Isolation of a Kawasaki disease-associated bacterial sequence from peripheral blood leukocytes

Background: The clinical and epidemiologic features of Kawasaki disease (KD ) suggest an infectious etiology, but the agent(s) remains unknown. We aime d to isolate the causative bacterial gene from peripheral blood leukocytes of patients with acute KD. Methods: Nested polymerase chain reaction (PCR) assay was used to amplify t he bacterial 16S ribosomal RNA gene (rDNA). The amplified DNA were cloned i nto a plasmid vector and sequenced. Phylogenetic analysis was performed wit h clustal W program and the neighbour-joining method. Results: First, the PCR reagents were examined by the PCR assay using conse rvative primers and we found more than IO 16S rDNA sequences contaminating the reagents. We then examined five KD patients using the PCR assay, exclud ing the contaminated sequences, and obtained five 16S rDNA sequences as pos sible KD-associated sequences. The primers specific to each 16S rDNA sequen ce were synthesized and used for specific PCR assays. Only the PCR assay sp ecific to the 16S rDNA sequence termed 16S71-33 did not show any false posi tives with the control DNA from non-KD patients. The 16S71-33 sequence was positive in three of 20 patients with acute KD before gamma-globulin therap y, but it became negative after therapy. The phylogenetic analysis showed a new species of the genus Corynebacterium as the origin of the 16S71-33 seq uence. Conclusions: These data show that an infectious KD agent is traced in perip heral leukocytes and that a new Corynebacterium species may be responsible for KD in some cases. The true frequency and the role of the new Corynebact erium in KD would be clarified by measuring specific antibodies to it.