Self-swabbing versus assisted swabbing for viral detection by qRT-PCR: the experience from SARS-CoV-2 based on a meta-analysis of six prospectively designed evaluations conducted in a UK setting.

PURPOSE: In April 2020, the UK Government implemented NHS Test and Trace to provide SARS-CoV-2 quantitative reverse transcription polymerase chain reaction (qRT-PCR) testing for the public, with nose-and-throat swabbing for samples performed by trained staff. Self-swabbing (SS) would allow rapid scale-up of testing capacity and access. Six studies were undertaken to determine whether SS was as effective for detecting SARS-CoV-2 as swabbing performed by trained staff. METHODS: Six prospective studies were conducted between April-October 2020, using six swab/media combinations. Differences between assisted swabbing (AS) and SS were evaluated for concordance, positivity, sensitivity, cycle threshold (Ct) values and void rates. Statistical analysis was performed using 95% confidence intervals (CIs), paired t-tests and model-based methods. RESULTS: Overall, 3,253 individuals were recruited (median age 37 years, 49% female), with 2,933 having valid paired qRT-PCR results. Pooled concordance rate was 98% (95% CI: 96%, 99%). Positivity rate differences for SS (8.1%) and AS (8.4%) and differences in pooled sensitivities between SS (86%; 95% CI: 78%, 92%) and AS (91%; 95% CI: 78%, 96%) were nonsignificant. Both types of swabbing led to pooled void rates below 2% and strongly correlated Ct values. Age, sex and previous swabbing experience did not have a significant impact on concordance or sensitivity. CONCLUSION: The UK adopted a policy to promote self-testing for SARS-CoV-2 based on data demonstrating equivalence of SS versus AS. Positive outcomes with SS are likely generalisable to testing for other respiratory pathogens, and we consider self-sampling and self-testing essential for future pandemic preparedness.

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.

BACKGROUND: The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. RESULTS: In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. CONCLUSIONS: These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.

Double testing with lateral flow antigen test devices for COVID-19: does a second test in quick succession add value?

BACKGROUND/OBJECTIVES: We investigated if performing two lateral flow device (LFD) tests, LFD2 immediately after LFD1, could improve diagnostic sensitivity or specificity for detecting severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) antigen. STUDY DESIGN: Individuals aged ≥16 years attending UK community testing sites (February-May 2021) performed two successive LFD tests and provided a nose-and-throat sample for a polymerase chain reaction (PCR) test. Using the PCR result as the reference diagnosis, we assessed whether improvements could be achieved in sensitivity (by counting a positive result in either LFD as a positive overall test result) or specificity (by using LFD2 as confirmatory test). RESULTS: Overall, 2231 participants were included with 159 (7 %) having a positive PCR test. Of 2223 participants who completed both LFD tests, LFD results were highly concordant both with each other and with PCR tests (>97 %). The proportion of discord LFD results decreased significantly over the study period. Combined LFD usage achieved a sensitivity of 68.6 %, versus 67.1 % for either LFD individually. The specificity increased from 99.5 % to 99.8 % when using LFD2 as confirmatory test. Observed increases in sensitivity and specificity were not statistically significant. Void results were recorded for 31 (1.4 %) LFD1s, 19 (0.9 %) LFD2s and 6 (0.3 %) combined LFD tests. CONCLUSIONS: LFD tests were highly reproducible even when they were performed by untrained users following only written instructions and without supervision. While performing two LFD tests of the same type in quick succession marginally increased sensitivity or specificity, statistically significant improvements were not detected in our study.

Rapid antigen testing for SARS-CoV-2 by lateral flow assay: A field evaluation of self- and professional testing at UK community testing sites.

