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Reply to “Detection of Human Herpesviruses (HHVs) DNA in blood samples: A true marker of Fever of Unknown Origin (FUO)?”
Journal of Clinical Virology 61 (2014) 619–620
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Letter to the Editor
Reply to “Detection of Human Herpesviruses (HHVs) DNA in blood samples: A true marker of Fever of Unknown Origin (FUO)?” Fever of unknown origin (FUO) in pediatrics is a challenging diagnostic dilemma [1]. Infection is by far the most commonly identified etiology of FUO in all pediatric studies [1,2]. Viral aetiologies for FUO, particularly human herpes virus (HHV) infection, are more common in developed countries [2,3]. When exploring the possible role of HHVs in classical pediatric FUO patients, it can be of value to evaluate DNA for different HHV types and compare the epidemiological and clinical profiles between patients and a matched healthy population. Therefore, we applied a previously validated multiplex PCR assay for detecting HHV prevalence [4] using blood samples from FUO patients and afebrile pediatric patients. Our data showed that the prevalences of Epstein–Barr virus and HHV6 were significantly higher in FUO patients than in the afebrile cohort [5], which concurs with the literature [1–3,6]. Broccolo and Toniolo [7] suggested that the interpretation of the clinical significance of minute HHV DNA amounts in blood requires the use of a complete panel of calibrated quantitative real-time PCR (cQPCR) assays for measuring the viral load in plasma/serum together with a QPCR assay for the quantitative detection of genomic DNA. These procedures require one to consider potential errors introduced by contamination due to cell lysis or to the presence of chromosomally integrated HHV6. Whole blood (WB) is obtained and transported easily, is stable when frozen, and does not require lengthy cell separation procedures, reducing the potential for contamination during subsequent processes [8]. In our assay system, EDTA-anticoagulated WB samples were centrifuged at 2000 × g for 20 min to remove cell-associated viruses (either latent or replicating), as well as cellular DNA. This was followed by treatment with DNase I to remove unencapsidated viral DNA. These pre-treatment procedures before DNA extraction prevent contamination due to cell lysis or to the presence of chromosomally integrated HHVs [9]. Moreover, our data showed that the frequency with which most HHV DNA, except CMV, is detected in blood is very low (<4.9%) in a normal afebrile cohort based on our multiplex PCR protocol [5]. Therefore, we suggest that the detection of HHV DNA in WB samples using our multiplex PCR protocol is a sensitive and specific procedure that can be performed with minimal sample processing, independent of the patient’s peripheral blood leukocyte count, and with low assay variability. In summary, our qualitative multiplex PCR protocol was used to compare the prevalence (not the load) of HHV DNA in FUO patients versus afebrile pediatric patients, to establish a http://dx.doi.org/10.1016/j.jcv.2014.09.011 1386-6532/© 2014 Elsevier B.V. All rights reserved.
