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Does NGS on HIV-1 DNA improve resistance assessment in highly treatment-experienced and multi-resistant individuals under virological control? An experience from the PRESTIGIO Registry

Background: This study aimed to clarify whether NGS might be useful for resistance assessment in virologically suppressed highly treatment-experienced (HTE) individuals with multidrug resistance (MDR).

Methods: Ninety-one HTE MDR individuals from the PRESTIGIO registry were analysed. HIV-1 DNA PR/RT/IN and V3 sequences were obtained through NGS on MiSeq platform. Major resistance mutations (MRM) and APOBEC editing estimation (APOBEC mutations [APO-M]; stop codons) were evaluated through HIVdb algorithm. NGS cut-offs at ≥1%, ≥5% and ≥20% were tested. Minority MRM with frequency ranging 1-5% (mV1%) and 5-20% (mV5%) and majority MRM (frequency >20%, mV20%) were compared to historical-GRT (H-GRT). Variants distribution was compared between individuals who experienced virological rebound after NGS-GRT and those who maintained virological control.

Results: At NGS-GRT, individuals had a median (IQR) cART exposure of 23 (21-25) years, had been virologically suppressed since 3 (2-5) years and had a total HIV-DNA of 2,377 (1,274-4,949) copies/106 CD4+ cells. X4 tropism was detected in 61.5% of individuals.
A total of 1,772 MRM were detected. Around half of MRM detected by NGS were already found in H-GRT; on the other hand, NGS-GRT detected a considerable number of additional mutations never observed before (Figure 1). The highest detection rate of historical MRM was obtained by setting NGS at 1% (Figure 2).
NGS set at 1% showed poor reliability, although associated with the highest detection rate of historical MRM. In fact, mV1% (N=337) were frequently detected in samples with stop codons (94.4%) or APO-M (97.4%) providing potential misleading resistance assessment.
Differently, among mV5% (N=370), a substantial proportion of cases was not affected by APOBEC editing and contributed in expanding detection of historical MRM (25.9%) or detecting new MRM (18.6%).
Regarding majority variants, mV20% (N=704) were marginally detected in samples with stop codons (2.9%) or APO-M (5.3%), and mostly contributed to detect (69.4%) historical MRM or detect new MRM (25.4%).
After NGS-GRT, 21 individuals underwent virological rebound with a median (IQR) viremia of 365 (98-7,840) copies/mL. Among them, only the median (IQR) number of mV5% detected exclusively by NGS-GRT was higher (2 [1-3]) compared to those who maintained virological control (1 [0-2], p=0.030, Figure 3). The number of mV5% newly detected by NGS in failing individuals positively correlated with plasma HIV-RNA levels detected at virological rebound (Spearman test, Rho=0.474, P=0.030).

Conclusions: In HTE MDR virologically suppressed individuals, NGS-GRT on HIV-1 DNA allows detection of around 60-70% historical MRM and detects considerable new resistance. Our results confirm that setting NGS at 5% might be a good choice to obtain reliable sequence data. At this setting, an increased number of minority species correlates with loss of virological control and with viremia levels at virological rebound.

Dated Fri Jun 16 2023

Authors: D.Armenia, V.Spagnuolo, M.C.Bellocchi, L.Galli, L.Duca, G.Marchegiani, T.Clemente, L.Carioti, R.Lolatto, L.Calza, B.M.Celesia, A.Cascio, D.Francisci, A.Saracino, C.Torti, M.Zazzi, A.Castagna, M.M.Santoro, on behalf of the PRESTIGIO Registry

Affiliations: UniCamillus, Saint Camillus International University of Health Sciences, Rome, Italy, 2University of Rome “Tor Vergata”, Rome, Italy, 3Laboratory of Clinical Microbiology, Virology and Bioemergencies, ASST Fatebenefratelli Sacco - University of Milan, Milan, Italy, 4Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Turin, Italy, 5Azienda Ospedaliera Universitaria Policlinico "P. Giaccone" - Universita' degli Studi di Palermo, Palermo, Italy, 6Hygiene Unit, Ospedale Policlinico San Martino, Genoa, Italy, 7Section of Experimental and Clinical Pathology, Department of Precision and Regenerative Medicine and Jonic Area, University of Bari, Bari, Italy, 8Microbiology Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy, 9Virology Unit, Polyclinic of “Tor Vergata”, Rome , Italy, 10Virology Unit, AOU Pisana, Pisa, Italy, 11Microbiology and Virology Unit, Diagnostic Department, AOU Sassari, Sassari, Italy, 12Department of Medicine and Surgery, University of Insubria, Varese, Italy, 13Microbiology and Virology Unit, Policlinico Riuniti Foggia Hospital, Foggia, Italy, 14U.O.C. Microbiologia e Virologia, P.O. "D.Cotugno" - AO dei Colli - Napoli, Italy, 15Department of Medical Biotechnologies, University of Siena, Siena, Italy, 16Infectious Disease Department, USL SUDEST, Toscana, Misericordia Hospital, Grosseto, Italy, 17IPRO-InformaPRO S.r.l., Rome, Italy, EuResist Network GEIE, Rome, Italy