- Email: [email protected]
Febrile Seizures and Primary Human Herpesvirus 6 Infection
Febrile Seizures and Primary Human Herpesvirus 6 Infection Ioanna Laina, MD*, Vassiliki P. Syriopoulou, MD*, George L. Daikos, MD†, Eleftheria S. Roma, MD*, Foteini Papageorgiou, PhD*, Talia Kakourou, MD*, and Maria Theodoridou, MD* Primary human herpesvirus 6 infection is acquired mainly during the first two years of life and is often associated with febrile seizures. The aim of the present study was to investigate in Greece the frequency and clinical characteristics of primary human herpesvirus 6 (HHV-6) infection in hospitalized children with febrile seizures. Children aged from 6 months to 5 years without known neurologic disease were examined for primary HHV-6 infection, by real-time polymerase chain reaction in acute-phase plasma and by indirect immunofluorescent assay for antibody titers in acute and convalescent serum. Of 65 children included in the analysis, 55 experienced the first febrile episode of seizures and 10 the second. Primary HHV-6 infection was verified in 10 of 55 children with a first febrile episode (18%), whereas none of the 10 children with a second episode of seizures had primary HHV-6 infection. Eight children were infected with HHV-6 type B and two with type A. None of the 85 control subjects had primary HHV-6 infection, but 49% had immunoglobulin G antibodies against the virus. These findings suggest that primary HHV-6 infection is frequently associated with febrile seizures in children in this geographic region and should be considered, especially for a first episode of febrile seizures. Ó 2010 by Elsevier Inc. All rights reserved. Laina I, Syriopoulou VP, Daikos GL, Roma ES, Papageorgiou F, Kakourou T, Theodoridou M. Febrile seizures and primary human herpesvirus 6 infection. Pediatr Neurol 2010;42:28-31.
From the *First Department of Pediatrics, Aghia Sophia Children’s Hospital, Athens University, and †First Department of Propaedeutic Medicine, Laiko General Hospital, Athens University, Athens, Greece.
28 PEDIATRIC NEUROLOGY Vol. 42 No. 1
Introduction Human herpesvirus 6 (HHV-6), first described in 1986, is an ubiquitous human herpesvirus, acquired in approximately 70% of infants by 24 months of age [1-3]. Two variants are classified on the basis of immunologic and molecular biologic analysis: type A (HHV-6A) and type B (HHV-6B). These are very closely related and display >90% nucleotide identity, but pathogenesis and clinical manifestations differ substantially [4]. Primary HHV-6B infection usually appears as exanthema subitum, but neurologic complications such as febrile seizures and encephalitis-encephalopathy or epilepsy can also occur [3]. In contrast, HHV-6A has been isolated predominantly from immunocompromised hosts, such as human immunodeficiency virus-acquired immune deficiency syndrome patients [5]. In association with human immunodeficiency virus infection, geographic variations exist between HHV-6A and HHV-6B. HHV-6A has been identified more frequently in a region in Zambia, accounting for 44% of HHV-6 cases of nonspecific illness among infants [6]. After primary infection, the virus remains latent not only in saliva, but also in the central nervous system [7-9], and may occasionally reactivate systematically under immunodeficient status. In immunocompetent hosts, HHV-6 may reactivate during pregnancy or in coinfection with HHV-7, measles, or dengue virus. In recent years, attention has focused on febrile seizures due to HHV-6 infection. Febrile seizures are an age- dependent condition, occurring in children from 6 months to 5 years old. The current understanding is that HHV-6 plays a causative role in febrile seizures, based on the neurotropic
Communications should be addressed to: Dr. Syriopoulou; First Department of Pediatrics; Aghia Sophia Children’s Hospital; Thivon and Levadias Street; Athens 11527, Greece. E-mail: [email protected] Received March 4, 2009; accepted July 22, 2009.
