Search Results (3,010)

Affordable, flexible surveillance tools are needed to detect SARS-CoV-2 and other pathogens in wildlife. Standard nucleic acid amplification tests (NAATs) are reliable but restricted to predefined targets, limiting their ability to detect co-infections or emerging pathogens. To address this, we adapted a targeted next-generation sequencing (tNGS) panel for mesocarnivores to the Oxford Nanopore Technologies (ONT) MinION platform and combined it with a SARS-CoV-2 whole-genome sequencing assay. Merging both assays before library preparation enables simultaneous SARS-CoV-2 detection, variant identification, and broader pathogen screening. The MinION platform also improves turnaround time because sequencing can begin immediately on small numbers of samples, reducing costs in low-volume workflows. We converted our validated carnivore tNGS panel from the Ion Torrent system to MinION, optimizing amplification conditions, primer pools, and barcoding for multiplexing. Analytical sensitivity was measured using contrived wildlife samples spiked with serial dilutions of SARS-CoV-2 and tested in parallel with a commercial NAAT. Diagnostic sensitivity was assessed using contrived positives, and specificity was evaluated using NAAT-negative wildlife samples and in silico analyses. All 161 wildlife samples were NAAT-negative. MinION tNGS detected SARS-CoV-2 down to Ct 34 and produced ≥ 99% genome coverage for Ct ≤ 24 while simultaneously identifying additional pathogens. Diagnostic sensitivity and specificity were 96.7% and 100%. This workflow offers a low-cost, scalable approach for comprehensive wildlife pathogen surveillance. Full article

Background: Memory B cells (B_mem) facilitate the generation of renewed and rapid antigen-specific antibody responses long after the initial antigen exposure, at a time when circulating serum antibodies may have declined. As the generation and/or recruitment of B_mem is at the core of most vaccination strategies, the assessment of antigen-specific B_mem is highly informative for forecasting and profiling the elicited B cell immune response. Methods: The two prevalent techniques used to detect antigen-specific B_mem cells at single-cell resolution are probe-based flow cytometry and B cell ImmunoSpot, while the measurement of B cell-derived antibodies in culture supernatants of stimulated B cells offers a semi-quantitative alternative. To the best of our knowledge, a direct side-by-side comparison of these assay systems has not yet been reported using the same starting PBMC material in a blinded fashion to test all three assays simultaneously. Results: These three assay systems were run in parallel to detect SARS-CoV-2 Wuhan-1 strain Spike-specific IgG⁺ B_mem in peripheral blood mononuclear cell (PBMC) samples obtained from well-defined cohorts comprising pre-COVID-19 era “naïve” individuals (negative controls), individuals shortly after recovery from a PCR-verified SARS-CoV-2 infection (positive controls), and a cohort of donor PBMCs isolated in 2024 (the experimental group). Each assay was able to discern Spike-exposed individuals from naïve , with ImmunoSpot suggesting superior sensitivity and specificity. ImmunoSpot and flow cytometry results were closely correlated. Conclusions: The study demonstrates that all three assays are suited for the detection of specific B_mem in antigen-primed individuals when such B_mem occur in the mid- to high-frequency range, and that they broadly concur. Strengths and weaknesses of the three test systems are discussed. Full article

Background/Objectives: The purpose of this study was to identify shared and differing characteristics of individuals testing for both SARS-CoV-2 and MPXV in 2022 in the greater Houston metro area. Methods: Data from the Houston Electronic Disease Surveillance System (HEDSS) identified 7,754,198 SARS-CoV-2 PCR lab results and 1246 MPXVV PCR lab results in 2022. Three cohorts for analysis were created where tests were performed, as follows: those positive for both viruses, those negative for COVID-19 but positive for MPXV, and those positive for COVID-19 but negative for MPXV. Results: We identified 88 individuals positive for both viral infections, those negative for COVID-19 but positive for MPXV (n = 38), and those positive for COVID-19 but negative for MPXV (n = 96). While groups were generally similar in regard to demographics (age, sex, and race) and risk factors reported, key differences in timing of testing and risk factors were reported. Notably, there was statistically significant difference in the time between t-tests for dual-infected individuals (99 days) compared to MPXV-positive only (58 days, p < 0.01) or COVID-19 positive only (63 days, p < 0.01). Conclusions: In the setting of multiple disease outbreaks, the characteristics of infected patients may be largely similar. Some people with dual infection may show unusual test results or symptom patterns compared with those with only one infection. Large public health studies with robust reporting systems and laboratory screening are vital for early detection of dual infections. Public health strategies to educate providers and outreach teams enhance response during concurrent outbreaks. Further research is needed on behavior and risk factors in communities with simultaneous outbreaks. Full article

