key: cord-0877251-ijpj41us authors: Brown, Benjamin; O'Hara, Robert William; Guiver, Malcolm; Davies, Emma; Birtles, Andrew; Farooq, Hamzah; Verma, Arpana; Guo, Hui; Hayden, Katharine; Machin, Nicholas title: Evaluation of a Novel Direct RT-LAMP Assay for the Detection of SARS Cov-2 from Saliva Samples in Asymptomatic Individuals date: 2022-03-24 journal: J Clin Virol Plus DOI: 10.1016/j.jcvp.2022.100074 sha: 8325dec1c5364d1abbb5d471b8edea8810f1b3b9 doc_id: 877251 cord_uid: ijpj41us Large scale screening of health care workers and the general population for asymptomatic COVID-19 infection requires modalities that are amenable to testing at scale while retaining acceptable levels of sensitivity and specificity. This study evaluated a novel COVID-19 Direct-RT LAMP assay using saliva samples in asymptomatic individuals by comparison to RT-PCR. Additional studies were performed using VTM collected from routine diagnostic testing. Analytical sensitivity was determined for Direct RT-LAMP assay using the WHO International Standard. Finally, quantified results from RT-PCR testing of 9177 nose and throat swabs obtained from routine diagnostic testing were used to estimate the sensitivity of Direct RT-LAMP using the limit of detection curve obtained from the analytical sensitivity data. Results from saliva testing demonstrated a sensitivity of 40.91% and a specificity of 100% for Direct RT-LAMP. The sensitivity and specificity for nose and throat swabs were 44.85% and 100% respectively. The 95% limit of detection (LOD) for Direct RT-LAMP was log 7.13 IU/ml (95% 6.9-7.5). The estimated sensitivity for Direct-RT LAMP based on the results of 9117 nose and throat swabs was 34% and 45% for saliva and VTM respectively. The overall diagnostic sensitivity of Direct RT-LAMP was low compared to RT-PCR. Testing of nose and throat swabs and estimating the sensitivity based on a large cohort of clinical samples demonstrated similar results. This study highlights the importance of utilising the prospective collection of samples from the intended target population in the assessment of diagnostic sensitivity. It is now established that SARS-CoV-2 can be detected in asymptomatic individuals with estimates as high as 30.8% 1 . Throughout the pandemic there has been considerable concern around asymptomatic COVID-19 contributing to nosocomial infection. One screening pilot in a large UK teaching hospital found that 3% of staff had asymptomatic infection, of which 57% did not go on to develop symptoms 2 . There is also evidence to support the fact that asymptomatic infection in health care workers is associated with transmission in these settings 3, 4 . There has therefore been a compelling case for COVID -19 screening in asymptomatic healthcare workers to reduce the risk of transmission to patients and in England this has been reflected in national guidance 5 . Large scale testing requires modalities that are amenable to testing at scale while retaining acceptable levels of sensitivity and specificity. Recent studies have shown that SARS-CoV-2 RNA can be detected in saliva, with viral loads peaking during the first week of symptoms 6 . Some studies have shown that viral loads in saliva are comparable to or higher than nasopharyngeal swabs 7, 8 while others have observed lower viral loads 9 . The aim of this study was to evaluate a novel Direct-RT LAMP assay for the detection of SARS-CoV-2 in asymptomatic individuals using saliva samples. The study involved two phases; the first was conducted in the general population and was part of multicentre evaluation in collaboration with the UK Department of Health and Social Care (data from which has previous been published 10 ) and the second focused on asymptomatic health care workers in secondary care as a continued evaluation of the performance of the assay. OptiGene IFU for Direct RT-LAMP lists nose and throat swabs collected in Virocult® media alongside saliva samples as an accepted sample type 11 . Specimens submitted for routine testing were collected over a period of three weeks, concurrent with phase 2 of the study. Previous positive patients were excluded from the assessment and then randomly selected before being unlinked and anonymised. Blinded testing was performed by a second operator within 8 hours of receipt and before freezing. Saliva samples were inactivated by mixing 300µl of sample with 300µl Qiagen AL buffer (Qiagen, Hilden, Germany) and heated at 80°C for 15 minutes. RT-PCR was split over 2 workstreams using CDC N1 and N2 primers and probes 12 To investigate the variability of Ct value caused by different gene targets, further testing was performed on nucleic acid extracts of positive samples using the genesig ® SARS-CoV-2 winterplex assay (Primerdesign