Transitioning from Triton X-100 to Tergitol 15-S-9: impacts on diagnostic assays using viral PCR sample solution.

In 2019, the European Union banned Triton X-100, a detergent widely used in laboratory diagnostics, including the Viral PCR Sample Solution (VPSS), and urged manufacturers to find environmentally sustainable alternatives. Tergitol 15-S-9 (VPSS2) has been proposed as an alternative surfactant. This multicenter study evaluated the effectiveness of VPSS2, a Tergitol-based viral solution, as a replacement for VPSS. Our results show the equivalent performance of VPSS2 to VPSS for nucleic acid extraction and viral stability over time at different temperatures. The new VPSS formulation was also tested against external quality assurance panels and clinical samples. The results of this work support adopting this modified viral PCR sample solution to replace Triton X-100-containing viral transport solutions.

Adeno-associated virus 2 infection in children with non-A-E hepatitis.

An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland1 in April 2022 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4+ T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10-12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a 'helper virus' to support AAV2 replication) and disease susceptibility related to HLA class II status.

Impact of swab removal in the detection of SARS-CoV-2 weakly-positive specimens.

Removing the swab after collection can speed up diagnosis and improve the quality of laboratory procedures. This study investigates the impact of swab removal on SARS-CoV-2 detection in clinical specimens with a focus on high Cycle threshold (Ct) samples (Cts≥32). The method assessed pairs of SARS-CoV-2 samples mimicking combined throat and nose swabs and tested them on two real-time-PCR platforms; the Applied Biosystems 7500 and the Abbott Alinity. Swab removal did not significantly affect detection rates of SARS-CoV-2 samples with Ct values<32, regardless of the PCR platform. However, reduced reproducibility was seen at the endpoint limit of detection of the platforms, which meant that fewer samples with Ct values≥32 were detected in the swab removal group.

Viral Coinfections in Hospitalized Coronavirus Disease 2019 Patients Recruited to the International Severe Acute Respiratory and Emerging Infections Consortium WHO Clinical Characterisation Protocol UK Study.

Background: We conducted this study to assess the prevalence of viral coinfection in a well characterized cohort of hospitalized coronavirus disease 2019 (COVID-19) patients and to investigate the impact of coinfection on disease severity. Methods: Multiplex real-time polymerase chain reaction testing for endemic respiratory viruses was performed on upper respiratory tract samples from 1002 patients with COVID-19, aged <1 year to 102 years old, recruited to the International Severe Acute Respiratory and Emerging Infections Consortium WHO Clinical Characterisation Protocol UK study. Comprehensive demographic, clinical, and outcome data were collected prospectively up to 28 days post discharge. Results: A coinfecting virus was detected in 20 (2.0%) participants. Multivariable analysis revealed no significant risk factors for coinfection, although this may be due to rarity of coinfection. Likewise, ordinal logistic regression analysis did not demonstrate a significant association between coinfection and increased disease severity. Conclusions: Viral coinfection was rare among hospitalized COVID-19 patients in the United Kingdom during the first 18 months of the pandemic. With unbiased prospective sampling, we found no evidence of an association between viral coinfection and disease severity. Public health interventions disrupted normal seasonal transmission of respiratory viruses; relaxation of these measures mean it will be important to monitor the prevalence and impact of respiratory viral coinfections going forward.

Characterisation of a Hepatitis C Virus Subtype 2a Cluster in Scottish PWID with a Suboptimal Response to Glecaprevir/Pibrentasvir Treatment.

