Bryony Armson

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse (TM), followed by dilution in 10 % (w/v) Chelex(C) 100 Resin and a 98 degrees C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected noninvasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.

Rapid, high throughput diagnostics are a valuable tool, allowing the detection of SARS-CoV-2 in populations, in order to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as reverse transcriptase quantitative PCR (RT-qPCR), particularly throughout the first months of the COVID-19 pandemic. We investigated the use of LamPORE, where loop mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS-CoV-2 in symptomatic and asymptomatic populations. In an asymptomatic prospective cohort, for three weeks in September 2020 health care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily. These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza like illness from March 2020 – June 2020, were similarly tested from nasopharyngeal swabs. In the asymptomatic cohort a total of 1200 participants supplied 23,427 samples (3,966 swab, 19,461 saliva) over a three-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% (reducing to ∼ 98% when clustered estimation was used) in both swab and saliva asymptomatic samples when compared to the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. LamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR.

Previous studies have described reverse-transcription loop-mediated isothermal amplification (RTLAMP) for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal/oropharyngeal swab and saliva samples. This multisite clinical evaluation describes the validation of an improved sample preparation method for extraction-free RT-LAMP and reports clinical performance of four RT-LAMP assay formats for SARS-CoV-2 detection. 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 samples from asymptomatic and symptomatic individuals across health care and community settings. For direct RT-LAMP, overall diagnostic sensitivity (DSe) was 70.35% (95% CI, 63.48%-76.60%) on swabs and 84.62% (95% CI, 79.50%-88.88%) on saliva, with diagnostic specificity of 100% (95% CI, 98.98%-100.00%) on swabs and 100% (95% CI, 99.72%-100.00%) on saliva, compared with quantitative RT-PCR (RT-qPCR); analyzing samples with RT-qPCR ORF1ab C-T values of 25 and < 33, DSe values were 100% (95% CI, 96.34%-100%) and 77.78% (95% CI, 70.99%-83.62%) for swabs, and 99.01% (95% CI, 94.61%-99.97%) and 87.61% (95% CI, 82.69%-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and diagnostic specificity were 96.06% (95% CI, 92.88% -98.12%) and 99.99% (95% CI, 99.95%-100%) for swabs, and 80.65% (95% CI, 73.54%-86.54%) and 99.99% (95% CI, 99.95%-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use cases, including frequent, interval-based direct RT-LAMP of saliva from asymptomatic individuals who may otherwise be missed using symptomatic testing alone. (J Mol Diagn 2022, 24: 320-336; https://doi.org/10.1016/j.jmoldx.2021.12.007)

•Novel rapid RT-LAMP assay with diagnostic sensitivity and specificity of 97% and 99%.•Development of an RNA extraction free direct detection method for SARS-CoV-2.•Use case modelling for rapid direct RT-LAMP in near-patient clinical practice.•Developing diversity of testing modalities within a diagnostic laboratory during a pandemic. The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 101 and 1 × 102 copies per reaction 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 rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) 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, CT < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

Peste des petits ruminants (PPR) is a major constraint to the productivity of small ruminants in Nigeria. Understanding of the current epidemiological status of PPR is crucial to its effective control. A review of the epidemiology of PPR in Nigeria was performed and research gaps were identified. Thirty-seven eligible articles were reviewed: these presented information from 30 of the 36 states of Nigeria. Most studies focused on goats and/or sheep (n = 33) but camels (n = 4), cattle (n = 1) and wild ruminants (n = 2) were also considered. Fourteen (37.8%) of the articles reported seroprevalence in small ruminants, which varied from 0.0% to 77.5% where more than 10 animals were sampled. Molecular characterization and phylogenetic analysis were performed in 6 studies, with lineages II and IV, detected in sheep and goats. In one study in small ruminants, sequences clustering into lineage I showed a similarity to the vaccine strain, Nigeria 75/1, based on phylogenetic analysis of F gene sequences. However, if the preferred method of sequencing the N gene had been performed, this isolate would have been grouped into lineage II. According to N gene phylogenetic analysis in the other studies, sequences were identified that clustered with clade II-NigA, II-NigB (closely related to the Nigeria 75/1 vaccine strain), and others which were well separated, suggesting a high diversity of PPRV in Nigeria. Five articles reported the detection of lineage IV in 22/36 states, with IV-NigA and IV-NigB detected, highlighting its widespread distribution in Nigeria. Risk factors for PPRV seropositivity were reported in 10/37 (27.0%) articles, with a higher seroprevalence observed in female animals, although differing results were observed when considering species and age separately. There were inconsistencies in study design and data reporting between studies which precluded conduct of a meta-analysis. Nevertheless, several research gaps were identified including the need to investigate the low uptake of PPRV vaccine, and the economic benefits of PPR control measures to small ruminant farmers. Such data will inform PPR control strategies in Nigeria and subsequently contribute to the global 2030 PPR eradication strategy.