Ó 2010 by Elsevier Inc. All rights reserved. doi:10.1016/j.pediatrneurol.2009.07.016 0887-8994/08/$—see front matter
properties of this virus and the similarity of the groups in which febrile seizures and HHV-6 infection occur [10]. The aim of this study was to describe the clinical characteristics of children with febrile seizures during primary HHV6 infection and to investigate the frequency of primary HHV-6 infection in children with febrile seizures in Greece.
HHV-6A from HHV-6B by binding over a single base mismatch between the two viruses [11]. Primer and probe sequences are given in Table 1. For the real-time polymerase chain reaction assay, 10 mL of extracted sample was added to 40 mL of master mix. The master mix consisted of 5 mmol/L of each primer, 3.33 mmol/L probe LC 640, 6.675 mmol/L fluorescent label probe FL, 5 mL reaction mix, 4 mmol/L MgCl2, 10.995 mL water. The Light Cycler was programmed as following: initial denaturation at 95 C for 10 minutes, followed by 45 amplification cycles; each cycle consisted of denaturation at 95 C for 10 seconds, annealing at 47 C for 15 seconds, and elongation at 72 C for 20 seconds. The temperature was raised slowly (0.2 C/second) and the fluorescence signal was set to be monitored continuously. Differentiation of HHV-6 variants A and B was accomplished by postamplification melting curve analysis [11]. Validation runs contained controls for both HHV-6 variants A and B (Advanced Biotechnologies, Columbia, MD) and yielded consistent melting peaks. The melting temperatures Tm for the two variants are close, because of the high degree of homology; for HHV-6A, Tm = 61.16 C and for HHV-6, Tm = 60.35 C). Real-time polymerase chain reaction assays were performed twice, to check the initial findings. The antibody titers to HHV-6 were determined by immune fluorescence assay using the commercially available Biognost antibody assay (Bios, Munich, Germany). All immunofluorescence results were read by the same investigator and were performed twice.
Materials and Methods
Results
Patients
A total of 130 children with febrile seizures were hospitalized during the study period, of whom 98 agreed to participate in the study. Of these, 65 returned for a follow-up visit and were included in the analysis; 36 were boys and 29 were girls, with a mean age of 21.35 months. Of the 65 patients, 55 experienced febrile seizures for the first time and 10 were hospitalized for their second episode (Table 2). Primary HHV-6 infection was verified in 10/65 patients (15%). Mean age of the HHV-6 infected patients was 17.2 months. Of note, HHV-6 DNA was not detected in three cerebrospinal fluid samples that were examined. Eight HHV-6 strains were identified as type B and two as type A, as determined by postamplification melting curve analysis. In addition, three patients had seroconversion for HHV-6 but HHV-6 DNA was not detected in serum; these patients were considered to have possible HHV-6 primary infection. None of the control subjects had primary HHV6 infection, but 49% had immunoglobulin G anti-HHV-6 antibodies. All children with primary HHV-6 infection had febrile seizures for the first time (10/55, or 18%). Of these 10 HHV-6 positive children, 6 (60%) had typical roseola, which in one case was complicated with acute otitis media, 6 presented with respiratory or gastrointestinal infection, and 1 (7%) had no other symptoms than fever and seizures. Two of 10 children with primary HHV-6 infection and 8 of 52 children without the infection had complicated seizures; the difference was not statistically significant.
Table 1. Primers and hybridization probes used in real-time polymerase chain reaction for the detection of human herpesvirus 6 (HHV-6) Oligonucleotide Sequence (50 / 30 ) LC-HHV-6 P1 GTC GTG TTT GAT TTT CAA AGT TTA TAT CC LC-HHV-6 P2 ATA AAC ACA CAA TCC GTA TCA CCA TAA ATA ACC T H6 FL AAC ATA ATC CAC CGT GGA ACA AAG CAT C-FL H6 LC LCRed 640 CTC TTC CAA GAC ACG TTA CAG AAG CAC C Abbreviation: FL = Fluorescein label
The study was conducted in a tertiary children’s hospital in Athens, Greece, from November 2002 to December 2005. Consecutive children aged 6 months to 5 years who presented at the emergency room with febrile seizures without known neurologic disease were evaluated. A standard questionnaire was given to the parents, and they were informed about the purpose of the study. Pertinent information was recorded on a predesigned form. Febrile seizures were defined as seizures associated with fever but without evidence of any definitive causative disease, such as central nervous system infection, metabolic abnormality, or intoxication. Thus, both simple and complicated seizures were included. The control group comprised 85 children hospitalized for elective surgery during the same time period and at the same hospital. Children were considered to have primary HHV-6 infection if they had both detection of virus DNA in acute-phase plasma by real-time polymerase chain reaction and seroconversion or at least a fourfold increase of anti-HHV-6 immunoglobulin G antibody titers. The children who had only seroconversion or fourfold increase of immunoglobulin G antibody titers were considered to have possible HHV-6 infection. The study was approved by the Institutional Review Board.
