Culture-free whole genome sequencing analysis of Haemophilus ducreyi from genital ulcer disease cases in Malawi, 2020-2022: a retrospective genomic surveillance study

Summary Background Chancroid, aetiological agent Haemophilus ducreyi, prevalence has declined globally. However, H. ducreyi remains causing genital ulcer disease in some sub-Saharan African countries and is causing non-genital cutaneous ulcers in tropical settings. Genomic data on H. ducreyi are exceedingly scarce, limiting understanding of transmission dynamics, lineage structure, and antimicrobial resistance (AMR). We examined H. ducreyi circulating in Malawi using whole-genome sequencing (WGS). Methods We performed culture-free WGS using target-enrichment from PCR-positive chancroid samples (n=118) collected in Malawi (2020-2022). Eighty-seven H. ducreyi genomes were generated and analysed alongside 55 publicly available genomes. Phylogenomic reconstruction, recombination, genomic and pan-genome analysis, and genome-wide association studies characterised population structure, clinical associations, and AMR determinants. Findings All Malawian genomes were assigned as class I and formed an extremely clonal lineage, with a mean pairwise distance of 2.6 SNPs and lack of recombination, consistent with a recent ongoing outbreak. All Malawian genomes carried plasmid-associated AMR determinants including blaTEM-1, tet(B), tet(32), and sul2, indicating multidrug resistance to previously used antibiotics. No determinants associated with resistance to fluoroquinolones, macrolides, or ceftriaxone were identified. Interpretation A clonal expansion of one H. ducreyi strain is driving the contemporary chancroid outbreak in Malawi, highlighting the continued public health relevance of this pathogen. No AMR determinants associated with resistance to fluoroquinolones azithromycin, or ceftriaxone were detected, supporting continued use of recommended first-line therapies while highlighting the importance of continued AMR surveillance. Continued genomic H. ducreyi surveillance is essential to monitor transmission, support early detection, and timely detection of emerging AMR.