Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from human saliva

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sample collection. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service (UoNATS) protocol simplifies sample collection and bypasses the need for RNA extraction, additives, or extraneous processing steps, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service (UKAS). We observed a sensitivity of 1 viral copy per microlitre of saliva and demonstrated a concordance of >99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium with surprising longevity, and allows for a highly precise, repeatable, and robust testing method.

Prospective examination of mental health in university students during the COVID-19 pandemic

BackgroundThe impact of changing social restrictions on the mental health of students during the COVID-19 pandemic warrants exploration. AimsTo prospectively examine changes to university students mental health during the pandemic. MethodsStudents completed repeated online surveys at three time points (October 2020 (baseline), February 2021, March 2021) to explore relationships between demographic and psychological factors (loneliness and positive mood) and mental health outcomes (depression, anxiety, and stress). ResultsA total of 893 students participated. Depression and anxiety levels were higher at all timepoints than pre-pandemic normative data (p<.001). Scores on all mental health measures were highest in February, with depression and anxiety remaining significantly higher in March than baseline. Female students and those with previous mental health disorders were at greatest risk of poor mental health outcomes. Lower positive mood and greater loneliness at baseline were associated with greater depression and anxiety at follow-ups. Baseline positive mood predicted improvement of depression and anxiety at follow-ups. ConclusionDepression and anxiety were significantly higher than pre-pandemic norms, with female students and those with previous mental health difficulties being at greatest risk. Given these elevated rates, universities should ensure adequate support is available to meet potentially increased demand for services.

Experiences of the COVID-19 pandemic: cross-sectional analysis of risk perceptions and mental health in a student cohort

ObjectiveThis study examined the COVID-19 risk perceptions and mental health of university students on returning to campus in the midst of the COVID-19 pandemic. MethodsAn online survey was completed during the first four weeks of the academic year (October 2020) by 897 university students. The survey included demographics and measures of experiences of COVID-19 testing, self-isolation, shielding, perceived risk, mental health and indices capturing related psychological responses to the pandemic. ResultsWe observed higher levels of depression and anxiety, but not stress, in students compared with pre- pandemic normative data, but lower than levels reported earlier in the pandemic in other similar cohorts. Depression, anxiety and stress were independently associated with greater loneliness and reduced positive mood. Greater worry about COVID-19 was also independently associated with anxiety and stress. Female students and those with pre-existing mental health disorders were at greatest risk of poor mental health outcomes. ConclusionAlthough students perceived themselves at only moderate risk of COVID-19, the prevalence of depression and anxiety among university students should remain a concern. Universities should provide adequate support for students mental health during term-time. Interventions to reduced loneliness and worry, and improve mood, may benefit students overall mental well-being.

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

The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At 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 hours is required in order to facilitate timely infection control measures. 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), and asked if detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits. Using purified RNA, we found the CerTest VIASURE kit to be comparable to 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 1-step qRT-PCR mix and 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). 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.