Laboratory Investigation In addition to routine laboratory testing (complete blood cell count, urinalysis, electrolyte determinations, and cultures), acute-phase plasma was examined for HHV-6 by qualitative real-time polymerase chain reaction and acute and convalescent (2-4 weeks later) serum for anti-HHV-6 antibody titers by indirect immunofluorescent assay. When clinically indicated, mainly in cases with complicated febrile seizures, lumbar puncture was performed and cerebrospinal fluid was examined for HHV-6 DNA. Briefly, HHV-6 DNA was extracted from blood samples using a commercially available high pure nucleic acid kit (Roche Diagnostics-Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. Real-time polymerase chain reaction in blood sera was performed with a Light Cycler System 2.0 (Roche Diagnostics) and primers specific for the herpesvirus polymerase gene, as described previously [11]. Fluorescent hybridization probes were designed to bind inside the 533-bp polymerase gene fragment generated. The hybridization probes H6FL/ H6LC were designed by TIB Molbiol (Berlin, Germany) to differentiate
Discussion In the present study, it was observed that the two main clinical characteristics of children with primary HHV-6 infection were exanthema subitum and febrile seizures. The majority of the HHV-6 infections were caused by type B
Laina et al: HHV-6 Infection and Febrile Seizures 29
Table 2. Characteristics of children with febrile seizures according to etiology Characteristic
Primary HHV-6 Infection
Other Infection*
Possible HHV-6 Infection
P Value†
Sample size Mean age, months Sex, male/female, no. Febrile seizures, no. First episode Second episode Complicated Exanthem subitum, no. Laboratory findings, no. HHV-6 DNA detected Plasma CSF Serology IgM positive IgG positive
n = 10 17.2 7/3
n = 52 24.16 28/24
n=3 12.4 1/2
0.32 —
10 0 2 6
42 10 8 0
— — 1 —
0.61 0.08 0.60 0.00
10 0/3‡
0 —
0 —
0.00 —
5 10
0 37
0 3
1.00 1.00
* Other infection: 2 children with urinary tract infections (Escherichia coli), 5 with otitis media, 1 with encephalitis, 3 with gastroenteritis (1 Campylobacter, 1 Salmonella), 3 with upper respiratory infection, and 1 with pneumococcal bacteremia. The remaining 37 had possible viral infections other than HHV-6. † Comparisons were made between patients with primary HHV-6 infection and other infection. ‡ Cerebrospinal fluid was examined in only 3 of the 10 cases of primary HHV-6 infection. Abbreviations: CSF = Cerebrospinal fluid Ig = Immunoglobulin
viruses, as expected, and only two infections were caused by HHV-6 type A. The association between HHV-6 and febrile seizures has been reported previously [1]. Although in the present study a cause-and-effect relationship between HHV-6 and febrile seizure could not be established, all children with primary HHV-6 infection experienced their first episode of febrile seizures. Moreover, in 18% of children with a first episode of febrile seizures, the seizures were attributed to primary HHV-6 infection. In contrast, the second episode of febrile seizures was not associated with primary HHV-6 infection, as has been noted by others [12]. Several studies have examined the role of primary HHV6 infection in febrile seizures. Among children with a first episode of febrile seizures, primary HHV-6 was considered the cause of seizure in 20-43% of children, a percentage similar to the present findings [2,13-15]. On the other hand, 8-20% of children with primary HHV-6 infection had febrile seizures as a main manifestation [1,16]. In contrast to those reports, in the present study all 10 of the children) with primary HHV-6 infection experienced febrile seizures. This difference between the present findings and previous reports could be attributed to differences in methodology, study design, or heterogeneity of study populations. Moreover, virus strains isolated from diverse geographic location may have different biologic properties. Notably, the majority of infections in the present study were caused by type B virus, which is more frequently associated with primary infection or febrile seizures [12,17]. Divergent opinions have been expressed on what are the best assays to distinguish active from latent HHV-6 infec-
