Search Results (30)

Hybrid capture-based target enrichment prior to sequencing has been shown to significantly improve the sensitivity of detection for genetic regions of interest. In the context of One Health relevant pathogen detection, we present a hybrid capture-based sequencing method that employs an optimized probe set consisting of 149,990 probes, targeting 663 viruses associated with humans and animals. The detection performance was initially assessed using viral reference materials in a background of human nucleic acids. Compared to standard metagenomic next-generation sequencing (mNGS), our method achieved substantial read enrichment, with increases ranging from 143- to 1126-fold, and enhanced detection sensitivity by lowering the limit of detection (LoD) from 10³–10⁴ copies to as few as 10 copies based on whole genomes. This method was further validated using infectious samples from both animals and humans, including bovine rectal swabs and throat swabs from SARS-CoV-2 patients across various concentration gradients. In both sample types, our hybrid capture-based sequencing method exhibited heightened sensitivity, increased viral genome coverage, and more comprehensive viral identification and characterization. Our method bridges a critical divide between diagnostic detection and genomic surveillance. These findings illustrate that our hybrid capture-based sequencing method can effectively enhance sensitivity to as few as 10 viral copies and genome coverage to >99% in medium-to-high viral loads. This dual capability is particularly impactful for emerging pathogens like SARS-CoV-2, where early detection and genomic characterization are equally vital, thereby addressing the limitations of metagenomics in the surveillance of emerging infectious diseases in complex samples. Full article

The aim of this study was to analyse the trends in HIV infection, including diagnostic ones, in the Republic of Crimea in the period of 2014–2023. The source of data for this study was various statistical forms and reports. The findings revealed a significant downward trend in HIV incidence and a significant upward trend in HIV prevalence. The mortality rate was stable. The coverage of HIV testing and antiretroviral therapy increased over time. The number of patients with a suppressed viral load in the Republic fluctuated in different years of observation and reached 81% in 2023. In the second part of this study, we analysed the demographic and clinical laboratory characteristics of newly diagnosed patients with HIV. A predominance of men was noted. The proportion of injection drug users (IDUs) decreased, and the proportion of new HIV infection cases through heterosexual contacts increased. An increase in the median age of patients was also noted. Analysis of CD4 cell counts revealed significant differences between subgroups by gender, age, and route of infection. The longest time to disease detection was typical for IDUs. A comprehensive assessment of HIV infection trends in Crimea allows us to evaluate the effectiveness of measures and decisions taken on the issue of HIV infection. Full article

Background. The combined or multivalent vaccines are actively used in pediatric practice and offer a series of advantages, including a reduced number of injections and visits to the doctor, simplicity of the vaccination schedule and minimization of side effects, easier vaccine monitoring and storage, and lower vaccination costs. The practice of widespread use of the combined vaccines has shown the potential to increase vaccination coverage against single infections. The mRNA platform has been shown to be effective against the COVID-19 pandemic and enables the development of combined vaccines. There are currently no mRNA-based combined vaccines approved for use in humans. Some studies have shown that different mRNA components in a vaccine can interact to increase or decrease the immunogenicity and efficacy of the combined vaccine. Objectives. In the present study, we investigated the possibility of combining the mRNA vaccines, encoding seasonal influenza and SARS-CoV-2 antigens. In our previous works, both vaccine candidates have shown excellent immunogenicity and efficacy profiles in mice. Methods. The mRNA-LNPs were prepared by microfluidic mixing, immunogenicity in mice was assessed by hemagglutination inhibition assay, enzyme-linked immunoassay and virus neutralization assay. Immunological efficacy was assessed in a mouse viral challenge model. Results. In this work, we demonstrated that the individual mRNA components of the combined vaccine did not affect the immunogenicity level of each other. The combined vaccine demonstrated excellent protective efficacy, providing a 100% survival rate when mice were infected with the H1N1 influenza virus and reducing the viral load in the lungs. Four days after the challenge with SARS-CoV-2 EG.5.1.1., no viable virus and low levels of detectable viral RNA were observed in the lungs of vaccinated mice. Conclusions. The combination does not lead to mutual interference between the individual vaccines. We believe that such a combined mRNA-based vaccine could be a good alternative to separated human vaccinations for the prevention of COVID-19 and influenza. Full article