Ltd, Chandler's Ford, UK) according to the manufacturer's IFU. In brief 8 ul of previously extracted sample was added to master mix composed of 10µl of onestep master mix and 2µl of Orf 1a/b/S primer and probe mix followed by RT-PCR run on a Fast 7500 Fast PCR system with cycling parameters of 55°C for 10 minutes, 95°C for 2 minutes and 45 cycles of 95°C 10 secs and 60°C for 60 secs. Samples for the nose and throat swab validation were tested using the Cobas SARS-CoV-2 assay (Roche, Basel, Switzerland) according to the manufacturer's instructions. The assay targets both Orf1ab and E genes, any single target or >Ct 35 positive results were confirmed using the Biofire SARS-CoV-2 assay. Samples were inactivated by mixing 50µl of sample with 50µl of RapiLyze buffer (OptiGene, Horsham, UK) in a 96 well MicroAmp Optical Reaction Plate (Thermofisher) and held at 98°C for two minutes on an Applied Biosystems 9600 Thermal Cycler (Thermofisher). A negative extract control containing Rapilyze alone was included with each sample set. During phase 1 of the study Direct SARS-CoV-2 RT-LAMP was performed according to the manufacturer's protocol v1.1. All Direct RT-LAMP tests were performed on inactivated saliva samples within 30 minutes post-inactivation. All samples were tested in singles. Master mix comprised 17.5µl of Direct RT-LAMP master mix and 2.5µl of 10X COVID 19 (OptiGene) to which 5µl of inactivated sample was added and mixed. Direct RT-LAMP was performed on a Genie HT platform (OptiGene), using an amplification stage of 65°C for 20 minutes followed by anneal curve analysis, 98°C for 1 minute then cooled to 80°C at a rate of 0.05°C/s. In addition to the Rapilyze extraction control, a nuclease free water NTC, a DNA CD-COV19-100 positive control (OptiGene) and an additional SARS-CoV-2 whole genome RNA positive control were included on each run. Samples were identified as positive automatically by the Genie HT platform through detection of increased fluorescence combined with an anneal peak within the defined range. All sample amplification plots were manually checked to ensure any potential signals were not missed. Direct RT-LAMP in phase 2 of the study was performed using the Covid 19 DirectPlus RT-LAMP protocol. The modified DirectPlus RT-LAMP thermal protocol was provided by the manufacturer. The modification from phase 1 was an alteration to the interpretive software, directed on the final anneal analysis. A total of 1383 samples were collected during the two phases of the saliva sample study. The percentage of samples rejected due to issues with viscosity was 7%. The results from testing multiple replicates of each dilution are listed in Table 1 . The Probit analysis results are listed in Table 3 . The 95% LOD for detection of SARS-CoV-2 RNA from saliva samples was log 7.13 IU/ml (95% 6.9-7.5), and for Virocult ® VTM was log 6.46 IU/ml (95%CI 6.3-6.8). A total of 22 samples were positive by RT-PCR (Table 2) . Nine samples were positive by Direct RT-LAMP, with an overall sensitivity of 40.91% and a specificity of 100% (Table 3 ). In phase 1, two RT-PCR positive samples were characterised as negative by the Direct RT-LAMP instrument but showed strong amplification below Tp 10:00 and produced peaks with anneal temp analysis around the correct temperature and so were recorded as positive. SARS Cov-2 samples positive by the N1/N2 PCR assay were confirmed in 21 of the 22 specimens by the Wintreplex assay (Table 4) , with a single sample with a Ct of 34.7 recorded as negative. The Ct values produced by both assays were similar, with a mean difference in Ct of 0.9. The sensitivity of Direct RT-LAMP is closely related to the Ct value produced by the RT-PCR assay, as seen in figure 1. Sensitivity was 100% on samples with a Ct <25, representing samples with a high viral burden. Sensitivity drops off rapidly above this level as the viral load declines. Results from testing nose and throat swabs by Direct RT-LAMP are listed in Table 5 . The overall sensitivity in nose and throat swabs was similar to saliva at 44.85% (95%CI 36.32 -53.61%) with a specificity of 100%. Figure 2 shows the spread of Ct values producing true positive and false negative results by Direct RT-LAMP. The same pattern of high sensitivity at Ct values ≤25 and a reduced sensitivity ≥25 is displayed. The overall diagnostic sensitivity of Direct RT-LAMP for detection of SARS-CoV-2 directly from saliva samples was 40.91% combined with a specificity of 100%. Direct RT-LAMP failed to detect most samples that were positive above Ct 25 by RT-PCR. These results were obtained prospectively from combined data across two phases of asymptomatic testing in Manchester. Therefore, this study provides a representative insight into the diagnostic sensitivity of the