30 PEDIATRIC NEUROLOGY Vol. 42 No. 1
tions in specific clinical circumstances. In the present study, a real-time polymerase chain reaction assay in plasma was used to detect cell free viremia, because active, replicating infection is demonstrated only if viral DNA is detected in noncellular specimens such as cerebrospinal fluid, serum, or plasma [18]. The age spectrum of the patients studied here was infancy and childhood, the period of first acquisition of HHV-6, and qualitative real-time polymerase chain reaction in plasma was deemed to provide a rapid and accurate assay for diagnosing primary HHV-6 infection in young children. Monitoring viral loads could also help distinguish active from latent infection, but this assay is performed mainly in immunocompromised hosts, increases the cost of testing, and is not routinely available. Three patients were classified as having possible HHV-6 infection, because they had seroconversion only. A long period of storage (6 months) and the instability of the virus might be the main reasons for not detecting HHV-6 DNA. Although primary HHV-6 infection is found with high frequency among children with febrile seizures, whether a causal association truly exists remains to be proven. A case-control study of 35 children with febrile seizures and 33 control subjects did not establish an association between HHV-6 and febrile seizures [14]. Nevertheless, an increasing number of studies support the opposite conclusion. Thus, in the medical literature it is considered that primary HHV-6 infection is an important factor in the pathogenesis of febrile seizures [10]. Further research is still needed to determine whether this association is causal. In conclusion, the present findings indicate that, in Greece, primary HHV-6 infection is frequently associated
with febrile seizures in young children and should be taken into consideration, especially in patients experiencing their first episode. Primary HHV-6 infection does not appear to be a risk factor for recurrent febrile seizures. The fact that 18% of children seen in the emergency department with first episode of febrile seizures experience primary HHV-6 infection supports including HHV-6 detection among routine laboratory tests. This study was cofunded by the European Social Fund and National Resource EPEAEK II (Operational Program for Education and Initial Vocational Training)-Herakleitus.
References [1] Asano Y, Yoshikawa T, Suga S, et al. Clinical features of infants with primary human herpesvirus 6 infection (exanthem subitum, roseola infantum). Pediatrics 1994;93:104-8. [2] Hall CB, Long CE, Schnabel KC, et al. Human herpesvirus-6 infection in children: a prospective study of complications and reactivation. N Engl J Med 1994;331:432-8. [3] Theodore WH, Epstein L, Gaillard WD, Shinnar S, Wainwright MS, Jacobson S. Human herpes virus 6B: a possible role in epilepsy? Epilepsia 2008;49:1828-37. [4] Clark DA. Human herpesvirus 6. Rev Med Virol 2000;10:155-73. [5] Carrigan DR, Harrington D, Knox KK. Variant A human herpesvirus six as a cofactor in the pathogenesis of AIDS. J Acquir Immune Defic Syndr Hum Retrovirol 1996;13:97-8. [6] Kasolo FC, Mpabalwani E, Gompels UA. Infection with AIDS-related herpesviruses in human immunodeficiency virus-negative infants and endemic childhood Kaposi’s sarcoma in Africa. J Gen Virol 1997;78:847-55.
