Detection of Adenovirus DNA in Throat Swabs and Blood by SYBR Green Real-Time PCR Assay in Patients with Adenovirus-Associated Tonsillitis

Human adenovirus causes a variety of diseases including upper respiratory tract infection, acute conjunctivitis, cystitis and gastroenteritis. Acute febrile exudative tonsillitis is caused by various agents, including group A β-hemolytic streptococci and viruses. Group A β-streptococcal tonsillitis is most common in children 6 years of age or older; by contrast, viral tonsillitis is most common in children younger than 3 years of age (1). Adenovirus is the most frequent agent among viral tonsillitis. Adenovirus-associated exudative tonsillitis induces high grade and prolonged fever, sore throat and poor appetite; hospitalization is often required for such patients. Strong inflammatory responses such as leukocytosis with neutrophilia and high C-reactive protein (CRP) values are usually observed. These laboratory findings are different from those of common viral infections, making it difficult to distinguish adenovirus respiratory infection from bacterial infection. However, the mechanisms of these findings induced by adenovirus infection have not been well elucidated. Realtime polymerase chain reaction (PCR) methods (2) are thought to be useful in the diagnosis and monitoring of Epstein-Barr virus (EBV) (3) and cytomegalovirus (CMV) infections (4). We have reported SYBR Green real-time PCR assay for the quantitative detection of adenovirus DNA (5). Measurement of adenovirus load in clinical samples from adenovirus infections may provide important information for analyzing the pathogenesis of various adenovirus infections. In this study, we evaluated the adenovirus DNA load in patients with adenovirus-associated exudative tonsillitis and analyzed the correlation between the viral load and clinical findings. Thirty patients at Nakano Children’s Hospital, Osaka, Japan, between June 2003 and October 2004 were clinically diagnosed as having acute adenovirus-associated tonsillitis based on their high fever (>38.0°C) and exudative tonsillitis, and were admitted for fluid transfusion because of poor fluid intake. These patients did not have chronic tonsillitis as an underlying illness. Etiologic diagnosis was confirmed using an immunochromatographic rapid diagnostic kit (Adenoclone; TFB, Tokyo, Japan) with throat swabs. Patients included 20 boys and 10 girls aged from 8 months to 8 years (mean age, 4 years and 11 months). Forty-one throat swabs from 30 patients and 30 whole blood samples from 23 patients were collected. Throat swabs were collected by swabbing the tonsils of all patients. Throat swabs from 10 patients and blood from 6 patients were collected in the acute phase (within 4 days of the onset of fever) and recovery phase (between 5 and 10 days after the onset of fever), respectively. Throat swabs were collected and suspended in 1 ml of normal saline, and blood was collected in EDTA-coated tubes. Plasma was collected after centrifugation at 5,000 rpm for 5 min. Informed consent was obtained from the parents or guardians of all patients. DNA was extracted from 200 μl of throat swabs, whole blood and plasma using a High Pure Viral Nucleic Acid Kit (Roche Applied Science, Mannheim, Germany), according to the manufacturer’s instructions. DNA was eluted in 50 μl of distilled water and stored at –20°C until use. Adenovirus DNA was tested by quantitative SYBR Green real-time PCR