapies, remains low, especially among treatment-naïve individuals.

Keywords: GRT; HIV; NGS; genotypic resistance test; mutations; next generation sequencing; resistance; subtypes.

Figure 1

The absolute distribution of GRTs across Sicily, for a total of 383 GRTs, is shown. Most of the samples were collected in Palermo (PA), which alone accounts for 239 GRTs, representing 62.4% of the total. Ragusa (RG) follows with 52 GRTs (13.6%), while both Messina (ME) and Siracusa (SR) report 26 GRTs each, corresponding to 6.8% of the total. Trapani (TP) and Caltanissetta (CL) each produced 17 GRTs (4.4%), whereas Agrigento (AG) produced 3 (0.8%). Enna (EN) and Catania (CT) show the lowest frequencies, with only 2 GRTs (0.5%) and 1 GRT (0.3%), respectively.

Figure 2

The bar chart illustrates the distribution of HIV-1 subtypes in the study population, expressed as percentages. U: unknown.

Figure 3

Distribution of HIV-1 subtypes among treatment-naïve (a) and treatment-experienced (b) individuals, respectively.

Figure 4

Absolute frequency of NRTI mutations according to treatment status: overall (a), treatment-naïve (b), treatment-experienced (c). Treatment-naïve: M184 (0.4%), D67N (0.4%), K219 (0.4%), T215 (0.4%), L210 (2.9%), L74 (0.4%), M14L (0.4%), M41L (1.7%). Treatment-experienced: M184 (13.5%), T69 (1.9%), A62V (2.9%), K65 (2.9%), D67N (0.9%), K219 (1.9%), T215 (6.7%), K70 (2.9%), L210 (6.7%), L74 (2.9%), M41L (1.9%), V75 (0.9%), Y115 (0.9%). * Notation used to indicate any amino acid substitution.

Figure 5

Absolute frequency of NNRTI mutations according to treatment status: overall (a), treatment-naïve (b), treatment-experienced (c). Treatment-naïve: G190 (0.4%), V106 (6.7%), V108 (0.8%), Y188 (0.8%), L100 (0.4%), K103 (4.2%), E138 (8.0%), A98G (0.8%), V179 (0.8%), N348I (1.3%), K101 (0.4%). Treatment-experienced: G190 (2.9%), V106 (6.7%), V108 (1.9%), Y188 (1.0%), K103 (7.7%), E138 (7.7%), Y318 (1.9%), F227 (1.9%), A98G (2.9%) K10E (1.0%), N348I (1.0%), Y181 (1.0%). * Notation used to indicate any amino acid substitution.

Figure 6

Absolute frequency of PI mutations according to treatment status: overall (a), treatment-naïve (b), treatment-experienced (c). Treatment-naïve: L33F (0.4%), M46I (0.4%), I54 (0.4%), L90M (1.3%), Q58E (2.1%), K43T (1.3%), K20T (0.4%), L10 (0.8%), V32 (0.4%), I47V (0.4%), L32I (0.4%), G73 (0.4%), L89V (0.4%). Treatment-experienced: L33F (1.9%), M46I (1.9%), I54 (1.0%), V82 (1.0%), L90M (1.0%), Q58E (1.0%), L10 (1.9%), V11 (1.0%), L76V (1.0%), I50V (1.0%), T74P (1.0%). * Notation used to indicate any amino acid substitution.

Figure 7

Absolute frequency of INI mutations according to treatment status: overall (a), treatment-naïve (b), treatment-experienced (c). Treatment-naïve: E157Q (2.9%), S30R (0.4%), Q146P (0.4%), Q148 (0.8%), L74 (0.4%), T97A (0.4%). Treatment-experienced: N155 (3.8%), E157Q (7.7%), Y143 (4.8%), S147 (2.9%), Q146P (5.8%), G140 (2.9%), Q148 (5.8%), L74 (1.0%), E92Q (1.0%), T97A (1.9%), E138K (1.9%), G149A (1.0%), Q95K (1.9%), H51Y (1.0%). * Notation used to indicate any amino acid substitution.

Figure 8

Cumulative antiretroviral resistance for all cases (a), naive (b), and experienced (c).

Figure 9

The diagram presents a network visualization of HIV drug resistance according to antiretroviral drug classes. The size of the ovals with the name of the drug class is proportional to the total number of associated resistances: NNRTI (31), NRTI (23), INI (13), and PI (7). The frequency with which mutations for the different classes are associated with each other is indicated by the number placed between the segments that join the two ovals. The size of the numbers and of the segment is proportional to the frequency of the association.