Rapid detection of SARS-CoV-2 with a mobile device based on pulse controlled amplification.

In the past, vast research has been conducted on biosensors and point-of-care (PoC) diagnostics. Despite rapid advances especially during the SARS-CoV-2 pandemic in this research field a low-cost molecular biosensor exhibiting the user-friendliness of a rapid antigen test, and also the sensitivity and specificity of a PCR test, has not been developed yet. To this end we developed a novel microfluidics based and handheld PoC device, that facilitates viral detection at PCR sensitivity and specificity in less than 40 min, including 15 min sample preparation. This was attained by incorporation of pulse controlled amplification (PCA), a method which uses short electrical pulses to rapidly increase the temperature of a small fraction of the sample volume. In this work, we present a low-cost PCA device with a microfluidic consumable intended for the use in a decentralized or home-setting. We used finite element analysis (FEA) simulations to display the fundamental principle and highlight the critical parameter dependency of PCA, such as pulse length and resistor shape. Furthermore, we integrated a simple and fast workflow for sample preparation and evaluated the limit of detection (LoD) for SARS-CoV-2 viral RNA, which is 0.88 copies/µL (=44 copies/reaction), and thus, comparable to conventional RT-qPCR. Additionally, target specificity of the device was validated. Our device and PCA approach enables cost-effective, rapid and mobile molecular diagnostics while remaining highly sensitive and specific.

Evaluation of Cross-Immunity to the Mpox Virus Due to Historic Smallpox Vaccination.

When the Mpox virus (MPXV) began spreading globally in 2022, it became critical to evaluate whether residual immunity from smallpox vaccination provided cross-protection. To assess the cross-immune response to MPXV, we collected serum samples (n = 97) and PBMCs (n = 30) from healthy-donors, either born before 1974 and reporting smallpox vaccination during childhood or born after 1975 and not vaccinated with Vaccinia virus (VACV)-based vaccines. We evaluated the levels of anti-MPXV IgG and neutralizing antibodies (Nabs) and the presence of a T cell response against MPXV. We found anti-MPXV IgG and Nabs in 60 (89.6%) and 40 (70.1%) vaccinated individuals, respectively. We observed a T cell response to Orthopoxviruses and MPXV peptide pools in 30% of vaccinated individuals. We thus show that a high proportion of subjects who received the smallpox vaccine 40 to 60 years ago have humoral cross-immunity, while the T-cell-specific response against MPXV was observed in a smaller group (30%) of vaccinated individuals. This study, combined with information on immunity developed during natural infection or the administration of current vaccines, will contribute to a better understanding of humoral and cellular responses against MPXV.

Serum anti-Spike immunoglobulin G levels in random blood donors in Italy: High-titre convalescent plasma is easier than ever to procure.

BACKGROUND AND OBJECTIVES: COVID-19 convalescent plasma (CCP) has retained potency and clinical efficacy against SARS-CoV-2 and is currently of utmost value for seronegative immunocompromised patients. Since most of the effect is due to the vaccine boost of infection-elicited antibodies, there is a theoretical concern that the frequency of suitable donors is declining. MATERIALS AND METHODS: In this single-institution serosurvey, we screened 599 consecutive donors attending our area in two different seasons (300 in November 2022 and 299 in February 2023) using the Abbott Alinity® anti-Spike immunoglobulin G assay. RESULTS: More than 80% of random donors qualify according to the FDA criteria for high-titre CCP (>4350 AU/mL), with a stable trend. CONCLUSION: Despite reduced anti-Spike vaccine boost deployment in the general population, we have shown here that high-titre CCP units are easier than ever to procure. This finding also has implications for the derivation of standard immunoglobulins, which are finally approaching the potency of hyperimmune serum and could soon represent an alternative to CCP.

Profiling the acute phase antibody response against mpox virus in patients infected during the 2022 outbreak.

Information on the immune response during the mpox virus (MPXV) infection is still scarce or limited to past studies when cross-reactive immunity from smallpox vaccination was predominant. Here, we describe the short-term kinetics of the antibody response in patients with acute MPXV infection during the 2022 multicountry outbreak. A total of 64 samples from 18 MPXV-positive patients were longitudinally collected from the day of symptom onset (DSO) up to 20 days after and tested for anti-MPXV immunoglobulin G (IgG), IgM, IgA, and neutralizing antibodies (nAb) using the whole-live virus isolated in May 2022. IgG, IgM, and IgA were detected as early as 4 DSO (median time of seroconversion 7.5 DSO for IgG, 8 DSO for IgM and IgA). Anti-MPXV nAb were detectable in samples collected as early as 1 week after symptoms, with stable levels up to 20 DSO. After 2 weeks, IgG and nAb reached high titers. No significant differences were observed regardless of status of smallpox vaccination, human immunodeficiency virus positivity, or disease severity. Significant lower levels of IgM and IgG were observed in the patients treated with antivirals. These results contribute to extending the knowledge of the MPXV infection and the antibody response in a population with no historic smallpox vaccination.

