Mpox, caused by the Monkeypox virus (MPV), is a global public health threat. Virus isolation is the gold standard to confirm MPV infection, but this process can face many challenges. As an alternative, a new method was developed in in vitro settings using 50 µM of propidium monoazide (PMAxx, a DNA-binding agent) coupled with digital droplet PCR (ddPCR). Frozen clinical samples analyzed by PMAxx-ddPCR had a median of 0.8 copies/µL, while untreated samples had a median of 29.8 copies/µL. Since a substantial percentage of reduction was observed in these samples (>80%), it was verified whether this reduction could be due to the freezing process. This hypothesis was confirmed both in vitro and using clinical samples. A gradual increase in the mean percentage of reduction was observed after freezing–thawing cycles of MPV-isolate (59.5−81.4%). Moreover, a different percentage of reduction was observed before (68.2%) and after freezing (97.4%) the specimens, suggesting that the freezing process could reduce the number of complete viral particles. Our study shows strong evidence of the usefulness of PMAxx in clinical settings. PMAxx ensures the detection of intact MPV particles, which improves the accuracy of MPV load measurements. This method not only increases the reliability of MPV diagnosis but also overcomes virus isolation limitations.

Torquetenovirus (TTV) is a ubiquitous, non-pathogenic DNA virus that has been suggested as a biomarker of immune competence, with the viral load correlating with the level of immunosuppression. However, by detecting non-intact viral particles, standard PCR-based quantification may overestimate the TTV viremia. To improve the clinical relevance of TTV quantification, in this study, we investigated the use of PMAxx™, a virion viability dye that selectively blocks the amplification of compromised virions. Serum samples from 10 Hepatitis C Virus-positive (HCV+) individuals, 81 liver transplant recipients (LTRs), and 40 people with HIV (PWH) were treated with PMAxx™ and analyzed for TTV DNA loads by digital droplet PCR (ddPCR). Furthermore, anti-SARS-CoV-2 IgG levels and neutralizing antibody (nAbs) titers were measured post-COVID-19 vaccination. Using ddPCR, the PMAxx™ treatment significantly reduced the TTV DNA levels in all the groups (mean reduction: 0.66 Log copies/mL), indicating the abundant presence of non-intact, circulating viral genomes. However, correlations between TTV DNA and SARS-CoV-2 IgG or nAbs were weak or absent in both PMAxx™-treated and untreated samples. These findings suggest that while PMAxx™ enhanced the specificity of TTV quantification, it did not improve the predictive value of TTV viremia at assessing vaccine-induced humoral responses.

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.

Background: Torquetenovirus (TTV) viremia is increasingly recognized as a biomarker of host immune competence. We assessed the association between baseline TTV DNA levels and immune responses to the Mpox virus (MPXV) and dengue virus (DGV) vaccines in two prospective cohorts. Methods: A total of 248 individuals were enrolled, and TTV DNA was quantified before vaccination. Humoral and cellular responses to MVA-BN (for MPXV) and QDENGA (for DGV) vaccines were measured by using serology, neutralization assays, and interferon-γ ELISpot, and correlations with TTV viremia were investigated. Results: TTV DNA was detected in 81.2% of individuals, with a significantly higher prevalence and viral loads in the Mpox-Vac group than in the DGV-Vac group. Between both groups, the only significant association observed was an inverse correlation between pre-vaccination TTV load and DGV neutralizing antibody titers in the DGV-Vac group and was limited to the subset of TTV-positive individuals; no additional correlations with antibody and T responses were identified. For the Mpox-Vac group, stratified analyses in people living with HIV (PLWH) confirmed this lack of association. Conclusions: TTV viremia does not predict vaccine immunogenicity in immunocompetent or mildly immunosuppressed individuals. These results, which derive from within-cohort analyses and do not rely on direct comparisons between heterogeneous vaccine populations, support the role of TTV as a marker of immune status along a continuum of immunosuppression, with predictive value likely confined to populations with more severe immune impairment.