Retrospective screening of routine respiratory samples revealed undetected community transmission and missed intervention opportunities for SARS-CoV-2 in the United Kingdom.

In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on individuals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected individual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV-2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea - also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger sample of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.

Enterovirus D68 epidemic, UK, 2018, was caused by subclades B3 and D1, predominantly in children and adults, respectively, with both subclades exhibiting extensive genetic diversity.

Enterovirus D68 (EV-D68) has recently been identified in biennial epidemics coinciding with diagnoses of non-polio acute flaccid paralysis/myelitis (AFP/AFM). We investigated the prevalence, genetic relatedness and associated clinical features of EV-D68 in 193 EV-positive samples from 193 patients in late 2018, UK. EV-D68 was detected in 83 (58 %) of 143 confirmed EV-positive samples. Sequencing and phylogenetic analysis revealed extensive genetic diversity, split between subclades B3 (n=50) and D1 (n=33), suggesting epidemiologically unrelated infections. B3 predominated in children and younger adults, and D1 in older adults and the elderly (P=0.0009). Clinical presentation indicated causation or exacerbation of respiratory distress in 91.4 % of EV-D68-positive individuals, principally cough (75.3 %), shortness of breath (56.8 %), coryza (48.1 %), wheeze (46.9 %), supplemental oxygen required (46.9 %) and fever (38.9 %). Two cases of AFM were observed, one with EV-D68 detectable in the cerebrospinal fluid, but otherwise neurological symptoms were rarely reported (n=4). Both AFM cases and all additional instances of intensive care unit (ICU) admission (n=5) were seen in patients infected with EV-D68 subclade B3. However, due to the infrequency of severe infection in our cohort, statistical significance could not be assessed.

Human parainfluenza 2 & 4: Clinical and genetic epidemiology in the UK, 2013-2017, reveals distinct disease features and co-circulating genomic subtypes.

BACKGROUND: Human Parainfluenza viruses (HPIV) comprise of four members of the genetically distinct genera of Respirovirus (HPIV1&3) and Orthorubulavirus (HPIV2&4), causing significant upper and lower respiratory tract infections worldwide, particularly in children. However, despite frequent molecular diagnosis, they are frequently considered collectively or with HPIV4 overlooked entirely. We therefore investigated clinical and viral epidemiological distinctions of the relatively less prevalent Orthorubulaviruses HPIV2&4 at a regional UK hospital across four autumn/winter epidemic seasons. METHODS: A retrospective audit of clinical features of all HPIV2 or HPIV4 RT-PCR-positive patients, diagnosed between 1st September 2013 and 12th April 2017 was undertaken, alongside sequencing of viral genome fragments in a representative subset of samples. RESULTS: Infection was observed across all age groups, but predominantly in children under nine and adults over 40, with almost twice as many HPIV4 as HPIV2 cases. Fever, abnormal haematology, elevated C-reactive protein and hospital admission were more frequently seen in HPIV2 than HPIV4 infection. Each of the four seasonal peaks of either HPIV2, HPIV4 or both, closely matched that of RSV, occurring in November and December and preceding that of Influenza A. A subset of viruses were partially sequenced, indicating co-circulation of multiple subtypes of both HPIV2&4, but with little variation between each epidemic season or from limited global reference sequences. CONCLUSIONS: Despite being closest known genetic relatives, our data indicates a potential difference in associated disease between HPIV2 and HPIV4, with more hospitalisation seen in HPIV2 mono-infected individuals, but a greater overall number of HPIV4 cases.

Comparative effects of viral-transport-medium heat inactivation upon downstream SARS-CoV-2 detection in patient samples.

Introduction. The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.Hypothesis. Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.Aim. We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).Methodology. We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.Results. Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically (n=127; R2=0.9259).Conclusion. Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.

Human Bocavirus infection and respiratory tract disease identified in a UK patient cohort.

BACKGROUND: Since its first isolation in 2005, Human Bocavirus (HBoV) has been repeatedly associated with acute respiratory tract infections, although its role in pathogenicity remains unclear due to high co-infection rates. OBJECTIVES: To assess HBoV prevalence and associated disease in a cohort of respiratory patients in the East Midlands, UK between 2015 and 2019. STUDY DESIGN: We initially investigated the undiagnosed burden of HBoV in a retrospective paediatric cohort sampled between 2015 and 2017 using an in-house PCR assay. HBoV was subsequently incorporated into the standard respiratory diagnostic pathway and we audited a calendar year of HBoV positive results between 2018 and 2019. RESULTS: Our retrospective PCR screening of previously routine diagnostic-negative samples from juvenile patients identified a 9% (n = 30) prevalence of HBoV type 1. These apparent HBoV1 mono-infections were frequently associated with respiratory tract symptoms, often severe requiring ventilation, oxygen and steroid intervention with 31% (n = 9) of individuals requiring intensive care. When HBoV screening was subsequently adopted into the routine respiratory diagnostic pathway, year-round infections were observed in both children and adults peaking in February. 185 of 9098 (2.03%) individuals were found to be HBoV positive with children aged 12-24 months the principally infected group. However, HBoV infection was also observed in patients aged over 60, predominantly as a mono-infection. 23% of the 185 unique patients were HBoV monoinfected and persistent low-level DNA positivity was observed in 15 individuals up to 6-months after initial presentation. CONCLUSION: HBoV1 is a prevalent respiratory infection in the UK capable of causing serious monoinfections.

Enterovirus subtyping in a routine UK laboratory setting between 2013 and 2017.

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.