Our study demonstrates evidence of notable B. holmesii circulation in the India subcontinent, underscoring this pathogen’s potential role in co-infection and also as a primary etiologic agent, as demonstrated by the presence of this bacterium in unvaccinated PT IgG–negative case-patients (despite limitations in detailed clinical characterization of pertussis-like symptoms [e.g., cough duration, paroxysms] observed uniformly across cases). This evidence concerns the gene F2 and pertussis.