Torquetenovirus Viremia Quantification Using Real-Time PCR Developed on a Fully Automated, Random-Access Platform.

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.

The Emerging Tool of the Human Anellovirome.

The blood virome is dominated by the Anelloviridae family, which emerges early in life; the anellome, which represents the variety of anelloviruses within an individual, stabilizes by adulthood [...].

MPXV DNA kinetics in bloodstream and other body fluids samples.

Since spring 2022, the global epidemiology of the monkeypox virus (MPXV) has changed. The unprecedented increase of human clade II MPXV cases worldwide heightened concerns about this emerging zoonotic disease. We analysed the positivity rates, viral loads, infectiousness, and persistence of MPXV DNA for up to 4 months in several biological samples from 89 MPXV-confirmed cases. Our data showed that viral loads and positivity rates were higher during the first two weeks of symptoms for all sample types. Amongst no-skin-samples, respiratory specimens showed higher MPXV DNA levels and median time until viral clearance, suggesting their usefulness in supporting MPXV diagnosis, investigating asymptomatic patients, and monitoring viral shedding. Infectious virus was cultured from respiratory samples, semen, and stools, with high viral loads and collected within the first 10 days. Notably, only one saliva and one semen were found positive for viral DNA after 71 and 31 days from symptoms, respectively. The focus on bloodstream samples showed the best testing sensitivity in plasma, reporting the overall highest MPXV DNA detection rate and viral loads during the 3-week follow-up as compared to serum and whole-blood. The data here presented can be useful for MPXV diagnostics and a better understanding of the potential alternative routes of its onward transmission.

Effect of Cigarette Smoking on Clinical and Molecular Endpoints in COPD Patients.

Cigarette smoking is a primary contributor to mortality risks and is associated with various diseases. Among these, COPD represents a significant contributor to global mortality and disability. The objective of this study is to investigate the effect of smoking on a selected battery of variables, with an emphasis on DNA damage. A total of 87 elderly patients diagnosed with COPD, divided into three groups based on their smoking history (current, former, never-smokers), were evaluated using a cross-sectional approach. Clinical features including mortality and inflammatory/oxidative parameters (Lymphocytes/Monocytes, Neutrophils/Lymphocytes, Platelets/Lymphocytes ratio), SII, MDA, 8-Oxo-dG, and IL6 (ELISA assay), as well as DNA damage (comet assay), were investigated. Virus infection, i.e., influenza A virus subtype H1N1, JC polyomavirus (JCPyV), BK polyomavirus (BKPyV), and Torquetenovirus (TTV), was also tested. Current smokers exhibit higher levels of comorbidity (CIRS; p < 0.001), Platelets/Lymphocytes ratio (p < 0.001), systemic immune inflammation (p < 0.05), and DNA damage (p < 0.001). Former smokers also showed higher values for parameters associated with oxidative damage and showed a much lower probability of surviving over 5 years compared to never- and current smokers (p < 0.0017). This study showed a clear interaction between events which are relevant to the oxidative pathway and cigarette smoking. A category of particular interest is represented by former smokers, especially for lower survival, possibly due to the presence of more health problems. Our findings raise also the attention to other parameters which are significantly affected by smoking and are useful to monitor COPD patients starting a program of pulmonary rehabilitation (DNA damage, inflammation parameters, and selected viral infections).

Rapid Determination of SARS-CoV-2 Integrity and Infectivity by Using Propidium Monoazide Coupled with Digital Droplet PCR.