Direct-acting antivirals (DAAs) have revolutionised the treatment of Hepatitis C virus (HCV), allowing the World Health Organisation (WHO) to set a target of eliminating HCV by 2030. In this study we aimed to investigate glecaprevir and pibrentasvir (GP) treatment outcomes in a cohort of patients with genotype 2a infection. METHODS: Clinical data and plasma samples were collected in NHS Greater Glasgow & Clyde. Next generation whole genome sequencing and replicon assays were carried out at the MRC-University of Glasgow Centre for Virus Research. RESULTS: 132 cases infected with genotype 2a HCV were identified. The SVR rate for this group was 91% (112/123) following treatment with GP. An NS5A polymorphism, L31M, was detected in all cases of g2a infection, and L31M+R353K in individuals that failed treatment. The results showed that R353K was present in 90% of individuals in the Glasgow genotype 2a phylogenetic cluster but in less than 5% of all HCV subtype 2a published sequences. In vitro efficacy of pibrentasvir against sub-genomic replicon constructs containing these mutations showed a 2-fold increase in IC50 compared to wildtype. CONCLUSION: This study describes a cluster of HCV genotype 2a infection associated with a lower-than-expected SVR rate following GP treatment in association with the NS5A mutations L31M+R353K.

Seoul Virus Associated with Pet Rats, Scotland, UK, 2019.

We describe a case of hemorrhagic fever with renal syndrome caused by Seoul virus in a woman in Scotland, UK. Whole-genome sequencing showed the virus belonged to a lineage characterized by recent international expansion, probably driven by trade in pet rats.

Genetic epidemiology of SARS-CoV-2 transmission in renal dialysis units - A high risk community-hospital interface.

OBJECTIVES: Patients requiring haemodialysis are at increased risk of serious illness with SARS-CoV-2 infection. To improve the understanding of transmission risks in six Scottish renal dialysis units, we utilised the rapid whole-genome sequencing data generated by the COG-UK consortium. METHODS: We combined geographical, temporal and genomic sequence data from the community and hospital to estimate the probability of infection originating from within the dialysis unit, the hospital or the community using Bayesian statistical modelling and compared these results to the details of epidemiological investigations. RESULTS: Of 671 patients, 60 (8.9%) became infected with SARS-CoV-2, of whom 16 (27%) died. Within-unit and community transmission were both evident and an instance of transmission from the wider hospital setting was also demonstrated. CONCLUSIONS: Near-real-time SARS-CoV-2 sequencing data can facilitate tailored infection prevention and control measures, which can be targeted at reducing risk in these settings.

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland.

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity.

Prevalence of pre-treatment hepatitis C virus NS5A resistance associated amino-acid substitutions in genotype 1A infected patients in Scotland.

BACKGROUND: Hepatitis C (HCV) NS5A resistance associated amino-acid substitutions (RAS) can exist at baseline in treatment naïve individuals and have been shown to be associated with lower rates of sustained virological response (SVR) for patients infected with HCV genotype 1A (G1A) following treatment with NS5A inhibitors. OBJECTIVES: The aim of this study was to measure the prevalence of baseline NS5A resistance in Scotland. STUDY DESIGN: The study population consisted of 531 treatment naïve, G1A infected patients. The patient samples were collected between March and September 2017. The NS5A region was amplified and sequenced. RESULTS: Baseline NS5A resistance in Scotland is high (16.8%) and is comparable to rates reported by a number of previously published studies. The high rate of baseline RAS, together with the high cost of direct-acting antivirals (DAAs), supports resistance testing to guide current patient treatment. However, given the rate at which new DAAs are currently being licensed with ever broader genotype efficacy and higher SVR rates, baseline resistance testing may not be required in the near future. CONCLUSIONS: Baseline NS5A inhibitor resistance is high. The results of the present study support performing resistance testing at baseline for current regimens.

Integrating patient and whole-genome sequencing data to provide insights into the epidemiology of seasonal influenza A(H3N2) viruses.