[7] Chan PK, Ng HK, Hui M, Cheng AF. Prevalence and distribution of human herpesvirus 6 variants A and B in adult human brain. J Med Virol 2001;64:42-6. [8] Donati D, Akhyani N, Fogdell-Hahn A, et al. Detection of human herpesvirus-6 in mesial temporal lobe epilepsy surgical brain resections. Neurology 2003;61:1405-11. [9] Caserta MT, Hall CB, Schnabel K, et al. Neuroinvasion and persistence of human herpesvirus 6 in children. J Infect Dis 1994;170:1586-9. [10] Millichap JG, Millichap JJ. Role of viral infections in the etiology of febrile seizures. Pediatr Neurol 2006;35:165-72. [11] Safronetz D, Humar A, Tipples GA. Differentiation and quantitation of human herpesviruses 6A, 6B and 7 by real-time PCR. J Virol Methods 2003;112:99-105. [12] Jee SH, Long CE, Schnabel KC, Sehgal N, Epstein LG, Hall CB. Risk of recurrent seizures after a primary human herpesvirus 6-induced febrile seizure. Pediatr Infect Dis J 1998;17:43-8. [13] Barone SR, Kaplan MH, Krilov LR. Human herpesvirus-6 infection in children with first febrile seizures. J Pediatr 1995;127: 95-7. [14] Hukin J, Farrell K, MacWilliam LM, et al. Case-control study of primary human herpesvirus 6 infection in children with febrile seizures. Pediatrics 1998;101:E3. [15] Suga S, Suzuki K, Ihira M, et al. Clinical characteristics of febrile convulsions during primary HHV-6 infection. Arch Dis Child 2000;82: 62-6. [16] Ward KN, Gray JJ. Primary human herpesvirus-6 infection is frequently overlooked as a cause of febrile fits in young children. J Med Virol 1994;42:119-23. [17] Chua KB, Lam SK, AbuBakar S, Koh MT, Lee WS. The incidence of human herpesvirus 6 infection in children with febrile convulsion admitted to the University Hospital, Kuala Lumpur. Med J Malaysia 1997; 52:335-41. [18] Leach CT. Human herpesvirus-6 and -7 infections in children: agents of roseola and other syndromes. Curr Opin Pediatr 2000;12:269-74.
Laina et al: HHV-6 Infection and Febrile Seizures 31
From the *First Department of Pediatrics, Aghia Sophia Children’s Hospital, Athens University, and †First Department of Propaedeutic Medicine, Laiko General Hospital, Athens University, Athens, Greece.
28 PEDIATRIC NEUROLOGY Vol. 42 No. 1
Introduction Human herpesvirus 6 (HHV-6), first described in 1986, is an ubiquitous human herpesvirus, acquired in approximately 70% of infants by 24 months of age [1-3]. Two variants are classified on the basis of immunologic and molecular biologic analysis: type A (HHV-6A) and type B (HHV-6B). These are very closely related and display >90% nucleotide identity, but pathogenesis and clinical manifestations differ substantially [4]. Primary HHV-6B infection usually appears as exanthema subitum, but neurologic complications such as febrile seizures and encephalitis-encephalopathy or epilepsy can also occur [3]. In contrast, HHV-6A has been isolated predominantly from immunocompromised hosts, such as human immunodeficiency virus-acquired immune deficiency syndrome patients [5]. In association with human immunodeficiency virus infection, geographic variations exist between HHV-6A and HHV-6B. HHV-6A has been identified more frequently in a region in Zambia, accounting for 44% of HHV-6 cases of nonspecific illness among infants [6]. After primary infection, the virus remains latent not only in saliva, but also in the central nervous system [7-9], and may occasionally reactivate systematically under immunodeficient status. In immunocompetent hosts, HHV-6 may reactivate during pregnancy or in coinfection with HHV-7, measles, or dengue virus. In recent years, attention has focused on febrile seizures due to HHV-6 infection. Febrile seizures are an age- dependent condition, occurring in children from 6 months to 5 years old. The current understanding is that HHV-6 plays a causative role in febrile seizures, based on the neurotropic
Communications should be addressed to: Dr. Syriopoulou; First Department of Pediatrics; Aghia Sophia Children’s Hospital; Thivon and Levadias Street; Athens 11527, Greece. E-mail: [email protected] Received March 4, 2009; accepted July 22, 2009.
