Log10 Copies Research Articles

P-442. Quantification of TTV in Saliva and Plasma of People Living With HIV

Abstract Background Torque tenovirus (TTV) viral load in blood is now accepted as a marker of immunocompetence in immunocompromised patients such as transplantation recipients and people living with HIV (PLWH). TTV has been shown to predominantly infect T cells, which are vital for the control of TTV. In PLWH the level of TTV has been shown to correlate with the CD4 count. Recently, TTV has also been detected in saliva of PLWH. However, at this time it is not known if salivary TTV can be used as a non-invasive alternative to blood TTV, or as an alternative to a CD4 count. In this study, we explore the relationship between TTV in blood and in saliva with T cell subsets, including CD4+ T cells, to determine the clinical potential for this novel marker in PLWH. Methods 80 PLWH visiting the outpatient clinic of our center were recruited. Inclusion criteria were age ≥ 18 years, use of combination antiretroviral therapy for ≥ 6 months and < 40 copies/mL in the last two viral load measurements. The CE-marked TTV R-Gene® kit (bioMérieux, Lyon, France) was used to detect and quantify TTV in plasma and saliva. Results are expressed in log copies/ml, with a cut off of 2.4 log copies. Absolute numbers of CD4+ T cells were measured using the MultiTest TruCount method (Becton Dickinson). HIV viral load was performed on EDTA plasma samples using the Abbott Real-Time HIV-1 assay. Plasma TTV correlates with salivary TTV, R2 0.89, p<0.01. Results Individuals were predominantly male (86%), had a mean age of 51. The mean CD4+ count was 670 (447-844) and 25% had a low detectable HIV load (Table 1). TTV was detectable in 24% of plasma and 20% of saliva samples with an average of 3.86 log copies/ml and 4.01 log copies/ml respectively. Plasma TTV and saliva TTV correlate well (R2 0.89, p< 0.01) (figure 1). There is no relationship between plasma TTV (R2 0.004, p = 0.58), (figure 2) or saliva TTV and the CD4+ count (R2 0.004, p = 0.58) (figure 3). Plasma TTV does not correlate with CD4 count, R2 0.004, p = 0.58. Conclusion Whilst neither salivary nor plasma levels of TTV correlate with the CD4 count of HIV patients in our study, salivary TTV correlates with TTV detected in blood. With the increasing interest in TTV in transplantation as a marker for immunosuppression, the fact that measurements of TTV can be performed on the saliva, preventing or reducing the need to draw blood from patients deserves further exploration. Salivary TTV does not correlate with CD4 count, R2 0.004, p = 0.58. Disclosures All Authors: No reported disclosures

P-2134. Development and Validation of a Quantitative PCR Assay for the Detection of Mucorales in Human Serum Specimens

Abstract Background Mucormycosis are diseases with high morbidity and mortality caused by fungal species belonging to the order Mucorales. The intended use of a Mucorales qPCR assay is as an adjunct to microbiological, clinical, and radiologic means of diagnosing invasive mucormycosis (IM). Serum collection is a non-invasive alternative to BAL and biopsy. Our design process included sequences from all species within the Mucorales genera: Cunninghamella, Lichtheimia, Absidia, Apophysomyces, Mucor, Rhizomucor, Rhizopus and Saksenaea. The Mucorales qPCR assay is designed to detect and quantify Mucorales DNA extracted from human serum. Methods Primers and probes were designed to target the 18S regions of Mucorales species. Nucleic acid was extracted using the MagMAX™ Cell-Free DNA Isolation Kit and KingFisher™ Flex system. Amplification and detection were performed using TaqMan™ Fast Universal Master Mix and the Applied Biosystems™ QuantStudio 7 Pro. Quantification was performed using Rhizopus oryzae target-bearing linearized plasmid standards with results reported in copies/mL. Analytical validation was performed by spiking negative human serum with four different Mucorales species’ DNA: Rhizopus oryzae, Rhizomucor miehei, Lichtheimia corymbifera, and Cunninghamella bertholletiae. Results The limit of detection was 181 copies/mL, and the lower limit of quantification was determined to be 253 copies/mL as shown in Table 1. Linear regression of dilution data produced an R2=0.997, slope=1.029, and y-intercept=0.212 as show in Figure 1. The Mucorales target detection was 100% (60 of 60) of blinded positive samples with all observed log10 copies/mL results within ±0.5 log10 copies of expected values as shown in Table 2. All (20 of 20) blinded negative samples were negative for Mucorales and positive for Internal Control (IC). Intra-assay precision ranged from 5 – 22 %CV and inter-assay precision ranged from 12 – 17 %CV as Shown in Table 3. Conclusion The validated assay described here demonstrates excellent sensitivity, linearity, precision, and accuracy, providing a reliable means of detecting Mucorales DNA in human serum. This assay represents a non-invasive, rapid, and sensitive method to guide clinical decisions on patient populations at high risk of IM. Disclosures Jerod Davidson, B.A., Eurofins Viracor: Employee James Grantham, B.S., Eurofins Viracor: Employee Clayton Thomas, B.S., Eurofins Viracor: Employee Jamie Nutt, n/a, Eurofins Viracor: Employee Pam Morris, M.S., PMP, Eurofins Viracor: Employee Zachary Kockler, PhD, Eurofins Viracor: Employee Steve Kleiboeker, PhD, Eurofins: employee|Eurofins: Stocks/Bonds (Public Company)

