From clinical fluids to environmental matrices: quantitative monitoring of infectious monkeypox virus based on PMA-qPCR during the Guangdong outbreak.

BackgroundMpox, caused by monkeypox virus (MPXV), remains a public health priority. Standard qPCR detects viral DNA but cannot distinguish infectious virions from residual genetic material of inactivated virus, complicating transmission risk assessment. This study aimed to develop a rapid assay for quantifying infectious MPXV across clinical and environmental matrices in Guangdong Province, China.MethodsWe developed a propidium monoazide-assisted qPCR (PMA-qPCR) assay that selectively amplifies DNA from intact virions, and embedded it within the Guangdong provincial Center for Disease Control and Prevention surveillance network. We examined 23 clinical fluid specimens from 15 mpox patients, 337 surface swabs and 53 domestic wastewater samples from patient living environments, and 498 terminal wastewater samples from flights and sewage treatment plants collected between June 2023 and August 2024. Decay kinetics were evaluated at 25 °C and 30 °C under varying pH, with initial viral loads of 103 and 104 PFU/mL.ResultsPMA-qPCR discriminated infectious from non-infectious virus. Co-incubation with 50 μM PMA and 0.005% SDS effectively blocked amplification of non-infectious DNA, and the assay showed quantitative concordance with the standard plaque assay. Infectious MPXV loads were highest in skin lesions and anal swabs, exceeding throat swabs by more than 100-fold. Among surface samples, 41.54% (140/337) tested positive for MPXV DNA by qPCR, but only 25.71% (36/140) of these contained infectious virus at low loads (126.79 ± 93.57 PFU/mL). For wastewater, 11.32% (6/53) of domestic samples from patient living environments tested positive by qPCR, yet no infectious MPXV was detected. No MPXV DNA was detected in 498 terminal wastewater samples from flights or sewage treatment plants. Decay experiments demonstrated that when the initial concentration was 103 to 104 PFU/mL, infectious MPXV decreased by more than 99% within 48 h, with accelerated decay under acidic pH and elevated temperature, particularly at lower initial concentrations.ConclusionsPMA-qPCR provides a practical tool for rapid cross-matrix quantification of infectious MPXV. Direct contact with skin lesions or anal secretions likely dominates transmission, whereas environmental persistence is limited. Testing for infectious virus within 48 h of collection is recommended, as infectious MPXV decays by over 99% within this window.