Genetic surveillance of seasonal influenza is largely focused on sequencing of the haemagglutinin gene. Consequently, our understanding of the contribution of the remaining seven gene segments to the evolution and epidemiological dynamics of seasonal influenza is relatively limited. The increased availability of next-generation sequencing technologies allows rapid and economic whole-genome sequencing (WGS) of influenza virus. Here, 150 influenza A(H3N2) positive clinical specimens with linked epidemiological data, from the 2014/15 season in Scotland, were sequenced directly using both Sanger sequencing of the HA1 region and WGS using the Illumina MiSeq platform. Sequences generated by the two methods were highly correlated, and WGS provided on average >90 % whole genome coverage. As reported in other European countries during 2014/15, all strains belonged to genetic group 3C, with subgroup 3C.2a predominating. Multiple inter-subgroup reassortants were identified, including three 3C.3 viruses descended from a single reassortment event, which had persisted in the population. Cases of severe acute respiratory illness were significantly clustered on phylogenies of multiple gene segments indicating potential genetic factors warranting further investigation. Severe cases were also more likely to be associated with reassortant viruses and to occur later in the season. These results suggest that WGS provides an opportunity to develop our understanding of the relationship between the influenza genome and disease severity and the epidemiological consequences of within-subtype reassortment. Therefore, increased levels of WGS, linked to clinical and epidemiological data, could improve influenza surveillance.

The importance of set up time and temperature in real-time PCR; an essential reminder.

BACKGROUND: Non-specific amplification can arise in real-time PCR when temperatures are above 4°C during PCR set up. Pressure of high throughput tests, particularly in a clinical setting, can lead to short cuts being taken during PCR set up. OBJECTIVES: This study set out to evaluate the outcome of exposing a real-time PCR assay to increasing durations of room temperature prior to PCR amplification. STUDY DESIGN: A real-time PCR assay was exposed to increasing durations of room temperature prior to PCR amplification. RESULTS: We found that reactions left at room temperature for 30min or more produced non-specific traces in the negative controls which could be mistaken for weak positive traces. In addition we found that the fluorescence of positive control traces was significantly reduced indicating reduced reaction efficiency, however the Ct valves were comparable between all reactions highlighting that control Ct monitoring alone would not have detected this issue. CONCLUSIONS: This study acts as a reminder for PCR users to set up reactions on ice/chill blocks prior to PCR amplification.

Prevalence of HCV NS3 pre-treatment resistance associated amino acid variants within a Scottish cohort.

BACKGROUND: Protease inhibitors (PI) including boceprevir, telaprevir and simeprevir have revolutionised HCV genotype 1 treatment since their introduction. A number of pre-treatment resistance associated amino acid variants (RAVs) and polymorphisms have been associated with reduced response to treatment. OBJECTIVES: We measured the prevalence of RAVs/polymorphisms in a PI treatment-naïve HCV genotype 1 Scottish cohort using Sanger sequencing. STUDY DESIGN: Chronically infected, treatment-naïve, HCV genotype 1 patients (n = 146) attending NHS Greater Glasgow and Clyde clinics were investigated for RAVs/polymorphisms to the PIs boceprevir, telaprevir and simeprevir. The NS3/4A region was amplified by nested polymerase chain reaction. The 1.4 kb amplified product was sequenced using an ABI 3710XL DNA sequencer. Sequence analysis was performed using web-based ReCall (beta 2.10). Amino acid positions 36, 41, 43, 54, 55, 80, 109, 122, 155, 156, 168 and 170 were analysed for RAVs/polymorphisms. RESULTS: Overall, 23.29% (34/146) of patients had an RAV or polymorphism detected. Overall, 13.69% (20/146) of patients had HCV virus that contained the Q8 K polymorphism. Other RAVs detected were: V36 M 0.70% (1/146), V36L 0.70% (1/146), T54S 6.85% (10/146), V55A 3.42% (5/146) and V/I170A 0.68% (1/146). Four patients had dual combinations of mutations (T54S+V36L; T54S+V55A and 2 patients with T54S+Q80K). CONCLUSIONS: Q80K was the most prevalent baseline polymorphism detected in the Scottish cohort. Simeprevir treatment is not recommended in patients infected with the Q80K genotype 1a variant. This highlights the need for baseline sequencing prior to administration of this drug in this population.

From sidaway to pearce and beyond: is the legal regulation of consent any better following a quarter of a century of judicial scrutiny?

