Hannah C Howson-Wells1, Stephen Winckles2, Camille Aliker2, Alexander W Tarr3, William L Irving4, Gemma Clark1, C Patrick McClure5. 1. Clinical Microbiology, Nottingham University Hospitals NHS Trust, Nottingham, NG7 2UH, United Kingdom. 2. Life Sciences, University of Nottingham, Nottingham, NG7 2UH, United Kingdom. 3. Life Sciences, University of Nottingham, Nottingham, NG7 2UH, United Kingdom; NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, University of Nottingham, United Kingdom. 4. Clinical Microbiology, Nottingham University Hospitals NHS Trust, Nottingham, NG7 2UH, United Kingdom; Life Sciences, University of Nottingham, Nottingham, NG7 2UH, United Kingdom; NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, University of Nottingham, United Kingdom. 5. Life Sciences, University of Nottingham, Nottingham, NG7 2UH, United Kingdom; NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, University of Nottingham, United Kingdom. Electronic address: patrick.mcclure@nottingham.ac.uk.
Abstract
BACKGROUND: Human enteroviruses (EV) are the leading cause of viral meningitis. EV genotyping is predominantly performed through amplification and sequencing of viral capsid protein-1 (VP1), frequently by national reference laboratories (NRLs). OBJECTIVE: To determine the frequency of genotyping failure in our NRL-submitted samples and apply a superior alternative assay to resolve untyped specimens. STUDY DESIGN: We initially audited genotyping data received for a cohort of patients in the East Midlands, UK by the NRL between 2013 and 2017, then identified an alternative RT-PCR typing method by literature review and evaluated primers from both assays in silico against comprehensive publicly available genomic data. The alternative assay was further optimised and applied to archived nucleic acids from previously untypable samples. RESULTS: Genotyping data showed a significant increase in untypable EV strains through the study period (p = 0.0073). Typing failure appeared unrelated to sample type or viral load. In silico analyses of 2,201 EV genomes showed high levels of mismatch between reference assay primers and clinically significant EV-species, in contrast to a selected alternative semi-nested RT-PCR VP1-typing assay. This alternative assay, with minor modifications, successfully genotyped 23 of 24 previously untypable yet viable archived specimens (EV-A, n = 4; EV-B, n = 19). Phylogenetic analyses identified no predominant strain within NRL untypable isolates, suggesting sub-optimal reference assay sensitivity across EV species, in agreement with in silico analyses. CONCLUSION: This modified highly sensitive RT-PCR assay presents a suitable alternative to the current English national reference VP1-typing assay and is recommended in other settings experiencing typing failure.
BACKGROUND:Human enteroviruses (EV) are the leading cause of viral meningitis. EV genotyping is predominantly performed through amplification and sequencing of viral capsid protein-1 (VP1), frequently by national reference laboratories (NRLs). OBJECTIVE: To determine the frequency of genotyping failure in our NRL-submitted samples and apply a superior alternative assay to resolve untyped specimens. STUDY DESIGN: We initially audited genotyping data received for a cohort of patients in the East Midlands, UK by the NRL between 2013 and 2017, then identified an alternative RT-PCR typing method by literature review and evaluated primers from both assays in silico against comprehensive publicly available genomic data. The alternative assay was further optimised and applied to archived nucleic acids from previously untypable samples. RESULTS: Genotyping data showed a significant increase in untypable EV strains through the study period (p = 0.0073). Typing failure appeared unrelated to sample type or viral load. In silico analyses of 2,201 EV genomes showed high levels of mismatch between reference assay primers and clinically significant EV-species, in contrast to a selected alternative semi-nested RT-PCR VP1-typing assay. This alternative assay, with minor modifications, successfully genotyped 23 of 24 previously untypable yet viable archived specimens (EV-A, n = 4; EV-B, n = 19). Phylogenetic analyses identified no predominant strain within NRL untypable isolates, suggesting sub-optimal reference assay sensitivity across EV species, in agreement with in silico analyses. CONCLUSION: This modified highly sensitive RT-PCR assay presents a suitable alternative to the current English national reference VP1-typing assay and is recommended in other settings experiencing typing failure.
Authors: Hannah C Howson-Wells; Theocharis Tsoleridis; Izzah Zainuddin; Alexander W Tarr; William L Irving; Jonathan K Ball; Louise Berry; Gemma Clark; C Patrick McClure Journal: Microb Genom Date: 2022-05