Next-generation sequencing (NGS) has improved HIV-1 genotypic resistance testing (GRT) by enabling the detection of minority drug-resistance variants, although interpretation of low-frequency mutations remains challenging because of sequencing artifacts. This study compared the analytical performance and workflow efficiency of two Ion Torrent platforms, GeneStudio S5 (S5) and Genexus (GX), for routine HIV-1 GRT. A total of 134 plasma samples from people with HIV were prospectively collected, and 100 samples successfully sequenced on both platforms were included in the comparative analysis. Overall concordance for resistance-associated mutations was 88.0%, with agreement rates of 97.0% for protease, 90.0% for reverse transcriptase, and 100% for integrase. Both platforms generated clinically interpretable resistance profiles; however, 13 discordant mutations were identified. Application of a standardized confirmation algorithm, integrating Stanford HIVdb analysis with manual read-level inspection in Geneious software (version 2025.2.2), reclassified several discordant mutations as low-confidence or non-confirmed variants. Operationally, GX provided a fully automated workflow with approximately 24 h turnaround time and minimal hands-on processing, whereas S5 required approximately 72 h and substantially greater operator involvement. These findings support both platforms for routine HIV-1 GRT while emphasizing the importance of standardized bioinformatic review for reliable variant interpretation.

Human endogenous retroviruses (HERVs) originate from ancient retroviral integration into the primate germline. Although most are defective proviruses, the most recently endogenized groups, like the HERV-K family, retain intact ORFs encoding retroviral proteins. HERVs usually remain transcriptionally silent, yet this status is reversible. Multiple HIV-HERV interactions, mainly mediated by the HIV Tat protein, lead to HERV transcription and protein production. The present study investigates HERV-K transcription in particular of Human MMTV-like (HML) group-2 and 6 in peripheral blood of people with HIV (PWH). Using different experimental approaches—such as single-cell and plasma transcriptomics-, we found that HERV-K transcripts may be detected during both acute and chronic phases of the infection, with HML-6 showing higher expression compared to HML-2, predominantly within myeloid cells. Effective combined antiretroviral therapy (cART) was able to significantly reduce HML-6 transcription, regardless of whether the treatment was initiated in the acute or late chronic phases of HIV infection. Notably, chronic infections showed higher HML-6 transcript levels compared to acute infections in both naïve and successfully cART-treated subjects, potentially associated with persistent immune dysregulation observed in chronic HIV infection, although a direct causal role of HML-6 expression remains to be established.

The extensive genetic diversity of HIV-1, also represented by the circulation of multiple subtypes and circulating recombinant forms (CRFs), poses significant challenges for accurate subtype classification, especially when sequencing is limited to partial genomic regions. This study evaluated the performance of four commonly used automated subtyping tools (Stanford HIVdb, COMET, REGA, and Geno2pheno) by comparing their outputs with molecular phylogenetic analysis (Mphy), considered the gold standard, using three NGS-derived sequence data sets: protease-reverse transcriptase (PR-RT), pol, and near full-length (NFL). One hundred plasma samples were processed to generate sequences of increasing length, which were analyzed to assess concordance, sensitivity, and specificity. NFL-based Mphy identified a higher proportion of circulating recombinant forms (51.6%) than PR-RT and pol (44.1%) and enabled the reclassification of 13 samples as more complex CRFs. Automated tools displayed good concordance with Mphy for PR-RT and pol, particularly for pure subtypes, whereas concordance decreased considerably for NFL sequences, especially among non-B subtypes and CRFs. Sensitivity varied substantially across tools and subtypes, while specificity remained consistently high. Overall, the findings indicate that whole genome or NFL sequencing enhances the detection of CRFs and that the accuracy of automated tools is strongly influenced by the completeness and updating of their reference databases.

The mechanisms underlying post-acute sequelae of SARS-CoV-2 infection (PASC) are a topic of debate. This study examined the presence of SARS-CoV-2 microRNA (miRNA)-like small RNAs in extracellular fluids and their potential link to PASC by using a quantitative stem-loop RT-PCR MiRNA assay. Initially, it was demonstrated that three previously identified SARS-CoV-2 miRNA-like small RNAs, specifically svRNA 1 and 2 and miR-07a, were significantly expressed in infected cells in vitro and released into the supernatant following infection by different SARS-CoV-2 variants. Then, the expression of three SARS-CoV-2 small RNAs was studied in both nasopharyngeal swabs (NPS) and sera from 24 patients at their initial COVID-19 diagnosis (T0) and in sera collected 91 to 193 days post-diagnosis (T1). Notably, 11 out of 24 patients (46%) reported PASC consequences. All NPS samples showed SARS-CoV-2 small RNA expression with an altered cytokine network during acute infection, but it did not correlate with PASC outcomes. Serum samples had similar small RNA statuses, though PASC patients, notably at T1, but not at T0, displayed reduced overall positivity compared to those without PASC. The host target expression of SARS-CoV-2 small RNAs was not significantly different between groups. This suggests a need for further research into SARS-CoV-2 small RNA and its role in viral behavior and PASC consequences.