Background/Objectives: Notwithstanding the declaration by the World Health Organization in May 2023 regarding the conclusion of the COVID-19 pandemic, new cases of this potentially lethal infection continue to be documented globally, exerting a sustained influence on the worldwide economy and social structures. Contemporary SARS-CoV-2 variants, while associated with a reduced propensity for severe acute pathology, retain the capacity to induce long-term post-COVID syndrome, including in ambulatory patient populations. This clinical phenomenon may be attributable to potential autoimmune reactions hypothetically triggered by antiviral antibodies, thereby underscoring the need for developing novel, universal vaccines against COVID-19. The nucleocapsid protein (N), being one of its most conserved and highly immunogenic components of SARS-CoV-2, presents a promising target for such investigative efforts. However, the protective role of anti-N antibodies, generated during natural infection or through immunization with N-based vaccines, alongside the potential adverse effects associated with their production, remains to be fully elucidated. In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits, and hamsters. Methods: We employed Western blot analysis of lysates from human cell lines (MCF7, HEK293T, THP-1, CaCo2, Hep2, T98G, A549) coupled with mass spectrometric identification to assess the cross-reactivity of polyclonal and monoclonal antibodies generated against recombinant SARS-CoV-2 N protein with human self-antigens. Results: We showed that anti-N antibodies developed in mice and rabbits exhibit pronounced immunoreactivity towards specific components of the human proteome. In contrast, anti-N immunoglobulins from hamsters showed no non-specific cross-reactivity with either hamster or human proteomic extracts because of the lack of autoreactivity or immunogenicity differences. Subsequent mass spectrometric analysis of the immunoreactive bands identified principal autoantigenic targets, which were predominantly heat shock proteins (including HSP90-beta, HSP70, mitochondrial HSP60, and HSPA8), histones (H2B, H3.1–3), and key metabolic enzymes (G6PD, GP3, PKM, members of the 1st family of aldo-keto reductases). Conclusions: The results obtained herein highlight the differences in the development of anti-N humoral responses in humans and in the Syrian hamster model. These data provide a foundational basis for formulating clinical recommendations to predict possible autoimmune consequences in COVID-19 convalescents and are of critical importance for the rational design of future N protein-based, cross-protective vaccine candidates against novel coronavirus infections. Full article

The emergence and spread of coronavirus infections have created a necessity to develop serological methods for assessing population immunity. The enzyme-linked immunosorbent assay (ELISA) remains one of the most accessible and informative approaches for these purposes. The choice of recombinant proteins plays an important role in the sensitivity and specificity of the test system, and in this regard, the creation of a domestic ELISA based on the chimeric SM protein to the SARS-CoV-2 virus is relevant. In this work, a recombinant chimeric SM protein expressed in the E. coli system and purified using metal-affinity chromatography on Ni-NTA agarose was constructed and presented for the first time. An ELISA test system was developed and tested using panels of positive and negative sera, including samples obtained before the COVID-19 pandemic. The obtained sensitivity (90.48%) and specificity (93.65%) indicators with a ROC curve AUC = 0.9623 (OD450 = 0.213) indicate the diagnostic accuracy of the test system. The positive diagnostic ratio (LR+) = 14.25.0 indicates the reliability of a positive result. The domestically developed ELISA test system can be used for serological monitoring and assessment of the immune status of the population. Full article

Accurate and sensitive antibody detection remains critical for advanced COVID-19 diagnostics and monitoring SARS-CoV-2 immunity. This study presents a highly sensitive technique for detecting anti-SARS-CoV-2 antibodies in human serum using an integrated photonic sensing platform. The platform utilizes disposable one-dimensional photonic crystal biochips engineered to sustain Bloch Surface Waves. The biochips are integrated into a custom-made optical set-up, which is capable of dual-mode detection: label-free refractometry and label-based fluorescence. Tests on human serum, including negative controls and positive samples from a recovered COVID-19 patient, confirmed the platform’s effective performance. In fluorescence mode, clear discrimination between positive and negative samples was achieved down to a 1:10⁴ serum dilution, with an optimal operating range centered around 1:10³ dilution. These results demonstrate the potential of the technique as a highly sensitive and versatile platform for antibody detection, with significant relevance for advanced COVID-19 diagnostics. Full article