Porcine circovirus type 2 (PCV2) is strongly linked to a group of syndromes referred to as porcine-circovirus-associated diseases (PCVADs), which are controlled through vaccination; however, this does not induce sterilizing immunity but is instead involved in the evolution of the virus and is considered a factor in vaccine failure. This study sampled 84 herds (167 pigs) vaccinated against PCV2 and with clinical signs of PCVADs in five provinces across Colombia. PCV2 was identified and further characterized at the molecular level via genotyping and phylogenetic reconstructions. In addition, PCV2-associated lesions were examined via histopathology. Furthermore, the PCV2-Cap sequences retrieved were compared with three vaccines via the EpiCC tool and T cell epitope coverage. The prevalence of PCV2 was 82% in pigs and 92.9% in herds. The highest viral loads were identified in lymphoid tissue, and PCV2d emerged as the most predominant in pigs and herds (93.4% and 92.3%). Sequences for PCV2-ORF2 (n = 57; 55 PCV2d and 2 PCV2a) were determined, and PCV2d sequences were highly similar. The most common pneumonia pattern was suppurative bronchopneumonia, while the most common lung lesion was exudation in the airways; in lymphoid tissue, there was lymphoid depletion. The bivalent vaccine (PCV2a and PCVb) exhibited a higher EpiCC score (8.36) and T cell epitope coverage (80.6%) than monovalent PCV2a vaccines. In conclusion, PCV2d currently circulates widely in Colombia. Despite vaccination, there are clinical cases of PCV2, and immunoinformatic analyses demonstrate that bivalent vaccines improved the average coverage. Full article

During the COVID-19 pandemic, antibody-based vaccines targeting the SARS-CoV-2 spike glycoprotein were the focus for development because neutralizing antibodies were associated with protection against the SARS-CoV-2 infection pre-clinically and in humans. While deploying these spike-based vaccines saved millions of lives worldwide, it has become clear that the immunological mechanisms of protection against severe disease are multifaceted and involve non-neutralizing antibody components. Here, we describe a novel pan-sarbecovirus T-cell vaccine, ChAdOx1.COVconsv12, designed to complement and broaden the protection of spike vaccines. The vaccine immunogen COVconsv12 employs the two regions in the viral proteome most conserved among sarbecoviruses, which are delivered by replication-deficient vector ChAdOx1. It directs T cells towards epitopes shared among sarbecoviruses including evolving SARS-CoV-2 variants. Here, we show that ChAdOx1.COVconsv12 induced broad T-cell responses in the BALB/c and C57BL/6 mice. In the Syrian hamster challenge model, ChAdOx1.COVconsv12 alone did not protect against the SARS-CoV-2 infection, but when co-administered with 1/50th of the ChAdOx1 nCoV-19 spike vaccine protective dose, faster recovery and lower oral swab viral load were observed. Induction of CD8⁺ T cells may decrease COVID-19 severity and extend the T-cell response coverage of variants to match the known (and as yet unknown) members of the β-coronavirus family. Full article

The amount of SARS-CoV-2 in a sample is often measured using Ct values. However, the same Ct value may correspond to different viral loads on different platforms and assays, making them difficult to compare from study to study. To address this problem, we developed ct2vl, a Python package that converts Ct values to viral loads for any RT-qPCR assay/platform. The method is novel in that it is based on determining the maximum PCR replication efficiency, as opposed to fitting a sigmoid (S-shaped) curve relating signal to cycle number. We calibrated ct2vl on two FDA-approved platforms and validated its performance using reference-standard material, including sensitivity analysis. We found that ct2vl-predicted viral loads were highly accurate across five orders of magnitude, with 1.6-fold median error (for comparison, viral loads in clinical samples vary over 10 orders of magnitude). The package has 100% test coverage. We describe installation and usage both from the Unix command-line and from interactive Python environments. ct2vl is freely available via the Python Package Index (PyPI). It facilitates conversion of Ct values to viral loads for clinical investigators, basic researchers, and test developers for any RT-qPCR platform. It thus facilitates comparison among the many quantitative studies of SARS-CoV-2 by helping render observations in a natural, universal unit of measure. Full article