Human cytomegalovirus (HCMV) coinfection is associated with a faster HIV disease progression and adverse clinical outcomes. HCMV-encoded miRNA expression, in individuals acutely infected with HIV (AHI), compared to those with HCMV monoinfection, was investigated in relation to viral replication and inflammation/immune activation. Sixteen individuals with AHI coinfected with HCMV were analyzed at serodiagnosis (T0) and after 6 (T1) and 12 (T2) months of antiretroviral therapy initiated within one week from serodiagnosis. Fourteen HCMV monoinfected subjects were also studied. Plasma RNA was reverse-transcribed and amplified with a panel designed to detect 14 different HCMV-microRNAs (miRNAs). VEGF-A and IP-10 plasma levels were quantified using ELISA. Except for hcmv-miR-70-3p, detected in all subjects, hcmv-miR-UL112-3p, hcmv-miR-US25-1-5p, hcmv-miR-US25-2-3p, hcmv-miR-US4-5p, hcmv-miR-US5-1, hcmv-miR-US5-2-3p, hcmv-miR-UL36-3p, and hcmv-miR-UL36-5p were significantly more frequently detected when HCMV DNA was present (lytic infection). In latent HCMV infection, hcmv-miR-UL22A-5p and hcmv-miR-UL148D were more frequently observed in HIV/HCMV-coinfected individuals, compared to mono-HCMV infection. Hcmv-miR-UL22A-5p and hcmv-miR-US33-5p showed a direct correlation with HIV-1 RNA. Notable positive correlations between hcmv-miR-UL22A-5p and the interferon-gamma-inducible protein 10 (IP-10), as well as between hcmv-miR-UL148D and the vascular endothelial growth factor A (VEGF-A), were also observed. HCMV-miRNA expression varies between lytic and latent infection and differs in HIV coinfection. In HCMV/HIV coinfection, increased levels of hcmv-miR-UL148D, associated with VEGF-A production, seem to be less linked to HIV viremia with respect to hcmv-miR-UL22A-5p and hcmv-miR-US33-5p. A deeper understanding of HCMV-encoded miRNA biology may facilitate the comprehension of HCMV/HIV coinfection pathogenetic mechanisms.

Next-generation sequencing (NGS) of near full-length HIV genomes was performed to investigate natural resistance to Fostemsavir (FTR) and Lenacapavir (LEN) at the quasispecies level in nine naïve primary HIV infections harboring different HIV subtypes and recombinant forms. Reconstructed genomes provided a median (IQR) coverage for gag and env of 1710 (750–6063) and 1768 (871–5270), respectively. In the gp120 encoding region, the M426R variant was found with a frequency of 100% in two HIV subtypes B: one of these also showed the A204T variant at 100%. In the more conserved capsid coding region no mutations possibly related to LEN natural resistance were observed.

Resistant/refractory Cytomegalovirus (CMV) infection is an important threat in the management of solid organ transplant recipients, often associated with poor outcomes. We describe the case of a heart-transplanted patient who developed primary CMV infection with fever and subsequent appearance of resistance to ganciclovir and maribavir during antiviral treatment, and with important toxicity due to foscarnet. The outcome was good with reduced-dose continuous infusion of foscarnet. Further research is needed to improve the management of difficult-to-treat CMV infection.

Human and viral microRNAs (miRNAs) are involved in the regulation of gene transcription, and the establishment of their profiles in acute (AHI) and chronic (CHI) HIV infections may shed light on the pathogenetic events related to different phases of HIV disease. Next-generation sequencing (NGS) of miRNA libraries was performed, and the reads were used to analyze miRNA differential expression in the plasma with AHI and CHI. Functional analysis was then undertaken to investigate the biological processes characterizing the two phases of HIV infection. Except for hsa-miR-122-5p, which was found in 3.39% AHI vs. 0.18% CHI, the most represented human miRNAs were similarly represented in AHI and CHI. However, when considering the overall detected miRNAs in AHI and CHI, 15 displayed differential expression (FDR p < 0.05). Functional analysis identified 163 target mRNAs involved in promoting angiogenesis activation in AHI versus CHI through the action of hsa-miR10b-5p, hsa-miR1290, hsa-miR1-3p, and hsa-miR296-5p. The viral miRNAs detected, all belonging to herpesviruses, accounted for only 0.014% of total reads. The present data suggest that AHI patients exhibit strong innate immune activation through the upregulation of hsa-miR-122-5p and early activation of angiogenesis. More specific investigations are needed to study the role of viral miRNAs in HIV pathogenesis.

