Molecular Detection of Non‐Polio Enteroviruses and Emergence of Novel Enterovirus D111 in Stool Samples From Children Reported With Acute Flaccid Paralysis Symptoms During 2021 in Pakistan

This study explores the molecular detection and genetic diversity of non-polio enteroviruses (NPEVs) reported with symptoms of acute flaccid paralysis (AFP) cases (referring to individual patients) in Pakistan during 2021. A total of 2337 stool samples were analyzed through WHO-recommended intratypic differentiation (ITD) using real-time PCR. Among these, 134 samples detected positive for NPEVs, corresponding to 66 individuals AFP cases. From these, 21 representative samples-selected based on sample quality and geographical distribution across Punjab, Sindh, and Khyber Pakhtunkhwa province-underwent partial VP1 gene sequencing. Thirteen distinct enterovirus serotypes were identified, with Enterovirus B (EV-B) being predominant (95.24%). Echovirus 13 (14.29%) was most frequent, followed by E-7, CV-B2, E-11, E-12, E-20, and EV-B75 (each 9.52%). Punjab contributed the highest proportion (52.38%) of sequenced cases. Most affected children were under 5 years old, with the highest proportion (42.8%) between 13 and 24 months. A significant finding was the first detection of novel Enterovirus D111 (EV-D111) in Pakistan and Asia, identified in the stool sample of a 72-month-old child reported with the symptoms of acute flaccid paralysis. However, due to limited clinical data and the absence of confirmatory neurological evaluationsno definitive causal relationship could be set up between EV-D111 and the observed AFP symptoms. Confirmation was achieved via Sanger sequencing. Phylogenetic analysis indicated that the EV-D111 strain clustered closely with strains from Central Africa and retained conserved capsid motifs. These findings highlight the substantial genetic diversity of NPEVs in Pakistani AFP cases and amphasize the importance of enhanced molecular surveillance to detect emerging and rare strains like EV-D111, which are critical for strengthening AFP monitoring and public health preparedness in the region.