With the increase in uptake of multi-month antiretroviral therapy dispensing (MMD) for children, little is known about consistency of MMD receipt over time and its association with virological outcomes. This analysis aims to assess the uptake of 3-month MMD among children, consistent receipt of MMD after uptake, and clinical outcomes following transition to MMD in 16 health facilities in Gaza and Inhambane Provinces. This is a secondary analysis involving children <15 years living with HIV with clinical visits during the period from September 2019 to August 2020. Of 4383 children, 82% ever received MMD (at least one pickup of a 3-month MMD supply) during the study period but only 40% received it consistently (defined as MMD at every visit during the study period). Consistent MMD was most common among older children and children without indications of clinical instability. Overall viral load (VL) coverage was 40% (733/1851). Consistent MMD was significantly associated with lower odds of having a VL (0.78, 95% CI: 0.64–0.95). In conclusion, while receipt of a multi-month supply was common particularly during the early days of the COVID-19 pandemic, only a minority of children received consistent MMD; however, there is a need to ensure children with fewer visits still receive timely VL monitoring. Full article

(1) Background: We aimed to determine the prevalence of hepatitis B virus (HBV) resistance-associated mutations (RAMs) in people with HBV and human immunodeficiency virus (HBV/HIV) in Botswana. (2) Methods: We sequenced HBV deoxyribonucleic acid (DNA) from participants with HBV/HIV from the Botswana Combination Prevention Project study (2013–2018) using the Oxford Nanopore GridION platform. Consensus sequences were analyzed for genotypic and mutational profiles. (3) Results: Overall, 98 HBV sequences had evaluable reverse transcriptase region coverage. The median participant age was 43 years (IQR: 37, 49) and 66/98 (67.4%) were female. Most participants, i.e., 86/98 (87.8%) had suppressed HIV viral load (VL). HBV RAMs were identified in 61/98 (62.2%) participants. Most RAMs were in positions 204 (60.3%), 180 (50.5%), and 173 (33.3%), mostly associated with lamivudine resistance. The triple mutations rtM204V/L180M/V173L were the most predominant (17/61 [27.9%]). Most participants (96.7%) with RAMs were on antiretroviral therapy for a median duration of 7.5 years (IQR: 4.8, 10.5). Approximately 27.9% (17/61) of participants with RAMs had undetectable HBV VL, 50.8% (31/61) had VL < 2000 IU/mL, and 13/61 (21.3%) had VL ≥ 2000 IU/mL. (4) Conclusions: The high prevalence of lamivudine RAMs discourages the use of ART regimens with 3TC as the only HBV-active drug in people with HIV/HBV. Full article

Zambia’s adult HIV prevalence is high at 11% and faces challenges in achieving UNAIDS 95-95-95 targets for HIV, with a national viral load suppression of 86.2% falling short of the required 95%. North-Western Province has the lowest viral load suppression at 77.5%. Our study investigated the role of an integrated sample referral system in optimizing HIV viral load coverage and Early Infant Diagnosis turnaround time in the province. Using electronic data from the DISA Laboratory Information System and Smartcare, a retrospective cross-sectional analysis was conducted, involving 160,922 viral load and Early Infant Diagnosis results. The chi-square test and multiple linear regression were used for analysis. Following the implementation of the integrated sample referral system, viral load coverage consistently increased monthly (p < 0.001), Early Infant Diagnosis turnaround time improved by 47.7%, and sample volume increased by 25%. The study identifies associations between various factors and testing outcomes. These findings demonstrate improvements in viral load coverage and the Early Infant Diagnosis turnaround time and suggest targeting modifiable factors to further optimize the referral system. We recommend continued strengthening of the referral system and more deliberate demand-creation implementation strategies. Full article

