Use of next-generation sequencing on HIV-1 DNA to assess archived resistance in highly treatment-experienced people with multidrug-resistant HIV under virological control: data from the PRESTIGIO Registry.

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), P = 0.030] and positively correlated with viraemia levels at rebound (rho = 0.474, P = 0.030). 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.

Frequency of Atypical Mutations in the Spike Glycoprotein in SARS-CoV-2 Circulating from July 2020 to July 2022 in Central Italy: A Refined Analysis by Next Generation Sequencing.

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.

First Case of a COVID-19 Patient Infected by Delta AY.4 with a Rare Deletion Leading to a N Gene Target Failure by a Specific Real Time PCR Assay: Novel Omicron VOC Might Be Doing Similar Scenario?

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.