potential association between FUO and primary or reactivated HHV infection based on statistical analysis in combination with epidemiological and clinical profiles [5]. It does not require extensive calibration technologies or quantitative assays. Indeed, the main limitation of our study was addressed [5], and measurements of HHV DNA using appropriate quantitative assays [10] with large numbers of samples from a healthy cohort might support the association between FUO and the active replication of different HHV types. Funding None. Competing interests The authors declare that there is no conflict of interest. Ethical approval Not required. References [1] Chow A, Robinson JL. Fever of unknown origin in children: a systematic review. World J Pediatr 2011;7(1):5–10. [2] Shi XC, Liu XQ, Zhou BT, Zhang LF, Ma XJ, Deng GH, et al. Major causes of fever of unknown origin at Peking Union medical college hospital in the past 26 years. Chin Med J 2013;126(5):808–12. [3] Akpede GO, Akenzua GI. Management of children with prolonged fever of unknown origin and difficulties in the management of fever of unknown origin in children in developing countries. Paediatr Drugs 2001;3(4): 247–62. [4] Tanaka T, Kogawa K, Sasa H, Nonoyama S, Furuya K, Sato K. Rapid and simultaneous detection of 6 types of human herpes virus (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus6A/B, and human herpes virus 7) by multiplex PCR assay. Biomed Res 2009;30(5):279–85. [5] Zhou W, Tan X, Li Y, Tan W. Human Herpes Viruses are associated with classic Fever of Unknown Origin (FUO) in Beijing patients. PLOS ONE 2014;9:e101619. [6] Pasic S, Minic A, Djuric P, Micic D, Kuzmanovic M, Sarjanovic L, et al. Fever of unknown origin in 185 paediatric patients: a single-centre experience. Acta Paediatr 2006;95(4):463–6. [7] Broccolo F, Toniolo A. Detection of Human Herpesviruses (HHVs) DNA in blood samples: a true marker of Fever of Unknown Origin (FUO)? J Clin Virol 2014;61:617–8. [8] Nesbitt SE1, Cook L, Jerome KR. Cytomegalovirus quantitation by real-time PCR is unaffected by delayed separation of plasma from whole blood. J Clin Microbiol 2004;42(March (3)):1296–7. [9] Sergerie Y, Boivin G. Evaluation of susceptibility of human herpesvirus 8 to antiviral drugs by quantitative real-time PCR. J Clin Microbiol 2003;41:3897–900. [10] Michelin BD, Hadzisejdic I, Bozic M, Grahovac M, Hess M, Grahovac B, et al. Detection of cytomegalovirus (CMV) DNA in EDTA whole-blood samples: evaluation of the quantitative artus CMV LightCycler PCR kit in conjunction with automated sample preparation. J Clin Microbiol 2008;46(April (4)):1241–5.
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Letter to the Editor / Journal of Clinical Virology 61 (2014) 619–620
Weimin Zhou Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China Xinyi Tan Beijing No. 2 High School, Dongcheng District, Beijing 100010, China Yamin Li Wenjie Tan ∗ Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China
∗ Corresponding author at: National Institute for Viral Disease Control and Prevention, China CDC, 155 Changbai Road, Changping District, Beijing 102206, China. Tel.: +86 10 5890 0878; fax: +86 10 5890 0878. E-mail address: [email protected] (W. Tan)
9 September 2014
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Letter to the Editor
Reply to “Detection of Human Herpesviruses (HHVs) DNA in blood samples: A true marker of Fever of Unknown Origin (FUO)?” Fever of unknown origin (FUO) in pediatrics is a challenging diagnostic dilemma [1]. Infection is by far the most commonly identified etiology of FUO in all pediatric studies [1,2]. Viral aetiologies for FUO, particularly human herpes virus (HHV) infection, are more common in developed countries [2,3]. When exploring the possible role of HHVs in classical pediatric FUO patients, it can be of value to evaluate DNA for different HHV types and compare the epidemiological and clinical profiles between patients and a matched healthy population. Therefore, we applied a previously validated multiplex PCR assay for detecting HHV prevalence [4] using blood samples from FUO patients and afebrile pediatric patients. Our data showed that the prevalences of Epstein–Barr virus and HHV6 were significantly higher in FUO patients than in the afebrile cohort [5], which concurs with the literature [1–3,6]. Broccolo and Toniolo [7] suggested that the interpretation of the clinical significance of minute HHV DNA amounts in blood requires the use of a complete panel of calibrated quantitative real-time PCR (cQPCR) assays for measuring the viral load in plasma/serum together with a QPCR assay for the quantitative detection of genomic DNA. These procedures require one to consider potential errors introduced by contamination due to cell lysis or to the presence of chromosomally integrated HHV6. Whole blood (WB) is obtained and transported easily, is stable when frozen, and does not require lengthy cell separation procedures, reducing the potential for contamination during subsequent processes [8]. In our assay system, EDTA-anticoagulated WB samples were centrifuged at 2000 × g for 20 min to remove cell-associated viruses (either latent or replicating), as well as cellular DNA. This was followed by treatment with DNase I to remove unencapsidated viral DNA. These pre-treatment procedures before DNA extraction prevent contamination due to cell lysis or to the presence of chromosomally integrated HHVs [9]. Moreover, our data showed that the frequency with which most HHV DNA, except CMV, is detected in blood is very low (<4.9%) in a normal afebrile cohort based on our multiplex PCR protocol [5]. Therefore, we suggest that the detection of HHV DNA in WB samples using our multiplex PCR protocol is a sensitive and specific procedure that can be performed with minimal sample processing, independent of the patient’s peripheral blood leukocyte count, and with low assay variability. In summary, our qualitative multiplex PCR protocol was used to compare the prevalence (not the load) of HHV DNA in FUO patients versus afebrile pediatric patients, to establish a http://dx.doi.org/10.1016/j.jcv.2014.09.011 1386-6532/© 2014 Elsevier B.V. All rights reserved.
