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

BackgroundCurrent 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. Alternative sample types, such as saliva, which enable earlier detection could bolster the mpox response by mitigating transmission and facilitating access to antiviral treatments. MethodsWe evaluated five PCR assays and compared their detection of mpox DNA extracted from 30 saliva specimens collected in Spectrum SDNA-1000 tubes. We sequenced seven mpox-positive samples and assessed concordance with the primers and probes of the PCR assays. Following, we incorporated these PCR assays into a simplified, extraction-free protocol to evaluate its feasibility for testing raw (unsupplemented) saliva samples. To further explore the potential of this approach, we investigated the stability of mpox detection in raw saliva diluted 1:10 and 1:100 in mpox-negative saliva, after storage at 4{degrees}C, room temperature ([~]19{degrees}C), 30{degrees}C, and 40{degrees}C for 72 hours and through simulated shipping conditions. ResultsDespite identifying three nucleotide substitutions in the CDCs Monkeypox virus Generic Real-Time PCR Tests primer sequences, we observed no difference in the mean Ct-values generated between assays. We successfully incorporated each assay into our saliva-based extraction-free PCR protocol. Detection in raw saliva following storage at 4{degrees}C, [~]19{degrees}C, and 30{degrees}C remained relatively stable for 24-48 hours and following simulated shipping conditions. ConclusionsThis pilot investigation supports a flexible, saliva-based, extraction-free PCR test as a promising approach for diagnosis, outbreak response or ongoing surveillance of mpox. With detection in raw saliva remaining stable for 24-48 hours and through simulated shipping temperatures, saliva-based sampling and simplified testing could reduce diagnostic costs, increase access to testing and address hurdles in low- and middle-income countries.