Single-dose suraxavir marboxil for acute uncomplicated influenza in adults and adolescents: a multicenter, randomized, double-blind, placebo-controlled phase 3 trial.

Suraxavir marboxil (GP681) is an antiviral drug inhibiting the polymerase acidic protein (PA) of RNA polymerase, of influenza. It has shown therapeutic activity against influenza A and B virus infections in preclinical studies. In this multicenter randomized, double-blind, placebo-controlled, phase 3 trial, we aimed to investigate the efficacy and safety of single-dose suraxavir marboxil (40-mg oral dose) in otherwise healthy outpatients aged 5-65 years with uncomplicated influenza unaccompanied by severe issues. From 28 July 2022 to 31 October 2023, 591 outpatients aged 5-65 years with uncomplicated influenza underwent randomization in 46 research centers in China and were randomly assigned in a 2:1 ratio to receive suraxavir marboxil (40 mg) or placebo within 2 days of symptom onset. The primary outcome was time to alleviation of influenza symptoms (TTAS) (from the start of treatment until body temperature returned to 37.2 °C or less and all seven influenza symptoms (cough, sore throat, headache, nasal congestion, feverishness or chills, muscle or joint pain, and fatigue) resolved for at least 21.5 h) within 15 days by treatment. The secondary endpoints included virological indicators, system and respiratory symptoms, PA variant mutation and adverse events. The median TTAS was significantly shorter in the group that received suraxavir marboxil compared to the placebo group (42.0 h versus 63.0 h, P = 0.002). Suraxavir marboxil was associated with more rapid decrease in viral load from baseline than placebo by 1 day after administration, with a mean change of -2.2 ± 1.3 compared to -1.3 ± 1.7 log10 copies per ml (P < 0.001) in the placebo group. Adverse events were reported in 28.4% (112 of 395) of suraxavir marboxil recipients and 23.3% (45 of 193) of placebo recipients, most of which were mild or moderate. The incidences of PA variants with the I38T mutation in the H1N1pdm and H3N2 subtypes were 0.7% (1 of 138) and 0.9% (2 of 213), respectively. Low acquired drug resistance was observed. In this trial, timely single-dose suraxavir marboxil was effective in shortening TTAS and reducing the influenza viral load in patients aged 5-65 years with uncomplicated influenza safely. ClinicalTrials.gov registration: NCT05474755 .

Accumulation of microbial hazards and assessment of food hygiene associated with broiler chicken processing at open air food markets in Maputo, Mozambique

The burden of foodborne disease due to the consumption of animal-sourced foods is substantial in low- and middle-income countries (LMICs). Open air markets, while providing fresh and affordable foods, often have unhygienic practices that may contribute to contamination during the slaughter and processing of chicken meat. This study examines whether and how the common practice of rinse water (stored water used for rinsing broiler carcasses during processing) reuse leads to accumulation of pathogens, with potential cross contamination of chicken meat.To assess the accumulation of Campylobacter jejuni/coli, Salmonella spp., and the indicator of fecal contamination, Escherichia coli, in rinse water used during the slaughtering process at open air food markets in Maputo, Mozambique. We conducted a time-series study at three open air food markets. In a first experiment, we collected paired rinse water (N = 70), water used for chicken processing, and broiler chicken carcass (N = 60) samples from 10 vendors at 75-min intervals starting prior to any processing activity. In a second experiment, we collected 100, 50 mL rinse water samples, immediately before and after processing, from 10 vendors. Chicken processing activity and associated hygiene practices were captured through direct observation. Vendors processed 24 chickens per day, on average. In the first experiment, C. jejuni/coli and E. coli were detected in 30 % and 80 % of rinse water samples, respectively, prior to processing (baseline), and no Salmonella was detected. After the first carcass rinse, C. jejuni/coli and E. coli were detected in 100 % of samples, and Salmonella spp. was detected in 42 % of rinse water samples and 48 % of carcass samples. C. jejuni/coli showed an average 0.1 log10 copies (95 % CI 0.0, 0.2) increase in rinse water and carcass samples every 75 min. In the second experiment, no C. jejuni/coli or Salmonella spp. were detected in baseline rinse water samples, and E. coli were detected in 78 % of baseline rinse water samples. After processing the first carcass, C. jejuni/coli were detected in 100 % of remaining samples, Salmonella spp. were detected in 28 % of pre-final rinse and 36 % of post-final rinse samples, and E. coli were detected in 81 % of pre-final rinse and 100 % of post-final rinse samples. Our results reveal that consumers are at a high risk of purchasing chicken meat contaminated with human enteropathogens. Once contaminated, rinse water stays contaminated throughout the day. Low-cost and feasible interventions implemented at the carcass wash step are needed to reduce microbial hazards on chicken meat before purchase.