BACKGROUND: The advent of lateral flow devices (LFDs) for SARS-CoV-2 detection enabled widespread use of rapid self-tests during the pandemic. While self-testing using LFDs is now common, whether self-testing provides comparable performance to professional testing was a key question that remained important for pandemic planning. METHODS: Three prospective multi-centre studies were conducted to compare the performance of self- and professional testing using LFDs. Participants tested themselves or were tested by trained (professional) testers at community testing sites in the UK. Corresponding qRT-PCR test results served as reference standard. The performance of Innova, Orient Gene and SureScreen LFDs by users (self) and professional testers was assessed in terms of sensitivity, specificity, and kit failure (void) rates. Impact of age, sex and symptom status was analysed using logistic regression modelling. RESULTS: 16,617 participants provided paired tests, of which 15,418 were included in the analysis. Self-testing with Innova, Orient Gene or SureScreen LFDs achieved sensitivities of 50 %, 53 % or 72 %, respectively, compared to qRT-PCR. Self and professional LFD testing showed no statistically different sensitivity with respect to corresponding qRT-PCR testing. Specificity was consistently equal to or higher than 99 %. Sex and age had no or only marginal impact on LFD performance while sensitivity was significantly higher for symptomatic individuals. Sensitivity of LFDs increased strongly to up to 90 % with higher levels of viral RNA measured by qRT-PCR. CONCLUSIONS: Our results support SARS-CoV-2 self-testing with LFDs, especially for the detection of individuals whose qRT-PCR tests showed high viral concentrations.

Genomic attributes of airway commensal bacteria and mucosa.

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance.

Swab pooling enables rapid expansion of high-throughput capacity for SARS-CoV-2 community testing.

BACKGROUND: The challenges of rapid upscaling of testing capacity were a major lesson from the COVID-19 pandemic response. The need for process adjustments in high-throughput testing laboratories made sample pooling a challenging option to implement. OBJECTIVE: This study aimed to evaluate whether pooling samples at source (swab pooling) was as effective as qRT-PCR testing of individuals in identifying cases of SARS-CoV-2 in real-world community testing conditions using the same high-throughput pipeline. METHODS: Two cohorts of 10 (Pool10: 1,030 participants and 103 pools) and 6 (Pool6: 1,284 participants and 214 pools) samples per pool were tested for concordance, sensitivity, specificity, and Ct value differences with individual testing as reference. RESULTS: Swab pooling allowed unmodified application of an existing high-throughput SARS-Cov-2 testing pipeline with only marginal loss of accuracy. For Pool10, concordance was 98.1% (95% Confidence interval: 93.3-99.8%), sensitivity was 95.7% (85.5-99.5%), and specificity was 100.0% (93.6-100.0%). For Pool6, concordance was 97.2% (94.0-99.0%), sensitivity was 97.5% (93.7-99.3%), and specificity was 96.4% (87.7-99.6%). Differences of outcomes measure between pool size were not significant. Most positive individual samples, which were not detected in pools, had very low viral concentration. If only individual samples with a viral concentration > 400 copies/ml (i.e. Ct value < 30) were considered positive, the overall sensitivity of pooling increased to 99.5%. CONCLUSION: The study demonstrated high sensitivity and specificity by swab pooling and the immediate capability of high-throughput laboratories to implement this method making it an option in planning of rapid upscaling of laboratory capacity for future pandemics.

Airway microbial communities, smoking and asthma in a general population sample.

BACKGROUND: Normal airway microbial communities play a central role in respiratory health but are poorly characterized. Cigarette smoking is the dominant global environmental influence on lung function, and asthma has become the most prevalent chronic respiratory disease worldwide. Both conditions have major microbial components that are incompletely defined. METHODS: We investigated airway bacterial communities in a general population sample of 529 Australian adults. Posterior oropharyngeal swabs were analyzed by sequencing of the 16S rRNA gene. The microbiota were characterized according to their prevalence, abundance and network memberships. FINDINGS: The microbiota were similar across the general population, and were strongly organized into co-abundance networks. Smoking was associated with diversity loss, negative effects on abundant taxa, profound alterations to network structure and expansion of Streptococcus spp. By contrast, the asthmatic microbiota were selectively affected by an increase in Neisseria spp. and by reduced numbers of low abundance but prevalent organisms. INTERPRETATION: Our study shows that the healthy airway microbiota in this population were contained within a highly structured ecosystem, suggesting balanced relationships between the microbiome and human host factors. The marked abnormalities in smokers may contribute to chronic obstructive pulmonary disease (COPD) and lung cancer. The narrow spectrum of abnormalities in asthmatics encourages investigation of damaging and protective effects of specific bacteria. FUNDING: The study was funded by the Asmarley Trust and a Wellcome Joint Senior Investigator Award to WOCC and MFM (WT096964MA and WT097117MA). The Busselton Healthy Ageing Study is supported by the Government of Western Australia (Office of Science, Department of Health) the City of Busselton, and private donations.