Ó 2010 by Elsevier Inc. All rights reserved. doi:10.1016/j.pediatrneurol.2009.07.016 0887-8994/08/$—see front matter
properties of this virus and the similarity of the groups in which febrile seizures and HHV-6 infection occur [10]. The aim of this study was to describe the clinical characteristics of children with febrile seizures during primary HHV6 infection and to investigate the frequency of primary HHV-6 infection in children with febrile seizures in Greece.
HHV-6A from HHV-6B by binding over a single base mismatch between the two viruses [11]. Primer and probe sequences are given in Table 1. For the real-time polymerase chain reaction assay, 10 mL of extracted sample was added to 40 mL of master mix. The master mix consisted of 5 mmol/L of each primer, 3.33 mmol/L probe LC 640, 6.675 mmol/L fluorescent label probe FL, 5 mL reaction mix, 4 mmol/L MgCl2, 10.995 mL water. The Light Cycler was programmed as following: initial denaturation at 95 C for 10 minutes, followed by 45 amplification cycles; each cycle consisted of denaturation at 95 C for 10 seconds, annealing at 47 C for 15 seconds, and elongation at 72 C for 20 seconds. The temperature was raised slowly (0.2 C/second) and the fluorescence signal was set to be monitored continuously. Differentiation of HHV-6 variants A and B was accomplished by postamplification melting curve analysis [11]. Validation runs contained controls for both HHV-6 variants A and B (Advanced Biotechnologies, Columbia, MD) and yielded consistent melting peaks. The melting temperatures Tm for the two variants are close, because of the high degree of homology; for HHV-6A, Tm = 61.16 C and for HHV-6, Tm = 60.35 C). Real-time polymerase chain reaction assays were performed twice, to check the initial findings. The antibody titers to HHV-6 were determined by immune fluorescence assay using the commercially available Biognost antibody assay (Bios, Munich, Germany). All immunofluorescence results were read by the same investigator and were performed twice.
Materials and Methods
Results
Patients
A total of 130 children with febrile seizures were hospitalized during the study period, of whom 98 agreed to participate in the study. Of these, 65 returned for a follow-up visit and were included in the analysis; 36 were boys and 29 were girls, with a mean age of 21.35 months. Of the 65 patients, 55 experienced febrile seizures for the first time and 10 were hospitalized for their second episode (Table 2). Primary HHV-6 infection was verified in 10/65 patients (15%). Mean age of the HHV-6 infected patients was 17.2 months. Of note, HHV-6 DNA was not detected in three cerebrospinal fluid samples that were examined. Eight HHV-6 strains were identified as type B and two as type A, as determined by postamplification melting curve analysis. In addition, three patients had seroconversion for HHV-6 but HHV-6 DNA was not detected in serum; these patients were considered to have possible HHV-6 primary infection. None of the control subjects had primary HHV6 infection, but 49% had immunoglobulin G anti-HHV-6 antibodies. All children with primary HHV-6 infection had febrile seizures for the first time (10/55, or 18%). Of these 10 HHV-6 positive children, 6 (60%) had typical roseola, which in one case was complicated with acute otitis media, 6 presented with respiratory or gastrointestinal infection, and 1 (7%) had no other symptoms than fever and seizures. Two of 10 children with primary HHV-6 infection and 8 of 52 children without the infection had complicated seizures; the difference was not statistically significant.