Background: Rapid pathogen detection is crucial for the timely containment of outbreaks, particularly for respiratory infectious diseases which are highly transmissible and possess high epidemic potential. Methods: We developed a sensitive reverse transcription recombinase-aided amplification (RT-RAA) assay for the rapid detection of six common respiratory viruses: respiratory syncytial virus type A (RSV A), influenza A virus (Flu A), influenza B virus (Flu B), human parainfluenza virus (HPIV), SARS-CoV-2 and adenovirus (ADV). The assay employs a single, standardized protocol for the on-demand detection of any one of the six targets. Its performance was validated using nucleic acid standards and clinical pharyngeal swab specimens. Results: The assay enables rapid detection within 20 min at 39 °C using a portable, self-powered device. It demonstrated high sensitivity, with detection limits below 10³ copies/mL for all targets and as low as 10¹ copies/mL for ADV. Cross-reactivity testing with 21 other pathogens confirmed excellent specificity. Validation with 85 clinical samples showed 100% concordance with RT-PCR, while offering significantly faster results and enhanced portability compared to RT-PCR. Conclusions: This sensitive, specific, and user-friendly RT-RAA assay provides a robust tool for rapid detection of respiratory viruses, particularly suitable for deployment in resource-limited settings and point-of-care testing during outbreaks. Full article

Background/Objectives: SARS-CoV-2 infection has been linked to long-term neurological sequelae and structural brain alterations. Previous analyses, including baseline results from the COVIMMUNE-Clin study, showed brain volume reductions in COVID-19 patients. Longitudinal data on progression are scarce. This study examined brain volume changes 12 months after baseline MRI in individuals who have recovered from mild or severe COVID-19 compared with controls. Methods: In this IRB-approved cohort study, 112 out of 172 recruited age- and sex-matched participants (38 controls, 36 mild/asymptomatic 38 severe COVID-19) underwent standardized brain MRI 12 months after baseline. Volumetric analysis was performed using AI-based software (mdbrain). Regional volumes were compared between groups with respect to absolute and normalized values. Multivariate regression controlled for demographics. Results: After 12 months, a significant decline in right hippocampal volume was observed across all groups, most pronounced in severe COVID-19 (SEV: Δ = −0.32 mL, p = 0.001). Normalized to intracranial volume, the reduction remained significant (SEV: Δ = −0.0003, p = 0.001; ASY: Δ = −0.0001, p = 0.001; CTL: minimal reduction, Δ ≈ 0, p = 0.005). Minor reductions in frontal and parietal lobes (e.g., right frontal SEV: Δ = −1.35 mL, p = 0.001), largely fell within physiological norms. These mild regional changes are consistent with expected ageing-related variability and do not suggest pathological progression. No widespread progressive atrophy was detected. Conclusions: This study demonstrates delayed, severity-dependent right hippocampal atrophy in recovered COVID-19 patients, suggesting long-term vulnerability of this memory-related region. In contrast, no progression of atrophy in other areas was observed. These findings highlight the need for extended post-COVID neurological monitoring, particularly of hippocampal integrity and its cognitive relevance. Full article

Background: The continuous emergence of SARS-CoV-2 variants represents a major public health concern. Next-generation sequencing (NGS) enables genomic surveillance, facilitating the detection and monitoring of mutations that impact viral evolution. Methods: In this study, full-length SARS-CoV-2 genomes were analyzed between February 2022 and March 2024 as part of routine genomic surveillance conducted in Verona, Italy. Mutations in the envelope (E), membrane (M), and nucleocapsid (N) structural proteins were investigated. Only substitutions with a total prevalence of greater than 1% in the study dataset were considered. Results: A total of 178 mutations were identified across the three proteins (E: 16; M: 33; N: 129), of which 18 met the inclusion threshold (E: 3; M: 5; N: 10). Mutations were classified according to temporal dynamics as fixed, emerging, or transient. Throughout the study period, fixed mutations were consistently prevalent, emerging mutations appeared later but persisted with an ascending trend, while transient mutations displayed a single frequency peak before disappearing. Several mutations were reported with potential structural or functional relevance based on the existing literature, while others remain of unknown significance. Conclusions: The mutational patterns detected in this study broadly reflect global evolutionary trends of SARS-CoV-2. These findings emphasize the importance of continued genomic surveillance and underline the need for integrated experimental approaches to clarify the biological and epidemiological impact of poorly characterized mutations. Full article