SARS-CoV-2 is a highly infectious virus responsible for the COVID-19 pandemic. Therefore, it is important to assess the risk of SARS-CoV-2 infection, especially in persistently positive patients. Rapid discrimination between infectious and non-infectious viruses aids in determining whether prevention, control, and treatment measures are necessary. For this purpose, a method was developed and utilized involving a pre-treatment with 50 µM of propidium monoazide (PMAxx, a DNA intercalant) combined with a digital droplet PCR (ddPCR). The ddPCR method was performed on 40 nasopharyngeal swabs (NPSs) both before and after treatment with PMAxx, revealing a reduction in the viral load at a mean of 0.9 Log copies/mL (SD ± 0.6 Log copies/mL). Furthermore, six samples were stratified based on the Ct values of SARS-CoV-2 RNA (Ct < 20, 20 < Ct < 30, Ct > 30) and analyzed to compare the results obtained via a ddPCR with viral isolation and a negative-chain PCR. Of the five samples found positive via a ddPCR after the PMAxx treatment, two of the samples showed the highest post-treatment SARS-CoV-2 loads. The virus was isolated in vitro from both samples and the negative strand chains were detected. In three NPS samples, SARS CoV-2 was present post-treatment at a low level; it was not isolated in vitro, and, when detected, the strand was negative. Our results indicate that the established method is useful for determining whether the SARS-CoV-2 within positive NPS samples is intact and capable of causing infection.

COVID-19 therapeutics.

SUMMARYSince the emergence of COVID-19 in 2020, an unprecedented range of therapeutic options has been studied and deployed. Healthcare providers have multiple treatment approaches to choose from, but efficacy of those approaches often remains controversial or compromised by viral evolution. Uncertainties still persist regarding the best therapies for high-risk patients, and the drug pipeline is suffering fatigue and shortage of funding. In this article, we review the antiviral activity, mechanism of action, pharmacokinetics, and safety of COVID-19 antiviral therapies. Additionally, we summarize the evidence from randomized controlled trials on efficacy and safety of the various COVID-19 antivirals and discuss unmet needs which should be addressed.

HIV-1 RNA monitoring with a dual-target diagnostic assay: A case report.

In a restricted subset of people living with HIV-1 (PLWH) on antiretroviral therapy (ART) with persistent suppressed viral load (i.e., pol-based HIV-RNA repeatedly undetected), a dual-target (pol and LTR) diagnostic assay for HIV-RNA monitoring can measure quantifiable levels of viral loads (VL) above 30 copies/mL exclusively through the amplification of the LTR region, while the pol target results undetected. We report a patient who shows high levels of HIV-RNA detected exclusively through amplification of the LTR region while undetected by the pol region, during a long monitoring period, from 2018 to date. In this follow-up, the ART was modified without reaching LTR-based undetected HIV-RNA values. Immunological and virological parameters remained optimal with a progressive and steady gain of the CD4/CD8 ratio. The clinical history of this patient, shows that LTR-based viremia above 50 copies/mL can be found occasionally or persistently in the plasma of PLWH under suppressive ART, even at high levels. Based on previous studies, VL detected and quantified exclusively through the amplification of the LTR region corresponds to partial or incomplete HIV-RNA transcripts, which cannot trigger new infections. Interestingly, changes in ART do not eliminate repeated findings of these unusual viral elements.

Online dashboards for SARS-CoV-2 wastewater-based epidemiology.

Aim: Wastewater-based epidemiology (WBE) is increasingly used to monitor pandemics. In this manuscript, we review methods and limitations of WBE, as well as their online dashboards. Materials & methods: Online dashboards were retrieved using PubMed and search engines, and annotated for timeliness, availability of English version, details on SARS-CoV-2 sublineages, normalization by population and PPMoV load, availability of case/hospitalization count charts and of raw data for export. Results: We retrieved 51 web portals, half of them from Europe. Africa is represented from South Africa only, and only seven portals are available from Asia. Conclusion: WBS provides near-real-time cost-effective monitoring of analytes across space and time in populations. However, tremendous heterogeneity still persists in the SARS-CoV-2 WBE literature.

Monitoring the amount of a virus in the sewage system provides a way to work out the circulation of the virus among a population at a given time. Standard procedures are needed to produce data that can be compared across countries. Timely sharing of wastewater surveillance data across publicly accessible web portals is important to inform researchers and plan public health policies. This study shows that we are still far away from standardization and timely and transparent reporting.

Molnupiravir increases SARS-CoV-2 genome diversity and complexity: A case-control cohort study.

Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed during the COVID-19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS-CoV-2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS-CoV-2 whole-genome sequencing on 38 molnupiravir-treated persistently positive COVID-19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab-treated outpatients served as controls. Mutational analyses confirmed that SARS-CoV-2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G->A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies.

