ABSTRACT
There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.
Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Hot Temperature , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Inactivation , COVID-19/epidemiology , COVID-19/virology , Epidemics/prevention & control , Humans , Nasopharynx/virology , Reagent Kits, Diagnostic , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/physiology , Sensitivity and Specificity , Specimen Handling/methods , WorkflowABSTRACT
Containing the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral flow device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or presymptomatic individuals rapidly. While LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or presymptomatic carriers may wrongly be assumed to be noninfectious, posing a significant risk of further spread in the community. Here, we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample. We quantified significant immobilized antigen-antibody-labeled conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to â¼10â¯000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely, being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance both to public health monitoring during the Coronavirus Disease 2019 (COVID-19) pandemic and to the rapid refinement of these tools for immediate and future applications.
Subject(s)
COVID-19/diagnosis , COVID-19/virology , Immunoassay/instrumentation , Severe acute respiratory syndrome-related coronavirus/isolation & purification , Animals , Chlorocebus aethiops , Microscopy, Electron, Transmission , Real-Time Polymerase Chain Reaction , Reference Standards , Severe acute respiratory syndrome-related coronavirus/ultrastructure , Sensitivity and Specificity , Spectrometry, X-Ray Emission , Vero CellsABSTRACT
There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna ® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP (PHE guidelines). All RNA extraction methods provided similar results. FastVirus and Luna proved most sensitive. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrate that treatment of nasopharyngeal swabs with 70 degrees for 10 or 30 min, or 90 degrees for 10 or 30 min (both original variant and B 1.1.7) inactivates SARS-CoV-2 employing plaque assays, and that it has minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable to settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/ .
ABSTRACT
During the first wave of the global COVID-19 pandemic the clinical utility and indications for SARS-CoV-2 serological testing were not clearly defined. The urgency to deploy serological assays required rapid evaluation of their performance characteristics. We undertook an internal validation of a CE marked lateral flow immunoassay (LFIA) (SureScreen Diagnostics) using serum from SARS-CoV-2 RNA positive individuals and pre-pandemic samples. This was followed by the delivery of a same-day named patient SARS-CoV-2 serology service using LFIA on vetted referrals at central London teaching hospital with clinical interpretation of result provided to the direct care team. Assay performance, source and nature of referrals, feasibility and clinical utility of the service, particularly benefit in clinical decision-making, were recorded. Sensitivity and specificity of LFIA were 96.1% and 99.3% respectively. 113 tests were performed on 108 participants during three-week pilot. 44% participants (n = 48) had detectable antibodies. Three main indications were identified for serological testing; new acute presentations potentially triggered by recent COVID-19 e.g. pulmonary embolism (n = 5), potential missed diagnoses in context of a recent COVID-19 compatible illness (n = 40), and making infection control or immunosuppression management decisions in persistently SARS-CoV-2 RNA PCR positive individuals (n = 6). We demonstrate acceptable performance characteristics, feasibility and clinical utility of using a LFIA that detects anti-spike antibodies to deliver SARS-CoV-2 serology service in adults and children. Greatest benefit was seen where there is reasonable pre-test probability and results can be linked with clinical advice or intervention. Experience from this pilot can help inform practicalities and benefits of rapidly implementing new tests such as LFIAs into clinical service as the pandemic evolves.
Subject(s)
COVID-19 Serological Testing , COVID-19 , Pandemics , SARS-CoV-2/metabolism , Adult , COVID-19/blood , COVID-19/complications , COVID-19/diagnosis , COVID-19/epidemiology , Female , Humans , Male , SyndromeABSTRACT
Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10-15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection.
Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/pathology , Female , Humans , Kinetics , Longitudinal Studies , Male , Middle Aged , Seroconversion , Severity of Illness Index , Young AdultABSTRACT
There is a clear requirement for an accurate SARS-CoV-2 antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. We therefore evaluated the performance of a variety of antibody testing technologies and their potential use as diagnostic tools. Highly specific in-house ELISAs were developed for the detection of anti-spike (S), -receptor binding domain (RBD) and -nucleocapsid (N) antibodies and used for the cross-comparison of ten commercial serological assays-a chemiluminescence-based platform, two ELISAs and seven colloidal gold lateral flow immunoassays (LFIAs)-on an identical panel of 110 SARS-CoV-2-positive samples and 50 pre-pandemic negatives. There was a wide variation in the performance of the different platforms, with specificity ranging from 82% to 100%, and overall sensitivity from 60.9% to 87.3%. However, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over 95% seen in several tests when assessing samples from more than 20 days post onset of symptoms. Furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. Rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of SARS-CoV-2 infections.
Subject(s)
Antibodies, Viral/analysis , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Point-of-Care Systems , Serologic Tests/methods , Adult , Aged , Betacoronavirus , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Community Health Services , Coronavirus Nucleocapsid Proteins , Enzyme-Linked Immunosorbent Assay , Female , Hospitals , Humans , Immunoassay , Luminescent Measurements , Male , Middle Aged , Nucleocapsid Proteins/immunology , Pandemics , Phosphoproteins , SARS-CoV-2 , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
An evaluation of a rapid portable gold-nanotechnology measuring SARS-CoV-2 IgM, IgA and IgG antibody concentrations against spike 1 (S1), spike 2 (S) and nucleocapsid (N) was conducted using serum samples from 74 patients tested for SARS-CoV-2 RNA on admission to hospital, and 47 historical control patients from March 2019. 59 patients were RNA(+) and 15 were RNA(-). A serum (±) classification was derived for all three antigens and a quantitative serological profile was obtained. Serum(+) was identified in 30% (95% CI 11-48) of initially RNA(-) patients, in 36% (95% CI 17-54) of RNA(+) patients before 10 days, 77% (95% CI 67-87) between 10 and 20 days and 95% (95% CI 86-100) after 21 days. The patient-level diagnostic accuracy relative to RNA(±) after 10 days displayed 88% sensitivity (95% CI 75-95) and 75% specificity (95% CI 22-99), although specificity compared with historical controls was 100% (95%CI 91-100). This study provides robust support for further evaluation and validation of this novel technology in a clinical setting and highlights challenges inherent in assessment of serological tests for an emerging disease such as COVID-19.
Subject(s)
Antibodies, Viral/analysis , Betacoronavirus/immunology , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Serologic Tests/methods , Adult , Aged , Aged, 80 and over , Antibodies, Viral/immunology , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Cohort Studies , Coronavirus Infections/blood , Coronavirus Nucleocapsid Proteins , False Negative Reactions , Female , Gold/chemistry , Humans , Immunoglobulin A/analysis , Immunoglobulin A/immunology , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Immunoglobulin M/analysis , Immunoglobulin M/immunology , Male , Metal Nanoparticles/chemistry , Middle Aged , Nucleocapsid Proteins/immunology , Pandemics , Phosphoproteins , Pneumonia, Viral/blood , SARS-CoV-2 , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology , Young AdultABSTRACT
Crohn's disease (CD) and ulcerative colitis (UC), the 2 main clinical phenotypes of inflammatory bowel disease (IBD), are diseases that result from a dysregulated immune response to gut microbiota in genetically susceptible hosts. This aberrant immune response may intrinsically predispose IBD patients to infectious complications. Moreover, immunosuppressive medications used to treat IBD including corticosteroids, thiopurines, methotrexate, calcineurin inhibitors, anti-tumor necrosis factor (anti-TNF) agents and other biologics, further increase patients' susceptibility to opportunistic infections. Herpes zoster (HZ), also known as shingles, is an opportunistic viral reactivation often observed in IBD patients with several case reports demonstrating complicated or disseminated disease in those on immunosuppression. While HZ vaccination is recommended in all immunocompetent adults aged ≥60 years, as a live virus vaccine, it is currently contraindicated in IBD patients on anti-TNF therapy and in other significantly immunocompromised patient groups. While caution is still warranted in these circumstances, recent clinical data has emerged which has prompted us to review and examine the universal approach to HZ vaccination in the immunosuppressed IBD population. In the following narrative review, we will discuss and provide an overview of the clinical manifestations, incidence, management and prevention of HZ in the IBD patient.
