<b>EPIDEMIOLOGICAL</b><b> </b><b>AND</b><b> </b><b>MOLECULAR</b><b> </b><b>STUDY</b><b> </b><b>OF</b><b> </b><b>DIFFERENT STRAINS

Measles remains a significant cause of morbidity and mortality among children in Pakistan despite the availability of safe and effective vaccines. District Swabi in Khyber Pakhtunkhwa has consistently reported measles cases, but comprehensive data on the epidemiological characteristics and circulating viral strains in the district have been lacking. This study aimed to determine the epidemiological characteristics, vaccination coverage, clinical outcomes, and molecular diversity of measles virus strains circulating in District Swabi. A cross-sectional study was conducted from January to December 2024 at major healthcare facilities across all union councils of District Swabi. A total of 185 suspected measles cases meeting the WHO case definition were enrolled. Demographic and clinical data were collected using structured questionnaires, and vaccination histories were verified through vaccination cards or parental recall. Throat swabs were collected from all participants and transported in viral transport medium under cold chain conditions. Viral RNA was extracted and real-time RT-PCR was performed to confirm measles virus infection. Positive samples were subjected to conventional RT-PCR targeting the 450-nucleotide carboxyl-terminal region of the nucleoprotein (N-450) gene for genotyping. Amplified products were sequenced using Sanger sequencing, and phylogenetic analysis was performed using MEGA software with the neighbor-joining method and 1000 bootstrap replicates. Data were analyzed using SPSS version 25. 185 suspected cases, 144 (77.8%) were confirmed positive for measles virus by RT-PCR. Infants aged 0-12 months were the most affected group (36.2%), followed by toddlers aged 13-36 months (31.4%). Males constituted 58.4% of cases, and 61.1% of patients resided in rural areas. Vaccination status revealed that 53.0% of confirmed cases were completely unvaccinated, 23.2% were partially vaccinated, and only 16.8% were fully vaccinated with two doses. Complications occurred in 57.3% of patients, with pneumonia being the most common (25.9%), followed by diarrhea (20.0%). Hospitalization was required for 34.6% of cases, and four deaths (2.2%) were recorded, all in unvaccinated children. Fully vaccinated individuals had significantly lower rates of complications (16.1% vs 65.3%, p<0.001) and hospitalization (12.9% vs 49.0%, p<0.001) compared to unvaccinated individuals. Seasonal analysis showed peaks in late spring (May-June) and late autumn (November-December). Phylogenetic analysis of 98 successfully sequenced isolates revealed that 90.8% belonged to genotype B3, while 9.2% showed other genotypes. Three distinct phylogenetic sub-clusters were identified within genotype B3: Swabi-Cluster A (42 isolates, 87% bootstrap) showed close genetic relatedness to sequences from Afghanistan and Iran; Swabi-Cluster B (31 isolates, 82% bootstrap) clustered with sequences from other Pakistani provinces including Punjab and Sindh; and Swabi-Cluster C (25 isolates, 79% bootstrap) showed relatedness to sequences from Russia, the United Kingdom, and China. Temporal analysis revealed sequential appearance of clusters, with Cluster A dominating in spring, Cluster B in summer, and Cluster C in autumn. Geographic distribution showed clustering of specific lineages in different union councils. Mutation analysis of the hemagglutinin gene identified several non-synonymous substitutions compared to the vaccine strain, including conserved changes at positions 174, 211, 260, 335, and 481 present in all isolates. The estimated district-wide vaccination coverage was 54%, far below the WHO-recommended 95% threshold for herd immunity.