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Background and Objectives: Bordetella holmesii is associated with a pertussis-like respiratory syndrome in healthy individuals and also a rare cause of septicaemia, endocarditis, pneumonia, and septic arthritis, mostly in immunocompromised patients. Culture technique and real-time PCR are 2 methods used to detect Bordetella spp. Materials and Methods: In this study, 435 nasopharyngeal specimens of patients with suspected whooping cough were checked for the presence of B. holmesii using 2 methods of culture technique and real-time PCR. Results: In this study, we detected hIS1001 and IS481 of B. holmesii in 2 infants suspected of having pertussis-like syndrome. Conclusion: Our observations demonstrate that accurate diagnosis is needed to discriminate between B. holmesii and B. pertussis infections among pertussis cases; otherwise, it could lead to misestimating pertussis rate and vaccine efficacy.