BACKGROUND:Transmitted drug resistance (TDR) is still a critical aspect for the management of individuals living with HIV-1. Thus, its evaluation is crucial to optimize HIV care. METHODS:Overall, 2386 HIV-1 protease/reverse transcriptase and 1831 integrase sequences from drug-naïve individuals diagnosed in north and central Italy between 2015 and 2021 were analysed. TDR was evaluated over time. Phylogeny was generated by maximum likelihood. Factors associated with TDR were evaluated by logistic regression. RESULTS:Individuals were mainly male (79.1%) and Italian (56.2%), with a median (IQR) age of 38 (30-48). Non-B infected individuals accounted for 44.6% (N = 1065) of the overall population and increased over time (2015-2021, from 42.1% to 51.0%, P = 0.002). TDR prevalence to any class was 8.0% (B subtype 9.5% versus non-B subtypes 6.1%, P = 0.002) and remained almost constant over time. Overall, 300 transmission clusters (TCs) involving 1155 (48.4%) individuals were identified, with a similar proportion in B and non-infected individuals (49.7% versus 46.8%, P = 0.148). A similar prevalence of TDR among individuals in TCs and those out of TCs was found (8.2% versus 7.8%, P = 0.707).By multivariable analysis, subtypes A, F, and CFR02_AG were negatively associated with TDR. No other factors, including being part of TCs, were significantly associated with TDR. CONCLUSIONS:Between 2015 and 2021, TDR prevalence in Italy was 8% and remained almost stable over time. Resistant strains were found circulating regardless of being in TCs, but less likely in non-B subtypes. These results highlight the importance of a continuous surveillance of newly diagnosed individuals for evidence of TDR to inform clinical practice.

Signatures of neurodegeneration in clinical samples from a subject with multiple sclerosis (MS) acutely infected with HIV were investigated with single-cell transcriptomics using 10X Chromium technology. Sequencing was carried out on NovaSeq-TM, and the analysis was performed with Cell Ranger software (v 7.1.0) associated with a specifically established bioinformatic pipeline. A total of 1446 single-cell transcriptomes in cerebrospinal fluid (CSF) and 4647 in peripheral blood mononuclear cells (PBMCs) were obtained. In the CSF, many T-cell lymphocytes with an enriched amount of plasma cells and plasmacytoid dendritic (pDC) cells, as compared to the PBMCs, were detected. An unsupervised cluster analysis, putting together our patient transcriptomes with those of a publicly available MS scRNA-seq dataset, showed up-regulated microglial neurodegenerative gene expression in four clusters, two of which included our subject’s transcriptomes. A few HIV-1 transcripts were found only in the CD4 central memory T-cells of the CSF compartment, mapping to the gag-pol, vpu, and env regions. Our data, which describe the signs of neurodegenerative gene expression in a very peculiar clinical situation, did not distinguish the cause between multiple sclerosis and HIV infection, but they can give a glimpse of the high degree of resolution that may be obtained by the single-cell transcriptomic approach.

Torquetenovirus (TTV) is the most abundant component of the human blood virome and its replication is controlled by a functioning immune system. In this study, TTV replication was evaluated in 21 people with acute HIV infection (AHI) and immune reconstitution following antiretroviral therapy (ART). PBMC-associated TTV and HIV-1 DNA, as well as plasma HIV-1 RNA, were measured by real-time PCR. CD4 and CD8 differentiation, activation, exhaustion, and senescence phenotypes were analyzed by flow cytometry. Thirteen healthy donors (HD) and twenty-eight chronically infected HIV individuals (CHI), late presenters at diagnosis, were included as control groups. TTV replication in AHI seems to be controlled by the immune system being higher than in HD and lower than in CHI. During ART, a transient increase in TTV DNA levels was associated with a significant perturbation of activation and senescence markers on CD8 T cells. TTV loads were positively correlated with the expansion of CD8 effector memory and CD57+ cells. Our results shed light on the kinetics of TTV replication in the context of HIV acute infection and confirm that the virus replication is strongly regulated by the modulation of the immune system.