Loop-Mediated Isothermal Amplification Assay for the Detection of Citrus Canker Causing Bacterial Variant, Xanthomonas citri pv. citri Aw Strain.

Citrus canker, a highly transmissible bacterial disease, has three major types, with Asiatic canker (Canker A), caused by Xanthomonas citri pv. citri (Xcc A), being the most widespread and severe, affecting most citrus varieties. Xcc A has two mild variants, Xcc A* and Aw with a limited host range, reported in Southwest Asia and Florida, respectively. Since 2015, the canker caused by Xcc Aw has been being reported in the Rio Grande Valley of South Texas where the Texas commercial citrus industry is located. In 2016, a more severe Canker A was reported in the upper Texas gulf coast region, north of the Rio Grande Valley, posing a potential threat to the Texas citrus industry. Given that existing diagnostic methods cannot reliably distinguish Xcc Aw from Xcc A, we developed a loop-mediated isothermal amplification (LAMP) assay specific to Xcc Aw (LAMP-Aw) for rapid, field-based identification of this bacterial variant. The detection limit of LAMP-Aw was ~4.52 Log10 copies of the target molecule. This study also evaluated the field applicability of the LAMP-Aw assay by coupling the LAMP-Aw assay with a lateral flow immunoassay system.

Ziresovir in Hospitalized Infants with Respiratory Syncytial Virus Infection

BackgroundRespiratory syncytial virus (RSV) is a leading cause of severe illness in infants, with no effective treatment. Results of a phase 2 trial suggested that ziresovir may have efficacy in the treatment of infants hospitalized with RSV infection.MethodsIn a phase 3, multicenter, double-blind, randomized, placebo-controlled trial conducted in China, we enrolled participants 1 to 24 months of age who were hospitalized with RSV infection. Participants were randomly assigned, in a 2:1 ratio, to receive ziresovir (at a dose of 10 to 40 mg, according to body weight) or placebo, administered twice daily, for 5 days. The primary end point was the change from baseline to day 3 (defined as 48 hours after the first administration) in the Wang bronchiolitis clinical score (total scores range from 0 to 12, with higher scores indicating greater severity of signs and symptoms). The intention-to-treat population included all the participants with RSV-confirmed infection who received at least one dose of ziresovir or placebo; the safety population included all the participants who received at least one dose of ziresovir or placebo.ResultsThe intention-to-treat population included 244 participants, and the safety population included 302. The reduction from baseline in the Wang bronchiolitis clinical score at day 3 was significantly greater with ziresovir than with placebo (−3.4 points [95% confidence interval {CI}, −3.7 to −3.1] vs. −2.7 points [95% CI, −3.1 to −2.2]; difference, −0.8 points [95% CI, −1.3 to −0.3]; P=0.002). The reduction in the RSV viral load at day 5 was greater in the ziresovir group than in the placebo group (−2.5 vs. −1.9 log10 copies per milliliter; difference, −0.6 log10 copies per milliliter [95% CI, −1.1 to −0.2]). Improvements were observed in prespecified subgroups, including in participants with a baseline bronchiolitis score of at least 8 and in those 6 months of age or younger. The incidence of adverse events related to the drug or placebo was 16% with ziresovir and 13% with placebo. The most common adverse events that were assessed by the investigator as being related to the drug or placebo were diarrhea (in 4% and 2% of the participants, respectively), an elevated liver-enzyme level (in 3% and 3%, respectively), and rash (in 2% and 1%). Resistance-associated mutations were identified in 15 participants (9%) in the ziresovir group.ConclusionsZiresovir treatment reduced signs and symptoms of bronchiolitis in infants and young children hospitalized with RSV infection. No safety concerns were identified. (Funded by Shanghai Ark Biopharmaceutical; AIRFLO ClinicalTrials.gov number, NCT04231968.)