This study investigated the potential of pooled milk as an alternative sample type for foot-and-mouth disease (FMD) surveillance. Real-time RT-PCR (rRT-PCR) results of pooled milk samples collected weekly from five pooling facilities in Nakuru County, Kenya, were compared with half-month reports of household-level incidence of FMD. These periodic cross-sectional surveys of smallholder farmers were powered to detect a threshold household-level FMD incidence of 2.5% and collected information on trends in milk production and sales. FMD virus (FMDV) RNA was detected in 9/219 milk samples, and using a type-specific rRT-PCR, serotype SAT 1 was identified in 3/9 of these positive samples, concurrent with confirmed outbreaks in the study area. Four milk samples were FMDV RNA-positive during the half-months when at least one farmer reported FMD; that is, the household-level clinical incidence was above a threshold of 2.5%. Additionally, some milk samples were FMDV RNA-positive when there were no reports of FMD by farmers. These results indicate that the pooled milk surveillance system can detect FMD household-level incidence at a 2.5% threshold when up to 26% of farmers contributed milk to pooling facilities, but perhaps even at lower levels of infection (i.e., below 2.5%), or when conventional disease reporting systems fail. Further studies are required to establish a more precise correlation with estimates of household-level clinical incidence, to fully evaluate the reliability of this approach. However, this pilot study highlights the potential use of this non-invasive, routinely collected, cost-effective surveillance tool, to address some of the existing limitations of traditional surveillance methods.

Pooled milk is used for the surveillance of several diseases of livestock. Previous studies demonstrated the detection of foot-and-mouth disease virus (FMDV) in the milk of infected animals at high dilutions, and consequently, the collection of pooled milk samples could be used to enhance FMD surveillance. This study evaluated pooled milk for FMDV surveillance on a large-scale dairy farm that experienced two FMD outbreaks caused by the A/ASIA/G-VII and O/ME-SA/Ind-2001d lineages, despite regular vaccination and strict biosecurity practices. FMDV RNA was detected in 42 (5.7%) of the 732 pooled milk samples, and typing information was concordant with diagnostic reports of clinical disease. The FMDV positive milk samples were temporally clustered around reports of new clinical cases, but with a wider distribution. For further investigation, a model was established to predict real-time RT-PCR (rRT-PCR) C T values using individual cattle movement data, clinical disease records and virus excretion data from previous experimental studies. The model explained some of the instances where there were positive results by rRT-PCR, but no new clinical cases and suggested that subclinical infection occurred during the study period. Further studies are required to investigate the effect of vaccination on FMDV excretion in milk, and to evaluate more representative sampling methods. However, the results from this pilot study indicate that testing pooled milk by rRT-PCR may be valuable for FMD surveillance and has provided evidence of subclinical virus infection in vaccinated herds that could be important in the epidemiology of FMD in endemic countries where vaccination is used.

An owner's ability to detect changes in the behavior of a dog afflicted with osteoarthritis (OA) may be a barrier to presentation, clinical diagnosis and initiation of treatment. Management of OA also relies upon an owner's ability to accurately monitor improvement following a trial period of pain relief. The changes in behavior that are associated with the onset and relief of pain from OA can be assessed to determine the dog's health-related quality of life (HRQOL). HRQOL assessments are widely used in human medicine and if developed correctly can be used in the monitoring of disease and in clinical trials. This study followed established guidelines to construct a conceptual framework of indicators of HRQOL in dogs with OA. This generated items that can be used to develop a HRQOL assessment tool specific to dogs with OA. A systematic review was conducted using Web of Science, PubMed and Scopus with search terms related to indicators of HRQOL in dogs with osteoarthritis. Eligibility and quality assessment criteria were applied. Data were extracted from eligible studies using a comprehensive data charting table. Resulting domains and items were assessed at a half-day workshop attended by experts in canine osteoarthritis and quality of life. Domains and their interactions were finalized and a visual representation of the conceptual framework was produced. A total of 1,264 unique articles were generated in the database searches and assessed for inclusion. Of these, 21 progressed to data extraction. After combining synonyms, 47 unique items were categorized across six domains. Review of the six domains by the expert panel resulted in their reduction to four: physical appearance, capability, behavior, and mood. All four categories were deemed to be influenced by pain from osteoarthritis. Capability, mood, and behavior were all hypothesized to impact on each other while physical appearance was impacted by, but did not impact upon, the other domains. The framework has potential application to inform the development of valid and reliable instruments to operationalize measurement of HRQOL in canine OA for use in general veterinary practice to guide OA management decisions and in clinical studies to evaluate treatment outcomes.