Design and Validation of Multiplexed Respiratory RT-LAMP assay for the differentiation of SARS-CoV-2, Influenza A and Respiratory Syncytial Virus (RSV) in COVID-19 Pandemic Samples from Western Kenya.

The COVID-19 pandemic highlighted the importance of widespread, accessible viral surveillance to ensure pandemic-preparedness. The rapid diagnosis of respiratory infections is crucial not only for effective patient management but also to inform ongoing surveillance for the detection of outbreaks. Reverse transcriptase-Loop mediated isothermal amplification (RT-LAMP) assays are an attractive alternative to reverse transcriptase polymerase chain reaction (RT-PCR) for rapid, decentralised testing in resource limited settings due being rapid and low cost. Here we present the design and evaluation of a multiplexed RT-LAMP assay for the simultaneous detection of SARS-CoV-2, Influenza A and Respiratory Syncytial Virus via the incorporation of fluorescently labelled LAMP probes. Mid-nasal swabs collected from 175 participants on the day of enrolment in the MALCOV Cohort study in Kisumu, Kenya were used in the evaluation of the RT-LAMP assay. Overall, 114 SARS-CoV-2, six Influenza A, two Influenza B and zero RSV samples were identified in the cohort by the Allplex SARS-CoV-2/FluA/FluB/RSV RT-PCR kit (Seegene Inc) reference test, in addition to 53 negatives. Sensitivity and specificity of the RT-LAMP assay (SARS-CoV-2/FluA/RSV) when compared to RT-PCR was 82.64% [CI95%:74.70-88.92%] and 100% [CI95%:93.40-100%] at 40 minutes for all samples respectively. The mean time to a positive result was 11minutes, highlighting the speed of this technology compared to the more commonly used RT-PCR. RT-LAMP enables rapid multi-pathogen detection with comparable diagnostic accuracy to RT-PCR.