The effects of irrigation on the survival of Clostridium sporogenes in the phyllosphere and soil environments of lettuce

This study aimed to address the gap in knowledge regarding the fate of foodborne pathogens within agro-ecosystems. It specifically focused on the surrogate microorganism Clostridium sporogenes, which was introduced into lettuce-producing environments via surface and spray irrigation methods, respectively. The concentration of C. sporogenes in the rhizosphere, phyllosphere, and non-rhizosphere soil was quantified by quantitative polymerase chain reaction (qPCR) over a 42-day trial. The surface irrigation method exhibited a more noticeable contamination effect on the soil environments, compared to the phyllosphere. The results indicated a noticeable increase in C. sporogenes concentrations during the initial 22 days, with a 10.4-fold rise (0.39–4.05 log copy numbers/g soil) in the rhizosphere and 1.9-fold increase (2.97–5.59 log copy numbers/g soil) in the non-rhizosphere. However, concentrations in both soil environments subsequently decreased, falling below the initial inoculum concentration by the end of the trial. In contrast, the spray irrigation method resulted in most of the contamination being localised on the lettuce phyllosphere, with a high C. sporogenes concentration of 9.09 log copy numbers/g leaves on day 0. This concentration exponentially decreased to a minimal 0.019 log copy numbers/g leaves by day 32. Although concentrations in both soil environments decreased over time, trace concentrations of C. sporogenes were detectable at the end of the trial, posing a potential hazard to the microbiological safety of postharvest produce. These findings shed light on the dynamics of C. sporogenes in agro-ecosystems and underscore the importance of irrigation practices that ensure the safety of those who consume fresh produce.

Active diagnostic ingredients (ADIs) for PCR: A mini-bioreactor producing dNTPs with silica immobilized R5-kinases.

Low availability of routine nucleic acid amplification testing (NAAT) during infection outbreaks, especially in less resourced environments, was highlighted by the Covid pandemic. One of the barriers lies with the supply chain and cost of the active diagnostic ingredients (ADIs) that are the reagents for NAATs. This work explores a novel synthesis method to produce a key NAAT reagent, namely the 2'-deoxynucleoside 5'-triphosphate (dNTPs), via a reusable enzyme bioreactor, that can be integrated into a NAAT workflow. A self-immobilizing R5-silaffin kinase fusion enzyme was designed for immobilization on silica, converting dNMPs to their respective dNTP ADIs for PCR in a R5-kinase mini-bioreactor, designed to be implemented in a reusable device, stable over 2 months, when stored at 4°C. The performance is demonstrated for PCR reactions of the lambda genome and showed successful amplification up to 7.5 kb. In comparison with commercial dNTPs, in Plasmodium malariae NAATs, a high linear correlation was shown between the Ct value and the log(Copy Number), with lower incidence of false positives than with the commercial dNTPs. Overall a pathway to generate deoxynucleotides from monophosphate precursors was demonstrated, and an immobilized enzyme mini-bioreactor investigated as a proof-of-principle for work-flow integration with NAAT in low-resource research and diagnostics labs.

Hercules: Enabling Atomic Durability for Persistent Memory with Transient Persistence Domain

Persistent memory (pmem) products bring the persistence domain up to the memory level. Intel recently introduced the eADR feature that guarantees to flush data buffered in CPU cache to pmem on a power outage, thereby making the CPU cache a transient persistence domain . Researchers have explored how to enable the atomic durability for applications’ in-pmem data. In this article, we exploit the eADR-supported CPU cache to do so. A modified cache line, until written back to pmem, is a natural redo log copy of the in-pmem data. However, a write-back due to cache replacement or eADR on a crash overwrites the original copy. We accordingly developed Hercules, a hardware logging design for the transaction-level atomic durability, with supportive components installed in CPU cache, memory controller (MC), and pmem. When a transaction commits, Hercules commits on-chip its data staying in cache lines. For cache lines evicted before the commit, Hercules asks the MC to redirect and persist them into in-pmem log entries and commits them off-chip upon committing the transaction. Hercules lazily conducts pmem writes only for cache replacements at runtime. On a crash, Hercules saves metadata and data for active transactions into pmem for recovery. Experiments show that, by using CPU cache for both buffering and logging, Hercules yields much higher throughput and incurs significantly fewer pmem writes than state-of-the-art designs.

