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 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 gamma-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.
Shibata
M
M
Department of Pediatrics, Chukyo Hospital, Nagoya, Japan. shibatam@med.nagoya-u.ac.jp
Ezaki
T
T
Hori
M
M
Nagashima
M
M
Morishima
T
T
D001483:Base Sequence; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D003352:Corynebacterium / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D007223:Infant; D007962:Leukocytes / Q000382:microbiology*; D008297:Male; D008969:Molecular Sequence Data; D009080:Mucocutaneous Lymph Node Syndrome / Q000382:microbiology*; D010802:Phylogeny
D004269:DNA, Bacterial; D004275:DNA, Ribosomal