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Journal of Clinical Microbiology, May 2007, p. 1426-1432, Vol. 45, No. 5
0095-1137/07/$08.00+0     doi:10.1128/JCM.01515-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Simultaneous Quantification of Epstein-Barr Virus, Cytomegalovirus, and Human Herpesvirus 6 DNA in Samples from Transplant Recipients by Multiplex Real-Time PCR Assay{triangledown}

Kaoru Wada,1 Naomi Kubota,1 Yoshinori Ito,2 Hiroshi Yagasaki,2 Koji Kato,3 Tetsushi Yoshikawa,4 Yasuyuki Ono,5 Hisami Ando,5 Yasuhiro Fujimoto,6 Tetsuya Kiuchi,6 Seiji Kojima,2 Yukihiro Nishiyama,1 and Hiroshi Kimura1*

Departments of Virology,1 Pediatrics,2 Pediatric Surgery,5 Transplant Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan,6 Department of Pediatrics, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan,3 Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Japan4

Received 22 July 2006/ Returned for modification 31 October 2006/ Accepted 12 March 2007

We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.

* Corresponding author. Mailing address: Department of Virology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan. Phone: 81-52-744-2207. Fax: 81-52-744-2452. E-mail: hkimura{at}med.nagoya-u.ac.jp

{triangledown} Published ahead of print on 21 March 2007.

Journal of Clinical Microbiology, May 2007, p. 1426-1432, Vol. 45, No. 5
0095-1137/07/$08.00+0     doi:10.1128/JCM.01515-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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