Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from 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 sampling. 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 protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, 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 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. 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 that allows for a highly precise, repeatable, and robust testing method.

Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples.

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal 'testing fatigue' and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.

A complex interplay between the extracellular matrix and the innate immune response to microbial pathogens.

The role of the host extracellular matrix (ECM) in infection tends to be neglected. However, the complex interactions between invading pathogens, host tissues and immune cells occur in the context of the ECM. On the pathogen side, a variety of surface and secreted molecules, including microbial surface components recognizing adhesive matrix molecules and tissue-degrading enzymes, are employed that interact with different ECM proteins to effectively establish an infection at specific sites. Microbial pathogens can also hijack or misuse host proteolytic systems to modify the ECM, evade immune responses or process biologically active molecules such as cell surface receptors and cytokines that direct cell behaviour and immune defence. On the host side, the ECM composition and three-dimensional ultrastructure undergo significant modifications, which have a profound impact on the specific signals that the ECM conveys to immune cells at the forefront of infection. Unexpectedly, activated immune cells participate in the remodelling of the local ECM by synthesizing ECM glycoproteins, proteoglycans and collagen molecules. The close interplay between the ECM and the innate immune response to microbial pathogens ultimately affects the outcome of infection. This review explores and discusses recent data that implicate an active role for the ECM in the immune response to infection, encompassing antimicrobial activities, microbial recognition, macrophage activation, phagocytosis, leucocyte population balance, and transcriptional and post-transcriptional regulation of inflammatory networks, and may foster novel antimicrobial approaches.