Wastewater-based surveillance is an effective method to monitor community health by detecting pathogens excreted from large populations. Traditionally, wastewater analyses are considered privacy-preserving since samples are pooled. However, advances in molecular resolution may challenge this assumption. This study investigated whether a rare SARS-CoV-2 variant detected in a hospital sewershed could be linked to a specific patient. During 2023, wastewater samples were collected weekly and analyzed for SARS-CoV-2 concentrations and variant composition using droplet digital PCR (ddPCR) and tiled sequencing. During the same period, nasal swab samples from hospital patients were sequenced for comparison. While wastewater was dominated by Omicron lineages, several weeks showed a sharp increase in SARS-CoV-2 concentrations, with over 80% belonging to the Delta lineage AY.25—rare globally since 2022. Nasal swab sequencing identified one patient carrying AY.25, whose admission and discharge coincided with the emergence and disappearance of AY.25 in the hospital’s wastewater. The temporal pattern and sequence similarity suggest that the Delta signal might have originated from this single patient, with sequence differences potentially reflecting intrahost divergence. These findings indicate that, under certain conditions, wastewater signals can be attributed to individuals, highlighting the need to keep in mind the ethical considerations surrounding privacy and data use while benefiting the community through wastewater surveillance. Full article

Introduction: The contribution of SARS-CoV-2 infection to the severity of placental alterations in preeclampsia remains unclear. This study aimed to evaluate the morphological changes in placentas of women who experienced COVID-19 during pregnancy, with a focus on the presence or absence of preeclampsia. Materials and Methods: The study included placentas from: (1) patients with both COVID-19 during pregnancy and preeclampsia (n = 20, 2022); (2) patients with COVID-19 during pregnancy without preeclampsia (n = 20, 2022); (3) patients with preeclampsia but without COVID-19 (n = 5, 2019); (4) patients with physiological pregnancies without COVID-19 or gestational complications (n = 5, 2019). Histological and immunohistochemical examinations of the placentas were performed using antibodies against the SARS-CoV-2 spike protein, DPP4 (CD26), and VEGF. Results: Placentas from patients with both COVID-19 and preeclampsia demonstrated the most pronounced stromal and vascular alterations, including pseudo-infarctions and villous fibrosis. Chorangiosis, excessive fibrinoid deposition in the intervillous space, and accelerated villous maturation with an increased number of syncytial knots were more common in the preeclampsia groups, regardless of prior COVID-19 infection. Symptomatic forms of coronavirus infection were associated with more severe manifestations of malperfusion. Expression of the SARS-CoV-2 spike protein was detected in 78% of syncytiotrophoblast cells and 37% of decidual cells in 28 of 40 placentas from women with previous COVID-19, while its presence in the vascular endothelium, macrophages, and villous fibroblasts was focal, as was CD26 expression. VEGF expression did not differ significantly between patients with and without COVID-19. Conclusions: COVID-19 is associated with more pronounced stromal–vascular alterations in the placenta; however, not all of these changes are directly caused by the virus itself but rather reflect the severe course of preeclampsia. Inflammatory alterations are nonspecific for COVID-19, even though CD26 and the SARS-CoV-2 spike protein are detectable in nearly all placental structures, whereas VEGF levels remain comparable to those observed in placentas prior to the coronavirus pandemic. Full article

Background: Acute Respiratory Infections are a common reason for Pediatric Emergency Department (PED) visits. Differentiating bacterial and viral infections may be challenging and might result in incorrect antibiotic prescriptions and exacerbation of antimicrobial resistance. This study evaluated the impact of new diagnostic tests in PED. Methods: A retrospective cohort of 4882 acute febrile respiratory infection cases presenting to the PED was analyzed, comparing two periods: Period 1 (October 2016–March 2017, n = 2181) and Period 2 (October 2023–March 2024, n = 2701). During Period 1, Group A Streptococcus and Respiratory Syncytial Virus rapid antigen detection tests were available. During Period 2, new point-of-care tests (POCTs), including rapid C-reactive protein and rapid antigen detection for Influenza A, Influenza B, and SARS-CoV-2, and a multiplex PCR nasal swab, were introduced. Results: In Period 2, antibiotic prescriptions decreased by 28.4%, along with a reduction in broad-spectrum antibiotic use. A significant correlation was observed between reduced antibiotic prescription and the use of new POCTs and multiplex PCR tests. Performance of blood tests and chest radiographs also decreased. Conclusions: Implementing novel diagnostic tests in PED helps clinicians select more appropriate management options with an impact on reduced stress and radiation exposure and antibiotic prescription. Full article