Subject(s)
Herpes Zoster Vaccine/therapeutic use , Herpes Zoster/prevention & control , Immunosuppressive Agents/adverse effects , Inflammatory Bowel Diseases/virology , Opportunistic Infections/prevention & control , Adult , Contraindications, Drug , Female , Herpes Zoster/chemically induced , Humans , Immunocompromised Host , Inflammatory Bowel Diseases/drug therapy , Male , Middle Aged , Opportunistic Infections/virologyABSTRACT
UNLABELLED: Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpes virus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE: Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.
Subject(s)
Herpesvirus 3, Human/genetics , Recombination, Genetic , Adult , Child , Child, Preschool , Cluster Analysis , DNA, Viral/chemistry , DNA, Viral/genetics , Genetic Variation , Genome, Viral , Herpesvirus 3, Human/isolation & purification , Humans , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Sequence HomologyABSTRACT
BACKGROUND: It is increasingly recognized that human rhinoviruses (HRV) can be associated with severe infections. However, conflicting results have been reported on the relative prevalence and severity of the three HRV species. OBJECTIVES: The relative prevalence and clinical characteristics of HRV-A, B and C, in children attending a South London teaching hospital were investigated retrospectively. STUDY DESIGN: Children aged<16 years with episodes of respiratory tract infections and detectable entero/rhinovirus RNA in respiratory samples between November 2009 and December 2010 were investigated. Retrospective case review was performed and patients' characteristics recorded. RESULTS: Entero/rhinoviruses were the commonest viral pathogens (498/2316; 21.5%). Amongst 204 infection episodes associated with entero/rhinovirus, 167 were typed HRV, HRV-C was the most prevalent (99/167, 59.3%) followed by HRV-A (60/167; 35.9%) and HRV-B (8/167, 4.8%). The severity spectrum of HRV-A and HRV-C infections were similar and affected all parts of the respiratory tract. Co-pathogens were observed in 54 (26.5%) episodes. Severity was increased in patients with non-viral co-pathogens and those with an underlying respiratory condition. Univariate and multiple regression analyses of potential prognostic variables including age, co-pathogens and underlying respiratory illnesses showed that mono-infection with HRV-C, as compared with other HRV species, was associated with more severe disease in young children<3 years. CONCLUSIONS: HRV-C was the most prevalent species and on its own was associated with severe disease in children<3 years. The association between infection with HRV species and clinical presentation is complex and affected by many confounding factors.
Subject(s)
Picornaviridae Infections/epidemiology , Picornaviridae Infections/pathology , Respiratory Tract Infections/epidemiology , Respiratory Tract Infections/pathology , Rhinovirus/classification , Rhinovirus/isolation & purification , Child, Preschool , Enterovirus/genetics , Enterovirus/isolation & purification , Enterovirus Infections/epidemiology , Enterovirus Infections/virology , Female , Humans , Infant , Infant, Newborn , London/epidemiology , Male , Picornaviridae Infections/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Respiratory Tract Infections/virology , Retrospective Studies , Rhinovirus/genetics , Severity of Illness IndexABSTRACT
BACKGROUND: Primary BK virus (BKV) infection is probably acquired by the respiratory route in childhood, and latent virus persists principally in the urinary tract. BKV reactivation is implicated in late onset haemorrhagic cystitis (HC) post Haematopoietic Stem Cell Transplant (HSCT). There is emerging evidence that BKV can cause life-threatening pneumonitis in immunocompromised individuals. OBJECTIVES: To describe the first known case of BKV pneumonitis in an adult HSCT recipient. STUDY DESIGN/RESULTS: A 19-year old male underwent an ABO-incompatible, volunteer unrelated donor allogeneic HSCT for high risk AML. The post-transplant period was complicated by moderate-severe cutaneous and gut acute graft-versus-host disease (aGVHD) and severe HC, attributable to BKV. Treatment encompassed intensification of immunosupression for aGVHD and weekly intravenous (IV) cidofovir (2.5mg/Kg) for BK viruria. He was readmitted with presumed septic shock and acute renal failure. After a transient improvement on broad spectrum antibacterials, he suffered significant respiratory deterioration. CT imaging revealed diffuse 'ground-glass' attenuation. Cytopathological assessment of a broncho-alveolar sample (BAL) was consistent with polyomavirus pneumonitis. No other cause was found to account for the respiratory deterioration. He did not respond to therapy and died of multi-organ failure. CONCLUSIONS: BKV is implicated in haemorrhagic cystitis in HSCT recipients but not routinely considered as a cause of pneumonitis. There are just 5 other cases in the literature, including 3 patients with AIDS. BKV should be considered as a possible cause of pneumonitis in HSCT recipients.