In 1987, following a period of increasing judicial activity, Margaret Brazier published her insightful article on the legal regulation of consent: Patient autonomy and consent to treatment: the role of the law? In her article, she exposed the flaws in the law following the House of Lords case of Sidaway. She considered the strengths and weaknesses of the alternative standards of disclosure: the professional or Bolam standard, the reasonable patient standard, and the particular patient standard. After noting that all of these standards have their problems, she suggested that the best way forward was for a national law and ethics commission to explore the issues before revising the law by legislation. Almost a quarter of a century following her article, Professor Brazier's criticisms remain aposite. In this article, I explain her view of the law in 1987 and then I examine the current law through the lens of her article and conclude that her recommendations still have strength.

The validation of a real-time RT-PCR assay which detects influenza A and types simultaneously for influenza A H1N1 (2009) and oseltamivir-resistant (H275Y) influenza A H1N1 (2009).

Influenza A H1N1 (2009) was declared by the World Health Organisation (WHO) as the first influenza pandemic of the 21st century. Rapid detection of influenza A and differentiation of influenza A H1N1 (2009) and seasonal influenza A is beneficial. In addition the rapid detection of antiviral resistant strains of influenza A H1N1 (2009) would be useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and type simultaneously for influenza A H1N1 (2009) and oseltamivir resistant (H275Y) influenza A H1N1 (2009). This multiplex assay will allow laboratories to screen respiratory samples for all types of influenza A, influenza A H1N1 (2009) virus and oseltamivir resistant (H275Y) influenza A H1N1 (2009) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. Since most virology laboratories already offer a molecular service for influenza A this assay could easily be implemented into most areas at little cost therefore increasing local access to resistance testing.

Development of a multiplex real-time RT-PCR that allows universal detection of influenza A viruses and simultaneous typing of influenza A/H1N1/2009 virus.

On June 11, 2009, the World Health Organization declared that the influenza A/H1N1/2009 virus had become the first influenza pandemic of the 21st century. Rapid detection and differentiation from seasonal and avian influenza would be beneficial for patient management and infection control. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and offer simultaneous typing for influenza A/H1N1/2009. This would be a useful addition to existing diagnostic protocols for influenza A. Its routine use would allow laboratories to screen out influenza A/H1N1/2009 positive samples rapidly and would reduce overall testing costs.

Development of a real-time RT-PCR for the detection of swine-lineage influenza A (H1N1) virus infections.

BACKGROUND: A novel influenza A virus, subtype H1N1 of swine-lineage (H1N1 swl) has transmitted rapidly to many regions of the world with evidence of sustained transmission within some countries. Rapid detection and differentiation from seasonal influenza is essential to instigate appropriate patient and public health management and for disease surveillance. OBJECTIVES: To develop a rapid and sensitive real-time reverse transcriptase polymerase chain reaction (rtRT-PCR) for confirmation of H1N1 swl. STUDY DESIGN: A one-step rtRT-PCR approach was employed to target the matrix gene of the novel influenza A/H1N1 swl and validated against a panel of seasonal influenza A (H1N1 and H3N2), swine influenza A/H1N1 and avian influenza A/H5N1 viruses. The assay following validation was then used prospectively to detect H1N1 swl positive specimens from the recent outbreaks in the UK and the Republic of Ireland. RESULTS: The one-step H1N1 swl matrix rtRT-PCR successfully detected H1N1 swl clinical specimens and did not cross-react with seasonal influenza A, subtypes H1N1 and H3N2 viruses and swine influenza A (H1N1). The H1N1 swl matrix assay did cross react with H5N1. The H1N1 swl matrix assay was then compared to two other assays using a dilution series and a panel of untyped influenza A positive clinical samples. These experiments found the assay to have a comparable sensitivity to the established universal influenza A rtRT-PCR and was more sensitive than the H1N1 swl specific assay that targeted the H1 region. CONCLUSIONS: The results demonstrate that the rtRT-PCR is sensitive and should be used alongside existing universal influenza A assays to rapidly detect the novel H1N1 swl virus.