The Impact of Chloroform on Viability of Temperate Bacteriophage Lambda and Survival of its Host, Escherichia coli

Background: This study investigates the effect of chloroform, an organic compound with anti-bacterial properties, produced both naturally and due to anthropogenic activities on the viability of temperate bacteriophage lambda (phage lambda) and its host, Escherichia coli (E. coli). The study was initiated while repurposing phage lambda for testing efficacy and efficiency of disinfectants. Results: Plaque assays and Polymerase Chain Reaction (PCR) using primers specific to the Lambda Tail Protein (LTP) demonstrated a decrease in plaque-forming units and the log copy number of phage lambda following chloroform treatment of phage lysates. Interestingly, PCR with 16 S rRNA primers detected the presence of bacteria in both chloroform-treated and untreated lysates, suggesting that E. coli lysogens, small enough to pass through 0.2 μm filters, which is commonly used for sterilization, were present in the phage lysates. The presence of lysogens in the phage lysates was further supported by the absence of a 16 S rRNA amplicon in lysates prepared without phage lambda infection and the presence of an amplicon in DNase-treated lysates. Additionally, PCR and microscopy revealed that chloroform treatment of phage lysates increased the lysogen copy number, decreased lysogen size, and altered lysogens phage-producing ability. Conclusion: In summary, while chloroform had a slight effect on the viability of phage lambda, most of the phage remained resistant to chloroform because they lack a lipid coat. Phage lambda transitioned to a lysogenic life cycle under sub-optimal growth conditions, leading to the formation of E. coli lysogens. These lysogens were more resistant to stresses like chloroform, ensuring the survival of both the phage and the host during adverse conditions

Real-time PCR-based quantitative microbiome profiling elucidates the microbial dynamic succession in backslopping fermentation of Taiwanese pickled cabbage.

Microbiota succession determines the flavor and quality of fermented foods. Quantitative PCR-based quantitative microbiome profiling (QMP) has been applied broadly for microbial analysis from absolute abundance perspectives, transforming microbiota ratios into counts by normalizing 16S ribosomal RNA (16S rRNA) gene sequencing data with gene copies quantified by quantitative PCR. However, the application of QMP in fermented foods is still limited. QMP elucidated microbial succession of Taiwanese pickled cabbage. In the spontaneous first-round fermentation (FR), the 16S rRNA gene copies of total bacteria increased from 6.1 to 10 log copies mL-1. The dominant lactic acid bacteria genera were successively Lactococcus, Leuconostoc and Lactiplantibacillus. Despite the decrease in the proportion of Lactococcus during the succession, the absolute abundance of Lactococcus still increased. In the backslopping second-round fermentation (SR), the total bacteria 16S rRNA gene copies increased from 7.6 to 9.9 log copies mL-1. The addition of backslopping starter and vinegar rapidly led to a homogenous microbial community dominated by Lactiplantibacillus. The proportion of Lactiplantibacillus remained consistently around 90% during SR, whereas its absolute abundance exhibited a continuous increase. In SR without vinegar, Leuconostoc consistently dominated the fermentation. The present study highlights that compositional analysis would misinterpret microbial dynamics, whereas QMP reflected the real succession profiles and unveiled the essential role of vinegar in promoting Lactiplantibacillus dominance in backslopping fermentation of Taiwanese pickled cabbage. Quantitative microbiome profiling (QMP) was found to be a more promising approach for the detailed observation of microbiome succession in food fermentation compared to compositional analysis. © 2024 Society of Chemical Industry.

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.

Quantitative fecal source characterization of urban municipal storm sewer system outfall ‘wet’ and ‘dry’ weather discharges

Urban areas are built environments containing substantial amounts of impervious surfaces (e.g., streets, sidewalks, roof tops). These areas often include elaborately engineered drainage networks designed to collect, transport, and discharge untreated stormwater into local surface waters. When left uncontrolled, these discharges may contain unsafe levels of fecal waste from sources such as sanitary sewage and wildlife even under dry weather conditions. This study evaluates paired measurements of host-associated genetic markers (log10 copies per reaction) indicative of human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), canine (DG3), and avian (GFD) fecal sources, 12-hour cumulative precipitation (mm), four catchment land use metrics determined by global information system (GIS) mapping, and Escherichia coli (MPN/100 ml) from seven municipal separate storm sewer system outfall locations situated at the southern portion of the Anacostia River Watershed (District of Columbia, U.S.A.). A total of 231 discharge samples were collected twice per month (n = 24 sampling days) and after rain events (n = 9) over a 13-month period. Approximately 50 % of samples (n = 116) were impaired, exceeding the local E. coli single sample maximum of 2.613 log10 MPN/100 ml. Genetic quality controls indicated the absence of amplification inhibition in 97.8 % of samples, however 14.7 % (n = 34) samples showed bias in DNA recovery. Of eligible samples, quantifiable levels were observed for avian (84.1 %), human (57.4 % for HF183/BacR287 and 40 % for HumM2), canine (46.7 %), and ruminant (15.9 %) host-associated genetic markers. Potential links between paired measurements are explored with a recently developed Bayesian qPCR censored data analysis approach. Findings indicate that human, pet, and urban wildlife all contribute to storm outfall discharge water quality in the District of Columbia, but pollutant source contributions vary based on ‘wet’ and ‘dry’ conditions and catchment land use, demonstrating that genetic-based fecal source identification methods combined with GIS land use mapping can complement routine E. coli monitoring to improve stormwater management in urban areas.

