Biology subjects

Hayes, P.

Publications and source records attributed to Hayes, P..

4 recordsLinked to original sources

bioRxiv2025

Development and qualification of an enzyme-linked immunosorbent assay to detect human serum immunoglobulin G reactive to multiple lineages of Lassa virus nucleoprotein

Lassa fever is a severe, often fatal febrile illness endemic to West Africa caused by Lassa virus (LASV). The viral nucleoprotein (NP) is a target antigen for serological assays to identify previous exposure to LASV. To our knowledge, there is no commercially available assay that reliably quantifies anti-LASV-NP IgG antibodies in human serum. We report the development and qualification of an ELISA designed to detect and quantify anti-LASV-NP IgG in human serum samples. The assay employs recombinant Lineage IV LASV-NP immobilized on microwells to capture NP-specific IgG antibodies, which are then detected using horseradish peroxidase-conjugated anti-human IgG followed by TMB substrate and reading of optical densities. Optimal assay reagent concentrations, incubation times and temperature were determined along with assay positivity criteria and dynamic range. A reference standard prepared from pooled sera from donors in endemic Lassa fever regions was established and calibrated to the first WHO international standard for LASV antibodies. High and low positive controls for assay quality control were generated. Naive human serum was used as a negative control. Following assay optimization, performance was assessed through assay qualification. The assay positivity criteria, lower limit of detection, upper and lower limits of quantitation, inter-assay precision, selectivity and dilutional linearity were determined. Our anti-LASV-NP IgG ELISA was shown to reliably measure anti-LASV-NP IgG levels in human serum. The method demonstrated sensitivity, intra- and inter-assay precision, dilution linearity across its analytical range and specificity for anti-LASV-NP IgG. Establishing this assay represents an essential step toward strengthening LASV epidemiology research and supporting urgently needed development of a vaccine to prevent Lassa Fever.

immunology↗

bioRxiv2025

Population structure, antimicrobial resistance, and virulence factors of diabetic foot-associated E. coli

Diabetic foot infections (DFI) are a major complication of diabetes, often leading to lower limb amputations. Escherichia coli is a predominant Gram-negative pathogen in DFI, yet its genomic and pathogenic features remain poorly characterised. Here, we present a whole genome sequence-based analysis of diabetic foot-associated E. coli (DFEC) isolates from diverse geographical locations. Phylogenetic reconstruction revealed substantial diversity, with strains spanning seven phylogroups and 28 sequence types. Capsule biosynthesis loci linked to invasive infections, such as K1, K2ab, and K5, were also detected. The DFEC pangenome comprised 18,263 gene clusters, indicating high genomic plasticity. The plasmid repertoire was also varied and contributed to the genomic diversity of the strains. Approximately 78% of isolates were multidrug-resistant (MDR) or extensively drug-resistant (XDR), with resistance to last-resort antibiotics such as colistin and carbapenems also observed. High frequencies of virulence factors involved in host cell adherence, iron metabolism, serum survival, as well as toxins and type 3 secretion system (T3SS) genes were also detected. In contrast, metabolic modelling showed conserved biochemical profiles. Clustering based on accessory metabolic functions did not mirror phylogeny, suggesting metabolic convergence among distinct lineages. Collectively, these findings reveal that DFEC are versatile pathogens with a repertoire of antimicrobial resistance and virulence determinants. These traits make them functionally distinct from commensal E. coli strains and highlight the potential of DFEC to cause severe and invasive infections. ImportanceThis study presents the first multisite genomic characterisation of diabetic foot-associated Escherichia coli (DFEC). Our findings reveal that DFEC strains are phylogenetically diverse and span multiple lineages. The high prevalence of multidrug-resistant and extensively drug-resistant genotypes underscores the underestimated antimicrobial resistance (AMR) threat posed by DFEC. We detect high frequencies of virulence factors commonly associated with extraintestinal pathogenic E. coli (ExPEC), which indicates that DFEC might have the potential to cause severe complications, such as sepsis. The large accessory genome and evidence of metabolic convergence across distinct lineages highlight the adaptive versatility of DFEC in the polymicrobial and inflammatory environment of chronic wounds. These insights advance our understanding of DFEC pathobiology and support the development of targeted diagnostics, AMR surveillance, and therapeutic strategies to improve clinical outcomes for diabetic patients.

microbiology↗

bioRxiv2022

The Free Association Sessions: Perspectives on a Novel Teaching Platform by Final Year Medical Students and Basic Specialist Trainees in Psychiatry.

