Absence of parvoviral genomes in endothelial cells of Kawasaki disease patients with coronary artery lesions.

To the Editors: Kawasaki disease (KD) is the most common systemic vasculitis syndrome primarily affecting small and medium-sized arteries, particularly the coronary artery. It is widely believed that KD is due to one or more common infectious agents. Since its initial description, identification of a definitive infectious agent has been elusive, including contradictory data on the role of parvovirus B19 (PVB19). Parvovirus infection of the cardiac vascular endothelium has been associated with impaired coronary microcirculation and with diastolic dysfunction and coexisting endothelial dysfunction. Human bocavirus (HBoV) is a new member of the Parvoviridae family and appears to be a common cause of acute upper and lower respiratory tract infection in children. Catalano-Pons et al recently reported the detection of HBoV DNA in nasopharyngeal, serum, or stool samples from 5 of 16 patients with KD, suggesting that this emerging virus may also play a pathogenic role in KD. One of the major limiting factors in efforts to identify etiologic agents in patients with KD is the lack of cardiac tissue samples; most studies have used peripheral samples to look for pathogens. We have previously shown that the number of circulating endothelial cells is higher in KD patients with coronary artery lesions (CAL). Therefore, we speculated that parvovirus infection of the coronary endothelium may result in the development of CAL leading to increased endothelial cell shedding into the circulation. In this investigation we studied 22 patients (10 boys and 12 girls), median age being 1.8 years (0.7–5.9 years), with acute KD. The patients were further classified as those with (n 18) or without CAL (n 4). From these 22 patients, 39 blood samples were collected up to 4 weeks after disease onset for cell preparations (mean 1.70 0.69). Endothelial cell smears were prepared and quantitated, as previously described. The peripheral blood samples from acute KD patients had elevated numbers of circulating endothelial cells at the time of diagnosis (1.21 3.13/mL). They continued to increase, even after IVIG administration (15.1 14.1/mL), and peaked at 2 weeks (44.2 29.4/mL), which is the time when CAL usually develops, before falling (4.77 2.89/mL; 4 weeks after treatment). The cells from each slide were resuspended in phosphate-buffered saline and then an aliquot was amplified by whole genome amplification using a REPLI-g kit (Qiagen, Valencia, CA). After purification, the DNA was amplified by nested PCR, as described previously for PVB19; primers and amplification conditions available on request. Verification of the presence of amplifiable DNA extracted was performed by single step PCR amplification of a fragment of a single copy gene. Although DNA was isolated for each of the samples, none of the 39 samples was positive for PVB19 or HBoV DNA. These data suggest that persistent infection of coronary endothelial cells with the parvoviruses PVB19 or HBoV does not cause endothelial cell shedding in KD patients. However, it is still not possible to definitively exclude PVB19 or HoBV as causative agents of KD. First, we have not proven that these are derived from the coronary endothelium. Second, if these viruses infected coronary endothelial cells, triggering the events that lead to cell shedding, it is possible that this infection was transient and the viral genome was already cleared. Finally, it is possible that the number of viral genomes present in the cell smears is below the detection limit of the assay. However, the combination of whole genome amplification and nested PCR increased sensitivity to less that 5 genome copies per sample.