Federica Forbici

HIV-1 proviral integration into the human genome dictates viral and host gene expression, as well as HIV reservoir persistence. This study analyzed insertion site (IS) landscapes in three groups of people with HIV (PWH): acute (AHI) naïve (n = 8), chronic (CHI) naïve (n = 12), and CHI on long-term antiretroviral therapy (cART, n = 12). Host DNA was digested with restriction enzymes, ligated to a linker, and amplified by a PCR targeting both the linker and the HIV LTR. Amplicons were sequenced on the Ion Torrent S5 system. A bioinformatics pipeline was designed to detect ISs on the basis of a set of pre-defined parameters. Of all the 388 different ISs obtained, 58.5% were in gene regions. The highest number of ISs was observed in the CHI naïve group, followed by CHI on cART and AHI, showing a statistically significant difference in the distributions. IS patterns varied among groups, eg., chr8:102,815,760 was absent in the AHI group but was found in 92% of the CHI naïve group, reaching 100% in CHI on cART subjects. Although proving causality remains complex, these findings add new data to the literature on this topic, suggesting that active/passive mechanisms for specific IS enrichment may operate during HIV infection and antiretroviral therapy.

The mitigation measures adopted to reduce the spread of COVID-19 have not only impacted the transmission of respiratory infections but also other infectious diseases. In this study, we explored the effect of the pandemic on the epidemiology of Central Nervous System (CNS) infections by conducting a retrospective analysis of diagnostic data obtained from cerebrospinal fluids (CSFs) collected before, during, and after the pandemic from patients with acute-suspected neuro-infectious syndrome. The samples were analyzed using a meningitis/encephalitis molecular syndromic panel. A total of 4203 CSFs were split into three groups: pre-pandemic (August 2018–February 2020), pandemic (March 2020–March 2022) and post-pandemic (April 2022–March 2024). Each pathogen was statistically assessed individually, comparing one group vs. another. Results showed that viral pathogens were the most frequently detected across all groups; however, Streptococcus pneumoniae was the single most identified pathogen. Comparison of the pre-pandemic and pandemic groups by statistical analysis showed a significant reduction in neuro-infections caused by enterovirus, S. pneumoniae and N. meningitidis, while the other pathogens were not affected. In conclusion, a reduction in CNS infections caused by respiratory and close-contact-transmitted pathogens, including both viral and bacterial agents, was observed during the period when COVID-19 containment measures were in effect.

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.

We report the case of a 48-year-old man admitted with severe pneumonia, profound immunosuppression and multiple co-infections, showing unusual pattern of HIV-1 viremia. With the Aptima HIV-1 Quant Dx assay, a dual-target diagnostic assay for monitoring of viral RNA in people living with HIV-1 (PWH), the patient showed viral RNA consistently detected exclusively through the pol target, while the LTR signal remained absent in all samples during one year of follow-up on antiretroviral therapy. Despite this persistent atypical viral load (pol+/LTR-), near full-length next-generation sequencing of HIV-1 RNA confirmed an almost intact viral genome, including the LTR region and no resistance-associated mutations. Several mechanisms may account for explaining the persistent lack of LTR detection, such as defective quasi-species, RNA structural rearrangements, or epi-transcriptomic modifications interfering with primers annealing, and further studies are currently underway to clarify the biological origins and the clinical implications of the detection patterns of atypical HIV-1 RNA. The case described here is an example of a few PWH undergoing antiretroviral therapy who demonstrated single-target HIV-1 viremia with the Aptima dual-target assay. These particular clinical situations with single-target viremia, even at high levels of HIV-1 RNA, should be carefully considered in clinical management as they would indicate the presence of atypical viral RNA, for which, in some cases, switching antiretroviral therapy could be not necessary.

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.

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.

