Background: Kawasaki disease (KD), the most common multisystem vasculitis of childhood, is a febrile illness of unknown cause. Epidemiological data supports a role for infections in KD’s pathogenesis yet the relationship between KD and infections is unclear. To date, no studies have applied next-generation sequencing to investigate known pathogens in serum from children with KD. We therefore performed pan-microbial interrogation of serum from children with acute KD and healthy controls using shotgun metagenomic sequencing.
Methods: Serum samples underwent unbiased metagenomic sequencing with host depletion using Oxford Nanopore technology. Sequencing reads were filtered for quality and host contamination, followed by taxonomic classification and reference-based alignment for pathogen identification. To reduce false-positive detections, a conservative minimum read length of 500 base pairs (bp) was applied, with significance defined as ≥1,500 base pairs of unique alignment.
Results: Serum from 39 children with acute KD (56% male, median age 1.9 years) and 18 healthy children (61% male, median age 2.7 years) was sequenced. In total, 45 taxonomically classifiable microbial detections, encompassing both pathogenic and non-pathogenic taxa, met the predefined significance threshold. Of these, 44 were detected in 15/39 (38%) children with KD, compared with a single detection (1/18; 5%) heathy controls. Among detections in the KD cohort, 4/44 (9%) were known human pathogens: coxsackie virus, adenovirus, rhinovirus and rotavirus. Adenoviral and rhinoviral cases corresponded with positive nasopharyngeal PCR results, whereas coxsackievirus and rotavirus were not identified on routine clinical testing. Non- pathogenic microbes present at threshold level in KD samples included ripaflec virus (5/12), anelloviridae sp, human pegivirus, TTV-like minivirus, torque-teno midi virus, and low-abundance bacterial taxa. The single microbial detection in healthy controls was a non-pathogenic Streptococcus species.
Interpretation: Almost 40% of children with acute KD had detectable microbial sequences in serum by deep metagenomic sequencing, although only a minority represented known pathogens. Few microbes were detected in healthy controls from the same setting. Ongoing work will expand this cohort, explore novel pathogens, integrate matched respiratory sampling, and examine whether microbial detection is associated with clinical or immunological features relevant to KD pathogenesis and outcomes.