RT-LAMP has high accuracy for detecting SARS-CoV-2 in saliva and naso/oropharyngeal swabs from asymptomatic and symptomatic individuals

Previous studies have described RT-LAMP methodology for the rapid detection of SARS-CoV-2 in nasopharyngeal (NP) and oropharyngeal (OP) swab and saliva samples. This study describes the validation of an improved sample preparation method for extraction free RT-LAMP and defines the clinical performance of four different RT-LAMP assay formats for detection of SARS-CoV-2 within a multisite clinical evaluation. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva from asymptomatic and symptomatic individuals across healthcare and community settings. For Direct RT-LAMP, overall diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva was observed, with diagnostic specificity (DSp) of 100% (98.98-100.00%) on swabs and 100% (99.72-100.00%) on saliva when compared to RT-qPCR; analysing samples with RT-qPCR ORF1ab CT values of [≤]25 and [≤]33, DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs were observed, and 99.01% (94.61-99.97%) and 87.61% (82.69-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and DSp were 96.06% (92.88-98.12%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use-cases, including frequent, interval-based testing of saliva with Direct RT-LAMP from asymptomatic individuals that may otherwise be missed using symptomatic testing alone.

A prospective diagnostic study to measure the accuracy of detection of SARS-CoV-2 Variants Of Concern (VOC) utilising a novel RT-PCR GENotyping algorithm in an In silico Evaluation (VOC-GENIE)

BackgroundSARS-CoV-2 variants of concern (VOCs) have been associated with higher rate of transmission, and evasion of immunisation and antibody therapeutics. Variant sequencing is widely utilized in the UK. However, only 0.5% (~650k) of the 133 million cumulative positive cases worldwide were sequenced (in GISAID) on 08 April 2021 with 97% from Europe and North America and only ~0.25% (~320k) were variant sequences. This may be due to the lack of availability, high cost, infrastructure and expert staff required for sequencing. Public health decisions based on a non-randomised sample of 0.5% of the population may be insufficiently powered, and subject to sampling bias and systematic error. In addition, sequencing is rarely available in situ in a clinically relevant timeframe and thus, is not currently compatible with diagnosis and treatment patient care pathways. Therefore, we investigated an alternative approach using polymerase chain reaction (PCR) genotyping to detect the key single nucleotide polymorphisms (SNPs) associated with increased transmission and immune evasion in SARS-CoV-2 variants. MethodsWe investigated the utility of SARS-CoV-2 SNP detection with a panel of PCR-genotyping assays in a large data set of 640,482 SARS-CoV-2 high quality, full length sequences using a prospective in silico trial design and explored the potential impact of rapid in situ variant testing on the COVID-19 diagnosis and treatment patient pathway. ResultsFive SNPs were selected by screening the published literature for a reported association with increased transmission and / or immune evasion. 344881 sequences contained one or more of the five SNPs. This algorithm of SNPs was found to be able to identify the four variants of concern (VOCs) and sequences containing the E484K and L452R escape mutations. InterpretationThe in silico analysis suggest that the key mutations and variants of SARS-CoV-2 may be reliably detected using a focused algorithm of biologically relevant SNPs. This highlights the potential for rapid in situ PCR genotyping to compliment or replace sequencing or to be utilized instead of sequences in settings where sequencing is not feasible, accessible or affordable. Rapid detection of variants with in situ PCR genotyping may facilitate a more effective COVID-19 diagnosis and treatment patient pathway. FundingThe study was funded by Primer Design (UK), with kind contributions from all academic partners.

C-reactive protein guided use of procalcitonin in COVID-19

Low procalcitonin (PCT) concentrations (<0.5ng/mL) can facilitate exclusion of bacterial co-infection in viral infections, including COVID-19. However, costs associated with PCT measurement preclude universal adoption, indicating a need to identify settings where PCT provides clinical information beyond that offered by other inflammatory markers, such as C-reactive protein (CRP) and white cell count (WCC). In an unselected cohort of 299 COVID-19 patients, we tested the hypothesis that PCT<0.5ng/mL was associated with lower levels of CRP and WCC. We demonstrated that CRP values below the geometric mean of the entire patient population had a negative predictive value for PCT<0.5ng/mL of 97.6% and 100% at baseline and 48 hours into admission respectively, and that this relationship was not confounded by intensive care admission or microbiological findings. CRP-guided PCT testing algorithms can reduce costs and support antimicrobial stewardship strategies in COVID-19.

A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 within nasopharyngeal and oropharyngeal swabs at Hampshire Hospitals NHS Foundation Trust

The COVID-19 pandemic has illustrated the importance of rapid, accurate diagnostic testing for the effective triaging and cohorting of patients and timely tracking and tracing of cases. However, a surge in diagnostic testing quickly resulted in worldwide competition for the same sample preparation and real-time RT-PCR diagnostic reagents (rRT-PCR). Consequently, Hampshire Hospitals NHS Foundation Trust, UK sought to diversify their diagnostic portfolio by exploring alternative amplification chemistries including those that permit direct testing without RNA extraction. This study describes the validation of a SARS-CoV-2 RT-LAMP assay, which is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed on extracted RNA (RNA RT-LAMP) or directly from swab (Direct RT-LAMP). Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1[x]101 and 1[x]102 copies when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care (SoC) rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a very low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct-RT-LAMP was 67% and 97%, respectively. When setting CT cut-offs of [≤]33 and [≤]25, the DSe increased to 75% and 100%, respectively. Time from swab-to-result for a strong positive sample (CT < 25) was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increase in throughput, whereas Direct RT-LAMP could be used as a screening tool for triaging patients into appropriate hospitals wards, at GP surgeries and in care homes, or for population screening to identify super shedders. Direct RT-LAMP could also be used during times of high prevalence to save critical extraction and rRT-PCR reagents by screening out those strong positives from diagnostic pipelines.

SARS-CoV-2 genomics beyond the consensus sequence: evidence for circulating mixed viral populations

Extensive global sampling and whole genome sequencing of the pandemic virus SARS-CoV-2 have enabled researchers to characterise its spread, and to identify mutations that may increase transmission or enable the virus to escape therapies or vaccines. Two important components of viral spread are how frequently variants arise within individuals, and how likely they are to be transmitted. Here, we characterise the within-host diversity of SARS-CoV-2, and the extent to which genetic diversity is transmitted, by quantifying variant frequencies in 1390 clinical samples from the UK, many from individuals in known epidemiological clusters. We show that SARS-CoV-2 infections are characterised by low levels of within-host diversity across the entire viral genome, with evidence of strong evolutionary constraint in Spike, a key target of vaccines and antibody-based therapies. Although within-host variants can be observed in multiple individuals in the same phylogenetic or epidemiological cluster, highly infectious individuals with high viral load carry only a limited repertoire of viral diversity. Most viral variants are either lost, or occasionally fixed, at the point of transmission, consistent with a narrow transmission bottleneck. These results suggest potential vaccine-escape mutations are likely to be rare in infectious individuals. Nonetheless, we identified Spike variants present in multiple individuals that may affect receptor binding or neutralisation by antibodies. Since the fitness advantage of escape mutations in highly-vaccinated populations is likely to be substantial, resulting in rapid spread if and when they do emerge, these findings underline the need for continued vigilance and monitoring.