African swine fever (ASF) is a lethal contagious viral disease of domestic pigs and wild boars caused by the African swine fever virus (ASFV). The pandemic spread of ASF has caused severe effects on the global pig industry. Whole-genome sequencing provides crucial information for virus strain characterization, epidemiology analysis and vaccine development. Here, we evaluated the performance of next-generation sequencing (NGS) in generating ASFV genome sequences from clinical samples. Thirty-four ASFV-positive field samples including spleen, lymph node, lung, liver and blood with a range of Ct values from 14.73 to 25.95 were sequenced. For different tissue samples collected from the same sick pigs, the proportion of ASFV reads obtained from the spleen samples was 3.69–9.86 times higher than other tissues. For the high-viral-load spleen samples (Ct < 20), a minimum of a 99.8% breadth of ≥10× coverage was revealed for all the samples. For the spleen samples with Ct ≥ 20, 6/12 samples had a minimum of a 99.8% breadth of ≥10× coverage. A high average depth of sequencing coverage was also achieved from the blood samples. According to our results, high-quality ASFV whole-genome sequences could be obtained from the spleen or blood samples with Ct < 20. The high-quality ASFV genome sequence generated in this study was further used for the high-resolution phylogenetic analysis of the ASFV genomes in the early stage of the ASF epidemic in China. Our study demonstrates that NGS may act as a useful tool for efficient ASFV genome characterization, providing valuable information for disease control. Full article

Russia remains one of the areas most affected by HIV in Eastern Europe and Central Asia. The aim of this study was to analyze HIV infection indicators and study trends in Russia using data from the Federal Statistic Form No. 61 “Information about HIV infection”. HIV incidence, prevalence, HIV testing and mortality rates (from 2011 to 2022), and treatment success rates (from 2016 to 2022) were analyzed. These indicators were compared across different federal districts (FDs) of Russia. The findings revealed a significant downward trend in HIV incidence, while a significant upward trend was observed for HIV prevalence. The mortality rate has stabilized since 2018. The coverage of HIV testing and antiretroviral therapy increased over time. The number of people living with HIV-1 (PLWH) with a suppressed viral load in Russia as a whole varied between 72% and 77% during the years under observation. The Siberian and Ural federal districts recorded the highest HIV incidence, while the North Caucasian FD reported the lowest. An increase in HIV testing coverage was observed across all FDs. This comprehensive evaluation of HIV infection indicators within the regional context contributes to the timely implementation of measures aimed at preventing the spread of HIV. Full article

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCV_PA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCV_PA and the wild-type subgroup 3 tobamovirus. TVCV_PA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCV_PA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCV_PA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta₂O₅ sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCV_PA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes. Full article