Simple and Reliable Method to Quantify the Hepatitis B Viral Load and Replicative Capacity in Liver Tissue and Blood Leukocytes.

BACKGROUND: A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients. OBJECTIVES: The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples. PATIENTS AND METHODS: Clinical samples from a cohort of CHB patients, including liver biopsies in some, were collected for the analysis of intracellular HBV molecular markers using novel molecular assays. RESULTS: A plasmid construct, including sequences from the HBV genome and from the human gene hTERT, was generated as an isomolar multi-standard for HBV quantitation and normalization to the cellular contents. The specificity of the real-time assay for the cccDNA was assessed using Dane particles isolated on a density gradient. A comparison of liver tissue from 6 untreated and 6 treated patients showed that the treatment deeply reduced the replicative capacity (total DNA/cccDNA), but had limited impact on the parenchymal colonization. The peripheral blood mononuclear cells (PBMCs) and granulocytes from the treated and untreated patients were also analyzed. CONCLUSIONS: A straightforward method for the quantification of intracellular HBV molecular parameters in clinical samples was developed and validated. The widespread use of such versatile assays could better define the prognosis of CHB, and allow a more rational approach to time-limited tailored treatment strategies.

a statistical model based on serological parameters for predicting occult HBV infection: implications for organ/ blood donations.

The transmission of hepatitis B virus by donors with occult HBV infection (OBI) is a threat for blood transfusion and organ/tissue transplantation. The risk of carrying HBV DNA is currently not predictable by simple serologic markers, while HBV DNA testing is not universally deployed. This study evaluated an integrated serologic approach for assessing this risk. Anti-HBc positive subjects (461 HIV-negative, 262 HIV-positive) were selected for the study. Serology was analyzed by a commercial CMIA technique. HBV DNA was analyzed by both commercial and home-brew real-time amplification assays. A penalized maximum likelihood logistic approach was used to analyze the data. In HBsAg-negative subjects (HIV-negative), anti-HBc signal/cut off values, the presence of anti-HBc IgM, the absence of anti-HBsAg, and the absence of anti-HCV were correlated to the probability of finding circulating HBV DNA. A model for predicting HBV DNA presence by 4 serological parameters is therefore proposed. The predictive value of the logistic model based on simple serologic markers may represent a reasonable tool for the assessment of HBV transmission risk by transfusion or organ/tissue donation in the context of limited resources and where nucleic acid testing is not performed. In addition, it may be helpful for assessing the risk of reactivation in immunosuppressed OBI patients.

Improving clinical laboratory efficiency: a time-motion evaluation of the Abbott m2000 RealTime and Roche COBAS AmpliPrep/COBAS TaqMan PCR systems for the simultaneous quantitation of HIV-1 RNA and HCV RNA.

BACKGROUND: Diagnostic laboratories need automation that facilitates efficient processing and workflow management to meet today's challenges for expanding services and reducing cost, yet maintaining the highest levels of quality. METHODS: Processing efficiency of two commercially available automated systems for quantifying HIV-1 and HCV RNA, Abbott m2000 system and Roche COBAS Ampliprep/COBAS TaqMan 96 (docked) systems (CAP/CTM), was evaluated in a mid/high throughput workflow laboratory using a representative daily workload of 24 HCV and 72 HIV samples. Three test scenarios were evaluated: A) one run with four batches on the CAP/CTM system, B) two runs on the Abbott m2000 and C) one run using the Abbott m2000 maxCycle feature (maxCycle) for co-processing these assays. Cycle times for processing, throughput and hands-on time were evaluated. RESULTS: Overall processing cycle time was 10.3, 9.1 and 7.6 h for Scenarios A), B) and C), respectively. Total hands-on time for each scenario was, in order, 100.0 (A), 90.3 (B) and 61.4 min (C). CONCLUSIONS: The interface of an automated analyzer to the laboratory workflow, notably system set up for samples and reagents and clean up functions, are as important as the automation capability of the analyzer for the overall impact to processing efficiency and operator hands-on time.

Snail controls differentiation of hepatocytes by repressing HNF4alpha expression.

Epithelial-to-mesenchymal transition (EMT) is a coordinated process, occurring both during morphogenesis and tumor progression, that allows epithelial cells to dissociate from initial contacts and migrate to secondary sites. The transcriptional repressors of the Snail family induce EMT in different epithelial cell lines and their expression is strictly correlated with EMT during the development and progression of carcinomas. We have previously shown that EMT in hepatocytes correlates with the downregulation of hepatic differentiation key factors HNFs (hepatocyte nuclear factors), and in particular of HNF4alpha. Here, we demonstrate that Snail overexpression is sufficient (i) to induce EMT in hepatocytes with conversion of morphology, downregulation of several epithelial adhesion molecules, reduction of proliferation and induction of matrix metalloproteinase 2 expression and, (ii) most relevantly, to repress the transcription of the HNF4alpha gene through a direct binding to its promoter. These finding demonstrate that Snail is at the crossroads of the regulation of EMT in hepatocytes by a dual control of epithelial morphogenesis and differentiation.

Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis.

Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.