The role of FGL2 in vascular smooth muscle cell dysfunction in coronary artery bypass vein graft failure

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): British Heart Foundation Rationale The sudden exposure of venous endothelial cells (vECs) to arterial fluid shear stress (FSS) is thought to be a major contributor to coronary artery bypass vein graft failure (VGF). However, the acute effects of arterial FSS on the vEC secretome and the corresponding effects on underlying vascular smooth muscle cells (VSMCs) remains poorly characterised. Methods &amp; Results Human umbilical vein endothelial cells (HUVECs; n=6) pre-conditioned to venous FSS (18h; 1.5 dynes/cm2) were further exposed to venous or arterial FSS (24h; 15 dynes/cm2) using an Ibidi pump system. Analysis of the EC secretome using Tandem Mass Tagging proteomics detected significantly increased fibroleukin (FGL2) in conditioned media from ECs exposed to arterial FSS compared to venous FSS (fold-change 1.97±0.72; P=0.0078). In addition, RT-qPCR analysis of FGL2 production showed a significant increase in vECs exposed to arterial FSS (2.11±0.27 vs. 0.05±0.04 log(copy number/mg); P=0.0312). Subsequently, pooled human saphenous vein VSMCs (3 donors per pool) were treated with conditioned media from ECs exposed to arterial FSS (n=6) which showed a significant increase in apoptosis, measured by active cleaved caspase-3 (CC3) immunocytochemistry (27±1% vs 22±1%; P=0.0391), and a significant decrease in the contractile markers smoothelin and desmin quantified by Western blotting (fold-change 0.76±0.06 and 0.44±0.11; P=0.004 and P=0.0421, respectively), in comparison to VSMCs treated with conditioned media from ECs exposed to venous FSS. Treatment of VSMCs (n=6) with recombinant FGL2 (20 ng/mL) for 72h likewise resulted in increased apoptosis (27±4% vs. 16±3%; P=0.0169) and decreased desmin and smoothelin (fold-change 0.68±0.03 and 0.61±0.07; P=0.0322 and P=0.0479, respectively). Conclusions The exposure of vECs to arterial FSS results in increased production and release of FGL2 which induced VSMC apoptosis and de-differentiation and therefore potentially contributes to VGF. Consequently, targeting FGL2 production or release may provide a therapeutic target to improve vein graft patency.

Perioperative remedial antiviral therapy in hepatitis B virus-related hepatocellular carcinoma resection: How to achieve a better outcome.

Although the benefits of antiviral therapy for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) have been proven, researchers have not confirmed the differences in patient outcomes between patients who received preoperative antiviral therapy for a period of time (at least 24 wk) and patients who received remedial antiviral therapy just before radical resection for HBV-related HCC. To investigate the efficacy of perioperative remedial antiviral therapy in patients with HBV-related HCC. A retrospective study of patients who underwent radical resection for HBV-related HCC at the First Affiliated Hospital of Xi'an Jiaotong University from January 2016 to June 2019 was conducted. Considering the history of antiviral therapy, patients were assigned to remedial antiviral therapy and preoperative antiviral therapy groups. Kaplan-Meier analysis revealed significant differences in overall survival (P < 0.0001) and disease-free survival (P = 0.035) between the two groups. Multivariate analysis demonstrated that a history of preoperative antiviral treatment was independently related to improved survival (hazard ratio = 0.27; 95% confidence interval: 0.08-0.88; P = 0.030). In patients with HBV-related HCC, it is ideal to receive preoperative long-term antiviral therapy, which helps patients tolerate more extensive hepatectomy; however, remedial antiviral therapy, which reduces preoperative HBV-DNA levels to less than 4 Log10 copies DNA/mL, can also result in improved outcomes.

