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Isolation of a Kawasaki disease‐associated bacterial sequence from peripheral blood leukocytes
Author(s) -
Shibata MOTOHIRO,
Ezaki TAKAYUKI,
Hori MITSUHIRO,
Nagashima MASAMI,
Morishima TSUNEO
Publication year - 1999
Publication title -
pediatrics international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.49
H-Index - 63
eISSN - 1442-200X
pISSN - 1328-8067
DOI - 10.1046/j.1442-200x.1999.01115.x
Subject(s) - polymerase chain reaction , 16s ribosomal rna , sequence analysis , nested polymerase chain reaction , microbiology and biotechnology , biology , nucleic acid sequence , ribosomal rna , kawasaki disease , virology , medicine , gene , genetics , surgery , artery
Background : The clinical and epidemiologic features of Kawasaki disease (KD) suggest an infectious etiology, but the agent(s) remains unknown. We aimed 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 the bacterial 16S ribosomal RNA gene (rDNA). The amplified DNA were cloned into a plasmid vector and sequenced. Phylogenetic analysis was performed with clustal W program and the neighbour‐joining method. Results : First, the PCR reagents were examined by the PCR assay using conservative primers and we found more than 10 16S rDNA sequences contaminating the reagents. We then examined five KD patients using the PCR assay, excluding the contaminated sequences, and obtained five 16S rDNA sequences as possible KD‐associated sequences. The primers specific to each 16S rDNA sequence were synthesized and used for specific PCR assays. Only the PCR assay specific to the 16S rDNA sequence termed 16S71–33 did not show any false positives with the control DNA from non‐KD patients. The 16S71–33 sequence was positive in three of 20 patients with acute KD before γ‐globulin therapy, but it became negative after therapy. The phylogenetic analysis showed a new species of the genus Corynebacterium as the origin of the 16S71–33 sequence. Conclusions : These data show that an infectious KD agent is traced in peripheral leukocytes and that a new Corynebacterium species may be responsible for KD in some cases. The true frequency and the role of the new Corynebacterium in KD would be clarified by measuring specific antibodies to it.