Background: Rapid and high-throughput diagnostic methods are essential for controlling the spread of infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Lateral flow immunoassay (LFIA) strips provide a cost-effective and user-friendly platform for point-of-care testing. However, the sensitivity of conventional LFIA kits is often limited by the performance of their detection probes. This study reports a highly sensitive LFIA strip for detecting the SARS-CoV-2 nucleocapsid (NP) protein using platinum-decorated poly(2-vinylpyridine) nanoparticles (Pt-P2VPs) as probes. Methods: Monoclonal antibodies against SARS-CoV-2 NP were conjugated with Pt-P2VPs and incorporated into LFIA strips. The test line was coated with anti–SARS-CoV-2 NP monoclonal antibody, and the control line with goat anti-mouse IgG. Recombinant proteins, viral strains, and nasopharyngeal swab specimens from patients were used to evaluate assay performance, with reverse transcription polymerase chain reaction (RT-PCR) as the reference standard. Diagnostic accuracy was assessed using nonparametric statistical tests. Results: Pt-P2VP-based LFIA strips enabled sensitive detection of recombinant NP and inactivated SARS-CoV-2, with minimal cross-reactivity. In 200 clinical specimens (100 PCR-negative and 100 PCR-positive), the assay achieved 74% sensitivity and 100% specificity, with strong correlation to viral RNA load. Compared with conventional LFIA kits, Pt-P2VP strips demonstrated superior sensitivity at lower viral loads. Conclusions: Pt-P2VPs represent a promising probe material for enhancing LFIA performance and may facilitate the development of rapid, sensitive, and scalable immunoassays for infectious disease diagnostics in biomedical applications. Full article

During the COVID-19 pandemic, the global focus on SARS-CoV-2 overshadowed the epidemiology of other respiratory pathogens. This study aimed to characterize the circulation of endemic human coronaviruses (HCoVs) in Brazil. We retrospectively analyzed results from 22,472 PCR tests for HCoVs (from 5183 patients) and 601,278 tests for SARS-CoV-2 (from 475,856 patients) between November 2019 and June 2021. HCoVs were detected in 160 patients (3.09%), with HCoV-NL63 as the most frequent species. HCoV circulation was intermittent, with positivity peaks up to 4% but also periods of up to six months with an absence of detections in 2020, contrasting with the sustained high positivity of SARS-CoV-2 (22.37%). Co-infections were frequent: 26.25% of HCoV-positive patients were co-infected with at least one other respiratory pathogen, most commonly Rhinovirus/Enterovirus, and cases involving up to five pathogens were observed, seven patients had co-infections between HCoVs and SARS-CoV-2. These findings reveal the persistent, often cryptic, circulation of HCoVs during the pandemic and highlight their role as key components in complex multi-pathogen infections. This underscores the critical importance of implementing comprehensive molecular diagnostic panels in routine respiratory surveillance to ensure accurate etiology, guide appropriate clinical management, and fully assess the public health burden of non-SARS-CoV-2 coronaviruses. Full article

The coronavirus disease 2019 (COVID-19) pandemic highlighted the critical need for scalable, timely, and unbiased methods to monitor disease transmission at the population level. Wastewater-based epidemiology (WBE) provides an effective method for monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission by detecting viral RNA shed into the sewage system. Because it does not rely on individual testing, WBE can offer timely, cost-effective, and community-level insights into infection trends. In this study, we present a hierarchical Beta-Binomial model that integrates SARS-CoV-2 RNA concentration in wastewater with reported COVID-19 case counts to enhance the monitoring of community-level transmission dynamics. The model incorporates wastewater viral loads as a predictor and reported cases as the response, while adjusting for testing volume to account for biases introduced by fluctuations in testing practices. This approach enables reliable estimation of the effective reproduction number ($R_t$), even in the absence of consistent reporting of clinical data. Applied to twenty counties in California, our modeling framework demonstrates the potential of wastewater surveillance to inform public health decision making, particularly in locations with sparse clinical data. Full article