Rapid and accurate diagnosis of infections is fundamental to containment of disease. Several monkeypox virus (MPV) real‐time diagnostic assays have been recommended by the CDC; however, the specificity of the primers and probes in these assays for the ongoing MPV outbreak has not been investigated. We analyzed the primer and probe sequences present in the CDC recommended MPV generic real‐time PCR assay by aligning those sequences against 1730 MPV complete genomes reported in 2022 worldwide. Sequence mismatches were found in 99.08% and 97.46% of genomes for the MPV generic forward and reverse primers, respectively. Mismatch‐corrected primers were synthetized and compared to the generic assay for MPV detection. Results showed that the two primer‐template mismatches resulted in a ~11‐fold underestimation of initial template DNA in the reaction and 4‐fold increase in the 95% LOD. We further evaluated the specificity of seven other real‐time PCR assays used for MPV and orthopoxvirus (OPV) detection and identified two assays with the highest matching score (>99.6%) to the global MPV genome database in 2022. Genetic variations in the primer‐probe regions across MPV genomes could indicate the temporal and spatial emergence pattern of monkeypox disease. Our results show that the current MPV real‐time generic assay may not be optimal to accurately detect MPV, and the mismatch‐corrected assay with full complementarity between primers and current MPV genomes could provide a more sensitive and accurate detection of MPV.
Domestic wastewater, when collected and evaluated appropriately, can provide valuable health-related information for a community. As a relatively unbiased and non-invasive approach, wastewater surveillance may complement current practices towards mitigating risks and protecting population health. Spurred by the COVID-19 pandemic, wastewater programs are now widely implemented to monitor viral infection trends in sewersheds and inform public health decision-making. This review summarizes recent developments in wastewater-based epidemiology for detecting and monitoring communicable infectious diseases, dissemination of antimicrobial resistance, and illicit drug consumption. Wastewater surveillance, a quickly advancing Frontier in environmental science, is becoming a new tool to enhance public health, improve disease prevention, and respond to future epidemics and pandemics.
The current monkeypox outbreak as a global health emergency now spreads into more than 90 counties. Rapid and accurate diagnosis of the infections is fundamental for the viral disease containment. Here, we analyzed the primer and probe sequences in the CDC recommended monkeypox virus (MPV) generic real-time PCR assay by aligning against 683 reported MPV genomes worldwide. Sequence mismatches were found in more than 92% of genomes for the MPV generic forward and reverse primers. We also evaluated the specificity of seven other real-time PCR assays for MPV and orthopoxvirus (OPV) detection, and identified two assays (MPV_F3L and OPV_F8L) with the highest matching score (>99.4%) to the global MPV genome database. Genetic variations on the MPV genomes corresponding with the real-time PCR primer and probe regions were further specified and used to indicate the temporal and spatial emergence pattern of monkeypox disease. Our results show that the current MPV real-time generic assay may be unsuitable to accurately detect MPV highlighting the need to develop new detection assays.
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