Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2.

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

Assessment of online self-testing and self-sampling service providers for sexually transmitted infections against national standards in the UK in 2020.

OBJECTIVES: Online testing for STIs may help overcome barriers of traditional face-to-face testing, such as stigma and inconvenience. However, regulation of these online tests is lacking, and the quality of services is variable, with potential short-term and long-term personal, clinical and public health implications. This study aimed to evaluate online self-testing and self-sampling service providers in the UK against national standards. METHODS: Providers of online STI tests (self-sampling and self-testing) in the UK were identified by an internet search of Google and Amazon (June 2020). Website information on tests and associated services was collected and further information was requested from providers via an online survey, sent twice (July 2020, April 2021). The information obtained was compared with British Association for Sexual Health and HIV and Faculty of Sexual and Reproductive Healthcare guidelines and standards for diagnostics and STI management. RESULTS: 31 providers were identified: 13 self-test, 18 self-sample and 2 laboratories that serviced multiple providers. Seven responded to the online survey. Many conflicts with national guidelines were identified, including: lack of health promotion information, lack of sexual history taking, use of tests licensed for professional-use only marketed for self-testing, inappropriate infections tested for, incorrect specimen type used and lack of advice for postdiagnosis management. CONCLUSIONS: Very few online providers met the national STI management standards assessed, and there is concern that this will also be the case for service provision aspects that were not covered by this study. For-profit providers were the least compliant, with concerning implications for patient care and public health. Regulatory change is urgently needed to ensure that all online providers are compliant with national guidelines to ensure high-quality patient care, and providers are held to account if non-compliant.

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

Persistent SARS-CoV-2 infection in immunocompromised patients facilitates rapid viral evolution: Retrospective cohort study and literature review.

Background: Most patients with SARS-CoV-2 are non-infectious within 2 weeks, though viral RNA may remain detectable for weeks. However there are reports of persistent SARS-CoV-2 infection, with viable virus and ongoing infectivity months after initial detection. Beyond individuals, viral evolution during persistent infections may be accelerated, driving emergence of mutations associated with viral variants of concern. These patients often do not meet inclusion criteria for clinical trials, meaning clinical and virologic characteristics, and optimal management strategies are poorly evidence-based. Methods: We analysed cases of SARS-CoV-2 infection from a regional testing laboratory in South-West England between March 2020 and December 2021, with at least two SARS-CoV-2 positive samples separated by ≥ 56 days were identified. Excluding those with confirmed or likely re-infection, we identified patients with persistent infection, characterised by an ongoing clinical syndrome consistent with COVID-19 alongside monophyletic viral lineage of SARS-CoV-2. We examined clinical and virologic characteristics, treatment, and outcome. We further performed a literature review investigating cases of persistent SARS-CoV-2 infection, reviewing patient characteristics and treatment. Results: We identified six patients with persistent SARS-CoV-2 infection. All were hypogammaglobulinaemic and had underlying haematological malignancy, with four having received B-cell depleting therapy. Evidence of viral evolution, including accrual of mutations associated with variants of concern, was demonstrated in five cases. Four patients ultimately cleared SARS-CoV-2. In two patients, clearance followed treatment with casirivimab/imdevimab. Both survived beyond thirty days following viral clearance, having experienced infections of 305- and 269-days duration respectively, after failed attempts at clearance with alternative therapies. We found 60 cases of confirmed persistent infection in the literature, with a further 31 probable cases. Of those, 80% of patients treated with monoclonal antibodies cleared SARS-CoV-2, and none died. Conclusion: Haematological malignancy and patients receiving B-cell depleting therapies represent key groups at risk of persistent SARS-CoV-2 infection. Throughout persistent infection, SARS-CoV-2 can evolve rapidly, giving rise to significant mutations, including those implicated in variants of concern. Monoclonal antibodies appear to be a promising therapeutic option, potentially in combination with antivirals, crucial for individuals, and for public health.

Prospective Study of the Performance of Parent-Collected Nasal and Saliva Swab Samples, Compared with Nurse-Collected Swab Samples, for the Molecular Detection of Respiratory Microorganisms.

Respiratory tract infections (RTIs) are ubiquitous among children in the community. A prospective observational study was performed to evaluate the diagnostic performance and quality of at-home parent-collected (PC) nasal and saliva swab samples, compared to nurse-collected (NC) swab samples, from children with RTI symptoms. Children with RTI symptoms were swabbed at home on the same day by a parent and a nurse. We compared the performance of PC swab samples as the test with NC swab samples as the reference for the detection of respiratory pathogen gene targets by reverse transcriptase PCR, with quality assessment using a human gene. PC and NC paired nasal and saliva swab samples were collected from 91 and 92 children, respectively. Performance and interrater agreement (Cohen's κ) of PC versus NC nasal swab samples for viruses combined showed sensitivity of 91.6% (95% confidence interval [CI], 85.47 to 95.73%) and κ of 0.84 (95% CI, 0.79 to 0.88), respectively; the respective values for bacteria combined were 91.4% (95% CI, 86.85 to 94.87%) and κ of 0.85 (95% CI, 0.80 to 0.89). In saliva samples, viral and bacterial sensitivities were lower at 69.0% (95% CI, 57.47 to 79.76%) and 78.1% (95% CI, 71.60 to 83.76%), as were κ values at 0.64 (95% CI, 0.53 to 0.72) and 0.70 (95% CI, 0.65 to 0.76), respectively. Quality assessment for human biological material (18S rRNA) indicated perfect interrater agreement. At-home PC nasal swab samples performed comparably to NC swab samples, whereas PC saliva swab samples lacked sensitivity for the detection of respiratory microbes. IMPORTANCE RTIs are ubiquitous among children. Diagnosis involves a swab sample being taken by a health professional, which places a considerable burden on community health care systems, given the number of cases involved. The coronavirus disease 2019 (COVID-19) pandemic has seen an increase in the at-home self-collection of upper respiratory tract swab samples without the involvement of health professionals. It is advised that parents conduct or supervise swabbing of children. Surprisingly, few studies have addressed the quality of PC swab samples for subsequent identification of respiratory pathogens. We compared NC and PC nasal and saliva swab samples taken from the same child with RTI symptoms, for detection of respiratory pathogens. The PC nasal swab samples performed comparably to NC samples, whereas saliva swab samples lacked sensitivity for the detection of respiratory microbes. Collection of swab samples by parents would greatly reduce the burden on community nurses without reducing the effectiveness of diagnoses.

Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2.

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in 4 infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein, with a corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ- and/or TNF-α-producing T cells is comparable between infants and adults. On principal-component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19.

Novel multi-virus rapid respiratory microbiological point-of-care testing in primary care: a mixed-methods feasibility evaluation.

BACKGROUND AND OBJECTIVES: Rapid multi-viral respiratory microbiological point-of-care tests (POCTs) have not been evaluated in UK primary care. The aim of this study was to evaluate the use of a multi-viral microbiological POCT for suspected respiratory tract infections (RTIs). METHODS: In this observational, mixed-methods feasibility study practices were provided with a POCT machine for any patient aged ≥3 months with suspected RTI. Dual throat/nose swabs tested for 17 respiratory viruses and three atypical bacteria, 65 minutes per sample. RESULTS: Twenty clinicians recruited 93 patients (estimated 1:3 of all RTI cases). Patient's median age was 29, 57% female, and 44% with co-morbidities. Pre-test diagnoses: upper RTI (48%); lower RTI (30%); viral/influenza-like illness (18%); other (4%). Median set-up time was 2.72 minutes, with 72% swabs processed <4 hours, 90% <24 hours. Tests detected ≥1 virus in 58%, no pathogen 37% and atypical bacteria 2% (3% inconclusive). Antibiotics were prescribed pre-test to 35% of patients with no pathogen detected and 25% with a virus. Post-test diagnoses changed in 20%, and diagnostic certainty increased (P = 0.02), more so when the test was positive rather than negative (P < 0.001). Clinicians predicted decreased antibiotic benefit post-test (P = 0.02). Interviews revealed the POCT has clear potential, was easy to use and well-liked, but limited by time-to-result and the absence of testing for typical respiratory bacteria. CONCLUSIONS: This POCT was acceptable and appeared to influence clinical reasoning. Clinicians wanted faster time-to-results and more information about bacteria. Randomized trials are needed to understand the safety, efficacy and patient perceptions of these POCTs.

The UK government has called for the introduction of rapid diagnostics to curb overuse of antibiotics for common infections. Multi-viral respiratory 'point-of-care' tests (POCTs) are available but have not been used in UK primary care before. These POCTs use samples from the nose or back of the throat and give results quickly, to see if viruses or bacteria are there. In this study, four GP practices were given POCT machines for 6 weeks to see how they were used. Of the 93 patient samples tested, 3% were inconclusive, 37% tested negative, 58% had at least one virus and only 2% had a bacterial infection. Clinicians were more certain of patient diagnoses after testing especially when a virus or bacterium was detected and they were also less likely to predict the patient would benefit from antibiotics. Clinical diagnoses changed in 20% of patients after testing but less than 10% were contacted to change their treatment plan. During interviews, clinicians revealed they liked the test finding it easy-to-use but wanted faster time-to-results and testing for more bacteria. Clinical trials are needed to see if these POCTs can safely and cost-effectively reduce antibiotic prescribing in primary care.

Modelling patient flows and resource use within a sexual health clinic through discrete event simulation to inform service redesign

OBJECTIVES: Continuous improvement in the delivery of health services is increasingly being demanded in the UK at a time when budgets are being cut. Simulation is one approach used for understanding and assessing the likely impact of changes to the delivery of health services. However, little is known about the usefulness of simulation for analysing the delivery of sexual health services (SHSs). We propose a simulation method to model and evaluate patient flows and resource use within an SHS to inform service redesign. METHODS: We developed a discrete event simulation (DES) model to identify the bottlenecks within the Unity SHS (Bristol, UK) and find possible routes for service improvement. Using the example of the introduction of an online service for sexually transmitted infection (STI) and HIV self-sampling for asymptomatic patients, the impact on patient waiting times was examined as the main outcome measure. The model included data such as patient arrival time, staff availability and duration of consultation, examination and treatment. We performed several sensitivity analyses to assess uncertainty in the model parameters. RESULTS: We identified some bottlenecks under the current system, particularly in the consultation and treatment queues for male and female walk-in patients. Introducing the provision of STI and HIV self-sampling alongside existing services decreased the average waiting time (88 vs 128 min) for all patients and reduced the cost of staff time for managing each patient (£72.64 vs £88.74) compared with the current system without online-based self-sampling. CONCLUSIONS: The provision of online-based STI and HIV self-sampling for asymptomatic patients could be beneficial in reducing patient waiting times and the model highlights the complexities of using this to cut costs. Attributing recognition for any improvement requires care, but DES modelling can provide valuable insights into the design of SHSs ensuing in quantifiable improvements. Extension of this method with the collection of additional data and the construction of more informed models seems worthwhile.