Table 1. Primers and hybridization probes used in real-time polymerase chain reaction for the detection of human herpesvirus 6 (HHV-6) Oligonucleotide Sequence (50 / 30 ) LC-HHV-6 P1 GTC GTG TTT GAT TTT CAA AGT TTA TAT CC LC-HHV-6 P2 ATA AAC ACA CAA TCC GTA TCA CCA TAA ATA ACC T H6 FL AAC ATA ATC CAC CGT GGA ACA AAG CAT C-FL H6 LC LCRed 640 CTC TTC CAA GAC ACG TTA CAG AAG CAC C Abbreviation: FL = Fluorescein label
The study was conducted in a tertiary children’s hospital in Athens, Greece, from November 2002 to December 2005. Consecutive children aged 6 months to 5 years who presented at the emergency room with febrile seizures without known neurologic disease were evaluated. A standard questionnaire was given to the parents, and they were informed about the purpose of the study. Pertinent information was recorded on a predesigned form. Febrile seizures were defined as seizures associated with fever but without evidence of any definitive causative disease, such as central nervous system infection, metabolic abnormality, or intoxication. Thus, both simple and complicated seizures were included. The control group comprised 85 children hospitalized for elective surgery during the same time period and at the same hospital. Children were considered to have primary HHV-6 infection if they had both detection of virus DNA in acute-phase plasma by real-time polymerase chain reaction and seroconversion or at least a fourfold increase of anti-HHV-6 immunoglobulin G antibody titers. The children who had only seroconversion or fourfold increase of immunoglobulin G antibody titers were considered to have possible HHV-6 infection. The study was approved by the Institutional Review Board.
Laboratory Investigation In addition to routine laboratory testing (complete blood cell count, urinalysis, electrolyte determinations, and cultures), acute-phase plasma was examined for HHV-6 by qualitative real-time polymerase chain reaction and acute and convalescent (2-4 weeks later) serum for anti-HHV-6 antibody titers by indirect immunofluorescent assay. When clinically indicated, mainly in cases with complicated febrile seizures, lumbar puncture was performed and cerebrospinal fluid was examined for HHV-6 DNA. Briefly, HHV-6 DNA was extracted from blood samples using a commercially available high pure nucleic acid kit (Roche Diagnostics-Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. Real-time polymerase chain reaction in blood sera was performed with a Light Cycler System 2.0 (Roche Diagnostics) and primers specific for the herpesvirus polymerase gene, as described previously [11]. Fluorescent hybridization probes were designed to bind inside the 533-bp polymerase gene fragment generated. The hybridization probes H6FL/ H6LC were designed by TIB Molbiol (Berlin, Germany) to differentiate
Discussion In the present study, it was observed that the two main clinical characteristics of children with primary HHV-6 infection were exanthema subitum and febrile seizures. The majority of the HHV-6 infections were caused by type B
Laina et al: HHV-6 Infection and Febrile Seizures 29
Table 2. Characteristics of children with febrile seizures according to etiology Characteristic
Primary HHV-6 Infection
Other Infection*
Possible HHV-6 Infection
P Value†
Sample size Mean age, months Sex, male/female, no. Febrile seizures, no. First episode Second episode Complicated Exanthem subitum, no. Laboratory findings, no. HHV-6 DNA detected Plasma CSF Serology IgM positive IgG positive
n = 10 17.2 7/3
n = 52 24.16 28/24
n=3 12.4 1/2
0.32 —
10 0 2 6
42 10 8 0
— — 1 —
0.61 0.08 0.60 0.00
10 0/3‡
0 —
0 —
0.00 —
5 10
0 37
0 3
1.00 1.00
* Other infection: 2 children with urinary tract infections (Escherichia coli), 5 with otitis media, 1 with encephalitis, 3 with gastroenteritis (1 Campylobacter, 1 Salmonella), 3 with upper respiratory infection, and 1 with pneumococcal bacteremia. The remaining 37 had possible viral infections other than HHV-6. † Comparisons were made between patients with primary HHV-6 infection and other infection. ‡ Cerebrospinal fluid was examined in only 3 of the 10 cases of primary HHV-6 infection. Abbreviations: CSF = Cerebrospinal fluid Ig = Immunoglobulin