CROCuS, a Phase II Study Evaluating the Antiviral Activity, Clinical Outcomes, and Safety of Rilematovir in Children Aged ≥28 Days and ≤3 Years with Acute Respiratory Tract Infection Due to Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) causes significant morbidity and mortality in children aged ≤5 years and adults aged ≥60 years worldwide. Despite this, RSV-specific therapeutic options are limited. Rilematovir is an investigational, orally administered inhibitor of RSV fusion protein-mediated viral entry. To establish the antiviral activity, clinical outcomes, safety, and tolerability of rilematovir (low or high dose) in children aged ≥28days and ≤3years with RSV disease. CROCuS was a multicenter, international, double-blind, placebo-controlled, randomized, adaptive phase II study, wherein children aged ≥28 days and ≤3 years with confirmed RSV infection who were either hospitalized (Cohort 1) or treated as outpatients (Cohort 2) were randomized (1:1:1) to receive rilematovir (low or high dose) or placebo. Study treatment was administered daily as an oral suspension from days 1 to 7, with dosing based on weight and age groups. The primary objective was to establish antiviral activity of rilematovir by evaluating the area under the plasma concentration-time curve of RSV viral load in nasal secretions from baseline through day 5. Severity and duration of RSV signs and symptoms and the safety and tolerability of rilematovir were also assessed through day 28 (±3). In total, 246 patients were randomized, treated, and included in the safety analysis population (Cohort 1: 147; Cohort 2: 99). Of these, 231 were included in the intent-to-treat-infected analysis population (Cohort 1: 138; Cohort 2: 93). In both cohorts, demographics were generally similar across treatment groups. In both cohorts combined, the difference (95% confidence interval) in the mean area under the plasma concentration-time curve of RSV RNA viral load through day 5 was -1.25 (-2.672, 0.164) and -1.23 (-2.679, 0.227) log10 copies∙days/mL for the rilematovir low-dose group and the rilematovir high-dose group, respectively, when compared with placebo. The estimated Kaplan-Meier median (95% confidence interval) time to resolution of key RSV symptoms in the rilematovir low-dose, rilematovir high-dose, and placebo groups of Cohort 1 was 6.01 (4.24, 7.25), 5.82 (4.03, 8.18), and 7.05 (5.34, 8.97) days, respectively; in Cohort 2, estimates were 6.45 (4.81, 9.70), 6.26 (5.41, 7.84), and 5.85 (3.90, 8.27) days, respectively. A similar incidence of adverse events was reported in patients treated with rilematovir and placebo in Cohort 1 (rilematovir: 61.9%; placebo: 58.0%) and Cohort 2 (rilematovir: 50.8%; placebo: 47.1%), with most reported as grade 1 or 2 and none leading to study discontinuation. The study was terminated prematurely, as the sponsor made a non-safety-related strategic decision to discontinue rilematovir development prior to full recruitment of Cohort 2. Data from the combined cohort suggest that rilematovir has a small but favorable antiviral effect of indeterminate clinical relevance compared with placebo, as well as a favorable safety profile. Safe and effective therapeutic options for RSV in infants and young children remain an unmet need. EudraCT Number: 2016-003642-93; ClinicalTrials.gov Identifier: NCT03656510. First posted date: 4 September, 2018.

Viral clearance as a surrogate of clinical efficacy for COVID-19 therapies in outpatients: a systematic review and meta-analysis

Surrogates of antiviral efficacy are needed for COVID-19. We aimed to investigate the relationship between the virological effect of treatment and clinical efficacy as measured by progression to severe disease in outpatients treated for mild-to-moderate COVID-19. In this systematic review and meta-analysis, we searched PubMed, Scopus, and medRxiv from database inception to Aug 16, 2023, for randomised placebo-controlled trials that tested virus-directed treatments (ie, any monoclonal antibodies, convalescent plasma, or antivirals) in non-hospitalised individuals with COVID-19. We only included studies that reported both clinical outcomes (ie, rate of disease progression to hospitalisation or death) and virological outcomes (ie, viral load within the first 7 days of treatment). We extracted summary data from eligible reports, with discrepancies resolved through discussion. We used an established meta-regression model with random effects to assess the association between clinical efficacy and virological treatment effect, and calculated I2 to quantify residual study heterogeneity. We identified 1718 unique studies, of which 22 (with a total of 16 684 participants) met the inclusion criteria, and were in primarily unvaccinated individuals. Risk of bias was assessed as low in 19 of 22 studies for clinical outcomes, whereas for virological outcomes, a high risk of bias was assessed in 11 studies, some risk in ten studies, and a low risk in one study. The unadjusted relative risk of disease progression for each extra log10 copies per mL reduction in viral load in treated compared with placebo groups was 0·12 (95% CI 0·04-0·34; p<0·0001) on day 3, 0·20(0·08-0·50; p=0·0006) on day 5, and 0·53 (0·30-0·94; p=0·030) on day 7. The residual heterogeneity in our meta-regression was estimated as low (I2=0% [0-53] on day 3, 0% [0-71] on day 5, and 0% [0-43] on day 7). Despite the aggregation of studies with differing designs, and evidence of risk of bias in some virological outcomes, this review provides evidence that treatment-induced acceleration of viral clearance within the first 5days after treatment is a potential surrogate of clinical efficacy to prevent hospitalisation with COVID-19. This work supports the use of viral clearance as an early phase clinical trial endpoint of therapeutic efficacy. Australian Government Department of Health, Medical Research Future Fund, and Australian National Health and Medical Research Council.