potential association between FUO and primary or reactivated HHV infection based on statistical analysis in combination with epidemiological and clinical profiles [5]. It does not require extensive calibration technologies or quantitative assays. Indeed, the main limitation of our study was addressed [5], and measurements of HHV DNA using appropriate quantitative assays [10] with large numbers of samples from a healthy cohort might support the association between FUO and the active replication of different HHV types. Funding None. Competing interests The authors declare that there is no conflict of interest. Ethical approval Not required. References [1] Chow A, Robinson JL. Fever of unknown origin in children: a systematic review. World J Pediatr 2011;7(1):5–10. [2] Shi XC, Liu XQ, Zhou BT, Zhang LF, Ma XJ, Deng GH, et al. Major causes of fever of unknown origin at Peking Union medical college hospital in the past 26 years. Chin Med J 2013;126(5):808–12. [3] Akpede GO, Akenzua GI. Management of children with prolonged fever of unknown origin and difficulties in the management of fever of unknown origin in children in developing countries. Paediatr Drugs 2001;3(4): 247–62. [4] Tanaka T, Kogawa K, Sasa H, Nonoyama S, Furuya K, Sato K. Rapid and simultaneous detection of 6 types of human herpes virus (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus6A/B, and human herpes virus 7) by multiplex PCR assay. Biomed Res 2009;30(5):279–85. [5] Zhou W, Tan X, Li Y, Tan W. Human Herpes Viruses are associated with classic Fever of Unknown Origin (FUO) in Beijing patients. PLOS ONE 2014;9:e101619. [6] Pasic S, Minic A, Djuric P, Micic D, Kuzmanovic M, Sarjanovic L, et al. Fever of unknown origin in 185 paediatric patients: a single-centre experience. Acta Paediatr 2006;95(4):463–6. [7] Broccolo F, Toniolo A. Detection of Human Herpesviruses (HHVs) DNA in blood samples: a true marker of Fever of Unknown Origin (FUO)? J Clin Virol 2014;61:617–8. [8] Nesbitt SE1, Cook L, Jerome KR. Cytomegalovirus quantitation by real-time PCR is unaffected by delayed separation of plasma from whole blood. J Clin Microbiol 2004;42(March (3)):1296–7. [9] Sergerie Y, Boivin G. Evaluation of susceptibility of human herpesvirus 8 to antiviral drugs by quantitative real-time PCR. J Clin Microbiol 2003;41:3897–900. [10] Michelin BD, Hadzisejdic I, Bozic M, Grahovac M, Hess M, Grahovac B, et al. Detection of cytomegalovirus (CMV) DNA in EDTA whole-blood samples: evaluation of the quantitative artus CMV LightCycler PCR kit in conjunction with automated sample preparation. J Clin Microbiol 2008;46(April (4)):1241–5.
620
Letter to the Editor / Journal of Clinical Virology 61 (2014) 619–620
Weimin Zhou Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China Xinyi Tan Beijing No. 2 High School, Dongcheng District, Beijing 100010, China Yamin Li Wenjie Tan ∗ Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China
∗ Corresponding author at: National Institute for Viral Disease Control and Prevention, China CDC, 155 Changbai Road, Changping District, Beijing 102206, China. Tel.: +86 10 5890 0878; fax: +86 10 5890 0878. E-mail address: [email protected] (W. Tan)
9 September 2014