Wastewater Surveillance of Enteroviruses Potentially Associated with Hand, Foot, and Mouth Disease in Nepal

Enteroviruses (EVs), including those linked with hand, foot, and mouth disease (HFMD), are commonly transmitted via the fecal-oral route. Considering the underreporting of HFMD-associated EVs and Nepal's significant burden of enteric disease, wastewater surveillance presents a vital tool for monitoring community-level viral transmission. Through this study, we intended to detect EVs, including EVA serotypes that are often associated with HFMD, in wastewater to support public health surveillance. Raw influent wastewater samples (n = 25) collected previously during April-October 2022 from a wastewater treatment plant in Kathmandu, Nepal, were tested for the presence of EVs and four serotypes of EV type A (EVA), namely EVA71, Coxsackievirus A types 6 (CVA6), 10 (CVA10), and 16 (CVA16), by reverse transcription-quantitative polymerase chain reaction. In all samples, EVs were detected with concentrations of 7.4 ± 0.4 log₁₀ copies/L. In addition, EVA71 was detected in 64% (16/25) of the samples, with concentrations of 5.3 ± 0.5 log₁₀ copies/L, suggesting frequent detection with more prominent peaks around the pre-monsoon (75%, April and May) and monsoon seasons (71%, June-September). CVA6, CVA10, and CVA16 were not detected in any of the tested samples. The present findings demonstrated the high prevalence of EVs in wastewater in Kathmandu. This study is the first to indicate the presence of EVA71 in the wastewater of Nepal. The co-detection of EVs and EVA71, along with respiratory viruses (severe acute respiratory syndrome coronavirus 2, influenza A virus, and respiratory syncytial virus) and hepatitis virus, detected in our previous studies, was also explored in the present study. Except for respiratory syncytial virus, all tested viruses were more frequently co-detected in 36% (9/25) of the samples, especially during the monsoon (43%, 6/14) and pre-monsoon (38%, 3/8) seasons. The co-detection of multiple viruses underscores the necessity for integrated viral surveillance and calls for further investigation into the clinical implications of co-infections.