Antibiofilm Properties and Demineralization Suppression in Early Enamel Lesions Using Dental Coating Materials.

This study aimed to investigate the effects of dental coating materials on Streptococcus mutans biofilm formation. The test materials were PRG Barrier Coat (PRG), BioCoat Ca (BioC), and FluorDental Jelly (FluorJ). Bovine enamel specimens were demineralized to mimic early enamel lesions. The biofilm was developed on a specimen treated with one of the materials by using a modified Robbins device flow-cell system. Scanning electron and fluorescence confocal laser scanning microscopy, viable and total cell counts, and gene expression assessments of the antibiofilm were performed. Ion incorporation was analyzed using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. All materials allowed biofilm formation but reduced its volume. FluorJ was the only material that inhibited biofilm accumulation and had a bactericidal effect, revealing 0.66 log CFU in viable cells and 1.23 log copy reduction in total cells compared with the untreated group after 24 h of incubation. The ions released from PRG varied depending on the element. BioC contributed to enamel remineralization by supplying calcium ions while blocking the acid produced from the biofilm. In summary, the dental coating materials physically prevented acid attacks from the biofilm while providing ions to the enamel to improve its mechanical properties.

Oral Simnotrelvir for Adult Patients with Mild-to-Moderate Covid-19

BackgroundSimnotrelvir is an oral 3-chymotrypsin–like protease inhibitor that has been found to have in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential efficacy in a phase 1B trial.MethodsIn this phase 2–3, double-blind, randomized, placebo-controlled trial, we assigned patients who had mild-to-moderate coronavirus disease 2019 (Covid-19) and onset of symptoms within the past 3 days in a 1:1 ratio to receive 750 mg of simnotrelvir plus 100 mg of ritonavir or placebo twice daily for 5 days. The primary efficacy end point was the time to sustained resolution of symptoms, defined as the absence of 11 Covid-19–related symptoms for 2 consecutive days. Safety and changes in viral load were also assessed.ResultsA total of 1208 patients were enrolled at 35 sites in China; 603 were assigned to receive simnotrelvir and 605 to receive placebo. Among patients in the modified intention-to-treat population who received the first dose of trial drug or placebo within 72 hours after symptom onset, the time to sustained resolution of Covid-19 symptoms was significantly shorter in the simnotrelvir group than in the placebo group (180.1 hours [95% confidence interval {CI}, 162.1 to 201.6] vs. 216.0 hours [95% CI, 203.4 to 228.1]; median difference, −35.8 hours [95% CI, −60.1 to −12.4]; P=0.006 by Peto–Prentice test). On day 5, the decrease in viral load from baseline was greater in the simnotrelvir group than in the placebo group (mean difference [±SE], −1.51±0.14 log10 copies per milliliter; 95% CI, −1.79 to −1.24). The incidence of adverse events during treatment was higher in the simnotrelvir group than in the placebo group (29.0% vs. 21.6%). Most adverse events were mild or moderate.ConclusionsEarly administration of simnotrelvir plus ritonavir shortened the time to the resolution of symptoms among adult patients with Covid-19, without evident safety concerns. (Funded by Jiangsu Simcere Pharmaceutical; ClinicalTrials.gov number, NCT05506176.)

Inactivation of Human Norovirus GII.4's Infectivity in Fresh Oysters (Crassostrea gigas) through Thermal Treatment in Association with Propidium Monoazide.

The current study investigated the effects of heat treatment (85 °C or 100 °C for 5-20 min) on human norovirus (HuNoV) GII.4's capsid stability in fresh oysters. In addition, propidium monoazide (PMA) was used in viral samples to distinguish infectious viruses and evaluated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Further, we explored the effect of the heat treatment on oyster quality (Hunter color and hardness). The titer of HuNoV for oysters significantly (p < 0.05) decreased to 0.39-1.32 and 0.93-2.27 log10 copy number/μL in the non-PMA and PMA-treated groups, respectively, after heat treatment. HuNoV in oysters not treated with PMA showed a decrease of <1.5 - log10, whereas in PMA-treated oysters, a decrease of >1 - log10 was observed after treatment at 85 °C for 10 min. Treatments for both 15 min and 20 min at 100 °C showed a >99% log10 reduction using PMA/RT-qPCR. In the Hunter color, an increase in heat temperature and duration was associated with a significant decrease in 'L' (brightness+, darkness-) and an increase in 'a' (redness+, greenness-) and 'b' (yellowness+, blueness-) (p < 0.05). Our findings confirmed that the hardness of oyster meat significantly increased with increasing temperature and time (p < 0.05). This study demonstrated that PMA/RT-qPCR was effective in distinguishing HuNoV viability in heat-treated oysters. The optimal heat treatment for oysters was 10 min at 85 °C and 5 min at 100 °C.