viruses, as expected, and only two infections were caused by HHV-6 type A. The association between HHV-6 and febrile seizures has been reported previously [1]. Although in the present study a cause-and-effect relationship between HHV-6 and febrile seizure could not be established, all children with primary HHV-6 infection experienced their first episode of febrile seizures. Moreover, in 18% of children with a first episode of febrile seizures, the seizures were attributed to primary HHV-6 infection. In contrast, the second episode of febrile seizures was not associated with primary HHV-6 infection, as has been noted by others [12]. Several studies have examined the role of primary HHV6 infection in febrile seizures. Among children with a first episode of febrile seizures, primary HHV-6 was considered the cause of seizure in 20-43% of children, a percentage similar to the present findings [2,13-15]. On the other hand, 8-20% of children with primary HHV-6 infection had febrile seizures as a main manifestation [1,16]. In contrast to those reports, in the present study all 10 of the children) with primary HHV-6 infection experienced febrile seizures. This difference between the present findings and previous reports could be attributed to differences in methodology, study design, or heterogeneity of study populations. Moreover, virus strains isolated from diverse geographic location may have different biologic properties. Notably, the majority of infections in the present study were caused by type B virus, which is more frequently associated with primary infection or febrile seizures [12,17]. Divergent opinions have been expressed on what are the best assays to distinguish active from latent HHV-6 infec-
30 PEDIATRIC NEUROLOGY Vol. 42 No. 1
tions in specific clinical circumstances. In the present study, a real-time polymerase chain reaction assay in plasma was used to detect cell free viremia, because active, replicating infection is demonstrated only if viral DNA is detected in noncellular specimens such as cerebrospinal fluid, serum, or plasma [18]. The age spectrum of the patients studied here was infancy and childhood, the period of first acquisition of HHV-6, and qualitative real-time polymerase chain reaction in plasma was deemed to provide a rapid and accurate assay for diagnosing primary HHV-6 infection in young children. Monitoring viral loads could also help distinguish active from latent infection, but this assay is performed mainly in immunocompromised hosts, increases the cost of testing, and is not routinely available. Three patients were classified as having possible HHV-6 infection, because they had seroconversion only. A long period of storage (6 months) and the instability of the virus might be the main reasons for not detecting HHV-6 DNA. Although primary HHV-6 infection is found with high frequency among children with febrile seizures, whether a causal association truly exists remains to be proven. A case-control study of 35 children with febrile seizures and 33 control subjects did not establish an association between HHV-6 and febrile seizures [14]. Nevertheless, an increasing number of studies support the opposite conclusion. Thus, in the medical literature it is considered that primary HHV-6 infection is an important factor in the pathogenesis of febrile seizures [10]. Further research is still needed to determine whether this association is causal. In conclusion, the present findings indicate that, in Greece, primary HHV-6 infection is frequently associated
with febrile seizures in young children and should be taken into consideration, especially in patients experiencing their first episode. Primary HHV-6 infection does not appear to be a risk factor for recurrent febrile seizures. The fact that 18% of children seen in the emergency department with first episode of febrile seizures experience primary HHV-6 infection supports including HHV-6 detection among routine laboratory tests. This study was cofunded by the European Social Fund and National Resource EPEAEK II (Operational Program for Education and Initial Vocational Training)-Herakleitus.
References [1] Asano Y, Yoshikawa T, Suga S, et al. Clinical features of infants with primary human herpesvirus 6 infection (exanthem subitum, roseola infantum). Pediatrics 1994;93:104-8. [2] Hall CB, Long CE, Schnabel KC, et al. Human herpesvirus-6 infection in children: a prospective study of complications and reactivation. N Engl J Med 1994;331:432-8. [3] Theodore WH, Epstein L, Gaillard WD, Shinnar S, Wainwright MS, Jacobson S. Human herpes virus 6B: a possible role in epilepsy? Epilepsia 2008;49:1828-37. [4] Clark DA. Human herpesvirus 6. Rev Med Virol 2000;10:155-73. [5] Carrigan DR, Harrington D, Knox KK. Variant A human herpesvirus six as a cofactor in the pathogenesis of AIDS. J Acquir Immune Defic Syndr Hum Retrovirol 1996;13:97-8. [6] Kasolo FC, Mpabalwani E, Gompels UA. Infection with AIDS-related herpesviruses in human immunodeficiency virus-negative infants and endemic childhood Kaposi’s sarcoma in Africa. J Gen Virol 1997;78:847-55.
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