Expansion of a low-cost, saliva-based PCR test for the detection of mpox virus

Abstract

ABSTRACTBackgroundCurrent recommendations for the diagnosis of mpox rely on lesion-swabs as the gold-standard specimen type even though many patients experience symptoms prior to lesion-onset. Earlier detection could bolster the mpox response by mitigating transmission and facilitating access to antiviral treatments.MethodsWe first compared five PCR assays for their detection of mpox DNA extracted from 30 saliva specimens in collection devices with a stabilizing buffer. Next, we investigated the stability of mpox detection in five raw, unsupplemented saliva samples diluted 1:10 in mpox-negative saliva, after storage at 4°C, room temperature (∼19°C), 30°C, and 40°C for 72 hours. We also investigated the stability of virus detection through simulated shipping conditions. Lastly, we performed amplicon sequencing on seven saliva samples and assessed concordance of the PCR assays against mpox virus sequences.ResultsDespite identifying three different substitutions in the CDC’s Monkeypox Virus Generic Real-Time PCR Test’s forward and reverse primers, we observed no difference in the mean cycle threshold values generated between assays. However, one gene target for one assay performed better for overall detection when validated. Detection following storage at 4°C, ∼19°C, and 30°C remained relatively stable for 24-48 hours but this declined by 72 hours. At 40°C, detection was stable at 24 hours but declined by 48 hours. Detection following simulated summer and winter shipping temperature profiles also remained stable.ConclusionsFindings of this pilot investigation support a flexible, saliva-based, extraction-free PCR test as a promising approach for the low-cost detection of mpox virus. With stability observed for 24-48 hours as well as over simulated shipping temperatures, saliva-based sampling and simplified testing could reduce mpox diagnostic costs, increase access to testing and address hurdles in low- and middle-income countries. Future studies should build upon this and assess the temporal dynamics of mpox virus in saliva.