RESUMO
BACKGROUND: To clarify whether next-generation sequencing (NGS) can be useful for resistance assessment in virologically suppressed highly treatment-experienced (HTE) individuals with MDR HIV. METHODS: Ninety-one participants from the PRESTIGIO Registry were included. NGS was performed on HIV-DNA at 1%, 5% and 20% cut-offs; major drug resistance mutations (DRMs) were evaluated and compared with those detected in historical plasma genotypic resistance testing (h-GRT). APOBEC editing was also characterized. RESULTS: Participants had a complex and long treatment history [median 23 (IQR 21-25) years of ART exposure) and had been virologically suppressed since a median of 3 (IQR 2-5) years. Among all major DRMs detected by HIV-DNA NGS and/or h-GRT, 30% were exclusively found through NGS. The highest detection rate of historical major DRMs was reached with NGS set at 1%, but unusual substitutions and extensive APOBEC hypermutations suggest technical issues and poor clinical relevance in the 1%-5% interval. At NGS set at 5%, 67.2% of historical major DRMs were detected. The number of major DRMs detected exclusively by DNA-NGS as minority variants (frequency 5%-20%) was significantly higher in individuals who later experienced virological rebound compared with those who maintained virological control [median 2 (IQR 1-3) versus 1 (0-2), Pâ=â0.030] and positively correlated with viraemia levels at rebound (rhoâ=â0.474, Pâ=â0.030). CONCLUSIONS: In non-viraemic people with an MDR virus, HIV-1 DNA NGS set at 5% is an acceptable technical cut-off that might help to reveal mutations with a potential clinical relevance. Moreover, the number of minority resistance mutations additionally detected by NGS might be associated with loss of virological control.
Assuntos
DNA Viral , Farmacorresistência Viral Múltipla , Infecções por HIV , HIV-1 , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Humanos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , HIV-1/efeitos dos fármacos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Farmacorresistência Viral Múltipla/genética , DNA Viral/genética , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/farmacologia , Genótipo , Sistema de Registros , Terapia Antirretroviral de Alta AtividadeRESUMO
In this study, we provided a retrospective overview in order to better define SARS-CoV-2 variants circulating in Italy during the first two years of the pandemic, by characterizing the spike mutational profiles and their association with viral load (expressed as ct values), N-glycosylation pattern, hospitalization and vaccination. Next-generation sequencing (NGS) data were obtained from 607 individuals (among them, 298 vaccinated and/or 199 hospitalized). Different rates of hospitalization were observed over time and among variants of concern (VOCs), both in the overall population and in vaccinated individuals (Alpha: 40.7% and 31.3%, Beta: 0%, Gamma: 36.5% and 44.4%, Delta: 37.8% and 40.2% and Omicron: 11.2% and 7.1%, respectively, both p-values < 0.001). Approximately 32% of VOC-infected individuals showed at least one atypical major spike mutation (intra-prevalence > 90%), with a distribution differing among the strains (22.9% in Alpha, 14.3% in Beta, 41.8% in Gamma, 46.5% in Delta and 15.4% in Omicron, p-value < 0.001). Overall, significantly less atypical variability was observed in vaccinated individuals than unvaccinated individuals; nevertheless, vaccinated people who needed hospitalization showed an increase in atypical variability compared to vaccinated people that did not need hospitalization. Only 5/607 samples showed a different putative N-glycosylation pattern, four within the Delta VOC and one within the Omicron BA.2.52 sublineage. Interestingly, atypical minor mutations (intra-prevalence < 20%) were associated with higher Ct values and a longer duration of infection. Our study reports updated information on the temporal circulation of SARS-CoV-2 variants circulating in Central Italy and their association with hospitalization and vaccination. The results underline how SARS-CoV-2 has changed over time and how the vaccination strategy has contributed to reducing severity and hospitalization for this infection in Italy.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Sequenciamento de Nucleotídeos em Larga Escala , SARS-CoV-2/genética , Estudos Retrospectivos , COVID-19/epidemiologia , Mutação , Itália/epidemiologia , GlicoproteínasRESUMO
Herein, we report a case of an Italian male infected by Delta sublineage AY.4 harboring an atypical deletion, leading to a N gene target failure (NGTF) by a commercial molecular assay for SARS-CoV-2 diagnosis (AllplexTM SARS-CoV-2 Assay, Seegene). A 59-year-old unvaccinated patient was hospitalized for pulmonary embolism, with first negative results obtained by both molecular and antigen tests. After several days of viral negativity, he presented positive results for E and RdRP/S genes, but negative in N gene. Negativity in N gene was repeatedly confirmed in the following days. Suspecting an infection by the Omicron variant, SARS-CoV-2 genome sequencing was rapidly performed from nasopharyngeal swab by MiSeq and revealed the presence of the Delta sublineage AY.4 variant with an atypical deletion of six nucleotides, leading to G214-G215 deletion in the Nucleocapsid, thus responsible for NGTF. The analysis of GISAID sequences (N = 2,618,373 12 January 2022) showed that G214-G215 deletion is rarely occurring in most circulating Delta lineages and sublineages in the globe and Europe, with an overall prevalence never exceeding 0.2%. Hence, this study highlights the importance to perform SARS-CoV-2 sequencing and to characterize novel mutations/deletions that could jeopardize the proper interpretation of molecular diagnostic tests. Based on these assumptions, the role of deletions in the recently identified Omicron variant deserves further investigation.