The Burden and Genomic Characterization of Shigella -Associated Diarrhea in Children Under Five in Lusaka, Zambia: A Prospective Cohort Study
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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.
Research in context
Evidence before this study
Despite Shigella being a leading cause of bacterial diarrheal mortality globally, there is a critical lack of up-to-date data on its burden and associated antimicrobial resistance (AMR) in high-risk settings, such as sub-Saharan Africa (SSA). We searched PubMed for studies published between 2000 and May 2025, using the terms “ Shigella ,” “antimicrobial resistance,” and “sub-Saharan Africa.” Only three studies (GEMS, MAL-ED, VIDA) involving extensive surveillance in SSA countries were identified, all conducted before 2018. None integrated disease burden, genomic characterization of circulating strains, and both phenotypic and genotypic AMR profiling. In Zambia, we found no published surveillance data on Shigella or resistance patterns in children.
The added value of the study
We present an up-to-date, integrated assessment of the burden, genomic diversity, and AMR profiles among Shigella isolates in children under five in SSA, specifically in a high-risk peri-urban setting in Lusaka, Zambia. By prospectively following a large cohort of 1,400 children and combining culture with WGS, we provide detailed insights into the disease burden and epidemiology of circulating Shigella strains and the relevance of candidate vaccine antigens. We reveal a high prevalence of multidrug resistance (MDR), including plasmid-mediated and phenotypic resistance to ciprofloxacin, the first-line treatment for Shigella . We further complement genotypic AMR with phenotypic AMR testing to predict potential resistance genes while measuring antibiotic susceptibility in real clinical settings.
Implications of all the available evidence
We demonstrate that Shigella is genomically diverse and an important etiology of moderate to severe gastroenteritis in Zambian children. Risk factors include increasing age and poor WASH. We observed plasmid-mediated resistance to ciprofloxacin and MDR, which threatens the efficacy of current Shigella treatment and risks population-level AMR spread. These results highlight the need for improved WASH, antibiotic stewardship, and the development of effective vaccines, supported by ongoing genomic surveillance, to facilitate disease monitoring, inform treatment guidelines, guide vaccine antigen selection, and inform evidence-based antibiotic stewardship.
