Amplification Free Detection of SARS-CoV-2 Using Multi-Valent Binding.

We present the development of electrochemical impedance spectroscopy (EIS)-based biosensors for sensitive detection of SARS-CoV-2 RNA using multi-valent binding. By increasing the number of probe-target binding events per target molecule, multi-valent binding is a viable strategy for improving the biosensor performance. As EIS can provide sensitive and label-free measurements of nucleic acid targets during probe-target hybridization, we used multi-valent binding to build EIS biosensors for targeting SARS-CoV-2 RNA. For developing the biosensor, we explored two different approaches including probe combinations that individually bind in a single-valent fashion and the probes that bind in a multi-valent manner on their own. While we found excellent biosensor performance using probe combinations, we also discovered unexpected signal suppression. We explained the signal suppression theoretically using inter- and intra-probe hybridizations which confirmed our experimental findings. With our best probe combination, we achieved a LOD of 182 copies/µL (303 aM) of SARS-CoV-2 RNA and used these for successful evaluation of patient samples for COVID-19 diagnostics. We were also able to show the concept of multi-valent binding with shorter probes in the second approach. Here, a 13-nt-long probe has shown the best performance during SARS-CoV-2 RNA binding. Therefore, multi-valent binding approaches using EIS have high utility for direct detection of nucleic acid targets and for point-of-care diagnostics.

User acceptability of saliva and gargle samples for identifying COVID-19 positive high-risk workers and household contacts.

Throughout the COVID-19 pandemic nasopharyngeal or nose and/or throat swabs (NTS) have been the primary approach for collecting patient samples for the subsequent detection of viral RNA. However, this procedure, if undertaken correctly, can be unpleasant and therefore deters individuals from providing high quality samples. To overcome these limitations other modes of sample collection have been explored. In a cohort of frontline health care workers we have compared saliva and gargle samples to gold-standard NTS. 93% of individuals preferred providing saliva or gargle samples, with little sex-dependent variation. Viral titers collected in samples were analyzed using standard methods and showed that gargle and saliva were similarly comparable for identifying COVID-19 positive individuals compared to NTS (92% sensitivity; 98% specificity). We suggest that gargle and saliva collection are viable alternatives to NTS swabs and may encourage testing to provide better disease diagnosis and population surveillance.

Rapid detection of SARS-CoV-2 variants using allele-specific PCR.

Tracking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants through whole genome sequencing (WGS) is vital for effective infection control and prevention (IPC) measures, but can be time-consuming and resource-heavy. We describe an in-house validation of an allele-specific polymerase chain reaction (ASP) variant assay to detect variants of concern (VOC). ASP sensitivity for detecting Delta, Alpha and Beta was 99.45 %, 100 %, and 66.67 %, respectively, compared with WGS. Specificity was 100 % in detecting all three VOC. ASP generated results 1.3 days faster compared with WGS. These findings suggest using variant assays such as ASP may enhance epidemiological surveillance and IPC measures.

Sensitivity of RT-PCR testing of upper respiratory tract samples for SARS-CoV-2 in hospitalised patients: a retrospective cohort study [version 2;peer review: 2 approved]

Background: This study aimed to determine the sensitivity and specificity of reverse transcription PCR (RT-PCR) testing of upper respiratory tract samples from hospitalised patients with coronavirus disease 2019 (COVID-19), compared to the gold standard of a clinical diagnosis. Methods: All RT-PCR testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in NHS Lothian, Scotland, United Kingdom between the 7 th of February and 19 th April 2020 (inclusive) was reviewed, and hospitalised patients were identified. All upper respiratory tract RT-PCR tests were analysed for each patient to determine the sequence of negative and positive results. For those who were tested twice or more but never received a positive result, case records were reviewed, and a clinical diagnosis of COVID-19 allocated based on clinical features, discharge diagnosis, and radiology and haematology results. For those who had a negative RT-PCR test but a clinical diagnosis of COVID-19, respiratory samples were retested using a multiplex respiratory panel, a second SARS-CoV-2 RT-PCR assay, and a human RNase P control. Results: Compared to the gold standard of a clinical diagnosis of COVID-19, the sensitivity of a single upper respiratory tract RT-PCR for COVID-19 was 82.2% (95% confidence interval 79.0-85.1%).   The sensitivity of two upper respiratory tract RT-PCR tests increased sensitivity to 90.6% (CI 88.0-92.7%). A further 2.2% and 0.9% of patients who received a clinical diagnosis of COVID-19 were positive on a third and fourth test;this may be an underestimate of the value of further testing as the majority of patients 93.0% (2999/3226) only had one or two RT-PCR tests. Conclusions: The sensitivity of a single RT-PCR test of upper respiratory tract samples in hospitalised patients is 82.2%. Sensitivity increases to 90.6% when patients are tested twice.  A proportion of cases with clinically defined COVID-19 never test positive on RT-PCR despite repeat testing.