Quantification of Torquetenovirus (TTV) viremia is becoming important for evaluating the status of the immune system in solid organ transplant recipients, monitoring the appearance of post-transplant complications, and controlling the efficacy of maintenance immunosuppressive therapy. Thus, diagnostic approaches able to scale up TTV quantification are needed. Here, we report on the development and validation of a real-time PCR assay for TTV quantification on the Hologic Panther Fusion^® System by utilizing its open-access channel. The manual real-time PCR previously developed in our laboratories was optimized to detect TTV DNA on the Hologic Panther Fusion^® System. The assay was validated using clinical samples. The automated TTV assay has a limit of detection of 1.6 log copies per ml of serum. Using 112 samples previously tested via manual real-time PCR, the concordance in TTV detection was 93% between the assays. When the TTV levels were compared, the overall agreement between the methods, as assessed using Passing–Bablok linear regression and Bland–Altman analyses, was excellent. In summary, we validated a highly sensitive and accurate method for the diagnostic use of TTV quantification on a fully automated Hologic Panther Fusion^® System. This will greatly improve the turnaround time for TTV testing and better support the laboratory diagnosis of this new viral biomarker.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by fast evolution with the appearance of several variants. Next-Generation Sequencing (NGS) technology is considered the gold standard for monitoring known and new SARS-CoV-2 variants. However, the complexity of this technology renders this approach impracticable in laboratories located in areas with limited resources. We analyzed the capability of the ThermoFisher TaqPath COVID-19 RT-PCR (TaqPath) and the Seegene Novaplex SARS-CoV-2 Variant assay (Novaplex) to detect Omicron variants; the Allplex VariantII (Allplex) was also evaluated for Delta variants. Sanger sequencing (SaS) was the reference method. The results obtained with n = 355 nasopharyngeal samples were: negative with TaqPath, although positive with other qualitative molecular assays (n = 35); undetermined (n = 40) with both the assays; negative for the ∆69/70 mutation and confirmed as the Delta variant via SaS (n = 100); positive for ∆69/70 and confirmed as Omicron BA.1 via SaS (n = 80); negative for ∆69/70 and typed as Omicron BA.2 via SaS (n = 80). Novaplex typed 27.5% of samples as undetermined with TaqPath, 11.4% of samples as negative with TaqPath, and confirmed 100% of samples were Omicron subtypes. In total, 99/100 samples were confirmed as the Delta variant with Allplex with a positive per cent agreement (PPA) of 98% compared to SaS. As undermined samples with Novaplex showed RdRp median Ct values (Ct = 35.4) statistically higher than those of typed samples (median Ct value = 22.0; p < 0.0001, Mann–Whitney test), the inability to establish SARS-CoV-2 variants was probably linked to the low viral load. No amplification was obtained with SaS among all 35 negative TaqPath samples. Overall, 20% of samples which were typed as negative or undetermined with TaqPath, and among them, twelve were not typed even by SaS, but they were instead correctly identified with Novaplex. Although full-genome sequencing remains the elected method to characterize new strains, our data show the high ability of a SNP-based assay to identify VOCs, also resolving samples typed as undetermined with TaqPath.

The definitive diagnosis of the Rift Valley fever virus (RVFV) requires a form of testing that is available only in reference laboratories. It includes indirect immunofluorescence assay (IFA), the serum neutralization assay (NA), and real-time PCR. Therefore, often, no attempts are made to detect it, even among travelers from endemic areas. In this study, the presence of anti-RVFV IgG and IgM was retrospectively screened in stored serum samples from people who were admitted with arbovirus symptoms at the National Institute for Infectious Diseases (INMI) L. Spallanzani, Rome, Italy. Overall, 80 residual serum samples were anonymized, and sub-aliquots were prepared and tested for anti-RVFV IgG and IgM. A serum neutralization assay was used as a confirmatory test. There was a positive result in eight out of 80 samples (10%) for anti-RVFV IgG, with titers ranging from 1:40 up to 1:1280. Three of eight (2.6%) samples were confirmed as seropositive through an in-house serum neutralization assay, with antibody titers ranging from 1:10 to 1:160. All samples resulted negative for anti-RVFV IgM and RVFV RNA when tested by IFA and real-time RT-PCR, respectively. Our data highlight that several RVFV infections can possibly escape routine virological diagnosis, which suggests RVFV testing should be set up in order to monitor virus prevalence.

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.

Rift Valley Fever virus (RVFV) is a mosquito-borne virus with high pathogenic potential in ruminants and humans. Due to its high potential for spreading, it is considered a priority pathogen, and it is included in the Bluepoint list of the World Health Organization (WHO). Given the high pathogenic potential of the virus, it is crucial to develop a rapid heat-mediated inactivation protocol to create a safer working environment, particularly in medical facilities that lack a biosafety level 3 laboratory required for direct handling of RVFV. Our results reveal the broad tissue tropism of RVFV, showing the virus’s capacity for replication in various cell lines. In terms of the thermal stability of RVFV, our findings showed that a 70 °C heat treatment did not fully inactivate the virus within 15 min. However, when exposed to 80 °C and 95 °C, the virus was completely inactivated after 15 min and 5 min, respectively. Additionally, our results indicated that heat-treatment only slightly decreased the integrity of the RVFV genome whether there is a high or low number of viral RNA copies. Overall, the study established a straightforward protocol for heat inactivation that may be beneficial in handling clinical and research samples of RVFV.