IntroductionNotwithstanding the many advantages of outcomes-based education within Psychiatry placements in Medical School and Basic Specialist Training within the College of Psychiatry of Ireland, there is limited protected time for broad Reflective Practice that appreciates the complexity of working in the Mental Health setting. Furthermore, there are limitations to the current model of restricting Reflective Practice sessions to the Balint Group Format. MethodsA novel programme of structured Reflective Practice was offered to students in the School of Medicine in The University of Limerick and trainees in the Mid-West Deanery. Six student and fourteen trainee participants were subsequently invited to provide perspectives on the programme via Focus Groups. The study employed an inductive latent phenomenological approach for analysis of qualitative data. ResultsFive major themes emerged: These related to the teaching environment, personal identity, complexity, awareness of cognitive dissonance and the structure of the sessions. There were a number of different perceptions, relating to the participants stage of training. ConclusionsThere was evidence that the structured reflective sessions created a comfortable environment, addressed hierarchy issues and facilitated wide-ranging opportunities for reflective practice, with an observed increased appreciation for complexity in Psychiatry. There was apparent tension between controlling content and facilitating the form of group process. Nonetheless, the structure appeared more approachable for medical students and those early in training.

scientific communication and education↗

bioRxiv2018

Performance of an IAVI-African Network of Clinical Research Laboratories in Standardized ELISpot and Peripheral Blood Mononuclear Cell Processing in Support of HIV Vaccine Clinical Trials

Immunological assays performed in different laboratories participating in multi-centre clinical trials must be standardized in order to generate comparable and reliable data. This entails standardized procedures for sample collection, processing, freezing and storage. The International AIDS Vaccine Initiative (IAVI) partnered with local institutions to establish Good Clinical Laboratory Practice (GCLP)-accredited laboratories to support clinical trials in Africa, Europe and Asia. Here we report on the performance of seven laboratories based in Africa and Europe in the interferon-gamma enzyme-linked immunospot (IFN-{gamma} ELISpot) assay and peripheral blood mononuclear cell (PBMC) processing over four years. Characterized frozen PBMC samples from 48 volunteer blood packs processed at a central laboratory were sent to participating laboratories. For each stimulus, there were 1751 assays performed over four years. 98% of these ELISpot data were within acceptable ranges with low responses to mock stimuli. There were no significant differences in ELISpot responses at five laboratories actively conducting immunological analyses in support of IAVI sponsored clinical trials or HIV research. In a separate study, 1,297 PBMC samples isolated from healthy HIV-1 negative participants in clinical trials of two prophylactic HIV vaccine candidates were analysed for PBMC yield from fresh blood and cell recovery and viability following freezing and thawing. 94 % and 96 % of samples had fresh PBMC viabilities and cell yields within the pre-defined acceptance criteria while for frozen PBMC, 99 % and 96 % of samples had acceptable viabilities and cell recoveries respectively, along with acceptable ELISpot responses in 95%. These findings demonstrate the competency of laboratories across different continents to generate comparable and reliable data in support of clinical trials.\n\nImportanceThere is a need for the establishment of an African network of laboratories to support large clinical trials across the continent to support and further the development of vaccine candidates against emerging infectious diseases such as Ebola, Zika and dengue viruses and the continued HIV-1 pandemic. This is particularly true in sub-Saharan Africa where the HIV-1 pandemic is most severe. In this report we have demonstrated by using standardized SOPs, training, equipment and reagents that GCLP-accredited clinical trial laboratories based in Africa and Europe can process clinical trial samples and maintain cell integrity and functionality demonstrated by IFN-{gamma} ELISpot testing, producing comparable and reliable data.

immunology↗