Background: Drug-resistance mutations were mostly detected using capillary electrophoresis sequencing, which does not detect minor variants with a frequency below 20%. Next-Generation Sequencing (NGS) can now detect additional mutations which can be useful for HIV-1 drug resistance interpretation. The objective of this study was to evaluate the performances of CE-IVD assays for HIV-1 drug-resistance assessment both for target-specific and whole-genome sequencing, using standardized end-to-end solution platforms. Methods: A total of 301 clinical samples were prepared, extracted, and amplified for the three HIV-1 genomic targets, Protease (PR), Reverse Transcriptase (RT), and Integrase (INT), using the CE-IVD DeepChek^® Assays; and then 19 clinical samples, using the CE-IVD DeepChek^® HIV Whole Genome Assay, were sequenced on the NGS iSeq100 and MiSeq (Illumina, San Diego, CA, USA). Sequences were compared to those obtained by capillary electrophoresis. Quality control for Molecular Diagnostics (QCMD) samples was added to validate the clinical accuracy of these in vitro diagnostics (IVDs). Nineteen clinical samples were then tested with the same sample collection, handling, and measurement procedure for evaluating the use of NGS for whole-genome HIV-1. Sequencing analyzer outputs were submitted to a downstream CE-IVD standalone software tailored for HIV-1 analysis and interpretation. Results: The limits of range detection were 1000 to 10⁶ cp/mL for the HIV-1 target-specific sequencing. The median coverage per sample for the three amplicons (PR/RT and INT) was 13,237 reads. High analytical reproducibility and repeatability were evidenced by a positive percent agreement of 100%. Duplicated samples in two distinct NGS runs were 100% homologous. NGS detected all the mutations found by capillary electrophoresis and identified additional resistance variants. A perfect accuracy score with the QCMD panel detection of drug-resistance mutations was obtained. Conclusions: This study is the first evaluation of the DeepChek^® Assays for targets specific (PR/RT and INT) and whole genome. A cutoff of 3% allowed for a better characterization of the viral population by identifying additional resistance mutations and improving the HIV-1 drug-resistance interpretation. The use of whole-genome sequencing is an additional and complementary tool to detect mutations in newly infected untreated patients and heavily experienced patients, both with higher HIV-1 viral-load profiles, to offer new insight and treatment strategies, especially using the new HIV-1 capsid/maturation inhibitors and to assess the potential clinical impact of mutations in the HIV-1 genome outside of the usual HIV-1 targets (RT/PR and INT). Full article

Introduction: The therapeutic impact of molnupiravir in the Omicron variant phase is unknown. The goal of the current meta-analysis was to compare the real-world clinical outcomes of molnupiravir for the treatment of mild to moderate COVID-19 during the dominance of the Omicron variant in adult patients to that of a placebo. Methods: To be included, studies had to directly compare the clinical effectiveness of molnupiravir in treating adult COVID-19 patients to that of a placebo. Studies were included based on the following outcomes: all-cause mortality, composite outcome of disease progression, hospitalization rate, and viral load. Results: The current meta-analysis included six studies that indicated that the risk of mortality was reduced by 34%, and the risk of composite outcome of disease progression was reduced by 37% among patients who received molnupiravir. Molnupiravir was associated with faster reduction in viral loads than the placebo. There was no clinical benefit of reducing all-cause mortality in mild to moderate COVID-19 patients with high COVID-19 vaccination coverage. Conclusion: The clinical effectiveness of molnupiravir was associated with COVID-19 vaccination coverage in COVID-19 patients. There is a lack of detailed data on its effectiveness in vaccinated patients, especially those with low COVID-19 vaccination coverage. Full article

Rift Valley fever (RVF) is a febrile vector-borne disease endemic in Africa and continues to spread in new territories. It is a climate-sensitive disease mostly triggered by abnormal rainfall patterns. The disease is associated with high mortality and morbidity in both humans and livestock. RVF is caused by the Rift Valley fever virus (RVFV) of the genus Phlebovirus in the family Phenuiviridae. It is a tripartite RNA virus with three genomic segments: small (S), medium (M) and large (L). Pathogen genomic sequencing is becoming a routine procedure and a powerful tool for understanding the evolutionary dynamics of infectious organisms, including viruses. Inspired by the utility of amplicon-based sequencing demonstrated in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Ebola, Zika and West Nile viruses, we report an RVFV sample preparation based on amplicon multiplex polymerase chain reaction (amPCR) for template enrichment and reduction of background host contamination. The technology can be implemented rapidly to characterize and genotype RVFV during outbreaks in a near-real-time manner. To achieve this, we designed 74 multiplex primer sets covering the entire RVFV genome to specifically amplify the nucleic acid of RVFV in clinical samples from an animal tissue. Using this approach, we demonstrate achieving complete RVFV genome coverage even from samples containing a relatively low viral load. We report the first primer scheme approach of generating multiplex primer sets for a tripartite virus which can be replicated for other segmented viruses. Full article