In this large prospective controlled study, the performance of a rapid RT-LAMP assay performed with crude saliva samples directly after saliva collection was analyzed. We used a CE marked assay specifically designed for saliva samples and for a point-of-care use. The test was authorized in France on November 2020 in symptomatic individuals for whom nasopharyngeal sampling was impossible or difficult. Our results showed, in a real-life rigorous evaluation, a low sensitivity of this method (34%) compared to nasopharyngeal RT-PCR. Its sensitivity remained low whatever the reference test considered (saliva RT-PCR, nasopharyngeal Ag test), ranging between 28 to 37%.
Our results differed strongly with the sensitivity of 86% (95CI 78%-94%) reported in Santos Schneider et al. study 27, and whatever the reference test used, nasopharyngeal RT-PCR or any other composite reference test including saliva RT-PCR and antigen test. In Santos Schneider et al. study, the authors evaluated the EasyCOV® assay in a central laboratory and tested each sample in triplicate. A sample was considered positive if at least two replicates out of three were positive. In our study, we tested all samples once directly in screening centers and according to manufacturer instructions, and to its expected use in routine conditions.
No difference in RT-LAMP sensitivity between symptomatic or asymptomatic participants was reported. Median time of testing was 3 days after symptom onset or 6 days after last contact of confirmed case. In 103 subjects already diagnosed for COVID-19, Nagura-Ikeda et al. reported, with another RT-LAMP assay, sensitivity results on saliva that differed according to the clinical state 23. The RT-LAMP assay was performed with nucleic acid extract of saliva instead of crude saliva. Overall sensitivity was of 71% compared to nasopharyngeal RT-PCR, with a higher sensitivity (85%) in patients tested within 9 days after symptom onset than after 9 days (44%). In asymptomatic individuals, the sensitivity was 60%.
According to other studies evaluating RT-LAMP tests, the critical step for sensitivity seemed to be the RNA extraction 13,23,25,28. A high level of concordance between RT-PCR on nasopharyngeal samples and RT-LAMP on saliva was observed when an automated extraction step (i.e. Qiasymphony RNA kit) was used. In a limited series of 34 positive samples (17 nasopharyngeal swabs and 17 saliva) tested by RT-PCR, Taki et al. reported a sensitivity of a RT-fluorescence LAMP assay performed with nucleic acid extracts of 97% and 100% in nasopharyngeal and saliva samples, respectively 25. Without RNA extraction on the same samples sensitivities decreased respectively to 71% and 47%, suggesting that RNA extraction process may be critical for the SARS-CoV-2 RNA detection by RT-LAMP especially for saliva samples. In our study, the RT-LAMP assay is an extraction-free test based on a 10 minutes heating at 80°C for virus inactivation and viral RNA release. This quick step suitable for a point-of-care test might be not optimal for RT-LAMP reaction with saliva samples and results may depend on miscellaneous factors according to the quality of saliva (volume, pH, viscosity, food by-products). The participants did not drink, eat or smoke within 30 minutes before saliva sampling. In addition, we did not find any significant effect of cigarettes or alcohol consumption within 2 or 24 hours. Another hypothesis is the impact of viral load. As we showed, Ct values of nasopharyngeal RT-PCR were lower in RT-LAMP true positive samples than in RT-LAMP false negative samples (26 vs 28), suggesting an impact of viral load on saliva RT-LAMP efficacy. However when considering only high or significant SARS-CoV-2 loads in nasopharyngeal samples, saliva RT-LAMP sensitivity remained low. Thus, the viral load per se does not explain the weak performance of the assay.
Finally, our study confirmed, as previously 19, the good performance of saliva RT-PCR and nasopharyngeal antigen testing as reliable alternative strategies to detect SARS-CoV-2 in both symptomatic and asymptomatic individuals in the ambulatory setting. Further work is needed to optimize an assay combining collected saliva and rapid point-of-care isothermal detection of SARS-CoV-2 RNA.