Amyotrophic lateral sclerosis stratification: unveiling patterns with virome, inflammation, and metabolism molecules.

Amyotrophic lateral sclerosis (ALS) is an untreatable and clinically heterogeneous condition primarily affecting motor neurons. The ongoing quest for reliable biomarkers that mirror the disease status and progression has led to investigations that extend beyond motor neurons' pathology, encompassing broader systemic factors such as metabolism, immunity, and the microbiome. Our study contributes to this effort by examining the potential role of microbiome-related components, including viral elements, such as torque tenovirus (TTV), and various inflammatory factors, in ALS. In our analysis of serum samples from 100 ALS patients and 34 healthy controls (HC), we evaluated 14 cytokines, TTV DNA load, and 18 free fatty acids (FFA). We found that the evaluated variables are effective in differentiating ALS patients from healthy controls. In addition, our research identifies four unique patient clusters, each characterized by distinct biological profiles. Intriguingly, no correlations were found with site of onset, sex, progression rate, phenotype, or C9ORF72 expansion. A remarkable aspect of our findings is the discovery of a gender-specific relationship between levels of 2-ethylhexanoic acid and patient survival. In addition to contributing to the growing body of evidence suggesting altered peripheral immune responses in ALS, our exploratory research underscores metabolic diversity challenging conventional clinical classifications. If our exploratory findings are validated by further research, they could significantly impact disease understanding and patient care customization. Identifying groups based on biological profiles might aid in clustering patients with varying responses to treatments.

An update on the anti-spike monoclonal antibody pipeline for SARS-CoV-2.

BACKGROUND: Anti-spike monoclonal antibodies represent one of the most tolerable prophylaxis and therapies for COVID-19 in frail and immunocompromised patients. Unfortunately, viral evolution in Omicron has led all of them to failure. OBJECTIVES: We review here the current pipeline of anti-spike mAb's, discussing in detail the most promising candidates. SOURCES: We scanned PubMed, ClinicalTrials.gov and manufacturers' press releases for clinical studies on anti-spike monoclonal antibodies. CONTENT: We present state-of-art data clinical progress for AstraZeneca's AZD3152, Invivyd's VYD222, Regeneron's REGN-17092 and Aerium Therapeutics' AER-800. IMPLICATIONS: The anti-spike monoclonal antibody clinical pipeline is currently limited to few agents (most being single antibodies) with unknown efficacy against the dominant JN.1 sublineage. The field of antibody-based therapies requires boosting by both manufacturers and institutions.

Passive immunotherapies for the next influenza pandemic.

Influenzavirus is among the most relevant candidates for a next pandemic. We review here the phylogeny of former influenza pandemics, and discuss candidate lineages. After briefly reviewing the other existing antiviral options, we discuss in detail the evidences supporting the efficacy of passive immunotherapies against influenzavirus, with a focus on convalescent plasma.

Poor durability of the neutralizing response against XBB sublineages after a bivalent mRNA COVID-19 booster dose in persons with HIV.

We estimated the dynamics of the neutralizing response against XBB sublineages and T cell response in persons with HIV (PWH) with previous AIDS and/or CD4 < 200/mm3 receiving the bivalent original strain/BA.4-5 booster dose in fall 2022. Samples were collected before the shot (Day 0), 15 days, 3, and 6 months after. PWH were stratified by immunization status: hybrid immunity (HI; vaccination plus COVID-19) versus nonhybrid immunity (nHI; vaccination only). Fifteen days after the booster, 16% and 30% of PWH were nonresponders in terms of anti-XBB.1.16 or anti-EG.5.1 nAbs, respectively. Three months after, a significant waning of anti-XBB.1.16, EG.5.1 and -XBB.1 nAbs was observed both in HI and nHI but nAbs in HI were higher than in nHI. Six months after both HI and nHI individuals displayed low mean levels of anti-XBB.1.16 and EG.5.1 nAbs. Regarding T cell response, IFN-γ values were stable over time and similar in HI and nHI. Our data showed that in PWH, during the prevalent circulation of the XBB.1.16, EG.5.1, and other XBB sublineages, a mRNA bivalent vaccine might not confer broad protection against them. With a view to the 2023/2024 vaccination campaign, the use of the monovalent XBB.1.5 mRNA vaccine should be urgently warranted in PWH to provide adequate protection.

