Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna ® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP (PHE guidelines). All RNA extraction methods provided similar results. FastVirus and Luna proved most sensitive. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrate that treatment of nasopharyngeal swabs with 70 degrees for 10 or 30 min, or 90 degrees for 10 or 30 min (both original variant and B 1.1.7) inactivates SARS-CoV-2 employing plaque assays, and that it has minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable to settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/ .

Estimates of the rate of infection and asymptomatic COVID-19 disease in a population sample from SE England.

BACKGROUND: Understanding of the true asymptomatic rate of infection of SARS-CoV-2 is currently limited, as is understanding of the population-based seroprevalence after the first wave of COVID-19 within the UK. The majority of data thus far come from hospitalised patients, with little focus on general population cases, or their symptoms. METHODS: We undertook enzyme linked immunosorbent assay characterisation of IgM and IgG responses against SARS-CoV-2 spike glycoprotein and nucleocapsid protein of 431 unselected general-population participants of the TwinsUK cohort from South-East England, aged 19-86 (median age 48; 85% female). 382 participants completed prospective logging of 14 COVID-19 related symptoms via the COVID Symptom Study App, allowing consideration of serology alongside individual symptoms, and a predictive algorithm for estimated COVID-19 previously modelled on PCR positive individuals from a dataset of over 2 million. FINDINGS: We demonstrated a seroprevalence of 12% (51 participants of 431). Of 48 seropositive individuals with full symptom data, nine (19%) were fully asymptomatic, and 16 (27%) were asymptomatic for core COVID-19 symptoms: fever, cough or anosmia. Specificity of anosmia for seropositivity was 95%, compared to 88% for fever cough and anosmia combined. 34 individuals in the cohort were predicted to be Covid-19 positive using the App algorithm, and of those, 18 (52%) were seropositive. INTERPRETATION: Seroprevalence amongst adults from London and South-East England was 12%, and 19% of seropositive individuals with prospective symptom logging were fully asymptomatic throughout the study. Anosmia demonstrated the highest symptom specificity for SARS-CoV-2 antibody response. FUNDING: NIHR BRC, CDRF, ZOE global LTD, RST-UKRI/MRC.

Application of a recombinase polymerase amplification (RPA) assay and pilot field testing for Giardia duodenalis at Lake Albert, Uganda.

BACKGROUND: Giardia duodenalis is a gastrointestinal protozoan causing 184 million cases of giardiasis worldwide annually. Detection is by microscopy or coproantigen assays, although sensitivity is often compromised by intermittent shedding of cysts or trophozoites, or operator expertise. Therefore, for enhanced surveillance field-applicable, point-of-care (POC), molecular assays are needed. Our aims were to: (i) optimise the recombinase polymerase amplification (RPA) assay for the isothermal amplification of the G. duodenalis ß-giardin gene from trophozoites and cysts, using published primer and probes; and (ii) perform a pilot field validation of RPA at a field station in a resource-poor setting, on DNA extracted from stool samples from schoolchildren in villages around Lake Albert, Uganda. Results were compared to an established laboratory small subunit ribosomal RNA (SSU rDNA) qPCR assay with additional testing using a qPCR targeting the triose phosphate isomerase (tpi) DNA regions that can distinguish G. duodenalis of two different assemblages (A and B), which are human-specific. RESULTS: Initial optimisation resulted in the successful amplification of predicted RPA products from G. duodenalis-purified gDNA, producing a double-labelled amplicon detected using lateral flow strips. In the field setting, of 129 stool samples, 49 (37.9%) were positive using the Giardia/Cryptosporidium QuikChek coproantigen test; however, the RPA assay when conducted in the field was positive for a single stool sample. Subsequent molecular screening in the laboratory on a subset (n = 73) of the samples demonstrated better results with 21 (28.8%) RPA positive. The SSU rDNA qPCR assay resulted in 30/129 (23.3%) positive samples; 18 out of 73 (24.7%) were assemblage typed (9 assemblage A; 5 assemblage B; and 4 mixed A+B). Compared with the SSU rDNA qPCR, QuikChek was more sensitive than RPA (85.7 vs 61.9%), but with similar specificities (80.8 vs 84.6%). In comparison to QuikChek, RPA had 46.4% sensitivity and 82.2% specificity. CONCLUSIONS: To the best of our knowledge, this is the first in-field and comparative laboratory validation of RPA for giardiasis in low resource settings. Further refinement and technology transfer, specifically in relation to stool sample preparation, will be needed to implement this assay in the field, which could assist better detection of asymptomatic Giardia infections.