Enteroviruses (EVs) are ubiquitous viruses that circulate worldwide, causing sporadic or epidemic infections, typically during the summer and fall. They cause a broad spectrum of illnesses, ranging from an unspecified febrile clinical presentation to a severe illness. EVs are recognized to be the most frequent etiological agents of aseptic meningitis in children. However, as the infection is usually mild and self-limiting, it remains underestimated, and the epidemiology of EVs is poorly understood. To date, no vaccine or effective therapy for all types of enteroviruses is available, and EVs constitute a public health concern. Here, we investigated the molecular epidemiology of EV strains circulating in the Lazio region over a 10-year time span (2012–2023) by using a sequence-typing approach and phylogenetic analysis. The epidemiological trend of EV infection has undergone changes during the SARS-CoV-2 pandemic (2020–2021), which resulted in a modification in terms of the number of diagnosed cases and seasonality. From 2022, the circulation of EVs showed a behavior typical of the pre-pandemic period, although changes in predominantly circulating strains have been noted. Both epidemic and sporadic circulation events have been characterized in the Lazio region. Further analyses are needed to better characterize any strain with higher potential pathogenic power and to identify possible recombinant strains.

Progressive Multifocal Leukoencephalopathy (PML) is a demyelinating disease occurring in advanced HIV infection, caused by the reactivation of poliomavirus JC (JCV). The use of pembrolizumab for treatment is based on the inhibition of programmed cell death protein 1 (PD-1), potentially improving the anti JCV-specific response. We used pembrolizumab with combined antiretroviral treatment (cART) on a compassionate-use basis. At each administration, clinical evaluation, MRI and laboratory testing, including CD3, CD4, CD8, PD-1 markers, HIV-RNA and JCV-DNA in cerebrospinal fluid (CSF)/plasma pairs, were performed. The JCV-specific T cell response was analysed by Elispot assay. This study included five HIV patients: four male, median age 43 years (29–52), median CD4 and CD8 count 150 (15–158) and 973 (354–1250) cell/mm³, respectively; median JCV-DNA and HIV-RNA in CSF/plasma pairs 9.540/1.503 cps/mL and 2.230/619 cp/mL, respectively. Overall, patients received between two and seven doses of pembrolizumab. After treatment, we observed JCV-DNA reduction and PD-1 down-regulation both in CSF and in plasma (high in circulating CD4 and CD8 at baseline), which remained stable at low levels in all patients. Three out of five patients showed stability of clinical picture and neuroimaging, while two others died. More data are needed in order to identify predictors of response to therapy.

The optimal therapeutic approach for primary HIV infection (PHI) is still debated. We aimed to compare the viroimmunological response to a four- versus a three-drug regimen, both INSTI-based, in patients with PHI. This was a monocentric, prospective, observational study including all patients diagnosed with PHI from December 2014 to April 2018. Antiretroviral therapy (ART) was started, before genotype resistance test results, with tenofovir/emtricitabine and either raltegravir plus boosted darunavir or dolutegravir. Cumulative probability of virological suppression [VS] (HIV-1 RNA< 40 cp/mL), low-level HIV-1 DNA [LL-HIVDNA] (HIV-1 DNA < 200 copies/10⁶PBMC), and CD4/CD8 ratio ≥1 were estimated using Kaplan–Meier curves. Factors associated with the achievement of VS, LL-HIVDNA, and CD4/CD8 ≥ 1 were assessed by a Cox regression model. We enrolled 144 patients (95.8% male, median age 34 years): 110 (76%) started a four-drug-based therapy, and 34 (24%) a three-drug regimen. Both treatment groups showed a comparable high probability of achieving VS and a similar probability of reaching LL-HIVDNA and a CD4/CD8 ratio ≥1 after 48 weeks from ART initiation. Higher baseline HIV-1 RNA and HIV-1 DNA levels lowered the chance of VS, whereas a better preserved immunocompetence increased that chance. Not statistically significant factors associated with LL-HIVDNA achievement were found, whereas a higher baseline CD4/CD8 ratio predicted the achievement of immune recovery. In PHI patients, the rapid initiation of either an intensified four-drug or a standard three-drug INSTI-based regimen showed comparable responses in terms of VS, viral reservoir size, and immunological recovery.