Subject(s)
BK Virus/isolation & purification , Hematopoietic Stem Cell Transplantation/adverse effects , Pneumonia/diagnosis , Polyomavirus Infections/diagnosis , Tumor Virus Infections/diagnosis , Bronchoalveolar Lavage Fluid/cytology , Cytological Techniques , Humans , Immunocompromised Host , Male , Pneumonia/pathology , Polyomavirus Infections/pathology , Transplantation , Tumor Virus Infections/pathology , Young AdultABSTRACT
Enterovirus 68 (EV68) was first isolated in 1962. Very few cases of EV68 infection were described over the ensuing 40 years. However, in the past few years, an increase in severe respiratory tract infections associated with EV68 has been reported. We identified two clusters of EV68 infection in South London, UK, one each in the autumn/winters of 2009 and 2010. Sequence comparison showed significant homology of the UK strains with those from other countries including the Netherlands, Japan and the Philippines, which reported EV68 outbreaks between 2008 and 2010. Phylogenetic analysis of all available VP1 sequences indicated the presence of two modern EV68 lineages. The 2010 UK strains belonged to lineage 2. Lineage 1 could be further divided into two sub-lineages: some Japanese and Dutch strains collected between 2004 and 2010 form a distinct sub-lineages (sub-lineage 1.1), whereas other strains from the UK, Japan, Netherlands and Philippines collected between 2008 and 2010 represent sub-lineage 1.2. The UK 2009 strains together with several Dutch and Japanese strains from 2009/2010 represents one variant (1.2.1), whereas those from the Philippines a second variant (1.2.2). Based on specific deletions and substitutions, we suggest rules for the assignment of lineages and sub-lineages. Molecular epidemiological analysis indicates rapid recent evolution of EV68 and this may explain the recent findings of a global resurgence of EV68. Continuous global monitoring of the clinical and molecular epidemiology of EV68 is recommended.
Subject(s)
Disease Outbreaks , Enterovirus D, Human/genetics , Enterovirus Infections/virology , Genetic Variation , 5' Untranslated Regions , Adolescent , Amino Acid Sequence , Base Sequence , Capsid Proteins/genetics , Child , Child, Preschool , Enterovirus Infections/epidemiology , Female , Humans , Male , Molecular Sequence Data , Molecular Typing , Multiplex Polymerase Chain Reaction , Phylogeny , Sequence Analysis, DNA , Sequence Deletion , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
The European Union Tissues and Cells Directive requires screening of tissue and cell donors for infective organisms to prevent inter-patient transmission. The Directive includes the unique term partner donation, which refers to "donation of reproductive cells between a man and a woman who declare that they have an intimate physical relationship". In line with the Directive, partners undergoing Assisted Reproductive Technology (ART) now require screening before each treatment, regardless of the time interval between consecutive cycles. Evidence to support this recommendation is lacking. Therefore, we conducted a retrospective study of all virology screening tests undertaken over a three year period for individuals attending an assisted conception unit serving a high risk inner city population. We ascertained prevalence and seroconversion rates for HIV, hepatitis B and C and estimated the additional cost of implementing the Directive fully in our unit. With more than 3910 ART individuals screened between January 2007 and December 2009, the prevalence of HIV, hepatitis B and C was 0.6, 1.7 and 0.4%, respectively. A total of 422 individuals had a second screening test during the three year period and none seroconverted. This study suggests that increasing the frequency of screening individuals undergoing ART to less than 12 months would not confer added benefit and has significant cost implications.