OptiGene LAMP assay as a function of the range of viral loads present in an asymptomatic population. This study was limited by a small number of positive samples due to the relatively low prevalence at the time. To address this, randomly selected, first-time positive nose and throat swabs from routine diagnostic testing were re-tested with the Direct RT-LAMP assay. The results showed a moderately increased sensitivity of 44.85% with an upper confidence level limit of 53.61%. The slightly increased sensitivity in comparison to saliva samples suggests that the VTM did not inhibit the LAMP reaction. Probit analysis from the analytical sensitivity study demonstrated an estimated 95% limit of detection of log 7.13 IU/ml for Direct RT-LAMP from saliva samples and log 6.46 IU/ml from Virocult ® samples. Other studies using RT-LAMP technology have also recorded analytical sensitivities of between log 5 -5.8 log c/ml 13,14 however, the DHSC report for OptiGene Direct RT-LAMP states an analytical sensitivity of 1000 c/ml 15 To rule out any possibility that the gene used in our saliva RT-PCR assay was a contributing factor to the observed sensitivity, we compared Ct values from positive samples obtained by the in-house N1/N2 PCR with the Winterplex assay, which targets the same ORF 1ab gene used in the Direct RT-LAMP assay. There was good concordance between the Ct values of both assays with a mean difference of only 0.90, indicating minimal variation in sensitivity. We therefore conclude that either assay could be used as a comparator for Direct LAMP. The use of saliva samples offers some advantages for ease of collection and our study has shown a similar sensitivity to nose and throat swabs. However, direct testing of samples without nucleic extraction presents technical challenges for enzymatic amplification of RNA by RT-PCR or LAMP methods which may be inhibited by clinical samples. Saliva samples are heterogenous and our experience was that saliva specimens may often be highly viscous making the use of automated sample processing extremely difficult. MHRA TPP guidance for point of care 19 and laboratory based 20 assays recommends the use of an internal control but the OptiGene assay does not currently include this, making it impossible to identify the presence of inhibitory factors in the sample. The overall diagnostic sensitivity of Direct RT-LAMP in this real-world prospective study was low compared to RT-PCR. Testing of a larger set of randomly collected nose and throat swabs confirmed a similar sensitivity with a similar range of viral loads. The analytical sensitivity data and projection on to the viral loads observed in a large cohort of clinical samples tested throughout the COVID-19 pandemic is also consistent with the low diagnostic sensitivity that was observed. This study highlights the importance of utilising the prospective collection of samples from the intended target population in the assessment of diagnostic sensitivity. ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Evidence of SARS-CoV-2 Infection in Returning Travelers from Wuhan Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission Presymptomatic SARS-CoV-2 Infections and Transmission in a Skilled Nursing Facility Genomic and healthcare dynamics of nosocomial sars-cov-2 transmission Overview | COVID-19 rapid guideline: haematopoietic stem cell transplantation | Guidance | NICE. COVID-19 rapid guideline: haematopoietic stem cell transplantation Viral dynamics of SARS-CoV-2 in saliva from infected patients Comparison of SARS-CoV-2 detection in nasopharyngeal swab and saliva Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs Saliva as a Noninvasive Specimen for Detection of SARS-CoV-2 RT-LAMP has high accuracy for detecting SARS-CoV-2 in saliva and naso/oropharyngeal swabs from asymptomatic and symptomatic individuals OptiGene Limited Instructions For Use COVID-19_RNA RT-LAMP KIT-500 Real-time RT-PCR Primers and Probes for COVID-19 | CDC Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP) Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples Rapid evaluation of OptiGene RT-LAMP assay (direct and RNA formats) -GOV Concerning the OptiGene Direct LAMP assay, and it's use in at-risk groups and hospital staff A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection Department of Health and Social Care. Technical Validation of OptiGene RT LAMP Assay (Direct and RNA Formats) Medicines and Healthcare products Regulatory Agency. Target Product Profile: Point of Care SARS-CoV-2 detection tests -GOV Medicines and Healthcare products Regulatory Agency. Target Product Profile: Laboratory-Based SARS-CoV-2 Viral Detection tests -GOV Analytical sensitivity of the Direct RT-LAMP assay was determined using a dilution of the 1st