Human and Viral microRNA Expression in Acute and Chronic HIV Infections.

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.

B-cell-depleted patients with persistent SARS-CoV-2 infection: combination therapy or monotherapy? A real-world experience.

Objectives: The aim of the study was to describe a cohort of B-cell-depleted immunocompromised (IC) patients with prolonged or relapsing COVID-19 treated with monotherapy or combination therapy. Methods: This is a multicenter observational retrospective study conducted on IC patients consecutively hospitalized with a prolonged or relapsing SARS-CoV-2 infection from November 2020 to January 2023. IC COVID-19 subjects were stratified according to the monotherapy or combination anti-SARS-CoV-2 therapy received. Results: Eighty-eight patients were enrolled, 19 under monotherapy and 69 under combination therapy. The study population had a history of immunosuppression (median of 2 B-cells/mm3, IQR 1-24 cells), and residual hypogammaglobulinemia was observed in 55 patients. A reduced length of hospitalization and time to negative SARS-CoV-2 molecular nasopharyngeal swab (NPS) in the combination versus monotherapy group was observed. In the univariable and multivariable analyses, the percentage change in the rate of days to NPS negativity showed a significant reduction in patients receiving combination therapy compared to those receiving monotherapy. Conclusion: In IC persistent COVID-19 patients, it is essential to explore new therapeutic strategies such as combination multi-target therapy (antiviral or double antiviral plus antibody-based therapies) to avoid persistent viral shedding and/or severe SARS-CoV-2 infection.

Rift Valley Fever Virus: An Overview of the Current Status of Diagnostics.

Rift Valley fever is a vector-borne zoonotic disease caused by the Rift Valley fever virus (Phlebovirus genus) listed among the eight pathogens included in the Bluepoint list by the WHO. The transmission is mainly vehicled by Aedes and Culex mosquito species. Symptoms of the disease are varied and non-specific, making clinical diagnosis often challenging, especially in the early stages. Due to the difficulty in distinguishing Rift Valley fever from other viral hemorrhagic fevers, as well as many other diseases that cause fever, an early diagnosis of the infection is important to limit its spread and to provide appropriate care to patients. To date, there is no validated point-of-care diagnostic tool. The virus can only be detected in the blood for a brief period, suggesting that molecular methods alone are not sufficient for case determination. For this, it is preferable to combine both molecular and serological tests. The wide distribution of competent vectors in non-endemic areas, together with global climate change, elicit the spread of RVFV to continents other than Africa, making surveillance activities vital to prevent or to limit the impact of human outbreaks and for a rapid identification of positive cases, making diagnosis a key factor for this achievement.

Concomitant Syndromic Diagnosis of Mpox and Other Vesicular Viruses in Patients with Skin and Genital Lesions.

The recent multi-country outbreak of the zoonotic monkeypox virus (MPXV) infection in humans without an epidemiological link with endemic areas has raised concerns about the route of transmission. Since the infection spread largely among men who have sex with men who, in most cases, presented primary lesions of the genital and oral mucosa, sexual transmission has been proposed. In the present study, we retrospectively evaluated specimens of vesicular lesions collected from the skin and genital tract of 35 patients (23 positive and 12 negative) presenting at our Institute for monkeypox (mpox) diagnosis by using a novel molecular syndromic vesicular virus panel (VVP) assay. All MPXV-positive samples but one was confirmed; however, the viral syndromic analysis revealed that 8.6% of them were coinfected with one or more viruses, and 17% had at least a virus different from the MPXV. The percentage of coinfections increased to more than 25% when nonviral pathogens, such as gonorrhea and syphilis, were also considered. These results show the usefulness of syndromic diagnosis in cases where MPXV is suspected (and vice versa) and at the same time highlight that the broader screening of sexually transmitted infections in the population with high-risk sexual behavior is critical to ensure a complete etiology and appropriate treatment.