Inhaled corticosteroid suppression of cathelicidin drives dysbiosis and bacterial infection in chronic obstructive pulmonary disease.

Bacterial infection commonly complicates inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). The mechanisms of increased infection susceptibility and how use of the commonly prescribed therapy inhaled corticosteroids (ICS) accentuates pneumonia risk in COPD are poorly understood. Here, using analysis of samples from patients with COPD, we show that ICS use is associated with lung microbiota disruption leading to proliferation of streptococcal genera, an effect that could be recapitulated in ICS-treated mice. To study mechanisms underlying this effect, we used cellular and mouse models of streptococcal expansion with Streptococcus pneumoniae, an important pathogen in COPD, to demonstrate that ICS impairs pulmonary clearance of bacteria through suppression of the antimicrobial peptide cathelicidin. ICS impairment of pulmonary immunity was dependent on suppression of cathelicidin because ICS had no effect on bacterial loads in mice lacking cathelicidin (Camp -/-) and exogenous cathelicidin prevented ICS-mediated expansion of streptococci within the microbiota and improved bacterial clearance. Suppression of pulmonary immunity by ICS was mediated by augmentation of the protease cathepsin D. Collectively, these data suggest a central role for cathepsin D/cathelicidin in the suppression of antibacterial host defense by ICS in COPD. Therapeutic restoration of cathelicidin to boost antibacterial immunity and beneficially modulate the lung microbiota might be an effective strategy in COPD.

Longitudinal assessment of sputum microbiome by sequencing of the 16S rRNA gene in non-cystic fibrosis bronchiectasis patients.

BACKGROUND: Bronchiectasis is accompanied by chronic bronchial infection that may drive disease progression. However, the evidence base for antibiotic therapy is limited. DNA based methods offer better identification and quantification of microbial constituents of sputum than standard clinical culture and may help inform patient management strategies. Our study objective was to determine the longitudinal variability of the non-cystic fibrosis (CF) bronchiectasis microbiome in sputum with respect to clinical variables. Eighty-five patients with non-CF bronchiectasis and daily sputum production were recruited from outpatient clinics and followed for six months. Monthly sputum samples and clinical measurements were taken, together with additional samples during exacerbations. 16S rRNA gene sequencing of the sputum microbiota was successful for 381 samples from 76 patients and analysed in conjunction with clinical data. RESULTS: Microbial communities were highly individual in composition and stability, usually with limited diversity and often containing multiple pathogens. When compared to DNA sequencing, microbial culture had restricted sensitivity in identifying common pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae, Moraxella catarrhalis. With some exceptions, community characteristics showed poor correlations with clinical features including underlying disease, antibiotic use and exacerbations, with the subject showing the strongest association with community structure. When present, the pathogens mucoid Pseudomonas aeruginosa and Haemophilus influenzae may also shape the structure of the rest of the microbial community. CONCLUSIONS: The use of microbial community analysis of sputum added to information from microbial culture. A simple model of exacerbations driven by bacterial overgrowth was not supported, suggesting a need for revision of principles for antibiotic therapy. In individual patients, the management of chronic bronchial infection may be improved by therapy specific to their microbiome, taking into account pathogen load, community stability, and acute and chronic community responses to antibiotics.