Development of a lateral flow recombinase polymerase assay for the diagnosis of Schistosoma mansoni infections.

Infection with Schistosoma mansoni causes intestinal schistosomiasis, a major health problem across Africa. The accurate diagnosis of intestinal schistosomiasis is vital to inform surveillance/control programs. Diagnosis mainly relies on microscopic detection of eggs in faecal samples but many factors affect sensitivity. Molecular diagnostics are sensitive and specific but application is limited as necessary infrastructure, financial resources and skilled personnel are often lacking in endemic settings. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is practical in nearly any setting. Here we developed a RPA lateral flow (LF) assay targeting the 28S rDNA region of S. mansoni. The 28S LF-RPA assay's lower limit of detection was 10pg DNA with the lower test parameters permitting sufficient amplification being 6 min and 25°C. Optimal assay parameters were 40-45°C and 10 min with an analytical sensitivity of 102 copies of DNA. Additionally the PCRD3 lateral flow detection cassettes proved more robust and sensitive compared to the Milenia HybriDetect strips. This 28S LF-RPA assay produces quick reproducible results that are easy to interpret, require little infrastructure and is a promising PON test for the field molecular diagnosis of intestinal schistosomiasis.

HLA-DPB1 associations differ between DRB1*03 positive anti-Jo-1 and anti-PM-Scl antibody positive idiopathic inflammatory myopathy.

OBJECTIVE: The HLA 8.1 ancestral haplotype (HLA-B*08/DRB1*03/DQA1*05/DQB1*02) is associated with adult/juvenile idiopathic inflammatory myopathy (IIM), but confers a greater strength of association in patients possessing anti-Jo-1 or anti-PM-Scl antibodies. The HLA-DPB1 gene is centromeric to other HLA class II loci and separated by a recombination hotspot. We investigated whether HLA-DPB1 associations differ between anti-Jo-1 and anti-PM-Scl antibody-positive IIM cases. METHODS: Two hundred and thirty-three adult IIM patients (73% females, 49.4 +/- 13.6 years) with PM (n = 89), DM (n = 88) and myositis associated with another CTD (n = 55) and 85 juvenile DM patients (75% females, 6.2 +/- 3.6 years) were compared with 678 UK Caucasian controls. Patients/controls were genotyped for HLA-DPB1 and DRB1 alleles. Myositis-specific and associated antibodies were identified in cases using immunoprecipitation. RESULTS: HLA-DPB1*0101 was associated with IIM overall [22 vs 13% controls, corrected probability (P(corr)) = 2 x 10(-03); odds ratio (OR) 2.0; 95% CI 1.4, 2.9], PM (P(corr) = 7 x 10(-03); OR 2.5; 95% CI 1.5, 4.4) and anti-Jo-1 (P(corr) = 3 x 10(-5); OR 4.1; 95% CI 2.1, 7.8). No significant DPB1*0101 difference was present between anti-PM-Scl cases and controls. The HLA-DPB1*0101 association in IIM overall cases was dependent on the presence of DRB1*03. A number of HLA-DRB1*03/DPB1 haplotypes were identified, but only DRB1*03/DPB1*0101 was associated with anti-Jo-1 antibody-positive cases. CONCLUSIONS: The HLA-DRB1*03/DPB1*0101 haplotype is a risk factor for anti-Jo-1 antibody-positive IIM. Thus, although DRB1*03 is strongly associated with possession of either anti-Jo-1 or anti-PM-Scl, differing antibody associations are observed at the HLA-DPB1 locus.