The Burden and Genomic Characterization of Shigella-Associated Diarrhea in Children Under Five in Lusaka, Zambia: A Prospective Cohort Study

Background: Shigella is a leading cause of childhood diarrhea in low- and middle-income countries and is increasingly resistant to first-line antibiotics. We conducted a surveillance study to determine the incidence, genomic characteristics, and AMR profiles of Shigella infections in children under five with moderate to severe diarrhea (MSD) in Lusaka, Zambia. Methods: Between 15 September 2020 and 30 November 2021, a prospective cohort study of 1,400 children under five was enrolled during a community census in a peri-urban setting and passively followed for 9.5 months for MSD. During enrollment, socio-demographic data were collected using electronic questionnaires, while clinical data were collected through the DHIS platform. The main outcome, Shigella in diarrheal stool in under 5 children, was detected using culture and Loop-mediated Isothermal Amplification (LAMP) targeting the ipaH gene. Cox proportional hazards models were used to assess the incidence and risk factors of Shigella (ipaH) infections. Whole-genome sequencing (WGS) was used to characterize the genomic diversity and antimicrobial resistance genes, complemented by phenotypic antibiotic susceptibility testing. Results: There were 230 first episodes of Shigella over a follow-up time of 9,581.7 child-months, yielding an incidence of 24.0 (95% CI 21.1-27.3) cases per 1,000 child-months, with the highest incidence among 2 to 3-year-olds. The key risk factors identified were the water source (p=0.025) and age group (p=0.014). Genotypic characterization revealed 10 S. flexneri, 9 S. sonnei, and 3 S. boydii. The S. sonnei isolates formed two clusters, differing in virulence factors and plasmid profiles, indicating two possible circulating strains. Shigella isolates exhibited phenotypic and genotypic multidrug resistance, including against trimethoprim, aminoglycosides, and beta-lactams. Plasmid-mediated quinolone resistance (qnrS1) was identified in four S. flexneri isolates, with these genes located on the IncFIB(K) plasmid, highlighting the potential for horizontal transmission and spread of quinolone resistance in this region. No phenotypic and genotypic resistance to macrolides, the first-line treatment for Shigella in Zambia, was observed. Interpretation: We report a high burden of Shigella with multidrug resistance, including resistance to fluoroquinolones. These findings highlight the increasing resistance of Shigella to first-line antibiotics and underscore the importance of developing safe and effective vaccines, improving WASH conditions, and ongoing AMR surveillance. Funding: The EDCTP2 program, supported by the European Union, the Faculty for the Future Foundation (FFTF), the Netherlands Organization for Health Research and Development (ZonMw), and Health-Holland AMR-Global, Gloria, and Track-AMR.